Stem cell modeling of mitochondrial parkinsonism reveals key functions of OPA1.

OBJECTIVE: Defective mitochondrial function attributed to optic atrophy 1 (OPA1) mutations causes primarily optic atrophy and, less commonly, neurodegenerative syndromes. The pathomechanism by which OPA1 mutations trigger diffuse loss of neurons in some, but not all, patients is unknown. Here, we used a tractable induced pluripotent stem cell (iPSC)-based model to capture the biology of OPA1 haploinsufficiency in cases presenting with classic eye disease versus syndromic parkinsonism. METHODS: iPSCs were generated from 2 patients with OPA1 haploinsufficiency and 2 controls and differentiated into dopaminergic neurons. Metabolic profile was determined by extracellular flux analysis, respiratory complex levels using immunoblotting, and complex I activity by a colorimetric assay. Mitochondria were examined by transmission electron microscopy. Mitochondrial DNA copy number and deletions were assayed using long-range PCR. Mitochondrial membrane potential was measured by tetramethylrhodamine methyl ester uptake, and mitochondrial fragmentation was assessed by confocal microscopy. Exome sequencing was used to screen for pathogenic variants. RESULTS: OPA1 haploinsufficient iPSCs differentiated into dopaminergic neurons and exhibited marked reduction in OPA1 protein levels. Loss of OPA1 caused a late defect in oxidative phosphorylation, reduced complex I levels, and activity without a significant change in the ultrastructure of mitochondria. Loss of neurons in culture recapitulated dopaminergic degeneration in syndromic disease and correlated with mitochondrial fragmentation. INTERPRETATION: OPA1 levels maintain oxidative phosphorylation in iPSC-derived neurons, at least in part, by regulating the stability of complex I. Severity of OPA1 disease associates primarily with the extent of OPA1-mediated fusion, suggesting that activation of this mechanism or identification of its genetic modifiers may have therapeutic or prognostic value. Ann Neurol 2018;83:915-925.

Amyotrophic lateral sclerosis patient iPSC-derived astrocytes impair autophagy via non-cell autonomous mechanisms.

Amyotrophic lateral sclerosis, a devastating neurodegenerative disease, is characterized by the progressive loss of motor neurons and the accumulation of misfolded protein aggregates. The latter suggests impaired proteostasis may be a key factor in disease pathogenesis, though the underlying mechanisms leading to the accumulation of aggregates is unclear. Further, recent studies have indicated that motor neuron cell death may be mediated by astrocytes. Herein we demonstrate that ALS patient iPSC-derived astrocytes modulate the autophagy pathway in a non-cell autonomous manner. We demonstrate cells treated with patient derived astrocyte conditioned medium demonstrate decreased expression of LC3-II, a key adapter protein required for the selective degradation of p62 and ubiquitinated proteins targeted for degradation. We observed an increased accumulation of p62 in cells treated with patient conditioned medium, with a concomitant increase in the expression of SOD1, a protein associated with the development of ALS. Activation of autophagic mechanisms with Rapamycin reduces the accumulation of p62 puncta in cells treated with patient conditioned medium. These data suggest that patient astrocytes may modulate motor neuron cell death by impairing autophagic mechanisms, and the autophagy pathway may be a useful target in the development of novel therapeutics.

In vitro and ex vivo models of multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune disorder of the central nervous system (CNS). Current therapies suppress a misdirected myelin-destructive immune response. To combat the progressive, neurodestructive phase of MS, the therapeutic research focus is currently on compounds that might boost the endogenous potential of the brain to remyelinate axons, thereby achieving lesion repair. Here, we describe the testing of fingolimod on cultures of oligodendrocytes (OLs) and organotypic brain slices. We detail the protocols, pros, and cons of these in vitro and ex vivo approaches, along with the potential benefit of exploiting skin-punch biopsies from patients with MS, before concluding with a summary of future developments.