Giorgio Vivacqua

The role of alpha-synuclein in neurotransmission and synaptic plasticity

Alpha-synuclein (α-syn), a synaptic protein richly expressed in the central nervous system, has been implicated in several neurodegenerative diseases, such as Alzheimers disease, Parkinsons disease, multiple system atrophy, and dementia with Lewy bodies, which are collectively known as synucleinopathies. By contrast to the clear evidence for the involvement of α-syn in synucleinopathies, its physiological functions remain elusive, which becomes an impediment for revelation of its pathological mechanism. Since α-syn is richly expressed in presynaptic terminals and associated with synaptic vesicles, a large number of studies have been focused on revealing the potential functions of this protein in neurotransmission and synaptic plasticity. In this review article, we summarized recent advances for the role of α-syn in synaptic vesicle recycling, neurotransmitter synthesis and release, and synaptic plasticity. We discussed the possible relevance between the loss of normal α-syn functions in disease conditions and the onset of some neurodegenerative diseases.

Abnormal Salivary Total and Oligomeric Alpha-Synuclein in Parkinson’s Disease

In Parkinson’s disease (PD), alpha-synuclein (a-syn) can be detected in biological fluids including saliva. Although previous studies found reduced a-syn total (a-syntotal) concentration in saliva of PD patients, no studies have previously examined salivary a-syn oligomers (a-synolig) concentrations or assessed the correlation between salivary a-syntotal, a-synolig and clinical features in a large cohort of PD patients. Is well known that a-synolig exerts a crucial neurotoxic effect in PD. We collected salivary samples from 60 PD patients and 40 age- and sex-comparable healthy subjects. PD was diagnosed according to the United Kingdom Brain Bank Criteria. Samples of saliva were analyzed by specific anti-a-syn and anti-oligomeric a-syn ELISA kits. A complete clinical evaluation of each patient was performed using MDS-Unified Parkinsons Disease Rating Scale, Beck Depression Inventory, Montreal Cognitive Assessment and Frontal Assessment Battery. Salivary a-syntotal was lower, whereas a-synolig was higher in PD patients than healthy subjects. The a-synolig/a-syntotal ratio was also higher in patients than in healthy subjects. Salivary a-syntotal concentration negatively correlated with that of a-synolig and correlated with several patients’ clinical features. In PD, decreased salivary concentration of a-syntotal may reflect the reduction of a-syn monomers (a-synmon), as well as the formation of insoluble intracellular inclusions and soluble oligomers. The combined detection of a-syntotal and a-synolig in the saliva might help the early diagnosis of PD.

Immunolocalization of alpha-synuclein in the rat spinal cord by two novel monoclonal antibodies

This study provides the first immunohistochemical evidence of the presence and distribution patterns in the rat spinal cord of alpha-synuclein (alpha-Syn), a soluble acidic protein, widely expressed in the CNS and closely associated to the pathogenesis of neurodegenerative conditions such as Parkinsons and Alzheimers diseases. We used two novel homemade monoclonal antibodies (2E3 and 3D5) recognizing two different epitopes of alpha-Syn. Both antibodies localized alpha-Syn within the nerve terminals, whereas 3D5 alone also localized it within the neuronal nuclei. alpha-Syn-immunoreactive nervous elements were widely recognized throughout rat spinal cord and in almost all the gray matter laminae. However, they appeared particularly concentrated within laminae I, II, VII and X and more scattered in the others. Double immunofluorescent labeling showed that alpha-Syn colocalized with synaptophysin in the presynaptic nerve terminals, with neuropeptide Y (NPY) in lamina I, II, IX and X, and had close relationships with tyrosine hydroxylase (TH) immunoreactive neurons in laminae VII and X. Interestingly, the alpha-Syn-immunoreactive nerve elements, in lamina X, contained little of calbindin-28KD and calretinin-31KD. Our findings could help in understanding the genesis of some early clinical symptoms of Parkinsons disease (PD), such as pain and dysautonomic disorders, and indicate the spinal cord as their probable starting point, according to the ascending theory of PD, proposed by Braak.

Spinal cord and parkinsonism: Neuromorphological evidences in humans and experimental studies

The involvement of the spinal cord in parkinsonism is becoming more and more evident based on human autopsies and on experimental models, obtained using specific neurotoxins or genetic manipulations. Besides Parkinson disease, other degenerative disorders characterized by parkinsonism, involve the spinal cord, and multiple neurotransmitters, apart dopamine, are altered in parkinsonism, also in their spinal projections. In the present review we discuss spinal cord pathology of different genetic or toxic experimental models of parkinsonism, as well as the neuropathological reports from autoptic cases of sporadic Parkinson disease and of other neurodegenerative conditions, overlapping with parkinsonism. Furthermore, anatomical distribution of alpha-synuclein in the spinal cord and coeruleo-spinal projections are reviewed, at the light of their possible involvement in spinal neurons degeneration. All these evidences call for an anatomical stemmed novel approach to understand specific features of parkinsonism, which might be due to such an involvement of the spinal cord. Moreover they suggest a common neurodegenerative process, underlying distinct neurodegenerative disorders, to which spinal neurons could be the more sensible.

Localization of α-Synuclein in Teleost Central Nervous System: Immunohistochemical and Western Blot Evidence by 3D5 Monoclonal Antibody in the Common Carp, Cyprinus carpio

Alpha synuclein (α‐syn) is a 140 amino acid vertebrate‐specific protein, highly expressed in the human nervous system and abnormally accumulated in Parkinsons disease and other neurodegenerative disorders, known as synucleinopathies. The common occurrence of α‐syn aggregates suggested a role for α‐syn in these disorders, although its biological activity remains poorly understood. Given the high degree of sequence similarity between vertebrate α‐syns, we investigated this proteins in the central nervous system (CNS) of the common carp, Cyprinus carpio, with the aim of comparing its anatomical and cellular distribution with that of mammalian α‐syn. The distribution of α‐syn was analyzed by semiquantitative western blot, immunohistochemistry, and immunofluorescence by a novel monoclonal antibody (3D5) against a fully conserved epitope between carp and human α‐syn. The distribution of 3D5 immunoreactivity was also compared with that of choline acetyltransferase (ChAT), tyrosine hydroxylase (TH), and serotonin (5HT) by double immunolabelings. The results showed that a α‐syn‐like protein of about 17 kDa is expressed to different levels in several brain regions and in the spinal cord. Immunoreactive materials were localized in neuronal perikarya and varicose fibers but not in the nucleus. The present findings indicate that α‐syn‐like proteins may be expressed in a few subpopulations of catecholaminergic and serotoninergic neurons in the carp brain. However, evidence of cellular colocalization 3D5/TH or 3D5/5HT was rare. Differently, the same proteins appear to be coexpressed with ChAT by cholinergic neurons in several motor and reticular nuclei. These results sustain the functional conservation of the α‐syn expression in cholinergic systems and suggest that α‐syn modulates similar molecular pathways in phylogenetically distant vertebrates. J. Comp. Neurol. 523:1095–1124, 2015.

Choline acetyltransferase of the common type immunoreactivity in the rat brain following different heroin treatments: A pilot study

Previous studies suggest that behavioral consequences of heroin treatment depend on the drug history of the animals and that cholinergic neurotransmission is involved in both behavioral and motor sensitization induced by heroin and other drugs of abuse. Immunohistochemistry, using a recently developed antiserum, specific for choline acetyl-transferase of the common type (cChAT), was applied to four different groups of rats, differing in drug regimens. Two groups of rats were submitted to the same schedule of heroin sensitization and then challenged for vehicle or heroin before sacrifice, obtaining two distinct groups, namely heroin-vehicle (HV) and heroin-heroin (HH). The same challenge was applied to another group of rats, previously submitted to a treatment with vehicle, obtaining other two groups, vehicle-vehicle (VV) and vehicle-heroin (VH), respectively. The number of cChAT-positive neurons is significantly increased (p<0.05) in the diagonal band nuclei (with a consequent increase of cChAT positive fibers in the dentate gyrus) and notably, even not significantly (p>0.05), increased in the nucleus accumbens core of heroin-sensitized rats (HV, HH). Instead, acute heroin treatment significantly increase (p<0.05) the number of cChAT-positive cells in the nucleus accumbens shell of both heroin-naïve (VH) and heroin-sensitized (HH) rats. In heroin-sensitized rats (HV, HH), moreover, staining intensity of cChAT-positive fibers is significantly increased in the dorsal striatum, and basolateral amygdala (p<0.05). Unlikely, cChAT positive fibers in the central amygdala are significantly increased (p<0.05) by acute heroin treatments (VH, HH). The increase of cholinergic fibers in the dentate gyrus of the heroin sensitized rats (HV, HH) seems accompanied by a evident reduction in calretinin immunoreactive neurons in the same area. Our results, in a small group of animals, support the view that cholinergic mechanisms are intimately associated with the development of addictive phenotype. Furthermore, they suggest that cholinergic system is differentially engaged, following different heroin treatments.

Expression of brain derivated neurotrophic factor and of its receptors: TrKB and p75NT in normal and bile duct ligated rat liver.

Cholangiocytes are the cells lining the biliary tree from canals of Hering to larger bile ducts. By morphology, they are divided in small and large cholangiocytes, which result heterogeneous at functional and proliferative levels. Proliferating cholangiocytes acquire a neuroendocrine phe- notype, modulated by several factors including neurotrophins. Brain Derivated Neurotrophic Factor (BDNF) is a neurotrophin expressed in the nervous system and also in different types of epithelial and progenitor cells. The aim of the present study is to detect the expression of BDNF and of its two receptors (TrKB and p75NT, or p75NTR) in normal and bile duct ligated (BDL) rat livers. In normal and BDL livers, BDNF and its receptors are expressed by small and large cholan- giocytes and by hepatic progenitors cells. In cholangiocytes, the expression of BDNF and of its receptors changes after different BDL timing. After one or two weeks of BDL, both BDNF and TrKB and p75NT receptors are highly expressed, while after BDL for three weeks BDNF expression is drastically reduced and p75NT receptor prevails on TrKB. The expression of BDNF and of its receptors correlates with the proliferation rate of biliary tree during BDL. Indeed, after one or two weeks of BDL, proliferation prevails on apoptosis, whereas after BDL for three weeks, apoptosis prevails on proliferation. Our morphological results strongly suggest that BDNF plays a role in the remodeling of biliary tree during cholestasis and that it may be involved in the pathophysiology of cholestasic liver diseases.

Detection of α-Synuclein in Saliva: The Importance of Preanalytical Assessment: α-Synuclein in Saliva

patients in our cohort may have had MSA or, conversely, that some PD patients had been erroneously misdiagnosed as MSA and therefore excluded from this cohort as a result of early autonomic dysfunction. However, a review of our data showed that 37/104 (35.6%) of the patients included in our cohort had early autonomic impairment, suggesting that the latter possibility, if not fully ruled out, is improbable. We do agree that autonomic dysfunction represents one of “the hidden game changers in PD” because it is an underrecognized and, therefore, undertreated source of PD disability, particularly orthostatic hypotension (OH). For instance, a phase III, randomized, placebo-controlled, double-blind clinical trial of 224 nondemented PD patients with OH showed a reduction in the rate of falls by 295% during an 8-week observational period in those receiving droxidopa compared to placebo. In a chart review of 316 PD patients, we found that OH increased health care utilization independently from age, disease duration, motor severity, dopaminergic treatment, and cognitive function. Importantly, even “asymptomatic” OH (ie, not endorsing postural lightheadedness) yields similar impairment in quality of life and rate of falls than symptomatic OH, suggesting that traditional screening questionnaires may not be sufficient to uncover this treatable complication. Multiple studies, including the recent publication from De Pablo-Fernandez and colleagues titled “Association of Autonomic Dysfunction With Disease Progression and Survival in Parkinson Disease,” have shown that autonomic dysfunction may affect patients with PD from the very early stages or even in premotor stages. In that publication, the authors further noted that the earlier appearance of autonomic dysfunction increased the risk of reaching milestones of PD disability and shortened survival despite the lack of correlation with a-synuclein pathologic staging. Although confirming the key role of dysautonomia as a determinant of functional disability in PD, these data suggest that our current pathological model fails to recapitulate the complexity of autonomic dysfunction involved in the dysautonomia of MSA and PD. In conclusion, we agree with the central premise from De Pablo-Fernandez and Warner that “the possibility of either clinical misdiagnosis or inappropriate exclusion of patients must be considered in design and data interpretation, as it may lead to potential bias affecting the conclusions of the study.” We also advocate for the institution of a cohort employing comprehensive autonomic assessments as well as skin biopsies, peripheral biomarkers, and autopsy material to assist the next generation of biomarker development efforts and, ultimately, ushering the era of precision medicine in PD. Data Access and Responsibility Statement

Spinal cord pathology during chronic exposure to MPTP in mice

MPTP (1-metyl 4-phenyl 1,2,3,6-tetrahydro-piridin) treatment is a validated toxic model to produce experimental parkinsonism. Chronic exposure to little amounts of MPTP has been reported to reproduce neuropathological features characterizing dopaminergic neurons in Parkinson disease (1). Recent studies by our group have found that MPTP, besides dopaminergic neurons, affects also spinal motor neurons (2), thus reproducing also the neuropathology of spinal cord in Parkinson’s disease. The aim of the present study is to investigate, through immune-histochemistry, the involvement of spinal motor neurons after chronic administration of MPTP. Chronic MPTP treatment is able to induce motor neurons loss in the lumbar spinal cord. Increased immune-reactivity of the autophagy protein MAP LC3β is detectable in motor neurons which indicates the activation of autophagy. Immune-staining for alpha-synuclein, the hallmark protein for Parkinson’s disease, was altered in spinal motor neurons of MPTP treated mice, as detected by two novel homemade monoclonal antibodies. Furthermore, immunohistochemical alterations have been detected in the Renshaw cells and in tyrosine-hydroxylase-containing axons in line with the alterations of noradrenergic projections in Parkinsons’s disease (3). Our results suggest that spinal cord may contribute to motor symptoms occurring in Parkinson’s disease.

The involvement of Spinal Cord in MPTP-induced neurotoxicity: morphological evidence

Parkinson Disease (PD) is the prototype of synucleinopathies, a group of neurodegenerative disorders characterized by the accumulation of alpha-synuclein (α-syn). The involvement of spinal cord (SC) in PD is described in human autopsies, clinical reports and experimental studies [1]. The neurotoxin MPTP reproduces to a remarkable extent neuropathology of PD [2]. Furthermore, recent studies have shown that MPTP, besides dopaminergic neurons, affects also spinal Motor Neurons (MNs) [3]. To detect the effects of different MPTP treatments on the neuropathology in the SC at different ages and in different animal species. Following MPTP, neurotoxicity involves the whole thoraco-lumbar spinal cord. A model-dependent variation in the pattern of α-syn immunostaining has been detected. MNs, sympathetic pre-ganglionic neurons and calbindin immunoreactive (IR) cells of lamina VII are mostly affected, whereas TH positive fibers are spared. Alterations in α-syn positive fibers occur in the posterior laminae (I, II and III) and correlate with a significant loss of Met-Enk, SP and calbindin 28 kDa. A-syn accumulates in the affected neurons and in glial cells. Our results indicate that the SC represents a target of the parkinsonism-inducing neurotoxin MPTP. These data suggest that altered α-syn distribution associates with MPTP-induced neurotoxicity.