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.

Neurodegeneration-associated instability of ribosomal DNA.

Homologous recombination (HR)-mediated instability of the repetitively organized ribosomal DNA (rDNA) has been proposed as a mediator of cell senescence in yeast triggering the DNA damage response. High individual variability in the content of human rDNA suggests that this genomic region remained relatively unstable throughout evolution. Therefore, quantitative real-time polymerase chain reaction was used to determine the genomic content of rDNA in post mortem samples of parietal cortex from 14 young and 9 elderly individuals with no diagnosis of a chronic neurodegenerative/neurological disease. In addition, rDNA content in that brain region was compared between 10 age-matched control individuals and 10 patients with dementia with Lewy bodies (DLB) which involves neurodegeneration of the cerebral cortex. Probing rRNA-coding regions of rDNA revealed no effects of aging on the rDNA content. Elevated rDNA content was observed in DLB. Conversely, in the DLB pathology-free cerebellum, lower genomic content of rDNA was present in the DLB group. In the parietal cortex, such a DLB-associated instability of rDNA was not accompanied by any major changes of cytosine-phosphate-guanine methylation of the rDNA promoter. As increased cerebro-cortical rDNA content was previously reported in Alzheimer's disease, neurodegeneration appears to be associated with instability of rDNA. The hypothetical origins and consequences of this phenomenon are discussed including possibilities that the DNA damage-induced recombination destabilizes rDNA and that differential content of rDNA affects heterochromatin formation, gene expression and/or DNA damage response. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.

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.

Pharmacological inhibition of spinal cord injury-stimulated ribosomal biogenesis does not affect locomotor outcome.

After unresolved endoplasmic reticulum stress, recovery of protein synthesis including increased expression of ribosomal components and translation factors may induce cell death. Using a mouse model of moderate contusive spinal cord injury (SCI) at the T9 level, upregulation of ribosomal biogenesis was observed in the injury epicenter at 24h after trauma. Such upregulation coincided with endoplasmic reticulum stress response as previously reported in this model. It was also accompanied by changes in expression of many other genes associated with translational regulation. Systemic treatment with a pharmacological inhibitor of RNA-Polymerase-1, BMH-21 reduced rRNA transcription in the spinal cord. Moreover, in the injury epicenter, treatment with BMH-21 increased expression of oligodendrocyte-specific transcripts including Mbp and Cldn11 at 3days post injury. Although such findings may suggest at least transient reduction of oligodendrocyte death, locomotor outcome was mostly unaffected except slightly accelerated recovery of hindlimb function at week 2 post-injury. Therefore, at least in mice, RNA-Polymerase-1 does not appear to be a robust target for therapies to protect spinal cord tissue after contusion. However, these findings raise an interesting possibility that altered rate of ribosomal biogenesis contributes to the apparent translational reprogramming after contusive SCI. Such a reprogramming could be a major regulator of SCI-induced gene expression.