Increased pituitary adenylate cyclase-activating polypeptide in the central bed nucleus of the stria terminalis in mood disorders in men.

The mood disorders major depressive disorder (MDD) and bipolar disorder (BD) are highly prevalent worldwide. Women are more vulnerable to these psychopathologies than men. The bed nucleus of the stria terminalis (BNST), the amygdala, and the hypothalamus are the crucial interconnected structures involved in the stress response. In mood disorders, stress systems in the brain are put into a higher gear. The BNST is implicated in mood, anxiety, and depression. The stress-related neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is highly abundant in the central BNST (cBNST). In this study, we investigated alterations in PACAP in the cBNST of patients with mood disorders. Immunohistochemical (IHC) staining of PACAP and in situ hybridization (ISH) of PACAP mRNA were performed on the cBNST of post-mortem human brain samples. Quantitative IHC revealed elevated PACAP levels in the cBNST in both mood disorders, MDD and BD, but only in men, not in women. The PACAP ISH was negative, indicating that PACAP is not produced in the cBNST. The results support the possibility that PACAP innervation of the cBNST plays a role in mood disorder pathophysiology in men.

Rat Group IIA Secreted Phospholipase A2 Binds to Cytochrome c Oxidase and Inhibits Its Activity: A Possible Episode in the Development of Alzheimer's Disease.

Alzheimer's disease (AD), a progressive form of dementia, is characterized by the increased expression of secreted phospholipase A2 group IIA (GIIA) in the affected tissue and the dysfunction of neuronal mitochondria, similar to that induced by an orthologous GIIA from snake venom, ß-neurotoxic ammodytoxin (Atx), in the motor neurons. To advance our knowledge about the role of GIIA in AD, we studied the effect of rat GIIA on the neuronal mitochondria and compared it with that of the Atx. We produced recombinant rat GIIA (rGIIA) and its enzymatically inactive mutant, rGIIA(D49S), and demonstrated that they interact with the subunit II of cytochrome c oxidase (CCOX-II) as Atx. rGIIA and rGIIA(D49S) bound to this essential constituent of the respiratory chain complex with an approximately 100-fold lower affinity than Atx; nevertheless, both rGIIA molecules potently inhibited the CCOX activity in the isolated rat mitochondria. Like Atx, rGIIA was able to reach the mitochondria in the PC12 cells from the extracellular space, independent of its enzymatic activity. Consistently, the inhibition of the CCOX activity in the intact PC12 cells and in the rat's brain tissue sections was clearly demonstrated using rGIIA(D49S). Our results show that the effects of mammalian and snake venom ß-neurotoxic GIIA on the neuronal mitochondria have similar molecular backgrounds. They suggest that the elevated extracellular concentration of GIIA in the AD tissue drives the translocation of this enzyme into local neurons and their mitochondria to inhibit the activity of the CCOX in the respiratory chain. Consequently, the process of oxidative phosphorylation in the neurons is attenuated, eventually leading to their degeneration. Atx was thus revealed as a valuable molecular tool for further investigations of the role of GIIA in AD.

Development of potent reversible selective inhibitors of butyrylcholinesterase as fluorescent probes.

Brain butyrylcholinesterase (BChE) is an attractive target for drugs designed for the treatment of Alzheimer's disease (AD) in its advanced stages. It also potentially represents a biomarker for progression of this disease. Based on the crystal structure of previously described highly potent, reversible, and selective BChE inhibitors, we have developed the fluorescent probes that are selective towards human BChE. The most promising probes also maintain their inhibition of BChE in the low nanomolar range with high selectivity over acetylcholinesterase. Kinetic studies of probes reveal a reversible mixed inhibition mechanism, with binding of these fluorescent probes to both the free and acylated enzyme. Probes show environment-sensitive emission, and additionally, one of them also shows significant enhancement of fluorescence intensity upon binding to the active site of BChE. Finally, the crystal structures of probes in complex with human BChE are reported, which offer an excellent base for further development of this library of compounds.

Upregulation and axonal transport of synaptotagmin-IV in the direct-pathway medium spiny neurons in hemi-parkinsonian rats induced by dopamine D1 receptor stimulation.

Synaptotagmin-IV (Syt-IV) may function as a regulator of Ca(2+) -dependent synaptic transmission. In the hemi-parkinsonian rats with unilateral lesions of dopaminergic nigrostriatal neurons Syt-IV and substance-P (SP) mRNAs could be upregulated within the dopaminergically hypersensitive striatum of the lesioned brain hemisphere via the stimulation of striatal dopamine D1 (D1-R), but not D2 receptors. The hypersensitive D1-R-mediated transmission may be the culprit for the undesired expression of levodopa-induced dyskinesia, implying the involvement of Syt-IV and SP in the process. First, striatal cellular phenotypes expressing Syt-IV were determined. It was found to be expressed in all striatal neurons and a small population of astrocytes. Then it was examined, if the D1-R-mediated upregulation of Syt-IV mRNA may result in the upregulation of the translated protein. It was found that, after acute stimulation with a selective D1 agonist, (±)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF-82958), Syt-IV was elevated within the SP-expressing striatal neurons of the lesioned side. This was followed by the upregulation of Syt-IV, but not of its mRNA, within the ipsilateral target nuclei of the direct-pathway medium spiny neurons, indicating axonal transport of de novo synthesized protein to their SP-positive synaptic terminals. However, despite the striatal upregulation of SP and Syt-IV following a similar time-course, their subcellular co-localization within the axonal terminals was not found. It was therefore suggested that Syt-IV may regulate the hypersensitive striatal synaptic transmission, although via a SP-independent mechanism.

Behavioral sensitization and tolerance induced by repeated treatment with ketamine enantiomers in male Wistar rats.

Ketamine has gained significant attention as a fast-acting antidepressant. However, ketamine is also associated with undesirable side effects. In our preclinical study, we explored the behavioral effects of ketamine enantiomers at subanesthetic doses. During repeated intermittent treatment, we examined locomotor stimulation and sensitization, ataxia, and expression of natural behaviors (grooming and rearing). Male Wistar rats were subcutaneously treated repeatedly with either 5 mg/kg of R-ketamine or S-ketamine, 15 mg/kg of R-ketamine, S-ketamine or racemic ketamine, 30 mg/kg of racemic ketamine or saline every third day for three weeks (seven treatments overall). After the first treatment, only 15 mg/kg of S-ketamine induced locomotor stimulation, and both 15 mg/kg of S-ketamine and 30 mg/kg of racemic ketamine induced ataxia. Upon repeated administration, doses of 15 mg/kg of R-ketamine, S-ketamine, and racemic ketamine, as well as 30 mg/kg of racemic ketamine, stimulated locomotion. 15 mg/kg of R-ketamine, S-ketamine, and racemic ketamine additionally resulted in locomotor sensitization. The last administration of 15 mg/kg of S-ketamine, 15 mg/kg of racemic ketamine, and 30 mg/kg of racemic ketamine resulted in ataxia. In the case of 15 mg/kg of S-ketamine, ataxic effects were significantly weaker in comparison to the effects from the first administration, indicating tolerance. Natural behaviors were attenuated after 5 and 15 mg/kg of S-ketamine and 15 and 30 mg/kg of racemic ketamine. Neither of the R-ketamine doses produced such an effect. We conclude that S-ketamine has a stronger behavioral effect than R-ketamine.

Magnetic resonance imaging for rapid screening for the nephrotoxic and hepatotoxic effects of microcystins.

In vivo visualization of kidney and liver damage by Magnetic Resonance Imaging (MRI) may offer an advantage when there is a need for a simple, non-invasive and rapid method for screening of the effects of potential nephrotoxic and hepatotoxic substances in chronic experiments. Here, we used MRI for monitoring chronic intoxication with microcystins (MCs) in rat. Male adult Wistar rats were treated every other day for eight months, either with MC-LR (10 µg/kg i.p.) or MC-YR (10 µg/kg i.p.). Control groups were treated with vehicle solutions. T1-weighted MR-images were acquired before and at the end of the eight months experimental period. Kidney injury induced by the MCs presented with the increased intensity of T1-weighted MR-signal of the kidneys and liver as compared to these organs from the control animals treated for eight months, either with the vehicle solution or with saline. The intensification of the T1-weighted MR-signal correlated with the increased volume density of heavily injured tubuli (R2 = 0.77), with heavily damaged glomeruli (R2 = 0.84) and with volume density of connective tissue (R2 = 0.72). The changes in the MR signal intensity probably reflect the presence of an abundant proteinaceous material within the dilated nephrons and proliferation of the connective tissue. T1-weighted MRI-is a valuable method for the in vivo screening of kidney and liver damage in rat models of intoxication with hepatotoxic and nephrotoxic agents, such as microcystins.

Up-regulation of Synaptotagmin IV within amyloid plaque-associated dystrophic neurons in Tg2576 mouse model of Alzheimer's disease.

AIM: To investigate the involvement of the vesicular membrane trafficking regulator Synaptotagmin IV (Syt IV) in Alzheimer's disease pathogenesis and to define the cell types containing increased levels of Syt IV in the ß-amyloid plaque vicinity. METHODS: Syt IV protein levels in wild type (WT) and Tg2576 mice cortex were determined by Western blot analysis and immunohistochemistry. Co-localization studies using double immunofluorescence staining for Syt IV and markers for astrocytes (glial fibrillary acidic protein), microglia (major histocompatibility complex class II), neurons (neuronal specific nuclear protein), and neurites (neurofilaments) were performed in WT and Tg2576 mouse cerebral cortex. RESULTS: Western blot analysis showed higher Syt IV levels in Tg2576 mice cortex than in WT cortex. Syt IV was found only in neurons. In plaque vicinity, Syt IV was up-regulated in dystrophic neurons. The Syt IV signal was not up-regulated in the neurons of Tg2576 mice cortex without plaques (resembling the pre-symptomatic conditions). CONCLUSIONS: Syt IV up-regulation within dystrophic neurons probably reflects disrupted vesicular transport or/and impaired protein degradation occurring in Alzheimer's disease and is probably a consequence but not the cause of neuronal degeneration. Hence, Syt IV up-regulation and/or its accumulation in dystrophic neurons may have adverse effects on the survival of the affected neuron.

Differences between inflammatory cells infiltrated into tunica intima, media, and adventitia of ascending aortic aneurysms within diabetic and hypertensive patients.

The risk factors that are the most significant for the development of most cardiovascular diseases are arterial hypertension (AH), type 2 diabetes (DM), and inflammation. However, for the development of aortic aneurysms, DM is not one of them. Our study aimed to evaluate the difference between inflammatory infiltration in three individual layers of the ascending aortic aneurysm within diabetic and hypertensive patients. Forty-five patients aged 36 to 80 were divided into a group with diabetic patients without AH (group DM, N=8) and hypertensive patients without DM (group AH, N=37). For the histological analysis, aortic aneurysms were stained with hematoxylin eosin and Movat. We used immunochemical methods to detect pro- (M1), anti-inflammatory (M2) macrophages, T-helper, T-killer cells, B cells, and plasma cells. Statistical analysis was done by independent-samples Kruskal-Wallis test adjusted by Bonferroni correction for multiple tests (P<0.05). We found no difference in the volume density of collagen, elastin, vascular smooth muscle cells (VSMC), and ground substance between groups. In the DM group, there were significantly fewer M2, T-helpers, and T-killers in the media than in the intima and the adventitia (P<0.05). There were no significant differences in the number of M1, B, and plasma cells between all three vascular layers (P<0.05). In the AH group, there were significantly fewer B and plasma cells, T-helper, T-killer cells, M1, and M2 in the media than in the intima and adventitia (P<0.05). Our results conclude that the tunica media in the aneurismal wall of the AH group retained immune privilege. In contrast, in the DM group, all three layers were immune-privileged.

Pseudo-irreversible butyrylcholinesterase inhibitors: Structure-activity relationships, computational and crystallographic study of the N-dialkyl O-arylcarbamate warhead.

Alongside reversible butyrylcholinesterase inhibitors, a plethora of covalent butyrylcholinesterase inhibitors have been reported in the literature, typically pseudo-irreversible carbamates. For these latter, however, most cases lack full confirmation of their covalent mode of action. Additionally, the available reports regarding the structure-activity relationships of the O-arylcarbamate warhead are incomplete. Therefore, a follow-up on a series of pseudo-irreversible covalent carbamate human butyrylcholinesterase inhibitors and the structure-activity relationships of the N-dialkyl O-arylcarbamate warhead are presented in this study. The covalent mechanism of binding was tested by IC50 time-dependency profiles, and sequentially and increasingly confirmed by kinetic analysis, whole protein LC-MS, and crystallographic analysis. Computational studies provided valuable insights into steric constraints and identified problematic, bulky carbamate warheads that cannot reach and carbamoylate the catalytic Ser198. Quantum mechanical calculations provided further evidence that steric effects appear to be a key factor in determining the covalent binding behaviour of these carbamate cholinesterase inhibitors and their duration of action. Additionally, the introduction of a clickable terminal alkyne moiety into one of the carbamate N-substituents and in situ derivatisation with azide-containing fluorophore enabled fluorescent labelling of plasma human butyrylcholinesterase. This proof-of-concept study highlights the potential of this novel approach and for these compounds to be further developed as clickable molecular probes for investigating tissue localisation and activity of cholinesterases.

Modulation by Estradiol of L-Dopa-Induced Dyskinesia in a Rat Model of Post-Menopausal Hemiparkinsonism.

Treatment with levodopa (L-dopa) in Parkinson's disease (PD) leads to involuntary movements termed L-dopa-induced dyskinesia (LID). There are contradictory data about the influence of hormone therapy in female PD patients with LID and of 17-ß-estradiol (E2) on animal correlates of LID-abnormal involuntary movements (AIMs). Our aim was to characterize the influence of E2 on motor impairment and AIMs in ovariectomized 6-hydroxydopamine (6-OHDA) rat model of PD. Half of the rats received empty and the other half implants filled with E2. Following the 6-OHDA surgery, the rats received daily treatment with either L-dopa or saline for 16 days. They were assessed for AIMs, contralateral rotations, and FAS. In the L-dopa-treated rats, E2 intensified and prolonged AIMs and contralateral rotations. On the other hand, it had no effect on motor impairment. Postmortem tyrosine hydroxylase immunostaining revealed an almost complete unilateral lesion of nigrostriatal dopaminergic neurons. E2 partially prevented the upregulation of striatal ΔFosB caused by dopamine depletion. L-dopa potentiated the upregulation of ΔFosB within the dopamine-depleted striatum and this effect was further enhanced by E2. We speculate that the potentiating effects of E2 on AIMs and on contralateral rotations could be explained by the molecular adaptations within the striatal medium spiny neurons of the direct and indirect striatofugal pathways.

Effects of L-dopa on expression of prolactin and synaptotagmin IV in 17-beta-estradiol-induced prolactinomas of ovariectomized hemiparkinsonian rats.

Parkinson's disease (PD) is a long-term degenerative disorder of the central nervous system that mainly affects the motor system. Dopamine precursor levodopa (L-dopa) is used as the first-line treatment for PD. Evidence suggests neuroprotective effects of estrogens in PD. Since both 17b-estradiol (E2) and L-dopa act as regulators of prolactin (PRL) secretion from the pituitary gland, we investigated their effect on the expression of PRL in prolactinomas that developed in ovariectomized hemiparkinsonian rats treated with E2. We also investigated the effect of E2 and L-dopa on the expression of synaptotagmin IV (Syt IV), an immediate early gene whose product is abundant in the pituitary gland and was found to be highly co-expressed with PRL in lactotrophs (>90%). The hemiparkinsonian rat model was obtained by unilateral lesioning of dopaminergic nigrostriatal neurons. Rats received silastic tubing implants with E2 and were treated with L-dopa. Enzyme-linked immunosorbent assay and immunohistochemistry were used to assess the serum concentrations of PRL and E2 and expression of PRL and Syt IV in the tissue of adenohypophysis, respectively. We found that high levels of serum E2 were associated with the upregulation of Syt IV and PRL in PRL-ir cells, while treatment with L-dopa decreased the size of prolactinomas and downregulated Syt IV but had no effect on PRL expression or serum concentrations.

Evolution of Nova-dependent splicing regulation in the brain.

A large number of alternative exons are spliced with tissue-specific patterns, but little is known about how such patterns have evolved. Here, we study the conservation of the neuron-specific splicing factors Nova1 and Nova2 and of the alternatively spliced exons they regulate in mouse brain. Whereas Nova RNA binding domains are 94% identical across vertebrate species, Nova-dependent splicing silencer and enhancer elements (YCAY clusters) show much greater divergence, as less than 50% of mouse YCAY clusters are conserved at orthologous positions in the zebrafish genome. To study the relation between the evolution of tissue-specific splicing and YCAY clusters, we compared the brain-specific splicing of Nova-regulated exons in zebrafish, chicken, and mouse. The presence of YCAY clusters in lower vertebrates invariably predicted conservation of brain-specific splicing across species, whereas their absence in lower vertebrates correlated with a loss of alternative splicing. We hypothesize that evolution of Nova-regulated splicing in higher vertebrates proceeds mainly through changes in cis-acting elements, that tissue-specific splicing might in some cases evolve in a single step corresponding to evolution of a YCAY cluster, and that the conservation level of YCAY clusters relates to the functions encoded by the regulated RNAs.

Neuroinflammation-Induced Upregulation of Glial Cathepsin X Expression and Activity in vivo.

Neuroinflammation is an important factor in the pathogenesis of neurodegenerative diseases. Microglia-derived lysosomal cathepsins have been increasingly recognized as important inflammatory mediators that trigger signaling pathways that aggravate neuroinflammation. In vitro, a contribution to neuroinflammation processes has been shown for cathepsin X: however, the expression patterns and functional role of cathepsin X in neuroinflammatory brain pathology remain elusive. In this study we analyzed the expression, activity, regional distribution and cellular localization of cathepsin X in the rat brain with neuroinflammation-induced neurodegeneration. The unilateral injection of lipopolysaccharide (LPS) induced a strong upregulation of cathepsin X expression and its activity in the ipsilateral striatum. In addition to the striatum, cathepsin X overexpression was detected in other brain areas such as the cerebral cortex, corpus callosum, subventricular zone and external globus pallidus, whereas the upregulation was mainly restricted to activated microglia and reactive astrocytes. Continuous administration of the cathepsin X inhibitor AMS36 indicated protective effects against LPS-induced striatal degeneration, as seen by the attenuated LPS-mediated dilation of the lateral ventricles and partial decreased extent of striatal lesion. Taken together, our results indicate that cathepsin X plays a role as a pathogenic factor in neuroinflammation-induced neurodegeneration and represents a potential therapeutic target for neurodegenerative diseases associated with neuroinflammation.

Prolonged treatment with donepezil increases acetylcholinesterase expression in the central nervous system.

OBJECTIVE: Acetylcholinesterase (AChE), an enzyme catalysing rapid hydrolysis of acetylcholine is the major enzyme in the metabolism of this neurotransmitter in the central nervous system and in the skeletal and smooth muscles. Donepezil is a reversible, primarily non-competitive, selective inhibitor of AChE used in patients with Alzheimer's disease for the improvement of cognitive deficits. We hypothesized that prolonged treatment with donepezil could increase AChE mRNA levels and AChE activity in the central nervous system. METHODS: The levels of AChE mRNA and AChE activity in the brain sections of 6 animals treated for 28 days with donepezil (2 mg/kg s.c.) were visualized by an autoradiographic method of in situ hybridization and by Koelle histochemical staining, respectively, and compared with 6 control animals treated with physiologic saline. The images of autoradiograms and of AChE-stained brain sections were densitometrically analysed with a computerized imaging analysis system. RESULTS: We observed that 28-day treatment with donepezil in comparison to control treatment increased hippocampal AChE mRNA levels and AChE activity. CONCLUSIONS: Our data suggest that AChE up-regulation induced by prolonged treatment with AChE inhibitors may be the rationale for up-titration of AChE inhibitors during the treatment of AD. Further preclinical and clinical data are needed to evaluate the relative impact of the up-regulation of AChE activity on the outcome of prolonged treatment of AD patients with donepezil.

Upregulation of Cysteine Protease Cathepsin X in the 6-Hydroxydopamine Model of Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of midbrain dopaminergic neurons in the substantia nigra pars compacta (SNc). In vitro, a contribution to neuroinflammation and neurotoxicity has been shown for the lysosomal protease cathepsin X; however, its expression and its role in PD remain unknown. Therefore, the current study was designed to address the regional, cellular, and subcellular localization and activity of cathepsin X in hemi-parkinsonian rats with 6-hydroxydopamine (6-OHDA)-induced excitotoxicity in the unilateral medial forebrain bundle (MFB) lesion. We report for the first time that cathepsin X expression and activity are rapidly increased in the ipsilateral SNc after injection of 6-OHDA into the MFB reaching a maximum after 12 h but seem to stay strongly upregulated after 4 weeks after injection. At early time points of 6-OHDA injection into the MFB, the increased cathepsin X is localized in the lysosomes in the neuronal, predominantly tyrosine hydroxylase-positive dopaminergic cells. After 12 h of 6-OHDA induced lesion, only a few activated microglial cells are positive for cathepsin X whereas, in 4 weeks post-lesion accompanied with complete loss of dopaminergic neurons, there is persistent cathepsin X upregulation restricted to activated glia cells. Taken together, our results demonstrate that cathepsin X upregulation in the lesioned dopaminergic system may play a role as a pathogenic factor in PD. Moreover, inhibition of cathepsin X expression or activity may be useful in protecting the nigrostriatal dopaminergic projection in the PD.

The Magic of Crystal Structure-Based Inhibitor Optimization: Development of a Butyrylcholinesterase Inhibitor with Picomolar Affinity and in Vivo Activity.

The enzymatic activity of butyrylcholinesterase (BChE) in the brain increases with the progression of Alzheimer's disease, thus classifying BChE as a promising drug target in advanced Alzheimer's disease. We used structure-based drug discovery approaches to develop potent, selective, and reversible human BChE inhibitors. The most potent, compound 3, had a picomolar inhibition constant versus BChE due to strong cation-π interactions, as revealed by the solved crystal structure of its complex with human BChE. Additionally, compound 3 inhibits BChE ex vivo and is noncytotoxic. In vitro pharmacokinetic experiments show that compound 3 is highly protein bound, highly permeable, and metabolically stable. Finally, compound 3 crosses the blood-brain barrier, and it improves memory, cognitive functions, and learning abilities of mice in a scopolamine model of dementia. Compound 3 is thus a promising advanced lead compound for the development of drugs for alleviating symptoms of cholinergic hypofunction in patients with advanced Alzheimer's disease.

Upregulation of synaptotagmin IV protein in kainate-induced seizures.

Synaptotagmin IV is a product of immediate early-response gene. It is involved in the regulated neurosecretion in the brain. Its putative role, however, in vesicular transport and localization in secretor y vesicles is still a matter of debate. Here we followed the spatiotemporal pattern of synaptotagmin IV protein upregulation in the hippocampus, caudate putamen, nucleus accumbens, nucleus amygdalae, piriform and entorhinal cortices of rats with kainate-induced seizures. We found that upregulation pattern paralleled the direction of depolarization through the hippocampus and also reflecting seizure activity spreading to other brain regions. We speculate that synaptotagmin IV may have a role in the vesicular transport of the upregulated peptides and proteins involved in the plasticity and/or neurodegeneration provoked by the kainate.

Effect of apomorphine on striatal synaptotagmin 7 mRNA levels in reserpinized rats.

Synaptotagmin 7 (Syt 7) is a Ca2+ sensor implicated in the regulation of membrane fusion in vesicular transport, but its precise role in neurons is still a matter of controversy. Dopaminergic drugs have been shown to modulate its expression in the striatum. Here we investigate whether dopamine receptor agonist-up-regulation of Syt 7 mRNA is specifically involved in the pathophysiological adaptations of hypersensitive striatum by analyzing other dopaminergic neurons containing brain regions. We treated rats with systemic reserpine injections that rapidly depletes dopamine throughout the brain, but leaves dopaminergic neurons spared from destruction. We analyzed the effects of apomorphine, a D1 and D2 receptor agonist on Syt 7 mRNA expression in caudate putamen, nucleus accumbens, cingulate cortex, substantia nigra compacta, ventral tegmental area and hippocampus. The treatment with reserpine resulted in akinesia, catalepsy and rigidity and up-regulation of proenkephalin and down-regulation of preprotachykinin mRNA in caudate putamen, indicating a severe depletion. By acute treatment with apomorphine proenkephalin mRNA was down-regulated and preprotachykinin mRNA up-regulated in the caudate putamen of reserpinized rats. Apomorphine increased Syt 7 mRNA levels only in striatum (caudate putamen and nucleus accumbens) of reserpinized rats, while in other brain regions it did not have such effect. The reserpinization and/or apomorphine treatment had no effect on Syt 1 mRNA expression in caudate putamen. It may be concluded, that in the striatum depleted of biogene amines, such as occurs after reserpine treatment, the up-regulation of Syt 7 could play a specific role as part of hypersensitive response to dopaminergic agonists.

Synaptotagmin 7 and SYNCRIP proteins are ubiquitously expressed in the rat brain and co-localize in Purkinje neurons.

Synaptotagmin 7 (SYT7) is ubiquitously expressed calcium sensor, involved in neuronal membrane trafficking. Immunoprecipitation experiments demonstrated that SYT7 interacts with Synaptotagmin-binding, cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a component of mRNA granules, which are transported to dendrites and are prerequisite for synaptic plasticity. Given the potential significance of SYT7 regulation in processes of neurodegeneration, which are characterized by high level of synaptic vulnerability, we aimed to analyse and compare the distribution of SYT7 and SYNCRIP proteins in the adult rat striatum, hippocampus, cerebral and cerebellar cortex. We investigated the degree of SYT7-SYNCRIP co-localization in order to examine possible functional interaction of these two proteins. We found that SYT7 is abundantly distributed in neuropil of all examined anatomical areas of the brain, most prominently in axons. On the contrary, SYNCRIP had cytoplasmic somatodendritic pattern of expression, which was most prominent in the hippocampus and cerebellum. In the striatum, hippocampus and cerebral cortex SYT7 and SYNCRIP immunofluorescent signals were mutually excluded, thus diminishing the probability for their physiological interaction. In somata of Purkinje neurons in the cerebellar cortex, both SYT7 and SYNCRIP were expressed and partially co-localized suggesting possible functional connection between SYT7 and SYNCRIP proteins in Purkinje neurons.

Modelling FUS Mislocalisation in an In Vitro Model of Innervated Human Muscle.

Degeneration of distal axons and neuromuscular junctions is an early feature in the pathology of amyotrophic lateral sclerosis (ALS), which culminates in motor neuron loss due to axon retraction and muscle atrophy. The complex interactions in the pathogenesis of ALS between motor neurons, muscle cells and accompanying glia require an appropriate experimental model. Here, we have defined a co-culture model based on human myotubes innervated by neurons from embryonic rat spinal cord explants to investigate the pathology and treatment of ALS. This model was first characterised for endogenous expression and distribution of ALS-related proteins TDP-43 and FUS. Then, wild-type FUS and its mutants were introduced into these co-cultures to determine how FUS defects in nuclear transport modulate the pathological conditions. FUS-bearing plasmids were introduced by classical transfection and electroporation, as novel approaches to deliver plasmids into explants, and their cellular distributions were characterised. Endogenous nuclear expression of TDP-43 and FUS was observed in explants and myoblasts/myotubes. After transfection, wild-type FUS was expressed in nuclei of myoblasts, myotubes and explants, although with low transfection rates. Following successful electrotransfection into explants, the localisation of wild-type FUS was nuclear, and it was detected in neurons, astrocytes, Schwann cells and oligodendrocyte precursors, whereas the FUS∆Y, FUSY526A and FUSY526E mutants were cytoplasmic, and the FUSY526F mutant was nuclear and cytoplasmic. This co-culture model is applicable to the study of neuronal and non-neuronal cell contributions to ALS and other neurodegenerative diseases, and it can be used to investigate drug targets amenable to intervention.