Loss of HDAC6, a novel CHIP substrate, alleviates abnormal tau accumulation

The abnormal accumulation of the microtubule-binding protein tau is associated with a number of neurodegenerative conditions, and correlates with cognitive decline in Alzheimer's disease. The ubiquitin ligase carboxy terminus of Hsp70-interacting protein (CHIP) and the molecular chaperone Hsp90 are implicated in protein triage decisions involving tau, and have consequently been targeted for therapeutic approaches aimed at decreasing tau burden. Here, we present evidence that CHIP binds, ubiquitinates and regulates expression of histone deacetylase 6 (HDAC6). As the deacetylase for Hsp90, HDAC6 modulates Hsp90 function and determines the favorability of refolding versus degradation of Hsp90 client proteins. Moreover, we demonstrate that HDAC6 levels positively correlate with tau burden, while a decrease in HDAC6 activity or expression promotes tau clearance. Consistent with previous research on Hsp90 clients in cancer, we provide evidence that a loss of HDAC6 activity augments the efficacy of an Hsp90 inhibitor and drives client degradation, in this case tau. Therefore, our current findings not only identify HDAC6 as a critical factor for the regulation of tau levels, but also indicate that a multi-faceted treatment approach could more effectively arrest tau accumulation in disease

Truncated stathmin-2 is a marker of TDP-43 pathology in frontotemporal dementia.

No treatment for frontotemporal dementia (FTD), the second most common type of early-onset dementia, is available, but therapeutics are being investigated to target the 2 main proteins associated with FTD pathological subtypes: TDP-43 (FTLD-TDP) and tau (FTLD-tau). Testing potential therapies in clinical trials is hampered by our inability to distinguish between patients with FTLD-TDP and FTLD-tau. Therefore, we evaluated truncated stathmin-2 (STMN2) as a proxy of TDP-43 pathology, given the reports that TDP-43 dysfunction causes truncated STMN2 accumulation. Truncated STMN2 accumulated in human induced pluripotent stem cell-derived neurons depleted of TDP-43, but not in those with pathogenic TARDBP mutations in the absence of TDP-43 aggregation or loss of nuclear protein. In RNA-Seq analyses of human brain samples from the NYGC ALS cohort, truncated STMN2 RNA was confined to tissues and disease subtypes marked by TDP-43 inclusions. Last, we validated that truncated STMN2 RNA was elevated in the frontal cortex of a cohort of patients with FTLD-TDP but not in controls or patients with progressive supranuclear palsy, a type of FTLD-tau. Further, in patients with FTLD-TDP, we observed significant associations of truncated STMN2 RNA with phosphorylated TDP-43 levels and an earlier age of disease onset. Overall, our data uncovered truncated STMN2 as a marker for TDP-43 dysfunction in FTD

TDP-1/TDP-43 Regulates Stress Signaling and Age-Dependent Proteotoxicity in <em>Caenorhabditis elegans</em>

<div><p>TDP-43 is a multifunctional nucleic acid binding protein linked to several neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia. To learn more about the normal biological and abnormal pathological role of this protein, we turned to <em>Caenorhabditis elegans</em> and its orthologue TDP-1. We report that TDP-1 functions in the Insulin/IGF pathway to regulate longevity and the oxidative stress response downstream from the forkhead transcription factor DAF-16/FOXO3a. However, although <em>tdp-1</em> mutants are stress-sensitive, chronic upregulation of <em>tdp-1</em> expression is toxic and decreases lifespan. ALS–associated mutations in TDP-43 or the related RNA binding protein FUS activate the unfolded protein response and generate oxidative stress leading to the <em>daf-16</em>–dependent upregulation of <em>tdp-1</em> expression with negative effects on neuronal function and lifespan. Consistently, deletion of endogenous <em>tdp-1</em> rescues mutant TDP-43 and FUS proteotoxicity in <em>C. elegans</em>. These results suggest that chronic induction of wild-type TDP-1/TDP-43 by cellular stress may propagate neurodegeneration and decrease lifespan.</p> </div

Urine levels of the polyglutamine ataxin-3 protein are elevated in patients with spinocerebellar ataxia type 3

Introduction: Accumulation of polyglutamine (polyQ) ataxin-3 (ATXN3) contributes to the pathobiology of spinocerebellar ataxia type 3 (SCA3). Recently, we showed that polyQ ATXN3 is elevated in the plasma and cerebrospinal fluid (CSF) of SCA3 patients, and has the potential to serve as a biological marker for this disease [1]. Based on these findings, we investigated whether polyQ ATXN3 can also be detected in urine samples from SCA3 patients. Methods: We analyzed urine samples from 30 SCA3 subjects (including one pre-symptomatic subject), 35 subjects with other forms of ataxia, and 37 healthy controls. To quantify polyQ ATXN3 protein levels, we used our previously developed immunoassay. Results: PolyQ ATXN3 can be detected in the urine of SCA3 patients, but not in urine samples from healthy controls or other forms of ataxia. There was a significant statistical association between polyQ ATXN3 levels in urine samples and those in plasma. Further, the levels of polyQ ATXN3 urine associated with an earlier age of SCA3 disease onset. Conclusion: As clinical trials for SCA3 advance, urine polyQ ATXN3 protein has potential to be a useful, non-invasive and inexpensive biomarker for SCA3

Casein kinase II induced polymerization of soluble TDP-43 into filaments is inhibited by heat shock proteins.

Trans-activation Response DNA-binding Protein-43 (TDP-43) lesions are observed in Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Degeneration with ubiquitin inclusions (FTLD-TDP) and 25-50% of Alzheimer's Disease (AD) cases. These abnormal protein inclusions are composed of either amorphous TDP-43 aggregates or highly ordered filaments. The filamentous TDP-43 accumulations typically contain clean 10-12 nm filaments though wider 18-20 nm coated filaments may be observed. The TDP-43 present within these lesions is phosphorylated, truncated and ubiquitinated, and these modifications appear to be abnormal as they are linked to both a cellular heat shock response and microglial activation. The mechanisms associated with this abnormal TDP-43 accumulation are believed to result in a loss of TDP-43 function, perhaps due to the post-translational modifications or resulting from physical sequestration of the TDP-43. The formation of TDP-43 inclusions involves cellular translocation and conversion of TDP-43 into fibrillogenic forms, but the ability of these accumulations to sequester normal TDP-43 and propagate this behavior between neurons pathologically is mostly inferred. The lack of methodology to produce soluble full length TDP-43 and recapitulate this polymerization into filaments as observed in disease has limited our understanding of these pathogenic cascades.The protocols described here generate soluble, full-length and untagged TDP-43 allowing for a direct assessment of the impact of various posttranslational modifications on TDP-43 function. We demonstrate that Casein Kinase II (CKII) promotes the polymerization of this soluble TDP-43 into 10 nm diameter filaments that resemble the most common TDP-43 structures observed in disease. Furthermore, these filaments are recognized as abnormal by Heat Shock Proteins (HSPs) which can inhibit TDP-43 polymerization or directly promote TDP-43 filament depolymerization.These findings demonstrate CKII induces polymerization of soluble TDP-43 into filaments and Hsp90 promotes TDP-43 filament depolymerization. These findings provide rational for potential therapeutic intervention at these points in TDP-43 proteinopathies

Integrated model for stress-induced TDP-1 expression.

<p>TDP-1 has multiple contributions to lifespan and the cellular stress response. (A) Under normal conditions TDP-1 may function in the Insulin/IGFP pathway upstream from DAF-16 to regulate lifespan. (B) A variety of cellular stresses induce TDP-1 expression. TDP-1 induction by oxidative stress and/or the Insulin/IGF pathway is dependent on DAF-16, while induction by osmotic stress is independent of DAF-16. Misfolded proteins activate the unfolded protein response and a secondary consequence of this is the generation of oxidative stress that in turn can induce TDP-1 expression via DAF-16. The downstream consequences of <i>tdp-1</i> expression are dependent on the length and strength of induction. <i>tdp-1</i> mutants are sensitive to stress suggesting that TDP-1 is essential for protection against acute stress. Genetically encoded proteotoxicity from proteins like mutant TDP-43 leads to chronic induction of TDP-1 expression with negative consequences including enhanced neurodegeneration. This model also suggests that mutant proteins may act as a seed for the induction of pathological TDP-1 expression.</p

TDP-1 regulates mutant TDP-43 and FUS proteotoxicity in <i>C. elegans</i>.

<p>(A) <i>TDP-43[A315T];TDP-1::GFP</i> transgenics had shorter lifespans than transgenics expressing mutant TDP-43 alone (P<0.01). (B) <i>FUS[S57Δ];TDP-1::GFP</i> transgenics had shorter lifespans than transgenics expressing mutant FUS alone (P<0.001). Transgenics expressing (C) mutant TDP-43 or (D) mutant FUS along with TDP-1::GFP had accelerated rates of paralysis compared to transgenics expressing either mutant TDP-43 or mutant FUS alone (P<0.001 for mutant TDP-43 or FUS compared to mutant TDP-43;TDP-1::GFP or mutant FUS;TDP-1::GFP respectively). (E) Transgenics expressing mutant TDP-43 or FUS show age-dependent paralysis that is greatly reduced in worms harboring a deletion mutation of endogenous <i>tdp-1</i> (P<0.001). (F) Age-dependent motor neuron degeneration was reduced in <i>tdp-1(ok803);TDP-43[A315T]</i> and <i>tdp-1(ok803);FUS[S57Δ]</i> strains compared <i>to TDP-43[A315T]</i> or FUS[S57Δ] transgenics respectively (*, **P<0.001). Deletion of <i>tdp-1</i> did not affect the proportion of insoluble (G) mutant TDP-43 or (H) FUS proteins in extracts from whole worms. Please also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002806#pgen.1002806.s009" target="_blank">Tables S1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002806#pgen.1002806.s011" target="_blank">S3</a>.</p

Mutant TDP-43 induces HSP-4/BiP expression.

<p>(A to D) are low-resolution photographs of young adult transgenic worms. The images have been converted to black and white and photo-reversed to aid visualization. Difficult to see worms are outlined. (A) Representative image of a young adult worm containing an integrated <i>hsp-4p::GFP</i> transgene under non-stressed conditions. (B) <i>hsp-4p::GFP</i> expression was induced by the chemical ER stressor tunicamycin. (C) <i>hsp-4p::GFP</i> expression was not induced by wild type TDP-43 in a double transgenic <i>TDP-43 WT;hsp-4p::GFP</i> strain. (D) <i>hsp-4p::GFP</i> expression was induced by mutant TDP-43 in a double transgenic <i>TDP-43[A315T];hsp-4p::GFP</i> strain.</p