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.

Kufor-Rakeb syndrome (KRS) is caused by loss-of-function mutations in ATP13A2 (PARK9) and characterized by juvenile-onset parkinsonism, pyramidal signs, and cognitive decline. Previous studies suggested that PARK9 deficiency causes lysosomal dysfunction and ␣-synuclein (␣-syn) accumulation, whereas PARK9 overexpression suppresses toxicity of ␣-syn. However, the precise mechanism of PARK9 effect on lysosomes and ␣-syn has been unknown. Here, we found that overexpressed PARK9 localized to multivesicular bodies (MVBs) in the human H4 cell line. The results from patient fibroblasts showed that loss of PARK9 function leads to decreased number of the intraluminal vesicles in MVBs and diminished release of exosomes into culture media. By contrast, overexpression of PARK9 results in increased release of exosomes in H4 cells and mouse primary cortical neurons. Moreover, loss of PARK9 function resulted in decreased secretion of ␣-syn into extracellular space, whereas overexpressed PARK9 promotes secretion of ␣-syn, at least in part via exosomes. Finally, we found that PARK9 regulates exosome biogenesis through functional interaction with the endosomal sorting complex required for transport machinery. Together, these data suggest the involvement of PARK9 in the biogenesis of exosomes and ␣-syn secretion and raise a possibility that disruption of these pathways in patients with KRS contributes to the disease pathogenesis.

Alcohol use disorder (AUD) is highly comorbid with depression. Withdrawal from chronic alcohol drinking results in depression and understanding brain molecular mechanisms that drive withdrawal-related depression is important for finding new drug targets to treat these comorbid conditions. Here, we performed RNA sequencing of the rat hippocampus during withdrawal from chronic alcohol drinking to discover key signaling pathways involved in alcohol withdrawal-related depressive-like behavior. Data were analyzed by weighted gene co-expression network analysis to identify several modules of co-expressed genes that could have a common underlying regulatory mechanism. One of the hub, or highly interconnected, genes in module 1 that increased during alcohol withdrawal was the transcription factor, signal transducer and activator of transcription 3 (Stat3), a known regulator of immune gene expression. Total and phosphorylated (p)STAT3 protein levels were also increased in the hippocampus during withdrawal after chronic alcohol exposure. Further, pSTAT3 binding was enriched at the module 1 genes Gfap, Tnfrsf1a, and Socs3 during alcohol withdrawal. Notably, pSTAT3 and its target genes were elevated in the postmortem hippocampus of human subjects with AUD when compared with control subjects. To determine the behavioral relevance of STAT3 activation during alcohol withdrawal, we treated rats with the STAT3 inhibitor stattic and tested for sucrose preference as a measure of anhedonia. STAT3 inhibition alleviated alcohol withdrawal-induced anhedonia. These results demonstrate activation of STAT3 signaling in the hippocampus during alcohol withdrawal in rats and in human AUD subjects, and suggest that STAT3 could be a therapeutic target for reducing comorbid AUD and depression.

Alcohol use disorder (AUD) has a complex pathogenesis, making it a difficult disorder to treat. Identifying relevant signaling pathways in the brain may be useful for finding new pharmacological targets to treat AUD. The receptor tyrosine kinase anaplastic lymphoma kinase (ALK) activates the transcription factor STAT3 in response to ethanol in cell lines. Here, we show ALK activation and upregulation of known STAT3 target genes (Socs3, Gfap and Tnfrsf1a) in the prefrontal cortex (PFC) and ventral hippocampus (HPC) of mice after 4 days of binge‐like ethanol drinking. Mice treated with the STAT3 inhibitor stattic drank less ethanol than vehicle‐treated mice, demonstrating the behavioral importance of STAT3. To identify novel ethanol‐induced target genes downstream of the ALK and STAT3 pathway, we analyzed the NIH LINCS L1000 database for gene signature overlap between ALK inhibitor (alectinib and NVP‐TAE684) and STAT3 inhibitor (niclosamide) treatments on cell lines. These genes were then compared with differentially expressed genes in the PFC of mice after binge‐like drinking. We found 95 unique gene candidates, out of which 57 had STAT3 binding motifs in their promoters. We further showed by qPCR that expression of the putative STAT3 genes Nr1h2, Smarcc1, Smarca4 and Gpnmb were increased in either the PFC or HPC after binge‐like drinking. Together, these results indicate activation of the ALK‐STAT3 signaling pathway in the brain after binge‐like ethanol consumption, identify putative novel ethanol‐responsive STAT3 target genes, and suggest that STAT3 inhibition may be a potential method to reduce binge drinking in humans.

Background Greater circulating levels of the steroid hormone 17β‐estradiol (E2) are associated with higher levels of binge drinking in women. In female mice, estrogen receptors in the ventral tegmental area, a dopaminergic region of the brain involved in the motivation to consume ethanol, regulate binge‐like ethanol intake. We recently developed a brain‐penetrant selective estrogen receptor degrader (SERD), YL3‐122, that could be used to test the behavioral role of brain estrogen receptors. We hypothesized that treating female mice with this compound would reduce binge‐like ethanol drinking. Methods Female C57BL/6J mice were treated systemically with YL3‐122 and a related SERD with low brain penetrance, XR5‐27, and tested for binge‐like ethanol consumption in the drinking in the dark (DID) test. Mice were also tested for sucrose and water consumption and blood ethanol clearance after treatment with the SERDs. Finally, the effect of ethanol exposure on Esr1 gene expression was measured in the ventral tegmental area (VTA), prefrontal cortex (PFC), and ventral hippocampus (vHPC) of male and female mice by quantitative real‐time PCR after 4 DID sessions. Results YL3‐122 reduced ethanol consumption when mice were in diestrus but not estrus. YL3‐122 also decreased sucrose consumption but did not alter water intake or blood ethanol clearance. XR5‐27 did not affect any of these measures. Binge‐like ethanol drinking resulted in increased Esr1 transcript in the VTA of both sexes, male vHPC, and female PFC. Conclusions These results indicate that SERD treatment can decrease binge‐like ethanol drinking in female mice. Thus, it could be a novel strategy to reduce binge drinking in women, with the caveat that effectiveness may depend on menstrual cycle phase. In addition, Esr1 transcript is increased by binge ethanol exposure in both sexes but in a brain region‐specific manner.