Alpha- synuclein immunoreactivity in the enteric nervous system of human small intestine

Alpha-synuclein (α-syn) is a 140 amino acid protein, belonging to the synuclein family, expressed in mammalian neurons. Structural alterations of α-syn as well as its overexpression have been related to the onset and the progression of several human neurodegenerative diseases, as Parkinson’s diseases (PD). Indeed, α-syn aggregates are the main component of the Lewy bodies (Lbs), considered as pathological hallmarks of neurodegenerative diseases [1-2], known as synucleinopathies. PD is a multicentric neurodegenerative process that affects several neuronal structures in the central and peripheral nervous system, among which is the enteric nervous system (ENS). Remarkably, recent reports have shown that the lesions in the ENS occurred at very early stage of the disease, even before the involvement of the central nervous system. So, the ENS could be critical in the pathophysiology of PD [3-4] and the pathological alterations within the ENS could be involved in the gastrointestinal dysfunction frequently encountered by parkinsonian patients. Although at present Lbs, as well as α-syn pathological aggregates, have been evidenced throughout the autonomic nervous system projecting to the gut of patients affected by PD or other neurodegenerative diseases, however data on the distribution of α-syn in human normal ENS are lacking. Our study focused on the immunohistochemical distribution of α-syn in the ENS of proximal tract of human normal small intestine. Surgical specimens of duodenum and proximal jejunum, collected from patients submitted to a pancreaticoduodenectomy, were fixed and paraffin embedded. Intestinal slices underwent immunohistochemical procedure using monoclonal anti α-syn antibody. Alpha-syn immunoreactive (ir) structures were detected along both myenteric and submucosal plexuses as well as in the circular and longitudinal muscular layers. We found perivascular α-syn-ir fibers in the submucosa and a dense ir periglandular network projecting up to the axis of the villi in the mucosa. The immunohistochemical distribution pattern of α-syn has been compared with that of major enteric neurotransmitters. Our preliminary observations confirm a physiological role of α-syn in the ENS, and may contribute to clarify its role in the peripheral nervous system. References [1] Spillantini et al, Nature 1997; 388:839-40. [2] Arima et al, Brain Res 1998; 808:93-100. [3] Braak et al, Neurosci Lett 2006; 396:67-72. [4] Wakabayashi et al, Acta Neuropathol 2010; 120:1-12

Distribution of choline acetyltransferase (ChAT) immunoreactivity in the brain of the teleost cyprinus carpio

Cholinergic systems play a role in basic cerebral functions and its dysfunction is associated with deficit in neurodegenerative disease. Mechanisms involved in human brain diseases, 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 sections of the brain and the spinal cord were immunostained for ChAT. Results showed that positive neurons are present in several nuclei of the forebrain, the midbrain, the hindbrain and the spinal cord. Moreover, ChAT-positive neurons were detected in the synencephalon 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 cholinergic distribution pattern in the Cyprinus carpio central nervous system has revealed similarities but also some interesting differences with other cyprinids. Our results provide additional information on the cholinergic system from a phylogenetic point of view and may add new perspectives to physiological roles of cholinergic system during evolution and the neuroanatomical basis of neurological diseases

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

Endocrine cells distribution in human proximal small intestine: an immunohistochemical and morphometrical study

Atrophy of the pancreatic remnant after pancreaticoduodenectomy might be consequent to deregulation of pancreatic endocrine stimuli after duodenal removal. Relative technical surgical solution could be the anastomosis of the 1st jejunal loop to the stomach and the 2nd to the pancreatic stump. Data on the distribution of endocrine cells within the proximal intestine might represent the lacking tile of the problem. Our aims were to investigate the distribution pattern of serotonin, cholecystokinin and secretin cells in the duodenum, the 1st and 2nd jejunal loops of humans. Bowel specimens of ten patients submitted to pancreaticoduodenectomy were collected; immunohistochemical reactions and morphometric analyses were performed. A general ab-oral decrease of enteroendocrine cells was found. The rate of serotonin cells showed a significant 30.67±8.13% reduction starting from the 1st jejunal loop versus duodenum. The rate of both cholecystokinin and secretin cells in the duodenum was superimposable to that in the 1st jejunal loop, with a significant 62.88±4.80% loss of cholecystokinin and 39.5±9.31% of secretin cells in the 2nd loop. After removal of duodenum, preservation of the 1st jejunal loop could impact the function of pancreatic remnant maintaining the physiological enteroendocrine stimulus for pancreatic secretion that can compensate, at least in part for the abolished duodenal hormonal release

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 Parkinson's 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 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 ChAT, TH and 5HT by double immunolabelings. Results show that 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. Present findings indicate that α-syn-like proteins may be expressed in 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 co-expressed 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. This article is protected by copyright. All rights reserved

Distribution and characterization of α-syn and VIP immunoreactivity in the enteric nervous system of human small intestine

The enteric nervous system (ENS), our “second brain”, consists of a complex network of neurons and glial cells located within the gastrointestinal (GI) tract. ENS interacts through numerous neurotransmitters, and it can work independently of the central nervous system (CNS), modulating several functions within the GI tract (1). Th e ENS has two ganglionated plexuses, the myenteric and submucosal plexuses. Th ere is increasing evidence that several neurodegenerative diseases are not confi ned to the CNS but also to the peripheral nervous system (PNS), including the ENS (2). In 2003, Braak et al. theorized that the biological process underlying Parkinson Disease (PD) may originate in the ENS and may lead to CNS impairment (3). Alpha-synuclein (α-syn), a small presynaptic protein involved in the pathogenesis of PD, is abundantly expressed in the CNS, but also in the peripheral nerves of PD patients (4). In the normal gut, distribution of α-syn has been reported in the nerve fi bers of the lamina propria, sub-mucosa, and in the ganglia (5). Dysfunctions of vasoactive intestinal peptide (VIP) have been associated with impaired motility in infl ammatory bowel diseases (6). Th e present study aims to characterize the presence of α-syn and VIP in the normal human jejunum. Specimens of proximal jejunum were collected from patients and sections underwent immunohistochemical procedure using antibodies for α-syn and VIP. Alpha-syn immunoreactive (ir) structures were detected along both plexuses as well as in the circular and longitudinal muscular layers. We found perivascular α-syn-ir fi bers in the submucosa and a dense ir periglandular network projecting in the axis of the villi. Th e distribution pattern of α-syn and VIP has been compared. Our preliminary observations of codistribution of α-syn and VIP may elucidate their physiological role in the ENS and can shed light on how their structural alterations could contribute to the visceral pathogenesis of neurodegenerative disease. (1) Furness JB. Th e enteric nervous system and neurogastroenterology. Nat Rev Gastroenterol Hepatol. 2012 Mar 6;9(5):286-94. doi: 10.1038/nrgastro. 2012.32. PMID: 22392290. (2) Lebouvier, T., Chaumette, T., Paillusson, S., Duyckaerts, C., Bruley des Varannes, S., Neunlist, M., et al., 2009. Th e second brain and Parkinson’s disease. Eur. J. Neurosci. 30, 735–741. (3) Braak H, Del Tredici K, Rub U, de Vos RA, Jansen Steur EN, Braak E. Staging of brain pathology related to sporadic Parkinson’s disease. Neurobiol Aging. 2003;24(2):197-211. (4) Travagli RA, Browning KN, Camilleri M. Parkinson disease and the gut: new insights into pathogenesis and clinical relevance. Nat Rev Gastroenterol Hepatol. 2020 Nov;17(11):673-685. doi: 10.1038/s41575- 020-0339-z. Epub 2020 Jul 31. PMID: 32737460. (5) Casini A, Mancinelli R, Mammola CL, Pannarale L, Chirletti P, Onori P, et al. Distribution of alpha-synuclein in normal human jejunum and its relations with the chemosensory and neuroendocrine system. Eur J Histochem. 2021;65 (6) Tomita, R., Tanjoh, K., Fujisaki, S., Fukuzawa, M., 2000. Peptidergic nerves in the colon of patients with ulcerative colitis. Hepato. Gastroenterology 47 (32), 4000–4

Glucosylceramidase Mass and Subcellular Localization Are Modulated by Cholesterol in Niemann-Pick Disease Type C

Niemann-Pick disease type C (NPC) is characterized by the accumulation of cholesterol and sphingolipids in the late endosomal/lysosomal compartment. The mechanism by which the concentration of sphingolipids such as glucosylceramide is increased in this disease is poorly understood. We have found that, in NPC fibroblasts, the cholesterol storage affects the stability of glucosylceramidase (GCase), decreasing its mass and activity; a reduction of cholesterol raises the level of GCase to nearly normal values. GCase is activated and stabilized by saposin C (Sap C) and anionic phospholipids. Here we show by immunofluorescence microscopy that in normal fibroblasts, GCase, Sap C, and lysobisphosphatidic acid (LBPA), the most abundant anionic phospholipid in the endolysosomal system, reside in the same intracellular vesicular structures. In contrast, the colocalization of GCase, Sap C, and LBPA is markedly impaired in NPC fibroblasts but can be re-established by cholesterol depletion. These data show for the first time that the level of cholesterol modulates the interaction of GCase with its protein and lipid activators, namely Sap C and LBPA, regulating the GCase activity and stability

Distribution of Tyrosine hydroxylase 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