Gail V. Johnson - US grants

The process, rate and extent of phosphorylation of cytoskeletal proteins are of fundamental importance in normal neuronal function and are likely candidates for pathological dysfunction in certain neurodegenerative disorders, especially Alzheimer's disease. The first goals of this proposed investigation are: 1) to identify the sites on the heat-stable microtubule-associated proteins (MAPs), MAP-2 and tau, that are phosphorylated in vivo, 2) to determine the in vivo rates at which the phosphates turnover on these MAPs, and 3) access the physiological relevance of certain phosphate-transferring enzymes by comparing the sites on MAP-2 and tau that are phosphorylated in vivo with those sites acted upon in vitro by catalytic unit of cyclic AMP-dependent protein kinase, insulin receptor kinase and phosphatase 2B (calcineurin). The importance of this study lies in the more complete delineation of the features of in vivo phosphorylation and is accomplished by using a unique combination of methods recently applied in this laboratory. These techniques include: radiolabeling of phosphoproteins in vivo by intracerebroventricular (icv) administration of 32Pi, focused-beam microwave irradiation (a procedure that rapidly inactivates brain enzymes in situ) to sacrifice animals and isolation of the MAPs by immunoprecipitation with monoclonal antibodies. Subsequent to characterization of the in vivo phosphorylation of MAP-2 and tau, the relationship between specific phosphorylation states of these MAPs and their function(s) will be determined. This objective will be approached by measuring the ability of MAP-2 and tau in defined states of phosphorylation to bind to microtubules and to promote microtubule assembly. The results obtained will provide crucial information about the normal in vivo phosphorylation of MAP-2 and tau and help clarify the role of aberrant cytoskeletal protein phosphorylation in pathological conditions.

DESCRIPTION (Investigator's abstract): Calcium plays a fundamental role in the maintenance and modulation of the neuronal cytoskeleton. Disruptions in the calcium homeostatic mechanisms are likely to contribute to the pathological dysfunction of the cytoskeleton in certain neurodegenerative diseases, especially Alzheimer's disease. The microtubule-associated protein, tau is a critically important phosphorylated cytoskeletal protein necessary for the maintenance of neuronal structure and function. Tau is also the predominant protein of paired helical filaments, which are the primary components of neurofibrillary tangles (NFTs) in Alzheimer's disease. Tau in the NFTs is in an extremely insoluble form and is also abnormally phosphorylated. The overall goals of this proposal are to use in vitro and in situ approaches to examine the modification of tau by two calcium-dependent enzymes, the protease, calpain, and the cross-linking enzyme, transglutaminase, and to determine how these processes are regulated by site specific phosphorylation of tau. Our comprehensive working hypothesis is that disruption of calcium homeostasis contributes to the pathology of Alzheimer's disease by disregulating these enzymes leading to dysfunctional modifications of tau, resulting in abnormal cytoskeletal structure and function. The specific goals of this proposal are to test the following hypothesis: (1) Tau is a substrate for the calcium-activated, cross-linking enzyme, transglutaminase, which leads to the formation of SDS-insoluble Alz-50 positive, filamentous polymers of tau, (2) transglutaminase is present and can be activated in cultured cells expressing a neuronal phenotype and the enzyme is modulated in situ, (3) transglutaminase-catalyzed cross-linking of tau is modulated by site-specific phosphorylation, (4) tau is a substrate for calpain in situ, and this is modulated by site- specific phosphorylation, (5) the calpain-induced breakdown products of tau are not substrates for transglutaminase, and (6) tau which has been crosslinked by transglutaminase is no longer susceptible to calpain hydrolysis. This proposal represents an extension of our highly successful previous studies examining the phosphorylation of tau by specific protein kinases and determining how these site-specific phosphorylation modulate the susceptibility of tau to proteolysis by calpain. In addition, we reported the novel finding that transglutaminase catalyzes the formation of SDS-insoluble, Alz-50 reactive, filamentous polymers of bovine tau. The studies in this proposal will extend those findings using human tau and purified recombinant human tau isoforms to examine transglutaminase- mediated crosslinking. By examining the interrelationships between phosphorylation, calpain proteolysis and the transglutaminase-mediated crosslinking of tau, we will obtain important new information and be able to form specific hypotheses as to the mechanisms involved in the formation of insoluble NFTs in Alzheimer's disease.

In order for the neuronal cytoskeleton to function properly, intracellular calcium levels and oxidative states must by maintained within specific limits. Disruptions in the calcium homeostatic mechanisms, or conditions of heightened oxidative stress are likely to contribute tot he pathological dysfunction of the cytoskeleton in certain neurodegenerative diseases, especially Alzheimer's disease (AD). These abnormal conditions have been suggested to result in the disregulation of the activities of specific enzymes, including specific protein phosphatases and kinases, which ultimately contribute to abnormal cytoskeletal rearrangements and the death of the neuron. The microtubule-associated protein tau is a critically important phosphorylated cytoskeletal protein necessary for the maintenance of neuronal structure and function. Tau, in a hyperphosphorylated form, is also the predominant protein of paired helical filaments (PHFs) which are found in Alzheimer brain. The overall goals of this proposal are to examine the modulation of the phosphorylation state of tau by calcium- dependent processes, and to determine the role of oxidative/reductive conditions in mediating the metabolism and function of tau. Our overall hypothesis is that oxidative stress and disrupted calcium homeostasis contribute to the pathology of AD by altering the activities of specific enzymes which leads to modifications of tau which are pathological. The goals of this proposal are to test the following hypotheses: (1) During programmed cell death, specific calcium-mediated alterations occur in the phosphorylation state of tau as a part of the cascade leading to cell death, (2) heightened oxidative stress results in alterations in the phosphorylation state of tau, (3) compromised mitochondrial function, which produces an increase in intracellular calcium levels, results in alterations in the phosphorylation state of tau, (4) after transient calcium-stimulated dephosphorylation, tau becomes more extensively hyperphosphorylated and (5) specific mechanisms eliciting increases in intracellular calcium and heightened oxidative stress result in calcium- mediated alterations in the localization of tau within the cell. In preliminary studies, treatment of NGF-differentiated PC12 cells with low levels of the calcium ionophore, A23187 resulted in an increase ina the phosphorylation of tau, while increasing oxidative stress by depleting reduced glutathione resulted in a dephosphorylation of tau. Additionally, treatment of rat brain cortical slices with an inhibitor of oxidative phosphorylation or the excitatory amino acid, N-methyl-D-aspartate resulted in a site-selective dephosphorylation of tau by the calcium/calmodulin- dependent phosphatase, calcineurin (phosphatase 2B). These and other preliminary findings clearly demonstrate that the studies outlined in this proposal will yield important new information on how the phosphorylation state of tau is modulated in response to calcium and oxidative stress, and the functional consequences of these alterations.

DESCRIPTION: (Verbatim from the Applicant's Abstract) During apoptosis the cytoskeleton of the cell undergoes dynamic alterations which result in the characterisitic morphological changes common to most apoptotic cells. Recently, using the classical paradigm of inducing apoptosis differentiated PC12 cells by withdrawal of serum and nerve growth factor (NGF), we demonstrated that the neuronal cytoskeletal protein tau is hyperphosphorylated at specific epitopes during apoptosis. Further, there are associated functional changes, as the microtubule-bindng capacity of tau from apoptotic cells is significantly reduced, and it is restored after dephosphorylation. This demonstrates directly that the increased phosphorylation of tau in cells undergoing apoptosis impairs the function of tau, and thus may contribute to the microtubule instability and the cytoskeletal based morphological changes of apoptotic cells. These findings are exciting both for the insight they provide for understanding the drastic morphological changes associated with apoptosis, and for the potential links between apoptosis in Alzheimer's disease and hyperphosphorylated tau. There is increasing evidence that apoptotic-like processes may contribute to the neuronal death in Alzheimer's disease, as well as other neurodegenerative disorders. In Alzheimer's disease brain, extensively hyperphosphorylated tau forms paired helical filaments (PHFs). In addition, the microtubule binding of PHF-tau is impaired, but can be restored at least partially by dephosphorylation. Thus, apoptosis during Alzheimer's disease may contribute to the formation of hyperphosphorylated tau that accumulated in this disease, thereby further emphasising the need to clarify the mechanisms that control tau phosphorylation in these conditions. In AD brain, cdc2, casein kinase1d (CK1d ) and cdk5 are elevated, and the investigators have found them to be increased during apoptosis as well. Considering these and other findings, the comprehensive working hypothesis is that during apoptosis tau is hyperphosphorylated at specific sites by specific protein kinases and this hyperphosphorylation results in compromised tau function, which contributes to the structural changes that occur during apoptosis. Elucidation of the changes in tau phosphorylation that occur during apoptosis will contribute towards the understanding of the processes that result in the hyperphosphorylation of tau in AD and other neurodegenerative disorders. The specific aims of this proposal are to test the hypotheses that: (1) during apoptosis tau is phosphorylated at specific sites and the increases in the activities of cdc2, CK1d and cdk5 are essential components of this process, (2) that the specific sites on tau that are phosphorylated in apoptotic cells modulate tau function and localization, and (3) that during apoptosis, tau with frontal temporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17) mutations is differentially phosporylated and localized compared to wild type tau.

Tissue transglutaminase (tTG) is a novel, dual function protein that is both a calcium-dependent transamidating enzyme and a signal transducing GTP-binding protein (Galpha h). As a transamidating enzyme, tTG catalyzes the formation of isopeptide bonds between specific substrate proteins to produce insoluble polymeric structures. A defining characteristic of Alzheimer's disease brain is the presence of intracellular (neurofibrillary tangles [NFTs]) and extracellular (senile plaques) filamentous proteinaceous aggregates that are highly insoluble. Studies from this, and other laboratories, have demonstrated that tau, the major protein of the NFTs, and Abeta (1-40), a primary peptide of the senile plaques, are both excellent in vitro substrates of tTG. Recent studies from the applicants laboratory have demonstrated that in cerebral cortex, where NFTs and senile plaques are prevalent, but in cerebellum which is virtually devoid of these lesions, tTG levels and TG activity are elevated significantly in Alzheimer's disease brain compared to age-matched controls. In addition, it has been hypothesized that tTG maybe involved in the neurodegeneration of codon reiteration diseases, such as Huntington's disease, by facilitating the formation of insoluble neuronal inclusions. These and other findings indicate that tTG could contribute to the formation of the insoluble, pathological lesions in certain neurodegenerative disorders. The focus of this proposal, which is a competing continuation, is on investigating the direct and indirect in situ regulation of tTG, predominantly by calcium and GTP, and how these processes may be disrupted, especially in conditions associated with Alzheimer's disease. This focus on the modulation of tTG, in situ represents a significant advance compared to the many previous in vitro studies. The applicants comprehensive working hypothesis is that in situ tTG is tightly regulated, and that perturbations of these regulatory processes results in inappropriate increases in the levels and transamidating activity of tTG and this contributes to the neurodegenerative processes of Alzheimer's disease. In this proposal the majority of experiments will be carried out in human neuroblastoma cells, although primary cell cultures of rat cerebral cortical neurons, as well as hippocampal neurons, will also be used in some studies. The goals of this proposal are to test the following hypotheses (1) that GTP and calcium work in concert to regulate tTG activity through direct and indirect mechanisms, (2) that receptor-mediated mobilization of calcium from the endoplasmic reticulum (ER) plays a significant role in modulating the transamidating activity of tTG, (3) that activation of the transamidating activity of tTG results in the modification of tau, and these modifications are associated with specific alterations in the metabolism, function and subcellular distribution of tau, (4) that GTP modulates tTG interactions with specific proteins which direct the localization and determine the function of tTG (i.e., as a transamidating enzyme or signal transducing G protein), and (5) that Abeta and/or Alzheimer's presenilin mutants increase the transamidating activity of tTG by direct and/or indirect mechanisms. These studies will increase our understanding of the regulation of tTG significantly and are likely to provide insight into its putative role in neurodegenerative processes.

DESCRIPTION (provided by applicant): Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder that is caused by a pathological expansion of CAG repeats within the gene encoding for a 350 kD protein called huntingtin. This polyglutamine expansion within huntingtin is fundamental to the pathogenesis of HD, however the mechanisms by which this mutation causes the disease are unknown. One of the leading hypotheses of the etiology of HD is that mutant huntingtin directly or indirectly compromises mitochondrial function resulting in impairment of energy metabolism, increased oxidative damage and eventually neuronal death. Indeed, a marked reduction in the activity of mitochondrial complexes II and Ill, and to a lesser extent complex IV, has been detected in the striatum of subjects with HD. Further, an N-terminal fragment of mutant huntingtin has been localized to the nucleus, and there is data to suggest that mutant huntingtin can alter gene expression. Considering these and other findings, it is of fundamental importance to determine how mutant huntingtin affects mitochondrial function, and further how these changes modulate the cellular toxicity of mutant huntingtin. Our overall working hypothesis is that mutant huntingtin compromises the function of mitochondria which results in altered cellular functions and an increased sensitivity of the neurons to specific stressors. The specific aims of this proposal are to: (1) test the hypothesis that mutant huntingtin impairs mitochondrial function which sensitizes the cells to specific stressors, (2) test the hypothesis that expression of mutant huntingtin results in selective alterations in the expression of mitochondrial proteins involved in energy metabolism and (3) test the hypothesis that impaired mitochondrial function compromises proteasome activity and this results in alterations in huntingtin processing. Further, we hypothesize that this process is exacerbated in cells expressing mutant huntingtin. For these studies we will establish immortalized striatal neurons that inducibly express wild type and mutant huntingtin constructs. These studies will provide critically important data on the effects of mutant huntingtin on mitochondrial function and energy metabolism and provide insight into the mechanisms by which mutant huntingtin impairs mitochondrial function and contributes to the neurodegenerative processes in HD.

DESCRIPTION (provided by applicant): Transglutaminases (TGs) are highly regulated, calcium-dependent enzymes that likely play key roles in several critical processes in the nervous system. TGs catalyze a transamidating reaction that results in the incorporation of polyamines into specific glutatmine residues within proteins, or if a protein-bound lysine is the amine donor, the tTG-catalyzed reaction results in the formation of a crosslink between the glutamine and lysine residues in proteins. This is highly specific reaction and only a limited number of proteins have been identified as in situ substrates of TG. Tissue TG (tTG), a member of this family, is the most abundant TG in the human brain and is found within neurons. The levels of tTG and TG activity are increased in neurodegenerative conditions such as Alzheimer's disease and Huntington's disease, and increases in tTG can facilitate neuronal cell death in response to specific stressors. Further, in a recent study it was found that treatment with cystamine, a drug that inhibits transglutaminase in vitro, prolonged surviv approximately 8-10% in a mouse model of Huntington's disease. These and other results suggest that transglutaminase inhibitors may be beneficial in studying the pathological mechanisms and in the treatment of neurodegenerative diseases such as Huntington's Disease and Alzheimer's Disease. However, there are numerous problems with using cystamine as a drug to treat human neurodegenerative diseases, including the fact that it is a non-specific drug with a very steep toxicity curve. Further, it is a charged molecule that likely does not efficiently cross the blood brain barrier. Therefore it is clear that more selective and efficacious TG inhibitors need to be developed for the study and treatment of neurodegenerative disorders. This laboratory has a longstanding interest in the regulation and function of TGs, and therefore is optimally suited to develop TG assays, both in vitro and in situ, that can be used in high throughput drug screening to identify and develop TG inhibitors that are neuroprotective. The aims of this proposal are to: 1. To develop an assay to measure TG activity for a 96 well format that is robust, reproducible and has a simple readout. 2. To develop an assay to measure TG activity in a cellular model using a 96 well format that is reproducible and easily measured. 3. To validate the assays using pharmacological standards, and then use these assays in an initial screen of a small but diverse collection of FDA approved drugs to demonstrate feasibility.

Tau is a microtubule-associated protein that plays a pivotal role in the pathogenesis of Alzheimer's disease (AD). Site-specific phosphorylation regulates tau function and in AD brain tau is abnormally phosphorylated, is functionally impaired, is abnormally cleaved and accumulates as filamentous structures, events that likely impair neuronal function. Although it is clear that all these changes in tau take place in AD brain, the sequence of events and their contribution to neuronal cell death in AD has not been clearly delineated. Our long range goal is to fully elucidate the sequence of pathological processes that result in abberant posttranslational processing of tau and how these events compromise neuronal survival in AD brain so that effective therapeutics can be developed. The objective of this project is to determine how the phosphorylation of specific sites on tau affect tau-microtubule interactions, tau oligomerization, tau interaction with molecular chaperones (and thus its functional state), and tau turnover through the ubiquitin-proteasome system.The central hypothesis of this application is that a cascade of tau phosphorylation events in which glycogen synthase kinase 3 (GSK3) plays a key role, initially results in impaired microtubule binding and subsequently, in conjunction with caspase-cleavage, results in tau aggregation. These processes disrupt the ability of chaperones to regulate tau function and turnover and ultimately result in increased cellular toxicity. The rationale for these studies is that once it is known how specific posttranslational processes negatively impact tau function and contribute to neuronal dysfunction and death, then therapeutic targets for the treatment of AD can be identified. The objectives of this project will be accomplished through three specific aims that test the hypotheses: (1) that phosphorylation of key sites on tau impairs tau function, increases tau-tau interactions and plays a central role in tau's facilitation of cell death processes, (2) that caspase cleavage of tau increases its propensity to self-associate and decrease cell survival, and that these effects are exacerbated by phosphorylation of specific sites on tau, and (3) that chaperones and the E3 ubiquitin ligase CHIP control tau conformation and proteasome targeting, that these interactions are regulated by site-specific tau phosphorylation and that tau cleaved by caspases accumulates because it is no longer an efficient substrate of the proteasome system. Overall these are important and timely studies that will clearly define the role of site-specific phosphorylation, caspase cleavage and the impact of chaperones and the ubiquitin-proteasome system on pathological changes in tau that contribute to the disease processes in AD.

DESCRIPTION: The Molecular Detection Core (MDC) is designed to fill an unmet need in the UAB neuroscience community for sensitive, automated immunohistochemistry (IHC) and in situ hybridization (ISH) detection. The centerpiece of the facility will be a Ventana Discovery1 System capable of fully automated IHC and ISH applications. This system, combined with well-trained technical support staff, will produce unparaheled high-throughput, reproducible, molecular localization results for both novice and experienced IHC/ISH investigators. The Discovery System is the only commercially-available automated detection instrument capable of ISH and multi-label localization procedures and the MDC will be the first facility of any type at UAB with these capabilities. The MDC will offer both conventional and ultrasensitive Tyramide Signal Amplification-Plus (TSA -Plus) detection procedures developed by Dr. Kevin A. Roth, core director, in collaboration with scientists at Perkin-Elmer Life Sciences. A novel procedure for sensitive and photostable immunofluorescence detection using combined TSA and quantum dot labeling, recently developed by Dr. Roth's laboratory, will also be available. The MDC will also offer both chromogenic and fluorescent detection options as well as perform multi-labeling IHC and ISH procedures. For inexperienced users, MDC personnel will assist investigators in determining optimal fixation conditions, tissue processing techniques, and experimental designs for detection of nervous system-associated molecules of interest. For experienced IHC/ISH investigators, the MDC will provide consistent, high quality labeled samples and assist in the development and implementation of novel detection procedures. The MDC will maintain a website where protocols can be accessed, images shared, and questions asked of other core users. An annual mini-symposium will be sponsored for investigators to present their results to other core users and to help facilitate collaborative research efforts. The overall goal of this core is to provide NINDS supported investigators, and other UAB neuroscience investigators, with state of the art molecular detection techniques so as to more effectively and efficiently achieve their research objectives. We expect the MDC to be an important bridge between pre-existing UAB campus facilities. Thus, we will receive specimen input from any of several tissue processing core laboratories and produce IHC/ISH labeled slides that can be visualized at any of several imaging facilities (both standard and confocal microscopy facilities are readily available). Alternatively, investigators may submit cell and/or tissue sections prepared in their own laboratory or with MDC-associated tissue processing equipment for IHC/ISH labeling with subsequent visualization performed using microscopes available in the MDC or in their own laboratories. This large degree of experimental flexibility will permit each investigator to "customize" their MDC usage to best meet their individual needs.

[unreadable] DESCRIPTION (provided by applicant): Tau is a microtubule-associated protein that plays a pivotal role in the pathogenesis of Alzheimer's disease (AD). Site-specific phosphorylation regulates tau function and in AD brain tau is abnormally phosphorylated, is functionally impaired, is abnormally cleaved and accumulates as filamentous structures, events that likely impair neuronal function. Although it is clear that all these changes in tau take place in AD brain, the sequence of events and their contribution to neuronal cell death in Alzheimer's disease has not been clearly delineated. Our long range goal is to fully elucidate the sequence of pathological processes that result in aberrant posttranslational processing of tau and how these events compromise neuronal survival in AD brain so that effective therapeutics can be developed. The objective of this project is to determine how the phosphorylation of specific sites on tau affect tau-microtubule interactions, tau oligomerization, tau interaction with molecular chaperones (and thus its functional state), and tau turnover through the ubiquitin-proteasome system. The central hypothesis of this application is that a cascade of tau phosphorylation events in which glycogen synthase kinase 3 (GSK3) plays a key role, initially results in impaired microtubule binding and subsequently, in conjunction with caspase-cleavage, results in tau aggregation. These processes disrupt the ability of chaperones to regulate tau function and turnover and ultimately result in increased cellular toxicity. The rationale for these studies is that once it is known how specific posttranslational processes negatively impact tau function and contribute to neuronal dysfunction and death, then therapeutic targets for the treatment of Alzheimer's disease can be identified. The objectives of this project will be accomplished through three Specific Aims that test the hypotheses: (1) that phosphorylation of key sites on tau impairs tau function, increases tau-tau interactions and plays a central role in tau's facilitation of cell death processes, (2) that caspase cleavage of tau increases its propensity to self-associate and decrease cell survival, and that these effects are exacerbated by phosphorylation of specific sites on tau, and (3) that chaperones and the E3 ubiquitin ligase CHIP control tau conformation and proteasome targeting, that these interactions are regulated by site-specific tau phosphorylation and that tau cleaved by caspases accumulates because it is no longer an efficient substrate of the proteasome system. Overall these are important and timely studies that will clearly define the role of site-specific phosphorylation, caspase cleavage and the impact of chaperones and the ubiquitin-proteasome system on pathological changes in tau that contribute to the disease processes in Alzheimer's disease. [unreadable] [unreadable] [unreadable]

The focus of this application is on elucidating the molecular mechanisms by which transglutaminase 2 (TG2) regulates hypoxia inducible factor (HIF) signaling and attenuates ischemic-induced cell death. In response to ischemia there is an upregulation of genes that can facilitate either cell survival or cell death. Whether cell survival or delayed cell death is the final outcome is dependent on the complement of ischemia-induced genes expressed, which can differ depending on variables such as duration and severity of the insult and the presence of regulatory proteins. HIF, which is composed of the oxygen sensitive HIF1 subunit and the constitutively expressed HIF12, is the transcription factor that is primarily responsible for the upregulation of hypoxic responsive genes. We have demonstrated that TG2 binds HIF12, attenuates HIF signaling, attenuates the expression of specific HIF- responsive genes and protects against ischemic insult. Furthermore, we have compelling data demonstrating that neuronal expression of human TG2 significantly reduces infarct volume in a mouse stroke model. The central hypothesis of this application is that TG2 is a regulator of hypoxic-induced transcriptional signaling, and that TG2 plays a fundamental role in protecting neurons against ischemia-induced cell death. The objectives of this proposal will be met through three specific aims that test the hypotheses: (1) that the TG2- mediated suppression of HIF signaling is dependent on the localization of TG2 to the nucleus, independent of transamidating activity and requires interaction with HIF12, (2) that the protective effects of TG2 against ischemic-induced cell death are independent of transamidating activity, but require nuclear localization and further, that the interaction of TG2 with HIF1¿ is necessary for the protective role of TG2 against ischemic insult, and (3) that TG2 attenuates the delayed cell death that occurs in response to stroke in vivo. Overall, this is an integrated, mechanistic and holistic proposal. In our studies we will be using recombinant proteins to define the interacting domains between TG2 and HIF1¿, cell models to understand how TG2 modulates HIF dependent transcriptional events and attenuates ischemia-induced cell death, and mouse models to delineate the role of neuronal TG2 in decreasing stroke damage. We believe that by using this full complement of approaches we will be able to more completely understand the role of TG2 in ameliorating ischemia-induced cell death. These are exciting and innovative studies in that they not only define a new framework for understanding the function of TG2, but that they also are providing new insights into the regulation of ischemia-induced cell death processes.

DESCRIPTION (provided by applicant): The focus of this proposal is on determining the role of autophagy in the selective clearance of pathological tau and the role of the Nrf2 pathway in regulating this process. We will also be exploring potential therapeutic strategies to both increase the clearance of pathological tau and protect against cellular dysfunction caused by toxic forms of tau. In Alzheimer disease (AD) brain tau is abnormally truncated at Asp421 (tau-¿C), as well as being abnormally phosphorylated, and both of these modifications likely facilitate the formation of toxic conformations that result in compromised neuronal function. Therefore strategies that result in selective clearance of these pathological forms of tau may provide a potential therapeutic approach for the treatment of AD. Previously we provided evidence that full length-tau is preferentially degraded by the proteasome, while tau-¿C is cleared predominantly by macroautophagy. There are also findings suggesting that tau phosphorylated at Ser262/356 may be preferentially degraded through the autophagy pathway, and there is evidence that the autophagy system may be compromised in AD brain which could be a contributing factor to the accumulation of pathological forms of tau. The Nrf2 pathway plays a central role in regulating the expression of cell survival genes. Nrf2 is activated by oxidative stress, as well as other stressors, which results in the expression of cytoprotective genes, including proteins involved in the autophagy pathway. We found that basal Nrf2 activity is lower in cells expressing tau-¿C compared to cells expressing full-length tau; however Nrf2 can still be significantly activated in the tau-¿C cells. In preliminary experiments we found that activation of the Nrf2 pathway resulted in decreased levels of tau-¿C but not of full-length tau, suggesting an activation of autophagy. Intriguingly, we also found that in Nrf2-/- mice there was an accumulation of abnormally phosphorylated tau, as well as insoluble tau species. In AD brain nuclear Nrf2 levels are decreased, and expression of exogenous Nrf2 or activation of the Nrf2 pathway in AD mouse models attenuates learning deficits. Considering these and other studies our overall hypothesis is that pathologically modified forms of tau are preferentially degraded by autophagy, and that activation of the Nrf2 pathway is likely to have beneficial effects in AD in part by facilitating the degradation of pathological forms of tau. The specific aims of this proposal are t test the following hypotheses: 1. That specific pathological forms of tau are preferentially targeted to the autophagy pathway for degradation. 2. That the Nrf2 pathway plays a role in facilitating the degradation of pathological forms of tau. 3. That activation of the Nrf2 or autophagy pathway results in increased survival of cells that express pathological forms of tau.

? DESCRIPTION (provided by applicant): This revised application is for the FOA, Promoting Research in Basic Neuroscience (PAS-15-029). Therefore this is a fundamental basic research proposal that is focused on understanding the role of Bcl-2-associated anthogene 3 (BAG3) in mediating selective autophagy and the turnover of tau in neurons and how these processes change during aging. Selective autophagy is part of a cell's protein quality control (PQC) system, which is the collection of cellular pathways that sense damaged proteins and facilitate their removal. PQC is essential for the maintenance of the appropriate, functional complement of proteins in a cell. Tau is a neuronally enriched multifunctional protein. Given the importance of tau in neuronal function, understanding the PQC processes which regulate phospho-tau species, as well as total tau levels, is of critical importance. There is a growing awareness that selective autophagy, i.e., the specific recognition and targeting of client proteins to the developing autophagosome is essential for effective PQC. Chaperones and co-chaperones, along with the autophagic machinery, are key PQC players in the process of recognizing and removing damaged proteins. The co-chaperone BAG3 is an important player in selective autophagy and thus a pivotal player in the PQC system. Recently we made the exciting discovery that in neurons BAG3 plays a significant role in directing endogenous, soluble tau to autophagy. This is of fundamental importance because in healthy neurons autophagy is constitutively active and plays a significant role in maintaining a functional proteome. BAG3 is a stress-induced protein that increases during normal aging. It has been suggested that during aging the change in cellular demands increases the use of autophagy to maintain protein homeostasis, and that BAG3 plays an essential role in this process. Indeed, it has been documented that during aging BAG3 levels increase in the rodent brain concurrent with an increased dependence on autophagy for proteostasis. Therefore, understanding the role of BAG3 in autophagy and how it functions to facilitate the clearance of soluble tau species is of fundamental significance. The overall hypothesis of this proposal is that selective autophagy plays a significant role in the clearance of tau and that BAG3 plays a central role in facilitating the degradation of soluble tau species through this mechanism. The specific aims of this proposal are: (1) To test the hypothesis that BAG3 modulates autophagy and tau turnover in situ and in vivo, and that during aging the dependence on BAG3 to facilitate autophagy and tau clearance increases, (2) To identify essential features of the BAG3-chaperone complex that mediate tau clearance, and (3) To characterize the mechanisms that regulate BAG3 expression and test their role in autophagy-based clearance of tau in neurons. Currently little is known about the role of BAG3 in mediating autophagy in neurons, as well as how it facilitates the turnover of tau; this application addresses these gaps in our knowledge.

Tau is a central player in the pathogenesis of numerous age-related neurodegenerative diseases, with Alzheimer disease (AD) being the best example. Tau from AD brain is defined by aberrant posttranslational modifications (PTMs), including increases in phosphorylation and acetylation at specific epitopes. The UNDERLYING PREMISE of this proposal is that many of these aberrant PTMs increase tau self-association and toxicity. While the formation of insoluble fibrillary structures is influenced by PTMs, data strongly indicate that soluble forms of abnormally modified tau are the mediators of neuronal toxicity. A CRITICAL KNOWLEDGE GAP is how these modified tau species pathologically impact neuronal function. Additionally, the underlying mechanisms responsible for the increased presence of phosphorylated and acetylated forms in AD have not been fully elucidated. It has been suggested that alterations in how pathologically modified forms of tau are targeted to the autophagic machinery and degraded could be a contributing factor, as well as the fact that they may impair selective autophagy processes. Indeed, there is data indicating that overexpression of AD-relevant forms of tau results in increased levels of fragmented, dysfunctional mitochondria, which may be due to in part due to impaired mitophagy, as well as other perturbations of mitochondrial quality control mechanisms. The OVERALL HYPOTHESIS of this R21 proposal is that AD relevant tau modifications slow tau turnover, impair mitochondrial quality control mechanisms and thus increase the presence of less functional mitochondria with a concomitant increase in oxidative stress and neuronal dysfunction that result in an earlier onset of an aged neuron phenotype. The NOVELTY of this project stems from its use of the model organism C. elegans and its vast repertoire of genetic, transgenic and genomic resources, which has been used extensively to investigate the molecular underpinnings of AD, as well as other tauopathies. Young and older animals will be used to delineate how the presence of these pathological relevant tau species alters these processes as a function of age. The aims of this proposal are to test the hypotheses that: (1) tau acetylated at K274 and K281, tau phosphorylated at T231, or both are not as efficiently turned over as wild type tau and impair mitophagy. C. elegans single copy transgenic models and fluorescent biosensors will be used to address this hypothesis, and (2) tau acetylated at K274 and K281, tau phosphorylated at T231, or both, exacerbate toxicity through chronic mitochondrial stress responses resulting in increased oxidative stress. The relative contribution of these responses to neuronal age-dependent deficits will be further tested using unique genetic resources available in worms. The IMPACT of these studies will be to provide crucial new insights into the mechanisms by which pathological tau species compromise neuronal health and function. They will also provide the foundation for future studies delineating the mechanisms by which specifically modified forms of tau impair neuronal processes and identifying new targets for the development of therapeutics for AD.

Tau is a central player in the pathogenesis of numerous age-related neurodegenerative diseases, with Alzheimer?s disease (AD) being the best example. Tau from AD brain is defined by aberrant posttranslational modifications (PTMs), including increases in phosphorylation and acetylation at specific epitopes. The UNDERLYING PREMISE of this proposal is that specific, disease relevant PTMs impair tau function which negatively impacts neuronal health. While the formation of insoluble fibrillary structures is influenced by PTMs, data strongly indicate that soluble forms of abnormally modified tau are the mediators of neuronal toxicity. There are data indicating that overexpression of AD-relevant forms of tau results in increased levels of fragmented, dysfunctional mitochondria, which may be due to in part due to impaired mitophagy, as well as other perturbations of mitochondrial quality control (MQC) mechanisms. However, a CRITICAL KNOWLEDGE GAP is how these modified tau species, when present at physiologically relevant levels, influence mitochondrial and neuronal health. The OVERALL HYPOTHESIS of this proposal is that tau with AD-relevant PTMs exerts toxic effects through impairing MQC mechanisms. An impaired ability to resolve mitochondrial stress would increase the presence of less functional mitochondria with a concomitant increase in oxidative stress and neuronal dysfunction. The overall result would be an earlier onset of an aged neuron phenotype. The NOVELTY of this project stems in part from its use of single-copy transgenic tau models that avoid overexpression, as well as the inclusion of age as a variable in a genetic model organism. The nematode C. elegans benefits from a vast repertoire of genetic, transgenic and genomic resources that will be leveraged to investigate the molecular underpinnings of AD and to define the precise mechanism through which tau PTMs compromise mitochondrial function and accelerate neuronal aging. Our preliminary data support this approach as worms with single copy expression of tau with specific AD-relevant PTMs in mechanosensory neurons show a significant increase in age-dependent neurodegeneration and a suppression of stress-induced mitophagy. These and other preliminary data provide a strong foundation for the studies in this application. The aims of this proposal are: (1) To determine the impact of AD relevant tau PTMs on mitochondrial stress responses and how this influences healthy aging of neurons, (2) To test the hypothesis that tau with AD relevant PTMs impairs mitochondrial dynamics and mitophagy, and (3) To address whether enhancing MQC is a viable therapeutic avenue. The relative contribution of these responses to neuronal age-dependent deficits will be tested using unique genetic resources available in worms. The IMPACT of these studies will be to provide crucial new insights into the mechanisms by which pathological tau species compromise mitochondrial function and neuronal health.

Protein quality control systems are essential for maintaining neuronal health, with vacuolar dependent pathways playing a primary role in these systems. The endolysosome system is a major contributor to the maintenance of the neuronal proteome, and dysfunction of this system occurs early in the pathogenesis of Alzheimer?s disease (AD) , and likely contributes to the accumulation and mislocalization of tau, which plays a key role in disease pathogenesis. Recent data provide compelling evidence that the stress responsive, multi-domain protein, Bcl-2- associated anthogene 3 (BAG3) plays a role in maintaining protein homeostasis and neuronal health. The expression of BAG3 in specific neuronal populations positively correlates with resistance to the development of tau pathology in AD. Further, our preliminary data indicate that BAG3 is an upstream regulator of vacuolar dependent pathways. The UNDERLYING PREMISE of this proposal is that in neurons, BAG3 plays a critical role in mediating vacuolar dependent pathways, and thus proteostasis and neuronal integrity. The importance of BAG3 in mediating proteostasis is illustrated by the fact that BAG3 not only plays an important role in protecting neurons from the accumulation of pathological tau species, but also likely supports synaptic structure. Nonetheless, our understanding of the mechanisms by which BAG3 regulates vacuolar dependent processes is very limited. Although previous studies have implicated BAG3 as a mediator of autophagy, we are the first to present evidence that BAG3 is a regulator of the endolysosome pathway. Further, our preliminary findings implicate BAG3 as an important modulator of endosomal sorting required for transport (ESCRT) machinery. BAG3 interacts withTBC1D10B, the primary GTPase activating protein (GAP) for Rab35, which facilitates the recruitment of the ESCRT machinery and protein clients to the endosome, as well as mediating other vacuolar dependent processes. The conceptual framework that BAG3 acts upstream of vacuolar pathways through its regulation of Rab35 activity is novel and innovative. The OVERALL HYPOTHESIS of this proposal is that the BAG3-TBC1D10B-Rab35 signaling axis regulates endosome-lysosome function and neuronal health. In the context of this overall hypothesis the specific aims of this proposal are: to test the hypotheses that: (1) the BAG3-TBC1D10B-Rab35 signaling axis regulates ESCRT and the endolysosome pathway, (2) in vivo BAG3 and Rab35 coordinate to mediate neuronal ESCRT/vacuolar processes, and contribute to the maintenance synaptic integrity, and (3) the BAG3-TBC1D10B-Rab35 axis plays a critical role in regulating the clearance of pathological tau species. These studies will be carried out using mouse models and primary neuron cultures. The IMPACT of these studies is that they will provide crucial new insights into the mechanisms by which BAG3 acts as an upstream mediator of the endolysosome systems to maintain a healthy neuron. Overall these studies represent a new, unexplored area of investigation that will increase our understanding of the factors that are essential to maintain a healthy, functional neuronal proteome.

Astrocytes play an indispensable role in maintaining a healthy environment for neuronal function, and in mediating the response of the CNS to injury. Following an injury astrocytes become ?reactive? and mediate both helpful and harmful outcomes depending on their gene expression profile. However the molecular mechanisms that regulate gene expression in astrocytes and thus their response to injury has not been fully delineated. One protein that plays a key role in regulating the response of astrocytes to insults is transglutaminase 2 (TG2). Deletion or depletion of TG2 results in astrocytes taking on a more ?helpful? phenotype, however the underlying molecular mechanisms are unknown. TG2 is a multifunctional protein; it catalyzes a calcium dependent transamidation reaction, binds and hydrolyzes GTP and can function as a scaffold or linker protein. TG2 undergoes large conformational changes mediated by calcium and GTP binding, and its conformation can dictate its function independent of its enzymatic activities. These conformational states are key a factors in determining cell survival/cell death outcomes. There is a growing awareness that TG2 likely regulates gene expression, however the mechanisms of TG2-mediated regulation of gene expression in astrocytes has not been fully explored. One possible mechanism may be by TG2 interacting with, and modulating the function of, a protein that plays a pivotal role in regulating gene expression. One factor that plays a key role in regulating chromatin accessibility and the activity of specific transcription factors is Zbtb7a. Intriguingly, preliminary data indicate that TG2 interacts with Zbtb7a, and binding sites for these transcription factors are in the majority of pro-survival genes that are upregulated in TG2-/- astrocytes. The UNDERLYING PREMISE of this proposal is that TG2 plays a role in regulating gene expression in astrocytes, and thus how they respond to injury. However, a CRITICAL KNOWLEDGE GAP is how TG2 regulates gene expression. The OVERALL HYPOTHESIS of this application is that TG2, in a conformational dependent manner, moderates chromatin accessibility and the gene expression landscape, which contributes to how astrocytes respond to injury. The NOVELTY of this project is that we will be using an integrated ?omic? approach (ATAC-seq, RNA-seq, ChIP-seq) and both in vitro and in vivo models of TG2+/+ and TG2-/- astrocytes to establish the mechanisms by which TG2 in a specific conformation regulates gene expression. The specific aims of this proposal are to test the hypothesis that: (1) the conformation of TG2 play an essential role in determining its ability to regulate chromatin accessibility and gene expression, and (2) TG2 mediates gene expression in astrocytes in part by regulating the function of Zbtb7a. The data generated from these novel and exploratory studies will provide the basis for a future R01 grant application focused on delineating the molecular mechanisms and pathways regulated by TG2 in astrocytes to direct outcomes following CNS injury.