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

Up-regulation of Synaptotagmin IV within amyloid plaqueassociated 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 β-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

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

Distribution and role of metals in sclerotic hippocampi of patients with mesial temporal lobe epilepsy

The accumulating evidence on the relation between the disturbed metal homeostasis and epilepsy urges the need for data regarding the total metal concentrations, as well as metal distribution in the brain itself, in order to indicate where to direct the potential therapy, to metal supplementation or chelation. This paper summarizes our results on the measurements of some important essential metals in hippocampi of patients with mesial temporal lobe epilepsy (mTLE) who underwent amigdalohippocampectomy. The key findings point out that levels of copper and manganese are deficient in hippocampi of mTLE patients, and that their concentrations correlated positively with neuronal loss in affected regions of sclerotic hippocampus. In addition, the Cu concentration was decreased in the areas of total neuronal loss. Iron and zinc total hippocampal levels were neither accumulated nor deficient compared to control. Our results contribute to deeper insight of metals biology in the epilepsy and may represent the initial point of new and non-invasive therapy of drug resistant epilepsy

Relationship Between Regional Distributions of Cytochrome C Oxidase and Copper-Delivering Chaperones in Sclerotic Hippocampi of Epilepsy Patients

Aims: A drop in copper level and the loss of energy homeostasis are both portrayed in mesial temporal lobe epilepsy (mTLE) with hippocampal sclerosis (HS) patients. Cytochrome c oxidase (COX) represents a crossroad of energy and copper metabolism; it is a key component of mitochondrial machinery and contains two copper centers. Our aim here was to examine the link between COX activity and the copper transporting system in HS. COX activity and the levels of mRNA of selected chaperones - COX11, COX17, Sco1 and Sco2 were determined in 13 anatomically distinct hippocampal regions. Methods: Study was performed on seven hippocampal samples, four of which had been acquired during the course of amygdalohippocampectomy treatment of medically intractable epilepsy and three control postmortem samples. Adjacent slices were used for Nissl staining, COX activity assay and mRNA in situ hybridization with autoradiography. Densitometry was performed using ImageJ. Results: Overall COX activity was decreased in HS compared to controls (P = 0.0003). However, 5 regions showed significantly lower COX activity in HS and 8 did not. Subiculum showed slightly higher activity in HS. The levels of mRNA levels were lowered in HS in 6 regions for COX11, 10 regions for COX17, two regions for Sco1 and 11 regions for Sco2. Conclusions: Our findings suggest the loss of energy homeostasis in HS may be related to pathological changes in specific components of copper delivery to COX, and that the impact may vary between different hippocampal regions

The importance of copper in pathology of mesial temporal lobe epilepsy

More and more studies are identifying the regulation of metal homeostasis as one of the key points of central nervous system’s well-being. Epilepsy is a particularly interesting neurological condition when viewed in terms of the correlation between the amount of metals and the development of a seizure. This lecture will present contribution of our group to the field of metal biology in epilepsy by mapping brain metals in sclerotic hippocampus resected from drug resistant mesial temporal lobe epilepsy (mTLE) patients as surgical therapeutic approach. Direct insight into this epileptogenic area, by two powerful techniques, optical emission and mass spectrometry, has led us to investigation of copper turnover. Namely, among the examined metals, we found the deficiency of copper in sclerotic hippocampus on two levels: (i) in whole structure (ii) and locally in the areas of neuronal loss, with significant correlation between copper concentration and neuron density. Furthermore, analysis of copper metalloproteins showed: (i) significant increase or decrease in levels of protein that is participating in copper transport into the cell (CTR1) depending on the degree of hippocampal neuronal loss; (ii) and lower activity of an enzyme in which copper is part of the active site, cytochrome c oxidase, in sclerotic hippocampi of patients compared to control tissue. In our further investigations it remained to be determined whether changes in copper concentrations and copper metalloproteins are causal to pathology of mTLE or they represent epiphenomenon

Differential Patterns of Synaptotagmin7 mRNA Expression in Rats with Kainate- and Pilocarpine-Induced Seizures

Previous studies in rat models of neurodegenerative disorders have shown disregulation of striatal synaptotagmin7 mRNA. Here we explored the expression of synaptotagmin7 mRNA in the brains of rats with seizures triggered by the glutamatergic agonist kainate (10 mg/kg) or by the muscarinic agonist pilocarpine (30 mg/kg) in LiCl (3 mEq/kg) pre-treated (24 h) rats, in a time-course experiment (30 min - 1 day). After kainate-induced seizures, synaptotagmin7 mRNA levels were transiently and uniformly increased throughout the dorsal and ventral striatum (accumbens) at 8 and 12 h, but not at 24 h, followed at 24 h by somewhat variable upregulation within different parts of the cerebral cortex, amigdala and thalamic nuclei, the hippocampus and the lateral septum. By contrast, after LiCl/pilocarpine-induced seizures, there was a more prolonged increase of striatal Synaptotagmin7 mRNA levels (at 8, 12 and 24 h), but only in the ventromedial striatum, while in some other of the aforementioned brain regions there was a decline to below the basal levels. After systemic post-treatment with muscarinic antagonist scopolamine in a dose of 2 mg/kg the seizures were either extinguished or attenuated. In scopolamine post-treated animals with extinguished seizures the striatal synaptotagmin7 mRNA levels (at 12 h after the onset of seizures) were not different from the levels in control animals without seizures, while in rats with attenuated seizures, the upregulation closely resembled kainate seizures-like pattern of striatal upregulation. In the dose of 1 mg/kg, scopolamine did not significantly affect the progression of pilocarpine-induced seizures or pilocarpine seizures-like pattern of striatal upregulation of synaptotagmin7 mRNA. In control experiments, equivalent doses of scopolamine per se did not affect the expression of synaptotagmin7 mRNA. We conclude that here described differential time course and pattern of synaptotagmin7 mRNA expression imply regional differences of pathophysiological brain activation and plasticity in these two models of seizures

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-β-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

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. T$_1$-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 T$_1$-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 T$_1$-weighted MR-signal correlated with the increased volume density of heavily injured tubuli (R$^2$ = 0.77), with heavily damaged glomeruli (R$^2$ = 0.84) and with volume density of connective tissue (R$^2$ = 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. T$_1$-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