Theme 05 - Human Cell Biology and Pathology (including iPSC studies)

Background: TDP-43 pathology is the hallmark of ALS found in 98% of cases. While mutations in the gene encoding TDP43, TARDBP, are a rare cause of ALS, the deposition of TDP-43 positive cytoplasmic inclusions remains a common neuropath- ology for the majority of cases. Recent reports have shown that mitochondrial dysfunction plays a significant role in motor neuron degeneration in ALS and TDP-43 was found to modu- late several mitochondrial transcripts. Identifying dysfunctional mitochondrial pathways in neurons from TDP-43 patients would significantly contribute to our understanding of disease mechanisms and potential new therapeutic targets. Objectives: The aim of this study is to determine how TDP- 43 mutations affect mitochondrial function and intracellular transport using iPS-derived MNs from patients. Methods: In this study, we differentiated patient motor neurons derived from induced pluripotent stem cells (iPSCs) carrying mutations in TDP-43 (M337V and I383T). Seahorse XFe was used to assess mitochondrial respiration, ATP production and spare respiratory capacity and live calcium imaging was used to determine mitochondrial calcium buffering. Neurons were grown on microfluidic chambers for studying axonal transport and MitoTracker movement was quantified during live imaging in the microgrooves. Results: We found that TDP-43-M337V and TDP-43-I383T MNs show reduced mitochondrial basal respiration and ATP production at baseline and reduced spare respiratory capacity when ER stress was induced by thapsigargin. Furthermore, we also detect significantly reduced mitochondrial length and surface area in patient iPS-MNs, indicating increased fragmentation. RNA sequencing and immunoblot- ting confirmed that mutant TDP-43 modulated the expression of key molecules involved in ATP production and respiration, such as ATP synthase and COX5A. Moreover, colocalization studies showed that TDP-43 directly binds to ATPB. Imaging of axonal transport revealed reduced speed of retrograde mitochondrial transport in TDP-43-M337V and TDP-43-I383T as well as reduced endosomal transport, which correlated with an imbalance of motor proteins, such as KIF5A, DNAH and dynactin-1. Conclusions: This study shows that ALS iPS-derived MNs with mutations in TDP-43 have deficiencies in essential mitochondrial functions, such as respiration and ATP production, as well as reduced intracellular transport of mitochondria and endosomes. and altered lipid are of amyotrophic laterals sclerosis (ALS) and critical components of ALS disease pathology. In healthy individuals, inflammation is resolved through the Methods: We used flow cytometry in combination with a cus-tomized panel to profile monocytes in peripheral blood mono-nuclear cells from ALS patients at three-time points ( n at visit 1 ¼ 40, n at visit 2 ¼ 18 and n at visit 3 ¼ 12). We used unsuper-vised clustering methods to identify monocyte cell populations. Differential state analysis was performed comparing the FPRL1 abundance in each cluster and a cluster containing all monocytes at the second and third visit against the first visit. Results: Longitudinal analysis of ALS patients identified depletion of FPRL1 in monocytes at the second visit compared to the first visit (adjusted p -value ¼ 0.027, fold change ¼ (cid:3) 1.850). FPRL1 further depleted in monocytes at the third visit compared to the first visit (adjusted p -value ¼ 0.012, fold change ¼(cid:3) 2.337). visit (cid:3) 1.899) at the third visit. Conclusion: Our longitudinal analysis identified depletion of FPRL1 in monocytes, particularly in subpopulations of CD11b þ monocytes, in the second and third visits compared to the first visit. FPRL1 depletion may exacerbate the already known inflammatory state of CD11b þ monocytes. Previous studies have shown that the recruitment of inflammatory monocytes to the spinal cord plays a pathological role in ALS. The depletion of FPRL1 in inflammatory monocyte subpopulations may amplify their pathological role in ALS. Our results so far provide evidence for the use of FPRL1 as a biomarker for disease stratification and a potential new therapeutic target to treat ALS. in they consist of FUS determine the extent of Tyr526-FUS phosphorylation in and in in mouse and Methods: We used plasmids of GFP-FUS and kinases of the Src-family for transfection of HEK293T cells in our overexpres- sion studies coupled with siRNA silencing, phosphatase Additionally, immunohistochemical staining was performed with mouse brain sections and human postmortem tissues of FTD and control patients. Results: Tyr526 phosphorylation of FUS by Src-family kinases indicated impaired nucleocytoplasmic distribution and aggregation of FUS in cell models, and pronounced phospho-Tyr526 co- localization with pSrc/pAbl was detected in mouse brain. Brain region-specific phospho-Tyr526 FUS co-localization with active pSrc/pAbl kinases in mice pointed to preferential involvement of cAbl in cytoplasmic phospho-Tyr526 FUS mislocalization in cortical neurons. Final analysis of the detail patterns of active cAbl kinase and phospho-Tyr526 FUS in the neurons of human postmortem frontal cortex brain tissue demonstrated the increased cytoplasmic phospho-Tyr526 FUS distribution in the cortical neurons of ALS/FTD patients as compared to FTD. Discussion: Considering the overlapping patterns of cAbl activity and phospho-Tyr526 FUS distribution in cortical neurons, we propose that cAbl kinase is involved in mediating cytoplasmic toxic FUS mislocalization in FTD and ALS/FTD patients, likely leading to differences in disease progression. Background: Cytoplasmic mislocalization and aggregation of the RNA-binding protein TDP-43 constitutes the neuropathological hallmark of ALS. These pathological changes known as TDP-43 pathology are detected in nearly all patients, suggesting a convergent mechanism of TDP-43 dysfunction in both familial and sporadic disease (1). Furthermore, mutations in Background: Mislocalization of the normally nuclear transac- tive response DNA-binding protein 43 (TDP-43) and its formation of cytoplasmic ubiquitinated aggregates are a hallmark of Amyotrophic Lateral Sclerosis (ALS). The biochemical signature of TDP-43 pathology includes the presence of low molecular weight species generated through alternative splicing Background: A central molecular signature of chronic neuro- degenerative diseases, including Motor Neuron Disease (MND), is inappropriate protein aggregation. However, deca- des of research on protein aggregates have not yet determined the cause-effect relationship and overall role of aggregates in disease pathology and progression. This is in part due to the low abundance and high heterogeneity of aggregate species which has made their quantitation and study challenging. As a consequence, it remains unclear how Objectives: Establish a single-molecule method to quantify and characterize aggregate particles containing MND-associ- ated proteins, then compare aggregate particles extracted from patient-derived tissues with those present in induced pluripotent stem cell (iPSC) models of MND from various genetic backgrounds. Methods: Here, we report a novel single-molecule immuno- assay for sensitive and specific detection of small soluble oligo-meric aggregates. Using the MND-associated aggregation-prone protein TAR DNA-binding protein 43 (TDP-43), we show this assay can detect and characterize aggregate particles ranging from purified recombinant fragments (RRM1-RRM2) to complex whole-proteome mixtures. Finally, we demonstrate the applica-tion of this assay to patient-derived extracts. Results: Surprisingly, we did not observe a difference in the number of aggregate particles extracted from disease-derived tissues ( n ¼ 5) when compared with age-matched healthy controls ( n ¼ 5). However, we find differences in the physico-chemical properties of these aggregates. TDP-43 aggregate particles were on average larger, and the proportion adorned with the post-translational modification phospho-serine 409/ 410 was higher, among disease-derived extracts. Discussion: This knowledge contributes to the growing body of research targetting the fundamental biology of MND and may improve our understanding of the relationship between protein aggregation and disease progression to inform future early diagnosis efforts. In addition, we hope quantitative evaluation of existing cellular models will empower research- ers to select the most appropriate model system when investigating aggregate pathology, paving the way for more robust early-stage therapeutic research. Background: Amyotrophic lateral sclerosis (ALS) is a non-cell autonomous disorder as many cell types contribute to motor neurons death. The lack of effective treatments is probably due to the absence of a realistic model that can recapitulate pathogenic mechanisms. Cerebral organoids are pluripotent stem cell-derived self-organizing structures that allow in vitro generation of the tissues. We developed a new method for the generation of spinal cord organoids (SCOs) that can be used for the study of pathogenic mechanisms in ALS. Objectives: Aim of the work was to characterize a 3D orga- noid model for the study of ALS pathogenesis. Methods: We started from iPSCs obtained from healthy controls and sporadic ALS (sALS) patients. We differentiated iPSCs into neural stem cells (NSCs). We dissociated NCSs using StemPro Accutase and a cell strainer. Then, we plated NSCs on low-attachment plates and we cultured them in floating conditions using an orbital shaker. We differentiated NSCs to generate SCOs. We then characterized cells by phase-contrast and confocal microscopy. Results: We found that SCOs derived from sALS patients were smaller and with irregular morphology compared to healthy controls. Using the GFAP marker, we found that sALS organoids have a thicker glial layer compared to healthy con- trols. We also found that healthy controls organoids show longer neurites compared to