The accumulation of misfolded proteins is a common pathological feature of many neurodegenerative disorders, including synucleinopathies such as Parkinson's disease (PD), which is characterized by the presence of ␣-synuclein (␣-syn)-containing Lewy bodies. However, although recent studies have investigated ␣-syn accumulation and propagation in neurons, the molecular mechanisms underlying ␣-syn transmission have been largely unexplored. Here, we examined a monogenic form of synucleinopathy caused by loss-offunction mutations in lysosomal ATP13A2/PARK9. These studies revealed that lysosomal exocytosis regulates intracellular levels of ␣-syn in human neurons. Loss of PARK9 function in patient-derived dopaminergic neurons disrupted lysosomal Ca 2ϩ homeostasis, reduced lysosomal Ca 2ϩ storage, increased cytosolic Ca 2ϩ , and impaired lysosomal exocytosis. Importantly, this dysfunction in lysosomal exocytosis impaired ␣-syn secretion from both axons and soma, promoting ␣-syn accumulation. However, activation of the lysosomal Ca 2ϩ channel transient receptor potential mucolipin 1 (TRPML1) was sufficient to upregulate lysosomal exocytosis, rescue defective ␣-syn secretion, and prevent ␣-syn accumulation. Together, these results suggest that intracellular ␣-syn levels are regulated by lysosomal exocytosis in human dopaminergic neurons and may represent a potential therapeutic target for PD and other synucleinopathies.

Besides a reduction of L-type Ca2+-currents (Ca(V)1), muscarine and the peptidic M1-selective agonist, MT-1, reduced currents through Ca(V)2.1 (P/Q) and Ca(V)2.2 (N) Ca2+ channel types. This modulation was strongly blocked by the peptide MT-7, a specific muscarinic M1-type receptor antagonist but not significantly reduced by the peptide MT-3, a specific muscarinic M4-type receptor antagonist. Accordingly, MT-7, but not MT-3, blocked a muscarinic reduction of the afterhyperpolarizing potential (AHP) and decreased the GABAergic inhibitory postsynaptic currents (IPSCs) produced by axon collaterals that interconnect spiny neurons. Both these functions are known to be dependent on P/Q and N types Ca2+ channels. The action on the AHP had an important effect in increasing firing frequency. The action on the IPSCs was shown to be caused presynaptically as it coursed with an increase in the paired-pulse ratio. These results show: first, that muscarinic M1-type receptor activation is the main cholinergic mechanism that modulates Ca2+ entry through voltage-dependent Ca2+ channels in spiny neurons. Second, this muscarinic modulation produces a postsynaptic facilitation of discharge together with a presynaptic inhibition of the GABAergic control mediated by axon collaterals. Together, both effects would tend to recruit more spiny neurons for the same task.

SummaryA major challenge for clinical application of pluripotent stem cell therapy for Parkinson's disease (PD) is large-scale manufacturing and cryopreservation of neurons that can be efficiently prepared with minimal manipulation. To address this obstacle, midbrain dopamine neurons were derived from human induced pluripotent stem cells (iPSC-mDA) and cryopreserved in large production lots for biochemical and transplantation studies. Cryopreserved, post-mitotic iPSC-mDA neurons retained high viability with gene, protein, and electrophysiological signatures consistent with midbrain floor-plate lineage. To test therapeutic efficacy, cryopreserved iPSC-mDA neurons were transplanted without subculturing into the 6-OHDA-lesioned rat and MPTP-lesioned non-human-primate models of PD. Grafted neurons retained midbrain lineage with extensive fiber innervation in both rodents and monkeys. Behavioral assessment in 6-OHDA-lesioned rats demonstrated significant reversal in functional deficits up to 6 months post transplantation with reinnervation of the host striatum and no aberrant growth, supporting the translational development of pluripotent cell-based therapies in PD.

Although it is well established that many glutamatergic neurons sequester Zn2+ within their synaptic vesicles, the physiological significance of synaptic Zn2+ remains poorly understood. In experiments performed in a Zn2+-enriched auditory brainstem nucleus -- the dorsal cochlear nucleus -- we discovered that synaptic Zn2+ and GPR39, a putative metabotropic Zn2+-sensing receptor (mZnR), are necessary for triggering the synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). The postsynaptic production of 2-AG, in turn, inhibits presynaptic probability of neurotransmitter release, thus shaping synaptic strength and short-term synaptic plasticity. Zn2+-induced inhibition of transmitter release is absent in mutant mice that lack either vesicular Zn2+ or the mZnR. Moreover, mass spectrometry measurements of 2-AG levels reveal that Zn2+-mediated initiation of 2-AG synthesis is absent in mice lacking the mZnR. We reveal a previously unknown action of synaptic Zn2+: synaptic Zn2+ inhibits glutamate release by promoting 2-AG synthesis.

. The modulatory effect of D 2 dopamine receptor activation on calcium currents was studied in neostriatal projection neurons at two stages of rat development: postnatal day (PD)14 and PD40. D 2 -class receptor agonists reduced whole cell calcium currents by about 35% at both stages, and this effect was blocked by the D 2 receptor antagonist sulpiride. Nitrendipine partially occluded this modulation at both stages, indicating that modulation of Ca V 1 channels was present throughout this developmental interval. Nevertheless, modulation of Ca V 1 channels was significantly larger in PD40 neurons. -Conotoxin GVIA occluded most of the Ca 2ϩ current modulation in PD14 neurons. However, this occlusion was greatly decreased in PD40 neurons. -Agatoxin TK occluded a great part of the modulation in PD40 neurons but had a negligible effect in PD14 neurons. The data indicate that dopaminergic D 2 -mediated modulation undergoes a change in target during development: from Ca V 2.2 to Ca V 2.1 Ca 2ϩ channels. This change occurred while Ca V 2.2 channels were being down-regulated and Ca V 2.1 channels were being up-regulated. Presynaptic modulation mediated by D 2 receptors reflected these changes; Ca V 2.2 type channels were used for release in young animals but very little in mature animals, suggesting that changes took place simultaneously at the somatodendritic and the synaptic membranes.