Biology subjects

Birsa, N.

Publications and source records attributed to Birsa, N..

9 recordsLinked to original sources

bioRxiv2025

Characterisation of LAMP1- and LAMP2A-positive organelles in neurons

LAMP1 and LAMP2A are abundant proteins of late endosomal/lysosomal compartments, which are often used interchangeably to label what is thought to be the same pool of organelles, potentially obscuring their unique physiological roles. Here, we characterised the transport dynamics of LAMP1- and LAMP2A-positive compartments in human iPSC-derived cortical neurons. We found that axonal LAMP1-positive organelles move more slowly in the retrograde direction, pause more frequently, and show a broader velocity distribution in the anterograde direction than LAMP2A-positive vesicles, suggesting they are distinct compartments with differential trafficking behaviour. To explore the molecular mechanism underlying these differences, we characterised with high spatiotemporal precision, the protein interactomes of LAMP1 and LAMP2A-positive compartments through proximity labelling, using full-length LAMP1 or LAMP2A fused to the light-activated biotin ligase LOV-Turbo. We identified and validated the endosomal protein, ZFYVE16, as a novel member of LAMP1 and LAMP2A interactomes. We suggest that LAMP2A-positive organelles represent a subset of LAMP1-positive compartments, which are surprisingly enriched in synaptic vesicle proteins. Summary statementLAMP1- and LAMP2A-positive organelles have different axonal transport dynamics and form distinct organelle pools characterised by specific protein compositions.

neuroscience↗

bioRxiv2025

Impaired BDNF-TrkB trafficking and signalling in Down syndrome basal forebrain neurons

Brain derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase B (TrkB) play crucial roles in neuronal development, synaptic transmission, and neuroplasticity. Deficits in BDNF/TrkB signalling and trafficking have been identified in several neurodegenerative diseases, including Alzheimers disease (AD). Individuals with Down syndrome (DS) are at an increased risk of developing AD compared to the general population. Basal forebrain neurons (BFNs) are among the first to degenerate in AD and DS, but the mechanisms underlying their vulnerability remain unclear. Using BFNs derived from the Dp1Tyb mouse model of DS, we investigated neurotrophic signalling and trafficking deficits in AD-DS. We found enlarged early endosomes and elevated levels of active Rab5, a GTPase critical for early endosome formation, in Dp1Tyb BFNs. These abnormalities were associated with impaired transport of internalised TrkB from axon terminals to the soma. Using microfluidic devices, we demonstrated that axonal BDNF stimulation enhanced signalling endosome dynamics in wild-type but not Dp1Tyb BFNs, which is likely due to impaired axonal ERK1/2 signalling. Our findings establish a link between Rab5 hyperactivation, endosomal dysfunction, and impaired ERK1/2 signalling, highlighting the interplay between trafficking and neurotrophic signalling, and underscore the importance of targeting endolysosomal and signalling pathways to mitigate neuronal dysfunction in AD-DS.

neuroscience↗

bioRxiv2025

Innervation and cargo-specific axonal transport impairments in FUS-ALS mice with gain and loss of function

Mutations in the RNA-binding protein FUS lead to nuclear depletion and cytoplasmic mislocalisation of the protein and cause amyotrophic lateral sclerosis (ALS). Using a novel FUS-ALS mouse model, we found that adult mutant mice develop loss of function transcriptomic alterations, along with aberrant cytoplasmic partitioning associated with translatome deficits. Neuromuscular junction innervation was selectively impaired in FUS-ALS females; however, reinnervation following sciatic nerve crush was equally perturbed in both sexes. Additionally, we observed cargo-specific axonal transport alterations, a process critical for neuronal maintenance. In vivo mitochondrial transport was impaired across FUS-ALS mice, whereas the transport of signalling endosomes was selectively disrupted in mutant females. Altogether, our findings identify broader and sex-dependent motor neuron dysfunction in FUS-ALS, emphasise the link between endosomal transport impairments and denervation in disease, and establish FUS-ALS mice as a valuable model for investigating early cellular impairments driving ALS pathology.

neuroscience↗

bioRxiv2024

TDP-43 loss induces extensive cryptic polyadenylation in ALS/FTD

Nuclear depletion and cytoplasmic aggregation of the RNA-binding protein TDP-43 is the hallmark of ALS, occurring in over 97% of cases. A key consequence of TDP-43 nuclear loss is the de-repression of cryptic exons. Whilst TDP-43 regulated cryptic splicing is increasingly well catalogued, cryptic alternative polyadenylation (APA) events, which define the 3 end of last exons, have been largely overlooked, especially when not associated with novel upstream splice junctions. We developed a novel bioinformatic approach to reliably identify distinct APA event types: alternative last exons (ALE), 3UTR extensions (3Ext) and intronic polyadenylation (IPA) events. We identified novel neuronal cryptic APA sites induced by TDP-43 loss of function by systematically applying our pipeline to a compendium of publicly available and in house datasets. We find that TDP-43 binding sites and target motifs are enriched at these cryptic events and that TDP-43 can have both repressive and enhancing action on APA. Importantly, all categories of cryptic APA can also be identified in ALS and FTD post mortem brain regions with TDP-43 proteinopathy underlining their potential disease relevance. RNA-seq and Ribo-seq analyses indicate that distinct cryptic APA categories have different downstream effects on transcript and translation. Intriguingly, cryptic 3Exts occur in multiple transcription factors, such as ELK1, SIX3, and TLX1, and lead to an increase in wild-type protein levels and function. Finally, we show that an increase in RNA stability leading to a higher cytoplasmic localisation underlies these observations. In summary, we demonstrate that TDP-43 nuclear depletion induces a novel category of cryptic RNA processing events and we expand the palette of TDP-43 loss consequences by showing this can also lead to an increase in normal protein translation.

neuroscience↗

bioRxiv2020

FUS-ALS mutants alter FMRP phase separation equilibrium and impair protein translation

Mutations in the RNA binding protein (RBP) FUS cause amyotrophic lateral sclerosis (ALS) and result in its nuclear depletion and cytoplasmic mislocalisation, with cytoplasmic gain of function thought to be crucial in pathogenesis. Here, we show that expression of mutant FUS at physiological levels drives translation inhibition in both mouse and human motor neurons. Rather than acting directly on the translation machinery, we find that mutant FUS forms cytoplasmic condensates that promote the phase separation of FMRP, another RBP associated with neurodegeneration and robustly involved in translation regulation. FUS and FMRP co-partition and repress translation in vitro. In our in vivo model, FMRP RNA targets are depleted from ribosomes. Our results identify a novel paradigm by which FUS mutations favour the condensed state of other RBPs, impacting on crucial biological functions, such as protein translation.

neuroscience↗

bioRxiv2020

RNA-binding protein network alteration causes aberrant axon branching and growth phenotypes in FUS ALS mutant motoneurons

Mutations in the RNA-binding protein (RBPs) FUS have been genetically associated with the motoneuron disease amyotrophic lateral sclerosis (ALS). Using both human induced pluripotent stem cells and mouse models, we found that FUS-ALS causative mutations affect the activity of two relevant RBPs with important roles in neuronal RNA metabolism: HuD/ELAVL4 and FMRP. Mechanistically, mutant FUS leads to upregulation of HuD protein levels through competition with FMRP for HuD mRNA 3UTR binding. In turn, increased HuD levels overly stabilize the transcript levels of its targets, NRN1 and GAP43. As a consequence, mutant FUS motoneurons show increased axon branching and growth upon injury, which could be rescued by dampening NRN1 levels. Since similar phenotypes have been previously described in SOD1 and TDP-43 mutant models, increased axonal growth and branching might represent broad early events in the pathogenesis of ALS.

neuroscience↗

bioRxiv2019

FUS ALS-causative mutations impact FUS autoregulation and the processing of RNA-binding proteins through intron retention

Mutations in the RNA-binding protein FUS cause amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease in which the loss of motor neurons induces progressive weakness and death from respiratory failure, typically only 3-5 years after onset. FUS plays a role in numerous aspects of RNA metabolism, including mRNA splicing. However, the impact of ALS-causative mutations on splicing has not been fully characterised, as most disease models have been based on FUS overexpression, which in itself alters its RNA processing functions. To overcome this, we and others have recently created knock-in models, and have generated high depth RNA-sequencing data on FUS mutants in parallel to FUS knockout. We combined three independent datasets with a joint modelling approach, allowing us to compare the mutation-induced changes to genuine loss of function. We find that FUS ALS-mutations induce a widespread loss of function on expression and splicing, with a preferential effect on RNA binding proteins. Mutant FUS induces intron retention changes through RNA binding, and we identify an intron retention event in FUS itself that is associated with its autoregulation. Altered FUS regulation has been linked to disease, and intriguingly, we find FUS autoregulation to be altered not only by FUS mutations, but also in other genetic forms of ALS, including those caused by TDP-43, VCP and SOD1 mutations, supporting the concept that multiple ALS genes interact in a regulatory network.

neuroscience↗

bioRxiv2018

Miro ubiquitination is critical for efficient damage-induced PINK1/Parkin-mediated mitophagy

Clearance of mitochondria following damage is critical for neuronal homeostasis. Here, we investigate the role of Miro proteins in mitochondrial turnover by the PINK1 / Parkin mitochondrial quality control system in vitro and in vivo. We find that upon mitochondrial damage, Miro is promiscuously ubiquitinated on multiple lysine residues. Combined knockout of both Miro1 and Miro2 or block of Miro ubiquitination and subsequent degradation, lead to slowed mitophagy. In cultured neurons, Miro1 knockout also leads to delayed Parkin translocation onto damaged mitochondria and reduced mitochondrial clearance. In vivo, postnatal knockout of Miro1 in hippocampus and cortex disrupts mitophagy and leads to a dramatic age dependent upregulation of the mitofusin mitochondrial fusion machinery. Fluorescence imaging of aged neurons conditionally knocked out for Miro1 and expressing mitoDendra to label mitochondria in vivo, reveals that Mfn1 / Mfn2 upregulation leads to enlarged and hyperfused somatic mitochondria. Our results provide new insights into the role of Miro in PINK1/Parkin dependent mitophagy and further suggest that disruption of this regulation may be implicated in human neurological pathology.

neuroscience↗

bioRxiv2017

The mitochondrial Rho-GTPase, Miro, is resident at peroxisomes and regulates peroxisomal trafficking and morphology

Peroxisomes are essential for a number of cellular functions, including reactive oxygen species metabolism, fatty acid {beta}-oxidation and lipid biosynthesis. To ensure optimal functionality of peroxisome-dependent processes throughout the cell they must be trafficked; however, peroxisomal transport remains poorly characterised. Here we show that Miro1 and Miro2, outer mitochondrial membrane proteins essential for mitochondrial trafficking, are also localised to peroxisomes. Peroxisomal localisation of Miro1 is negatively regulated by its first GTPase domain and is mediated by an interaction through its transmembrane domain with the peroxisomal-membrane protein chaperone, Pex19. By using Miro1/2 double knockout mouse embryonic fibroblasts (MEFs) we find that the loss of Miro1/2 leads to a significant reduction in short-range microtubule-independent peroxisomal motility. Additionally, Miro regulates peroxisomal size and morphology. Our results contribute to the fundamental understanding of peroxisomal trafficking and morphology, supporting a complex crosstalk between peroxisomal and mitochondrial biology.

cell biology↗