A new non-aggregative splicing isoform of human Tau is decreased in Alzheimer's disease.

Tauopathies, including Alzheimer's disease (AD) and frontotemporal lobar degeneration with Tau pathology (FTLD-tau), are a group of neurodegenerative disorders characterized by Tau hyperphosphorylation. Post-translational modifications of Tau such as phosphorylation and truncation have been demonstrated to be an essential step in the molecular pathogenesis of these tauopathies. In this work, we demonstrate the existence of a new, human-specific truncated form of Tau generated by intron 12 retention in human neuroblastoma cells and, to a higher extent, in human RNA brain samples, using qPCR and further confirming the results on a larger database of human RNA-seq samples. Diminished protein levels of this new Tau isoform are found by Westernblotting in Alzheimer's patients' brains (Braak I n = 3; Braak II n = 6, Braak III n = 3, Braak IV n = 1, and Braak V n = 10, Braak VI n = 8) with respect to non-demented control subjects (n = 9), suggesting that the lack of this truncated isoform may play an important role in the pathology. This new Tau isoform exhibits similar post-transcriptional modifications by phosphorylation and affinity for microtubule binding, but more interestingly, is less prone to aggregate than other Tau isoforms. Finally, we present evidence suggesting this new Tau isoform could be linked to the inhibition of GSK3ß, which would mediate intron 12 retention by modulating the serine/arginine rich splicing factor 2 (SRSF2). Our results show the existence of an important new isoform of Tau and suggest that further research on this less aggregation-prone Tau may help to develop future therapies for Alzheimer's disease and other tauopathies.

Huntington's disease-specific mis-splicing unveils key effector genes and altered splicing factors.

Correction of mis-splicing events is a growing therapeutic approach for neurological diseases such as spinal muscular atrophy or neuronal ceroid lipofuscinosis 7, which are caused by splicing-affecting mutations. Mis-spliced effector genes that do not harbour mutations are also good candidate therapeutic targets in diseases with more complex aetiologies such as cancer, autism, muscular dystrophies or neurodegenerative diseases. Next-generation RNA sequencing (RNA-seq) has boosted investigation of global mis-splicing in diseased tissue to identify such key pathogenic mis-spliced genes. Nevertheless, while analysis of tumour or dystrophic muscle biopsies can be informative on early stage pathogenic mis-splicing, for neurodegenerative diseases, these analyses are intrinsically hampered by neuronal loss and neuroinflammation in post-mortem brains. To infer splicing alterations relevant to Huntington's disease pathogenesis, here we performed intersect-RNA-seq analyses of human post-mortem striatal tissue and of an early symptomatic mouse model in which neuronal loss and gliosis are not yet present. Together with a human/mouse parallel motif scan analysis, this approach allowed us to identify the shared mis-splicing signature triggered by the Huntington's disease-causing mutation in both species and to infer upstream deregulated splicing factors. Moreover, we identified a plethora of downstream neurodegeneration-linked mis-spliced effector genes that-together with the deregulated splicing factors-become new possible therapeutic targets. In summary, here we report pathogenic global mis-splicing in Huntington's disease striatum captured by our new intersect-RNA-seq approach that can be readily applied to other neurodegenerative diseases for which bona fide animal models are available.

Pathogenic SREK1 decrease in Huntington's disease lowers TAF1 mimicking X-linked dystonia parkinsonism.

Huntington's disease and X-linked dystonia parkinsonism are two monogenic basal ganglia model diseases. Huntington's disease is caused by a polyglutamine-encoding CAG repeat expansion in the Huntingtin (HTT) gene leading to several toxic interactions of both the expanded CAG-containing mRNA and the polyglutamine-containing protein, while X-linked dystonia parkinsonism is caused by a retrotransposon insertion in the TAF1 gene, which decreases expression of this core scaffold of the basal transcription factor complex TFIID. SRSF6 is an RNA-binding protein of the serine and arginine-rich (SR) protein family that interacts with expanded CAG mRNA and is sequestered into the characteristic polyglutamine-containing inclusion bodies of Huntington's disease brains. Here we report decreased levels of the SRSF6 interactor and regulator SREK1-another SR protein involved in RNA processing-which includes TAF1 as one of its targets. This led us to hypothesize that Huntington's disease and X-linked dystonia parkinsonism pathogeneses converge in TAF1 alteration. We show that diminishing SRSF6 through RNA interference in human neuroblastoma cells leads to a decrease in SREK1 levels, which, in turn, suffices to cause diminished TAF1 levels. We also observed decreased SREK1 and TAF1 levels in striatum of Huntington's disease patients and transgenic model mice. We then generated mice with neuronal transgenic expression of SREK1 (TgSREK1 mice) that, interestingly, showed transcriptomic alterations complementary to those in Huntington's disease mice. Most importantly, by combining Huntington's disease and TgSREK1 mice we verify that SREK1 overexpression corrects TAF1 deficiency and attenuates striatal atrophy and motor phenotype of Huntington's disease mice. Our results therefore demonstrate that altered RNA processing upon SREK1 dysregulation plays a key role in Huntington's disease pathogenesis and pinpoint TAF1 as a likely general determinant of selective vulnerability of the striatum in multiple neurological disorders.

The dependence receptor TrkC regulates the number of sensory neurons during DRG development.

The development of the sensory nervous system is the result of fine-tuned waves of neurogenesis and apoptosis which control the appropriate number of precursors and newly generated neurons and orient them toward a specific lineage. Neurotrophins and their tyrosine-kinase receptors (RTK) orchestrate this process. They have long been in the scope of the neurotrophic theory which established that a neuron is committed to die unless a trophic factor generated by its target provides it with a survival signal. The neural death has thus always been described as a "default" program, survival being the major player to control the number of cells. New insights have been brought by the gain of function studies which recently demonstrated that TrkC (NTRK3) is a "dependence receptor" able to actively trigger apoptosis in absence of its ligand NT-3. In order to address the role of TrkC pro-apoptotic activity in the control of sensory neurons number, we generated a TrkC gene-trap mutant mice. We found out that this new murine model recapitulates the sensory phenotype of TrkC constitutive mutants, with reduced DRG size and reduced number of DRG neurons. We engineered these mice strain with a lacZ reporter in order to follow the fate of neurons committed to a TrkC lineage and observed that they are specifically protected from NT-3 mediated apoptosis in NT-3/TrkC double knock-out embryos. Finally, using a chicken model we demonstrated that silencing NT-3 emanating from the ventral neural tube induced apoptosis in the DRG anlage. This apoptosis was inhibited by silencing TrkC. This work thus demonstrates that, during in vivo DRG development, TrkC behaves as a two-sided receptor transducing positive signals of neuronal survival in response to NT-3, but actively inducing neuronal cell death when unbound. This functional duality sets adequate number of neurons committed to a TrkC identity in the forming DRG.

Isolation, Purification, and Culture of Primary Murine Sensory Neurons.

Cultured primary neurons have been of extraordinary value for the study of neuronal anatomy, cell biology, and physiology. While use of neuronal cell lines has ease and utility, there are often caveats that arise due to their mitotic nature. This methods article presents detailed methodology for the preparation, purification, and culture of adult murine sensory neurons for the study of herpes simplex virus lytic and latent infections. While virology is the application for our laboratory, these cultures also have broad utility for neurobiologists and cell biologists. While these primary cultures have been highly informative, the methodology is challenging to many investigators. Through publication of this highly detailed protocol, it is our hope that the use of this culture system can spread in the field to allow more rapid progress in furthering our understanding of neurotropic virus infection.

Correction to: Isolation, Purification, and Culture of Primary Murine Sensory Neurons.

In chapter 15, section 2.3, the unit "180 µg/ml mouse laminin in HBSS. Prepare 150 µl per coverslip" is corrected to "18 µg/ml mouse laminin in HBSS. Prepare 150 µl per coverslip."

Faulty splicing and cytoskeleton abnormalities in Huntington's disease.

Huntington's disease (HD) is caused by a CAG-repeat encoding a polyglutamine (polyQ) tract in the huntingtin protein. There is plenty of evidence of polyQ-driven toxicity. However, CAG repeat RNA-driven alteration of splicing has recently been proposed in analogy to CUG-repeat diseases. Here we review the reported alteration of the CAG-repeat associated splicing factor SRSF6 in brains of HD patients and mouse models and how this correlates with altered splicing of, at least, two microtubule-associated proteins in HD, namely MAPT (tau) and MAP2. Regarding tau, altered splicing of exon 10 has been reported, along with increased levels and 4R/3R-tau ratio and detection of tau in a new nuclear rod-shaped histopathological hallmark termed tau nuclear rod (TNR) or tau nuclear indentation (TNI). These findings, together with an attenuation of HD phenotype in R6/1 mice with tau deficiency and subsequent studies showing increased phosphorylation in mouse models and increased levels in CSF of patients, has led to proposing HD as a tauopathy. Regarding MAP2, an increase in its juvenile form and a decrease in total MAP2 together with redistribution from dendrites to soma is observed in HD patients, which may contribute to the dendritic atrophy in HD. Furthermore, MAP2 positive structures filling nuclear indentations have occasionally been found and co-localized with tau. Therefore, altered MAP function with imbalance in tau/MAP2 content could contribute to HD striatal atrophy and dysfunction. Besides, TNIs might be indicative of such MAP abnormalities. TNIs are also found in early pathology Alzheimer's disease and in tauopathy mice over-expressing mutant 4R-tau. This indicates that tau alteration is sufficient for TNI detection, which becomes a marker of increased total tau and/or altered 4R/3R-tau ratio and reporter of pathology-associated nuclear indentations. Altogether, these recent studies suggest that correcting the SRSF6-driven missplicing and/or microtubule-associated imbalance might be of therapeutic value in HD.

Huntington's disease is a four-repeat tauopathy with tau nuclear rods.

An imbalance of tau isoforms containing either three or four microtubule-binding repeats causes frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) in families with intronic mutations in the MAPT gene. Here we report equivalent imbalances at the mRNA and protein levels and increased total tau levels in the brains of subjects with Huntington's disease (HD) together with rod-like tau deposits along neuronal nuclei. These tau nuclear rods show an ordered filamentous ultrastructure and can be found filling the neuronal nuclear indentations previously reported in HD brains. Finally, alterations in serine/arginine-rich splicing factor-6 coincide with tau missplicing, and a role of tau in HD pathogenesis is evidenced by the attenuation of motor abnormalities of mutant HTT transgenic mice in tau knockout backgrounds.

RET modulates cell adhesion via its cleavage by caspase in sympathetic neurons.

RET is a tyrosine kinase receptor involved in numerous cellular mechanisms including proliferation, neuronal navigation, migration, and differentiation upon binding with glial cell derived neurotrophic factor family ligands. RET is an atypical tyrosine kinase receptor containing four cadherin domains in its extracellular part. Furthermore, it has been shown to act as a dependence receptor. Such a receptor is active in the absence of ligand, triggering apoptosis through a mechanism that requires receptor intracellular caspase cleavage. However, different data suggest that RET is not always associated with the cell death/survival balance but rather provides positional information. We demonstrate here that caspase cleavage of RET is involved in the regulation of adhesion in sympathetic neurons. The cleavage of RET generates an N-terminal truncated fragment that functions as a cadherin accessory protein, modifying cadherin environment and potentiating cadherin-mediated cell aggregation. Thus, the caspase cleavage of RET generates two RET fragments: one intracellular domain that can trigger cell death in apoptotic permissive settings, and one membrane-anchored ectodomain with cadherin accessory activity. We propose that this latter function may notably be important for the adequate development of the superior cervical ganglion.