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OPEN ACCESS Review article Lead (Pb2+) neurotoxicity: Ion-mimicry with calcium (Ca2+) impairs synaptic transmission A Review with Animated Illustrations of the Pre- and Post-synaptic Effects of Lead

1 Universität Trier, Fachbereich Ana-Maria Florea1, Jasmin Taban, Elizabeth Varghese2, Blane T. Alost, VI, Umwelttoxikologie, 2* Universitätsring 15, 54296 Trier, Stacy Moreno, and Dietrich Büsselberg Germany 2 Weill Cornell Medical College in Qatar, Qatar Foundation – Education City, P.O. Box 24144, Doha, Qatar Publication date: 22 April 2013 * Corresponding author: Dietrich Büsselberg [email protected] Abstract Lead (Pb2+) is ubiquitously distributed in the environment and shows significant health effects in humans, especially in the nervous system. In this review we illustrate how Pb2+ neurotoxicity is associated with its ability to partially mimic the function of Ca2+ and modifies synaptic transmission pre- and post-synaptically. As Pb2+ binds to calcium-binding sites it alters their functionality, ranging from reduced currents through voltage and receptor gated channels, to modulation of ion-transporters and alterations of calcium-dependent signaling pathways. Overall Pb2+ exposure not only reduces pre-synaptically the transmitter release, but also post- synaptically the likelihood to generate a new action potential. This review will highlight the major-interactions with the different targets in schemes and short animated sequences to allow a general understanding of lead neurotoxicity to a wider audience; therefore, not all possible mechanisms will be mentioned or discussed.

Publisher’s note: This article should be read in conjunction with the animated figures available online at: http://www.authorsqscience.com/custom-assets/custom-enriched-articles/jlghs.2013.4-Animations.pdf

http://dx.doi.org/10.5339/ jlghs.2013.4

© 2013, Florea, Taban, Varghese, Alost, Moreno, Büsselberg, licensee Bloomsbury Qatar Foundation Journals. This is an open access article distributed under the terms of the Creative Commons Attribution license CC BY 3.0, which permits unrestricted use, distribution and reproduction in any medium, provided the original work is properly cited.

Cite this article as: Florea A, Taban J, Varghese E, Alost BT, Moreno S, and Büsselberg D. Lead (Pb2+) neurotoxicity: Ion-mimicry with calcium (Ca2+) impairs synaptic transmission. A review with animated illustrations of the pre- and post-synaptic effects of lead, Journal of Local and Global Health Science, 2013:4 http://dx.doi.org/10.5339/jlghs.2013.4 2 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

1 Historical Background Lead is a chemical element found ubiquitously in the environment, while lead poisoning is one of the earliest identified and most well-known occupational diseases. As lead (Pb2+) has favorable properties for many man-made applications, it has been used for centuries. Pb2+ use dates back to the beginning of Roman Empire, where large amounts of Pb2+ were used in the construction of the famous aqueducts (during the reign of Emperor Titus more than 40,000 people were employed in the mining of lead). Furthermore, lead acetate was used to sweeten and refine wines, therefore the first documented lead intoxications are from this time (Riva et al., 2012).

Lead intoxication was recognized as early as 2000 BC and the widespread use of lead has been a cause of endemic chronic plumbism (saturnism) in several societies throughout history (Ibels and Pollock, 1986). Over 2000 years ago the Greek physician Dioscorides wrote, “Lead makes the mind give way”. While lead poisoning existed and was recognized at the time it was forgotten, at least in the literature, until the end of the Middle Ages (Hernberg, 2000; Ibels and Pollock, 1986).

In the 14th century, European lead production substantially increased with the high demand for gun powder and more powerful weapons, while a century later Pb2+ was used in printing materials such as inks and dyes. In the 17th century Pb2+ was used in roofing (e.g. the cathedral in Cologne). Even more Pb2+ was used with the invention of the rechargeable battery in 19th century, but though it had multiple applications in industry, not much was understood about its detrimental effects in the human body. For a more comprehensive historical background see the review of Riva et al. (2012).

In the 19th century lead toxicity reached epidemic dimensions during the period of industrialization, while clinical understanding of lead poisoning began as late as the 20th century, together with its preventive efforts. Furthermore, lead environmental pollution caused by the use of tetraethyl lead in gasoline became an alarming public health problem. While its use as an anti-knock additive became restricted in the 1980s; lead pollution declined, but the effects are today still evident. Research often focuses on the effects of low exposure, frequently with the aim of defining threshold concentrations (Hernberg, 2000).

While for millennia Pb2+ has been revered as an intensely useful product in commercial, industrial, and residential settings, it also has become known as a highly toxic element for children and adults alike, as it has no known biological function. Generally, human intoxication occurs through the air, skin, and water e.g. by inhaling polluted air or eating contaminated food.

The clinical features of lead intoxication are non-specific and often go unrecognized. The early manifestations are largely neuropsychiatric, followed by more significant disturbances of the central and peripheral nervous systems, symptomatic gastrointestinal, musculoskeletal, haematological and endocrine abnormalities. The association of lead poisoning with renal disease is well documented and must be considered, particularly if there is associated hypertension and/or gout. Unfortunately, the frequently cited blood lead concentrations are an unreliable predictor of body lead stores, as they are indicative only of recent exposure (Ibels and Pollock, 1986).

Lead can be absorbed through many different conduits, but the ability of lead to pass through the blood-brain barrier is due in large part to its ability to substitute for calcium ions. Within the brain, lead-induced damage in the prefrontal cerebral cortex, hippocampus, and cerebellum can lead to a variety of neurologic disorders. At the molecular level, lead interferes with the regulatory action of calcium on cell functions and disrupts many intracellular biological activities (Sanders et al., 2009). 3 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

While lead affects multiple organ systems, this review will focus on the well documented effects on the nervous system. Numerous developmental and behavioral studies strongly correlate Pb2+ exposure to deficits in neural plasticity, learning and memory (e.g. Bellinger, 2007; Müller et al., 2008; Sanders et al., 2009). Pb2+ affects both the retrieval of memory, as well as the storage of new information through learning and memory formation (Bleecker et al., 2007). The processes that facilitate the development of essential neural circuitry in corresponding neural substrate are degraded and impeded by Pb2+ exposure. In conjunction with the previous, it is widely accepted that learning and memory happens by highly complex and organized mechanisms. The process most attributed to it is known as long-term potentiation (LTP). In LTP, the efficiency to generate a post-synaptic depolarization which is sufficient to trigger an action potential, is enhanced. Different pre- and postsynaptic processes are involved, mediated with the help of a variety of proteins like membrane channels, ATPase and the modulation of intracellular pathways.

As Pb2+-ions have some similarities with Ca2+-ions, they can interfere with the multiple intracellular actions of this ion. This includes, but is not limited to: binding to and altering calcium-binding sites, decreasing calcium conductance, alterations in calcium-dependent signaling pathways, vesicular mobilization and vesicular formation. Furthermore, these interactions allow lead to affect different biologically-significant processes, including metal transport, energy metabolism, apoptosis, ionic conduction, cell adhesion, inter- and intracellular signaling, diverse enzymatic processes, protein maturation, and genetic regulation (Garza et al., 2006). In the nervous system this results in a systematic reduction of signal transduction from the pre- to post-synaptic terminals (Garza et al., 2006; Goyer, 1997; Wiemann et al., 1999, for review see: Sadiq et al., 2012).

This review presents and pictures some major (simplified) mechanisms of Pb2+ at the pre- and postsynaptic terminal, including membrane channels, transporters and important second messengers which are modified by Pb2+. It contrasts the effects of lead by describing and illustrating the normal function and the impairment which occurs when lead is present. The main aim of this image- and animation-orientated presentation is not to review the latest aspects of lead toxicity, nor to highlight the numerous effects in detail, but to give a basic understanding of the main underlying mechanisms which are involved in Pb2+ neurotoxicity to a wider audience (Bleecker et al., 2007; Singh and Jiang, 1997; Neal and Guilarte, 2010).

2 Impairment of Synaptic Transmission by Lead (Pb2+) To facilitate the understanding of how lead affects synaptic transmission at the chemical synapse, the next section (2.1) will highlight the different pre-synaptic targets and, thereafter, the post-synaptic effects (section 2.2) on the generation of postsynaptic action potentials (section 2.2.1) and its effects on long-term potentiation (section 2.2.2) will be discussed.

2.1 Pre-Synaptic Effects of Pb2+: Impairment of Transmitter Release On the pre-synaptic side, lead has multiple effects on the mechanisms which are involved in the release of neurotransmitter(s) (NT) release. An overview of the basic pre-synaptic mechanisms is depicted in Figure 1 and will be discussed in the upcoming chapters (Fig. 1). 4 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

Figure 1. Overview of the pre-synaptic targets of lead (Pb2+): Lead is a potent neurotoxin, it affects multiple presynaptic targets in neurons including voltage-gated Ca2+-channels, Ca2+-ATPases, PKC, Calmodulin, signal cascades like the PLC system and the pathway of neurotransmitter release, where red arrows indicate inhibitory effects and black arrows activation.

1A

Mitochondria

IP3 Endoplasmic PLC Re/culum

Ca2+ release

CaM Kinase II

Calmodulin

Pb2+

PKC

1B Endoplasmic reAculum/ mitochondria

Calmodulin

Ca2+/calmodulin-­‐dependent protein kinase II (CaM Kinase II)

Protein kinase C

PLC Phospholipase C

Ca2+ ATPase

NeurotransmiHer re-­‐uptake pump

Voltage-­‐gated T-­‐type 2+ Ca Channel

SynapAc vesicle

AcAn skeleton

At the pre-synaptic terminal, the incoming action-potential(s) will be converted to the release to the neurotransmitter, which is stored in the pre-synaptic vesicles. As a general rule: the more action potentials arrive, the more neurotransmitter will be released.

For the “conversion” of the signal, which is coded by the sequence of action potentials to a gradual release of transmitter(s), multiple steps are required and modulations of the intracellular calcium concentration play a crucial role in the processes involved (Fig. 2A). As the lead ion – Pb2+ -- has a high affinity to calcium binding sites (Chekunova and Minkina, 1989), even low (nano- to micro-molar) concentrations of this metal can interfere with various calcium binding sites, resulting in malfunction of the processes involved (Fig. 2B and C).

The following sections describe the main steps under normal conditions (left side) and when lead is present (right side) (compare Figs. 1 to 8): 5 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

Figure 2. Pre-synaptic transmitter release is reduced by lead:

A) Multiple steps are involved in the release of neurotransmitter under normal conditions: When an action potential reaches the axon terminal voltage-gated Mitochondria 2+ Endoplasmic Ca -channels are opened allowing the Re4culum influx of calcium into the cell. Binding of 2+ Ca2+ calcium to synaptotagmin I, a Ca sensor-

Ca2+ protein in the vesicle membrane evokes Ca2+ fusion of synaptic vesicles with the presynaptic membrane and the release

Ca2+ of neurotransmitter. Another pathway of Ca2+ Ca2+ Ca2+ CaM Kinase II calcium for neurotransmitter release is Ca2+ 2+ Ca2+ Ca 2+ Ca2+ the activation of calmodulin. Ca -binding Ca2+ Ca2+ Ca2+ to calmodulin induces activation of the Ca2+ Ca2+ Calmodulin Ca2+ CaM Kinase II which phosphorylates Ca2+ synaptic associated proteins such as

2+ Ca Ca2+ 2+ 2+ 2+ 2+ Ca synapsin I. Synaptic vesicles are released Ca Ca Ca NT NT NT NT Ca2+ Ca2+ NT NT NT from the reserve pool to a readily Ca2+ Ca2+ 2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ 2+ 2+ 2+ 2+ releasable pool of vesicles. Ca binding Ca Ca Ca Ca Ca2+ Ca2+ Ca2+ Ca2+ results in fusion and neurotransmitter 2+ Ca2+ Ca release into the synaptic cleft.

B) Lead blocks voltage gated calcium channels and eventually enters the pre-synaptic terminal: While lead binds to the selectivity filter of voltage gated calcium channels it impairs the calcium current through these channels. While the binding affinity of lead is high to this binding site eventually some Pb2+ might enter the cell through these channels.

C) Intracellular lead modifies multiple functions which are related to the release to neurotransmitters: Elevated intracellular lead levels result in substitution of Ca2+ in cellular processes. Binding of lead to the synaptic vesicle associated protein synaptotagmin I prevents membrane fusion and therefore neurotransmitter release from synaptic vesicles. Lead also affects the activity of the CaM Kinase II in a concentration-dependent manner. In this way, conversion of the reserve pool to a readily releasable pool of synaptic vesicles is inhibited. The activity of Ca2+-ATPases is reduced by lead while the reuptake of the neurotransmitter from the synaptic cleft is enhanced. 6 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

Normal Conditions When Lead is Present

Step 1: Activation of Voltage Activated Channels Allows Calcium Entry in the Pre-synaptic Neuron As the membrane is depolarized by the incoming action potential(s), voltage gated calcium channels (VGCCs: mainly L-, P/Q-, N-, and to some extent R-type) will be opened (Figs. 1, 2A and 3A). Due to the large concentration gradient of calcium (extracellular 1-1.5 mM and intracellular ~100 µM) the ion will follow the driving force generated by the concentration gradient thereby generating a membrane current though the VGCCs. While calcium enters the cell the intracellular calcium 2+ concentration ([Ca ]i) will be elevated (Figs. 1, 2A and 3A).

Figure 3. Impairment of voltage-gated Ca2+-channel currents by lead:

A) As a depolarization of the presynaptic terminal (by an action potential) opens voltage gates calcium channels, Ca2+ is following its concentration gradient and enters the cell through this calcium selective channel. A selectivity filter composed of four glutamate (E) residues, called the EEEE locus exhibits a high affinity for Ca2+-ions and hence pretends the influx of other cations, such as sodium and potassium.

B) As Pb2+-ions have a higher affinity for the EEEE locus than Ca2+-ions they bind considerably longer to this binding side preventing Ca2+-ions to pass through the channel.

Pb2+ reduces the evoked release of the neurotransmitters GABA (gamma-amino-butyric-acid) and glutamate in a concentration dependent manner, pointing to a common mechanism in which Pb2+ affects the release of NT i.e. VGCCs (Braga et al., 1999). 7 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

As Pb2+ binds to structures of the VGCCs it blocks the channel and therefore reduces the current through the channel (Büsselberg et al., 1990, 1994, 1995; Evans et al., 1991; Sadiq et al., 2012; Hegg et al., 1996; Shafer et al., 1996; Atchison, 2003; Audesirk and Audesirk, 1991; Audesirk and Audesirk, 1993) (Fig. 2B and C), while voltage gated sodium and potassium channels are only affected with higher concentrations. Pb2+ is effective in the low micro-molar range, as total blockade (>80%) in this cellular model is obtained with >1μM of Pb2+ (Büsselberg et al., 1994). The block is only partially reversible when Pb2+ is removed (Singh and Jiang, 1997).

Interference of Pb2+ with VGCC is most likely mediated through the selectivity filter. With a higher affinity for 2+Pb than for Ca2+, the firm binding of the lead ion will therefore prevent calcium ions from passing through the channel (Neal and Guilarte, 2010) (Fig. 3B).

As less calcium will pass through the channel, the intracellular rise of calcium will be reduced. VGCCs also may serve as a gate for Pb2+ entry. While this was controversially discussed (Domann et al., 1997), they actually may allow for the voltage-independent passage of Pb2+ in the intracellular compartment (Tomsig and Suszkiw, 1991). As eventually lead ions pass through the channel, the intracellular concentration of this ion increases (Figs. 1, 2B and 3B).

The reduction of voltage gated calcium channel membrane currents are also described for other metals (e.g. zinc, aluminum or mercury) (Büsselberg et al., 1994a and b; Platt and Büsselberg, 1994; Platt et al., 1995; Pekel et al., 1993, Leonhard et al., 1996). While the mechanisms of action might be different, nevertheless some additive effects (in reducing the membrane currents though voltage gated channels) have been described when different metals were applied subsequently or simultaneously, broadening the concern for environmental exposure (Platt and Büsselberg, 1994).

The variations in the expression and distribution of different types of voltage gated calcium channels (pre- as well as post-synaptically) has been recently reviewed by Vacher and colleagues (2008). As lead has different binding affinities for the different channel subtypes (Peng et al., 2002) and their distribution varies in different brain structures, differences of lead actions in different tissues (neurons) are likely to occur.

Step 2: Vesicular Mobilization and Docking The mobilization of synaptic vesicles to the pre- synaptic terminal includes an intricate synchronization of several mechanisms and is inalienable for synaptic 8 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

transmission (Fig. 1, 2A, 4A and 5A). It involves multiple proteins including calmodulin, CaM Kinase II (CaMKII), synapsin, and VGCCs. A systematic rise in cytoplasmic 2+ 2+ Ca ([Ca ]i) is essential for vesicular mobilization and is mediated through VGCCs (as described in step 1). Initially, synaptic vesicles are held in a readily-available pool by the interaction of the vesicle anchor protein, synapsin, with the actin cytoskeleton (Fon and Edwards, 2001) (Fig. 2A). Calcium will bind to and activate calmodulin-mediated signaling cascades and the degree to which these processes fortify 2+ synaptic transmission are due to the rise in [Ca ]i (Fig. 4A). Furthermore, activation of CaMKII will then phosphorylate synapsin in order to release synaptic vesicles from the actin cytoskeleton (Suszkiw, 2004). At this point, synaptic vesicles are “free” to mobilize via microtubular transport to the pre-synaptic membrane and dock through the interaction of v-SNAREs (vesicle proteins) and t-SNAREs (plasma membrane proteins) (Fon and Edwards, 2001) (Figs. 2A, 4A and 5A).

Pb2+ has the capability of interacting with several mechanisms involved in vesicular mobilization and docking, due to its ability to bind to Ca2+ binding sites (Figs 2B and 2C). Initially, Pb2+ competes with the binding site for Ca2+ in calmodulin. Gill and co-workers (2003) indicated that in vitro Pb2+ exposure mediated the phosphorylation of synaptic vesicle by disrupting calmodulin (Fig. 4B, C and 5B). Lead exhibits different effects over a variety of concentrations. For example, incubation with 10μM Pb2+ stimulates a calmodulin-dependent protein phosphorylation resulting in a possible neurotransmitter release while a ten times higher concentration inhibited calmodulin-dependent protein phosphorylation and consequently synaptic transmission (Gill et al., 2003) (compare Fig. 4B to Fig. 4C). In vivo Pb2+ exposure stimulated calmodulin-dependent synaptic vesicle protein phosphorylation by 22.8% (Gill et al., 2003).

Furthermore, Westerink and colleagues (2002) used dexamethasone-differentiated PC12 cells to measure neurotransmitter release. With 1µM Pb2+ a gradual increase of vesicle released was observed. With cells exposed to KN- 62 (a drug inhibitor of CaM Kinase II) and Pb2+, there was a significant decrease in the number of vesicles released and there was a delay of Pb2+ induced exocytosis. Cells exposed to W7 (a calmodulin inhibitor) reduced the onset of Pb2+ induced exocytosis (Westerink et al., 2002).

CamKII is a target for Pb2+ (Fig. 2C, 4 B and C). Chronic Pb2+ exposed rats showed a 41% decrease in the maximal velocity of CaMKII in the presence of calcium and calmodulin and an increase (22%) in affinity of enzyme for

substrate (Km) (Toscano et al., 2005). However, hippocampal extracts exposed to 10µM Pb2+ acetate showed a 20% 9 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

decrease in the Km in control but not in Pb2+-exposed

rats. Thus, lead did not affect the Vmax of the hippocampal CaMKII. These results suggest that Pb2+ interferes with CaMKII function. The experiments with Pb2+ exposed rats indicate that CamKII is already saturated with Pb2+ due to in vivo exposure and that exogenious addition of Pb2+ would have no apparent effect on Km. The authors conclude Pb2+ exposure modulates CaMKII, a component of calcium signaling associated with synaptic plasticity, learning, and memory (Toscano et al., 2005) (see also chapter 2.2).

Step 3: Neurotransmitter Release Fusion of the plasma membrane (PM) with the synaptic vesicle (SV) is the key element of the release of neurotransmitter (Atlas, 2001). Vesicles dock to the plasma membrane through v-SNARES and t-SNARES. The specific proteins that interact are synaptobrevin (v-SNARE) and SNAP-25 and syntaxin (t-SNAREs). Neurotransmitter release 2+ is regulated with a rise of [Ca ]i which is mediated mainly through Ca2+ entry via VGCC (see “step 1”). Synaptotagmin I acts as a calcium sensor which then drives the fusion of the SV with the plasma membrane (Fig. 6A). Synaptotagmin contains two calcium sensing domains referred to as the C2A and C2B domains which bind 3 Ca2+ and 2 Ca2+ ions, respectively. The C2A domain interacts with the phospholipids and syntaxin, while the C2B domain stimulates synaptotagmin dimerization (Atlas, 2001). The 2+ activation of synaptotagmin through [Ca ]i signaling, promotes fusion of the plasma membrane and the vesicle to release neurotransmitter (Fig. 6A). Lead not only competes for the calcium binding sites at the C2A and C2B domains and therefore prevents the binding to the phospholipids and the syntaxin, but also the reduced level of intracellular calcium (due to the impaired calcium entry discussed above) reduces the likelihood that the vesicle fuses with the membrane and reduces the transmitter (Fig. 6B).

2+ Step 4: Restoration of [Ca ]i The previously described mechanisms have increased 2+ [Ca ]i in order to trigger the transmitter release. Active Ca2+ transport mechanisms have to be activated in order to 2+ reestablish the original [Ca ]i level in the synaptic button. 2+ This is important, as an elevated [Ca ]i-level would trigger a continuous release of neurotransmitter and the neuron would be unable to specifically react to future action potentials. Therefore, Ca2+-ATPases, at the endoplasmic reticulum, the mitochondria and the plasma membrane, continuously transport Ca2+ against its concentration 2+ gradient out of the cytosol in order to reduce [Ca ]i (Levy et al., 2003) (Figs. 2A and 7A). 10 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

Figure 4. Calmodulin, CaK-Kinase-II activation and vesicle recruitment is modulated by lead in a concentration dependent manner:

A) Calmodulin (CaM) is a calcium-binding messenger protein which detects changes in Ca2+-concentration and transduces Ca2+-signals in activation of Ca2+-/CaM-dependent proteins such as the CaM Kinase II which phosphorylates synapsin I. This process allows the dissociation of synaptic vesicles from actin in the cytoskeleton and may promote their translocation to the readily releasable pool of synaptic vesicles. Binding of calcium to synaptotagmin I on these vesicles induces neurotransmitter release.

B) A low intracellular Pb2+-concentration substitutes Ca2+-ions and enhances calmodulin activity which leads to an increased synaptic vesicle protein phosphorylation (syntaxin I) by CaM II. This results in an elevated number of readily releasable synaptic vesicles whereby binding of Pb2+ to synaptotagmin I induces inhibition of membrane fusion and therefore a decreased neurotransmitter release.

C) A high intracellular Pb2+-concentration has an inhibitory effect on the activity of calmodulin. Hence the conversion of synaptic vesicles from the reserved to the readily releasable pool as well as the neurotransmitter release by exocytosis is suppressed. 11 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

Figure 5. Lead interferes with the PLC-system and transmitter release:

A) Binding of calcium to G protein- coupled receptors induces activation of Phospholipase C (PLC) which is responsible for signal transduction processes. PLC cleaves membrane phosphatidyl inositol biphosphate (PIP2) to inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 triggers Ca2+ release from the endoplasmatic reticulum while DAG translocates the protein kinase C (PKC) to the membrane which is activated by Ca2+ binding. Activated PKC then phosphorylates target proteins that are involved in neurotransmitter release.

CaPb2+ 2+

PLC

(Splits PIR2)

B) In the presence of lead, PLC as well as PKC activity is enhanced whereas 2+ high concentrations of Pb results in DAG IP inhibition of these enzymes. 3 CaPb2+

PKC Ca2+ release from ER

Phosphorylates Increased Modulate substrate transmi1er transmiDer proteins release 12 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

Figure 6. Fusion of a synaptic vesicle with the presynaptic membrane: Calcium binding to the C2A (3 Ca2+-ions) and C2B (2 Ca2+-ions) domains of the vesicle membrane protein synaptotagmin I triggers interaction with T-Snares syntaxin, Snap25 and V-Snare synaptobrevin, forming a SNARE-complex. This exocytosis machinery mediates vesicle fusion and therefore neurotransmitter release.

A) illustrates the normal conditions

B) depicts the situation when lead competes with the calcium binding sites. As Pb2+ does not induce assembly of the SNARE-complex it reduces the chance of the vesicles to fuse with the membrane and therefore reduces the release of the neurotransmitter. 13 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

Figure 7. Lead inhibits the intracellular regulation of Ca2+ by interfering with the Ca2+-ATPase:

A) As calcium is an important second messenger its intracellular concentration is tightly regulated by Ca2+-ATPases which transport calcium against its concentration gradient to the extracellular space or the intracellular stores. The transport process of calcium is ATP-dependent and provided by two conformations (E1 and E2) which exhibit different affinities for calcium. Binding of two intracellular Ca2+-ions to the Ca2+-ATPase induces the exchange of ADP for ATP on the nucleotide binding domain, whereby the ATPase is phosphorylated and undergoes a change from the E1 to the E2 conformation. Therefore, the ATPase opens to the extracellular site and releases the two Ca2+-ions because of low affinity. Instead, two protons are pumped into the cytosol.

B) Lead inhibits the enzyme activity by blocking the interaction of Ca2+-ions with the Ca2+-ATPase. This inhibitory effect prevents the regulation of the intracellular Ca2+-concentration which results in an unusual high intracellular concentration of calcium. 14 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

Lead at micromolar concentrations significantly inhibits the Ca2+-ATPase of synaptic plasma membranes and microsomes of cerebral cortex and cerebellum in a concentration-dependent manner (Bettaiya et al., 1996) (Figs. 2C and 7B). Other investigations also found that in vitro lead (0.25-2 µM) inhibits the inositol 1,4,5-triphosphate-mediated Ca2+ uptake and release in microsomes of rat cerebellum (Vig et al., 1994) (Fig. 5B).

Step 5: Endocytosis of the Synaptic Vesicles After the release of the neurotransmitter, the vesicle membrane is readily endocytosed in order to maintain a normal surface area at the synaptic cleft. This process is dependent on a clathrin coat. Synaptotagmin will bind the protein AP-2 (which is a clathrin adapter protein) that will also recruit other AP3 complexes which will promote clathrin coat assembly. Dynamin will initiate endocytosis by the invagination and pinching off of the coated membrane. The protein Calcineurin is a phosphatase that is activated by Ca2+/Calmodulin. Calcineurin dephosphorylates Dynamin in order for it to initiate endocytosis of the vesicle (Haucke and De Camilli, 1999; Martensen et al., 1989) (Fig. 8A).

In vivo as well as in vitro experiments have demonstrated that lead activates the re-uptake of glutamate from the synaptic cleft (Zhang et al., 1997). The authors conclude that lead accelerates the clearance of the transmitter and therefore has inhibitive effects on synaptic transmission (Fig. 8B). 15 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

Figure 8. Synaptic vesicle endocytosis is reduced by lead:

8A A) The total number of synaptic vesicles in a nerve terminal is limited. To maintain the vesicle pool, a recycling process is required which involves clathrin- mediated endocytosis. The tetrameric adaptor protein Ap2 is recruited to the membrane by binding to the synaptic vesicle protein synaptotagmin I. In this way, the assembly of the clathrin coat can start with binding of the AP2 to the membrane. Parallel, activation of the calcium-dependent phosphatase calcineurin induces dephosphorylation of the GTPase dynamin I in nerve terminals. After bud formation, activated dynamin I supports the fission of the coated vesicle from the plasma membrane, thus creating a free clathrin-coated vesicle. Finally, uncoating of the clathrin- coated vesicle occurs, when the ATPase Hsc70 is recruited to the coat. Then, the synaptic vesicle can be refilled with neurotransmitters for another round of exocytosis.

8B SV Endocytosis

AP2 + Rise Pb2+ in Pb2+ 2+ Synaptotagmin [Ca ]i

Recruite AP3 Calcineurin Complex B) High concentration of Pb2+ results in inhibition of calcineurin and synaptotagmin I by which clathrin-mediated endocytosis is disturbed and decreases. Dephosphorylates Clathrin coat Dynamin assembly

Decreased Endocytosis 16 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

2.2 Post-Synaptic Effects of Pb2+ Postsynaptically, the neurotransmitter molecules bind to the receptors, which in turn will activate receptor gated channels, resulting in an ion flux through the membrane. Ionotropic glutamate receptors are most important to initiate the post-synaptic depolarization. The glutamate receptors are mainly the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor, the kainate receptor, and the N-methyl-D-aspartate (NMDA) receptor (Fig. 9 and 10).

Two different scenarios will be discussed: 1. How lead interferes with the regular synaptic transmission: this section discusses whether the postsynaptic depolarization (with/without lead) is sufficient to reach the threshold potential and trigger an action potential (section 2.2.1). 2. How lead modifies the synaptic transmission after a high frequency stimulation resulting/not resulting in the induction of long term potentiation (LTP) (section 2.2.2).

Figure 9. Overview of Postsynaptic processes which could be altered by lead: Hyperlinks to the different postsynaptic processes which could be impaired in the presence of lead.

9 POSTSYNAPTIC ALTERATIONS

N-­‐Methyl-­‐D-­‐Aspartate Receptor/Channel Pb2+ Impaired N-­‐Methyl-­‐D-­‐Aspartate Receptor/Channel Normal PostsynapDc PotenDals Pb2+ Impaired PostsynapDc PotenDals PostsynapDc PotenDals with MulDple Signals Pb2+ Impaired PostsynapDc PotenDals with MulDple Signals Normal Long-­‐Term Poten.a.on (LTP) Pb2+ Impaired Long-­‐Term Poten.a.on (LTP) SynapDc Transmission aHer InducDon of LTP SynapDc Transmission aHer InducDon of LTP with Pb2+ PostsynapDc Pathways PostsynapDc Pathways with 2+ Pb 17 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

Figure 10. Lead binding sites at the post-synaptic neuron

10 A Postsynap)c effects

+ 2+ + + Ca2+ Na Ca Na Na Ca2+

G Gly G G G Pb2+

Pb2+ Pb2+

Pb2+ CaM Kinase II Calmodulin

PKA Adenyl Cyclase

cAMP MAPK 2+ CREB Pb Nucleus X

10 B N-­‐methyl-­‐D-­‐asparHc (NMDA) acid Receptor/Channel Complex

α-­‐amino-­‐3-­‐hydroxy-­‐5-­‐methyl-­‐4-­‐isoxazolepropionic acid (AMPA) Receptor/Channel Complex Different receptor channel/complexes as well as calcium-dependent proteins Kainate Receptor/Channel Complex such as calmodulin are a possible Voltage-­‐gated T-­‐type 2+ Ca Channel target for lead whereby postsynaptic signaling cascades are deregulated Voltage-­‐gated + Na Channel which results in disturbance of the postsynaptic potential and Voltage-­‐gated + K Channel synaptic plasticity. G Glutamate Gly Glycine Calmodulin

Adenyl Cyclase

Cyclic adenosine monophosphate (cAMP) Protein kinase A (PKA)

Ca2+/calmodulin-­‐dependent protein kinase II (CaM Kinase II)

cAMP response element binding (CREB)

Mitogen acHvated protein kinase (MAPK)

2.2.1 Lead Interferes with the Generation of Excitatory (Inhibitory) Post- Synaptic Potentials (EPSPs and IPSPs) and the Generation of Action Potentials As described before, the pre-synaptic cell releases neurotransmitter, thus activating receptor- gated channels at the postsynaptic terminal. Primarily the transmitter binds to AMPA and Kainate receptors, activating the associate channels and thereby initiating (mainly) excitatory post- synaptic potentials (EPSPs).

The spatial (the number of other pre-synaptic neurons which project to the same postsynaptic button and fire while the postsynaptic potential is still depolarized) and temporal (the number of action potentials are which are fired in a specific time interval and how much neurotransmitter was released during the sequence of action potentials in the pre-synaptic 18 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

cell). Overall the additive changes of the (summarization) of these EPSPs will determine to which degree the post-synaptic cell will be depolarized (in some cases hyperpolarized) and whether this depolarization is sufficient to reach the threshold potential (compare Figs. 11A and 12A) and to generate a new action potential.

Only when the postsynaptic depolarization is sufficient to reach the threshold potential (at this potential the depolarizing current though voltage gated sodium channels will activate more voltage gated sodium channels – generating a larger current and an increasing depolarization - resulting in an avalanche-like activation of basically all voltage gated channels) a new action potential will be generated (Fig. 12 A) (and the voltage gated sodium channel will be inactivated and no longer open for sodium to pass through).

Therefore, the combination of the activation of receptor and voltage gated channels determines whether an action potential will be generated post-synaptically.

Figure 11. Lead interferes with the magnesium (Mg2+) and zinc (Zn2+) binding sites of the NMDA-receptor/channel-complex:

A) NMDA-receptors (N-methyl-D- aspartate receptors) are glutamate- gated cation channels which plays an important role in synaptic plasticity. At resting membrane potential, the NMDA-receptor channel is blocked by Mg2+. The NMDA-receptor is co-activated by L-glutamate and glycine which bind to the modulatory (NR2) and structural (NR1) subunit, respectively. An excitatory postsynaptic potential induces strong depolarization of the postsynaptic cell which could remove the Mg2+-block in the channel and allows the passage of Na+ and Ca2+ into the cell and K+ out of the cell. Importantly, opening of the NMDA-receptor requires both, simultaneous glycine/glutamate- binding as well as depolarization of the membrane.

B) Pb2+ prevents the passage of cations through the channel by binding to the Zn2+-binding side at the NMDA-receptor. 19 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

Figure 12. Lead reduces “normal” synaptic transmission:

A) When an excitatory action potential depolarizes the presynaptic membrane, L-glutamate is released from presynaptic vesicles that diffuses across the synaptic cleft and binds to postsynaptic AMPA/kainite and NMDA receptors (ionotropic glutamate receptors). Activation of AMPA/kainite receptors leads Na+ flow across the postsynaptic membrane while the NMDA receptor is still blocked by the Mg2+ ion. Whether the resulting postsynaptic excitatory potential is sufficient to generate an action potential depends on multiple factors. In the figure shown the EPSP does not reach the threshold potential and no action potential is generated.

B) In the presence of lead, presynaptic L-glutamate release is reduced that results in a decreased transmitter release and therefore in a decreased activation of the postsynaptic receptors and consequently in a reduced Na+-ion flow across the postsynaptic membrane through AMPA/kainite receptors. Thus, the postsynaptic potential is decreased compared to the situation without lead (as shown in part (A)). 20 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

Normal Conditions When Lead is Present

Involvement of Voltage Gated Channels Post-synaptically, as in any other neuron, the activation of voltage gated channels generates an action potential and voltage gated sodium calcium and potassium channels are involved. To conduct calcium, different types of VGCC are expressed. However, the low-voltage activated, rapidly inactivating T-type and high-voltage activated, slowly inactivating L-type channels play a major role for the regeneration of the post-synaptic action potential (Vacher et al., 2008).

As discussed in the previous section, lead reduces the amount of pre-synaptically released transmitter. As fewer transmitters are released in the synaptic cleft, they bind to a smaller number of postsynaptic receptors and therefore, the strength of the EPSP as well as IPSP is reduced (Braga et al., 1999). The consequence is that the total postsynaptic depolarization and the likelihood to trigger an action potential is smaller (Fig. 11B).

Furthermore, post-synaptically, the currents through voltage gated channels (primarily voltage gated calcium channels) will be reduced by lead, due to the same mechanisms as discussed pre-synaptically. Therefore, even when low voltage gated calcium channels (basically T-type) will be activated by the receptor gated induced depolarization, the reduced current through these channels will further decrease the likelihood to trigger an action potential (compare Fig. 3).

As pre-synaptically voltage activated sodium and potassium channels will only be affected by lead at higher concentrations (Busselberg et al., 1994).

Involvement of Receptor Gated Channels Three major glutamate receptors are found at the post- synaptic membrane: NMDA (N-Methyl-D-Aspartate; which will be discussed in the next chapter) and AMPA (2-amino-3- (5-methyl-3-oxo-1,2-oxazol-4-yl)propanoic acid) and kinate receptors (Fig. 9-13 and 15-16).

The activation of AMPA and kinate receptors and the opening of the associated ion channels will generally result in a depolarization of the postsynaptic cell (mostly due to sodium entry through these channels). When the threshold potential is reached an action potential will be triggered due to the mechanisms described above (Fig. 11A and 12A). While the agonist might also bind to the NMDA receptor, ions will not be able to pass through the channel due a to block of the channel by an magnesium ion (Fig. 14A). 21 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

Compared to the NMDA receptor/channel complex, both Kainate and AMPA receptors are only affected by relatively high concentrations of lead (Uteshev et al., 1993), therefore, lead actions at these receptors will not be further discussed.

2.2.2 Lead Interferes with the Induction of Long Term Potentiation (LTP) Long term potentiation (LTP) is defined as a long-lasting enhancement of synaptic transmissions after a high frequency stimulation. It was discovered by Lomo in 1966 (reviewed in Lomo, 2003). LTP demonstrates the ability of a synapse to modify its strength and is therefore regarded as a crucial element for synaptic plasticity and as a key element for higher functions such as learning and memory.

The LTP can be divided in different stages (LTP1, LTP2 and LTP3) depending on the activation of different intracellular mechanisms. The induction of LTP1, also named “Early-LTP”, depends on the transient activation of CaMKII and PKC, while its maintenance will require their continuous activation. LPT2 is defined by the activation of gene transcription in the postsynaptic cell and LTP3 by activation of gene transcription and protein synthesis. LTP2 and LTP3 are also known as “Late-LTP”.

Normal Conditions When Lead is Present

Lead Changes the Early-LTP (LTP1): Involvement of the NMDA Receptor/Channel Complex The activation of the NMDA receptor/channel complex is crucial to induce the early-LTP. NMDA receptors, as stated earlier, are an ionotropic glutamate receptor/ channel subtype that allows Ca2+ to enter the postsynaptic cell when activated. The rise of the intracellular calcium concentration activates the CaMKII, the first crucial step to induce LTP1 (Fig. 10 and 16A).

The NMDA receptor/channel complex has different binding sites for various types of compounds and free ions (Fig. 14A). The NMDA receptor/channel complex is composed of four subunits which vary in structure, depending on their location and use (Barria and Malinow, 2002; Mierau et al., 2004; Philpot et al., 2001; Quilan et al., 2004). However, all contain a mandatory NR1 structural subunit and two different types of subunits, In addition to the essential NR1, at least one of the four different NR2 types (named A, B, C and D), or one of the two forms of NR3 have to be present. The NR1 and NR2 subunits are significantly dense in the hippocampal region of the brain, which has its implications in learning and memory.

High frequency stimulation (multiple incoming action potentials) of the pre-synaptic neuron will release a high amount of transmitter and activate the postsynaptic receptors. Activation of AMPA and Kinate receptors will depolarize the postsynaptic membrane for a prolonged period of time, while initially (at resting membrane 22 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

potential) the pore of the NMDA receptor/channel complex is blocked by an Mg2+-ion within the channel (Fig. 14-16).

A. The Mg2+-binding Site of the NMDA- Receptor/Channel-Complex: Due to intracellular depolarization this Mg2+-ion is driven out of the channel (Fig. 14 and 15). Therefore, the channel pore is open for other ions (calcium, as well as sodium and potassium to some degree).

As the NMDA receptor is activated by glutamate (as the AMPA and kinate receptors) an additional post- synaptic conductance (channel) is operational after removal to the Mg2+-block. As the most important conductance to this receptor/channel-complex is for calcium ions, this additional depolarizing current, after the removal of the magnesium block, will be activated when neurotransmitter is released. The additional current will facilitate the postsynaptic depolarization. Overall, the likelihood to reach the crucial threshold potential in order to trigger an action-potential is enhanced. Furthermore, the elevated intracellular calcium concentration will activate multiple intracellular pathways initiating late-LTP.

B. The Zn2+-binding Site of the NMDA- Receptor/Channel-Complex: Zn2+ is an essential metal that has important neurological functions including synaptic development and clipping. The importance of the Zn2+-binding site of the NMDA receptor is still a matter of debate. It is located on the NR2 subunit and modulates both excitatory and inhibitory pathways involved in LTP and LTD however; the concentration ranges appear to indicate that these effects happen at similar concentrations (Fig. 14).

Zn2+ potentiates currents through NMDA channels (and LTP) in the low micro-molar range (5μM) but inhibits NMDA function (and LTP) at higher concentrations (30µM) in hippocampal CA1 synapses (Takeda et al., 2009).

Furthermore, it is discussed that Zn2+ plays a role in receptor/channel-complex densities on the post- synaptic side, as well as in NMDA receptor subunit composition, by acting as a mediator between the structural and modulatory subunits. 23 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

Figure 13. Spatial or temporal summation of multiple signals postsynaptic potentials without and with lead:

A) When multiple action potentials depolarize the presynaptic membrane in a short time interval, more L-glutamate is released that leads to the activation of AMPA/ kainite receptors and allows an increased passage of Na+ ions which trigger opening of voltage- gated Na+-, Ca2+- and K+-channels. This results in an increased influx of positive charged ions into the postsynaptic cell. Summation of these presynaptic impulses causes sufficient depolarization of the postsynaptic membrane to overcome the threshold and to generate an excitatory postsynaptic action potential.

B) Pb2+ exposure results in a decreased release of L-glutamate that less channels in the postsynaptic membrane and even with a subsequent activation the membrane potential might not reach the threshold potential as illustrated. 24 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

Figure 14. Lead impairs the induction of Long Term Potentiation:

A) Long term potentiation (LTP) is a synaptic enhancement that represents the synaptic basis of learning and memory. Induction of LTP depends on the stimulation of the presynaptic cell with synchronous high- frequency impulses which leads to release of L-glutamate from the presynaptic cell to depolarize the postsynaptic neuron sufficiently to release the magnesium (Mg2+) block of the NMDA receptor/channel complex resulting in calcium entry into the postsynaptic cell. Postsynaptically the calcium triggers not only depolarizes the neuron but also triggers signaling cascades which result in long lasting synaptic changes.

B) Induction of Long term potentiation (LTP) is suppressed when lead is present. Although depolarization of the postsynaptic membrane produced by high frequency stimulus might be strong enough to remove the Mg2+-block, the NMDA channel is alternatively blocked by Pb2+ that prevents Ca2+ entry into the postsynaptic cell. 25 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

Figure 15. Lead reduces the excitatory postsynaptic potentials after induction of Long Term Potentiation:

A) When LTP has been induced – the NMDA receptor/channel complex is not blocked by Mg2+ - postsynaptic depolarization is facilitated due to the additional conductance through the NMDA receptor/channel complex.

B) Because NMDA receptors mediate the basis of regulation of synaptic development and function as well as the majority of excitatory synaptic transmission, they represent one of the most important targets for lead. Blockage of the NMDA receptor by lead inhibits the passage of Na+ and Ca2+ ions through the postsynaptic membrane after a presynaptic stimulus, thus reducing excitatory postsynaptic potentials (EPSPs) and therefore the likelihood for generating an action potential. 26 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

It has been suggested that the NMDA receptor/ channel-complex is impaired by Pb2+ (Büsselberg et al., 1994, Uteschev et al., 1993, Yamada et al., 1995; for review see Guilarte, 1997). The reduction of the current could be the result of two different and independent mechanisms:

A. Possible Pb2+ interactions with the Mg2+- binding Site within the Channel. It was discussed whether Pb2+ as a divalent cation has an affinity to the Mg2+ binding site within the channel and therefore decreases the current though the channel, thus reconfiguring the situation as if the Mg2+-block would be present. But, as the effect of Pb2+ is not voltage dependent (Alkondon et al., 1990; Ujihara and Albuquerque, 1992; Büsselberg et al., 1994) it is more likely that Pb2+ does not occupy the Mg-binding site of the pore (Fig. 14B and 15B).

B. Pb2+ Interferes with the Zn2+-binding Site at the NMDA Receptor/Channel-Complex. Pb2+ affects the Zn2+-binding site (Fig. 14B) as well as the Ca2+ dependent proteins and pathways responsible for LTP, protein synthesis, and gene transcription (Zarei and Dani, 1995; Sheng and Kim, 2002; Petrović et al., 2005). The interaction of Pb2+ at (or close to) the Zn-binding site in the NMDA receptor N terminal domain is considered to be one of the most significant and specific interaction of this heavy metal in the postsynaptic membrane, and could also have presynaptic relevance, as NMDA receptor channels are present also in the presynaptic membrane (Berretta and Jones, 1996). Pb2+ action partially overlaps with that of Zn2+, but precise coincidence with Zn2+ binding site is debated (Lasley and Gilbert, 1999; Gavazzo et al. 2008).

The effects of Pb2+ are specific for subunits of the NMDA-receptor/channel-complex (Omle-chenko et al., 1996), as rats exposed to 1500ppm of Pb2+ have significant decreases in gene and protein expression of the NR1 subunit (Nihei et al. 2001). Furthermore, complete removal of the NR1 subunit of adult mice demonstrate deficits in LTP and spatial learning (Tsien et al., 1996) which are congruent with findings where rats have been exposed to 1500ppm of inorganic Pb2+ (Guilarte and McGlothan, 2003). The NR2 subunit directs the delivery of receptors to synapses with specific rules for its various forms (Barria and Malinow, 2002), while the NR2A and NR2B subunits diverge in different circumstances (Kash and Winder, 2007). Most notably, they vary in their kinetic features and decay rates (Vicini et al., 1998). 27 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

Specifically, blockade of the NR2A and NR2B abolishes the induction of LTP and LTD, respectively (Liu et al., 2004). This is in agreement with the finding that chronic Pb2+ exposure particularly reduces NR2A protein levels (Nihei and Guilarte, 1999).

As the NMDA receptor/channel complex has a different composition in different brain regions also the effects induced by Pb2+ vary (Nihei and Guilarte, 2001) as Pb2+ has different binding affinities for diverse types of NR2 subunits. In general, Pb2+ affects the NR2A receptor the most followed by the NR2C and then the NR2D (Omelchenko et al., 1997). The NR2 subunit can also be combinations of the A, B, C, and D forms and unlike the typical inhibitory effect of Pb2+, low micro-molar concentrations of the ion can activate the NR2A/C subunit type. This fact makes the ions’ toxicity even more puzzling and elusive (Guilarte and McGlothan, 2003).

Zn2+ is an important cofactor in NMDA activation. Zn2+-uptake in LTP is associated with augmenting changes in gene transcription and protein synthesis for increasing signal transduction efficiency (Vallee and Falchuk, 1993).

Block-out and residue studies have determined that the Zn2+-binding site contains multiple points for interaction and that this may be responsible for the variety in results. Therefore, Pb2+ interaction may not necessarily be in the exact same spot, but rather there is an overlap of interaction. It is purported that this is related to changes in downstream targets including those that alter gene transcription and protein synthesis.

Overall, deficits in LTP are correlated to changes in gene expression and protein synthesis of overall NMDA composition. NMDA receptor/channel complexes are arguably most important for their close relationship to signaling pathways involving the mitogen-activated protein kinase (MAPK) family (reviewed in Haddad, 2005). But, the question remains how increased synaptic NMDA receptor activity enhances neurotransmission and further transcription of LTP-related receptor complexes (Lee et al., 2005).

Lead changes the Late-LTP (LTP2 and LTP3): Gene Transcription and Protein Synthesis CaMKII activity is important in synaptic plasticity in learning and memory (Cammorato et al. 1998). Rises in intracellular Ca2+ levels initiate expression of CAMKII through binding of the Ca2+/calmodulin complex (Hudmon and Schulman 2002). Furthermore, CaMKII expression could be mediated through NMDA receptor activation (Murray et al., 2003) (Fig. 16A). 28 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

Figure 16. The activation of postsynaptic genes and protein synthesis in the Late-LTP is impaired by lead:

A) LTP is induced by the activation of the NMDA receptor that permits Ca2+-entry into the postsynaptic cell which triggers activation of calmodulin. The increase of intracellular Ca2+ concentration leads to stimulation of the Ca2+/ CaM-dependent protein kinase II (CaMKII) that activates the mitogen-activated protein kinase (MAPK) by phosphorylation. A second pathway is also involved in LTP that leads to calmodulin- dependent activation of the cyclic adenosine monophosphate (cAMP)/ protein kinase A (PKA) that is stimulated by cAMP through the adenyl cyclase. PKA is responsible for the phosphorylation of the transcription factor, cAMP response element-binding protein (CREB). Activated CREB as well as MAPK are translocated into the cell nucleus and initiate gene expression. Thereby protein synthesis maintains LTP and is required for synaptic plasticity which leads to an increase in synaptic strength.

B) As lead mimics the cellular function of calcium it impairs the activation the signaling pathways in the postsynaptic cell and therefore inhibits gene expression and protein synthesis. Thus, a long term enhancement of synaptic transmission will not be established. 29 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

In terms of heavy metal toxification, Pb2+ acts as an NMDA inhibitor which means that CaMKII activation is inhibited as well (Fig. 16 B). This translates into significant cognitive deficiencies (Bejar et al., 2002; Hardingham et al., 2003). Pb2+ modifies the signaling proteins calmodulin, Ca2+/ calmodulin-dependent protein kinase II (CaMKII), protein kinase A (PKA) and adenyl-cyclase as well as transcription factors from the mitogen activated protein kinase (MAPK), extracellular receptor kinase (ERK) and cAMP responsive element binding (CREB) family of proteins. CaMKII activity happens almost immediately after stimulation, whereas ERK activation occurs only after a prolonged period of stimulation. When the stimulation ceases, ERK activity drops off quickly while the CaMKII activity declines more slowly (Murphy et al., 1994). This could be due to the fact that the NR2B NMDA subunit reportedly has the ability to bind and activate CaMKII (Barria and Malinow, 2005).

Adenylate-Cylase has an outstanding role for Late- LTP. Modulation of this protein depends on intracellular Ca2+ levels and the activation of the Ca2+/calmodulin complex. Adenylate cyclase is responsible for the resulting production of cAMP followed by PKA. PKA activation is dependent upon cAMP levels mediated by adenylate cyclase and subsequent metabolism of cAMP (Taylor et al., 2004) (Fig. 16A). Pb2+ decreases in adenylate-cylase activity and in turn decreases PKA activation (Sandhir and Gill 1994). In later phases of LTP, PKA can no longer activate MAPK or CREB family transcription factors in the presence of Pb2+ (Fig. 16B).

Additional factors primarily involved in LTP-related transcription are ERK 1, ERK 2 and p38 (Adams and Swdatt, 2002; Thomas and Huganir, 2004).

Unlike most processes that are inhibited during Pb2+ toxification, ERK 1/2 and p38 activity has been shown to increase at 50 ppm (Ramesh et al., 2001; Leal et al., 2002). The possible association of NMDA inhibition by Pb2+ and ERK1/2 activation is extensively discussed by Cordova and colleagues (2004). This activation has been correlated to NMDA receptor inhibition by Pb2+ and could be a possible defense mechanism under such conditions (Chandler et al., 2001; Friguls et al., 2002; Paul et al., 2003). Furthermore, Pb2+ has been shown to increase NR1/NR2B subunit containing NMDA receptors (Toscano et al., 2002) which could be the conduit by which ERK levels increase considering that the NR2B subunit is coupled directly to CaMKII (Krapivinsky et al., 2003). 30 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

CREB, activation can be both neuroprotective and excitotoxic. CREB has an essential role in learning and memory (Deisseroth et al., 1996; Tao et al., 1998). In terms of Late-LTP, CREB is critical in the maintenance of LTP and CREB phosphorylation increases after LTP-induction and learning (Impey et al., 1996: Mizuno et al., 2002; Viola et al., 2000). The amount of phosphorylated CREB depends on the amount of Ca2+ influx through activated NMDA channels (Hardingham and Bading, 2003). In conjunction, PKA, CaMKII, and MAPK have been revealed to activate CREB by phosphorylation at Serine-133—all of which are mediated by Ca2+ influx (Wu et al., 2001; Lonze and Ginty, 2002) (Fig. 16A).

Pb2+ alters CREB binding and activation (Toscano et al., 2003). This could be due to direct interaction with CREB or through Pb2+ inhibition of the NMDA receptor/channel complex and subsequent Ca2+ blockade (Fig. 16B).

3 Concluding Remarks In most parts of the world lead neurotoxicity is a serious public health problem. While the actual lead blood levels may vary depending on the local circumstances, worldwide little is done to prevent intoxication with this metal. In 1991 the U.S. Centers of Disease Control and Prevention (CDC) lowered the “acceptable” blood lead level in children to 10µg/dL, but scientific reports clearly indicate that this concentration is too high to be regarded to be safe. Thus, Gilbert and Weiss (2006) have suggested lowering this value to 2µg/dL, but, unfortunately no action has been taken. Clearly, even with “only” 2µg/dL lead affects the nervous system. Furthermore, there is evidence that Pb2+ effects persist even at low dosages, as well as low blood levels, while the impairments are only slightly reversible and differ from childhood exposure to adulthood (Bellinger et al., 1991; Bellinger and Stiles, 1993; White et al., 1993).

Over the last two decades it has been discussed whether compensatory mechanisms might occur at long term exposure to Pb2+. Hegg and co-workers (1997) exposed rat neurons to lead (0 to 50µM) for up to 12 weeks. Chronic exposure for one month did not modify the peak or the sustained calcium current amplitudes in lead-treated neuronal cells when compared to control cultures. However, after two-month exposure of either 25 or 50µM lead, an increased peak and sustained calcium current amplitude was observed, which remained increased in the third month of exposure. It has been argued that this increase is the result of calcium channel up regulation and therefore a compensatory mechanism, but the final conclusion is not yet clear (Hegg and Miletic 1997). Other indications for such mechanisms are shown by Singh and Jiang (1997) who found that chronic low-level lead exposure during early development and adulthood may decrease the synthesis of VGCCs but not their antagonist binding-affinity in both the neonatal and the adult rats (Singh and Jiang, 1997).

Developmental aspects In development, Pb2+ alters the compositional switch in NMDA receptors from the natal to the mature brain. In adult brains such changes result in decreased synaptic efficiency and LTP including significant behavioral diminutions. Studying granule cell neurogenesis and morphology, Verina and co-workers (2007) found significant changes at environmentally relevant concentrations of Pb2+. The authors conclude that these changes “provide a cellular and morphological basis for the deficits in synaptic plasticity and spatial learning documented 31 of 38 pages Florea et al, Journal of Local and Global Health Science 2013:4

in Pb2+-exposed animals”. Thus, these findings may give concerns in regard to lead exposure during fetal development especially since the blood lead concentration of the mother might rise as her body recruits calcium from the bones, where lead could have been as well stored. These observations resulted in the hypothesis that the amount of maternal bone lead stores is the relevant parameter for predicting the level of neurotoxicity in the fetus. Ronchetti and colleagues (2006) argue that strategies could be developed for dietary supplementations for the mother which can reduce bone reabsorption and lead mobilization during pregnancy and therefore reducing the lead burden for the unborn. Up until now, too little is still known in regard to the neurotoxicity of lead in children. While accumulating data are suggesting that there are toxicological effects with behavioral concomitants at exceedingly low levels of exposure (Lidsky and Schneider, 2003).

Human exposure to lead and other metal ions interfere with some of the functions described in this overview e.g.: zinc (Büsselberg et al., 1994a and b), aluminum (Platt and Büsselberg, 1994, Platt et al., 1995) or mercury (Pekel et al., 1993, Leonhard et al., 1996). Multiple reviews describe their general actions (e.g. Florea and Büsselberg, 2006) their action on voltage gated channels (e.g. Büsselberg, 1995) or, more specifically, metal interactions at pre- and postsynaptic processes (Sadiq et al., 2012). Additionally it has to be considered that these metal interactions occur simultaneously. As this question is difficult to tackle, only limited data are available, but a study of Platt and Büsselberg (1994) clearly demonstrated additive effects of different metals in reducing the current through voltage gated calcium channels.

Overall, the potency of Pb2+ induced neurotoxicity relies in its ability to mimic the action of the second messenger, Ca2+, thereby impairing the physiological function of Ca2+. In order to avoid the Pb2+ induced neurotoxicity it is important to prevent the environmental contamination and to minimize occupational and accidental exposure. Hopefully this review will help to understand some of the major underlying mechanisms of lead neurotoxicity to a wider audience and help to reduce environmental exposure and to find alternatives for the use of lead and heavy metals in general.

Acknowledgement: This review was made possible by the Biomedical Research Program (BMRP) of Qatar Foundation. The statements made herein are solely the responsibility of the authors.

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