We investigated the role of mitochondria in the regulation of intracellular Ca 2ϩ ([Ca 2ϩ ] i) and excitability of myenteric neurons in guinea pig ileum, using microelectrodes and fura-2 [Ca 2ϩ ] i measurements. In AH/Type-II neurons, action potentials evoke ryanodine-sensitive increases in [Ca 2ϩ ] i that activate Ca 2ϩ-dependent K ϩ channels and slow afterhyperpolarizations (AH) lasting ϳ15 sec. Exposure to the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP; 1 M) had no significant effect on the membrane potential or resting [Ca 2ϩ ] i. However, action potentials elicited in the presence of FCCP triggered a sustained (Ͼ5 min) increase in [Ca 2ϩ ] i and a compound hyperpolarization (13.4 Ϯ 1.5 mV). The respiratory chain blockers antimycin A and rotenone (10 M) had similar effects that developed more slowly. Depletion of the intracellular Ca 2ϩ stores with thapsigargin (2 M) or ryanodine (10 M) greatly attenuated the hyperpolarization caused by FCCP. S/Type-I neurons that do not have AH were hyperpolar-ized by mitochondrial inhibition independently of action potentials. Blockade of the F 0 F 1 ATPase by oligomycin (10 M) had variable effects on myenteric neurons. The majority of AH/ Type-II neurons were hyperpolarized by oligomycin, most likely by activating ATP-dependent K ϩ channels. This hyperpolarization was not triggered by action potential firing and not accompanied by an increase in [Ca 2ϩ ] i. MitoTracker staining revealed a dense mitochondrial network particularly in myenteric AH/ Type-II neurons, supporting the importance of mitochondrial Ca 2ϩ buffering in this subset of neurons. The data indicate that mitochondrial uptake of Ca 2ϩ released from the endoplasmic reticulum sets [Ca 2ϩ ] i and the activity of Ca 2ϩ-dependent conductances, thus regulating the excitability of myenteric neurons.

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Hexanucleotide repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we use human induced pluripotent stem cell-derived motor neurons to show that C9orf72 repeat expansions impair microtubule-based transport of mitochondria, a process critical for maintenance of neuronal function. Cargo transport defects are recapitulated by treating healthy neurons with the arginine-rich dipeptide repeat proteins (DPRs) that are produced by the hexanucleotide repeat expansions. Single-molecule imaging shows that these DPRs perturb motility of purified kinesin-1 and cytoplasmic dynein-1 motors along microtubules in vitro. Additional in vitro and in vivo data indicate that the DPRs impair transport by interacting with both microtubules and the motor complexes. We also show that kinesin-1 is enriched in DPR inclusions in patient brains and that increasing the level of this motor strongly suppresses the toxic effects of arginine-rich DPR expression in a Drosophila model. Collectively, our study implicates an inhibitory interaction of arginine-rich DPRs with the axonal transport machinery in C9orf72-associated ALS/FTD and thereby points to novel potential therapeutic strategies. INTRODUCTIONA GGGGCC (G4C2) repeat expansion in the C9orf72 gene is the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9-ALS/FTD) (1,2). Since the association between the hexanucleotide repeat expansions (HREs) and these neurodegenerative diseases was discovered, three non-mutually exclusive pathological mechanisms have been proposed. The first is a loss-of-function scenario due to decreased expression of C9orf72 transcript and protein observed in C9-ALS/FTD patients (1,3). The second is an RNA gain-of-function mechanism caused by the accumulation of expanded repeat transcripts that sequester numerous RNA-binding proteins (4-9). The third proposed mechanism is a protein gain-of-function via the generation of pathological dipeptide repeat proteins (DPRs) originating from non-ATG mediated translation of the expanded repeat transcripts (10-13).This repeat-associated non-ATG (RAN) translation occurs in all reading frames of sense and antisense transcripts resulting in five DPR proteins: poly-GR and poly-GA exclusively from the sense transcript, poly-PR and poly-PA exclusively from the antisense transcript, and poly-GP from both transcripts (10-13). DPRs are found in cytoplasmic inclusions in C9-ALS/FTD post-mortem brain and spinal cord tissue, and also have been detected in motor neurons differentiated from patient-derived induced pluripotent stem cells (iPSCs) (5,10,11,(14)(15)(16)(17)(18). The arginine-rich DPRs -poly-PR and poly-GR -are potently toxic in numerous disease models (14,(19)(20)(21)(22)(23)(24)(25)(26)(27), and have been shown to cause mitochondrial (28,29) and endoplasmic reticulum stress (26), as well as disturbances...

Parkinson's disease (PD) is a neurodegenerative disease with motor and non-motor symptoms, including constipation. Therefore, several studies have investigated the gastrointestinal tract, and more specifically the enteric nervous system (ENS), in search of an early biomarker of PD. Besides α-synuclein aggregation, mitochondrial dysfunction and dysregulation of intracellular Ca2+ concentration probably contribute to the pathogenesis of PD. Here we assessed neuronal and mitochondrial functioning in primary enteric neurons of PD patients and their healthy partners as controls. Using a unique combination of live microscopy techniques, applied to routine duodenum biopsies, we were able to record neuronal Ca2+ responses and mitochondrial membrane potential in these nerve tissues. We found that submucous neurons were not affected in PD patients, which suggests that these neurons are not involved in the pathogenesis or the gastrointestinal symptoms of PD. Our study provides for the first time functional information on live neurons in PD patients.DOI: http://dx.doi.org/10.7554/eLife.26850.001

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