Von Recklinghausen neurofibromatosis (NF1) is a common autosomal dominant disorder characterized by abnormalities in multiple tissues derived from the neural crest. No reliable cellular phenotypic marker has been identified, which has hampered direct efforts to identify the gene. The chromosome location of the NF1 gene has been previously mapped genetically to 17q11.2, and data from two NF1 patients with balanced translocations in this region have further narrowed the candidate interval. The use of chromosome jumping and yeast artificial chromosome technology has now led to the identification of a large (approximately 13 kilobases) ubiquitously expressed transcript (denoted NF1LT) from this region that is definitely interrupted by one and most likely by both translocations. Previously identified candidate genes, which failed to show abnormalities in NF1 patients, are apparently located within introns of NF1LT, on the antisense strand. A new mutation patient with NF1 has been identified with a de novo 0.5-kilobase insertion in the NF1LT gene. These observations, together with the high spontaneous mutation rate of NF1 (which is consistent with a large locus), suggest that NF1LT represents the elusive NF1 gene.

Summary Mutations causing amyotrophic lateral sclerosis (ALS) often affect the condensation properties of RNA-binding proteins (RBPs). However, the role of RBP condensation in the specificity and function of protein-RNA complexes remains unclear. We created a series of TDP-43 C-terminal domain (CTD) variants that exhibited a gradient of low to high condensation propensity, as observed in vitro and by nuclear mobility and foci formation. Notably, a capacity for condensation was required for efficient TDP-43 assembly on subsets of RNA-binding regions, which contain unusually long clusters of motifs of characteristic types and density. These “binding-region condensates” are promoted by homomeric CTD-driven interactions and required for efficient regulation of a subset of bound transcripts, including autoregulation of TDP-43 mRNA. We establish that RBP condensation can occur in a binding-region-specific manner to selectively modulate transcriptome-wide RNA regulation, which has implications for remodeling RNA networks in the context of signaling, disease, and evolution.

TAR DNA-binding protein 43 (TDP-43) inclusions are a pathological hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), including cases caused by G4C2 repeat expansions in the C9orf72 gene (c9FTD/ALS). Providing mechanistic insight into the link between C9orf72 mutations and TDP-43 pathology, we demonstrated that a glycine-arginine repeat protein [poly(GR)] translated from expanded G4C2 repeats was sufficient to promote aggregation of endogenous TDP-43. In particular, toxic poly(GR) proteins mediated sequestration of full-length TDP-43 in an RNA-independent manner to induce cytoplasmic TDP-43 inclusion formation. Moreover, in GFP-(GR)200 mice, poly(GR) caused the mislocalization of nucleocytoplasmic transport factors and nuclear pore complex proteins. These mislocalization events resulted in the aberrant accumulation of endogenous TDP-43 in the cytoplasm where it co-aggregated with poly(GR). Last, we demonstrated that treating G4C2 repeat–expressing mice with repeat-targeting antisense oligonucleotides lowered poly(GR) burden, which was accompanied by reduced TDP-43 pathology and neurodegeneration, including lowering of plasma neurofilament light (NFL) concentration. These results contribute to clarification of the mechanism by which poly(GR) drives TDP-43 proteinopathy, confirm that G4C2-targeted therapeutics reduce TDP-43 pathology in vivo, and demonstrate that alterations in plasma NFL provide insight into the therapeutic efficacy of disease-modifying treatments.

Highlights d Importins directly bind to arginine-rich dipeptide repeat proteins (R-rich DPRs) d R-rich DPRs induce importin and TDP-43 condensation d Poly-GR impairs TDP-43 nuclear import d Elevated importin levels shield R-rich DPRs from pathological interactions

We reported previously that stem cells associated with adult rat testis seminiferous tubules are able to give rise to differentiated Leydig cells in vitro. The regulatory mechanisms by which they do so, however, are uncertain. Herein, we hypothesized that the proliferation and differentiation of Leydig cell stem cells (stem Leydig cells, SLCs) depend upon locally produced factors from the seminiferous tubules. Microarray analysis revealed that platelet-derived growth factor receptor alpha (PDGFRalpha) is up-regulated and PDGFRbeta is down-regulated with postnatal differentiation of SLCs. This suggested that their ligands, PDGF-AA and PDGF-BB, respectively, might have important roles in SLC proliferation and differentiation. To test this, we developed a unique in vitro culture system in which SLCs proliferate on the surfaces of cultured seminiferous tubules largely during Week 1 of culture and their progeny subsequently differentiate to testosterone-forming Leydig cells during Weeks 2 through 4. Using this system, seminiferous tubules from adult rat testes were cultured with PDGF-AA or PDGF-BB for up to 4 wk. Both ligands stimulated SLC proliferation during the first week of culture, with PDGF-BB significantly more potent than PDGF-AA. Furthermore, PDGF-AA had a stimulatory effect on SLC differentiation from Weeks 2 through 4 of culture. In contrast, PDGF-BB, which stimulated cell proliferation during Week 1, had a significant inhibitory effect on differentiation during Weeks 2 through 4. These findings, made possible by the development of the seminiferous tubule culture system, reveal distinct roles by locally produced PDGFs in SLC regulation.