Tdp-43 cryptic exons are highly variable between cell types

Background: TDP-43 proteinopathy is a prominent pathological feature that occurs in a number of human diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and inclusion body myositis (IBM). Our recent finding that TDP-43 represses nonconserved cryptic exons led us to ask whether cell type-specific cryptic exons could exist to impact unique molecular pathways in brain or muscle. Methods: In the present work, we investigated TDP-43’s function in various mouse tissues to model disease pathogenesis. We generated mice to conditionally delete TDP-43 in excitatory neurons or skeletal myocytes and identified the cell type-specific cryptic exons associated with TDP-43 loss of function. Results: Comparative analysis of nonconserved cryptic exons in various mouse cell types revealed that only some cryptic exons were common amongst stem cells, neurons, and myocytes; the majority of these nonconserved cryptic exons were cell type-specific. Conclusions: Our results suggest that in human disease, TDP-43 loss of function may impair cell type-specific pathways

Untersuchungen von Motorik, Kognition und Verhalten bei Mausmodellen von Motoneuronerkrankungen

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease, characterized by degeneration of upper and lower motor neurons. Most cases (90%) are classified as sporadic ALS, 10 % are inherited and referred to as familial ALS. In about 15-20% of those cases, a mutation in the gene encoding the antioxidant enzyme Cu/Zn dismutase 1 (SOD1) has been found. There is an increasing recognition of clinical overlap between frontotemporal dementia (FTD) and ALS in some ALS patients. Following the current discussion of ALS and FTD overlap, the aim of the current study was to explore the effects of mutations in mouse models. The investigated effects refer to motor functions, behaviour, and cognition. The classical ALS mouse model SOD1, the Cra1 and the double-transgenic Cra1/SOD1 animals, were to be investigated on their validity of ALS models. Age matched wild type animals with the identical genetic background served as control as well as the P301L model. This is a transgenic mouse, characterized by a mutation in the protein tau, and therefore a FTD model. The behavioural tests were complemented by histomorphometric characterisation of the musculus quadrizeps. The most important results concerned the Cra1 animals. They showed deficits in agility, motor coordination and muscular strength, distinct hyperactivity, no disturbance of exploration and anxiety as P301L mice as well as a normal cognition, no neurogenic changes in skeletal muscles. These results suggest that Cra1 mice are neither an ALS model nor a tauopathie model. Therefore the present classification of the Cra1 mice, in terms of an ALS model, has to be challenged. Similar characteristics with regard to hyperactivity and deficits in motor coordination in Cra1 mice can be found in mouse models with neurodegeneration at the corpus striatum. Therefore, I can conclude that Cra1 mice resemble an animal model with degenerations in the striatum

Intraventricular Delivery of siRNA Nanoparticles to the Central Nervous System

Alzheimer's disease (AD) is a progressive neurodegenerative disease currently lacking effective treatment. Efficient delivery of siRNA via nanoparticles may emerge as a viable therapeutic approach to treat AD and other central nervous system disorders. We report here the use of a linear polyethyleneimine (LPEI)-g-polyethylene glycol (PEG) copolymer-based micellar nanoparticle system to deliver siRNA targeting BACE1 and APP, two therapeutic targets of AD. Using LPEI-siRNA nanoparticles against either BACE1 or APP in cultured mouse neuroblastoma (N2a) cells, we observe selective knockdown, respectively, of BACE1 or APP. The encapsulation of siRNA by LPEI-g-PEG carriers, with different grafting degrees of PEG, leads to the formation of micellar nanoparticles with distinct morphologies, including worm-like, rod-like, or spherical nanoparticles. By infusing these shaped nanoparticles into mouse lateral ventricles, we show that rod-shaped nanoparticles achieved the most efficient knockdown of BACE1 in the brain. Furthermore, such knockdown is evident in spinal cords of these treated mice. Taken together, our findings indicate that the shape of siRNA-encapsulated nanoparticles is an important determinant for their delivery and gene knockdown efficiency in the central nervous system

Loss of TDP-43 function and rimmed vacuoles persist after T cell depletion in a xenograft model of sporadic inclusion body myositis.

Sporadic inclusion body myositis (IBM) is the most common acquired muscle disease in adults over age 50, yet it remains unclear whether the disease is primarily driven by T cell–mediated autoimmunity. IBM muscle biopsies display nuclear clearance and cytoplasmic aggregation of TDP-43 in muscle cells, a pathologic finding observed initially in neurodegenerative diseases, where nuclear loss of TDP-43 in neurons causes aberrant RNA splicing. Here, we show that loss of TDP-43–mediated splicing repression, as determined by inclusion of cryptic exons, occurs in skeletal muscle of subjects with IBM. Of 119 muscle biopsies tested, RT-PCR–mediated detection of cryptic exon inclusion was able to diagnose IBM with 84% sensitivity and 99% specificity. To determine the role of T cells in pathogenesis, we generated a xenograft model by transplanting human IBM muscle into the hindlimb of immunodeficient mice. Xenografts from subjects with IBM displayed robust regeneration of human myofibers and recapitulated both inflammatory and degenerative features of the disease. Myofibers in IBM xenografts showed invasion by human, oligoclonal CD8+ T cells and exhibited MHC-I up-regulation, rimmed vacuoles, mitochondrial pathology, p62-positive inclusions, and nuclear clearance and cytoplasmic aggregation of TDP-43, associated with cryptic exon inclusion. Reduction of human T cells within IBM xenografts by treating mice intraperitoneally with anti-CD3 (OKT3) suppressed MHC-I up-regulation. However, rimmed vacuoles and loss of TDP-43 function persisted. These data suggest that T cell depletion does not alter muscle degenerative pathology in IBM

Neuroprotective Function of Cellular Prion Protein in a Mouse Model of Amyotrophic Lateral Sclerosis

Transgenic mice expressing human mutated superoxide dismutase 1 (SOD1) linked to familial forms of amyotrophic lateral sclerosis are frequently used as a disease model. We used the SOD1G93A mouse in a cross-breeding strategy to study the function of physiological prion protein (Prp). SOD1G93APrp−/− mice exhibited a significantly reduced life span, and an earlier onset and accelerated progression of disease, as compared with SOD1G93APrp+/+ mice. Additionally, during disease progression, SOD1G93APrp−/− mice showed impaired rotarod performance, lower body weight, and reduced muscle strength. Histologically, SOD1G93APrp−/− mice showed reduced numbers of spinal cord motor neurons and extended areas occupied by large vacuoles early in the course of the disease. Analysis of spinal cord homogenates revealed no differences in SOD1 activity. Using an unbiased proteomic approach, a marked reduction of glial fibrillary acidic protein and enhanced levels of collapsing response mediator protein 2 and creatine kinase were detected in SOD1G93APrp−/− versus SOD1G93A mice. In the course of disease, Bcl-2 decreases, nuclear factor-κB increases, and Akt is activated, but these changes were largely unaffected by Prp expression. Exclusively in double-transgenic mice, we detected a significant increase in extracellular signal-regulated kinase 2 activation at clinical onset. We propose that Prp has a beneficial role in the SOD1G93A amyotrophic lateral sclerosis mouse model by influencing neuronal and/or glial factors involved in antioxidative defense, rather than anti-apoptotic signaling

A point mutation in the dynein heavy chain gene leads to striatal atrophy and compromises neurite outgrowth of striatal neurons

The molecular motor dynein and its associated regulatory subunit dynactin have been implicated in several neurodegenerative conditions of the basal ganglia, such as Huntington's disease (HD) and Perry syndrome, an atypical Parkinson-like disease. This pathogenic role has been largely postulated from the existence of mutations in the dynactin subunit p150Glued. However, dynactin is also able to act independently of dynein, and there is currently no direct evidence linking dynein to basal ganglia degeneration. To provide such evidence, we used here a mouse strain carrying a point mutation in the dynein heavy chain gene that impairs retrograde axonal transport. These mice exhibited motor and behavioural abnormalities including hindlimb clasping, early muscle weakness, incoordination and hyperactivity. In vivo brain imaging using magnetic resonance imaging showed striatal atrophy and lateral ventricle enlargement. In the striatum, altered dopamine signalling, decreased dopamine D1 and D2 receptor binding in positron emission tomography SCAN and prominent astrocytosis were observed, although there was no neuronal loss either in the striatum or substantia nigra. In vitro, dynein mutant striatal neurons displayed strongly impaired neuritic morphology. Altogether, these findings provide a direct genetic evidence for the requirement of dynein for the morphology and function of striatal neurons. Our study supports a role for dynein dysfunction in the pathogenesis of neurodegenerative disorders of the basal ganglia, such as Perry syndrome and HD