Merari F. R. Ferrari

Protein aggregation containing beta-amyloid, alpha-synuclein and hyperphosphorylated tau in cultured cells of hippocampus, substantia nigra and locus coeruleus after rotenone exposure

BackgroundProtein aggregates containing alpha-synuclein, beta-amyloid and hyperphosphorylated tau are commonly found during neurodegenerative processes which is often accompanied by the impairment of mitochondrial complex I respiratory chain and dysfunction of cellular systems of protein degradation. In view of this, we aimed to develop an in vitro model to study protein aggregation associated to neurodegenerative diseases using cultured cells from hippocampus, locus coeruleus and substantia nigra of newborn Lewis rats exposed to 0.5, 1, 10 and 25 nM of rotenone, which is an agricultural pesticide, for 48 hours.ResultsWe demonstrated that the proportion of cells in culture is approximately the same as found in the brain nuclei they were extracted from. Rotenone at 0.5 nM was able to induce alpha-synuclein and beta amyloid aggregation, as well as increased hyperphosphorylation of tau, although high concentrations of this pesticide (over 1 nM) lead cells to death before protein aggregation. We also demonstrated that the 14kDa isoform of alpha-synuclein is not present in newborn Lewis rats.ConclusionRotenone exposure may lead to constitutive protein aggregation in vitro, which may be of relevance to study the mechanisms involved in idiopathic neurodegeneration.

Change in the expression of NPY receptor subtypes Y1 and Y2 in central and peripheral neurons related to the control of blood pressure in rats following experimental hypertension

Neuropeptide Y (NPY) is known to participate in central mechanisms of blood pressure control. However, variations on the expression of its receptors in response to a hypertensive challenge are not well defined, specially when considering that Y1 and Y2 often mediate opposite responses. In this study we have employed in situ hybridization to analyze changes in mRNA expression of NPY receptor subtypes Y1 and Y2 in the nucleus tractus solitarii (NTS), paraventricular nucleus of the hypothalamus (PVN) and petrosal and nodose ganglions 2 h, 3 and 7 days after aortic coarctation induced hypertension. Quantification by image analysis showed significant differences between sham-operated and aortic-coarcted hypertensive rats. Y1 receptor mRNA expression was increased (39%) in petrosal ganglion, 3 days after surgery. Y2 receptor mRNA expression was increased (143%) in the NTS of hypertensive compared with sham rats 2 h after surgery. Y2 receptor mRNA was decreased (62%) in the nodose ganglion of hypertensive compared with sham rats 2 h after surgery. No change was seen in Y1 and Y2 mRNA expression in the PVN in any analyzed period. The data suggest that NPY Y1 and Y2 receptors might participate in the mechanisms involved in the establishment/maintenance of hypertension induced by aortic coarctation. Acute changes seem to be involved with the adaptation to the new hypertensive state.

Pericytes Extend Survival of ALS SOD1 Mice and Induce the Expression of Antioxidant Enzymes in the Murine Model and in IPSCs Derived Neuronal Cells from an ALS Patient

AbstractAmyotrophic Lateral Sclerosis (ALS) is one of the most common adult-onset motor neuron disease causing a progressive, rapid and irreversible degeneration of motor neurons in the cortex, brain stem and spinal cord. No effective treatment is available and cell therapy clinical trials are currently being tested in ALS affected patients. It is well known that in ALS patients, approximately 50% of pericytes from the spinal cord barrier are lost. In the central nervous system, pericytes act in the formation and maintenance of the blood-brain barrier, a natural defense that slows the progression of symptoms in neurodegenerative diseases. Here we evaluated, for the first time, the therapeutic effect of human pericytes in vivo in SOD1 mice and in vitro in motor neurons and other neuronal cells derived from one ALS patient. Pericytes and mesenchymal stromal cells (MSCs) were derived from the same adipose tissue sample and were administered to SOD1 mice intraperitoneally. The effect of the two treatments was compared. Treatment with pericytes extended significantly animals survival in SOD1 males, but not in females that usually have a milder phenotype with higher survival rates. No significant differences were observed in the survival of mice treated with MSCs. Gene expression analysis in brain and spinal cord of end-stage animals showed that treatment with pericytes can stimulate the host antioxidant system. Additionally, pericytes induced the expression of SOD1 and CAT in motor neurons and other neuronal cells derived from one ALS patient carrying a mutation in FUS. Overall, treatment with pericytes was more effective than treatment with MSCs. Our results encourage further investigations and suggest that pericytes may be a good option for ALS treatment in the future. Graphical Abstractᅟ

Chronic nicotine administration. Analysis of the development of hypertension and glutamatergic neurotransmission.

Among numerous neurotransmitters involved in central cardiovascular control, glutamate is one of the most studied transmitters that are related to nicotine considering its release and its postsynaptic regulation. However, there are no conclusive studies about nicotine effects on glutamatergic system and its relevance on hypertension development, which can help to understand the role of these two systems in that pathology. In this context, the objective of the present study is to evaluate the effects of systemic chronic nicotine exposure on hypertension development as well as the interaction between nicotine and the glutamatergic system in normotensive and neurogenic hypertensive rats. By means of high performance liquid chromatograph, immunohistochemistry, in situ hybridization and binding techniques, glutamatergic system was evaluated in SHR and Wistar Kyoto (WKY) rats treated with nicotine, delivered subcutaneously through nicotine pellets, for 8 weeks. The most important findings in this study were that (1) moderate doses of nicotine accelerated the onset and increased blood pressure in SHR but not in WKY rats, (2) the nicotine dosage and time of treatment employed did not affect body weight, (3) chronic nicotine treatment differentially affected glutamatergic system in normotensive and hypertensive rats, and (4) spontaneously hypertensive rats seem to be more sensitive to peripherally administered nicotine than Wistar Kyoto rats considering blood pressure and glutamatergic neurotransmission changes. In conclusion, we have demonstrated that a moderate dose of nicotine accelerates the onset and exacerbates hypertension in the SHR and that might be, at least in part, related to the modulation of glutamatergic neurotransmission.

Quantitative autoradiography of adrenergic, neuropeptide Y and angiotensin II receptors in the nucleus tractus solitarii and hypothalamus of rats with experimental hypertension

Catecholamines, neuropeptide Y (NPY) and angiotensin II (Ang II) are known to participate in the central control of blood pressure. However, the modulation of these neurotransmitter receptors in response to a hypertensive stimulus is not appropriately established. The purpose of the present study was to examine binding parameters of alpha(2)-adrenergic, NPY and Ang II receptors in the nucleus tractus solitarii (NTS) and paraventricular hypothalamic nucleus (PVN) following a hypertensive stimulus in the aortic-coarcted rat by means of quantitative receptor autoradiography. No changes were seen in binding parameters of alpha(2)-adrenergic and NPY receptors in the NTS of the hypertensive rat compared to control. However, an increased affinity (54%) of noradrenaline competing for 3H-PAC was seen in the PVN. Moreover, an increased binding (49%) of 125I-PYY was also observed in the PVN. The affinity of Ang II for 125I-Sar(1)Ile(8)-Ang II binding sites was also increased (57%) in the NTS of the hypertensive rat. No changes in the binding parameters of radioactive Ang II were observed in the PVN. The results suggest that systems involved with hypertension like Ang II in the NTS and catecholamines in the PVN might collaborate in the development/maintenance of high blood pressure in the aortic-coarcted rat.

Plasticity of Opioid Receptors in the Female Periaqueductal Gray: Multiparity-Induced Increase in the Activity of Genes Encoding for Mu and Kappa Receptors and a Post-Translational Decrease in Delta Receptor Expression

The periaqueductal gray (PAG) has been reported as a potential site for opioid regulation of behavioral selection. Opioid-mediated behavioral and physiological responses differ between nulliparous and multiparous females. This study addresses the effects of multiple reproductive experiences on μ-, κ- and δ-opioid receptor (Oprm1, Oprk1, and Oprd1 respectively) gene activity and μ, κ and δ protein expression (MOR, KOR and DOR respectively) in the PAG of the female rats. This was done by evaluating the opioid gene expression using real-time (RT-PCR) and quantification of each protein receptor by Western blot analysis. The RT-PCR results show that multiple reproductive experiences increase Oprm1 and Oprk1 gene expression. Western blot analysis revealed increased MOR and KOR while DOR protein was decreased in multiparous animals. Taken together, these data suggest that multiple reproductive experiences influence both gene activity and opioid receptor expression in the PAG. Post-translational mechanisms seem particularly relevant for DOR expression. Thus, opioid transmission in the PAG might be modulated by different mechanisms of multiparity-induced plasticity according to the opioid receptor type.

Modulation of Tyrosine Hydroxylase, Neuropeptide Y, Glutamate, and Substance P in Ganglia and Brain Areas Involved in Cardiovascular Control after Chronic Exposure to Nicotine

Considering that nicotine instantly interacts with central and peripheral nervous systems promoting cardiovascular effects after tobacco smoking, we evaluated the modulation of glutamate, tyrosine hydroxylase (TH), neuropeptide Y (NPY), and substance P (SP) in nodose/petrosal and superior cervical ganglia, as well as TH and NPY in nucleus tractus solitarii (NTS) and hypothalamic paraventricular nucleus (PVN) of normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) after 8 weeks of nicotine exposure. Immunohistochemical and in situ hybridization data demonstrated increased expression of TH in brain and ganglia related to blood pressure control, preferentially in SHR, after nicotine exposure. The alkaloid also increased NPY immunoreactivity in ganglia, NTS, and PVN of SHR, in spite of decreasing its receptor (NPY1R) binding in NTS of both strains. Nicotine increased SP and glutamate in ganglia. In summary, nicotine positively modulated the studied variables in ganglia while its central effects were mainly constrained to SHR.

Decreases in the expression of CGRP and galanin mRNA in central and peripheral neurons related to the control of blood pressure following experimental hypertension in rats

Calcitonin gene-related peptide (alpha CGRP) and galanin (GAL) are peptides known to participate in central mechanisms of blood pressure control. Nonetheless, variations in the synthesis of the peptides in response to a hypertensive challenge are not well described, specially using a model, which allows acute and chronic analyses. In this study, we have employed in situ hybridization to analyse changes in mRNA expression of alpha CGRP and GAL in the nucleus tractus solitarii (NTS), hypothalamic paraventricular nucleus (PVN) as well as petrosal and nodose ganglia after aortic coarctation-induced hypertension in rats. Acute (2h) and chronic (3 and 7 days) analyses were performed in order to evaluate the involvement of both peptides in different periods of hypertension. The analysis of relative mRNA levels showed significant differences between sham-operated and aortic coarcted hypertensive rats. alpha CGRP mRNA expression was decreased 2h (40%) and 3 days (42%) in nodose and petrosal ganglia, respectively, after coarctation. No changes in CGRP mRNA signal were seen in the NTS and PVN in the analysed periods. GAL mRNA expression was decreased in the NTS (19%) and PVN (55%), 3 and 7 days, respectively, after coarctation-induced hypertension. No changes in GAL mRNA expression were observed in petrosal and nodose ganglia following aortic coarctation. Data suggest that alpha CGRP and GAL may participate in the mechanisms involved in the establishment/maintenance of hypertension induced by aortic coarctation. Acute changes might be involved with the adaptation to the hypertensive state, while changes at the chronic phase might be related to counteraction of hypertension.

Rotenone-Dependent Changes of Anterograde Motor Protein Expression and Mitochondrial Mobility in Brain Areas Related to Neurodegenerative Diseases

The presence of protein aggregates is common in neurodegenerative disorders; however, the real cause and effect of these aggregates during neurodegeneration is still a matter of investigation. We hypothesize that impairment of intracellular traffic may appear in the absence of protein inclusions and might trigger protein aggregation. In the present study, we aimed to evaluate mitochondria mobility as well as protein and messenger RNA expression of KIF1B and KIF5 that are molecular motors for neuronal anterograde traffic, in hippocampus, substantia nigra, and locus coeruleus of 10-month-old Lewis rats and cultured cells, from these same areas, following exposure to low doses of rotenone that do not lead to protein inclusions. The present study showed alteration in KIF1B and KIF5 expression, as well as in mitochondria mobility prior to protein aggregation involved in neurodegenerative disorders. These findings suggest that change in intracellular trafficking might be critical and one of the primary events for impairment of cell physiology during neurodegeneration associated with protein inclusions.

Alpha2-adrenergic receptor distribution and density within the nucleus tractus solitarii of normotensive and hypertensive rats during development☆ , ☆☆

The nucleus tractus solitarii (NTS), located in the brainstem, is one of the main nuclei responsible for integrating different signals in order to originate a specific and orchestrated autonomic response. Antihypertensive drugs are well known to stimulate alpha(2)-adrenoceptor (alpha(2R)) in brainstem cardiovascular regions to induce reduction in blood pressure. Because alpha(2R) impairment is present in several models of hypertension, the aim of the present study was to investigate the distribution and density of alpha(2R) binding within the NTS of Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats during development (1,15,30 and 90 day-old) by an in vitro autoradiographical study. The NTS shows heterogeneous distribution of alpha(2R) in dorsomedial/dorsolateral, subpostremal and medial/intermediate subnuclei. Alpha(2R) increased from rostral to caudal dorsomedial/dorsolateral subnuclei in 30 and 90 day-old SHR but not in WKY. Alpha(2R) decreased from rostral to caudal subpostremal subnucleus in 15, 30 and 90 day-old SHR but not in WKY. Medial/intermediate subnuclei did not show any changes in alpha(2R) according to NTS levels. Furthermore, alpha(2R) are decreased in SHR as compared with WKY in all NTS subnuclei and in different ages. Surprisingly, alpha(2R) impairment was also found in pre-hypertensive stages, specifically in subpostremal subnucleus of 15 day-old rats. Finally, alpha(2R) decrease from 1 to 90 day-old rats in all subnuclei analyzed. This decrease is different between strains in rostral dorsomedial/dorsolateral and caudal subpostremal subnuclei within the NTS. In summary, our results highlight the importance of alpha(2R) distribution within the NTS regarding the neural control of blood pressure and the development of hypertension.