Activation of an Endogenous Suicide Response after Perturbation of rRNA Synthesis Leads to Neurodegeneration in Mice (original) (raw)
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Inhibition of nucleolar transcription as a trigger for neuronal apoptosis
Journal of Neurochemistry, 2008
In postmitotic 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, BDNF-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, shRNA-mediated inhibition of a RNA-Polymerase-I co-factor, TIF-IA, attenuated rRNA transcription causing nucleolar stress and p53-dependent neuronal apoptosis. The protein synthesis inhibitor cycloheximide blocked apoptosis that was induced by overexpressed 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 postmitotic neurons as sensors of DNA damage coupling reduced rRNA transcription to p53-mediated apoptosis that requires denovo expression of protein-coding genes. Thus, rDNA selectivity of DNA damage may determine its ability to induce neuronal apoptosis.
Impaired rRNA synthesis triggers homeostatic responses in hippocampal neurons
Frontiers in Cellular Neuroscience, 2013
Decreased rRNA synthesis and nucleolar disruption, known as nucleolar stress, are primary signs of cellular stress associated with aging and neurodegenerative disorders. Silencing of rDNA occurs during early stages of Alzheimer's disease (AD) and may play a role in dementia. Moreover, aberrant regulation of the protein synthesis machinery is present in the brain of suicide victims and implicates the epigenetic modulation of rRNA. Recently, we developed unique mouse models characterized by nucleolar stress in neurons. We inhibited RNA polymerase I by genetic ablation of the basal transcription factor TIF-IA in adult hippocampal neurons. Nucleolar stress resulted in progressive neurodegeneration, although with a differential vulnerability within the CA1, CA3, and dentate gyrus (DG). Here, we investigate the consequences of nucleolar stress on learning and memory. The mutant mice show normal performance in the Morris water maze and in other behavioral tests, suggesting the activation of adaptive mechanisms. In fact, we observe a significantly enhanced learning and re-learning corresponding to the initial inhibition of rRNA transcription. This phenomenon is accompanied by aberrant synaptic plasticity. By the analysis of nucleolar function and integrity, we find that the synthesis of rRNA is later restored. Gene expression profiling shows that 36 transcripts are differentially expressed in comparison to the control group in absence of neurodegeneration. Additionally, we observe a significant enrichment of the putative serum response factor (SRF) binding sites in the promoters of the genes with changed expression, indicating potential adaptive mechanisms mediated by the mitogen-activated protein kinase pathway. In the DG a neurogenetic response might compensate the initial molecular deficits. These results underscore the role of nucleolar stress in neuronal homeostasis and open a new ground for therapeutic strategies aiming at preserving neuronal function.
A neuroprotective phase precedes striatal degeneration upon nucleolar stress
Cell Death and Differentiation, 2013
The nucleolus is implicated in sensing and responding to cellular stress by stabilizing p53. The pro-apoptotic effect of p53 is associated with several neurodegenerative disorders, including Huntington's disease (HD), which is characterized by the progressive loss of medium spiny neurons (MSNs) in the striatum. Here we show that disruption of nucleolar integrity and function causes nucleolar stress and is an early event in MSNs of R6/2 mice, a transgenic model of HD. Targeted perturbation of nucleolar function in MSNs by conditional knockout of the RNA polymerase I-specific transcription initiation factor IA (TIF-IA) leads to late progressive striatal degeneration, HD-like motor abnormalities and molecular signatures. Significantly, p53 prolongs neuronal survival in TIF-IA-deficient MSNs by transient upregulation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor that inhibits mammalian target of rapamycin signaling and induces autophagy. The results emphasize the initial role of nucleolar stress in neurodegeneration and uncover a p53/PTEN-dependent neuroprotective response.
Brain Pathology
The Purkinje cell (PC) degeneration (pcd) phenotype results from mutation in nna1 gene and is associated with the degeneration and death of PCs during the postnatal life. Although the pcd mutation is a model of the ataxic mouse, it shares clinical and pathological characteristics of inherited human spinocerebellar ataxias. PC degeneration in pcd mice provides a useful neuronal system to study nuclear mechanisms involved in DNA damage-dependent neurodegeneration, particularly the contribution of nucleoli and Cajal bodies (CBs). Both nuclear structures are engaged in housekeeping functions for neuronal survival, the biogenesis of ribosomes and the maturation of snRNPs and snoRNPs required for pre-mRNA and pre-rRNA processing, respectively. In this study, we use ultrastructural analysis, in situ transcription assay and molecular markers for DNA damage, nucleoli and CB components to demonstrate that PC degeneration involves the progressive accumulation of nuclear DNA damage associated with disruption of nucleoli and CBs, disassembly of polyribosomes into monoribosomes, ribophagy and shut down of nucleolar and extranucleolar transcription. Microarray analysis reveals that four genes encoding repressors of nucleolar rRNA synthesis (p53, Rb, PTEN and SNF2) are upregulated in the cerebellum of pcd mice. Collectively, these data support that nucleolar and CB alterations are hallmarks of DNA damage-induced neurodegeneration.
Molecular Pharmacology
We describe a new molecular mechanism of cell death by excitotoxicity mediated through nuclear transcription factor B (NFB) in rat embryonic cultures of dopaminergic neurons. Treatment of mesencephalic cultures with ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) resulted in a number of changes that occurred selectively in dopaminergic neurons, including persistent elevation in intracellular Ca 2ϩ monitored with Fura-2, and a significant increase in intramitochondrial oxidation of dihydrorhodamine 123, probably asso-ciated with transient increase of mitochondrial permeability, cytochrome c release, nuclear translocation of NFB, and transcriptional activation of the oncogene p53. Interruption of any of these steps by specific antagonists prevented neurite pruning and programmed cell death. In contrast, cell death was not prevented by caspase antagonists and only partly prevented by nitric-oxide synthase inhibitors. This signal transduction pathway might be a contributing mechanism in ongoing neuronal death in Parkinson disease.
Molecular Cell, 2005
cell growth and proliferation, transcription of ribosomal RNA genes by RNA polymerase I (Pol I) is efficiently 1 Division of Molecular Biology of the Cell II 2 Division of Molecular Biology of the Cell I regulated in response to changes in growth and specific environmental challenges, such as growth factors, 3 Division of Cellular and Molecular Pathology German Cancer Research Center drugs, stress, or nutrient availability (for review, see Grummt, 2003). The key factor that transduces extra-Im Neuenheimer Feld 280 D-69120 Heidelberg cellular signals to the Pol I transcription apparatus is the transcription initiation factor TIF-IA, the mammalian Germany homolog of yeast Rrn3p (Schnapp et al., 1990; Yamamoto et al., 1996; Bodem et al., 2000). TIF-IA interacts Summary with both Pol I and the TBP-containing factor TIF-IB/ SL1, and these interactions are required to recruit Pol Growth-dependent regulation of rRNA synthesis is I to the rDNA promoter and to generate a productive mediated by TIF-IA, a basal transcription initiation transcription initiation complex (Miller et al., 2001; Yuan factor for RNA polymerase I. We inactivated the muet al., 2002). TIF-IA is phosphorylated at multiple sites rine TIF-IA gene by homologous recombination in by a complex network of protein kinases. External sigmice and embryonic fibroblasts (MEFs). TIF-IA −/− emnals that affect cell growth and proliferation alter the bryos die before or at embryonic day 9.5 (E9.5), disphosphorylation pattern of TIF-IA, modulate TIF-IA acplaying retardation of growth and development. In tivity, and regulate Pol I transcription (Zhao et al., 2003; MEFs, Cre-mediated depletion of TIF-IA leads to dis-Mayer et al., 2004). Thus, TIF-IA plays a key role in the ruption of nucleoli, cell cycle arrest, upregulation of signaling networks that control cell growth and prolifer-p53, and induction of apoptosis. Elevated levels of ation. p53 after TIF-IA depletion are due to increased bind-To elucidate the mechanisms that link TIF-IA activity ing of ribosomal proteins, such as L11, to MDM2 and to ribosome synthesis and cell proliferation, we disdecreased interaction of MDM2 with p53 and p19 ARF . rupted the TIF-IA gene in mouse embryonic stem cells RNAi-induced loss of p53 overcomes proliferation arby homologous recombination and generated mice rest and apoptosis in response to TIF-IA ablation. The harboring the mutant TIF-IA allele. Mice that are homostriking correlation between perturbation of nucleolar zygous for the mutant TIF-IA allele die before or at E9.5. function, elevated levels of p53, and induction of cell TIF-IA −/− embryos are extremely small, with a retarded suicide supports the view that the nucleolus is a and discordant development and an increased rate of stress sensor that regulates p53 activity. apoptosis. Cre-mediated depletion of TIF-IA in embryonic fibroblasts (MEFs) not only abrogates Pol I
Dna Repair, 2008
According to a long-standing hypothesis, aging is mainly caused by accumulation of nuclear (n) DNA damage in differentiated cells such as neurons due to insufficient nDNA repair during lifetime. In line with this hypothesis it was until recently widely accepted that neuron loss is a general consequence of normal aging, explaining some degree of decline in brain function during aging. However, with the advent of more accurate procedures for counting neurons, it is currently widely accepted that there is widespread preservation of neuron numbers in the aging brain, and the changes that do occur are relatively specific to certain brain regions and types of neurons. Whether accumulation of nDNA damage and decline in nDNA repair is a general phenomenon in the aging brain or also shows cell-type specificity is, however, not known. It has not been possible to address this issue with the biochemical and molecular-biological methods available to study nDNA damage and nDNA repair. Rather, it was the introduction of autoradiographic methods to study quantitatively the relative amounts of nDNA damage (measured as nDNA single-strand breaks) and nDNA repair (measured as unscheduled DNA synthesis) on tissue sections that made it possible to address this question in a cell-type-specific manner under physiological conditions. The results of these studies revealed a formerly unknown inverse relationship between age-related accumulation of nDNA damage and age-related impairment in nDNA repair on the one hand, and the age-related, selective, loss of neurons on the other hand. This inverse relation may not only reflect a fundamental process of aging in the central nervous system but also provide the molecular basis for a new approach to understand the selective neuronal vulnerability in neurodegenerative diseases, particularly Alzheimer's disease.
Erratic expression of DNA polymerases by beta-amyloid causes neuronal death
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 2002
An ectopic reentrance into the cell cycle with ensuing DNA replication is required for neuronal apoptosis induced by beta-amyloid. Here, we investigate the repertoire of DNA polymerases expressed in beta-amyloid-treated neurons, and their specific role in DNA synthesis and apoptosis. We show that exposure of cultured cortical neurons to beta-amyloid induces the expression of DNA polymerase-beta, proliferating cell nuclear antigen, and the p49 and p58 subunits of DNA primase. Induction requires the activity of cyclin-dependent kinases. The knockdown of the p49 primase subunit prevents beta-amyloid-induced neuronal DNA synthesis and apoptosis. Similar effects are observed by knocking down DNA polymerase-beta or by using dideoxycytidine, a preferential inhibitor of this enzyme. Thus, the reparative enzyme DNA polymerase-beta unexpectedly mediates a large component of de novo DNA synthesis and apoptotic death in neurons exposed to beta-amyloid. These data indicate that DNA polymerases b...