RRM adjacent TARDBP mutations disrupt RNA binding and enhance TDP-43 proteinopathy

Amyotrophic lateral sclerosis (ALS) presents with focal muscle weakness due to motor neuron degeneration that becomes generalized,leading to death from respiratory failure within 3–5 years from symptom onset. Despite the heterogeneity of aetiology, TDP- 43 proteinopathy is a common pathological feature that is observed in 495% of ALS and tau-negative frontotemporal dementia(FTD) cases. TDP-43 is a DNA/RNA-binding protein that in ALS and FTD translocates from being predominantly nuclear to formdetergent-resistant, hyperphosphorylated aggregates in the cytoplasm of affected neurons and glia. Mutations in TARDBP accountfor 1–4% of all ALS cases and almost all arise in the low complexity C-terminal domain that does not affect RNA binding andprocessing. Here we report an ALS/FTD kindred with a novel K181E TDP-43 mutation that is located in close proximity to the RRM1 domain. To offer predictive gene testing to at-risk family members, we undertook a series of functional studies to characterizethe properties of the mutation. Spectroscopy studies of the K181E protein revealed no evidence of significant misfolding.Although it is unable to bind to or splice RNA, it forms abundant aggregates in transfected cells. We extended our study to includeother ALS-linked mutations adjacent to the RRM domains that also disrupt RNA binding and greatly enhance TDP-43 aggregation,forming detergent-resistant and hyperphosphorylated inclusions. Lastly, we demonstrate that K181E binds to, and sequesters, wild-type TDP-43 within nuclear and cytoplasmic inclusions. Thus, we demonstrate that TDP-43 mutations that disrupt RNAbinding greatly enhance aggregation and are likely to be pathogenic as they promote wild-type TDP-43 to mislocalize andaggregate acting in a dominant-negative manner. This study highlights the importance of RNA binding to maintain TDP-43solubility and the role of TDP-43 aggregation in disease pathogenesis

HMGA2 protects induced pluripotent stem cell chromosomes against hydroxyurea-induced DNA damage.

High Motility Group A-T hook 2 (HMGA2) is a transcriptional regulator that binds to short AT rich sequences and is involved in global chromatin reorganization. HMGA2 is normally expressed during early development and is involved in specification of mesoderm-derived tissue. However, HMGA2 is re-expressed in many aggressive neoplasias and correlated with poor patient outcomes. Our previous research has shown an involvement of HMGA2 in Base Excision Repair (BER) and in stabilizing stalled replication forks. Cancer cells could hijack these properties of HMGA2 to suppress chromosomal instabilities. In this study, we use murine induced Pluripotent Stem Cells (iPSC) to investigate whether overexpression of HMGA2 provides protection against DNA damage. The chemotherapeutic Hydroxyurea (HU) was used to disrupt DNA synthesis, create stalled forks and induce DNA damage. Chromosomal instabilities were investigated via Giemsa staining of metaphase spreads. Our results clearly show fewer chromosomal aberrations for cells that express exogenous HMGA2 in comparison to the control. We also found overexpression of HMGA2 could increase cell survival at a high HU concentration, and that it reduces the rate of cell proliferation. These results are in excellent agreement with our previous findings and demonstrate a key role for HMGA2 in the cell cycle.Bachelor of Science in Biological Science

Fully automated leg tracking of Drosophila neurodegeneration models reveals distinct conserved movement signatures.

Some neurodegenerative diseases, like Parkinsons Disease (PD) and Spinocerebellar ataxia 3 (SCA3), are associated with distinct, altered gait and tremor movements that are reflective of the underlying disease etiology. Drosophila melanogaster models of neurodegeneration have illuminated our understanding of the molecular mechanisms of disease. However, it is unknown whether specific gait and tremor dysfunctions also occur in fly disease mutants. To answer this question, we developed a machine-learning image-analysis program, Feature Learning-based LImb segmentation and Tracking (FLLIT), that automatically tracks leg claw positions of freely moving flies recorded on high-speed video, producing a series of gait measurements. Notably, unlike other machine-learning methods, FLLIT generates its own training sets and does not require user-annotated images for learning. Using FLLIT, we carried out high-throughput and high-resolution analysis of gait and tremor features in Drosophila neurodegeneration mutants for the first time. We found that fly models of PD and SCA3 exhibited markedly different walking gait and tremor signatures, which recapitulated characteristics of the respective human diseases. Selective expression of mutant SCA3 in dopaminergic neurons led to a gait signature that more closely resembled those of PD flies. This suggests that the behavioral phenotype depends on the neurons affected rather than the specific nature of the mutation. Different mutations produced tremors in distinct leg pairs, indicating that different motor circuits were affected. Using this approach, fly models can be used to dissect the neurogenetic mechanisms that underlie movement disorders