Pathological tau drives ectopic nuclear speckle scaffold protein SRRM2 accumulation in neuron cytoplasm in Alzheimer's disease.

Several conserved nuclear RNA binding proteins (sut-1, sut-2, and parn-2) control tau aggregation and toxicity in C. elegans, mice, and human cells. MSUT2 protein normally resides in nuclear speckles, membraneless organelles composed of phase-separated RNAs and RNA-binding proteins that mediate critical steps in mRNA processing including mRNA splicing. We used human pathological tissue and transgenic mice to identify Alzheimer's disease-specific cellular changes related to nuclear speckles. We observed that nuclear speckle constituent scaffold protein SRRM2 is mislocalized and accumulates in cytoplasmic lesions in AD brain tissue. Furthermore, progression of tauopathy in transgenic mice is accompanied by increasing mislocalization of SRRM2 from the neuronal nucleus to the soma. In AD brain tissue, SRRM2 mislocalization associates with increased severity of pathological tau deposition. These findings suggest potential mechanisms by which pathological tau impacts nuclear speckle function in diverse organisms ranging from C. elegans to mice to humans. Future translational studies aimed at restoring nuclear speckle homeostasis may provide novel candidate therapeutic targets for pharmacological intervention.

AlphaScreen Identifies MSUT2 Inhibitors for Tauopathy-Targeting Therapeutic Discovery.

Tauopathies are neurological disorders characterized by intracellular tau deposits forming neurofibrillary tangles, neuropil threads, or other disease-specific aggregates composed of the protein tau. Tauopathy disorders include frontotemporal lobar degeneration, corticobasal degeneration, Pick's disease, and the largest cause of dementia, Alzheimer's disease. The lack of disease-modifying therapeutic strategies to address tauopathies remains a critical unmet need in dementia care. Thus, novel broad-spectrum tau-targeted therapeutics could have a profound impact in multiple tauopathy disorders, including Alzheimer's disease. Here we have designed a drug discovery paradigm to identify inhibitors of the pathological tau-enabling protein, MSUT2. We previously showed that activity of the RNA-binding protein MSUT2 drives tauopathy, including tau-mediated neurodegeneration and cognitive dysfunction, in mouse models. Thus, we hypothesized that MSUT2 inhibitors could be therapeutic for tauopathy disorders. Our pipeline for MSUT2 inhibitory compound identification included a primary AlphaScreen, followed by dose-response validation, a secondary fluorescence polarization orthogonal assay, a tertiary specificity screen, and a preliminary toxicity screen. Our work here serves as a proof-of-principle methodology for finding specific inhibitors of the poly(A) RNA-binding protein MSUT2 interaction. Here we identify 4,4'-diisothiocyanostilbene-2,2'-sulfonic acid (DIDS) as a potential tool compound for future work probing the mechanism of MSUT2-induced tau pathology.

Distinct Poly(A) nucleases have differential impact on sut-2 dependent tauopathy phenotypes.

Aging drives pathological accumulation of proteins such as tau, causing neurodegenerative dementia disorders like Alzheimer's disease. Previously we showed loss of function mutations in the gene encoding the poly(A) RNA binding protein SUT-2/MSUT2 suppress tau-mediated neurotoxicity in C. elegans neurons, cultured human cells, and mouse brain, while loss of PABPN1 had the opposite effect (Wheeler et al., 2019). Here we found that blocking poly(A) tail extension with cordycepin exacerbates tauopathy in cultured human cells, which is rescued by MSUT2 knockdown. To further investigate the molecular mechanisms of poly(A) RNA-mediated tauopathy suppression, we examined whether genes encoding poly(A) nucleases also modulated tauopathy in a C. elegans tauopathy model. We found that loss of function mutations in C. elegans ccr-4 and panl-2 genes enhanced tauopathy phenotypes in tau transgenic C. elegans while loss of parn-2 partially suppressed tauopathy. In addition, loss of parn-1 blocked tauopathy suppression by loss of parn-2. Epistasis analysis showed that sut-2 loss of function suppressed the tauopathy enhancement caused by loss of ccr-4 and SUT-2 overexpression exacerbated tauopathy even in the presence of parn-2 loss of function in tau transgenic C. elegans. Thus sut-2 modulation of tauopathy is epistatic to ccr-4 and parn-2. We found that human deadenylases do not colocalize with human MSUT2 in nuclear speckles; however, expression levels of TOE1, the homolog of parn-2, correlated with that of MSUT2 in post-mortem Alzheimer's disease patient brains. Alzheimer's disease patients with low TOE1 levels exhibited significantly increased pathological tau deposition and loss of NeuN staining. Taken together, this work suggests suppressing tauopathy cannot be accomplished by simply extending poly(A) tails, but rather a more complex relationship exists between tau, sut-2/MSUT2 function, and control of poly(A) RNA metabolism, and that parn-2/TOE1 may be altered in tauopathy in a similar way.

Targeting Pathological Tau by Small Molecule Inhibition of the Poly(A):MSUT2 RNA-Protein Interaction.

Neurofibrillary tangles composed of aberrantly aggregating tau protein are a hallmark of Alzheimer's disease and related dementia disorders. Recent work has shown that mammalian suppressor of tauopathy 2 (MSUT2), also named ZC3H14 (Zinc Finger CCCH-Type Containing 14), controls accumulation of pathological tau in cultured human cells and mice. Knocking out MSUT2 protects neurons from neurodegenerative tauopathy and preserves learning and memory. MSUT2 protein functions to bind polyadenosine [poly(A)] tails of mRNA through its C-terminal CCCH type zinc finger domains, and loss of CCCH domain function suppresses tauopathy in Caenorhabditis elegans and mice. Thus, we hypothesized that inhibiting the poly(A):MSUT2 RNA-protein interaction would ameliorate pathological tau accumulation. Here we present a high-throughput screening method for the identification of small molecules inhibiting the poly(A):MSUT2 RNA-protein interaction. We employed a fluorescent polarization assay for initial small molecule discovery with the intention to repurpose hits identified from the NIH Clinical Collection (NIHCC). Our drug repurposing development workflow included validation of hits by dose-response analysis, specificity testing, orthogonal assays of activity, and cytotoxicity. Validated compounds passing through this screening funnel will be evaluated for translational effectiveness in future studies. This preclinical drug development pipeline identified diverse FDA approved drugs duloxetine, saquinavir, and clofazimine as potential repurposing candidates for reducing pathological tau accumulation.

Synergistic toxicity between tau and amyloid drives neuronal dysfunction and neurodegeneration in transgenic C. elegans.

Aggregates of Aß peptide and the microtubule-associated protein tau are key molecular hallmarks of Alzheimer's disease (AD). However, the interaction between these two pathologies and the mechanisms underlying disease progression have remained unclear. Numerous failed clinical trials suggest the necessity for greater mechanistic understanding in order to refine strategies for therapeutic discovery and development. To this end, we have generated a transgenic Caenorhabditis elegans model expressing both human Aß1-42 peptide and human tau protein pan-neuronally. We observed exacerbated behavioral dysfunction and age-dependent neurodegenerative changes in the Aß;tau transgenic animals. Further, these changes occurred in the Aß;tau transgenic animals at greater levels than worms harboring either the Aß1-42 or tau transgene alone and interestingly without changes to the levels of tau expression, phosphorylation or aggregation. Functional changes were partially rescued with the introduction of a genetic suppressor of tau pathology. Taken together, the data herein support a synergistic role for both Aß and tau in driving neuronal dysfunction seen in AD. Additionally, we believe that the utilization of the genetically tractable C. elegans model will provide a key resource for dissecting mechanisms driving AD molecular pathology.

Genome wide analysis reveals heparan sulfate epimerase modulates TDP-43 proteinopathy.

Pathological phosphorylated TDP-43 protein (pTDP) deposition drives neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). However, the cellular and genetic mechanisms at work in pathological TDP-43 toxicity are not fully elucidated. To identify genetic modifiers of TDP-43 neurotoxicity, we utilized a Caenorhabditis elegans model of TDP-43 proteinopathy expressing human mutant TDP-43 pan-neuronally (TDP-43 tg). In TDP-43 tg C. elegans, we conducted a genome-wide RNAi screen covering 16,767 C. elegans genes for loss of function genetic suppressors of TDP-43-driven motor dysfunction. We identified 46 candidate genes that when knocked down partially ameliorate TDP-43 related phenotypes; 24 of these candidate genes have conserved homologs in the human genome. To rigorously validate the RNAi findings, we crossed the TDP-43 transgene into the background of homozygous strong genetic loss of function mutations. We have confirmed 9 of the 24 candidate genes significantly modulate TDP-43 transgenic phenotypes. Among the validated genes we focused on, one of the most consistent genetic modifier genes protecting against pTDP accumulation and motor deficits was the heparan sulfate-modifying enzyme hse-5, the C. elegans homolog of glucuronic acid epimerase (GLCE). We found that knockdown of human GLCE in cultured human cells protects against oxidative stress induced pTDP accumulation. Furthermore, expression of glucuronic acid epimerase is significantly decreased in the brains of FTLD-TDP cases relative to normal controls, demonstrating the potential disease relevance of the candidate genes identified. Taken together these findings nominate glucuronic acid epimerase as a novel candidate therapeutic target for TDP-43 proteinopathies including ALS and FTLD-TDP.

Activity of the poly(A) binding protein MSUT2 determines susceptibility to pathological tau in the mammalian brain.

Brain lesions composed of pathological tau help to drive neurodegeneration in Alzheimer's disease (AD) and related tauopathies. Here, we identified the mammalian suppressor of tauopathy 2 (MSUT2) gene as a modifier of susceptibility to tau toxicity in two mouse models of tauopathy. Transgenic PS19 mice overexpressing tau, a model of AD, and lacking the Msut2 gene exhibited decreased learning and memory deficits, reduced neurodegeneration, and reduced accumulation of pathological tau compared to PS19 tau transgenic mice expressing Msut2 Conversely, Msut2 overexpression in 4RTauTg2652 tau transgenic mice increased pathological tau deposition and promoted the neuroinflammatory response to pathological tau. MSUT2 is a poly(A) RNA binding protein that antagonizes the canonical nuclear poly(A) binding protein PABPN1. In individuals with AD, MSUT2 abundance in postmortem brain tissue predicted an earlier age of disease onset. Postmortem AD brain tissue samples with normal amounts of MSUT2 showed elevated neuroinflammation associated with tau pathology. We observed co-depletion of MSUT2 and PABPN1 in postmortem brain samples from a subset of AD cases with higher tau burden and increased neuronal loss. This suggested that MSUT2 and PABPN1 may act together in a macromolecular complex bound to poly(A) RNA. Although MSUT2 and PABPN1 had opposing effects on both tau aggregation and poly(A) RNA tail length, we found that increased poly(A) tail length did not ameliorate tauopathy, implicating other functions of the MSUT2/PABPN1 complex in tau proteostasis. Our findings implicate poly(A) RNA binding proteins both as modulators of pathological tau toxicity in AD and as potential molecular targets for interventions to slow neurodegeneration in tauopathies.

Constitutive XBP-1s-mediated activation of the endoplasmic reticulum unfolded protein response protects against pathological tau.

To endure over the organismal lifespan, neurons utilize multiple strategies to achieve protein homeostasis (proteostasis). Some homeostatic mechanisms act in a subcellular compartment-specific manner, but others exhibit trans-compartmental mechanisms of proteostasis. To identify pathways protecting neurons from pathological tau protein, we employed a transgenic Caenorhabditis elegans model of human tauopathy exhibiting proteostatic disruption. We show normal functioning of the endoplasmic reticulum unfolded protein response (UPRER) promotes clearance of pathological tau, and loss of the three UPRER branches differentially affects tauopathy phenotypes. Loss of function of xbp-1 and atf-6 genes, the two main UPRER transcription factors, exacerbates tau toxicity. Furthermore, constitutive activation of master transcription factor XBP-1 ameliorates tauopathy phenotypes. However, both ATF6 and PERK branches of the UPRER participate in amelioration of tauopathy by constitutively active XBP-1, possibly through endoplasmic reticulum-associated protein degradation (ERAD). Understanding how the UPRER modulates pathological tau accumulation will inform neurodegenerative disease mechanisms.

Pathological phosphorylation of tau and TDP-43 by TTBK1 and TTBK2 drives neurodegeneration.

BACKGROUND: Progressive neuron loss in the frontal and temporal lobes of the cerebral cortex typifies frontotemporal lobar degeneration (FTLD). FTLD sub types are classified on the basis of neuronal aggregated protein deposits, typically containing either aberrantly phosphorylated TDP-43 or tau. Our recent work demonstrated that tau tubulin kinases 1 and 2 (TTBK1/2) robustly phosphorylate TDP-43 and co-localize with phosphorylated TDP-43 in human postmortem neurons from FTLD patients. Both TTBK1 and TTBK2 were initially identified as tau kinases and TTBK1 has been shown to phosphorylate tau epitopes commonly observed in Alzheimer's disease and other tauopathies. METHODS: To further elucidate how TTBK1/2 activity contributes to both TDP-43 and tau phosphorylation in the context of the neurodegeneration seen in FTLD, we examined the consequences of elevated human TTBK1/2 kinase expression in transgenic animal models of disease. RESULTS: We show that C. elegans co-expressing tau/TTBK1 tau/TTBK2, or TDP-43/TTBK1 transgenes in combination exhibit synergistic exacerbation of behavioral abnormalities and increased pathological protein phosphorylation. We also show that C. elegans co-expressing tau/TTBK1 or tau/TTBK2 transgenes in combination exhibit aberrant neuronal architecture and neuron loss. Surprisingly, the TTBK2/TDP-43 transgenic combination showed no exacerbation of TDP-43 proteinopathy related phenotypes. Additionally, we observed elevated TTBK1/2 protein expression in cortical and hippocampal neurons of FTLD-tau and FTLD-TDP cases relative to normal controls. CONCLUSIONS: Our findings suggest a possible etiology for the two most common FTLD subtypes through a kinase activation driven mechanism of neurodegeneration.

The phosphatase calcineurin regulates pathological TDP-43 phosphorylation.

Detergent insoluble inclusions of TDP-43 protein are hallmarks of the neuropathology in over 90 % of amyotrophic lateral sclerosis (ALS) cases and approximately half of frontotemporal dementia (FTLD-TDP) cases. In TDP-43 proteinopathy disorders, lesions containing aggregated TDP-43 protein are extensively post-translationally modified, with phosphorylated TDP-43 (pTDP) being the most consistent and robust marker of pathological TDP-43 deposition. Abnormally phosphorylated TDP-43 has been hypothesized to mediate TDP-43 toxicity in many neurodegenerative disease models. To date, several different kinases have been implicated in the genesis of pTDP, but no phosphatases have been shown to reverse pathological TDP-43 phosphorylation. We have identified the phosphatase calcineurin as an enzyme binding to and catalyzing the removal of pathological C-terminal phosphorylation of TDP-43 in vitro. In C. elegans models of TDP-43 proteinopathy, genetic elimination of calcineurin results in accumulation of excess pTDP, exacerbated motor dysfunction, and accelerated neurodegenerative changes. In cultured human cells, treatment with FK506 (tacrolimus), a calcineurin inhibitor, results in accumulation of pTDP species. Lastly, calcineurin co-localizes with pTDP in degenerating areas of the central nervous system in subjects with FTLD-TDP and ALS. Taken together, these findings suggest calcineurin acts on pTDP as a phosphatase in neurons. Furthermore, patient treatment with calcineurin inhibitors may have unappreciated adverse neuropathological consequences.

The tau tubulin kinases TTBK1/2 promote accumulation of pathological TDP-43.

Pathological aggregates of phosphorylated TDP-43 characterize amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP), two devastating groups of neurodegenerative disease. Kinase hyperactivity may be a consistent feature of ALS and FTLD-TDP, as phosphorylated TDP-43 is not observed in the absence of neurodegeneration. By examining changes in TDP-43 phosphorylation state, we have identified kinases controlling TDP-43 phosphorylation in a C. elegans model of ALS. In this kinome-wide survey, we identified homologs of the tau tubulin kinases 1 and 2 (TTBK1 and TTBK2), which were also identified in a prior screen for kinase modifiers of TDP-43 behavioral phenotypes. Using refined methodology, we demonstrate TTBK1 and TTBK2 directly phosphorylate TDP-43 in vitro and promote TDP-43 phosphorylation in mammalian cultured cells. TTBK1/2 overexpression drives phosphorylation and relocalization of TDP-43 from the nucleus to cytoplasmic inclusions reminiscent of neuropathologic changes in disease states. Furthermore, protein levels of TTBK1 and TTBK2 are increased in frontal cortex of FTLD-TDP patients, and TTBK1 and TTBK2 co-localize with TDP-43 inclusions in ALS spinal cord. These kinases may represent attractive targets for therapeutic intervention for TDP-43 proteinopathies such as ALS and FTLD-TDP.

MicroRNA-based single-gene circuits buffer protein synthesis rates against perturbations.

Achieving precise control of mammalian transgene expression has remained a long-standing, and increasingly urgent, challenge in biomedical science. Despite much work, single-cell methods have consistently revealed that mammalian gene expression levels remain susceptible to fluctuations (noise) and external perturbations. Here, we show that precise control of protein synthesis can be realized using a single-gene microRNA (miRNA)-based feed-forward loop (sgFFL). This minimal autoregulatory gene circuit consists of an intronic miRNA that targets its own transcript. In response to a step-like increase in transcription rate, the network generated a transient protein expression pulse before returning to a lower steady state level, thus exhibiting adaptation. Critically, the steady state protein levels were independent of the size of the stimulus, demonstrating that this simple network architecture effectively buffered protein production against changes in transcription. The single-gene network architecture was also effective in buffering against transcriptional noise, leading to reduced cell-to-cell variability in protein synthesis. Noise was up to 5-fold lower for a sgFFL than for an unregulated control gene with equal mean protein levels. The noise buffering capability varied predictably with the strength of the miRNA-target interaction. Together, these results suggest that the sgFFL single-gene motif provides a general and broadly applicable platform for robust gene expression in synthetic and natural gene circuits.

Direct measurement of oxygen consumption rates from attached and unattached cells in a reversibly sealed, diffusionally isolated sample chamber.

Oxygen consumption is a fundamental component of metabolic networks, mitochondrial function, and global carbon cycling. To date there is no method available that allows for replicate measurements on attached and unattached biological samples without compensation for extraneous oxygen leaking into the system. Here we present the Respiratory Detection System, which is compatible with virtually any biological sample. The RDS can be used to measure oxygen uptake in microliter-scale volumes with a reversibly sealed sample chamber, which contains a porphyrin-based oxygen sensor. With the RDS, one can maintain a diffusional seal for up to three hours, allowing for the direct measurement of respiratory function of samples with fast or slow metabolic rates. The ability to easily measure oxygen uptake in small volumes with small populations or dilute samples has implications in cell biology, environmental biology, and clinical diagnostics.