Cell-autonomous immune dysfunction driven by disrupted autophagy in C9orf72-ALS iPSC-derived microglia contributes to neurodegeneration.

Although microglial activation is widely found in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the underlying mechanism(s) are poorly understood. Here, using human-induced pluripotent stem cell-derived microglia-like cells (hiPSC-MG) harboring the most common ALS/FTD mutation (C9orf72, mC9-MG), gene-corrected isogenic controls (isoC9-MG), and C9orf72 knockout hiPSC-MG (C9KO-MG), we show that reduced C9ORF72 protein is associated with impaired phagocytosis and an exaggerated immune response upon stimulation with lipopolysaccharide. Analysis of the C9ORF72 interactome revealed that C9ORF72 interacts with regulators of autophagy and functional studies showed impaired initiation of autophagy in mC9-MG and C9KO-MG. Coculture studies with motor neurons (MNs) demonstrated that the autophagy deficit in mC9-MG drives increased vulnerability of mC9-MNs to excitotoxic stimulus. Pharmacological activation of autophagy ameliorated both cell-autonomous functional deficits in hiPSC-MG and MN death in MG-MN coculture. Together, these findings reveal an important role for C9ORF72 in regulating immune homeostasis and identify dysregulation in myeloid cells as a contributor to neurodegeneration in ALS/FTD.

Dysregulation in Subcellular Localization of Myelin Basic Protein mRNA Does Not Result in Altered Myelination in Amyotrophic Lateral Sclerosis.

Pathological hallmarks of amyotrophic lateral sclerosis (ALS), including protein misfolding, are well established in oligodendrocytes. More recently, an RNA trafficking deficit of key myelin proteins has been suggested in oligodendrocytes in ALS but the extent to which this affects myelination and the relative contribution of this to disease pathogenesis is unclear. ALS autopsy research findings showing demyelination contrasts with the routine clinical-pathological workup of ALS cases where it is rare to see white matter abnormalities other than simple Wallerian degeneration secondary to widespread neuronal loss. To begin to address this apparent variance, we undertook a comprehensive evaluation of myelination at an RNA, protein and structural level using human pathological material from sporadic ALS patients, genetic ALS patients (harboring C9orf72 mutation) and age- and sex-matched non-neurological controls. We performed (i) quantitative spatial profiling of the mRNA transcript encoding myelin basic protein (MBP), (ii) quantification of MBP protein and (iii) the first quantitative structural assessment of myelination in ALS post-mortem specimens by electron microscopy. We show no differences in MBP protein levels or ultrastructural myelination, despite a significant dysregulation in the subcellular trafficking of MBP mRNA in ALS patients compared to controls. We therefore confirm that whilst there are cell autonomous mRNA trafficking deficits affecting oligodendrocytes in ALS, this has no effect on myelin structure.

iPSC-derived myelinoids to study myelin biology of humans.

Myelination is essential for central nervous system (CNS) formation, health, and function. Emerging evidence of oligodendrocyte heterogeneity in health and disease and divergent CNS gene expression profiles between mice and humans supports the development of experimentally tractable human myelination systems. Here, we developed human iPSC-derived myelinating organoids ("myelinoids") and quantitative tools to study myelination from oligodendrogenesis through to compact myelin formation and myelinated axon organization. Using patient-derived cells, we modeled a monogenetic disease of myelinated axons (Nfasc155 deficiency), recapitulating impaired paranodal axo-glial junction formation. We also validated the use of myelinoids for pharmacological assessment of myelination-both at the level of individual oligodendrocytes and globally across whole myelinoids-and demonstrated reduced myelination in response to suppressed synaptic vesicle release. Our study provides a platform to investigate human myelin development, disease, and adaptive myelination.

Transactive response DNA-binding protein-43 proteinopathy in oligodendrocytes revealed using an induced pluripotent stem cell model.

Oligodendrocytes are implicated in amyotrophic lateral sclerosis pathogenesis and display transactive response DNA-binding protein-43 (TDP-43) pathological inclusions. To investigate the cell autonomous consequences of TDP-43 mutations on human oligodendrocytes, we generated oligodendrocytes from patient-derived induced pluripotent stem cell lines harbouring mutations in the TARDBP gene, namely G298S and M337V. Through a combination of immunocytochemistry, electrophysiological assessment via whole-cell patch clamping, and three-dimensional cultures, no differences in oligodendrocyte differentiation, maturation or myelination were identified. Furthermore, expression analysis for monocarboxylate transporter 1 (a lactate transporter) coupled with a glycolytic stress test showed no deficit in lactate export. However, using confocal microscopy, we report TDP-43 mutation-dependent pathological mis-accumulation of TDP-43. Furthermore, using in vitro patch-clamp recordings, we identified functional Ca2+-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor dysregulation in oligodendrocytes. Together, these findings establish a platform for further interrogation of the role of oligodendrocytes and cellular autonomy in TDP-43 proteinopathy.

Generation of pure monocultures of human microglia-like cells from induced pluripotent stem cells.

Microglia are resident tissue macrophages of the central nervous system (CNS) that arise from erythromyeloid progenitors during embryonic development. They play essential roles in CNS development, homeostasis and response to disease. Since microglia are difficult to procure from the human brain, several protocols have been developed to generate microglia-like cells from human induced pluripotent stem cells (hiPSCs). However, some concerns remain over the purity and quality of in vitro generated microglia. Here, we describe a new protocol that does not require co-culture with neural cells and yields cultures of 100% P2Y12+ 95% TMEM119+ ramified human microglia-like cells (hiPSC-MG). In the presence of neural precursor cell-conditioned media, hiPSC-MG expressed high levels of human microglia signature genes, including SALL1, CSF1R, P2RY12, TMEM119, TREM2, HEXB and SIGLEC11, as revealed by whole-transcriptome analysis. Stimulation of hiPSC-MG with lipopolysaccharide resulted in downregulation of P2Y12 expression, induction of IL1B mRNA expression and increase in cell capacitance. HiPSC-MG were phagocytically active and maintained their cell identity after transplantation into murine brain slices and human brain spheroids. Together, our new protocol for the generation of microglia-like cells from human iPSCs will facilitate the study of human microglial function in health and disease.

FMRP Interacts with C/D Box snoRNA in the Nucleus and Regulates Ribosomal RNA Methylation.

FMRP is an RNA-binding protein that is known to localize in the cytoplasm and in the nucleus. Here, we have identified an interaction of FMRP with a specific set of C/D box snoRNAs in the nucleus. C/D box snoRNAs guide 2'O methylations of ribosomal RNA (rRNA) on defined sites, and this modification regulates rRNA folding and assembly of ribosomes. 2'O methylation of rRNA is partial on several sites in human embryonic stem cells, which results in ribosomes with differential methylation patterns. FMRP-snoRNA interaction affects rRNA methylation on several of these sites, and in the absence of FMRP, differential methylation pattern of rRNA is significantly altered. We found that FMRP recognizes ribosomes carrying specific methylation patterns on rRNA and the recognition of methylation pattern by FMRP may potentially determine the translation status of its target mRNAs. Thus, FMRP integrates its function in the nucleus and in the cytoplasm.

Neurexins 1-3 Each Have a Distinct Pattern of Expression in the Early Developing Human Cerebral Cortex.

Neurexins (NRXNs) are presynaptic terminal proteins and candidate neurodevelopmental disorder susceptibility genes; mutations presumably upset synaptic stabilization and function. However, analysis of human cortical tissue samples by RNAseq and quantitative real-time PCR at 8-12 postconceptional weeks, prior to extensive synapse formation, showed expression of all three NRXNs as well as several potential binding partners. However, the levels of expression were not identical; NRXN1 increased with age and NRXN2 levels were consistently higher than for NRXN3. Immunohistochemistry for each NRXN also revealed different expression patterns at this stage of development. NRXN1 and NRXN3 immunoreactivity was generally strongest in the cortical plate and increased in the ventricular zone with age, but was weak in the synaptogenic presubplate (pSP) and marginal zone. On the other hand, NRXN2 colocalized with synaptophysin in neurites of the pSP, but especially with GAP43 and CASK in growing axons of the intermediate zone. Alternative splicing modifies the role of NRXNs and we found evidence by RNAseq for exon skipping at splice site 4 and concomitant expression of KHDBRS proteins which control this splicing. NRXN2 may play a part in early cortical synaptogenesis, but NRXNs could have diverse roles in development including axon guidance, and intercellular communication between proliferating cells and/or migrating neurons.