Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a familial progressive degenerative disorder and is caused by mutations in NOTCH3 gene. Previous study reported that mutant NOTCH3 is more prone to form aggregates than wild-type NOTCH3 and the mutant aggregates are resistant to degradation. We hypothesized that aggregation or accumulation of NOTCH3 could be due to impaired lysosomal-autophagy machinery in VSMC. Here, we investigated the possible cause of accumulation/aggregation of NOTCH3 in CADASIL using cerebral VSMCs derived from control and CADASIL patients carrying NOTCH3(RI33C) mutation. Thioflavin-S-staining confirmed the increased accumulation of aggregated NOTCH3 in VSMCR133C compared to VSMCWT. Increased levels of the lysosomal marker, Lamp2, were detected in VSMCR133C, which also showed co-localization with NOTCH3 using double-immunohistochemistry. Increased level of LC3-II/LC3-I ratio was observed in VSMCR133C suggesting an accumulation of autophagosomes. This was coupled with the decreased co-localization of NOTCH3 with LC3, and Lamp2 and, further, increase of p62/SQSTM1 levels in VSMCR133C compared to the VSMCWT. In addition, Western blot analysis indicated phosphorylation of p-ERK, p-S6RP, and p-P70 S6K. Altogether, these results suggested a dysfunction in the autophagy-lysosomal pathway in VSMCR133C. The present study provides an interesting avenue of the research investigating the molecular mechanism of CADASIL.

Cerebral dopamine neurotrophic factor (CDNF) is neuroprotective for nigrostriatal dopamine neurons and restores dopaminergic function in animal models of Parkinson's disease (PD). To understand the role of CDNF in mammals, we generated CDNF knockout mice (Cdnf(-/-)), which are viable, fertile, and have a normal life-span. Surprisingly, an age-dependent loss of enteric neurons occurs selectively in the submucosal but not in the myenteric plexus. This neuronal loss is a consequence not of increased apoptosis but of neurodegeneration and autophagy. Quantitatively, the neurodegeneration and autophagy found in the submucosal plexus in duodenum, ileum and colon of the Cdnf(-/-) mouse are much greater than in those of Cdnf(+/+) mice. The selective vulnerability of submucosal neurons to the absence of CDNF is reminiscent of the tendency of pathological abnormalities to occur in the submucosal plexus in biopsies of patients with PD. In contrast, the number of substantia nigra dopamine neurons and dopamine and its metabolite concentrations in the striatum are unaltered in Cdnf(-/-) mice; however, there is an age-dependent deficit in the function of the dopamine system in Cdnf(-/-) male mice analyzed. This is observed as D-amphetamine-induced hyperactivity, aberrant dopamine transporter function, and as increased D-amphetamine-induced dopamine release demonstrating that dopaminergic axon terminal function in the striatum of the Cdnf(-/-) mouse brain is altered. The deficiencies of Cdnf(-/-) mice, therefore, are reminiscent of those seen in early stages of Parkinson's disease.

The European autophagy consortium Driving next-generation autophagy researchers towards translation (DRIVE) held its kick-off meeting in Groningen on the 14(th) and 15(th) of June 2018. This Marie Sklodowska-Curie Early Training Network was approved under the European Union's Horizon 2020 Research and Innovation Program and is funded for 4 years. Within DRIVE, 14 European research teams from academia and industry will train 15 PhD students through applied, cross-disciplinary and collaborative macroautophagy/autophagy research. The goal of DRIVE is to stimulate applied approaches in autophagy research and provide training towards translation, while advancing our knowledge on autophagy in specific physiological and pathological states. The strong focus on translation will prepare the PhD students to be at the forefront to exploit autophagy for the development of therapies directly benefitting patients. Thereby, DRIVE will contribute to filling the educational gap that currently exists between academia and industry, and will prepare its PhD students for alternative and highly flexible professional paths.

Dry age-related macular degeneration (AMD) is a currently untreatable vision threatening disease. Impaired proteasomal clearance and autophagy in the retinal pigment epithelium (RPE) and subsequent photoreceptor damage are connected with dry AMD, but detailed pathophysiology is still unclear. In this paper, we discover inhibition of cytosolic protease, prolyl oligopeptidase (PREP), as a potential pathway to treat dry AMD. We showed that PREP inhibitor exposure induced autophagy in the RPE cells, shown by increased LC3-II levels and decreased p62 levels. PREP inhibitor treatment increased total levels of autophagic vacuoles in the RPE cells. Global proteomics was used to examine the phenotype of a commonly used cell model displaying AMD characteristics, oxidative stress and altered protein metabolism, in vitro. These RPE cells displayed induced protein aggregation and clear alterations in macromolecule metabolism, confirming the relevance of the cell model. Differences in intracellular target engagement of PREP inhibitors were observed with cellular thermal shift assay (CETSA). These differences were explained by intracellular drug exposure (the unbound cellular partition coefficient, Kpuu). Importantly, our data is in line with previous observations regarding the discrepancy between PREP's cleaving activity and outcomes in autophagy. This highlights the need to further explore PREP's role in autophagy so that more effective compounds can be designed to battle diseases in which autophagy induction is needed. The present work is the first report investigating the PREP pathway in the RPE and we predict that the PREP inhibitors can be further optimized for treatment of dry AMD.

Osteosarcoma (OS) is a rare malignant bone tumor but is one leading cause of cancer mortality in childhood and adolescence. Cancer metastasis accounts for the primary reason for treatment failure in OS patients. The dynamic organization of the cytoskeleton is fundamental for cell motility, migration, and cancer metastasis. Lysosome Associated Protein Transmembrane 4B (LAPTM4B) is an oncogene participating in various biological progress central to cancer biogenesis. However, the potential roles of LAPTM4B in OS and the related mechanisms remain unknown. Here, we established the elevated LAPTM4B expression in OS, and it is essential in regulating stress fiber organization through RhoA-LIMK-cofilin signaling pathway. In terms of mechanism, our data revealed that LAPTM4B promotes RhoA protein stability by suppressing the ubiquitin-mediated proteasome degradation pathway. Moreover, our data show that miR-137, rather than gene copy number and methylation status, contributes to the upregulation of LAPTM4B in OS. We report that miR-137 is capable of regulating stress fiber arrangement, OS cell migration, and metastasis via targeting LAPTM4B. Combining results from cells, patients' tissue samples, the animal model, and cancer databases, this study further suggests that the miR-137-LAPTM4B axis represents a clinically relevant pathway in OS progression and a viable target for novel therapeutics.

Glioblastoma Multiforme (GBM) is the most common and aggressive types of glioma in adults. Autophagy allows degradation and recycling of cellular components such as damaged proteins and dysfunctional organelles to sustain the metabolism and homeostasis of rapidly growing cells. Recent reports suggest that autophagy may promote tumor cell survival under stress conditions and can be an emerging target for cancer therapy. Autophagy inhibitor combined with TMZ can induce glioblastoma cell death and improve the radiotherapy efficacy. The Neuropilin-1 (NRP1) is 120 kDa-130 kDa type I integral transmembrane protein. It was originally identified as a co-receptor for the class 3 semaphorins (SEMA3) involved in axon guidance and found to interact with VEGF/VEGFR2 to promote angio-genesis. Recent studies have revealed its much broader roles on tumor progression. In various types of human cancers, NRP1 is often up-regulated and associated with aggressive clinical tumor behaviour. NRP1 overexpression is independently correlated with poor prognosis in human glioma and contributes to balance the glioblastoma cell proliferation and survival. In cancer cells, it interacts with diverse growth factor receptors including TGFR, c-Met, FGFR, EGFR as well as PDGFR to promote their signal-ling pathways in tumor cell survival, proliferation, migration and invasion. Although these functions of NRP1 mainly rely on its ectodomain, the cytoplasmic domain of NRP1 has been recently found to be essential for the internalization of NRP1-binding complex. In addition, the C-terminal SEA sequence on its cytoplasmic domain have potential to bind intracellular PDZ domain-containing molecules. Tyrosine phosphorylation of p130Cas has been identified to regulate the downstream pathways of NRP1, which is dependent on NRP1 intracellular domain. However, the downstream trafficking of NRP1 is poorly understood and its tumor-promoting function relevance remains ambiguous. The p62/Sequestosome 1, encoded by SQSTM1 gene, is an intracellular protein commonly found in inclusion bodies. It is asso-iated with protein aggregation diseases in liver and brain. Owing to its ability to interact with multiple important cellular intermediates, it works as a 'hub' adaptor linked to nuclear factor-kappaB (NF-κB) activation, protein aggregates formation, selective autophagy, adipogenesis and tumorigenesis. The p62 has a critical role on autophagy via regulating the collection and delivery of ubiquitinylated cargos to the autophagosome via its PB1, UBA and LIR domains. On the other hand, recent study has revealed a new role of p62 as a negative regulator in the autophagy regulation. High level of p62 is able to suppress the autophagy by pro-moting mTORC1 activation. This route forms a feed-forward loop for increasing level of p62 due to the reduced autophagy. Thus, p62 plays a critical role in regulation of autophagy. Here we observed that suppression of NRP1 in glioblastoma cell clearly exhibits a defected autophagy accompanied by marked accumulation of p62, the autophagic adaptor. Overexpression of NRP1 by glioblastoma cells shows enhanced autophagy flux. These data suggests the role of NRP1 in autophagy promotion. In addition, we mapped out that p62 binds to the cytoplasmic domain of NRP1 mediating its pro-autophagy effects. PB1 domain of p62 overexpression enhances the p62-positive aggregates and NRP1/p62 interaction. Taken together, our results define a novel role of NRP1 in the regulation of autophagy through its association with p62. In summary, our present results provide novel insights into the molecular basis of the emerging interplay between NRP1 and autophagy, the identification of a new cytoplasmic protein that binding to intracellular domain of NRP1 and the implications of the p62-mediated signalling loop for NRP1-promoted autophagy in GBMs. Since efforts to inhibit autophagy to improve GBM therapy have thereby attracted great interest, our findings may provide valuable clues for future cancer therapeutic strategies.

Growing evidence emphasizes insufficient clearance of pathological alpha-synuclein (alpha SYN) aggregates in the progression of Parkinson's disease (PD). Consequently, cellular degradation pathways represent a potential therapeutic target. Prolyl oligopeptidase (PREP) is highly expressed in the brain and has been suggested to increase alpha SYN aggregation and negatively regulate the autophagy pathway. Inhibition of PREP with a small molecule inhibitor, KYP-2407, stimulates autophagy and reduces the oligomeric species of alpha SYN aggregates in PD mouse models. However, whether PREP inhibition has any effects on intracellular alpha SYN fibrils has not been studied before. In this study, the effect of KYP2407 on alpha SYN preformed fibrils (PFFs) was tested in SH-SY5Y cells and human astmcytes. Immunostaining analysis revealed that both cell types accumulated alpha SYN PFFs intracellularly but KYP-2047 decreased intracellular alpha SYN deposits only in SH-SY5Y cells, as astrocytes did not show any PREP activity. Western blot analysis confirmed the reduction of high molecular weight alpha SYN species in SH-SY5Y cell lysates, and secretion of aSYN from SH-SY5Y cells also decreased in the presence of KYP-2407. Accumulation of alpha SYN inside the SH-SY5Y cells resulted in an increase of the auto-lysosomal proteins p62 and LC3BII, as well as calpain 1 and 2, which have been shown to be associated with PD pathology. Notably, treatment with KYP-2407 significantly reduced p62 and LC3BII levels, indicating an increased autophagic flux, and calpain 1 and 2 levels returned to normal in the presence of KYP-2407. Our findings indicate that PREP inhibition can potentially be used as therapy to reduce the insoluble intracellular alpha SYN aggregates.

RAB24 belongs to a family of membrane traffic controlling RAB proteins and has been implicated to function in autophagy. Here we confirm the intracellular localization of RAB24 to autophagic vacuoles with immuno electron microscopy and cell fractionation, and show that prenylation and guanine nucleotide binding are necessary for the targeting of RAB24 to autophagic compartments. Further, we show that RAB24 plays a role in the maturation and/or clearance of autophagic compartments under nutrient-rich conditions, but not during short amino acid starvation. Quantitative electron microscopy showed an increase in the numbers of late autophagic compartments in cells silenced for RAB24, and mRFP-GFP-LC3 probe and autophagy flux experiments indicated that this was due to a hindrance in their clearance. Formation of autophagosomes was shown to be unaffected by RAB24 silencing with siRNA. A defect in aggregate clearance in the absence of RAB24 was also shown in cells forming polyglutamine aggregates. This study places RAB24 function in the termination of the autophagic process under nutrient-rich conditions.

Previous studies have shown that prolyl oligopeptidase (PREP) negatively regulates autophagy and increases the aggregation of alpha-synuclein (alpha Syn), linking it to the pathophysiology of Parkinson's disease. Our earlier results have revealed that the potent small molecular PREP inhibitor KYP-2047 is able to increase autophagy and decrease dimerization of alpha Syn but other PREP inhibitors have not been systematically studied for these two protein-protein interaction mediated biological functions of PREP. In this study, we characterized these effects for 12 known PREP inhibitors with IC50-values ranging from 0.2 nM to 1010 nM. We used protein-fragment complementation assay (PCA) to assess alpha Syn dimerization and Western Blot of microtubule-associated protein light chain 3B II (LC3B-II) and a GFP-LC3-RFP expressing cell line to study autophagy. In addition, we tested selected compounds in a cell-free alpha Syn aggregation assay, native gel electrophoresis, and determined the compound concentration inside the cell by LC-MS. We found that inhibition of the proteolytic activity of PREP did not predict decreased alpha Syn dimerization or increased autophagy, and we also confirmed that this result did not simply reflect concentration differences of the compounds inside the cell. Thus, PREP ligands regulate the effect of PREP on autophagy and alpha Syn aggregation through a conformational stabilization of the enzyme that is not equivalent to inhibiting its proteolytic activity.