Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons

Abstract

Summary An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but its pathogenic mechanism remains unclear. Here we use human induced motor neurons (iMNs) to show that repeat-expanded C9ORF72 is haploinsufficient in ALS. We show that C9ORF72 interacts with endosomes and is required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduces C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms leads to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescues patient neuron survival and ameliorates neurodegenerative processes in both gain- and loss-of function C9ORF72 mouse models. Thus, modulating vesicle trafficking can rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN, and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS/FTD.

Mutant C9orf72 human iPSC‐derived astrocytes cause non‐cell autonomous motor neuron pathophysiology

“…C9orf72 is believed to cause disease by three putative mechanism(s); haploinsufficiency, sequestration of RNA binding proteins by RNA foci and/or di‐peptide repeat (DPR) mediated toxicity (Mizielinska & Isaacs, ; Shi et al, ; Tabet et al, ). As intranuclear RNA foci are observed in astrocytes in post‐mortem derived material from C9orf72 patients (Lagier‐Tourenne et al, ), we first used fluorescent in situ hybridization (FISH) to confirm the presence of abundant intranuclear RNA foci in mutant astrocytes that were absent in controls (Figure a).…”

Stem Cells, Genome Editing, and the Path to Translational Medicine

“…One strategy to robustly control in vitro differentiation is directed conversion of hPSCs to pure and functionally mature neurons driven by forced expression of cell fate specifying neuronal transcription factors such as NGN2 to induce cortical excitatory neurons (iNs) (Zhang et al, 2013) or NGN2, ISL1, LHX3, NEUROD1, BRN2, ASCL1 and MYT1L to induce motor neurons (iMNs) (Shi et al, 2018). This approach allowed dissection of the molecular effects of distinct ApoE isoforms, a strong risk factor for Alzheimer’s disease, amyloid-beta precursor protein (APP) expression and disease susceptibility (Huang et al, 2017).…”

“…An alternate strategy selects specific cell types within a heterogeneous population by genetically inserting cell-type specific selection or fluorescent marker proteins. Using longitudinal tracking of the fluorescent motor neuron specific reporter Hb9-RFP in hiPSCs derived from patients with GGGGCC repeat expansion in C9ORF72, which underlies amyotrophic lateral sclerosis and frontotemporal dementia, Shi and colleagues observed a robust degeneration of patient-derived cells and identified disruption of vesicle trafficking as the pathogenic molecular mechanism (Shi et al, 2018). …”

RNA versus protein toxicity in C9orf72 ALS/FTLD

“…Homozygous knockout of C9orf72 causes a variable (lupuslike) immune phenotype due to strong expression of C9orf72 in the myeloid lineage, but the mice show no overt neurodegeneration [1,20]. However, C9orf72 haploinsufficiency sensitizes patient-derived motoneurons to DPR toxicity and other stressors, suggesting it may contribute to neurodegeneration in C9orf72 ALS/FTD [28]. Thus, lowering C9orf72 expression to treat gain-of-function mechanisms may be detrimental.…”

“…Human iPSC-derived neurons are widely popular, but DPR expression is very low in most differentiation protocols and robust TDP-43 pathology has not been reported. Thus, iPSC models may be most useful to study RNA toxicity and the consequences of reduced C9orf72 protein expression [28]. Human neuropathology clearly remains the gold standard.…”