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

Chemistry and Technology of Wine Aging with Oak Chips

The use of wood chips is a common winemaking practice that has been permitted in Europe since the early 2000s. The use of oak chips, or other wood alternative products, has not always been favorably viewed by both producers and wine consumers. Beyond possible misuse, however, wood chips are a useful tool for the optimal achievement of numerous oenological objectives, including the extraction of certain volatile odor compounds from oak wood chips as well as compounds that will improve wine quality. This chapter deals with the main oenological uses of oak wood chips, the chemical transformations that underlie this practice and the effect of their utilization on wine quality. A final aspect concerns the main compositional and sensory differences between wines aged in barrel and those aged with alternative products, as well as the discriminative analytical methods used for this purpose

Synthesis and Biological Evaluation of a New Structural Simplified Analogue of cADPR, a Calcium-Mobilizing Secondary Messenger Firstly Isolated from Sea Urchin Eggs

Herein, we reported on the synthesis of cpIPP, which is a new structurally-reduced analogue of cyclic ADP-ribose (cADPR), a potent Ca2+-releasing secondary messenger that was firstly isolated from sea urchin eggs extracts. To obtain cpIPP the "northern" ribose of cADPR was replaced by a pentyl chain and the pyrophosphate moiety by a phophono-phosphate anhydride. The effect of the presence of the new phosphono-phosphate bridge on the intracellular Ca2+release induced by cpIPP was assessed in PC12 neuronal cells in comparison with the effect of the pyrophosphate bridge of the structurally related cyclic N1-butylinosine diphosphate analogue (cbIDP), which was previously synthesized in our laboratories, and with that of the linear precursor of cpIPP, which, unexpectedly, revealed to be the only one provided with Ca2+release properties

Characterisation of refined marc distillates with alternative oak products using different analytical approaches

The use of oak barrel alternatives, including oak chips, oak staves and oak powder, is quite common in the production of spirits obtained from the distillation of vegetal fermented products such as grape pomace. This work explored the use of unconventional wood formats such as peeled and sliced wood. The use of poplar wood was also evaluated to verify its technological uses to produce aged spirits. To this aim, GC-MS analyses were carried out to obtain an aromatic characterisation of experimental distillates treated with these products. Moreover, the same spirits were studied for classification purposes using NMR, NIR and e-nose. A significant change in the original composition of grape pomace distillate due to sorption phenomena was observed; the intensity of this effect was greater for poplar wood. The release of aroma compounds from wood depended both on the toasting level and wood assortment. Higher levels of xylovolatiles, namely, whisky lactone, were measured in samples aged using sliced woods. Both the NIR and NMR analyses highlighted similarities among samples refined with oak tablets, differentiating them from the other wood types. Finally, E-nose seemed to be a promising alternative to spectroscopic methods both for the simplicity of sample preparation and method portability

T cell activation induces CuZn Superoxide Dismutase (SOD)-1) intracellular re-localization, production and secretion

Reactive Oxygen Species (ROS) behave as second messengers in signal transduction for a series of receptor/ligand interactions. A major regulatory role is played by hydrogen peroxide (H2O2), more stable and able to freely diffuse through cell membranes. CuZn Superoxide dismutase (SOD)-1 is a cytosolic enzyme involved in scavenging oxygen radicals to H2O2 and molecular oxygen, thus representing a major cytosolic source of peroxides. Previous studies suggested that superoxide anion and H2O2 generation are involved in T Cell Receptor (TCR)-dependent signaling. Here, we describe that antigen-dependent activation of human T lymphocytes significantly increased extracellular SOD-1 levels in lymphocyte cultures. This effect was accompanied by the synthesis of SOD-1-specific mRNA and by the induction of microvesicle SOD-1 secretion. It is of note that SOD-1 increased its concentration specifically in T cell population, while no significant changes were observed in the “non T” cell counterpart. Moreover, confocal microscopy showed that antigen-dependent activation was able to modify SOD-1 intracellular localization in T cells. Indeed, was observed a clear SOD-1 recruitment by TCR clusters. The ROS scavenger N-acetylcysteine (NAC) inhibited this phenomenon. Further studies are needed to define whether SOD-1-dependent superoxide/peroxide balance is relevant for regulation of T cell activation, as well as in the functional cross talk between immune effectors

Prolonged NCX activation prevents SOD1 accumulation, reduces neuroinflammation, ameliorates motor behavior and prolongs survival in a ALS mouse model.

Abstract Imbalance in cellular ionic homeostasis is a hallmark of several neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS). Sodium-calcium exchanger (NCX) is a membrane antiporter that, operating in a bidirectional way, couples the exchange of Ca2+ and Na + ions in neurons and glial cells, thus controlling the intracellular homeostasis of these ions. Among the three NCX genes, NCX1 and NCX2 are widely expressed within the CNS, while NCX3 is present only in skeletal muscles and at lower levels of expression in selected brain regions. ALS mice showed a reduction in the expression and activity of NCX1 and NCX2 consistent with disease progression, therefore we aimed to investigate their role in ALS pathophysiology. Notably, we demonstrated that the pharmacological activation of NCX1 and NCX2 by the prolonged treatment of SOD1G93A mice with the newly synthesized compound neurounina: (1) prevented the reduction in NCX activity observed in spinal cord; (2) preserved motor neurons survival in the ventral spinal horn of SOD1G93A mice; (3) prevented the spinal cord accumulation of misfolded SOD1; (4) reduced astroglia and microglia activation and spared the resident microglia cells in the spinal cord; (5) improved the lifespan and mitigated motor symptoms of ALS mice. The present study highlights the significant role of NCX1 and NCX2 in the pathophysiology of this neurodegenerative disorder and paves the way for the design of a new pharmacological approach for ALS

Location and function of TDP-43 in platelets, alterations in neurodegenerative diseases and arising considerations for current plasma biobank protocols

The TAR DNA Binding Protein 43 (TDP-43) has been implicated in the pathogenesis of human neurodegenerative diseases and exhibits hallmark neuropathology in amyotrophic lateral sclerosis (ALS). Here, we explore its tractability as a plasma biomarker of disease and describe its localization and possible functions in the cytosol of platelets. Novel TDP-43 immunoassays were developed on three different technical platforms and qualified for specificity, signal-to-noise ratio, detection range, variation, spike recovery and dilution linearity in human plasma samples. Surprisingly, implementation of these assays demonstrated that biobank-archived plasma samples yielded considerable heterogeneity in TDP-43 levels. Importantly, subsequent investigation attributed these differences to variable platelet recovery. Fractionations of fresh blood revealed that ≥ 95% of the TDP-43 in platelet-containing plasma was compartmentalized within the platelet cytosol. We reasoned that this highly concentrated source of TDP-43 comprised an interesting substrate for biochemical analyses. Additional characterization of platelets revealed the presence of the disease-associated phosphoserine 409/410 TDP-43 proteoform and many neuron- and astrocyte-expressed TDP-43 mRNA targets. Considering these striking similarities, we propose that TDP-43 may serve analogous functional roles in platelets and synapses, and that the study of platelet TDP-43 might provide a window into disease-related TDP-43 dyshomeostasis in the central nervous system

The Cu,Zn-superoxide dismutase (SOD1) exerts neuroprotective effects in Amyotrophic Lateral Sclerosis through the Ca2+/ERK1/2/Akt prosurvival pathway

Amyotrophic lateral sclerosis (ALS) is a human adult-onset neurodegenerative disease characterized by progressive weakness, muscles atrophy, spasticity and paralysis resulting in respiratory arrest. These symptoms are related to degeneration and loss of upper motor neurons in cerebral cortex and lower motor neurons in brainstem and spinal cord. Most of cases of ALS are sporadic (sALS), while in the 5-10% of cases the disease is familiar (fALS) with autosomic dominant inheritance. In particular, in more than 15% of cases, the fALS is caused by mutations in the gene coding for the Cu,Zn-superoxide dismutase SOD1. Today, more than 125 different mutations in this gene are known, but how SOD1 caused motor neurons degeneration is unclear. Interestingly, most of mutants partially preserve their enzymatic activity, suggesting that SOD1 mutations are associated with a gain of toxic function rather than a loss of function. For instance, G93A mutation, that has been studied very intensely, leaves the enzyme activity intact. At last, while wt SOD1 secretion is compromise in ALS, mutant SOD1 is not secreted and accumulates in motor neurons within the endoplasmic reticulum and Golgi apparatus. Furthermore, after secretion, SOD1, independently from its dismutase activity, induces phospholipase C (PLC)/ protein kinase C (PKC) transductional pathway. Very interestingly, SOD1 is released by microglial cells and protect against 6-hydroxy-dopamine (6OHDA) toxin. Accordingly, wt SOD1 administration in transgenic SOD1G93A mice ameliorates motor symptoms of disease with an unknown mechanism. Thus, starting from the hypothesis that the neuroprotective effects of SOD1 could be due to its transductional property, we studied the effects of SOD1 and ApoSOD, which lacks dismutase activity, in both NSC-34 motor neurons and primary motor neurons exposed to the cicad neurotoxin L-beta-methylaminoalanine (L-BMAA), a model to reproduce ALS in vitro, and in NSC-34 motor neurons exposed to chemical hypoxia, a model of Ca2+ and ROS-dependent neurodegeneration. We showed that the exogenous administration of both SOD1 and ApoSOD, but not of human recombinant SOD1G93A, prevented cell death induced by the exposure to 300 µM L-BMAA for 48 hrs or to 45 min chemical hypoxia. On the other hand, MnTMPyP pentachloride, a SOD mimetic drug, failed to protect neurons, thus suggesting that SOD1 exerts neuroprotection independently from its most common enzymatic function. Interestingly both the pharmacological inhibition and the knocking down of MEK by PD98059 and siMEK, respectively, counteracted SOD1-induced neuroprotection. Analogously, the well known inhibitor of PI3'K LY294002 and the dominant negative form of Akt prevented SOD1-induced neuroprotective effect on neuronal death induced by L-BMAA and chemical hypoxia. Accordingly, wt SOD1 and ApoSOD elicited a phosphorylation of ERK1/2 and Akt in motor neurons through an early increase of intracellular Ca2+ concentration, thus suggesting that a Ca2+-dependent activation of ERK1/2 and Akt is mainly involved in SOD1-induced neuroprotective effects. Finally, SOD1 prevents ER stress induced by both L-BMAA and chemical hypoxia, as observed by a reduction of GRP78, CHOP, caspase-12 and caspase-3 levels in motor neurons pre-incubated with SOD1 and then exposed to toxic stimuli. Collectively, our data suggest that SOD1 is able to activate Ca2+/ERK1/2/Akt pro-survival pathway in ALS thus exerting a neuroprotective effects independently from its enzymatic function

Calcium Dyshomeostasis and Lysosomal Ca2+ Dysfunction in Amyotrophic Lateral Sclerosis

Recent findings in the understanding of amyotrophic lateral sclerosis (ALS) revealed that alteration in calcium (Ca2+) homeostasis may largely contribute to motor neuron demise. A large part of these alterations is due to dysfunctional Ca2+-storing organelles, including the endoplasmic reticulum (ER) and mitochondria. Very recently, lysosomal Ca2+ dysfunction has emerged as an important pathological change leading to neuronal loss in ALS. Remarkably, the Ca2+-storing organelles are interacting with each other at specialized domains controlling mitochondrial dynamics, ER/lysosomal function, and autophagy. This occurs as a result of interaction between specific ionic channels and Ca2+-dependent proteins located in each structure. Therefore, the dysregulation of these ionic mechanisms could be considered as a key element in the neurodegenerative process. This review will focus on the possible role of lysosomal Ca2+ dysfunction in the pathogenesis of several neurodegenerative diseases, including ALS and shed light on the possibility that specific lysosomal Ca2+ channels might represent new promising targets for preventing or at least delaying neurodegeneration in ALS

Ca2 + dysregulation in the pathogenesis of amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease without appropriate cure. One of the main reasons for the lack of a proper pharmacotherapy in ALS is the narrow knowledge on the molecular causes of the disease. In this respect, the identification of dysfunctional pathways in ALS is now considered a critical medical need. Among the causative factors involved in ALS, Ca2 + dysregulation is one of the most important pathogenetic mechanisms of the disease. Of note, Ca2 + dysfunction may induce, directly or indirectly, motor neuron degeneration and loss. Interestingly, both familial (fALS) and sporadic ALS (sALS) share the progressive dysregulation of Ca2 + homeostasis as a common noxious mechanism. Mechanicistically, Ca2 + dysfunction involves both plasma membrane and intracellular mechanisms, including AMPA receptor (AMPAR)-mediated excitotoxicity, voltage-gated Ca2 + channels (VGCCs) and Ca2 + transporter dysregulation, endoplasmic reticulum (ER) Ca2 + deregulation, mitochondria-associated ER membranes (MAMs) dysfunction, lysosomal Ca2 + leak, etc. Here, a comprehensive analysis of the main pathways involved in the dysregulation of Ca2 + homeostasis has been reported with the aim to focus the attention on new putative druggable targets