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Lysosomes require an acidic lumen between pH 4.5 and 5.0 for effective digestion of macromolecules. This pH optimum is maintained by proton influx produced by the V-ATPase and efflux through an unidentified "H + leak" pathway. Here we show that TMEM175, a genetic risk factor for Parkinson's disease (PD), mediates the lysosomal H + leak by acting as a proton-activated, proton-selective channel on the lysosomal membrane (LyPAP). Acidification beyond the normal range potently activated LyPAP to terminate further acidification of lysosomes. An endogenous polyunsaturated fatty acid and synthetic agonists also activated TMEM175 to trigger lysosomal proton release. TMEM175 deficiency caused lysosomal over-acidification, impaired proteolytic activity, and facilitated α-synuclein aggregation in vivo. Mutational and pH normalization analyses indicated that the channel's H + conductance is essential for normal lysosome function. Thus, modulation of LyPAP by cellular cues may dynamically tune the pH optima of endosomes and lysosomes to regulate lysosomal degradation and PD pathology., Competing Interests: Declaration of interests H.X. is the scientific co-founder and a partial owner of Lysoway Therapeutics Inc (Boston, MA)., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Loss-of-function mutations in Parkin are the most common causes of autosomal recessive Parkinson's disease (PD). Many putative substrates of parkin have been reported; their pathogenic roles, however, remain obscure due to poor characterization, particularly in vivo. Here, we show that synaptotagmin-11, encoded by a PD-risk gene SYT11, is a physiological substrate of parkin and plays critical roles in mediating parkin-linked neurotoxicity. Unilateral overexpression of full-length, but not C2B-truncated, synaptotagmin-11 in the substantia nigra pars compacta (SNpc) impairs ipsilateral striatal dopamine release, causes late-onset degeneration of dopaminergic neurons, and induces progressive contralateral motor abnormalities. Mechanistically, synaptotagmin-11 impairs vesicle pool replenishment and thus dopamine release by inhibiting endocytosis. Furthermore, parkin deficiency induces synaptotagmin-11 accumulation and PD-like neurotoxicity in mouse models, which is reversed by SYT11 knockdown in the SNpc or knockout of SYT11 restricted to dopaminergic neurons. Thus, PD-like neurotoxicity induced by parkin dysfunction requires synaptotagmin-11 accumulation in SNpc dopaminergic neurons.

Embryonic stem cell-based therapies exhibit great potential for the treatment of Parkinson's disease (PD) because they can significantly rescue PD-like behaviors. However, whether the transplanted cells themselves release dopamine in vivo remains elusive. We and others have recently induced human embryonic stem cells into primitive neural stem cells (pNSCs) that are self-renewable for massive/transplantable production and can efficiently differentiate into dopamine-like neurons (pNSC-DAn) in culture. Here, we showed that after the striatal transplantation of pNSC-DAn, (i) pNSC-DAn retained tyrosine hydroxylase expression and reduced PD-like asymmetric rotation; (ii) depolarization-evoked dopamine release and reuptake were significantly rescued in the striatum both in vitro (brain slices) and in vivo, as determined jointly by microdialysis-based HPLC and electrochemical carbon fiber electrodes; and (iii) the rescued dopamine was released directly from the grafted pNSC-DAn (and not from injured original cells). Thus, pNSC-DAn grafts release and reuptake dopamine in the striatum in vivo and alleviate PD symptoms in rats, providing proof-of-concept for human clinical translation.