Examining Responses of RNA Binding Proteins to ALS and Stress in Human Motor Neurons

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by rapid progression and relatively selective motor neuron loss. RNA dysregulation and abnormal proteostasis have been shown to play a key role in ALS pathogenesis. In particular, RNA binding protein (RBP) nuclear-to-cytoplasmic mislocalisation and accumulation have been identified as a pathological hallmark of the disease. RBPs are essential contributors to cellular stress responses, highlighting one important intersect between stress and ALS. Here, I have used healthy human induced pluripotent stem cell (hiPSC)-derived motor neurons to model the cellular stress responses. I have observed the assembly and disassembly of stress granules (SGs) in response to various stressors, and identified recurrent heat stress insults does not result in subsequent SG formation. When examining the nucleocytoplasmic localisation of key ALS-linked RBPs in response to stress, I identified stress- and RBP- specific responses. This was also true when examining the kinetics of nuclear relocalisation upon recovery, with TDP-43 and FUS, two of the most recognised RBPs in ALS pathogenesis, as exhibiting delayed nuclear relocalisation following stress. To model ALS, I have used hiPSC-derived motor neurons from patients carrying ALS-causing mutations in valosin containing protein (VCP). I identified TDP-43, FUS and SFPQ nuclear-to-cytoplasmic mislocalisation in VCP mutant motor neurons, with closer analysis finding this mislocalisation extends to within neurites. I showed VCP D2-ATPase inhibition robustly rescued TDP-43 and FUS mislocalisation, suggesting a gain-in-function in the D2 ATPase drives mislocalisation. To gain further insight into possible disease mechanisms, I examined the cellular stress response in terms of SG dynamics and RBP nucleocytoplasmic localisation responses in VCP mutant motor neurons. Across multiple stressors and recovery, there were no aberrant stress responses in VCP mutant motor neurons, suggesting that these are not cellular pathways contributing to early disease pathogenesis in motor neurons. Together, these data provide insights into the pathogenesis of ALS, highlighting possible molecular events for therapeutic intervention

Topics in need of more attention in Premarital Counseling

Abstract Premarital counseling (PMC) is a program designed to equip couples with strategies that can facilitate healthy marriages and relationships. This research sought to examine topics that participants who received premarital counseling thought were not adequately covered for them and topics participants believed would help strengthen their marriage. Data was collected from 241 Ghanaian participants who identified as being married, separated, or widowed. Each participant was allowed up to three open-ended responses. There were a total of 531 responses out of a maximum of 723 potential responses. Participants for this study were either living in Ghana (74%) or were living abroad (26%). The majority of the participants were female (61%) with an average age of 36 years (Range 31-76). At the time of the study, the majority of the participants were married (69%), Cohabitating (28%), Divorced (2%), or Widowed (1%); however all participants had taken part in PMC at some point in time before marriage. The qualitative data were coded using the Thematic Analytic approach (Maguire & Delahunt, 2017). Two researchers coded for the question “Name three topics that were not adequately covered for you” and two different researchers coded for the question “Which topics would help strengthen your ongoing marriage?”. After the coding was completed it was reviewed by a fifth researcher to determine the reliability among coders for the questions. Results showed that 14 themes emerged from the coded data. The top three topics that participants reported as receiving insufficient training on doing PMC were sex/intimacy, finances, and parenting difficulties. The top three topics proposed for in-depth information during PMC were values in marriage, finances, and sex/intimacy. Findings from this study show empirical evidence for the importance of coaching couples about these topics to prepare and promote healthy behaviors in intimate relationships and marriages. Findings from this study will benefit marriage counselors by providing empirical data on topics to prioritize during premarital counseling sessions. Secondly, findings will help develop/improve current premarital counseling training manuals in Ghana. Future Cross-cultural research is recommended. Further research and coding are currently being completed on the remaining questions from the survey. Key Words: Premarital Counseling, Ghana, Marriage, Relationships, Intimacy, Thematic Analytic Approach, Qualitative Researc

Amplifying the Heat Shock Response Ameliorates ALS and FTD Pathology in Mouse and Human Models

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are now known as parts of a disease spectrum with common pathological features and genetic causes. However, as both conditions are clinically heterogeneous, patient groups may be phenotypically similar but pathogenically and genetically variable. Despite numerous clinical trials, there remains no effective therapy for these conditions, which, in part, may be due to challenges of therapy development in a heterogeneous patient population. Disruption to protein homeostasis is a key feature of different forms of ALS and FTD. Targeting the endogenous protein chaperone system, the heat shock response (HSR) may, therefore, be a potential therapeutic approach. We conducted a preclinical study of a known pharmacological amplifier of the HSR, called arimoclomol, in mice with a mutation in valosin-containing protein (VCP) which causes both ALS and FTD in patients. We demonstrate that amplification of the HSR ameliorates the ALS/FTD-like phenotype in the spinal cord and brain of mutant VCP mice and prevents neuronal loss, replicating our earlier findings in the SOD1 mouse model of ALS. Moreover, in human cell models, we demonstrate improvements in pathology upon arimoclomol treatment in mutant VCP patient fibroblasts and iPSC-derived motor neurons. Our findings suggest that targeting of the HSR may have therapeutic potential, not only in non-SOD1 ALS, but also for the treatment of FTD

Physiological intron retaining transcripts in the cytoplasm abound during human motor neurogenesis

Intron retention (IR) is now recognized as a dominant splicing event during motor neuron (MN) development, however the role and regulation of intron-retaining transcripts (IRTs) localized to the cytoplasm remain particularly understudied. Here we show that IR is a physiological process that is spatiotemporally regulated during MN lineage restriction and that IRTs in the cytoplasm are detected in as many as 13% (n=2297) of the genes expressed during this process. We identify a major class of cytoplasmic IRTs, which are not associated with reduced expression of their own genes, but instead show a high capacity for RNA-binding protein and miRNA occupancy. Finally, we show that ALS-causing VCP mutations lead to a selective increase in cytoplasmic abundance of this particular class of IRTs, which in turn temporally coincides with an increase in the nuclear expression level of predicted miRNA target genes. Altogether, our study identifies a previously unrecognized class of cytoplasmic intronic sequences with potential regulatory function beyond gene expression

Nucleocytoplasmic mRNA redistribution accompanies RNA binding protein mislocalization in ALS motor neurons and is restored by VCP ATPase inhibition

Amyotrophic lateral sclerosis (ALS) is characterized by nucleocytoplasmic mislocalization of the RNA-binding protein (RBP) TDP-43. However, emerging evidence suggests more widespread mRNA and protein mislocalization. Here, we employed nucleocytoplasmic fractionation, RNA sequencing, and mass spectrometry to investigate the localization of mRNA and protein in induced pluripotent stem cell-derived motor neurons (iPSMNs) from ALS patients with TARDBP and VCP mutations. ALS mutant iPSMNs exhibited extensive nucleocytoplasmic mRNA redistribution, RBP mislocalization, and splicing alterations. Mislocalized proteins exhibited a greater affinity for redistributed transcripts, suggesting a link between RBP mislocalization and mRNA redistribution. Notably, treatment with ML240, a VCP ATPase inhibitor, partially restored mRNA and protein localization in ALS mutant iPSMNs. ML240 induced changes in the VCP interactome and lysosomal localization and reduced oxidative stress and DNA damage. These findings emphasize the link between RBP mislocalization and mRNA redistribution in ALS motor neurons and highlight the therapeutic potential of VCP inhibition

Progressive Motor Neuron Pathology and the Role of Astrocytes in a Human Stem Cell Model of VCP-Related ALS.

Motor neurons (MNs) and astrocytes (ACs) are implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), but their interaction and the sequence of molecular events leading to MN death remain unresolved. Here, we optimized directed differentiation of induced pluripotent stem cells (iPSCs) into highly enriched (> 85%) functional populations of spinal cord MNs and ACs. We identify significantly increased cytoplasmic TDP-43 and ER stress as primary pathogenic events in patient-specific valosin-containing protein (VCP)-mutant MNs, with secondary mitochondrial dysfunction and oxidative stress. Cumulatively, these cellular stresses result in synaptic pathology and cell death in VCP-mutant MNs. We additionally identify a cell-autonomous VCP-mutant AC survival phenotype, which is not attributable to the same molecular pathology occurring in VCP-mutant MNs. Finally, through iterative co-culture experiments, we uncover non-cell-autonomous effects of VCP-mutant ACs on both control and mutant MNs. This work elucidates molecular events and cellular interplay that could guide future therapeutic strategies in ALS

LEARN: A multi-centre, cross-sectional evaluation of Urology teaching in UK medical schools

OBJECTIVE: To evaluate the status of UK undergraduate urology teaching against the British Association of Urological Surgeons (BAUS) Undergraduate Syllabus for Urology. Secondary objectives included evaluating the type and quantity of teaching provided, the reported performance rate of General Medical Council (GMC)-mandated urological procedures, and the proportion of undergraduates considering urology as a career. MATERIALS AND METHODS: LEARN was a national multicentre cross-sectional study. Year 2 to Year 5 medical students and FY1 doctors were invited to complete a survey between 3rd October and 20th December 2020, retrospectively assessing the urology teaching received to date. Results are reported according to the Checklist for Reporting Results of Internet E-Surveys (CHERRIES). RESULTS: 7,063/8,346 (84.6%) responses from all 39 UK medical schools were included; 1,127/7,063 (16.0%) were from Foundation Year (FY) 1 doctors, who reported that the most frequently taught topics in undergraduate training were on urinary tract infection (96.5%), acute kidney injury (95.9%) and haematuria (94.4%). The most infrequently taught topics were male urinary incontinence (59.4%), male infertility (52.4%) and erectile dysfunction (43.8%). Male and female catheterisation on patients as undergraduates was performed by 92.1% and 73.0% of FY1 doctors respectively, and 16.9% had considered a career in urology. Theory based teaching was mainly prevalent in the early years of medical school, with clinical skills teaching, and clinical placements in the later years of medical school. 20.1% of FY1 doctors reported no undergraduate clinical attachment in urology. CONCLUSION: LEARN is the largest ever evaluation of undergraduate urology teaching. In the UK, teaching seemed satisfactory as evaluated by the BAUS undergraduate syllabus. However, many students report having no clinical attachments in Urology and some newly qualified doctors report never having inserted a catheter, which is a GMC mandated requirement. We recommend a greater emphasis on undergraduate clinical exposure to urology and stricter adherence to GMC mandated procedures

Stress-Specific Spatiotemporal Responses of RNA-Binding Proteins in Human Stem Cell-Derived Motor Neurons

RNA-binding proteins (RBPs) have been shown to play a key role in the pathogenesis of a variety of neurodegenerative disorders. Amyotrophic lateral sclerosis (ALS) is an exemplar neurodegenerative disease characterised by rapid progression and relatively selective motor neuron loss. Nuclear-to-cytoplasmic mislocalisation and accumulation of RBPs have been identified as a pathological hallmark of the disease, yet the spatiotemporal responses of RBPs to different extrinsic stressors in human neurons remain incompletely understood. Here, we used healthy induced pluripotent stem cell (iPSC)-derived motor neurons to model how different types of cellular stress affect the nucleocytoplasmic localisation of key ALS-linked RBPs. We found that osmotic stress robustly induced nuclear loss of TDP-43, SPFQ, FUS, hnRNPA1 and hnRNPK, with characteristic changes in nucleocytoplasmic localisation in an RBP-dependent manner. Interestingly, we found that RBPs displayed stress-dependent characteristics, with unique responses to both heat and oxidative stress. Alongside nucleocytoplasmic protein distribution changes, we identified the formation of stress- and RBP-specific nuclear and cytoplasmic foci. Furthermore, the kinetics of nuclear relocalisation upon recovery from extrinsic stressors was also found to be both stress- and RBP-specific. Importantly, these experiments specifically highlight TDP-43 and FUS, two of the most recognised RBPs in ALS pathogenesis, as exhibiting delayed nuclear relocalisation following stress in healthy human motor neurons as compared to SFPQ, hnRNPA1 and hnRNPK. Notably, ALS-causing valosin containing protein (VCP) mutations did not disrupt the relocalisation dynamics of TDP-43 or FUS in human motor neurons following stress. An increased duration of TDP-43 and FUS within the cytoplasm after stress may render the environment more aggregation-prone, which may be poorly tolerated in the context of ALS and related neurodegenerative disorders. In summary, our study addresses stress-specific spatiotemporal responses of neurodegeneration-related RBPs in human motor neurons. The insights into the nucleocytoplasmic dynamics of RBPs provided here may be informative for future studies examining both disease mechanisms and therapeutic strategy

TDP-43 and FUS mislocalization in VCP mutant motor neurons is reversed by pharmacological inhibition of the VCP D2 ATPase domain

RNA binding proteins have been shown to play a key role in the pathogenesis of amyotrophic lateral sclerosis (ALS). Mutations in valosin-containing protein (VCP/p97) cause ALS and exhibit the hallmark nuclear-to-cytoplasmic mislocalization of RNA binding proteins (RBPs). However, the mechanism by which mutations in VCP lead to this mislocalization of RBPs remains incompletely resolved. To address this, we used human-induced pluripotent stem cell-derived motor neurons carrying VCP mutations. We first demonstrate reduced nuclear-to-cytoplasmic ratios of transactive response DNA-binding protein 43 (TDP-43), fused in sarcoma/translocated in liposarcoma (FUS) and splicing factor proline and glutamine rich (SFPQ) in VCP mutant motor neurons. Upon closer analysis, we also find these RBPs are mislocalized to motor neuron neurites themselves. To address the hypothesis that altered function of the D2 ATPase domain of VCP causes RBP mislocalization, we used pharmacological inhibition of this domain in control motor neurons and found this does not recapitulate RBP mislocalization phenotypes. However, D2 domain inhibition in VCP mutant motor neurons was able to robustly reverse mislocalization of both TDP-43 and FUS, in addition to partially relocalizing SFPQ from the neurites. Together these results argue for a gain-of-function of D2 ATPase in VCP mutant human motor neurons driving the mislocalization of TDP-43 and FUS. Our data raise the intriguing possibility of harnessing VCP D2 ATPase inhibitors in the treatment of VCP-related ALS

Topics needing more attention in Premarital Counseling

Abstract Premarital counseling (PMC) is a program designed to equip couples with strategies that can facilitate healthy marriages and relationships. This research sought to examine topics that participants who received premarital counseling thought were not adequately covered for them and topics participants believed would help strengthen their marriage. Data was collected from 241 Ghanaian participants who identified as being married, separated, or widowed. Each participant was allowed up to three open-ended responses. There were a total of 531 responses out of a maximum of 723 potential responses. Participants for this study were either living in Ghana (74%) or were living abroad (26%). The majority of the participants were female (61%) with an average age of 36 years (Range 31-76). At the time of the study, the majority of the participants were married (69%), Cohabitating (28%), Divorced (2%), or Widowed (1%); however all participants had taken part in PMC at some point in time before marriage. The qualitative data were coded using the Thematic Analytic approach (Maguire & Delahunt, 2017). Two researchers coded for the question “Name three topics that were not adequately covered for you” and two different researchers coded for the question “Which topics would help strengthen your ongoing marriage?”. After the coding was completed it was reviewed by a fifth researcher to determine the reliability among coders for the questions. Results showed that 14 themes emerged from the coded data. The top three topics that participants reported as receiving insufficient training on doing PMC were sex/intimacy, finances, and parenting difficulties. The top three topics proposed for in-depth information during PMC were values in marriage, finances, and sex/intimacy. Findings from this study show empirical evidence for the importance of coaching couples about these topics to prepare and promote healthy behaviors in intimate relationships and marriages. Findings from this study will benefit marriage counselors by providing empirical data on topics to prioritize during premarital counseling sessions. Secondly, findings will help develop/improve current premarital counseling training manuals in Ghana. Future Cross-cultural research is recommended. Further research and coding are currently being completed on the remaining questions from the survey. Key Words: Premarital Counseling, Ghana, Marriage, Relationships, Intimacy, Thematic Analytic Approach, Qualitative Researc