Tau-RNA complexes inhibit microtubule polymerization and drive disease-relevant conformation change.

Alzheimer's disease and related disorders feature neurofibrillary tangles and other neuropathological lesions composed of detergent-insoluble tau protein. In recent structural biology studies of tau proteinopathy, aggregated tau forms a distinct set of conformational variants specific to the different types of tauopathy disorders. However, the constituents driving the formation of distinct pathological tau conformations on pathway to tau-mediated neurodegeneration remain unknown. Previous work demonstrated RNA can serve as a driver of tau aggregation, and RNA associates with tau containing lesions, but tools for evaluating tau/RNA interactions remain limited. Here, we employed molecular interaction studies to measure the impact of tau/RNA binding on tau microtubule binding and aggregation. To investigate the importance of tau/RNA complexes (TRCs) in neurodegenerative disease, we raised a monoclonal antibody (TRC35) against aggregated tau/RNA complexes. We showed that native tau binds RNA with high affinity but low specificity, and tau binding to RNA competes with tau-mediated microtubule assembly functions. Tau/RNA interaction in vitro promotes the formation of higher molecular weight tau/RNA complexes, which represent an oligomeric tau species. Coexpression of tau and poly(A)45 RNA transgenes in Caenorhabditis elegans exacerbates tau-related phenotypes including neuronal dysfunction and pathological tau accumulation. TRC35 exhibits specificity for Alzheimer's disease-derived detergent-insoluble tau relative to soluble recombinant tau. Immunostaining with TRC35 labels a wide variety of pathological tau lesions in animal models of tauopathy, which are reduced in mice lacking the RNA binding protein MSUT2. TRC-positive lesions are evident in many human tauopathies including Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration and Pick's disease. We also identified ocular pharyngeal muscular dystrophy as a novel tauopathy disorder, where loss of function in the poly(A) RNA binding protein (PABPN1) causes accumulation of pathological tau in tissue from post-mortem human brain. Tau/RNA binding drives tau conformational change and aggregation inhibiting tau-mediated microtubule assembly. Our findings implicate cellular tau/RNA interactions as modulators of both normal tau function and pathological tau toxicity in tauopathy disorders and suggest feasibility for novel therapeutic approaches targeting TRCs.

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.

Adult onset pan-neuronal human tau tubulin kinase 1 expression causes severe cerebellar neurodegeneration in mice.

The kinase TTBK1 is predominantly expressed in the central nervous system and has been implicated in neurodegenerative diseases including Alzheimer's disease, frontotemporal lobar degeneration, and amyotrophic lateral sclerosis through its ability to phosphorylate the proteins tau and TDP-43. Mutations in the closely related gene TTBK2 cause spinocerebellar ataxia, type 11. However, it remains unknown whether altered TTBK1 activity alone can drive neurodegeneration. In order to characterize the consequences of neuronal TTBK1 upregulation in adult brains, we have generated a transgenic mouse model with inducible pan-neuronal expression of human TTBK1. We find that these inducible TTBK1 transgenic mice (iTTBK1 Tg) exhibit motor and cognitive phenotypes, including decreased grip strength, hyperactivity, limb-clasping, and spatial memory impairment. These behavioral phenotypes occur in conjunction with progressive weight loss, neuroinflammation, and severe cerebellar degeneration with Purkinje neuron loss. Phenotype onset begins weeks after TTBK1 induction, culminating in average mortality around 7 weeks post induction. The iTTBK1 Tg animals lack any obvious accumulation of pathological tau or TDP-43, indicating that TTBK1 expression drives neurodegeneration in the absence of detectable pathological protein deposition. In exploring TTBK1 functions, we identified the autophagy related protein GABARAP to be a novel interacting partner of TTBK1 and show that GABARAP protein levels increase in the brain following induction of TTBK1. These iTTBK1 Tg mice exhibit phenotypes reminiscent of spinocerebellar ataxia, and represent a new model of cerebellar neurodegeneration.

Cell Cycle Analysis of Hematopoietic Stem and Progenitor Cells by Multicolor Flow Cytometry.

Maintenance of hematopoietic stem cell (HSC) quiescence is critical for self-renewal and differentiation into mature lineages. Therefore, the ability to reliably detect abnormal HSC cycling is essential for experiments that seek to investigate abnormalities of HSC function. The ability to reproducibly evaluate cell cycle status in a rare cell subset requires careful optimization of multiple parameters during cell preparation and sample processing. Here, we describe a method where data acquisition parameters and fluorochrome combination for long-term HSC staining have been specifically designed for concurrent use with DAPI and Ki-67 antibodies. © 2018 by John Wiley & Sons, Inc.