Brian C. Kraemer, Ph.D. - US grants

DESCRIPTION (provided by applicant): The lesions seen in the degenerating neurons of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U) consist primarily of the TDP-43 protein. How aggregated TDP-43 protein causes neuronal dysfunction and neurodegeneration is poorly understood. However, mutations in the gene encoding TDP-43 cause some forms of familial ALS proving that abnormal TDP-43 causes neurodegeneration. Furthermore, TDP-43 positive pathological lesions appear in postmortem samples from several different neurodegenerative disorders including FTLD-U, ALS, Alzheimer's disease (AD), Parkinson's disease, dementia with Lewy bodies, and Guam amyotrophic lateral sclerosis/Parkinson's dementia. To model TDP-43 neurotoxicity in a simple animal model, we have transgenically expressed human TDP-43 protein in the neurons of the nematode worm, C. elegans. Expression of human TDP-43 in worm neurons causes neuronal dysfunction and accumulation of nuclear inclusions consisting of aggregated insoluble TDP-43 protein. We have previously reported a C. elegans model for the tau pathology seen in human tauopathy disorders including and AD. We propose to use the same methodologies to characterize the new model for TDP-43 proteinopathy with a focus on the mechanisms of TDP-43 mediated neurotoxicity and how the localization of TDP-43 protein alters its toxicity. The neuronal consequences of tau and TDP-43 protein expression will be investigated by profiling the transcriptional and post-translational responses to neurotoxicity. Transcriptional changes will be monitored using an mRNA tagging/microarray hybridization approach to identify genes up-regulated and down-regulated in response to neurotoxicity. The, genetic pathways that normally act to protect against the neurotoxic effects of TDP-43 will be identified using a genome-wide RNAi screening approach. Genes normally required for tau neurotoxicity will also be tested for their ability to modify the neurotoxicity of TDP-43. The long term goal of this work is to develop neuroprotective strategies for neurodegenerative disorders with TPD-43 and tau protein deposits. PUBLIC HEALTH RELEVANCE: Deposits of abnormally folded aggregated protein are found in a number of disorders affecting the nervous system including Alzheimer's disease and amyotrophic lateral sclerosis. We have generated different animal models for the nerve cell death seen in these disorders using the worm, C. elegans to understand how protein misfolding can causes disease. Our current work focuses on the newly identified aggregating protein TDP-43, a protein that forms misfolded protein deposits in many different nervous system disorders.

DESCRIPTION (provided by applicant): Developing neuroprotective strategies for proteinopathy. The lesions seen in the degenerating neurons of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin positive inclusions (FTLD-U) consist primarily of abnormal TDP-43 protein. Pathological TDP-43 containing deposits associated with motor neuron neurodegeneration are the hallmark pathology in over 90% of ALS cases, including both familial and sporadic types. How aggregated, ubiquitinated and phosphorylated TDP-43 protein causes neuronal dysfunction and neurodegeneration remains incompletely understood. This work focuses on extending previous studies to complete the molecular dissection of the mechanisms causing neurodegeneration in ALS and FTLD. In the previous funding period we characterized a C. elegans model of ALS mutation driven TDP-43 proteinopathy and investigated the molecular, cellular, and genomic basis of TDP-43 neurotoxicity. We identified phosphorylation of TDP-43 at serines 409/410 as a critical molecular species driving neurotoxicity, and identified kinases modulating neurodegeneration by controlling the accumulation of phosphorylated TDP-43. The specific aims of this competitive renewal are: 1) Determine the relative toxicity of phosphorylated wild type TDP-43 and the role of kinase activation in the genesis of phosphorylated TDP-43; 2) Identify the cellular machinery responsible for detoxifying phosphorylated TDP-43 3) Dissect the mechanisms by which Ubiquilin mediates TDP-43 neuropathology and neurodegeneration. The development of neuroprotective strategies for TDP-43 related neuropathology in ALS and FTLD is the long term objective of this work. By completing the proposed experiments we will construct additional models of sporadic ALS/FTLD, address the critical question of whether or not pS409/410 TDP-43 is a neurotoxic species in mammals, dissect the molecular mechanism mediating TDP-43 toxicity and capitalize on this information to develop new translationally relevant neuroprotective strategies for targeting TDP-43 neurotoxicity.

? DESCRIPTION (provided by applicant): Neuronal lesions containing abnormal aggregated tau protein are one of the diagnostic hallmarks of Alzheimer's disease (AD), related tauopathy disorders, and advanced aging of the brain. How aggregated tau leads to the dysfunction and loss of neurons in AD patients remains enigmatic, although neuronal dysfunction and loss clearly causes dementia. To better understand how abnormal tau contributes to neurodegeneration in AD and other tauopathies, we established a transgenic model in C. elegans for neurodegeneration driven by human tau aggregation. Through investigation of the genes involved in tau neurotoxicity, we have identified XBP1, the master transcriptional regulator of the unfolded protein response (UPR). ER stress and activation of the UPR have clearly been implicated in human tauopathy disorders by other laboratories although the functional consequences of UPR activation on tau pathology remain unclear. We have leveraged our C. elegans model of tauopathy to dissect the functional role of the UPR in tau pathology. While C. elegans lacking XBP1 function are viable under normal conditions, when challenged with ER stress they die. Likewise C. elegans expressing human tau but lacking XBP1 are not viable. These findings suggest tau pathology induces ER stress, and UPR activation protects against tauopathy. To test this hypothesis, we upregulated the UPR in the absence of ER stress specifically in neurons using a constitutively active XBP1 transgene. Preliminary data indicates constitutively active XBP1 suffices to protect against tauopathy related phenotypes bolstering the argument for the UPR acting to protect against tauopathy. The data gathered to date do not address the mechanism of how UPR activation protects against tauopathy. Given the high level of conservation of the UPR system between mammals and C. elegans, we propose to utilize the existing model and transgenes to dissect the mechanism by which the UPR protects against tau neurotoxicity. While the mechanism appears to be mediated through XBP1 target genes, the molecular underpinnings remain unclear. The Specific Aims of this project are to: identify which XBP1 target genes modulate tau mediated neuronal dysfunction and neurodegeneration, dissect the mechanism by which these target genes regulate tauopathy, and determine whether or not there is comprehensive engagement of all three branches of the UPR in protecting against tau toxicity. Completion of the project as proposed will identify specific genes and pathways normally functioning to detoxify tau pathology. Identification of new regulators of tauopathy will provide the field with additional points of intervention in tauopathy which will set the stage for future translational studies of ta mediated neurodegeneration and UPR mediated neuroprotection in mouse models.

Abstract: Neuronal lesions containing abnormal aggregated tau protein constitute one of the diagnostic hallmarks of Alzheimer's disease, related tauopathy disorders, and advanced aging of the brain. In Alzheimer's disease, tau neuropathology correlates with severity of dementia. However the mechanisms by which aggregated tau leads to the dysfunction and loss of neurons in Alzheimer's disease patients remain enigmatic. We previously demonstrated that a conserved gene called sut- 2/MSUT2 controls tau aggregation and toxicity in C. elegans and human cells. Preliminary studies have demonstrated that MSUT2 controls neuronal susceptibility to tau toxicity in the mammalian brain. The proposed work will verify these findings and explore the molecular underpinnings by which MSUT2 acts to modulate tauopathy disease mechanisms. Although MSUT2 appears to bind RNA, the molecular mechanisms of MSUT2 modulation of tauopathy remains unclear. The Specific Aims of this project are to: 1) Characterize the consequences of MSUT2 knockout in mouse models of tauopathy. 2) Determine the effect of increased MSUT2 activity on tau neuropathology and behavioral phenotypes in mice. 3) Dissect the molecular mechanisms of MSUT2 modulation of tauopathy. Completion of the project as proposed will demonstrate the importance of MSUT2 in tauopathy. We will also gain significant understanding of the molecular mechanisms involved in MSUT2 modulation of tau pathology in diverse organisms ranging from C. elegans to humans. This knowledge will set the stage for future translational studies by providing novel candidate therapeutic targets for pharmacological intervention.

Neuronal lesions containing either abnormal pathological tau or abnormal pathological TDP-43 protein characterize frontotemporal lobar degeneration (FTLD). How aggregated, ubiquitinated and phosphorylated TDP-43 protein causes neuronal dysfunction and neurodegeneration is unknown. Likewise, the mechanisms of tau toxicity remain incompletely understood, but hyperphosphorylated, aggregated, and oligomeric tau species exhibit neurotoxicity. This work focuses on extending our previous studies of TDP-43 proteinopathy to complete the molecular genetic dissection of the mechanisms causing neurodegeneration in FTLD-tau and FTLD-TDP. In the previous funding period we showed activation of tau tubulin kinase 1 (TTBK1) stimulates the production of phosphorylated TDP-43, which drives neurodegeneration. We also demonstrated that Calcineurin is the major TDP-43 phosphatase responsible for detoxifying phosphorylated TDP-43. Surprisingly, both TTBK1 and calcineurin play a similar role in the genesis of pathological tau protein in disease. Taken together these findings support the premise of this application that common molecular mechanisms may underpin both TDP-43 and tau pathology in FTLD. The specific aims of this competitive renewal are: 1) Identify genes with protective variants preventing tau or TDP-43 proteinopathy; 2) Conduct comparative analysis of protective genetic variants for impact in molecular mechanisms of neurodegeneration; 3) Assess protective variants for translational potential as therapeutic targets. Completion of the proposed work will enable the development of neuroprotective strategies targeting pathological tau and TDP-43 in FTLD. The experiments proposed here will define new genes and pathways mediating tau and TDP-43 toxicity. The work will also delineate the relationships between the identified genes and pathways to inform development of new therapies for FTLD.

In Alzheimer's disease (AD), tau neuropathology correlates with severity of dementia. However, interventions for AD and related dementias are limited to treatment of symptoms that do not directly alter tau pathology or the resultant neurodegeneration. This underscores the need for tau-targeted disease-modifying therapeutics. Furthermore, the results from amyloid-targeted clinical trials in AD patients suggest that achieving cognitive preservation in AD may require tau-targeted therapy in conjunction with the removal of amyloid. Our work has demonstrated that MSUT2 controls neuronal susceptibility to tau toxicity in the mammalian brain. The mechanism of MSUT2 modulation of tauopathy appears to involve MSUT2 binding to poly(A) RNA and its modulation of RNA polyadenylation. The identification of small molecules that inhibit MSUT2 from binding to poly(A) RNA will provide a pharmacological means of intervening against tauopathy. We hypothesize that small- molecule antagonism of MSUT2/poly(A) RNA-binding after onset of pathological tau deposition will reverse the toxic consequences of pathological tau. The specific aims of this proposal will identify and develop potent and specific brain-penetrant MSUT2 inhibitors and use them to dissect the temporal and mechanistic relationship between MSUT2 activity and tauopathy. Completion of the project as proposed will also further demonstrate the importance of MSUT2 in Alzheimer's disease. This knowledge will set the stage for future translational studies by both generating MSUT2 specific tool compounds and validating a novel candidate therapeutic target for pharmacological intervention in tauopathy disorders.

Pathological deposition of abnormal aggregated tau protein in neurons is one of the diagnostic hallmarks of Alzheimer's disease (AD) and related dementia (ADRD). How pathological tau causes dysfunction and degeneration of neurons remains an enigma. However, neuronal dysfunction and neurodegeneration clearly cause dementia. To understand how abnormal tau contributes to neurodegeneration in AD and ADRD, we established a transgenic model in C. elegans for neurodegeneration driven by human tau aggregation. In our previous work, we identified XBP-1, the master transcriptional regulator of the unfolded protein response (UPR), as a critical regulator of pathological tau accumulation and toxicity. ER stress and activation of the UPR have clearly been implicated in human tauopathy disorders by other laboratories although the functional consequences of UPR activation on tau pathology remain unclear. We have leveraged our C. elegans model of tauopathy to dissect the functional role of the UPR in tau pathology. We have found that tau pathology can induce ER stress, and that UPR activation protects against tauopathy through XBP-1s. We hypothesize that XBP-1s target genes can modulate accumulation and clearance of pathological tau. To test this hypothesis, we upregulated the UPR in neurons in the absence of ER stress, using a constitutively active XBP-1s expressing transgene. Transcriptomic studies of this system have revealed key XBP-1s target genes that modulate tauopathy and cross talk with other regulatory branches of the UPR (ATF6 and PERK branches). Given the high level of conservation of the UPR system between mammals and C. elegans, we propose to utilize the existing model and transgenes to dissect the mechanism by which the UPR protects against tau neurotoxicity. The Specific Aims of this project are to: 1) Identify the molecular mechanisms of XBP1s mediated suppression of tauopathy; 2) Examine UPR branch crosstalk contributing to tau clearance and ER associated degradation, 3) Address the disease relevance of XBP-1s target genes to neurodegeneration in both human disease and mouse models of tauopathy. Completion of the project as proposed will inform the molecular mechanisms by which the UPR participates in tauopathy. We will also explore the neuroprotective translational potential of XBP-1s mediated tau clearance in the mammalian brain.