SummaryMotor 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.
The human genome contains thousands of natural antisense transcripts (NAT) that can regulate epigenetic state, transcription, RNA stability, or translation of their overlapping genes 1,2 . We describe MAPT-AS1, a primate-conserved, brain-enriched NAT containing an embedded mammalian-wide interspersed repeat (MIR), which represses tau translation by competing with rRNA pairing to MAPT mRNA internal ribosome entry site (IRES) 3 . Tau, a neuronal intrinsically disordered protein (IDP), stabilises axonal microtubules. Hyperphosphorylated, aggregation-prone tau forms the hallmark inclusions of tauopathies 4 . MAPT mutations cause familial frontotemporal dementia (FTLD-tau), and common variation forming the MAPT H1 haplotype is a significant risk factor in many tauopathies 5 , and Parkinson's disease. Notably, expression of MAPT-AS1 or its minimal essential sequences including MIR reduces, whereas silenced MAPT-AS1 increases neuronal tau, and is correlated with tau pathology in human brain. Moreover, we identified hundreds additional NATs with embedded MIRs (MIR-NATs), which are overrepresented at coding genes linked to neurodegeneration, and/or encoding IDPs, and confirmed MIR-NAT-mediated translational control of one such gene, PLCG1. Collectively, we present the importance of MAPT-AS1 for tauopathies, while also uncovering a potentially broad contribution of MIR-NATs to the tightly controlled translation of IDPs 6 , with particular relevance for proteostasis in neurodegeneration.
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.
The discovery of mutations within genes associated with autosomal recessive Parkinson’s disease allowed for the identification of PINK1/Parkin regulated mitophagy as an important pathway for the removal of damaged mitochondria. While recent studies suggest that AKT-dependent signalling regulates Parkin recruitment to depolarised mitochondria, little is known as to whether this can also regulate PINK1 mitochondrial accumulation and downstream mitophagy. Here, we demonstrate that inhibition of AKT signalling decreases endogenous PINK1 accumulation in response to mitochondria depolarisation, subsequent Parkin recruitment, phosphorylation of ubiquitin, and ultimately mitophagy. Conversely, we show that upon stimulation of AKT signalling via insulin, the mitophagy pathway is increased in SHSY5Y cells. These data suggest that AKT signalling is an upstream regulator of PINK1 accumulation on damaged mitochondria. Importantly, we show that the AKT pathway also regulates endogenous PINK1-dependent mitophagy in human iPSC-derived neurons.
In the process of generating organoids, basement membrane extracts or Matrigel are often used to encapsulate cells but they are poorly defined and contribute to reproducibility issues. While defined hydrogels are increasingly used for organoid culture, the effects of replacing Matrigel with a defined hydrogel on neural progenitor growth, neural differentiation, and maturation within organoids are not well‐explored. In this study, the use of alginate hydrogels as a Matrigel substitute in spinal cord organoid generation is explored. It is found that alginate encapsulation reduces organoid size variability by preventing organoid aggregation. Importantly, alginate supports neurogenesis and gliogenesis of the spinal cord organoids at a similar efficiency to Matrigel, with mature myelinated neurons observed by day 120. Furthermore, using alginate leads to lower expression of non‐spinal markers such as FOXA2, suggesting better control over neural fate specification. To demonstrate the feasibility of using alginate‐based organoid cultures as disease models, an isogenic pair of induced pluripotent stem cells discordant for the ALS‐causing mutation TDP43G298S is used, where increased TDP43 mislocalization in the mutant organoids is observed. This study shows that alginate is an ideal substitute for Matrigel for spinal cord organoid derivation, especially when a xeno‐free and fully defined 3D culture condition is desired.
RNA binding proteins fulfil a wide number of roles in gene expression. Multiple mechanisms of RNA binding protein dysregulation have been implicated in the pathomechanisms of several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Oxidative stress and mitochondrial dysfunction also play important roles in these diseases. In this review, we highlight the mechanistic interplay between RNA binding protein dysregulation, oxidative stress and mitochondrial dysfunction in ALS. We also discuss different potential therapeutic strategies targeting these pathways.
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