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PAGE 1 This is Your Mouse on Celastrol: The Behavioral Effects of Celastrol on a Transgenic Mouse Model of Alzheimers Disease BY AMELIA MARCH A Thesis Submitted to the Division of Natural Sciences New College of Florida In partial fulfillment of the requirements for the degree Bachelor of Arts Under the Sponsorship of Dr. Alfred Beulig, Jr. Sarasota, FL May, 2010 PAGE 2 ii Acknowledgments I would like to thank Dr. Fiona Crawford and Dr Daniel Paris for their assistance and for allowing me to do my thesis at t he Roskamp Institute; I would also like to thank David, Jim, Chris and all of my other friends at the Institute who helped me when I needed it and made my experience t here even better. I would also like to thank my advisor, Dr. Alfr ed Beulig, for his support, supervision, and useful suggestions during my thesi s, and for the Bocas del Toro experience. I would like to thank my family, who believed i n me and pushed me to graduate with the New College incoming class of 06 even when I didnt think I could. Thank you for being my best friend, Lucy. Si sters for life! These four years at New College have transforme d me more than I can say. I dedicate this thesis to everyone who has touched me and been a part of my New College experience: Eric, Nikki, Ben, Monica, Danie lle, Michelle, Gretchen, Mike, inhabitants of 10(death), Lissa, Christina, Eric Q. inhabitants and frequenters of the Beverly House, Jack, Geordie, Allie and Marisol to name a few: when I think of New College, I think of you. Thank you. Amelia March PAGE 3 iii Table of Contents 1. Title . i 2. Acknowledgements .ii 3. Table of Contents ...iii 4. Lists of Figures and Graphs ... ...v 5. Abstract ....vi 6. Introduction ...1 6.1 Alzheimers disease ...1 6.2 Tau and Neurofibrillary Tangles ...3 6.3 Amyloid -peptide and the Amyloid Precursor Protein . 8 6.4 Neurotoxicity of A ...14 6.5 The genetics of Alzheimers disease 19 6.6 NFB and A . 26 6.7 Celastrol .32 7. Materials and Methods ..36 7.1 Animals ..36 7.2 Morris Water Maze performance test ...37 7.3 Y-Maze ..40 7.4 Rota Rod ...42 7.5 Mouse Sacrificing ....43 7.6 Brain A Quantification ...44 7.7 Statistical Analysis ...45 7.8 Acute Treatment Study .. . 45 PAGE 4 iv 7.9 Powder Food Study .....46 8. Results .49 8.1 Powder Food Study . 49 8.1a Weights ..49 8.1b Y-Maze .. 52 8.1c Rota Rod 53 8.1d Morris Water Maze performance test 54 8.1e Brain A Quantification 56 8.1f Observations ..57 8.2 Acute Study ... 58 8.2a Y-Maze ..58 8.2b Morris Water Maze performance test 59 8.2c Observations 60 9. Discussion 61 10. References ..70 PAGE 5 v List of Figures and Graphs Figures 1. Proteolytic processing of APP by the secretases 9 2. The chemical structure of Celastrol .33 3. Morris Water Maze Diagram .40 4. Diagram of Y-Maze 42 5. Diagram of Rota Rod .43 Graphs 1. Effects of orally administered Celastrol on mous e weight over time ..51 2. Effects of orally administered Celastrol on perc ent alternation ...53 3. Effects of orally administered Celastrol on late ncy to fall .54 4. Effects of orally administered Celastrol on MWM latencies .55 5. Effects of orally administered Celastrol on MWM NE quadrant duration..56 6. Effects of orally administered Celastrol on Brai n A (1-40) and (1-42) .57 7. Effects of IP injected Celastrol on MWM latency ..59 8. Effects of IP injected Celastrol on MWM NE quadr ant duration .60 PAGE 6 vi Abstract The NFB inhibiting compound Celastrol has been shown to l ower levels of A, an amino acid-peptide known to be a major compone nt of the senile plaques seen in the brains of individuals with Alzheimers disease, both in vivo and in vitro Celastrol has also been found to reduce the produ ction of CTF and sAPP, which both result from cleavage of the APP ec todomain by the protease -secretase (BACE-1) at the N-terminus of A. Thus, one likely method by which Celastrol lowers brain A burden is by decreasing the expression of BACE-1, in turn reducing the expression of the substrate CTF, which results in A production after proteolytic processing by secretase. The memory and abilitybased performance of two strains of mice (Tg APPsw and Tg PS1/APPsw) administered Celastrol or a placebo were tested by the Morris Water Maze, Ymaze, and Rota Rod tests. The Tg APPsw strain overe xpresses both human A (1-42) and A (1-40), and the Tg PS1/APPsw strain has an increas e in the A (142) to A (1-40) ratio. Brain A levels of these mice were also quantified after a long term and an acute study. These studies reveale d that Celastrol is likely to be most useful as a prophylactic treatment rather than a therapeutic one. ______________________ __ Alfred Beulig, Jr. Division of Natura l Sciences PAGE 7 Introduction Alzheimers disease Alzheimers disease is a progressive, hetero geneous and cognitively debilitating illness of the elderly that appears to include a number of diverse, sporadic and hereditary neurodegenerative diseases which share common clinical and pathological features. It is a common and devastating disorder, currently affecting more than 5.2 million Americans (Welsh-Bohmer 2009). Alzheimers disease typically leads to forgetfulnes s of new and recent events, and eventually progresses to difficulty finding wor ds and reasoning, and completing normal activities. By the end of their l ives, those who suffer from Alzheimers often rely almost completely on others to function. The first recorded case of Alzheimers disease was diagnosed in the early 1900s (Dahm 2006). Alois Alzheimer (1864-1915) was working as an assistant physician at the psychiatric institution in the Ger man city of Frankfurt when he first encountered Auguste D. His careful observatio n of Auguste, admitted by her husband on November 25th, 1901, would eventually lead Alzheimer to discover the neurodegenerative disease which bears his name. Auguste exhibited symptoms which accurately summarize the range of pr ogressive changes observed in many Alzheimers patients today: a dete riorating memory (especially in the recollection of recent events), disorientati on, a decreased ability to speak coherently, problems understanding and judging situ ations, and restless and erratic behavior. Over time, Auguste D.s speech be came unintelligible, until she PAGE 8 2 stopped talking completely. She became unable to fe ed herself, and spent most of her time in bed until her death in 1906. After Augustes death, Dr. Alzheimer was able t o examine the extraordinary morphology of her brain tissue using several histol ogical staining methods. Alzheimer identified pronounced atrophy in many are as of her brain, in addition to thick fibrils staining many of the remaining neu rons. Deposits of plaques were also identified throughout the cerebral cortex. The se abnormalities were, to a certain extent, similar to the degenerative changes seen in senile dementia. However, patients with senile dementia generally be gin to display signs of dementia in their 70s or 80s. Auguste D. began exhi biting signs of dementia when she was only 51. Additionally, the pathologica l changes in Auguste D.s brain were much more dramatic than those Alzheimer had seen in patients suffering from senile dementia. These factors convi nced Alzheimer that he had discovered something new (Dahm 2006). The pathological changes Alzheimer observe d in Auguste D.s brain are considered today to be key diagnostic features of a n Alzheimers diseased brain. The brain of an individual with Alzheimers disease can be characterized by a few factors. The brain of a patient with Alzheimers is likely to include visible shrinkage in the areas related to memory, particula rly the hippocampus and the entorhinal cortex (Welsh-Bohmer 2009). This atrophy due to synaptic loss and neuronal death, can be clearly seen under a microsc ope, and may also be present in the outer gray matter, or the cerebral c ortex, resulting in a visibly smaller brain. Of course, this is no reason to co rrelate the size of the brain with PAGE 9 3 the likelihood that a person has Alzheimers. A key diagnostic neuropathologic feature of Alzheimers is the buildup of extracellu lar amyloid plaques or senile plaques found in the brain, along with intracellul ar neurofibrillary tangles which result from the buildup of an abnormal form of the protein tau (). Another diagnostic feature of Alzheimers disease is congop hilic amyloid angiopathy, in which amyloid deposits form in the walls of the blo od vessels of the central nervous system (CNS) (Welsh-Bohmer 2009). These sen ile plaques are usually surrounded by dead or dying neurons and inflammator y cells. Tau and Neurofibrillary Tangles Neurofibrillary tangles (NFTs) are one of the t hree major types of neurofibrillary lesions which correlate closely wit h AD. Although not restricted to AD, NFTs and other neurofibrillary lesions have bee n connected to the severity of dementia in AD (Selkoe 1989). The two other majo r types of neurofibrillary lesions besides NFTs are neuropil threads (NTs) and dystrophic plaqueassociated neurites, or neurites that surround amyl oid-rich senile plaque cores (Trojanowski 1994). The classification of the neuro fibrillary lesion depends on the location of the lesion in the neuron. At the struct ural level, fibrils 10-12 nm in diameter twist around each other to form paired hel ical filaments (PHFs) which have a period of about 80 nm. Straight filaments (c omposed of the same subunit proteins as PHFs) are common to each of these lesio n types (Goedert 1993). In 1991, Lee et al. demonstrated that altered f orms of tau () proteins known as PHF (or A68), which have been improperly phosphorylate d at Ser-396 PAGE 10 4 relative to their normal counterparts, are major su bunits of PHFs. Normal adult human CNS (CNS) consists of 6 isoforms which range from 352 to 441 amino acids, each with 3 or 4 microtubule (MT)-binding re peats of 31 or 32 amino acids arranged in tandem near the carboxyl terminus of th e protein. This repeat region of is known to function as the MT binding domain (Goe dert 1993). Additionally, the C-terminal tail of tubulin is thought to represent the binding domain of tubulin. Thus, consists of a carboxy-terminus microtubule-binding domain and an amino-terminus projection domain. All six of these adult human isoforms are known to contribute to the formation of PHFs (Goedert 1993). Though some studies (Wille 1992) have demonstrated that the microtubule (MT) binding repeat domain of was enough to form PHFlike structures in vitro without the requisite phosphorylation, the phospho rylation state is necessary to regulate the ability of the p roteins to bind MTs (Goedert 1993). So it is possible that phosphorylation might still alter the normal function of in a way that negatively affects neuronal function just as the generation of PHF affects neuronal function. The protein family of low-molecular-weight, microtubul e-associated proteins normally has several functions, such as regulating microtubule assembly and promoting the stabilization of polymerized microtub ules; microtubules assembled in the presence of show arms projecting from the surface (Hirokawa 19 88). The binding of to microtubules reduces their dynamic instability by increasing the rate of association and decreasing the rate of disa ssociation of tubulin molecules at the growing end, inhibiting the transition to th e catastrophic shortening phase PAGE 11 5 (Drechsel 1992). While the tandem 31 or 32 amino-ac id repeat region of plays a critical role in microtubule binding (Goedert 1993) the inclusion of sequences flanking the repeats (in particular, the proline-ri ch region upstream of the repeats, which includes a cluster of serine/threonine-prolin e phosphorylation sites) is essential for binding (Kanai 1992). The protein is very highly developmentally regulated. In the a dult brain, multiple isoforms are produced from a single gene through a lternative mRNA splicing (Goedert 1993). However, in the immature b rain only the transcript encoding the shortest isoform with three tandem ami no acid repeats is expressed (Goedert 1989). Additionally, phosphorylation of is developmentally regulated, thus, from the immature brain is phosphorylated at more sites than from the adult brain (at six to eight sites on the shortest isoform in the immature brain, compared to two to three sites on all six isoforms in the adult brain)(KsiezakReding 1992). Several specific phosphorylation sites have bee n identified through the use of mass spectroscopy, and the identified sites suggest that protein kinases with specificity for seryl-proline and threonyl-proline are responsible for the phosphorylation of in the normal brain (Goedert 1993). Several kinase s have been discovered to phosphorylate on some of these residues in vivo, such as mitogen-activated protein (MAP) kinase, which phosp horylates up to 12-14 residues in recombinant and glycogen synthase kinase-3, which phosphorylates up to four residues (Goedert 1993, H anger 1992). For a protein, the percentage that is phosphorylated depends on th e relative amount of protein PAGE 12 6 kinase and protein phosphatase activity, and phosphorylated by MAP is dephosphorylated only by protein phosphatase 2A (Go edert 1993). It has been well established that forms an integral part of the PHFs common to the lesion types seen in Alzheimers disease. Th e sequences obtained from pronase-treated PHFs extracted from tangle fragment s have shown that the three and four amino acid repeat region of contributes to the core of the PHF and three of the repeats offer protection from pron ase digestion (Jakes 1991). Although the same region of the molecule which is known to function as the microtubule binding domain has also been seen to ac t as the PHF-core binding domain, there is no evidence that the inner core of the PHF contains tubulin. In 1993, a study by Lee et al. involving the purificat ion of dispersed PHFs to homogeneity provided strong evidence that is the major component of PHF. These studies involving either dispersed PHFs o r PHFs extracted from tangle fragments have brought to light the natural history of PHF after assembly. In Alzheimers disease, the amino-terminus and part of the carboxy-terminus of are lost in the transition from intrato extrace llular tangles, just as the pronasetreated PHFs in the 1991 study by Jakes et al. indi cated that the amino-and the carboxyterminal regions are lost after digestion in vitro (while the -core association in the three-amino-acid repeat region o f offers pronase protection). Dispersed PHFs, in contrast, contain the whole of which indicates that dispersed PHFs constitute an earlier stage of PHFs than the bulk o f filaments isolated from tangle fragments (Goedert 1993). Furt hermore, the majority of the tangle fragments are ubiquitinated, while the dispe rsed PHFs are not. One study PAGE 13 7 by Morishima-Kawashima in 1993 identified several l ysine residues in the amino acid repeat region of that ubiquitin becomes attached to, which indicate s that these residues are on the surface of the PHF. Thus, the natural history of the PHF after assembly may be traced from an initial gu anidine-soluble intracellular form to an insoluble, proteolysed, ubiquitinated an d cross-linked extracellular form, with the possibility of a multitude of interm ediate steps. The abnormal phosphorylation of in the PHF was demonstrated by Goedert et al. in 1992. protein extracted from PHF runs as three bands of 60, 64 and 68 kDa, with a variable amount of background smear res ulting from proteolysed and crosslinked PHFs. These bands run more slowly on ge ls than normal or recombinant and contain the whole of (this is demonstrated by the fact that they stain with antibodies directed against the ami noand carboxytermini of ). After treatment with alkaline phosphatase (which de phosphorylated the proteins) at high temperatures, these three PHFbands become six bands which align with the six recombinant isoforms. This not only demonstrated the abnormal phophorylation state of in the PHF, but the inclusion of all six isoforms in the PHF. Phosphorylation negatively regulates the abilit y of to bind to microtubules; in fact, phosphorylated by MAP kinase has one-tenth the abi lity of nonphosphorylated to bind to microtubules (Drechsel 1992). This impa irment results entirely from abnormal phosphorylation, sin ce dephosphorylated PHFbinds as well to microtubules as normal The conversion of normal adult brain proteins into PHF via the abnormal phosphorylation of proteins, coupled with PAGE 14 8 reduced normal could lower levels of MT binding enough that MTs a re destabilized, disrupting axonal transport to and fr om the cell body of neurons (Drechsel 1992). This disruption could result in th e dysfunction and degeneration of neurons, as seen in Alzheimers. Additionally, t he sequestration of in PHFs aggregated into perikaryal NFTs or in dystrophic pr ocesses would cause the disruption of axonal transport by occupying space w ithin neurons, including their processes, and physically displacing or deforming o ther neuronal organelles (Trojanowski 1993). Amyloid -peptide and the Amyloid Precursor Protein A major component of these senile plaques i s a 38-43 amino acid-peptide known as the amyloid -peptide or A (Kang 1987).Increased A accumulation has been associated with all familial forms of Alzh eimers disease. A is also present in the brains of age-matched controls, but does not reach the same concentrations or result in the extensive depositio n and plaque formation seen in an AD brain (Evin 2003). In 1987 the cloning of the type-1 integral membrane protein amyloid precursor protein, or APP, establis hed that A is a proteolytic fragment derived from the protein (Kang 1987). APP is a member of a gene family of homologous integral membrane proteins, an d undergoes several posttranslational modifications. After synthesis, it be comes Nand Oglycosylated, sulfated, and phosphorylated (Weidemann 2001). Addi tionally, APP can be proteolytically processed by a set of proteases. AP P is integrated into the PAGE 15 9 membrane through a single transmembrane domain, wit h a large ectodomain which is shed from the membrane during proteolytic processing. Figure 1. Proteolytic processing of APP by the secr etases. A schematic depiction of the two alternative cellular pathways by which the large ectodomain of APP is shed, beginning with cleavage by either or secretase (Evin 1993). Several studies suggest that in the normally fu nctioning brain, APP may play an important role in the regulation of nerve proces s outgrowth and synaptogenesis. For instance, it was found that APP expression increases in the embryonic chick brain between days 6 and 9, which c orrelates with the period of maximal neurite outgrowth development (Small 1992). Additionally, the expression of APP in the central and peripheral ner vous systems is consistent with a role in the formation or maintenance of syna pses. Schubert et al. (1991) found that in cultured cells an abundance of APP co rrelates positively with increased surface activities such as membrane vesic ulation and the formation of cellular extensions during growth. APP expression i ncreases during development in vivo; Arai et al. (1994) showed the expression of full le ngth APP isoforms PAGE 16 10 extending from amino to carboxyl terminus in both w hite and gray matter in the early stages of spinal cord development, as well as diminished expression with maturation. As demonstrated by Fukuchi et al. (199 2), APP expression increases during the differentiation of murine embr yonal carcinoma cells in vitro. It also increases in hippocampal neurons and PC12 c ells grown in the presence of a nerve growth factor (Clarris 1994, Refolo 1989 ). A study by Clarris in 1995 on the expression of APP in the rat olfactory system strongly suggests a role played by APP in process o utgrowth and synaptogenesis. This study showed that APP is first expressed in olfactory neuroepithelial stem cells at embryonic day 16, soo n after they begin to differentiate into olfactory receptor neurons by ex tending neurites towards the olfactory bulb. As the neurites begin to arrive in the olfactory bulb, mitral cells within the bulb begin to express high levels of APP Around birth, high levels of APP can be detected in the olfactory glomeruli, whi ch are highly specialized regions of synaptic neuropil formed between primary olfactory axons and dendrites of olfactory bulb neurons. Glomeruli norm ally experience high levels of synaptogenesis because of the continual turnover of olfactory receptor neurons (Mackay-Sim 2006). APP expression in the glomeruli decreases after birth, correlating with the lowered levels of synaptogensi s as the process of receptor neuron turnover continues into adulthood. This obse rvation suggests that APP may have an important function in synaptogenesis. It is also likely that APP serves an important function in wound repair. This idea is supported by several studies, such as the 1 988 study by Salbaum, which PAGE 17 11 showed the presence of acute-phase elements (elemen ts which respond to inflammation) in the APP promoter. APP is expressed in high levels in cells associated with wound healing, such as platelets (B ush 1990), and fibroblasts, which was demonstrated in 1992 by Roch et al. who o bserved APP to stimulate the mitogenesis of a fibroblast cell line. Addition ally, Abe et al. (1991) found that APP expression increases following consistent focal ischemia, or neuronal damage, in a rat cerebral cortex, suggesting that A PP expression may be induced in the reactive astrocytes or microglia. Ba nati in 1995 found that APP expression increases in microglia following the ind uction of experimental allergic encephalomyelitis, or inflammation of the brain and spinal cord. Several studies have found APP to be a marker of early axonal injur y; lesions to the spinal cord produce an increase in APP expression both within a xons and in astrocytes which lie adjacent to the lesion (Chauvet 1997). Proteolytic processing of APP is achieved by tw o alternative pathways. Both pathways are considered to be by-products of normal cellular metabolism of APP. The most commonly occurring pathway in periphe ral cells is nonamyloidogenic and involves the protease termed -secretase (Evin 2003). Secretases have been shown to be members of the ADA M (a disintegrin and metalloprotease) family (Lammich et al., 1999). The cellular enzyme -secretase cleaves the protein between amino acids 16 and 17 o f the A sequence, that is, inside the A sequence (Sinha 1999). This endoproteolytic cleav age generates secreted APP (known as -sAPP) and a corresponding C-terminal fragment, CTF. By definition, this pathway could not produce the A peptide. PAGE 18 12 The alternative, amyloidogenic pathway for prot eolytic processing of APP is minor in peripheral cell lines, but predominant in neuronal cells (Evin 2003). This process involves cleavage of the APP ectodomain by the protease -secretase at the N-terminus of A, resulting in a C-terminal fragment of 99 peptides containing the A sequence (known as C99 or CTF), as well as secreted APP known as -sAPP. Several research groups have identified the cellular enzyme -secretase as the transmembrane aspartyl protease -APP cleaving enzyme, or BACE. Hussain et al. (1999) demonstrated that when the two active-site aspartic residues of BACE were mutated, the expression of th e mutant proteins did not result in an increase in -sAPP secretion or accumulation of CTF, as did the expression of the wild-type proteins. Sinha et al. (1999) purified the enzyme BACE after finding it to be the predominant source of cleavage activity in the brain, and showed that it has all of the properties predicted for -secretase, such as an acidic pH and high levels of activity in the cells and tissue of CNS origin. Subsequent processing of both and C-terminal fragments is carried out by the -secretase/presenilin proteolytic complex. The iden tity of -secretase remains unknown, but it has been well established t hat polytopic membrane protein homologs presenilin (PS) 1 and 2 play an es sential role in -cleavage (Wolfe 2001). Using a solubilized -secretase assay which faithfully reproduced the properties of the protease observed in cells (i ncluding the ratio of A-40 to A-42 production), Li et al. (2000) separated the sol ubilized material by sizeexclusion chromatography. After separation, -secretase activity coeluted with PAGE 19 13 two subunits of PS1. In the same study, immunopreci pitated PS1 heterodimers produced A from the cellular substrate. Direct evidence that presenilins are the cataly tic components of a large secretase complex also include affinity labeling st udies using transition-state analog inhibitors targeting the active site of the protease. Shearman et al. (2000) identified a peptidomimetic that blocks -secretase activity in the protease assay, and contains a transition-state mimicking functiona l group. Photoactivatable versions of this compound bound covalently to prese nilin subunits exclusively. The proteolytic processing of the and C-terminal fragments by the secretase/presinilin complex results in p3 and A peptides, respectively (Evin 2003). The biological significance of p3 remains ob scure (Nunan 2000). The secretase complex processes the C-terminal fragment at different sites; after Val40 to produce A (1-40) (the more common A species), and after Ala42 to produce A (1-42) (a less common but more aggregating species which constitutes the beginnings of senile plaque formati on) (Evin 2003). In 2001, Weidemann et al. found a novel cleavage site within the transmembrane domain (termed the -cleavage site) after Leu49 which releases CTF (also known as C50, or the APP intracellular domain, AICD) into th e cytosol. Like -cleavage, cleavage requires presenilins, and is sensitive to the same class of inhibitors as -cleavage. Simons et al. (1996) also described a cl eavage site at Thr584 called -cleavage, which is generated in neurons by an unid entified -secretase. PAGE 20 14 Neurotoxicity of A The neurotoxicity of A fibrils is supported by several studies. Exposure to A has been shown to be toxic to a variety of differen t cell lines in culture, including hippocampal and cortical cells (Frautschy 1991, Pik e 1991). An 11-amino acid residue sequence within the A peptide, A 25-35, is homologous to the conserved region in peptides of the tachykinin fami ly, and is reportedly the toxic region of the peptide (Yankner 1990). This region i s able to form fibrils similar to those formed by intact A (Pike 1995). Several suggestions have been made about the cause of neurotoxicity in this region; fo r instance, Joslin et al. (1991) found that A, along with several tachykinins, compete for bindi ng to serpin (SEC) receptors. Thus, A may sequester SEC receptors, allowing proteolytic damage to occur to the cell. It has also been sugge sted that the neurotoxic effects of A may be mediated through binding to a receptor for advanced glycation endproducts (RAGE receptor), which would effectively tether the peptide to the cell membrane where it could exert i ts oxidant effects in close proximity to cellular structures, evading antioxida nt mechanisms common to extracellular space (Shi Du et al., 1996, Shelat et al. 2008). Another suggestion about the neurotoxic effects of A is that they are mediated by a disruption of calcium homeostasis, re sulting in increased neuronal intracellular calcium levels. Increased levels of A would allow accumulation and aggregation of the peptide, and neurons exposed to A would be more exposed to excitotoxicity. Additionally, decreased levels o f -sAPP would deprive neurons of a neuroprotective substance that can stabilize c alcium levels (Mattson 1993). PAGE 21 15 The reason for this disruption of homeostasis on in tracellular calcium by A may be due to the opening of L-type voltage-gated calci um channels. Ueda et al. (1997) suggested that A 25-35 enhanced ionic permeation through L-type channels, or increased the number of these channels in cultured neurons. A also causes a reduction in Na+/K+-ATPase activity, which may cause a Na+ influx that could lead to membrane depolarization a nd Ca2+ influx through voltage-dependent channels (Mark 1995). Additionall y, it was found that cytochalasin D, a compound which depolymerises acti n microfilaments and inhibits calcium entry, selectively protects hippoc ampal neurons from A neurotoxicity (Furukawa 1995). It has also been proposed that the liberation o f oxygen species plays a role in the neurotoxicity of A. The disturbance in redox activity may disrupt cal cium homeostasis, as reactive oxygen species can impair ATPase activities (Shi et al. 2010, Mark 1995). A can induce lipid peroxidation in cultured neurons, and can increase the activity of hydrogen peroxide, which c an increase the cells susceptibility to oxidative stress (Behl 1994, Caf 1996). The idea that the neurotoxic effects of A are due to a disturbance in redox potential by rea ctive oxygen species is supported by the observation that the neurotoxic effects of A are reduced by treatment with free radical scavenge rs (Bruce 1996). The presence of activated microglia within amyl oid plaque cores has led to proposals that the effect of A on glial cells may result in an inflammatory reaction which could influence the pathogenesis of AD, since microglia and astrocytes have been identified as brain-derived so urces of growth factors and PAGE 22 16 inflammatory mediators (Casoli et al. 2010). The co nsistent finding that antiinflammatory drugs have a beneficial effect on the progression of the disease suggests the importance of the inflammatory compone nt of the disease mechanism (Pasinetti 1996). Glial cells have been f ound to be closely associated with neuritic plaques, suggesting the degree to whi ch they are involved with the disease process. A was found to modulate the secretion of cytokine, a n inflammatory mediator, from human astrocytoma cells in a conformationdependent manner (Gitter 1995). It may also influen ce microglial function as it has been found to bind to and be internalized by sc avenger receptors on macrophages. The uptake of A by a scavenger receptor may play a role in the clearance of A (Parasce 1996). Amyloid is not deposited in all regions of the brain. The formation of amyloid plaques and neurofibrillary tangles occurs in a spe cific spatio-temporal pattern which precedes any cognitive decline. Regions of th e brain such as the cortex and the hippocampus have been found to exhibit many more of the pathologic features of AD, like plaques or tangles, than regio ns such as the cerebellum (Games 1995). This indicates that the overexpressio n of A (1-42), which presumably occurs in all cells which express APP (a nd, in turn, in both the regions of the brain which do and do not exhibit ma ny of the pathologic features of AD), is not sufficient to induce amyloid plaque formation or neurodegeneration. Therefore, other susceptibility factors must be inv olved. While the overproduction of the amyloidogenic A (1-42) species may be an early event in the diseas e PAGE 23 17 pathogenesis, it is the cellular response to that o verproduction which drives amyloid deposition and determines whether neurodege neration occurs. Small et al. (1998) proposes an explanation for the specific spatio-temporal pattern of amyloid deposition. The model by Small e t al. assumes that the production of amyloid fibrils is the underlying mec hanism that triggers neurodegeneration, and that the expression of APP i s upregulated following nerve damage, which might be expected to occur foll owing amyloid fibril formation. According to the model, the presence of amyloid fibrils in the neuropil sets off a series of inflammatory reactions, neurit ic sprouting and other tissue repair responses, which then leads to the upregulat ion of APP expression, following by increased A production. The cycle begins again, as the increas ed A production leads to further amyloid fibril formati on. The initial increase in the production of amyloidogenic forms of A may be caused by an environmental factor, head injury, or a mutation in the presenili n or APP genes (Bush 1994, Nicoll 1995). This model provides an explanation for the loca lization of the AD neuropathology to regions in the brain such as the cortex and the hippocampus. Amyloid deposition, according to the model, will co ntinue only if a positive feedback loop develops. This positive feedback loop requires an upregulation of APP expression, which may be caused by neuritic spr outing. In the adult brain, mechanisms exist that regulate neurite outgrowth du ring development. As the adult brain develops and as myelination occurs, the expression of neurite outgrowth inhibitors such as oligodendrocyte-synthe sized, myelin-associated PAGE 24 18 glycoprotein and chondroitin sulfate proteoglycans increases (Li 1996, Small 1996). At the same time, levels of neurite outgrowt h-promoting substances such as laminin and heparin sulfate proteoglycans decrea se. Neuritic outgrowth may also be suppressed by electric activity as neurotra nsmission begins and neurons start to fire after making synaptic contact; Cohan et al. (1987) found that electrical stimulation to the neuronal cell body ca used a rise in the calcium levels of the cells growth cones. Calcium levels have bee n suggested to have a regulatory role in the motility of many different c ell types, and this rise in calcium levels may suppress neuritic outgrowth. However, neurite outgrowth does continue in som e regions of the brain. Synapses remodel in the cortex, where such growth l ikely plays a role in the formation of long term memory. Bailey (1982) found that even learning experiences such as habituation and sensitization c an modify the functional expression of neural connections in Aplysia The fact that synaptic remodeling is required for the formation of long term memory coul d be the reason why disorders of memory occur early in AD. Synaptic gro wth and remodeling also occurs in the olfactory system and hippocampus, whe re neurons can remodel synaptic connections with cells in the dentate gyru s (Moulton 1974, Masliah, 1991). These locations of continued neurite outgrow th coincide with the regions of the brain that have been found to exhibit many o f the pathologic features of AD. For instance, olfactory nerve degeneration is a n early event in AD (Esiri 1984). PAGE 25 19 Also in support of this model is that fact that neurofibrillary changes inversely recapitulate development, that is, the regions whic h are the last to be myelinated are the first to degenerate in AD (Braak 1996). Thi s is consistent with the hypothesis that myelin proteins inhibit neuritic gr owth (via the synthesis of myelin-associated glycoprotein as discussed earlier ) and thereby suppress APP expression. From these observations, it can be pred icted that neurons undergoing synaptic remodeling degenerate early in the disease. Olfactory nerves provide an example for this hypothesis: Esir i et al. (1984) found significant tangle formation and cell loss in the anterior olfa ctory nuclei, and Clarris et al. (1995) showed a correlation between APP expression in the mitral cells and the invasion of olfactory axons into the bulb. Their ea rly degeneration in AD may be linked to the fact that olfactory receptor neurons turn over often, and make new synaptic connections with the cells of the olfactor y bulb well into adulthood. Proteins known to have neurite outgrowth-promoting effects such as laminin, heparin sulfate proteoglycan, fibronectin, apolipro tein E and APP have all been observed in association with A deposits (Small 1998). The genetics of Alzheimers disease Clinical and pathological evidence for heteroge neity in Alzheimers disease has been documented for some time. Alzheimers dise ase has been linked to several factors, including trisomy 21, the mutation of the presenilin genes on chromosome 14, the APO E4 allele, as well as a numb er of different early-onset familial Alzheimers disease mutations in the -amyloid peptide or the amyloid PAGE 26 20 precursor protein (Pericak-Vance 1991, Ropper 1980, Schellenberg 1992). It is also clear that the disease develops in patients in which the aforementioned genetic factors do not play a role. Thus, AD is a h eterogeneous disorder in which pathology can be initiated or accelerated by a numb er of genetic or environmental factors. All factors linked to the di sease have one common element, and that is their ability to enhance patho logy that is provoked by the rate-limiting elements (such as amyloid deposition) of the pathological changes that produce AD (namely, the senile plaques, the ne urofibrillary tangles, and the associated neuron loss). The most compelling evidence for the role of AP P and A in the pathogenesis of AD comes from genetic studies on the familial fo rms of the disease. Several point mutations have been identified on the APP gen e, all clustering around the region that encodes the A sequence. The mutations around the A sequence include mutations at codon 717 which result in subs titutions of a valine (located three residues toward the carboxyl direction from t he A sequence) residue for isoleucine, phenylalanine, or glycine (Naruse 1991, Murrell 1991, Chartier-Harlin 1991). Another mutation of the A sequence, the Swedish double mutation, occurs at codons 670 and 671 and involves the subst itution of two amino acid residues, an asparagine and a leucine, for a methio nine and aspartate at the Nterminus of the A sequence (Tanzi 1991). Both of these early-onset, familial Alzheimers disease (FAD) mutations in the APP gene have been found to cause an increase in th e production of A (1-42). Of the two species of A, A (1-40) is normally the more commonly produced PAGE 27 21 species, while A (1-42) is the more amyloidogenic.The mutations at codon 717 cause an increase in the ratio of A (1-42):A (1-40) (Suzuki 1994), whereas the Swedish mutation causes an increase in the producti on of both A (1-42) and A (1-40); in fact, it increases the secretion of 4-kD A six fold in cultured cells (Cai, 1993). The effect of both mutations, however, is to increase the production of the more amyloidogenic form of A. These findings strongly favor the hypothesis that amyloid deposition is a critical element in the dev elopment of AD. The APP gene defects account for only a small p ortion (5-7%) of all FAD cases (Small 1998). Two other FAD genes called pres enilins are known to be involved in the familial form of the disease. Prese nilin 1 (PS1) and Presenilin 2 are both located on chromosome 14, and encode homol ogous proteins of approximately 50 kDA (Cook 1996). PS1 and PS2 mRNAs are expressed primarily in neurons. PS1 and PS2 contain between s ix and nine transmembrane domains and are localized in association with the e ndoplasmic reticulum, dendrites, and Golgi membranes, where they undergo proteolysis (Cook 1996). APP has been shown to first be delivered to axons a nd subsequently to dendrites by a transcytosis process (Simons 1996), which sugg ests a possible role for the presenilins in amyloid protein delivery. The idea of presenilins playing a role in amylo id protein delivery is supported homologies between the presenilins and proteins wit h known biological roles. Though their exact functions remain unclear, the ho mologies between the presenilins and the Caenorhabditis elegans proteins sel-12 (about 50 percent amino acid identity) and spe-4 (about 25 percent am ino acid identity) have led to PAGE 28 22 the suggestion that the presenilins have an importa nt role in cellular trafficking (Levitan 1995, LHernault 1992). Sel-12, for exampl e, plays a role in facilitating the localization and recycling of lin-12, a member of the Notch family of receptors (Levitan 1995). Given these homologies along with t he localization of both PS1 and APP to neuronal dendrites, it is possible that PS1 plays a role in delivering APP to or from the dendritic processes. Another pos sibility is that presenilin missense mutations may alter membrane protein traff icking in a way that enhances exposure of APP to the secretase that cleaves at A (1-42), thereby increasing A (1-42) generation; presenilin is required for the secretase cleavage of APP (Esler 2001). More than 35 FAD mutations have been reported i n the presenilins (Small 1998). The effect of the presenilin mutations is si milar to the effect of mutations in the codon 717 mutations; that is, the presenilin mu tations also cause an increase in the proportion of A (1-42) to A (1-40) production (Scheuner 1996). Because A is deposited early and selectively in the senile p laques that are an invariant feature of all forms of AD, this effect is likely t o be directly related to the pathogenesis of AD. Additionally, this provides str ong support that overproduction of the amyloidogenic A (1-42) in particular is a key event in the pathogenesis of AD. This makes reduction of A concentration and prevention of A deposition attractive as a therapeutic target for treatment of Alzheimers disease. The pathology of late-onset Alzheimers disease can be linked to genetic factors just as early-onset AD can. The e4 allele o f the apoliprotein E (apoE) PAGE 29 23 gene on chromosome 19 is a major susceptibility fac tor for late-onset familial and sporadic AD. ApoE is a 299-amino acid glycoprotein, and one of almost a dozen constituents of plasma lipoproteins that serve vari ous functions, including the maintenance of the structure of the lipoprotein par ticles and the regulation of the metabolism of several different lipoproteins (Mahle y 1988). It is a constituent of liver-synthesized, very low density lipoproteins, w hich function in the transport of triglyceride from the liver to peripheral tissues, as well as a subclass of high density lipoproteins, which function in cholesterol redistribution among cells. Its major function is thus the transport of lipids amon g various cells of the body from the site of synthesis or absorption to the sites of utilization or excretion. Though the largest quantity of Apoliprotein E m RNA may be found in the liver, the second largest quantity is found in the brain ( approximately one third the level seen in the liver) (Mahley 1988). ApoE exists predo minantly as a lipoprotein in the brain. It is produced and secreted in the CNS b y astrocytes and microglial cells, and its synthesis in the nervous system is i ncreased following injury and is implicated in the growth and repair of the nervous system during development or after injury (Strittmatter 1993). The role of ApoE in neuronal injury may involve not just facilitation of cholesterol transport, but also interactions that result in the targeting or the sequestering of proteins. ApoE is also increased in several chronic neuro degenerative disorders, including Cruetzfeld-Jakob disease in which it is b ound to the prion protein containing amyloid plaque, and AD in which it is fo und in vascular endothelial cells, neurons and neuritic processes (Schmechel 19 96). In AD, ApoE is seen PAGE 30 24 bound to extracellular senile plaques, to intracell ular neurofibrillary tangles, and at the sites of cerebral vessel congophilic angiopa thy (Strittmatter 1993); in fact, is it increased sevenfold in the brains of patients with Alzheimers disease (Namba 1991). In 1993, Strittmatter demonstrated high-avidity binding of ApoE in CSF to the A peptide. A function in ApoE involving the binding, transport, or targeting of the A peptide may link diagnostic lesions and genetic su sceptibility. The 3 common isoforms of apoE are the genetically heterogeneous apoE 2, 3, and 4. These isoforms are encoded by alleles e2, e3 and e4, resp ectively (Mahley 1988). The e4 allele frequency is greatly increased in individ uals with AD (Strittmatter 1993). Additionally, individuals with more copies of the e 4 allele have larger amounts of A immunoreactivity in their brains (Schmechel 1993). This implies that there may be allele-specific functions that contribute to the molecular mechanism of the disease expression, or that there exists anothe r intragenic polymorphism on an ApoE4 background that confers susceptibility to the disease. It also explains much of the variability in the extent of amyloid de position noted in late-onset AD patients (Reiman 2009). Though these relationships are known to exist, the exact link between the apoE4 isoform and A is not known for certain. Because apoE4 is more li kely to readily promote amyloid fibril formation than the o ther apoE isoforms (Schmechel 1993), one hypothesis to explain the link between a poE isoforms and AD is that apoE4 promotes A aggregation. However, studies have also found that apoE4 binds less to A than both the ApoE 2 and 3 isoforms, and have demo nstrated a PAGE 31 25 20-fold difference between the level of ApoE3/A complex and Apoe4/A complex (Yang 1997). These results seem to be in direct contradictio n to the findings that the density of A deposition is directly related to the inheritance of an e4 allele (Schmechel 1994). However, this discrepancy may be explained b y ApoE isoforms like ApoE3 efficiently binding to A and clearing it from extracellular space, while th e apoE4 isoform is less capable of clearing A from the neuropil than the other isoforms, leading to the accumulation and depositio n of A in the extracellular space. The varied preparations of ApoE and its lipi d composition used in these various studies complicate the interpretation of th eir results because the amount and type of lipid bound to apoE may influence the w ay the protein binds to A. ApoE exists primarily in the brain as a lipoprotein with a protein bound to it; however, there have been no studies on A aggregation using apoE-enriched lipoprotein particles purified from the brain. Another reason the role of apoE isoforms in the promotion of AD pathology is not clear comes from the results on the presenilin mutations. While the presence of e4 has been found to increase the risk of late-o nset AD and FAD caused by mutations in the APP gene, it does not increase the risk of FAD caused by mutations within the presenilin genes (Sherrington 1996). Even though the PS1 and APP codon 717 mutations both increase the ratio of A(1-42) to A(1-40) species produced, the presence of the e4 allele onl y increases the risk for individuals with mutations in the APP gene. The rea son for the dependence of the increase of risk on the location of the mutatio n remains unknown. It is PAGE 32 26 possible that the functional effects of the PS1 and PS2 mutations are either remote from the functional effect of the Apoe4 alle le, or affect a biochemical pathway leading to AD which is different from that influenced by Apoe4. Thus, the exact mechanism by which apoE4 increases the ri sk for AD remains unknown. NFB and A NFB is a family of transcription factors with the uni que property of being sequestered in the cytoplasm in association with in hibitory proteins called IB. The activation and regulation of NFB is largely controlled by the IB proteins. IB proteins are able to mask the nuclear localizatio n signals of NFB through noncovalent association, preventing NFB nuclear translocation. Using X-ray crystallographic analyses of bacterially expressed p50 homodimer bound to its cognate DNA binding sites, Muller et al. (1995) fou nd that the IB binding site on NFB is separated from the DNA and dimer interface, bu t because IB is large enough it could extend from the groove between the dimerization domains to the region of DNA contact and interfere with binding di rectly. When stimulated with signaling molecules such as tumor necrosis factor (TNF ) or lipopolysaccaride (LPS), NFB is released from IB and translocated to the nucleus where it regulates the transcription of many genes, includin g cytokines, immunoreceptors, IL6, adhesion molecules, COX-2 and iNOS, and neurop eptides (Verma et al. 1995). Stimulation and activation of NFB transcription factors does not require protein synthesis, thus allowing for rapid and effi cient activation of target genes PAGE 33 27 (Collins et al. 1995). This system of rapid activat ion is particularly useful in immune, inflammatory, and acute phase responses whe re the rapid activation of defense genes is critical for the survival of the o rganism following exposure to pathogens such as bacteria and viruses. NFB is also implicated in cellular proliferation and programmed cell death, and the de regulation of NFB activity is directly associated with cellular transformation (V erma et al. 1995). NFB was originally discovered as a lymphoid-specific protein that bound to a decameric oligonucleotide present in the intronic e nhancer element of the immunoglobulin light chain (Ig) gene (Sen 1986). Members of the NFB family of transcription factors include p50 (NFB1), p65 (RelA), NFB2 (p52), and RelB (Verma 1995). All members of the Rel/NFB family of proteins share an aminoterminal ~300-amino-acid domain called the Rel homo logy domain (RHD). The RHD includes DNA binding and dimerization domains a nd the nuclear translocation signal (NLS), which is most likely th e binding site for IB (Miyamoto 1995). Most members of the Rel/NFB family of proteins can form homoand heterodimers in vitro with other Rel/NFB members (Verma 1995). The DNA binding and dimerization domains in the RHD are distinct from other motifs, and the structural assignment for these fun ctional domains was accomplished by Ghoush et al. (1995) using x-ray cr ystallography. Ghoush et al. found that the Rel homology region folds into two d istinct domains, similar to those in the immunoglobulin superfamily. Members of the immunoglobulin superfamily play important roles in vertebrate immu ne and neural systems by mediating specific interactions with cell surface m olecules. PAGE 34 28 The DNA base recognition in the RHD is mediated by peptide loops entering major grooves of the target DNA (Ghoush et al. 1995 ). Many backbone phosphate groups are also involved in stabilization of this protein-DNA complex of an unusually high affinity, with a dissociation constant of ~10^-12 M (Baeuerle 1991). The base pairs and phosphate groups of the DNA B site are recognized by conserved residues throughout the RHD of p50. P5 0 dimerization is exclusively mediated by a ~100-amino-acid domain in the carboxyl terminus of the RHD and is stabilized by a series of -sheets involving core hydrophobic and peripheral hydrophilic residues. Because the majori ty of amino acid residues involved in DNA and dimer contacts are conserved in the Rel/NFB family of proteins (Verma et al. 1995), the specific DNA and dimer partner preferences must be determined by slight amino acid differences in the DNA-binding and dimerization domains. These critical residues for s pecific DNA binding lie in the amino-terminal DNA recognition loop (Muller 1995) Subtle differences in the dimerization domain also play a critical role in de termining dimer partner specificity (Ganchi 1993). An interesting aspect of the NFB/IB system is the number and types of agents that can induce NFB activity in various types of cells. There is no obvious common secondary messenger that can be iden tified from the list of known inducers (Verma et al. 1995), which implies t hat NFB/IB directly responds to many independent signaling molecules. N FB behaves exactly like an oxidative stress response factor: all of the kno wn NFB activating signals can be inhibited by antioxidants. Hydrogen peroxide, a major reactive oxygen PAGE 35 29 intermediate produced by the cell, activates NFB. The activation of NFB by PMA is also enhanced by hydrogen peroxide (Baeuerle 1994). Another feature of NFB regulation is the direct transcriptional activati on of the inhibitor gene IB by NFB, creating an autoregulatory loop. After the p50-p 65 complex is released from the complex with IB, it traverses the nucleus and binds to cognate B binding sites. The promoter of IB contains B binding sites and thus is a target of the NFB complex (Chiao 1994). The newly synthesized IB protein will immediately bind up free cytoplasmic NFB and inactivate its potential nuclear translocation. When the amount of IB exceeds the capacity of NFB substrates to anchor it in the cytoplasm, the exc ess IB migrates into the nucleus to remove active NFB from DNA and terminates its activity (ArenzanaSeisdedos 1995). The nuclear NFB/ IB complex may be transported back to the cytoplasm or degraded in the nucleus. Circumven tion of this continuous negative regulation is possible only when continuou s stimulation is provided, resulting in sustained NFB activation. IB phosphorylation is an integral and obligatory ste p in NFB activation. In vitro studies using partially purified IB showed that direct phosphorylation of IB by various kinases, including PK-C, PK-A, and hemeregulated eIF-2, resulted in the inactivation of IB activity (Ghosh 1990). When the substrate from these kinases was the NFB/IB complex, phosphorylation of IB resulted in the release of NFB and the appearance of NFB-DNA binding activity. Through the use of antibodies and phosphatase inhibitors, phosp horylation of IB is observed in many cell types following stimulation ( Verma 1995). This PAGE 36 30 phosphorylation event precedes NFB activation. IB undergoes complete degradation following stimulation but prior to NFB activation (Chiao 1994). The process of IB degradation is efficient and extensive, lasting ~1 0 minutes and producing no obvious intermediates (Verma et al. 19 95). The phosphorylation of IB probably occurs at Ser-32 and Ser-36 (Brown 1995). It is followed by multiubiquination at residues Lys-21 and Lys-22, de gradation of IB by 26S proteasome, and, finally, liberation of free NFB to translocate into the nucleus. NFB is widely expressed in the nervous system and in synaptic terminals. Increased nuclear translocation of NFB has been observed close to synaptic sites in the hippocampus and cerebellar granule neu rons as a result of activity (Guerrini 1995). These observations suggest the pre sence of a synapse-tonucleus signaling system in which transcriptional f actors activated by local synaptic signals transmit this signal to the nucleu s after retrograde transport. NFB has been detected in astrocyte and glial cell lin es, and it is also thought to play a role in immune and inflammatory response (O Neill 1997). It has been shown in a number of model systems that NFB levels increase as a consequence of brain injury. It is possible that in jury leads to an increase in production of signaling molecules such as IL-1 or T NF, which then activates NFB and leads to the induction of the expression of a range of pro-inflammatory genes. Evidence is accumulating for a role for NFB in neurodegenerative disease. A was found to activate NFB in neuroblastoma cells and in cerebellar granule cells (Kaltschmidt 1997). Studies of postmortem bra in tissue in patients with AD PAGE 37 31 have shown increased NFB immunoreactivity in neurons and astrocytes surrounding -amyloid plaques (Ferrer 1998). This activation of NFB by A is dependent on the formation of reactive oxygen inter mediates like hydrogen peroxide. A similar mechanism has been shown to be operative in nanomolecular concentrations in the activation of NFB via glycosylated proteins; as a consequence of glycosylation, PHF-generates free oxygen radicals, activating transcription via NFB (Yan 1995). Some of the mechanisms of this activation have been clarified; for instance, in 1996, Yan et al. showed that the RAGE receptor, whi ch has been found to signal the activation of NFB, is also a receptor for A. They found that expression of RAGE in COS (CV-1, or simian) cells resulted in cyt otoxicity with nanomolar amounts of A. In this system Kaltschmidt (1997) showed that NFB was activated in regions around early plaque stages in AD patients. Many genes newly induced in AD are under immediate-early trans criptional control of NFB; the gene for APP has been identified as an NFB target, explaining the upregulation of this gene by IL-1 (Grilli 1995). This system generates a positive feedback loop, because IL-1 is able to act as a sig naling molecule for the stimulation of the release of NFB from IB (Kaltschmidt 1997). In 2004, Sung et al. showed that the non-steroi dal anti-inflammatory drug indomethacin leads to a reduction in the level of A peptides and NFB in the brains of a transgenic mouse model of AD, whereas a nother anti-inflammatory compound, nimesulide, had no effect of the levels o f either A peptides or NFB. This suggests that an NFB activation blockade could reduce amyloid patholog y PAGE 38 32 in the transgenic mouse model of AD. In 2007, Paris et al. showed that the inhibition of I kappa B kinase (IKK-2) significantl y inhibits both A (1-40) and A (1-42) production. Inhibition of IKK-2 indirectly b locks NFB activation, suggesting that secretion and/or production of A (1-40) and A (1-42) is NFB dependent. Celastrol Inhibition of NFB has been shown to lead to a reduction in amyloid pathology, and thus compounds which effectively inhibit the tr anscription factor may prove useful in reducing Alzheimers disease pathology. O ne such compound has been found in the family Celastraceae. Members of the fa mily Celastraceae produce various sesquiterpene polyol esters and alkaloids. Root extracts from the Celastraceae family, or Thunder of God Vine, have been used as an antiinflammatory remedy for centuries in China. Some ha ve been shown to exhibit effects similar to insect antifeedants (substances which reduce consumption by insects), while others exhibit antitumor activity a nd reversal of multidrug resistance (Tu, 1990, Tu (2) 1990, Kim 1999). The C elastraceae family produces diand triterpene compounds such as celaphanol and Celastrol. Celastrol has been shown to have antitumor acti vity, antitumor promoting activity, and inhibitory effects on IL1 release in lipopolysaccharide (LPS) stimulated human peripheral mononuclear cells (Ngas saoa 1994). In 2002, Jin et al. showed that the methanol extract of the roots o f Celastrus orbiculatus (Celastraceae) showed potent inhibition of NFB in cells transfected with NFB PAGE 39 33 reporter construct. Of the eight compounds isolated from the roots of Celastrus orbiculatus that Jin et al. tested, Celastrol showed the most potent inhibitory activity in the reporter gene expression, with an I C50 value of 0.27 M. Figure 2. The chemical structure of Celastrol. The chemical structure of Celastrol, a compound isolated from the roots of Celastrus orbic ulatus found to inhibit NF B (Paris et al. 2010). Cleren et al. (2005) showed that Celastrol was able to cross the blood brain barrier, and that it exerted a strong neuroprotecti ve effect in a model of neurodegeneration, almost completely preventing 3-N P-induced striatal lesions and strongly reducing 3-NP-induced astrogliosis. Cl eren et al. also showed that the compound can decrease brain NFB immunostaining. In 2007, Paris et al. investigated the effects of various NFB inhibitors on the production of A peptides using Chinese Hamster ovary cells transfec ted with wildtype APP 751 (7W CHO), overproducing human A. Among the NFB inhibitors tested, PAGE 40 34 Celastrol appeared to be the most potent, with more than 90% inhibition of A (140) and A (1-42) using 500nM in 7W CHO cells. It has been suggested that A itself can trigger BACE-1 expression in glial cells via NFB activation. BACE-1 is the rate limiting enzyme re sponsible for the production of A, and NFB has been shown to regulate its expression level (Bourne 2007). Glia and astrocytes in particular ma y produce significant amounts of BACE-1 and A, especially during inflammation. Because glia outn umber neurons by a factor of 10, a slight increase in gli al BACE-1 expression might contribute substantially to cerebral A levels, promoting AD pathology. Exploring the effect of NFB inhibition on A levels and BACE-1 expression may show that NFB inhibition reduces A production by targeting the -cleavage of APP, which could lead to a potential therapy for AD patients. Thus, Paris et al. (2010) studied whether NFB inhibition could lead to an inhibition of A accumulation and reduced BACE-1 levels both in vit ro and in vivo. They showed that Celastrol reduced the produc tion of APP-CTF and sAPP in 7W CHO cells in vitro, confirming that Cela strol impacted the -cleavage of APP, and that the compound appeared to reduce BA CE-1 expression and oppose BACE-1 up regulation induced by NFB stimulation. Chronic treatment with Celastrol in Tg PS1/APPsw mice overexpressing levels of human A reduced brain levels of soluble A (1-38), A (1-40), and A (1-42) by approximately 50%, and the levels of the insoluble forms of these peptides by 60%. The evaluation of activated microglia revealed that microgliosis was significantly reduced in Celastrol-treated animals (Paris et al. 2010). PAGE 41 35 This thesis details the progression and results of two studies in which Celastrol was administered to transgenic mice exhibiting huma n Alzheimers disease symptoms and the effects on behavior and performanc e were observed; a long term study in which the drug was administered enter ally through the powdered food consumed by the mice, and an acute study in wh ich the drug was administered parenterally through intraperitoneal i njection. To date, there have been no behavioral studies performed on the transge nic mouse model of Alzheimers disease treated with the NFB inhibiting compound Celastrol, and so these studies incorporated the Morris Water Maze, Y -maze, and Rota Rod performance tests. In order to evaluate the effects of the drug on brain levels of A (1-40) and A (1-42), ELISAs were used to quantify brain A after the behavioral and performance tests. The results of th ese studies begin to highlight the effectiveness of Celastrol as a long term treat ment rather than a short term one, and could serve as an initial step in establis hing the drugs effectiveness as a prophylactic treatment rather than a therapeutic one. PAGE 42 36 Materials and Methods Animals All the experiments involving mice were approve d by the Institutional Animal Care and Use Committee of the Roskamp Institute. Be tween the acute treatment and powdered food experiments used to assess the ef fect of Celastrol on behavior and brain A levels, the following transgenic (Tg) mice were us ed: Tg mice overexpressing the human APP695sw (K670N, M671 L) (line Tg 2576) mutation and the human presenilin-1 (M146L) (line 6 .2) mutation (Tg PS1/APPsw), Tg mice overexpressing the human APP695 sw mutation alone (Tg APPsw), and Tg mice overexpressing the presenilin-1 mutation alone (Tg PS1). The presenilin-1 mutation increases cleaving of APP CTF secretase, resulting in an increased production of A peptides from the APP CTF substrate; specifically, an increase in the ratio o f A (1-42) to A (1-40) production (Scheuner 1996). This increase in the A (1-42) to A (1-40) ratio favors the aggregation of A peptides. ELISA data have shown that mutant PS1 mice have an approximate twofold elevation in endog enous A (1-42) levels, but not A (1-40) (Duff et al. 1996). In both the acute treat ment and powdered food experiments, the Tg PS1 mice are used as controls, in combination with the wild type (WT) mice. Without the APP695sw mutation, the mice do not produce an APP molecule more suitable for cleavage by secretase (BACE-1), and thus the mice Tg PS1 alone produce no substrate for secretase, which is the molecule affected by the PS1 mutation. Mice with the PS1 mut ation alone therefore do not PAGE 43 37 experience an increase in the A (1-42) to A (1-40) ratio, and are used as controls, along with WT mice. The APP695sw, renders the APP molecule more sui table for cleavage by secretase (BACE-1). Thus, this mutation results in the production of more substrate for secretase to cleave, leading to an increase in the production of both A (1-42) and A (1-40) (Cai, 1993). This mutation is known as the Swedish mutation, and was the first identified mutation whi ch causes Alzheimers disease (Mullan 1992). The doubly transgenic PS1/APPsw mice experience d an overproduction of human APP and A, and also experience an elevation in endogenous A (1-42) levels, but not A (1-40). These Tg mice may be generated by the cros sing of hemizygous transgenic mice that express the APP695s w (K670N, M671L) (line Tg 2576) mutation and of hemizygous lines of PS1 mi ce that express human mutant PS1 (M146L) (line 6.2). Tg PS1/APPsw mice ty pically start to develop A deposits by 4 months of age and display a significa nt amount of A by 6 months. Morris Water Maze performance test Spatial learning and memory were evaluated in t he Celastrol-treated and control mice by using the Morris Water Maze (MWM) p erformance test (Morris 1981). The MWM test is a memory test based on the c apacity of animals to rescue themselves from a pool of moderately cool wa ter by reaching a hidden goal platform. The MWM test was performed in a circ ular water-maze tank (diameter, 2 m and height, 40.4 cm) filled with wat er (temperature, 17 degrees PAGE 44 38 Celsius) that had been opacified by adding powdered milk until the water level was 33.3 cm high. A transparent Plexiglass platform (diameter, 20 cm and height 30.3) was submerged 3 cm below the water surface an d placed in the NE quadrant. It remained in this position across the a cquisition trials. The maze was located in the center of a dimly lit room (the brig htness of the lighting was kept constant during the experiment) with pictorial and symbolic visuals cues on each of the four walls (30 cm from the edge of the pool) corresponding with the four cardinal directions (Figure 3). The swimming path of each mouse was monitored b y an overhead video camera connected to a personal computer and was ana lyzed by the automated tracking system Ethovision XT 5.0.216 (Noldus Infor mation Technology, Sterling, VA, USA). During the 9 day training (acquisition) p eriod, the animals were required to locate the hidden platform, which remai ned in the same quadrant, in relation to external visual cues. The training was carried out in 1 block of 4 trials per day. To begin each trial, the mice were placed in th e water by the maze wall in one of the four cardinal directions. The daily order of entry into the maze from each direction for the mice was as follows: South, West, North, East. Each trial ended once the animal found the platform; if the mouse wa s unable to find the platform within 90 seconds, they were guided towards it by a n experimenter. If the animal found the platform within 90 seconds, it was requir ed to stay on the platform for a period of 3 seconds in order to stop the timer and complete the trial. After an additional period of 15 seconds on the platform, th e mice were dried and PAGE 45 39 returned to their respective cages. The individual mice were not immediately replaced at a new directional starting position after each trial; instead, all mice were tested from one direction in order of ID numbers be fore being tested from another direction. In each trial, the time taken for the mouse to reach the platform (escape latency in seconds, which could not exceed 90 secon ds), the length of the swim path (distance in centimeters) and the swimming spe ed (centimeters/second) was measured. On day 10 of the performance test, af ter the 9-day acquisition period, probe trials were conducted in the absence of the platform (all other environmental factors were kept constant). The mice were released from the SW quadrant, opposite where the platform was located d uring the acquisition period, and were allowed to swim for 60 seconds in absence of the platform. The percentage of time the mice spent in the NE quadran t was measured, as well as the length of the swim path (distance in centimeter s), the swimming speed (centimeters/second), and the latency to find the N E quadrant. PAGE 46 40 Figure 3. Morris Water Maze Diagram Spatial learning and memory are tested as a mouse locates the hidden platform in order to exit the maze. (Medical College of Georgia, 2006) Y-Maze Short-term memory and the ability of the mice t o discern a novel situation were evaluated in the Celastrol-treated and control mice by using the Y-maze. The Ymaze is a maze with three connected branches labele d A, B and C in the shape of a Y (Figure 2) and no external openings. The wal ls of the Y-maze branches were black, made of plastic and 0.5 cm thick. Each branch measured 30.5 cm long, 13.2 cm high. A thin layer of bedding was spr ead over the floor of the Ymaze, and all three of the branches were otherwise empty. The bedding was not replaced between mice. The maze was placed in a sou nd-attenuated room under dim illumination (the brightness of the lighting wa s kept constant during the experiment). Pictorial and symbolic visual cues wer e present on each of the four walls. PAGE 47 41 Each mouse received one trial, in the course of which the animal was placed in a starting branch (this branch was the same for each mouse) and allowed free exploration of the maze for 10 minutes. An overhead video camera connected to a television monitor recorded the activity of each mouse in the Y-maze, and the branches visited by the mouse in the 10 minute time period were recorded (in order) by an experimenter in a separate room. The v ery center of the Y-maze was considered a neutral zone; nothing was record ed while the mouse remained in the center of the Y-maze until the mous e left the center to visit a branch. A branch visit was recorded when the entire body of the mouse moved into the branch; the inclusion of the tail was not necessary. Similarly, only when the entire body of the mouse moved into the center of the Y-maze was the mouse considered to have left a branch. Repeated vi sits to the same branch were recorded; for instance, if the mouse left bran ch A, entered the neutral zone, and re-entered branch A, A was recorded t wice. The Y-Maze thus tested the ability of the mouse to remember the two branches which it had most recently visited, and to choose to visit the branch which it had visited least recently; the third, no vel branch. So, for example, a mouse which performs ideally in the Y-Maze would vi sit the branches in the following order: ABCABCABC PAGE 48 42 Figure 4. Diagram of Y-Maze. The three branches are labeled A, B, and C. Each branch visited by the mouse in the 10 minute time p eriod is recorded. (Rat Behavior and Biology, 2004) Rota Rod The Rota Rod (Ugo Basile, Comerio, VA, Italy) t ests the physical endurance and motor coordination of each mouse. It consists o f a rod, 1.3 cm in diameter, divided into five segments of equal length by six d iscs (24.1 cm in diameter). The discs served to minimize lateral movements and redu ce attempts by the mice to change directions while on the rod. The rod was pow ered by a motor with continuous variable speed adjustment (0-100 rpm) an d was linked to a tachometer to measure accuracy in speed control. Each Rota Rod trial consisted of placing an ind ividual mouse on a rod rotating at 5 rpm, which, after 10 seconds, began to acceler ate. If the subject failed to remain on the rod or changed directions on the rod before rotation began, it was correctly placed on the rod. The rod accelerated to 40 rpm over a period of 1 PAGE 49 43 minute. The time which elapsed from the onset of ac celeration until the subject fell from the rod served as a measure of performanc e known as the latency to fall. A fallen mouse landed on a fall sensor, which stopped the timer. The distance from the rod to the fall sensor was 16 cm. Figure 5. Diagram of Rota Rod Schematic representation of a standard rotarod apparatus for testing motor coordination (Carter 20 01). Mouse Sacrificing The mice were placed, one at a time, in a conta iner which received a constant flow of 1 liter of oxygen per minute, as well as is oflurane, adjusted to 3% of the total gas flow. After the mice were observed to be asleep for 5 minutes, they were placed on an operating table, and their heads were placed in a 50 ml PAGE 50 44 Falcon tube containing an isoflurane-soaked cotton ball. An incision was made on the underside of the mouse, below the liver. The incision was followed up to the diaphragm, without harming any of the internal organs, and the diaphragm was punctured. While the mice were still alive, nee dle was inserted into the right ventricle of the heart, and blood was drawn directl y from the heart. The blood was centrifuged at 4000 rpm for 4 minutes at room t emperature (RT) to obtain the plasma. Afterwards, the cerebellum and cerebrum together were carefully removed from the mice. One brain hemisphere from ea ch subject was snap frozen in liquid nitrogen and stored at -80 degrees Celcius, whereas the other hemisphere was fixed in 7 ml of 4% paraformaldehyde Brain A Quantification Mice brains were homogenized by sonication in 7 00 l of ice cold lysis buffer. Each 20 ml of lysis buffer was comprised of 20 ml M -Per plus 40 l PMSF, 200 l of 100x cocktail protease inhibitors, and 200 l of EDTA (Pierce protein research product, IL, USA). Brain homogenates were centrifug ed at 4 degrees Celsius for 30 minutes at 14,000 rpm. One hundred ml of the sup ernatant containing soluble A (GS) was collected and treated with 100 ml 5 M gua nidine isothiocyanate for 1 hour at room temperature, and then vortexed for 1 5 minutes and stored at -80 degrees Celsius. One hundred ml of the pellet conta ining insoluble A (GI) was dissolved and denatured in 100 ml 5 M guanidine iso thiocyanate for 1 hour at room temperature, and then vortexed for 15 minutes and stored at -80 degrees Celsius. Protein concentrations were estimated in b oth fractions by the BCA PAGE 51 45 method (Pierce, IL, USA). A (1-40) and A (1-42) were quantified by Enzymelinked immunosorbent assay (ELISA) according to the manufacturers recommendations (Invitrogen, CA, USA). A concentrations were calculated in pg/mg of protein. Statistical Analysis Statistical analysis was completed using SAS fo r Windows release (SAS 9.2 TS Level 2M2). The MWM test, Y-maze, Rota Rod, mous e weight and ELISA results were analyzed using 2-way ANOVA, repeated m easures ANOVA, t-tests, correlation tests and post hoc tests where appropri ate. The Y-maze tests resulted in a spontaneous percent alternation score for each mouse, as well as the total number of branches visited. Spontaneous a lternation, expressed as a percentage, refers to ratio of arm choices differin g from the previous two choices to the total number of arm entries. Spontaneous alt ernation was calculated using a program created by Venkat Mathura at The Roskamp Institute. Acute Treatment Study Sixteen Tg APPsw mice at 14 months of age were administered a short term, 3 week treatment of once daily intraperitoneal inje ctions of either a solution of Celastrol diluted with PBS, or DMSO diluted with PB S. The treatment was administered to the underside of each mouse, around the area of the small intestine. Initially, each mouse was injected with 0.1 ml of either a 1:1 dilution of 600 l Celastrol and 600 l PBS, or 600 l DMSO and 600 l PBS (resulting in a PAGE 52 46 Celastrol intake of 2 mg/kg of mouse). Two days aft er the beginning of this treatment regimen, the dilutions of the solutions w ere changed to 3:1 (either 900 l Celastrol and 300 l PBS, or 900 l DMSO and 300 l PBS), so that each mouse received only 1 mg/kg of Celastrol. After 7 days of treatment, the subjects were al l tested once using the Y-maze. Treatment continued on this day. After 8 days of tr eatment, the subjects were all tested using the Morris Water Maze performance test The subjects underwent 9 days of acquisition trials, followed by a probe tes t on the 10th day. Treatment continued during the Morris Water Maze testing peri od. Shortly after the probe trial, the mice were killed and tissue samples were taken. Powder Food Study Twenty Tg PS1/APPsw, 20 Tg APPsw, and 20 Tg PS1 and WT mice at 13-16 months of age were administered a long term, 5 mont h treatment of either Celastrol mixed into regular powdered food, or powd ered food alone. The food remained in their cages at all times. The mice were acclimated to the powdered food alone for 28 days before half of the subjects were given the mix containing Celastrol. The mice were first administered a 0.01% mix of Celastrol and powdered food (50 mg Celastrol/500 g food) based on the observation that the mice ate 20 mg of powdered food per kg of mouse. Gi ven a 10% rate of body absorption, the mice would absorb 2 mg/kg of the mi xture. After 4 days, the mice were given a 0.02% mix o f Celastrol and powdered food (100 mg Celastrol/500 g food). Seven days later, th e mice were given a 0.05% PAGE 53 47 mix of Celastrol and powdered food (250 mg Celastro l/500 g food). Seventeen days later, all mice were given powdered food alone for one week. The mice then continued their treatment with a 0.03% mix of Celas trol and powdered food (150 mg Celastrol/500g food) for 7 days, followed by a 0 .02% mix. After one month, the Tg PS1/APPsw mice were all given powdered food alone. Ten days later, all mice in the study were given powdered food alone. F inally, a week later, the mice were administered a 0.02% mix for the remainder of the study. These changes were made in response to drastically the fluctuatin g weights of the mice over the course of the study. The mice were first weighed 2 days before first being administered the Celastrol/powdered food mixture. Then, beginning 1 month after the start of the treatment, the mice were weighed once a week for th e remainder of the study. The mice were each tested once using the Y-maze aft er 2 months of treatment. Additionally, beginning after two months of treatme nt, the mice were tested using the Rota Rod once every two days for three weeks. E ach mouse was tested only once a day. After 5 months of treatment, the mice w ere all tested using the Morris Water Maze performance test. The subjects un derwent 9 days of acquisition trials, followed by a probe test on the 10th day. Treatment continued during the Morris Water Maze testing period. Shortly after the probe trial, one brain hemisp here from each subject was snap frozen in liquid nitrogen and stored at -80 degrees Celsius, whereas the other hemisphere, as well as liver, kidney, intestine, an d stomach samples, were fixed in 7 ml paraformaldehyde. The brain hemispheres wer e homogenized, and A PAGE 54 48 (1-42) and (1-40) concentrations were quantified in both the GS and GI fractions by ELISAs. PAGE 55 49 Results Powder Food Study Weights To assess the toxicity and, to some extent, the bioavailability of enterally administered Celastrol on Tg PS1/APPsw, Tg APPsw, a nd Tg PS1 and WT mice, the mice were weighed 2 days before first being adm inistered the Celastrol/powdered food mixture. Then, beginning 1 month after the start of the treatment, the mice were weighed once a week for th e remainder of the study. The weights of the mice fed Celastrol fluctuated gr eatly during the study compared to the weights of the mice receiving powde red food alone. Graph 1a shows the average weights of the mice administered Celastrol over the entirety of the study, and Graph 1b shows the weights of the mice receiving powdered food alone during the study. In Graph 1a, the sharpest decline is seen betwe en 5/15 and 6/5, when the amount of Celastrol mixed with the powdered food wa s raised from 50 mg Celastrol/500 g food to 100 mg Celastrol/500 g food to 250 mg Celastrol/500 g food in a short period of time (See Materials and M ethods). A recovery is seen between 6/16 and 6/22, during a time when no mice r eceived Celastrol. As soon as the mice were put back on the drug (a 150 mg Cel astrol/500g food mix), they experienced another sharp decline in weight (6/22-6 /29). When the dosage was lowered to 100 mg Celastrol/500 g food again, the w eight levels off (after 6/29). At 7/27-8/3, during a time when the Tg PS1/APPsw mi ce were given powdered PAGE 56 50 food alone, an increase in weight is seen. At 8/10 when the other genotypes are given regular powdered food, their weights increase as well (additionally, the weight of Tg PS1/APPsw mice levels off). At 8/17, t he dose is brought back to 100 mg Celastrol/500 g, and the weights of all geno types are seen to decline. Repeated measures ANOVA revealed a significant difference in the weight means between all three genotypes (P<0.0001), and b etween the paired weights (weights measured on the same day) of all three gen otypes (P<0.0001) administered Celastrol. A Tukey post hoc test showe d significant differences between each of the genotypes pairs, with the P val ues for Tg PS1/APPsw vs. Tg APPsw, Tg PS1/APPsw vs. WT and PS1, and Tg APPsw vs. WT and PS1 of <0.01, <0.001, and <0.05, respectively. A Spearmans rank correlation test showed signi ficant negative correlation between the milligrams of Celastrol per 500 grams o f powdered food fed to mice, and the weights of the mice, for all genotypes. The Spearman r value for the correlation between the amount of Celastrol in the powdered food and the mouse weight for Tg PS1/APPsw, Tg APPsw and WT and PS1 wa s -0.8249, -0.6673, and -0.6062, respectively, and the P values for the se correlations were 0.0002, 0.0047, and 0.0099, respectively. PAGE 57 51 A Weights (g) over time20 22 24 26 28 30 32 34 36 38 40 5 / 1 5 /09 6 / 1 6 /09 6 / 2 9 /0 9 7 / 1 3 /0 9 7 / 2 7 /0 9 8 / 1 0/ 0 9 8 / 2 4/ 0 9 9/8/ 09 9 / 2 1/09 DatesWeight (g) PSAPP APP PS1 and WT B Weights (g) over time20 22 24 26 28 30 32 34 36 38 5/15 /0 9 6/16 /0 9 6/29 /0 9 7/13/ 0 9 7/27/ 0 9 8/10/ 0 9 8/24/09 9 / 8/09 9/21/09 DatesWeight (g) PSAPP APP PS1 and WT Graph 1. Effects of orally administered Celastrol o n mouse weight over time A) Line graph showing the fluctuating weights of the m ice receiving Celastrol during the powdered food study. B) Line graph showing the weig hts of the placebo mice during the powdered food study. PAGE 58 52 Y-Maze To assess the effect of orally administered Cel astrol on the memory and the ability of the mice to discern a novel situation, a Y-maze test was conducted on Tg PS1/APPsw, Tg APPsw, and WT and PS1 mice after 2 months of treatment with a 0.02% mix of Celastrol and powdered food. Th e Y-maze tests resulted in spontaneous percent alternation scores for each mou se (See Statistical Analysis under Materials and Methods section), whi ch were calculated and quantified using the program created by Venkat Math ura at The Roskamp Institute. Two way ANOVA revealed a significant inc rease in the quantified alternation values for the mice administered Celast rol compared to the powdered food alone (P=0.0289), however the genotype and the genotype/dosage interaction was not significant (P=0.5262 and 0.651 6, respectively) (Graph 2). A Bonferroni post hoc test showed no significant diff erence between percent alternation for any genotypes pairs, with or withou t Celastrol (P> 0.05). PAGE 59 53 drug no drug 0 10 20 30 40 50 60 70PSAPP APP WT DoseAlternation (seconds) Graph 2. Effects of orally administered Celastrol o n percent alternation. Histogram representing the effect of drug administration and genotype on Y-maze percent alternation. Rota Rod The physical endurance and motor coordination o f Tg PS1/APPsw, Tg APPsw, and WT and PS1 mice treated with orally administere d Celastrol after two months of treatment was assessed using the Rota Rod test once every two days for three weeks. The Rota Rod tests resulted in a l atency to fall score, the time which elapsed from the onset of acceleration until the subject fell from the accelerating rod. The improvement in latency scores from testing day 1 to day 7 were compared using two way ANOVA. This test reveal ed that the genotype/dosage interaction and that the genotype a lone acted as a significant source of variation for the quantified latency to f all values (P=0.0190 and 0.0003, respectively), however the dosage alone did not (Gr aph 3). A Bonferroni post hoc test showed a significant difference of latency per iods between the PSAPP and control and the APP and control pairs (P<0.001, P<0 .01 respectively) given PAGE 60 54 Celastrol, but no significant effect between these same pairs given regular powdered food. The post hoc test also showed a sign ificant increase in the latency period of PSAPP mice compared to APP mice g iven regular powdered food (P<0.05), but not Celastrol. drug no drug 0 5 10 15 20 25 30 35PSAPP APP WT DoseLatency Graph 3. Effects of orally administered Celastrol o n latency to fall. Histogram representing the effect of drug administration and genotype on the change in time which elapsed from the onset of acceleration until the su bject fell from the Rota Rod between testing days 1 and 7. Morris Water Maze performance test Spatial learning and memory of Tg PS1/APPsw, Tg APPsw, and WT and PS1 mice treated with orally administered Celastrol aft er 5 months of treatment was assessed using the MWM performance test for 9 days of acquisition trials, followed by a probe test on the 10th day. The MWM performance test resulted in an escape latency score which could not exceed 90 s econds from the acquisition trials, as well as the percentage of time the mice spent in the NE quadrant from the probe trial. The improvements in latency scores between day 1 and day 9 of the acquisition period were examined using two way ANOVA. This test revealed PAGE 61 55 significant variation in the latency improvements b etween the dosages and the dosage/genotype interaction (P=0.0299 and 0.0277, r espectively), but not between the genotypes alone. The Bonferroni post ho c test showed significant increase in the latency improvement of the WT and P S1 mice that received Celastrol compared to the Tg PS1/APPsw (P < 0.05), but not between any other genotype/dosage interactions (Graph 4). drug no drug 0 10 20 30 40 50APP WT PSAPP DoseAcq Improvement (seconds) Graph 4. Effects of orally administered Celastrol on MWM lat encies. Histogram representing the effect of drug administration and genotype on the change in latency between acquisition days 1 and 9. The duration of time spent by the mice in the N E quadrant in the probe trial was examined using two way ANOVA. This tested revea led no significant variation between the duration periods of any of th e genotypes, nor between the dosage types or the genotype/dosage interactions (G raph 5). PAGE 62 56 drug no drug 0 5 10 15 20PSAPP APP WT DoseDuration (seconds) Graph 5. Effects of orally administered Celastrol o n MWM NE quadrant duration. Histogram representing the effect of drug administr ation and genotype on the change in duration in the NE quadrant during the MWM probe tr ial. Brain A Quantification Soluble and insoluble forms of A (1-40) and A (1-42) in the Tg PS1/APPsw and Tg APPsw mice in the powdered food study were q uantified by ELISAs according to the manufacturers recommendations (In vitrogen, CA, USA). A concentrations were calculated in pg/mg of protein. These concentrations were examined using two way ANOVA. This test found signi ficance in the variation of concentration of insoluble A 1-42 and 1-40 between the genotypes (P<0.0001 for both 1-42 and 1-40), but not between the dosage s or the dosage/genotype interaction (histogram not pictured). The test foun d significance in the variation of concentration of soluble A 1-42 and 1-40 between the genotypes (P<0.0001 for both 1-42 and 1-40), the dosages (P=0.0016 for 1-40 and 0.0366 for 1-42), and the dosage/genotype interaction (P= 0.0009 for 1-40 and 0.0239 for 1-42) (Graph 6). PAGE 63 57 A PSAPP APP 0 1000000 2000000 3000000 4000000 5000000 6000000 7000000 8000000Drug Placebo GenotypeConcentration (pg/ml) B PSAPP APP 0 5000000 10000000 15000000 20000000Drug Placebo GenotypeConcentration (pg/ml) Graph 6. Effects of orally administered Celastrol o n Brain A (1-40) and (1-42). A) Histogram showing representing the effect of drug a dministration and genotype on soluble A (1-40) concentration in mouse brains. B) Histogram showing representing the effect of drug administration and genotype on solub le A (1-42) concentration in mouse brains. Observations The mice in the powdered food study were checke d daily and were seen to eat the powdered food, even after the drug was added to the food. The weights of the mice receiving Celastrol in their powdered food fluctuated during the study PAGE 64 58 (Graph 1a) as the Celastrol dosages were modified, and the most drastic weight loss was observed in the Tg PS1/APPsw mice when rec eiving large dosages of Celastrol. The mice experiencing the most weight lo ss also behaved irregularly; they were less active, stayed in a hunched back pos ition, and kept their ears down. These symptoms, as well as weight loss, were alleviated as the Celastrol dosages were lowered. During the Rota Rod tests, t he mice receiving Celastrol were observed to have a more calm temperament than the mice receiving powdered food only. Upon dissection, all Tg PS1/APP sw mice receiving Celastrol were observed to possess a bloated intestine, as wa s one Tg APPsw mouse receiving Celastrol. This was not observed in any o f the WT and PS1 mice. Acute Study Y-maze To assess the effect of Celastrol administered over a short period of time via intraperitoneal (IP) administration on the memory a nd the ability of the mice to discern a novel situation, a Y-maze test was conduc ted on Tg APPsw mice after 7 days of treatment with 1 mg/kg of Celastrol daily The resulting percent alternation scores for the mice were examined using a t-test. The test revealed no significant difference between the percent alter nation for the Celastrol and placebo mice (histogram not pictured). PAGE 65 59 Morris Water Maze performance test Spatial learning and memory of Tg APPsw mice tr eated with Celastrol administered over a short period of time via IP adm inistration was assessed after 8 days of treatment with 1 mg/kg of Celastrol daily using the MWM performance test for 9 days of acquisition trials, followed by a probe test on the 10th day. The improvements in latency scores between day 1 and da y 9 of the acquisition period were examined using a t-test. This test reve aled no significance between the improvements in latencies of the Celastrol trea ted mice and the placebo mice (Graph 7). drug no drug 0 10 20 30DoseImprovement (seconds) Graph 7. Effects of IP injected Celastrol on MWM la tency. Histogram representing the effect of drug administration on the improvemen t in latency between acquisition days 1 and 9 in Tg APPsw mice administered Celastrol or placebo IP. The duration of time spent by the mice in the N E quadrant in the probe trial was examined using a t-test. This test revealed sig nificant increase in the duration spent by the placebo mice in the NE quadra nt (P=0.0068) (Graph 8). PAGE 66 60 Placebo Drug 0 10 20 30DoseDuration (seconds) Graph 8. Effects of IP injected Celastrol on MWM NE quadrant duration. Histogram representing the effect of drug administration on t he change in duration in the NE quadrant during the MWM probe trial in Tg APPsw mic e administered Celastrol or placebo IP. Observations Before the dosage of Celastrol received daily b y IP injection was lowered from 2 mg/kg of mouse to 1 mg/kg of mouse, the mice were observed to move slowly, hunch their backs, and keep their eyes nearly shut following injections. The mice were relatively nonresponsive, as well. This effect wore off after enough time had passed since the last injection, and was not observ ed in the placebo mice. PAGE 67 61 Discussion The NFB inhibiting compound Celastrol has been found to r educe both A (140) and A (1-42) species and to dose dependently reduce the production of APP-CTF and -sAPP in vitro (Paris et al 2007, 2010). It has als o been shown to reduce brain levels of soluble A (1-38), A (1-40), and A (1-42) by approximately 50%, and the levels of the insoluble forms of these peptides by 60% in vivo (Paris et al 2010). However, there have been no behavioral studies performed on the transgenic mouse model of Alzheime rs disease treated with the compound Celastrol until now. The behavioral an d performance-related effects of Celastrol on Tg mouse models of human Al zheimers disease were tested in both a long term study in which the drug was administered enterally through the powdered food consumed by the mice, and an acute study in which the drug was administered parenterally through intr aperitoneal injection. Throughout the powdered food study, there were no differences in the performance and behavior of the WT and the Tg PS1 m ice, confirming that neither group experienced abnormal levels of amyloi d deposition. The weights of the mice in the powdered food study were measured 2 days before first being administered the Celastrol/powdered food mixture, a nd then, beginning 1 month after the start of the treatment, once a week for t he remainder of the study. The weight results showed a strong correlation between an increased amount of Celastrol mixed in with the powdered food, and weig ht loss in all genotypes (Figure 4). This suggests that the levels of Celast rol absorbed by the mice eating the Celastrol/powdered food mix reached toxic level s. Additionally, the weights of PAGE 68 62 all genotype groups during the study were shown to be significantly different, with the Tg PSAPPsw mice weighing significantly less tha n the Tg APPsw and WT/PS1 mice, and the Tg APPsw weighing significantl y less than the WT/PS1 mice. These results suggest that Celastrol has a mo re toxic effect on the transgenic mice than the wild type mice and the Tg PS1 mice which do not overproduce human A. The mice maintained a steady weight at around 50100 mg Celastrol/500 g food, and increased concentratio ns of Celastrol are advised against in future studies. A Y-maze test was conducted on the mice after 2 months of treatment with a 0.02% mix of Celastrol and powdered food in order t o assess the effect of orally administered Celastrol on the memory and the abilit y of the mice to discern a novel situation. The Y-Maze accomplished this by te sting the ability of mice to choose to enter a novel branch; one that they do not remember having recently visited (See Y-Maze under Materials and Methods). This ability is quantified by the percent alternation value, or the ratio of arm choices differing from the previous two choices to the total number of arm ent ries. The Y-maze test showed significant increase in the quantified alternation values for the mice administered Celastrol compared to the powdered food alone, but no significant variation in the quantified alternation values for genotype alone, o r the genotype/dosage interaction. A Y-maze test was also conducted on mi ce administered Celastrol over a short period of time via intraperitoneal (IP ) administration after 7 days of treatment with 1 mg/kg of Celastrol daily, but show ed no significant difference in the percent alternation between the mice receiving Celastrol and the mice PAGE 69 63 receiving placebo, suggesting that the effect of Ce lastrol on memory depends on the length of the period of Celastrol intake; Celas trol administered orally over a long period of time has an effect on memory, but Ce lastrol administered in large amounts through IP injection over a short time peri od does not. A Morris Water Maze performance test was conduc ted on the mice after 5 months of treatment in order to assess the spatial learning and memory of mice receiving a 0.02% mix of Celastrol and powdered foo d. Each of the three mouse genotype groups demonstrated significant improvemen t in latency periods over the nine acquisition days independent of dosage (Fi gure 7), which demonstrates learning in all of the mice and the validity of the acquisition trials as a training period. The latency scores of the WT mice, independ ent of dosage, were significantly lower than those of the Tg PS1/APPsw mice in the third block of the acquisition trial period, and the improvement in la tency scores between day 1 and day 9 showed significant increase in the latenc y improvement of the WT/PS1 mice that received Celastrol compared to the Tg PS1 /APPsw that did. These findings demonstrate a clear debilitation in the Tg PS1/APPsw compared to the WT/PS1 mice, which suggests that Celastrol administ ered orally over a long period of time has no effect on the spatial learnin g and memory in mice expressing an increased ratio of A (1-42) to A (1-40). A MWM test was also performed on Tg APPsw mice after 7 days of treatment with 1 mg/kg of Celastrol injected IP daily. The im provements in latencies of the Celastrol treated mice and the placebo mice over th e 9 acquisition days were not significantly different from one another, and the d uration of time spent by the PAGE 70 64 Celastrol treated mice in the NE quadrant of the pr obe trial was not significantly greater than the duration of time spent there by th e placebo mice, which suggests that Celastrol administered in large amoun ts through IP injection over a short time period has no effect on spatial learning and memory in mice overproducing A (1-42) and A (1-40). A Rota Rod test was conducted on the mice in th e powdered food study after two months of treatment in order to assess the phys ical endurance and motor coordination of mice receiving a 0.02% mix of Celas trol and powdered food. The Rota Rod test showed that the improvement in latenc y to fall between days 1 and 7 for WT/PS1 was significantly greater than both th e Tg PS1/APPsw and the Tg APPsw mice given Celastrol (Figure 6), which sugges ts that Celastrol may hinder the physical endurance of mice overexpressing human A, and mice expressing an increased ratio of A (1-42) to A (1-40). The effect of Celastrol on motor coordination in mice overexpressing human A cannot be properly assessed by this study, because the clear debilitation in the p hysical endurance of these mice compromised their ability to demonstrate good motor coordination. The results of the Rota Rod and MWM tests in th e powdered food study show clear debilitation in the Tg PS1/APPsw mice receivi ng Celastrol, and, to a lesser extent, debilitation in the Tg APPsw mice receiving Celastrol. During the Rota Rod tests, unusually calm demeanors were observed i n the PS1/APPsw mice. This may have affected the results of the test, and could be related to adverse physical effects of the drug on the Tg PS1/APPsw (w hich may have occurred, to a lesser extent, in the Tg APPsw mice compared to t he WT/PS1 mice). The PAGE 71 65 results from the Y-Maze test did not show a signifi cant increase in the performance of WT mice over Tg PS1/APPsw or Tg APPs w mice given the drug like the MWM and Rota Rod tests did. This may be be cause the Y-maze did not test the physical endurance and motor coordination of the mice like the Rota Rod and, by its nature, the MWM test (which involves mi ce swimming relatively large distances in addition to finding the hidden platfor m). Thus, this difference in performance trends between the Y-Maze results and t he MWM/Rota Rod results may be due to adverse physical effects of the drug on the Tg PS1/APPsw and Tg APPsw mice compared to the WT/PS1 mice. The idea of adverse physical effects of the dru g on the transgenic mice is supported by the daily observation of the mice in t he powdered food study, which showed that the mice receiving Celastrol were less active, stayed in a withdrawn, hunched back position, and kept their ears down. These are typical signs of a sick mouse. Weight loss was also observed in all mi ce in the powdered food study as the dosage was increased. The Tg PS1/APPsw mice receiving Celastrol experienced lower weight levels than the other geno types receiving Celastrol for nearly the entirety of the study (Figure 4). After 5 months of treatment, upon dissection, all of the Tg PS1/APPsw mice that recei ved Celastrol were observed to possess a bloated intestine. These observations suggest a toxic effect of Celastrol on mice at a certain dosage, especially t hose mice which overproduce human APP and A and express an increased ratio of A (1-42) to A (1-40). Administration of extracts of the family Celastrace ae have previously been shown to have negative gastro-intestinal effects. Tripterygium wilfordii Hook F. is PAGE 72 66 a perennial vine belonging to Celastraceae, which h as a long history in traditional Chinese herbal medicine. A refined extract from the vines root xylem, called GTW is commonly used to treat rheumatoid arthritis, chronic nephritis and various skin disorders, and the side effects at dos es of 60 to 90 mg/day are gastro-intestinal disturbances, including including nausea, vomiting, anorexia, epigastric burning, xerostomia, diarrhea and consti pation (Li 2000). Additionally, in U.S. Pat. No. 5500340, Lipsky and Tao disclosed the administration of an extract containing Celastrol to patients with rheum atoid arthritis, and reported that the side effects of the treatment included dia rrhea and abdominal pain. The side effects in these cases may be related to the i ntestinal bloating seen in the transgenic mice and possibly the poor performance i n the tests of physical endurance by the transgenic mice administered Celas trol. Soluble and insoluble forms of A (1-40) and A (1-42) in the mice receiving a 0.02% mix of Celastrol and powdered food were quant ified by the ELISA method after 5 months of treatment. The results suggested that Celastrol caused a significant decrease in the concentration of solubl e A 1-42 and 1-40 in the brain, however, Celastrol had no significant effect on the levels of insoluble A 1-42 and 1-40, the forms of A associated with amyloidosis in Alzheimers disease (Wang et al 1999). The results of the brain A quantification in the powder food study mice, compared to the results from a similar study conduc ted by Paris et al (2010) could serve to highlight the importance of Celastro l as a prophylactic treatment rather than a therapeutic treatment. In the Paris e t al study, 5 month old Tg PAGE 73 67 PS1/APPsw mice received Celastrol in the form of cu stom made biodegradable pellets implanted subcutaneously ensuring a continu ous release of 2.5 mg/Kg of body weight/day for one month. In this study, brain A quantification showed that Celastrol reduced brain levels of soluble A (1-38), A (1-40), and A (1-42) by approximately 50%, and the levels of the insoluble forms of these peptides by 60%. In contrast, the powdered food study used mice at 13-16 months of age, and showed no significant effect of Celastrol on insolu ble forms of A. Because Tg PS1/APPsw mice start to develop A deposits by 4 months of age and display a significant amount of A by 6 months, the results the present study may be due to the fact that Celastrol was first administered t o the transgenic mice long after they had begun to form A deposits. Because of the inconsistencies between t he two studies in terms of mode and length of treatmen t, a study of long term enteral administration of Celastrol in young (4-5 month old ) Tg PS1/APPsw, Tg APPsw, and WT/PS1 mice would be useful in order to verify the importance of Celastrol as a prophylactic drug. If Celastrol is found to work effectively as a prophylactic drug against A burden in the brain, it would not be the first to d o so. Nonviral A DNA vaccines administered before the initial A deposition in mouse models (Tg APP23) of Alzheimers disease resulted in reduced A burden to 15.5% and 38.5% of that found in untreated mice at 7 and 18 months of age ( Okura et al 2006). These vaccines are effective because they cause A proteins to be released in the extracellular space, inducing antibodies against th ese proteins The release of PAGE 74 68 proteins into extracellular space is triggered when naked plasmid DNAs encoding proteins are taken into the cell after being inject ed into the muscle or skin, where they produce the proteins in a small amount for a r elatively long period of time. When considering the results of the study in wh ich Celastrol was administered to the mouse models via a Celastrol/powdered food m ix, the absorption of the drug in the bodies of the mice should be taken into account. The powdered food study incorporated no method of measuring the amoun t of Celastrol absorbed by the mice. When initially calculating the amount of drug to be mixed into the powdered food, a rate of absorption of 10% was assu med, and from there the amount of food eaten by the mice each day was used to determine the amount of powdered Celastrol to be added to the food. If the powdered food study is to be replicated, it should be done so after a suitable amount of pharmacokinetic (PK) data about Celastrol has been obtained in order to determine the bioavailability of this d rug orally when administered. Bioavailability, or the fraction of administered dr ug that reaches the systemic circulation after enteral administration, is affect ed by many drug absorption barriers, including solubility, stability, permeabi lity, active efflux and metabolism. An ideal PK study to determine the bioavailability of orally administered Celastrol would involve obtaining blood and cerebrospinal flu id samples from animals at different points in time following a single dose of Celastrol, administered enterally and parenterally. The plasma or cerebrospinal fluid samples could then be analyzed by liquid chromatography-mass spectrometry to determine the concentration of Celastrol absorbed in the blood or cerebrospinal fluid, and the PAGE 75 69 ratio of the area under the curve (AUC) of the bloo d or cerebrospinal fluid concentration versus time curve after enteral admin istration divided by the AUC of the blood or cerebrospinal fluid concentration v ersus time curve after parenteral administration would act as a quantitiat ive measure of absolute bioavailability. A PK study measuring the bioavailability of Cel astrol would also help to ensure that the in the event that the powdered food study is replicated, the drug would be administered to the mice at levels that are nontoxic at all times. This would prevent drastic weight fluctuation and adverse effe cts in the transgenic mice, which likely contributed to the results of the beha vioral and performance tests conducted on these mice. Celastrol did not show a significant overall ef fect in the improvement of memory and ability-based performance in mice receiv ing a therapeutic treatment of the drug, both enterally and parenterally. Howev er, these studies did serve to highlight the effectiveness of the drug over long p eriods of continuous treatment rather than shorter periods of treatment with a lar ge amount of the drug. Further studies with Celastrol are likely to be more effect ive if Celastrol is used as a prophylactic treatment. 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