Sp3 and sp4 transcription factor levels are increased in brains of patients with Alzheimer's disease.

BACKGROUND/AIMS: Alzheimer's disease (AD) is characterized by extracellular Abeta peptide deposition originating from amyloid precursor protein cleavage and intracellular neurofibrillary tangles resulting from pathological tau protein aggregation. These processes are accompanied by dramatic neuronal losses, further leading to different cognitive impairments. Neuronal death signalings involve gene expression modifications that rely on transcription factor alterations. Herein, we investigated the fate of the Sp family of transcription factors in postmortem brains from patients with AD disease and in different contexts of neuronal death. METHODS/RESULTS: By immunohistochemistry we found that the Sp3 and Sp4 levels were dramatically increased and associated with neurofibrillary tangles and pathological tau presence in neurons from the CA1 region of the hippocampus, as well as the entorhinal cortex of AD patient brains. The Sp transcription factor expression levels were further analyzed in cortical neurons in which death is induced by amyloid precursor protein signaling targeting. While the Sp1 levels remained constant, the Sp4 levels were slightly upregulated in response to the death signal. The Sp3 isoforms were rather degraded. Interestingly, when overexpressed by transfection experiments, the three Sp family members induced neuronal apoptosis, Sp3 and Sp4 being the most potent proapoptotic factors over Sp1. CONCLUSION: Our data evidence Sp3 and Sp4 as new hallmarks of AD in postmortem human brains and further point out that Sp proteins are potential triggers of neuronal death signaling cascades.

HP1alpha guides neuronal fate by timing E2F-targeted genes silencing during terminal differentiation.

A critical step of neuronal terminal differentiation is the permanent withdrawal from the cell cycle that requires the silencing of genes that drive mitosis. Here, we describe that the alpha isoform of the heterochromatin protein 1 (HP1) protein family exerts such silencing on several E2F-targeted genes. Among the different isoforms, HP1alpha levels progressively increase throughout differentiation and take over HP1gamma binding on E2F sites in mature neurons. When overexpressed, only HP1alpha is able to ensure a timed repression of E2F genes. Specific inhibition of HP1alpha expression drives neuronal progenitors either towards death or cell cycle progression, yet preventing the expression of the neuronal marker microtubule-associated protein 2. Furthermore, we provide evidence that this mechanism occurs in cerebellar granule neurons in vivo, during the postnatal development of the cerebellum. Finally, our results suggest that E2F-targeted genes are packaged into higher-order chromatin structures in mature neurons relative to neuroblasts, likely reflecting a transition from a 'repressed' versus 'silenced' status of these genes. Together, these data present new epigenetic regulations orchestrated by HP1 isoforms, critical for permanent cell cycle exit during neuronal differentiation.

Pituitary adenylyl cyclase-activating polypeptide 38 reduces astroglial proliferation by inhibiting the GTPase RhoA.

Pituitary adenylyl cyclase-activating polypeptide 38 (PACAP38) plays an important role in the proliferation and differentiation of neural cells. In the present study, we have investigated how PACAP38 inhibits the proliferation of cultured neocortical astroglial cells. When applied to synchronized cells during the G(1) phase of the cell cycle, PACAP38 diminished the subsequent nuclear uptake of bromodeoxyuridine. When applied for 2 days, it reduced the cell number. PACAP38 did not exert its antiproliferative effect by activating protein kinase A. It also did not reduce the activity of mitogen-activated protein kinases essential for G(1) phase progression. Instead, PACAP38 acted on a member of the Rho family of small GTPases. It reduced the activity of RhoA as was shown with a Rhotekin pull-down assay. The decrease in endogenous RhoA activity induced by treatment of the cells with C3 exotoxin or by expression of dominant negative RhoA also reduced the nuclear uptake of bromodeoxyuridine. In contrast, expression of constitutively active RhoA prevented the effect of PACAP38. Our data show a novel signal transduction pathway by which the neuropeptide influences cell proliferation.

HDAC-3 participates in the repression of e2f-dependent gene transcription in primary differentiated neurons.

Activation of e2f-1 gene expression is an event that has been now established in many models of neuronal apoptosis. Accumulated E2F-1 protein has also been observed in post mortem brains obtained from patients suffering from different neurodegenerative diseases. We have previously shown in primary neuronal cultures that e2f-1 gene transcription was actively repressed in neuroprotective conditions through HDAC-dependent regulation on the E2F-responsive elements (E2F-REs) located in the e2f-1 gene promoter. Here, we further investigated the protein complex bound to these sites by gel shift analysis. We found that the specific protein binding to E2F-REs is altered in apoptotic conditions compared to neuroprotective conditions, suggesting that the proteic constituents of the complex are likely to be modified upon apoptosis onset. Indeed, Western blot analysis showed a time-dependent degradation of the Rb/E2F binding protein HDAC-3 during apoptosis, a degradation that is caspase-dependent. Altogether, these data point to HDAC-3 as a good candidate involved in the active e2f-1 repression necessary for neuroprotection.

Rho GTPases and phosphoinositide 3-kinase organize formation of branched dendrites.

Neurons receive information from other neurons via their dendritic tree. Dendrites and their branches result from alternating outgrowth and retraction. The Rho GTPases Rac and Cdc42 (cell division cycle 42) facilitate the outgrowth of branches, whereas Rho attenuates it. The mechanism of neurite retraction is unknown. Because the adenylyl cyclase activator forskolin causes numerous branched extensions in NG108-15 cells, we have investigated the underlying mechanism in this cell line. In additional studies, we used cultured hippocampal neurons in which forskolin induces branched dendrites. In both cell types, forskolin enhanced the activity of Cdc42, but not that of Rac, although both GTPases were necessary for the formation of branched extensions. Time lapse microscopy showed that forskolin did not increase the rate of addition of new extensions or branches, but it reduced the rate of the retraction so that more branched extensions persisted. Inhibition of phosphoinositide 3-kinase activity by wortmannin or LY294002 also reduced the rate of retraction and thus facilitated dendritic arborization. Forskolin diminished the activity of phosphoinositide 3-kinases. Inhibitors of phosphoinositide 3-kinases not only reduced the retraction but also the addition of new dendrites and branches. This reduction was no longer present when Rho kinase was simultaneously inactivated, suggesting an interaction of phosphoinositide 3-kinases and Rho kinase. The present results show a central role of phosphoinositide 3-kinases in dendrite formation. In neuronal cells, increased levels of cAMP can support dendritic arborization by modulating the activity of the lipid kinase.

Critical loss of CBP/p300 histone acetylase activity by caspase-6 during neurodegeneration.

By altering chromatin structure, histone acetyltransferases (HATs) act as transcriptional regulators. We observed in a model of primary neurons that histone acetylation levels decreased at the onset of apoptosis. The CREB-binding protein (CBP) is a HAT of particular interest because it also acts as a co-activator controlling, among others, CREB-dependent transcriptional activity. It has been demonstrated that CREB exerts neuroprotective functions, but the fate of CBP during neuronal apoptosis remained unclear till now. This work provided evidence that CBP is specifically targeted by caspases and calpains at the onset of neuronal apoptosis, and CBP was futher identified as a new caspase-6 substrate. This ultimately impinged on the CBP/p300 HAT activity that decreased with time during apoptosis entry, whereas total cellular HAT activity remained unchanged. Interestingly, CBP loss and histone deacetylation were observed in two different pathological contexts: amyloid precursor protein-dependent signaling and amyotrophic lateral sclerosis model mice, indicating that these modifications are likely to contribute to neurodegenerative diseases. In terms of function, we demonstrated that fine-tuning of CBP HAT activity is necessary to ensure neuroprotection.

The protein kinase A inhibitor H89 acts on cell morphology by inhibiting Rho kinase.

The small GTPase RhoA can retract cell extensions by acting on two Rho kinases (ROCKs). Activated protein kinase A (PKA) inhibits RhoA and induces extensions. The isoquinoline H89 inhibits PKA and thus should prevent the inactivation of RhoA. In kinase assays, H89 has been recently found to inactivate a ROCK-II also. Because H89 may be able to exert opposite effects on cell extensions, we have studied the effects of H89 on neurite formation in the neuroblastoma-glioma line NG 108-15, which expresses ROCK-I and ROCK-II. We found that H89 can indeed inhibit ROCKs and PKA. Because ROCKs act downstream of RhoA, the inhibitory effect of H89 on ROCKs is most prominent. The data indicate that H89 may not be used as an antagonist of PKA in systems in which ROCKs play a role.