BackgroundRibosomal deficits are documented in mild cognitive impairment (MCI), which often represents an early stage Alzheimer's disease (AD), as well as in advanced AD. The nucleolar rRNA genes (rDNA), transcription of which is critical for ribosomal biogenesis, are regulated by epigenetic silencing including promoter CpG methylation.Methodology/Principal FindingsTo assess whether CpG methylation of the rDNA promoter was dysregulated across the AD spectrum, we analyzed brain samples from 10 MCI-, 23 AD-, and, 24 age-matched control individuals using bisulfite mapping. The rDNA promoter became hypermethylated in cerebro-cortical samples from MCI and AD groups. In parietal cortex, the rDNA promoter was hypermethylated more in MCI than in advanced AD. The cytosine methylation of total genomic DNA was similar in AD, MCI, and control samples. Consistent with a notion that hypermethylation-mediated silencing of the nucleolar chromatin stabilizes rDNA loci, preventing their senescence-associated loss, genomic rDNA content was elevated in cerebrocortical samples from MCI and AD groups.Conclusions/SignificanceIn conclusion, rDNA hypermethylation could be a new epigenetic marker of AD. Moreover, silencing of nucleolar chromatin may occur during early stages of AD pathology and play a role in AD-related ribosomal deficits and, ultimately, dementia.

In post‐mitotic neurons, the mechanisms of the apoptotic checkpoint that is activated by DNA damage remain unclear. Here we show that in cultured cortical neurons, the DNA damaging agent camptothecin (CPT) reduced transcription of rRNA and disrupted nucleolar staining for B23/nucleophosmin suggesting DNA damage‐induced nucleolar stress. Although CPT activated the pro‐apoptotic protein p53, the CPT‐induced nucleolar stress was unaffected by p53 inhibition. In addition, brain‐derived neurotrophic factor‐mediated protection from CPT‐induced apoptosis prevented neither nucleolar stress nor p53 activation. Therefore, inhibition of rRNA transcription might be upstream of the pro‐apoptotic p53 activity. Indeed, short hairpin RNA‐mediated inhibition of a RNA‐Polymerase‐I co‐factor, transcription initiation factor IA, attenuated rRNA transcription causing nucleolar stress and p53‐dependent neuronal apoptosis. The protein synthesis inhibitor cycloheximide blocked apoptosis that was induced by over‐expressed shTIF‐IA or active form of p53. Also, the general transcription inhibitor actinomycin D triggered nucleolar stress and activated p53. However, it did not induce apoptosis except at the low concentration of 0.05 μg/mL with stronger inhibitory activity against nucleolar than extranucleolar transcription. Hence, nucleolar stress‐activated apoptosis requires extranucleolar transcription. This study identifies the nucleoli of post‐mitotic neurons as sensors of DNA damage coupling reduced rRNA transcription to p53‐mediated apoptosis that requires de novo expression of protein‐coding genes. Thus, rDNA selectivity of DNA damage may determine its ability to induce neuronal apoptosis.

Serum response factor (SRF)-mediated transcription contributes to developmental and adult brain plasticity. Therefore, we investigated the role of a newly identified SRF coactivator, MKL1, in the regulation of SRF-driven transcription in rat forebrain neurons. MKL1 expression was found in newborn rat cortical or hippocampal neurons in culture as well as in adult rat forebrain. Immunostaining demonstrated constitutive nuclear localization of MKL1 in the CA1 region of the hippocampus, in the deep layers of the neocortex, and in cultured neurons. Overexpression of MKL1 in primary cortical neurons elevated SRF-driven transcription and enhanced its stimulation by BDNF. In addition, inhibition of endogenous MKL1 by overexpression of a dominant-negative MKL1 mutant or by small interfering RNA reduced BDNF activation of SRF-driven transcription. In neurons, endogenous MKL1 was associated with SRF-regulated chromatin regions of several endogenous genes including c-fos, JunB, Srf, and Cyr61. BDNF activation of MKL1/SRF-driven transcription was dependent on the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, which also led to MKL1 phosphorylation. Finally, synaptic activity stimulation of SRF-driven transcription was reduced by inhibition of endogenous MKL1. Conversely, synaptic activity enhanced transcription by overexpressed MKL1. MKL1 regulation by synaptic activity was mediated through the NMDA receptor-activated ERK1/2. These results suggest that neuronal MKL1 contributes to SRF-regulated gene expression induced by BDNF or synaptic activity. In addition, MKL1 appears as a novel mediator of the signaling between ERK1/2 and SRF. Moreover, MKL1 is a likely regulator of SRF-driven transcription programs that underlie neuronal plasticity.

During cortical development, when NR2B subunit is the major component of the NMDA glutamate receptors (NMDARs), moderate NMDAR activity supports neuronal survival at least in part by regulating gene transcription. We report that, in cultured cortical neurons from newborn rats, the NMDARs activated the calcium-responsive transcription regulator nuclear factor of activated T cells (NFAT). Moreover, in developing rat cortex, the NFAT isoforms c3 and c4 (NFATc3 and NFATc4) were expressed at relatively higher levels at postnatal day 7 (P7) than P21, overlapping with the period of NMDAR-dependent survival. In cultured cortical neurons, NFATc3 and NFATc4 were regulated at least in part by the NR2B NMDAR. Conversely, knockdown of NFATc4 but not NFATc3 induced cortical neuron apoptosis. Likewise, NFATc4 inhibition prevented antiapoptotic neuroprotection in response to exogenous NMDA. Expression of the brain-derived neurotrophic factor (BDNF) was reduced by NFATc4 inhibition. NFATc4 regulated transcription by the NMDARresponsive bdnf promoter IV. In addition, NMDAR blockers including NR2B-selective once reduced BDNF expression in P7 cortex and cultured cortical neurons. Finally, exogenous BDNF rescued from the proapoptotic effects of NFATc4 inhibition. These results identify bdnf as one of the target genes for the antiapoptotic signaling by NMDAR-NFATc4. Thus, the previously unrecognized NMDARNFATc4 -BDNF pathway contributes to the survival signaling network that supports cortical development.

N-methyl-D-aspartate receptors (NMDARs) are critical for neuronal plasticity and survival, whereas their excessive activation produces excitotoxicity and may accelerate neurodegeneration. Here, we report that stimulation of NMDARs in cultured rat hippocampal or cortical neurons and in the adult mouse brain in vivo disinhibited glycogen synthase kinase 3␤ (GSK3␤) by protein phosphatase 1(PP1)-mediated dephosphorylation of GSK3␤ at the serine 9 residue. NMDA-triggered GSK3␤ activation was mediated by NMDAR that contained the NR2B subunit. Interestingly, GSK3␤ inhibition reduced inhibitory phosphorylation of the PP1 inhibitor 2 (I2) and attenuated serine 9 dephosphorylation by PP1. These data suggest existence of a feedback loop between GSK3␤ and PP1 that results in amplification of PP1 activation by GSK3␤. In addition, GSK3␤ inhibition decreased PP1-mediated dephosphorylation of the cAMPresponse element-binding protein (CREB) at the serine 133 residue in NMDA-stimulated neurons. Conversely, overexpression of GSK3␤ abolished non-NR2B-mediated activation of CRE-driven transcription. These data suggest that cross-talk between GSK3␤ and PP1 contributes to NR2B NMDAR-induced inhibition of CREB signaling by non-NR2B NMDAR. The excessive activation of NR2B-PP1-GSK3␤-PP1 circuitry may contribute to the deficits of CREBdependent neuronal plasticity in neurodegenerative diseases.