Alessandro Negro

Full length α-synuclein is present in cerebrospinal fluid from Parkinson's disease and normal subjects

Several clues suggest that α-synuclein, a presynaptic protein, plays a central role in the pathogenesis of idiopathic Parkinsons disease (PD). To search a peripheral marker of PD, we analyzed presence and amount of α-synuclein in CSF from 12 PD patients and 10 neurologically normal subjects. The protein was extracted from CSF samples through immunoprecipitation and immunoblotting with different specific anti-α-synuclein antibodies. We identified a 19 kDa band that corresponds to monomeric α-synuclein, given its comigration with homologue human recombinant peptide as well as with the protein extracted from cerebral cortex of normal subjects. The amount of CSF 19 kDa α-synuclein did not significantly vary in PD and normal cases. These findings have two implications: (a) full length α-synuclein is released by neurons in the extracellular space; (b) α-synuclein does not appear a peripheral marker of PD pathology.

{alpha}-Synuclein and Parkinson's disease

Alpha‐synuclein (α‐syn) is a small soluble protein expressed primarily at presynaptic terminals in the central nervous system. Interest in α‐syn has increased dramatically after the discovery of a relationship between its dysfunction and several neurodegenerative diseases, including Parkinsons disease (PD). The physiological functions of α‐syn remain to be fully defined, although recent data suggest a role in regulating membrane stability and neuronal plasticity. Various trigger factors, either environmental or genetic, can lead to a cascade of events involving misfolding or loss of normal function of α‐syn. In dopaminergic neurons, this may promote a vicious cycle in which elevation in cytoplasmic dopamine, oxidative stress, α‐syn dysfunction, and disruption of vesicle function lead to dopaminergic cell loss and PD. α‐Syn dysfunction appears to be a common feature of all forms of PD. The mechanism by which α‐syn induces neuronal cell toxicity may invoke multiple pathways, such as aggregation or interaction with other proteins and molecules, including synphilin‐1, chaperone 14‐3‐3 protein, and dopamine itself. This complexity has hindered the development of models to study PD. The available animal models of PD, each present distinct advantages and limits. Findings to date suggest that α‐syn‐based models represent a paradigm, which is closest to the human pathology.— Recchia, A., Debetto, P., Negro, A., Guidolin, D., Skaper, S. D., Giusti, P. α‐Synuclein and Parkinsons disease.

Neurotrophins rescue cerebellar granule neurons from oxidative stress-mediated apoptotic death: selective involvement of phosphatidylinositol 3-kinase and the mitogen-activated protein kinase pathway.

Abstract: Cerebellar granule neurons maintained in medium containing serum and 25 mM K+ reliably undergo an apoptotic death when switched to serum‐free medium with 5 mM K+. New mRNA and protein synthesis and formation of reactive oxygen intermediates are required steps in K+ deprivation‐induced apoptosis of these neurons. Here we show that neurotrophins, members of the nerve growth factor gene family, protect from K+/serum deprivation‐induced apoptotic death of cerebellar granule neurons in a temporally distinct manner. Switching granule neurons, on day in vitro (DIV) 4, 10, 20, 30, or 40, from high‐K+ to low‐K+/serum‐free medium decreased viability by >50% when measured after 30 h. Treatment of low‐K+ granule neurons at DIV 4 with nerve growth factor, brain‐derived neurotrophic factor (BDNF), neurotrophin‐3, or neurotrophin‐4/5 (NT‐4/5) demonstrated concentration‐dependent (1–100 ng/ml) protective effects only for BDNF and NT‐4/5. Between DIV 10 and 20, K+‐deprived granule neurons showed decreasing sensitivity to BDNF and no response to NT‐4/5. Cerebellar granule neuron death induced by K+ withdrawal at DIV 30 and 40 was blocked only by neurotrophin‐3. BDNF and NT‐4/5 also circumvented glutamate‐induced oxidative death in DIV 1–2 granule neurons. Granule neuron death caused by K+ withdrawal or glutamate‐triggered oxidative stress was, moreover, limited by free radical scavengers like melatonin. Neurotrophin‐protective effects, but not those of antioxidants, were blocked by selective inhibitors of phosphatidylinositol 3‐kinase or the mitogen‐activated protein kinase pathway, depending on the nature of the oxidant stress. These observations indicate that the survival‐promoting effects of neurotrophins for central neurons, whose cellular antioxidant defenses are challenged, require activation of distinct signal transduction pathways.

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.

Tyrosine and serine phosphorylation of α-synuclein have opposing effects on neurotoxicity and soluble oligomer formation

Mutations in the neuronal protein alpha-synuclein cause familial Parkinson disease. Phosphorylation of alpha-synuclein at serine 129 is prominent in Parkinson disease and influences alpha-synuclein neurotoxicity. Here we report that alpha-synuclein is also phosphorylated at tyrosine 125 in transgenic Drosophila expressing wild-type human alpha-synuclein and that this tyrosine phosphorylation protects from alpha-synuclein neurotoxicity in a Drosophila model of Parkinson disease. Western blot analysis of fly brain homogenates showed that levels of soluble oligomeric species of alpha-synuclein were increased by phosphorylation at serine 129 and decreased by tyrosine 125 phosphorylation. Tyrosine 125 phosphorylation diminished during the normal aging process in both humans and flies. Notably, cortical tissue from patients with the Parkinson disease-related synucleinopathy dementia with Lewy bodies showed less phosphorylation at tyrosine 125. Our findings suggest that alpha-synuclein neurotoxicity in Parkinson disease and related synucleinopathies may result from an imbalance between the detrimental, oligomer-promoting effect of serine 129 phosphorylation and a neuroprotective action of tyrosine 125 phosphorylation that inhibits toxic oligomer formation.

α-Synuclein Controls Mitochondrial Calcium Homeostasis by Enhancing Endoplasmic Reticulum-Mitochondria Interactions.

Background: α-Synuclein has a central role in Parkinson disease, and it has been proposed to regulate mitochondrial morphology and autophagic clearance. Results: α-Synuclein overexpression augments mitochondrial Ca2+ transients by enhancing endoplasmic reticulum-mitochondria interactions. Conclusion: Physiological levels of α-synuclein are required to sustain mitochondrial function and morphology integrity. Significance: Mitochondrial Ca2+ represents an important site where α-synuclein can modulate cell bioenergetics and survival. α-Synuclein has a central role in Parkinson disease, but its physiological function and the mechanism leading to neuronal degeneration remain unknown. Because recent studies have highlighted a role for α-synuclein in regulating mitochondrial morphology and autophagic clearance, we investigated the effect of α-synuclein in HeLa cells on mitochondrial signaling properties focusing on Ca2+ homeostasis, which controls essential bioenergetic functions. By using organelle-targeted Ca2+-sensitive aequorin probes, we demonstrated that α-synuclein positively affects Ca2+ transfer from the endoplasmic reticulum to the mitochondria, augmenting the mitochondrial Ca2+ transients elicited by agonists that induce endoplasmic reticulum Ca2+ release. This effect is not dependent on the intrinsic Ca2+ uptake capacity of mitochondria, as measured in permeabilized cells, but correlates with an increase in the number of endoplasmic reticulum-mitochondria interactions. This action specifically requires the presence of the C-terminal α-synuclein domain. Conversely, α-synuclein siRNA silencing markedly reduces mitochondrial Ca2+ uptake, causing profound alterations in organelle morphology. The enhanced accumulation of α-synuclein into the cells causes the redistribution of α-synuclein to localized foci and, similarly to the silencing of α-synuclein, reduces the ability of mitochondria to accumulate Ca2+. The absence of efficient Ca2+ transfer from endoplasmic reticulum to mitochondria results in augmented autophagy that, in the long range, could compromise cellular bioenergetics. Overall, these findings demonstrate a key role for α-synuclein in the regulation of mitochondrial homeostasis in physiological conditions. Elevated α-synuclein expression and/or eventually alteration of the aggregation properties cause the redistribution of the protein within the cell and the loss of modulation on mitochondrial function.

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.

Enhanced parkin levels favor ER-mitochondria crosstalk and guarantee Ca2 + transfer to sustain cell bioenergetics

Loss-of-function mutations in PINK1 or parkin genes are associated with juvenile-onset autosomal recessive forms of Parkinson disease. Numerous studies have established that PINK1 and parkin participate in a common mitochondrial-quality control pathway, promoting the selective degradation of dysfunctional mitochondria by mitophagy. Upregulation of parkin mRNA and protein levels has been proposed as protective mechanism against mitochondrial and endoplasmic reticulum (ER) stress. To better understand how parkin could exert protective function we considered the possibility that it could modulate the ER-mitochondria inter-organelles cross talk. To verify this hypothesis we investigated the effects of parkin overexpression on ER-mitochondria crosstalk with respect to the regulation of two key cellular parameters: Ca(2+) homeostasis and ATP production. Our results indicate that parkin overexpression in model cells physically and functionally enhanced ER-mitochondria coupling, favored Ca(2+) transfer from the ER to the mitochondria following cells stimulation with an 1,4,5 inositol trisphosphate (InsP(3)) generating agonist and increased the agonist-induced ATP production. The overexpression of a parkin mutant lacking the first 79 residues (ΔUbl) failed to enhance the mitochondrial Ca(2+) transients, thus highlighting the importance of the N-terminal ubiquitin like domain for the observed phenotype. siRNA-mediated parkin silencing caused mitochondrial fragmentation, impaired mitochondrial Ca(2+) handling and reduced the ER-mitochondria tethering. These data support a novel role for parkin in the regulation of mitochondrial homeostasis, Ca(2+) signaling and energy metabolism under physiological conditions.

Protein misfolding in Alzheimer's and Parkinson's disease: Genetics and molecular mechanisms

The accumulation of altered proteins is a common pathogenic mechanism in several neurodegenerative disorders. A causal role of protein aggregation was originally proposed in Alzheimers disease (AD) where extracellular deposition of beta-amyloid (Abeta) is the main neuropathological feature. It is now believed that intracellular deposition of aggregated proteins may be relevant in Parkinsons disease (PD), amyotrophic lateral sclerosis and polyglutamine disorders. An impairment of ubiquitin-proteasome system (UPS) appears directly involved in these disorders. We reviewed the results on the role of protein misfolding in AD and PD and the influence of mutations associated with these diseases on the expression of amyloidogenic proteins. Results of genetic screening of familial cases of AD and PD are summarized. In the familial AD population (70 subjects) we found several mutations of the presenilin 1 (PS1) gene with a frequency of 12.8% and one mutation in the gene encoding the protein precursor of amyloid (APP) (1.4%). One mutation of Parkin in the homozygous form and two in the heterozygous form were identified in our PD population. We also reported data obtained with synthetic peptides and other experimental models, for evaluation of the pathogenic role of mutations in terms of protein misfolding.

Multiple phosphorylation of α-synuclein by protein tyrosine kinase Syk prevents eosin-induced aggregation

The presence of aggregated α‐synuclein molecules is a common denominator in a variety of neurodegenerative disorders. Here, we show that α‐synuclein (α‐syn) is an outstanding substrate for the protein tyrosine kinase p72syk(Syk), which phosphorylates three tyrosyl residues in its C‐terminal domain (Y‐125, Y‐133, and Y‐136), as revealed from experiments with mutants where these residues have been individually or multiply replaced by phenylalanine. In contrast, only Y‐125 is phosphorylated by Lyn and c‐Fgr. Eosin‐induced multimerization is observed with wildtype α‐syn, either phosphorylated or not by Lyn, and with all its Tyr to Phe mutants but not with the protein previously phosphorylated by Syk. Syk‐mediated phosphorylation also counteracts α‐syn assembly into filaments as judged from the disappearance of α‐syn precipitated upon centrifugation at 100,000 × g. We also show that Syk and α‐syn colocalize in the brain, and upon cotransfection in Chinese hamster ovary cells, α‐syn becomes Tyr‐phosphorylated by Syk. Moreover, Syk and α‐syn interact with each other as judged from the mammalian two‐hybrid system approach. These data suggest that Syk or tyrosine kinase(s) with similar specificity may play an antineurodegenerative role by phosphorylating α‐syn, thereby preventing its aggregation.