Alterations in autophagy and the ubiquitin proteasome system (UPS) are commonly implicated in protein aggregation and toxicity which manifest in a number of neurological disorders. In fact, both UPS and autophagy alterations are bound to the aggregation, spreading and toxicity of the so-called prionoid proteins, including alpha synuclein (α-syn), amyloid-beta (Aβ), tau, huntingtin, superoxide dismutase-1 (SOD-1), TAR-DNA-binding protein of 43 kDa (TDP-43) and fused in sarcoma (FUS). Recent biochemical and morphological studies add to this scenario, focusing on the coordinated, either synergistic or compensatory, interplay that occurs between autophagy and the UPS. In fact, a number of biochemical pathways such as mammalian target of rapamycin (mTOR), transcription factor EB (TFEB), Bcl2-associated athanogene 1/3 (BAG3/1) and glycogen synthase kinase beta (GSk3β), which are widely explored as potential targets in neurodegenerative proteinopathies, operate at the crossroad between autophagy and UPS. These biochemical steps are key in orchestrating the specificity and magnitude of the two degradation systems for effective protein homeostasis, while intermingling with intracellular secretory/trafficking and inflammatory pathways. The findings discussed in the present manuscript are supposed to add novel viewpoints which may further enrich our insight on the complex interactions occurring between cell-clearing systems, protein misfolding and propagation. Discovering novel mechanisms enabling a cross-talk between the UPS and autophagy is expected to provide novel potential molecular targets in proteinopathies.

Fragments of mitochondrial DNA (mtDNA) are released outside the cell and they appear to persist in extracellular fluids as circulating, cell-free, mtDNA (ccf-mtDNA). When compared to nuclear DNA, such a double stranded mtDNA is more resistant to nuclease degradation. In fact, it is stable extracellularly where it can be detected in both plasma and cerebrospinal fluid (CSF), here acting as a potential biomarker in various disorders. In neurological diseases (Alzheimer's disease, Parkinson's disease and end-stage progressive Multiple Sclerosis), a decreased amount of CSF ccf-mtDNA is related with progressive cell dysfunction. This suggests an alteration in neuronal mtDNA levels (mtDNA replication, degradation and depletion) in vulnerable brain regions at early stages of neurodegeneration leading to reduced mtDNA release, which takes place before actual cell death occurs. On the other hand, elevated CSF ccf-mtDNA levels are reported in acute phases of relapsing-remitting Multiple Sclerosis (RRMS). This occurs during acute inflammation, which anticipates the neurodegenerative process. Thus, an increase in inflammatory cells in the affected regions is expected to add on mtDNA release into the CSF. In addition, similarly to bacterial DNA, the non-methylated CpG sites of mtDNA, which activate innate immunity and inflammation, are likely to participate in the molecular mechanisms of disease. Thus, ccf-mtDNA may represent a powerful biomarker for disease screening and prognosis at early stage, although its biological role may extend to generating the neurobiology of disease. The present manuscript discusses recent experimental findings in relationship with clinical evidence comparing neuro-immunological features of neurodegenerative disorders with frankly neuro-infectious diseases.

Protein clearing pathways named autophagy (ATG) and ubiquitin proteasome (UP) control homeostasis within eukaryotic cells, while their dysfunction produces neurodegeneration. These pathways are viewed as distinct biochemical cascades occurring within specific cytosolic compartments owing pathway-specific enzymatic activity. Recent data strongly challenged the concept of two morphologically distinct and functionally segregated compartments. In fact, preliminary evidence suggests the convergence of these pathways to form a novel organelle named autophagoproteasome. This is characterized in the present study by using a cell line where, mTOR activity is upregulated and autophagy is suppressed. This was reversed dose-dependently by administering the mTOR inhibitor rapamycin. Thus, we could study autophagoproteasomes when autophagy was either suppressed or stimulated. The occurrence of autophagoproteasome was shown also in non-human cell lines. Ultrastructural morphometry, based on the stochiometric binding of immunogold particles allowed the quantitative evaluation of ATG and UP component within autophagoproteasomes. The number of autophagoproteasomes increases following mTOR inhibition. Similarly, mTOR inhibition produces overexpression of both LC3 and P20S particles. This is confirmed by the fact that the ratio of free vs. autophagosome-bound LC3 is similar to that measured for P20S, both in baseline conditions and following mTOR inhibition. Remarkably, within autophagoproteasomes there is a slight prevalence of ATG compared with UP components for low rapamycin doses, whereas for higher rapamycin doses UP increases more than ATG. While LC3 is widely present within cytosol, UP is strongly polarized within autophagoproteasomes. These fine details were evident at electron microscopy but could not be deciphered by using confocal microscopy. Despite its morphological novelty autophagoproteasomes appear in the natural site where clearing pathways (once believed to be anatomically segregated) co-exist and they are likely to interact at molecular level. In fact, LC3 and P20S co-immunoprecipitate, suggesting a specific binding and functional interplay, which may be altered by inhibiting mTOR. In summary, ATG and UP often represent two facets of a single organelle, in which unexpected amount of enzymatic activity should be available. Thus, autophagoproteasome may represent a sophisticated ultimate clearing apparatus.

BackgroundMicroRNAs are highly conserved, noncoding RNAs involved in post-transcriptional gene silencing. They have been shown to participate in a wide range of biological processes, including myogenesis and muscle regeneration. The goal of this study is to test the hypothesis that myo-miRs (myo = muscle + miR = miRNA) expression is altered in muscle from patients affected by myotonic dystrophy type 1 (DM1), the most frequently inherited neuromuscular disease in adults. In order to gain better insights about the role of miRNAs in the DM1 pathogenesis, we have also analyzed the muscular expression of miR-103 and miR-107, which have been identified in silico as attractive candidates for binding to the DMPK mRNA.MethodsTo this aim, we have profiled the expression of miR-133 (miR-133a, miR-133b), miR-1, miR-181 (miR-181a, miR-181b, miR-181c) and miR-206, that are specifically induced during myogenesis in cardiac and skeletal muscle tissues. miR-103 and miR-107, highly expressed in brain, heart and muscle have also been included in this study. QRT-PCR experiments have been performed on RNA from vastus lateralis biopsies of DM1 patients (n = 7) and control subjects (n = 4). Results of miRNAs expression have been confirmed by Northern blot, whereas in situ hybridization technique have been performed to localize misexpressed miRNAs on muscle sections from DM1 and control individuals.ResultsOnly miR-206 showed an over-expression in 5 of 7 DM1 patients (threshold = 2, fold change between 1.20 and 13.22, average = 5.37) compared to the control group. This result has been further confirmed by Northern blot analysis (3.37-fold overexpression, R2 = 0.89). In situ hybridization localized miR-206 to nuclear site both in normal and DM1 tissues. Cellular distribution in DM1 tissues includes also the nuclear regions of centralized nuclei, with a strong signal corresponding to nuclear clumps.ConclusionsThis work provides, for the first time, evidences about miRNAs misexpression in DM1 muscle tissues, adding a new element in the pathogenesis of this complex genetic disease.

The mammalian Target of Rapamycin (mTOR) is a molecular complex equipped with kinase activity which controls cell viability being key in the PI3K/PTEN/Akt pathway. mTOR acts by integrating a number of environmental stimuli to regulate cell growth, proliferation, autophagy, and protein synthesis. These effects are based on the modulation of different metabolic pathways. Upregulation of mTOR associates with various pathological conditions, such as obesity, neurodegeneration, and brain tumors. This is the case of high-grade gliomas with a high propensity to proliferation and tissue invasion. Glioblastoma Multiforme (GBM) is a WHO grade IV malignant, aggressive, and lethal glioma. To date, a few treatments are available although the outcome of GBM patients remains poor. Experimental and pathological findings suggest that mTOR upregulation plays a major role in determining an aggressive phenotype, thus determining relapse and chemoresistance. Among several activities, mTOR-induced autophagy suppression is key in GBM malignancy. In this article, we discuss recent evidence about mTOR signaling and its role in normal brain development and pathological conditions, with a special emphasis on its role in GBM.

Autophagy (ATG) and the Ubiquitin Proteasome (UP) are the main clearing systems of eukaryotic cells, in that being ultimately involved in degrading damaged and potentially harmful cytoplasmic substrates. Emerging evidence implicates that, in addition to their classic catalytic function in the cytosol, autophagy and the proteasome act as modulators of neurotransmission, inasmuch as they orchestrate degradation and turnover of synaptic vesicles (SVs) and related proteins. These findings are now defining a novel synaptic scenario, where clearing systems and secretory pathways may be considered as a single system, which senses alterations in quality and distribution (in time, amount and place) of both synaptic proteins and neurotransmitters. In line with this, in the present manuscript we focus on evidence showing that, a dysregulation of secretory and trafficking pathways is quite constant in the presence of an impairment of autophagy-lysosomal machinery, which eventually precipitates synaptic dysfunction. Such a dual effect appears not to be just incidental but it rather represents the natural evolution of archaic cell compartments. While discussing these issues, we pose a special emphasis on the role of autophagy upon dopamine (DA) neurotransmission, which is early affected in several neurological and psychiatric disorders. In detail, we discuss how autophagy is engaged not only in removing potentially dangerous proteins, which can interfere with the mechanisms of DA release, but also the fate of synaptic DA vesicles thus surveilling DA neurotransmission. These concepts contribute to shed light on early mechanisms underlying intersection of autophagy with DA-related synaptic disorders.

Recurrent pancreatitis in children has several etiologies. Genetic testing confirms the high frequency of CFTR mutations. This suggests that it is of some value to identify patients with late-onset CF and CFTR-related disorders.

Angiotensin-converting enzyme 2 (ACE2) has been recognized as the entry receptor of the novel severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2). Structural and sequence variants in ACE2 gene may affect its expression in different tissues and determine a differential response to SARS-Cov-2 infection and the COVID-19-related phenotype. The present study investigated the genetic variability of ACE2 in terms of single nucleotide variants (SNVs), copy number variations (CNVs), and expression quantitative loci (eQTLs) in a cohort of 268 individuals representative of the general Italian population. The analysis identified five SNVs (rs35803318, rs41303171, rs774469453, rs773676270, and rs2285666) in the Italian cohort. Of them, rs35803318 and rs2285666 displayed a significant different frequency distribution in the Italian population with respect to worldwide population. The eQTLs analysis located in and targeting ACE2 revealed a high distribution of eQTL variants in different brain tissues, suggesting a possible link between ACE2 genetic variability and the neurological complications in patients with COVID-19. Further research is needed to clarify the possible relationship between ACE2 expression and the susceptibility to neurological complications in patients with COVID-19. In fact, patients at higher risk of neurological involvement may need different monitoring and treatment strategies in order to prevent severe, permanent brain injury.