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There is an increasing body of literature pointing to cytoskeletal proteins as spatial organizers and interactors of organelles. In this study, we identified protein 600 (p600) as a novel microtubule-associated protein (MAP) developmentally regulated in neurons. p600 exhibits the unique feature to interact with the endoplasmic reticulum (ER). Silencing of p600 by RNA interference (RNAi) destabilizes neuronal processes in young primary neurons undergoing neurite extension and containing scarce staining of the ER marker Bip. Furthermore, in utero electroporation of p600 RNAi alters neuronal migration, a process that depends on synergistic actions of microtubule dynamics and ER functions. p600-depleted migrating neurons display thin, crooked, and "zigzag" leading process with very few ER membranes. Thus, p600 constitutes the only known MAP to associate with the ER in neurons, and this interaction may impact on multiple cellular processes ranging from neuronal development to neuronal maturation and plasticity. [ABSTRACT FROM AUTHOR]

Abnormal aggregation of α-Synuclein (αS) drives cell loss and produces intracellular inclusions in synucleinopathies, which may be due to insufficient degradation resulting from either a dysfunctional ubiquitin-proteasome system (UPS) or a constant production of mutated αS that overwhelms the UPS. Recent in vitro studies suggest that the autophagylysosome pathway is also involved in αS degradation. On the other hand, synucleinopathies are associated with microglial activation and infiltration. In this postmortem study, we aimed to investigate the relationship between microglial activity and αS disposition in serial patients with cerebral infarct-induced Wallerian degeneration (CI-WD) of the corticospinal fibers. Immunoreactivity for αS was found in activated microglias localized in the CI-WD regions of seven patients (mean 65.6 ± 9.3 years), but not five neuropathologically normal subjects (mean 70.4 ± 8.0 years). No immunoreactivity for ubiquitin or neurofilament protein was present in the microglias. Confocal analysis of double-immunofluorescence staining confirmed the colocalization of αS with the microglial marker. Our results suggest endocytosis (phagocytosis and/or pinocytosis) of αS into activated microglias during the process of CI-WD. This finding indicates that αS may alternatively undergo microglia-associated lysosomal proteolysis for degradation. [ABSTRACT FROM AUTHOR]

Background: Gene editing in induced pluripotent stem (iPS) cells has been hailed to enable new cell therapies for various monogenetic diseases including dystrophic epidermolysis bullosa (DEB). However, manufacturing, efficacy and safety roadblocks have limited the development of genetically corrected, autologous iPS cell-based therapies., Methods: We developed Dystrophic Epidermolysis Bullosa Cell Therapy (DEBCT), a new generation GMP-compatible (cGMP), reproducible, and scalable platform to produce autologous clinical-grade iPS cell-derived organotypic induced skin composite (iSC) grafts to treat incurable wounds of patients lacking type VII collagen (C7). DEBCT uses a combined high-efficiency reprogramming and CRISPR-based genetic correction single step to generate genome scar-free, COL7A1 corrected clonal iPS cells from primary patient fibroblasts. Validated iPS cells are converted into epidermal, dermal and melanocyte progenitors with a novel 2D organoid differentiation protocol, followed by CD49f enrichment and expansion to minimize maturation heterogeneity. iSC product characterization by single cell transcriptomics was followed by mouse xenografting for disease correcting activity at 1 month and toxicology analysis at 1-6 months. Culture-acquired mutations, potential CRISPR-off targets, and cancer-driver variants were evaluated by targeted and whole genome sequencing., Findings: iPS cell-derived iSC grafts were reproducibly generated from four recessive DEB patients with different pathogenic mutations. Organotypic iSC grafts onto immune-compromised mice developed into stable stratified skin with functional C7 restoration. Single cell transcriptomic characterization of iSCs revealed prominent holoclone stem cell signatures in keratinocytes and the recently described Gibbin-dependent signature in dermal fibroblasts. The latter correlated with enhanced graftability. Multiple orthogonal sequencing and subsequent computational approaches identified random and non-oncogenic mutations introduced by the manufacturing process. Toxicology revealed no detectable tumors after 3-6 months in DEBCT-treated mice., Interpretation: DEBCT successfully overcomes previous roadblocks and represents a robust, scalable, and safe cGMP manufacturing platform for production of a CRISPR-corrected autologous organotypic skin graft to heal DEB patient wounds., Competing Interests: DECLARATION OF INTERESTS The authors declare no competing or conflicting interests.