Michal Hetman

DNA fragmentation in rat brain after intraperitoneal administration of kainate.

Cell death occurs in many neuropathological conditions. However, the mechanisms governing this process(es) remain generally unknown. In this report we studied whether excitotoxic neuronal death evoked by kainic acid (KA) in rat brain is associated with ladder-like DNA fragmentation. DNA was isolated from hippocampi, entorhinal and sensory cortices at various times following intraperitoneal KA (10 mg kg-1) injections. Typical oligonucleosome-sized DNA fragmentation was observed in all three structures at 18 h and 72 h following KA administration. These findings were further confirmed by in situ nick-translation. DNA fragmentation is believed to be diagnostic for apoptosis. The clear ladders of DNA fragmentation appeared after 18 h, although slight degradation was observed as early as 12 h after KA administration.

Ciliary Neurotrophic Factor Mediates Dopamine D2 Receptor-Induced CNS Neurogenesis in Adult Mice

Neurogenesis continues in the adult forebrain subventricular zone (SVZ) and the dentate gyrus of the hippocampal formation. Degeneration of dopaminergic projections in Parkinsons disease and animals reduces, whereas ciliary neurotrophic factor (CNTF) promotes, neurogenesis. We tested whether the dopaminergic system promotes neurogenesis through CNTF. Astrocytes of the SVZ and dentate gyrus expressed CNTF and were close to dopaminergic terminals. Dopaminergic denervation in adult mice reduced CNTF mRNA by ∼60%, whereas systemic treatment with the D2 agonist quinpirole increased CNTF mRNA in the SVZ and hippocampal formation, and in cultured astrocytes by 1.5–5 fold. The effect of quinpirole in vitro was blocked by the D2 antagonist eticlopride and did not cause astroglial proliferation or hypertrophy. Systemic quinpirole injections increased proliferation in wild-type mice by ∼25–75% but not in CNTF−/− littermates or in the SVZ of mice infused with CNTF antibodies. Quinpirole increased the number of neuroblasts in wild-type but not in CNTF−/− littermates. Neurogenesis was reduced by ∼20% in CNTF−/− mice, confirming the endogenous role of CNTF. Nigrostriatal denervation did not affect SVZ proliferation in CNTF−/− mice, suggesting that the dopaminergic innervation normally regulates neurogenesis through CNTF. Quinpirole acted on postsynaptic receptors as it reversed the reduced proliferation seen after dopaminergic denervation in wild-type mice. Thus, CNTF mediates dopaminergic innervation- and D2 receptor-induced neurogenesis in the adult forebrain. Because CNTF is predominantly expressed in the nervous system, this mechanism and the ability to pharmacologically modulate it have implications for Parkinsons disease and cell-replacement therapies for other disorders.

Survival signaling pathways activated by NMDA receptors.

N-methyl-D-aspartate receptors (NMDAR) have a recognized role in neuronal plasticity while their excessive activation results in excitotoxic death. Therefore, NMDAR antagonists are considered for neuroprotective interventions. However, there is also an emerging role of NMDAR in supporting neuronal survival. Thus, during CNS development, basal NMDAR activity suppresses neuronal apoptosis while moderate NMDAR activation may, at least under some conditions, protect against excitotoxic/ischemic insults. These suggest that while protecting from excitotoxicity, NMDAR antagonists would also reduce pro-survival activity of NMDAR. Hence, the identification of the switches controlling pro-survival vs. pro-excitotoxic outcome of NMDAR stimulation may lead to development of NMDAR antagonists that selectively block the excitotoxicity while enhancing the protective NMDAR signaling. On the other hand, the existence of anti-apoptotic/pro-proliferative NMDAR signaling in transformed cells may result in new strategies to attack cancer. This review focuses on the emerging field of neuroprotective signaling mediators that are implicated in pro-survival activity of NMDAR. We discuss the evidence implicating either NR2B or nonNR2B NMDAR in mediating the protection. We also present the reports linking NMDAR-mediated protection to the activation of survival signaling kinases including ERK and Akt, or suppression of a pro-apoptotic kinase, GSK-3beta. The protective role of transcription factors is also discussed. Finally, we review the existing evidence suggesting that NMDAR support survival by regulating the pro-survival trophic factor signaling and/or the cell death machinery. Although NMDAR provide a major survival input to CNS neurons, the NMDAR-activated protective signaling is poorly understood and, therefore, deserves further research effort.

Epigenetic Silencing of Nucleolar rRNA Genes in Alzheimer's Disease

Background Ribosomal deficits are documented in mild cognitive impairment (MCI), which often represents an early stage Alzheimers 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 Findings To 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/Significance In 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.

Emerging roles of the neuronal nucleolus

Although, the nucleolus has been observed for almost 200 years in neurons, studies that directly address the neuronal roles of this subnuclear structure have appeared only recently. The aim of this review is to discuss recent progress and identify some critical questions that remain to be answered. As expected for the cellular center of ribosome biogenesis, the nucleolus is essential for the growth of developing neurons, including neurite morphogenesis and long-term maintenance of mature neurons. In addition, the nucleolus contributes to neuronal stress responses, including the regulation of apoptosis. Hence, disrupted neurodevelopment or neurodegeneration are among the likely consequences of nucleolar dysfunction. Conversely, the presence of active nucleoli may determine the potential for neurorepair.

Role of Megakaryoblastic Acute Leukemia-1 in ERK1/2-Dependent Stimulation of Serum Response Factor-Driven Transcription by BDNF or Increased Synaptic Activity

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.

NEURONAL EXCITATION-DRIVEN AND AP-1-DEPENDENT ACTIVATION OF TISSUE INHIBITOR OF METALLOPROTEINASES-1 GENE EXPRESSION IN RODENT HIPPOCAMPUS

Understanding of biological function of AP-1 transcription factor in central nervous system may greatly benefit from identifying its target genes. In this study, we present several lines of evidence implying AP-1 in regulating expression of tissue inhibitor of metalloproteinases-1 (timp-1) gene in rodent hippocampus in response to increased neuronal excitation. Such a notion is supported by the findings that timp-1 mRNA accumulation occurs in the rat hippocampus after either kainate- or pentylenetetrazole-evoked seizures with a delayed, in comparison with AP-1 components, time course, as well as with spatial overlap with c-Fos protein (major inducible AP-1 component) expression. Furthermore, AP-1 sequence derived from timp-1 promoter is specifically bound by hippocampal AP-1 proteins after treating the rats with either pro-convulsive agent. Finally, timp-1 promoter responds to excitatory activation both in vivo, in transgenic mice harboring the timp-LacZ gene construct, and in vitro in neurons of the hippocampal dentate gyrus cultures. These findings suggest that the AP-1 transcription factor may exert its role in the brain through affecting extracellular matrix remodeling.

Attenuating the Endoplasmic Reticulum Stress Response Improves Functional Recovery After Spinal Cord Injury

Activation of the unfolded protein response (UPR) is involved in the pathogenesis of numerous CNS myelin abnormalities; yet, its direct role in traumatic spinal cord injury (SCI)‐induced demyelination is not known. The UPR is an evolutionarily conserved cell defense mechanism initiated to restore endoplasmic reticulum homeostasis in response to various cellular stresses including infection, trauma, and oxidative damage. However, if uncompensated, the UPR triggers apoptotic cell death. We demonstrate that the three signaling branches of UPR including the PERK, ATF6, and IRE1α are rapidly initiated in a mouse model of contusive SCI specifically at the injury epicenter. Immunohistochemical analyses of the various UPR markers revealed that in neurons, the UPR appeared at 6 and 24‐h post‐SCI. In contrast, in oligodendrocytes and astroglia, UPR persisted at least for up to 3 days post‐SCI. The UPR‐associated proapoptotic transcriptional regulator CHOP was among the UPR markers upregulated in neurons and oligodendrocytes, but not in astrocytes, of traumatized mouse spinal cords. To directly analyze its role in SCI, WT and CHOP null mice received a moderate T9 contusive injury. Deletion of CHOP led to an overall attenuation of the UPR after contusive SCI. Furthermore, analyses of hindlimb locomotion demonstrated a significant functional recovery that correlated with an increase in white‐matter sparing, transcript levels of myelin basic protein, and Claudin 11 and decreased oligodendrocyte apoptosis in CHOP null mice in contrast to WT animals. Thus, our study provides evidence that the UPR contributes to oligodendrocyte loss after traumatic SCI.

A Positive Feedback Loop between Glycogen Synthase Kinase 3β and Protein Phosphatase 1 after Stimulation of NR2B NMDA Receptors in Forebrain Neurons

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 3β (GSK3β) by protein phosphatase 1(PP1)-mediated dephosphorylation of GSK3β at the serine 9 residue. NMDA-triggered GSK3β activation was mediated by NMDAR that contained the NR2B subunit. Interestingly, GSK3β 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 GSK3β and PP1 that results in amplification of PP1 activation by GSK3β. In addition, GSK3β inhibition decreased PP1-mediated dephosphorylation of the cAMP-response element-binding protein (CREB) at the serine 133 residue in NMDA-stimulated neurons. Conversely, overexpression of GSK3β abolished non-NR2B-mediated activation of CRE-driven transcription. These data suggest that cross-talk between GSK3β and PP1 contributes to NR2B NMDAR-induced inhibition of CREB signaling by non-NR2B NMDAR. The excessive activation of NR2B-PP1-GSK3β-PP1 circuitry may contribute to the deficits of CREB-dependent neuronal plasticity in neurodegenerative diseases.

Inhibition of nucleolar transcription as a trigger for neuronal apoptosis

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.