Proteins In Rat Cerebellum Research Articles

Antidepressant effects of aqueous extract of saffron and its effects on CREB, P-CREB, BDNF, and VGF proteins in rat cerebellum.

ObjectiveThe role of BDNF (brain-derived neurotrophic factor), CREB (cAMP response element binding) and VGF neuropeptide has been proved in antidepressant activity of long term saffron administration in the rat hippocampus. In this study we evaluated the role of these proteins in antidepressant activity of saffron in long term administration in the rat cerebellum.MethodsSaffron aqueous extract (40 and 80 mg/kg/day) and imipramine (10 mg/kg/day) were administered intraperitoneally for 21 days to rats. At the end of experiment, animals were sacrificed and cerebellums were separated. The protein levels of BDNF, VGF, CREB and P- CREB in the rat cerebellum were evaluated using western blot analysis.ResultsSaffron aqueous extract (80mg/kg/day) caused significant increase in protein level of P-CREB in long term treatment in the rat cerebellum. The increases in the protein levels of VGF, CREB and BDNF were not significant.ConclusionIn summary, our results showed that antidepressant effect of saffron in rat cerebellum might be due to the enhanced phosphorylation of CREB.

Native Presynaptic Metabotropic Glutamate Receptor 4 (mGluR4) Interacts with Exocytosis Proteins in Rat Cerebellum

The eight pre- or/and post-synaptic metabotropic glutamatergic receptors (mGluRs) modulate rapid excitatory transmission sustained by ionotropic receptors. They are classified in three families according to their percentage of sequence identity and their pharmacological properties. mGluR4 belongs to group III and is mainly localized presynaptically. Activation of group III mGluRs leads to depression of excitatory transmission, a process that is exclusively provided by mGluR4 at parallel fiber-Purkinje cell synapse in rodent cerebellum. This function relies at least partly on an inhibition of presynaptic calcium influx, which controls glutamate release. To improve the understanding of molecular mechanisms of the mGluR4 depressant effect, we decided to identify the proteins interacting with this receptor. Immunoprecipitations using anti-mGluR4 antibodies were performed with cerebellar extracts. 183 putative partners that co-immunoprecipitated with anti-mGluR4 antibodies were identified and classified according to their cellular functions. It appears that native mGluR4 interacts with several exocytosis proteins such as Munc18-1, synapsins, and syntaxin. In addition, native mGluR4 was retained on a Sepharose column covalently grafted with recombinant Munc18-1, and immunohistochemistry experiments showed that Munc18-1 and mGluR4 colocalized at plasma membrane in HEK293 cells, observations in favor of an interaction between the two proteins. Finally, affinity chromatography experiments using peptides corresponding to the cytoplasmic domains of mGluR4 confirmed the interaction observed between mGluR4 and a selection of exocytosis proteins, including Munc18-1. These results could give indications to explain how mGluR4 can modulate glutamate release at parallel fiber-Purkinje cell synapses in the cerebellum in addition to the inhibition of presynaptic calcium influx.

Comparative Proteomic Profiling of Membrane Proteins in Rat Cerebellum, Spinal Cord, and Sciatic Nerve

Proteomics is an increasingly powerful technology that can provide in-depth insights into entire proteomes and their variation upon disease. Large-scale proteomics today enables identification and measurement of changes of thousands of proteins from minute amount of tissues. Here, we provide a proteomic profile of three distinct parts of the murine nerve system: cerebellum, spinal cord, and sciatic nerve. We focus on membrane proteins as the key regulators of neural transmission and memory. Rat tissues were homogenized and extracted to remove nonmembrane proteins and the resulting membranes were solubilized with detergents. Proteins were fractionated by size exclusion chromatography, depleted for detergents, digested and analyzed by LC-MS/MS using the LTQ-Orbitrap instrument. With the application of stringent identification criteria, in total, 4124 proteins were identified. Of these proteins, 3528, 3290, and 1649 were mapped to cerebellum, spinal cord, and sciatic nerve, respectively, allowing in-depth mapping of neurotransmitter receptors, ion channels, and transporter proteins. This work is the most in-depth proteomic analysis of nerve tissues to date and provides the first unbiased insights into the proteomes of anatomically and functionally distinct parts of the membrane proteome of the central and peripheral nerve systems. The methods applied here can be directly applied to studying nerve systems and their disorders.

Cocaine induces the expression of homer 1b/c, homer 3a/b, and hsp 27 proteins in rat cerebellum

The long homer proteins 1b/c, 2a/b, and 3a/b play an important role in postsynaptic neurons by clustering glutamate receptors and by coupling the receptors with various intracellular effectors. Using immunohistochemistry and Western-blot analysis, this study shows that the expression of the long homer isoforms 1b/c and 3a/b was induced in rat cerebellum in response to cocaine administration. Acute treatment produced a very robust induction of both constitutive isoforms, whereas repeated treatment for 10 days induced a large expression of homer 1b/c and a more modest increase in the expression of the 3a/b isoform. The heat shock protein hsp 27 was also considerably induced in the cerebellum of cocaine-treated rats, suggesting that it participates in assisting the correct folding of proteins, and by counteracting oxidative stress mechanisms triggered by the psychostimulant. In addition of being expressed in Purkinje neurons, homer 3a/b and hsp 27, but not homer 1b/c, were localized within Bergmann glial cells and in their extensions, which surround Purkinje cells, as assessed by coimmunoreactivity with glial fibrillary acidic protein. Cocaine was also found to induce both proteins in the Bergmann glial cells. Since we found that homer 3a/b colocalized with the mGluR1 receptor in Purkinje cells, the data suggest that the long homer isoforms are involved in the cocaine-induced neuroplasticity that takes place in the cerebellum, by reshaping postsynaptic densities in Purkinje cell dendrites.

Cellular and sub-cellular localisation of GABA B1 and GABA B2 receptor proteins in the rat cerebellum

Following the recent discovery that GABA B receptors expressed in cell lines are only functional when both GABA B1 and GABA B2 are expressed, the present study reports on the development of polyclonal antisera specific for carboxyl-terminal portions of the two related GABA B receptor components respectively. Western blotting indicated the specificity of affinity-purified antibodies for native or recombinant expressed GABA BR1 and GABA BR2, with no cross-reactivity, both antisera detecting the heterodimer in rat cerebellar membranes. Immunohistochemistry revealed a distinct distribution of both receptor proteins in rat cerebellum. GABA B1 immunoreactivity was primarily located in the granule cell layer and Purkinje cells, with discrete immuno-positive cell bodies being present in the molecular layer. GABA B2 staining revealed intense immunoreactivity in the molecular layer, with weaker staining in the granule cell layer. Purkinje cell bodies were less intensely immuno-positive for GABA B2. Co-localisation of both receptor proteins was observed using double immunofluorescence techniques, consistent with the notion that both proteins are required for the formation of functional GABA B receptors in vivo. Immunofluorescence also indicated that GABA B receptors did not co-localise with glial fibrillary acid protein, confirming a neuronal localisation for GABA B receptors. Electron microscopic analysis of the molecular layer revealed that the distribution of immunolabelling for both GABA B1 and GABA B2 was mainly located on the membrane of Purkinje cell dendrites and spines and in parallel fibre terminals.

The Effect of Age and Testosterone on the Expression of Glial Fibrillary Acidic Protein in the Rat Cerebellum

Testosterone reversed the age-related increase in glial fibrillary acidic protein (GFAP) in the male rat cerebellum, a brain region not generally associated with gonadal steroid hormone sensitivity. This supports the hypothesis that a decrease in circulating testosterone contributes to age-related increase in GFAP. These data also suggest that reductions in circulating gonadal steroids during aging could render the brain more susceptible to neurodegeneration and that hormone replacement therapy might have value in neurodegenerative disease intervention.

Developmental expression of neurofilament and glial filament proteins in rat cerebellum.

Neurofilament protein (NFP) consists of three subunits: NF200, NF150 and NF68. Several studies on expression of NFP in developing brain have shown that NF200 appears later than NF150 and NF68. However, there are some reports on simultaneous appearance of these subunits in development. The present study is an attempt to resolve this controversy. Rat cerebellum was chosen as most of its development takes place during the first three weeks of postnatal period. Cytoskeletal and NFP preparations from newborn (P0), postnatal day 8 (P8), P15, P21, P30 and adult (3 months) rat cerebella were subjected to electrophoresis on 7.5% SDS-PAGE. All the NFP subunits were present from P0 onwards and there was an increase in NFP content and glial fibrillary acidic protein (GFAP) with age as revealed by the densitometric scanning. Immunoblots of NFP preparations confirmed the presence of NF200 in the early postnatal cerebellum. In vivo phosphorylation studies indicated the presence of phosphorylated NF subunits from P8 onwards, which was confirmed by staining in immunoblots by SMI31. Immunohistochemical studies on Bouin's fixed tissues revealed that in P0 cerebella, the deeper neurones (soma and processes) expressed all the NFP subunits while from P8 onwards they were negative for NF200. Similarly, Purkinje cells (soma) expressed transiently NF200 subunits on P8 and ceased to express them from P15 onwards. The white matter was immunopositive for NF200 and NF150 on P0 and the intensity of staining increased progressively. Astrocytes expressing GFAP were seen in cerebellar white matter from P8 onwards and the staining in radial glia could be detected from P15 onwards.(ABSTRACT TRUNCATED AT 250 WORDS)

Prominent location of a K + channel containing the α subunit K V 1.2 in the basket cell nerve terminals of rat cerebellum

A panel of monoclonal antibodies specific for a family of voltage-dependent, fast-activating K + channels, raised against α-dendrotoxin acceptors purified from bovine brain, were used to probe the distribution of these important proteins in rat cerebellum. All the antibodies reacted with their antigens in the folial white matter, the granular cell layer and the basket cell nerve termini within the Purkinje cell layer. However, a very intense staining pattern was exhibited by only one monoclonal that reacts exclusively with K v 1.2 α subunit, the predominant isoform present in α-dendrotoxin sensitive K + channels. Double-labelling procedures with neuronal and glial markers were used to verify this discrete antibody staining of the basket cell terminals that synapse with the base of Purkinje cell bodies in a readily recognizable and characteristic fashion. This is the first direct demonstration, using a monoclonal antibody, of a presynaptic location for a voltage-activated K + channel; its discrete distribution in the basket cell pinceau suggests that it could control release of the inhibitory transmitter GABA and, thereby, influence excitability of Purkinje cells in the cerebellum.

Distinct cellular expression of pertussis toxin-sensitive GTP-binding proteins in rat cerebellum

Immunohistochemical localization of pertussis toxin-sensitive GTP-binding proteins in rat cerebellum was investigated using antibodies raised against purified G αo and synthetic peptides corresponding to specific sequences of G αi1, G αi2 and G αi3. G αo was detected mostly in the molecular layer but not in the cell body of the Purkinje cells. G αi1 and G αi2 were found predominantly in the molecular layer and in addition in the cell body of the Purkinje cells. G αi3 was not detected in rat cerebellum. Immunoblot analysis demonstrated that in the cerebellum membrane G α0 was most abundant and surprisingly the amount of G αi2 was much higher than that of G αi1. Thus, each pertussis toxin substrate was found to be expressed differently in rat cerebellum. The different patterns of expression imply that each subtype of GTP-binding proteins may have a specific function in modulating the neuronal activity in rat cerebellum.

Brain calbindin D28k and an Mr 29,000 calcium binding protein in cerebellum are different but related proteins: evidence obtained from sequence analysis by tandem mass spectrometry

A calcium binding protein of Mr 29,000 which cross-reacts with antibodies raised against chick calbindin-D28k was previously reported to be present in rat cerebellum [Pochet, R., Parmentier, M., Lawson, D. E. M., & Pasteels, J. L. (1985) Brain Res. 345, 251-254]. It was suggested that the Mr 29,000 protein represents another form of calbindin-D28k. In our laboratory we were able to identify Mr 28,000 and 29,000 proteins in rat, human, and chick cerebellum by their ability to bind 45Ca in a 45Ca blot assay. Two calcium binding proteins of Mr 27,680 and 29,450 were isolated from rat cerebelli by the use of gel permeation chromatography and preparative gel electrophoresis. After reverse-phase high-performance liquid chromatography (HPLC) the proteins were sequenced. Sequence analysis by tandem mass spectrometry indicated only 52% identity between the rat cerebellar Mr 28,000 and 29,000 proteins. Thus they are not different forms of the same protein, as previously suggested. Eighty-nine percent identity was observed between the rate cerebellar Mr 29,000 protein and chick calretinin [Rogers, J. H. (1987) J. Cell Biol. 105, 1343-1353]. The difference in identity between the rat cerebellar Mr 29,000 protein and chick calretinin may be due to species differences, and thus this protein is most likely rat calretinin. However, barely detectable levels of mRNA and weak immunoreactivity have been reported for calretinin in cerebellum. Since we found the Mr 29,000 calcium binding protein to be an abundant protein in rat cerebellum, these results suggest either posttranscriptional regulation of calretinin in cerebellum or species differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes of mitochondrial membrane proteins in rat cerebellum during aging.

Qualitative and quantitative changes of mitochondrial membrane proteins during aging were investigated. Free (non-synaptic) mitochondria were purified from rat cerebellum at different ages (4, 8, 12, 16, 20, and 24 months). Mitochondrial outer membrane (OM), inner membrane (IM) and matrix (MX) were separated and the proteins were extracted and analyzed by gel-electrophoresis. After staining, the gels were scanned densitometrically to quantify the proteins. No significant changes in the quantity of OM or MX protein subunits were observed, while several statistically significant quantitative changes in IM proteins with age were found. These age-dependent modifications of inner membrane mitochondrial proteins may play an important role in energy transduction, transport systems and regulatory enzymatic activities in mitochondria.

Improved separation at low temperature of glycoproteins by Con A-Sepharose affinity chromatography in the presence of sodium dodecyl sulfate (SDS).

The Con A-Sepharose affinity chromatography of glycoproteins was even more effective at 4 degrees C than that at room temperature (26-28 degrees C) in the presence of sodium dodecyl sulfate (SDS). Application of this methodology to the separation of several glycoproteins from SDS-solubilized membrane proteins in rat cerebellum, including a glycoprotein characteristic of the Purkinje cells, was successful.

Effects of undernutrition and thyroid state on the ontogenetic changes of D1, D2, and D3 brain-specific proteins in rat cerebellum.

Disturbances in metabolic balance brought about by alterations in thyroid state and undernutrition during early life had a marked effect on the concentrations of the brain-specific proteins, D1, D2, and D3 in the developing rat cerebellum. In normal rats, the concentrations of D1 and D3 increased and that of D2 decreased during the first 3 weeks after birth. In the hyperthyroid state a small but consistent advancement was observed in the developmental curves of these proteins. The hypothyroid state caused a marked retardation in the maturational pattern of D1 and D2 but not of D3. In undernutrition, at 6 days the concentrations of D1 and D3 proteins were higher than in controls, but thereafter the developmental increase was markedly delayed for D1 only. The concentration of D2 was normal at 6 days, but after the first week a marked retardation was observed in the maturational pattern of this protein in undernourished rats. In addition, the "anodic-immature" form of D2 predominated in 6-day-old controls, but this was gradually replaced by a "cathodic-mature" form which progressively became the dominant form of D2 in 35-day-old rat cerebellum. The developmental switch in terms of the two forms was also advanced in hyperthyroidism and retarded in thyroid deficiency and undernutrition. Furthermore, daily treatment of hypothyroid rats with physiological doses of thyroxine from birth restored the concentrations of D1 and D2 to normal, but that of D3 was increased above control levels, indicating differences between the proteins in their sensitivity to mechanisms of control by thyroid hormone. Also, the overall effects of undernutrition were markedly different from those of hypothyroidism.

An ultrastructural immunocytochemical study of nerve-specific protein in rat cerebellum.

The ultrastructural localization of the neuron-specific enolase (14-3-2 protein) has been investigated in the cerebellum of the adult rat using the indirect antibody immunohistochemical method. The protein was found exclusively in neurons: perikaryal cytoplasm, axons and dendrites were labelled while nuclei were not. Reaction product was found to be attached to intracytoplasmic membranes, the surface membranes of mitochondria and microtubules in addition to its dispersion as a flocculent material throughout the cytoplasm. All classes of cerebellar neurons were found to be labelled though large variations in the level of labelling between different types of neuron were noted. Purkinje cells appeared to have a much lower cytoplasmic concentration of this protein than other neurons.