Research Projects INVESTIGATION OF PATHOGENETIC MECHANISMS OF NEUROLOGICAL DISORDERS IN HUMAN CELLULAR MODELS
The main interest of our group consists in the integration of clinical and basic scientific research in order to understand the pathogenic mechanisms involved in some of the most relevant neurological disorders, such as Alzheimer disease, Parkinson disease, Amyotrophic Lateral Sclerosis, and Stroke. The final goal is to identify new strategies for diagnosis, treatment, and prevention. In particular our work focuses on excitotoxicity, oxidative stress and protein misfolding mechanisms and their relationship to genetic, epigenetic and environmental risk factors, both in central and peripheral cellular models. All the quoted works represent selected examples of the scientific products of our research group.
ALZHEIMER DISEASE
Alzheimer disease is the most common form of dementia in the aged population and is characterized by an increased production of the neurotoxic β-amyloid peptide (Aβ) deriving from an altered metabolism of the amyloid precursor protein (APP). Aβ can directly or indirectly (through reactive oxygen species or mitochondrial failure) impair glutamate transporters, leading to the accumulation of extracellular glutamate, stimulation of excitatory amino acid receptors and subsequent neuronal death (a phenomenon known as excitotoxicity). Moreover, exposure to low chronic levels of A may elicit adaptive responses such as the activation of stress-activated protein kinase pathways. Our group has characterized several of these alterations in peripheral cells obtained from AD patients; in particular, we showed a dysfunction of glutamate uptake (Ferrarese et al. 2000), an alteration of glutamate transporters (Zoia et al. 2004; 2005) and an increased susceptibility to oxidative stress (Begni et al. 2005) in platelets and/or fibroblasts from AD patients. We are currently investigating Aβ effects on the intracellular redox balance, mitochondrial function, and MAPK pathways both in human neuroblastoma cell lines and peripheral cells obtained from patients and controls (Facheris et al. 2004; Tremolizzo et al. 2004).
Moreover, although AD has traditionally been considered a neurodegenerative condition in which vascular dysfunction plays a marginal role, it is not clear if this type of dementia also shares pathogenic mechanisms with the cerebrovascular disease. In fact, recent reports show that A has also harmful effects on vessels, indicating that vascular damage could be involved in the pathogenesis of AD. Furthermore, there are many evidences that also endothelial dysfunction may be involved in the pathogenesis of this disease. Based on these data, we hypothesize that Aβ may induce endothelial dysfunction, thus promoting ischemic damage, which may in turn affect APP processing and Abeta production. This reciprocal interaction may provide an explanation to the pathogenic link between these two conditions. The study of the effect of vascular risk factors on Aβ processing could help to elucidate whether vascular disease has only an additive effect on cognitive performance or it is also intrinsic to the pathogenesis of AD.
PARKINSON DISEASE
Parkinson disease is a neurodegenerative disorder characterized by loss of dopaminergic neurons and Lewy body presence in the substantia nigra. Alfa-synuclein (α-syn), a synaptic protein of unknown function, is a major component of Lewy bodies and its aggregation in vitro has been associated to increased oxidative stress. Oxidative stress from dopamine metabolism and dopamine auto-oxidation may contribute to selectively increase the vulnerability of dopaminergic cells to mitochondrial toxins and dysfunctional protein degradation pathways, since most oxidized proteins get degraded by the ubiquitin-proteasome system. Dopaminergic cell vulnerability is also linked to the glutamatergic input to the substantia nigra and to oxidative stress-induced excitotoxic mechanisms. Following the demonstration of systemic mitochondrial impairment in PD patients, and our observation of decreased glutamate transport in platelets (Ferrarese et al. 2001), we are currently investigating oxidative stress markers and 20S and 26S proteasome activities in peripheral cells obtained from familial and sporadic PD patients, in order to explore their possible relationship with genetic and environmental risk factors and L-DOPA assumption (Prigione et al. 2006).
Another line of work is related to the assessment of soluble and insoluble α-syn expression levels in lymphomonocytes from sporadic PD patients and their relation to the α-syn promoter polymorphisms, oxidative status and epigenetic factors (such as DNA methylation levels). Furthermore, in collaboration with the Mayo Clinic of Rochester (USA), we are investigating the role of the genetic variability in the susceptibility to PD development. Micro-array technology have been used to perform haplotype analysis of linkage-derived and whole genome association- derived candidate genes, in order to evaluate which combinations of polymorphisms seem to be more frequently associated to PD and their influence on the clinical phenotype (Elbaz et al. 2006; Maraganore et al. 2006). The next step we are into consists in assessing in peripheral cells from patients the pathophysiologic significance of the most interesting data emerging from the association studies.
Moreover, we are presently studying the role and the expression levels of the vesicular monoamine transporters (VMATs) in platelets from PD patients. In fact, VMATs represent key proteins for the correct maintenance of the dopaminergic homeostasis, and we are trying to also address the putative impact of anti-PD drugs on the expression of this transporter.
AMYOTROPHIC LATERAL SCLEROSIS
Amyotrophic Lateral Sclerosis is a progressive neurological disorder characterized by preferential degeneration of motor neurons, resulting in few years in marked disability and respiratory failure leading to the death of the patients. Glutamate transport alterations have been shown in patients affected by amyotrophic lateral sclerosis (ALS) and in animal models of this disease. Cu, Zn superoxide dismutase (SOD1) mutations typical of familial ALS (FALS) are able to oxidatively inactivate glutamate transporters: excitotoxic mechanisms and oxidative stress could converge and concur to generate motor neuron injury. Both peripheral models consisting of platelets and fibroblast cultures from ALS patients, and human neuroblastoma SH-SY5Y expressing either wild type SOD1 or mutant G93A SOD1 are currently employed by our group to study correlation between oxidative stress and excitotoxicity (Ferrarese et al. 2001; Beretta et al. 2003; Sala et al. 2005). Since the excitotoxic hypothesis of ALS pathogenesis is mainly related to a decreased expression of the glutamate transporter EAAT2 due to post-translational mechanisms, a better comprehension of the regulation of EAAT2 expression may be extremely useful for defining a new line of intervention. For instance, promising evidences come from the availability of epigenetic drugs, such as valproate, able to change gene transcription levels through changes in the structure of the chromatin (Tremolizzo et al. 2002; 2005).
STROKE
Stroke, the third leading cause of death among Western population, is usually due to cerebral arterial occlusion. Inflammation and excitotoxicity are well established mechanisms that play a pathogenetical role during cerebral ischemia. Indeed glutamate levels, both in cerebrospinal fluid (CSF) and in peripheral blood, are elevated after stroke. Moreover, C reactive protein (a paradigmatic protein for inflammatory pathways) is a predictive marker for cerebrovascular risk and its plasmatic levels are higher during brain ischemia. Our laboratory has recently depicted a transient glutamatergic dysfunction in patients affected by stroke (Aliprandi et al. 2005). In particular, higher glutamate plasma levels within the first two weeks after cerebral ischemia were described and a long lasting impairment of platelet glutamate uptake was shown, with a complete normalization nine months after the disease. Furthermore, a long lasting alteration of cytokines release from peripheral leukocytes of stroke patients was previously described (Ferrarese et al. 1999). For these reasons, we are now investigating a possible link between the glutamatergic dysfunction and the “chronic” inflammatory background in stroke patients.
MITOCHONDRIAL DISEASES
MELAS and LHON represent two major diseases due to mutations of the mitochondrial DNA. Recently our group demonstrated a decrease of glutamate uptake in cybrids created from patients affected by these disorders (Beretta et al. 2004; 2006), reinforcing the idea of the existence of a link between the mitochondrial oxphos chain dysfunction and the malfunctioning of glutamate transporters. Differences in free radicals production (DiFrancesco et al. 2007) and in the reversibility of the uptake defect following antioxidant exposure (Sala et al. 2007) are explained in terms of differences in the pathogenesis of these two disorders, affecting different oxphos chain mitochondrial complexes.
Facilities include: Clinical Department of General Neurology with 40 beds, including 8 beds in a Semi-intensive Stroke Unit Care, Laboratory of Neurobiology, Laboratory of Neurophysiology, and Laboratory of Neuropsychology.
Staff includes: 14 staff neurologists, 6 biologists, 36 residents in neurology, 5 PhD students in neuroscience, 2 laboratory technicians.
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