Role of the lysosomal channel Transient Receptor Potential Mucolipin 1 (TRPML1) in the functional coupling between endoplasmic reticulum and lysosomes in experimental models of brain ischemia

A growing interest has been recently devoted to the role of intracellular Ca2+ stores in brain ischemia. For instance, disturbances of Ca2+ content in the endoplasmic reticulum (ER), the main intracellular Ca2+ store, have been reported as one of the main mechanisms underlying this neurological disease. Interestingly, lysosomes are emerging as other important Ca2+-storing organelles, cooperating with the ER in the handling of intracellular Ca2+ concentration ([Ca2+]i). One of the main regulators of lysosomal Ca2+ homeostasis is represented by Transient Receptor Potential Mucolipin 1 (TRPML1), a non-selective cation channel releasing lysosomal Ca2+ into the cytosol. In this study we investigated the role of ER/lysosome Ca2+ coupling and the contribution of TRPML1 in brain ischemia. Our results showed that under physiological conditions TRPML1 activation induced by its specific agonist ML-SA1 or by lysosomal v-ATPase inhibitor bafilomycin A1 significantly increased [Ca2+]i in cortical neurons. ML-SA1-induced Ca2+ leak from lysosomes strongly reduced ER Ca2+ content, whereas the TRPML1 inhibitor trans-Ned19 or channel knocking down increased ER Ca2+ levels. However, this interplay was disrupted under hypoxic conditions produced by exposing cortical neurons to oxygen and glucose deprivation (OGD) followed by reoxygenation (Rx). Indeed, during OGD/Rx both ER and lysosomal Ca2+ levels were significantly impaired. Interestingly, the administration of trans-Ned19 during the reoxygenation phase prevented dysfunctional lysosomal Ca2+ homeostasis and neuronal death. In consideration of the role played by lysosomes in autophagy regulation, we showed that trans-Ned19 hampered the autophagic flux during hypoxia thus protecting neurons. Moreover, we found that in ischemic rats subjected to the transient occlusion of the middle cerebral artery (tMCAO) the intracerebroventricular (icv) administration of this drug at the onset of reperfusion was able to reduce the brain ischemic volume, ameliorated the general and focal deficits, and promoted a protective blockade of the autophagic flux. Collectively, the results presented in my PhD thesis demonstrate the detrimental role of TRPML1 dysfunction in the neurodegeneration associated to brain ischemia, thus identifying a novel potential therapeutic target that could be pharmacologically modulated, together with other relevant mechanisms, to induce neuroprotection

The activation of Mucolipin TRP channel 1 (TRPML1) protects motor neurons from L-BMAA neurotoxicity by promoting autophagic clearance

Cellular clearance mechanisms including the autophagy-lysosome pathway are impaired in amyotrophic lateral sclerosis (ALS). One of the most important proteins involved in the regulation of autophagy is the lysosomal Ca2+ channel Mucolipin TRP channel 1 (TRPML1). Therefore, we investigated the role of TRPML1 in a neuronal model of ALS/Parkinson-dementia complex reproduced by the exposure of motor neurons to the cyanobacterial neurotoxin beta-methylamino-L-alanine (L-BMAA). Under these conditions, L-BMAA induces a dysfunction of the endoplasmic reticulum (ER) leading to ER stress and cell death. Therefore we hypothesized a dysfunctional coupling between lysosomes and ER in L-BMAA-treated motor neurons. Here, we showed that in motor neuronal cells TRPML1 as well as the lysosomal protein LAMP1 co-localized with ER. In addition, TRPML1 co-immunoprecipitated with the ER Ca2+ sensor STIM1. Functionally, the TRPML1 agonist ML-SA1 induced lysosomal Ca2+ release in a dose-dependent way in motor neuronal cells. The SERCA inhibitor thapsigargin increased the fluorescent signal associated with lysosomal Ca2+ efflux in the cells transfected with the genetically encoded Ca2+ indicator GCaMP3-ML1, thus suggesting an interplay between the two organelles. Moreover, chronic exposure to L-BMAA reduced TRPML1 protein expression and produced an impairment of both lysosomal and ER Ca2+ homeostasis in primary motor neurons. Interestingly, the preincubation of ML-SA1, by an early activation of AMPK and beclin 1, rescued motor neurons from L-BMAA-induced cell death and reduced the expression of the ER stress marker GRP78. Finally, ML-SA1 reduced the accumulation of the autophagy-related proteins p62/SQSTM1 and LC3-II in L-BMAA-treated motor neurons. Collectively, we propose that the pharmacological stimulation of TRPML1 can rescue motor neurons from L-BMAA-induced toxicity by boosting autophagy and reducing ER stress

The impact of the 'Mis-Peptidome' on HLA Class I-Mediated Diseases: contribution of ERAP1 and ERAP2 and effects on the immune response

The strong association with the Major Histocompatibility Complex (MHC) class I genes represents a shared trait for a group of autoimmune/autoinflammatory disorders having in common immunopathogenetic basis as well as clinical features. Accordingly, the main risk factors for Ankylosing Spondylitis (AS), prototype of the Spondyloarthropathies (SpA), the Behçet's disease (BD), the Psoriasis (Ps) and the Birdshot Chorioretinopathy (BSCR) are HLA-B*27, HLA-B*51, HLA-C*06:02 and HLA-A*29:02, respectively. Despite the strength of the association, the HLA pathogenetic role in these diseases is far from being thoroughly understood. Furthermore, Genome-Wide Association Studies (GWAS) have highlighted other important susceptibility factors such as Endoplasmic Reticulum Aminopeptidase (ERAP) 1 and, less frequently, ERAP2 that refine the peptidome presented by HLA class I molecules to CD8+ T cells. Mass spectrometry analysis provided considerable knowledge of HLA-B*27, HLA-B*51, HLA-C*06:02 and HLA-A*29:02 immunopeptidome. However, the combined effect of several ERAP1 and ERAP2 allelic variants could generate an altered pool of peptides accounting for the "mis-immunopeptidome" that ranges from suboptimal to pathogenetic/harmful peptides able to induce non-canonical or autoreactive CD8+ T responses, activation of NK cells and/or garbling the classical functions of the HLA class I molecules. This review will focus on this class of epitopes as possible elicitors of atypical/harmful immune responses which can contribute to the pathogenesis of chronic inflammatory diseases

Laparoscopic R1 vascular hepatectomy for hepatocellular carcinoma (with video)

Surgical resection is considered the standard of treatment for hepatocellular carcinoma (HCC), when realized with negative margins (R0)1. Not infrequently, R0 resection is unachievable, thus the concept of R1 vascular hepatectomy has been introduced and has been defined as exposure of the tumor on the specimen surface due to its detachment from vascular structure

Size-Based Effects of Anthropogenic Ultrafine Particles on Lysosomal TRPML1 Channel and Autophagy in Motoneuron-like Cells

An emerging body of evidence indicates an association between anthropogenic particulate matter (PM) and neurodegeneration. Although the historical focus of PM toxicity has been on the cardiopulmonary system, ultrafine PM particles can also exert detrimental effects in the brain. However, only a few studies are available on the harmful interaction between PM and CNS and on the putative pathomechanisms