Reduced Expression of Oligodendrocyte Linage-Enriched Transcripts During the Endoplasmic Reticulum Stress/Integrated Stress Response.

Endoplasmic reticulum (ER) stress in oligodendrocyte (OL) linage cells contributes to several CNS pathologies including traumatic spinal cord injury (SCI) and multiple sclerosis. Therefore, primary rat OL precursor cell (OPC) transcriptomes were analyzed using RNASeq after treatments with two ER stress-inducing drugs, thapsigargin (TG) or tunicamycin (TM). Gene ontology term (GO) enrichment showed that both drugs upregulated mRNAs associated with the general stress response. The GOs related to ER stress were only enriched for TM-upregulated mRNAs, suggesting greater ER stress selectivity of TM. Both TG and TM downregulated cell cycle/cell proliferation-associated transcripts, indicating the anti-proliferative effects of ER stress. Interestingly, many OL lineage-enriched mRNAs were downregulated, including those for transcription factors that drive OL identity such as Olig2. Moreover, ER stress-associated decreases of OL-specific gene expression were found in mature OLs from mouse models of white matter pathologies including contusive SCI, toxin-induced demyelination, and Alzheimer's disease-like neurodegeneration. Taken together, the disrupted transcriptomic fingerprint of OL lineage cells may facilitate myelin degeneration and/or dysfunction when pathological ER stress persists in OL lineage cells.

The ER stress response compromises the transcriptomic identity of the OL lineage. Therefore, persistent, pathological ER stress may have a negative impact on structural and/or functional integrity of the white matter.

Opposite modulation of functional recovery following contusive spinal cord injury in mice with oligodendrocyte-selective deletions of Atf4 and Chop/Ddit3.

The integrated stress response (ISR)-activated transcription factors ATF4 and CHOP/DDIT3 may regulate oligodendrocyte (OL) survival, tissue damage and functional impairment/recovery in white matter pathologies, including traumatic spinal cord injury (SCI). Accordingly, in OLs of OL-specific RiboTag mice, Atf4, Chop/Ddit3 and their downstream target gene transcripts were acutely upregulated at 2, but not 10, days post-contusive T9 SCI coinciding with maximal loss of spinal cord tissue. Unexpectedly, another, OL-specific upregulation of Atf4/Chop followed at 42 days post-injury. However, wild type versus OL-specific Atf4-/- or Chop-/- mice showed similar white matter sparing and OL loss at the injury epicenter, as well as unaffected hindlimb function recovery as determined by the Basso mouse scale. In contrast, the horizontal ladder test revealed persistent worsening or improvement of fine locomotor control in OL-Atf4-/- or OL-Chop-/- mice, respectively. Moreover, chronically, OL-Atf-/- mice showed decreased walking speed during plantar stepping despite greater compensatory forelimb usage. Therefore, ATF4 supports, while CHOP antagonizes, fine locomotor control during post-SCI recovery. No correlation between those effects and white matter sparing together with chronic activation of the OL ISR suggest that in OLs, ATF4 and CHOP regulate function of spinal cord circuitries that mediate fine locomotor control during post-SCI recovery.

Role of circadian rhythms in pathogenesis of acute CNS injuries: Insights from experimental studies.

A wide range of physiological processes show circadian oscillations that are critical for organismal homeostasis. Consequently, disruption of such rhythmicity contributes to the pathogenesis of various chronic diseases. The occurrence, severity, and resolution of acute injuries to the central nervous system may also be modulated by circadian rhythms and/or anti-rhythmic disruptions. Mechanistically, circadian rhythmicity originates from the intrinsic circadian activity of the clock pathway transcription factors that regulate gene expression in a cycle of about 24 h. In addition, their activity is synchronized by external time cues including light, sleep or feeding to produce diurnal rhythms of 24 h. The pathogenic significance of circadian rhythms can be tested experimentally by determining the effects of (i) natural diurnal/circadian time, (ii) time cue manipulations that perturb the rhythmicity, (iii) drugs that target the clock pathway, and (iv) genetic manipulations to inactivate key mediators of the clock pathway. This review summarizes emerging evidence from all those strategies that supports a role of circadian and/or diurnal rhythms in rodent models of stroke, traumatic brain or spinal cord injury, status epilepticus and encephalomyelitis. Potential clinical implications are also considered, including pathogenic effects of the chronodisruptive environment or time of day variability in response to therapeutic interventions. Well-controlled animal studies avoid effects of confounding factors that may complicate interpretation of epidemiological data. They can also help to identify mechanisms that mediate the circadian modulation of a CNS pathology.

Limited changes in locomotor recovery and unaffected white matter sparing after spinal cord contusion at different times of day.

The circadian gene expression rhythmicity drives diurnal oscillations of physiological processes that may determine the injury response. While outcomes of various acute injuries are affected by the time of day at which the original insult occurred, such influences on recovery after spinal cord injury (SCI) are unknown. We report that mice receiving moderate, T9 contusive SCI at ZT0 (zeitgeber time 0, time of lights on) and ZT12 (time of lights off) showed similar hindlimb function recovery in the Basso mouse scale (BMS) over a 6 week post-injury period. In an independent study, no significant differences in BMS were observed after SCI at ZT18 vs. ZT6. However, the ladder walking test revealed modestly improved performance for ZT18 vs. ZT6 mice at week 6 after injury. Consistent with those minor effects on functional recovery, terminal histological analysis revealed no significant differences in white matter sparing at the injury epicenter. Likewise, blood-spinal cord barrier disruption and neuroinflammation appeared similar when analyzed at 1 week post injury at ZT6 or ZT18. Therefore, locomotor recovery after thoracic contusive SCI is not substantively modulated by the time of day at which the neurotrauma occurred.

Improved locomotor recovery after contusive spinal cord injury in Bmal1-/- mice is associated with protection of the blood spinal cord barrier.

The transcription factor BMAL1/ARNTL is a non-redundant component of the clock pathway that regulates circadian oscillations of gene expression. Loss of BMAL1 perturbs organismal homeostasis and usually exacerbates pathological responses to many types of insults by enhancing oxidative stress and inflammation. Surprisingly, we observed improved locomotor recovery and spinal cord white matter sparing in Bmal1-/- mice after T9 contusive spinal cord injury (SCI). While acute loss of neurons and oligodendrocytes was unaffected, Bmal1 deficiency reduced the chronic loss of oligodendrocytes at the injury epicenter 6 weeks post SCI. At 3 days post-injury (dpi), decreased expression of genes associated with cell proliferation, neuroinflammation and disruption of the blood spinal cord barrier (BSCB) was also observed. Moreover, intraspinal extravasation of fibrinogen and immunoglobulins was decreased acutely at dpi 1 and subacutely at dpi 7. Subacute decrease of hemoglobin deposition was also observed. Finally, subacutely reduced levels of the leukocyte marker CD45 and even greater reduction of the pro-inflammatory macrophage receptor CD36 suggest not only lower numbers of those cells but also their reduced inflammatory potential. These data indicate that Bmal1 deficiency improves SCI outcome, in part by reducing BSCB disruption and hemorrhage decreasing cytotoxic neuroinflammation and attenuating the chronic loss of oligodendrocytes.

Ribosomal biogenesis as an emerging target of neurodevelopmental pathologies.

Development of the nervous system is carried out by complex gene expression programs that are regulated at both transcriptional and translational level. In addition, quality control mechanisms such as the TP53-mediated apoptosis or neuronal activity-stimulated survival ensure successful neurogenesis and formation of functional circuitries. In the nucleolus, production of ribosomes is essential for protein synthesis. In addition, it participates in chromatin organization and regulates the TP53 pathway via the ribosomal stress response. Its tight regulation is required for maintenance of genomic integrity. Mutations in several ribosomal components and trans-acting ribosomal biogenesis factors result in neurodevelopmental syndromes that present with microcephaly, autism, intellectual deficits and/or progressive neurodegeneration. Furthermore, ribosomal biogenesis is perturbed by exogenous factors that disrupt neurodevelopment including alcohol or Zika virus. In this review, we present recent literature that argues for a role of dysregulated ribosomal biogenesis in pathogenesis of various neurodevelopmental syndromes. We also discuss potential mechanisms through which such dysregulation may lead to cellular pathologies of the developing nervous system including insufficient proliferation and/or loss of neuroprogenitors cells, apoptosis of immature neurons, altered neuronal morphogenesis, and neurodegeneration.

RNA Polymerase 1 Is Transiently Regulated by Seizures and Plays a Role in a Pharmacological Kindling Model of Epilepsy.

Ribosome biogenesis, including the RNA polymerase 1 (Pol1)-mediated transcription of rRNA, is regulated by the pro-epileptogenic mTOR pathway. Therefore, hippocampal Pol1 activity was examined in mouse models of epilepsy including kainic acid- and pilocarpine-induced status epilepticus (SE) as well as a single seizure in response to pentylenetetrazole (PTZ). Elevated 47S pre-rRNA levels were present acutely after induction of SE suggesting activation of Pol1. Conversely, after a single seizure, 47S pre-rRNA was transiently downregulated with increased levels of unprocessed 18S rRNA precursors in the cornu Ammonis (CA) region. At least in the dentate gyrus (DG), the pilocarpine SE-mediated transient activation of Pol1 did not translate into long-term changes of pre-rRNA levels or total ribosome content. Unaltered hippocampal ribosome content was also found after a 20-day PTZ kindling paradigm with increasing pro-convulsive effects of low dose PTZ that was injected every other day. However, after selectively deleting the essential Pol1 co-activator, transcription initiation factor-1A (Tif1a/Rrn3) from excitatory neurons, PTZ kindling was impaired while DG total ribosome content was moderately reduced and no major neurodegeneration was observed throughout the hippocampus. Therefore, Pol1 activity of excitatory neurons is required for PTZ kindling. As seizures affect ribosome biogenesis without long-term effects on the total ribosome content, such a requirement may be associated with a need to produce specialized ribosomes that promote pro-epileptic plasticity.

Proapoptotic Requirement of Ribosomal Protein L11 in Ribosomal Stress-Challenged Cortical Neurons.

While impaired ribosomal biogenesis is observed in neurodegenerative diseases, its pathogenic contributions are not clear. For instance, it is well established that in rodent neurons, genetic inhibition of RNA-polymerase 1 that transcribes rRNA results in structural disruption of the nucleolus, neuronal apoptosis, and neurodegeneration. However, in most neurodegenerative diseases, nucleolar morphology is unaffected. It is reported here that in primary cortical neurons from newborn rats, inhibition of ribosomal biogenesis by shRNA-mediated knockdowns of several ribosomal proteins including S6, S14, or L4 resulted in p53-mediated apoptosis despite absence of structural disruption of the nucleolus. Conversely, knockdown of the RP L11, which in nonneuronal systems mediates p53 activation downstream of ribosomal stress, protected neurons against inhibition of ribosomal biogenesis but not staurosporine. Moreover, overexpression of L11 enhanced p53-driven transcription and increased neuronal apoptosis. In addition, inhibition of p53, or L11 knockdown, blocked apoptosis in response to the RNA analog 5-fluorouridine which perturbed nucleolar structure, inhibited ribosomal synthesis, and activated p53. Although the DNA double-strand break (DSB) inducer etoposide activated p53, nucleolar structure appeared intact. However, by activating the DNA damage response kinase ATM, etoposide increased 47S pre-rRNA levels, and enhanced nucleolar accumulation of nascent RNA, suggesting slower rRNA processing and/or increased Pol1 activity. In addition, shL11 reduced etoposide-induced apoptosis. Therefore, seemingly normal morphology of the neuronal nucleolus does not exclude presence of ribosomal stress. Conversely, targeting the ribosomal stress-specific signaling mediators including L11 offers a novel approach to uncover neurodegenerative contributions of deregulated ribosomal synthesis as exemplified in DSB-challenged neurons.

Ribosomal stress and Tp53-mediated neuronal apoptosis in response to capsid protein of the Zika virus.

We report here that in rat and human neuroprogenitor cells as well as rat embryonic cortical neurons Zika virus (ZIKV) infection leads to ribosomal stress that is characterized by structural disruption of the nucleolus. The anti-nucleolar effects were most pronounced in postmitotic neurons. Moreover, in the latter system, nucleolar presence of ZIKV capsid protein (ZIKV-C) was associated with ribosomal stress and apoptosis. Deletion of 22 C-terminal residues of ZIKV-C prevented nucleolar localization, ribosomal stress and apoptosis. Consistent with a casual relationship between ZIKV-C-induced ribosomal stress and apoptosis, ZIKV-C-overexpressing neurons were protected by loss-of-function manipulations targeting the ribosomal stress effector Tp53 or knockdown of the ribosomal stress mediator RPL11. Finally, capsid protein of Dengue virus, but not West Nile virus, induced ribosomal stress and apoptosis. Thus, anti-nucleolar and pro-apoptotic effects of protein C are flavivirus-species specific. In the case of ZIKV, capsid protein-mediated ribosomal stress may contribute to neuronal death, neurodevelopmental disruption and microcephaly.

Nucleolar Enrichment of Brain Proteins with Critical Roles in Human Neurodevelopment.

To study nucleolar involvement in brain development, the nuclear and nucleolar proteomes from the rat cerebral cortex at postnatal day 7 were analyzed using LC-MS/iTRAQ methodology. Data of the analysis are available via ProteomeXchange with identifier PXD002188. Among 504 candidate nucleolar proteins, the overrepresented gene ontology terms included such cellular compartmentcategories as "nucleolus", "ribosome" and "chromatin". Consistent with such classification, the most overrepresented functional gene ontology terms were related to RNA metabolism/ribosomal biogenesis, translation, and chromatin organization. Sixteen putative nucleolar proteins were associated with neurodevelopmental phenotypes in humans. Microcephaly and/or cognitive impairment were the most common phenotypic manifestations. Although several such proteins have links to ribosomal biogenesis and/or genomic stability/chromatin structure (e.g. EMG1, RPL10, DKC1, EIF4A3, FLNA, SMC1, ATRX, MCM4, NSD1, LMNA, or CUL4B), others including ADAR, LARP7, GTF2I, or TCF4 have no such connections known. Although neither the Alazami syndrome-associated LARP7nor the Pitt-Hopkins syndrome-associated TCF4 were reported in nucleoli of non-neural cells, in neurons, their nucleolar localization was confirmed by immunostaining. In cultured rat hippocampal neurons, knockdown of LARP7 reduced both perikaryal ribosome content and general protein synthesis. Similar anti-ribosomal/anti-translation effects were observed after knockdown of the ribosomal biogenesis factor EMG1 whose deficiency underlies Bowen-Conradi syndrome. Finally, moderate reduction of ribosome content and general protein synthesis followed overexpression of two Pitt-Hopkins syndrome mutant variants of TCF4. Therefore, dysregulation of ribosomal biogenesis and/or other functions of the nucleolus may disrupt neurodevelopment resulting in such phenotypes as microcephaly and/or cognitive impairment.

Requirement of Neuronal Ribosome Synthesis for Growth and Maintenance of the Dendritic Tree.

The nucleolus serves as a principal site of ribosome biogenesis but is also implicated in various non-ribosomal functions, including negative regulation of the pro-apoptotic transcription factor p53. Although disruption of the nucleolus may trigger the p53-dependent neuronal death, neurotoxic consequences of a selective impairment of ribosome production are unclear. Here, we report that in rat forebrain neuronal maturation is associated with a remarkable expansion of ribosomes despite postnatal down-regulation of ribosomal biogenesis. In cultured rat hippocampal neurons, inhibition of the latter process by knockdowns of ribosomal proteins S6, S14, or L4 reduced ribosome content without disrupting nucleolar integrity, cell survival, and signaling responses to the neurotrophin brain-derived neurotrophic factor. Moreover, reduced general protein synthesis and/or formation of RNA stress granules suggested diminished ribosome recruitment to at least some mRNAs. Such a translational insufficiency was accompanied by impairment of brain-derived neurotrophic factor-mediated dendritic growth. Finally, RNA stress granules and smaller dendritic trees were also observed when ribosomal proteins were depleted from neurons with established dendrites. Thus, a robust ribosomal apparatus is required to carry out protein synthesis that supports dendritic growth and maintenance. Consequently, deficits of ribosomal biogenesis may disturb neurodevelopment by reducing neuronal connectivity. Finally, as stress granule formation and dendritic loss occur early in neurodegenerative diseases, disrupted homeostasis of ribosomes may initiate and/or amplify neurodegeneration-associated disconnection of neuronal circuitries.

S100A6 competes with the TAZ2 domain of p300 for binding to p53 and attenuates p53 acetylation.

S100A6 is a calcium binding protein that, like some other members of the S100 protein family, is able to bind p53. This interaction may be physiologically relevant considering the numerous connotations of S100 proteins and of S100A6, in particular, with cancer and metastasis. In this work, we show that the interaction with S100A6 is limited to unmodified or phosphorylated p53 and is inhibited by p53 acetylation. Using in vitro acetylation assay, we show that the presence of S100A6 attenuates p53 acetylation by p300. Furthermore, using ELISA, we show that S100A6 and the TAZ2 domain of p300 bind p53 with similar affinities and that S100A6 effectively competes with TAZ2 for binding to p53. Our results add another element to the complicated scheme of p53 activation.

Tubulin binding protein, CacyBP/SIP, induces actin polymerization and may link actin and tubulin cytoskeletons.

CacyBP/SIP, originally identified as a S100A6 target, was shown to interact with some other S100 proteins as well as with Siah-1, Skp1, tubulin and ERK1/2 kinases (reviewed in Schneider and Filipek, Amino Acids, 2010). Here, we show that CacyBP/SIP interacts and co-localizes with actin in NB2a cells. Using a zero-length cross-linker we found that both proteins bound directly to each other. Co-sedimentation assays revealed that CacyBP/SIP induced G-actin polymerization and formation of unique circular actin filament bundles. The N-terminal fragment of CacyBP/SIP (residues 1-179) had similar effect on actin polymerization as the entire CacyBP/SIP protein, while the C-terminal one (residues 178-229) had not. To check the influence of CacyBP/SIP on cell morphology as well as on cell adhesion and migration, a stable NIH 3T3 cell line with an increased level of CacyBP/SIP was generated. We found that the adhesion and migration rates of the modified cells were changed in comparison with the control ones. Interestingly, the co-sedimentation and proximity ligation assays indicated that CacyBP/SIP could simultaneously interact with tubulin and actin, suggesting that CacyBP/SIP might link actin and tubulin cytoskeletons.

S100A6 (calcyclin) deficiency induces senescence-like changes in cell cycle, morphology and functional characteristics of mouse NIH 3T3 fibroblasts.

S100A6 (calcyclin) is a calcium binding protein with two EF-hand structures expressed mostly in fibroblasts and epithelial cells. We have established a NIH 3T3 fibroblast cell line stably transfected with siRNA against S100A6 to examine the effect of S100A6 deficiency on non-transformed cell physiology. We found that NIH 3T3 fibroblasts with decreased level of S100A6 manifested altered cell morphology and proliferated at a much slower pace than the control cells. Cell cycle analysis showed that a large population of these cells lost the ability to respond to serum and persisted in the G0/G1 phase. Furthermore, fibroblasts with diminished S100A6 level exhibited morphological changes and biochemical features of cellular senescence as revealed by beta-galactosidase and gelatinase assays. Also, S100A6 deficiency induced changes in the actin cytoskeleton and had a profound impact on cell adhesion and migration. Thus, we have shown that the S100A6 protein is involved in multiple aspects of fibroblast physiology and that its presence ensures normal fibroblast proliferation and function.

S100A6 - new facts and features.

S100A6 (calcyclin) is a 10.5kDa Ca(2+)-binding protein that belongs to the S100 protein family. S100A6 contains two EF-hand motifs responsible for binding of Ca(2+). It also binds Zn(2+) through not yet identified structures. Binding of Ca(2+) induces a conformational change in the S100A6 molecule which in consequence increases its overall hydrophobicity and allows for interaction with target proteins. S100A6 was found in different mammalian and avian (chicken) tissues. A high level of S100A6 is observed in epithelial cells, fibroblasts and in different kinds of cancer cells. The function of S100A6 is not clear at present, but it has been suggested that it may be involved in cell proliferation, cytoskeletal dynamics and tumorigenesis. Additionally, S100A6 might have some extracellular activities. This review presents new facts and features concerning the S100A6 protein.

S100A6 binds p53 and affects its activity.

S100A6 (calcyclin) is a calcium-binding protein implicated in many cellular processes and often up-regulated in cancer. Its various biological effects possibly originate from the fact that it may bind to other proteins and modulate their function by inducing conformational changes or interfering with posttranslational modifications. Thus, to elucidate the biological role of S100A6 it is important to identify its targets. Here, we report, based on affinity chromatography and co-immunoprecipitation results that S100A6 interacts with p53 in the presence of calcium ions. We investigated functional implications of the S100A6-p53 interaction by comparing various aspects of p53 activity in HEp-2 cells with either unaltered or diminished S100A6 content due to stable expression of siRNA. We found that the presence of S100A6 results in higher p53 transcriptional activity which is also reflected by higher cell susceptibility to apoptosis evoked by hydrogen peroxide. As revealed by electrophoretic mobility shift assay (EMSA) S100A6 does not affect p53 binding to DNA. On the other hand, we observed that the presence of S100A6 coincides with more efficient nuclear accumulation of p53 under stress conditions. Collectively, our results indicate that S100A6 interacts with p53 and affects its biological activity.

A putative role of the Amyloid Precursor Protein Intracellular Domain (AICD) in transcription.

Amyloid Precursor Protein (APP) Intracellular Domain (AICD) is the product of APP processing realized by alpha- or beta-secretases and gamma-secretase. It was shown that AICD is able to interact with several proteins which regulate its stability and cellular localization. The Fe65 adaptor protein translocates AICD into nucleus where the APP-Fe65-Tip60 ternary complex may activate transcription of target genes. In the light of recent studies AICD seems to be another product of APP proteolysis endowed with important biological functions that may contribute to Alzheimer's disease pathology.

Epigenetic control of the S100A6 (calcyclin) gene expression.

S100A6 (calcyclin) is a calcium-binding protein of cell-specific expression whose gene is clustered with other S100 genes within the epidermal differentiation complex, on human chromosome 1q21. Many S100 proteins, including S100A6, are expressed in human epidermis at various stages of differentiation and their expression is often deregulated in skin and epithelial cancers. To gain insight into the mechanism of regulation of S100A6 expression, we examined epigenetic marks, that is DNA methylation and histone modifications along the S100A6 gene. Sequencing of bisulfite-modified DNA within a 3,247 bp long genomic region encompassing the promoter/first exon CpG island, the coding sequence of the S100A6 gene and a downstream region showed that it is almost entirely methylation-free in S100A6 expressing human epidermoid carcinoma (Hep-2) cells and lymphocytes and methylated in S100A6-negative embryonic epithelial (HEK293) cells. Chromatin immunoprecipitation revealed profound differences in the level of histone H3 acetylation and methylation and in the in vivo binding of upstream regulatory factor (USF), to the S100A6 gene promoter in S100A6-negative and -positive cells. These data demonstrate that cell-specific S100A6 expression is under control of epigenetic mechanisms.