A Dissertation Entitled Development of a Novel Pharmacological Model Of

A Dissertation

entitled

Development of a Novel Pharmacological Model of Okadaic Acid-induced Alzheimer’s

Disease in Zebrafish and its use in Drug Discovery

Daniel M Koehler

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Doctor of Philosophy Degree in

Experimental Therapeutics

______Dr. Frederick Williams, Committee Chair

______Dr. Zahoor Shah, Committee Member

______Dr. Jeffrey Sarver, Committee Member

______Dr. Caren L. Steinmiller, Committee Member

______Dr. Amanda Bryant-Friedrich, Dean College of Graduate Studies

The University of Toledo August 2018

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author.

An Abstract of

Development of a Novel Pharmacological Model of Okadaic Acid-induced Alzheimer’s Disease in Zebrafish and its use in Drug Discovery

Daniel M Koehler

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Experimental Therapeutics

The University of Toledo August 2018

Alzheimer’s disease (AD) is a progressive neurodegenerative disease hallmarked by the presence of amyloid beta deposition forming plaques and neurofibrillary tangles

(NFTs) that are comprised mainly of hyperphosphorylated tau protein. AD is the most common form of dementia and is currently the sixth leading cause of death in the United

States. Even though AD has been heavily studied in the past several decades, no cure exists and the exact etiology of the disease is unknown and the factors that are essential for its progression are also not well understood.

Many animal models of AD have been developed and have contributed to the understanding of the disease. However, these animal models are not ideal with many of the transgenic models only manifesting a portion of the disease pathology, developing motor impairments (which make cognition testing difficult), and being cost and time inefficient. In addition, the current AD animal models have yet to yield a successful drug to treat AD. Therefore, it is crucial that new animal models to study the AD pathology and to do preclinical testing of drugs are developed. The studies presented here utilized a newly developed AD model in zebrafish. A protein phosphatase inhibitor, okadaic acid iii

(OKA), was dissolved in water and the zebrafish were housed in the water for a total of 9 days. This treatment resulted in the formation of many AD pathological hallmarks including cognitive decline, phosphorylation of tau, the formation of plaques, and the increase in kinase expression. The studies presented here were designed to further characterize the model and simultaneously use it as a drug screening tool. The first study showed that TDZD-8, a glycogen synthase kinase 3β (GSK3β) inhibitor, is able to treat the OKA-induced AD pathology by rescuing cognitive function, downregulating active

GSK3β, downregulating pTau (Ser199), and normalizing protein phosphatase 2A activity. GSK3β has been established as a target for treating AD because of its role in phosphorylating tau. This study served as a proof of concept that the OKA-induced AD model in zebrafish can be utilized as a drug screen tool. The last studies demonstrated that a novel therapeutic, lanthionine ketimine-5-ethyl ester (LKE), is able to treat OKA insults by rescuing cognitive function and upregulating the BDNF/Akt/CREB pathway and reducing apoptosis in the zebrafish dorsal lateral pallium which is homologous to the human hippocampus.

Acknowledgements

I would like to thank Dr. Frederick Williams for being a patient advisor over the course of my dissertation. Your clear perspective and guidance throughout my troubles during my time at the University of Toledo helped me tremendously and kept me grounded, focused, and confident. Thank you for making me a better student, researcher, and person. I also would like to thank my committee members; Dr. Zahoor Shah, Dr.

Jeffrey Sarver, and Dr. Caren Steinmiller. All 3 of you were a tremendous help. You were always there for me when I was seeking advice, even when it came to matters outside of research, and for that, I thank you dearly. I would like to thank my lab partner,

Alexander Wisner, who has been in the lab with me during my entire Ph.D. study. You were the one who taught me everything from the beginning and always kept me even.

More than a lab partner, you are a great lifelong friend. I would like to thank my family.

You were honest with me and kept me strong and focused during my struggles and you celebrated my achievements in a way that inspired me. I love you all. Holly Helminski.

Words cannot express the gratitude that I have for you. You have been beyond wonderful, and someone whom I will remember forever. Lastly, I would like to thank the entire Department of Pharmacology and Experimental Therapeutics at the University of

Toledo for being accommodating and for allowing me this great opportunity. It has been a journey that has been difficult but has served me in becoming a better human.

Table of Contents

Abstract ...... iii

Acknowledgements ...... v

Table of Contents ...... vi

List of Tables ...... x

List of Figures ...... xi

List of Abbreviations ...... xii

List of Symbols ...... xv

1 General Introduction: Modeling Alzheimer’s Disease in Zebrafish to Aid in Drug

Discovery...... 1

1.1 Alzheimer’s Disease ...... 2

1.2 Amyloid Hypothesis…...... 2

1.3 Tau Hypothesis ...... 3

1.4 Interplay between Amyloid and Tau ...... 4

1.5 Tau Protein Kinases ...... 5

1.6 Tau Protein Phosphatases ...... 7

1.7 Using Okadaic Acid to Study Neurodegeneration ...... 7

1.8 Modeling Alzheimer’s Disease in Zebrafish.…...... 9

1.9 Utilizing Okadaic Acid in Zebrafish ...... 11

References…………… ...... ………………………..13

2 The GSK3β inhibitor, TDZD-8, Rescues Cognition in a Zebrafish Model of

Okadaic Acid-induced Alzheimer’s Disease ...... 28

2.1 Introduction ...... 29

2.2 Materials and Methods…...... 31

2.2.1 Animals ...... 31

2.2.2 Drug Treatment ...... 32

2.2.3 Learning and Memory Test ...... 32

2.2.4 PP2A Activity Assay ...... 33

2.2.5 Western Blotting ...... 34

2.2.6 Statistical Analysis ...... 34

2.3 Results……...... 35

2.3.1 Treatment with TDZD-8 reduced mortality induced by OKA ...... 35

2.3.2 TDZD-8 rescues the OKA induced cognition impairments ...... 37

2.3.3 Pre-treatment cognition results ...... 37

2.3.4 Post-treatment cognition results ...... 38

2.3.5 OKA treated zebrafish exhibit reduced activity of PP2A ...... 43

2.3.6 OKA treated zebrafish exhibit reduced expression of PP2A ...... 45

2.3.7 TDZD-8 reduces the tau kinase, GSK3β, active: inactive expression

levels in OKA treated zebrafish………… ...... 45

2.3.8 TDZD-8 reduces the expression of phosphorylated tau in OKA

treated zebrafish tau kinase……… ...... 45

2.4 Discussion...... 48

References…… ...... 53 vii

3 Lanthionine Ketimine-5-Ethyl Ester Provides Neuroprotection in a Zebrafish

Model of Okadaic Acid-induced Alzheimer’s Disease....…...... 59

3.1 Introduction ...... 61

3.2 Materials and Methods…...... 62

3.2.1 Animals ...... 62

3.2.2 Drug Treatment ...... 62

3.2.3 Learning and Memory Test ...... 63

3.2.4 Western Blotting ...... 64

3.2.5 TUNEL Assay ...... 65

3.2.6 Statistical Analysis ...... 66

3.3 Results……...... 66

3.3.1 LKE Rescues the OKA Induced Memory Impairments ...... 66

3.3.2 Pre-treatment results ...... 67

3.3.3 Post-treatment results ...... 67

3.3.4 Decreased Apoptosis in LKE treated Zebrafish ...... 72

3.3.5 The Neurotrophic Factor BDNF is increased in the LKE Treated

Fish…………………………...... 75

3.3.6 The Survival Protein, pAkt (Ser473), is increased in LKE treated

Zebrafish ………… ...... 75

3.3.7 The Cognitive Enhancer pCREB is increased in LKE treated Fish..76

3.4 Discussion...... 79

References…… ...... 85

viii

4 Lanthionine Ketimine-5-Ethyl Ester Rescues Cognition in a Zebrafish Model of

Okadaic Acid-induced Alzheimer’s Disease....…...... 91

4.1 Introduction ...... 92

4.2 Materials and Methods…...... 93

4.2.1 Animals ...... 93

4.2.2 Drug Treatment ...... 94

4.2.3 Learning and Memory Test ...... 95

4.2.6 Statistical Analysis ...... 95

4.3 Results……...... 96

4.3.1 Treatment with LKE reduced mortality induced by OKA ...... 96

4.3.2 LKE Rescues the OKA Induced Memory Impairments ...... 98

4.3.3 Pre-treatment results ...... 98

4.3.4 Post-treatment results ...... 98

4.4 Discussion...... 103

References…… ...... 105

5 Summary and Future Directions....…...... 107

5.1 Summary…...... 108

5.2 Future Directions…...... 110

References…… ...... 112

References…… ...... 114

List of Tables

3.1 Antibodies Used for Western Blot Analysis ...... 65

4.1 Mortality Rate of Certain Treatment Aspects ...... 97

List of Figures

2-1 Mortality Rate and Necropsy Observations of Treated Fish (TDZD-8

Experiment)...... 36

2-2 Learning Data for Control Zebrafish ...... 39

2-3 Learning Data for TDZD-8 treated Zebrafish ...... 40

2-4 Learning Data for TDZD-8 + OKA treated Zebrafish ...... 41

2-5 Learning Data for OKA treated Zebrafish ...... 42

2-6 PP2A Activity Analysis after Treatment ...... 44

2-7 Western Blot Analysis after TDZD-8 Treatment ...... 46

3-1 Learning Data for Control Zebrafish ...... 68

3-2 Learning Data for OKA treated Zebrafish ...... 69

3-3 Learning Data for LKE + OKA treated Zebrafish ...... 70

3-4 TUNEL Assay Analysis after LKE Treatment ...... 73

3-5 Western Blot Analysis after LKE Treatment ...... 77

4-1 Experiment Design for LKE + OKA Treated Zebrafish ...... 94

4-2 Necropsy Pictures of OKA Treated Zebrafish ...... 97

4-3 Learning Data for Control Zebrafish ...... 99

4-4 Learning Data for OKA treated Zebrafish ...... 100

4-5 Learning Data for LKE + OKA treated Zebrafish ...... 101

List of Abbreviations

Aβ ...... Amyloid Beta AD ...... Alzheimer’s Disease AGD ...... Argyrophilic Grain Disease Akt...... Protein Kinase B ANOVA ...... Analysis of Variance APOE ...... Apolipoprotein Variant APP ...... Amyloid Precursor Protein ATP ...... Adenosine Triphosphate

BAD ...... Bcl-2-Associated Death Promotor BBB...... Blood-Brain-Barrier BDNF ...... Brain-derived Neurotrophic Factor BSA ...... Bovine Serum Albumin

Cat ...... Catalog Ce ...... Cerebellum CBD ...... Corticobasal Degeneration Cdk5 ...... Cyclic-dependent Kinase 5 CNS ...... Central Nervous System CREB ...... cAMP Response Element Binding Protein CRMP2 ...... Collapsin Response Mediator Protein 2

Dc ...... Dorsal Central Pallium Dl...... Dorsal Lateral Pallium Dm...... Dorsal Medial Pallium Dp ...... Dorsal Posterior Pallium DRP2/DPYSL2 ...... Dhydropyrimidinase-like Protein 2 DS ...... Down Syndrome DSP ...... Diarrheic Shellfish Poisoning

FAD...... Familial Alzheimer’s Disease FTDP-17 ...... Frontotemporal Dementia with Parkinsonism Linked to Chromosome 17

GABA ...... γ-Aminobutyric Acid GSK3β ...... Glycogen Synthase 3β Beta xii

HCl ...... Hydrochloric Acid hr ...... Hour HRP ...... Horseradish Peroxidase

JNK ...... c-Jun N-terminal Kinases

L ...... Liter LanCL1 ...... Lanthionine Synthetase-like Protein 1 LK ...... Lanthionine Ketimine LKE ...... Lanthionine Ketimine-5-Ethyl Ester LTP ...... Long-term Potentiation

MAPK ...... Mitogen-Activated Protein Kinase MAPT ...... Microtubule-associated Protein Tau mRNA ...... Messenger Ribonucleic Acid min ...... Minute mg ...... Milligram µL ...... Microliter mL ...... Milliliter

NaCl ...... Sodium Chloride NFT ...... Neurofibrillary Tangles nM ...... Nanomolar

OB ...... Olfactory Bulb OKA ...... Okadaic Acid

PHF ...... Paired Helical Filament 3- PO4 ...... Phosphate Group PP ...... Protein Phosphatase PSEN ...... Presenilin PSP ...... Progressive Supranuclear Palsy PVDF ...... Polyvinylidine Difluoride PBS ...... Phosphate Buffered Saline PI3K ...... Phosphatidylinositol-4,5-bisphosphate 3-Kinase

RPM ...... Revolutions Per Minute ROS ...... Reactive Oxygen Species RT ...... Room Temperature

SAD……...... Sporadic Alzheimer’s STXBP1…… ...... Syntaxin-binding Protein SEM ...... Standard Error of the Mean Ser ...... Serine SDS ...... Sodium Dodecyl Sulfate xiii

SNP ...... Sodium Nitroprusside

TDZD-8...... 4-benzyl-2-methyl-1, 2, 4-thiadiazolidine-3, 5-dione Tel ...... Telencephalon TO ...... Tectum Opticum Tris ...... Tris(hydroxymethyl)aminomethane TrkB ...... Tropmyosin Receptor Kinase B/Tyrosine Receptor Kinase B Tyr ...... Tyrosine

xiv

List of Symbols

α ...... Alpha β ...... Beta μ ...... Micro n...... Nano - ...... Minus (Negative) °C ...... Degrees Celsius #...... Number > ...... Greater Than % ...... Percentage

Chapter 1

General Introduction: Modeling Alzheimer’s Disease in Zebrafish to Aid in Drug Discovery

Daniel Koehler

1.1 Alzheimer’s Disease

First identified and presented in 1907 by Alois Alzhiemer [1], Alzheimer’s disease is a progressive neurodegenerative disease [2]. Alzheimer’s disease (AD) is the most common form of dementia and currently 5.7 million Americans are living with the disease. AD is classified as being familial Alzheimer’s disease (FAD) or sporadic

Alzheimer’s (SAD). FAD, which accounts for less than 10% of AD cases, occurs before the age of 65 and SAD occurs after the age of 65. Not only is AD the 6th leading cause of death in the United States, but it also has tremendous effects on the economy by costing the American people $277 billon. It is estimated that by the year 2050, 14-15 million people in the United States will suffer from AD and that it will cost the United States

$1.1 trillion dollars in healthcare expenses[3]. AD’s main clinical characterization is progressive memory loss and the main pathological features include extracellular amyloidβ (Aβ) plaques and intracellular neurofibrillary tangles (NFTs) [4].

1.2 Amyloid Hypothesis

In 1984 George Glenner isolated and purified a unique protein from amyloid fibrils (plaques) found in cerebrovascular amyloidosis from patients with AD [5]. This

4,200 dalton protein was termed amyloid β protein (Aβ) (Now known was Aβ42 since various Aβ fragments exist) and was also found to exist in Down syndrome (DS) patients.

Many patients with DS develop AD therefore the relationship between the two has been and still is a highly researched topic [5, 6]. A year later, it was found that the amyloid plaques found in the Alzheimer’s brain itself (as opposed to the cerebrovascular) comprised mainly of this Aβ protein [7]. Shortly thereafter by using cDNA to Aβ, a gene encoding the amyloid precursor protein (APP) was isolated, characterized, and localized. 2

Localization of the APP on human chromosome 21 suggested a genetic link between AD and DS [8-11] since DS is caused by the presence of a third copy of the 21st chromosome

[12]. It was also a possible explanation why so many patients with DS develop AD. In

1991, a discovery was made that a mutation in the APP gene caused autosomal dominant

AD [13]. These early studies established the “amyloid hypothesis” of AD which was first formally articulated in 1991/1992 [14, 15]. The “amyloid hypothesis” states that the buildup of amyloid β protein is the primary impetus of AD and its related pathogenesis, including the formation of neurofibrillary tangles, synapse loss, and neuronal cell death directly follow the deposition of Aβ.

The “amyloid hypothesis” was bolstered by several subsequent discoveries.

Mutations in the Presenilin 1 (PSEN 1) and Presenilin 2 (PSEN 2) genes caused autosomal dominant AD [16-18] and these mutations increased Aβ42 production and aggregation [19, 20]. In addition, apolipoprotein variant 4 (APOE4) was found to be a high risk factor for sporadic (late onset) AD [21, 22]. Humans possess three APOE alleles: APOE2, APOE3, and APOE4 [23]. APOE2 is considered protective against AD while APOE3 is considered neutral (i.e. not associated with AD) [24]. APOE appears to be important in the function of clearing Aβ, with APOE4 being the least effective at clearing Aβ [25, 26]. The “amyloid hypothesis” has led to new insights of AD, but it has failed to lead to a clinical application for AD patients therefore deterring many researchers from pursuing the “amyloid hypothesis” any further [27, 28].

1.3 Tau Hypothesis

Tau proteins are microtubule associated proteins localized in the axon of neurons and are primarily responsible for the assembly and stabilization of microtubules by 3

binding to tubulin [29-32]. Through its interaction with neural microtubules, tau is also involved in neurite outgrowth and regulating axonal transport of vesicles, mitochondria, and endoplasmic reticulum [33-35]. In its native form, tau is found unfolded with little tendency to aggregate. Though under pathological conditions tau aggregates into paired helical filaments (PHFs) and subsequently into neurofibrillary tangles (NFTs), which are both mainly comprised of hyperphosphorylated tau, resulting in various neurodegenerative diseases altogether known as tauopathies [32, 36]. The “tau hypothesis” of AD states that neurofibrillary tangles are the causative driver of the disease [37]. Tau pathology in AD starts in the entorhinal cortex, then moves across synapes into the hippocampus, progresses throughout the limbic system and eventually progresses into the neocortex [38-40].

The initial studies that formed the “tau hypothesis” demonstrated that NFTs but not the plaques correlated with AD severity and progression [41, 42]. Also, 20-30% of older yet clinically healthy patients showed Aβ deposition levels equal to or greater than the levels used to diagnose AD while the same cannot be stated for NFTs [43]. Other tauopathies such as Pick’s disease, progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic grain disease (AGD), and frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) occur with the absence of pathological Aβ deposition demonstrating that tau dysfunction by itself is capable of causing and/or driving neurodegeneration [36, 44, 45].

1.4 Interplay between Amyloid and Tau

Many studies suggest that there is a strong interplay between Aβ and tau in AD.

The first evidence that linked functionally working between Aβ and tau together showed 4

that P301L transgenic mouse overexpressing human tau have accelerated NFT formation when injected with Aβ [46]. A second study showcased that when the P301L transgenic mice were crossed with Aβ plaque forming transgenic mice, an increased number of

NFTs without the increase of Aβ plaques, were formed in the hybrid than in the parental

P301L transgenic [47]. These studies provided evidence that Aβ deposition is upstream of tau in AD pathogenesis and that it triggers the formation of dysfunctional tau. Another study showed that reducing endogenous tau in a mouse model expressing human APP reduced cognitive deficiencies and excitotoxicity without changing Aβ levels [48]. This indicates that aggregates of tau protein are not just a byproduct of Aβ deposition, but rather that aggregation of tau protein is required for Aβ toxicity. A report opposing the notion that Aβ deposition is upstream of toxic tau stated that reducing endogenous tau in

APP/PS1 mutant mice rescued neuronal loss, synapse loss, and memory deficits while reducing the Aβ deposition and fragments [49]. This, combined with the earlier studies showing that Aβ increases the formation of NFTs, indicates that there is a possible feedback loop, instead of a one way stream, that exists between Aβ and tau. This exact mechanistic process has yet to be elucidated.

1.5 Tau Protein Kinases

Protein kinases are enzymes responsible for catalyzing the transfer of a phosphate

3- group (PO4 ) from adenosine triphosphate (ATP) or guanosine triphosphate (GTP) to their respective substrate [50]. Tau has 85 putative phosphorylation sites, and due to the irregular phosphorylation and consequently the aggregation of tau causing pathological properties in AD brains, research efforts to treat AD have focused on the development of specific kinase inhibitors [51-53]. These efforts have been directed against a variety of 5

kinases, but the main kinase targets in current AD therapeutic research are glycogen synthase 3β (GSK3β), cyclin-dependent kinase 5 (Cdk5), c-Jun N-terminal kinases

(JNK), and p38α mitogen-activated protein kinase (MAPK).

Even though many kinases have implications in AD, GSK3β has proven to be one of the strongest drug target candidates [54, 55]. GSK3, a proline directed serine/threonine kinase, was originally discovered as a regulatory kinase in glycogen metabolism. GSK3 was found to phosphorylate and consequently inactivate glycogen synthase [56, 57].

Since then, GSK3 has been found to play a role in many other various processes including cell signaling, cellular transport, apoptosis, proliferation, and intracellular communication [58, 59]. GSK3 exists in two isoforms; GSK3α and GSK3β [60]. GSK3β is constitutively active and is rendered active when phosphorylated at its tyrosine 216 position and rendered inactive when phosphorylated at its serine 9 position [59, 61]. The interest in GSK3β as a therapeutic target for AD began with the discovery that tau protein kinase I, which was shown to induce PHF [62], was identical to GSK3β [63]. Following in vitro studies demonstrated that GSKβ played a major role in AD pathogenesis [64, 65].

Overexpression of GSK3β in a mouse model induced tau phosphorylation and neurodegeneration [66]. Cell lines overexpressing GSK3β were subject to increased apoptotic cell death [67, 68]. Furthermore, GSK3β has been found co-localized with NFT and its active form is increased in the AD brain [69-71]. Inhibiting GSK3β or restoring its levels to normal has been proven effective in models of AD and neurotoxicity [72-76].

Altogether these findings strongly suggest GSK3β is a strong candidate for treating AD.

1.6 Tau Protein Phosphatases

Protein phosphatases are enzymes responsible for catalyzing the transfer of a phosphate group from a phosphoprotein to a water molecule (dephosphorylation) [50].

Several phosphatases have exhibited dephosphorylation activity on tau. These include protein phosphatase 1 (PP1), PP2A, PP2B, PP2C, and PP5 [32, 77]. Amongst these,

PP2A is the most significant as it accounts for approximately 71% of the tau phosphatase activity in the human brain [77, 78]. Direct evidence that PP2A plays a role in AD pathogenesis stems from studies showing that when PP2A is inhibited in cultured cells and in rodents, tau becomes hyperphosphorylated and cognitive impairments emerge

([79-82]. In addition, the activity and expression of PP2A is significantly decreased in

AD therefore contributing to the hyperphosphorylation of tau and cognitive decline [83-

85]. These findings suggest that PP2A plays a significant role in the regulation of tau phosphorylation in AD.

1.7 Using Okadaic Acid to Study Neurodegeneration

Okadaic acid (OKA) is a derivative of a C38 fatty acid and was originally isolated from two marine sponges, Halichondria okadaii and Halichondria melanodocia [86].

After its isolation, it was found that OKA is produced by several species of dinoflagellates ([87, 88]. OKA is consumed by humans through its accumulation in mussels, and the human consumption of OKA leads to a syndrome known as diarrheic shellfish poisoning (DSP) [89]. OKA is a selective inhibitor of PP1 and PP2A [90]. Due to its inhibitory actions on PP1 and PP2A, OKA is used as a tool to study the role of protein phosphorylation in cellular processes [91, 92]. Since the expression and activity of PP2A is decreased in the AD brain, OKA has been successfully used as a tool to study 7

AD pathology [93, 94]. First reports of OKA producing neurotoxic effects came from a study that dosed non-neuronal cells and cerebellar neurons. It revealed that non-neuronal cells were unaffected by concentrations that were lethal to the neuronal cells [95].

Treatment of human brain slices with OKA induced hyperphosphorylated forms of tau in a dose-dependent manner [96]. The first in vivo experiment using OKA to alter protein phosphorylation dynamics in the rat brain showed that a single injection of OKA into the basal nucleus results in the hyperphosphorylation of tau, formation of PHFs, increased

APP immunoreactivity, and memory deficits [97]. The experiment was the first proof-of- concept study that showed that OKA could be used to model the pathology of AD. The same group went on to conduct a series of experiments of chronic intraventricular infusion of OKA into the rat brain with similar results and even went on to describe the administration of OKA into the rat brain as a “non-transgenic model of Alzheimer’s disease” [98-101]. These studies also revealed that PP2A inhibition by OKA induced the deposition of Aβ plaque-like structures [98, 101]. Since then numerous studies have been conducted using OKA to help clarify the pathomechanistic profile of OKA neurodegeneration and to investigate new ways to protect against the OKA induced pathology. Several studies have demonstrated that GSK3β plays a role in the hyperphosphorylation of tau in the OKA models [102-104]. Progressive cholinergic dysfunction is another characteristic attributed to AD, and it has been proven that OKA also induces cholinergic dysfunction by reducing levels of acetylcholine, reducing messenger ribonucleic acid (mRNA) expression of α7-nicotinic receptor, and reducing acetylcholine esterase activity [105, 106]. Agonist for α7-nicotinic acetylcholine receptor and α2-nicotinic acetylcholine receptor provided neuroprotection against OKA [104]. 8

Lastly, OKA administration also results in increased neuroinflammation, increased oxidative-stress, increased caspase activity, and decreased brain glucose uptake, which are all characteristics of AD [104, 107-110]. Hence, the OKA-induced AD-like pathology model is capable of aiding in the discovery process for AD (and potentially other related neurodegenerative disorders) therapeutics.

1.8 Modeling Alzheimer’s Disease in Zebrafish

Goats, sheep, chimpanzees, cats, and dogs are of the few species known to spontaneously develop pathological symptoms similar to AD [111-114]. However the use of these animals for translational research is limited by their availability, high costs, and ethical reasons. Therefore a variety of pharmacological to transgenic AD animal models have been produced. Transgenic mice have been the most abundantly used AD animal model [115]. These models have generated important advancements in deciphering the pathology of AD. Unfortunately the preclinical success of many once promising AD drugs in these transgenic models has not translated to clinical success. Many transgenic models are hindered by only exhibiting fractions of AD pathology, especially neuronal loss [115-117]. Also it has been argued that transgenic models only represent the familial form of the disease and that the two forms (sporadic and familial) might have separate neuropathological processes [117]. To date, no ideal animal model of AD has been developed and with an aging population it is imperative that new animal models are developed to accelerate the drug discovery process.

This is where the zebrafish (Danio rerio) has emerged as a promising tool to study neurodegenerative diseases. Zebrafish are vertebrates therefore they are more applicable to understanding human biology and in drug discovery against human diseases 9

than invertebrate models such as Drosophila melanogaster and Caenorhabditis elegans.

The nervous system of invertebrates is smaller and contains fewer neurons therefore trying to decipher physiological and behavioral relevance to humans is more appropriate by using vertebrates. Overall, the nucleotide sequence of zebrafish genes shows about

70% homology with that of human genes, and 84% of genes that are known to be associated with human disease have a zebrafish counterpart [118]. This supports the translational value of zebrafish models. The basic zebrafish brain structure has a high conservation when compared to the mammalian brain. The mammalian and zebrafish brain are both organized into a hindbrain, midbrain, and forebrain while further organizing them into the divisions of telencephalon, mesencephalon, metencephalon, and myelencephalon. Within these specified regions are many similarly defined areas including but not limited to the olfactory bulbs, hypothalamus, and cerebellum [118-120].

Since AD manifest in the hippocampus of the human brain, it is important to note that the dorsal lateral pallium of the zebrafish is its homolog to the mammalian hippocampus. The zebrafish blood-brain-barrier (BBB) is also similar in structure and function to the mammalian BBB allowing for novel neuro-drugs to be assessed for their permeability of the BBB. Zebrafish also possess the main genes involved in AD which are microtubule- associated protein tau (MAPT), (APOE), (APP), (PSEN1), and (PSEN2). The major neurotransmitter systems, such as dopaminergic, serotoninergic, cholinergic, glutamatergic, glycinergic, and GABAergic systems are all present in the zebrafish brain

[120]. Various cognitive paradigms are also in place to test the learning and memory ability of the zebrafish. These include but are not limited to a two-chamber spatial alternation task, a three-chamber spatial alternation task, conditioned placed preference, 10

associated learning in a plus maze, and tap-elicited startle reflex response [121-124]. In fact, many of the same behavioral tests done with rodents can be conducted on the zebrafish [118].

Several transgenic zebrafish models mimicking parts of the pathologies seen in

AD exist, but they only simulate a part of the AD pathology while memory deficiencies are seldom exhibited [120, 125, 126]. A pharmacological model using adult zebrafish and administering OKA has recently been developed [127].

1.9 Utilizing Okadaic Acid in Zebrafish

A novel and robust AD model utilizing adult zebrafish and OKA was recently developed. The advantages that this model provides are its ease of implementation and that it provides all the major molecular hallmarks of AD while maintaining cost effectiveness and efficiency. The initial development of the model comprised of testing various doses ranging from 10 nM to 1 μM OKA (with the inclusion of a control group), evaluating the expression changes of ptau, tau, Aβ fragments, pGSK3α/β, and the formation of senile plaques whilst also subjecting the various groups to a spatial alternation learning and memory test. When compared to the controls, each of the different dosing groups had significant increases in the aforementioned tested molecular hallmarks of AD indicating that OKA does induce AD-like pathology in the zebrafish.

The results of the learning and memory also demonstrated that OKA induces a cognitive decline in zebrafish. The learning and memory paradigm is a two-choice design therefore

50% correct is mathematically deemed as random chance. Animals who demonstrate functional learning and memory ability in this paradigm would progress from around

50% to around 75-90% correct towards the end of the testing period. In this study, the 11

control zebrafish were able to correctly execute the task 75% or more of the time towards the end of the testing period. Each of the OKA treated groups was not able to perform above 50% indicating that learning and memory are not evident. The initial testing proved that at 100 nM OKA the maximum number of fish survived while still inducing the major molecular hallmarks of AD [127].

An interesting observation was made during the initial dosing study. The fish that did not survive died of hemorrhaging in the brain region. It is reported that approximately

80% of AD patients also suffer from some form of cerebral amyloid angiopathy (CAA)

[128]. CAA is characterized by the buildup of amyloid deposits in the blood vessels of the brain and at its most severe stage, causes intracerebral hemorrhaging [128]. It needs to be further explored if this bleeding was indeed caused by amyloid deposition in the vasculature of the zebrafish central nervous system [127].

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Chapter 2

The GSK3β inhibitor, TDZD-8, Rescues Cognition in a Zebrafish Model of Okadaic Acid-induced Alzheimer’s Disease

Daniel Koehler a, Zahoor A. Shah b, and Frederick E. Williams *a

Address correspondence to: Dr. Frederick Williams University of Toledo, College of Pharmacy and Pharmaceutical Sciences Department of Pharmacology and Experimental Therapeutics Health Science Campus, 3000 Arlington Avenue, HEB 274C Toledo, OH 43614. USA E-mail: [email protected] Tel: 419-383-1991 (Office)

Abstract

Currently, no treatments exist that are able to directly treat against Alzheimer’s disease (AD), and we are facing an inevitable increase in the near future of the amount of patients who will suffer from AD. Most animal models of AD are limited by not being able to recapitulate the entire pathology of AD. Recently an AD model in zebrafish was established by using the protein phosphatase 2A inhibitor, okadaic acid (OKA).

Administering OKA to zebrafish was able to recapitulate most of the neuropathology associated with AD, therefore, providing a drug discovery model for AD that is also time and cost efficient. This study was designed to investigate the effects of GSK3β inhibition by 4-benzyl-2-methyl-1, 2, 4-thiadiazolidine-3, 5-dione (TDZD-8) on this newly developed AD model. Fish were divided into 4 groups. Group #1= negative control,

Group #2= 1μM TDZD-8, Group #3= 1μM TDZD-8 + 100nM OKA, and Group #4=

100nM OKA. Administering the GSK3β inhibitor to zebrafish concomitantly with OKA proved to be protective. TDZD-8 treatment reduced the mortality rate, active: inactive

GSK3β, pTau (Ser199), and restored PP2A activity. This further corroborates the use of

GSKβ inhibitors in the treatment against AD and also bolsters the use of the OKA- induced AD-like zebrafish model for drug discovery.

2.1 Introduction

Alzheimer’s disease (AD) is a chronic neurodegenerative disease with the two main neuropathological hallmarks being amyloid-β (Aβ) aggregates and neurofibrillary tangles (NFTs) [1, 2]. According to the 2018 Alzheimer’s Association Report, 5.7 million

Americans are suffering from Alzheimer’s disease (AD) and this is expected to grow to

13.8 million Americans by the year 2050 [3]. To date, the therapeutic standards of care 29

for AD do not modify disease progression. They merely mask the cognitive symptoms of

AD and even this is poorly accomplished, as the average delay of symptom progression is only 6 to 12 months [4]. In fact, of the top 10 causes of death in the United States, AD is the only one that cannot be prevented, cured, nor delayed.

For the near future, due to the growing number of people suffering from AD and the lack of treatment, it is apparent that novel medical interventions against AD are dire and our current methodologies for discovery of those interventions are not sufficient.

Current commonly used animal models used do not provide us with quick or cost- effective in vivo research designs in screening for potential drug candidates. This is where the zebrafish has been a promising emerging in vivo tool to model and combat AD.

Zebrafish have highly conserved genetics and behavioral traits when compared to rodents. They also exhibit sensitivity to all major neurotropic drugs and respond to them in a similar fashion as humans [5]. This marks them as an organism with high utilization in neuropharmacological research while having significantly reduced costs associated with their care. Several AD models including pharmacological (effecting cholinergic, glutamatergic, and GABAergic systems) and transgenic models (APP, PSEN1, PSEN2, and TAU) have been utilized, but none of them are able to recreate most of the molecular hallmarks and behavioral hallmarks of AD [6, 7]. Recently, a pharmacological model was established that was able to recapitulate both the traditional pathological hallmarks of

Alzheimer’s disease and cognitive decline. Exposing zebrafish to the protein phosphatase

1 (PP1) and 2A (PP2A) inhibitor, okadaic acid (OKA), resulted in memory impairments,

Aβ fragment deposition, senile plaque induction, hyperphosphorylated tau protein, and cell loss [8, 9]. Furthermore, this model was used to test the potential neuroprotective role 30

of an experimental drug, lanthionine ketimine-5-ethyl ester (LKE), demonstrating this model’s worth in as a screening tool in neurodegenerative drug discovery [8].

However, this model needs to be further implemented in order to gain acceptance as a commonly used AD model. In this report, it was demonstrated that a commercially available compound, 4-benzyl-2-methyl-1, 2, 4-thiadiazolidine-3, 5-dione (TDZD-8), was effective in ameliorating the AD-like pathology that OKA induces in zebrafish. Again, this further demonstrates the potential of this newly developed OKA-induced

Alzheimer’s disease model in zebrafish. Glycogen synthase kinase 3 β (GSK3β) has been a strongly studied drug target in the CNS to combat AD [10-12]. Dosing the fish with a

GSK3β inhibitor and having successful endpoints supports, both that the OKA-induced

AD in zebrafish model is effective to employ in drug discovery, and that GSK3β inhibitors are a potential therapeutic against AD.

2.2 Materials and Methods

2.2.1 Animals

All animal experiments were approved by the University of Toledo Health

Science Campus Institutional Animal Care and Use Committee. AB zebrafish (Danio rerio) used in the various experiments were between the ages of twelve to fifteen months.

The fish were divided into 4 groups, and each group contained 4 male and 4 female fish.

They were housed at 26-28˚C with a 14:10 h light/dark cycle with feeding twice a day.

The fish were purchased from the Zebrafish International Resource Center (Eugene, OR)

(Catalog ID: ZL1).

2.2.2 Drug Treatment

Okadaic acid (OKA) sodium salt (product # O-5857) ˃98% pure was purchased from LC Laboratories (Woburn, MA, USA). The OKA was dissolved in 95% ethanol and further diluted in fish water to a concentration of 100nM. 2-methyl-4-(phenylmethyl)-

1,2,4-thiadizolidine-3,5-dione (TDZD-8) (item # 16287) was purchased from Cayman

Chemical (Ann Arbor, MI, USA). TDZD-8 was dissolved in 95% ethanol, and diluted in fish water to a final concentration of 1μM. 4 different groups of fish were established;

Group #1= negative control, Group #2= 1μM TDZD-8, Group #3= 1μM TDZD-8 +

100nM OKA, and Group #4= 100nM OKA. For the negative control group, an ethanol volume equivalent to that used in dissolving the OKA and TDZD-8 was added to the water. The exposure period lasted for 9 days and the water along with the various treatments were refreshed every other day as described previously [4]. Before and after the treatments were conducted, the fish were subject to a learning & memory function test. After the last learning and memory test, the fish were then euthanized by being immersed into ice cold water (0-4˚C). The telencephalon region of the zebrafish forebrains were then re moved and snap frozen and stored at -80˚C until use.

2.2.3 Learning and Memory Test

Pre-treatment (learning) and post-treatment (memory) tests were performed as described previously [13]. Briefly, the fish were placed into individual 10 liter aquariums

(N=1 per aquarium) and assigned random numbers to assure that testing personnel were blinded. Each 10 liter aquarium was filled with 26-28˚C DI/RO water with 60 mg/L of

Instant Ocean® sea salt (Instant Ocean, Blacksburg, VA). Each aquarium was divided into two equal sections by a central opaque divider that allows for adequate space for the 32

fish to swim from one side to the other side of the aquarium. One end of the aquarium is colored red as a means for visually distinguishing the two sections of the aquarium as zebrafish have the ability to see red [9]. Before the test is to begin, the fish have their diet restricted for 48-72 hours and are introduced to their respective aquariums for at least 48 hours prior to testing. Trials were initiated with a light tap (discriminative stimulus) at the center of the aquarium. After the light tap, there was a 5 second delay followed by food presentation. To avoid satiation and to keep the fish positively motivated, only a small amount of food (approximately 5 brine shrimp nauplii) was dispensed per trial. In 20 minute intervals, food presentation continued on alternating sides for a total of 28 trials

(14 trials per side). A response was analyzed as correct if the fish was physically present on the side of food presentation within 5 seconds of the discriminative stimulus.

Zebrafish are usually deemed to have learned the task when 75% of the responses are correct [8].

2.2.4 PP2A Activity Assay

For analysis of PP2A activity, brain tissue was lysed in RIPA buffer plus 1x protease inhibitor cocktail (ThermoFisher, cat # 88266) and incubated for 30 minutes on ice. The samples were then centrifuged at 14000 rpm (4˚C) for 10 minutes, and the supernatant was mixed with phosphate buffer ((40 mM Tris–HCl, pH 8.4, 34 mM

MgCl2, 4 mM EDTA and 4 mM DTT) and assayed for PP2A activity by the p-

Nitrophenyl Phosphate colorimetric assay (pNPP)[14]. Absorbance readings were taken at 405 nM.

2.2.5 Western Blotting

For Western blot analysis, brain tissue was lysed in tissue extraction reagent

(ThermoFisher, cat # FNN0071) plus with a 1x protease inhibitor cocktail

(ThermoFisher, cat # 88266) and incubated for 30 minutes on ice. The samples were then centrifuged at 14000 rpm (4˚C) for 10 minutes, and the supernatant was assayed for protein concentration by the Bradford method [15]. Equal amounts of protein were mixed with reducing sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate

[SDS], 10% glycerol, 0.002% bromophenol blue, and 5% β-mercaptoethanol) and boiled for 5 min. at 98˚C. Proteins were subject to electrophoresis across 10-15% SDS- polyacrylamide gels and transferred to polyvinylidine difluoride (PVDF) membranes

(pore size 0.45 μM, and cat. # IPVH00010 respectively). The blots were blocked for 1 h at room temperature (RT) in Tris-buffered saline blocking buffer (50 mM Tris-HCl, 150 mM NaCl) containing 5% bovine serum albumin followed by incubation overnight at 4˚C with primary antibodies for rabbit anti GSK3β (Cell Signaling, cat. #12456), rabbit anti phospho-GSK3β (Tyr216) (LifeSpan Biosciences, LS-C335917), rabbit anti phospho-

GSK3β (Ser9) (Cell Signaling, cat. #9336, rabbit anti phospho-Tau (Ser199) (GenScript,

A00894-40), mouse anti PP2A (Santa Cruz, sc-80665), rabbit anti β-Actin (Cell

Signaling, cat. #4967), and rabbit anti α-Tubulin (Cell Signaling, cat. #2125). Finally, the blots were incubated in HRP-conjugated secondary antibody for 1 hr at RT and visualized using enhanced chemiluminescence reagents (Bio-Rad, cat # 1705060).

2.2.6 Statistical Analysis

All data are presented as means ± SEM. Western blot analysis was carried out using one-way ANOVA with a Newman-Keuls post-hoc test. A value of p < 0.05 was 34

reported as significant. Zebrafish learning and memory was analyzed as previously described [16]. The mathematical model for learning was formulated to measure the probability, P, of a correct response and the formula used is:

푡 5 푏 ( ) 푃 = 0.5 + 푐 푡 5 1 + (푐)

Where “b” is the amount of learning that takes place, “c” is the number of trials it takes to reach half-maximum learning, and “t” is the trial number. These parameters, “b” and “c”, were measured using the SAS nonlinear modeling procedure NLIN. The parameter “b” will be referenced in the paper as “maximum learning”. Plots of the final prediction for P and the success frequency as functions of trial for each treated group were overlaid and used to display the fit of the estimated model.

2.3 Results

2.3.1 Treatment with TDZD-8 reduced mortality induced by OKA

Exposure to 100 nM OKA resulted in a mortality rate of 25%, while the group co- treated with 1 μM TDZD-8 + 100nM OKA resulted in a mortality rate of 8.3% (Fig. 2-

1A). Necropsy observations demonstrated that mortality was caused by brain and peripheral hemorrhaging (Fig. 2-1B).

Figure 2-1. Mortality rate and necropsy observations of cause of death: A. No fish died during the control and TDZD-8 treatments. 1 fish died during TDZD-8 + OKA treatment.

3 fish died during OKA treatment. B. Hemorrhage is observed to be cause of death for all fish that died during the experiment. Ruptured vessels are marked by black boxes and arrows.

2.3.2 TDZD-8 rescues the OKA induced cognition impairments

A pre-treatment test was conducted on all 4 groups, and then 10 days later, a post- treatment test was conducted. Even though Group #1 did not receive any treatment, a pre and post-treatment was still conducted on the group as a control. The 100nM OKA treated zebrafish (Group #4) showed no ability to remember; whereas the control zebrafish (Group #1), 1 μM TDZD-8 only (Group #2), and the 1 μM TDZD-8 + 100nM

OKA (Group #3) treated zebrafish all demonstrated evidence of memory (Fig.2-2, Fig. 2-

3, Fig. 2-4). The ability for Group #2 to demonstrate the ability to remember, exhibits that TDZD-8, at least at a concentration of 1 μM, is not toxic to the cognitive function of the zebrafish.

2.3.3 Pre-treatment cognition results

Group #1 demonstrated a pre-test maximum learning of about 79% which is 29% above the initial random chance of 50%. Measurement of half-maximal, which indicates a change in learning or memory of the fish, was established at around the 10th trial (Fig.

2-2A). Group #2 demonstrated a pre-test maximum learning of about 76% which is 26% above the initial random chance of 50%. Half-maximal learning was found to be at about the 8th trial (Fig. 2-3A). Group #3 had a maximum learning of about 82% which is 32% above the initial random chance of 50%. Half-maximal learning for Group #3 pre-test occurred around the 13th trial (Fig. 2-4A). Group #4 had a maximum learning of about

85% which is 35% above the initial random chance of 50%. Half-maximal learning for

Group #4 pre-test occurred around the 18th trial (Fig. 2-5A).

2.3.4 Post-treatment cognition results

Group #1 started the post-test at a 60% success rate which was already 10% higher than the pre-treatment test’s determined random chance of 50%. Group #1 performed at a maximum learning success rate of 85% and half of maximum learning

(from the starting point of 60%) started at around the 7th trial (Fig. 2-2B). Group #2 started the post-treatment test at the random success rate of 50% which was the same as the pre-treatment starting point. Group #2 performed at a maximum success rate of around 85% with half maximum performance being around the 8th trial (Fig. 2-3B).

Group #3 started the post-treatment test at a 50% success rate which was the same as the pre-treatment starting point. Group #3, during the post-treatment test, established a maximum success rate of about 79% and half of maximum performance was calculated to be around the 10th trial (Fig. 2-4B). Group #4 never reached a maximum performance of at least 70-75%, and therefore, it was concluded that memory was not demonstrated.

Group #4 started the post-treatment test at the random success rate of 50% and performed at a maximum success rate of around 50%. Therefore, the half maximum performance was unable to be calculated. Group #4 never reached a maximum performance of at least

70-75% and therefore it was concluded that memory was not demonstrated (Fig. 2-5B).

Figure 2-2. Learning (pre-treatment) and memory (post-treatment) data of control zebrafish. The dots on each graph represent the group’s running average at each trial point. The curved line represents a non-linear least-squares regression curve of the probability correct responses. A. Zebrafish were subject to the spatial alteration paradigm before being treated. The control group demonstrated the ability to learn by reaching 75% correct. B. After receiving treatment, the zebrafish were again subject to the spatial alteration paradigm. The control group demonstrated the ability to remember by starting the behavioral task at 60% instead of the random chance probability of 50% and by reaching the 75% mark at an accelerated rate. n= 12 (6 male and 6 female for pre- treatment control), n=12 (6 male and 6 female for post-treatment control)

Figure 2-3. Learning (pre-treatment) and memory (post-treatment) data of TDZD-8 treated zebrafish. The dots on each graph represent the group’s running average at each trial point. The curved line represents a non-linear least-squares regression curve of the probability correct responses. A. Zebrafish were subject to the spatial alteration paradigm before being treated. The TDZD-8 group demonstrated the ability to learn by reaching

75% correct. B. After receiving treatment, the zebrafish were again subject to the spatial alteration paradigm. The TDZD-8 group demonstrated the ability to learn by reaching

75% correct. n= 12 (6 male and 6 female for pre-treatment TDZD-8), n=12 (6 male and 6 female for post-treatment TDZD-8)

Figure 2-4. Learning (pre-treatment) and memory (post-treatment) data of TDZD-8 +

OKA treated zebrafish. The dots on each graph represent the group’s running average at each trial point. The curved line represents a non-linear least-squares regression curve of the probability correct responses. A. Zebrafish were subject to the spatial alteration paradigm before being treated. The TDZD-8 + OKA group demonstrated the ability to learn by reaching 75% correct. B. After receiving treatment, the zebrafish were again subject to the spatial alteration paradigm. The TDZD-8 + OKA group demonstrated the ability to learn by reaching 75% correct. n= 12 (6 male and 6 female for pre-treatment

TDZD-8), n=11 (5 male and 6 female for post-treatment TDZD-8)

Figure 2-5. Learning (pre-treatment) and memory (post-treatment) data of OKA treated zebrafish. The dots on each graph represent the group’s running average at each trial point. The curved line represents a non-linear least-squares regression curve of the probability correct responses. A. Zebrafish were subject to the spatial alteration paradigm before being treated. The OKA group demonstrated the ability to learn by reaching 75% correct. B. After receiving treatment, the zebrafish were again subject to the spatial alteration paradigm. The OKA group did not demonstrate memory by starting the post- treatment paradigm at random chance of 50% and never reaching a successful response of 75%. n= 12 (6 male and 6 female for pre-treatment OKA), n=9 (4 male and 5 female for post-treatment OKA)

2.3.5 OKA treated zebrafish exhibit reduced activity of PP2A

Protein phosphatase 2A is responsible for dephosphorylating serine/threonine motifs and has been shown to be significantly decreased in patients diagnosed with AD, ultimately leading to the hyperphosphorylation of tau protein [17]. PP2A activity in the zebrafish forebrain is significantly decreased (p<0.05) by 39% when compared to the control. TDZD-8 by itself did not significantly affect the activity of PP2A. There was also no significant difference between the control and the 1 μM TDZD-8 + 100 nM OKA group, indicating that TDZD-8 treatment was able to normalize the PP2A activity (Fig. 2-

6).

Figure 2-6. Okadaic acid lowers PP2A activity in zebrafish forebrain. PP2A activity was analyzed using tissue taken from the telencephalon region of the zebrafish. PP2A activity was significantly reduced in the OKA treated zebrafish when compared to the control and

TDZD-8 group. No significant difference of PP2A activity was determined between the control and TDZD-8 + OKA treated zebrafish. The bar graphs are presented as means ±

SEM; *p<0.05, n= 3 (1 male and 2 female for control), n= 3 (1 male and 2 female for

TDZD-8), n= 3 (1 male and 2 female for TDZD-8 + OKA), n=3 (1 male and 2 female for

OKA).

2.3.6 OKA treated zebrafish exhibit reduced expression of PP2A

PP2A expression is decreased by 44% (p<0.001) in the OKA group when compared to the control. 1 μM TDZD-8 only and 1 μM TDZD-8 + 100 nM OKA do not alter the levels of PP2A expression when compared to the control. Compared to each respective group, PP2A expression is significantly decreased (p<0.001) in the OKA group (Fig. 2-7A).

2.3.7 TDZD-8 reduces the tau kinase, GSK3β, active: inactive expression levels in

OKA treated zebrafish

GSK3β, also known as Tau Kinase I, is a proline directed serine/threonine kinase that has been extensively studied and has strong implications in the pathogenesis of AD

[18]. Its activated form, pGSKβ (Tyr216), is increased in the AD brain [19]. When compared to the control, OKA significantly increased (p<0.001) the ratio of active pGSKβ (Tyr216) to inactive pGSKβ (Ser9). Compared to each respective group, the active to inactive ratio of pGSK3β is significantly increased (p<0.001) in the OKA group. 1 μM of TDZD-8 does not alter the expression level of active: inactive pGSK3β when compared to the control, but 1 μM TDZD-8 when concomitantly added to 100 nM

OKA seems to normalize the level of active: inactive pGSK3β back to the amount of the control (Fig. 2-7B).

2.3.8 TDZD-8 reduces the expression of phosphorylated tau in OKA treated zebrafish

Tau protein is mainly expressed in neurons and plays an essential role in stabilizing the microtubules. Phosphorylated tau disassembles from the microtubule and 45

forms deposits correlating with cognitive decline [20, 21]. When compared to the control, the expression level of pTau (Ser199) in the OKA treated group was significantly increased (p<0.001). The concomitant treatment of zebrafish with 1 μM TDZD-8 + 100 nM OKA normalized the pTau (Ser199) amount (Fig. 2-7C).

Figure 2-7. TDZD-8 lowers Alzheimer’s related protein expression levels that are increased by okadaic acid. Western blotting was done on tissue taken from the telencephalon region of the zebrafish forebrain. A. Immunoblotting for PP2A shows a decrease of PP2A expression in the OKA treated group when compared to the control and the TDZD-8 group. Reduction in PP2A expression appears in the TDZD-8 + OKA group, but no significant difference was found. B. Immunoblotting for active pGSK3β (Tyr216) and inactive pGSK3β (Ser9) shows an increase in the ratio of active to inactive pGSK3β in OKA treated zebrafish when compared to all other groups. No difference was in the ratio of active to inactive pGSK3β between the control, TDZD-8, and TDZD-8 + OKA treated zebrafish. C. Immunoblotting for pTau (Ser199) shows an increase in pTau expression of OKA treated zebrafish when compared to all other groups. No difference was found in pTau expression between the control, TDZD-8, and TDZD-8 + OKA treated zebrafish. The bar graphs are presented as means ± SEM; *p<0.05 **p<0.01,

***p<0.001, n= 6 (3 male and 3 female for control), n= 6 (3 male and 3 female for

TDZD-8), n= 6 (3 male and 3 female for TDZD-8 + OKA), n=6 (3 male and 3 female for

OKA)

2.4 Discussion

In this study, it was established that TDZD-8 renders protection against OKA- induced Alzheimer’s like pathology in zebrafish. These protective effects, of TDZD-8, included decreased lethality, improved cognitive function, and decreased expression of several pathological hallmarks of Alzheimer’s disease, including p-tau and p-GSK3β.

This is the first time that the effects of the GSK3β inhibitor, TDZD-8, have been demonstrated in the zebrafish OKA-induced AD model. A 1 μM dose of TDZD-8 simultaneously administered with a 100 nM dose of OKA proved to be an effectual preventive treatment against OKA-induced AD.

Currently no direct treatments for AD exist and the last FDA approved

Alzheimer’s drug was Namzaric® in 2014, and which is not a novel drug as it is a combination pill of previously approved drugs for Alzheimer’s disease. The last true new drug for Alzheimer’s disease was approved in 2003 [22]. Even though AD is an important disease that will continue to affect a greater number of the population in the near future, we continue to be have difficulty combating this disease. A contribution to this problem is the lack of ideal animal AD models as most are only able to mimic a partial set of symptoms [23]. Recent studies have demonstrated that when zebrafish are exposed to OKA they undergo learning and memory dysfunction and molecular changes associated with AD such as increased expression of phosphorylated tau, the deposition of

Aβ-fragment, plaque formation, and cell death [8, 9].

OKA is a protein phosphatase 1 and 2A inhibitor [24]. Recent therapeutic strategies for AD have shifted from reducing Aβ deposition to reducing abnormal phosphorylation of tau [25]. This is influenced by the many clinical failures of Aβ 48

directed treatments and the stronger correlation between cognitive decline and phosphorylated tau [26, 27]. One of the major factors in the phosphorylation state of tau is PP2A which has been discovered to account for approximately 71% of the total tau phosphatase activity in the human brain [28, 29]. Not only a key component to the phosphorylation state of tau in the healthy brain, PP2A is also implicated in the AD brain as its expression and activity levels are significantly decreased in those with AD. These changes in PP2A are thought to contribute to the hyperphosphorylation of tau and the associated cognitive decline [29-31]. Therefore OKA, with it being a PP2A inhibitor, has been used to study various neurodegenerative diseases including AD [32, 33]. The administration of OKA in vitro and in vivo leads to pathologies observed in AD including

Aβ deposition, tau hyperphosphorylation, oxidative stress, inflammation, neurodegeneration, and cognitive impairments [34-38]. A limitation of many transgenic animal models in assessing learning and memory is the occurrence of motor dysfunction within these models. However, the use of OKA to study neurodegeneration has failed to show impairments in motor function indicating that cognitive defects are attributed to learning and memory defects and not motor dysfunction [38].

The effect on cognitive function of OKA, TDZD-8, and TDZD-8 + OKA was studied by a spatial alternation paradigm [13] (Fig. 2-2, 2-3, 2-4, 2-5). In relation to the learning paradigm utilized, zebrafish are deemed to have learned the task if the correct choice percentage is ≥75%. Long-term memory is assessed in this behavioral paradigm by removing the fish from the testing apparatus for 10 days after proving their ability to learn in the initial testing phase (in this case before treatment), and then after having removed them for 10 days, they are put through the paradigm for another round of 49

testing. Memory retention is determined by the fact that the fish begin the second testing phase above the random chance success rate of 50% and reach the ≥75% success rate in fewer trials than in the initial testing period [13].

Here group #4, before treatment with OKA, demonstrated the ability to learn by reaching a maximum probability correct of 85%. Their linear regression curve resembled the pre-treatment curves of group #1, group #2, and group #3. After the 9 days of OKA treatment, group #4 never reached a probability correct higher than 50%, indicating an inability to retain memory and proving that OKA causes a cognitive defect.

Deciphering the memory retention capacity of group #3 (TDZD-8 + OKA) is a bit complicated. It is apparent that their capability to learn remains, but if they retained memory of the initial testing is questionable. Group #3 post-treatment spatial alternation task demonstrated the ability to learn by starting the task at 50% (random chance) and performing to a maximum correct rate of 79%. This group’s pre-treatment results showed a maximum learning of 82% which is higher than the maximum learning rate of the post- treatment test. However, that difference is not statistically significant. The post-treatment test indicates that half-maximal learning occurred around the 10th trial, and for the pre- treatment test half-maximal learning occurred around the 13th trial. This exhibits that the fish were able to learn at a quicker rate after the initial testing period. So it is difficult to determine if memory was retained with the treatment of TDZD-8 + OKA, but the capacity to learn unequivocally remained. Therefore it is determined that TDZD-8, when administered simultaneously with OKA, does provide protection against OKA induced cognitive impairment.

TDZD-8 is a selective non-ATP competitive glycogen synthase kinase 3β

(GSK3β) inhibitor initially designed as a potential treatment of Alzheimer’s disease [39].

Several reports indicate that TDZD-8 is an effective protectant against neuronal injury following ischemia and the administration of the neurotoxin 6-OHDA by increasing

GSK3β (Ser9) phosphorylation and subsequently inactivating GSK3β [40-42]. The

Ser199 position of tau is phosphorylated by various kinases including GSK3β.

Phosphorylation of tau at the Ser199 position might prove to be an effective biomarker for AD, and has been determined to be an early event in the development of the pathogenesis of AD [43-46]. As mentioned previously, there has been shift in the strategies in thwarting AD by targeting hyperphosphorylated tau. Consequently, inhibiting certain kinases such as GSK3β will reduce the phosphorylation of tau [10-12].

In the present report, TDZD-8 was able to render protection against OKA in zebrafish by decreasing the ratio of active: inactive GSK3β which decreased the amount of pTau

(Ser199). Whether acted upon directly or indirectly, TDZD-8 also increased the expression and activity of PP2A which has additional potential to contribute to the decreased levels of pTau.

In summary, TDZD-8 treatment given simultaneously with OKA is able to protect against OKA induced AD pathology. TDZD-8 was able to restore cognitive dysfunction, reduce mortality, increase the expression and activity of PP2A, decrease the activity of

GSK3β, and reduce the expression of pTau. It is paramount to note that the molecular analysis was done on the zebrafish forebrain, specifically the telencephalon. The telencephalon is responsible for learning and memory in teleost fish [6, 47]. Showing the effectiveness of TDZD-8 in preventing OKA-induced Alzheimer’s disease in zebrafish, 51

further demonstrates the effectiveness of using the zebrafish model of OKA-induced

Alzheimer’s disease in drug discovery.

Disclosure

The authors declare no conflict of interest

Funding:

The study was supported by American Heart Association grant

#17AIREA33700076/ZAS/2017 to ZAS

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Chapter 3

Lanthionine Ketimine-5-Ethyl Ester Provides Neuroprotection in a Zebrafish Model of Okadaic Acid-induced Alzheimer’s Disease

Daniel Koehler a, Zahoor A. Shah b, Kenneth Hensley c and Frederick E. Williams *a

a Department of Pharmacology and Experimental Therapeutics, b Department of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, University of Toledo, Toledo, Ohio, USA c Department of Biochemistry, Molecular and Cell Science, Arkansas College of Osteopathic Medicine, Fort Smith, Arkansas, USA

(Note: This chapter has been published as it in Neurochemistry International (2018), Volume 115, Pages 61-68)

Abstract

Okadaic acid (OKA) is a protein phosphatase-2A inhibitor that is used to induce neurodegeneration and study disease states such as Alzheimer’s disease (AD).

Lanthionine ketimine-5-ethyl ester (LKE) is a bioavailable derivative of the naturally occurring brain sulfur metabolite, lanthionine ketimine (LK). In previously conducted studies, LKE exhibited neuroprotective and neurotrophic properties in murine models but its mechanism of action remains to be clarified. In this study, a recently established zebrafish OKA-induced AD model was utilized to further elucidate the neuroprotective and neurotrophic properties of LKE in the context of an AD-like condition. The fish were divided into 3 groups containing 8 fish per group. Group #1= negative control, Group

#2= 100nM OKA, Group #3= 100nM OKA + 500μM LKE. OKA caused severe cognitive impairments in the zebrafish, but concomitant treatment with LKE protected against cognitive impairments. Further, LKE significantly and substantially reduced the number of apoptotic brain cells, increased brain-derived neurotrophic factor (BDNF), and increased phospho-activation of the pro-survival factors pAkt (Ser 473) and pCREB

(Ser133). These findings clarify the neuroprotective and neurotrophic effects of LKE by highlighting particular survival pathways that are bolstered by the experimental therapeutic LKE.

Keywords: Zebrafish; Okadaic acid; Lanthionine ketimine-5-ethyl-ester; BDNF; CREB;

PKB/Akt

3.1 Introduction

Alzheimer’s disease (AD) is a chronic neurodegenerative disease associated with progressive cognitive decline. Research has shown that the main neuropathological hallmarks of AD are the formation of amyloid plaques and neurofibrillary tangles (NFTs)

[1]. Current animal models used in the research of AD do not provide us with quick nor cost-effective approaches to collecting data and screening for potential drug candidates.

Alternative animal models need to be utilized so that the research on AD is more efficient in this way. The zebrafish has emerged as a promising tool in modeling AD. Several AD models using zebrafish have been created but none of them are able to recreate both molecular and behavioral hallmarks of AD [2]. To fill this need, we have been exploring the use of the protein phosphatase 1 (PP1) and 2A (PP2A) inhibitor, okadaic acid (OKA)

[3], to induce AD-like pathology in zebrafish. Using carefully treated OKA zebrafish we were able to demonstrate many of the phenotypes of AD in zebrafish. Zebrafish treatment with OKA resulted in learning and memory impairments, increased Aβ fragment deposition, senile plaque induction, and hyperphosphorylated tau protein [4].

Furthermore, we used this OKA-treatment as a paradigm to test the efficacy of an experimental therapeutic, lanthionine-ketimine-5-ethyl-ester (LKE), to reduce AD-like molecular and behavioral effects in the zebrafish model.

Lanthionine ketimine (LK) is a natural sulfur amino acid metabolite which is formed through alternative reactions of the transsulfuration pathway followed by subsequent transamination [5, 6]. The exact physiological function(s) of LK is unknown and was perceived as being metabolic waste. However, recent findings suggest that LK has neurotophic and neuroprotective properties through its interaction with collapsin 61

response mediator protein 2 (CRMP2). A synthetic derivative of LK, LKE, designed to be cell-penetrating and readily bioavailable also demonstrated neurotrophic and neuroprotective properties [6, 7].

The present study was designed to investigate the therapeutic potential and mechanism of LKE in the OKA-induced AD model in zebrafish. LKE was able to rescue the cognitive decline caused by OKA which was corroborated by a decrease in apoptotic cells. LKE also induced a significant increase in the expression of brain-derived neurotrophic factor (BDNF), the phosphorylation/activation of protein kinase B

(PKB/Akt), and the phosphorylation/activation of cAMP response element binding protein (CREB).

3.2 Materials and Methods

3.2.1 Animals

All animal experiments were approved by the University of Toledo Health

Science Campus Institutional Animal Care and Use Committee. AB zebrafish (Danio rerio) used in the various experiments were between the ages of twelve to fifteen months.

The fish were divided into 3 groups, and each group contained 4 male and 4 female fish.

They were housed at 26-28˚C with a 14:10 h light/dark cycle with feeding twice a day.

The fish were purchased from Zebrafish International Resource Center (Eugene, OR)

(Catalog ID: ZL1).

3.2.2 Drug Treatment

University of Toledo (Toledo, OH, USA) as previously described [7]. LKE was dissolved in water, and diluted to the final concentrations of 500μM. 3 different groups of fish were established; Group #1= negative control, Group #2= 100nM OKA, Group #3= 100nM

OKA + 500μM LKE. For the negative control group, an ethanol volume equivalent to that used in dissolving the OKA was added to the water. The exposure period lasted for 9 days and the water along with the various treatments were refreshed every other day as described previously [4]. Before and after the treatments were conducted, the fish were subject to a learning & memory function test.

3.2.3 Learning and Memory Test

Pre-treatment (learning) and post-treatment (memory) tests were performed as described previously [8]. Briefly, the fish were placed into individual 10 liter aquariums

(N=1 per aquarium) and assigned random numbers to assure that testing personnel were blinded. Each 10 liter aquarium was filled with 26-28˚C DI/RO water with 60 mg/L of

Instant Ocean® sea salt (Instant Ocean, Blacksburg, VA). Each aquarium was divided into two equal sections by a central opaque divider that allows for adequate space for the fish to swim from one side to the other side of the aquarium. One end of the aquarium is colored red as a means for visually distinguishing the two sections of the aquarium as zebrafish have the ability to see red [9]. Before the test is to begin, the fish have their diet restricted for 48-72 hours and are introduced to their respective aquariums for at least 48 hours prior to testing. Trials were initiated with a light tap (discriminative stimulus) at the center of the aquarium. After the light tap, there was a 5 second delay followed by food presentation. To avoid satiation and to keep the fish positively motivated, only a small amount of food (approximately 5 brine shrimp nauplii) was dispensed per trial. In 20 63

minute intervals, food presentation continued on alternating sides for a total of 28 trials

(14 trials per side). A response was analyzed as correct if the fish was physically present on the side of food presentation within 5 seconds of the discriminative stimulus.

Zebrafish are deemed to have learned the task when 75% of the responses are correct [8].

3.2.4 Western Blotting

For Western blot analysis, brain tissue was lysed in tissue extraction reagent

(ThermoFisher, cat # FNN0071) plus 1x protease inhibitor cocktail (ThermoFisher, cat #

88266) and incubated for 30 minutes on ice. The samples were then centrifuged at 14000 rpm (4˚C) for 10 minutes, and the supernatant was assayed for protein concentration by the Bradford method [10]. Equal amounts of protein were mixed with reducing sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate [SDS], 10% glycerol,

0.002% bromophenol blue, and 5% β-mercaptoethanol) and boiled for 5 min. at 98˚C.

Proteins were subject to electrophoresis across 10-15% SDS-polyacrylamide gels and transferred to polyvinylidine difluoride membranes (pore size 0.2 μM & 0.45 μM, cat #

ISEQ00010 and cat # IPVH00010 respectively). The blots were blocked for 1 h at room temperature (RT) in Tris-buffered saline blocking buffer (50 mM Tris-HCl, 150 mM

NaCl) containing 5% bovine serum albumin, and incubated in different primary antibodies (listed in Table 3.1) overnight at 4˚C. Finally, the blots were incubated in

HRP-conjugated secondary antibody for 1 hr at RT and visualized using enhanced chemiluminescence (Bio-Rad, cat # 1705060).

Table 3.1. Antibodies Used In This Study

Antibody Source Dilution Supplier Catalog Number BDNF Rabbit 1:500 Bioss bs-4989R β-Actin Rabbit 1:2000 Cell Signaling #4967 Phospho-Akt Rabbit 1:2000 Cell Signaling #4060 Akt Rabbit 1:1000 Cell Signaling #4691 Phospho-CREB Rabbit 1:1000 Cell Signaling #9198 CREB Rabbit 1:1000 Cell Signaling #9197

3.2.5 TUNEL Assay

After their respective exposures and behavioral task completion, the zebrafish were euthanized by placing in <4˚C water and then into 4% paraformaldehyde overnight at 4˚C. The brains of the zebrafish were then removed with the aid of a dissecting microscope and LED lighting. After washing with 1x PBS, the brains were processed for paraffin-embedded tissue sectioning and sectioned at 6 μM increments. Sections were then dewaxed by immersing in xylene for 5 minutes at room temperature and rehydrated sequentially by immersing slides through graded ethanol washes (100%, 95%, 70%, and

50%) for 5 minutes each at room temperature. After rehydration the sections were washed in 1x PBS for 5 minutes at room temperature, and allowed to permeabilize in proteinase K for 15 minutes. Lastly, the sections were incubated with TUNEL reaction mixture at 37˚C for 60 minutes and stained with DAPI. Analysis of TUNEL results was conducted on the dorsal lateral pallium of the zebrafish. The dorsal lateral pallium of the zebrafish is homologous to the mammalian hippocampus and is responsible for the formation of memories [11-13].

3.2.6 Statistical Analysis

All data are presented as means ± SEM. Western blot and TUNEL assay statistical analysis was carried out using one-way ANOVA with a Newman-Keuls post- hoc test. A value of p < 0.05 was reported as significant. Zebrafish learning and memory was analyzed as previously described [14]. The mathematical model for learning was formulated to measure the probability, P, of a correct response and the formula used is:

푡 5 푏 ( ) 푃 = 0.5 + 푐 푡 5 1 + (푐)

3.3 Results

3.3.1 LKE Rescues the OKA Induced Memory Impairments

A pre-treatment test was conducted on all 3 groups, and then 10 days later, a post- treatment test was conducted. Even though Group #1 did not receive any treatment, a pre and post-treatment was still conducted on the group as a control. The 100nM OKA treated zebrafish (Group #2) showed no ability to remember; whereas the control zebrafish (Group #1) and the 500μM + 100nM OKA treated zebrafish (Group #3) both demonstrated evidence of memory (Fig. 3-1, 3-2, 3-3).

3.3.2 Pre-treatment results

Group #1 demonstrated a pre-test maximum learning of about 77% which is 32% above the initial random chance of 45%. Measurement of half-maximal, which indicates a change in learning or memory of the fish, was established at around the 10th trial (Fig.

3-1A). Group #2 demonstrated a pre-test maximum learning of about 72% which is 22% above the initial random chance of 50%. Half-maximal learning was found to be at about the 14th trial (Fig. 3-2A). Group #3 had a maximum learning of about 77% which is 27% above the initial random chance of 50%. Half-maximal learning for Group #3 pre-test occurred around the 18th trial (Fig. 3-3A).

3.3.3 Post-treatment results

Group #1 started the post-test at a 65% success rate which was already 20% higher than the pre-treatment test’s determined random chance of 45%. Group #1 performed at a maximum learning success rate of 86% and half of maximum learning

(from the starting point of 65%) started at around the 7th trial (Fig. 3-1B). Group #2 started the post-treatment test at the random success rate of 50% and performed at a maximum success rate of around 58% with half maximum performance being around the

6th trial. Group #2 never reached a maximum performance of at least 70-75% and therefore it was concluded that memory was not demonstrated (Fig. 3-2B). Group #3 started the post-treatment test at a 65% success rate which was 15% higher than the pretest established random chance of 50%. Group #3, during the post-treatment test, established a maximum success rate of about 89% and half of maximum performance

(from the starting point of 65%) was calculated to be around the 24th trial (Fig. 3-3B).

Figure 3-1. Learning (pre-treatment) and memory (post-treatment) data of control zebrafish. The dots on each graph represent the group’s running average at each trial point. The curved line represents a non-linear least-squares regression curve of the probability correct responses. A. Zebrafish were subject to the spatial alteration paradigm before being treated. The control group demonstrated the ability to learn by reaching 70-

75% correct. B. After receiving their respective treatment, the zebrafish were again subject to the spatial alteration paradigm. The control group demonstrated the ability to remember by starting the behavioral task at 65% instead of the random chance probability of 50%. In addition to memory demonstration, the control zebrafish increased performance demonstrated by reaching 85%-90% correct. n= 8 (4 male and 4 female for both pre and post-treatment control)

Figure 3-2. Learning (pre-treatment) and memory (post-treatment) data of OKA treated zebrafish. The dots on each graph represent the group’s running average at each trial point. The curved line represents a non-linear least-squares regression curve of the probability correct responses. A. Zebrafish were subject to the spatial alteration paradigm before being treated. The control group demonstrated the ability to learn by reaching 70-

75% correct. B. After receiving treatment, the zebrafish were again subject to the spatial alteration paradigm. The OKA group did not demonstrate memory retention by starting the post-treatment paradigm at random chance of 50%. They also did not increase performance, demonstrated by their max correct response of 55-60%. n= 8 (4 male and 4 female for both pre and post-treatment OKA)

Figure 3-3. Learning (pre-treatment) and memory (post-treatment) data of LKE + OKA treated zebrafish. The dots on each graph represent the group’s running average at each trial point. The curved line represents a non-linear least-squares regression curve of the probability correct responses. A. Zebrafish were subject to the spatial alteration paradigm before being treated. The LKE + OKA group demonstrated the ability to learn by reaching 70-75% correct. B. After receiving their respective treatment, the zebrafish were again subject to the spatial alteration paradigm. The LKE + OKA group demonstrated the ability to remember by starting the behavioral task at 65% instead of the random chance probability of 50%. In addition to memory demonstration, the LKE+OKA zebrafish

increased performance demonstrated by reaching about 90% correct. n= 8 (4 male and 4 female for both pre and post-treatment LKE+OKA)

3.3.4 Decreased Apoptosis in LKE treated Zebrafish

Apoptosis, an indicator of cell death, was examined by conducting a TUNEL assay on the dorsal lateral pallium of the zebrafish. There was a 38% significant increase

(p<0.01) of the percentage of apoptotic cells in the zebrafish treated with 100nM OKA when compared to the control. In contrast, there was not a significant difference (p>0.05) between the zebrafish treated with 500 μM LKE + 100nM OKA and the control zebrafish. When comparing the 500 μM LKE + 100nM OKA to the 100nM OKA, a significant decrease (p<0.05) of 39% of apoptotic cells was observed. The TUNEL assay concludes that LKE is protecting the zebrafish dorsal lateral pallium from OKA induced cell death (Fig. 3-4 A, B).

Figure 3-4. Pictures were taken of the dorsal lateral pallium located within the telencephalon of the zebrafish and analyzed by TUNEL assay. A. DAPI stain (blue) and

TUNEL stain (green) and the overlay images taken using a 20X objective. B. DAPI stain

(blue) and TUNEL stain (green) and the overlay images taken using a 100X objective.

The images show an increase in apoptosis in the zebrafish dorsal lateral pallium treated with OKA which was analyzed and shown in C. Apoptosis was significantly increased in the dorsal lateral pallium of OKA treated zebrafish compared to the control and 73

LKE+OKA treated zebrafish. No difference was found between the LKE+OKA and control group. The bar graphs are presented as means ± SEM; *p<0.05 **p<0.01, n= 4 (2 male and 2 female for control), n= 4 (2 male and 2 female for LKE+OKA), n= 4 (2 male and 2 female for OKA). D. Schematic overview of the whole adult zebrafish brain with sectioning scheme of the telencephalon. E. Schematic coronal section of the telencephalon indicating the specific area of interest, the dorsal lateral pallium. Zebrafish brain illustrations were adapted and modified from [48]. OB, Olfactory bulb; Ce, cerebellum; Dc, dorsal central pallium; Dl, dorsal lateral pallium; Dm, dorsal medial pallium; Dp, dorsal posterior pallium Tel, telencephalon; TO, tectum opticum.

3.3.5 The Neurotrophic Factor BDNF is increased in the LKE Treated Fish

BDNF is a neurotrophic factor that stimulates neuronal growth, differentiation, survival, regeneration, and repair. Its role in AD has been extensively studied, and it is believed that BDNF might protect against the progression of AD [15]. BDNF expression appears to be decreased by 40% in the OKA group when compared to the control, however, the post-hoc test determined there was no significant difference (p>0.05).

When the 500 μM LKE + 100 nM OKA group was compared to the control group, there is a 94% significant increase (p<0.05) of BDNF expression. A 135% significant increase

(p<0.01) in BDNF expression when the 500 μM LKE + 100 nM OKA group was compared to the 100 nM group (Fig. 3-5A).

3.3.6 The Survival Protein, pAkt (Ser473), is increased in LKE treated Zebrafish

Akt, also known as protein kinase B, is a serine/threonine kinase that has a role in cell survival by inhibiting apoptosis. When the Ser473 epitope of Akt is phosphorylated,

Akt is active and has the ability to further phosphorylate proteins such as Bcl-2- associated death promotor (BAD) resulting in a loss of pro-apoptotic function [16, 17]. It appears that expression of pAkt (Ser473) is reduced in the OKA group when compared to the control, however, the post-hoc test determined there was no significant difference

(p>0.05). When compared to the control, the 500 μM LKE + 100 nM OKA group had a

191% significant increase (p<0.05) in pAkt (Ser473) expression, and when compared to the 100 nM OKA group it had a significant increase (p<0.01) in pAkt (Ser473) expression of 255% (Fig. 3-5B).

3.3.7 The Cognitive Enhancer pCREB is increased in LKE treated Fish

CREB is activated upon phosphorylation of Ser133 which signals for neuronal survival and long-term potentiation (LTP) which are both important in keeping and forming memories respectively [18, 19]. Just as the previous results were for BDNF and pAkt expression, it appears that pCREB (Ser133) expression is decreased by 54% in the

100 nM OKA group when compared to the control, however post-hoc analysis determined there to be no significant difference (p>0.05). The LKE treated fish (500 μM

LKE + 100 nM OKA) showed a 147% significant increase (p<0.01) when compared to the 100 nM OKA group and a 93% significant increase when compared to the control

(p<0.05) (Fig. 3-5C).

Figure 3-5. Zebrafish forebrain showcases an increase in neurotrophic signaling expression by western blot analysis. A. Immunoblotting with an anti-BDNF antibody shows an increase in the BDNF expression of LKE+OKA zebrafish when compared to the control and the OKA zebrafish. OKA zebrafish show a reduction of BDNF but no significant difference was found. B. Immunoblotting for pAkt (Ser473) with an anti- pAkt(Ser473) shows an increase in pAkt expression of LKE+OKA zebrafish when compared to the control and the OKA zebrafish. No significant difference found between

the control group and OKA group even though pAkt seems to be reduced in the OKA group. C. Immunoblotting with an anti-pCREB (Ser133) antibody shows an increase in pCREB of LKE+OKA zebrafish when compared to the control and OKA treated zebrafish. No significant difference found between the control group and OKA group even though it appears that pCREB is reduced in the OKA group. The bar graphs are presented as means ± SEM; *p<0.05 **p<0.01, n= 4 (2 male and 2 female for control), n= 4 (2 male and 2 female for LKE+OKA), n=4 (2 male and 2 female OKA)

3.4 Discussion

The present study, we were able to demonstrate that LKE exhibited neuroprotection against the behavioral and pathological symptoms of OKA-induced AD zebrafish model. These protective effects included improved cognitive function. This is the first time that the effects of LKE have been demonstrated in the zebrafish OKA- induced AD model. A 500 μM dose of LKE concomitantly administered with a 100 nM dose of OKA proved to be an effective prophylactic agent against OKA-induced neurotoxicity.

OKA is a protein phosphatase (PP) inhibitor, particularly of PP2A and PP1 [3].

Even though it is not classified as a neurotoxin, OKA is being used to induce neurotoxicity to study various neurodegenerative diseases including AD [20]. Just as the pathology observed in AD, OKA is able to trigger neurodegeneration, tau hyperphosphorylation, the accumulation of NFTs, Aβ deposition, oxidative stress, neuronal inflammation, and learning and memory impairment [21-23]. A major advantage of using OKA to study neurodegenerative diseases that cause cognitive deficiencies is that OKA does not change motor function [23, 24], therefore, the cognitive deficiencies revealed after OKA administration can be attributed to learning and memory impairments and not motor impairments. A recent report revealed that when zebrafish are exposed to 100 nM of OKA they sustain learning and memory impairment in addition to increased expression of phosphorylated tau, the deposition of Aβ-fragment, and plaque formation [4].

The effect on cognitive function of OKA and LKE+OKA was studied by a spatial alternation paradigm [8]. Before treatment, the to-be-OKA-treated-fish did demonstrate 79

evidence of learning by having their probability correct reach just under 75% and by having a linear regression curve that resembled both (pre-treatment) the control group and LKE + OKA group. Long-term memory was assessed in the behavioral paradigm by removing the fish from the testing apparatus for 10 days after proving their ability to learn in the initial testing round (before treatment). After 9 days of treatment the fish would be reintroduced to the testing apparatus for a second round of testing (after treatment). If the zebrafish remember the task from 10 days earlier, then they would start the task at a higher probability correct than the random probability of around 50%. Also the number of trials needed to reach 70-75% would be reduced in comparison to the initial test [8]. After 9 days of treatment with OKA, their linear regression curve stayed steadily around 55-60%, indicating their inability to remember after OKA exposure.

The LKE+OKA fish demonstrated evidence of learning and memory that strikingly resembled the control group. This shows that LKE, when concomitantly administered with OKA, is able to inhibit the cognitive impairment in zebrafish induced by OKA treatment. TUNEL assay results demonstrating a difference in the amount of cells undergoing apoptosis in the dorsal lateral pallium of OKA treated zebrafish versus

LKE + OKA treated zebrafish supports the idea that LKE reduces OKA generated effects on the dorsal lateral pallium of the zebrafish. Not only did the OKA treated fish exhibit a higher fraction of apoptotic cells when compared to the LKE+OKA fish, but the LKE +

OKA fish fraction of apoptotic cells resembled that of the control. The TUNEL assay analysis substantiates the spatial alternation paradigm data analysis. The observed

TUNEL assay changes were done in the dorsal lateral pallium area of the zebrafish. The dorsal lateral pallium of the zebrafish is homologous to the mammalian hippocampus 80

[11-13]. The hippocampus plays vital roles in forming long-term memories from short- term memory information, long-term-potentiation, and in spatial memory. AD manifests itself in the entorhinal cortex and the hippocampus and brain imaging analysis shows severe shrinking of the hippocampus in patients suffering from AD [25-27]. Therefore, it can be reasonably hypothesized that the improved cognitive function exhibited by the

LKE + OKA treated fish is attributable to the decreased cell death in the dorsal lateral pallium of the LKE + OKA treated fish.

LKE is reported to be an effective protectant in several neurodegenerative disease models including 3x Tg-AD mouse modeling AD, permanent ischemia brain injury in mice, SOD1G93A mouse model of familial amyotrophic lateral sclerosis (ALS), and an encephalomyelitis treated mice to model multiple sclerosis [6, 28-30]. However, its mechanism of action is not well understood. A proteomic study concluded that LK binds to 3 proteins selectively: Collapsin response mediator protein 2/dhydropyrimidinase-like protein 2 (CRMP2/DRP2/DPYSL2), syntaxin-binding protein (STXBP1/Munc18/nSec1), and lanthionine synthetase-like protein 1 (LanCL1) [7]. CRMP2, specifically, has been a central focus of investigation on how LKE is able to elicit its effects. CRMP2 has been an emerging target of discussion in regards to neurodegenerative diseases due to its role in axonal pathfinding and neuron polarization [31] and differential expression rates in disease states such as AD [32]. Therefore, most of the attributes or therapeutic benefits of LKE have been tied back to pCRMP2/CRMP2 in the literature. These investigations have determined an altered state of pCRMP2/CRMP2 expression after LKE intervention.

However, it is possible that other partners of LKE exist. The previous proteomic analysis on LKE (mentioned above) would likely not have identified cell surface membrane 81

receptors nor membrane associated proteins. Other brain protein binding partners could possibly exist that were not effectively canvassed by proteomics techniques thus far. Cell surface membrane receptors and strongly membrane associated proteins, in particular, likely would not have been identified in previous investigations [6]. Therefore, it is important to explore other potential mechanisms of action.

We were able to provide a possible mechanism behind the neuroprotective effects of LKE against OKA-induced neurodegeneration in the zebrafish. In the zebrafish group treated with OKA, LKE augmented the expression of the neurotrophic factor BDNF, the anti-apoptotic kinase pAkt (Ser473), and the transcription factor pCREB (Ser133). It is interesting to note that the levels of BDNF, pAkt, and pCREB were not significantly lowered in the OKA treated fish when compared to the control fish. This leads to the conclusion that LKE does not ameliorate OKA-induced neurodegeneration directly but rather through an indirect route. Several studies, including anecdotal evidence, show that

LKE has the tendency to work better in systems under stress [33]. It is plausible, but needs to be further explored, that LKE has nootropic effects that act in a positive feedback loop when it is administered to a system that has been given an insult. This could be attributed to LKE being a derivative of the natural metabolite lanthionine ketimine, therefore, having minimal effects in a normal homeostatic state. This is corroborated by work done in 5 day old zebrafish exposed to up to 500 μM LKE and no changes were observed (data not shown). In addition, a study of LKE and its neuroprotective qualities in an in vitro model of oxidative stress where LKE was given alone proved to have no effects [30].

Activation of neurotrophic signaling pathways promotes cognitive function through synapse formation, synaptic plasticity, neuronal survival, and neurogenesis [34,

35]. The investigation into neurotrophic pathways and their modulation in neurodegenerative diseases has been an increasing focal point as many other investigational leads have led to clinical failures, especially in combating AD.

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophic family that promotes neuronal growth, differentiation, survival, regeneration, and repair by primarily interacting with tropmyosin receptor kinase B/tyrosine receptor kinase B

(TrkB) [34-36]. This interaction stimulates the phosphatidylinositol 3-kinase (PI3K)-Akt pathway [37]. Akt, also known as protein kinase B, is a serine/threonine kinase that has a well-established role in numerous cellular functions including proliferation and survival.

Akt activation is governed by phosphorylation at Ser473 and Thr308 which leads to Akt phosphorylating many subsequent downstream targets including the proapoptotic proteins

BAD, MDM2, and caspase-9. The increase in pAkt (Ser473) seen in animals dosed with

500 μM LKE + 100 nM OKA group is not attributed to OKA even though OKA is a

PP2A inhibitor. PP2A dephosphorylates Thr308 while PH domain and leucine rich repeat protein phosphatases (PHLPP) dephosphorylate Ser473 of Akt [16, 17]. In addition, it was observed that OKA reduces the expression of pAkt (Ser473) in our studies which is consistent with other studies involving OKA and pAkt [38, 39].

The direct phosphorylation, by Akt, of BAD, MDM2, and caspase-9, negatively regulates their pro-apoptotic function leading to cellular survival [40]. Another important target of Akt is the transcription factor cAMP response element binding protein (CREB) which is phosphorylated at the Ser133 position [41]. Upon activation, CREB is able to 83

promote neuron survival and long-term potentiation via regulating gene expression [18,

19]. Interestingly enough, CREB is able to regulate the expression of BDNF [42], suggesting that BDNF and CREB could be operating in a positive feedback loop.

Many reports state that the activation of BDNF/TrkB/CREB pathway enhances neuroprotection from multiple insults, especially within the hippocampus [19, 43-47]. We report that the expression levels of BDNF, pAkt (Ser473), and pCREB (Ser133) were all increased in the LKE+OKA treated fish. The activation of the BDNF/TrkB/CREB pathway by LKE reduced the cell death in the dorsal lateral pallium of the zebrafish which could lead to their normal cognitive function.

Disclosure

Dr. Hensley is inventor on a patent concerning composition and use of LKE for medical purposes, and holds equity in XoNovo Ltd., a company engaged in development of the compound.

Acknowledgements

We would like to thank Alexander Wisner and Kevin Nash for their intellectual support during the experimental set-up and data collection.

Funding

This work was supported in part by a grant from the National Institutes of Health

(NS093594, KH)

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Chapter 4: A Partial Study

Lanthionine Ketimine-5-Ethyl Ester Rescues Cognition in a Zebrafish Model of Okadaic Acid-induced Alzheimer’s Disease

Daniel Koehler and Megan Muhleman

Abstract

The zebrafish okadaic acid (OKA) –induced Alzheimer’s disease (AD) model has been subject to various drug treatments. These drug treatments proved to be effective against the AD-like effects of OKA. However these drug treatments were implemented at the same time period as OKA exposure. This only proves that these drugs are effective as prophylactic agents, and therefore the next step would be to test these drugs in a more clinical relevant scenario. In this study, the fish were divided into 3 groups; Group #1= negative control, Group #2= 100nM OKA, Group #3= 100nM OKA + 500μM lanthionine ketimine-5-ethyl ester (LKE). The various groups were treated for 9 days.

The drug treatment, LKE, was not introduced until after 4 days of OKA exposure. OKA proved fatal by itself, but once LKE was administered, no further fish suffered fatally.

OKA caused severe cognitive impairments that were rescued by treatment with LKE.

4.1 Introduction

Alzheimer’s disease (AD) is a neurodegenerative disease that causes progressive cognitive and behavioral decline [1]. Despite the many years of extensive research using rodent models to study Alzheimer’s disease (AD) no cure or disease halting drug exists.

A rapidly increasing number of people are suffering from the disease and a therapeutic intervention is in desperate need. Therefore, it is necessary to have complementary models to aid in drug discovery. The zebrafish animal model is emerging as a valuable model for the investigation of AD and neurodegenerative drug discovery. Recently an

AD model was established by administering okadaic acid (OKA), a protein phosphatase inhibitor, to zebrafish [2]. This model has been utilized to study the efficacy of two drugs,

lanthionine ketimine-5-ethyl ester (LKE) and 4-benzyl-2-methyl-1, 2, 4-thiadiazolidine-3,

5-dione (TDZD-8).

However, these studies only proved that LKE and TDZD-8 are effective prophylactic agents because treatments commenced simultaneously with OKA treatment.

Therefore, the “disease” induced by OKA was never able to progress before drug treatment started. This is not relevant to the clinical setting because AD starts its pathological changes in the brain 10-20 years before any overt symptoms are observed [3,

4]. The present study was designed to investigate the therapeutic potential of LKE in a more clinically relevant OKA-induced AD model in zebrafish. To enable a more clinically relevant animal study, the OKA was administered to the fish for a period of 4 days before treatment with 500 μM LKE started. After the initial period of 4 days of

OKA exposure, the exposure continued for another 4 days with OKA + LKE. LKE was able to treat against the cognitive impairments caused by OKA. Additionally, once LKE was administered to the zebrafish, no further deaths that were cause by OKA ensued.

4.2 Materials and Methods

4.2.1 Animals

All animal experiments were approved by the University of Toledo Health

Science Campus Institutional Animal Care and Use Committee. AB zebrafish (Danio rerio) used in the various experiments were between the ages of twelve to fifteen months.

The fish were divided into 3 groups, and each group contained 4 male and 4 female fish.

They were housed at 26-28˚C with a 14:10 h light/dark cycle with feeding twice a day.

The fish were purchased from the Zebrafish International Resource Center (Eugene, OR)

(Catalog ID: ZL1). 93

4.2.2 Drug Treatment

University of Toledo (Toledo, OH, USA) as previously described [5]. LKE was dissolved in water, and diluted to the final concentrations of 500μM. 3 different groups of fish were established; Group #1= negative control, Group #2= 100nM OKA, Group #3= 100nM

OKA + 500μM LKE. For the negative control group, an ethanol volume equivalent to that used in dissolving the OKA was added to the water. The exposure period lasted for 9 days and the water along with the various treatments were refreshed every other day as described previously [2]. However, LKE was added on day 5 of the exposure period while OKA was administered on day 1 (Fig. 4-1). Before and after the treatments were conducted, the fish were subject to a learning & memory function test.

Figure 4-1. Treatment regimen of the LKE + OKA group.

4.2.3 Learning and Memory Test

Pre-treatment (learning) and post-treatment (memory) tests were performed as described previously [6]. Briefly, the fish were placed into individual 10 liter aquariums

(N=1 per aquarium) and assigned random numbers to assure that testing personnel were blinded. Each 10 liter aquarium was filled with 26-28˚C DI/RO water with 60 mg/L of

Instant Ocean® sea salt (Instant Ocean, Blacksburg, VA). Each aquarium was divided into two equal sections by a central opaque divider that allows adequate space for the fish to swim from one side to the other side of the aquarium. One end of the aquarium is colored red as a means for visually distinguishing the two sections of the aquarium as zebrafish have the ability to see red [7]. Before the test is to begin, the fish have their diet restricted for 48-72 hours and are introduced to their respective aquariums for at least 48 hours prior to testing. Trials were initiated with a light tap (discriminative stimulus) at the center of the aquarium. After the light tap, there was a 5 second delay followed by food presentation. To avoid satiation and to keep the fish positively motivated, only a small amount of food (approximately 5 brine shrimp nauplii) was dispensed per trial. In 20 minute intervals, food presentation continued on alternating sides for a total of 28 trials

(14 trials per side). A response was analyzed as correct if the fish was physically present on the side of food presentation within 5 seconds of the discriminative stimulus.

Zebrafish are deemed to have learned the task when 75% of the responses are correct [6].

4.2.4 Statistical Analysis

Zebrafish learning and memory was analyzed as previously described [8]. The mathematical model for learning was formulated to measure the probability, P, of a correct response and the formula used is: 95

푡 5 푏 ( ) 푃 = 0.5 + 푐 푡 5 1 + (푐)

4.3 Results

4.3.1 Treatment with LKE reduced mortality induced by OKA

The first 5 days of exposure to 100 nM OKA resulted in a mortality rate of 32%.

Once the LKE was administered to the group, no further deaths ensued. The group treated with 100nM OKA resulted in a total mortality rate of 25%, with 8.3% of fatalities occurring on days 1-4 and 16.7% of fatalities occurring on days 5-9 (Table 4.1).

Necropsy observations demonstrated that mortality was caused by brain swelling (Fig. 4-

2).

Table 4.1. Mortality rate on various days of the treatment regimen.

Drug Treatment Day 1-4 Day 5-9

Control 0% 0%

100 nM OKA 8.3% 16.7%

32% 0% 500 μM LKE + (100 nM (500 μ LKE + 100 nM OKA OKA) OKA)

Figure 4-2. 32% of Group #3 died on day 4 of the experiment after administration of

100nM OKA (1 day before the administration of LKE). Once the fish started treatment with LKE + OKA on day 5, no further fatalities ensued. The fish that succumbed to the

OKA exposure experienced swelling of the brain to the point of protrusion through the skull.

4.3.2 LKE Rescues the OKA Induced Memory Impairments

A pre-treatment test was conducted on all 3 groups, and then 10 days later, a post- treatment test was conducted. Even though Group #1 did not receive any treatment, a pre and post-treatment test was still conducted as a control. The 100nM OKA treated zebrafish (Group #2) showed no ability to remember; whereas the control zebrafish

(Group #1) and the 100nM OKA + 500 μM LKE treated zebrafish (Group #3) both demonstrated evidence of learning and/or memory (Fig. 4-3, 4-4, 4-5).

4.3.3 Pre-treatment results

Group #1 demonstrated a pre-test maximum learning of about 76% which is 26% above the initial random chance of 50%. Measurement of half-maximal, which indicates a change in learning or memory of the fish, was established at around the 7th trial (Fig. 4-

3A). Group #2 demonstrated a pre-test maximum learning of about 79% which is 24% above the initial starting success rate of 55%. Half-maximal learning was found to be at about the 6th trial (Fig. 4-4A). Group #3 had a maximum learning of about 86% which is

31% above the initial starting success rate of 55%. Half-maximal learning for Group #3 pre-test occurred around the 12th trial (Fig. 4-5A).

4.3.4 Post-treatment results

(from the starting point of 60%) started at around the 7th trial (Fig. 4-3B). Group #2 started the post-treatment test at the random success rate of 50% and performed at a 98

maximum success rate of around 56% with half maximum performance being around the

5th trial. Group #2 never reached a maximum performance of at least 75% and therefore it was concluded that memory or learning was not demonstrated (Fig. 4-4B). Group #3 started the post-treatment test at a 50% success rate. Group #3, during the post-treatment test, established a maximum success rate of about 80% and half of maximum performance was calculated to be around the 9th trial (Fig. 4-5B).

Figure 4-3. Learning (pre-treatment) and memory (post-treatment) data of control zebrafish (Group #1). The dots on each graph represent the group’s running average at each trial point. The curved line represents a non-linear least-squares regression curve of the probability correct responses. A. Zebrafish were subject to the spatial alteration paradigm before being treated. The control group demonstrated the ability to learn by

reaching 75% correct. B. After receiving their respective treatment, the zebrafish were again subject to the spatial alteration paradigm. The control group demonstrated the ability to remember by starting the behavioral task at 60% instead of the random chance probability of 50%. In addition to memory demonstration, the control zebrafish increased performance demonstrated by reaching 83% correct. n= 12 (6 male and 6 female for both pre and post-treatment control)

Figure 4-4. Learning (pre-treatment) and memory (post-treatment) data of OKA treated zebrafish (Group #2). The dots on each graph represent the group’s running average at each trial point. The curved line represents a non-linear least-squares regression curve of the probability correct responses. A. Zebrafish were subject to the spatial alteration paradigm before being treated. The OKA group demonstrated the ability to learn by

100

reaching 75% correct. B. After receiving treatment, the zebrafish were again subject to the spatial alteration paradigm. The OKA group did not demonstrate memory retention by starting the post-treatment paradigm at random chance of 50%. They also did not increase performance, demonstrated by their max correct response of 55%. n= 12 (6 male and 6 female for both pre and post-treatment OKA) n= 12 (6 male and 6 female for pre- treatment OKA), n=9 (5 male and 4 female for post-treatment OKA)

Figure 4-5. Learning (pre-treatment) and memory (post-treatment) data of LKE + OKA treated zebrafish (Group #3). The dots on each graph represent the group’s running average at each trial point. The curved line represents a non-linear least-squares regression curve of the probability correct responses. A. Zebrafish were subject to the spatial alteration paradigm before being treated. The LKE + OKA group demonstrated

101

the ability to learn by reaching 75% correct. B. After receiving their respective treatment, the zebrafish were again subject to the spatial alteration paradigm. The LKE + OKA group did not demonstrate the ability to remember by starting the behavioral task at random chance of 50%. However, the LKE + OKA did demonstrate the ability to learn by reaching about 80% correct. n= 16 (8 male and 8 female for pre-treatment LKE +

OKA), n=11 (5 male and 6 female for post-treatment LKE + OKA)

102

4.4 Discussion

Previously it was demonstrated that LKE, when concomitantly exposed to OKA, exhibits neuroprotection against behavioral and pathological abrasions induced by OKA

[9]. However, since LKE was administered at the same time as OKA, this work only demonstrates that LKE has potential as a preventative treatment against OKA-induced

Alzheimer’s disease. The present study involved an approach that is more relevant to a clinical setting. Twelve to fifteen month old zebrafish were treated with LKE after 5 days of OKA exposure. The OKA exposure continued as the zebrafish were being treated with

LKE. This exposure/treatment design allows for the “disease” to manifest and progress prior to pharmacotherapy. This holds more clinical relevance because the pathological changes in Alzheimer’s disease manifest 10-20 years before clinical symptoms appear [3,

4].

This study showed that LKE is able to rescue cognitive function in the zebrafish

OKA-induced AD model after 4 days of OKA exposure followed by 5 days of OKA +

LKE exposure. The cognitive function of the various zebrafish treatment groups was studied by using a spatial alternation paradigm [6]. Long-term memory was assessed in the behavioral paradigm by removing the fish from the testing apparatus for 10 days after proving their ability to learn in the initial testing round (pre-treatment). After 9 days of treatment the fish would be reintroduced to the testing apparatus for a second round of testing (post-treatment). If the zebrafish remember the task from 10 days earlier, then they would start the task at a higher probability correct than the random probability of around 50%. Also the number of trials needed to reach 75% would be reduced in comparison to the initial test [6]. 103

Before treatment, Group #2 did demonstrate evidence of learning by having their probability correct reach just under 80%. The linear regression curve of the pre-treatment assessment for Group #2 resembled the pre-treatment curves for Group #1 and Group #3.

After 9 days of treatment with 100 nM OKA, the linear regression curve stayed around

56%, indicating their inability to remember or learn after OKA exposure. The previous study, when LKE was concomitantly administered with OKA, the fish demonstrated evidence of learning and memory that resembled the control group. However, in this follow up study, the fish exposed to 500 μM LKE after 5 days of 100 nM OKA exposure only showed evidence of learning. There was no evidence of memory because the post- treatment (100 nM OKA + 500 μM) results show that the curve resembles that of the pre- treatment results. The fish did not start the post-treatment cognition test at a higher probability correct, they did not reach a higher probably of success, and the rate of learning was the same. Therefore it can only be concluded that group #3, after exposure, was able to retain the ability to learn but not remember.

Further studies investigating the protective mechanism(s) of LKE, in this more clinically relevant experimental design, are currently in progress. Previously, LKE provided neuroprotection by increasing the expression of BDNF, pAkt, and pCREB [9].

It is suspected that LKE would elicit neuroprotection in the same manner, but the current investigation has yet to reach a conclusion.

Disclosure

Dr. Hensley is inventor on a patent concerning composition and use of LKE for medical purposes, and holds equity in XoNovo Ltd., a company engaged in development of the compound. 104

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metabolite lanthionine ketimine (LK) and documentation of LK effects on

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132.

105

7. Avdesh, A., et al., Evaluation of color preference in zebrafish for learning and

memory. J Alzheimers Dis, 2012. 28(2): p. 459-69.

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exposures affect zebrafish learning. Neurotoxicol Teratol, 2010. 32(2): p. 246-55.

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2018. 115: p. 61-68.

106

Chapter 5

Summary and Future Directions

Daniel Koehler

107

5.1 Summary

A common theme across these studies is the observed effect that OKA has on cognition and causing death. Fatalities by OKA were observed to be caused by brain hemorrhaging and/or edema. Micro-bleeds often accompany late-stage AD, further adding to morphological hallmarks observed using this model. Specifically, cerebral amyloid angiopathy (CAA) is a condition where amyloid deposits in the blood vessels of the brain. CAA is a common co-occurrence with AD and can result in intracranial hemorrhaging [1, 2]. It needs to be further explored and validated if the intracranial hemorrhaging is indeed caused by CAA. The fish being exposed to OKA, without additional treatment, resulted in impaired cognition to the extent of being unable to perform the spatial alternation task above random chance. The three various drug treatment regimens undergone by the zebrafish resulted in rescued cognition.

Zebrafish subject to concurrent exposure of TDZD + OKA exhibited learning capabilities while their memory function could not be determined. TDZD-8 is a GSK3β inhibitor and treatment with TDZD-8 reduced the levels of active/inactive TDZD-8 when compared to the OKA group. GSK3β is known to phosphorylate tau [3] and the reduction of overall GSK3β activity by TDZD-8 led to decreased levels of pTau. Concurrent exposure of LKE + OKA resulted in retained memory and ability to learn that resembled the control group. The neuroprotective properties of LKE against OKA was verified by reduced TUNEL staining in the dorsal lateral pallium of the LKE + OKA group (similar amount of staining to the control) when compared to the OKA only group. Western blot quantitative analysis showed that the telencephalon of the LKE + OKA zebrafish brain had significantly elevated levels of pAkt, pCREB, and BDNF. Studies show that the 108

activation of the BDNF/Akt/CREB promotes regeneration of neurons and stimulates survival [4-6]. The follow-up study that implemented an experimental design that allowed for more clinical relevance, demonstrated that LKE is still able to rescue cognition after OKA induced neurotoxicity. However, this study only showcased that the animals retained the ability to learn but their long-term memory capability was deemed inconclusive.

The studies here highlight the use of the newly developed OKA-induced

Alzheimer’s model in zebrafish. The model serves as a drug screening paradigm for drugs designed to enhance or retain cognition. Further exploration into the mechanism of action behind a successful cognition test run is also possible with the described model.

The model recapitulates most of the prominent molecular hallmarks of AD, allowing for investigation into the drug’s mechanism of action. Previous studies have concluded that

OKA can be used in cell culture and/or in rodents as an experimental tool to study the cellular and molecular mechanism of Alzheimer’s disease pathology [7]. Our studies indicate that zebrafish can be included as an animal model in OKA-induced Alzheimer’s pathology. This model is advantageous to other in vivo models using OKA to study AD because using zebrafish is cost and time-effective while still maintaining conservation to the human condition.

Currently Alzheimer’s is the 6th leading cause of death, and it is quite possible that it will rise over the years due to the current aging population. The aging segment of the population is growing quickly and it is believed that the amount of people suffering from AD in the United States will triple by the year 2050 [8]. The typical drug costs

(including the cost of failures) $2.6-2.8 billion dollars to develop [9]. The estimated cost 109

(including the cost of failures) of developing drug therapies for AD, in a 2014 analysis, was around $5.7 billion [10]. Preclinical testing accounts for 29% of the cost. Current preclinical testing for AD drug development takes an average of 50 months and accounts for 31% of the total time it would take for a newly developed AD drug to go through the clinical process including post phase III regulatory review [10]. An opportunity exists with the OKA-induced AD model in zebrafish to reduce the time and the financial risk of

AD drug development leading to more innovation and increasing the success rate of the

AD drug development pipeline.

5.2 Future Directions

It is necessary to validate that LKE does activate the BDNF/Akt/CREB pathway.

ANA-12 is an antagonist of TrkB, the receptor of BDNF [11]. Administering ANA-12 concomitantly with OKA and LKE would allow for further investigation into the mechanism of action of LKE. If ANA-12 were to reduce or eliminate the protective effects and the upregulated expression of BDNF/Akt/CREB induced by LKE, then it can be concluded that LKE does work by directly activating BDNF. Additionally, proving that TDZD-8 mechanistically acts by inhibiting GSK3β can be done by administrating sodium nitroprusside (SNP). SNP is a known GSK3β activator [12]. If concomitant administration of SNP, TDZD-8, and OKA were to reverse the cognitive protection that

TDZD-8 offered while also upregulating active GSK3β and the phosphorylation of tau, then it can be concluded that TDZD-8 mechanistically acts by inhibiting GSK3β.

Further studies need to be conducted on the OKA-induced AD model in zebrafish to enhance its validity as an Alzheimer’s model.

110

Studies that are currently underway are looking into the production of reactive oxygen species (ROS) and anti-oxidant activity in the brain of OKA exposed zebrafish.

Oxidative stress plays a role in AD and previous studies showed that OKA does promote oxidative stress [7]. If OKA were to cause oxidative stress in the zebrafish brain, then it would strengthen the validity of the OKA-induced Alzheimer’s model in the zebrafish as it would encompass further hallmarks of Alzheimer’s disease.

It would also be beneficial to characterize the timeline of the model. Currently the exposure period, to OKA, is set at 9 days and all characterization of the model has been conducted after the 9 day exposure. In the clinical setting, AD is broken down into different phases/stages depending on the pathological process. It is believed that, in part, many potential AD drugs fail in phase 3 clinical trials because the drugs were tested in the early progressions of the disease in nonclinical testing and during clinical testing they are administered during later stages of the disease. In order to make adequate interpretation(s) of potential drugs (to treat AD) screened using the zebrafish model, the actual disease progression before treatment would start would need to be determined.

Also, the suspected cause of death needs to be evaluated. Necropsy observations conclude cause of death to be brain hemorrhaging/swelling. As previously mentioned

CAA is a common co-occurrence with AD and is the cause of brain bleeds in late stages of AD. It needs to be determined whether the brain bleeds in the OKA zebrafish AD model is due to amyloid deposits in the vasculature. If this is the case, then this would be one of the few documented AD animal models that includes CAA.

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