Characterization of Inositol Transporters as a Method for Drug Delivery to the Central Nervous System
by
Daniela Fenili
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Laboratory Medicine and Pathobiology University of Toronto
© Copyright by Daniela Fenili 2010
Characterization of Inositol Transporters as a Method for Drug Delivery to the Central Nervous System
Daniela Fenili
Doctor of Philosophy
Department of Laboratory Medicine and Pathobiology University of Toronto
2010
ABSTRACT
A challenge in the treatment of central nervous system (CNS) diseases is the transport of drug candidates into the brain. Inositol stereoisomers have show promise as therapeutic agents for
CNS disorders. scyllo-Inositol was an effective prophylactic and therapeutic for Alzheimer’s disease (AD) in TgCRND8 mice, a model of AD. This suggests inositol stereoisomers have excellent CNS bioavailability. They enter the brain through inositol transporters, of which there are three: one hydrogen myo-inositol transporter (HMIT) and two sodium myo-inositol transporters (SMIT1, SMIT2). HYPOTHESIS: Given the high CNS bioavailability of inositol stereoisomers, it may be possible to use inositol transporters to shuttle other compounds into the
CNS. OBJECTIVES: 1. To confirm the CNS bioavailability of the two main inositol stereoisomers, myo- and scyllo-inositol, in both TgCRND8 and wild-type mice. 2. To examine inositol transporter expression in the brains, as a function of time and disease pathology, in both groups. 3. To evaluate the flexibility of the inositol transporters for transporting compounds by determining the substrate structural features required for active transport. RESULTS: myo-Inositol and scyllo-inositol accumulated in the brain following oral administration.
Disease pathology did not alter baseline inositol levels or uptake. Brain subregional transporter expression was unaltered as a function of age or disease pathology. In vitro cell culture
ii experiments found HMIT inactive and therefore not a contender for drug transport. In contrast
SMIT1 and SMIT2 were both active and competitive transport assays, revealed distinct criteria for active transport through each system. However, both were stringent in the substitutions to the structure of myo-inositol possible to maintain active transport. CONCLUSION: Active transport through the inositol transporters is very sensitive to changes in the structure of myo- inositol and only conservative changes are possible. Therefore, these transporters would not make effective shuttling systems for drug transport into the brain.
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ACKNOWLEDGEMENTS
I would like to thank JoAnne for her help and guidance throughout my PhD, both towards designing and interpreting my experiments and during the preparation of my thesis.
I would like to thank my PhD committees, both past and present for their helpful suggestions and questions.
I’d like to acknowledge my fellow lab members, both past and present for their help with interpreting and understanding my experiments, as well as for creating a great working environment full of fun, laughter and inappropriate music (you know who you are).
Finally, I would like to thank my family and friends for their support and understanding whenever my experiment and thesis took away from our quality time.
Without all of your support, this thesis would not have been possible.
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TABLE OF CONTENTS
ABSTRACT...... ii LIST OF TABLES ...... vii LIST OF FIGURES ...... vii LIST OF ABBREVIATIONS...... ix CHAPTER 1 ...... 1 Introduction 1.1 Brain Barriers...... 2 1.1.1 The blood-brain barrier ...... 2 1.1.2 The Blood-CSF Barrier...... 5 1.2 Strategies for transport of drugs across brain barriers ...... 6 1.2.1 Barrier Circumvention ...... 7 1.2.2 Barrier Navigation...... 9 1.3 Inositol Transporters as a Therapeutic Strategy...... 13 1.3.1 Inositol in Health and Disease...... 13 1.3.2 scyllo-Inositol as a Therapeutic for Alzheimer’s Disease...... 16 1.3.3 The Inositol Stereoisomers...... 23 1.3.4 Inositol in Nature...... 25 1.3.5 Inositol Synthesis and Degradation Pathways ...... 25 1.3.6 The Inositol Transporters ...... 28 1.3.7 Inositol Efflux ...... 34 1.3.8 Inositol Pools...... 35 CHAPTER 2 ...... 36 Rationale, Hypothesis and Objectives 2.1 Rationale ...... 37 2.2 Hypothesis...... 37 2.3 Objectives...... 38 CHAPTER 3 ...... 39 Materials and Methods...... 39 CHAPTER 4 ...... 53 myo- and scyllo-Inositol Levels and Equilibrium in the Brain Abstract ...... 54 Introduction...... 55 Results...... 58 Discussion ...... 77 CHAPTER 5 ...... 81 Quantification of Inositol Transporter Expression Levels Abstract ...... 82 Introduction...... 83 Results...... 86 Discussion ...... 100
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CHAPTER 6 ...... 104 Substrate Structural Requirements for Inositol Transport Abstract ...... 105 Introduction...... 106 Results...... 110 Discussion ...... 141 CHAPTER 7 ...... 145 Discussion, Conclusions and Future Directions Discussion ...... 146 Conclusions...... 162 Future Directions...... 163 REFERENCES...... 168
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LIST OF TABLES
Table 3.1 Mouse QPCR primers...... 45 Table 3.2 Human QPCR primers...... 45 Table 6.1 Comparison of the protein homology of the three inositol transporters to each other in humans, mice and rats...... 117 Table 6.2 Analysis of the SMIT1 transport model based on the structure of the scyllo-inositol derivatizes that were transported or not...... 139
LIST OF FIGURES
Figure 1.1 The inositol stereoisomers...... 24 Figure 1.2 Endogenous sources of scyllo-inositol...... 27 Figure 4.1 Derivatization...... 59 Figure 4.2 Gas chromatography method development...... 61 Figure 4.3 Selected ion recording...... 63 Figure 4.4 Selected ion recording for plasma samples...... 64 Figure 4.5 Examples of myo-inositol and scyllo-inositol concentration curves...... 66 Figure 4.6 Baseline brain myo- and scyllo-inositol levels...... 67 Figure 4.7 The effects of myo-inositol treatment on brain myo- and scyllo-inositol...... 69 Figure 4.8 Brain and CSF myo- and scyllo-inositol levels following scyllo-inositol treatment.71 Figure 4.9 Plasma myo- and scyllo-inositol concentrations following scyllo-inositol treatment...... 74 Figure 4.10 scyllo-Inositol incorporation into phosphatidylinositol...... 76 Figure 5.1 Concentration and dissociation curves for the inositol transporters primers...... 87 Figure 5.2 Microarray, concentration and dissociation curves for each of the control genes....89 Figure 5.3 HMIT expression as a function of age in TgCRND8 mice and their wild-type littermates...... 91 Figure 5.4 SMIT1 expression as a function of age in TgCRND8 mice and their wild-type littermates...... 92
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Figure 5.5 SMIT2 expression as a function of age in TgCRND8 mice and their wild-type littermates...... 93 Figure 5.6 Relative mRNA expression of the three inositol transporters in the brain...... 95 Figure 5.7 A comparison of regional expression for each of the inositol transporters...... 97 Figure 5.8 Kidney inositol transporter expression...... 99 Figure 6.1 Structures of the inositol stereoisomers, derivatives and related compounds...... 109 Figure 6.2 Interspecies protein alignment for HMIT...... 111 Figure 6.3 Interspecies protein alignment for SMIT1...... 113 Figure 6.4 Interspecies protein alignment for SMIT2...... 115 Figure 6.5 HMIT transport activation...... 119 Figure 6.6 Measurement of HMIT myo-inositol transport in primary cells...... 121 Figure 6.7 QPCR quantification of inositol transporter expression levels in HEK293 cells...122 Figure 6.8 myo-Inositol-(2-3H) and scyllo-inositol-(2-3H) transport in HEK 293 cells...... 123 Figure 6.9 Basic structural model for SMIT1 transport...... 125 Figure 6.10 Basic structural model for SMIT2 transport...... 126 Figure 6.11 Substrates not transported by SMIT1...... 128 Figure 6.12 Substrates not transported by SMIT2...... 129 Figure 6.13 myo-Inositol and scyllo-inositol transport kinetics in HEK293 cells...... 131 Figure 6.14 A comparison of D- and L-chiro-inositol transport in HEK293 cells...... 134 Figure 6.15 L-fucose-(5,6-3H) transport...... 136 Figure 6.16 Transport of scyllo-inositol derivatives via SMIT1...... 138
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LIST OF ABBREVIATIONS
AD Alzheimer’s disease
BBB blood-brain barrier
CSF cerebral spinal fluid
CNS central nervous system
Gapdh glyceraldehyde-3-phosphate dehydrogenase
GC gas chromatography
GC/MS gas chromatography/mass spectrometry
HEK293 human endothelial kidney cell-line
HMIT hydrogen myo-inositol transporter
MRS magnetic resonance spectroscopy
PBS phosphate buffered saline
PCR polymerase chain reaction
QPCR quantitative polymerase chain reaction
SMIT1 sodium myo-inositol transporter 1
SMIT2 sodium myo-inositol transporter 2
Tbp TATA box binding protein
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CHAPTER 1
Introduction
Inositol Transporters and Drug Development
Portions of this section have been previously published in: Fenili D, Ma K, McLaurin J (2010) scyllo-Inositol, a Potential Therapeutic for Alzheimer’s Disease. Emerging Drugs & Targets for AD. Royal Society of Chemistry. Cambridge, UK
1 2 Characterization of inositol transporters as a method for drug delivery to the CNS
For diseases of the central nervous system (CNS), the primary challenge for drug design is frequently not creating drugs to target and treat the disease, but the successful transportation of drug candidates into the brain. While certain drugs can be administered via intracerebral implantation of pumps or through the direct injection of drugs into the brain or the cerebral spinal fluid (CSF), these methods of administration are very invasive and concerns arise over how often the drugs need to be administered, how well they perfuse to areas of disease pathology, whether they reach therapeutically relevant concentrations in those areas and whether the method of administration will cause significant inflammation and/or cell death. Whenever possible, the preferred method of drug delivery to the CNS is oral, especially if treatment of the disease is projected to be long-term. One reason why drug delivery to the CNS is so challenging is because of the presence of the blood-brain and blood-CSF barriers.
1.1 Brain Barriers
1.1.1 The blood-brain barrier
The blood-brain barrier (BBB) is a physiological barrier made up of endothelial cells and support cells that divide the brain from the rest of the body, in terms of drug transport. The presence of the BBB was first observed in the work of the 19th century bacteriologist Paul
Ehrlich, who noted that an intravenous injection of the dye coerulean-S into rats resulted in the staining of all the rat’s tissues, except the brain and spinal cord (Ehrlich, 1885). Thirty years later his student, Edwin Goldmann, performed the reverse experiment and found staining only in the spinal cord and brain and correctly concluded that there must be a barrier dividing the two regions (Goldmann, 1913). This and other experiments resulted in the term blood-brain barrier first being coined by Lina Stern at a meeting of the Medical Society of Geneva on April 21st,
1921 (reviewed in Vein, 2008). From her additional work using dyes, Stern concluded that the
3 BBB had three main purposes: 1. to protect the brain from any harmful substances present in the blood stream, 2. to allow the transport of substances needed for its function and 3. to maintain brain homeostasis (Stern, 1921). She proposed that the BBB was the rate-limiting factor determining the permeation of therapeutic drugs into the brain (Stern, 1921).
In the 1960s, with the advent of the electron microscope and the use of horseradish peroxidase to enzymatically amplify visual markers, it was determined that the key component of the BBB is endothelial cells, joined together in a monolayer by tight junctions (Reese and Karnovsky,
1967). Surrounding these endothelial cells are astrocytic endfeet, which induce the endothelial cells to form those tight junctions (Janzer and Raff, 1987). While the endothelial cells form tight junctions, astrocytic endfeet form gap junctions (Brightman and Reese, 1969). Therefore while astrocytic endfeet do surround the vasculature, they do not impose a physical barrier to drug transport (Brightman and Reese, 1969). There is a ~20 nm gap between the astrocytic endfeet that allows any compounds transported across endothelial cells to reach the brain and vice versa (Brightman and Reese, 1969). Pericytes also interact with and stabilize endothelial cells, both by surrounding the endothelial cells with processes to contribute to its mechanical stability and by forming specialized junctions with the endothelial cells, through which they influence their differentiation or quiescence (reviewed in von Tell et al, 2006). In addition, pericytes also appear to influence tight junction formation (Hori et al, 2004). Pericytes express angiopoetin, which induces endothelial cells to express occludin, a major component of tight junctions (Hori et al, 2004). The extracellular matrix of the basal lamina, alternatively known as the basement membrane, also acts to stabilize vascular structure, via the interaction of matrix proteins such as laminin with integrin receptors on endothelial cells (reviewed in Hynes, 1992).
In addition, the expression of matrix proteins such as type IV collagen, fibronectin and laminin, in the basal lamina also appears to influence tight junction expression (Tilling et al, 1998).
4 Completing this neurovascular unit, composed of endothelial cells, astrocytic endfeet, pericytes and the basal lamina are neurons that, via neuronal processes that innervate vascular endothelial cells and astrocytes, regulate cerebral blood flow and BBB permeability (reviewed in Hawkins and Davis, 2005).
Since the endothelial cells are responsible for providing a physical barrier to drug transport, they are of particular interest in drug design. They are polarized cells that appear to express certain transporters on either the luminal (blood) or abluminal (brain) side of their plasma membrane.
This was first noted by Betz and Goldstein (1978), who examined the transport of 14C-labelled
α–(methylamino)isobutyric acid and L-leucine in cerebral cortex capillaries isolated from adult rats. Using these two amino acids, which are transported by two different neutral amino acid transporter systems, they observed that the sodium-independent L system transporter was present on the luminal side, while the sodium-dependent A system transporter was present on the abluminal side of brain capillary endothelial cells (Betz and Goldstein, 1978). Therefore, large neutral amino acids, such as phenylalanine, leucine, tryptophan and methionine can be transported into the brain via the L system transporter, while small neutral amino acids, such as glycine, alanine, serine, proline and α–(methylamino)isobutyric acid can be transported out of the brain by the A system transporter. Glucose transporter 1, the only glucose transporter expressed at the BBB, is polarized in a 4:1 expression ratio between the abluminal and luminal membranes of endothelial cells (Farrell and Pardridge, 1991). Drug efflux transporters, such as
P-glycoprotein (Thiebaut et al, 1989), are also polar; they are present on the luminal side of endothelial cells and are responsible for preventing the transport of toxic substances from the blood into the brain. This transport across concentration gradients requires high levels of
5 energy, as evidenced by the fact that endothelial cells at the BBB contain five to six times as many mitochondria compared to endothelial cells elsewhere (Oldendorf et al, 1977). In order for a drug to be transported into the brain from the blood, it must navigate this highly selective brain barrier.
1.1.2 The Blood-CSF Barrier
In addition to the BBB, a second brain barrier exists, the blood-CSF barrier, through which drugs in the blood can be transported into the CSF. Lina Stern, along with a colleague of hers,
Constantin von Monakow, both concluded that the brain was supplied with nutrients by both the blood and the CSF and that the choroid plexus was responsible for functioning as a barrier, separating the blood from the CSF (Stern, 1921; Monakow, 1921). There are four choroid plexuses in humans, located in each of the ventricles: one in each of the lateral ventricles, one in the third and one in the fourth ventricle. The choroid plexus is composed of a network of capillaries, surrounded by interstitial fluid, separating those capillaries from a layer of epithelial cells, which act as a barrier to regulate entry into the CSF (reviewed in Johanson et al, 2005). In contrast to the structure of the BBB, the choroidal capillaries contain gap junctions (Dermietzel and Schunke, 1975), which allow the unimpeded movement of nutrients from the blood into the neighbouring interstitial fluid space (Johanson et al, 2005). Like the endothelial cells of the
BBB, the choroid plexus epithelial cells are also connected together by tight junctions, thus regulating the transport of compounds from the blood into the CSF (Brightman and Reese,
1969). Also like the BBB endothelial cells, the choroid plexus epithelial cells are polarized with different transporter expression between their apical and basolateral membranes. For example, the sodium myo-inositol transporter, SMIT1, is located only on the basolateral side of these cells
(Hakvoort et al, 1998).
6 Originally, the choroid plexus was thought to be less important for drug transport than the BBB because of the apparent size differential of the two structures (Pardridge et al, 1981). Now it has become increasingly apparent that these two barriers actually offer similar opportunities for drug transport, because of a number of anatomical features found in the choroid plexus. Research has shown that vascular perfusion into the choroid plexus is five to ten times higher than cerebral blood flow because of an increased number of capillaries in this region, thus resulting in an enhanced opportunity for nutrients to enter the brain via this barrier system (Johanson et al,
2005). In addition, unlike brain microvessels, which contain tight junctions to prevent nutrient transport, the choroidal capillary endothelium contains gap junctions (Dermietzel and Schunke,
1975), which allows for the ready movement of compounds into the interstitial fluid space
(Johanson et al, 2005). Furthermore, the epithelial cells that form the physical barrier between the blood and the CSF contain interdigitations on their basolateral membrane and a carpet of microvilli on their apical surface (Keep and Jones, 1990). Therefore, there is an increased opportunity for compounds to cross into the epithelial cells and an increased surface area on the apical membrane through which they can exit into the CSF (Keep and Jones, 1990). In the adult rat, the apical membrane surface area is 75 cm2, compared to 155 cm2 for cerebral capillaries
(Keep and Jones, 1990). The basolateral membrane surface area of the choroidal epithelial cells is 25 cm2, indicating the degree of extension offered by the microvilli, in comparison to the interdigitations (Keep and Jones, 1990). All these structural features make both the blood-brain and blood-CSF barriers, potentially important barriers for drug transport design.
1.2 Strategies for transport of drugs across brain barriers
In animal research, one common way to avoid the brain barriers when delivering drug candidates to the brain is through intracerebroventricular injection. This is an appealing strategy because the ependymal cells that line the ventricles contain gap junctions, which are permeable
7 to even macromolecules (discussed in Johanson et al, 2005). However, while this is an effective short-term solution, this is not an ideal method for drug delivery in humans. Therefore researchers have been searching for alternative solutions for the transport of drugs into the brain.
Some methods that have been/are being examined target circumvention of the barrier through the use of techniques, such as intranasal delivery, osmotic and biochemical disruption as well as barrier navigation strategies, such as designing drugs that will diffuse across the barriers or by aiding transport through the use of nanoparticle technology and Trojan horses.
1.2.1 Barrier Circumvention
1.2.1.1 Intranasal delivery Intranasal delivery of drugs has been suggested as one method of circumventing the brain barriers (reviewed in Hanson and Frey, 2008). First developed by Frey in 1989 for the administration of neurotrophic factors, such as nerve growth factor (27.5 kDa), to the CNS, it is now being tested in other drug delivery applications (Hanson and Frey, 2008). Using this technique, drugs are aerosolized in the nasal cavity and reach the CNS by flowing along the olfactory and trigeminal neural pathways to reach the brain within minutes. By bypassing the periphery and the brain barriers, a higher concentration of the drug is able to reach the brain and the risk of unrelated, systemic effects is reduced (Hanson and Frey, 2008). For antiepileptic medications, used to stop seizures, intranasal delivery of drugs offers an attractive alternative to intravenous drug delivery, allowing for faster, more convenient administration outside of a hospital setting, thereby reducing the duration of the seizures and the risk of brain damage
(Wermeling, 2009).
8 1.2.1.2 Osmotic disruption The ability of substances, such as urea, to alter osmolality and disrupt BBB integrity was first noted by Rapoport (1970) in cats injected with sodium bicarbonate with or without one of four solutions: sodium azide, sodium chloride, urea and ethanol. The rate of sodium bicarbonate transport correlated with each substances ability to alter osmolality. He hypothesized that these changes in osmolality might cause vascular endothelial cells to shrink and result in the formation of gaps between the cells, allowing for the passage of the sodium bicarbonate. The reversibility of this procedure was confirmed in a later experiment in rabbits, injected with an
Evans blue-albumin dye complex, and non-lipid soluble substances were concluded to be the most effective at reversibly opening the barrier (Rapoport et al, 1971). Next, the ability of animals to survive a temporary osmotic disruption in their BBB was studied in monkeys administered 2 M of urea through their left common carotid artery, followed by an intravenous injection of Evans blue dye (Rapoport et al, 1972). Monkeys were able to survive the procedure
(Rapoport et al, 1972) and following adjustments in their urea administration procedure to avoid compromising blood supply to the brain, no gross neurological deficits were observed (Rapoport and Thompson, 1973). Cresyl violet staining of the monkey brains, sacrificed 5 to 10 days following the procedure, showed no evidence of brain necrosis (Rapoport and Thompson,
1973). Electroencephalogram recordings of the brain, showed a temporary decrease in amplitude one day following the procedure, which disappeared by the time the animals were sacrificed (Rapoport and Thompson, 1973). Therefore, following additional adjustments, this procedure could be used to safely administer drugs to the CNS.
9 1.2.1.3 Biochemical disruption Another method for opening brain barriers is through biochemical disruption. One strategy is to use the kallikrein-kinin system, activated in ischemic stroke, to cause BBB disruption and brain edema, to reversibly open brain barriers (Wahl et al, 1983). For example, a low dose of the bradykinin analog, RMP-7 (0.1 µgram/kg/min) injected into the carotid artery in rats, selectively increased dextran (40 kD) transport 10-fold into RG2 glial tumors, when compared to the vehicle control group (Inamura et al, 1994). In contrast, transport into normal brain capillaries was unaltered by this low dose, which is important to limit the exposure of normal brain tissue to antitumor drugs (Inamura et al, 1994). In rats, RMP-7 pretreatment increased the uptake of the antitumor drug, carboplatin (371.3 daltons) into brain tumors (Elliot et al, 1996).
Barrier circumvention strategies offer viable options for drug entry into the brain. However, strategies such as osmotic and biochemical disruption open the brain barriers indiscriminately, not only to entry of the desired drugs but also to potential toxins or bacteria and viruses, therefore, more selective methods of transferring drugs into the brain might be more advantageous.
1.2.2 Barrier Navigation
Barrier navigation strategies aim to transport drugs through the brain barriers, rather than by disrupting those barriers. There is an estimated 600 meters of capillaries in the brain, which are
6-10 µm in diameter, creating a large surface area through which transport can occur (Begley and Brightman, 2003). If the brain barriers are disrupted, this creates a large surface area through which transport of undesired compounds can occur. By using barrier navigation strategies, such as diffusion, nanoparticle or Trojan horse delivery methods, this risk can be removed.
10 1.2.2.1 Diffusion Certain properties will influence whether a compound can diffuse across the BBB, including the oil/water partition ratio (Mayer et al, 1959), dissociation constant (Rall et al, 1959) and weight of the compound (discussed in Banks, 2009). When Mayer and colleagues (1959) examined the ability of different drugs to diffuse into the rabbit brain after injection through the femoral vein, they found diffusion into the brain was influenced by the oil/water distribution ratio of the compound at pH 7.4. Compounds such as thiopental and aniline showed the fastest diffusion rates, reaching equilibrium between CSF and plasma within 5 minutes, while N-acetyl-4- aminoantipyrine and salicylic acid were the slowest, failing to reach equilibrium within the 3 hour experiment window, but theoretically calculated to reach equilibrium within 16 and 32 hours, respectively (Mayer et al, 1959). The dissociation constant of a compound (pKa), described as the pH at which 50% of the compound is ionized, also influences whether a compound will cross the BBB. Compounds are more likely to cross into the brain if they are predominantly in unionized forms at a physiological pH (Rall et al, 1959). However, when
Brodie and colleagues (1960) compared the diffusion of 17 compounds, differing in their lipid solubility and their degree of ionization at physiological pH, they found that the oil/water partition coefficient of a compound, rather than the dissociation constant, was a stronger determinant of whether a compound would diffuse across the BBB. The dissociation constant was important for determining what level of the compound would be in an unionized form in the plasma, but lipid solubility ultimately determined whether the compound would cross into the brain in any form (Brodie et al, 1960). Perhaps the most important factor determining whether a compound will cross into the brain is weight. Generally, compounds must be less then 400-600
Daltons to be transported into the brain, however this is not a set rule (Banks, 2009). The largest compound reported to cross the BBB via transmembrane diffusion was cytokine-induced
11 neutrophil chemoattractant-1 (CINC-1), weighing 7,800 Daltons (Pan and Kastin, 2001; Banks,
2009). Designing drugs to conform to these three variables to transmembrane diffusion is one way to induce drug transport into the brain. However, this is not always a feasible, therefore other methods need to be considered.
1.2.2.2 Nanoparticle Technology Nanoparticle delivery is another mechanism under development for drug transport into the brain. Nanoparticles are macromolecule assemblies that range in size from 1 to 1000 nm.
Drugs are made to interact with these assemblies in one of three ways: they can either be 1. entrapped inside the particles, 2. associated with the exterior surface of the particles or 3. homogeneously dispersed throughout (reviewed in Lockman et al, 2002). There are several potential advantages of using nanoparticle technology. Through the use of surface ligands it is theoretically possible to directly deliver drugs to a desired tissue, removing the concern of systemic drug effects and decreasing the efficacy dose by minimizing non-specific binding
(reviewed in Provenzale and Silva, 2009). In addition, the temporal release of nanoparticles can theoretically be controlled through the use of light- or heat-labile liposomes (reviewed in
Gazeau et al, 2008; Huang et al, 2008). These two strategies are very appealing for diseases such as brain tumors, where antitumor drugs are very toxic and directed delivery of the drug and timing its release has the potential to greatly reduce side effects. The nanoparticle, polybutylcyanoacrylate, coated with polysorbate-80 to prolong its circulation by inhibiting scavenging by cells of the reticuloendothelial system, is an example of a nanoparticle that has shown promise as a drug delivery system to the brain. An intraperitoneal injection of nerve growth factor, normally unable to cross into the brain, absorbed onto the surface of polysorbate-
12 80 coated polybutylcyanoacrylate nanopoarticles was able to reduce parkinsonian symptoms in mice, in which a parkinsonian syndrome was chemically induced through an injection of 1- methyl-4-phenyl-1,2,3,6-tetrahydrophyrindine (Kurakhmaeva et al, 2008).
1.2.2.3 Trojan Horse A third option for barrier navigation is to conjugate the drug onto another compound that has access to the brain. Boado and colleagues (2009) describe molecular Trojan horses as endogenous peptides or peptidomimetic monoclonal antibodies that target endogenous brain barrier receptor-mediated transport systems. This technique was first developed in vivo by
Trowbridge and Domingo (1981) for the direct delivery of antitumor drugs to human melanoma cells in nude mice, in an effort to reduce undesired systemic effects. In this study, diphtheria fragment A from diphtheria toxin was conjugated to monoclonal antibodies against the transferrin receptor. Tranferrin receptors are preferentially expressed by actively proliferating tissues, such as tumors, which require higher amounts of iron (Gatter et al, 1983), thus allowing for directed drug delivery. Transferrin monoclonal antibodies were recently used to transfer therapeutic single chain Fv antibodies, normally unable to cross the BBB, into the mouse brain in a proof-of-principle experiment (Boado et al, 2009). By adapting a transport system found naturally at the brain barriers, into a drug shuttling system, the authors made it possible to convert a large molecule drug, normally unable to cross into the brain, into a viable CNS drug.
Therefore, barrier navigation strategies, such as diffusion, nanoparticle and Trojan horse drug delivery are all potentially effective methods for delivering compounds to the brain. In contrast to barrier circumvention strategies, these techniques try to work with the natural properties of the BBB to deliver drugs from the blood into the brain.
13 1.3 Inositol Transporters as a Therapeutic Strategy
Receptors, such as the transferrin receptor, appear to be an effective shuttling system for the delivery of molecules to the CNS (Boado et al, 2009). This is an example of a receptor- mediated transport system. Another potential option for drug transport is through a carrier- mediated transport system. An example of carrier-mediate transport is the inositol transporters
(Hager et al, 1995; Uldry et al, 2001; Coady et al, 2002; Bourgeois et al, 2005). Inositol stereoisomers, such as myo- and scyllo-inositol are transported from the blood into the brain using these transporters (Spector, 1988; Wiese et al, 1996; Hakvoort et al, 1998; Berry et al,
2003). The inositol stereoisomers have shown promise as therapeutic drugs in a number of CNS diseases, indicating the activity of this transport system (Benjamin et al, 1995; Levine et al,
1995; Fux et al, 1996; Einat et al, 1998; Bersudsky et al, 1999; Chengappa et al, 2000; Gelber et al, 2001; Palatnik et al, 2001; Eden et al, 2006; McLaurin et al, 2006). It might be possible to conjugate other compounds to the inositols to facilitate their transport into the brain or to alter the inositol stereoisomers into novel drug therapeutics.
1.3.1 Inositol in Health and Disease
Over the past 20+ years there has been interest in the ability of inositol stereoisomers to act as therapeutic drugs in the treatment of psychiatric disorders, such as depression (Levine et al,
1995), bipolar/affective disorder (Chengappa et al, 2000; Eden et al, 2006), obsessive- compulsive disorder (Fux et al, 1996), eating disorders (Gelber et al, 2001), panic disorder
(Benjamin et al, 1995; Palatnik et al, 2001) and anxiety (Einat et al, 1998; Bersudsky et al,
1999), as well as for the treatment of respiratory distress syndrome in premature infants
(Hallman et al, 1986; 1992), for preventing neural tube defects (Cogram et al, 2002) and to increase insulin sensitivity in polycystic ovary syndrome (Nestler et al, 1999; Gerli et al, 2003).
In 1978, Barkai and colleagues first noted a reduction in the levels of myo-inositol in the CSF of
14 people with affective disorder. This led to research into the levels of myo-inositol in psychiatric and other disorders and to the examination of inositol stereoisomers as potential therapeutic agents. A double-blind study, comparing 12 g/day of myo-inositol to a placebo, showed a significant improvement in Hamilton Depression Rating Scale scores in myo-inositol-treated patients, compared to placebo-treated patients, by the end of the four week study (Levine et al,
1995). When patients with bipolar depression were administered either 12 g/day of myo-inositol or a placebo for six weeks, 50% of the inositol-treated group showed a significant decrease in depressive symptoms, as measured using the Hamilton Depression Rating Scale and a Clinical
Global Improvement scale, compared to 30% of placebo controls (Chengappa et al, 2000).
Using the Montgomery-Asberg Depression Rating Scale as an outcome measure, 67% of inositol-treated patients showed a significant decrease in depressive symptoms, compared to
33% of placebo controls (Chengappa et al, 2000). In patients with obsessive-compulsive disorder, six weeks of treatment with 18 g/day of myo-inositol resulted in a significant decrease in obsessive-compulsive behaviour compared to the placebo treatment group in a double-blind, controlled crossover study (Fux et al, 1996). Similarly, in patients with bulimia nervosa and binge eating, six weeks of 18 g/day myo-inositol treatment resulted in significant improvements on the Global Clinical Impression Scale, the Visual Analogue Scale and the Eating Disorders
Inventory, when compared to a placebo treatment group in a double-blind crossover trial
(Gelber et al, 2001). For panic disorder, 12 g/ day myo-inositol treatment was significantly more effective than placebo at reducing the frequency and severity of panic attacks following four weeks of treatment (Benjamin et al, 1995). In a double-blind, controlled, crossover study comparing one month of 18 g/day myo-inositol to 150 mg/day fluvoxamine treatment, which is an accepted drug for the treatment of panic disorder, myo-inositol was significantly more effective at reducing the frequency of panic attacks, and patients reported less nausea or tiredness, when compared to the fluvoxamine-treated group (Palatnik et al, 2001).
15 In addition to acting on psychiatric disorders, myo-inositol supplementation has been proven to significantly decrease the rates of bronchopulmonary dysplasia and retinopathy of prematurity, in premature infants suffering from respiratory distress syndrome (Hallman et al, 1986; 1992).
Based on this research it has been determined that infants not receiving breast milk, who receive parenteral nutrition instead, which is inositol-free, require myo-inositol supplementation to significantly increase their chances of survival (Hallman et al, 1992).
A second inositol stereoisomer, epi-inositol, has shown promise in the treatment of anxiety disorder (Einat et al, 1998; Bersudsky et al, 1999). When anxiety in rats was compared following eleven daily intraperitoneal injectons of epi-inositol, myo-inositol or placebo on an elevated plus-maze model of anxiety, both epi- and myo-inositol were significantly more effective than placebo at reducing anxiety in the rats (Einat et al, 1998; Bersudsky et al, 1999). epi-Inositol treatment resulted in a stronger reduction in anxiety levels than myo-inositol, a finding that the authors suggested might result because epi-inositol metabolism may be slower, have a different mechanism of action, and because it is not a substrate of phosphatidylinositol synthase (Einat et al, 1998).
D-chiro-inositol has also been examined for disease treatment and it has shown promise at preventing neural tube defects in folate-resistant mice (Cogram et al, 2002) and as an insulin- sensitizing agent in women with polycystic ovary syndrome (Nestler et al, 1999; Gerli et al,
2003). Thirty percent of women who give birth to babies with neural tube defects are insensitive to folic acid treatment in early pregnancy as a preventative measure. myo-Inositol has shown some promise at reversing neural tube defects in the curly tail mouse model of folate- resistant neural tube defects (Greene and Copp, 1997). However, D-chiro-inositol appears to be more effective and in this same mouse model, D-chiro-inositol treatment resulted in a 73-86%
16 reduction in spina bifida rates, compared to a 53-56% reduction observed following myo- inositol treatment (Cogram et al, 2002). D-chiro-inositol also appears to improve insulin sensitivity, which is a concern in people with diabetes and those with polycystic ovary syndrome (Nestler et al, 1999). A 1200 mg per day dose of D-chiro-inositol for six to eight weeks, resulted in a significant improvement in insulin sensitivity, ovulatory function, blood pressure, androgen levels, and plasma triglycerides compared to the placebo group (Nestler et al,
1999). A randomized, double-blind study, comparing patients receiving 100 mg of D-chiro- inositol twice daily to those receiving a placebo, still resulted in a significant improvement in ovarian function and a significant increase in weight loss, in the D-chiro-inositol treatment group, despite the reduction in D-chiro-inositol concentration, highlighting the sensitivity of this treatment and the low concern for side effects (Gerli et al, 2003).
1.3.2 scyllo-Inositol as a Therapeutic for Alzheimer’s Disease
Alzheimer’s disease (AD) is the most common form of dementia and affects 12 million people worldwide (Citron, 2001). Clinically, the patient will exhibit memory impairments and can also display a loss of language function, sensory perception, exectutive function, as well as agitation, aggression, delusions, hallucinations and repetitive vocalizations (Sink et al, 2005; Chertkow,
2008).
Neuropathologically, AD is characterized by the appearance of extracellular Aβ deposits, as either diffuse or neuritic plaques, as well as intraneuronal neurofibrillary tangles of the hyperphosphorylated tau protein (Selkoe, 2001). Associated with Aβ neuritic plaques are distrophic neurites, activated microglia and reactive astrocytes, therefore plaques were initially found to be the more toxic form of aggregated Aβ (Selkoe, 2001). However, soluble Aβ has
17 now been shown to correlate better with AD severity (McLean et al, 1999) and soluble Aβ oligomers have been proven to be toxic to neurons in culture (Selkoe, 2002). Therefore, the majority of AD drug design has been focused on targeting either Aβ formation, aggregation, deposition and/or clearance, based on the hypothesis that limiting the levels of Aβ oligomers and/or fibrils will slow down disease progression and reduce cognitive and behavioural deficits in patients.
One potential disease therapeutic for AD is scyllo-inositol, which appears to bind and stabilize
Aβ (McLaurin et al, 1998), thereby inhibiting Aβ oligomerization and fibrillogenesis. The discovery of scyllo-inositol as a possible treatment for AD started with the investigation of Aβ- lipid interactions, as a mechanism for the promotion of fibril formation and as a possible mechanism for Aβ-mediated toxicity (McLaurin and Chakrabartty, 1996; 1997; Choo-Smith and
Surewicz, 1997; McLaurin et al, 1998; Kremer et al, 2000; Yip et al, 2001; Curtain et al, 2003).
Multiple lines of evidence suggest that one of the central events in the pathogenesis of AD is the accumulation of neurotoxic oligomeric/protofibrillar aggregates of Aβ (McLean et al, 1999;
Sinha, 2002). Acidic phospholipids were shown to induce a structural transition in Aβ40 and
Aβ42 to their more toxic β-form, with a concomitant disruption of the bilayer (McLaurin and
Chakrabartty, 1996; 1997). The most potent phospholipid at causing this transition, was phosphatidylinositol, therefore components of phosphatidylinositol, i.e. the headgroup, phosphorylation state and fatty acyl chains were examined to determine the crucial element(s) for β-structure induction (McLaurin et al, 1998). myo-Inositol, the headgroup of phosphatidylinositol, mediated a transition to β-structure for Aβ42, while the addition of a single phosphate group abolished this transition (McLaurin et al, 1998). This suggested that myo- inositol was responsible for inducing Aβ42 β-structural transition. The effect of myo-inositol
18 on this conformational change within Aβ42 was an immediate, not time-dependent, effect.
Interestingly, even though myo-inositol induced a β-structure formation, it did not induce fibrillization but maintained Aβ42 in a soluble form (McLaurin et al, 1998). Negative stain electron microscopy showed Aβ42 in buffer alone formed short thin fibrils whereas, no fibrils were observed in the presence of myo-inositol (McLaurin et al, 1998). Aβ42 - myo-inositol interactions suggested that even though β-structure is required for fibril formation, Aβ is also able to form stable non-fibrillar β-structures (McLaurin et al, 1998).
Since myo-inositol induced stable micelles of Aβ42, the effect of other inositol stereoisomers on
Aβ42 aggregation was similarly investigated (McLaurin et al, 2000). epi-Inositol, scyllo- inositol and chiro-inositol were examined as potential inhibitors of fibrillogenesis. These stereoisomers differ in the position of their hydroxyl groups; myo-inositol has five equatorial hydroxyl groups and one axial hydroxyl group. Compared to myo-inositol, epi-inositol and D- chiro-inositol have one or two extra hydroxyls in the axial position, respectively. scyllo-
Inositol, on the other hand, has all its hydroxyl groups in the equatorial position. epi-Inositol and scyllo-inositol but not chiro-inositol induced a random to β-structure transition in Aβ42, without formation of fibrils (McLaurin et al, 2000). Aβ42 incubated with chiro-inositol formed fibrils indistinguishable from Aβ42 in buffer alone, displaying an inactive isomer (McLaurin et al, 2000).
Analysis of Aβ42-inositol conformers in culture, found both PC-12 cells and primary neuronal cultures protected from any Aβ-mediated neuronal toxicity and death (McLaurin et al, 2000).
Normally, Aβ42 accumulates on the cell surface of cells. Aβ42 in the presence of myo-, scyllo-, and epi-inositol resulted in decreased cell surface Aβ accumulation, while incubation with chiro-inositol resulted in no change in the amount of Aβ accumulation (McLaurin et al, 2000).
19 Therefore, the ability of myo-, scyllo-, and epi-inositol to decrease Aβ accumulation on neuronal membranes offers a possible mechanism for the attenuation of Aβ-induced neurotoxicity.
In vitro studies showed that inositol stereoisomers stabilize Aβ conformers, inhibit Aβ fibril assembly, accelerate disassembly of preformed fibrils, and protect primary cultured neurons from Aβ-induced toxicity (McLaurin et al, 1998; 2000). The Aβ conformers stabilized by inositol were small β-structured spherical micelles that were non-toxic in vitro. These compounds exhibited stereoisomer-specific differences in their ability to inhibit Aβ aggregation and cytotoxicity. Aβ aggregation and toxicity were more efficiently inhibited by scyllo-inositol than by myo-inositol (McLaurin et al, 2000).
To determine the importance of this structure-function relationship, a series of scyllo-inositol derivatives were synthesized in which one or two hydroxyl groups were replaced with fluoro, chloro, methoxy or hydrogen substituents. This approach has been previously demonstrated to be an effective method to garner information about hydrogen bonding requirements of a given hydroxyl group in the carbohydrate binding sites of lectins or antibodies (Glaudemans, 1991;
Auzanneau et al, 1993; Swaminathan et al, 1997). The hydroxyl groups at positions C-1 and C-
4 were modified in light of evidence that vicinal diols at positions 1, 3, 4 and 6 are recognized by the epimerases that interconvert inositol stereoisomers (Hipps et al, 1977; Pak et al, 1992).
Therefore, replacement of epimerase-targeted hydroxyl groups at positions 1 and 4 with stable substituents was hypothesized to increase in vivo compound stability. These derivatives showed maintenance of Aβ activity with some substitutions and enhanced in vivo stability with respect to epimerization. The data on the effects of these compounds on Aβ-aggregation suggest that
20 only the most conservative single hydroxyl substitutions are tolerated, thus 1-deoxy-1-fluoro- scyllo-inositol behaved similarly to, but not as well as, the parent compound (Sun et al, 2008).
The potency of various inositol stereoisomers in vivo was investigated in a transgenic model of
AD, the TgCRND8 mouse (McLaurin et al, 2006). TgCRND8 mice express a human amyloid precursor protein transgene (APP695) bearing two missense mutations that cause AD in humans
(KM670/671NL and V717F). At about three months of age, the TgCRND8 mice display progressive spatial learning deficits that are accompanied both by rising cerebral Aβ levels and by increasing numbers of cerebral amyloid plaques (Chishti et al, 2001). By six months of age, the levels of Aβ and the morphology, density and distribution of the amyloid plaques in the brain of TgCRND8 mice are similar to those seen in the brains of humans with well-established
AD (Wang et al, 1999; Näslund et al, 2000; Li et al, 2004). As observed in patients with AD, the biochemical, behavioural and neuropathological features of this mouse are accompanied by accelerated mortality (Wang et al, 1999; Näslund et al, 2000; Li et al, 2004).
myo-Inositol had no beneficial effects, while epi-inositol had early effects that were not sustained with disease progression in this mouse model (McLaurin et al, 2006). In contrast, scyllo-inositol treatment of TgCRND8 mice increased the survival of treated mice from 42% to
72% at 6 months of age (p=0.02). This increase in survival was accompanied by the rescue of cognitive deficits observed using the Morris Water Maze test for spatial memory (McLaurin et al, 2006). scyllo-Inositol treatment decreased total Aβ40 (p<0.001) and Aβ42 (p<0.05) levels and a 25% reduction in Aβ42 concentrations was maintained over 6 months (p<0.05). Plaque load was uniformly decreased by 35% across the entire brain indicating that inositol action was not region specific (p<0.05). A similar reduction was seen in the percent brain area covered by
21 vascular amyloid and in the size of cerebrovascular Aβ deposits. This decrease in deposited Aβ was due to scyllo-inositol-induced alterations of Aβ species in treated transgenic mice.
Prophylactic treatment reduced soluble Aβ oligomers of mass greater than 40kDa by 40% at both 4 and 6 months of age. scyllo-Inositol treated 4-month-old TgCRND8 mice showed a significant decrease in high-molecular weight Aβ oligomers and an increase in trimeric and monomeric Aβ species.
This cognitive benefit was reflected in scyllo-inositol’s reduction of synaptic toxicity in transgenic animals, as evidenced by a 148% increase in synatophysin immunoreactive boutons and cell bodies by 6 months of age (McLaurin et al, 2006). Improvements in neuroinflammatory status were marked by a reduction in astrogliosis and microgliosis
(McLaurin et al, 2006). Both synaptic and inflammatory changes likely resulted from scyllo- inositol blockage of Aβ oligomer-induced toxicity.
Aβ oligomer-induced inhibition of long-term potentiation (LTP) was studied using mouse hippocampal slices and rescue of this phenotype was shown by pre-incubating these naturally occurring oligomers with scyllo-inositol prior to perfusing brain slices (Townsend et al, 2006).
Application of scyllo-inositol after Aβ perfusion did not confer LTP protection. This effect is due to the direct binding of scyllo-inositol to Aβ trimers and neutralization of toxicity. Taken together, binding and neutralizing Aβ trimers could explain the increased amount of trimeric Aβ species observed in scyllo-inositol treated TgCRND8 animals; stabilizing Aβ trimers would also cause the decrease in high-molecular weight Aβ oligomers observed in the same animals. Co- application of scyllo-inositol and Aβ oligomers also prevented oligomer-induced decreases in dendritic spine density (Shankar et al, 2007). The protective effect of scyllo-inositol on synaptic
22 dysfunction may be partially the result of preventing oligomer-induced reductions in phosphatidylinositol-4,5-bisphosphate levels (Berman et al, 2008).
These results demonstrate that selected inositols can significantly inhibit the development of
AD-like phenotype in TgCRND8 mice, when given prior to the onset of disease (McLaurin et al, 2006). However, most AD patients will seek treatment only after their disease state is significantly advanced - i.e. at a time when Aβ oligomerization, deposition, toxicity and plaque formation are already well advanced. To assess whether scyllo-inositol could abrogate an established disease state, treatment in TgCRND8 mice was delayed until five months of age
(McLaurin et al, 2006). At this age, TgCRND8 mice have significant behavioural deficits, accompanied by significant Aβ peptide and plaque burdens (Chishti et al, 2001). A 28-day course of scyllo-inositol treatment reduced brain levels of Aβ40 and Aβ42 (e.g. insoluble Aβ40 p<0.05; insoluble Aβ42 p<0.05) and significantly reduced plaque burden (p<0.05). The decrease in plaque burden was accompanied by a decrease in soluble, high molecular weight oligomers.
These results were comparable in effect size to those observed in the prophylactic studies.
Spatial learning in these mice was significantly improved when compared to untreated
TgCRND8 mice (p<0.02). The cognitive performance of these scyllo-inositol-treated animals was not significantly different from their non-transgenic littermates (p=0.11). This beneficial effect of inositol treatment was not due to non-specific effects because scyllo-inositol had no effect on the cognitive performance of non-transgenic mice (p=0.39).
In vivo studies in rats further confirmed the efficacy of scyllo-inositol in rescuing cognitive deficits caused by Aβ. Pre-incubation of scyllo-inositol with Aβ oligomers, prior to intracerebroventricular injection into rats improved delayed alternation and complex reference
23 memory, measured by the Lever Cyclic Ratio assay as switching and perseveration errors
(Townsend et al, 2006). Oral administration of scyllo-inositol via drinking water at least 3 days prior to intracerebroventricular Aβ injection in rats also restored switching and perseveration errors to baseline levels (Townsend et al, 2006). These combined results suggest that scyllo- inositol is effective in multiple model systems of AD.
Overall studies of inositols in health and disease, suggest that inositol stereoisomers, especially myo- and scyllo-inositol, demonstrate good CNS bioavailability. This suggests that if other compounds are designed for transport via the inositol transporters, they might show similar degrees of CNS bioavailability. In order to accomplish this, the inositol transporters and their system of transport need to be further studied.
1.3.3 The Inositol Stereoisomers myo-Inositol was first isolated in the mid-19th century from muscle extracts and was accordingly named inosit, from the Greek root word inos for muscle (Scherer, 1850). Eight years later, a second, related compound was isolated from the shark Scyllium canicula, the skates Raja batis and Raja clavataI, and was given the name Scyllit (Staedeler and Frerichs, 1858). These compounds were later renamed myo- and scyllo-inositol and identified as two of the nine possible stereoisomers of inositol (Bouveault, 1894).
The inositol isomers belong to the class of compounds known as cyclitols, 5-7 carbon ring compounds, containing at least 3 carbons with an attached hydroxyl group. Inositols are cyclohexanehexols and members of the polyol family. The inositol isomers are 6 carbon ring molecules, with a hydroxyl group attached to each carbon of the ring. The structure of scyllo- inositol was first determined by Posternak in 1941 (Posternak, 1941; 1942), followed by the structure of myo-inositol a year later (Dangschat, 1942). The 9 stereoisomers differ from each
24 other based on the spatial orientation of their hydroxyl groups along axial or equatorial planes
(Figure 1.1). Of the nine, seven are optically inactive, whereas D- and L-chiro-inositol are enantiomers. While nine stereoisomers are possible, only six are found in nature (myo-, scyllo-,
D-chiro, epi-, muco-, and neo-inositol).
Figure 1.1 The inositol stereoisomers. There are 9 possible stereoisomers for inositol, which differ from each other based on the spatial orientation of their hydroxyl groups along axial or equatorial planes.
25 1.3.4 Inositol in Nature
The biosynthesis of inositol is an evolutionarily conserved pathway in nature, found across phylogenic kingdoms, including plants, animals, parasites, bacteria and archaea (Majumder et al, 2003). The majority of inositol present in humans is in the form of myo-inositol (Fisher et al,
2002). The next most abundant isomer, scyllo-inositol, is present at 8-20% the concentration of myo-inositol (Michaelis et al, 1993; Seaquist and Gruetter, 1998). scyllo-Inositol has been documented in a number of other organisms, although dietary sources of scyllo-inositol are limited (Sherman et al, 1978; Sanz et al, 2004; Soria et al, 2009). In the skate (Raja erinacea), scyllo-inositol was recorded at higher concentrations than myo-inositol, with the highest levels reported in the kidney (Sherman et al, 1978). In fruit, scyllo-inositol has been found in trace amounts in grapes and citrus fruits, with the highest levels reported in grapefruit (Sanz et al,
2004). scyllo-Inositol has also been reported in the Apiaceae family of vegetables, which includes carrots, fennel, parsley and coriander (Soria et al, 2009). However, the parts per million levels of scyllo-inositol in dietary sources preclude the use of dietary intervention alone to significantly alter scyllo-inositol levels within the CNS. In humans, it is unclear what function scyllo-inositol serves, however, the presence of enzymes for the interconversion between scyllo-and myo-inositol would suggest that scyllo-inositol does have a function within the cell, if only as a reserve pool for myo-inositol.
1.3.5 Inositol Synthesis and Degradation Pathways
Inositol levels within the human body are maintained through a combination of diet, synthesis from D-glucose-6-phosphate, recycling from inositol phosphates and through interconversion between inositol derivatives (Hipps et al, 1976). The average human ingests 1 g/day of inositol in the diet, predominantly as myo-inositol and phytic acid (Holub, 1986) and can synthesize up to 4 g/day in the kidneys (Clements and Diethelm, 1979). myo-Inositol is synthesized from D-
26 glucose-6-phosphate, through the activity of two enzymes, myo-inositol-3-phosphate synthase and inositol monophosphatase. In the brain, myo-inositol-3-phosphate synthase is only expressed in the vasculature (Wong et al, 1987), therefore outside of this region inositol must be actively transported into the cells.
myo-Inositol can be recycled from inositol phosphates through the action of inositol polyphosphate phosphatases to generate inositol monophosphate, followed by the action of inositol monophosphatase to generate myo-inositol (for an extensive list of human inositol phosphatases see Caldwell et al, 2006). In the rat, Northern blot analysis showed strong inositol monophosphatase expression in all brain regions tested: cortex, hippocampus, striatum and cerebellum (McAllister et al, 1992), suggesting that unlike the synthesis from glucose-6- phosphate, which appears to only occur in the vasculature, the creation of myo-inositol from inositol phosphates can occur throughout the brain.
Enzymes that interconvert the inositol stereoisomers and related derivatives have also been identified within the CNS (Figure 1.2; Hipps et al, 1976; 1977). In bovine brain, two enzymes have been isolated that convert myo-inosose-2 into myo- and scyllo-inositol (Hipps et al, 1976).
The first, myo-inositol oxidoreductase, converts myo-inosose-2 to myo- or scyllo-inositol at a ratio of 10:1 with NADH as a cofactor and 1:1 with NADPH (Figure 1.2A; Hipps et al, 1976).
The second, scyllo-inositol oxidoreductase converts myo-inosose-2 to myo- or scyllo-inositol at a ratio of 1:3 with NADPH and showed no activity with NADH (Hipps et al, 1976). In addition, a
NADP+ dependent inositol epimerase, which was isolated from bovine brain, was also found to convert myo-inosose-2 to myo- or scyllo-inositol, at a ratio of 2:1, in the presence of NADP+
27 (Hipps et al, 1977). scyllo-Inositol and myo-inositol are also interconverted by the NADP+ dependent inositol epimerase (Hipps et al, 1977). This epimerase converts myo-inositol to either scyllo- or neo-inositol, at a ratio of 1:10, and converts scyllo-inositol back to myo-inositol
(Figure 1.2B).
Figure 1.2 Endogenous sources of scyllo-inositol. A) A number of enzymes have been isolated from bovine brain that convert myo-inositol to scyllo-inositol, via myo-inosose-2.22,28 The first, myo-inositol oxidoreductase, converts myo- inosose-2 to myo- or scyllo-inositol, using either NADH or NADPH as a cofactor. In contrast, scyllo-inositol oxidoreductase and inositol epimerase only function in the presence of NADPH to convert myo-inosose-2 to scyllo-inositol. B) In addition, another NADPH dependent epimerase has been isolated that converts myo-inositol to neo-inositol and scyllo-inositol and can convert scyllo-inositol back into myo-inositol.
28 Removal of inositol from the body occurs by direct excretion in the kidney or by degradation predominantly in the kidney and the liver. myo-Inositol oxygenase, which is the enzyme required for the first step in myo-inositol catabolism, is mainly expressed in the proximal tubular epithelial cells of the kidney cortex (Arner et al, 2006). Western blot analysis of mouse tissue, showed no detectable levels of myo-inositol oxygenase in the brain, heart, lung, liver, spleen, intestines or muscles (Arner et al, 2006). Trace amounts of the mRNA were detected in the sciatic nerve, liver and heart (Arner et al, 2006). This enzyme cleaves the 6-carbon ring of inositol to form D-glucuronate, which can be metabolized in the liver. These combined studies suggest that inositol is actively transported throughout the body for both function and degradation. Three inositol transporters have been identified and characterized in mammals, one proton-myo-inositol transporter (HMIT) and two sodium-myo-inositol transporters (SMIT1,
SMIT2).
1.3.6 The Inositol Transporters
1.3.6.1 H(+)-myo-inositol Transporter HMIT, alternatively known as solute carrier family 2 (facilitated glucose transporter), member
13, is a member of the major facilitator superfamily, a group of secondary transporters that includes uniporters, symporters and antiporters. These transporters are characterized by 12 transmembrane domains and N- and C-terminal tails located on the cytoplasmic side of the cellular membrane (Mueckler et al, 1985; reviewed by Zhao and Keating, 2007). HMIT is predominantly expressed in the brain, with limited expression also found in the adipose tissue and in the kidney (Uldry et al, 2001). In the brain, mRNA expression was observed in both neurons and glia with high expression observed in all brain regions examined: cerebral cortex, hippocampus, hypothalamus, cerebellum and brainstem (Uldry et al, 2001). Rat HMIT was shown to transport myo-inositol preferentially followed by scyllo-inositol > chiro-inositol >
29 muco-inositol at a 1:1 ratio with H+ (Uldry et al, 2001). allo-Inositol was not transported by
HMIT and no other inositols were examined. Despite being labeled a facilitated glucose transporter, rat HMIT did not transport D- or L-glucose or other related hexoses (galactose, fructose, mannose, 2-deoxy-glucose, glucosamine or maltose). Homology between rat and human HMIT is 90%, which suggests that similar transport activities are present.
HMIT is typically internalized in the cell and cell surface translocation and regulation of expression have been suggested to be stimulated by membrane depolarization, changes in protein kinase C, internal calcium concentrations and acidification of the extracellular environment (Uldry et al, 2001; 2004), all of which occur with synaptic activity (Chesler and
Kaila, 1992). In light of this, the primary role of HMIT may be to adjust the intracellular inositol pools involved in cellular signaling pathways or phosphatidylinositol synthesis, which are required in areas with high rates of signaling and endo/exocytosis (Uldry et al, 2004).
1.3.6.2 Na(+)-myo-inositol Transporter 1 SMIT1, also known as solute carrier family 5 (sodium/glucose cotransporter), member 3, is a member of the sodium/solute symporter family (Wright and Turk, 2004), which are characterized by 13-14 transmembrane domains, with an N-terminal domain located in the extracellular space and a C-terminal domain, either located in the cytoplasm in members with 13 transmembrane domains, or encased in the membrane as the 14th transmembrane domain, as is the case for SMIT1 (Wright and Turk, 2004). The human, mouse, rat, canine and bovine
SMIT1 amino acid sequences are more than 92% homologous (Kwon et al, 1992; Berry et al,
1995; Lubrich et al, 2000; McVeigh et al, 2000). In humans, SMIT1 mRNA expression was found in the kidney, brain, placenta, pancreas, heart, skeletal muscle and the lung, but not in the liver (Berry et al, 1995), although its expression has been reported in the HepG2 human liver
30 cell line (Ostlund et al, 1996). In brain, SMIT1 mRNA expression was highest in the choroid plexus (Inoue et al, 1996). High SMIT1 mRNA expression was also observed in the hippocampus, the locus coeruleus, the suprachiasmatic nucleus, the olfactory bulb and the
Purkinje and granule cell layers of the cerebellum (Inoue et al, 1996). Across the brain, SMIT1 was expressed in almost all neurons and small glia-like cells (Inoue et al, 1996). In the hippocampus, SMIT1 mRNA was localized to pyramidal cells in areas CA1 to CA3 and to granule cells in the dentate gyrus (Inoue et al, 1996). In the choroid plexus, SMIT1 has been specifically localized to the basolateral side of cells, indicating that it is responsible for transporting inositol from the blood into the epithelial cells (Hakvoort et al, 1998).
SMIT1 appears to be the main transporter responsible for inositol transport into cells, SMIT1-/- mice, show a 92% reduction in inositol levels in the brain and an 84% reduction in inositol levels in the periphery (Berry et al, 2003). Heterozygous mice show a 15% reduction in inositol levels in the frontal cortex and a 25% decrease in the hippocampus (Shaldubina et al, 2007), which would suggest a dependence on SMIT1 transport in these areas or that the demand for inositol is higher than elsewhere. When rat astrocytes from the cortex, hippocampus, cerebellum, diencephalon and tegmentum were cultured and compared, the cortical and hippocampal cultures had a lower Km and a higher Vmax for myo-inositol than those for the other three regions, supporting the view that these two regions require more SMIT1 transporters
(Lubrich et al, 2000). In addition, SMIT1 mRNA levels were 2.5-fold higher in the cortex than in the other regions (Lubrich et al, 2000).
SMIT1 is unique among the inositol transporters because it shows an equal affinity for myo- and scyllo-inositol, as demonstrated in Xenopus oocytes transfected with the canine SMIT1 gene
(Hager et al, 1995). Overall, the sugar selectivity of the transporter was myo-inositol = scyllo-
31 inositol > L-fucose > L-xylose > L-glucose = D-glucose = α-methyl-D-glucopyranoside > D- galactose = D-fucose = 3-O-methyl-D-glucose = 2-deoxy-D-glucose > D-xylose (Hager et al,
1995). The ability of scyllo-inositol to strongly compete out myo-inositol transport was confirmed in murine neuroblastoma, murine cerebral microvessel endothelial and bovine aortic endothelial cell-lines (Wiese et al, 1996).
Inositol transport through SMIT1 is dependent on the cotransport of sodium with a stochiometry ratio of two Na+ ions per molecule of myo-inositol (Hager et al, 1995). Not unexpectedly, this dependence on Na+ resulted in SMIT1 transport inhibition in the presence of the sodium dependent transport inhibitor, phlorizin (Hager et al, 1995). The two Na+ ions bind to the transporter first, followed by myo-inositol, this sequential binding may explain the sodium leak currents observed in the absence of substrate (Hager et al, 1995).
Cells react to changes in osmolality by adjusting the levels of compatible organic osmolytes in the cell. In cultured rat cortical astrocytes, myo-inositol accounts for 56-100% of the osmolyte(s) utilized for adaptation to hypertonicity (Strange et al, 1994). Its been shown that the accumulation of myo-inositol in the cell, as a response to hypertonicity, requires the presence of myo-inositol in the extracellular space, indicating that this accumulation is not due to synthesis from glucose-6-phosphate or conversion from other inositol derivatives or phosphates, but from the transportation of inositol into the cell (Nakanishi et al, 1989). SMIT1 activity is upregulated by hypertonicity (Nakanishi et al, 1989; Kwon et al, 1991; Paredes et al, 1992;
Cammarata et al, 1994a; 1994b; Miyai et al, 1995; Wiese et al, 1996; Mallee et al, 1997;
Denkert et al, 1998; Matskevitch et al, 1998; Yorek et al, 1998a; 1998b; Matsuoka et al, 1999;
Yorek et al, 1999; Lubrich et al, 2000) and downregulated by hypotonicity (Wiese et al, 1996).
32 SMIT1 mRNA levels and activity can also be regulated by TNF-α (Yorek et al, 1998a; 1998b), possibly via NFκB, protein kinase C and ceramide activation (Yorek et al, 1998b). Treatment of bovine aorta, pulmonary endothelial or cerebral microvessel endothelial cells with TNF-α caused a significant decrease in SMIT1 mRNA levels and activity (Yorek et al, 1998b). The effects of TNF-α on SMIT1 expression and activity required RNA synthesis and were inhibited by actinomycin D (Yorek et al, 1998a). TNF-α decreased SMIT1 Vmax without altering the Km, suggesting a reduction in SMIT1 transporters at the cell surface. In contrast, IGF-1, platelet- derived growth factor, TGF-β, IL-1α, IL-1β, IL-2 or IL-6 did not affect SMIT1 activity (Yorek et al, 1998a).
As shown for HMIT, SMIT1 activity is also regulated by changes in pH (Matskevitch et al,
1998; Eladari et al, 2002). However, unlike HMIT, SMIT1 is more active at physiological pH and inhibited when pH is reduced. This is the opposite pattern to that observed for HMIT
(Uldry et al, 2001) and is thought to be due to a reduction in the transporter’s affinity for sodium
(Eladari et al, 2002). SMIT1 activity was also reduced by depolarizing-concentrations of potassium (Wiesinger, 1991), again in direct opposition to HMIT regulation (Uldry et al, 2004).
Therefore, it would appear that the body has adapted multiple methods for regulating inositol concentrations at the cellular level.
1.3.6.3 Na(+)-myo-inositol Transporter 2 The last known member of the sodium/solute symporter family that transports inositol is
SMIT2, also known as solute carrier family 5 (sodium/glucose cotransporter), member 11.
When comparing the human amino acid sequences, SMIT2 is 43% homologous to SMIT1
(Coady et al, 2002) and like SMIT1, the SMIT2 amino acid sequence encodes for 14 transmembrane domains (Roll et al, 2002). In humans, SMIT2 expression is highest in the
33 kidney, liver, heart, skeletal muscles and placenta, with much lower expression observed in the brain and minimal levels detected in the spleen, small intestine, lungs and lymphocytes (Roll et al, 2002). Interestingly, in polarized cells, such as Madin-Darby canine kidney cells, SMIT1 is preferentially expressed at the basolateral membrane and SMIT2 is preferentially expressed at the apical membrane (Bissonnette et al, 2004; 2008). These studies suggest that SMIT1/2 might work in concert to regulate inositol within tissues.
In transport studies, SMIT2 displays an equal affinity for myo-inositol and its stereoisomer, D- chiro-inositol (Coady et al, 2002; Aouameur et al, 2007; Lin et al, 2009). SMIT2 in HepG2 human liver cells showed a transport preference for D-chiro-inositol and D-glucose over their L- stereoisomers (Ostlund et al, 1996) but did not transport α-methylglucose and L-fucose (Coady et al, 2002; Lahjouji et al, 2007). This is in contrast to SMIT1, which shows an equal preference for both D- and L-glucose stereoisomers63 and transports α–methylglucose and L-fucose (Coady et al, 2002; Lahjouji et al, 2007). The remaining inositol stereoisomers have not been examined for transport by SMIT2.
As observed for SMIT1, inositol transport by SMIT2 is dependent on the cotransport of two sodium ions per substrate molecule (Coady et al, 2002; Bourgeois et al, 2005). As was observed with the other members of the sodium/substrate symporter family, SMIT2 displays a sodium leak current in the absence of substrate (Coady et al, 2002), and SMIT2 activity is upregulated by hyperosmotic conditions (Bissonnette et al, 2004; 2008). This upregulation is the result of an increase in SMIT2 transcription and translation, since upregulation was inhibited by actinomycin B and cycloheximide treatment (Bissonnette et al, 2008). SMIT2 activity can also be regulated by the mitogen-activated protein kinases p38 and c-Jun amino-terminal kinase, but not the extracellular signal-regulated kinase (ERK; Bissonnette et al, 2008). Inhibiting p38 and
34 c-Jun amino-terminal kinase resulted in a 40% and 32% reduction in SMIT2 activity, respectively (Bissonnette et al, 2008). In addition, SMIT2 activity can be upregulated by insulin
(Lin et al, 2009). Insulin treatment for 24 h caused an 18-fold increase in D-chiro-inositol-3H transport in rat L6 skeletal muscle cells transiently transfected with human SMIT2 (Lin et al,
2009) and insulin treatment in diabetes has been linked to increased D-chiro-inositol levels
(reviewed in Larner, 2002). In contrast to HMIT and SMIT1, SMIT2 does not appear to be sensitive to changes in pH (Eladari et al, 2002).
The expression and activity of the inositol transporters both in the periphery and CNS place them in juxtiposition for the uptake of inositol as a therapeutic agent. In regards to AD, the high expression and activity of the transporters in the cortex and hippocampus further suggest that compounds utilizing these transporters could be preferentially targeted to these regions of the brain.
1.3.7 Inositol Efflux
Inositol efflux from cells is not a well understood process, although fast and slow efflux currents have been detected. Efflux of inositol may involve both volume-sensitive and volume- insensitive components (Cammarata et al, 1997; Karihaloo et al, 1997; Isaacks et al, 1999), which are both active following hypertonicity-induced cell swelling, though perhaps not under basal conditions. The fast component appears to be mediated by volume-sensitive organic osmolyte-anion channels (Jackson and Strange, 1993; Karihaloo et al, 1997; Isaacks et al, 1999;
Novak et al, 2000; Loveday et al, 2003). Both fast and slow myo-inositol efflux currents were observed in bovine lenses exposed to hypertonic solution (Cammarata et al, 1997). The fast efflux current was the result of the activation of a common anionic (chloride) channel. These two efflux currents were observed in primary astrocyte cultures, but only the fast component
35 was inhibitable by anion membrane transport inhibitors (Isaacks et al, 1999). In human teratocarcinoma-derived Ntera2/D1 neuron-like cells, inositol efflux was blocked by a Cl- channel blocker, supporting the hypothesis that the volume-sensitive organic osmolyte-anion channel is a common chloride channel (Novak et al, 2000).
1.3.8 Inositol Pools
Experimental data suggest that inositol in the cell exists as two or three separate pools (Diringer and Rott, 1977; Yorek et al, 1991). This was first suggested by Diringer and Rott (1977), who examined the effects of inositol metabolism in chick embryo cells and discovered differentially regulated inositol pools, one smaller pool and one or more larger pools. Only the smaller pool appeared to regulate the synthesis of phosphatidylinositol (Diringer and Rott, 1977). Behavioral evidence has also suggested the presence of inositol compartmentalization (Bersudsky et al,
1994). Lithium-pilocarpine administration to rats leads to seizures, through the combined actions of lithium, which prevents inositol resynthesis and pilocarpine, which increases the use of inositol for second messenger systems by stimulating cholinergic activity (Kofman et al,
1993). These effects could be prevented by intracerebroventricular administration of myo- inositol (Kofman et al, 1993) but not by osmotically-induced increases in inositol levels
(Bersudsky et al, 1994), suggesting that the inositol used to sustain phosphatidylinositol levels must be different from that used to maintain osmotic function, strengthening the argument of different inositol pools (Bersudsky et al, 1994).
Separate inositol pools have also been suggested by inositol uptake and efflux kinetics (Sigal et al, 1993; Wolfson et al, 2000). Uptake of myo-inositol-3H into the soluble fraction of cells showed no evidence of saturation, however incorporation into the lipid fraction had a Km of 0.28 mmol/L and was inhibited by phlorizin, a SMIT1/2 inhibitor. It was proposed that intracellular
36 inositol exists as one larger, metabolically inert, pool and one smaller pool that equilibrates with external inositol levels and is utilized for the synthesis of phosphoinositides (Sigal et al, 1993).
In astrocytes exposed to myo-inositol-3H, three inositol pools were identified; the largest showed slow efflux kinetics while the two smaller pools had faster efflux kinetics (Wolfson et al, 2000).
Unlike the findings in hepatocytes, the largest pool was membrane-associated and influenced the phosphatidylinositide second messenger system, while the two smaller pools were located in the cytosol and were thought to be involved in osmotic regulation (Wolfson et al, 2000). These results suggest that it may be possible to alter certain pools of inositol without altering signal transduction pathways.
CHAPTER 2
Rationale, Hypothesis and Objectives
36 37
2.1 Rationale
Transport of drug candidates into the brain is one of the key challenges for designing drug treatments for CNS diseases. Oral administration of drug candidates is the preferred method of drug delivery wherever possible because of the ease and accessibility of this procedure for the patient and their caregiver. However this method of drug delivery requires, in the case of CNS disorders, that drug candidates be able to cross the blood-brain and blood-CSF barriers. Many compounds fail in clinical trials because, while they are effective in vitro and in animal testing, they are unable to cross the brain barriers in humans to access their drug targets in the CNS.
Therefore, one goal in CNS drug design research is to generate non-invasive methods for delivering these otherwise promising compounds to the brain. One possible barrier navigation strategy is to try and use a compound that enters the brain for other purposes, not only as a drug candidate but as a drug carrier. For example, inositol stereoisomers, such as myo- and scyllo- inositol, have shown promise as therapeutic agents for the treatment of certain CNS disorders
(Hallman et al, 1986; Hallman et al, 1992; Benjamin et al, 1995; Levine et al, 1995; Fux et al,
1996; Chengappa et al, 2000; Gelber et al, 2001; Palatnik et al, 2001; Eden et al, 2006;
McLaurin et al, 2006). This is likely due to the presence of transporters for these inositols in the
CNS, more specifically the transporters HMIT, SMIT1 and SMIT2 (Berry et al, 1995; Inoue et al, 1996; Hakvoort et al, 1998; Uldry et al, 2001). The accessibility of inositol stereoisomers to the CNS and the presence of transporters for inositol in the brain, suggest that it may be possible to use these transporters as a shuttling system for the delivery of other compounds to the CNS.
2.2 Hypothesis
Inositol transporters represent an effective shuttling system for drug delivery into the CNS.
38
2.3 Objectives
1. Inositol levels: Determine the CNS bioavailability of the two main inositol stereoisomers,
myo- and scyllo-inositol, by comparing their baseline levels to their concentrations
following oral administration. These studies were conducted in TgCRND8 mice and their
wild-type littermates, to determine whether disease pathology would affect these outcome
measures.
2. Inositol transporter expression: Inositol transport was examined by quantification of
transporter expression levels in four regions of the brain: cortex, hippocampus, septum,
cerebellum, as well as the kidney as a function of age and disease pathology.
3. Inositol transport kinetics: The flexibility of the inositol transporters for the transportation
of compounds other than myo-inositol was evaluated by determining the structural features
required for active transport through each of the inositol transporters.
CHAPTER 3
Materials and Methods
Portions of this section have been previously published in: Fenili D, Brown M, Rappaport R, McLaurin J. Properties of scyllo-inositol as a therapeutic treatment of AD-like pathology. J Mol Med. 2007 Jun;85(6):603-11.
39 40
Mice
TgCRND8 mice were maintained on an outbred C3H/C57Bl6 background. These mice over express the human APP gene containing both the Swedish (KM670/671NL) and Indiana
(V717F) mutations under control of the Syrian hamster prion gene promoter (Chishti et al,
2001). Mice were kept on a 12-hour light/dark cycle and given water and standard rodent chow ad libitum. All experiments were performed according to the Canadian Council on Animal Care guidelines.
For gas chromatography/mass spectrometry studies, one group of mice was treated with 10 mg/ml myo- or scyllo–inositol ad libitum through their drinking water for one week and changes in inositol levels were quantified. Each mouse typically consumed between 2.5–3 ml per day, which is equivalent to a 25–30 mg dose of inositol per animal. A second group of animals were treated with a once-daily gavage dose of, either 10, 30 or 100 mg/kg/day of scyllo-inositol, for one month and sacrificed 8 hours following the final gavage treatment.
Materials
All reagents were purchased from Sigma (St. Louis, MO, USA) unless otherwise noted. epi-
Inositol, allo-inositol and cold scyllo–inositol was acquired from Transition Therapeutics
(Toronto, Ontario, Canada). Viburnitol (1-D-3-deoxy-myo-inositol; Cat #: FC-041), D-Ononitol
(1-D-4-O-methyl-myo-inositol; Cat #: FC-040), Sequoyitol (5-O-methyl-myo-inositol; Cat #:
FC-047) and D-Pinitol (3-O-methyl-D-chiro-inositol; Cat #: FC-026) were all
41 purchased from Industrial Research Ltd. (Lower Hutt, New Zealand). myo-Inositol-(2-3H) (Cat
#: ART 0116), scyllo-inositol-(2-3H) (Cat #: ART 0264) and L-fucose-(5,6-3H) (Cat #: ART
0106A) were all purchased from American Radiolabeled Chemicals Inc. (St. Louis, MO, USA).
Quantification of myo– and scyllo–inositol myo–Inositol and scyllo-inositol concentrations in the brain, CSF, and plasma, as well as synthetic stock solutions of both inositols were quantified using gas chromatography/mass spectrometry (GC/MS; Fenili et al, 2007). To increase the volatility and thermal stability of these compounds and to allow for peak separation, these samples were first derivatized. The derivatization protocol was adapted from Shetty et al. (1995). D-chiro-Inositol was added to all samples as an internal standard, at a concentration of 50 ng/µl for brain and plasma samples and
1 ng/µl for CSF and synthetic samples. Synthetic myo- and scyllo-inositol were used for initial method development and for the generation of concentration curves for sample analysis. For brain samples, one hemisphere was weighed and the tissue, along with D-chiro-inositol, was homogenized in 2 x 2 ml of methanol, and the resulting suspension was centrifuged for 5 min at
5,000 x g. A volume of supernatant equivalent to 30 mg of brain tissue (based on the weight of the tissue before homogenization) was analyzed. Similarly, for plasma, CSF and synthetic inositols, either 100 µl of plasma, 5 µl of CSF or different concentrations of synthetic inositols were mixed with 1 ml of methanol and D-chiro-inositol; the solution was allowed to stand at room temperature for 5 min, then centrifuged (5000 x g, 5 min) and the supernatant removed.
These samples were evaporated to dryness (Speedvac; 60°C); 100 µl of pyridine reagent (1 mg/ml 4 dimethylaminopyridine solution in pyridine) and 100 µl of acetic anhydride were added, and the tubes were flushed dry with nitrogen and heated (80°C, 30 min). After derivatization, the unreacted acetylating reagent was evaporated under a steady stream of
42 nitrogen. The derivatized products were re-dissolved (4 ml; hexane-ethyl acetate (80:20, v/v)) and washed with 1 ml of 5% sodium carbonate. After vortexing (5 min) and centrifugation (3 min, 1000 x g), the organic layer was transferred to a new tube and evaporated (Speedvac;
40°C). The residue was reconstituted (100 µl; ethyl acetate), and 1 µL of this was injected into the GC/MS system.
Initial GC method development was performed on a Perkin Elmer Autosystem XL, with a programmable split-splitless injector and a flame ionization detector. GC/MS was performed using a Perkin Elmer TurboMass Autosystem XL with a quadrupole mass spectrometer and electron ionization. For both machines, GC was performed using a 30 m x 0.25 mm x 0.25 mm
ZB 5 column (5% diphenyl/95% dimethylpolysiloxane, Phenomenex, Macclesfield, UK) and helium as the carrier gas (1 ml/min). Samples were injected with the split set to 50 at 1 min and
0 at 5 min; the injector temperature was set at 300°C. The initial optimized GC protocol consisted of a starting temperature of 190°C. This was increased by 2° increments to 210°C, held at this temperature for 4 minutes, then increased by 35° increments to 290°C and held at this temperature for 4 minutes. The final optimized GC/MS protocol, consisted of a GC protocol where, the initial oven temperature was set to 80°C. After a hold of 1 min, the temperature was increased by 25° increments to 190°C, then by 3° increments to 220°C and finally by 35° increments to 290°C, which was held for 1.5 minutes. Sample peaks were analyzed using selected-ion monitoring at m/z 168 for brain and CSF samples and m/z 373 for plasma samples.
Three selected-ion recording time points, corresponding to chiro-, myo- and scyllo-inositol
43 elution from the GC column, were programmed into the MS protocol for increased sampling accurracy: 11.9-12.3 minutes, 12.4-12.85 minutes and 13.25-13.52 minutes, respectively. The sample peak areas were compared to the standard concentration curves generated using synthetic inositols, which were analyzed using selected-ion monitoring at m/z 168.
Lipid extraction and hydrolysis
The method for lipid isolation and analysis was adapted from Kersting et al. (2003). One brain hemisphere was homogenized in 2 ml of dH2O, and 500 µl was used for lipid isolation. This was placed in a glass screw-cap tube containing 3.75 ml of chloroform/methanol/HCl (10:20:0.1, v/v) and vortexed. Chloroform (1.25 ml) and 0.1 M HCl (1.25 ml) were added and the solution revortexed. The samples were centrifuged (200 x g, 5 min) to separate the phases. The organic phase containing the lipids was dried under nitrogen gas and resuspended in 200 µl of chloroform/methanol (6:1, v/v) before streaking onto a silica gel 60 F254 plate (EM Industries,
Merck, Darmstadt, Germany). The plate was placed in hexane/ethyl ether/acetic acid (70:30:1, v/v). Once the solvent had migrated within 1 cm from the top of the plate, the plate was removed from the thin-layer chromatography tank and air dried. The origin containing phosphatidylinositol lipids was collected, and the lipids eluted using four 1 ml washes of chloroform/methanol/concentrated HCl (2:1:0.1, v/v). The lipids were dried under nitrogen, redissolved in 1 ml of 6 N HCl, and then acid hydrolyzed (110°C, 56 h), to release the inositols from inositol-containing phospholipids. The hydrolysate was dried under nitrogen and derivatized (as above) for GC/MS analysis. The sample peaks were analyzed using selected-ion monitoring, m/z 168.
44
Quantitation of inositol transporter expression
Control gene selection for the tissue studies:
Genes such as, tyrosine kinase receptor (Abl1), a RNA-dependent ATPase (Bat1), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), ribosomal protein (L32), nitric oxide synthase 2 (Nos2), proteosome 26S subunit ATPase 4 (PSMC4), mitochondrial ribosomal protein (S12), succinate dehydrogenase (SDHA), TATA box binding protein (Tbp) and the transferrin receptor (TFRC) were analyzed as potential control genes for this study. Potential control gene for this study were chosen based on listed commonly used control genes in QPCR control gene selection kits, such as, the Mouse Endogenous Control Gene Panel (Tataa
Biocenter, Göteborg, Sweden) and Validated qPCR Control Kits (Eurogentec, San Diego,
USA). Once potential control genes were selected, the National Center for Biotechnology
Information (NCBI), Gene Expression Omnibus website was used to scan mouse microarray data for each of these genes. Control genes were selected that demonstrated similar levels of expression in the kidney and in all four of the brain regions examined in this study. The control genes Gapdh and Tbp each had the most equivalent expression across these tissue regions, therefore they were selected for this study.
Primer design:
Potential primers for each of the five genes examined in this study were selected using the
Beacon Designer 7.5 software program for Mac OS X. Sequence amplification studies were conducted using a kidney RNA sample from a 2 month old, wild-type mouse. A concentration curve of RNA was used to test the amplification efficiency (slope) and accuracy (R2 value) of each primer pair. In addition, dissociation curve analysis was performed to determine whether one, or multiple, amplification products were generated by the primer pair under evaluation (see
45
Figures 5.1 and 5.2). Primers were selected if their slope was in the range 2.9-3.2, their R2 value was between 0.98-1 and if they produced one main amplification product with minimal, or preferably no, secondary amplification products (see Figures 5.1 and 5.2). The final primers chosen for mouse and human gene amplification are listed in Tables 3.1 and 3.2. β-actin was chosen as the control gene for human cell-line inositol transporter tests because primers for this gene had previously been chosen, courtesy of Mary Brown.
Table 3.1 Mouse QPCR primers. Protein Name Gene Accession # Primers TATA-box Binding Protein Tbp NM_013684 Forward: GCC TTC CAC CTT ATG CTC AG Reverse: GAG TAA GTC CTG TGC CGT AAG Glyceraldehyde 3-phosphate Gapdh XM_001473623 Forward: AAG AAG GTG GTG AAG CAG dehydrogenase GCA TC Reverse: CGA AGG TGG AAG AGT GGG AGT TG Hydrogen/myo-inositol transporter Slc2a13 NM_001033633 Forward: GTC ACC ATC AAC ACC CTC TTC Rerverse: ACC TCC ATC CAT CCT TCT GC Sodium/myo-inositol transporter 1 Slc5a3 NM_017391 Forward: CTG TGG TGC TGT GGG ATG ATG Reverse: CCT GCT GGG TCT GAA CTT TGC Sodium/myo-inositol transporter 2 Slc5a11 NM_146198 Forward: CAA GGT GGT GAG GGC TAT CC Reverse: CTA TGA CAG GTT CCG CTT TGC
Table 3.2 Human QPCR primers. Protein Name Gene Accession # Primers β-actin ACTB M10277 Forward: GCC GAG GAC TTT GAT TGC Reverse: GGA CTT GGG AGA GGA CTG G Hydrogen/myo-inositol transporter SLC2A13 NM_052885 Forward: TTG AAT CAC TCT TTG ACA AC Rerverse: CCT TTA CCC GAA TAT ATT CA Sodium/myo-inositol transporter 1 SLC5A3 NM_006933 Forward: AGC CTT CTC ACA CCA CCT C Reverse: CAG TTC TCC TTC ACC ACC AG Sodium/myo-inositol transporter 2 SLC5A11 NM_052944 Forward: TCC TGT GGC TCT GTG GAA TA Reverse: CGC AGA AAA TGA GGT TGA CG
46
Tissue RNA extraction:
Kidney, cortical, hippocampal, septal and cerebellar tissues were each placed in a homogenizer with 2 ml of Solution D (4 M guanidine thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl and 0.1 M 2-mercaptoethanol) and the tissue homogenized. The resulting suspension was transferred to a 50 ml falcon tube and 200 µl of 2 M sodium acetate (pH 4.0), 2 ml of water- equilibrated phenol and 400 µl of chloroform were added and the solution mixed vigorously for
10 seconds. The tubes were incubated on ice for 20 minutes, parafilmed and centrifuged (8000 rpm, 30 min, 4°C, Beckman Coulter Avanti J-20XP). The top, aqueous interface was removed and placed in a new Eppendorf tube with 12.5 µl of 1 N acetic acid and 250 µl of 100% ethanol added for each 0.5 ml of aqueous interface and the resulting solution was mixed thoroughly.
The solution was incubated (-20°C, 1 h), centrifuged (10,000 x g, 20 min, 4°C) and the supernatant discarded. The RNA pellet was dissolved in 0.3 mL of Solution D, after which 5 µl of 1 N acetic acid and 100 µl of 100% ethanol were added, the solution incubated (-20°C, 1 h) and then centrifuged (13,000 x g, 20 min, 4°C). The supernatant was discarded and the pellet washed three times in 0.5 mL of 70% ethanol (in DEPC-treated water) and centrifuged following each wash (13,000 x g, 20 min, 4°C). The supernatant was removed and the RNA pellet resuspended in a small volume of DEPC-treated water. The RNA concentration of the resulting solution was determined using a NanoDrop™ Spectrometer (Thermo Scientific,
Wilmington, USA).
Cell culture RNA extraction:
Trypsin was added to the cell culture dish and the cells incubated (3-5 min, 37°C) to harvest from the cell culture dish. Once the cells were lifted, culture medium, at twice the volume of trypsin, was added to inactivate the trypsin and the resulting suspension was transferred to a 15
47 mL falcon tube for centrifugation (1000 rpm, 5 min). The supernatant was removed and the cells washed once in ice cold phosphate buffered saline (PBS). The PBS was centrifuged (1000 rpm, 5 min), removed and replaced with 0.5 mL of Solution D (4 M guanidine thiocyanate, 25 mM sodium citrate, pH 7.0, 0.5% sarcosyl and 0.1 M 2-mercaptoethanol). The cells were re- suspended, transferred to an Eppendorf tube and homogenized using a 1 inch, 20 g needle attached to a 1 mL syringe. To this tube, 50 µl of 2M sodium acetate (pH 4.0), 500 µl of water- equilibrated phenol and 100 µl of chloroform were added. The final suspension was shaken vigorously for 10 seconds, incubated on ice for 20 minutes and centrifuged (10,000 x g, 20 min,
4°C). The top, aqueous interface was removed and the same procedure as tissue RNA extraction was followed from this point onward. The RNA concentration was determined using a NanoDrop™ Spectrometer.
DNase I Treatment:
RNA was treated with DNase I (Fermentas, Cat #: EN0521) to remove genomic DNA. RNase inhibitor (Fermentas, Cat #: EO0381) was added to the reaction mixture to prevent RNA degradation during DNase I treatment. To a RNase-free tube 500 ng of RNA was added plus enough DEPC-H2O to equal 1.75 µl and 3.25 µl of the following master mix: 4-parts 10X reaction buffer with MgCl2, 4-parts Deoxyribonuclease I (DNase I), RNase-free, 1-part
RiboLock™ RNase Inhibitor and 17-parts DEPC-treated water. Samples were incubated (37°C,
30 min), following which 25mM EDTA (0.5 µl) was added and the sample further incubated
(65°C, 10 min). Following DNase I treatment, concentrations were determined again using the
NanoDrop™ Spectrometer and DEPC-treated water was added to the samples to normalize their concentrations.
48
Reverse transcription:
For each sample, 3 cDNA reactions were performed, each one at a volume of 10 µl using a
SuperScript III First-Strand Synthesis SuperMix for qRT-PCR kit (Invitrogen, Cat #: 11752).
For this reaction, 5 µl of the kit provided 2X RT Reaction Mix, 1 µl of the RT Enzyme Mix and
60 ng of DNase-treated RNA were added to each tube on ice. Three cDNA tubes were created per sample. The tube contents were gently mixed and placed in a PCR machine and incubated at 25ºC for 10 min, 50ºC for 30 min and 85ºC for 5 min. Following this E. coli RNase H (2 U, 1
µl) was added and the mixture incubated (37ºC, 20 min) to remove any residual RNA. Next, autoclaved distilled water (43.6 µl) was added to each tube to dilute the samples for the quantitative polymerase chain reaction (QPCR).
Quantitative polymerase chain reaction:
For the QPCR step a SYBR® GreenER™ qPCR SuperMix Universal kit (Invitrogen, Cat #:
11762) was used. To each well of a MicroAmp™ Optical 96-Well Reaction Plate (Applied
Biosystems, Cat #: 4306737), 9.1 ml of diluted cDNA and 10.9 ml of the master mix consisting of: 10 µl of SYBR® GreenER™ qPCR SuperMix Universal, 10 µM of the forward primer (0.4
µl), 10 µM of the reverse primer (0.4 µl) and 0.1 µl of the ROX Reference Dye were added. A 2 month, non-transgenic, kidney mRNA sample, with ideal 260/280 and 260/230 ratios, was used as a between plate control. For analysis, relative quantification values for the three inositol transporters were adjusted to the two control genes, inter-plate calibrators and variations in primer efficiencies, then adjusted to an average expression level of 1 using the MultiD, GenEx software program (genex.gene-quantification.info/).
49
Protein homology analysis
Human, mouse and rat HMIT, SMIT1 and SMIT2 sequences were retrieved from the NCBI database (www.ncbi.nlm.nih.gov/pubmed/). Sequences were aligned using DNAMAN software
(www.lynnon.com/). Alignment was performed using the identity matrix, fast alignment protocol, with the following non-homology penalty parameters: gap penalty = 4, K-tuple = 2, gap open = 10 and gap extension = 0.1.
Substrate selectivity analysis
Cell culture:
Embryonic day 18 primary rat cortical neurons (Neuromics, PC35102) - The shipping medium was removed from the tube containing the tissue, saved and replaced with an enzymatic solution consisting of Hibernate-Ca medium (2 ml, supplied with kit), containing papain (2mgs/ml) and the tissue incubated (30°C, 30 min). The enzymatic solution was removed and 1 ml of the initial shipping medium was added back. The tissue was titurated using a blue plastic tip until the cells in the tissue were mostly dispersed. Undispersed tissue was allowed to settle by gravity for 1 min and the supernatant transferred to a 15 ml tube containing an additional 1ml of the initial shipping medium. The cells were gently mixed by swirling and centrifuged (1100 rpm, 1 min). The supernatant was discarded and the tube flicked a few times to loosen the cell pellet. Culture medium (NbActiv4 medium, Neuromics, M36107) was added and the cells resuspended by gentle pipetting. Viable cells were counted using a hemocytometer to a concentration of 32 x 103 cells/2 cm2 of substrate in 0.4 ml/2 cm2 substrate and plated. Half the medium was changed with fresh culture medium every 3-4 days and the cells used for competitive transport studies after 3 weeks in culture.
50
Cell-lines, human epithelial kidney (HEK293) cells and 1321 N1 astrocytoma cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were plated onto 24-well plates and allowed to grow to 90% confluency before the competitive transport assays were conducted.
Competitive transport assay:
Once 90% confluent, cells were washed in PBS, then incubated in PBS (pH 7.0) containing: myo-inositol-(2-3H) (3 µCi/mL, 100 µM), BSA (0.1%, used to reduce background radioactivity), with or without a substrate competitor. The substrate competitors consisted of 10 mM of either inositol stereoisomers (myo-, scyllo-, epi-, allo-, muco-, neo-, L-chiro-, or D-chiro-inositol) inositol derivatives (viburnitol, D-ononitol, sequoyitol, L-quebinchitol or D-pinitol) or structurally similar sugar substrates (D-glucose, L-glucose, D-galactose, D-mannose, D-fucose,
L-fucose, L-rhamnose, D-ribose, L-ribose or D-fructose). One well was dedicated to testing each competitor and triplicate plates were used for each experiment. Following incubation (3 h,
37°C), cells were washed twice with PBS containing 1 mM cold myo-inositol, to stop transporter activity, following which the cells were dissolved using 2% SDS and transferred to vials containing 5 mL scintillation fluid. Their radioactivity was then measured using a scintillation counter.
For competitor testing against scyllo-inositol transport, myo-inositol-(2-3H) was substituted with scyllo-inositol-(2-3H) at the same concentration and the same protocol as above was followed.
For competitor testing against L-fucose transport, myo-inositol-(2-3H) was substituted with L- fucose-(5,6-3H) at the same concentration. L-fucose transport was tested against the following
51 competitors: 1. 10 mM myo-inositol and 10, 50, 100, 150, 200 and 400 mM cold L-fucose. 2.
10 mM of cold L-fucose, D-fucose, myo-, scyllo-, allo-inositol, L-glucose and D-glucose. 3. 0.1 mM D-fucose and 10, 25, 50, 100 and 250 mM cold L-fucose.
For D- and L-chiro-inositol competitor testing, myo-inositol-(2-3H) transport was tested in the absence or presence of 10 mM cold myo-inositol, 1, 10, 20, 40, 80 or 160 mM of D- or L-chiro- inositol.
For scyllo-inositol derivative testing, myo-inositol-(2-3H) transport was tested in the absence or presence of 2 mM of the scyllo-inositol derivatives MN-001, MN-002, MN-003, MN-005, MN-
007, MN-010 or MN-012 (see Figure 6.16a for structures).
For the testing of cold myo- and scyllo-inositol concentration curves on myo-inositol-(2-3H) transport, transport was tested against a range of concentrations between 62.5 mM and 0.95 µM for myo-inositol and between 100 mM and 0.024 mM for scyllo-inositol. For the myo-inositol concentration curve experiments, incubation was limited to 15 minutes to better reflect the concentration versus transport ratio. In contrast, the scyllo-inositol concentration curve experiments were conducted with an incubation time of 3 hours, because of a slower uptake rate for scyllo-inositol.
For HMIT activation, four adjustments to the above competitive transport assay were made in different combinations to try and activate HMIT.
1. Cell surface depolarization, through an increase in external potassium levels – potassium
levels in the PBS solution, used for cell washes and the radioactivity incubations, was
52
increased from 2 g/L to 5.964 g/L (80 mM). This concentration was chosen based on the
concentration used by Uldry et al. (2004).
2. Acidification of the extracellular environment – the pH of the PBS solution, used to wash
cells and for the incubation solution, was lowered from 7.0 to 6.0.
3. Phloridzin induced SMIT1/2 inhibition – multiple mM concentrations of phloridzin were
tested and 2 mM chosen for SMIT1/2 transport inhibition. For these silencing experiments,
cells were washed in PBS and then incubated in 2 mM of phloridzin (37°C, 30 min) before
the radioactivity step. For the radioactivity step, 2 mM of phloridzin was added to the
solution to maintain the SMIT1/2 silencing.
4. Na-free PBS – The PBS solution, used for cell washes and the incubation solution, was
switched to a Na-free version, which consisted of (per L): 58 g LiCl, 2 g KCl, 2.4 g
KH2PO4 and 17.42 g K2HPO4, adjusted to pH 7.0.
Statistical analysis
Statistical analysis for the GC/MS and competition assay studies were performed using the
Statistical Package for the Social Sciences (SPSS) for Mac OS X. QPCR statistical analysis was conducted using the Statistical Analysis System (SAS) program (courtesy of Lillian Weng,
Sunnybrook Hospital). Groups were compared using a one-way ANOVA. If a significant F score was observed (p < 0.05), a Bonferroni post hoc test was used to compare the groups with the statistical significance set at p < 0.05.
CHAPTER 4
myo- and scyllo-Inositol Levels and Equilibrium in the Brain
Portions of this section have been previously published in: Fenili D, Brown M, Rappaport R, McLaurin J. Properties of scyllo-inositol as a therapeutic treatment of AD-like pathology. J Mol Med. 2007 Jun;85(6):603-11.
53 54
Abstract
Inositol stereoisomers have been examined as potential therapeutic agents with favorable results.
But while both myo- and scyllo-inositol are able to inhibit Aβ aggregation in vitro, only scyllo– inositol is effective at ameliorating disease pathology in vivo in the TgCRND8 mouse model of
AD. This disparity between in vitro and in vivo results is interesting and unexpected based on what is known about the transport of both inositols into the brain. Therefore, this project’s objectives were: 1. To design a gas chromatography/mass spectrometry (GC/MS) protocol to quantify myo- and scyllo-inositol. 2. To compare brain inositol levels between TgCRND8 mice and their wild-type littermates. 3. To examine inositol transport and equilibrium dynamics in these two groups by examining the effects of myo- and scyllo-inositol treatment on brain inositol levels. and 4. To determine whether an increase in scyllo-inositol levels would result in its incorporation into phosphatidylinositol. We conclude that brain inositol levels, and the effects of inositol administration on those levels, was not significantly altered by disease pathology. Ad libitum scyllo-inositol resulted in a significantly greater increase in its corresponding levels in the brain, than ad libitum myo-inositol, suggesting that myo-inositol is tightly regulated. Once- daily gavage treatment did not result in a sustained increase in CNS scyllo-inositol levels and increases in brain scyllo-inositol levels, did not result in incorporation into phosphatidylinositol lipids.
55
Introduction
Inositol is a simple polyol with nine stereoisomers, of which, the most commonly found in nature are myo-, D-chiro-, epi- and scyllo-inositol (Fisher et al, 2002). Inositol stereoisomers as a drug therapy have been extensively studied for more than 20 years. myo–Inositol has been successfully used in human studies to treat psychiatric disorders such as depression (Levine et al, 1995), bipolar/affective disorder (Chengappa et al, 2000; Eden et al, 2006), obsessive- compulsive disorder (Fux et al, 1996), eating disorders (Gelber et al, 2001) and panic disorder
(Benjamin et al, 1995; Palatnik et al, 2001), as well as for the treatment of respiratory distress syndrome in premature infants (Hallman et al, 1986; 1992). epi–Inositol has been successfully used in mice to treat anxiety (Einat et al, 1998; Bersudsky et al, 1999). D-chiro-inositol has been successfully used to prevent neural tube defects in folate-resistant mice (Cogram et al,
2002) and as an insulin-sensitizing agent in women with polycystic ovary syndrome (Nestler et al, 1999; Gerli et al, 2003). While these inositol stereoisomers have been extensively studied as potential disease therapeutics, our laboratory was the first to study scyllo–inositol as a therapeutic agent (McLaurin et al, 2006).
Both myo– and scyllo–inositol were tested as potential disease therapeutics in the TgCRND8 mouse model of AD (McLaurin et al, 2006). This mouse model of AD expresses a mutated form of the human amyloid precursor protein, which results in an increase in cerebral Aβ levels,
Aβ plaque formation and cognitive deficits by 3 months of age, and a pathology corresponding to humans in the advanced stages of AD by 6 months of age (Chishti et al, 2001). In vitro, myo– inositol successfully inhibited Aβ aggregation, but in vivo myo-inositol did not significantly increase survival or cognitive performance in the TgCRND8 animals (McLaurin et al, 2000;
2006). In contrast, scyllo–inositol successfully inhibited Aβ aggregation both in vitro and in
56 vivo (McLaurin et al, 2000; 2006). In vivo scyllo-inositol treatment significantly improved cognitive function, synaptic function and survival rates in the TgCRND8 mice (McLaurin et al,
2006). In addition, a significant decrease in Aβ40 and Aβ42 levels, vascular amyloid levels, plaque size and area were also observed (McLaurin et al, 2006). These positive effects occurred both in animals given scyllo–inositol prophylactically, before the visible onset of symptoms, and therapeutically at 5 months of age, once symptoms have fully developed. Therefore, scyllo– inositol is an effective treatment for decreasing disease pathology and improving behavioral correlates in the TgCRND8 model of AD (McLaurin et al, 2006).
However, it was unclear why myo-inositol would only be effective in vitro, while scyllo-inositol is effective both in vitro and in vivo. myo–Inositol is the most abundant isomer found in nature and is a ubiquitous component of all eukaryotic cells. It is a constituent of phosphatidylinositol, which is an important phospholipid in membranes and second messenger systems. scyllo-
Inositol is the next most abundant isomer and is found in humans at 8-20% of myo-inositol levels (Michaelis et al, 1993; Seaquist and Gruetter, 1998). The human brain has the highest concentration of inositol in the body, containing approximately 5 mM of myo–inositol and 0.5 mM of scyllo–inositol (Michaelis et al, 1993); values that are almost 100-fold greater than circulating myo- and scyllo-inositol levels (Palmano et al, 1977). Inositol content in the brain is either synthesized in situ from glucose or transported across the blood-brain and blood-CSF barriers (Spector, 1978; 1988). Endothelial cells, located at the BBB, are the only cells capable of producing inositol from glucose within the CNS; therefore, utilization of inositol by most cells in the CNS requires an active transport system. Inositol may enter the brain either by passive diffusion or through stereospecific saturable transport systems at the brain barriers
(Spector, 1988; Wiesinger, 1991; Rubin and Hale, 1993; Uldry et al, 2001; Coady et al, 2002).
57
myo–Inositol is critical for maintaining osmolarity and signal transduction pathways within the
CNS, and although the physiological concentrations of inositol in the brain and some mechanisms of its regulation have been reported, this understanding is still very general (Fisher et al, 2002). Overall, this study sought to better understand myo- and scyllo-inositol regulation within the brain and whether regulation changes with disease pathology in TgCRND8 mice. To address this, five objectives were set:
1. To develop a GC/MS method to quantify the levels of myo- and scyllo-inositol in the brain,
CSF and plasma of experimental animals.
2. To compare the brain levels of myo- and scyllo-inositol between TgCRND8 mice and their
wild-type littermates, to examine whether they change as a result of disease pathology.
3. To compare those findings to inositol levels following ad libitum myo-inositol treatment, as
a way to examine the effects of disease pathology on CNS inositol transport and the
regulation of inositol levels in the brain.
4. To examine the effects of scyllo-inositol dosing on CNS inositol levels, by treating animals
with one of four doses of scyllo-inositol and comparing brain inositol levels to an untreated
control group. CSF inositol levels were also examined to see if the same effects observed in
the brain would be reflected in the CSF.
5. Finally, incorporation of scyllo-inositol into phosphatidylinositol, following ad libitum
administration to animals, was examined.
58
Results
Gas chromatography/mass spectrometry method development
A GC/MS technique was developed to track inositol levels present in the brain, CSF and plasma of experimental animals. In order to increase the volatility of the inositol stereoisomers within the GC and to better chromatographically separate the different inositol stereoisomers, the compounds were first derivatized (Figure 4.1a). Derivatization consisted of replacing the hydroxyl groups of inositol with acetyl groups. Samples were derivatized in the presence of pyridine reagent and acetic anhydride at 80°C for 30 minutes, following which non-derivatized compounds and lipids were removed using organic extraction. The resulting derivatized compounds were dried and resolublized in ethyl acetate.
To confirm the effectiveness of the derivatization protocol, samples of synthetic inositol were derivatized and submitted for analysis using a matrix-assisted laser desorption time-of-flight mass spectrometer (Proteomic and Mass Spectrometry Centre, Medical Sciences Building). The resulting chomatograms, showing the peaks generated, were analyzed for the presence of the derivatization products (Figure 4.1b). The peak at mass 180 identifies the amount of unacetylated inositol present in the sample. While the peaks at mass 373 and 433 correspond to derivatized inositol, either in its more predominant form, where there are acetyl groups in place of 5 of the 6 hydroxyl groups and the final hydroxyl group has been replaced by a hydrogen (ion
373) or in a fully derivatized state, where all six carbons have an attached acetyl group (ion
433). While a small proportion of inositol was present in the sample at different degrees of acetylation, the majority of the inositol was found with either 5 or 6 acetyl groups attached.
These results confirmed that the derivatization protocol was effective.
59
Figure 4.1 Derivatization. In order to increase the volatility of the inositol stereoisomers so that they would vapourize when injected into the GC and to better separate when individual inositol stereoisomers would leave the GC column, the compounds were first derivatized. Derivatization consisted of replacing the hydroxyl groups of inositol with acetyl groups (A). Samples were derivatized in the presence of pyridine reagent and acetic anhydride, non-derivatized compounds and lipids were removed from the mixture using organic extraction and the remainder was dried and resolubilized in ethyl acetate. The derivatization protocol was tested by mass spectrometry analysis (B). The ions corresponding to inositol at each stage of acetylation are highlighted using red boxes. Most of the inositol was found in a form where the hydroxyl groups had been replaced by 5 or 6 acetyl groups.
60
Once the effectiveness of the derivatization protocol was confirmed, derivatized synthetic inositols were used for initial GC method development. For the GC method, the mobile gas phase used was helium and the stationary, liquid phase coating the column was 5% diphenyl/95% dimethylpolysiloxane. D-chiro-inositol was added to all samples, prior to derivatization, as an internal standard. By changing the temperature, to influence the partitioning behaviour of the inositol stereoisomers between the mobile and liquid phases, a method was developed to separate the stereoisomers into distinct peaks on the chromatogram
(Figure 4.2a). This method consisted of a starting temperature of 190°C, increased first by 2° increments to 210°C, held there for 4 minutes, then increased by 35° increments to 290°C, at which point the temperature was held for 4 minutes. This method resulted in excellent peak separation using synthetic compounds, but as expected, when organic samples were tested, background noise elevated thereby masking peak detection and preventing quantification.
Therefore, method development was switched to GC/MS instrumentation, which is superior at reducing background noise when analyzing organic samples. Once again helium and 5% diphenyl/95% dimethylpolysiloxane were used as the mobile and stationary phases, respectively. Attached to the GC was a quadrupole mass spectrometer, which allows the identification of compounds as they exit the GC column based on their ion fragmentation spectra. Changes in the GC method were made to better separate inositol peaks as a function of time and each of these peaks was scanned to ensure that it only contained the compound of interest. One of the most important adjustments made to the GC protocol was to change the starting temperature from 190°C to 80°C, which is just above the vaporization temperature of the solvent used, ethyl acetate. Starting at this temperature, rather than at a higher temperature,
61
Figure 4.2 Gas chromatography method development. Initial method development to separate myo-, scyllo- and D-chiro-inositol into distinct peaks on a chromatograph was initially conducted using a GC machine and synthetic inositols (A). Once peak separation was achieved, method development for organic samples was conducted using a GC/MS machine (B) and the method was further adjusted to reduce background noise (C).
62 resulted in the vaporization of only ethyl acetate and well derivatized compounds. After one minute the remaining, non-vaporized sample was flushed from the system. This resulted in a focusing of the sample onto the head of the column and a reduction in background noise (Figure
4.2b,c).
Once peak separation was optimized, a further change was made to the GC/MS protocol, from a total ion chromatography approach to sample analysis by selected ion recording. The distinct fragmentation pattern that exists for each compound (Figure 4.3a,b) was used to select certain ions for mass spectrometer detection. Using a selected ion recording protocol results in the mass spectrometer focusing its ion detection system away from the recording of every ion, to the quantification of selected ion(s). This increases the accuracy and detection limits of the machine. Total ion chromatography was used for the initial method development, to determine when compounds exit the column to be detected as peaks on the chromatogram (Figure 4.3c).
At this stage, mass spectrometry was used to identify which peaks contained the inositol stereoisomers of interest. Once these peaks were identified, each was carefully scanned to confirm the presence of only the one compound. Whenever a fusion of peaks was observed, changes were made to the GC method to improve peak separation. Once the GC protocol was optimized, the mass spectrometry protocol was modified to track only the selected ion, rather than the whole ion spectrum (Figure 4.3d). In addition, the mass spectrometry method was further adjusted to only track the chosen ion at the three elution times for the inositol compounds, thus further enhancing ion detection (Figure 4.3e). For brain and CSF samples, ion
168 was chosen, because it is present in the highest quantities in the mass spectra of the inositol
63
Figure 4.3 Selected ion recording. Samples derivatized and run on the GC/MS using total ion chromatography, resulted in a separation of myo-, scyllo- and D-chiro-inositol over time on the chromatograph. However, in organic samples these peaks were masked by background noise. By examining the mass spectrums for myo- (A), scyllo- (B) and D-chiro-inositol, selected ions specific to the inositol compounds, could be selected to focus mass spectrometry analysis, to enhance peak detection. This is evidenced when comparing inositol peaks (outlined by red box) in the total ion chromatograph (C) to those recorded using selected ion recording (D). The mass spectrometer method was further adjusted to quantify the selected ion (ion 168 for brain and CSF samples) at the three time points when the inositol compounds were recorded as exiting the GC column.
64 compounds. However, for plasma samples, because of a greater degree of background noise
(Figure 4.4a), masking the inositol peaks even following the selection of ion 168 (Figure 4.4b), further adjustments were made to the mass spectrometry methodology. Analysis of this region of the spectrum showed that the noise was being caused by large quantities of derivatized glucose and galactofuranose in plasma (Figure 4.4c,d). Ions 373 and 432 were examined as alternatives because they are components of the derivatized inositol mass spectra, but not those of derivatized glucose or galactofuranose. Ion 373 corresponds to the molecular weight of the predominate species of derivatized inositol with five acetyl groups, one of which
______
Figure 4.4 Selected ion recording for plasma samples. Plasma samples derivatized and run on the GC/MS using total ion chromatography, also showed myo-, scyllo- and D-chiro-inositol peaks masking by unrelated compounds (A). Selected ion recording for ion 168 still resulted in peak masking, in these samples, in the region of interest (B). Therefore, two other ions, 373 (C) and 432 (D), present in the mass spectra of the inositol compounds, were tested as alternatives. Based on this analysis, ion 373 was chosen for the selected ion recording of plasma samples. The red box highlights the region of the chromatograph corresponding to where the three inositol compounds exited the column.
65 is shared between two carbons, and ion 432, which corresponds to the molecular weight of fully derivatized inositol. Ion 432 was not present at quantifiable levels in our samples, whereas ion
373 was present and, effectively eliminated noise allowing for the accurate quantification of inositol levels in plasma samples.
Once method development was completed, inositol levels present in the brain, CSF and plasma samples were quantified by comparing the selected ion peaks generated to those resulting from the injection of derivatized synthetic inositols. Concentration curves, generated using these synthetic inositols, were used to quantify myo- and scyllo-inositol levels (Figure 4.5).
Baseline brain inositol levels in TgCRND8 mice and their wild-type littermates
Baseline brain levels of myo- and scyllo-inositol were quantified and compared between
TgCRND8 mice and their wild-type littermates. The brain levels of myo– and scyllo–inositol in the mice were comparable to those previously reported in the literature (Seaquist and Gruetter,
1998; Fisher et al, 2002). Brain myo-inositol concentrations have been reported in the range of
2 to 15 mM (Fisher et al, 2002), which is comparable to the 3 mM observed in this study. scyllo–Inositol concentrations between 8 and 20% of myo–inositol levels have been reported previously (Seaquist and Gruetter, 1998), which at an average of 14%, equals a concentration range for scyllo-inositol of 0.28 mM and 2.1 mM. In the present study, an average of 0.9 mM of scyllo-inositol was recorded in brain samples from untreated TgCRND8 mice and their wild- type littermates.
66
Figure 4.5 Examples of myo-inositol and scyllo-inositol concentration curves. Concentration curves were generated for myo- (A) and scyllo-inositol (B) using synthetic stock solutions, derivatized and analyzed using GC/MS. Curves such as these were generated for each experiment and used to calculate the concentrations of myo- and scyllo-inositol present in the organic samples under analysis. ______
A comparison of brain myo- and scyllo-inositol concentrations between TgCRND8 mice and their wild-type littermates, found no significant differences between the two groups (Figure 4.6; myo-inositol: F1,15 = 2.277, p = 0.152; scyllo-inositol: F1,15 = 0.980, p = 0.338). This suggests that basal myo- and scyllo-inositol levels present in the brain are not altered by disease pathology, such as that found in this mouse model of AD.
67
Figure 4.6 Baseline brain myo- and scyllo-inositol levels. myo-Inositol and scyllo-inositol levels were measured in brain homogenates from TgCRND8 mice and their wild-type littermates using GC/MS. A. No significant differences were observed in brain myo- and scyllo-inositol levels between the two groups. (p > 0.05). B. An analysis of the distribution of brain myo- and scyllo-inositol concentrations in both groups shows the wide range of baseline inositol concentrations present, from 2.3 to 3.5 mM for myo-inositol and from 0.3 to 1.5 mM for scyllo-inositol. (n = 17).
68
Brain inositol levels in TgCRND8 mice and their wild-type littermates following ad libitum myo-inositol treatment
To compare the effects of inositol administration on myo– and scyllo–inositol concentrations within the brain of TgCRND8 mice and their wild-type littermates, mice were administered myo–inositol ad libitum in their drinking water at a concentration of 10 mg/ml. The purpose of this test was to determine whether disease pathology would alter inositol transport into the brain or its equilibrium/regulation within the brain. myo-Inositol administration was chosen for this study because it is reported to be the primary substrate transported by all three inositol transporters. Therefore analysis of this inositol would give an accurate estimate of the effects of disease pathology, caused by cerebrovascular amyloid, on myo- and scyllo-inositol brain levels.
Ad libitum myo-inositol treatment caused a significant increase in brain myo-inositol levels and a significant decrease in scyllo-inositol levels in both groups, irrespective of disease phenotype
(Figure 4.7; myo-inositol: F3,28 = 6.647, p = 0.002; scyllo-inositol: F3,28 = 13.710, p < 0.001).
Both TgCRND8 mice and their wild-type littermates showed an approximate 17% increase in myo-inositol levels following myo-inositol treatment (p = 0.015 in wild-type animals versus p =
0.001 in TgCRND8 mice). scyllo-Inositol levels significantly decreased 5-fold in both groups following myo-inositol administration (p < 0.001 in wild-type animals versus p = 0.001 in
TgCRND8 mice). Therefore, disease pathology did not change inositol transport or its regulation within the brain. The decrease in scyllo–inositol levels, observed in both groups following myo–inositol administration, might reflect a shift in inositol equilibrium towards degradation pathways, in an effort to reestablish/maintain brain homeostasis.
69
Figure 4.7 The effects of myo-inositol treatment on brain myo- and scyllo-inositol. Both TgCRND8 mice and their wild-type littermates were administered 10 mg/ml of myo- inositol ad libitum in their drinking water for one week and brain myo- and scyllo-inositol concentrations were measured using GC/MS. myo-Inositol levels (A,B) were significantly increased following myo-inositol ad libitum treatment. In contrast, scyllo-Inositol levels (C,D) were significantly decreased. (*: p < 0.05; control group: n = 17, myo-inositol group: n = 15).
70
The effects of single dose versus continuous scyllo-inositol treatment on CNS inositol levels
Baseline brain myo- and scyllo-inositol levels were quantified and compared to their levels following either a once-daily gavage dose of 10, 30 or 100 mg/kg/day scyllo-inositol for one month, or an ad libitum dose of 10 mg/ml scyllo-inositol in their drinking water for one week.
These scyllo-inositol doses were chosen, based on previous research on the effects of scyllo- inositol treatment in TgCRND8 mice (McLaurin et al, 2006). Gavage doses of 0.3 to 30 mg/kg/day of scyllo-inositol, as well as an ad libitum dose of 10 mg/ml scyllo-inositol, showed a dose-dependent improvement in spatial memory and a corresponding dose-dependent decrease in brain Aβ oligomers and plaques (McLaurin et al, 2006). Plaque levels were significantly reduced following the administration of 1 mg/kg/day or higher doses of scyllo-inositol, soluble
Aβ oligomers were significantly reduced following the administration of 3.3 mg/kg/day or higher doses of scyllo-inositol and soluble Aβ42 levels were significantly reduced following the administration of 10 mg/kg/day or higher doses of scyllo-inositol (McLaurin et al, 2006). Ad libitum scyllo-inositol administration also significantly decreased soluble and insoluble Aβ40 and Aβ42 levels, plaque accumulation and improved spatial memory (McLaurin et al, 2006;
Fenili et al, 2007).
Overall, a significant difference in brain myo- and scyllo-inositol levels was observed between groups (Figure 4.8a,b; myo-inositol: F4,29 = 6.477, p = 0.001; scyllo-inositol: F4,29 = 87.370, p <
0.001). However, none of the gavaged doses of scyllo-inositol resulted in a significant change in either myo- or scyllo-inositol levels. Only ad libitum scyllo-inositol treatment resulted in a
71
Figure 4.8 Brain and CSF myo- and scyllo-inositol levels following scyllo-inositol treatment. scyllo-Inositol treatment consisted of either a once-daily gavage dose of either 10 mg/kg, 30 mg/Kg or 100 mg/kg scyllo-inositol for one month or ad libitum administration of 10 mg/ml scyllo-inositol in the animals drinking water for one week. Gavaged mice were sacrificed 8 hours following their last treatment, when brain scyllo-inositol levels are maximal (see Figure 4.9B). Gavage treatment did not significantly change either myo- or scyllo-inositol levels in the brain (A,B) or CSF (C,D). However, ad libitum treatment resulted in a significant decrease in brain myo-inositol levels (A) and a significant increase in scyllo-inositol levels (B). An examination of CSF inositol levels following ad libitum treatment showed no significant changes in myo-inositol levels (C) but a significant increase in scyllo-inositol levels (D). (*: p < 0.05; untreated group: n = 17 for brain and 10 for CSF, ad libitum group: n = 5 for both brain and CSF, once-daily groups: n = 5 per group for both brain and CSF).
72 significant 0.3-fold decrease in brain myo-inositol levels in comparison to the untreated control group (p < 0.001) and a significant 7-fold increase in brain scyllo-inositol levels in comparison to all other groups (p < 0.001).
Given the lack of change in brain scyllo-inositol levels following once-daily gavage treatments, yet a marked increase following ad libitum treatment, the corresponding changes in CSF levels were examined, to correlate with these findings. Once again, a significant difference was observed in scyllo-inositol levels but not in myo-inositol levels between groups (Figure 4.8c,d; myo-inositol: F4,28 = 2.603, p = 0.057; scyllo-inositol: F4,27 = 31.926, p < 0.001). As was found in the brain, none of the gavaged doses resulted in a significant increase in scyllo-inositol concentrations but ad libitum scyllo-inositol treatment raised CSF scyllo-inositol levels up to 10- fold in comparison to the other groups (p < 0.001). The 7.5- and 10-fold increases in brain and
CSF scyllo-inositol levels, respectively, observed following ad libitum treatment, indicates a shift in the inositol influx/efflux equilibrium pathways at the brain barriers in response to an increase in peripheral scyllo-inositol levels. This finding suggests that a sustained increase in scyllo-inositol levels in the periphery will be reflected in a subsequent sustained increase in brain scyllo-inositol levels. This effect was more pronounced following scyllo–inositol treatment than myo-inositol treatment, which may reflect a tighter control imposed to maintain myo-inositol equilibrium. These results further suggest that a once-daily dose of scyllo-inositol is insufficient to elevate scyllo-inositol levels in the CNS, but that more frequent administration would result in a sustained increase. These results are in agreement with cognitive testing of
TgCRND8 mice treated once- or twice-daily by gavage with scyllo-inositol, in which only twice-daily administration had beneficial effects (unpublished data).
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Plasma inositol levels as a function of time, post-scyllo-inositol gavage treatment
Preliminary research (courtesy of Mary Brown, Figure 4.9a,b), found a peak increase in scyllo- inositol-(2-3H) levels in plasma by 2 hours post-gavage treatment and a peak increase in brain scyllo-inositol-(2-3H) levels by 8 hours post-treatment. GC/MS analysis of brain samples at 8 hours post-gavage treatment, found scyllo-inositol levels were not significantly altered from control levels. Therefore, myo- and scyllo-inositol levels were tracked in plasma at 1.5, 2, 4 and
8 hours following scyllo-inositol gavage treatment, to track scyllo-inositol export from the plasma using GC/MS. A significant difference in both plasma myo- and scyllo-inositol levels was observed as a function of time post-gavage (Figure 9c,d; myo-inositol: F3,9 = 6.612, p =
0.012; scyllo-inositol: F3,9 = 5.674, p = 0.018). myo-Inositol was significantly decreased by 4 hours post-gavage (p = 0.030) and further decreased by 8 hours (p = 0.019). scyllo-Inositol levels were not significantly decreased by 4 hours post-gavage (p = 0.064) but were significantly decreased by 8 hours (p = 0.048). The significant decrease in scyllo-inositol levels observed in plasma by 8 hours post-gavage, supports the initial findings made using scyllo- inositol-(2-3H), that brain scyllo-inositol levels would be maximal by 8 hours post-gavage treatment. In addition, these results, along with the partial but not significant increase in brain scyllo-inositol levels observed at 8 hours post-gavage treatment, support our earlier suggestion that once-daily gavage treatments of scyllo-inositol are insufficient to significantly increase scyllo-inositol levels in the CNS. Multiple gavage treatments per day would be required to cause a sustained increase in brain scyllo-inositol levels.
74
Figure 4.9 Plasma myo- and scyllo-inositol concentrations following scyllo-inositol treatment. Bioavailability of scyllo-inositol in plasma (A) and brain (B), determined using orally administered scyllo-inositol-(2-3H) uptake studies, showed that scyllo-inositol levels increased rapidly, peaking at 2 hours post-administration in plasma and 8 hours post-administration in brain (experiment courtesy of Mary Brown). Based on these findings, mice were gavaged with 100 mg/kg scyllo-inositol and plasma myo- and scyllo-inositol levels were quantified at 1.5, 2, 4 and 8 hours post-gavage. There was a significant decrease in plasma myo-inositol levels (C) by 4 hours post-gavage and a significant decrease in scyllo-inositol levels (D) by 8 hours post- gavage. (§: p = 0.064. *: p < 0.05; 1h30, 4h and 8h groups: n = 3, 2h group: n = 4).
75 scyllo-Inositol Incorporation into Phosphatidylinositol
The phosphatidylinositol family of lipids are located at the cellular membrane and are important regulators of second messenger cell signaling pathways. The head group of phosphatidylinositol is usually myo-inositol, but scyllo-inositol incorporation into phosphatidylinositol has been reported to occur in lower organisms, such as mycobacteria and in barley seeds (Kinnard et al,
1995; Ryals et al, 1999; Salman et al, 1999; Riggs et al, 2007). Therefore, we examined whether scyllo-inositol incorporation into phosphatidylinositol lipids occurred in untreated and treated mice as a result of fluctuations in brain scyllo-inositol levels. For this purpose, brain phospholipids from both untreated and ad libitum scyllo-inositol treated animals were isolated using thin layer chromatography and their head group extracted for derivatization and analysis using GC/MS. scyllo–Inositol could not be detected in phosphatidylinositol lipids isolated from the brains of either control or scyllo–inositol-treated mice (Figure 4.10). scyllo–Inositol had an elution time of 18.2 min on the chromatograph, and a point by point examination of the signal between 17 and 19 minutes failed to detect scyllo–inositol. The lower limit of detection of the assay was determined to be approximately 0.25 ng/µl, therefore, although we cannot rule out minor concentrations of scyllo–inositol being present in brain phosphatidylinositol lipids, if this occurs, it occurs at negligible levels. Overall, these results suggest that scyllo–inositol does not become incorporated into phosphatidylinositol when present at elevated concentrations within the CNS. This suggests that increasing scyllo-inositol levels in the brain should not alter phosphatidylinositol levels and therefore should not alter signal transduction pathways.
76
Figure 4.10 scyllo-Inositol incorporation into phosphatidylinositol. GC/MS profiles, determining the levels of myo- and scyllo-inositol isolated from phosphatidylinositol in control mice versus those given scyllo-inositol. The phosphatidylinositol head groups were isolated through lipid isolation and hydrolysis. D-chiro- Inositol was added as an internal standard, the inositol compounds were derivatized and single mass ion 168 was used to track inositol levels. myo-Inositol was readily detected but scyllo- inositol could not be detected in any of the samples. This suggests that scyllo-inositol does not substitute myo-inositol as the head group for phosphatidylinositol. The insets show amplification of the regions of the chromatograph where scyllo-inositol should occur, a small arrow in the inset points out where the scyllo-inositol peak would be expected (n = 5 per group).
77
Discussion
It has previously been shown that the treatment of TgCRND8 mice with scyllo–inositol ad libitum resulted in improvements in cognition and a decrease in toxic soluble Aβ species within the CNS, while myo-inositol administration did not significantly improve these same disease outcome measures (McLaurin et al, 2006). Using GC/MS, the present study found that both myo- and scyllo-inositol administration resulted in a corresponding significant increase in their brain levels. The increases observed following myo-inositol treatment were more moderate and might explain why myo–inositol was not effective for the treatment of AD pathology in
TgCRND8 mice despite the observed in vitro efficacy (McLaurin et al, 2000; 2006). myo-
Inositol might be more tightly regulated than scyllo–inositol within the CNS, because it is important for osmolarity control and signal transduction pathways.
Since scyllo-inositol has no known functions within the brain, or the body, its levels appear to be less tightly regulated than those for myo-inositol. This allows for high CNS bioavailability, an important concern when designing drug therapeutics. Using various administration paradigms for scyllo-inositol it was found that ad libitum, but not single-dose scyllo–inositol administration, resulted in a significant elevation in brain scyllo–inositol levels. Drug efficacy is dependent on a sustained elevation of the drug in the brain for therapeutic benefit. The present study would suggest that a multiple dosing regiment might be more effective than a once-daily dose. In support of this suggestion, previous research examining the effects of different doses of scyllo-inositol in TgCRND8 mice on disease outcome measures found twice- daily oral gavage administration of scyllo–inositol more effective than once-daily treatment at reversing cognitive deficits in the animals (Unpublished results: McLaurin et al, 2006).
78
The inter-regulation of myo- and scyllo-inositol in the brain is of interest when examining inositol as a disease therapeutic. Rat, rabbit, and bovine brains are known to contain an epimerase that converts myo– to scyllo–inositol and vice versa (Sherman et al, 1968a; b; Hipps et al, 1977). myo–Inositol administration in women has demonstrated a 1% conversion to scyllo–inositol within the plasma (Groenen et al, 2003), and myo–inositol treatment in rats showed a 0.06% conversion to scyllo–inositol (Pak et al, 1992). An increase in inositol levels, with maintenance of the correlation between scyllo- and myo-inositol, has been reported to occur as a function of age within the white matter of healthy human subjects (Kaiser et al, 2005). A simultaneous fluctuation of scyllo- with myo-inositol has also been reported in patients with brain pathology (Griffith et al, 2007). In AD patients, increases in scyllo-inositol/creatine ratios were positively correlated with increases in myo-inositol/creatine ratios (Griffith et al, 2007).
Our study also found the levels of these two inositols to be inter-regulated. A significant (p <
0.05) decrease in brain scyllo-inositol levels was recorded following ad libitum myo-inositol administration and vice versa. The severe drop in scyllo-inositol levels that occurred following myo-inositol administration, compared to the smaller drop in myo-inositol levels that occurred following scyllo-inositol administration, supports our earlier statement that myo-inositol levels are more tightly regulated. The apparent inverse correlation between the two inositols following ad libitum administration, in comparison to the positive correlation observed in aging humans and as a result of disease pathology, likely results from early shifts in the levels of these inositols in an effort to re-establish equilibrium.
While scyllo-inositol has no known function in the body, we wanted to confirm that scyllo- inositol elevation in the brain would not result in competition with myo-inositol for incorporation into phosphatidylinositol, and thus alter cell signaling systems. In lower
79 organisms such as mycobacteria, tetrahymena cells and barley seeds, scyllo-inositol containing phosphatidylinositols, phosphatidylinositol-linked glycans and polyphosphoinositols have been observed (Kinnard et al, 1995; Ryals et al, 1999; Salman et al, 1999; Riggs et al, 2007).
However, scyllo-inositol incorporation into phosphatidylinositols has not been observed in higher organisms (Takenawa and Egawa, 1977). This may be due to the reduced binding affinity of phosphatidylinositol synthesizing enzymes for scyllo-inositol, even when levels are elevated (Takenawa and Egawa, 1977; Salman et al, 1999). The final step in the de novo synthesis of phosphatidylinositol is catalyzed by CDP-diglyceride:inositol transferase, which has been found to bind myo-inositol with a Km of 2.5 mM, a reaction for which scyllo-inositol does not appear to compete (Takenawa and Egawa, 1977). When phosphatidylinositol:inositol phosphatidyl transferase activity was examined in rat liver microsomes, scyllo-inositol inhibition of myo-inositol-3H incorporation into phosphatidylinositol was two orders of magnitude lower than that of myo-inositol and had no inhibitory effect if given at the micromolar concentrations found in most tissues (Irvine, 1998). These findings are supported by the present study, which found a lack of scyllo-inositol incorporation into phosphatidylinositols in mice, both under basal conditions and when scyllo-inositol levels were elevated in the brain. These combined results suggest that scyllo-inositol does not directly affect phosphatidylinositol pathways via replacement of myo-inositol and that an increase should not have deleterious effects. In fact, high levels of scyllo–inositol have been observed in a healthy subject with no apparent neurological problems, despite a myo– to scyllo–inositol ratio of 5:1 rather than the more typical 12:1 ratio (Seaquist and Gruetter, 1998).
80
Therefore, baseline myo- and scyllo-inositol levels are comparable between TgCRND8 mice and their wild-type littermate. Ad libitum myo-inositol administration resulted in a significant increase in brain myo-inositol levels in both groups, with no significant difference in uptake observed as a result of disease pathology. Similarly, ad libitum scyllo-inositol administration resulted in a significant increase in brain scyllo-inositol levels. This increase was significantly higher than the increase observed following myo-inositol administration. Both results indicate that myo- and scyllo-inositol, particularly scyllo-inositol, have excellent CNS bioavailability.
This suggests that compounds bound to scyllo-inositol or derivatives of scyllo-inositol that utilize the same transport systems might have similar CNS bioavailability, thus supporting my hypothesis.
CHAPTER 5
Quantification of Inositol Transporter Expression Levels
81 82
Abstract
GC/MS dose studies found both myo- and scyllo-inositol levels elevated in the brain following their oral administration, a finding that is particularly pronounced following scyllo-inositol administration. This elevation in inositol levels occurs across a concentration gradient as a result of active transport from the periphery. Three inositol transporters have been reported in the literature: one hydrogen/myo-inositol transporter (HMIT) and two sodium/myo-inositol transporters (SMIT1, SMIT2). Expression of all three transporters in the brain have been previously reported. However, limited information is available on their subregional expression, how their levels compare to each other and the effects of disease pathology and aging on their expression. Therefore, transporter mRNA levels were examined in four brain regions: the cortex, hippocampus, septum and cerebellum, all known to be affected by disease pathology in
AD, in TgCRND8 mice and control animals at 2, 4 and 6 months of age. The objectives of this study were to: 1. Develop a QPCR method to quantify the mRNA levels of HMIT, SMIT1 and
SMIT2, along with control genes in mouse tissues. 2. Examine the effects of age on transporter expression. 3. Examine the effects of disease pathology on expression, by comparing expression between TgCRND8 mice and their wild-type littermates. 4. Compare transporter expression levels to each other, to determine which transporter is present at the highest levels in each brain region. 5. Compare the expression of each transporter between brain regions. Aging and disease pathology did not alter inositol transporter levels. Overall, brain inositol transporter levels were found in the order HMIT > SMIT1 > SMIT2, however, regional differences in the expression profiles of the three transporters was observed.
83
Introduction
Inositol stereoisomers have shown promise as therapeutic agents in a number of diseases
(Hallman et al, 1986; Hallman et al, 1992; Benjamin et al, 1995; Levine et al, 1995; Fux et al,
1996; Einat et al, 1998; Bersudsky et al, 1999; Nestler et al, 1999; Chengappa et al, 2000;
Gelber et al, 2001; Palatnik et al, 2001; Cogram et al, 2002; Gerli et al, 2003; Eden et al, 2006;
McLaurin et al, 2006). In particular myo- and scyllo-inositol have shown promise as disease therapeutics in psychiatric disorders, respiratory distress syndrome in premature infants and in the TgCRND8 mouse model of AD (Hallman et al, 1986; Hallman et al, 1992; Benjamin et al,
1995; Levine et al, 1995; Fux et al, 1996; Chengappa et al, 2000; Gelber et al, 2001; Palatnik et al, 2001; Eden et al, 2006; McLaurin et al, 2006; Fenili et al. 2007). TgCRND8 mice show many of the hallmark features of AD, including an increase in cerebral Aβ levels, Aβ aggregation and plaque deposition, followed by cognitive deficits as the disease advances
(Chishti et al, 2001). While both myo- and scyllo-inositol were affective at preventing Aβ aggregation in vitro, only scyllo-inositol was found to significantly prevent aggregation in vivo
(McLaurin et al, 2000; 2006). This observed discrepancy between in vitro and in vivo data led to an examination of the effects of myo- and scyllo-inositol administration on their accumulation in the brains of both TgCRND8 mice and control animals (Chapter 4). GC/MS analysis found that scyllo-inositol administration caused a more pronounced increase in scyllo-inositol levels within the brain, when compared to the levels observed following administration of a similar dose of myo-inositol (Chapter 4). The discrepancy in effects of myo- and scyllo-inositol administration on the resulting brain concentrations, points to a potential tight regulation of myo-inositol equilibrium in the brain. This may result because myo-inositol is a key compound for the regulation of osmolarity in the brain and is a constituent of phosphatidylinositol; an
84 important membrane phospholipid and component of second messenger systems, while scyllo- inositol has no known function in the brain or in the periphery (Fisher et al, 2002).
scyllo-Inositol was a more effective treatment for AD in transgenic mice and levels were significantly elevated following its administration, however, both myo- and scyllo-inositol have shown promise as CNS disease therapeutic agents. Physiologically, these two stereoisomers are the most abundant inositol stereoisomers found in the body (Michaelis et al, 1993; Seaquist and
Gruetter, 1998). In the brain, 5 mM of myo–inositol and 0.5 mM of scyllo–inositol have been reported (Michaelis et al, 1993), values 100-fold greater than those found in the periphery
(Palmano et al, 1977). This elevation of inositol levels within the brain compared to the periphery, suggests the presence of active transport systems, in addition to simple diffusion, for the regulation of brain inositol levels. Therefore, the effectiveness of myo-inositol in the treatment of psychiatric ailments and respiratory distress syndrome (Hallman et al, 1986;
Hallman et al, 1992; Benjamin et al, 1995; Levine et al, 1995; Fux et al, 1996; Chengappa et al,
2000; Gelber et al, 2001; Palatnik et al, 2001; Eden et al, 2006) and scyllo-inositol in the treatment of AD in transgenic mice (McLaurin et al, 2006; Fenili et al., 2007) is probably due to ready access to the brain through these transporters.
Active transport of inositol stereoisomers, in particular myo- and scyllo-inositol, occurs via inositol transporters. Three inositol transporters have been reported in the literature, HMIT,
SMIT1 and SMIT2 (Kwon et al, 1992; Uldry et al, 2001; Roll et al, 2002). These inositol transporters exchange one hydrogen atom or two sodium atoms along their concentration gradients, to generate enough energy to actively transport myo-inositol across its concentration gradient (Hager et al, 1995; Uldry et al, 2001; Coady et al, 2002; Bourgeois et al, 2005). While
85 all three transporters are expressed in the brain (Berry et al, 1995; Inoue et al, 1996; Uldry et al,
2001; Roll et al, 2002), there is limited information on the subregional expression of the transporters and no information on how expression changes with disease pathology, such as that which occurs in the TgCRND8 mouse model of AD. In addition, how age affects their expression and how the levels of the three transporters compare to each other has not been previously examined. Therefore, to address these questions, several objectives were set:
1. To develop a QPCR method to quantify the mRNA levels of HMIT, SMIT1 and SMIT2,
standardized against control genes, in mouse tissues.
2. To examine the effects of age on inositol transporter mRNA expression levels, by
examining their expression at 2, 4 and 6 months of age in both TgCRND8 mice and their
wild-type littermates. Time points that correspond to different, key stages in AD pathology
in these animals.
3. To compare inositol transporter mRNA expression levels between TgCRND8 mice and
their wild-type littermates, in order to examine the effects of disease pathology on
expression.
4. To compare the levels of HMIT, SMIT1 and SMIT2 to each other, by quantifying their
expression in four brain regions: the cortex, hippocampus, septum and cerebellum, tissues
that are affected to varying degrees in AD and by Aβ aggregation in TgCRND8 mice.
5. To compare the expression of each inositol transporter between the four brain regions
examined.
86
Results
Inositol transporter primer design
Primers were designed, for each of the three inositol transporters, to quantify their mRNA levels using QPCR. To confirm the effectiveness of primers pairs for amplifying transporter cDNA, concentration curves of cDNA were used to test the amplification efficiency (slope) and accuracy (R2 value) of each primer pair (Figure 5.1a, c, e). In addition, dissociation curve analysis was performed to determine whether one, or multiple, amplification products were generated by each primer pair (Figure 5.1b, d, f). Primers were selected if their slope was between 2.9-3.2, their R2 value was between 0.98-1 and if they produced one main amplification product with minimal, or preferably no, secondary amplification products. The selected SMIT1 and SMIT2 primer pairs showed an excellent level of amplification efficiency and accuracy and only amplified one product. For HMIT, over 30 primer pairs were tested and the primer pair selected showed acceptable amplification efficiency and accuracy with only minor non-specific amplification.
Control gene selection and primer design
Several genes were examined as potential control genes for the QPCR study, based on a literature search of commonly used control genes, including tyrosine kinase receptor (Abl1), a
RNA-dependent ATPase (Bat1), glyceraldehyde-3-phosphate dehydrogenase (Gapdh), ribosomal protein (L32), nitric oxide synthase 2 (Nos2), proteosome 26S subunit ATPase 4
(PSMC4), mitochondrial ribosomal protein (S12), succinate dehydrogenase (SDHA), TATA box binding protein (Tbp) and the transferrin receptor (TFRC). The NCBI, Gene Expression
Omnibus website was used to examine mouse microarray data for each of the potential control genes. Expression of these genes was examined, with the goal of selecting control genes with
87
Figure 5.1 Concentration and dissociation curves for the inositol transporters primers. Concentrations curves (A,C,E) and dissociation curves (B,D,F) were analyzed for HMIT (A,B), SMIT1 (C,D) and SMIT2 (E,F). The concentration curves provided information on the amplification efficiency (slope) and accuracy (R2 value) of each primer set and the dissociation curves were used to check for the amplification of one, main product.
88 similar expression levels in all the brain regions in this study. This is important because inositol transporter gene amplification values are normalized using the control gene values, therefore, uneven control gene amplification between tissues can skew the relative expression values.
Based on an examination of the microarray data, two control genes, Gapdh and Tbp, were chosen (Figure 5.2a, d). Concentration curves were generated, with their corresponding slope and R2 values, for both of these control genes (Figure 5.2b, e). Dissociation curves were also examined to determine if only the product of interested was being generated (Figure 5.2c, f).
The Gapdh primers selected showed an excellent accuracy and efficiency of sequence amplification and the dissociation curve showed no nonspecific amplification. For Tbp primer design, 20 primer pairs were examined and the primer pair selected showed an acceptable level of sequence amplification accuracy and efficiency, with only minor nonspecific amplification.
As expected based on the microarray data, expression levels of the two control genes selected were similar across all the samples analyzed, indicating that they were appropriate control genes for this study. Therefore, transporter gene expression levels were normalized to both of the control genes, as well as within plate and between plate control samples. The resulting relative expression levels were normalized to an average expression of 1 and statistically analyzed.
QPCR was chosen, rather than PCR, for gene expression analysis because it is a more accurate method for quantifying mRNA expression levels. This is because QPCR, through the use of a dye that fluoresces when bound to double-stranded DNA and a machine to quantify fluorescence following each round of DNA amplification, allows the researcher to track DNA amplification and to quantify and compare gene expression when DNA amplification is in an
89
Figure 5.2 Microarray, concentration and dissociation curves for each of the control genes. Microarray data (A,D) from the NCBI, Gene Expression Omnibus website was used to select control genes with comparable expression levels in the regions of interest in the present study (highlighted using blue boxes). Concentration curves (B,E) and dissociation curves (C,F) were used to confirm the accuracy and efficiency of the primers. Based on these studies, Gapdh (A- C) and Tbp (D-F) were selected.
90 exponential growth phase for each gene under analysis. In contrast, PCR amplification only allows the researcher to try and quantify DNA levels after amplification is completed, when some gene amplification will have reached a plateau, while others will still be in an exponential amplification phase. This can result in the over/under estimation of DNA expression.
Inositol transporter expression as a function of age
The mRNA expression of the three inositol transporters was examined as a function of age, by examining their expression at 2, 4 and 6 months of age. No significant differences in the mRNA expression of any of the three inositol transporters was observed as a function of age, either in the TgCRND8 mice or in their wild-type littermates (Figure 5.3-5.5; p > 0.05). Therefore, mRNA expression of all three inositol transporters, remained stable across the time period examined.
Inositol transporter expression as a function of disease
The mRNA expression of the three inositol transporters was compared between TgCRND8 mice and their wild-type littermates at 2, 4 and 6 months of age, to examine the effects of disease pathology on inositol transporter expression. These time points were selected because they correspond to pre-plaque deposition, mid-stage AD and advanced stage AD in TgCRND8 mice
(Chishti et al, 2001). No significant differences were observed between TgCRND8 mice and their wild-type littermates, for any of the transporters, in any of the brain regions examined
(Figure 5.3-5.5; p > 0.05). These findings agree with GC/MS results, which found no differences in myo-inositol uptake between TgCRND8 mice and their wild-type littermates
(Figure 4.7).
91
Figure 5.3 HMIT expression as a function of age in TgCRND8 mice and their wild-type littermates. HMIT expression was examined in wild-type (A-D) and TgCRND8 mice (E-H) in the cortex (A,E), hippocampus (B,F), septum (C,G) and cerebellum (D,H) at 2, 4 and 6 months of age. No significant change in HMIT expression was observed as a function of time in either group. A comparison of HMIT expression between TgCRND8 mice and their wild-type littermates also found no significant differences. (p > 0.05; n = 5 animals per group, 3 wells per sample).
92
Figure 5.4 SMIT1 expression as a function of age in TgCRND8 mice and their wild-type littermates. SMIT1 expression was examined in wild-type (A-D) and TgCRND8 mice (E-H) in the cortex (A,E), hippocampus (B,F), septum (C,G) and cerebellum (D,H) at 2, 4 and 6 months of age. No significant change in SMIT1 expression was observed as a function of time in either group. A comparison of SMIT1 expression between TgCRND8 mice and their wild-type littermates also found no significant differences. (p > 0.05; n = 5 animals per group, 3 wells per sample).
93
Figure 5.5 SMIT2 expression as a function of age in TgCRND8 mice and their wild-type littermates. SMIT2 expression was examined in wild-type (A-D) and TgCRND8 mice (E-H) in the cortex (A,E), hippocampus (B,F), septum (C,G) and cerebellum (D,H) at 2, 4 and 6 months of age. No significant change in SMIT2 expression was observed as a function of time in either group. A comparison of SMIT2 expression between TgCRND8 mice and their wild-type littermates also found no significant differences. (p > 0.05; n = 5 animals per group, 3 wells per sample).
94
A comparison of brain inositol transporter gene expression
An examination of the mRNA expression levels of the three inositol transporters were compared in four regions of the brain: the cortex, hippocampus, septum and cerebellum. These four brain regions were examined because they are affected to varying degrees by Aβ plaque deposition in
AD (Thal et al, 2002). The earliest plaque deposition is observed in the cortex and hippocampus, followed by the septum and finally the cerebellum (Thal et al, 2002). Deposition in the cerebellum is only observed in the very advanced stages of AD and the patient often succumbs to the disease prior to deposition being observed (Thal et al, 2002). The brain mRNA expression levels of the three inositol transporters, in comparison to each other, was found to be significantly different, irrespective of the brain region examined (Figure 5.6; F2,117 = 468.1, p <
0.001). Overall, transporter mRNA expression levels in the brain were in the order HMIT >
SMIT1 > SMIT2. When analyzed in greater detail, HMIT mRNA expression was significantly higher than SMIT1 and SMIT2 expression in all regions of the brain tested (p < 0.001), except for the cerebellum, where HMIT levels were significantly higher than SMIT2 (p < 0.05), but not
SMIT1. SMIT1 mRNA expression was significantly higher than SMIT2 levels in the hippocampus and septum (p < 0.001), but expression of the two transporters was not significantly different in the cortex or the cerebellum.
Regional brain inositol transporter expression levels
An examination of each inositol transporter’s mRNA expression levels between the four brain regions examined was found to be significantly different (F3,86 = 54.87, p < 0.001). In addition, a significant correlation between brain region and the expression levels of each gene were observed (F6,354 = 39.48, p < 0.001). HMIT showed the highest mRNA expression in the cortex and septum and expression of the gene was not significantly different between the two
95
Figure 5.6 Relative mRNA expression of the three inositol transporters in the brain. mRNA expression of the three inositol transporters: HMIT, SMIT1 and SMIT2 were determined in the: A. cortex, B. hippocampus, C. septum and D. cerebellum. In all regions of the brain tested, except the cerebellum, expression occurred in the order HMIT > SMIT1 > SMIT2. (n = 30 animals; * = p < 0.05).
96 regions (Figure 5.7a). The next highest HMIT expression was observed in the hippocampus, which had a significantly lower expression of HMIT than the other two regions (p < 0.001).
Finally, HMIT mRNA expression in the cerebellum was significantly lower than the other three brain regions examined (p < 0.05). Therefore, the order of HMIT expression observed was cortex = septum > hippocampus > cerebellum (Figure 5.7a).
For SMIT1 mRNA, the highest levels of expression were in the septum, followed by the cerebellum, hippocampus and cortex (Figure 5.7b). SMIT1 cerebellular mRNA expression was significantly lower than septal SMIT1 expression (p < 0.05). Cortical and hippocampal SMIT1 mRNA expression were significantly lower than septal expression (p < 0.001) but not cerebellar
SMIT1 expression. Cortical and hippocampal SMIT1 expression were not significantly different from each other. Overall, SMIT1 expression occurred in the order septum > cerebellum ≥ cortex = hippocampus (Figure 5.7b).
In contrast, brain SMIT2 mRNA expression was highest in the cerebellum (Figure 5.7c). The next highest expression was in the septum, followed by the cortex and finally the hippocampus.
Septal and cortical SMIT2 mRNA expression levels were not significantly lower than cerebellar levels. Hippocampal SMIT2 levels were significantly lower than cerebellar and septal levels (p
< 0.005), but not cortical levels. Overall, SMIT2 expression occurred in the order cerebellum ≥ septum = cortex ≥ hippocampus (Figure 5.7c).
97
Figure 5.7 A comparison of regional expression for each of the inositol transporters. A comparison of the regional expression patterns of the three inositol transporters, HMIT (A), SMIT1 (B) and SMIT2 (C) in the brain showed a distinct pattern of expression for each transporter in the brain. While HMIT levels were highest in both the cortex and the septum, SMIT1 levels were highest in the septum alone and SMIT2 levels were highest in the cerebellum. (n = 30 animals; * = p < 0.05).
98
Kidney inositol transporter expression In addition to examining transporter expression levels in the brain, expression was examined in the kidney as a positive control tissue for the QPCR technique. Expression of all three transporters has been previously reported in the kidney (Berry et al, 1995; Uldry et al, 2001;
Roll et al, 2002). Similar to what was observed in the brain, disease pathology and age did not significantly alter mRNA expression for any of the three inositol transporters. However, in contrast to the brain, expression in the kidney occurred in the order SMIT2 > SMIT1 > HMIT
(Figure 5.8). SMIT2 levels were extremely high in the kidney, significantly higher than the expression of the other two transporters (p < 0.001). While HMIT levels were the lowest, significantly lower than the other two transporters (p < 0.001) and could almost be considered negligible.
99
Figure 5.8 Kidney inositol transporter expression. Examination of inositol transporter expression in the kidney showed expression in the order SMIT2 > SMIT1 > HMIT. Relative expression of SMIT2 (A) in the kidney was significantly higher than that of SMIT1 and HMIT (B). HMIT levels were extremely low in this tissue. (red = SMIT1, green = HMIT, n = 30 animals).
100
Discussion
Treatment of psychiatric and neurodegenerative disorders by inositol stereoisomers, requires their transport across the blood-brain and blood-CSF barriers to areas of the brain where they are needed. Since the concentrations of both myo- and scyllo-inositol are 100-fold higher in the brain than their concentrations in the periphery (Palmano et al, 1977), active transport of inositol into the brain is required. There are three inositol transporters that have been reported in the literature: HMIT, SMIT1 and SMIT2 (Kwon et al, 1992; Uldry et al, 2001; Roll et al, 2002), all of which are expressed in the brain (Berry et al, 1995; Inoue et al, 1996; Uldry et al, 2001; Roll et al, 2002). Therefore, active transport of inositol into the brain and between regions of the brain, would be expected to occur through any of these three transporters.
Oral administration of ad libitum myo- or scyllo-inositol to mice increased their corresponding levels in the brain (Chapter 4). Both TgCRND8 mice and their wild-type littermates showed a significant increase in myo-inositol levels in the brain following ad libitum administration
(Chapter 4). Baseline and inositol levels following treatment were comparable between the two groups (Chapter 4). These findings would suggest that TgCRND8 mice and wild-type mice express all three inositol transporters in the brain and that disease pathology does not alter inositol transporter expression levels. This hypothesis is supported by the observations made in the present study, which found matching HMIT, SMIT1 and SMIT2 expression profiles between
TgCRND8 mice and their wild-type littermates at all three of the time points tested. Since these time points correspond to pre-plaque formation, established disease pathology and advanced disease pathology in TgCRND8 mice (Chishti et al, 2001), this indicates that expression of all
101 three inositol transporters is not altered even when AD pathology is in its advanced stages. A stable expression pattern, irrespective of disease pathology, is an important factor for successful
CNS drug design, because it removes one potential confounding variable to shuttling drugs into the brain using these transporters.
In addition to examining the effects of disease pathology on transporter expression, the effect of age was also examined. A comparison of the transporter expression levels, at 2, 4 and 6 months was performed in both TgCRND8 mice and their wild-type littermates, to determine whether drug access to the brain, via these transport systems, would be altered as a function of age.
Fluctuations in inositol transporters with age would complicate drug testing and dose studies by adding another variable to consider. This study indicates that, at least within the age range tested, expression of the three inositol transporters is stable and unaffected by age.
Other variables to consider are the relative expression of the three transporters to each other and their subregional expression in the brain. Based on the literature it is known that all three inositol transporters are expressed in the brain (Berry et al, 1995; Inoue et al, 1996; Uldry et al,
2001; Roll et al, 2002), however, a more detailed examination of their expression profiles, allows for a better understanding of active inositol transport within the brain and between subregions. This study found differences in the relative expression levels of the three transporters. Overall, expression in the brain occurred in the order HMIT > SMIT1 > SMIT2.
Based on this information, compounds designed for transport via HMIT would be expected to reach the brain, and subregions of the brain, in higher quantities than compounds designed for transport through SMIT2. However, each transporter appears to have a distinct regional expression profile as well, which complicates this basic analysis. In the brain regions examined,
102
HMIT expression occurred in the order cortex = septum > hippocampus > cerebellum, while
SMIT1 expression occurred in the order septum > cerebellum ≥ cortex = hippocampus and
SMIT2 expression occurred in the order cerebellum ≥ septum = cortex ≥ hippocampus.
Therefore, if drug targeting to the cerebellum is required, than SMIT2 is the superior transporter, because brain expression levels were highest in the cerebellum, in comparison to the other three brain regions. In contrast, if targeted transport to the septum is required, then SMIT1 might be the better choice. In theory, depending on the transporter chosen, drug accumulation in selected subregions of the brain could be favored, an interesting prospect if increasing drug bioavailability to subregions of the brain were desired.
One caveate to using mRNA quantification to monitor transporter expression profiles is the herteogeneity of mRNA transcript half-lives that exist in the cell. Therefore, a combination of mRNA and protein quantification is usually preferred to add confidence to any conclusions reached regarding gene expression, transcription and translation. For this reason, the original project sought, in addition to monitoring mRNA levels of the transporters, to monitor protein concentrations, using both Western blots and immunohistochemistry. However, repeated efforts were made to create polyclonal antibodies for each of the transporters, without success. In addition, commercially available antibodies for each of the transporters were tested. In both instances, significant background and nonspecific labeling was observed and therefore, this aspect of the project was abandoned.
An additional variable that can be considered for CNS drug design is the rate at which a drug will be excreted from the body. In this study, kidney inositol transporter levels were examined as a positive control, because all three of the inositol transporters are expressed in the kidney
103
(Berry et al, 1995; Uldry et al, 2001; Roll et al, 2002). Since the kidney is the main avenue for inositol removal from the body (Arner et al, 2006), this additional information gives insights into which transporters are important regulators of inositol excretion. As was observed in the brain, inositol transporter expression levels were not altered with either disease pathology or as a function of age in the kidney, therefore suggesting that inositol excretion is unaltered by either variable. A comparison of the expression of the three inositol transporters, relative to each other, found expression in the order SMIT2 > SMIT1 > HMIT. This is the opposite of what was observed in the brain. QPCR analysis found a very high expression of SMIT2 in the kidney, 80- fold higher than the levels observed in the brain. A higher level of SMIT2 expression in the kidney, relative to the brain, has been previously reported in the literature (Roll et al, 2002). In contrast, HMIT levels were much lower in the kidney, relative to the brain, and its levels in the kidney were barely quantifiable. Uldry and colleagues (2001) also reported predominant HMIT expression in the brain, with limited expression in the kidney. This would suggest that targeting drugs to HMIT and SMIT1 would result in higher CNS bioavailability, than targeting drugs to
SMIT2.
In conclusion, inositol transporter expression in both the brain and the kidney is unaltered by disease pathology, as determined by comparing their expression profiles in TgCRND8 mice and their wild-type littermates, or as a function of age. Overall, inositol transporters in the brain were expressed in the order of HMIT > SMIT1 > SMIT2, while kidney inositol transporter expression occurred in the order SMIT2 > SMIT1 > HMIT. Both these findings would suggest that drug transport via HMIT or SMIT1 would have a greater likelihood of entering the brain in quantifiable amounts, than transport via SMIT2. Expression of all three transporters was found in all the brain regions examined, therefore, once a drug is transported into the brain using any
104 of these systems, the drug should be able to travel to any region of the brain. However, the regional expression profile of each transporter was unique. This feature might be exploitable to favorably target drug transport to particular subregions of the brain. An examination of the features of inositol required for transport would give a better indication of the degree to which each transporter system can be manipulated for targeted drug transport.
CHAPTER 6
Substrate Structural Requirements for Inositol Transport
104 105
Abstract
Inositol stereoisomers, such as myo- and scyllo-inositol, are known to cross into the brain and their ad libitum administration has been shown to result in an increase in their brain levels. This indicates that inositol stereoisomers, especially scyllo-inositol, have excellent CNS bioavailability. The main transport mechanism via which inositols enters the brain is the inositol transporters. Given the excellent CNS bioavailability that is possible through these transporters, it may be possible to use them to shuttle other compounds into the brain. To determine whether and to what extent substrates could be adapted and still transported into the brain using this system, the structural features of myo-inositol required for active transport were determined. A competitive transport assay was developed, to quantify myo- and scyllo-inositol transport, in the presence or absence of 23 potential competitive substrates. The substrates tested consisted of inositol stereoisomers, inositol derivatives and other related compounds. The results of these transport assays were then used to draw models of the basic structural features required for active transport. Based on these results, SMIT1 and SMIT2 appear to have different substrate structural requirements for active transport. Active transport through either appears to be very sensitive to changes in the structure of myo-inositol and it is concluded that only conservative changes are possible to maintain active transport.
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Introduction
One of the main challenges in the treatment of CNS diseases is transport of drugs across the brain barriers to regions of bioactivity. There are several strategies in which this can be accomplished. One possibility is to design drugs irrespective of this concern and try and use barrier circumvention strategies, such as intranasal delivery or osmotic disruption, to avoid the brain barriers entirely or temporarily open them to allow the drugs to get through. Alternatively, barrier navigation strategies, such as nanoparticle delivery or a Trojan horse delivery system can be used to convert a compound that cannot enter the brain into one that can. A final strategy is to try and use a compound, which enters the brain for other purposes, as a drug candidate and potentially as a drug carrier.
scyllo-Inositol is an example of a compound that is present in the body, crosses into the brain naturally and is an effective drug for the treatment of AD in TgCRND8 mice (Fisher et al, 2002;
McLaurin et al, 2006). In a previous chapter of this thesis, I showed that ad libitum administration resulted in a sustained increase in scyllo-inositol levels within the brain (Chapter
4). In addition, twice-daily scyllo-inositol treatment was an effective treatment of disease pathology in TgCRND8 mice (McLaurin et al, 2006). Both these results indicate that scyllo- inositol has excellent CNS bioavailability and suggests that derivatives of scyllo-inositol or compounds attached to scyllo-inositol might show the same rates of CNS bioavailability. In order for these compounds to be successfully transported into the brain, they need to cross the brain barriers. Three transporters have been reported in the literature as being inositol transporters: HMIT, SMIT1 and SMIT2, (Hager et al, 1995; Ostlund et al, 1996; Wiese et al,
1996; Lubrich et al, 2000; Uldry et al, 2001; Coady et al, 2002; Berry et al, 2003; Bissonnette et
107 al, 2004; Uldry et al, 2004; Bourgeois et al, 2005; Aouameur et al, 2007; Lahjouji et al, 2007;
Shaldubina et al, 2007; Bissonnette et al, 2008; Lin et al, 2009).
HMIT is mainly expressed in the brain, with limited expression in the kidney and adipose tissue
(Uldry et al, 2001). In the brain, mRNA expression was observed in both neurons and glia, with high levels found in all the brain regions examined: cerebral cortex, hippocampus, hypothalamus, cerebellum and brainstem (Uldry et al, 2001). In a previous chapter of this thesis, I recorded high brain HMIT mRNA expression levels in all four of the brain regions examined: cerebral cortex, hippocampus, septum and cerebellum (Chapter 5). Again, in agreement with previous reports, I found kidney HMIT mRNA expression levels to be extremely low (Chapter 5). In cells, HMIT is internalized because of three internalization sequences in the protein code of HMIT, but cell surface translocation has been reported to occur following membrane depolarization or a decrease in extracellular pH; conditions that are typical byproducts of synaptic activity (Uldry et al, 2001; 2004).
In contrast to HMIT, SMIT1 has a much wider mRNA expression pattern, with expression reported in the kidney, brain, placenta, pancreas, heart, skeletal muscle and in the lung (Berry et al, 1995). In the brain, SMIT1 mRNA expression is highest in the choroid plexus (Inoue et al,
1996). In addition, expression has been reported in the hippocampus, locus coeruleus, the suprachiasmatic nucleus, the olfactory bulb and in certain parts of the cerebellum (Inoue et al,
1996). In Chapter 5, I showed, using QPCR, expression of SMIT1 in all four of the brain regions studied: cerebral cortex, hippocampus, septum and cerebellum (Chapter 5). Based on
SMIT1 knockout studies, SMIT1 appears to be the main transporter responsible for maintaining myo-inositol levels both in the brain and in the periphery (Berry et al, 2003). Homozygous
108 knockout mice showed a 92% reduction in brain myo-inositol levels and an 84% reduction in myo-inositol levels in the periphery (Berry et al, 2003). Heterozygous mice showed a 15% reduction in frontal cortex myo-inositol levels and a 25% reduction in hippocampal myo-inositol levels, suggesting these regions are particularly dependent on SMIT1 activity (Shaldubina et al,
2007). This high dependence of myo-inositol levels on SMIT1 transport suggests that designing drugs to target this transporter might be a novel strategy for maximizing drug transport into the brain, particularly to the frontal cortex and hippocampus.
The final transporter, SMIT2, is part of the same sodium/solute symporter family as SMIT1.
Like SMIT1, SMIT2 shows a wide level of expression in the periphery, with the highest levels of expression reported in the kidney, liver, heart, skeletal muscles and placenta (Roll et al,
2002). However, unlike what is observed for SMIT1, SMIT2 expression in the brain is much lower (Roll et al, 2002). Using QPCR, I showed very high levels of SMIT2 mRNA expression in the mouse kidney and minimal expression in the brain (Chapter 5).
Based on the literature and my work presented in previous chapters of this thesis, it was concluded that all three of the inositol transporters are expressed in the brain and all three are potential portals for drug delivery to the CNS. Therefore, the objective of this study was to characterize the structural features of inositol required for active transport through HMIT,
SMIT1 and SMIT2. A competitive transport assay was developed, to quantify myo- and scyllo- inositol transport, in the presence or absence of potential competitive substrates, consisting of inositol stereoisomers, derivatives and related compounds (Figure 6.1). This technique allowed for an estimation of the structural features of inositol required for active transport by HMIT,
SMIT1 and SMIT2, with the caveate that transport channel blockers would also appear to be
109
Figure 6.1 Structures of the inositol stereoisomers, derivatives and related compounds. The structures of the inositol stereoisomers, derivatives and related compounds used for the initial competitive transport assays.
110 transported by this system. To alleviate the possibility that channel blockers would be misinterpreted as transported substrates, a model was drawn of the structural features required for active transport and a comparison was made between this model and both compounds apparently transported and not transported by this system. Using this technique, I was able to identify one SMIT transport channel blocker, L-fucose, and additional experiments were conducted to support this conclusion.
Results
Interspecies and intertransporter protein homology
Before examining the transport substrate specificity of each of the three transporters, interspecies protein alignment studies were conducted between the human, mouse and rat transporter sequences to see the degree of evolutionarily conservation. A large degree of variability in the protein sequences between species would complicate substrate transport studies, by requiring species-specific substrate transport models be designed. However, alignment of the protein sequences showed all three transporter sequences are very similar between the species examined (Figure 6.2-6.4). Overall, homology of the human, mouse and rat sequences was 90% for HMIT, 94% for SMIT1 and 89% for SMIT2. The HMIT protein was
87% homologous between rats and humans, 88% homologous between mice and humans and
95% homologous between rats and mice (Figure 6.2). The SMIT1 sequence was 92% homologous between rats and humans, 94% homologous between mice and humans and 96% homologous between rats and mice (Figure 6.3). Finally, the SMIT2 protein was 86% homologous between the human and both the rat and mouse sequences and 94% homologous between the rat and mouse sequences (Figure 6.4).
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Figure 6.2 Interspecies protein alignment for HMIT. An alignment of the human, mouse and rat HMIT proteins, to compare their amino acid sequences. Dots and dotted lines indicate gaps in the sequence between species, added for the purposes of an improved alignment. The consensus line provides information on where the three sequences match (wherever an amino acid is listed) and where a difference exists (blank spaces). Positive amino acids are shown in red, negative amino acids in bold, while the rest are neutral. The human sequence is 88% and 87% homologous with the mouse and rat sequences, respectively, while the mouse and rat sequences are 95% homologous with each other. The protein sequences were acquired using the NCBI database. Accession Numbers: human = NM_052885, mouse = NM_001033633 and rat = NM_133611.
Human MSRKASENVEYTLRSLSSLMGERRRKQPEPDAASAAGECS 40 Mouse MSRKASEdVEYTLRSLSSLMGERRRrQPEPgApg..GErS 38 Rat ...... MGERRRrQPEPgApg..GErS 19 Consensus mgerrr qpep a ge s
Human LLAAAESSTSLQSAGAGGGGVGDLERAARRQFQQDETPAF 80 Mouse LL.AAESaaSLQgAe...... LERAARRQFQrDETPAF 69 Rat LL.AAESaaSLQgAe...... LERAARRQFQrDETPAF 50 Consensus ll aaes slq a leraarrqfq detpaf
Human VYVVAVFSALGGFLFGYDTGVVSGAMLLLKRQLSLDALWQ 120 Mouse VYaaAaFSALGGFLFGYDTGVVSGAMLLLrRQmrLgAmWQ 109 Rat VYaaAaFSALGGFLFGYDTGVVSGAMLLLrRQmrLgAmWQ 90 Consensus vy a fsalggflfgydtgvvsgamlll rq l a wq
Human ELLVSSTVGAAAVSALAGGALNGVFGRRAAILLASALFTA 160 Mouse ELLVSgaVGAAAVaALAGGALNGalGRRsAILLASALcTv 149 Rat ELLVSgaVGAAAVaALAGGALNGalGRRsAILLASALcTv 130 Consensus ellvs vgaaav alaggalng grr aillasal t
Human GSAVLAAANNKETLLAGRLVVGLGIGIASMTVPVYIAEVS 200 Mouse GSAVLAAAaNKETLLAGRLVVGLGIGIASMTVPVYIAEVS 189 Rat GSAVLAAAaNKETLLAGRLVVGLGIGIASMTVPVYIAEVS 170 Consensus gsavlaaa nketllagrlvvglgigiasmtvpvyiaevs
Human PPNLRGRLVTINTLFITGGQFFASVVDGAFSYLQKDGWRY 240 Mouse PPNLRGRLVTINTLFITGGQFFASVVDGAFSYLQKDGWRY 229 Rat PPNLRGRLVTINTLFITGGQFFASVVDGAFSYLQKDGWRY 210 Consensus ppnlrgrlvtintlfitggqffasvvdgafsylqkdgwry
Human MLGLAAVPAVIQFFGFLFLPESPRWLIQKGQTQKARRILS 280 Mouse MLGLAAiPAVIQFlGFLFLPESPRWLIQKGQTQKARRILS 269 Rat MLGLAAiPAVIQFlGFLFLPESPRWLIQKGQTQKARRILS 250 Consensus mlglaa paviqf gflflpesprwliqkgqtqkarrils
Human QMRGNQTIDEEYDSIKNNIEEEEKEVGSAGPVICRMLSYP 320 Mouse QMRGNQTIDEEYDSIrNsIEEEEKEataAGPiICRMLSYP 309 Rat QMRGNQTIDEEYDSIrNsIEEEEKEasaAGPiICRMLSYP 290
112
Consensus qmrgnqtideeydsi n ieeeeke agp icrmlsyp
Human PTRRALIVGCGLQMFQQLSGINTIMYYSATILQMSGVEDD 360 Mouse PTRRALvVGCGLQMFQQLSGINTIMYYSATILQMSGVEDD 349 Rat PTRRALaVGCGLQMFQQLSGINTIMYYSATILQMSGVEDD 330 Consensus ptrral vgcglqmfqqlsgintimyysatilqmsgvedd
Human RLAIWLASVTAFTNFIFTLVGVWLVEKVGRRKLTFGSLAG 400 Mouse RLAIWLASiTAFTNFIFTLVGVWLVEKVGRRKLTFGSLAG 389 Rat RLAIWLASiTAFTNFIFTLVGVWLVEKVGRRKLTFGSLAG 370 Consensus rlaiwlas taftnfiftlvgvwlvekvgrrkltfgslag
Human TTVALIILALGFVLSAQVSPRITFKPIAPSGQNATCTRYS 440 Mouse TTVALIILALGFlLSAQVSPRvTFrPttPSdQNtTCTgYS 429 Rat TTVALtILALGFlLSAQVSPRvTFrPtAPSGQNATCTeYS 410 Consensus ttval ilalgf lsaqvspr tf p ps qn tct ys
Human YCNECMLDPDCGFCYKMNKSTVIDSSCVPVNKASTNEAAW 480 Mouse YCNECMLDPDCGFCYKiNgSaVIDSSCVPVNKASTtEAAW 469 Rat YCNECMLDPDCGFCYKiNsSaVIDSSCVPVNKASTNEAAW 450 Consensus ycnecmldpdcgfcyk n s vidsscvpvnkast eaaw
Human GRCENETKFKTEDIFWAYNFCPTPYSWTALLGLILYLVFF 520 Mouse GRCdNETKFKaEgahWAYsFCPTPYSWTALvGLvLYLVFF 509 Rat GRCENETKFKaEDvhWAYsFCPTPYSWTALvGLvLYLVFF 490 Consensus grc netkfk e way fcptpyswtal gl lylvff
Human APGMGPMPWTVNSEIYPLWARSTGNACSSGINWIFNVLVS 560 Mouse APGMGPMPWTVNSEIYPLWARSTGNACSaGINWIFNVLVS 549 Rat APGMGPMPWTVNSEIYPLWARSTGNACSaGINWIFNVLVS 530 Consensus apgmgpmpwtvnseiyplwarstgnacs ginwifnvlvs
Human LTFLHTAEYLTYYGAFFLYAGFAAVGLLFIYGCLPETKGK 600 Mouse LTFLHTAEYLTYYGAFFLYAGFAAVGLLFvYGCLPETKGK 589 Rat LTFLHTAEYLTYYGAFFLYAGFAAVGLLFvYGCLPETKGK 570 Consensus ltflhtaeyltyygafflyagfaavgllf ygclpetkgk
Human KLEEIESLFDNRLCTCGTSDSDEGRYIEYIRVKGSNYHLS 640 Mouse KLEEIESLFDhRLCsCGaaDSDEGRYIEYIRVKGSNYHLS 629 Rat KLEEIESLFDhRLCTCGTaDSDEGRYIEYIRVKGSNYHLS 610 Consensus kleeieslfd rlc cg dsdegryieyirvkgsnyhls
Human DNDASDVE 648 Mouse DNDASDVE 637 Rat DNDASDVE 618 Consensus dndasdve
113
Figure 6.3 Interspecies protein alignment for SMIT1. An alignment of the human, mouse and rat SMIT1 proteins, to compare their amino acid sequences. Dots and dotted lines indicate gaps in the sequence between species, added for the purposes of an improved alignment. The consensus line provides information on where the three sequences match (wherever an amino acid is listed) and where a difference exists (blank spaces). Positive amino acids are shown in red, negative amino acids in bold, while the rest are neutral. The human sequence is 94% and 92% homologous with the mouse and rat sequences, respectively, while the mouse and rat sequences are 96% homologous with each other. The protein sequences were acquired using the NCBI database. Accession Numbers: human = NM_006933, mouse = NM_017391 and rat = NM_053715.
Human MRAVLDTADIAIVALYFILVMCIGFFAMWKSNRSTVSGYF 40 Mouse MRAVLeaADIAvVALYFILVMCIGFFAMWKSNRSTVSGYF 40 Rat MRAVLeTADIAIVALYFvLVMCIGFFAMWKSNRSTVSGYF 40 Consensus mravl adia valyf lvmcigffamwksnrstvsgyf
Human LAGRSMTWVTIGASLFVSNIGSEHFIGLAGSGAASGFAVG 80 Mouse LAGRSMTWVaIGASLFVSNIGSEHFIGLAGSGAASGFAVG 80 Rat LAGRSMTWVaIGASLFVSNIGSEHFIGLAGSGAASGFAVG 80 Consensus lagrsmtwv igaslfvsnigsehfiglagsgaasgfavg
Human AWEFNALLLLQLLGWVFIPIYIRSGVYTMPEYLSKRFGGH 120 Mouse AWEFNALLLLQLLGWVFIPIYIRSGVYTMPEYLSKRFGGH 120 Rat AWEFNALLLLQLLGWVFIPIYIRSGVYTMPEYLSKRFGGH 120 Consensus awefnallllqllgwvfipiyirsgvytmpeylskrfggh
Human RIQVYFAALSLILYIFTKLSVDLYSGALFIQESLGWNLYV 160 Mouse RIQVYFAALSLlLYIFTKLSVDLYSGALFIQESLGWNLYV 160 Rat RIQVYFAALSLlLYIFTKLSVDLYSGALFIQESLGWNLYV 160 Consensus riqvyfaalsl lyiftklsvdlysgalfiqeslgwnlyv
Human SVILLIGMTALLTVTGGLVAVIYTDTLQALLMIIGALTLM 200 Mouse SVILLIGMTALLTVTGGLVAVIYTDTLQALLMIIGALTLM 200 Rat SVILLIGMTALLTVTGGLVAVIYTDTLQALLMIIGALTLM 200 Consensus svilligmtalltvtgglvaviytdtlqallmiigaltlm
Human IISIMEIGGFEEVKRRYMLASPDVTSILLTYNLSNTNSCN 240 Mouse vISmvkIGGFEEVKRRYMLASPDVaSILLkYNLSNTNaCm 240 Rat vISmMkvGGFEEVKRRYMLASPnVaSILLThNLSNTNSCm 240 Consensus is ggfeevkrrymlasp v sill nlsntn c
Human VSPKKEALKMLRNPTDEDVPWPGFILGQTPASVWYWCADQ 280 Mouse VhPKanALKMLRdPTDEDVPWPGFILGQTPASVWYWCADQ 280 Rat VhPKadALKMLRdPTDEDVPWPGFILGQTPASVWYWCADQ 280 Consensus v pk alkmlr ptdedvpwpgfilgqtpasvwywcadq
Human VIVQRVLAAKNIAHAKGSTLMAGFLKLLPMFIIVVPGMIS 320 Mouse VIVQRVLAAKNIAHAKGSTLMAGFLKLLPMFIIVVPGMIS 320 Rat VIVQRVLAAKNIAHAKGSTLMAGFLKLLPMFIIVVPGMIS 320
114
Consensus vivqrvlaakniahakgstlmagflkllpmfiivvpgmis
Human RILFTDDIACINPEHCMLVCGSRAGCSNIAYPRLVMKLVP 360 Mouse RIvFaDeIACINPEHCMqVCGSRAGCSNIAYPRLVMtLVP 360 Rat RILFvDDIACINPEHCMqVCGSRAGCSNIAYPRLVMeLVP 360 Consensus ri f d iacinpehcm vcgsragcsniayprlvm lvp
Human VGLRGLMMAVMIAALMSDLDSIFNSASTIFTLDVYKLIRK 400 Mouse VGLRGLMMAVMIAALMSDLDSIFNSASTIFTLDVYKLIRK 400 Rat VGLRGLMMAVMIAALMSDLDSIFNSASTIFTLDVYKLlRn 400 Consensus vglrglmmavmiaalmsdldsifnsastiftldvykl r
Human SASSRELMIVGRIFVAFMVVISIAWVPIIVEMQGGQMYLY 440 Mouse SASSRELMIVGRIFVAFMVVISIAWVPIIVEMQGGQMYLY 440 Rat nASSRELMIVGRIFVAFMVVISIAWVPIIVEMQGGQMYLY 440 Consensus assrelmivgrifvafmvvisiawvpiivemqggqmyly
Human IQEVADYLTPPVAALFLLAIFWKRCNEQGAFYGGMAGFVL 480 Mouse IQEVADYLTPPVAALFLLAIFWKRCNEQGAFYGGMAGFVL 480 Rat IQEVADYLTPPVAALFLLAIFWKRCNEQGAFYGGMAGFVL 480 Consensus iqevadyltppvaalfllaifwkrcneqgafyggmagfvl
Human GAVRLILAFAYRAPECDQPDNRPGFIKDIHYMYVATGLFW 520 Mouse GAVRLILAFtYRAPECDQPDNRPGFIKDIHYMYVATaLFW 520 Rat GAiRLILAFtYRAPECDQPDNRPsFIKDIHYMYVATaLFW 520 Consensus ga rlilaf yrapecdqpdnrp fikdihymyvat lfw
Human VTGLITVIVSLLTPPPTKEQIRTTTFWSKKNLVVKENCSP 560 Mouse iTGLITVIVSLLTPPPTKdQIRTTTFWSKKtLVtKEsCSq 560 Rat iTGLITVIVSLLTPPPTKdQIRTTTFWSKKtvVtKEsCSq 560 Consensus tglitvivslltppptk qirtttfwskk v ke cs
Human KEEPYQMQEKSILRCSENNETINHIIPNGKSEDSIKGLQP 600 Mouse KdEPYkMQEKSILqCSENsEvIsHtIPNGKSEDSIKGLQP 600 Rat KdEPYkMQEKSILRCSENsEvvsHtIPNGKSEDSIKGLQP 600 Consensus k epy mqeksil csen e h ipngksedsikglqp
Human EDVNLLVTCREEGNPVASLGHSEAETPVDAYSNGQAALMG 640 Mouse EDVNLLVTCREEGNPVASmGHSEAETPVDAYSNGQAALMG 640 Rat EDVNLLVTCREEGNPVASmGHSEAETPVDAYSNGQAALMG 640 Consensus edvnllvtcreegnpvas ghseaetpvdaysngqaalmg
Human EKERKKETDDGGRYWKFIDWFCGFKSKSLSKRSLRDLMEE 680 Mouse ErEReKETenrsRYWKFIDWFCGFKSKSLSKRSLRDLMdE 680 Rat EraReKETesrsRYWKFIDWFCGFKSrSLSKRSLRDsMdE 680 Consensus e r ket rywkfidwfcgfks slskrslrd m e
Human EAVCLQMLEETRQVKVILNIGLFAVCSLGIFMFVYFSL 718 Mouse EAVCLQMLEETpQVKVILNIGLFAVCSLGIFMFVYFSL 718 Rat EAVCLQMLEETprVrVILNIGLFAVCSLGIFMFVYFSL 718 Consensus eavclqmleet v vilniglfavcslgifmfvyfsl
115
Figure 6.4 Interspecies protein alignment for SMIT2. An alignment of the human, mouse and rat SMIT2 proteins, to compare their amino acid sequences. Dots and dotted lines indicate gaps in the sequence between species, added for the purposes of an improved alignment. The consensus line provides information on where the three sequences match (wherever an amino acid is listed) and where a difference exists (blank spaces). Positive amino acids are shown in red, negative amino acids in bold, while the rest are neutral. The human sequence is 86% homologous with both the mouse and rat sequences, while the mouse and rat sequences are 94% homologous with each other. The protein sequences were acquired using the NCBI database. Accession Numbers: human = NM_052944, mouse = NM_146198 and rat = NM_00100482.
Human MESGTSSPQPPQLDPLDAFPQKGLEPGDIAVLVLYFLFVL 40 Mouse MESaTiSPQPPQsDsLeAFPQKsmEPaDIAVLVLYFLFVL 40 Rat MEStTSSPQPPpsDaLeAFPQKsmEPaDIvVLVLYFLFVL 40 Consensus mes t spqpp d l afpqk ep di vlvlyflfvl
Human AVGLWSTVKTKRDTVKGYFLAGGDMVWWPVGASLFASNVG 80 Mouse AVGLWSTVrTKRDTVKGYFLAGGDMVWWPVGASLFASNVG 80 Rat AVGLWSTVrTKRDTVKGYFLAGGDMVWWPVGASLFASNVG 80 Consensus avglwstv tkrdtvkgyflaggdmvwwpvgaslfasnvg
Human SGHFIGLAGSGAATGISVSAYELNGLFSVLMLAWIFLPIY 120 Mouse SGHFIGLAGSGAAvGISVaAYELNGLFSVLMLAWvFLPIY 120 Rat SGHFIGLAGSGAAvGISVaAYELNGLFSVLMLAWIFLPIY 120 Consensus sghfiglagsgaa gisv ayelnglfsvlmlaw flpiy
Human IAGQVTTMPEYLRKRFGGIRIPIILAVLYLFIYIFTKISV 160 Mouse IAGQVTTMPEYLRrRFGGnRIsItLAVLYLFIYIFTKISV 160 Rat IAGQVTTMPEYLkrRFGGsRIPItLAVLYLFIYIFpilqV 160 Consensus iagqvttmpeyl rfgg ri i lavlylfiyif v
Human DMYAGAIFIQQSLHLDLYLAIVGLLAITAVYTVAGGLAAV 200 Mouse DMYAGAIFIQQSLHLDLYLAIVGLLAITAlYTVAGGLAAV 200 Rat DMYAGAIFIQQSLHLDLYLAIVGLLAvTAlYTVAGGLAAV 200 Consensus dmyagaifiqqslhldlylaivglla ta ytvagglaav
Human IYTDALQTLIMLIGALTLMGYSFAAVGGMEGLKEKYFLAL 240 Mouse IYTDALQTvIMLIGAfiLMGYSFAAVGGMEGLKdqYFLAL 240 Rat IYTDALQTvIMLIGAfiLMGYSFAAVGGMEGLKdqYFLAL 240 Consensus iytdalqt imliga lmgysfaavggmeglk yflal
Human ASNRSENSSCGLPREDAFHIFRDPLTSDLPWPGVLFGMSI 280 Mouse ASNRSENSSCGLPREDAFHIFRDPLTSDLPWPGiLFGMSI 280 Rat ASNRSENSSCGLPREDAFHIFRDPLTSDLPWPGiLFGMSI 280 Consensus asnrsensscglpredafhifrdpltsdlpwpg lfgmsi
Human PSLWYWCTDQVIVQRTLAAKNLSHAKGGALMAAYLKVLPL 320 Mouse PSLWYWCTDQVIVQRsLAAKNLSHAKGGsLMAAYLKVLPL 320 Rat PSLWYWCTDQVIVQRsLAAKNLSHAKGGsLMAAYLKVLPL 320
116
Consensus pslwywctdqvivqr laaknlshakgg lmaaylkvlpl
Human FIMVFPGMVSRILFPDQVACADPEICQKICSNPSGCSDIA 360 Mouse FlMVFPGMVSRvLFPDQVACAhPdICQrvCSNPSGCSDIA 360 Rat FlMVFPGMVSRILFPDQVACAhPdICQrvCSNPSGCSDIA 360 Consensus f mvfpgmvsr lfpdqvaca p icq csnpsgcsdia
Human YPKLVLELLPTGLRGLMMAVMVAALMSSLTSIFNSASTIF 400 Mouse YPKLVLELLPTGLRGLMMAVMVAALMSSLTSIFNSASTIF 400 Rat YPKLVLELLPTGLRGLMMAVMVAALMSSLTSIFNSASTIF 400 Consensus ypklvlellptglrglmmavmvaalmssltsifnsastif
Human TMDLWNHLRPRASEKELMIVGRVFVLLLVLVSILWIPVVQ 440 Mouse TMDLWNHiRPRASErELMIVGRiFVfaLVLVSILWIPiVQ 440 Rat TMDLWhHiRPRASErELMIVGRVFVLaLVLVSILWIPVVQ 440 Consensus tmdlw h rprase elmivgr fv lvlvsilwip vq
Human ASQGGQLFIYIQSISSYLQPPVAVVFIMGCFWKRTNEKGA 480 Mouse ASQGGQLFIYIQSISSYLQPPVAmVFIMGCFWKRTNEKGA 480 Rat ASQGGQLFIYIQSISSYLQPPVAVVFIMGCFWKRTNEKGA 480 Consensus asqggqlfiyiqsissylqppva vfimgcfwkrtnekga
Human FWGLISGLLLGLVRLVLDFIYVQPRCDQPDERPVLVKSIH 520 Mouse FsGLIlGLLLGLVRLiLDFvYaQPRCDQPDdRPavVKdvH 520 Rat FsGLIlGLLLGLVRLiLDFvYVQPRCDQPDdRPavVKdvH 520 Consensus f gli glllglvrl ldf y qprcdqpd rp vk h
Human YLYFSMILSTVTLITVSTVSWFTEPPSKEMVSHLTWFTRH 560 Mouse YLYFSMILSftTLITVvTVSWFTEtPSKEMVSrLTWFTRH 560 Rat YLYFSMILSstTLITVfTVSWFTEtPSKEMVSrLTWFTRH 560 Consensus ylyfsmils tlitv tvswfte pskemvs ltwftrh
Human DPVVQKEQAPPAAPLSLTLSQNGMPEASSSSSVQFEMVQE 600 Mouse ePVaQKdsAPPetPLSLTLSQNGttEApgtSiqlet.VQE 599 Rat ePVaQKdsvPPenPLSLTiSQNGttEAtgiSiqles.VQE 599 Consensus pv qk pp plslt sqng ea s vqe
Human NTSKTHSCDMTPKQSKVVKAILWLCGIQEKGKEELPARAE 640 Mouse sTtKacgdgvsPrhSKVVrAILWLCGm.EKnKEEpPskAE 638 Rat aTtKaHSdgvsPKQSKVlKAILWLCGm.EKdKEEpPskvE 638 Consensus t k p skv ailwlcg ek kee p e
Human AIIVSLEENPLVKTLLDVNLIFCVSCAIFIWGYFA 675 Mouse pvIVSLEENPLVKTLLDVNcIvCiSCAIFlWGYFA 673 Rat pvIVSLEENPLVKTLLDVNcIvCiSCAIFlWGYFA 673 Consensus ivsleenplvktlldvn i c scaif wgyfa
117
In addition to examining transporter homology between species, intertransporter homology was examined for each of the three species (Table 6.1). HMIT, SMIT1 and SMTI2 are all part of glucose transporter superfamilies; HMIT is a member of the solute carrier family 2, facilitated glucose transporter superfamily, while both SMIT1 and SMIT2 are part of the solute carrier family 5, sodium/glucose cotransporter superfamily. Therefore, we were curious to see how closely the three proteins would be homologous. Across the three species examined, HMIT was
6-11% homologous to SMIT1/2, while SMIT1 and SMIT2 were 40-43% homologous to each other. This indicates that, while the transporter protein sequences are very similar between species, the three inositol transporters are very different from each other and therefore would be predicted to have significantly different substrate structural requirements.
Table 6.1 Comparison of the protein homology of the three inositol transporters to each other in humans, mice and rats.
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To analyze the substrate structural requirements for active transport by each inositol transporter, competitive transport assays were developed. For these assays, cultures of cell-lines or primary cells were grown to 90% confluency, washed with PBS, and incubated in medium containing myo- or scyllo-inositol-(2-3H), without or with potential substrate competitors. Once the incubation period was completed, cells were washed with PBS to stop radioactive uptake and myo- and scyllo-inositol-(2-3H) levels were quantified using a liquid scintillation counter.
HMIT activation
The literature reports that HMIT is internalized in the cell and translocates to the cell surface in response to cellular changes such as depolarization of the cell membrane or acidification of the extracellular environment, both of which occur following synaptic activity (Uldry et al, 2001).
Based on these findings, four conditions were used to induce HMIT translocation to the cell surface, with the goal of studying its transporter kinetics. Two conditions were modeled on the paper by Uldry and colleagues (2001) and involved either cell surface depolarization, through an elevation in external potassium levels, and/or acidification of the extracellular environment.
The second two conditions try to induce HMIT activity through the inhibition of the sodium myo-inositol transporters. Since both SMIT1 and SMIT2 cotransport myo-inositol with two sodium ions, they are inhibited by phlorizin, a sodium-dependent transport inhibitor (Hager et al, 1995; Coady et al, 2002). Therefore, the effect of phlorizin-induced SMIT1/2 inhibition on
HMIT activity was examined (Figure 6.5a). Alternatively, SMIT1/2 silencing was induced, through a combination of prewashing cells in Na+-free medium, and using Na+-free medium during the competition transport assay (Figure 6.5b). While both of these techniques were effective at silencing SMIT1/2 activity, they did not result in HMIT activation (Figure 6.5c). In
119
Figure 6.5 HMIT transport activation. HMIT is internalized in cells and activation of the transporter requires translocation to the cell surface. This has been reported to occur following cell surface depolarization and/or acidification of the extracellular environment (Uldry et al, 2001). Based on these observations, multipe conditions to activate HMIT in HEK293 cells were examined, including silencing SMIT1/2 using phloridzin (A,C) and conducting the transport assay in sodium-free PBS (B). Based on an analysis of myo-inositol-(2-3H) transport in the presence of increasing concentrations of phloridzin (A), myo-inositol-(2-3H) competitive transport was tested in the presence of 2 mM phloridzin to test for activation (C). myo-Inositol-(2-3H) transport was eliminated by both phloridzin treatment and testing in sodium-free PBS, with only background radioactivity recorded in both conditions. (n = 3 wells per variable).
120 order to isolate HMIT activity during depolarization and acidification tests, these tests were conducted in the presence of phlorizin and/or Na+-free medium to silence SMIT1/2 activity.
Again, while both phlorizin and Na+-free medium were successful at silencing SMIT1/2 activity, none of the four conditions, even when combined, resulted in HMIT activation.
In order to rule out cell-dependent induction of HMIT activity, these tests were conducted in a number of cell lines including: 1321N1 human neuroblastoma cell-line, SH-SY5Y human astrocytoma cell-line, rat primary cortical neurons, as well as other cell-lines which, using
QPCR were found to express HMIT mRNA. In all the cells studied, no HMIT transport activity was ever observed. For example, in mouse cortical primary neurons, incubated in sodium-free medium (pH 6.0, 80 mM KCl) supplemented with myo-inositol-(2-3H) for 3 hours, only a small current, probably attributable to residual SMIT1/2 activity was observed (Figure 6.6a). When these cells were tested for competitive transport, again only background noise levels of radioactivity were observed and no competitive transport was found (Figure 6.6b). In agreement with my studies, a recent report by Di Daniel and colleagues (2009), conducted on both rat primary cortical neurons in culture and rat cortical slice cultures, also failed to observe
HMIT translocation to the cell surface following changes in pH or depolarization, despite initial reports to the contrary (Uldry et al, 2001; 2004). Therefore, these findings remove HMIT as a viable candidate for drug transport studies, despite the high levels of mRNA expression in the brain. Focus now shifted to SMIT1 and SMIT2 transport.
121
Figure 6.6 Measurement of HMIT myo-inositol transport in primary cells. An examination of HMIT transport in rat cortical primary neurons grown in culture for 3 weeks was conducted in sodium-free medium, supplemented with 80 mM of KCl to induce cell surface depolarization. No appreciable myo-inositol-(2-3H) transport was observed. A myo-inositol-(2- 3H) concentration curve showed only a small level of transport, likely due to residual SMIT1/2 activity (A) and no measurable competitive transport was observed in these cells (B). (n = 3 wells per variable).
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SMIT1 and SMIT2 substrate requirements
To examine SMIT1/2 substrate structural requirements, competition assays were conducted in the human endothelial kidney cell-line (HEK293), which was found, using QPCR, to express
SMIT1 and SMIT2 but not HMIT (Figure 6.7). The transport properties of both myo- and scyllo-inositol-(2-3H) were individually examined in these cells, in the presence or absence of 23 potential substrate competitors (Figure 6.1). Using this technique, different substrates were found to compete with myo- and scyllo-inositol-(2-3H) for transport. The substrates myo-, scyllo-, D-chiro-inositol, L-fucose, viburnitol, D-glucose, D-mannose, sequoyitol and D-pinitol competed with myo-inositol-(2-3H) transport, while only myo-, scyllo-, D-chiro-inositol, L- fucose and viburnitol competed with scyllo-inositol-(2-3H) transport (Figure 6.8). The literature reports that SMIT1 cotransports myo- and scyllo-inositol with equal preference (Hager et al,
______
Figure 6.7 QPCR quantification of inositol transporter expression levels in HEK293 cells. HMIT, SMIT1 and SMIT2 expression was analyzed in HEK293 cells using QPCR. Actin was used as an internal control for the QPCR assay. While SMIT1 and SMIT2 were present in these cells, no quantifiable levels of HMIT were found, as determined by tracking gene sequence amplification (Delta Rn) as a function of time (cycle number).
123
Figure 6.8 myo-Inositol-(2-3H) and scyllo-inositol-(2-3H) transport in HEK 293 cells. Transporter substrate specificity was examined by measuring myo-inositol-(2-3H) (A) and scyllo-inositol-(2-3H) (B) transport in the absence or presence of 23 competiting substrates. Since SMIT1, but not SMIT2 transports scyllo-inositol, a comparison of these two transport assays allowed for a comparison of compounds transported by SMIT1 alone, versus those transported by a combination of SMIT1 and SMIT2. Using this technique, basic transport models for each transporter were generated. (n = 3 wells per variable).
124
1995), while SMIT2 has never been shown to transport scyllo-inositol. This would explain the discrepancy between the myo- and scyllo-inositol-(2-3H) results and allowed us to isolate the individual substrate transport requirements of SMIT1 and SMIT2. In addition to only SMIT1 transporting scyllo-inositol, the literature reports that SMIT1, but not SMIT2, transports L- fucose, therefore allowing us to attribute its competitor activity to transport through SMIT1
(Hager et al, 1995; Coady et al, 2002). In addition, both SMIT1 and SMIT2 have been independently reported to transport D-chiro-inositol (Rubin and Hale, 1993; Ostlund et al,
1996). In bovine cardiac sarcolemmal vesicles, both a high-affinity and a low-affinity transport system for myo-inositol were observed (Rubin and Hale, 1993). The low-affinity transport system was unsaturable, suggesting diffusion, but the high-affinity system was Na+-dependent and showed an equal transport preference for myo- and scyllo-inositol, identifying it as SMIT1.
They showed that D-chiro-inositol could compete for this high-affinity transport, identifying it as a SMIT1 substrate (Rubin and Hale, 1993). In contrast, SMIT2 has been shown to transport both myo- and D-chiro-inositol with equal preference (Ostlund et al, 1996). This was determined in HepG2 cells, a human liver cell-line, using tritiated D-chiro-inositol. In the same set of experiments, tritiated D-chiro-inositol transport was also inhibited 30% by D-pinitol
(Ostlund et al, 1996). Therefore, with this additional information, it can be concluded that
SMIT1 transports myo- and scyllo-inositol with equal preference, followed by D-chiro-inositol and viburnitol. L-fucose, a competitive substrate for SMIT1 transport is theorized to be a putitive inhibitor for SMIT1 (see below). In contrast, SMIT2 transports myo-inositol and D- chiro-inositol with equal preference, followed by D-glucose, D-mannose, viburnitol, sequoyitol and D-pinitol. Based on these observations, two models of substrate structural requirements have been drawn (Figure 6.9, 6.10). For active transport SMIT1 requires that inositol derivatives contain four adjoining equatorial hydroxyl groups, while hydroxyl groups at the
125
Figure 6.9 Basic structural model for SMIT1 transport. The structures of the substrates transported by SMIT1 (A), along with the structures of substrates not transported by SMIT1, such as the putative inhibitor L-fucose (B), were used to design a basic structural model for SMIT1 transport (C).
126
Figure 6.10 Basic structural model for SMIT2 transport. The structures of the substrates transported by SMIT2 (A), along with the structures of substrates not transported by SMIT1 were used to design a basic structural model for SMIT2 transport (B).
127 remaining two carbons can be positioned in either an axial or equatorial orientation. The equatorial hydroxyl groups at positions 1 and 2 cannot be substituted with a methoxy group and still be transported. The hydroxyl group at position 5 can be substituted for a hydrogen group.
SMIT2, in contrast, has a different set of substrate structural requirements. The hydroxyls attached to carbons 1, 2, 5 and 6 must be in an equatorial orientation, while the hydroxyl group at position 4 must be in an axial orientation. The hydroxyl group at position 3 can be positioned either in an axial or equatorial orientation. The hydroxyl group at position 2 cannot be substituted for a methoxy group. The hydroxyl groups at positions 1, 5 and 6 can be substituted with other groups. At position 1, the hydroxyl group can be substituted with a methoxy group and still be transported. The hydroxyl group at position 5 can be substituted with a hydrogen group or the carbon with attached hydroxyl group can be substituted with an oxygen atom. At position 6, the hydroxyl group can be substituted with a hydroxymethyl group (Figure 6.10). To further test the proposed substrate transport models, substrates not transported by SMIT1 or
SMIT2 were analyzed to determine whether a structural feature could be identified to explain why each compound would not be transported based on the current models (Figure 6.11, 6.12).
This analysis further confirmed the proposed structural requirements for active transport through
SMIT1 and SMIT2. For example, a comparison of transported versus non-transported inositols, helps to determine which hydroxyl groups of inositol need to be in a fixed orientation for active transport to occur and which hydroxyl groups can be positioned either equatorially or axially. In addition, by comparing the structures of other compounds to these models, for example the structure of sequoyitol to the model for SMIT1, the model can be further refined to account for the importance of side chain composition for the maintenance of active transport. In the case of sequoyitol, transport through SMIT1 was abolished because the hydroxyl group at position 1 was substituted for a methoxy group.
128
Figure 6.11 Substrates not transported by SMIT1. The SMIT1 basic substrate model for transport (A) was compared to the structures of compounds not transported by SMIT1 (B). Structural differences, between the compounds not transported and the model, are highlighted using red boxes.
129
Figure 6.12 Substrates not transported by SMIT2. The SMIT2 basic substrate model for transport (A) was compared to the structures of compounds not transported by SMIT2 (B). Structural differences, between the compounds not transported and the model, are highlighted using red boxes.
130
Substrate saturation kinetics and testing of the model
The two compounds most readily transported by SMIT1/2 are myo- and scyllo-inositol, with
SMIT1 transporting both myo- and scyllo-inositol and SMIT2 only transporting myo-inositol.
To examine myo- and scyllo-inositol transport kinetics in greater detail, the effects of increasing concentrations of myo- and scyllo-inositol were examined in HEK293 cells (Figure 6.13). myo-
Inositol-(2-3H) transport was measured after 15 minutes of incubation, rather than at 3 hours as in the initial tests, to more accurately examine the rate of myo-inositol transport. In this study,
10 mM of cold myo-inositol, the concentration used in the competitive substrate transport studies, was found to produce a near maximal inhibition of myo-inositol-(2-3H) transport, while half-maximal inhibition (Km) was observed following the addition of approximately 200 µM of cold myo-inositol (Figure 6.13a). scyllo-Inositol-(2-3H) transport was examined in the presence of increasing concentrations of cold scyllo-inositol (Figure 6.13b). In contrast to myo-inositol transport, a 3 h incubation window was needed to obtain a similar level of radioactivity using scyllo-Inositol-(2-3H). This indicates a much slower transport rate for scyllo-inositol, likely due to the fact that it is only transported by SMIT1 and not by SMIT2. Once again, 10 mM of cold scyllo-inositol produced near maximal inhibition of scyllo-inositol-(2-3H) transport and a Km of approximately 50 µM was observed. These Km values for myo- and scyllo-inositol were within the range previously reported in the literature; ranging for myo-inositol from 55-117 µM for
SMIT1 to 120-348 µM for SMIT2 (Hager et al, 1995; Ostlund et al, 1996; Hakvoort et al, 1998;
Coady et al, 2002; Bissonnette et al, 2004; Aouameur et al, 2007; Klaus et al, 2008; Lin et al,
2009), while for scyllo-inositol, a Km of 53 µM had been previously reported (Hager et al,
1995). In Xenopus oocytes, transfected with canine SMIT1, a myo-inositol Km value of 55-59
µM was observed (Hager et al, 1995; Kaus et al, 2008). A similar scyllo-inositol Km value of 53
µM was also documented in these cells (Hager et al, 1995). In porcine choroid plexus cells in
131
Figure 6.13 myo-Inositol and scyllo-inositol transport kinetics in HEK293 cells. myo-Inositol testing was conducted in HEK293 cells, by incubating cells for 15 minutes in medium containing 100 µM of myo-inositol-(2-3H) in the presence of increasing concentrations of cold myo-inositol (A). Addition of 10 mM of cold myo-inositol, the concentration used in the competitive transport assays, resulted in a near maximal inhibition of myo-inositol-(2-3H) transport in these cells. scyllo-Inositol testing was conducted over a period of 3 hours in these cells, due to slower uptake rates (B). Once again, 10 mM of cold scyllo-inositol produced near maximal inhibition of scyllo-inositol-(2-3H). (n = 3 wells per concentration).
132
3 culture, which endogenously express SMIT1, a Km value of 117 µM for myo-inositol-(2- H) was reported (Hakvoort et al, 1998). Electrophysiological studies in Xenopus oocytes, transfected with rabbit SMIT2, showed a Km value of 120 µM for myo-inositol (Coady et al, 2002). Also using voltage-clamp recordings, a SMIT2 Km value for myo-inositol of 150 + 40 µM was observed in rat kidney brush border membrane vesicles (Aouameur et al, 2007). In contrast,
Xenopus oocytes, transfected with rat SMIT2, showed a Km for myo-inositol of 270 + 19 µM
(Aouameur et al, 2007). myo-Inositol competition against tritiated D-chiro-inositol transport, in
L6 myoblasts transfected with human SMIT2, showed a Km for myo-inositol of 158 µM (Lin et al, 2009). In the HepG2 human liver cell-line, a Km for myo-inositol of 348 µM was observed in competition against tritiated D-chiro-inositol (Ostlund et al, 1996) and finally, Km values of 120
µM and 334 µM, respectively, were found in Xenopus oocytes and in Madin-Darby canine kidney cells, both transfected with rabbit SMIT2 (Bissonnette et al, 2004). Therefore, our observed substrate saturation kinetics for myo- and scyllo-inositol are comparable to the range of values that has been reported in the literature.
The third most readily transported inositol stereoisomer, D-chiro-inositol, appears to be transported by both SMIT1 and SMIT2, however, its enantiomer, L-chiro-inositol, was not. We wanted to examine this discrepancy in the transport of these two compounds more closely, by examining the effects of increasing concentrations of these substrates on myo-inositol-(2-3H) transport. As expected, L-chiro-inositol was not transported by SMIT1/2, while D-chiro- inositol was transported (Figure 6.14). L-chiro-inositol transport was not observed even when its levels were increased 8-fold (Figure 6.14a). A drop in radioactivity was observed at the highest concentration (160 mM), likely as a result of L-chiro-inositol acting as a nonspecific blocking agent, since at that concentration a 1600-fold difference would exist between L-chiro-
133 inositol and tritiated myo-inositol (160 mM of L-chiro-inositol versus 100 µM of myo-inositol-
(2-3H)). In contrast, D-chiro-inositol competition for myo-inositol-(2-3H) transport did increase with an increase in its concentrations (Figure 6.14b). These results validate the conclusions made in the competitive transport studies and further point to the stereo-selectivity of the transporters.
Based on the models generated, SMIT1 has the most stringent substrate structural requirements.
Of the 23 compounds examined, the most structurally altered one apparently transported by
SMIT1 was L-fucose, in which two carbons are altered: one carbon with attached hydroxyl group is substituted with an oxygen atom and a second has a methyl group attached in place of its hydroxyl group (Figure 6.9). What makes L-fucose particularly intriguing however is that according to the SMIT1 model presented, L-fucose should not be transported by SMIT1, since one of the four equatorial hydroxyl groups, theorized to be needed for active transport, is oriented from an equatorial to an axial position. This would suggest that L-fucose might, in fact, be a SMIT1 transport blocker. Therefore, L-fucose transport activity was examined more closely. The initial experiments showed that 10 mM of L-fucose competed for myo-inositol-(2-
3H) transport, this was further examined in HEK-293 cells, using increasing concentrations of L- fucose. In agreement with the initial studies, increasing the concentration of L-fucose resulted in a reduction in myo-inositol-(2-3H) transport in these cells (Figure 6.15a). L-fucose and myo- inositol competitive transport was examined in a second set of experiments, in which myo- inositol inhibition of L-fucose-(5,6-3H) transport was examined. The theory behind this experiment was that if L-fucose was an actively transported substrate through SMIT1, then myo- inositol would be expected to compete for this transport. However, in contrast to this hypothesis, no prominent reductions in L-fucose-(5,6-3H) activity levels were observed upon the
134
Figure 6.14 A comparison of D- and L-chiro-inositol transport in HEK293 cells. A comparison of D- and L-chiro-inositol transport in HEK293 cells. Based on competitive substrate assays, D-chiro-inositol is transported by SMIT1/2, while L-chiro-inositol is not. This finding was examined more closely in HEK293 cells, by comparing the transport of myo- inositol-(2-3H) in the presence of increasing concentration of D-chiro-inositol (A), to transport observed in the presence of increasing concentrations of L-chiro-inositol (B). As expected myo- inositol-(2-3H) transport was inhibited by D-chiro-inositol in a concentration dependent manner. In contrast, L-chiro-inositol did not inhibit myo-inositol-(2-3H) transport, except at the highest concentration, 160 mM. This might result from L-chiro-inositol acting as a nonspecific blocking agent at this concentration. (n = 3 wells per variable).
135 addition of cold myo-inositol (Figure 6.15b). Even when L-fucose-(5,6-3H) transport was examined in the presence of increasing concentrations of cold L-fucose, no changes in the radioactivity levels were observed (Figure 6.15c). Therefore, we propose that L-fucose is not a
SMIT1 substrate competitor but that it is, in fact, a SMIT1 transport inhibitor/blocker. We hypothesize that L-fucose is only partially transported by SMIT1, whereupon it becomes lodged in the channel, thus preventing myo-inositol-(2-3H) transport and reducing radioactivity levels.
Inhibition of myo-inositol transport by L-fucose has been previously reported in the literature
(Yorek et al, 1992; Rubin and Hale, 1993; Hager et al, 1995). Yorek and colleagues (1992) also reported that myo-inositol competed for L-fucose-(3H) transport in neuroblastoma cells.
Neuroblastoma cells would be expected to express SMIT1 and possibly SMIT2, however in the study the authors only concluded that L-fucose was a competitive inhibitor of sodium- dependent, high-affinity myo-inositol transport, as SMIT1/2 had not yet been isolated when the study was published (Yorek et al, 1992). In contrast, Hager and colleagues (1995) specifically showed that L-fucose was a substrate competitor for myo-inositol transport in Xenopus oocytes transfected with canine SMIT1. However, they incorrectly identified L-fucose as a substrate for
SMIT1, rather than as a competitive inhibitor (Hager et al, 1995). Rubin and Hale (1993) labeled any compounds that competed for myo-inositol transport in cardiac sarcolemmal vesicles as transport inhibitors and did not specify whether each compound studied was a competitive substrate for inositol transport, or a transport channel blocker.
The effects of increasing concentrations of L-fucose on L-fucose-(5,6-3H) transport, not previously examined in the literature, was also determined in HEK-293 cells. No concentration dependent inhibition of L-fucose-(5,6-3H) transport was observed when cells were incubated in
L-fucose-(5,6-3H) with increasing concentrations of cold L-fucose (Figure 6.15c). This supports
136
Figure 6.15 L-fucose-(5,6-3H) transport. An analysis of competitive substrate transport found L-fucose to be a competitive inhibitor of myo- and scyllo-inositol-(2-3H) transport through SMIT1. However, a comparison of the structure of this compound to the SMIT1 basic structural transport model would indicate that L- fucose should not be transported by this system. This finding was further examined by: (A) examining myo-inositol-(2-3H) transport in the presence of increasing concentrations of L- fucose. A concentration-dependent reduction of myo-inositol-(2-3H) transport was observed. (B) An examination of L-fucose-(5,6-3H) transport in these cells, in the absence or presence of potential competitive substrates. Only background radioactivity was observed, with not active transport. (C) L-fucose-(5,6-3H) transport was examined in the absence or presence of D- fucose and increasing concentrations of cold L-fucose and again only backgroud radioactivity was observed. (n = 3 wells per variable).
137 the hypothesis that L-fucose is blocking SMIT1 activity in these cells. These experiments demonstrate that fact that substrate transport kinetic studies only suggest the features required for active transport into cells and the transport of any potential drug candidates must be confirmed through additional experiments.
The SMIT1 basic substrate transport model was further tested, using a set of scyllo-inositol derivatives, initially designed for an unrelated study, undertaken to examine the effects of scyllo-inositol derivatives on Aβ fibrillogenesis (Sun et al, 2008). Use of these compounds allowed a further testing and refinement of the SMIT1 basic transport model. In addition, it allowed an examination of the degree of change tolerated by this transporter system. Seven compounds were tested, in which either: one or two hydroxyl groups were removed, one hydroxyl group was exchanged for either a fluorine, azide, chlorine, or a hydroxy methyl group, or in the last compound, two hydroxyl groups were substituted for hydroxy methyl groups
(Figure 6.16a). Overall, each alteration resulted in very different degrees of transport into the cells (Figure 6.16b, Table 6.2). Transport was still observed following the removal of one hydroxyl group from scyllo-inositol, however, the removal of two hydroxyl groups stopped transport. This is in agreement with the SMIT1 model, which found that four adjoining hydroxyl groups, all in an equatorial position, are necessary for active transport to occur.
Similarly, one substitution of a hydroxyl group for a hydroxy methyl group still resulted in active transport, but two substitutions did not. When the effects of substituting one hydroxyl group with a fluorine, azide, chloride or hydroxy methyl group were examined, a hierarchy of
138
Figure 6.16 Transport of scyllo-inositol derivatives via SMIT1. Further testing of SMIT1 transport was conducted using derivatives of scyllo-inositol, in which one or two hydroxyl groups were removed or substituted (A). An analysis of myo-inositol-(2- 3H) transport in the presence of each of these compounds, found monodeoxy- but not dideoxy- scyllo-inositol competed for myo-inositol-(2-3H) transport. Single hydroxyl group substitutions were tolerated in the order chlorine > fluorine > azide > hydroxy methyl. (n = 3 wells per variable).
139
Table 6.2 Analysis of the SMIT1 transport model based on the structure of the scyllo-inositol derivatizes that were transported or not.
140 transport inhibition was observed in the following order: chlorine > fluorine > azide > hydroxy methyl. Based on how drastic the reduction in transport was following these relatively simple substitutions to scyllo-inositol, active transport through the SMIT1 transporter system appears to be very sensitive to any changes to the structure of the inositol backbone.
141
Discussion
There are three inositol transporters that have been reported in the literature: HMIT, SMIT1 and
SMIT2. Protein alignment between the human, rat and mouse sequences for each of these proteins found a high degree of interspecies homology. This finding points to the importance of each of these proteins for the maintenance of myo-inositol levels in the body. An analysis of intertransporter protein homology in the same three species, showed how distinct the three protein structures are from each other, with HMIT showing 11% or less homology to the two sodium myo-inositol transporters and SMIT1/2 showing only 43% or less homology with each other. Therefore, these three transporters are likely to have very distinct functions in the body.
My QPCR findings found that in the brain, HMIT mRNA was present at significantly higher concentrations than SMIT1 or SMIT2 mRNA, in nearly all the regions studied (Chapter 5).
However, while this is true, the present results suggest that HMIT is not active in cells. HMIT was first reported by Uldry et al (2001) to be a proton myo-inositol transporter, localized in the cytoplasm because of 3 internalization sequences in its protein structure, but reported to translocate to the cell surface under conditions that occur following synaptic activity. They reported that both cell depolarization and changes in extracellular pH would result in HMIT translocation to the cell surface and activation, resulting in myo-inositol transport (Uldry et al,
2001). Based on these findings, I tried a combination of four conditions to translocate HMIT to the cell surface, with the goal of studying substrate transporter kinetics in this system. These conditions included depolarization using elevated potassium levels, acidification of the extracellular environment, as well as two techniques to silence SMIT1/2 transport: phloridzin treatment and the use of Na+-free medium. However, none of these conditions resulted in
HMIT activation in any of the cells studied. A recent paper by Di Daniel et al (2009) supports
142 these findings. In both rat primary cortical neurons and brain slices, changes in pH or cell surface depolarization by either running a depolarizing current, increasing potassium or increasing glutamate levels, did not result in HMIT translocation to the cell surface (Di Daniel et al, 2009). Only mutation of the three internalization sequences resulted in HMIT translocation and activity at the cell surface (Uldry et al, 2001; Di Daniel et al, 2009). Therefore, an alternative function for HMIT has been suggested, as an inositol triphosphate transporter within the cell (Di Daniel et al, 2009). While interesting, these observations eliminate HMIT as a viable option for drug transport studies.
Next, I conducted substrate transport studies for SMIT1. QPCR tests showed SMIT1 to have the second highest brain mRNA expression levels of the three transporters studied (Chapter 5) and in contrast to HMIT, SMIT1 is constitutively expressed at the cell surface (Uldy et al,
2004), making it a better candidate as a drug transport system. Based on the competition assay results, SMIT1 transported myo- and scyllo-inositol with equal preference, followed by L- fucose, D-chiro-inositol, and viburnitol. These results differ from those found in Xenopus oocytes transfected with canine SMIT1, which found the following compounds transported: myo-inositol = scyllo-inositol > L-fucose > L-xylose > L-glucose = D-glucose = α-methyl-D- glucopyranoside > D-galactose = D-fucose = 3-O-methyl-D-glucose = 2-deoxy-D-glucose > D- xylose (Hager et al, 1995). This difference in substrate selectivity between the two studies could result from the current study being conducted on human SMIT1, while the Xenopus oocytes were transfected with canine SMIT1 (Hager et al, 1995). The human and canine SMIT1 amino acid sequences are more than 92% homologous (Kwon et al, 1992; Berry et al, 1995;
Lubrich et al, 2000; McVeigh et al, 2000). This level of homology is high, but since the sequences are not identical, this might account for the differences observed.
143
Strengthening this argument is the fact that in both studies, the top three compounds that competed for myo- and scyllo-inositol-(2-3H) transport remained the same, with myo- and scyllo-inositol being transported the most readily, and with equal preference, in both studies.
The ability of scyllo-inositol to strongly compete out myo-inositol transport has also been previously shown in murine neuroblastoma, murine cerebral microvessel endothelial and bovine aortic endothelial cell-lines (Wiese et al, 1996), indicating that this is a feature common to multiple species.
The present study found the third most competitive substrate for SMIT1 transport was L-fucose.
L-fucose might actually be a SMIT1 transport blocker, a conclusion reached based on the fact that myo-inositol and increasing concentrations of cold L-fucose did not reduce L-fucose-(5,6-
3H) radioactivity levels. Therefore any drug compounds, initially interpreted as being transported in cell-lines, need to be examined closely to ensure that they are in fact, transported by the shuttling system and not lodged within it. In addition, this finding that L-fucose, containing of a few minor substitutions in comparison to the structure of myo-inositol, becomes lodged in the SMIT1 transport channel, points to how rigid the SMIT1 transporter is to changes to the structure of myo-inositol.
Derivatives of inositol containing hydroxyl group deletions or substitutions, were used to further define SMIT1 transport selectivity. A preference of chlorine > hydroxyl > fluorine > azide > hydroxyl methyl, in place of a single hydroxl group was observed for SMIT1 transport.
Therefore, in agreement with the L-fucose observations, this indicates that SMIT1 is very
144 sensitive to changes to the structure of myo-inositol and that only drugs based on conservative substitutions to the structure of myo- or scyllo-inositol will likely be tolerated by this system.
The final inositol transporter, SMIT2, is also constitutively expressed at the cell surface, as was seen for SMIT1 (Uldry et al, 2004). Similar to SMIT1, expression of SMIT2 is observed in both the brain and the kidney, although expression in the kidney is significantly higher than expression in the brain (Chapter 5). My study found that SMIT2 transports a distinct group of compounds from SMIT1, specifically myo-inositol, D-chiro-inositol, D-glucose, D-mannose, viburnitol, sequoyitol and D-pinitol. The preference of SMIT2 for D-stereoisomers over L- stereoisomers has been previously reported in the literature (Ostlund et al, 1996). The HepG2 human liver cell-line was shown to prefer D-chiro-inositol and D-glucose over the L- stereoisomers (Ostlund et al, 1996). In the present study, both SMIT1 and SMIT2 showed a preference for D-chiro-inositol, over L-chiro-inositol, both in the initial substrate transport tests and following an examination of the effects of increasing concentrations of D- and L-chiro- inositol on myo-inositol-(2-3H) transport in HEK293 cells. Also in agreement with previously publications (Ostlund et al, 1996), SMIT2 showed a preference for D-glucose over the L- stereoisomer in HEK293 cells. Given the increased number of compounds transported by
SMIT2, this transporter would appear to be less stringent in substrate requirements than SMIT1.
However, given the expression pattern of SMIT2, drugs designed to target this transport system would preferentially be funneled out of the body, based on the high SMIT2 expression observed in the kidney.
In conclusion, the use of HMIT, SMIT1 or SMIT2 as a drug shuttling system appears unlikely to be effective, based on the results presented in my thesis. HMIT, while highly expressed in
145 the brain, is internalized in cells and therefore, unavailable as a drug shuttling system. SMIT1 and SMIT2 are both constitutively located at the cell surface membrane and are expressed in the brain, therefore, they are available for drug transport. However, they are both very selective in the compounds they will actively transport. SMIT1 especially appears to have very rigid substrate structural requirements for active transport and it is very sensitive to changes to the structure of myo- and scyllo-inositol, which are its preferred substrates. SMIT2 is slightly less stringent than SMIT1, but it is still only likely to act as an effective shuttling system for conservative derivatives of myo- or D-chiro-inositol, its two preferred substrates (Ostlund et al,
1996; Coady et al, 2002). Therefore, based on these observations, the inositol transporters would not make an effective shuttling system for drug transport into the brain, thus disproving my initial hypothesis.
CHAPTER 7
Discussion, Conclusions and Future Directions
145 146
Discussion
One significant challenge to the treatment of CNS diseases is shuttling drugs across the blood- brain and/or blood-CSF barriers to where they are needed. These barriers are very selective in the types of compounds they will let through. The BBB consists of a monolayer of endothelial cells joined together by tight junctions, separating the blood from the brain, supported on the brain side by astrocytic endfeet, pericytes, a basal lamina and neurons (reviewed in Hawkins and
Davis, 2005). The blood-CSF barrier, mainly consists of choroid plexuses located in each of the brain ventricles, which are composed of epithelial cells, joined together by tight junctions, separating a network of capillaries and surrounding interstitial fluid, from the CSF (reviewed in
Johanson et al, 2005). The main purposes of both these structures are to act as physiological barriers in order to protect the brain from any harmful substances that might be present in the blood stream, to transport any substances, such as glucose and amino acids, which are needed for proper brain function and to maintain homeostasis (Stern, 1921). A side effect of the rigorous control imposed by these barriers is that compounds that would prove beneficial to stopping CNS disease pathology, such as that which occurs in neurodegenerative diseases, are usually unable to access their drug targets in the brain. Therefore, strategies are needed to shuttle any drugs that cannot cross the brain barriers naturally, into the brain. Two strategies that are often employed in a laboratory setting to avoid these brain barriers are to either inject the potential drugs directly into the brain or implant a pump to slowly release drugs into the brain ventricles. However, both these methods are highly invasive and not ideal as long-term drug delivery strategies in humans. Therefore, alternative methods for drug delivery into the brain are being examined. One set of strategies use less invasive barrier circumvention strategies to bypass the brain barriers: these include avoiding the barriers entirely through the use of intranasal delivery or temporarily opening the barriers, through the use of techniques such
147 as osmotic disruption to allow the drug access (reviewed in Patel et al, 2009). Barrier navigation strategies are also being developed, such as the use of nanoparticles or a Trojan horse system for the delivery of compounds into the brain (reviewed in Patel et al, 2009). Both barrier circumvention and navigation strategies center on converting compounds that cannot enter the brain, into ones that can. An alternative technique that can be employed is to identify compounds that already enter the brain naturally and try to either use these compounds as disease therapeutics or attach other therapeutics to these compounds for transport into the brain.
myo-Inositol and scyllo-inositol are examples of compounds that are present in the body, enter the brain naturally and appear to act as disease therapeutics (Hallman et al, 1986; Hallman et al,
1992; Benjamin et al, 1995; Levine et al, 1995; Fux et al, 1996; Chengappa et al, 2000; Gelber et al, 2001; Palatnik et al, 2001; Eden et al, 2006; McLaurin et al, 2006). myo-Inositol has been successfully used in human studies to treat psychiatric disorders, such as depression (Levine et al, 1995), bipolar/affective disorder (Chengappa et al, 2000; Eden et al, 2006), obsessive- compulsive disorder (Fux et al, 1996), eating disorders (Gelber et al, 2001) and panic disorder
(Benjamin et al, 1995; Palatnik et al, 2001), as well as for the treatment of respiratory distress syndrome in premature infants (Hallman et al, 1986; 1992). scyllo-Inositol has been successfully used as an effective prophylactic and therapeutic for the treatment of AD in the
TgCRND8 mouse model of AD (McLaurin et al, 2006). Therefore, they are both naturally occurring compounds that show promise as disease therapeutics and it might be possible to alter or adapt them to either successfully treat other diseases or to transport other drug compounds into the brain.
148
In order to use these inositols as disease therapeutics or drug delivery systems, it is important to understand how carefully their concentrations are regulated within the brain, to determine to what extent additional transport of these compounds into the brain is possible. For example, in the TgCRND8 mouse model of AD, while scyllo-inositol was effective for the treatment of AD, both prophylactically, before the visible onset of symptoms, and therapeutically at 5 months of age, once symptoms had fully developed, myo-inositol did not significantly improve disease outcome measures in these animals (McLaurin et al, 2006). This is despite the fact that both compounds were equally effective at reducing Aβ aggregation, in vitro (McLaurin et al, 2000).
This discrepancy between in vitro and in vivo findings led us to examine myo- and scyllo- inositol regulation in the brain and to determine whether this regulation would change with disease pathology in TgCRND8 mice. Based on the study by McLaurin and colleagues (2006) on the effectiveness of the two compounds as therapeutic agents for AD, scyllo-inositol administration would be expected to reach higher levels in the brain, than myo-inositol administration. This was proven using GC/MS, which found that while oral ad libitum administration of either compound caused a significant increase in its corresponding levels in the brain, ad libitum scyllo-inositol administration resulted in a more significant increase in brain levels, 7-fold compared to 0.3-fold for myo-inositol. Therefore a tighter control of myo- inositol levels by the body appears to be in place. This could be because myo-inositol is critical for maintaining osmolarity in the body and it is a constituent of phosphatidylinositol, which is an important phospholipid in membranes and second messenger systems (Fisher et al, 2002). In contrast, scyllo-inositol has no known function either within the brain or in the periphery therefore, perhaps explaining the lack of tight regulation (Fisher et al, 2002).
149
Given the importance of myo-inositol and the presence of scyllo-inositol in the body, the inter- regulation of the two inositol isomers was tracked, by examining the effects of ad libitum myo- or scyllo-inositol administration on the other inositol stereoisomer. Rat, rabbit and bovine brains have been shown to contain an epimerase that can convert myo- to scyllo-inositol and vice versa (Sherman et al, 1968a; b; Hipps et al, 1977). In women a 1% conversion of myo- to scyllo-inositol within plasma has been reported and in rats a 0.06% conversion of myo- to scyllo- inositol has been observed (Groenen et al, 2003). These studies suggest that inter-regulation between the two isomers exists. This theory is supported by the present research, which found a reduction in scyllo-inositol levels following myo-inositol administration and vice versa. This decrease in the concentration of the non-administered isomer following either myo- or scyllo- inositol administration, might result from a shift in inositol equilibrium toward degradation pathways, in an effort to reestablish/maintain brain homeostasis. Degradation of both inositols occurs by the same pathway, therefore a shift towards degradation would occur for both inositols. The extent of this degradation becomes evident when the levels of the non- administered inositol are quantified between untreated and treated animals. A 5-fold decrease in scyllo-inositol levels was observed following myo-inositol administration, while only a 0.3-fold decrease in myo-inositol occurred following scyllo-inositol administration. This severe drop in scyllo-inositol levels observed following myo-inositol administration, compared to the smaller drop in myo-inositol levels observed following scyllo-inositol administration, supports the idea that myo-inositol levels are more tightly regulated. Therefore, these findings indicate that the body regulates both elevations in myo-inositol following ad libitum administration, but also
150 reductions in myo-inositol levels as a consequence of ad libitum scyllo-inositol administration.
This is an important finding, because it reduces the odds of ad libitum scyllo-inositol administration inadvertently causing a detrimental shift in either osmolarity or phosphatidylinositol levels in the body.
myo-Inositol is the most abundant inositol stereoisomer found in nature and it is a ubiquitous component of all eukaryotic cells (Fisher et al, 2002). scyllo-Inositol is the next most abundant inositol stereoisomer and its levels are usually 10% the levels of myo-inositol (Michaelis et al,
1993). While scyllo-inositol has no known function in the body, we wished to determine whether a 7-fold increase in its levels in the brain, as was observed following ad libitum administration, would result in its incorporation into phosphatidylinositol. This could alter second messenger systems and lead to undesired side effects. Incorporation of scyllo-inositol into phosphatidylinositol has been reported to occur in lower organisms, such as mycobacteria, tetrahymena cells and barley seeds (Kinnard et al, 1995; Ryals et al, 1999; Salman et al, 1999;
Riggs et al, 2007). However, scyllo-inositol incorporation into phosphatidylinositols has not been observed in higher organisms (Takenawa and Egawa, 1977). Therefore, the levels of scyllo-inositol incorporation into phosphatidylinositol, in both untreated mice and mice treated with ad libitum scyllo-inositol, were examined. No incorporation of scyllo-inositol into phosphatidylinositol was observed in either group. This could result because of a reduced binding affinity of phosphatidylinositol synthesizing enzymes for scyllo-inositol, which has been observed even when scyllo-inositol levels are elevated (Takenawa and Egawa, 1977;
Salman et al, 1999). For example, the final step in the de novo synthesis of phosphatidylinositol is catalyzed by CDP-diglyceride:inositol transferase and while it was found to bind myo-inositol with a Km of 2.5 mM, binding to scyllo-inositol was not observed (Takenawa and Egawa, 1977).
151
When the activity of another enzyme, phosphatidylinositol:inositol phosphatidyl transferase, was examined in rat liver microsomes, scyllo-inositol inhibition of myo-inositol-(3H) incorporation into phosphatidylinositol was two orders of magnitude lower than that of cold myo-inositol and nonexistent when examined using the micromolar concentrations of scyllo- inositol found endogenously in most tissues (Irvine, 1998). Therefore, an increase in the levels of scyllo-inositol should not result in any significant incorporation and increase in the levels of phosphatidylinositol, which could have deleterious effects on cell membrane composition and secondary messenger systems. In support of this hypothesis, significantly increased levels of scyllo-inositol have been previously observed in a human subject using MRS, with no apparent neurological problems, despite having twice the average concentration of scyllo-inositol in the brain (Seaquist and Gruetter, 1998).
The 7-fold increase in brain scyllo-inositol levels, observed following ad libitum oral administration to mice, would suggest a high degree of CNS bioavailability for scyllo-inositol.
This is an important factor when selecting or designing drug therapeutics. We examined the effects of once-daily administration of three different doses of scyllo-inositol versus ad libitum scyllo-inositol treatment, in order to further study scyllo-inositol CNS bioavailability. Once- daily administration of scyllo-inositol did not result in a sustained increase in brain scyllo- inositol, suggesting that a multiple dosing regiment would be more effective than a once-daily dose. This theory is supported by observations made using TgCRND8 mice, which found a twice-daily dose of scyllo-inositol more effective than a once-daily dose at treating AD
152 pathology (McLaurin et al, 2006). Therefore, while ad libitum scyllo-inositol administration resulted in a significant increase in brain scyllo-inositol levels, twice-daily scyllo-inositol administration was sufficient to achieve therapeutic doses of scyllo-inositol in the brain for effective disease modification.
While scyllo-inositol was an effective disease therapeutic against AD in TgCRND8 mice, myo- inositol was not (McLaurin et al, 2006). This finding was unexpected given, both the effectiveness of myo-inositol in vitro (McLaurin et al, 2000) and in other diseases in vivo
(Hallman et al, 1986; Hallman et al, 1992; Benjamin et al, 1995; Levine et al, 1995; Fux et al,
1996; Chengappa et al, 2000; Gelber et al, 2001; Palatnik et al, 2001; Eden et al, 2006). One possible reason why myo-inositol would be ineffective as a drug treatment in TgCRND8 mice could be because these animals display different myo-inositol regulation parameters than control animals. To test this theory, the effects of ad libitum myo-inositol administration on brain myo- and scyllo-inositol levels were compared between TgCRND8 mice and their wild-type littermates. No significant differences were observed between the two groups on the effects of myo-inositol administration on brain inositol levels. Both groups showed a similar, significant increase in brain myo-inositol levels and a similar, significant decrease in scyllo-inositol levels following ad libitum myo-inositol administration. Therefore, regulation of inositol levels in the brain appears to be comparable between the two groups and not quantifiably altered by disease pathology using this technique.
The similarity between TgCRND8 mice and their wild-type littermates was further examined by comparing the mRNA expression levels of the three inositol transporters, HMIT, SMIT1 and
SMIT2 between the two groups. Basal CNS myo- and scyllo-inositol levels are normally almost
153
100-fold higher than circulating plasma inositol levels (Palmano et al, 1977). This significant elevation in brain inositol levels in comparison to the periphery, points to the presence of active transport systems, in addition to simple diffusion, for the transport of inositol stereoisomers across the brain barriers (Spector, 1988; Wiesinger, 1991; Rubin and Hale, 1993; Uldry et al,
2001; Coady et al, 2002). There are three active inositol transporters that have been reported in the literature, HMIT, SMIT1 and SMIT2 (Kwon et al, 1992; Uldry et al, 2001; Roll et al, 2002) and all three are expressed in the brain (Berry et al, 1995; Inoue et al, 1996; Uldry et al, 2001;
Roll et al, 2002). In addition, because the endothelial cells located at the BBB are the only cells in the brain capable of synthesizing inositol from glucose (Wong et al, 1987), the majority of cells in the brain also require active transport to maintain their inositol levels. These inositol transporters function by using the cotransport of either a hydrogen atom or two sodium atoms along their concentration gradients, to generate enough energy to actively transport inositol across concentration gradients (Hager et al, 1995; Uldry et al, 2001; Coady et al, 2002;
Bourgeois et al, 2005). The mRNA expression of these three transporters was compared between TgCRND8 mice and their wild-type littermates in four brain regions: the cortex, hippocampus, septum and cerebellum, using QPCR. These four brain regions were chosen because they are regions that are affected to varying degrees by Aβ plaque deposition in AD
(Thal et al, 2002). The earliest plaque deposition in AD is observed in the cortex and hippocampus, followed by the septum and finally the cerebellum (Thal et al, 2002). Deposition in the cerebellum is only observed in advanced stages of AD (Thal et al, 2002). In addition to comparing transporter mRNA expression these two groups in the four brain regions listed, inositol transporter expression was also examined as a function of age, by examining expression at 2, 4 and 6 months of age. These time points were selected because they correspond to different stages in Aβ plaque deposition in TgCRND8 mice: 2 months of age corresponds to
154 pre-plaque deposition, 4 months of age corresponds to mid-stage AD and 6 months of age corresponds to advanced stage AD (Chishti et al, 2001). In agreement with the non-significant difference between TgCRND8 mice and wild-type animals observed in the GC/MS study following myo-inositol administration, disease pathology did not significantly change inositol transporter mRNA expression levels in any of the four brain regions examined, at any of the three ages selected. Inositol transporter mRNA expression was also not significantly changes as a function of age in either group of animals, indicating that transporter expression is stable with age, at least in the range of ages tested in this study. A stable expression pattern, irrespective of disease pathology or age, is an important factor for successful CNS drug design, because it removes potential confounding variables to successfully drug shuttling into the brain using these transporter systems.
It is known that all three inositol transporters are expressed in the brain (Berry et al, 1995; Inoue et al, 1996; Uldry et al, 2001; Roll et al, 2002), however, the relative expression of the three transporters to each other and their subregional expression in the brain have not been extensively studied. This information would provide a better understanding of active inositol transport within the brain and between subregions. Based on their relative expression and subregional distribution, individual transporters could be targeted in drug design studies to direct transport and subregional drug delivery. This study found that overall, relative expression of the transporters in the brain occurred in the order HMIT > SMIT1 > SMIT2. In addition, each transporter showed a distinct regional expression profile. HMIT mRNA subregional expression occurred in the order of concentration: cortex = septum > hippocampus > cerebellum. SMIT1 expression occurred in the order septum > cerebellum ≥ cortex = hippocampus and SMIT2 expression occurred in the order cerebellum ≥ septum = cortex ≥
155 hippocampus. If drug targeting to the cerebellum was required, than SMIT2 would be the best transporter to target, because its expression levels were highest in the cerebellum, in comparison to the other three brain regions examined. In contrast, if targeted drug transport to the septum was required, then SMIT1 would be the better choice, based on its expression profile.
Therefore, depending on the transporter chosen, drug accumulation could theoretically be preferentially directed to selected subregions of the brain.
In addition to examining CNS bioavailability, an additional variable that can be considered for
CNS drug design is the rate at which a drug will be excreted from the body. The kidney is where inositol excretion from the body occurs (Arner et al, 2006) and all three inositol transporters are expressed there (Berry et al, 1995; Uldry et al, 2001; Roll et al, 2002). An examination of mRNA expression of the three transporters in the kidney, which was used as a positive control tissue in QPCR experiments, found no changes in expression in any of the groups examined, therefore indicating that age and disease pathology do not affect inositol transporter expression in this tissue. A comparison of the relative expression levels of the inositol transporters in the kidney, found expression occurred in the order SMIT2 > SMIT1 >
HMIT, which is the opposite of what was observed in the brain. SMIT2 expression in the kidney was 80-fold higher than its expression in the brain. A higher level of SMIT2 expression in the kidney, relative to the brain, has been previously reported in the literature (Roll et al,
2002). This indicates that SMIT2 is particularly important for regulating inositol excretion in mice. In contrast, HMIT levels were much lower in the kidney, relative to the brain, which has also been previously reported in the literature (Uldry et al, 2001). This suggests that targeting drugs to SMIT1, rather than SMIT2, might result in higher CNS bioavailability, by reducing the rate of uptake and excretion by the kidney.
156
GC/MS found ad libitum myo-inositol and especially scyllo-inositol administration resulted in a sustained increase in their levels in the brain (Fenili et al, 2007). These findings indicate that scyllo-inositol has excellent CNS bioavailability and suggests that derivatives of scyllo-inositol or compounds attached to scyllo-inositol might show similar rates of CNS bioavailability. In order for these compounds to be successfully transported into the brain, they need to cross the brain barriers. The inositol transporters HMIT, SMIT1 and SMIT2 are expressed in the brain
(Berry et al, 1995; Inoue et al, 1996; Uldry et al, 2001; Roll et al, 2002) and QPCR found mRNA expression for all three transporters, in all four of the brain regions examined. This potentially creates three different avenues through which drugs can travel into the brain. By comparing the transport of tritiated myo- or scyllo-inositol in the absence or presence of 23 potential substrate competitors, the structural features required for active transport through each transporter can be characterized. This assay provides information on the basic structural features of inositol required for active transport and suggests how flexible the three transporters are to alterations to the base structure. This research helps determine whether derivatives of inositol can be transported or whether compounds can be attached to inositol and successfully shuttled into and within the brain, using these transporters.
The first transporter, HMIT, is mainly expressed in the brain, with limited expression in the kidney and adipose tissue (Uldry et al, 2001). In the brain, mRNA expression is observed in both neurons and glia, with high levels found in all the brain regions examined: cerebral cortex, hippocampus, hypothalamus, cerebellum and brainstem (Uldry et al, 2001). When the mRNA expression levels of the three transporters were compared in the brain and the kidney, HMIT showed the highest levels of expression in the brain and the lowest levels of expression in the
157 kidney. Based on these findings, HMIT would appear to have all the features required to be an effective drug shuttling system into the brain. One challenge to this conclusion is the fact that
HMIT is normally internalized in the cell, because of three internalization sequences located in the protein structure (Uldry et al, 2001). However, cell surface translocation of HMIT has been reported to occur following membrane depolarization or a decrease in extracellular pH (Uldry et al, 2001; 2004), conditions that are typical byproducts of synaptic activity (Chesler and Kaila,
1992). Since, HMIT might still be usable as a drug delivery system in regions where synaptic activity is present, an effort was made to study the transporter activity in vitro. Based on the cell surface translocation information published in the literature, two conditions: an elevation in external potassium levels and acidification of the extracellular environment were used to induce
HMIT translocation to the cell surface and activation. However, neither condition resulted in
HMIT activation, in any of the cells studied, which included the SH-SY5Y human neuroblastoma cell-line, the 1321N1 human astrocytoma cell-line and mouse primary cortical neurons. A recent study by Di Daniel and colleagues (2009), also did not observe HMIT translocation to the cell surface following changes in pH or depolarization, either in rat primary cortical neurons in culture or rat cortical slice cultures, despite initial reports to the contrary
(Uldry et al, 2001; 2004). Two additional conditions, centered on inducing HMIT translocation and activity through SMIT1/2 silencing, were also tested. Since both SMIT1 and SMIT2 cotransport inositol with two sodium ions, they are inhibited by phlorizin, a sodium-dependent transport inhibitor (Hager et al, 1995; Coady et al, 2002). Therefore, the effect of phlorizin- induced SMIT1/2 inhibition on HMIT activity was examined. In addition, prewashing cells in
Na+-free medium, and using Na+-free medium during the competition transport assay was tried as another method of silencing SMIT1/2 activity. Both of these techniques were effective in silencing SMIT1/2 activity, but they did not result in HMIT activation. Only mutation of the
158 three internalization sequences, located in the protein structure of HMIT, was found to cause translocation to the cell surface and activation (Uldry et al, 2001; Di Daniel et al, 2009). If
HMIT is not a cell surface inositol transporter, what is its function inside the cell? Di Daniel and colleagues (2009) suggest that HMIT regulates inositol triphosphate levels by acting as an inositol triphosphate transporter within the cytoplasm. Therefore, despite high levels of mRNA expression in the brain, HMIT does not appear to be a viable candidate for drug transport studies.
The next candidate for drug transport, SMIT1, is constitutively expressed at the cell surface
(Uldry et al, 2001). It has the second highest brain mRNA expression levels of the inositol transporters, with expression found in all four of the brain regions tested: the cortex, hippocampus, septum and cerebellum, using QPCR. Expression in the brain has also been observed in the locus coeruleus, the suprachiasmatic nucleus and the olfactory bulb (Inoue et al,
1996). However, the highest levels of SMIT1 in the brain are in the choroid plexus (Inoue et al,
1996), therefore, SMIT1 is very important for inositol transport across the blood-CSF barrier.
SMIT1 knockout studies would suggest that it is the main transporter responsible for maintaining inositol levels both in the brain and in the periphery (Berry et al, 2003).
Homozygous knockout mice showed a 92% and an 84% reduction in inositol levels in the brain and in the periphery, respectively (Berry et al, 2003). Heterozygous mice showed a 15% and a
25% reduction in frontal cortex and hippocampal inositol levels, respectively, suggesting these regions are particularly dependent on SMIT1 activity (Shaldubina et al, 2007). This high dependence of inositol levels on SMIT1, suggests that designing drugs to target this transporter might be an effective strategy for maximizing drug transport into the brain, particularly to the frontal cortex and hippocampus. Competition assay results found that SMIT1 transports myo-
159 and scyllo-inositol with equal preference, followed by L-fucose, D-chiro-inositol and viburnitol.
These findings contrast those of canine SMIT1 transfected into Xenopus oocytes, which transported the following compounds, in the order of preference: myo-inositol = scyllo-inositol >
L-fucose > L-xylose > L-glucose = D-glucose = α-methyl-D-glucopyranoside > D-galactose =
D-fucose = 3-O-methyl-D-glucose = 2-deoxy-D-glucose > D-xylose (Hager et al, 1995). This difference in substrate selectivity between the two studies is likely due to differences in the structures of the human and canine SMIT1 transporters. Despite the 92% homology between the human, mouse, rat, canine and bovine SMIT1 amino acid sequences (Kwon et al, 1992;
Berry et al, 1995; Lubrich et al, 2000; McVeigh et al, 2000), the differences that exist are reflected in the substrates transported by SMIT1 in each species. However, in both species, the top three compounds competing for myo-inositol-(2-3H) transport remained the same, with myo- and scyllo-inositol transported the most readily and with equal preference. This ability of scyllo-inositol to strongly compete out myo-inositol transport has also been shown in murine neuroblastoma, murine cerebral microvessel endothelial and bovine aortic endothelial cell-lines
(Wiese et al, 1996), indicating that this feature is common across multiple species.
Derivatives of inositol containing hydroxyl group omissions or substitutions were used to further define SMIT1 transport selectivity. Alterations in a single hydroxyl group resulted in substrate transport in the following order of substitution preference: chlorine > fluorine > hydroxyl > deoxy omission > azide > hydroxylmethyl. Based on the marked decrease in transport observed following these conservative changes to the structure of scyllo-inositol, it can be concluded that SMIT1 is very sensitive to alterations in the structure of inositol. Given this information, in addition to the initial observations that four neighboring hydroxyl groups need to
160 be in an equatorial position for transport and only the remaining two hydroxyl groups are flexible to reorientation, it would appear that only drugs resulting from very conservative substitutions to the structure of myo- or scyllo-inositol would be tolerated by this system.
This conclusion is supported by competitive transport studies using L-fucose. L-fucose contains three minor substitutions to the structure of myo-inositol: one carbon with its attached hydroxyl group is substituted with an oxygen, a second hydroxyl group is substituted with a methyl group and a third hydroxyl is reoriented from an equatorial to an axial position. These substitutions resulted in the partial transport of L-fucose by SMIT1, before becoming lodged in the transport channel. This conclusion was reached based on the observation that myo-inositol and increasing concentrations of cold L-fucose were not successful in reducing tritiated L-fucose radioactivity levels, suggesting that SMIT1 transport was blocked by L-fucose. Therefore, it can be concluded that carefully designed derivatives of myo- or scyllo-inositol will be transported by this system, but not complex compounds attached to inositol.
The third transporter, SMIT2, is also expressed at the cell surface (Uldry et al, 2004). Also like
SMIT1, it’s a member of the solute carrier family 5, sodium/glucose cotransporter superfamily, but the two transporters are only 40-43% homologous to each other. Therefore, the two would be expected to have very different substrate structural requirements. While SMIT2 is expressed in the brain, QPCR results show that expression in the brain is much lower than expression in the kidney. This lowers the chances of being an effective shuttling system for drugs to the brain, but its substrate structural requirements were examined, regardless. As expected, SMIT2 was found to transport a distinct group of compounds from SMIT1, specifically myo-inositol, D- chiro-inositol, D-glucose, D-mannose, viburnitol, sequoyitol and D-pinitol. A preference of
161
SMIT2 for D-stereoisomers over L-stereoisomers has been previously reported in the literature
(Ostlund et al, 1996). The HepG2 human liver cell-line was shown to prefer D-chiro-inositol and D-glucose rather than the L-stereoisomers (Ostlund et al, 1996). In the present study, both
SMIT1 and SMIT2 showed a preference for D-chiro-inositol, rather than L-chiro-inositol, both in the initial substrate transport tests and following an examination of the effects of increasing concentrations of D- and L-chiro-inositol on myo-inositol-(2-3H) transport in HEK293 cells.
Previously published data also observed a preference by SMIT2 for D-glucose over its L- stereoisomer (Ostlund et al, 1996). Of the 23 potential substrate competitors, SMIT2 transported more substrates than SMIT1 and it would appear to have less stringent substrate requirements. Like SMIT1, many of the hydroxyl groups on inositol need to be in a fixed position, however unlike SMIT1 many of these hydroxyl groups can be successfully substituted with other groups while still maintaining transport. Two hydroxyl groups can be substituted with a methoxy or hydroxymethyl, respectively. A third hydroxyl group can be substituted for a hydrogen or the carbon with attached hydroxyl group can be substituted for an oxygen. Overall, this results in SMIT2 being able to transport a wider range of structures than SMIT1, however, its substrate requirements are still relatively stringent and, like SMIT1, this system would probably only transport carefully designed derivatives of inositol. In addition, while all three transporters are expressed both in the brain and the kidney, the 80-fold higher SMIT2 expression levels observed in the kidney, relative to the brain, would suggest that drugs designed to target this transporter would be more likely to be excreted from the body prior to their transport into the brain.
162
Conclusions
In this thesis, experiments were conducted to quantify myo- and scyllo-inositol levels in the brain, to understand the effects of oral myo- or scyllo-inositol administration on these levels and to determine the expression of the inositol transporters in the brain and the substrate structural requirements for active transport using these transporters. These experiments were conducted to test the hypothesis that inositol transporters would make effective shuttling systems for drug delivery into the brain. A GC/MS study was conducted to quantify baseline brain myo- and scyllo-inositol levels and the effects of oral ad libitum administration, of either myo- or scyllo- inositol, on brain and CSF inositol concentrations. Using this technique, no differences were observed in either baseline brain myo- and scyllo-inositol levels or those levels following myo- inositol administration, when comparing TgCRND8 mice to their wild-type littermates. These findings led to the conclusion that myo- or scyllo-inositol concentrations and uptake into the brain are not significantly altered by disease pathology in these animals. An examination of inositol transporter mRNA expression using QPCR, conducted in four regions of the brain as well as the kidney, showed no significant changes in transporter expression as a function of either age or disease pathology. These findings are in agreement with the GC/MS conclusions.
QPCR analysis showed SMIT2 expression was very high in the kidney, with much lower expression levels in the brain, making it a less promising candidate for CNS drug transport. An examination of inositol transporter activity found HMIT to be inactive, with no evidence of cell surface transport and activity. HMIT was concluded to likely be an internal transporter for inositol derivatives and therefore, not a viable shuttling system for drugs into the brain. These findings placed SMIT1 as the primary candidate for drug transport into the brain. However, competitive substrate transport assays for both SMIT1 and SMIT2 were conducted and both were concluded to have very stringent requirements for the structural features required for
163 substrate transport. Therefore, it is concluded that inositol transporters would not make effective shuttling systems for drug transport into the brain, but only for the transport of certain inositol stereoisomers and derivatives, thus disproving the initial thesis hypothesis.
Future Directions scyllo-Inositol is an effective prophylactic and therapeutic against AD in TgCRND8 mice
(McLaurin et al, 2006) and it is currently in Phase II clinical trials for the treatment of AD
(www.clinicaltrials.gov). Additional research, to further understand how scyllo-inositol levels are regulated in both AD mice and in human subjects with or without AD, would further our understanding of the potential function of this compound and how it is transported into the brain. Towards this purpose, three future direction studies have been proposed. First, to compare basal myo- and scyllo-inositol concentrations and concentrations following scyllo- inositol treatment, in human patients with AD and control subjects. Second, to quantify mRNA levels for the inositol transporters in post-mortem tissue from both AD and control subjects, to determine where the transporters are expressed in human tissue and whether their expression is stable as a function of age and disease pathology (Chapter 5). Lastly, given the fact that SMIT1 appears primarily responsible for scyllo-inositol transport into the brain, to examine the effects of decreasing or increasing SMIT1 expression, through gene knockout or knockin studies, on scyllo-inositol transport into the brain of TgCRND8 mice and on its effectiveness as a prophylactic and therapeutic for AD.
In order to examine the levels of myo- and scyllo-inositol in human subjects in vivo, MRS can be used. Both myo- and scyllo-inositol have distinct resonance frequencies when examined using
MRS, allowing for the repetitive, non-invasive analysis of myo- and scyllo-inositol
164 concentrations as a function of age and disease pathology. Elevated levels of both myo- and scyllo-inositol have been reported when comparing human subjects with AD to age-matched control subjects (Valenzuela and Sachdev, 2001; Griffith et al, 2007). Increases in myo- and scyllo-inositol concentrations have also been reported in normal older subjects, compared to younger subjects (Kaiser et al, 2005). Therefore, both age and disease pathology are correlated with increases in brain myo- and scyllo-inositol concentrations (Valenzuela and Sachdev, 2001;
Kaiser et al, 2005; Griffith et al, 2007). An extensive examination of how myo- and scyllo- inositol levels change with age in both healthy subjects and patients with AD would help to clarify when these concentrations become altered. Sub-regional changes in myo- and scyllo- inositol levels, as a function of age and disease pathology, could also be monitored to help determine where changes in myo- and scyllo-inositol concentrations begin in these subjects and how these changes progress over time. In addition, MRS data from the Phase I and Phase II clinical trials for scyllo-inositol would provide information on the effects of twice-daily, oral administration of scyllo-inositol on brain myo- and scyllo-inositol levels, in AD patients and in older and younger healthy control subjects. These studies would provide additional information on the stability of scyllo- inositol levels within the brain and when these levels become altered.
While myo- and scyllo-inositol concentrations are not significantly different between TgCRND8 mice and their wild-type littermates, in human AD patients, these concentrations are elevated
(Valenzuela and Sachdev, 2001; Griffith et al, 2007). One possible reason for this increase might be a change in brain inositol transporter expression patterns in these patients, when compared to control subjects. Therefore, it would be interesting to analyze sub-regional inositol transporter expression in post-mortem tissue of AD patients and control subjects using QPCR.
In mice, no significant differences in SMIT1 or SMIT2 expression was observed in any of the
165 tissues examined, either as a function of age or disease pathology however, these studies have not been conducted on human tissue. This study would provide information on whether the elevations in brain myo- and scyllo-inositol concentrations observed in human subjects with AD and in older normal subjects, results from a corresponding elevation in SMIT1 and SMIT2 levels. These studies would help us gain greater insight into the process of inositol transport into the brain and in interpreting clinical trial findings.
In this thesis, SMIT1 was shown using competitive transport assays, to be responsible for scyllo-inositol transport across cellular membranes (Chapter 6). SMIT1 is expressed at both the blood-brain and blood-CSF barriers (Spector, 1988; Inoue et al, 1996) and as a result, it appears to be the main transporter responsible for myo- and scyllo-inositol entry into the brain. SMIT1-/- mice show a 92% reduction and an 84% reduction in myo-inositol levels in the brain and periphery, respectively, resulting in these mice expiring shortly after birth as a result of hypoventilation (Berry et al, 2003). A similar reduction in scyllo-Inositol levels would be expected to occur in the brains of these animals. In contrast, Ts65Dn mice, a mouse model of
Down syndrome, show a 38% increase in brain myo-inositol levels (Huang et al, 2000) and presumably scyllo-inositol levels. myo-Inositol levels in these mice are elevated in the frontal cortex, hippocampus and brainstem, but not in the cerebellum (Shetty et al, 2000). Therefore, by crossing these animals with TgCRND8 mice, studies could be conducted on the effects of decreasing or increasing SMIT1 expression, on scyllo-inositol transport into the brain and on its ability to alter disease pathology in the TgCRND8 mice. SMIT1 homozygous knockout mice expire soon after birth, however, TgCRND8 mice could be crossed with heterozygous SMIT1 knockout mice, which have a normal lifespan and show a more modest 15% and 25% reduction in frontal cortex and hippocampus myo-inositol levels, respectively (Shaldubina et al, 2007).
166
This would allow us to determine the effects of a reduction in SMIT1 activity on scyllo-inositol transport and on the treatment of AD. These affects could be compared to the effects of increasing the SMIT1 copy number scyllo-inositol transport and therapeutic activity.
Down syndrome mice have a triplication of a segment of chromosome 16, which corresponds to the region on chromosome 21 that is triplicated in the human condition and contains the SMIT1 gene (Gardiner et al, 2003). One potential concern with using Down syndrome mice as a method to triplicate the SMIT1 gene, is that this region of chromosome 16 also contains the
APP gene, which generates the amyloid precursor protein and following cleavage, the Aβ peptide, which is aggregated in AD (Kang et al, 1987). However, Ts1Cje mice (Sago et al,
1998), unlike Ts65Dn mice and other mouse models of Down syndrome, have a triplication of a shorter segment of chromosome 16 that does not include the APP gene, thus removing this potential confounding variable (Gardiner et al, 2003). The effects of SMIT1 gene copy number on baseline scyllo-inositol levels could be examined by comparing these two mouse models. In addition, prophylactic and therapeutic scyllo-inositol studies could be conducted in both sets of mice to examine the effects of changing the SMIT1 gene copy number on the effectiveness of scyllo-inositol as an AD therapeutic.
168
REFERENCES
Aouameur R, Da Cal S, Bissonnette P, Coady MJ, Lapointe JY. SMIT2 mediates all myo- inositol uptake in apical membranes of rat small intestine. Am J Physiol Gastrointest Liver Physiol. 2007 Dec;293(6):G1300-7. Arner RJ, Prabhu KS, Krishnan V, Johnson MC, Reddy CC. Expression of myo-inositol oxygenase in tissues susceptible to diabetic complications. Biochem Biophys Res Commun. 2006 Jan 20;339(3):816-20. Auzanneau FI, Hanna HR, Bundle DR. The synthesis of chemically modified disaccharide derivatives of the Shigella flexneri Y polysaccharide antigen. Carbohydr Res. 1993 Feb 24;240:161-81. Banks WA. Characteristics of compounds that cross the blood-brain barrier. BMC Neurol. 2009 Jun 12;9 Suppl 1:S3. Barkai AI, Dunner DL, Gross HA, Mayo P, Fieve RR. Reduced myo-inositol levels in cerebrospinal fluid from patients with affective disorder. Biol Psychiatry. 1978 Feb;13(1):65- 72.
Begley DJ, Brightman MW. Structural and functional aspects of the blood–brain barrier. Prog Drug Res. 2003; 61:39–78.
Benjamin J, Agam G, Levine J, Bersudsky Y, Kofman O, Belmaker RH. Inositol treatment in psychiatry. Psychopharmacol Bull. 1995;31(1):167-75. Berman DE, Dall'Armi C, Voronov SV, McIntire LB, Zhang H, Moore AZ, Staniszewski A, Arancio O, Kim TW, Di Paolo G. Oligomeric amyloid-beta peptide disrupts phosphatidylinositol-4,5-bisphosphate metabolism. Nat Neurosci. 2008 May;11(5):547-54. Berry GT, Mallee JJ, Kwon HM, Rim JS, Mulla WR, Muenke M, Spinner NB. The human osmoregulatory Na+/myo-inositol cotransporter gene (SLC5A3): molecular cloning and localization to chromosome 21. Genomics. 1995 Jan 20;25(2):507-13. Berry GT, Wu S, Buccafusca R, Ren J, Gonzales LW, Ballard PL, Golden JA, Stevens MJ, Greer JJ. Loss of murine Na+/myo-inositol cotransporter leads to brain myo-inositol depletion and central apnea. J Biol Chem. 2003 May 16;278(20):18297-302.
Bersudsky Y, Kaplan Z, Shapiro Y, Agam G, Kofman O, Belmaker RH. Behavioral evidence for the existence of two pools of cellular inositol. Eur Neuropsychopharmacol. 1994 Dec;4(4):463‐7. Bersudsky Y, Einat H, Stahl Z, Belmaker RH. Epi-inositol and inositol depletion: two new treatment approaches in affective disorder. Curr Psychiatry Rep. 1999 Dec;1(2):141-7. Betz AL, Goldstein GW. Polarity of the blood-brain barrier: neutral amino acid transport into isolated brain capillaries. Science. 1978 Oct 13;202(4364):225-7.
169
Bissonnette P, Coady MJ, Lapointe JY. Expression of the sodium-myo-inositol cotransporter SMIT2 at the apical membrane of Madin-Darby canine kidney cells. J Physiol. 2004 Aug 1;558(Pt 3):759-68. Bissonnette P, Lahjouji K, Coady MJ, Lapointe JY. Effects of hyperosmolarity on the Na+ - myo-inositol cotransporter SMIT2 stably transfected in the Madin-Darby canine kidney cell line. Am J Physiol Cell Physiol. 2008 Sep;295(3):C791-9. Boado RJ, Zhou QH, Lu JZ, Hui EK, Pardridge WM. Pharmacokinetics and Brain Uptake of a Genetically Engineered Bifunctional Fusion Antibody Targeting the Mouse Transferrin Receptor. Mol Pharm. 2009 Dec 3. [Epub ahead of print] Bourgeois F, Coady MJ, Lapointe JY. Determination of transport stoichiometry for two cation- coupled myo-inositol cotransporters: SMIT2 and HMIT. J Physiol. 2005 Mar 1;563(Pt 2):333- 43. Bouveault L. De l’isomérie optique dans les corps à chaines fermées. Bull la Société Chimique Paris. 1894;11:44–147.
Brightman MW, Reese TS. Junctions between intimately apposed cell membranes in the vertebrate brain. J Cell Biol. 1969 Mar;40(3):648-77. Brodie BB, Kurz H, Schanker LS. The importance of dissociaton constant and lipid-solubility in influencing the passage of drugs into the cerebrospinal fluid. J Pharmacol Exp Ther. 1960 Sep;130(1):20-5. Caldwell KK, Sosa M, Buckley CT. Identification of mitogen-activated protein kinase docking sites in enzymes that metabolize phosphatidylinositols and inositol phosphates. Cell Commun Signal. 2006 Jan 30;4:2. a. Cammarata PR, Chen HQ. Osmoregulatory alterations in myo-inositol uptake by bovine lens epithelial cells. Part 1: A hypertonicity-induced protein enhances myo-inositol transport. Invest Ophthalmol Vis Sci. 1994 Mar;35(3):1223-35. b. Cammarata PR, Chen HQ, Zhou C, Reeves R. Osmoregulatory alterations in myo-inositol uptake by bovine lens epithelial cells. III. Effects of cycloheximide and colchicine on Na(+)- myo-inositol cotransporter activity under hypertonic conditions, inhibition of a plasma membrane osmotic stress protein. Exp Eye Res. 1994 Jul;59(1):83-9. Cammarata PR, Xu GT, Huang L, Zhou C, Martin M. Inducible expression of Na+/myo-inositol cotransporter mRNA in anterior epithelium of bovine lens: affiliation with hypertonicity and cell proliferation. Exp Eye Res. 1997 May;64(5):745-57. Chengappa KN, Levine J, Gershon S, Mallinger AG, Hardan A, Vagnucci A, Pollock B, Luther J, Buttenfield J, Verfaille S, Kupfer DJ. Inositol as an add-on treatment for bipolar depression. Bipolar Disord. 2000 Mar;2(1):47-55. Chertkow H. Diagnosis and treatment of dementia: introduction. Introducing a series based on the Third Canadian Consensus Conference on the Diagnosis and Treatment of Dementia. CMAJ. 2008 Jan 29;178(3):316-21.
170
Chesler M, Kaila K. Modulation of pH by neuronal activity. Trends Neurosci. 1992 Oct;15(10):396-402. Chishti MA, Yang DS, Janus C, Phinney AL, Horne P, Pearson J, Strome R, Zuker N, Loukides J, French J, Turner S, Lozza G, Grilli M, Kunicki S, Morissette C, Paquette J, Gervais F, Bergeron C, Fraser PE, Carlson GA, George-Hyslop PS, Westaway D. Early-onset amyloid deposition and cognitive deficits in transgenic mice expressing a double mutant form of amyloid precursor protein 695. J Biol Chem. 2001 Jun 15;276(24):21562-70. Choo-Smith LP, Surewicz WK. The interaction between Alzheimer amyloid beta(1-40) peptide and ganglioside GM1-containing membranes. FEBS Lett. 1997 Feb 3;402(2-3):95-8. Citron M. Alzheimer's disease: treatments in discovery and development. Nat Neurosci. 2002 Nov;5 Suppl:1055-7. Clements RS Jr, Diethelm AG. The metabolism of myo-inositol by the human kidney. J Lab Clin Med. 1979 Feb;93(2):210-9. Coady MJ, Wallendorff B, Gagnon DG, Lapointe JY. Identification of a novel Na+/myo- inositol cotransporter. J Biol Chem. 2002 Sep 20;277(38):35219-24. Cogram P, Tesh S, Tesh J, Wade A, Allan G, Greene ND, Copp AJ. D-chiro-inositol is more effective than myo-inositol in preventing folate-resistant mouse neural tube defects. Hum Reprod. 2002 Sep;17(9):2451-8.
Curtain CC, Ali FE, Smith DG, Bush AI, Masters CL, Barnham KJ. Metal ions, pH, and cholesterol regulate the interactions of Alzheimer's disease amyloid-beta peptide with membrane lipid. J Biol Chem. 2003 Jan 31;278(5):2977-82.
Dangschat G. Acetonierung und konfiguration des meso-inosits. Naturwissenschaften. 1942;30:146.
Denkert C, Warskulat U, Hensel F, Häussinger D. Osmolyte strategy in human monocytes and macrophages: involvement of p38MAPK in hyperosmotic induction of betaine and myoinositol transporters. Arch Biochem Biophys. 1998 Jun 1;354(1):172-80. Dermietzel R, Schunke D. A complex junctional system in endothelial and connective tissue cells of the choroid plexus. Am J Anat. 1975 May;143(1):131-6. Di Daniel E, Mok MH, Mead E, Mutinelli C, Zambello E, Caberlotto LL, Pell TJ, Langmead CJ, Shah AJ, Duddy G, Kew JN, Maycox PR. Evaluation of expression and function of the H+/myo-inositol transporter HMIT. BMC Cell Biol. 2009 Jul 16;10:54.
Diringer H, Rott R. Different pools of free myoinositol in chick‐embryo cells as indicated by infection with Newcastle‐disease virus. Eur J Biochem. 1977 Oct 3;79(2):451‐7. Eden Evins A, Demopulos C, Yovel I, Culhane M, Ogutha J, Grandin LD, Nierenberg AA, Sachs GS. Inositol augmentation of lithium or valproate for bipolar depression. Bipolar Disord. 2006 Apr;8(2):168-74.
171
Ehrlich P. Das Sauerstoff-Bedürfnis des Organismus: Eine farbenanalytiche Studie. Hirschwald. Berlin. 1885 8:167. Einat H, Elkabaz-Shwortz Z, Cohen H, Kofman O, Belmaker RH. Chronic epi-inositol has an anxiolytic-like effect in the plus-maze model in rats. Int J Neuropsychopharmacol. 1998 Jul;1(1):31-34. Eladari D, Chambrey R, Pezy F, Podevin RA, Paillard M, Leviel F. pH dependence of Na+/myo-inositol cotransporters in rat thick limb cells. Kidney Int. 2002 Dec;62(6):2144-51. Elliott PJ, Hayward NJ, Dean RL, Blunt DG, Bartus RT. Intravenous RMP-7 selectively increases uptake of carboplatin into rat brain tumors. Cancer Res. 1996 Sep 1;56(17):3998- 4005. Farrell CL, Pardridge WM. Blood-brain barrier glucose transporter is asymmetrically distributed on brain capillary endothelial lumenal and ablumenal membranes: an electron microscopic immunogold study. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5779-83. Fenili D, Brown M, Rappaport R, McLaurin J. Properties of scyllo-inositol as a therapeutic treatment of AD-like pathology. J Mol Med. 2007 Jun;85(6):603-11. Fisher SK, Novak JE, Agranoff BW. Inositol and higher inositol phosphates in neural tissues: homeostasis, metabolism and functional significance. J Neurochem. 2002 Aug;82(4):736-54. Frey WH 2nd: [http:/ / www.wipo.int/ pctdb/ en/ wo.jsp?wo=1991007947&IA=WO199100794 7&DISPLAY=CLAIMS] (WO/1991/007947) Neurologic Agents for Nasal Administration to the Brain (priority date 5.12.89). Geneva, Switzerland: World Intellectual Property Organization; 1991. Fux M, Levine J, Aviv A, Belmaker RH. Inositol treatment of obsessive-compulsive disorder. Am J Psychiatry. 1996 Sep;153(9):1219-21. Gardiner K, Fortna A, Bechtel L, Davisson MT. Mouse models of Down syndrome: how useful can they be? Comparison of the gene content of human chromosome 21 with orthologous mouse genomic regions. Gene. 2003 Oct 30;318:137-47. Gatter KC, Brown G, Trowbridge IS, Woolston RE, Mason DY. Transferrin receptors in human tissues: their distribution and possible clinical relevance. J Clin Pathol. 1983 May;36(5):539-45. Gazeau F, Lévy M, Wilhelm C. Optimizing magnetic nanoparticle design for nanothermotherapy. Nanomed. 2008 Dec;3(6):831-44. Gelber D, Levine J, Belmaker RH. Effect of inositol on bulimia nervosa and binge eating. Int J Eat Disord. 2001 Apr;29(3):345-8. Gerli S, Mignosa M, Di Renzo GC. Effects of inositol on ovarian function and metabolic factors in women with PCOS: a randomized double blind placebo-controlled trial. Eur Rev Med Pharmacol Sci. 2003 Nov-Dec;7(6):151-9. Glaudemans CPJ. Mapping of subsites of monoclonal, anti-carbohydrate antibodies using deoxy and deoxyfluoro sugars. Chem Rev. 1991;91:25-33.
172
Goldmann EE. Vitalfärbung am Zentralnervensystem. Abh Preuss Adak Wiss Phys-Math 1913 1:1-60. Greene ND, Copp AJ. Inositol prevents folate-resistant neural tube defects in the mouse. Nat Med. 1997 Jan;3(1):60-6. Griffith HR, den Hollander JA, Stewart CC, Evanochko WT, Buchthal SD, Harrell LE, Zamrini EY, Brockington JC, Marson DC. Elevated brain scyllo-inositol concentrations in patients with Alzheimer's disease. NMR Biomed. 2007 Dec;20(8):709-16. Groenen PM, Merkus HM, Sweep FC, Wevers RA, Janssen FS, Steegers-Theunissen RP. Kinetics of myo-inositol loading in women of reproductive age. Ann Clin Biochem. 2003 Jan;40(Pt 1):79-85. Hager K, Hazama A, Kwon HM, Loo DD, Handler JS, Wright EM. Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus oocytes. J Membr Biol. 1995 Jan;143(2):103-13. Hakvoort A, Haselbach M, Galla HJ. Active transport properties of porcine choroid plexus cells in culture. Brain Res. 1998 Jun 8;795(1-2):247-56. Hallman M, Bry K, Hoppu K, Lappi M, Pohjavuori M. Inositol supplementation in premature infants with respiratory distress syndrome. N Engl J Med. 1992 May 7;326(19):1233-9. Hallman M, Järvenpää AL, Pohjavuori M. Respiratory distress syndrome and inositol supplementation in preterm infants. Arch Dis Child. 1986 Nov;61(11):1076-83. Hanson LR, Frey WH 2nd. Intranasal delivery bypasses the blood-brain barrier to target therapeutic agents to the central nervous system and treat neurodegenerative disease. BMC Neurosci. 2008 Dec 10;9 Suppl 3:S5. Hawkins BT, Davis TP. The blood-brain barrier/neurovascular unit in health and disease. Pharmacol Rev. 2005 Jun;57(2):173-85. Hipps PP, Eveland MR, Laird MH, Sherman WR. The identification of MYO-inositol:NAD(P)+ oxidoreductase in mammalian brain. Biochem Biophys Res Commun. 1976 Feb 23;68(4):1133- 8. Hipps PP, Holland WH, Sherman WR. Interconversion of myo- and scyllo-inositol with simultaneous formation of neo-inositol by an NADP+ dependent epimerase from bovine brain. Biochem Biophys Res Commun. 1977 Jul 11;77(1):340-6. Holub BJ. Metabolism and function of myo-inositol and inositol phospholipids. Annu Rev Nutr. 1986;6:563-97. Hori S, Ohtsuki S, Hosoya K, Nakashima E, Terasaki T. A pericyte-derived angiopoietin-1 multimeric complex induces occludin gene expression in brain capillary endothelial cells through Tie-2 activation in vitro. J Neurochem. 2004 Apr;89(2):503-13. Huang W, Galdzicki Z, van Gelderen P, Balbo A, Chikhale EG, Schapiro MB, Rapoport SI. Brain myo-inositol level is elevated in Ts65Dn mouse and reduced after lithium treatment. Neuroreport. 2000 Feb 28;11(3):445-8.
173
Huang Z, Xu H, Meyers AD, Musani AI, Wang L, Tagg R, Barqawi AB, Chen YK. Photodynamic therapy for treatment of solid tumors--potential and technical challenges. Technol Cancer Res Treat. 2008 Aug;7(4):309-20. Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11-25. Inamura T, Nomura T, Bartus RT, Black KL. Intracarotid infusion of RMP-7, a bradykinin analog: a method for selective drug delivery to brain tumors. J Neurosurg. 1994 Nov;81(5):752- 8. Inoue K, Shimada S, Minami Y, Morimura H, Miyai A, Yamauchi A, Tohyama M. Cellular localization of Na+/MYO-inositol co-transporter mRNA in the rat brain. Neuroreport. 1996 Apr 26;7(6):1195-8. Irvine RF. Manganese-stimulated phosphatidylinositol headgroup exchange in rat liver microsomes. Biochim Biophys Acta. 1998 Aug 28;1393(2-3):292-8. Isaacks RE, Bender AS, Kim CY, Shi YF, Norenberg MD. Effect of osmolality and anion channel inhibitors on myo-inositol efflux in cultured astrocytes. J Neurosci Res. 1999 Sep 15;57(6):866-71. Jackson PS, Strange K. Volume-sensitive anion channels mediate swelling-activated inositol and taurine efflux. Am J Physiol. 1993 Dec;265(6 Pt 1):C1489-500. Janzer RC, Raff MC. Astrocytes induce blood-brain barrier properties in endothelial cells. Nature. 1987 Jan 15-21;325(6101):253-7. Johanson CE, Duncan JA, Stopa EG, Baird A. Enhanced prospects for drug delivery and brain targeting by the choroid plexus-CSF route. Pharm Res. 2005 Jul;22(7):1011-37. Epub 2005 Jul 22. Kaiser LG, Schuff N, Cashdollar N, Weiner MW. Scyllo-inositol in normal aging human brain: 1H magnetic resonance spectroscopy study at 4 Tesla. NMR Biomed. 2005 Feb;18(1):51-5. Kang J, Lemaire HG, Unterbeck A, Salbaum JM, Masters CL, Grzeschik KH, Multhaup G, Beyreuther K, Müller-Hill B. The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor. Nature. 1987 Feb 19-25;325(6106):733-6.
Karihaloo A, Kato K, Greene DA, Thomas TP. Protein kinase and Ca2+ modulation of myo- inositol transport in cultured retinal pigment epithelial cells. Am J Physiol. 1997 Aug;273(2 Pt 1):C671-8. Keep RF, Jones HC. A morphometric study on the development of the lateral ventricle choroid plexus, choroid plexus capillaries and ventricular ependyma in the rat. Brain Res Dev Brain Res. 1990 Oct 1;56(1):47-53. Kersting MC, Boyette M, Massey JH, Ryals PE. Identification of the inositol isomers present in Tetrahymena. J Eukaryot Microbiol. 2003 May-Jun;50(3):164-8.
174
Kinnard RL, Narasimhan B, Pliska-Matyshak G, Murthy PP. Characterization of scyllo- inositol-containing phosphatidylinositol in plant cells. Biochem Biophys Res Commun. 1995 May 16;210(2):549-55. Klaus F, Palmada M, Lindner R, Laufer J, Jeyaraj S, Lang F, Boehmer C. Up-regulation of hypertonicity-activated myo-inositol transporter SMIT1 by the cell volume-sensitive protein kinase SGK1. J Physiol. 2008 Mar 15;586(6):1539-47. Kofman O, Bersudsky Y, Vinnitsky I, Alpert C, Belmaker RH. The effect of peripheral inositol injection on rat motor activity models of depression. Isr J Med Sci. 1993 Sep;29(9):580-6. Kremer JJ, Pallitto MM, Sklansky DJ, Murphy RM. Correlation of beta-amyloid aggregate size and hydrophobicity with decreased bilayer fluidity of model membranes. Biochemistry. 2000 Aug 22;39(33):10309-18. Kurakhmaeva KB, Voronina TA, Kapica IG, Kreuter J, Nerobkova LN, Seredenin SB, Balabanian VY, Alyautdin RN. Antiparkinsonian effect of nerve growth factor adsorbed on polybutylcyanoacrylate nanoparticles coated with polysorbate-80. Bull Exp Biol Med. 2008 Feb;145(2):259-62. Kwon HM, Yamauchi A, Uchida S, Preston AS, Garcia-Perez A, Burg MB, Handler JS. Cloning of the cDNa for a Na+/myo-inositol cotransporter, a hypertonicity stress protein. J Biol Chem. 1992 Mar 25;267(9):6297-301. Kwon HM, Yamauchi A, Uchida S, Robey RB, Garcia-Perez A, Burg MB, Handler JS. Renal Na-myo-inositol cotransporter mRNA expression in Xenopus oocytes: regulation by hypertonicity. Am J Physiol. 1991 Feb;260(2 Pt 2):F258-63. Lahjouji K, Aouameur R, Bissonnette P, Coady MJ, Bichet DG, Lapointe JY. Expression and functionality of the Na+/myo-inositol cotransporter SMIT2 in rabbit kidney. Biochim Biophys Acta. 2007 May;1768(5):1154-9. Larner J. D-chiro-inositol--its functional role in insulin action and its deficit in insulin resistance. Int J Exp Diabetes Res. 2002;3(1):47-60. Levine J, Barak Y, Gonzalves M, Szor H, Elizur A, Kofman O, Belmaker RH. Double-blind, controlled trial of inositol treatment of depression. Am J Psychiatry. 1995 May;152(5):792-4. Li R, Lindholm K, Yang LB, Yue X, Citron M, Yan R, Beach T, Sue L, Sabbagh M, Cai H, Wong P, Price D, Shen Y. Amyloid beta peptide load is correlated with increased beta-secretase activity in sporadic Alzheimer's disease patients. Proc Natl Acad Sci U S A. 2004 Mar 9;101(10):3632-7. Lin X, Ma L, Fitzgerald RL, Ostlund RE Jr. Human sodium/inositol cotransporter 2 (SMIT2) transports inositols but not glucose in L6 cells. Arch Biochem Biophys. 2009 Jan 15;481(2):197-201. Lockman PR, Mumper RJ, Khan MA, Allen DD. Nanoparticle technology for drug delivery across the blood-brain barrier. Drug Dev Ind Pharm. 2002 Jan;28(1):1-13.
175
Loveday D, Heacock AM, Fisher SK. Activation of muscarinic cholinergic receptors enhances the volume-sensitive efflux of myo-inositol from SH-SY5Y neuroblastoma cells. J Neurochem. 2003 Oct;87(2):476-86. Lubrich B, Spleiss O, Gebicke-Haerter PJ, van Calker D. Differential expression, activity and regulation of the sodium/myo-inositol cotransporter in astrocyte cultures from different regions of the rat brain. Neuropharmacology. 2000 Feb 14;39(4):680-90. Majumder AL, Chatterjee A, Ghosh Dastidar K, Majee M. Diversification and evolution of L- myo-inositol 1-phosphate synthase. FEBS Lett. 2003 Oct 9;553(1-2):3-10. Mallee JJ, Atta MG, Lorica V, Rim JS, Kwon HM, Lucente AD, Wang Y, Berry GT. The structural organization of the human Na+/myo-inositol cotransporter (SLC5A3) gene and characterization of the promoter. Genomics. 1997 Dec 15;46(3):459-65. Matskevitch J, Wagner CA, Risler T, Kwon HM, Handler JS, Waldegger S, Busch AE, Lang F. Effect of extracellular pH on the myo-inositol transporter SMIT expressed in Xenopus oocytes. Pflugers Arch. 1998 Nov;436(6):854-7. Matsuoka Y, Yamauchi A, Nakanishi T, Sugiura T, Kitamura H, Horio M, Takamitsu Y, Ando A, Imai E, Hori M. Response to hypertonicity in mesothelial cells: role of Na+/myo-inositol co- transporter. Nephrol Dial Transplant. 1999 May;14(5):1217-23. Mayer S, Maickel RP, Brodie BB. Kinetics of penetration of drugs and other foreign compounds into cerebrospinal fluid and brain. J Pharmacol Exp Ther. 1959 Nov;127(3):205- 211. McAllister G, Whiting P, Hammond EA, Knowles MR, Atack JR, Bailey FJ, Maigetter R, Ragan CI. cDNA cloning of human and rat brain myo-inositol monophosphatase. Expression and characterization of the human recombinant enzyme. Biochem J. 1992 Jun 15;284 ( Pt 3):749-54. McLaurin J, Chakrabartty A. Membrane disruption by Alzheimer beta-amyloid peptides mediated through specific binding to either phospholipids or gangliosides. Implications for neurotoxicity. J Biol Chem. 1996 Oct 25;271(43):26482-9. McLaurin J, Chakrabartty A. Characterization of the interactions of Alzheimer beta-amyloid peptides with phospholipid membranes. Eur J Biochem. 1997 Apr 15;245(2):355-63. McLaurin J, Franklin T, Chakrabartty A, Fraser PE. Phosphatidylinositol and inositol involvement in Alzheimer amyloid-beta fibril growth and arrest. J Mol Biol. 1998 Apr 24;278(1):183-94. McLaurin J, Golomb R, Jurewicz A, Antel JP, Fraser PE. Inositol stereoisomers stabilize an oligomeric aggregate of Alzheimer amyloid beta peptide and inhibit abeta -induced toxicity. J Biol Chem. 2000 Jun 16;275(24):18495-502. McLaurin J, Kierstead ME, Brown ME, Hawkes CA, Lambermon MH, Phinney AL, Darabie AA, Cousins JE, French JE, Lan MF, Chen F, Wong SS, Mount HT, Fraser PE, Westaway D, St George-Hyslop P. Cyclohexanehexol inhibitors of Abeta aggregation prevent and reverse Alzheimer phenotype in a mouse model. Nat Med. 2006 Jul;12(7):801-8.
176
McLean CA, Cherny RA, Fraser FW, Fuller SJ, Smith MJ, Beyreuther K, Bush AI, Masters CL. Soluble pool of Abeta amyloid as a determinant of severity of neurodegeneration in Alzheimer's disease. Ann Neurol. 1999 Dec;46(6):860-6. McVeigh KE, Mallee JJ, Lucente A, Barnoski BL, Wu S, Berry GT. Murine chromosome 16 telomeric region, homologous with human chromosome 21q22, contains the osmoregulatory Na(+)/myo-inositol cotransporter (SLC5A3) gene. Cytogenet Cell Genet. 2000;88(1-2):153-8. Michaelis T, Helms G, Merboldt KD, Hänicke W, Bruhn H, Frahm J. Identification of Scyllo- inositol in proton NMR spectra of human brain in vivo. NMR Biomed. 1993 Jan-Feb;6(1):105- 9. Miyai A, Yamauchi A, Nakanishi T, Sugita M, Takamitsu Y, Yokoyama K, Itoh T, Andou A, Kamada T, Ueda N, et al. Na+/myo-inositol cotransport is regulated by tonicity in cultured rat mesangial cells. Kidney Int. 1995 Feb;47(2):473-80. Monakow C. Der Kreislauf des Liquor cerebrospinalis. Scweiz Arch Neurol und Psychiatr. 1921; 8:233.
Mueckler M, Caruso C, Baldwin SA, Panico M, Blench I, Morris HR, Allard WJ, Lienhard GE, Lodish HF. Sequence and structure of a human glucose transporter. Science. 1985 Sep 6;229(4717):941-5. Nakanishi T, Turner RJ, Burg MB. Osmoregulatory changes in myo-inositol transport by renal cells. Proc Natl Acad Sci U S A. 1989 Aug;86(15):6002-6. Näslund J, Haroutunian V, Mohs R, Davis KL, Davies P, Greengard P, Buxbaum JD. Correlation between elevated levels of amyloid beta-peptide in the brain and cognitive decline. JAMA. 2000 Mar 22-29;283(12):1571-7. Nestler JE, Jakubowicz DJ, Reamer P, Gunn RD, Allan G. Ovulatory and metabolic effects of D-chiro-inositol in the polycystic ovary syndrome. N Engl J Med. 1999 Apr 29;340(17):1314- 20. Novak JE, Agranoff BW, Fisher SK. Regulation of Myo-inositol homeostasis in differentiated human NT2-N neurons. Neurochem Res. 2000 May;25(5):561-6. Oldendorf WH, Cornford ME, Brown WJ. The large apparent work capability of the blood- brain barrier: a study of the mitochondrial content of capillary endothelial cells in brain and other tissues of the rat. Ann Neurol. 1977 May;1(5):409-17. Ostlund RE Jr, Seemayer R, Gupta S, Kimmel R, Ostlund EL, Sherman WR. A stereospecific myo-inositol/D-chiro-inositol transporter in HepG2 liver cells. Identification with D-chiro-[3- 3H]inositol. J Biol Chem. 1996 Apr 26;271(17):10073-8.
Pak Y, Huang LC, Lilley KJ, Larner J. In vivo conversion of [3H]myoinositol to [3H]chiroinositol in rat tissues. J Biol Chem. 1992 Aug 25;267(24):16904-10.
Palatnik A, Frolov K, Fux M, Benjamin J. Double-blind, controlled, crossover trial of inositol versus fluvoxamine for the treatment of panic disorder. J Clin Psychopharmacol. 2001 Jun;21(3):335-9.
177
Palmano KP, Whiting PH, Hawthorne JN. Free and lipid myo-inositol in tissues from rats with acute and less severe streptozotocin-induced diabetes. Biochem J. 1977 Oct 1;167(1):229-35. Pan W, Kastin AJ. Changing the chemokine gradient: CINC1 crosses the blood-brain barrier. J Neuroimmunol. 2001 Apr 2;115(1-2):64-70.
Pardridge WM, Frank HJ, Cornford EM, Braun LD, Crane PD, Oldendorf WH. Neuropeptides and the blood-brain barrier. Adv Biochem Psychopharmacol. 1981;28:321-8.
Paredes A, McManus M, Kwon HM, Strange K. Osmoregulation of Na(+)-inositol cotransporter activity and mRNA levels in brain glial cells. Am J Physiol. 1992 Dec;263(6 Pt 1):C1282-8.
Patel MM, Goyal BR, Bhadada SV, Bhatt JS, Amin AF. Getting into the brain: approaches to enhance brain drug delivery. CNS Drugs. 2009;23(1):35-58.
Posternak T. Recherches dans la séries des cyclites V. Sur un inosose préparé par voie biochimique. Helv Chim Acta. 1941;24:1045-58.
Posternak T. Recherches dans la série des cyclites VI. Sur la configuration de la méso-inosite, de la scyllite et d'un inosose obtenu par voie biochimique (scyllo-ms-inosose). Helv Chim Acta. 1942;25:746-52.
Provenzale JM, Silva GA. Uses of nanoparticles for central nervous system imaging and therapy. AJNR Am J Neuroradiol. 2009 Aug;30(7):1293-301. Epub 2009 Jul 17. Rall DP, Stabenau JR, Zubrod CG. Distribution of drugs between blood and cerebrospinal fluid: general methodology and effect of pH gradients. J Pharmacol Exp Ther. 1959 Mar;125(3):185-93. Rapoport SI. Effect of concentrated solutions on blood-brain barrier. Am J Physiol. 1970 July;219(1):270-274. Rapoport SI, Bachman DS, Thompson HK. Chronic effects of osmotic opening of the blood- brain barrier in the monkey. Science. 1972 Jun 16;176(40):1243-5. Rapoport SI, Hori M, Klatzo I. Reversible osmotic opening of the blood-brain barrier. Science. 1971 Sep 10;173(4001):1026-8. Rapoport SI, Thompson HK. Osmotic opening of the blood-brain barrier in the monkey without associated neurological deficits. Science. 1973 Jun 1;180(89):971. Reese TS, Karnovsky MJ. Fine structural localization of a blood-brain barrier to exogenous peroxidase. J Cell Biol. 1967 Jul;34(1):207-17. Riggs BM, Lansley TA, Ryals PE. Phosphatidylinositol synthase of Tetrahymena: inositol isomers as substrates in phosphatidylinositol biosynthesis and headgroup exchange reactions. J Eukaryot Microbiol. 2007 Mar-Apr;54(2):119-24.
178
Roll P, Massacrier A, Pereira S, Robaglia-Schlupp A, Cau P, Szepetowski P. New human sodium/glucose cotransporter gene (KST1): identification, characterization, and mutation analysis in ICCA (infantile convulsions and choreoathetosis) and BFIC (benign familial infantile convulsions) families. Gene. 2002 Feb 20;285(1-2):141-8.
Rubin LJ, Hale CC. Characterization of a Mg-dependent, Na-inositol co-transport process in cardiac sarcolemmal vesicles. J Mol Cell Cardiol. 1993 Jun;25(6):721-31. Ryals PE, Kersting MC. Sodium-dependent uptake of [3H]scyllo-inositol by Tetrahymena: incorporation into phosphatidylinositol, phosphatidylinositol-linked glycans, and polyphosphoinositols. Arch Biochem Biophys. 1999 Jun 15;366(2):261-6.
Sago H, Carlson EJ, Smith DJ, Kilbridge J, Rubin EM, Mobley WC, Epstein CJ, Huang TT. Ts1Cje, a partial trisomy 16 mouse model for Down syndrome, exhibits learning and behavioral abnormalities. Proc Natl Acad Sci U S A. 1998 May 26;95(11):6256-61.
Salman M, Lonsdale JT, Besra GS, Brennan PJ. Phosphatidylinositol synthesis in mycobacteria. Biochim Biophys Acta. 1999 Jan 4;1436(3):437-50.
Sanz ML, Villamiel M, Martinez-Castro I. Inositols and carbohydrates in different fresh fruit juices. Food Chem. 2004 Sep; 87(3):325-8.
Scherer J. Ueber eine neue, aus dem Muskelfleische gewonnene Zuckerart. Liebigs Ann Chem. 1850;73:322–8.
Seaquist ER, Gruetter R. Identification of a high concentration of scyllo-inositol in the brain of a healthy human subject using 1H- and 13C-NMR. Magn Reson Med. 1998 Feb;39(2):313-6.
Selkoe DJ. Alzheimer's disease: genes, proteins, and therapy. Physiol Rev. 2001 Apr;81(2):741-66. Selkoe DJ. Alzheimer's disease is a synaptic failure. Science. 2002 Oct 25;298(5594):789-91. Shaldubina A, Buccafusca R, Johanson RA, Agam G, Belmaker RH, Berry GT, Bersudsky Y. Behavioural phenotyping of sodium-myo-inositol cotransporter heterozygous knockout mice with reduced brain inositol. Genes Brain Behav. 2007 Apr;6(3):253-9. Shankar GM, Bloodgood BL, Townsend M, Walsh DM, Selkoe DJ, Sabatini BL. Natural oligomers of the Alzheimer amyloid-beta protein induce reversible synapse loss by modulating an NMDA-type glutamate receptor-dependent signaling pathway. J Neurosci. 2007 Mar 14;27(11):2866-75. Sherman WR, Simpson PC, Goodwin SL. scyllo-inositol and myo-inositol levels in tissues of the skate Raja erinacea. Comp Biochem Physiol B. 1978;59(3):201-2. a. Sherman WR, Stewart MA, Kurien MM, Goodwin SL. The measurement of myo-inositol, myo-inosose-2 and scyllo-inositol in mammalian tissues. Biochim Biophys Acta. 1968 May;158(2):197-205.
179 b. Sherman WR, Stewart MA, Simpson PC, Goodwin SL. The identification of myo-inosose-2 and scyllo-inositol in mammalian tissues. Biochemistry. 1968 Feb;7(2):819-24. Shetty HU, Holloway HW, Rapoport SI. Capillary gas chromatography combined with ion trap detection for quantitative profiling of polyols in cerebrospinal fluid and plasma. Anal Biochem. 1995 Jan 1;224(1):279-85.
Shetty HU, Siarey RJ, Galdzicki Z, Stoll J, Rapoport SI. Ts65Dn mouse, a Down syndrome model, exhibits elevated myo-inositol in selected brain regions and peripheral tissues. Neurochem Res. 2000 Apr;25(4):431-5. Sigal SH, Yandrasitz JR, Berry GT. Kinetic evidence for compartmentalization of myo-inositol in hepatocytes. Metabolism. 1993 Mar;42(3):395-401. Sinha S. The role of beta-amyloid in Alzheimer's disease. Med Clin North Am. 2002 May;86(3):629-39. Sink KM, Holden KF, Yaffe K. Pharmacological treatment of neuropsychiatric symptoms of dementia: a review of the evidence. JAMA. 2005 Feb 2;293(5):596-608. Soria AC, Sanz ML, Villamiel M. Determination of minor carbohydrates in carrot (Daucus carota L.) by GC-MS. Food Chem. 2009 May 15;114(2):758-62. Spector R. The transport and metabolism of scyllo-inositol in the central nervous system. J Neurochem. 1978 Oct;31(4):1113-5. Spector R. Myo-inositol transport through the blood-brain barrier. Neurochem Res. 1988 Aug;13(8):785-7. Staedeler G, Frerichs FT. Ueber das vorkommen von harnstoff, taurin und scyllit in den organen den plagiostomen. J prakt Chem. 1858;73:48-55.
Stern L. Le liquide céfalo-rachidien au point de vue de ses rapports avec la circulation sanguine et avec les éléments nerveux de l’axe cérébrospinal. Schweiz.Arch.Neurol. und Psychiatr. 1921; 8(2): 215–232.
Strange K, Emma F, Paredes A, Morrison R. Osmoregulatory changes in myo-inositol content and Na+/myo-inositol cotransport in rat cortical astrocytes. Glia. 1994 Sep;12(1):35-43. Sun Y, Zhang G, Hawkes CA, Shaw JE, McLaurin J, Nitz M. Synthesis of scyllo-inositol derivatives and their effects on amyloid beta peptide aggregation. Bioorg Med Chem. 2008 Aug 1;16(15):7177-84. Swaminathan CP, Gupta D, Sharma V, Surolia A. Effect of substituents on the thermodynamics of D-galactopyranoside binding to winged bean (Psophocarpus tetragonolobus) basic lectin. Biochemistry. 1997 Oct 28;36(43):13428-34. Takenawa T, Egawa K. CDP-diglyceride:inositol transferase from rat liver. Purification and properties. J Biol Chem. 1977 Aug 10;252(15):5419-23.
180
Thal DR, Rüb U, Orantes M, Braak H. Phases of A beta-deposition in the human brain and its relevance for the development of AD. Neurology. 2002 Jun 25;58(12):1791-800. Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, Willingham MC. Immunohistochemical localization in normal tissues of different epitopes in the multidrug transport protein P170: evidence for localization in brain capillaries and crossreactivity of one antibody with a muscle protein. J Histochem Cytochem. 1989 Feb;37(2):159-64. Tilling T, Korte D, Hoheisel D, Galla HJ. Basement membrane proteins influence brain capillary endothelial barrier function in vitro. J Neurochem. 1998 Sep;71(3):1151-7. Townsend M, Cleary JP, Mehta T, Hofmeister J, Lesne S, O'Hare E, Walsh DM, Selkoe DJ. Orally available compound prevents deficits in memory caused by the Alzheimer amyloid-beta oligomers. Ann Neurol. 2006 Dec;60(6):668-76. Trowbridge IS, Domingo DL. Anti-transferrin receptor monoclonal antibody and toxin- antibody conjugates affect growth of human tumour cells. Nature. 1981 Nov 12;294(5837):171- 3. Uldry M, Ibberson M, Horisberger JD, Chatton JY, Riederer BM, Thorens B. Identification of a mammalian H(+)-myo-inositol symporter expressed predominantly in the brain. EMBO J. 2001 Aug 15;20(16):4467-77. Uldry M, Steiner P, Zurich MG, Béguin P, Hirling H, Dolci W, Thorens B. Regulated exocytosis of an H+/myo-inositol symporter at synapses and growth cones. EMBO J. 2004 Feb 11;23(3):531-40. Valenzuela MJ, Sachdev P. Magnetic resonance spectroscopy in AD. Neurology. 2001 Mar 13;56(5):592-8.
Vein AA. Science and Fate: Lina Stern (1878-1968), A Neurophysiologist and Biochemist, J Hist Neurosci. 2008; 17(2)195-206. von Tell D, Armulik A, Betsholtz C. Pericytes and vascular stability. Exp Cell Res. 2006 Mar 10;312(5):623-9. Wahl M, Young AR, Edvinsson L, Wagner F. Effects of bradykinin on pial arteries and arterioles in vitro and in situ. J Cereb Blood Flow Metab. 1983 Jun;3(2):231-7. Wang J, Dickson DW, Trojanowski JQ, Lee VM. The levels of soluble versus insoluble brain Abeta distinguish Alzheimer's disease from normal and pathologic aging. Exp Neurol. 1999 Aug;158(2):328-37. Wermeling DP. Intranasal delivery of antiepileptic medications for treatment of seizures. Neurotherapeutics. 2009 Apr;6(2):352-8. Wiese TJ, Dunlap JA, Conner CE, Grzybowski JA, Lowe WL Jr, Yorek MA. Osmotic regulation of Na-myo-inositol cotransporter mRNA level and activity in endothelial and neural cells. Am J Physiol. 1996 Apr;270(4 Pt 1):C990-7.
181
Wiesinger H. myo-inositol transport in mouse astroglia-rich primary cultures. J Neurochem. 1991 May;56(5):1698-704. Wolfson M, Bersudsky Y, Hertz E, Berkin V, Zinger E, Hertz L. A model of inositol compartmentation in astrocytes based upon efflux kinetics and slow inositol depletion after uptake inhibition. Neurochem Res. 2000 Jul;25(7):977-82. Wong YH, Kalmbach SJ, Hartman BK, Sherman WR. Immunohistochemical staining and enzyme activity measurements show myo-inositol-1-phosphate synthase to be localized in the vasculature of brain. J Neurochem. 1987 May;48(5):1434-42. Wright EM, Turk E. The sodium/glucose cotransport family SLC5. Pflugers Arch. 2004 Feb;447(5):510-8. Yip CM, Elton EA, Darabie AA, Morrison MR, McLaurin J. Cholesterol, a modulator of membrane-associated Abeta-fibrillogenesis and neurotoxicity. J Mol Biol. 2001 Aug 24;311(4):723-34. a. Yorek MA, Dunlap JA, Lowe WL Jr. Opposing effects of tumour necrosis factor alpha and hyperosmolarity on Na+/myo-inositol co-transporter mRNA levels and myo-inositol accumulation by 3T3-L1 adipocytes. Biochem J. 1998 Dec 1;336 ( Pt 2):317-25. Yorek MA, Dunlap JA, Lowe WL Jr. Osmotic regulation of the Na+/myo-inositol cotransporter and postinduction normalization. Kidney Int. 1999 Jan;55(1):215-24. b. Yorek MA, Dunlap JA, Thomas MJ, Cammarata PR, Zhou C, Lowe WL Jr. Effect of TNF- alpha on SMIT mRNA levels and myo-inositol accumulation in cultured endothelial cells. Am J Physiol. 1998 Jan;274(1 Pt 1):C58-71. Zhao FQ, Keating AF. Functional properties and genomics of glucose transporters. Curr Genomics. 2007 Apr;8(2):113-28.