MFGE-8 identified in fetal mesenchymal-stromal-cell-derived exosomes ameliorates acute hepatic failure pathology.

Liver transplantation is the gold-standard therapy for acute hepatic failure (AHF) with limitations related to organ shortage and life-long immunosuppressive therapy. Cell therapy emerges as a promising alternative to transplantation. We have previously shown that IL-10 and Annexin-A1 released by amniotic fluid human mesenchymal stromal cells (AF-MSCs) and their hepatocyte progenitor-like (HPL) or hepatocyte-like (HPL) cells induce liver repair and downregulate systemic inflammation in a CCl4-AHF mouse model. Herein, we demonstrate that exosomes (EXO) derived from these cells improve liver phenotype in CCl4-induced mice and promote oval cell proliferation. LC-MS/MS proteomic analysis identified MEFG-8 in EXO cargo that facilitates rescue of AHF by suppressing PI3K signaling. Administration of recombinant MFGE-8 protein also reduced liver damage in CCl4-induced mice. Clinically, MEFG-8 expression was decreased in liver biopsies from AHF patients. Collectively, our study provides proof-of-concept for an innovative, cell-free, less immunogenic, and non-toxic alternative strategy for AHF.

Quaternary structure of patient-homogenate amplified α-synuclein fibrils modulates seeding of endogenous α-synuclein.

Parkinson's disease (PD) and Multiple System Atrophy (MSA) are progressive and unremitting neurological diseases that are neuropathologically characterized by α-synuclein inclusions. Increasing evidence supports the aggregation of α-synuclein in specific brain areas early in the disease course, followed by the spreading of α-synuclein pathology to multiple brain regions. However, little is known about how the structure of α-synuclein fibrils influence its ability to seed endogenous α-synuclein in recipient cells. Here, we aggregated α-synuclein by seeding with homogenates of PD- and MSA-confirmed brain tissue, determined the resulting α-synuclein fibril structures by cryo-electron microscopy, and characterized their seeding potential in mouse primary oligodendroglial cultures. The combined analysis shows that the two patient material-amplified α-synuclein fibrils share a similar protofilament fold but differ in their inter-protofilament interface and their ability to recruit endogenous α-synuclein. Our study indicates that the quaternary structure of α-synuclein fibrils modulates the seeding of α-synuclein pathology inside recipient cells. It thus provides an important advance in the quest to understand the connection between the structure of α-synuclein fibrils, cellular seeding/spreading, and ultimately the clinical manifestations of different synucleinopathies.

Exosomes in Alpha-Synucleinopathies: Propagators of Pathology or Potential Candidates for Nanotherapeutics?

The pathological accumulation of alpha-synuclein governs the pathogenesis of neurodegenerative disorders, such as Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy, collectively termed alpha-synucleinopathies. Alpha-synuclein can be released in the extracellular space, partly via exosomes, and this extracellular protein pool may contribute to disease progression by facilitating the spread of pathological alpha-synuclein or activating immune cells. The content of exosomes depends on their origin and includes specific proteins, lipids, functional mRNAs and various non-coding RNAs. Given their ability to mediate intercellular communication via the transport of multilevel information, exosomes are considered to be transporters of toxic agents. Beyond neurons, glial cells also release exosomes, which may contain inflammatory molecules and this glia-to-neuron or neuron-to-glia transmission of exosomal alpha-synuclein may contribute to the propagation of pathology and neuroinflammation throughout the brain. In addition, as their content varies as per their originating and recipient cells, these vesicles can be utilized as a diagnostic biomarker for early disease detection, whereas targeted exosomes may be used as scaffolds to deliver therapeutic agents into the brain. This review summarizes the current knowledge regarding the role of exosomes in the progression of alpha-synuclein-related pathology and their potential use as biomarkers and nanotherapeutics in alpha-synucleinopathies.

Coordinated activation of TGF-ß and BMP pathways promotes autophagy and limits liver injury after acetaminophen intoxication.

Ligands of the transforming growth factor-ß (TGF-ß) superfamily, including TGF-ßs, activins, and bone morphogenetic proteins (BMPs), have been implicated in hepatic development, homeostasis, and pathophysiology. We explored the mechanisms by which hepatocytes decode and integrate injury-induced signaling from TGF-ßs and activins (TGF-ß/Activin) and BMPs. We mapped the spatiotemporal patterns of pathway activation during liver injury induced by acetaminophen (APAP) in dual reporter mice carrying a fluorescent reporter of TGF-ß/Activin signaling and a fluorescent reporter of BMP signaling. APAP intoxication induced the expression of both reporters in a zone of cells near areas of tissue damage, which showed an increase in autophagy and demarcated the borders between healthy and injured tissues. Inhibition of TGF-ß superfamily signaling by overexpressing the inhibitor Smad7 exacerbated acute liver histopathology but eventually accelerated tissue recovery. Transcriptomic analysis identified autophagy as a process stimulated by TGF-ß1 and BMP4 in hepatocytes, with Trp53inp2, which encodes a rate-limiting factor for autophagy initiation, as the most highly induced autophagy-related gene. Collectively, these findings illustrate the functional interconnectivity of the TGF-ß superfamily signaling system, implicate the coordinated activation of TGF-ß/Activin and BMP pathways in balancing tissue reparatory and regenerative processes upon APAP-induced hepatotoxicity, and highlight opportunities and potential risks associated with targeting this signaling system for treating hepatic diseases.

High content screening and proteomic analysis identify a kinase inhibitor that rescues pathological phenotypes in a patient-derived model of Parkinson's disease.

Combining high throughput screening approaches with induced pluripotent stem cell (iPSC)-based disease modeling represents a promising unbiased strategy to identify therapies for neurodegenerative disorders. Here we applied high content imaging on iPSC-derived neurons from patients with familial Parkinson's disease bearing the G209A (p.A53T) α-synuclein (αSyn) mutation and launched a screening campaign on a small kinase inhibitor library. We thus identified the multi-kinase inhibitor BX795 that at a single dose effectively restores disease-associated neurodegenerative phenotypes. Proteomics profiling mapped the molecular pathways underlying the protective effects of BX795, comprising a cohort of 118 protein-mediators of the core biological processes of RNA metabolism, protein synthesis, modification and clearance, and stress response, all linked to the mTORC1 signaling hub. In agreement, expression of human p.A53T-αSyn in neuronal cells affected key components of the mTORC1 pathway resulting in aberrant protein synthesis that was restored in the presence of BX795 with concurrent facilitation of autophagy. Taken together, we have identified a promising small molecule with neuroprotective actions as candidate therapeutic for PD and other protein conformational disorders.

A double-hit in vivo model of GBA viral microRNA-mediated downregulation and human alpha-synuclein overexpression demonstrates nigrostriatal degeneration.

Preclinical and clinical studies support a strong association between mutations in the GBA1 gene that encodes beta-glucocerebrosidase (GCase) (EC 3.2.1.45; glucosylceramidase beta) and Parkinson's disease (PD). Alpha-synuclein (AS), a key player in PD pathogenesis, and GBA1 mutations may independently and synergistically cause lysosomal dysfunction and thus, embody clinically well-validated targets of the neurodegenerative disease process in PD. However, in vivo models, recapitulating pathological features of PD that can be used to dissect the nature of the complex relationship between GCase and AS on the nigrostriatal axis, the region particularly vulnerable in PD, are direly needed. To address this, we implemented a bidirectional approach in mice to examine the effects of: 1) GCase overexpression (wild-type and mutant N370S GBA) on endogenous AS levels and 2) downregulation of endogenous GCase (Gba) combined with AS overexpression. Striatal delivery of viral-mediated GCase overexpression revealed minimal effects on cortical and nigrostriatal AS tissue levels and no significant effect on dopaminergic system integrity. On the other hand, microRNA (miR)-mediated Gba1 downregulation (miR Gba), combined with virus-mediated human AS overexpression (+AS), yields decreased GCase activity in the cortex, mimicking levels seen in GBA1 heterozygous carriers (30-40%), increased astrogliosis and microgliosis, decreased striatal dopamine levels (50% compared to controls) and loss of nigral dopaminergic neurons (~33%)- effects that were all reversible with miR rescue. Most importantly, the synergistic neurodegeneration of miR Gba + AS correlated with augmented AS accumulation and extracellular release in the striatum. Collectively, our results suggest that GCase downregulation alone is not sufficient to recapitulate key pathological features of PD in vivo, but its synergistic interplay with AS, via increased AS levels and extracellular release, drives nigrostriatal neurodegeneration. Furthermore, we report a novel double-hit GBA-AS model that can be used to identify putative mechanisms driving PD pathophysiology and can be subsequently used to test novel therapeutic approaches.

Autophagy mediates the clearance of oligodendroglial SNCA/alpha-synuclein and TPPP/p25A in multiple system atrophy models.

Accumulation of the neuronal protein SNCA/alpha-synuclein and of the oligodendroglial phosphoprotein TPPP/p25A within the glial cytoplasmic inclusions (GCIs) represents the key histophathological hallmark of multiple system atrophy (MSA). Even though the levels/distribution of both oligodendroglial SNCA and TPPP/p25A proteins are critical for disease pathogenesis, the proteolytic mechanisms involved in their turnover in health and disease remain poorly understood. Herein, by pharmacological and molecular modulation of the autophagy-lysosome pathway (ALP) and the proteasome we demonstrate that the endogenous oligodendroglial SNCA and TPPP/p25A are degraded mainly by the ALP in murine primary oligodendrocytes and oligodendroglial cell lines under basal conditions. We also identify a KFERQ-like motif in the TPPP/p25A sequence that enables its effective degradation via chaperone-mediated autophagy (CMA) in an in vitro system of rat brain lysosomes. Furthermore, in a MSA-like setting established by addition of human recombinant SNCA pre-formed fibrils (PFFs) as seeds of pathological SNCA, we thoroughly characterize the contribution of CMA and macroautophagy in particular, in the removal of the exogenously added and the seeded oligodendroglial SNCA pathological assemblies. We also show that PFF treatment impairs autophagic flux and that TPPP/p25A exerts an inhibitory effect on macroautophagy, while at the same time CMA is upregulated to remove the pathological SNCA species formed within oligodendrocytes. Finally, augmentation of CMA or macroautophagy accelerates the removal of the engendered pathological SNCA conformations further suggesting that autophagy targeting may represent a successful approach for the clearance of pathological SNCA and/or TPPP/p25A in the context of MSA.Abbreviations: 3MA: 3-methyladenine; ACTB: actin, beta; ALP: autophagy-lysosome pathway; ATG5: autophagy related 5; AR7: atypical retinoid 7; CMA: chaperone-mediated autophagy; CMV: cytomegalovirus; CTSD: cathepsin D; DAPI: 4',6-diamidino-2-phenylindole; DMEM: Dulbecco's modified Eagle's medium; Epox: epoxomicin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCIs: glial cytoplasmic inclusions; GFP: green fluorescent protein; HMW: high molecular weight; h: hours; HSPA8/HSC70: heat shock protein 8; LAMP1: lysosomal-associated membrane protein 1; LAMP2A: lysosomal-associated membrane protein 2A; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mcherry: monomeric cherry; MFI: mean fluorescence intensity; mRFP: monomeric red fluorescent protein; MSA: multiple system atrophy; OLN: oligodendrocytes; OPCs: oligodendroglial progenitor cells; PBS: phosphate-buffered saline; PC12: pheochromocytoma cell line; PD: Parkinson disease; PFFs: pre-formed fibrils; PIs: protease inhibitors; PSMB5: proteasome (prosome, macropain) subunit, beta type 5; Rap: rapamycin; RFP: red fluorescent protein; Scr: scrambled; SDS: sodium dodecyl sulfate; SE: standard error; siRNAs: small interfering RNAs; SNCA: synuclein, alpha; SQSTM1: sequestosome 1; TPPP: tubulin polymerization promoting protein; TUBA: tubulin, alpha; UPS: ubiquitin-proteasome system; WT: wild type.

Correction: Loss of ß-Glucocerebrosidase Activity Does Not Affect Alpha-Synuclein Levels or Lysosomal Function in Neuronal Cells.

[This corrects the article DOI: 10.1371/journal.pone.0060674.].

Neurons and Glia Interplay in α-Synucleinopathies.

Accumulation of the neuronal presynaptic protein alpha-synuclein within proteinaceous inclusions represents the key histophathological hallmark of a spectrum of neurodegenerative disorders, referred to by the umbrella term a-synucleinopathies. Even though alpha-synuclein is expressed predominantly in neurons, pathological aggregates of the protein are also found in the glial cells of the brain. In Parkinson's disease and dementia with Lewy bodies, alpha-synuclein accumulates mainly in neurons forming the Lewy bodies and Lewy neurites, whereas in multiple system atrophy, the protein aggregates mostly in the glial cytoplasmic inclusions within oligodendrocytes. In addition, astrogliosis and microgliosis are found in the synucleinopathy brains, whereas both astrocytes and microglia internalize alpha-synuclein and contribute to the spread of pathology. The mechanisms underlying the pathological accumulation of alpha-synuclein in glial cells that under physiological conditions express low to non-detectable levels of the protein are an area of intense research. Undoubtedly, the presence of aggregated alpha-synuclein can disrupt glial function in general and can contribute to neurodegeneration through numerous pathways. Herein, we summarize the current knowledge on the role of alpha-synuclein in both neurons and glia, highlighting the contribution of the neuron-glia connectome in the disease initiation and progression, which may represent potential therapeutic target for a-synucleinopathies.

Psychosis-Like Behavior and Hyperdopaminergic Dysregulation in Human α-Synuclein BAC Transgenic Rats.

BACKGROUND: Parkinson's disease psychosis is a prevalent yet underreported and understudied nonmotor manifestation of Parkinson's disease and, arguably, the most debilitating. It is unknown if α-synuclein plays a role in psychosis, and if so, this endophenotype may be crucial for elucidating the neurodegenerative process. OBJECTIVES: We sought to dissect the underlying neurobiology of novelty-induced hyperactivity, reminiscent of psychosis-like behavior, in human α-synuclein BAC rats. RESULTS: Herein, we demonstrate a prodromal psychosis-like phenotype, including late-onset sensorimotor gating disruption, striatal hyperdopaminergic signaling, and persistent novelty-induced hyperactivity (up to 18 months), albeit reduced baseline locomotor activity, that is augmented by d-amphetamine and reversed by classical and atypical antipsychotics. MicroRNA-mediated α-synuclein downregulation in the ventral midbrain rescues the hyperactive phenotype and restores striatal dopamine levels. This phenotype is accompanied by an abundance of age-, brain region- and gene dose-dependent aberrant α-synuclein, including hyperphosphorylation, C-terminal truncation, aggregation pathology, and mild nigral neurodegeneration (27%). CONCLUSIONS: Our findings demonstrate a potential role of α-synuclein in Parkinson's disease psychosis and provide evidence of region-specific perturbations prior to neurodegeneration phenoconversion. The reported phenotype coincides with the latest clinical findings that suggest a premotor hyperdopaminergic state may occur, while at the same time, premotor psychotic symptoms are increasingly being recognized. © 2020 International Parkinson and Movement Disorder Society.

In Search of Effective Treatments Targeting α-Synuclein Toxicity in Synucleinopathies: Pros and Cons.

Parkinson's disease (PD), multiple system atrophy (MSA) and Dementia with Lewy bodies (DLB) represent pathologically similar, progressive neurodegenerative disorders characterized by the pathological aggregation of the neuronal protein α-synuclein. PD and DLB are characterized by the abnormal accumulation and aggregation of α-synuclein in proteinaceous inclusions within neurons named Lewy bodies (LBs) and Lewy neurites (LNs), whereas in MSA α-synuclein inclusions are mainly detected within oligodendrocytes named glial cytoplasmic inclusions (GCIs). The presence of pathologically aggregated α-synuclein along with components of the protein degradation machinery, such as ubiquitin and p62, in LBs and GCIs is considered to underlie the pathogenic cascade that eventually leads to the severe neurodegeneration and neuroinflammation that characterizes these diseases. Importantly, α-synuclein is proposed to undergo pathogenic misfolding and oligomerization into higher-order structures, revealing self-templating conformations, and to exert the ability of "prion-like" spreading between cells. Therefore, the manner in which the protein is produced, is modified within neural cells and is degraded, represents a major focus of current research efforts in the field. Given that α-synuclein protein load is critical to disease pathogenesis, the identification of means to limit intracellular protein burden and halt α-synuclein propagation represents an obvious therapeutic approach in synucleinopathies. However, up to date the development of effective therapeutic strategies to prevent degeneration in synucleinopathies is limited, due to the lack of knowledge regarding the precise mechanisms underlying the observed pathology. This review critically summarizes the recent developed strategies to counteract α-synuclein toxicity, including those aimed to increase protein degradation, to prevent protein aggregation and cell-to-cell propagation, or to engage antibodies against α-synuclein and discuss open questions and unknowns for future therapeutic approaches.

Correction to: Endogenous oligodendroglial alpha-synuclein and TPPP/p25α orchestrate alpha-synuclein pathology in experimental multiple system atrophy models.

he original version of this article.

Endogenous oligodendroglial alpha-synuclein and TPPP/p25α orchestrate alpha-synuclein pathology in experimental multiple system atrophy models.

Multiple system atrophy (MSA) is characterized by the presence of distinctive glial cytoplasmic inclusions (GCIs) within oligodendrocytes that contain the neuronal protein alpha-synuclein (aSyn) and the oligodendroglia-specific phosphoprotein TPPP/p25α. However, the role of oligodendroglial aSyn and p25α in the formation of aSyn-rich GCIs remains unclear. To address this conundrum, we have applied human aSyn (haSyn) pre-formed fibrils (PFFs) to rat wild-type (WT)-, haSyn-, or p25α-overexpressing oligodendroglial cells and to primary differentiated oligodendrocytes derived from WT, knockout (KO)-aSyn, and PLP-haSyn-transgenic mice. HaSyn PFFs are readily taken up by oligodendroglial cells and can recruit minute amounts of endogenous aSyn into the formation of insoluble, highly aggregated, pathological assemblies. The overexpression of haSyn or p25α accelerates the recruitment of endogenous protein and the generation of such aberrant species. In haSyn PFF-treated primary oligodendrocytes, the microtubule and myelin networks are disrupted, thus recapitulating a pathological hallmark of MSA, in a manner totally dependent upon the seeding of endogenous aSyn. Furthermore, using oligodendroglial and primary cortical cultures, we demonstrated that pathology-related S129 aSyn phosphorylation depends on aSyn and p25α protein load and may involve different aSyn "strains" present in oligodendroglial and neuronal synucleinopathies. Importantly, this hypothesis was further supported by data obtained from human post-mortem brain material derived from patients with MSA and dementia with Lewy bodies. Finally, delivery of haSyn PFFs into the mouse brain led to the formation of aberrant aSyn forms, including the endogenous protein, within oligodendroglia and evoked myelin decompaction in WT mice, but not in KO-aSyn mice. This line of research highlights the role of endogenous aSyn and p25α in the formation of pathological aSyn assemblies in oligodendrocytes and provides in vivo evidence of the contribution of oligodendroglial aSyn in the establishment of aSyn pathology in MSA.

Autophagy dysfunction in peripheral blood mononuclear cells of Parkinson's disease patients.

BACKGROUND: Alpha-synuclein aggregation is considered one of the main causes of Parkinson's Disease (PD). Malfunction of autophagy-lysosomal pathways is believed to be an underlying mechanism of α-synuclein aggregation. Although such malfunction has been observed in PD brains, it is unclear whether it may also occur in extraneuronal tissues. OBJECTIVES: To assess lysosome-mediated protein degradation in cultured Peripheral Blood Mononuclear Cells (PBMCs) of PD patients and healthy controls. METHODS: Total protein degradation in cultured PBMCs was measured by labelling the cells with 3H-leucine using pulse-chase experiments. Different inhibitors were used to measure a range of autophagic pathways. RESULTS: Protein degradation through the main autophagic pathways is reduced in PD patients (n = 18) compared to age- and sex-matched healthy controls (n = 18), (macroautophagy, p = .018; Chaperone-Mediated autophagy, p = .04; and total lysosomal function, p = .007). CONCLUSIONS: Lysosomal dysfunction is present in cultured PBMCs of PD patients, suggesting that it may reflect a systemic feature of the disease.

Long Non-coding RNAs Associated With Neurodegeneration-Linked Genes Are Reduced in Parkinson's Disease Patients.

Transcriptome analysis has identified a plethora of long non-coding RNAs (lncRNAs) expressed in the human brain and associated with neurological diseases. However, whether lncRNAs expression levels correlate with Parkinson's disease (PD) pathogenesis remains unknown. Herein, we show that a number of lncRNA genes encompassing transcriptional units in close proximity to PD-linked protein-coding genes, including SNCA, LRRK2, PINK1, DJ-1, UCH-L1, MAPT and GBA1, are expressed in human dopaminergic cells and post-mortem material, such as cortex, Substantia Nigra and cerebellum. Interestingly, these lncRNAs are upregulated during neuronal differentiation of SH-SY5Y cells and of dopaminergic neurons generated from human fibroblast-derived induced pluripotent stem cells. Importantly, six lncRNAs are found under-expressed in the nigra and three in the cerebellum of PD patients compared to controls. Simultaneously, SNCA mRNA levels are increased in the nigra, while LRRK2 and PINK1 mRNA levels are decreased both in the nigra and the cerebellum of PD subjects compared to controls, indicating a possible correlation between the expression profile of the respective lncRNAs with their adjacent coding genes. Interestingly, all dysregulated lncRNAs are also detected in human peripheral blood mononuclear cells and four of them in exosomes derived from human cerebrospinal fluid, providing initial evidence for their potential use as diagnostic tools for PD. Our data raise the intriguing possibility that these lncRNAs may be involved in disease pathogenesis by regulating their neighboring PD-associated genes and may thus represent novel targets for the diagnosis and/or treatment of PD or related diseases.

Activation of both transforming growth factor-ß and bone morphogenetic protein signalling pathways upon traumatic brain injury restrains pro-inflammatory and boosts tissue reparatory responses of reactive astrocytes and microglia.

Various ligands and receptors of the transforming growth factor-ß superfamily have been found upregulated following traumatic brain injury; however, the role of this signalling system in brain injury pathophysiology is not fully characterized. To address this, we utilized an acute stab wound brain injury model to demonstrate that hallmarks of transforming growth factor-ß superfamily system activation, such as levels of phosphorylated Smads, ligands and target genes for both transforming growth factor-ß and bone morphogenetic protein pathways, were upregulated within injured tissues. Using a bone morphogenetic protein-responsive reporter mouse model, we showed that activation of the bone morphogenetic protein signalling pathway involves primarily astrocytes that demarcate the wound area. Insights regarding the potential role of transforming growth factor-ß superfamily activation in glia cells within the injured tissues were obtained indirectly by treating purified reactive astrocytes and microglia with bone morphogenetic protein-4 or transforming growth factor-ß1 and characterizing changes in their transcriptional profiles. Astrocytes responded to both ligands with considerably overlapping profiles, whereas, microglia responded selectively to transforming growth factor-ß1. Novel pathways, crucial for repair of tissue-injury and blood-brain barrier, such as activation of cholesterol biosynthesis and transport, production of axonal guidance and extracellular matrix components were upregulated by transforming growth factor-ß1 and/or bone morphogenetic protein-4 in astrocytes. Moreover, both ligands in astrocytes and transforming growth factor-ß1 in microglia shifted the phenotype of reactive glia cells towards the anti-inflammatory and tissue reparatory 'A2'-like and 'M0/M2'-like phenotypes, respectively. Increased expression of selected key components of the in vitro modulated pathways and markers of 'A2'-like astrocytes was confirmed within the wound area, suggesting that these processes could also be modulated in situ by the integrated action of transforming growth factor-ß and/or bone morphogenetic protein-mediated signalling. Collectively, our study provides a comprehensive comparative analysis of transforming growth factor-ß superfamily signalling in reactive astrocytes and microglia and points towards a crucial role of both transforming growth factor-ß and bone morphogenetic protein pathways in modulating the inflammatory and brain injury reparatory functions of activated glia cells.

Impairment of chaperone-mediated autophagy affects neuronal homeostasis through altered expression of DJ-1 and CRMP-2 proteins.

Chaperone-mediated autophagy (CMA) is a substrate-specific mode of lysosomal proteolysis, with multiple lines of evidence connecting its dysfunction to both ageing and disease. We have recently shown that CMA impairment through knock-down of the lysosomal receptor LAMP2A is detrimental to neuronal viability in vivo; however, it is not clear which subset of proteins regulated by the CMA pathway mediate such changes. In this study, we have manipulated CMA function through alterations of LAMP2A abundance in primary rat cortical neurons, to identify potential changes to the neuronal proteome occurring prior to neurotoxic effects. We have identified a list of proteins with significant, >2-fold change in abundance following our manipulations, of which PARK7/DJ-1 - an anti-oxidant implicated in hereditary forms of Parkinson's Disease (PD), and DPYSL2/CRMP-2 - a microtubule-binding phosphoprotein involved in schizophrenia pathogenesis - were both found to have measurable effects on neuronal homeostasis and phenotype. Taken together, this study describes alterations in the abundance of neuronal proteins involved in neuropsychiatric disorders upon CMA manipulation, and suggests that such alterations may in part be responsible for the neurodegeneration observed upon CMA impairment in vivo.

Autophagy orchestrates the regulatory program of tumor-associated myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) densely accumulate into tumors and potently suppress antitumor immune responses, promoting tumor development. Targeting MDSCs in tumor immunotherapy has been hampered by lack of understanding of the molecular pathways that govern MDSC differentiation and function. Herein, we identify autophagy as a crucial pathway for MDSC-mediated suppression of antitumor immunity. Specifically, MDSCs in patients with melanoma and mouse melanoma exhibited increased levels of functional autophagy. Ablation of autophagy in myeloid cells markedly delayed tumor growth and endowed antitumor immune responses. Notably, tumor-infiltrating autophagy-deficient monocytic MDSCs (M-MDSCs) demonstrated impaired suppressive activity in vitro and in vivo, whereas transcriptome analysis revealed substantial differences in genes related to lysosomal function. Accordingly, autophagy-deficient M-MDSCs exhibited impaired lysosomal degradation, thereby enhancing surface expression of MHC class II molecules, resulting in efficient activation of tumor-specific CD4+ T cells. Finally, targeting of the membrane-associated RING-CH1 (MARCH1) E3 ubiquitin ligase that mediates the lysosomal degradation of MHC II in M-MDSCs attenuated their suppressive function, and resulted in markedly decreased tumor volume followed by development of a robust antitumor immunity. Collectively, these findings depict autophagy as a molecular target of MDSC-mediated suppression of antitumor immunity.

Impairment of chaperone-mediated autophagy induces dopaminergic neurodegeneration in rats.

Chaperone-mediated autophagy (CMA) involves the selective lysosomal degradation of cytosolic proteins such as SNCA (synuclein α), a protein strongly implicated in Parkinson disease (PD) pathogenesis. However, the physiological role of CMA and the consequences of CMA failure in the living brain remain elusive. Here we show that CMA inhibition in the adult rat substantia nigra via adeno-associated virus-mediated delivery of short hairpin RNAs targeting the LAMP2A receptor, involved in CMA's rate limiting step, was accompanied by intracellular accumulation of SNCA-positive puncta, which were also positive for UBIQUITIN, and in accumulation of autophagic vacuoles within LAMP2A-deficient nigral neurons. Strikingly, LAMP2A downregulation resulted in progressive loss of nigral dopaminergic neurons, severe reduction in striatal dopamine levels/terminals, increased astro- and microgliosis and relevant motor deficits. Thus, this study highlights for the first time the importance of the CMA pathway in the dopaminergic system and suggests that CMA impairment may underlie PD pathogenesis.

Chaperone mediated autophagy in aging: Starve to prosper.

The major lysosomal proteolytic pathways essential for maintaining proper cellular homeostasis are macroautophagy, chaperone-mediated autophagy (CMA) and microautophagy. What differentiates CMA from the other types of autophagy is the fact that it does not involve vesicle formation; the unique feature of this pathway is the selective targeting of substrate proteins containing a CMA-targeting motif and the direct translocation into the lysosomal lumen, through the aid of chaperones/co-chaperones localized both at the cytosol and the lysosomes. CMA operates at basal conditions in most mammalian cell models analyzed so far, but it is mostly activated in response to stressors, such as trophic deprivation or oxidative stress. The activity of CMA has been shown to decline with age and such decline, correlating with accumulation of damaged/oxidized/aggregated proteins, may contribute to tissue dysfunction and, possibly, neurodegeneration. Herein, we review the recent knowledge regarding the molecular components, regulation and physiology of the CMA pathway, the contribution of impaired CMA activity to poor cellular homeostasis and inefficient response to stress during aging, and discuss the therapeutic opportunities offered by the restoration of CMA-dependent proteolysis in age-associated degenerative diseases.