Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative condition characterized by loss of motor neurons in the brain and spinal cord. Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9ORF72 gene are the most common cause of the familial form of ALS (C9-ALS), as well as frontotemporal lobar degeneration and other neurological diseases. How the repeat expansion causes disease remains unclear, with both loss of function (haploinsufficiency) and gain of function (either toxic RNA or protein products) proposed. Here, we report a cellular model of C9-ALS with motor neurons differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying the C9ORF72 repeat expansion. No significant loss of C9ORF72 expression was observed, and knockdown of the transcript was not toxic to cultured human motor neurons. Transcription of the repeat was increased leading to accumulation of GGGGCC repeat-containing RNA foci selectively in C9-ALS motor neurons. Repeat-containing RNA foci co-localized with hnRNPA1 and Pur-α, suggesting that they may be able to alter RNA metabolism. C9-ALS motor neurons showed altered expression of genes involved in membrane excitability including DPP6, and demonstrated a diminished capacity to fire continuous spikes upon depolarization compared to control motor neurons. Antisense oligonucleotides (ASOs) targeting the C9ORF72 transcript suppressed RNA foci formation and reversed gene expression alterations in C9-ALS motor neurons. These data show that patient-derived motor neurons can be used to delineate pathogenic events in ALS.

Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Decreased expression of C9orf72 is seen in expansion carriers, suggesting loss of function may play a role in disease. We find that two independent mouse lines lacking the C9orf72 ortholog (3110043O21Rik) in all tissues developed normally and aged without motor neuron disease. Instead, C9orf72 null mice developed progressive splenomegaly and lymphadenopathy with accumulation of engorged macrophage-like cells. C9orf72 expression was highest in myeloid cells, and loss of C9orf72 led to lysosomal accumulation and altered immune responses in macrophages and microglia, with age-related neuroinflammation similar to C9orf72 ALS but not sporadic ALS patient tissue. Thus, C9orf72 is required for normal function of myeloid cells, and altered microglial function may contribute to neurodegeneration in C9orf72 expansion carriers.

Summary Noncoding expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. Here we report transgenic mice carrying a bacterial artificial chromosome (BAC) containing the full human C9orf72 gene with either a normal allele (15 repeats) or disease-associated expansion (~100-1000 repeats; C9-BACexp). C9-BACexp mice displayed pathologic features seen in C9orf72 expansion patients, including widespread RNA foci and repeat associated non-ATG (RAN) translated dipeptides, which were suppressed by antisense oligonucleotides targeting human C9orf72. Nucleolin distribution was altered supporting that either C9orf72 transcripts or RAN dipeptides promote nucleolar dysfunction. Despite early and widespread production of RNA foci and RAN dipeptides in C9-BACexp mice, behavioral abnormalities and neurodegeneration were not observed even at advanced ages, supporting the hypothesis that RNA foci and RAN dipeptides occur presymptomatically, and are not sufficient to drive neurodegeneration in mice at levels seen in patients.

Figure 1. Impaired growth rate, sensorimotor function, grip strength, and vision in Thy1.2-MFN2 R94Q transgenic mice. (A) Schematic of the Thy1.2-MFN2 R94Q transgenic construct. The expression of human MFN2 R94Q or control MFN2 WT (N terminus tagged with Flag) was driven by the neuron-specific mouse Thy1.2 promoter. (B) Representative image of a nontransgenic (nTg) mouse, a Thy1.2-MFN2 WT mouse, and a Thy1.2-MFN2 R94Q mouse (5 months old). (C) Immunoblot of Flag-MFN2 WT or Flag-MFN2 R94Q transgene expression (14-month-old mice). Red arrow, Flag-MFN2; black arrow, endogenous mouse Mfn2. Expression levels of MFN2 WT or MFN2 R94Q transgenes were identical and slightly below endogenous Mfn2 levels. n = 3 mice/genotype. (D) Immunostaining of Flag-MFN2 WT or Flag-MFN2 R94Q protein expression and localization. Mouse cortex or spinal cord (5-month-old mice). Anti-Flag (red) and DAPI (blue). Scale bars: 50 μM. Punctate mitochondrial staining was observed in both transgenic lines, but only MFN2 R94Q mice showed mitochondrial accumulations in neuronal cytoplasm and proximal axons. n = 3 mice/genotype. (E) Body weight. Data are represented as mean ± SEM. n = 6-52 per genotype per time point. Student's 2-tailed t test (nTg vs. MFN2 R94Q). *P < 0.05. (F) Survival curve. nTg (n = 59), MFN2 WT (n = 29), MFN2 R94Q (n = 124). log-rank test with Bonferroni's correction. nTg vs. MFN2 WT , P > 0.05, not significant. nTg vs. MFN2 R94Q , P < 0.01. MFN2 WT vs. MFN2 R94Q , P < 0.05. (G) Open-field test (total activity) and (H) open-field test (rearing). Total activity was not significantly different between groups. nTg (n = 5), MFN2 WT (n = 5), MFN2 R94Q (n = 6) (3-month-old mice). (I) Rotarod testing. nTg (n = 5), MFN2 WT (n = 5), MFN2 R94Q (n = 6). (J) Grip-strength testing (forelimbs). nTg (n = 8-11), MFN2 WT (n = 5), MFN2 R94Q (n = 6). (K) Visual acuity measured by OKR. nTg (n = 6), MFN2 WT (n = 3), MFN2 R94Q (n = 7). In G-K, data are represented as mean ± SEM. Two-way ANOVA with Tukey's test was used for multiple comparison.

No abstract