Anna Maria Giuffrida-Stella

A Reduced Number of Metabotropic Glutamate Subtype 5 Receptors Are Associated with Constitutive Homer Proteins in a Mouse Model of Fragile X Syndrome

Fragile X (FRAX) syndrome is a common inherited form of mental retardation resulting from the lack of fragile X mental retardation protein (FMRP) expression. The consequences of FMRP absence in the mechanism underlying mental retardation are unknown. Here, we tested the hypothesis that glutamate receptor (GluR) expression might be altered in FRAX syndrome. Initial in situ hybridization and Western blotting experiments did not reveal differences in mRNA levels and protein expression of AMPA and NMDA subunits and metabotropic glutamate subtype 5 (mGlu5) receptors between control and Fmr1 knock-out (KO) mice during postnatal development. However, a detergent treatment (1% Triton X-100) revealed a selective reduction of mGlu5 receptor expression in the detergent-insoluble fraction of synaptic plasma membranes (SPMs) from KO mice, with no difference in the expression of NR2A, GluR1, GluR2/3, GluR4, and Homer proteins. mGlu5 receptor expression was also lower in Homer immunoprecipitates from Fmr1 KO SPMs. Homer, but not NR2A, mGlu5, and GluR1, was found to be less tyrosine phosphorylated in Fmr1 KO than control mice. Our data indicate that, in FRAX syndrome, a reduced number of mGlu5 receptors are tightly linked to the constituents of postsynaptic density and, in particular, to the constitutive forms of Homer proteins, with possible consequent alterations in synaptic plasticity.

OLIGODENDROGLIAL SURVIVAL FACTORS, PDGF-AA AND CNTF, ACTIVATE SIMILAR JAK/STAT SIGNALING PATHWAYS

Platelet‐derived growth factor (PDGF) and ciliary neurotrophic factor (CNTF) have pleiotropic actions on many cell types. In the presence of these factors, oligodendroglia respond by enhanced survival when deprived of trophic factors or in the presence of the cytotoxic cytokine, tumor necrosis factor‐α (TNF‐α). To determine whether these two oligodendroglial survival factors converge in their signaling cascades, we examined their JAK/STAT pathways in enriched oligodendrocyte (OL) progenitors and in the progenitor OL cell line, central glia‐4 (CG‐4). Cytokine pathways such as JAK/STAT have been characterized extensively in hematopoietic cells; however, it is increasingly evident that the same cytokines that play a role in hematopoiesis also play a role during development and injury of the central nervous system. This is the first study that clearly defines the presence and activation of JAK/STAT proteins in OL progenitors and compares the signal transduction pathway of two well‐known oligodendroglial survival factors. In this study, we report that PDGF‐ and CNTF‐induced OL progenitors responded with a rapid tyrosine phosphorylation of JAK1, JAK2, STAT1α/β, and STAT3. We feel that these identified JAK/STAT signaling molecules play a large role in the cellular response to these factors. Because both PDGF and CNTF enhance OL progenitor survival, these JAK/STATs may play a role in regulating this important cellular process. J. Neurosci. Res. 54:191–205, 1998.

Erratic expression of DNA polymerases by β-amyloid causes neuronal death

An ectopic reentrance into the cell cycle with ensuing DNA replication is required for neuronal apoptosis induced by β‐amyloid. Here, we investigate the repertoire of DNA polymerases expressed in β‐amyloid‐treated neurons, and their specific role in DNA synthesis and apoptosis. We show that exposure of cultured cortical neurons to β‐amyloid induces the expression of DNA polymerase‐β, proliferating cell nuclear antigen, and the p49 and p58 subunits of DNA primase. Induction requires the activity of cyclin‐dependent kinases. The knockdown of the p49 primase subunit prevents β‐amyloid‐induced neuronal DNA synthesis and apoptosis. Similar effects are observed by knocking down DNA polymerase‐β or by using dideoxycytidine, a preferential inhibitor of this enzyme. Thus, the reparative enzyme DNA polymerase‐β unexpectedly mediates a large component of de novo DNA synthesis and apoptotic death in neurons exposed to βamyloid. These data indicate that DNA polymerases become death signals when erratically expressed by differentiated neurons.

Differential Expression of Estrogen Receptors Alpha and Beta in the Spinal Cord during Postnatal Development: Localization in Glial Cells

Estrogens are recognized as neuroprotective and neurotrophic agents in the central nervous system. They are involved in neuronal differentiation and survival and promote neural development. Estrogen receptors alpha (ER-α) and beta (ER-β) are predominantly expressed in neurons, whereas their presence in glial cells in vivo is more controversial. Changes in their expression during development have been described in different brain areas, but little is known about their presence in the spinal cord. We have carried out an immunohistochemical study in an attempt to analyze the expression of both ERs in astrocytes and oligodendrocytes of the rat spinal cord and their modifications during postnatal development. RT-PCR analysis of whole spinal cord extracts from 4-, 12-, and 25-day-old and adult rats indicated changes in the expression of both receptors during maturation. Immunohistochemistry of slices of the lumbar tract revealed that in an area of the ventral spinal cord that does not contain neuronal cell bodies, but mainly fibers and glial cells, both ER-α and ER-β can be detected. Immunostaining is clearly nuclear, and, in the case of ER-α, both markedly positive and weakly labeled cells can be identified. ER-α is expressed during early development to progressively decline in the adult stage. In contrast, the ER-β signal is low and peaks at postnatal day 25, whereas it is almost undetectable at other ages. Colocalization studies revealed that, at postnatal day 25, ER-α and ER-β are expressed in astrocytes (identified by the specific marker glial fibrillar acidic protein) and oligodendrocytes (labeled by antimyelin 2′,3′-cyclic nucleotide 3′-phosphodiesterase). The present results confirm the expression of ER-α and ER-β in glial cells in vivo and suggest that, also in the spinal cord, glial cells may contribute to the effects of estrogen during development.

Glutamate Receptor-Driven Activation of Transcription Factors in Primary Neuronal Cultures

We have used primary neuronal cultures prepared from fetal cerebral hemispheres to investigate the effects of different glutamate receptor agonists and antagonists on the expression of transcription factor encoding genes, such as c-fos, fosB, c-jun, junB, junD, c-myc, and zif/268. The addition of glutamate (100 μM) to the culture medium rapidly activated c-fos, fosB, c-jun, junB and zif/268 gene expression, reaching the maximal level at 30–60 minutes for zif/268 and at 60 minutes for the other genes. The onset of fosB mRNA accumulation was slightly delayed in comparison to the other genes. No clear induction was found for junD and c-myc. Different glutamate receptor agonists, such as NMDA, kainate, quisqualate, trans-(±)-1-aminocyclopentane-1,3-dicarboxylic acid (t-ACPD) and α-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) were able to increase c-fos, c-jun, and zif-268 mRNA levels with rapid and transient kinetics similar to those observed after glutamate treatment. Similar results were obtained for junB and fosB after kainate and quisqualate stimulation. Pretreatment with MK-801, a non competitive NMDA antagonist, produced an almost complete inhibition of glutamate-driven expression of transcription factor genes, thus suggesting that NMDA receptor plays a major role in glutamate induced-gene expression. On the contrary the kainate/AMPA receptor antagonist, DNQX, did not influence glutamate induced-gene expression. Under the conditions used in the present study, NMDA was effective in inducing the simultaneous activation of several IEGs even when added to the culture medium containing millimolar concentration of magnesium. When experiments were performed in Krebs solution, NMDA was effective in stimulating zif/268 and c-fos mRNAs only in the absence of Mg2+, while glutamate activated c-fos and zif/268 both in the presence and absence of magnesium ions. As expected, NMDA effect was fully inhibited by MK-801. The level of AP-1 DNA binding activity, as measured by electrophoretic mobility shift assay, increased after addition of glutamate and NMDA to cultured neurons and such increase was antagonized by the pretreatment with MK-801.

Upregulation of neuronal nitric oxide synthase in in vitro stellate astrocytes and in vivo reactive astrocytes after electrically induced status epilepticus.

Neuronal nitric oxide synthase (nNOS) is a constitutively expressed and calcium-dependent enzyme. Despite predominantly expressed in neurons, nNOS has been also found in astrocytes, although at lower expression levels. We have studied the regulation of nNOS expression in cultured rat astrocytes from cortex and spinal cord by Western blotting and immunocytochemistry. nNOS was not detectable in cultured astrocytes grown in serum-containing medium (SCM), but was highly expressed after serum deprivation. Accordingly, calcium-dependent NOS activity and both intracellular nitrite levels and nitrotyrosine immunoreactivity after glutamate stimulation were higher in serum-deprived astrocytes than in cells grown in SCM. Serum deprivation induced a modification of astrocytes morphology, from flat to stellate. nNOS upregulation was also observed in reactive astrocytes of rat hippocampi after electrically induced status epilepticus, as demonstrated by double-labeling experiments. Thus, nNOS upregulation occurs in both in vitro stellate and in vivo reactive astrocytes, suggesting a possible involvement of glial nNOS in neurological diseases characterized by reactive gliosis.

A prolonged pharmacological blockade of type-5 metabotropic glutamate receptors protects cultured spinal cord motor neurons against excitotoxic death.

The causes of amyotrophic lateral sclerosis (ALS) are mostly undefined; however, excitotoxic injury and astrogliosis may contribute to motor neuron (MN) degeneration. Group I metabotropic glutamate (mGlu) receptors are over-expressed in reactive astrocytes in ALS, but the functional significance of this over-expression is presently unknown. We examined the role of group I mGlu receptors on excitotoxic death of spinal cord MNs grown in cultures enriched of astrocytes bearing a reactive phenotype. A prolonged exposure to the selective non-competitive mGlu5 receptor antagonist MPEP reduced AMPA-mediated toxicity and cobalt uptake in MNs. Expression levels of the GluR1 (but not GluR2) AMPA receptor subunit and levels of brain-derived neurotrophic factor (BDNF) were reduced in mixed spinal cord cultures pretreated with MPEP. In addition, neuroprotection by MPEP was less than additive with that produced by a neutralizing anti-BDNF antibody and a treatment with exogenous BDNF masked the protective effect of MPEP, suggesting that mGlu5 receptors and BDNF converge in facilitating excitotoxic MN death. The protective effect of MPEP was absent in cultures with a reduced number of astrocytes. We suggest that blocking astrocytic mGlu5 receptors is a potential therapeutic strategy in ALS.

Regulation of Cytochrome c Oxidase and FoF1-ATPase Subunits Expression in Rat Brain During Aging

In the present study we analyzed the age-dependent changes of mRNA levels for cytochrome c oxidase and F0F1-ATP synthase subunits in rat cerebral cortex and cerebellum. To establish whether the regulation of expression is transcriptional or post-transcriptional, the results were compared to those related to protein subunits levels, of the same enzymatic complexes, previously observed. The different patterns of age-related changes of mRNA subunits, in particular the lower increments, compared with those related to protein subunits, indicate that post-transcriptional mechanisms of regulation might be involved in the coordinated expression of the various subunits of each complex. Northern blotting analyses of RNA from the cerebellum of rats at the various ages, showed also differences in age-dependent patterns of transcription between cerebral cortex and cerebellum. Moreover, the major age-dependent changes of mitochondrial-encoded subunits, compared with the nuclear-encoded ones, previously observed at proteins level, occur also during transcription.

Effect of CDP-choline treatment on mitochondrial and synaptosomal protein composition in different brain regions during aging

Several age‐dependent modifications of inner mitochondrial membrane and synaptosomal plasma membrane proteins from different brain regions of 4‐, 12‐, 18‐ and 24‐month‐old male Wistar rats, were observed. Some proteins, identified by immunoblotting assay as various subunits of mitochondrial respiratory chain complexes and calmodulin, were particularly impaired. Chronic treatment with CDP‐choline at a dose of 20 mg/kg body weight per day for 28 days caused significant changes in the amounts of several of the above mentioned proteins. Most of the proteins, which decreased during aging, showed a significant increase after CDP‐choline treatment compared with the corresponding control values at the same age. The effect of CDP‐choline might be due to: — the increased availability of cytidylic nucleotides, which in the brain are present in limited amounts compared to the other nucleotides; — the increased content of total adenine nucleotides; —the improvement of brain energy metabolism.

Effect of hypoxia on protein composition of synaptic plasma membranes from cerebral cortex during aging

The effect of hypoxia on the protein composition of synaptic plasma membranes (SPM) isolated from cerebral cortex of rats at 4, 12, and 24 months of age was investigated. The proteins were separated by SDS polyacrilamide gel electrophoresis and the percent content was evaluated by measuring the optical density of the stained gels. After hypoxic treatment various proteins showed significant changes. Some proteins were only affected at 4 and 12 months of age and not at 24 months. The various modified porteins may be identified according to their molecular weight, as follows: the 18 kDa protein with calmodulin; the 23 kDa protein with D3 subunits; the 28 kDa protein could contain the δ subunit of the Ca2+ channel. The changes in the amount of some SPM proteins during hypoxia is consistent with the alteration in membrane polarization and neurotransmission observed in this condition. The effect of aging at the synaptosomal level seems to be a selective process; after hypoxia the age-related changes of many proteins are more pronounced.