miR-129-5p: A key factor and therapeutic target in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a relentless and fatal neurological disease characterized by the selective degeneration of motor neurons. No effective therapy is available for this disease. Several lines of evidence indicate that alteration of RNA metabolism, including microRNA (miRNA) processing, is a relevant pathogenetic factor and a possible therapeutic target for ALS. Here, we showed that the abundance of components in the miRNA processing machinery is altered in a SOD1-linked cellular model, suggesting consequent dysregulation of miRNA biogenesis. Indeed, high-throughput sequencing of the small RNA fraction showed that among the altered miRNAs, miR-129-5p was increased in different models of SOD1-linked ALS and in peripheral blood cells of sporadic ALS patients. We demonstrated that miR-129-5p upregulation causes the downregulation of one of its targets: the RNA-binding protein ELAVL4/HuD. ELAVL4/HuD is predominantly expressed in neurons, where it controls several key neuronal mRNAs. Overexpression of pre-miR-129-1 inhibited neurite outgrowth and differentiation via HuD silencing in vitro, while its inhibition with an antagomir rescued the phenotype. Remarkably, we showed that administration of an antisense oligonucleotide (ASO) inhibitor of miR-129-5p to an ALS animal model, SOD1 (G93A) mice, result in a significant increase in survival and improved the neuromuscular phenotype in treated mice. These results identify miR-129-5p as a therapeutic target that is amenable to ASO modulation for the treatment of ALS patients.

HSC70 expression is reduced in lymphomonocytes of sporadic ALS patients and contributes to TDP-43 accumulation.

Aim: The demonstration that chaperone-mediated autophagy (CMA) contributes to the degradation of TDP-43, the main constituent of cytoplasmic inclusions typically found in motor neurons of patients with sporadic amyotrophic lateral sclerosis (sALS), has pointed out a possible involvement of CMA in aggregate formation. To explore this possibility, in this study, we verified the presence of a possible systemic CMA alteration in sALS patients and its effect on TDP-43 expression. Materials and methods: Gene and protein expression of the cytosolic chaperone HSC70 and the lysosome receptor LAMP2A, the two pivotal mediators of CMA, was assessed in peripheral blood mononuclear cells (PBMCs) derived from 30 sALS patients and 30 healthy controls. The expression of TDP-43 and co-chaperones BAG1 and BAG3 was also analyzed. Results: We found reduced HSC70 expression in patient cells, with no change in LAMP2A, and increased insoluble TDP-43 protein levels, with an aberrant intracellular localization. We also observed an unbalanced expression of co-chaperones BAG1 and BAG3. HSC70 down-regulation was confirmed in immortalized lymphoblastoid cell lines derived from sporadic and TARDBP mutant ALS patients. Lastly, we demonstrated that HSC70 silencing directly increases TDP-43 protein levels in human neuroblastoma cells. Discussion: Our results do not support the existence of a systemic CMA alteration in sALS patients but indicate a direct involvement of HSC70 alterations in ALS pathogenesis.

Riluzole Selective Antioxidant Effects in Cell Models Expressing Amyotrophic Lateral Sclerosis Endophenotypes.

OBJECTIVE: Until recently, riluzole was the only drug licensed for amyotrophic lateral sclerosis (ALS). In spite of its efficacy, the mechanism of action remains elusive, and both blocking of glutamate release and antioxidant properties have been postulated. Here we characterized human SH-SY5Y neuroblastoma cell lines, taking advantage of their insensitivity to excitotoxic insults, in order to selectively assess the presence of a direct antioxidant effect of riluzole. METHODS: SH-SY5Y cells, either parental or overexpressing the G93A SOD1 mutation, were exposed for 24 hours to the selected stimuli. RESULTS: Riluzole (1‒10 µM) was able to counteract the effects of H2O2 exposure (200 µM/24 hr), limiting both cell death and whole-cell reactive oxygen species (ROS) increase. The same experiments were repeated using SH-SY5Y cells carrying the familial ALS-related G93A-SOD1 mutation and constitutively expressing two-fold increased whole-cell ROS levels with respect to wild-type cells: riluzole was ineffective in this paradigm. Analogously, riluzole was ineffective in preventing cell death induced by exposing SH-SY5Y cells to 3-morpholino-sydnonimine (SIN-1, 1.5 mM/24 hr), a reactive nitrogen species (RNS) donor. CONCLUSION: Our data support a direct antioxidant action of riluzole. Furthermore, the lack of efficacy of riluzole observed in the SOD1 cell model mirrors the lack of efficacy already demonstrated in cognate mouse models of ALS, plausibly reflecting differences in the underlying pathogenic mechanisms. Finally, riluzole inefficacy against nitrosative stress might support the idea that a combined therapeutic intervention may result more effective in ALS patients, as in the case of co-administration of edaravone, a drug known to reduce RNS.

Rotenone down-regulates HSPA8/hsc70 chaperone protein in vitro: A new possible toxic mechanism contributing to Parkinson's disease.

HSPA8/hsc70 (70-kDa heat shock cognate) chaperone protein exerts multiple protective roles. Beside its ability to confer to the cells a generic resistance against several metabolic stresses, it is also involved in at least two critical processes whose activity is essential in preventing Parkinson's disease (PD) pathology. Actually, hsc70 protein acts as the main carrier of chaperone-mediated autophagy (CMA), a selective catabolic pathway for alpha-synuclein, the main pathogenic protein that accumulates in degenerating dopaminergic neurons in PD. Furthermore, hsc70 efficiently fragments alpha-synuclein fibrils in vitro and promotes depolymerization into non-toxic alpha-synuclein monomers. Considering that the mitochondrial complex I inhibitor rotenone, used to generate PD animal models, induces alpha-synuclein aggregation, this study was designed in order to verify whether rotenone exposure leads to hsc70 alteration possibly contributing to alpha-synuclein aggregation. To this aim, human SH-SY5Y neuroblastoma cells were treated with rotenone and hsc70 mRNA and protein expression were assessed; the effect of rotenone on hsc70 was compared with that exerted by hydrogen peroxide, a generic oxidative stress donor with no inhibitory activity on mitochondrial complex I. Furthermore, the effect of rotenone on hsc70 was verified in primary mouse cortical neurons. The possible contribution of macroautophagy to rotenone-induced hsc70 modulation was explored and the influence of hsc70 gene silencing on neurotoxicity was assessed. We demonstrated that rotenone, but not hydrogen peroxide, induced a significant reduction of hsc70 mRNA and protein expression. We also observed that the toxic effect of rotenone on alpha-synuclein levels was amplified when macroautophagy was inhibited, although rotenone-induced hsc70 reduction was independent from macroautophagy. Finally, we demonstrated that hsc70 gene silencing up-regulated alpha-synuclein mRNA and protein levels without affecting cell viability and without altering rotenone- and hydrogen peroxide-induced cytotoxicity. These findings demonstrate the existence of a novel mechanism of rotenone toxicity mediated by hsc70 and indicate that dysfunction of both CMA and macroautophagy can synergistically exacerbate alpha-synuclein toxicity, suggesting that hsc70 up-regulation may represent a valuable therapeutic strategy for PD.

MEF2D and MEF2C pathways disruption in sporadic and familial ALS patients.

Amyotrophic lateral sclerosis (ALS) is a progressive neuro-muscular disease characterized by motor neuron loss. MEF2D and MEF2C are members of the myocyte enhancer factor 2 family (MEF2), a group of transcription factors playing crucial roles both in muscle and in neural development and maintenance; for this reason, a possible involvement of MEF2 in ALS context has been investigated. Since the transcriptional activity of each tissue specific MEF2 isoform is conserved in different cell types, we chose to assess our parameters in an easily accessible and widely used experimental tool such as peripheral blood mononuclear cells (PBMCs) obtained from 30 sporadic ALS patients (sALS), 9 ALS patients with mutations in SOD1 gene (SOD1+) and 30 healthy controls. Gene expression analysis showed a significant up-regulation of MEF2D and MEF2C mRNA levels in both sporadic and SOD1+ ALS patients. Although protein levels were unchanged, a different pattern of distribution for MEF2D and MEF2C proteins was evidenced by immunohistochemistry in patients. A significant down-regulation of MEF2 downstream targets BDNF, KLF6 and RUFY3 was reported in both sALS and SOD1+ ALS patients, consistent with an altered MEF2 transcriptional activity. Furthermore, the potential regulatory effect of histone deacetylase 4 and 5 (HDAC4 and HDAC5) on MEF2D and MEF2C activity was also investigated. We found that MEF2D and HDAC4 colocalize in PBMC nuclei, while HDAC5 was localized in the cytoplasm. However, the unchanged HDACs localization and protein levels between sALS and controls seem to exclude their involvement in MEF2 altered function. In conclusion, our results show a systemic alteration of MEF2D and MEF2C pathways in both sporadic and SOD1+ ALS patients, underlying a possible common feature between the sporadic and the familial form of disease. Although further analyses in other neuromuscular diseases are needed to determine the specificity of changes in these pathways to ALS, measuring MEF2 alterations in accessible biofluids may be useful as biomarkers for disease diagnosis and progression.

BDNF Serum Levels with Respect to Multidimensional Assessment in Amyotrophic Lateral Sclerosis.

BACKGROUND: The clinical presentation of amyotrophic lateral sclerosis (ALS) is characterized by high heterogeneity, the greatest part of which still remains unexplained. OBJECTIVE: To assess serum levels of brain-derived neurotrophic factor (BDNF) in ALS patients, implementing a multidimensional characterization focused on four a priori chosen elements of phenotypic variability: ALS bulbar/spinal subtype, cognitive impairment, mood dysfunction and disease progression speed. METHODS: Serum samples were obtained from 45 ALS outpatients (16% bulbar onset) and 22 healthy controls. Each patient underwent the Montreal Cognitive Assessment (MoCA) and the Beck Depression Inventory (BDI), and disease progression speed was estimated by calculating the decay of the ALSFRS-R score over time. RESULTS: BDNF serum levels did not differ between patients and controls, although ∼25% lower levels characterized those patients carrying a depressive trait. Finally, BDNF serum levels were significantly lower in ALS patients expressing lower ALSFRS-R scores (r = 0.39, p < 0.01). No differences were found when considering cognitive impairment, disease progression speed and site of onset. CONCLUSION: BDNF serum levels might mark and possibly contribute in part to ALS phenotypic variability.

Role of Chaperone-Mediated Autophagy Dysfunctions in the Pathogenesis of Parkinson's Disease.

Chaperone-mediated autophagy (CMA) represents a selective form of autophagy involved in the degradation of specific soluble proteins containing a pentapeptide motif that is recognized by a cytosolic chaperone able to deliver proteins to the lysosomes for degradation. Physiologically, CMA contributes to maintain crucial cellular functions including energetic balance and protein quality control. Dysfunctions in CMA have been associated to the pathogenesis of several neurodegenerative diseases characterized by accumulation and aggregation of proteins identified as CMA substrates. In particular, increasing evidence highlights the existence of a strong relationship between CMA defects and Parkinson's disease (PD). Several mutations associated with familial forms of PD (SNCA, LRRK2, UCHL1 and DJ-1) have been demonstrated to block or reduce the activity of CMA, the main catabolic pathway for alpha-synuclein (asyn). CMA dysfunctions also leads to a mislocalization and inactivation of the transcription factor MEF2D that plays a key-role in the survival of dopaminergic neurons. Furthermore, reduced levels of CMA markers have been observed in post mortem brain samples from PD patients. The aim of this review article is to provide an organic revision of evidence for the involvement of CMA dysfunctions in the pathogenesis of PD. Updated findings obtained in patient's specimens will be resumed, and results deriving from in vivo and in vitro studies will be discussed to evidence the current knowledge on the molecular mechanisms underlying CMA alterations in PD. Finally, the possibility of up-regulating CMA pathway as promising neuroprotective strategy will be considered.

Reduced expression of the chaperone-mediated autophagy carrier hsc70 protein in lymphomonocytes of patients with Parkinson's disease.

Chaperone-mediated autophagy (CMA) impairment is recognized to play a pathogenetic role in Parkinson's disease (PD). A reduced expression of lysosomal-associated membrane protein (lamp) 2A and heat shock cognate (hsc) 70 protein, the two key regulators of CMA, has been reported in brains of PD patients. To verify the existence of a possible systemic CMA dysfunction in PD, in this study the expression of hsc70 and lamp2A was assessed in peripheral blood mononuclear cells (PBMC) of patients with sporadic PD and compared to healthy subjects. The expression of myocyte enhancer factor 2D (MEF2D), a transcriptional factor implicated in neuronal survival and specific substrate of CMA, was also evaluated. Protein and gene expression was assessed by Western blot and real-time PCR, respectively, in PBMC obtained from 53 sporadic PD patients and 53 healthy subjects. A significant reduction of hsc70 levels was observed in PBMC of PD patients, both under basal conditions and after autophagy induction obtained with serum deprivation. No difference emerged in lamp2A and MEF2D expression between patients and controls. No influence of the clinical characteristics of patients emerged on hsc70, lamp2A and MEF2D expression. These results, despite being not suggestive of the existence of a CMA impairment in PBMC of PD patients, identify a systemic hsc70 reduction in PD patients. Further studies on specific mechanisms and biological significance of such alteration are needed to corroborate this finding that could lead to the identification of a new trait biomarker for PD.

Rotenone upregulates alpha-synuclein and myocyte enhancer factor 2D independently from lysosomal degradation inhibition.

Dysfunctions of chaperone-mediated autophagy (CMA), the main catabolic pathway for alpha-synuclein, have been linked to the pathogenesis of Parkinson's disease (PD). Since till now there is limited information on how PD-related toxins may affect CMA, in this study we explored the effect of mitochondrial complex I inhibitor rotenone on CMA substrates, alpha-synuclein and MEF2D, and effectors, lamp2A and hsc70, in a human dopaminergic neuroblastoma SH-SY5Y cell line. Rotenone induced an upregulation of alpha-synuclein and MEF2D protein levels through the stimulation of their de novo synthesis rather than through a reduction of their CMA-mediated degradation. Moreover, increased MEF2D transcription resulted in higher nuclear protein levels that exert a protective role against mitochondrial dysfunction and oxidative stress. These results were compared with those obtained after lysosome inhibition with ammonium chloride. As expected, this toxin induced the cytosolic accumulation of both alpha-synuclein and MEF2D proteins, as the result of the inhibition of their lysosome-mediated degradation, while, differently from rotenone, ammonium chloride decreased MEF2D nuclear levels through the downregulation of its transcription, thus reducing its protective function. These results highlight that rotenone affects alpha-synuclein and MEF2D protein levels through a mechanism independent from lysosomal degradation inhibition.