6 research outputs found
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Investigating the TrkB Interactome during Development and in Neurodegeneration
Brain-derived neurotrophic factor (BDNF) is a cardinal regulator of neuronal development, survival and synaptic plasticity, signalling via its major receptor, Tyrosine receptor kinase B (TrkB). In the developing brain, BDNF acts as a guidance cue influencing the direction of the axonal growth cone. In the mature brain, it ensures survival of neurons and synaptic plasticity.
Guidance cues remodel the nascent local proteome in the developing nervous system. Recently, guidance cue receptors DCC, Neuropilin-1 and Robo2 were shown to interact with the translation machinery. Both the protein and mRNA interactions of these receptors help them to define which proteins are locally synthesized in response to stimulation. BDNF likewise regulates the local nascent proteome with high spatiotemporal resolution via TrkB activation. While there is extensive data on expression level, trafficking and signalling of TrkB, little is known about its protein and RNA interactions. We hypothesised that like DCC, Neuropilin-1 and Robo-2, TrkB shows similar interaction with the translation machinery. Immunoprecipitation studies for TrkB were performed and protein interactors were analysed by LC-MS/MS, interacting mRNAs were sequenced. A specific set of ribosomal proteins, RNA-binding proteins, rRNAs and mRNAs interacting with TrkB were detected in the embryonic mouse brain. However, in the adult mouse hippocampus, where BDNF-induced local protein synthesis mediates synaptic plasticity, no receptor-ribosome interaction was detected. This suggests a remodelling of the TrkB interactome during development and that BDNF modulates the nascent proteome in adult brain via different mechanisms.
To elucidate possible new roles and regulatory mechanisms of TrkB in the mature brain, we screened for novel protein interactions in adult mouse hippocampus and cerebral synaptosomes. The TrkB protein interactome was largely composed of cytoskeleton and transport vesicle-related proteins, as well as transmembrane transporters. At the synapse, in particular, small molecule transporters interacted with TrkB, including ion and glutamate transporting excitatory amino acid transporter 1 (EAAT1). BDNF stimulation interrupts the TrkB-EAAT1 interaction and leads to increased EAAT1-dependent glutamate uptake in primary hippocampal cultures, elucidating a novel crosstalk between neurotrophin receptor and glutamate transporter.
The BDNF-TrkB axis is impaired in ageing and in neurodegeneration. We have poor understanding of the processes occurring in these conditions. We hypothesised that the TrkB interactome may vary in aging and neurodegenerative disease as a possible contributor to their impaired signalling. Interestingly, a screen of the interactomes in the mouse brain showed that major TrkB co-precipitating proteins are not affected by ageing. In murine prion disease, which results in extensive neurodegeneration, a TrkB-interactome screen revealed increased association of a distinct set of cell-adhesion proteins with TrkB, including Connexin-43, a gap-junction adhesion molecule which is associated with other neurodegenerative diseases. The identification of novel disease-related interactions may yield future insights into dysregulated BDNF signalling in neurodegeneration.
In conclusion, in this thesis, the interactome of TrkB was studied to increase our understanding of BDNF function in health and disease in the mouse brain. Novel interaction partners of the neurotrophin receptor TrkB were identified, during development, in adulthood and ageing, as well as in prion neurodegeneration. Specific interactors identified may, upon further investigation, represent novel mechanistic insights into BDNF signalling and possibly new targets to tackle dysregulated neurotrophic signalling in many pathological conditions.Doctoral Scholarship of the German Academic Scholarship Foundatio
2,4-Dichlorophenoxyacetic Acid Induces Degeneration of mDA Neurons In Vitro
Ruß T, Enders L, Zbiegly JM, Potru PS, Wurm J, Spittau B. 2,4-Dichlorophenoxyacetic Acid Induces Degeneration of mDA Neurons In Vitro. Biomedicines. 2023;11(11): 2882.Background: Parkinson’s disease (PD) affects 1–2% of the population over the age of 60 and the majority of PD cases are sporadic, without any family history of the disease. Neuroinflammation driven by microglia has been shown to promote the progression of midbrain dopaminergic (mDA) neuron loss through the release of neurotoxic factors. Interestingly, the risk of developing PD is significantly higher in distinct occupations, such as farming and agriculture, and is linked to the use of pesticides and herbicides. Methods: The neurotoxic features of 2,4-Dichlorophenoxyacetic acid (2,4D) at concentrations of 10 µM and 1 mM were analyzed in two distinct E14 midbrain neuron culture systems and in primary microglia. Results: The application of 1 mM 2,4D resulted in mDA neuron loss in neuron-enriched cultures. Notably, 2,4D-induced neurotoxicity significantly increased in the presence of microglia in neuron-glia cultures, suggesting that microglia-mediated neurotoxicity could be one mechanism for progressive neuron loss in this in vitro setup. However, 2,4D alone was unable to trigger microglia reactivity. Conclusions: Taken together, we demonstrate that 2,4D is neurotoxic for mDA neurons and that the presence of glia cells enhances 2,4D-induced neuron death. These data support the role of 2,4D as a risk factor for the development and progression of PD and further suggest the involvement of microglia during 2,4D-induced mDA neuron loss
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2,4-Dichlorophenoxyacetic Acid Induces Degeneration of mDA Neurons In Vitro.
Peer reviewed: TrueAcknowledgements: The authors thank Susanna Glaser and Ludmilla Butenko for excellent technical assistance. We acknowledge the support for the publication costs by the Open Access Publication Fund of the Bielefeld University.Publication status: PublishedBackground: Parkinson's disease (PD) affects 1-2% of the population over the age of 60 and the majority of PD cases are sporadic, without any family history of the disease. Neuroinflammation driven by microglia has been shown to promote the progression of midbrain dopaminergic (mDA) neuron loss through the release of neurotoxic factors. Interestingly, the risk of developing PD is significantly higher in distinct occupations, such as farming and agriculture, and is linked to the use of pesticides and herbicides. Methods: The neurotoxic features of 2,4-Dichlorophenoxyacetic acid (2,4D) at concentrations of 10 µM and 1 mM were analyzed in two distinct E14 midbrain neuron culture systems and in primary microglia. Results: The application of 1 mM 2,4D resulted in mDA neuron loss in neuron-enriched cultures. Notably, 2,4D-induced neurotoxicity significantly increased in the presence of microglia in neuron-glia cultures, suggesting that microglia-mediated neurotoxicity could be one mechanism for progressive neuron loss in this in vitro setup. However, 2,4D alone was unable to trigger microglia reactivity. Conclusions: Taken together, we demonstrate that 2,4D is neurotoxic for mDA neurons and that the presence of glia cells enhances 2,4D-induced neuron death. These data support the role of 2,4D as a risk factor for the development and progression of PD and further suggest the involvement of microglia during 2,4D-induced mDA neuron loss
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HNRNPH1 regulates the neuroprotective cold-shock protein RBM3 expression through poison exon exclusion
Enhanced expression of the cold-shock protein RNA binding motif 3 (RBM3) is highly neuroprotective both in vitro and in vivo. Whilst upstream signalling pathways leading to RBM3 expression have been described, the precise molecular mechanism of RBM3 cold induction remains elusive. To identify temperature-dependent modulators of RBM3, we performed a genome-wide CRISPR-Cas9 knockout screen using RBM3-reporter human iPSC-derived neurons. We found that RBM3 mRNA and protein levels are robustly regulated by several splicing factors, with heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) being the strongest positive regulator. Splicing analysis revealed that moderate hypothermia significantly represses the inclusion of a poison exon, which, when retained, targets the mRNA for nonsense-mediated decay. Importantly, we show that HNRNPH1 mediates this cold-dependent exon skipping via its thermosensitive interaction with a G-rich motif within the poison exon. Our study provides novel mechanistic insights into the regulation of RBM3 and provides further targets for neuroprotective therapeutic strategies.An Open Targets grant [OTAR2054], the UK Dementia Research Institute, which receives its funding from UK DRI Ltd, funded by the UK Medical Research Council (MRC), Alzheimer’s Society and Alzheimer’s Research UK
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HNRNPH1 regulates the neuroprotective cold-shock protein RBM3 expression through poison exon exclusion.
Funder: Alzheimer's Research UK (ARUK)Funder: Alzheimer's Society; doi: http://dx.doi.org/10.13039/501100000320Funder: UKRI | Medical Research Council (MRC); doi: http://dx.doi.org/10.13039/501100000265Enhanced expression of the cold-shock protein RNA binding motif 3 (RBM3) is highly neuroprotective both in vitro and in vivo. Whilst upstream signalling pathways leading to RBM3 expression have been described, the precise molecular mechanism of RBM3 cold induction remains elusive. To identify temperature-dependent modulators of RBM3, we performed a genome-wide CRISPR-Cas9 knockout screen using RBM3-reporter human iPSC-derived neurons. We found that RBM3 mRNA and protein levels are robustly regulated by several splicing factors, with heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) being the strongest positive regulator. Splicing analysis revealed that moderate hypothermia significantly represses the inclusion of a poison exon, which, when retained, targets the mRNA for nonsense-mediated decay. Importantly, we show that HNRNPH1 mediates this cold-dependent exon skipping via its thermosensitive interaction with a G-rich motif within the poison exon. Our study provides novel mechanistic insights into the regulation of RBM3 and provides further targets for neuroprotective therapeutic strategies.An Open Targets grant [OTAR2054], the UK Dementia Research Institute, which receives its funding from UK DRI Ltd, funded by the UK Medical Research Council (MRC), Alzheimer’s Society and Alzheimer’s Research UK
HNRNPH1 regulates the neuroprotective cold-shock protein RBM3 expression through poison exon exclusion.
Enhanced expression of the cold-shock protein RNA binding motif 3 (RBM3) is highly neuroprotective both in vitro and in vivo. Whilst upstream signalling pathways leading to RBM3 expression have been described, the precise molecular mechanism of RBM3 cold induction remains elusive. To identify temperature-dependent modulators of RBM3, we performed a genome-wide CRISPR-Cas9 knockout screen using RBM3-reporter human iPSC-derived neurons. We found that RBM3 mRNA and protein levels are robustly regulated by several splicing factors, with heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) being the strongest positive regulator. Splicing analysis revealed that moderate hypothermia significantly represses the inclusion of a poison exon, which, when retained, targets the mRNA for nonsense-mediated decay. Importantly, we show that HNRNPH1 mediates this cold-dependent exon skipping via its thermosensitive interaction with a G-rich motif within the poison exon. Our study provides novel mechanistic insights into the regulation of RBM3 and provides further targets for neuroprotective therapeutic strategies