Importantly, our work establishes a new approach for suppressing DPR protein toxicity and blocking C9ORF72 pathogenesis: restoring or replacing C9ORF72 activity. Although high levels of C9ORF72 isoform A may have slightly detrimental effects on control motor neuron survival, we have only observed this in neurons without C9ORF72 repeat expansion. Thus, we would not anticipate a harmful effect of forced C9ORF72 expression in C9ORF72 patients. In addition, a better understanding of the effects of forced C9ORF72 expression could inform safe development of this therapeutic strategy. For example, determining if C9ORF72 accelerates turnover of DPR aggregates by stimulating autophagy could lead help to identify new therapeutic targets.
An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the pathogenic mechanism of this repeat remains unclear. Using human induced motor neurons (iMNs), we found that repeat-expanded C9ORF72 was haploinsufficient in ALS. We found that C9ORF72 interacted with endosomes and was required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduced 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 led to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescued patient neuron survival and ameliorated neurodegenerative processes in both gain- and loss-of-function C9ORF72 mouse models. Thus, modulating vesicle trafficking was able to 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 and FTD.
The Li force is observable when it is employed. Unlike the Li force, Neijing is said to be invisible. The "pivot point" essential to Li combat is not necessary in Neijing. At the point of attack, one must ‘song’ (loosen) himself to generate all Neijing energy one possesses and direct this energy stream through one's contact point with an opponent.[5] The contact point only represents the gateway to conduct Neijing energy at the point of attack.[6]
Human lymphocytes from healthy subjects and ALS patients were obtained from the NINDS Biorepository at the Coriell Institute for Medical Research and reprogrammed into iPSCs as previously described using episomal vectors61. Briefly, mammalian expression vectors containing Oct4, Sox2, Klf4, L-Myc, Lin28, and a p53 shRNA were introduced into the lymphocytes using the Adult Dermal Fibroblast Nucleofector™ Kit and Nucleofector™ 2b Device (Lonza) according to the manufacturer’s protocol. The cells were then cultured on mouse feeders until iPSC colonies appeared. The colonies were then expanded and maintained on Matrigel (BD) in mTeSR1 medium (Stem Cell Technologies).
To confirm that glutamate receptor levels were increased on the surface of C9ORF72+/− and C9ORF72 patient iMNs, we used CRISPR/Cas9 editing to introduce a Dox-inducible polycistronic cassette containing NGN2, ISL1, and LHX3 into the AAVS1 safe-harbor locus of control, C9ORF72+/− and C9ORF72 patient iPSCs. This enabled large-scale production of iMNs that expressed motor neuron markers and had transcriptional profiles similar to 7F iMNs (Supplementary Fig. 11). Using this approach, we quantified the amount of surface-bound NR1 by immunoblotting after using surface protein biotinylation to isolate membrane-bound proteins. This confirmed that surface NR1 levels were higher on C9ORF72+/− and C9ORF72 patient iMNs (n=2 patients) than controls (n=3 controls)(Fig. 4e-h, Supplementary Fig. 5g, h).
The Li force is observable when it is employed. Unlike the Li force, Neijing is said to be invisible. The "pivot point" essential to Li combat is not necessary in Neijing. At the point of attack, one must ‘song’ (loosen) himself to generate all Neijing energy one possesses and direct this energy stream through one's contact point with an opponent.[5] The contact point only represents the gateway to conduct Neijing energy at the point of attack.[6]
The Li force is observable when it is employed. Unlike the Li force, Neijing is said to be invisible. The "pivot point" essential to Li combat is not necessary in Neijing. At the point of attack, one must ‘song’ (loosen) himself to generate all Neijing energy one possesses and direct this energy stream through one's contact point with an opponent.[5] The contact point only represents the gateway to conduct Neijing energy at the point of attack.[6]
Therapeutic strategies in development for C9ORF72 ALS/FTD target gain-of-function mechanisms. These include ASOs 6–8 and small molecules 13 that disrupt RNA foci formation. However, these approaches have not fully rescued neurodegeneration in human patient-derived neurons 6–8,13, indicating that replacing C9ORF72 function or new therapeutic targets may be required.
Therapeutic strategies in development for C9ORF72 ALS/FTD target gain-of-function mechanisms. These include ASOs 6–8 and small molecules 13 that disrupt RNA foci formation. However, these approaches have not fully rescued neurodegeneration in human patient-derived neurons 6–8,13, indicating that replacing C9ORF72 function or new therapeutic targets may be required.
To confirm that glutamate receptor levels were increased on the surface of C9ORF72+/− and C9ORF72 patient iMNs, we used CRISPR/Cas9 editing to introduce a Dox-inducible polycistronic cassette containing NGN2, ISL1, and LHX3 into the AAVS1 safe-harbor locus of control, C9ORF72+/− and C9ORF72 patient iPSCs. This enabled large-scale production of iMNs that expressed motor neuron markers and had transcriptional profiles similar to 7F iMNs (Supplementary Fig. 11). Using this approach, we quantified the amount of surface-bound NR1 by immunoblotting after using surface protein biotinylation to isolate membrane-bound proteins. This confirmed that surface NR1 levels were higher on C9ORF72+/− and C9ORF72 patient iMNs (n=2 patients) than controls (n=3 controls)(Fig. 4e-h, Supplementary Fig. 5g, h).
Immunostaining revealed that C9ORF72+/− and C9ORF72−/− iMNs contained elevated levels of NMDA (NR1) and AMPA (GLUR1) receptors on neurites and dendritic spines compared to control iMNs under basal conditions (Fig. 4a, c, d and Supplementary Fig. 5b and 10a, c-e, g, h, j, k). In addition, control iMNs treated with C9ORF72-specific ASOs displayed increased numbers of NMDA and AMPA receptors in their neurites (Supplementary Fig. 10l, m). C9ORF72 patient iMNs (n=3 patients) also showed elevated NR1 and GLUR1 levels compared to controls (n=3 controls), and forced expression of C9ORF72 isoform B reduced glutamate receptor levels in patient iMNs (n=3 patients) to that of controls (n=3 controls) (Fig. 4a-c and Supplementary Fig. 10a-h). mRNA levels of NR1 (GRIN1) and GLUR1 (GRIA1) were not elevated in flow-purified C9ORF72+/− iMNs, indicating that increased transcription could not explain the increased glutamate receptor levels (Supplementary Fig. 10n).
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