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The presynaptic proteins MUNC18-1, syntaxin-1, and SNAP25 drive SNARE-mediated synaptic vesicle fusion and are also required for neuronal viability. Their absence triggers rapid, cell-autonomous, neuron-specific degeneration, unrelated to synaptic vesicle deficits. The underlying cell death pathways remain poorly understood. Here, we show that hippocampi of munc18-1 null mice (unknown sex) express apoptosis hallmarks cleaved caspase 3 (CC-3) and phosphorylated p53, and have condensed nuclei. However, side-by-side in vitro comparison with classical apoptosis induced by camptothecin uncovered striking differences to syntaxin-1 and MUNC18-1 depleted neurons. First, live-cell imaging revealed consecutive neurite retraction hours before cell death in MUNC18-1 or syntaxin-1 depleted neurons, whereas all neurites retracted at once, directly before cell death in classical apoptosis. Second, CC-3 activation was observed only after loss of all neurites and cellular breakdown, whereas CC-3 is activated before any neurite loss in classical apoptosis. Third, a pan-caspase inhibitor and a p53 inhibitor both arrested classical apoptosis, as expected, but not cell death in MUNC18-1 or syntaxin-1 depleted neurons. Neuronspecific cell death, consecutive neurite retraction, and late CC-3 activation were conserved in syntaxin-1 depleted human neurons. Finally, no indications were observed for involvement of other established cell death pathways, including necroptosis, Wallerian degeneration, autophagic cell death, and pyroptosis. Together, these data show that depletion of presynaptic proteins MUNC18-1 or syntaxin-1 triggers an atypical, staged cell death pathway characterized by consecutive neurite retraction, ultimately leading to, but not driven by, apoptosis. [ABSTRACT FROM AUTHOR]

Aims Neurotropin® (NTP), an analgesic for chronic pain, has antihyperalgesic effects in specific alternation of rhythm in temperature (SART)-stressed rats. Previous studies have shown that SART stress induces hyperalgesia, as well as post-translational modification of proteins (including substrates for calpain, a calcium-dependent cysteine protease) in the mesencephalon of rats. To better understand the mechanism of action of NTP, we investigated whether SART stress activates calpain in the mesencephalon of rats and whether NTP inhibits this activation. Main methods Wistar rats were exposed to SART stress for 5 days. NTP (200 NU/kg/day) was administered intraperitoneally every day from the onset of SART stress. The mechanical pain threshold was measured using the Randall-Selitto test on the 6th day. Thereafter, the rat mesencephalon was immediately collected and calpain activity was examined using western blot analysis with a calpain cleavage site-specific antibody. Key findings SART stress induced hyperalgesia and increased the calpain activity in the mesencephalon of rats. In contrast, NTP treatment attenuated the hyperalgesia and prevented the increase in calpain activity in the mesencephalon of SART-stressed rats. Interestingly, a negative correlation was identified between calpain activity and mechanical pain threshold in SART-stressed rats treated with or without NTP. Furthermore, NTP inhibited calpain activity on mammalian uncoordinated-18 in rat mesencephalon homogenate and Ac-LLY-AFC as substrates in an in vitro cell-free system. Significance Our data demonstrate that NTP treatment prevents SART stress-induced calpain activation in the mesencephalon of rats and suggests that NTP-mediated antihyperalgesia is associated with an inhibition of calpain activity in the mesencephalon. [ABSTRACT FROM AUTHOR]

Adult-onset hypothyroidism induces various impairments in hippocampus-dependent cognitive function, in which numerous synaptic proteins and neurotransmitters are involved. Donepezil (DON), an acetylcholinesterase inhibitor, has been shown to be efficient in improving cognitive function. The aim of the present study was to investigate the effects of adult-onset hypothyroidism on the expression levels of the synaptic proteins syntaxin-1 and munc-18, as well as the content of the neurotransmitter acetylcholine (ACh) in the hippocampus. In addition, the study explored the effects of thyroxin (T4) and DON treatment on the altered parameters. The study involved 55 Sprague-Dawley rats that were randomly divided into five groups: Control, hypothyroid (0.05% 6-n-propyl-2-thiouracil; added to the drinking water), hypothyroid treated with T4 (6 μg/100 g body weight once daily; intraperitoneal injection), hypothyroid treated with DON (0.005%; added to the drinking water) and hypothyroid treated with a combination of the two drugs (6 μg/100 g T4 and 0.005% DON). The concentration of ACh was determined in the homogenized hippocampus of each animal by alkaline hydroxylamine colorimetry. The protein levels of syntaxin-1 and munc-18 were determined by immunohistochemistry. The results showed that the content of ACh in the hippocampi of the hypothyroid rats was significantly decreased compared with that in the controls and that T4 monotherapy and DON administration restored the ACh content to normal values. In the hippocampi of the hypothyroid group, munc-18 was expressed at significantly lower levels, while the expression levels of syntaxin-1 were increased compared with the levels in the control group. Treatment with T4 alone restored the expression of syntaxin-1 but failed to normalize munc-18 expression levels. The co-administration of T4 and DON returned the munc-18 levels to normal values. These observations indicate that adult-onset hypothyroidism induces alterations in the levels of munc-18, syntaxin-1 and ACh in the hippocampus. Syntaxin-1 and ACh levels were restored by T4 monotherapy while munc-18 levels were not. In addition, the co-administration of T4 and DON resulted in more effective restoration than either alone. The thyroid hormone has a direct effect on metabolism of hippocampal ACh in adult rats and DON is helpful for treatment of synaptic protein impairment induced by hypothyroidism. [ABSTRACT FROM AUTHOR]

Abstract: Prolactin induces maturation of insulin secretion in cultured neonatal rat islets. In this study, we investigated whether the improved secretory response to glucose caused by prolactin involves alteration in the expression, association and phosphorylation of several proteins that participate in these processes. Messenger RNA was extracted from neonatal rat islets cultured for 5 days in the presence of prolactin and reverse transcribed. Gene expression was analyzed by semi-quantitative RT-PCR and by Western blotting for proteins. The gene transcription and protein expression of kinesin and MAP-2 were increased in prolactin-treated islets compared to the controls. The association and phosphorylation of proteins was analyzed by immunoprecipitation followed by Western blotting, after acute exposure to prolactin. Prolactin increased the association between SNARE proteins and kinesin/MAP-2 while the association of munc-18/syntaxin 1A was decreased. Serine phosphorylation of SNAP-25, syntaxin 1A, munc-18, MAP-2 was significantly higher whereas kinesin phosphorylation was decreased in prolactin-treated islets. There was an increase in SNARE complex formation in islets stimulated with prolactin, 22mM glucose, 40mM K+, 200μM carbachol and 1μM PMA. The prolactin-induced increase in the formation of SNARE complex and syntaxin 1A phosphorylation was inhibited by PD098059 and U0126, inhibitors of the MAPK pathway. These findings indicate that prolactin primes pancreatic β-cells to release insulin by increasing the expression and phosphorylation/association of proteins implicated in the secretory machinery and the MAPK/PKC pathway is important for this effect. [Copyright &y& Elsevier]

Exocytosis is regulated by exocytotic proteins, which are present in insulin-secreting β-cells and play regulatory roles in insulin secretion. Non-insulin dependent diabetes mellitus (type 2 diabetes) is a disease characterized by impaired insulin secretion and insulin resistance. Exocytotic protein immunoreactivities were studied in pancreatic islets of type 2 diabetic Goto-Kakizaki (GK) rats using immunofluorescence histochemistry. The immunoreactivities for vesicle-associated membrane protein-2 (VAMP-2), synaptotagmin III, cysteine string protein (CSP), mammalian homologue of the unc-18 gene (Munc-18), α-soluble N-ethylmaleimide-sensitive attachment protein (α-SNAP), N-ethylmaleimide-sensitive factor (NSF) and synaptosomal-associated protein of 25 kDa (SNAP-25) exhibited weaker immunofluorescence intensity in islets of GK rats as compared to control Wistar rats. Insulin immunoreactivity was also decreased in GK rat β-cells, whereas no detectable alterations in the expression of actin immunoreactivity could be detected. The data suggest that reduced expression of exocytotic proteins and decreased insulin content may contribute to the diabetic syndrome in the GK rat. [Copyright &y& Elsevier]

Exocytosis is regulated by proteins which interact to promote docking and fusion of secretory granules with the plasma membrane. We have used in situ hybridization to study the mRNA expression for vesicle-associated membrane protein (VAMP) isoforms VAMP-1 and VAMP-2, synaptosomal-associated protein of 25-kDa (SNAP-25) isoforms SNAP-25a and SNAP-25b, mammalian homologue of unc-18 (munc-18) and Hrs-2 in neurosecretory neurons of the magnocellular paraventricular (PVN) and supraoptic (SON) nuclei of normal and osmotically challenged animals. In PVN and SON neurons of normal animals, strong labeling was demonstrated for VAMP-2 and SNAP-25a mRNA, whereas VAMP-1 or SNAP-25b mRNA could not be detected. Salt-loading (2% NaCl as drinking water), an animal model which increases the expression and secretion of hormones from hypothalamic magnocellular neurons, resulted in significantly increased mRNA levels for VAMP-2 (36%, 28%), munc-18 (74%, 68%) and SNAP-25a (59%, 77%) in the PVN and SON, respectively. There was no significant increase in Hrs-2 mRNA levels in the PVN, whereas a significant increase (22%) was observed in the SON. In the posterior pituitary, immunohistochemistry showed a marked decrease in numbers and intensity of vasopressin-immunoreactive (-IR) nerve endings after salt-loading. There were no obvious changes in numbers or intensity of VAMP-2-, munc-18-, Hrs-2- or SNAP-25-IR fibers. Large varicosities containing VAMP-2- and Hrs-2 immunocreactivity were seen in salt-loaded animals. The results show isoform-specific mRNA expression in neurosecretory neurons and an increased mRNA expression of proteins participating in the molecular regulation of exocytosis during an experimental situation characterized by increased secretion.Copyright © 1999 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]