A. Caruso

Mitotic signaling by β-amyloid causes neuronal death

Aggregates of β‐amyloid peptide (βAP), the main constituent of amyloid plaques in Alzheimers brain, kill neurons by a not yet defined mechanism, leading to apoptotic death. Here, we report that both full‐length βAP(j_40) or (1–42) and its active fragment βAP(25_35) act as proliferative signals for differentiated cortical neurons, driving them into the cell cycle. The cycle followed some of the steps observed in proliferating cells, including induction of cyclin Di, phosphorylation of retinoblastoma, and induction of cyclin E and A, but did not progress beyond S phase. Inactivation of cyclin‐dependent protein kinase‐4 or ‐2 prevented both the entry into S phase and the development of apoptosis in βAP(25_35)‐treated neurons. We conclude that neurons must cross the G1/S transition before succumbing to βAP signaling, and therefore multiple steps within this pathway may be targets for neuroprotective agents.—Copani, A., Condorelli, F., Caruso, A., Vancheri, C., Sala, A., Giuffrida Stella, A. M., Canonico, P. L., Nicoletti, F., Sortino, M. A. Mitotic signaling by β ‐amyloid causes neuronal death. FASEB J. 13, 2225–2234 (1999)

Ciliary Neurotrophic Factor Activates JAK/Stat Signal Transduction Cascade and Induces Transcriptional Expression of Glial Fibrillary Acidic Protein in Glial Cells

Abstract: In recent reports, ciliary neurotrophic factor (CNTF) has been implicated as an injury factor involved in regulating astrogliosis in the CNS. In this study, we used a rat oligodendroglial progenitor cell line that is highly responsive to CNTF to examine CNTF‐induced alterations that may play a role in activation of the glial fibrillary acidic protein (GFAP) gene. We determined that CNTF induces the transient translocation of Stat1α/p91 to the nucleus. This nuclear translocation was followed by GFAP promoter activation and an up‐regulation of GFAP mRNA and protein. Levels of CNTF‐α receptor mRNA, however, were unaffected by addition of the ligand. Transfection studies using an upstream 5′‐flanking, 1.9‐kb rat GFAP promoter linked to a luciferase reporter gene revealed CNTF‐induced transcriptional activation within 1 h of ligand exposure. Moreover, serial‐deleted constructs identified a distal (−1,857 to −1,546 bp) and a proximal (−384 to −106 bp) region as being important for CNTF‐induced GFAP promoter activation. These two regions showed a strong degree of overlap for CNTF‐ and serum‐induced activation of the GFAP gene. Analysis of the two regions revealed several cis‐elements that are thought to be involved in GFAP regulation and/or the regulation of other genes by members of the interleukin‐6 family of cytokines. Moreover, we are the first to report the presence of several putative CNTF‐responsive elements within our identified distal and proximal regions in the GFAP gene promoter.

Structural features of the rat GFAP gene and identification of a novel alternative transcript.

The glial fibrillary acidic protein (GFAP) is expressed in a cell‐specific manner and represents the major subunit of intermediate filaments of astroglial cells. The knowledge of the gene structure is an important step for further understanding the mechanisms of cell‐specific expression. In the present study, we report the complete sequence of the rat GFAP gene and provide evidence for the existence, in the rat brain, of a novel alternative transcript. Since three different transcripts, indicated as GFAPα, β, and γ, have been previously reported (Feinstein et al. [1992] J. Neurosci. Res. 32:1–14; Zelenika et al. [1995] Mol. Brain Res. 30:251–258), we called this novel mRNA isoform GFAPδ. It is generated by the alternative splicing of a novel exon located in the classic seventh intron. This alternative exon (called VII+) contains a 101‐bp coding sequence in frame with exon VII and interrupted by a stop codon TAA at position +5451. Therefore, the novel GFAPδ transcript encodes for an hypothetical GFAP where the forty‐two carboxy‐terminal amino acids encoded by exon VIII and IX are replaced by thirty‐three amino acids encoded by exon VII+. Northern blot analysis with a specific probe for exon VII+ revealed a 4.2‐kb mRNA, expressed in several brain areas, but absent in extracerebral tissues (lung, heart, kidney, liver, spleen). The previously discovered GFAP isoforms (α, β, and γ) produce hypothetical translation products differing in the aminoterminal Head domain. The present data suggest, for the first time, the possible existence of GFAP isoforms differing in the carboxy‐terminal Tail domain. J. Neurosci. Res. 56:219–228, 1999.

Metabotropic glutamate receptor expression in cultured rat astrocytes and human gliomas

In order to confirm the existence of metabotropic glutamate receptors in astroglial cultures and to provide information on different receptor subtypes, the expression of different mOIuRs was analysed in cultures highly enriched in rat astroglial cells. mRNA levels for mGluR1, 2, 3, 4, 7 were undetectable by Northern blot analysis in primary type-1 astroglial cultures derived from total cerebral hemispheres, cerebral cortex and striatum. Interestingly, these cultures expressed a low, but detectable, level of mGluR5 mRNA. The more sensitive technique Reverse Transcription-Polymerase Chain Reaction (RT-PCR) confirmed the presence of mGluR5 transcript in cultured astrocytes and, in addition, revealed the presence of mGluR3 mRNA. The lack of expression of mGluR5 in CG-4 cells, a rat cell line able to differentiate in type-2 astrocytes or oligodendrocytes depending on the culture conditions, suggested that the presence of mGluR5 was not a general feature of cells of glial origin. Moreover, all the examined mGluR transcripts were undetectable by RT-PCR in CG4 cells. In order to confirm the possible expression of mGluR5 in cell of glial origin we examined the mRNA levels for this receptor in tissue samples from human gliomas obtained after surgical resection of the tumors: only 1 sample (grade II astrocytoma), out of 8 examined, showed the presence of mGluR5 mRNA. In conclusion our data show that the only cloned metabotropic receptor linked to phosphoinositide hydrolysis, whose expression is detectable in cultured type-1 astrocytes, is mGluR5. It remains to be established if the low level of expression of mGluR3 could be responsible for the group II metabotropic glutamate receptor activity previously observed in cultured astroglial cells.

Effect of nitric oxide synthase induction on the expression of mitochondrial respiratory chain enzyme subunits in mixed cortical and astroglial cell cultures.

In the present study we evaluated the effects of NO synthase (NOS) induction on the regulation of cytochrome c oxidase (CO) and F0F1-ATPase subunit expression in astroglial and mixed cortical cell cultures. In mixed cortical cell cultures, 18 h of treatment with lipopolysaccharide (LPS, 0.1 microgram/mL) plus interferon-gamma (INF-gamma, 10 U/mL) caused an increase of mRNAs for CO-I, F0F1-ATPase 6 and also for iNOS at 20 DIV. The induction of both CO-I and F0F1-ATPase 6 was abolished by the NOS inhibitor N-monomethyl-L-arginine (NMMA) or by the enzymatic scavenger superoxide dismutase/catalase (SOD/CAT). In primary astroglial cell cultures, treatment for 18 h with increasing concentrations of LPS and INF gamma, produced an increase in the amount of mitochondrial encoded CO-I and -II subunits, with no significant modifications of nuclear encoded subunit IV. An increase was also observed at level of transcription for CO-I and -II, and F0F1-ATPase 6 mRNAs. These effects were abolished by addition of NMMA or SOD/CAT. mRNA induction of CO-I was higher in mixed cortical than in astroglial cell cultures while that of F0F1-ATPase 6 was similar in both cell types. These results suggest that the expression of mitochondrial encoded subunits (CO-I, CO-II and F0F1-ATPase 6) is up-regulated in response to oxygen and NO reactive species. The activity of cytochrome c oxidase decreased after LPS/INF gamma treatment in both astroglial and mixed cortical cultures. The activity of ATP synthase was unmodified, while ATP content drastically decreased after LPS/INF gamma treatment, in both astroglial and mixed cortical cultures. The enzymatic activities of catalase and Mn-SOD (mitochondrial) showed a significant increase after LPS/INF gamma treatment, which was abolished by NMMA.

GFAPβ mRNA expression in the normal rat brain and after neuronal injury

GFAPβ mRNA is an alternative transcript of the glial fibrillary acidic protein (GFAP) gene, whose transcriptional start site is located 169 nucleotides upstream to the classical GFAPα mRNA. By an RT-PCR method with primers on separate exons, we were able to confirm the presence of GFAP transcripts with a longer 5′ untraslated region in all the examined areas of rat brain and in primary cultures of astroglial cells. Northern blot analysis, using an oligoprobe specific for the 5′ region of GFAPβ, revealed a single hybridization band of 2.9 kb in all the brain regions examined and in primary cultures of astroglial cells. The availability of the quantitative Northern blot assay allowed further studies on the regulation of GFAPβ expression in vivo. Since it is well-known that neuronal brain injury is one of the most powerful inducers of GFAP, we examined the expression of GFAPα and β after a neurotoxic lesion in the rat hippocampus. Results obtained show a parallel increase in both GFAP transcripts with an identical time-course, suggesting that regulatory regions of the gene influence in similar way the rate of transcription at the two different start sites (α and β) or that a similar post-transcriptional mechanism is involved in regulating both mRNA isoforms.

A Neural-Specific Hypomethylated Domain in the 5' Flanking Region of the Glial Fibrillary Acidic Protein Gene

In the present study we examined the methylation status of the glial fibrillary acidic protein (GFAP) gene promoter, analyzing various CG sites in both the human and rat gene in GFAP-expressing and nonexpressing tissues. Moreover, we studied the methylation of specific CG sites in different rat brain areas during postnatal development, in cell cultures highly enriched in specific neural- or non-neural-cell types (fibroblasts), and in human gliomas. The obtained results do not support a simple correlation between demethylation and expression of the GFAP gene but help to identify a cluster of CG sites in the 5flanking region (from -1176 to -1471 in the rat) that are hypomethylated in neural cell types and localized in a region highly conserved between rat, mouse and human GFAP promoters. Neural-specific hypomethylation of this conserved zone can be observed also in the human GFAP gene both in normal brain tissue and neoplastic glial cells. A higher demethylation of the -1176 site at early stage of postnatal life was observed in specific rat brain areas, such as hippocampus and cerebellum. The most dramatic differences were observed in the cerebellum where a peak of demethylation of the -1176 site was detected at 15 days of postnatal life, followed by an intense remethylation of this site. Results of experiments in the CG4 glial progenitor cell line showed that demethylation of the -1176 site is already established before transcriptional activation of the GFAP gene. Moreover, results of experiments in primary cell cultures show that in neuronal cell types, such as cerebellar granule cells and embryonic cerebral hemisphere neurons, the level of demethylation of the -1176 site is comparable to that observed in cultured astrocytes. In contrast a high level of methylation can be observed in cultured non-neural cell types (fibroblasts). Such neural-specific hypomethylation could be established in a very early stage in the progression along the neural cell lineage and could play a role in maintaining a local open chromatin conformation which is then necessary to allow the interaction with specific regulatory factors present in astroglial cells.

Growth conditions differentially affect the constitutive expression of primary response genes in cultured cereballar granule cells

Cultured cerebellar granule cells underwent apoptotic degeneration when grown in medium containing 10 instead of 25 mM K+. Knowing that apoptosis is associated with changes in the expression of primary response genes, we have measured c-fos, zif/268, and c-jun mRNA levels during maturation of cultured granule cells grown in 10 or 25 mM K+. The constitutive expression of c-fos and zif/268 was differentially regulated by extracellular K+ concentration at 5 days of maturation in vitro (DIV), when cells grown under suboptimal conditions (i.e. in 10 mM K+) are committed to degenerate. At this stage, c-fos mRNA levels were detectable only in cultures grown in 25 mM K+, whereas zif/268 mRNA levels were dramatically elevated in cultures grown in 10 mM K+. This provides one of the few conditions in which c-fos and zif/268 are differentially regulated in nerve cells. Substantial changes in c-jun, or β-actin mRNA levels were detectable only at 7 DIV, when the percentage of apoptotic cells had already reached a plateau in ultures grown in 10 mM K+. We speculate that changes in the expression of zif/268 are important in the gene program associated with the induction of apoptosis by trophic deprivation in cultured neurons.