Theme 03 - In Vitro Experimental Models

The aggregation of amyloidogenic proteins/peptides is asso- ciated with the onset and progression of several amyloidoses, including neurodegenerative disorders, such as Alzheimer ‘ s or Parkinson ‘ s diseases. In recent years, it has been observed that the cross-interaction between these protein molecules may be one of the main influences for amyloid aggregate formation. This was first suggested by the presence of different proteins in amyloid plaques found in the nervous tissue of patients. While the cross-interaction between Alzheimer ‘ s disease-related amyloid-beta and tau protein is documented in numerous cases (1,2), there is a lack of information on other protein coaggregation. One such paring, which was examined in this work, was superoxide dismutase-1 (SOD1, related to amyotrophic lateral sclerosis) and prion protein (PrP, relation to prionopathies). Both of them share a localisation in vivo and there was only a single report regarding the influence that SOD1 had on the aggregation of PrP (3), which prompted this investigation. In order to investigate the influence of non-aggregated SOD1 on prion protein fibril formation, conditions were chosen which did not facilitate the aggregation SOD1, while actively promoting the fibrilization of PrP. A large number of samples was analyzed using different SOD1 concentrations to determine if there was a notable effect induced by the pro- tein cross-interaction. The resulting fibrils were examined using Fourier-transform infrared spectroscopy (FTIR) and atomic force microscopy (AFM). Parameters obtained from each assay were compared between each condition to determine the significance of SOD1 influence on PrP aggregation. It was discovered that the presence of SOD1 increased the lag time of PrP fibril formation and reduced their apparent rate of replication. There was also Background: The complement cascade is a critical compo-nent of the immune system, and its activation has been implicated in ALS (1). Furthermore, complement cascade components (e.g. C3 breakdown products) are reported to be deposited on the neuromuscular junction (NMJ) in people living with restored NMJ numbers to 76.1% of the ND control and fatigue index by 30.1% from hCS. In TDP-43 NMJs, hCS reduced NMJ numbers by 82.9% of the ND control and increased fatigue index by 22.8% (stimulation frequency of 2Hz), while the addition of pegcetacoplan restored NMJ numbers to 60.5% of the ND control and fatigue index by 9% from hCS. Furthermore, fidelity was increased in SOD-1 NMJs following treatment of pegcetacoplan and remained stable in TDP-43 NMJs. Conclusions: These data demonstrate that inhibiting C3 with pegcetacoplan in an inflammatory environment may improve overall NMJ survival and function. Background: Neuroinflammation plays a significant role in the onset and progression of amyotrophic lateral sclerosis (ALS), which presently has no effective treatment. Peptidylprolyl cis-/trans-isomerase A (PPIA), also as Background: RNA-binding proteins (RBPs) assemble into cytoplasmic complexes with mRNAs to control mRNA local translation and axonal transport. These processes are essential for maintaining neuronal survival and their impairment is implicated in the development of many neurodegenerative diseases, such as ALS (1). We have discovered that TDP-43 depletion, linked to ALS, drives the accumulation of an alter-natively spliced variant of heterogeneous nuclear ribonucleo- protein A1 (hnRNP A1), (2). This isoform, termed hnRNP A1B, has an elongated prion-like domain (PrLD) and is present in neuronal processes, while hnRNP A1 is absent (3). This finding supports a hypothesis that hnRNP A1B may have a cytosolic function in neurons that is not shared with hnRNP A1. In add- ition, hnRNP A1 and hnRNP A1B are mutated in rare cases of familial ALS with some mutation specific to hnRNP A1B. To date, the literature has mostly focused on the hnRNPA1 iso- form and little is known about hnRNP A1B function. Due to the key role of RNA binding proteins in mRNAs processes, a deeper understanding of their functions is needed to enlighten their contribution to physiology and pathology. Objectives: Identify and characterize hnRNP A1B cytoplasmic function in motoneurons and its potential impairment in ALS. Methods: Neuronal degeneration recognized as a predominant driver of disability and progression in central nervous system diseases such as sclerosis and Successful for these RNA binding hallmark pathological in these disorders, them as proteinopathies. Fused in sarcoma (FUS), a nucleus RBP, is to into physiological granules pathological inclusions which can cell homeostasis to neuronal cell death. Mutations in FUS its C-terminal nuclear localization autosomal dominant in and dis- its cytoplasmic signalling involved overexpressed in a model cell line. Methods: We used the BioID2 proximity labelling technique harnesses the ability of the enzyme biotin ligase to biotinylate proximal endogenous proteins. We prepared constructs of the FUS and FUSdNLS conjugated to BioID2 enzyme by a flexible linker and transiently expressed them in HEK293T cells. We cleared the biotinylated proteins from cell lysates and analyzed them by mass spectrometry. Results: Bioinformatic analyses of proteomic data identified interaction candidates involved in RNA processing and degradation, protein translation and various signal transduction pathways. Selected interactions were validated by pull-down assay and cell co-localization analyses in vitro. In vitro analyses NUDT21 and its nuclear expression, whereas NUDT21 interaction FUSdNLS, effects on 3 0 RNA cleavage and polyadenylation processing. has recognised as a hallmark of ALS. Motor neurons are particularly vulnerable to metabolic deficits due to their high energy demands, which are required for maintaining cellular processes including axonal transport. Mouse embryonic stem cell-derived motor neurons (mESC-MNs) provide an excellent model for high-throughput pheno- typic screening. Our group has previously developed a BAC transgenic mESC-MN model that expresses TDP-43WT or TDP-43 M337V at low levels. In this model, TDP-43 M337V mESC-MNs have previously been shown to demonstrate altered TDP-43 interactions, dysregulated stress granule char-acteristics and reduced survival in response to oxidative stress compared to TDP-43WT controls (1,2). Objectives: In this study, we aim to investigate cellular energy metabolism and axonal transport in TDP-43 M337V mESC-MNs compared to controls, and to examine the effects of a pro-survival drug on these pathways. Methods: Mouse ESCs were expanded as embryoid bodies and differentiated to MNs (1). Mitochondrial respiration and glycolysis were examined in unstressed and oxidative stress environments, using the Seahorse XF Analyser. To investigate axonal transport, mESCs were seeded into microfluidic cham- bers and incubated with MitoTracker, then imaged with live cell microscopy. To examine the effects of a pro-survival drug on cellular metabolism, mESC-MNs were treated with the drug at optimised concentrations 24-hours prior to analysis. Results: Analysis revealed no differences in cellular energy metabolism in unstressed conditions, however following oxidative stress we observed a significant reduction in glycolysis and mitochondrial spare respiratory capacity in TDP-43 M337V mESC-MNs relative to TDP-43WT controls. Following oxidative stress, treatment with a pro-survival drug signifi- cantly increased glycolysis and reduced mitochondrial respiration in TDP-43 M337V mESC-MNs. The speed of mitochondrial transport in TDP-43 M337V mESC-MNs was significantly reduced compared to TDP-43WT controls in unstressed conditions. Conclusions: Here we identify metabolic dysregulation following oxidative stress and impaired axonal transport in TDP- 43 M337V mESC-MNs. Treatment with a pro-survival drug leads to a significant increase in basal glycolysis accompanied by a significant decrease in basal respiration, suggesting this drug may induce neuroprotective effects through inducing a metabolic shift. Background: Heterozygous loss-of-function mutations in TANK-binding kinase 1 (TBK1) have been associated with sporadic and familial forms of ALS and FTD (1,2). TBK1 is a ubiquitously expressed serine/threonine protein kinase central to numerous cellular signalling pathways, including autophagy and immune signalling. Whilst cell autonomous dysfunction in neurons has been shown to contribute to ALS/FTD pathogenesis following TBK1 haploinsufficiency, the impact of TBK1 loss-of-function in microglia is less well understood. TBK1 is highly expressed in microglia, has an established role in the peripheral innate immune system, and interacts with other key ALS/FTD-associated genes, suggesting that disease-associated TBK1 mutations may cause microglial dysfunction in ALS/FTD. Objectives: We aim to investigate the molecular function of TBK1 in microglia and understand how ALS/FTD-associated TBK1 mutations may intrinsically impair microglial function to drive disease pathogenesis. Methods: To investigate the cell autonomous impact of TBK1 haploinsufficiency in microglia, we conducted TMT-based LC-MS/MS bulk proteomics of human HMC3 microglia treated TBK1 inhibitor GSK8612 Key dysregulated in human microglia, mouse BV2 microglia and human pluripotent stem cell-derived microglia. value Gene ontology enrichment and protein-protein interaction analysis of up- and downre- gulated protein sets highlighted significant dysregulation of interferon signalling, as well as deficits in phagocytosis, anti-gen presentation and the damage response. deficits in the type I interferon response confirmed, IRF3 and attenuated induction of IRF3-regulated genes (e.g. following TBK1 kinase inhibition. pHrodo-conjugated E. bioparticles to be impaired. We demonstrated that TBK1 loss of kinase function results in microglial dysregulation in a cell autono- mous manner and have shed light on novel dysregulated path