Sara Batelli

The SIRT1 activator resveratrol protects SK‐N‐BE cells from oxidative stress and against toxicity caused by α‐synuclein or amyloid‐β (1‐42) peptide

Human sirtuins are a family of seven conserved proteins (SIRT1‐7). The most investigated is the silent mating type information regulation‐2 homolog (SIRT1, NM_012238), which was associated with neuroprotection in models of polyglutamine toxicity or Alzheimer’s disease (AD) and whose activation by the phytocompound resveratrol (RES) has been described. We have examined the neuroprotective role of RES in a cellular model of oxidative stress, a common feature of neurodegeneration. RES prevented toxicity triggered by hydrogen peroxide or 6‐hydroxydopamine (6‐OHDA). This action was likely mediated by SIRT1 activation, as the protection was lost in the presence of the SIRT1 inhibitor sirtinol and when SIRT1 expression was down‐regulated by siRNA approach. RES was also able to protect SK‐N‐BE from the toxicity arising from two aggregation‐prone proteins, the AD‐involved amyloid‐β (1‐42) peptide (Aβ42) and the familiar Parkinson’s disease linked α‐synuclein(A30P) [α‐syn(A30P)]. Alpha‐syn(A30P) toxicity was restored by sirtinol addition, while a partial RES protective effect against Aβ42 was found even in presence of sirtinol, thus suggesting a direct RES effect on Aβ42 fibrils. We conclude that SIRT1 activation by RES can prevent in our neuroblastoma model the deleterious effects triggered by oxidative stress or α‐syn(A30P) aggregation, while RES displayed a SIRT1‐independent protective action against Aβ42.

DJ-1 Modulates α-Synuclein Aggregation State in a Cellular Model of Oxidative Stress: Relevance for Parkinson's Disease and Involvement of HSP70

BACKGROUND Parkinsons disease (PD) is a neurodegenerative pathology whose molecular etiopathogenesis is not known. Novel contributions have come from familial forms of PD caused by alterations in genes with apparently unrelated physiological functions. The gene coding for alpha-synuclein (alpha-syn) (PARK1) has been investigated as alpha-syn is located in Lewy bodies (LB), intraneuronal inclusions in the substantia nigra (SN) of PD patients. A-syn has neuroprotective chaperone-like and antioxidant functions and is involved in dopamine storage and release. DJ-1 (PARK7), another family-PD-linked gene causing an autosomal recessive form of the pathology, shows antioxidant and chaperone-like activities too. METHODOLOGY/PRINCIPAL FINDINGS The present study addressed the question whether alpha-syn and DJ-1 interact functionally, with a view to finding some mechanism linking DJ-1 inactivation and alpha-syn aggregation and toxicity. We developed an in vitro model of alpha-syn toxicity in the human neuroblastoma cell line SK-N-BE, influencing DJ-1 and alpha-syn intracellular concentrations by exogenous addition of the fusion proteins TAT-alpha-syn and TAT-DJ-1; DJ-1 was inactivated by the siRNA method. On a micromolar scale TAT-alpha-syn aggregated and triggered neurotoxicity, while on the nanomolar scale it was neuroprotective against oxidative stress (induced by H(2)O(2) or 6-hydroxydopamine). TAT-DJ-1 increased the expression of HSP70, while DJ-1 silencing made SK-N-BE cells more susceptible to oxidative challenge, rendering TAT-alpha-syn neurotoxic at nanomolar scale, with the appearance of TAT-alpha-syn aggregates. CONCLUSION/SIGNIFICANCE DJ-1 inactivation may thus promote alpha-syn aggregation and the related toxicity, and in this model HSP70 is involved in the antioxidant response and in the regulation of alpha-syn fibril formation.

Multidisciplinary perspectives for Alzheimer's and Parkinson's diseases: hydrogels for protein delivery and cell-based drug delivery as therapeutic strategies

This review presents two intriguing multidisciplinary strategies that might make the difference in the treatment of neurodegenerative disorders such as Alzheimers and Parkinsons diseases. The first proposed strategy is based on the controlled delivery of recombinant proteins known to play a key role in these neurodegenerative disorders that are released in situ by optimized polymer-based systems. The second strategy is the use of engineered cells, encapsulated and delivered in situ by suitable polymer-based systems, that act as drug reservoirs and allow the delivery of selected molecules to be used in the treatment of Alzheimers and Parkinsons diseases. In both these scenarios, the design and development of optimized polymer-based drug delivery and cell housing systems for central nervous system applications represent a key requirement. Materials science provides suitable hydrogel-based tools to be optimized together with suitably designed recombinant proteins or drug delivering-cells that, once in situ, can provide an effective treatment for these neurodegenerative disorders. In this scenario, only interdisciplinary research that fully integrates biology, biochemistry, medicine and materials science can provide a springboard for the development of suitable therapeutic tools, not only for the treatment of Alzheimers and Parkinsons diseases but also, prospectively, for a wide range of severe neurodegenerative disorders.

APOE epsilon‐4 allele and cytokine production in Alzheimer's disease

The APOE epsilon‐4 allele has consistently emerged as a susceptibility factor for Alzheimers disease (AD). Pro‐inflammatory cytokines are detectable at abnormal levels in AD, and are thought to play a pathophysiological role. Animal studies have shown dose‐dependent correlations between the number of APOE epsilon‐4 alleles and the levels of pro‐inflammatory cytokines. The aims of this study were to investigate the influence of APOE genotypes on TNF‐α, IL‐6, and IL‐1β secreted by peripheral blood mononuclear cells (PBMC) from human patients with AD and to analyze the correlation between cytokine production and AD clinical features.

Presenilin-1 mutation E318G and familial Alzheimer's disease in the Italian population.

Presenilin-1 (PSEN-1) is a component of the gamma-secretase complex involved in beta-amyloid precursor protein (betaAPP) processing. To date about 140 pathogenic mutations in the PSEN-1 gene have been identified and their main biochemical effect is to increase the production of the fibrillogenic peptide Abeta(1-42). An exception is the PSEN-1 [E318G] mutation that does not alter Abeta(1-42) generation and is generally considered a non-pathogenic polymorphism. Nevertheless, this mutation was reported to be a genetic risk factor for familial Alzheimers disease (FAD) in the Australian population. To independently confirm this indication, we performed a case-control association study in the Italian population. We found a significant association (p<0.05, Fishers exact test) between the presence of PSEN-1 [E318G] and FAD. In addition, on measuring the Abeta(1-42) and Abeta(1-40) concentrations in fibroblast-conditioned media cultured from PSEN-1 [E318G] carriers and PSEN-1 [wild type] controls we noted a significant decrease (p<0.05, Mann-Whitney test) in the Abeta(1-42)/Abeta(1-40) ratio in PSEN-1 [E318G] carriers, suggesting a peculiar biochemical effect of this mutation.

Early-onset Alzheimer disease in an Italian family with presenilin-1 double mutation E318G and G394V.

The genetic form of Alzheimer disease (FAD) accounts for about 5% of total Alzheimer disease (AD) cases, and the discovery of FAD-linked genes has shed new light on AD pathogenic mechanism. The presenilins genes (PSEN-1 and PSEN-2) carry the large majority of FAD-linked mutations. Here, we report an Italian kindred with FAD and PSEN-1 double mutation E318G+G394V that clearly cosegregates with the pathology. The E318G mutation has not an assessed pathogenic function, but some data have highlighted a role as a risk factor for AD in a predisposed familiar background. The G394V mutation was still described in association to an AD case with reported (but not demonstrated) familiar cosegregation. In an attempt to better characterize the biochemical effect of this double mutation, we assessed Aβ(1-40) and Aβ(1-42) concentrations in conditioned media from primary skin fibroblasts obtained from AD-affected and healthy family members. We did not find any modification of the Aβ(1-42)/Aβ(1-40) ratio, suggesting that this double mutation might be involved in early-onset AD etiopathogenesis by affecting a PSEN-1 function other than γ-secretase activity.

Macroautophagy and the proteasome are differently involved in the degradation of alpha-synuclein wild type and mutated A30P in an in vitro inducible model (PC12/TetOn).

Many data suggest that alpha synuclein (α-syn) aggregation is involved in Parkinsons disease (PD) neurotoxicity and is accelerated by the pathogenetic point mutation A30P. The triplication of α-syn gene has been linked to early-onset familial PD, suggesting that the cellular dosage of α-syn is an important modulator of its toxicity. To verify this point, we developed an inducible model of α-syn expression (both wild type [WT] and mutated A30P) in rat PC12/TetOn cells. At low expression level, both α-syn(WT) and (A30P) did not aggregate, were not toxic, and displayed a protective action against oxidative stress triggered by hydrogen peroxide (H2O2). By increasing α-syn expression, its antioxidant function was no longer detectable as for the A30P form, but again no aggregation and cell death were present both for the WT and the mutated protein. To clarify why α-syn did not accumulate at high expression level, we inhibited macroautophagy by 3-methyladenine (3-MA) and the proteasome by MG132. In presence of 3-MA, α-syn(WT) accumulated, A11 anti-oligomer antibody-positive aggregates were detectable, and cell toxicity was evident, while proteasome inhibition did not increase α-syn(WT) accumulation. Macroautophagy or proteasome inhibition slightly increased α-syn(A30P) toxicity, with no detectable aggregation. This model can provide useful details about α-syn function, aggregation, and degradation pathways.

Vitamin B12 Levels in Alzheimer's Disease: Association with Clinical Features and Cytokine Production

Alzheimers disease (AD) has been associated with up-regulation of pro-inflammatory cytokines (e.g., specific gene variants for TNF-alpha; IL-6; IFN-gamma) and low plasma levels of cyanocobalamin (vitamin B12). Our goal was to relate B12 levels to AD symptoms and to expression of pro-inflammatory cytokines. Clinical manifestations were investigated for a case series of fifty-five outpatients using the MMSE, Neuropsychiatric Inventory (NPI) and Cornell Scale for Depression in Dementia (CDDS). Plasma B12 levels were measured by radioligand binding assay. Basal and PMA-stimulated levels of IFN-gamma, TNF-alpha, and IL-6 were measured by ELISPOT (PBMC culture supernatant). 47 patients were genotyped for APOE. Ten patients (18%) had their B12 levels below < 250 pg/ml. They did not statistically differ from those 45 who had normal levels in most demographic and clinical features; their MMSE scores were lower (14.7 vs 19.6 p=0.03) but not after adjustment for disease duration. A greater basal production of IL-6 was reported in patients who had low B12 levels compared to normal B12 subjects (1333 pg/ml vs 976 p< 0.01); this association was confirmed after controlling for age of onset and APOE genotype. In conclusion, low B12 level is associated with greater production of IL-6 in peripheral blood mononuclear cells. Further research is warranted to elucidate whether this neuroinflammatory effect of cobalamin is implicated in the pathophysiology of AD.

Serotonin Transporter Gene Polymorphic Element 5-HTTLPR Increases the Risk of Sporadic Parkinson’s Disease in Italy

Parkinson’s disease (PD) is a neurodegenerative disorder causing muscular rigidity, resting tremor and bradykinesia. We conducted an association study assessing how PD risk in Italy was influenced by the serotonin transporter gene (SLC6A4) polymorphic region 5-HTTLPR, consisting of an insertion/deletion (long allele-L/short allele-S) of 43 bp in the SLC6A4 promoter region. The SLC6A4 promoter single nucleotide polymorphism rs25531(A→G) was evaluated too. We collected 837 independent subjects (393 PD, 444 controls). An association between the 5-HTTLPR polymorphism and risk of PD (S/S genotype OR [95% CI]: 1.7[1.2–2.5], p = 0.002) was found. The rs25531 and the haplotype 5-HTTLPR/rs25531 did not associate with risk of PD. Our data indicate that the 5-HTTLPR polymorphic element within the SLC6A4 promoter may govern the genetic risk of PD in Italians.

Interleukin-1 alpha and beta, TNF-alpha and HTTLPR gene variants study on alcohol toxicity and detoxification outcome.

Genetic factors may influence the liability to treatment outcome and medical complications in alcoholism. In the present study we investigated the IL-1A rs1800587, IL-1B rs3087258, TNF-alpha rs1799724 and the HTTLPR variants in a sample of 64 alcohol dependents and 47 relatives versus a set of clinical parameters and outcome measures. Alcohol dependents had a less favorable clinical profile compared to relatives (higher cholesterol, triglycerides, glucose, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, gamma-glutamyltransferase). After detoxification, all clinical indexes improved and hepatic enzyme levels were similar in alcohol dependents and relatives, except for the GGT that remained significantly higher in alcohol dependents. Alcoholic depressive and anxiety scores were significantly reduced after detoxification. IL-1A, IL-1B, TNF-alpha and HTTLPR variants were not associated with any baseline clinical index or change after detoxification. In our sample IL-1A, IL-1B, TNF-alpha and HTTLPR do not appear as liability factors for alcohol toxicity or detoxification outcome, however the small sample size may influence the observed results.