Neurological Disease Insights Through Translatomics
The dysregulation of translation in the central nervous system (CNS) has been linked to multiple neurodegenerative diseases, making translatomics a particularly important set of tools for learning and understanding more about neurological disease. Among the diseases in which translation has been implicated are amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), both of which can be promoted by TAR DNA-binding protein 43 (TDP-43) – an mRNA specific translational promoter that plays a role in mRNA transport. The neurological disease Fragile X syndrome is caused by the depletion of FMRP where translation becomes dysregulated leading to dendrite pathology in the brain. While the deletion of GTP-binding protein 2 (GTPBP2), which regulates stalling at the AGA codon, promotes a spontaneous ataxia-like neurodegenerative phenotype. Below is a discussion of four papers that exemplify the impact that translatomics can make in the area of neurological disease research.
Polysome Profiling Links Translational Control to the Radioresponse of Glioblastoma Stem-like Cells
Cancer Research, 2016; 76(10), pp.3078–3087
Wahba, A., Rath, B.H., Bisht, K., Camphausen, K. and Tofilon, P.J.,
Glioblastoma (GBM) is a type of cancer of the glial cells in the brain and is the most aggressive of all intracranial tumours. Radiotherapy is a highly cost-effective treatment for glioblastomas but the DNA damage induced by the radiation can trigger a signaling cascade that mediates radioresistance – a major cause of treatment failure in patients with GBM. The aim of this paper was to better understand the effect of ionising radiation (IR) on the human glioblastoma translatome using a set of human glioblastoma stem-like cell (GSC) lines and polysome profiling. Initially the authors conducted this study on established glioma cell lines 6 hours after exposure to 7Gy. The authors then carried out the investigation on three glioblastoma stem-like cell (GSC) lines (NSC11, 0923, and GBMJ1) and collected polysome-bound mRNA 1- 6 hours after exposure to 2 Gy. To determine whether or not the IR-induced changes observed are of biological significance, the authors then investigated the genes affected in terms of cellular processes and pathways.
- Radiation primarily modifies gene expression via translational control.
- DNA repair and cell cycle checkpoint regulation were among pathways that were upregulated after IR exposure.
- Cellular processes not traditionally associated with radioresponse such as activation of eIF4E and mTOR were activated by exposure to IR. This suggests cap-dependent translation is increased after exposure of GSC to IR.
- Mitochondrial response to IR has a high cell line specificity.
These data show that IR-induced translational control plays a significant role in the cellular response to IR in glioblastoma. This response influences cell survival and thus plays a role in radioresistance in this tumour type. Understanding more about how translational control of these genes responds to IR exposure offers as a target for glioblastoma radiosensitisation which could hopefully reduce relapse in patients treated for GBM.
Defects in mRNA Translation in LRRK2-Mutant hiPSC-Derived Dopaminergic Neurons Lead to Dysregulated Calcium Homeostasis
Cell Stem Cell, 2020; 27(4), pp.633-645.
Kim, J.W., Yin, X., Jhaldiyal, A., Khan, M.R., Martin, I., Xie, Z., Perez-Rosello, T., Kumar, M., Abalde-Atristain, L., Xu, J. and Chen, L.
Parkinson’s disease (PD) is the second most prevalent neurodegenerative disorder globally. The G2019S mutation in leucine-rich repeat kinase 2 (LRRK2) is a common cause of familial PD which causes dysregulated translation and results in the death of dopaminergic (DA) neurons. It is still unknown, however, how alterations in protein synthesis contribute to this neurodegeneration in humans. The authors hypothesised that dysregulated translation in G2019S LRRK2 may result in changes in the levels of highly regulated proteins that are essential for the long-term survival of DA neurons. The authors used LRRK2-mutant DA neurons derived from the human induced pluripotent stem cells (hiPSCs) of PD patients and investigated their translatome via ribosome profiling and RNA-seq profiling. The authors also differentiated isogenic (of the same genetic background) iPSC lines into human cortical neurons and measured their cytosolic Ca2+ levels in order to determine whether Ca2+ dysregulation caused by G2019S LRRK2 only existed in DA neurons, or whether DA neurons were more susceptible to Ca2+ dysregulation.
- mRNAs that have complex secondary structure in the 5′ untranslated region (UTR) are translated more efficiently in G2019S LRRK2 neurons
- A change in expression levels is observed for various genes involved in Ca2+ homeostasis in neurons, including voltage-gated Ca2+ channel (VGCC) subunits. This leads to an increase in Ca2+ influx, leading to elevated intracellular Ca2+ concentration in DA neurons.
- Ca2+ homeostasis is disrupted which potentially contributes to DA neurotoxicity in PD.
- Using a pair of isogenic iPSC lines, it was observed that correction of the G2019S LRRK2 mutation rescues elevated Ca2+ levels in human DA neurons. Notably, phosphorylation of the S15 T136 residue is reduced in human DA neurons derived from the mutation-corrected line.
The findings in this study has paved the way for future studies on the mechanisms involved in G2019S LRRK2 PD. The authors recommend that future studies aim at investigating the molecular crosstalk they observed between proteostasis and Ca2+ homeostasis, and in doing so this will provide a much more comprehensive insight into the neurodegenerative mechanisms in G2019S LRRK2 PD.
Ribosomal profiling during prion disease uncovers progressive translational derangement in glia but not in neurons
eLife, 2020; 9, p.e62911.
Scheckel, C., Imeri, M., Schwarz, P. and Aguzzi, A.
Prion diseases (PrD) are fatal neurodegenerative diseases that are caused by prions – a type of highly transmissible misfolded protein that causes healthy proteins in the brain to also misfold. Prions consist largely of PrPSc – pathological aggregates of the cellular prion protein (PrPC). In this study a murine model of PrD was used, which has previously been shown to represent most aspects of human PrD, including transcriptomic changes. The mouse strain used expressed a GFP-tagged version of a large ribosomal protein, RPL10a, in a Cre-dependent manner (loxP sites on either side of a stop codon placed upstream of a gene of interest prevents gene expression in the absence of Cre, but the stop codon is excised in the presence of Cre allowing gene expression to proceed). This was crossed with several Cre driver lines, expressing Cre recombinase under the control of the various promoters to induce the expression of GFP-tagged ribosomes in excitatory CamKIIa neurons, inhibitory parvalbumin (PV) neurons, astrocytes or microglia. A pull-down of the GFP tagged ribosomes allows a cell-type-specific determination of gene expression. Mice were intraperitoneally injected with RML6 prions or control brain homogenates and sacrificed at various time points during disease progression. The terminal time point for PrD in mice (31-32 weeks) was determined by onset of disease symptoms which included piloerection, hind limb clasping, kyphosis and ataxia. A combination of TRAP-seq and ribosome profiling was then used to characterise cell-type-specific molecular changes in PrD. TRAP-seq was used to identify which transcripts were translated in a given cell, while ribosome profiling determined the number of ribosome-protected fragments (RPFs), providing information on the translation rate.
- The authors observed a correlation in gene expression in both astrocytes and microglial cells and a high overlap at the final two time points (24 and 31-32 weeks post-inoculation (wpi)) especially among upregulated genes. From this it was shown that the pronounced translational changes in astrocytes and microglia become evident 2 months before the terminal PrD stage (31-32 wpi), which is long before any PrD-related symptoms manifest.
- The authors found that there was a weak correlation between the translational changes in the different cell types, i.e. that most prion-induced changes are cell-type-specific.
- The murine PrD model assessed here did not show a major decrease in neurons at the terminal stage. While this has been shown previously experimentally, it is in contrast with the pathological changes characteristic for human PrD patients.
- The authors observed that neurotrophic (A2) astrocyte genes and Gadd34 increase in astrocytes, suggesting that astrocytes might play a central role in limiting prion toxicity.
The data suggest that aberrant translation within glia (non-neuronal cells in the central nervous system) may suffice to cause severe neurological symptoms and may even be the primary driver of prion disease.
Mutant Huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease
Nature Communications, 2021; 12(1), pp.1-20.
Eshraghi, M., Karunadharma, P.P., Blin, J., Shahani, N., Ricci, E.P., Michel, A., Urban, N.T., Galli, N., Sharma, M., Ramírez-Jarquín, U.N. and Florescu, K
Huntington’s disease (HD) is a debilitative brain disorder that affects a region of the brain called the striatum. The genetic cause of the disease is a CAG codon expansion of HTT gene resulting in mutant HTT (mHTT) protein. The onset and severity of symptoms depend on the number of CAG repeats. The mechanism by which this is responsible for the HD has not been identified. However, altered ribosomal functions and association of HTT and mHTT with translating ribosomes were reported in HD model systems and HD patient-tissue. Evidence for either the role or mechanism of HTT in the regulation of protein synthesis is limited. The study investigated using striatal neuronal cells that express a targeted insertion of a chimeric human–mouse exon 1 with 7/7 CAG (STHdhQ7/Q7, control), 7/111 CAG (STHdhQ7/Q111, HD-het), and 111/111 CAG (STHdhQ111/Q111, HD-homo) repeats. The authors used polysome profiling as well as ribosome profiling under various conditions.
- A high polysome/monosome (PS/MS) ratio was found in the HD-homo compared to the HD-het or control cells. The authors tested their hypothesis that the high PS/MS ratio in HD-homo striatal cells could reflect a pause in ribosome movements using ribosome run-off experiments with harringtonine. If ribosomes are stalled, then the cells will have a higher PS/MS ratio than in cells in which ribosomes are not stalled.
- FMRP protein levels were significantly upregulated in the HD-homo cells, as well as in human HD patient striatum, whereas the total Fmr1 mRNA was differentially altered. Collectively, PS-RRA-mRNA-Seq data suggested that FMRP is upregulated in HD. Fmr1 depletion has no discernible effect on protein synthesis or ribosome stalling in HD cells.
- Global ribosome profiling reveals diverse 5′ and 3′ ribosome occupancy on mRNA transcripts in HD cells.
The data suggest that aberrant translation within glia (nonThis study found that wtHTT inhibits protein synthesis by inhibiting the speed of ribosomal translocation. This is exacerbated by mHTT, which further hinders protein synthesis and slows down ribosomal translocation. There are no effective therapies available that are directed toward inhibiting the abnormal functions of mHTT in HD. Therefore, outlining the mechanisms of mHTT in HD are an important part in developing effective therapies. This study provides a rich source of potential unknown targets for drug discovery in HD research.-neuronal cells in the central nervous system) may suffice to cause severe neurological symptoms and may even be the primary driver of prion disease.
