Philip C. Wong, PhD - US grants

Superoxide dismutase 1 (SOD1) Mutations cause approximately 20% of cases of familial amyotrophic lateral sclerosis (ALS), a fatal motor neuron disease and transgenic mice expressing mutant SOD1 develop a progressive motor neuron disease. It has been hypothesized that mutant SOD1 damage motor neurons by acquiring a toxic property by the following mechanism: the improperly folded mutant SOD1 catalyze, via peroxynitrites, the nitration of tyrosines; and mutant SOD1 possesses an enhanced peroxidase activity that generates elevated levels of hydroxyl radicals from hydrogen peroxide. In both hypotheses, copper (Cu) bound to mutant SOD1 is proposed to play a critical role, but for a variety of reasons it has been difficult to test the Cu hypothesis. An emerging view is that the delivery of Cu to specific proteins within various intracellular compartments is mediated through distinct Cu chaperones. Recently, it was shown that the delivery of Cu to SOD1 is mediated through a soluble yeast protein termed Lys7 and Cu chaperone for SOD1 (CCS in mammals) First, we will define the cellular distribution s and the axonal transport of Ccs in the nervous system, particularly in motor neurons. Second, to prove that CCS is necessary to charge SOD` with Cu, we will use gene-targeting to delete the gene encoding CCS and characterize the phenotype of CCS null mice. Third, since CCS colocalizes with SOD1, we will determine whether Ccs interacts physically with SOD1 using coimmunoprecipitation studies. Fourth, because CCS levels vary with abundance of SOD1, we will determine the mechanism whereby Ccs is coordinately regulated by SOD1 using a variety of cell culture systems. Our goal is to test whether the aberrant SOD1 Cu chemistry is directly responsible for mediating disease in mutant SOD1 transgenic mice. With information from each of these studies and the availability of CCS null mice, we will be in a unique position to examine directly this hypothesis. If Cu is the key effector of the disease, we predict that CCS null mice that express the FALS-linked mutant SOD1 will show significant amelioration of motor neuron disease. These studies will allow, the examination of Cu-based toxicity mechanisms in vivo; the results of this work, which have the potential to identify new therapeutic targets (i.e., CCS/SOD1 Cu trafficking pathway), will have important implications for the design of treatments for motor neuron disease.

DESCRIPTION: (Verbatim from the Applicant's Abstract) Cu/Zn superoxide dismutase (SOD1) mutations cause about 20 percent of cases of familial amyotrophic lateral sclerosis (FALS). Transgenic mice expressing mutant SOD1 develop a progressive motor neuron disease. A long term goal is to understand the molecular mechanisms by which SOD1 mutants cause selective motor neuron degeneration. Copper, an essential cofactor for SOD1 enzymatic activity, has been proposed to play a critical role in the pathogenesis of SOD1-linked ALS. Because free copper can be toxic in cells, the delivery of copper to specific proteins within various compartments of the cell is tightly regulated by specific copper chaperones like CCS. Previous efforts have demonstrated that CCS is necessary to deliver copper to SOD1, and CCS-deficient mice are viable and show no SOD1 activity. First, to test whether Cu in mutant SOD1 is required to cause motor neuron degeneration, CCS-deficient mice will be crossbred with a series of mutant SOD1 mice. If Cu participates in killing motor neurons, mutant SOD1 mice without CCS will show significant amelioration or rescue of motor neuron disease. Second, because 30 percent of SOD1 is not charged with Cu, transgenic mice overexpressing CCS will be generated to examine whether the level of active SOD1 can be increased in vivo by elevating the level of CCS. Finally, to determine the domains of CCS that are important for Cu delivery, a series of mutant CCS DNA constructs will be transfected into wild-type and CCS-deficient cells to identify CCS mutants that are able to inhibit the CCS Cu trafficking pathway. Taken together, these efforts will test Cu-based neurotoxicity mechanisms in vivo. The results will have the potential to identify novel therapeutic targets and may have implications for the design of drug treatments for motor neuron disease.

DESCRIPTION (provided by applicant): Alzheimer' s disease (AD), a progressive neurodegenerative disorder of the elderly, is characterized by the deposition of beta-amyloid (Abeta) and neurofibrillary tangles in the hippocampus and cerebral cortex. Endoproteolytic cleavages of amyloid precursor protein (APP) by beta-and gamma-secretases result in the generation of Abeta peptides that are believed to be neurotoxic. The formation of Abeta is precluded by the endoproteolytic cleavage of APP within the Abeta sequence by a-secretase. Following the discovery of two homologous transmembrane aspartyl proteases, termed BACE1 and BACE2, studies showed that BACE1 is the beta-secretase while BACE2 cleaves at sites within the Abeta domain to limit Abeta secretion. Recent studies indicated that BACE1 might be a susceptibility factor to brain amyloidosis and an excellent therapeutic target in Alzheimer's disease. The overall goals of this proposal are to assess the role of BACE 1/BACE2 as determinants of selective vulnerability of neuron/brain to amyloidosis and to evaluate BACE1 as a high priority therapeutic target in Alzheimer's disease. To begin to test the hypothesis that the abundance of BACE1 coupled to low levels of BACE2 activity is a major determinant of selective vulnerability of neurons to Abeta amyloidosis in the brain, we will first define the levels and distributions of BACE1 and BACE2 in neurons/brain and non-neural cells/organs; the axonal transport of BACE1 and BACE2 will also be determined. The levels/activities of these proteases will be determined in different cell types and the brain and other organs of normal mice and humans, mutant APP mice, and autopsied cases of Alzheimer's disease. Second, as a direct test of the hypothesis that BACE1 is a principal determinant of brain amyloidosis, we will determine whether Abeta deposition can be respectively ameliorated or accelerated in transgenic mouse models expressing reduced or increased levels of BACE1 in brain. Finally, towards the critical evaluation of BACE1 as a therapeutic target in Alzheimer's disease, the physiological roles of BACE1 and BACE2 will be determined through the generation and analysis of BACE1-null, BACE2-null, and BACE1; BACE2 double null mice. Results from these efforts will provide important information regarding why the brain is particularly susceptible to amyloid plaque deposition and will have important implications for the design of therapeutic strategy to inhibit BACE1 in efforts to ameliorate Abeta amyloidosis in Alzheimer's disease.

Alzheimer's disease (AD), a progressive neurodegenerative disorder of the elderly, is characterized by the deposition of 13-amyloid (AI_) and neurofibriUary tangles in the hippocampus and cerebral cortex. Endoproteolytic cleavages of amyloid precursor protein (APP) by _-and ?- secretases result in the generation of AI3 peptides that are believed to be neurotoxic. The presenilins (PS1 and PS2), which when mutated cause familial AD, are important for the intramembraneous proteolysis of several proteins, including APP and Notch1. The presenilins is part of a high molecular weight complex that contains the ?-secretase. Critical to this 7-secretase complex is another type I transmembrane glycoprotein, termed nicastrin that binds to both APP and Notch1 and is required for glp-l/notch signaling. Recent studies in nicastrin-deficient Drosophila demonstrate that the loss of nicastrin abolishes the presenilin-dependent intramembraneous proteolysis of Notch. Thus, we hypothesize that nicastrin is required for proteolytic processing and signaling of Notch1 in mammals. We plan first to determine whether reduced levels of nicastrin impact on presenilin-mediated Notch signaling in mammals by generating nicastrin.deficient mice and characterize the phenotypic consequences of reduction in nicastrin. Secondly, because studies indicated that nicastdn plays an important role in APP processing, we will test the role of nicastrin in presenilin-mediated 7-secretase cleavage of APP using nicastrin knockout cells. In addition, we will determine the mechanism whereby nicastrin influences presenilins to facilitate transmembrane cleavage of Notch and APP. Finally, to examine the in vivo role of nicastrin in adult brain, we will generate and characterize nicastrin conditional knockout mice. Taken together, outcomes from these efforts will have important implications toward our understanding of the mechanism whereby nicastrin influences presenilins in facilitating the presenilin-dependent 7-secretase activities on several critical pathways, including Notch and APP, as well as for nicastrin as a potential therapeutic target in AD.

With the discovery of BACE1 as the beta-secretase involved in the generation of beta-amyloid (Abeta) peptides in Alzheimer's disease (AD), we embarked on a series of studies to examine the functional roles of this transmembrane aspartal protease. We demonstrated that BACE1 is the principal beta-secretase in neurons and that it is essential to cleave APP to generate Abeta in the brain. Moreover, we have provided strong evidence to support our hypothesis that BACE1 and BACE2, along with APP are key determinants of selective vulnerability of brain to Abeta amyloidosis. Significantly, deletion of BACE1 in APPswe;PS1deltaE9 mice prevents both Abeta deposition and age-associated cognitive abnormalities that occur in this model of Abeta amyloidosis. In concert, these results suggest that inhibition of BACE1 should be effective in reducing the Abeta burden in AD. The overall goal is to evaluate critically BACE1 as a high priority therapeutic target for AD, particularly focusing on the reversibility of Abeta-induced abnormalities and the capacity of the brain for repair. In Aim 1, we will examine the role of BACE1 in the evolution of hypothesized Abeta related synaptic abnormalities in the perforant pathway by ultrastructural, immunocytochemical, and biochemical methods using APPswe-deltaE9 mice with varying gene dosage of BACE1. We then will examine in Aim 2 the reversibility of the structural and biochemical abnormalities in the perforant pathway by evaluating APPswe-PS1deltaE9 mice in which expression of BACE1 can be regulated via tet-off transgenic system or by lentiviral RNA interference methods. Results from our proposed studies will provide important information regarding the character and evolution of synaptic pathways, the reversibility of Ap-induced abnormalities, the capacity of the brain for repair in the perforant pathway, and the value of inhibition of BACE1 activity in efforts to ameliorate Abeta amyloidosis in AD.

DESCRIPTION (provided by applicant): Alzheimer's disease (AD), the most common cause of dementia of the elderly, is a progressive neurodegenerative disorder characterized by the deposition of amyloid-beta (Abeta) and neurofibrillary tangles in the brain. Endoproteolytic cleavages of APP by beta-and gamma-secretases result in the generation of Abeta peptides. The exciting discoveries of beta-secretase and components of the gamma-secretase complex over the past several years provided opportunities to examine the physiological roles of BACE1 and Nicastrin (NCT) and to evaluate these proteins as therapeutic targets for AD. We created BACE1 null mice and demonstrated that BACE1 is the principal beta-secretase necessary to cleave APR to generate Abeta. In addition, we generated NCT null (NCT-/-) mice and NCT-/- cells and established that NCT is an integral member of the gamma-secretase complex. Taking advantage of our multidisciplinary group of talented investigators, we plan to extend our beta and gamma-secretase program to address several key issues outlined in the following Aims: 1) To determine whether deficits in synaptic functions or cognitive performance occur in BACE1-/-, BACE2-/-, or BACE1-/-;BACE2-/- mice; 2) To develop a conditional tet-inducible BACE1 transgenic model to prospectively address the reversibility of Abeta induced abnormalities and the capacity of the brain to repair itself; 3) To determine whether Abeta burden can be reduced in brains of mutant PS1;APP mice by genetically modulating the levels of BACE1 and/or components of the gamma-secretase complex; 4) To test the hypothesis that Aph-1 and NCT are required to regulate the stability of each other to form a stable pre-complex for assembly of PS and Pen-2. In such a model, we suggest that the three mammalian Aph-1 homologues (Aph-1aL, Aph-1aS and Aph-1b) define a set of six distinct functional gamma-secretase complexes; 5) To determine physiological role of NCT during post-natal development, maturation, and aging outside the central nervous system, NCT transgenic mice will be generated, characterized and crossbred to NCT-/- mice to complement the developmental defects in NCT-/- mice, and 6) To determine the roles of Aph-1a and Aph-1b by generation and characterization of mice and cells deficient in these components of the gamma-secretase complex. In concert, the Projects in this proposal are designed not only to examine the roles of BACE1, BACE2, and components of the gamma-secretase complex, but also allow a critical evaluation of these proteins as therapeutic targets in efforts to ameliorate Abeta amyloidosis in individuals with AD. PROJECT 1 P.I.: Donald L. Price, M.D. Title: BACE1 and BACE2 in Cognition and Models of Aa Amyloidosis Description (provided by applicant) With the discovery of BACE1 as the a-secretase involved in the generation of a-amyloid (Aa) peptides in Alzheimer's disease (AD), we embarked on a series of studies to examine the functional roles of this transmembrane aspartyl protease. We have provided evidence to support our hypothesis that the distributions and levels of BACE1 and BACE2, along with APR, are key determinants of selective vulnerability of brain to Aa amyloidosis. Importantly, deletion of BACE1 abolished Aa deposition and prevented cognitive deficits occurring in brains of mutant APP;PS1 mice. Although BACE1 null mice do not exhibit overt developmental abnormalities, our recent studies show that these animals do manifest alterations in performance on tests of cognition and emotion. The goal of Project 1 is to assess the functional roles of BACE1 and BACE2 and to evaluate critically BACE1 as a high priority therapeutic target for treatment of AD. Thus, studies in Aim 1 are designed to examine whether deficits in synaptic functions or cognitive/behavioral abnormalities occur in BACE1-l-, BACE2-l-, or BACE1-l- , BACE2-l- mice. In Aim 2, we plan to examine the link between abnormal accumulations of Aa peptides and synaptic abnormalities occurring in APPswe;PSl?E9 mice. These studies are critical for Aim 3, which are designed to assess the degree of reversibility/recovery following experimental reductions of BACE1 at different stages of Aa amyloidosis and degeneration. We anticipate that novel mechanism-based treatments such as BACE1 inhibitors will become available in the future, and it is therefore important to prospectively address the issues of the reversibility of Aa induced abnormalities and the capacity of the brain to repair itself. Investigations in Aim 3 are designed to determine to what extent Aa deposition and associated abnormalities can be reversed following reduction of BACE1 activity at various times after the initiation of Aa deposition. Taken together, results from these studies will provide important information regarding the physiological roles of BACE1 and BACE2 and allow a critical evaluation of BACE1 as a therapeutic target in efforts to reduce Aa burden in individuals with AD. Furthermore, these studies provide important information regarding potential mechanism based toxicities associated with anti-BACE1 therapy in humans that should be carefully monitored in clinical trials in the future.

Presenilins (PS) form high molecular weight complexes with several other transmembrane proteins, termed NCT, Aph-1 and Pen-2 that are critical for generation of functional gamma-secretase complexes. However, the exact roles of these proteins, particularly for Aph-1 in mammals where two homologous genes exist, in regulation of gamma-secretase complex assembly remain uncertain. Although recent data support the notion that PS, NCT, Aph-1 and Pen-2 comprise the minimal gamma-secretase complex, the precise mechanism whereby these four components are assembled into the final active complex remain undefined. An interesting question is why there exist two mammalian Aph-1 genes, namely Aph-1a and Aph-1b, encoding three Aph-1 homologues called Aph-1aL, Aph-1aS and Aph-1b. Based on our recent find ings that the phenotype of Aph-1a null embryos resemble but not identical to those of Notch1 null or NCT null embryos, we hypothesize that Aph-1a is the principal mammalian Aph-1 homologue in presenilin-dependent gamma-secretase complexes required for embryonic development and that Aph-1 homologues are developmentally regulated. Thus, we plan in Aim 1 to address these issues by generation and characterization of Aph-1a null, Aph-1b null and Aph-1a+Aph-1b null mice. Based on our recent findings that the deletion of Aph-1a significantly reduces the levels of mature and immature NCT coupled with the finding that Aph-1 and NCT physically interact, we hypothesize that Aph-1 and NCT are required to regulate the stability of each other to form a stable precomplex for assembling PS and Pen-2. In such a model, we suggest that the three mammalian Aph-1 homologues (Aph-1aL, Aph-1aS and Aph-1b) define a set of six distinct functional gamma-secretase complexes. To test this model, we will generate and characterize a series of mice harboring different combination of Aph-1a and Aph-1b knockout allele and fibroblasts derived from these mice in Aim 2. Since we showed that Aph-1b null mice are viable and there is reduction in levels of PS and Pen-2 in brains of Aph-1-/- mice, we will test whether deletion of Aph-1b is sufficient to ameliorate Abeta deposition in brains of mutant APP;PS1 mice in Aim 3. Taken together, studies proposed here will address important mechanistic questions regarding physiological roles of mammalian Aph-1 homologues and critically evaluating Aph-1a and Aph-1b as therapeutic targets in efforts to ameliorate Abeta amyloidosis in AD.

Nicastrin (NCT), a type I transmembrane glycoprotein which forms high molecular weight complexes with presenilins (PS), is an essential component of the multimeric gamma-secretase complex required for both gamma-secretase activity and APP trafficking. Our recent studies suggest that NCT, with its large ectodomain, may be a potential therapeutic target for AD. Because PS/gamma-secretase activity is required for a variety of essential signaling pathways coupled with our recent finding that cognitive deficits occur in BACE1 null mice, complete inhibition of either secretase activity may not be completely free of mechanism-based toxicity. To minimize any potential toxicity, it may be beneficial to modulate both beta- and gamma-secretase activity such that a significant amelioration of Abeta deposition occurs without affecting other critical signaling pathways. Since our recent studies showed that Abeta deposits are sensitive to the dosage of both NCT and BACE1, we hypothesize that Abeta deposits could be ameliorated by decreasing levels of NCT and BACE1. As proof of principal, we will examine in Aim 1 whether partial reductions of NCT or both NCT and BACE1 will reduce Abeta burden in brains of mutant APP;PS1 mice. Based on our recent findings, we hypothesize that NCT and Aph-1 are required to regulate the stability of each other to form a stable pre-complex for assembly of PS and Pen-2. Since our recent studies indicated that PS1-/-;NCT+/- embryos exhibited a more severe phenotype as compared to PS1-/-;NCT+/- embryos, we hypothesize that this is due to reduced level of PS2-depenedant gamma-secretase complex in PST-/-;NCT+/- embryos. In Aim 2 we will to test whether PS2-dependent gamma-secretase activity is sensitive to the dosage of NCT in the absence of PS1. In addition, we plan to test whether partial reduction of both NCT and PS1 can further reduce the level of Abeta without altering Notch signaling. Positive outcomes from these studies provide support for the view that a combination of NCT- and PS-specific inhibitors may be a useful therapeutic strategy. In Aim 3, we will examine the impact of the absence of NCT in adult peripheral tissues by generation of transgenic mice expressing NCT under the control of the neuronal-specific Thy1 promoter to genetically complement the embryonic lethality of NCT-/- mice. Results from these studies will have important implications for understanding the biology of NCT and development of anti-Abeta therapeutic strategies in AD.

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS), a progressive neurodegenerative disease characterized by weakness, muscle atrophy and spasticity related to the selective loss of motor neurons in the cortex, brain stem and spinal cord, is the most common adult motor neuron disease. Approximately 5-10% of ALS cases are familial, and except for mutations in SOD1 that cause a subset of familial ALS, the etiology of ALS is largely unknown. The identification of specific genes causing ALS allows generation of animal models for studies of disease mechanisms to facilitate the design of rationale therapy for treatment of this devastating illness. Recently, a missense mutation (G59S) in a gene encoding the largest subunit of dynactin complex, termed p1509'U8d, was identified in a large family that associates with slowly progressive lower motor neuron disease. Although mutant dynactin p15CPlued alleles are inherited in an autosomal dominant fashion, suggesting that the disease may result from a toxic gain of function, initial in vitro studies indicated that disease linked mutant p150glued possessed a reduced binding efficiency to microtubules, indicating a partial loss of function. To begin to clarify the mechanism whereby mutant p1509lued causes selective motor neuron disease, we plan to take a genetic approach to first generate human wild type and mutant dynactin transgenic mice and characterize the consequences of expression of mutant p1509lued in mice. In addition, we plan to generate and characterize dynactin p150P'ued standard knockout mice by deleting the gene encoding dynactin p1509'UBd. We hypothesize that mutant, but not wild type dynactin p15CPlued mice will lead to clinical and neuropathological outcomes that are consistent with motor neuron disease whereas mice lacking one allele of dynactin p15QPlued are normal. Such outcomes would be consistent with the idea that ALS-linked mutant p1509'ued causes disease through a gain of function mechanism. Finally, since dynactin is involved in axonal transport, we will examine whether mutant dynactin p1509lued impacts on either anterograde or retrograde axonal transport in these wild type and mutant dynactin p15CPluf>d mice. Taken together, these efforts will clarify the mechanism whereby mutant dynactin p15CPlued causes motor neuron disease and will have the potential to identify novel therapeutic targets and allow design of drug treatments for motor neuron disease.

PROJECT 2 - PROJECT SUMMARY/ABSTRACT As estimates predict that AD is likely to become one of the most important global public health issues by 2050, the urgency to develop mechanism-based disease modifying therapies for AD is ever increasing. Over the past two decades, animal model systems have been instrumental in clarifying the molecular mechanisms and testing therapeutic strategies for AD. Notwithstanding advances made using current rodent models that exhibit robust Aß amyloidosis and tau pathology, success in translating preclinical drug discoveries to the clinic remains rather disappointing. A major limitation of current mouse models is the lack of robust age-dependent neuronal loss induced by the interaction of amyloid and tau aggregates in their brains, salient neuropathological hallmarks of individuals with AD. To overcome this problem, we propose to characterize a recently created mouse model that not only exhibits Aß amyloidosis and tauopathy, but also displays robust age-dependent neuronal loss. We will use our new mouse model to test our overarching hypothesis that A? induces tau aggregation to initiate synaptic dysfunction and promote neuronal loss. We believe that such a model system will provide the opportunity for the first time to evaluate A? amyloidosis and tauopathy dependent loss of neurons as the principal outcome measure in preclinical testing of mechanism-based therapies, such as those designed to target tauopathy and/or Aß amyloidosis. Toward this goal, we recently generated lines of transgenic mice expressing human four-repeat tau fragment containing the ?K280 mutation (Tau-4R?K280) in the forebrain that not only developed AD-like tau aggregates but displayed memory deficits, age-dependent neuronal loss and brain atrophy. By crossbreeding Tau4R-?K280 mice with a model of Aß amyloidosis, APPswe;PS1?E9 mice, we generated mice that develop both Ab and tau pathologies (Tau608-AP mice). Significantly, both the tauopathy and cell death observed in Tau4R-?K280 mice were greatly accelerated in Tau608-AP mice, which support a model that the presence of Aß could accelerate the development of AD-like tau pathologies and age-dependent neuronal loss. Our proposal thus will allow us to focus attention on several important questions. First, it is not completely clear how accumulation of tau aggregations leads to neuronal loss. Second, it is not known whether and how Aß interacts with tau aggregates to promote the early pathological alterations that leads to synaptic dysfunction and accelerates neuronal loss. Finally, it remains an open question as to whether early interventions designed to target tau will attenuate neuronal loss in the presence of amyloid pathology in AD. In Project 2, we will use our novel mouse models to directly address these important questions. Our proposed studies will identify the mechanisms underlying the interactions between tau and Aß pathology, and provide valuable information to evaluate the effectiveness of targeting tau pathologies.

? DESCRIPTION (provided by applicant): Amyotrophic Lateral Sclerosis (ALS), a fatal adult onset motor neuron disease characterized by selective loss of upper and lower motor neurons, and Fronto-Temporal Dementia (FTD), a common form of dementia characterized by a progressive deterioration in behavior, personality and/or language, share a common disease spectrum. The neuropathology involving Transactivation response element DNA-binding protein 43 (TDP-43) occurs in nearly all cases of ALS and large proportion of FTD, neurodegenerative diseases currently without effective therapy. The overall goals of this proposal are to clarify disease mechanism, validate a novel therapeutic strategy, and develop biomarkers for ALS-FTD. Our recent discovery established that TDP-43, a protein thought to be central in the pathogenesis of ALS-FTD, is a splicing suppressor of non-conserved cryptic exons and that loss of such function leads to down-regulation of a set of mRNA critical for cellular function via nonsense-mediated decay. Supporting the hypothesis that TDP-43 proteinopathy reflects a loss of TDP-43 function, we showed that in brains of ALS-FTD exhibiting TDP-43 proteinopathy, suppression of cryptic exon is impaired. We hypothesize that there exists neuron-specific TDP-43 dependent cryptic exons that would be relevant towards clarifying disease mechanisms to account for the selective vulnerability of neurons in ALS- FTD. We will address this critical question by identifying neuron specific cryptic exons in human neurons lacking TDP-43. We will then confirm whether suppression of such cryptic exons is also compromised in brains of cases of ALS-FTD. We further hypothesize that a specific set of cryptic exons predispose motor or frontal cortex in ALS or FTD cases. To test this possibility, we will determine whether a unique set of cryptic exons is linked either to cases of ALS or FTD. Importantly, we demonstrated in a cell model lacking Tdp-43 that these cryptic exons can be suppressed and cell death prevented by forced expression of a hybrid protein comprised of the N-terminal domain of TDP-43 fused to the splicing repressor domain of a well-characterized suppressor. Our findings offer a novel therapeutic strategy to suppress splicing of cryptic exons using this hybrid protein in an effort t ameliorate neurodegeneration in ALS-FTD. We propose to perform a series of preclinical proof-of- principal studies to validate the efficacy of this approach, information that will be critical or translating such a promising therapeutic strategy to the clinic. Biomarkers, particularly pre-symptomatic ones, for patient selection and monitoring of clinical trials remain a critical unmet need. We hypothesize that neoantigens against expressed cryptic exons represent novel biomarkers for ALS and FTD. We will generate monoclonal antisera to novel epitopes corresponding to several cryptic exons and evaluate their potential as pre-symptomatic biomarkers. Together, results from our proposed studies will have important implications for understanding disease mechanism, validating therapeutic strategy and developing functional biomarkers for ALS-FTD.

Alzheimer's Disease-Related Dementias (ADRD) is a group of progressive neurodegenerative disorders with mid to late life onset such as mixed etiology dementias (MED) including Alzheimer's disease (AD) with TDP-43 pathology. To clarify disease mechanisms and identify therapeutic targets, a new mouse models that replicate combinations of co-occurring pathological features of human dementia will be critical. It is well recognized that AD cases with TDP-43 pathology, as compared to those without, showed a greater decline in cognitive deficits. However, the molecular mechanisms underlying such contribution of TDP-43 remains elusive. We showed that TDP-43 pathology is due to loss of TDP-43's nuclear function, particularly its ability to repress cryptic exon splicing, that precedes formation of TDP-43 cytoplasmic aggregates. That splicing repression is a major role of TDP-43 in forebrain neurons led us to hypothesize that loss of TDP-43 repression exacerbates neurodegeneration and cognitive deficits. To address this question, we will take advantage of 1) our model lacking TDP- 43 in forebrain neurons which exhibits age-dependent neuron loss, cognitive deficits and defects in prelimbic cortical circuits; and 2) our tau model which show, in presence of amyloid plaques, tauopathy- dependent neuron loss, to develop a novel MED model that would exhibit beta-amyloidosis and tauopathy along with compromised TDP-43 repression in forebrain neurons, pathological features that mimic AD with loss of TDP-43 repression. By employing a comprehensive set of molecular, pathological, neuronal circuit and behavioural/cognitive approaches, we will rigorously characterize the MED mice across their lifespan, providing a highly innovative and instructive model to clarify disease mechanism and identify therapeutic targets.

Alzheimer's Disease-Related Dementias (ADRD) is a group of progressive neurodegenerative disorders with mid to late life onset such as FTLD-TDP or mixed etiology dementias (MED) including Alzheimer's disease (AD) with TDP-43 pathology. Human studies support the idea that loss of TDP-43 splicing repression underlies neuron loss in these disorders. We recently established that splicing repression is a major function of TDP-43 and validated TDP-43 repression as a promising therapeutic target for FTLD-TDP. By employing a comprehensive set of molecular, pathological, neuronal circuit and behavioral/cognitive approaches, we will functionally validate this type of AAV gene therapeutic strategy to complement TDP-43 repression using both in vivo mouse models and in vitro human iPSC derived neurons in the UG3 phase of the application. Upon meeting the Milestones for transition from UG3 to UH3 phase, we will determine: 1) the optimal dose of AAV gene expression the TDP-43 related repressor required to attenuate neuron loss while limiting any untoward side effects associated with long term exposure of this gene product; 2) the benefit of this AAV gene therapeutic strategy using our mouse model lacking TDP-43 in forebrain neurons in terms of attenuation of altered neuronal circuits, cognitive and behavioural deficits, and neurodegeneration; and 3) ability of AAV gene therapy to restore TDP-43 repression in cortical neurons derived from human iPSCs. Functional validation of TDP-43 repression will address a great unmet need for this type of ADRD.

PROJECT SUMMARY Sporadic Inclusion Body Myositis (IBM) is the most common muscle disease in adults over age 50, yet the cause of the disease is unknown, and there are no treatments. To better understand the pathogenesis of this disease and to identify therapeutic targets, we have developed two novel mouse models. First, by implanting human IBM biopsy tissue into immunocompromised mice, we have developed a human xenograft model that recapitulates key features of the disease including atrophy, endomysial inflammation, protein aggregates, and TDP-43 pathology. Second, by deleting TDP-43 specifically in skeletal muscle of mice, we replicated key IBM features including atrophy, rimmed vacuoles and protein aggregates. The goal of this proposal is to better understand the pathogenesis of IBM using these two mouse models and to validate therapeutic targets. We believe we have created the first clinically relevant mouse models of sporadic IBM. Such models have the potential to be useful for IBM studies including mechanistic studies and target validation. This project will independently and rigorously test both the role of the inflammatory response and the role of compromised TDP-43 splicing repression in IBM pathogenesis.