Mattia Toni

Fish Synucleins: An Update.

Synucleins (syns) are a family of proteins involved in several human neurodegenerative diseases and tumors. Since the first syn discovery in the brain of the electric ray Torpedo californica, members of the same family have been identified in all vertebrates and comparative studies have indicated that syn proteins are evolutionary conserved. No counterparts of syns were found in invertebrates suggesting that they are vertebrate-specific proteins. Molecular studies showed that the number of syn members varies among vertebrates. Three genes encode for α-, β- and γ-syn in mammals and birds. However, a variable number of syn genes and encoded proteins is expressed or predicted in fish depending on the species. Among biologically verified sequences, four syn genes were identified in fugu, encoding for α, β and two γ (γ1 and γ2) isoforms, whereas only three genes are expressed in zebrafish, which lacks α-syn gene. The list of “non verified” sequences is much longer and is often found in sequence databases. In this review we provide an overview of published papers and known syn sequences in agnathans and fish that are likely to impact future studies in this field. Indeed, fish models may play a key role in elucidating some of the molecular mechanisms involved in physiological and pathological functions of syn proteins.

Cellular, biochemical, and molecular characterization of nitric oxide synthase expressed in the nervous system of the prosobranch Stramonita haemastoma (Gastropoda, Neogastropoda)

Nitric oxide synthase (NOS) has been characterized in several opistobranchs and pulmonates but it was much less investigated in prosobranchs, which include more than 20,000 species and account for most of the gastropod diversity. Therefore, new data from this large group are needed for a better knowledge of the molecular evolution of NOS enzymes in molluscs. This study focused on NOS expressed in the nervous system of the prosobranch neogastropod Stramonita haemastoma. In this study we report compelling evidence on the expression of a constitutive Ca2+/CaM‐dependent neuronal NOS in the central and peripheral nervous system. The prevailing neuronal localization of NADPHd activity was demonstrated by NADPHd histochemistry in both central and peripheral nervous system structures. L‐arginine/citrulline assays suggested that Stramonita NOS is a constitutive enzyme which is both cytosolic and membrane‐bound. Molecular cloning of the full‐length Stramonita NOS (Sh‐NOS) by reverse‐transcription polymerase chain reaction (RT‐PCR) followed by 5′ and 3′ RACE showed that Sh‐NOS is a protein of 1,517 amino acids, containing a PDZ domain at the N‐terminus and sharing similar regulatory domains to the mammalian neuronal NOS (nNOS). Regional expression of the Sh‐NOS gene was evaluated by RT‐PCR. This analysis showed different expression levels in the nerve ring, the osphradium, the cephalic tentacles, the buccal tissues, and the foot, whereas NOS expression was not found in the salivary glands and the gland of Leiblein. The present data provide a solid background for further studies addressing the specific functions of NO in neogastropods. J. Comp. Neurol. 520:364–383, 2012.

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.

Synuclein expression in the lizard Anolis carolinensis

The synuclein (syn) family comprises three proteins: α-, β- and γ-syns. In humans, they are involved in neurodegenerative diseases such as Parkinson’s disease and in tumors. Members of the syn family were sequenced in representative species of all vertebrates and the comparative analysis of amino acid sequences suggests that syns are evolutionarily conserved, but information about their expression in vertebrate lineages is still scarce and completely lacking in reptiles. In this study, the expression of genes coding for α-, β- and γ-syns was analyzed in the green lizard Anolis carolinensis by semiquantitative RT-PCR and Western blot. Results demonstrate good expression levels of the three syns in the lizard nervous system, similarly to human syns. This, together with the high identity between lizard and human syns, suggests that these proteins fulfill evolutionarily conserved functions. However, differences between lizard and humans in the expression of syn variants (two different variants of γ-syn were detected in A. carolinensis) and differences in some amino acids in key positions for the regulation of protein conformation and affinity for lipid and metal ions also suggest that these proteins may have acquired different functional specializations in the two lineages.

Nitric Oxide Synthase in the Central Nervous System and Peripheral Organs of Stramonita haemastoma: Protein Distribution and Gene Expression in Response to Thermal Stress

Nitric oxide (NO) is generated via the oxidation of l-arginine by the enzyme NO synthase (NOS) both in vertebrates and invertebrates. Three NOS isoforms, nNOS, iNOS and eNOS, are known in vertebrates, whereas a single NOS isoform is usually expressed in invertebrates, sharing structural and functional characteristics with nNOS or iNOS depending on the species. The present paper is focused on the constitutive Ca2+/calmodulin-dependent nNOS recently sequenced by our group in the neogastropod Stramonita haemastoma (ShNOS). In this paper we provide new data on cellular distribution of ShNOS in the CNS (pedal ganglion) and peripheral organs (osphradium, tentacle, eye and foot) obtained by WB, IF, CM and NADPHd. Results demonstrated that NOS-like proteins are widely expressed in sensory receptor elements, neurons and epithelial cells. The detailed study of NOS distribution in peripheral and central neurons suggested that NOS is both intracellular and presynaptically located. Present findings confirm that NO may have a key role in the central neuronal circuits of gastropods and in sensory perception. The physiological relevance of NOS enzymes in the same organs was suggested by thermal stress experiments demonstrating that the constitutive expression of ShNOS is modulated in a time- and organ-dependent manner in response to environmental stressors.

Immunoreactivity and expression of synucleins in the South African clawed frog Xenopus laevis peripheral nervous system

Human synucleins (syns) genes coding for α-, β- and γ- isoforms are highly expressed in mammalian nervous system, in particular α-syn is implicated in several neurodegenerative diseases collectively named synucleinopathies, including Parkinson’s disease, frequently associated with motor impairment. The precise functions of syns remain elusive, but there are evidence indicating their involvement in the regulation of vesicular trafficking, exocytosis and synaptic function. Because of the high degree of conservation of syns among vertebrates, non-mammalian animal models may provide additional information on the evolution and the physiological role of these proteins [1,2]. Preliminary data are here reported on α- and β- syns expression and their morphological localization in different organs of adult specimens of the South African clawed frog Xenopus laevis, obtained by RT-qPCR, Western blot (WB) and immunohistochemistry (IHC). In WB and IHC experiments, two different commercial antibodies against mammalian α-, β- syns were used. Alpha- and β-syn immunoreactivities were differently distributed in the various tissues analyzed. Interestingly α -syn immunoreactivity was detected in both peripheral and autonomous nervous system respectively innervating skeletal muscles, cardiovascular system and gastrointestinal tract. Alpha-syn immunoreactive (IR) nerve fibers were found along skeletal muscle fibers, showing large varicosities typical of neuromuscular junctions. Moreover, both submucosal and myenteric plexuses of the gastrointestinal tract showed IR fibers. These preliminary observations suggest a conserved role for α-syn in synaptic vesicle trafficking in peripheral nerves and suggest that Xenopus laevis may be a promising model for the study of synucleinopathies.

Distribution of synuclein immunoreactivity in the central nervous system of the South African clawed frog Xenopus laevis

Alpha, β and γ synucleins (syns) have been identified in the nervous system of mammals and biochemical evidence suggest a crucial role for α-syn in the pathogenesis of several human neurodegenerative diseases. Our research is focused on the molecular expression and morphological localization of syns in the nervous system of representative species with the aim of understanding the evolutionary history of these proteins in vertebrates [1, 2]. Current model for our comparative analysis is the adult stage of the South African clawed frog Xenopus laevis. On the basis of previous studies on gene and protein expression of α- and β-syn in frog tissues, we have selected two antibodies immunoreactive for α- and β-syn for the immunohistochemical localization of Xenopus syns in the CNS. Double-immunohistostainings for ChAT , TH or serotonin were performed in order to analyze the distribution of syn immunoreactivity in cholinergic, catecholaminergic and serotoninergic areas. Both α- and β-syn were localized in the frog retina and in several brain regions with different patterns of distribution. Syn proteins are expressed in the retina of a wide range of vertebrates, including humans, and this suggests that retinal neurodegenerative diseases may be mediated by synucleinopathies. Strong α-syn immunoreactivity was also found in the visual projections and in the interpeduncolar nucleus, interspersed with cholinergic fibers, whereas β-syn immunoreactive axons formed a dense network in the ventral and dorsal striatum within the catecholaminergic plexus that plays a key role in the movement control in amphibians as in mammals. Present data are the background for further studies on physiological roles of syns during the vertebrate evolution.

Synuclein expression in the african clawed frog Xenopus laevis

The synuclein (syn) family comprises three proteins (α-, β- and γ- syns) encoded by different genes (snca, sncb and sncg). In mammals, α- and β- syn are primarily expressed in the brain where they are localized in pre-synaptic terminals while γ-syn is mainly expressed in the peripheral nervous system. In humans, synucleins are involved in neurodegenerative diseases such as Parkinson’s disease and in tumors. However, the normal cellular functions of the three syns have not yet been fully clarified. Members of the syn family were sequenced in representative species of all vertebrates and the comparative analysis of amino acid sequences suggests that syns are evolutionarily conserved, but information about their expression in vertebrate lineages is still scarce. Our research focused on the evolution of syns with the aim of analyzing their molecular and cellular expression in the CNS of representative vertebrates such as the carp Cyprinus carpio for teleost fish (1,2) and the green lizard Anolis carolinensis for reptiles (3). Current model of our comparative analysis for amphibians is the african clawed frog Xenopus laevis. The only information available on syn expression in this species relate to embryonic stages but data on syn expression in the adult are still lacking. At larval stages, amphibian snca is expressed in the brain, branchial arches and somites, and sncb signals were detected in the entire brain and spinal cord whereas sncg was only expressed in the peripheral nervous system including trigeminal nerve and dorsal root ganglion (4). Preliminary data are here reported on syn expression in adult specimen of X. laevis, obtained by RT-qPCR, Western blot and IHC. The results demonstrated that syns are expressed both in neuronal and non-neuronal tissues suggesting differences in the expression pattern between developmental and adult stages.

Distribution of Tyrosine hydroxylase (Th) immunoreactivity in the CNS of the common carp Cyprinus carpio

Catecholamines, including dopamine, are the principal neurotransmitters mediating a variety of functions in the CNS, such as motor control, cognition, emotion, memory processing, and endocrine modulation. Dysfunctional catecholamine neurotransmission is also implicated in neurologic and neuropsychiatric disorders. Human brain diseases, such as Parkinson’s disease (1), have been recently approached by using fish models, especially cyprinid teleosts, given basic similarities of the fish brain to that of mammals. The distribution of the catecholaminergic system has been studied in the forebrain of several teleosts, but relevant information are not available for the common carp, Cyprinus carpio, which is a model species in several studies. In this study, we have analyzed the distribution of catecholaminergic neurons in the carp brain by immunohistochemistry using a specific antibody to tyrosine hydroxylase (TH) on transverse serial frozen sections of the whole brain. In the carp brain, TH-immunoreactive (ir) neurons were present in several nuclei. In particular, positive neurons were detected in the ventral nucleus of the ventral telencephalic area. In addition, neuronal bodies and varicose fibers were stained for TH in the preoptic region, from the anterior to the posterior nuclei, in the suprachiasmatic nucleus, in the ventrolateral and ventromedial talamic nuclei. Moreover TH-ir neurons were also distributed in the periventricular pretectum and locus coeruleus. TH-ir structures were localized not only in recognizable catecholaminergic nuclei, corresponding to those of mammalian brain, but also in regions that are uniquely organized in teleosts, including the ventral telencephalon, the anterior and posterior preoptic region, the ventromedial thalamus, suggesting that they may be useful in elucidating homologies between fish and mammal brain. The present study partially confirmed TH distribution in other CNS of cyprinids (2), and provided more detailed information to a better understanding of the evolution of catecholaminergic system in vertebrates.

Distribution of choline acetyltransferase (ChAT) immunoreactivity in the CNS of the common carp Cyprinus carpio

Cholinergic systems play a role in basic cerebral functions and a number of human neurodegenerative disorders. Mechanisms involved in human brain diseases, including Parkinson’s disease (1), are often approached by using fish models, especially cyprinids, given basic similarities of the fish brain to that of mammals. In the present paper, the organization of central cholinergic systems have been described in the cyprinid Cyprinus carpio , the common carp, by using specific polyclonal antibodies against ChAT, the synthetic enzyme of acetylcholine, that is currently used as a specific marker for cholinergic neurons in all vertebrates. In this work, serial transverse and sagittal sections of the brain and the spinal cord were immunostained for ChAT. Results showed that positive neurons are present in several nuclei. In particular, ChAT-immunoreactive (ir) neurons were found in the forebrain (preoptic region, habenula), the midbrain (optic tectum, oculomotor nucleus, rostral tegmental nucleus), the hindbrain and the spinal cord (reticular formation, nucleus isthmi, secundary gustatory nucleus, cranial nerve motor nuclei from IV to X, spinal cord motoneurons). Moreover, ChAT-ir neurons were detected in the synencephalon (nucleus of the medial longitudinal fascicle) and in the cerebellum. In addition to neuronal bodies, afferent varicose fibers were stained for ChAT in the ventral telencephalon, the preoptic area, the hypothalamus and the posterior tuberculum. No neuronal cell bodies were present in the telencephalon. The comparison of ChAT-ir distribution observed in the present study with that reported in other CNS of cyprinids (2,3) has revealed a number of similarities and also some interesting differences. Our results provide additional information on the cholinergic system from a phylogenetic point of view, suggesting that cholinergic systems of the common carp show many primitive features that have been conserved during evolution, together with characteristics that are exclusive. In addition, the present study may add new perspectives to physiological roles of cholinergic system during evolution and the neuroanatomical basis of neurological diseases.