Age-dependent neurodegeneration and organelle transport deficiencies in mutant TDP43 patient-derived neurons are independent of TDP43 aggregation.

TAR DNA-binding protein 43 (TDP43) plays a significant role in familiar and sporadic amyotrophic lateral sclerosis (ALS). The diverse postulated mechanisms by which TDP43 mutations cause the disease are not fully understood. Human wildtype and TDP43 S393L and G294V mutant spinal motor neuron cultures were differentiated from patient-derived iPSCs. Mutant hTDP43 and wildtype motor neuron cultures did not differ in neuron differentiation capacity during early maturation stage. During aging we detected a dramatic neurodegeneration including neuron loss and pathological neurofilament abnormalities only in TDP43 mutant cultures. Additionally mitochondria and lysosomes of aging spinal motor neurons revealed robust TDP43 mutation dependent abnormal phenotypes in size, shape, speed and motility which all appeared without TDP43 mislocalization or aggregation formation. Furthermore, D-sorbitol - known to induce stress granules and cytoplasmic mislocalization of TDP43 - rescued axonal trafficking phenotypes without signs of TDP43 mislocalization or aggregation formation. Our data indicate TDP43 mutation-dependent but cytosolic aggregation-independent mechanisms of motor neuron degeneration in TDP43 ALS.

Methylation and Expression of Mutant FUS in Motor Neurons Differentiated From Induced Pluripotent Stem Cells From ALS Patients.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive disease leading to degeneration of motor neurons (MNs). Epigenetic modification of gene expression is increasingly recognized as potential disease mechanism. In the present study we generated motor neurons from induced pluripotent stem cells from ALS patients carrying a mutation in the fused in sarcoma gene (FUS) and analyzed expression and promoter methylation of the FUS gene and expression of DNA methyltransferases (DNMTs) compared to healthy control cell lines. While mutant FUS neural progenitor cells (NPCs) did not show a difference in FUS and DNMT expression compared to healthy controls, differentiated mutant FUS motor neurons showed significantly lower FUS expression, higher DNMT expression and higher methylation of the proximal FUS gene promoter. Immunofluorescence revealed perceived proximity of cytoplasmic FUS aggregates in ALS MNs together with 5-methylcytosin (5-mC). Targeting disturbed methylation in ALS may therefore restore transcriptional alterations and represent a novel therapeutic strategy.

Altered glutamate response and calcium dynamics in iPSC-derived striatal neurons from XDP patients.

X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disorder endemic to Panay Island (Philippines). Patients present with generalizing dystonia and parkinsonism. Genetic changes surrounding the TAF1 (TATA-box binding protein associated factor 1) gene have been associated with XDP inducing a degeneration of striatal spiny projection neurons. There is little knowledge about the pathophysiology of this disorder. Our objective was to generate and analyze an in-vitro model of XDP based on striatal neurons differentiated from induced pluripotent stem cells (iPSC). We generated iPSC from patient and healthy control fibroblasts (3 affected, 3 controls), followed by directed differentiation of the cultures towards striatal neurons. Cells underwent characterization of immunophenotype as well as neuronal function, glutamate receptor properties and calcium dynamics by whole-cell patch-clamp recordings and calcium imaging. Furthermore, we evaluated expression levels of AMPA receptor subunits and voltage-gated calcium channels by quantitative real-time PCR. We observed no differences in basic electrophysiological properties. Application of the AMPA antagonist NBQX led to a more pronounced reduction of postsynaptic currents in XDP neurons. There was a higher expression of AMPA receptor subunits in patient-derived neurons. Basal calcium levels were lower in neurons derived from XDP patients and cells with spontaneous calcium transients were more frequent. Our data suggest altered glutamate response and calcium dynamics in striatal XDP neurons.