Gastrointestinal symptoms are the first signs of fluoride (F) toxicity. In the present study, the jejunum of rats chronically exposed to F was evaluated by proteomics, as well as by morphological analysis. Wistar rats received water containing 0, 10 or 50 mgF/L during 30 days. HuC/D, neuronal Nitric Oxide (nNOS), Vasoactive Intestinal Peptide (VIP), Calcitonin Gene Related Peptide (CGRP), and Substance P (SP) were detected in the myenteric plexus of the jejunum by immunofluorescence. The density of nNOS-IR neurons was significantly decreased (compared to both control and 10 mgF/L groups), while the VIP-IR varicosities were significantly increased (compared to control) in the group treated with the highest F concentration. Significant morphological changes were seen observed in the density of HUC/D-IR neurons and in the area of SP-IR varicosities for F-treated groups compared to control. Changes in the abundance of various proteins correlated with relevant biological processes, such as protein synthesis, glucose homeostasis and energy metabolism were revealed by proteomics.
… Generally, many studies report Rab proteins as molecules present in the CNS and their specific roles. Although marked differences distinguish the neuronal function between the ENS [Enteric Nervous System] and CNS, their similarities allow the use of some principles established for the brain environment to be reapplied in the enteric context 38. Several cellular processes can be altered and promote the enteric neuronal alterations caused by F effects through mechanisms involving the Rab proteins, which are considered neuronal regulators involved in the traffic and signaling of different molecules that promote neurons homeostasis, such as the neurotrophins family of growth factors. The neurotrophins-receptors complexes trigger important signaling pathways that promote development, survival and other neuronal functions through intracellular transport mechanisms mediate by the Rab proteins 39.
Rab 1A is a regulator of specific vesicular trafficking from the ER to Golgi complex, and in dopaminergic neurons its expression presents a protective effect enhancing the control of motor function in surviving neurons of hemiparkinsonian animals 40. From the family of the Rab 3 proteins, 3 members were present in the 10 mgF/L group, Rab 3A, Rab 3C, and Rab 3D. The Rab 3 family is observed in different cell types with high exocytic function 41, in which they function as exocytosis regulators 42 correlated with neuronal traffic 39, and are present in synaptic vesicles, modulating the neurotransmitter release 42. Rab 3A is the most abundant isoform in the brain, where it presents a modulatory function in synaptic membrane fusion through a Ca 2 + -dependent manner 43. In the peripheral nervous system Rab 3A has increased expression in sciatic nerve lesion area associated to an increase in the expression of two other important proteins that contribute to neurotransmission, synaptophysin and synapsin I 44. Rab 3C is highly expressed in primary hippocampal neurons, mediating regulated exocytosis 45, while Rab 3D is present in secretory granules and vesicles of other cell types, such as adipocytes, exocrine glands, hematopoietic cells 46, and low levels of expression were already identified in the duodenum, confirming its presence in exocrine cells of the GIT 47.
The Rabs 4A and 4B were also identified as exclusive for the 10 mgF/L, and Rab4 is described as a regulator of early endosomes in the synapses, contributing to neurotransmitter receptor recycling through endosomes acting associated to other molecules in the later steps of the endocytic recycling pathway in dendrites, directing the neuronal membrane receptor trafficking 48. This process is extremely important for the delivery of neurotransmitter receptors into the synaptic membrane, determining the synaptic function and plasticity. Rab 5A presents a role in axonal and dendritic endocytosis, contributing to the biogenesis of synaptic vesicles 49. Rab 8 presents the same role as Rab 4, being required to direct into synapses neurotransmitter receptors as the AMPA-type glutamatergic receptors, presenting an important role in the control of synaptic function and plasticity at the postsynaptic membrane 50 …
… In the network comparing the 50 mgF/L vs. control groups (Fig. 6), some proteins with relevance for the neuronal homeostasis were expressed uniquely in the 50 mgF/L, such as Tektin-2 (Q6AYM2), Perforin-1 (Q5FVS5), and Mitochondrial fission 1 protein (Fis1 -P84817). The Tektins family has significant expression in adult brain and in embryonic stages of the choroid plexus, the forming retina, and olfactory receptor neurons, and can be considered a molecular target for the comprehension of neural development 67. Although not present in the subnetwork, Perforin participates in the CD8 + T cells response, promoting granule cytotoxicity leading to a fast cellular necrosis of the target cell in minutes 68 or apoptosis in a period of hours through a mechanism in which the target cell collaborates with perforin to deliver granzymes into the cytosol 69. Using these mechanisms perforin-dependent, CD8 + T cells promote neuronal damage in inflammatory CNS disorders 7.
… Mitochondrial fission is implicated in the cell death through a pathway that involves caspase activation 71, and Mitochondrial fission 1 protein (Fis1) is considered essential for mitochondrial fission 72. Overexpression of Fis1 caused increase of mitochondrial fragmentation, which conducted to apoptosis or triggered autophagy 73, 74, and neuroprotective effects are correlated with inhibition of Fis1 75.
The fact that these proteins presented increased expression in relation to the control group can reflect F neurotoxicity on the ENS with the concentration of 50 mgF/L, and could result in the decrease in the density of the general population of neurons since these 3 proteins are involved in pathways that conduct to cell death by distinct mechanisms…