Usp14 deficiency removes α-synuclein by regulating S100A8/A9 in Parkinson's disease.

Ubiquitin-proteasome system dysfunction triggers α-synuclein aggregation, a hallmark of neurodegenerative diseases, such as Parkinson's disease (PD). However, the crosstalk between deubiquitinating enzyme (DUBs) and α-synuclein pathology remains unclear. In this study, we observed a decrease in the level of ubiquitin-specific protease 14 (USP14), a DUB, in the cerebrospinal fluid (CSF) of PD patients, particularly females. Moreover, CSF USP14 exhibited a dual correlation with α-synuclein in male and female PD patients. To investigate the impact of USP14 deficiency, we crossed USP14 heterozygous mouse (USP14+/-) with transgenic A53T PD mouse (A53T-Tg) or injected adeno-associated virus (AAV) carrying human α-synuclein (AAV-hα-Syn) in USP14+/- mice. We found that Usp14 deficiency improved the behavioral abnormities and pathological α-synuclein deposition in female A53T-Tg or AAV-hα-Syn mice. Additionally, Usp14 inactivation attenuates the pro-inflammatory response in female AAV-hα-Syn mice, whereas Usp14 inactivation demonstrated opposite effects in male AAV-hα-Syn mice. Mechanistically, the heterodimeric protein S100A8/A9 may be the downstream target of Usp14 deficiency in female mouse models of α-synucleinopathies. Furthermore, upregulated S100A8/A9 was responsible for α-synuclein degradation by autophagy and the suppression of the pro-inflammatory response in microglia after Usp14 knockdown. Consequently, our study suggests that USP14 could serve as a novel therapeutic target in PD.

TSPAN1 inhibits metastasis of nasopharyngeal carcinoma via suppressing NF-kB signaling.

Nasopharyngeal carcinoma (NPC) originates in the epithelial cells of the nasopharynx and is a common malignant tumor in southern China and Southeast Asia. Metastasis of NPC remains the main cause of death for NPC patients even though the tumor is sensitive to radiotherapy and chemotherapy. Here, we found that the transmembrane protein tetraspanin1 (TSPAN1) potently inhibited the in vitro migration and invasion, as well as, the in vivo metastasis of NPC cells via interacting with the IKBB protein. In addition, TSPAN1 was essential in preventing the overactivation of the NF-kB pathway in TSPAN1 overexpressing NPC cells. Furthermore, reduced TSPAN1 expression was associated with NPC metastasis and the poor prognosis of NPC patients. These results uncovered the suppressive role of TSPAN1 against NF-kB signaling in NPC cells for preventing NPC metastasis. Its therapeutic value warrants further investigation.

Ulinastatin inhibits the metastasis of nasopharyngeal carcinoma by involving uPA/uPAR signaling.

Distant metastasis is the primary reason for treatment failure in patients with nasopharyngeal carcinoma (NPC). In this study, we investigated the effect of ulinastatin (UTI) on NPC metastasis and its underlying mechanism. Highly-metastatic NPC cell lines S18 and 58F were treated with UTI and the effect on cell proliferation, migration, and invasion were determined by MTS and Transwell assays. S18 cells with luciferase-expressing (S18-1C3) were injected into the left hind footpad of nude mice to establish a model of spontaneous metastasis from the footpad to popliteal lymph node (LN). The luciferase messenger RNA (mRNA) was measured by quantitative polymerase chain reaction (qPCR), and the metastasis inhibition rate was calculated. Key molecular members of the UTI-related uPA, uPAR, and JAT/STAT3 signaling pathways were detected by qPCR and immunoblotting. UTI suppressed the migration and infiltration of S18 and 5-8F cells and suppressed the metastasis of S18 cells in vivo without affecting cell proliferation. uPAR expression decreased from 24 to 48 h after UTI treatment. The antimetastatic effect of UTI is partly due to the suppression of uPA and uPAR. UTI partially suppresses NPC metastasis by downregulating the expression of uPA and uPAR.

Dietary intake of α-ketoglutarate ameliorates α-synuclein pathology in mouse models of Parkinson's disease.

Parkinson's disease (PD) is a progressive movement disorder characterized by dopaminergic (DA) neuron degeneration and the existence of Lewy bodies formed by misfolded α-synuclein. Emerging evidence supports the benefits of dietary interventions in PD due to their safety and practicality. Previously, dietary intake of α-ketoglutarate (AKG) was proved to extend the lifespan of various species and protect mice from frailty. However, the mechanism of dietary AKG's effects in PD remains undetermined. In the present study, we report that an AKG-based diet significantly ameliorated α-synuclein pathology, and rescued DA neuron degeneration and impaired DA synapses in adeno-associated virus (AAV)-loaded human α-synuclein mice and transgenic A53T α-synuclein (A53T α-Syn) mice. Moreover, AKG diet increased nigral docosahexaenoic acid (DHA) levels and DHA supplementation reproduced the anti-α-synuclein effects in the PD mouse model. Our study reveals that AKG and DHA induced microglia to phagocytose and degrade α-synuclein via promoting C1q and suppressed pro-inflammatory reactions. Furthermore, results indicate that modulating gut polyunsaturated fatty acid metabolism and microbiota Lachnospiraceae_NK4A136_group in the gut-brain axis may underlie AKG's benefits in treating α-synucleinopathy in mice. Together, our findings propose that dietary intake of AKG is a feasible and promising therapeutic approach for PD.

Cntnap4 partial deficiency exacerbates α-synuclein pathology through astrocyte-microglia C3-C3aR pathway.

Parkinson's disease (PD) is the most common progressive neurodegenerative movement disorder, which is characterized by dopaminergic (DA) neuron death and the aggregation of neurotoxic α-synuclein. Cntnap4, a risk gene of autism, has been implicated to participate in PD pathogenesis. Here we showed Cntnap4 lacking exacerbates α-synuclein pathology, nigrostriatal DA neuron degeneration and motor impairment, induced by injection of adeno-associated viral vector (AAV)-mediated human α-synuclein overexpression (AAV-hα-Syn). This scenario was further validated in A53T α-synuclein transgenic mice injected with AAV-Cntnap4 shRNA. Mechanistically, α-synuclein derived from damaged DA neuron stimulates astrocytes to release complement C3, activating microglial C3a receptor (C3aR), which in turn triggers microglia to secrete complement C1q and pro-inflammatory cytokines. Thus, the astrocyte-microglia crosstalk further drives DA neuron death and motor dysfunction in PD. Furthermore, we showed that in vivo depletion of microglia and microglial targeted delivery of a novel C3aR antagonist (SB290157) rescue the aggravated α-synuclein pathology resulting from Cntnap4 lacking. Together, our results indicate that Cntnap4 plays a key role in α-synuclein pathogenesis by regulating glial crosstalk and may be a potential target for PD treatment.

Correlated with better prognosis, CSTA inhibits metastasis of nasopharyngeal carcinoma cells via suppressing AKT signaling through promoting METTL3 degradation.

BACKGROUND: Metastasis is one of the main obstacles impeding the survival of nasopharyngeal carcinoma (NPC) patients, with the molecular mechanism underlying NPC metastasis still unclear. RESULTS: In this study, Cystatin A (CSTA) was found downregulated in NPC tissues with metastasis compared with those without metastasis. Shorter overall survival and distant metastasis-free survival were found in NPC patients with lower CSTA expression. Using functional assays, we found that CSTA prevented both the in vitro motility of NPC cells and their ability to metastasize in vivo. Transcriptome sequencing and western blot analysis revealed that CSTA inhibited the phosphorylation of AKT. Moreover, activating AKT using AKT agonist SG79 rescued the motility of CSTA-overexpressing NPC cells, whereas, treatment with AKT inhibitor MK2206 inhibited the motility of CSTA-knockdown NPC cells. Mechanically, immunoprecipitation coupled mass spectrometry found that CSTA interacted with the N6-adenosine-methyltransferase subunit METTL3 and promoted its ubiquitin-proteasome-mediated degradation following the upregulation of NKX3-1 and LHPP, which are negative regulators of AKT. Furthermore, knock-down of NKX3-1 and LHPP enhanced the motility of CSTA-overexpressing NPC cells. CONCLUSIONS: The inhibitory effect of CSTA upon NPC metastasis mainly depended on suppressing AKT signaling by the upregulation of NKX3-1 and LHPP expression resulting from the binding between CSTA and METLL3. Our study suggests that the CSTA-METLL3-NKX3-1/LHPP-AKT axis could be of therapeutic value for inhibiting NPC metastasis.

Lactobacillus reuteri normalizes altered fear memory in male Cntnap4 knockout mice.

BACKGROUND: Autism spectrum disorder (ASD) is a common neurodevelopmental disease, characterized by deficits in social communication, restricted and repetitive behaviours, and impaired fear memory processing. Severe gastrointestinal dysfunction and altered gut microbiome have been reported in ASD patients and animal models. Contactin associated protein-like 4 (CNTNAP4) has been suggested to be a novel risk gene, though its role in ASD remains unelucidated. METHODS: Cntnap4-/- mice were generated to explore its role in ASD-related behavioural abnormalities. Electrophysiological recording was employed to examine GABAergic transmission in the basolateral amygdala (BLA) and prefrontal cortex. RNA-sequencing was performed to assess underlying mechanisms. 16S rDNA analysis was performed to explore changes in faecal microbial composition. Male Cntnap4-/- mice were fed with Lactobacillus reuteri (L. reuteri) or faecal microbiota to evaluate the effects of microbiota supplementation on the impaired fear conditioning mediated by Cntnap4 deficiency. FINDINGS: Male Cntnap4-/- mice manifested deficiency in social behaviours and tone-cue fear conditioning. Notably, reduced GABAergic transmission and GABA receptor expression were found in the BLA but not the prefrontal cortex. In addition, gut Lactobacillus were less abundant in male Cntnap4-/- mice, and L. reuteri treatment or faecal microbiota transplantation rescued abnormal tone-cued fear memory and improved local GABAergic transmission in the BLA of male Cntnap4-/- mice. INTERPRETATION: Cntnap4 shapes GABAergic transmission of amygdala and fear conditioning, and microbial intervention represents a promising therapy in ASD intervention. FUNDING: National Natural Science Foundation of China, Science and Technology Planning Project of Guangzhou, Guangzhou Medical University, and China Postdoctoral Science Foundation.

Chaperone-mediated Autophagy Regulates Cell Growth by Targeting SMAD3 in Glioma.

Previous studies suggest that the reduction of SMAD3 (mothers against decapentaplegic homolog 3) has a great impact on tumor development, but its exact pathological function remains unclear. In this study, we found that the protein level of SMAD3 was greatly reduced in human-grade IV glioblastoma tissues, in which LAMP2A (lysosome-associated membrane protein type 2A) was significantly up-regulated. LAMP2A is a key rate-limiting protein of chaperone-mediated autophagy (CMA), a lysosome pathway of protein degradation that is activated in glioma. We carefully analyzed the amino-acid sequence of SMAD3 and found that it contained a pentapeptide motif biochemically related to KFERQ, which has been proposed to be a targeting sequence for CMA. In vitro, we confirmed that SMAD3 was degraded in either serum-free or KFERQ motif deleted condition, which was regulated by LAMP2A and interacted with HSC70 (heat shock cognate 71 kDa protein). Using isolated lysosomes, amino-acid residues 75 and 128 of SMAD3 were found to be of importance for this process, which affected the CMA pathway in which SMAD3 was involved. Similarly, down-regulating SMAD3 or up-regulating LAMP2A in cultured glioma cells enhanced their proliferation and invasion. Taken together, these results suggest that excessive activation of CMA regulates glioma cell growth by promoting the degradation of SMAD3. Therefore, targeting the SMAD3-LAMP2A-mediated CMA-lysosome pathway may be a promising approach in anti-cancer therapy.

Functional validation of a human GLUD2 variant in a murine model of Parkinson's disease.

Parkinson's disease (PD) is a common neurodegenerative disease characterized by Lewy body formation and progressive dopaminergic neuron death in the substantia nigra (SN). Genetic susceptibility is a strong risk factor for PD. Previously, a rare gain-of-function variant of GLUD2 glutamate dehydrogenase (T1492G) was reported to be associated with early onset in male PD patients; however, the function and underlying mechanism of this variant remains elusive. In the present study, we generated adeno-associated virus expressing GLUD2 and its mutant under the control of the glial fibrillary acidic protein promotor and injected the virus into the SN pars compacta of either untreated mice or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD model mice. Our results demonstrate that GLUD2 mutation in MPTP-induced PD mice exacerbates movement deficits and nigral dopaminergic neuron death and reduces glutamate transporters expression and function. Using GC-Q-TOF/MS-based metabolomics, we determined that GLUD2 mutation damages mitochondrial function by decreasing succinate dehydrogenase activity to impede the tricarboxylic acid cycle in the SN of MPTP-induced PD mice. Accordingly, GLUD2 mutant mice had reduced energy metabolism and increased apoptosis, possibly due to downregulation of brain-derived neurotrophic factor/nuclear factor E2-related factor 2 signaling in in vitro and in vivo PD models. Collectively, our findings verify the function of GLUD2 in PD and unravel a mechanism by which a genetic variant in human GLUD2 may contribute to disease onset.

CNTNAP4 deficiency in dopaminergic neurons initiates parkinsonian phenotypes.

Rationale: Contactin-associated protein-like 4 (CNTNAP4) belongs to the neurexin superfamily and has critical functions in neurological development and synaptic function. Loss of CNTNAP4 in interneurons has been linked to autism, schizophrenia, and epilepsy. CNTNAP4 is also highly enriched in dopaminergic (DA) neurons in the substantia nigra (SN), however, few studies have investigated the role of CNTNAP4 in DA neurons, and whether CNTNAP4 deficiency in DA neurons contributes to Parkinson's disease (PD) remains unclear. Methods: Effects of CNTNAP4 knockdown or overexpression on the DA MN9D cell line were assessed via Western blotting, immunocytochemistry, and RNA sequencing. An in vivo animal model, including CNTNAP4 knockout mice and stereotaxic injections of adeno-associated viral short-hairpin RNA with the tyrosine-hydroxylase promotor to silence CNTNAP4 in the SN, as well as the resulting physiological/behavioral effects, were evaluated via behavioral tests, Western blotting, immunohistochemistry, and transmission electron microscopy. Enzyme-linked immunosorbent assays (ELISAs) were performed to examine the cerebrospinal fluid (CSF) and plasma CNTNAP4 concentrations in PD patients. Results: We demonstrated that CNTNAP4 knockdown induced mitophagy and increased α-synuclein expression in MN9D cells. CNTNAP4 knockdown in the SN induced PD-like increases in SN-specific α-synuclein expression, DA neuronal degeneration, and motor dysfunction in mice. In addition, CNTNAP4 knockdown in SN-DA neurons increased autophagosomes and reduced synaptic vesicles in the SN. Furthermore, CNTNAP4 knockout mice showed movement deficits, nigral DA degeneration, and increased autophagy, which were consistent with the SN-specific knockdown model. We also found that CSF and plasma CNTNAP4 expression was increased in PD patients; in particular, plasma CNTNAP4 was increased in male PD patients compared with controls or female PD patients. Conclusion: Our findings suggest that CNTNAP4 deficiency may initiate phenotypes relevant to PD, of which we elucidated some of the underlying mechanisms.