Highlights
•HSPA8 knock-down induces HSPA1A overexpression.•HSPA8 and HSPA1A knock-down increases tau, SOD1 and α-synuclein protein levels.•HSPA8 knock-down or macro-autophagy inhibition increase ...high- and low-molecular weight forms of α-synuclein.•HSPA8 knock-down dysregulates α-synuclein gene transcription.
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Heat shock protein 70 family was demonstrated to play a critical role in protein homeostasis, a process profoundly impaired in neurodegenerative disorders. Neurodegenerative diseases are characterized by the accumulation of different kind of proteins and the formation of insoluble aggregates which are toxic for neurons. To explore the role of heat shock protein family 70 (in particular HSPA8 and HSPA1A) in the accumulation of proteins implied in neurodegeneration pathogenesis, in this study we verified in human SH-SY5Y neuroblastoma cells how HSPA8 or HSPA1A knock-down can affect protein levels of tau, superoxide dismutase 1 and α-synuclein. We found HSPA8 and HSPA1A reduction caused an increase of tau, superoxide dismutase 1 and α-synuclein protein levels. We also noticed HSPA8 knock-down increased α-synuclein oligomeric forms and mRNA expression. Our results suggest HSPA8 can play an important role in the homeostasis of tau, superoxide dismutase 1 and α-synuclein and in the balance between α-synuclein oligomeric and monomeric forms.
•NMR allows to identify amyloid ligands and inhibitors in complex food matrixes.•Green and roasted coffee extracts show a “multi-target” anti-amyloidogenic activity.•Coffee extracts hinder Aβ peptide ...aggregation and cytotoxicity in a human cell line.•Coffee extracts reduce oxidative stress and modulate autophagy in a human cell line.•Chlorogenic acids and melanoidins are the most active components of coffee extracts.
To identify food and beverages that provide the regular intake of natural compounds capable of interfering with toxic amyloidogenic aggregates, we developed an experimental protocol that combines NMR spectroscopy and atomic force microscopy, in vitro biochemical and cell assays to detect anti-Aβ molecules in natural edible matrices.
We applied this approach to investigate the potential anti-amyloidogenic properties of coffee and its molecular constituents.
Our data showed that green and roasted coffee extracts and their main components, 5-O-caffeoylquinic acid and melanoidins, can hinder Aβ on-pathway aggregation and toxicity in a human neuroblastoma SH-SY5Y cell line. Coffee extracts and melanoidins also counteract hydrogen peroxide- and rotenone-induced cytotoxicity and modulate some autophagic pathways in the same cell line.
Abstract 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.
Abstract Generation of reactive oxygen species during dopamine (DA) oxidation could be one of the factors leading to the selective loss of nigral dopaminergic neurons in Parkinson’s disease (PD). ...Vesicular monoamine transporter type 2 (VMAT2) proteins in nerve terminals uptake dopamine into synaptic vesicles, preventing its cytoplasmic accumulation and toxic damage to nigral neurons. Polymorphisms in VMAT2 gene and in its regulatory regions might therefore serve as genetic risk factors for PD. In the present study, we have analyzed 8 single-nucleotide polymorphisms (SNPs) located within/around the VMAT2 gene for association with PD in an Italian cohort composed of 704 PD patients and 678 healthy controls. Among the 8 SNPs studied, only the 2 located within the promoter region (rs363371 and rs363324) were significantly associated with PD. In the dominant model, odds ratios were 0.72 (95% confidence interval CI: 0.6–0.9, p < 0.005) for rs363371 and 0.76 (95% CI: 0.6–0.9, p = 0.01) for rs363324; in the additive model, odds ratios were 0.78 (95% CI: 0.65–0.94, p = 0.008) for rs363371 and 0.85 (95% CI: 0.7–20.92, p = 0.04) for rs363324. There were no significant relationships between the remaining SNPs (rs363333, rs363399, rs363387, rs363343, rs4752045, and rs363236) and the risk of sporadic PD in any genetic model. This study adds to the previous evidence suggesting that variability in VMAT2 promoter region may confer a reduced risk of developing PD, presumably via mechanisms of gene overexpression.
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.
The SPREAD Programme investigated prospectively the time trend from September 2002 through December 2005 of transmitted drug resistance (TDR) among 2793 patients in 20 European countries and in ...Israel with newly diagnosed human immunodeficiency virus type 1 (HIV-1) infection. The overall prevalence of TDR was 8.4% (225 of 2687 patients; 95% confidence interval CI, 7.4%–9.5%), the prevalence of nucleoside reverse-transcriptase inhibitor (NRTI) resistance was 4.7% (125 of 2687 patients; 95% CI, 3.9%–5.5%), the prevalence of nonucleoside reverse-transcriptase inhibitor (NNRTI) resistance was 2.3% (62 of 2687 patients; 95% CI, 1.8%–2.9%), and the prevalence of protease inhibitor (PI) resistance was 2.9% (79 of 2687 patients; 95% CI, 2.4%–3.6%). There was no time trend in the overall TDR or in NRTI resistance, but there was a statistically significant decrease in PI resistance (P=.04) and in NNRTI resistance after an initial increase (P=.02). We found that TDR appears to be stabilizing in Europe, consistent with recent reports of decreasing drug resistance and improved viral suppression in patients treated for HIV-1 infection
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.
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.
Despite advances in neuroimaging, the diagnosis of idiopathic Parkinson’s disease (PD) remains clinical. The identification of biological markers for an early diagnosis is of great interest to start ...a neuroprotective therapy aimed at slowing, blocking or reversing the disease progression. Vesicular monoamine transporter 2 (VMAT2) sequesters cytoplasmic dopamine into synaptic vesicles for storage and release. Thus, VMAT2 impairment can regulate intra- and extracellular dopamine levels, influencing oxidative stress and neuronal death. Because in vivo imaging studies have demonstrated a VMAT2 reduction in PD patients greater than would be explained by neuronal loss alone, as an exploratory study we assessed VMAT2 mRNA and protein levels in platelets from 39 PD patients, 39 healthy subjects and 10 patients with vascular parkinsonism (VP) to identify a possible peripheral biomarker for PD. A significant reduction (
p
< 0.05) of VMAT2 mRNA levels was demonstrated in PD patients versus healthy controls. Patients with VP showed VMAT2 mRNA levels similar to controls. No difference in VMAT2 mRNA levels was found in untreated versus treated patients. No correlation was observed between mRNA levels and demographic or clinical characteristics. Furthermore, eight SNPs tagging the VMAT2 gene did not show effects on VMAT2 mRNA levels. Western blot analysis did not allow the quantification of VMAT2 protein expression in blood platelets. Although further studies in a greater number of cases are needed to confirm our data, the reduction in VMAT2 mRNA in platelets from PD patients suggests the existence of a systemic impairment of this transporter possibly contributing to PD pathology.
The spread of drug-resistant HIV-1 might compromise the future success of current first-line regimens.
To analyse the extent and impact of transmission of drug-resistant HIV-1 variants in Europe.
The ...European prospective programme (SPREAD) collected demographic, clinical and virological data from 1245 HIV-1-infected individuals in 17 countries diagnosed in 2002-2003. The potential impact of transmitted drug resistance mutations (TDRMs) on therapy response was determined by using genotypic interpretation algorithms.
The overall prevalence of viruses with drug-resistance mutations was 9.1% 96/1050; 95% confidence interval: 7.5-11.1. The majority (71%) harboured only a single amino acid substitution with limited effect on predicted drug susceptibility. Mutations associated with resistance to nucleoside reverse transcriptase inhibitors were observed most frequently 57/1050 (5.4%), followed by mutations related to protease inhibitors 32/1050 (3.0%) and mutations related to non-nucleoside reverse transcriptase inhibitors (NNRTIs) 27/1050 (2.6%). In some cases, however, resistance was quite extensive. Four individuals were infected with viruses with reduced susceptibility to all nucleoside reverse transcriptase inhibitors, 3 to all protease inhibitors and 20 to both NNRTIs. Remarkably, in one individual, the resistance pattern was so extensive that none of the available current antiretroviral drugs was predicted to be fully active.
The prevalence of TDRM-HIV is quite prominent (9.1%) but did not increase in comparison with a large retrospective European study. Particularly the presence of single NNRTI mutations may impact the efficacy of the first-line regimens. Continuous prospective monitoring remains indicated to explore the patterns and factors contributing to the transmission of TDRMs as well as the potential clinical consequences.
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