bims-axbals Biomed News
on Axonal Biology and ALS
Issue of 2024‒10‒27
forty-two papers selected by
TJ Krzystek, ALS Therapy Development Institute
  1. Severe dynein dysfunction in cholinergic neurons exacerbates ALS-like phenotypes in a new mouse model.
  2. Dynactin-1 mediates rescue of impaired axonal transport due to reduced mitochondrial bioenergetics in amyotrophic lateral sclerosis motor neurons.
  3. A guide to selecting high-performing antibodies for PLC-gamma-2 for use in Western Blot, immunoprecipitation and immunofluorescence.
  4. Interference of nuclear speckles: A nexus of RNA foci, dipeptide repeats, and mis-splicing in C9ORF72 ALS/FTD.
  5. NDRG1 upregulation by ubiquitin proteasome system dysfunction aggravates neurodegeneration.
  6. A hydrophobic core stabilizes the residual structure in the RRM2 intermediate state of the ALS-linked protein TDP-43.
  7. Sleep Apnea and Amyotrophic Lateral Sclerosis: Cause, Correlation, Any Relation?
  8. The role of polo-like kinases 2 in the proteasomal and lysosomal degradation of alpha-synuclein in neurons.
  9. CSF and blood levels of Neurofilaments, T-Tau, P-Tau, and Abeta-42 in amyotrophic lateral sclerosis: a systematic review and meta-analysis.
  10. Recent Advances in Diagnostic Approaches for Mucormycosis.
  11. Validation of a functional human AD model with four AD therapeutics utilizing patterned ipsc-derived cortical neurons integrated with microelectrode arrays.
  12. Molecular mechanisms of polarized transport to the apical plasma membrane.
  13. Identification of nitric oxide-mediated necroptosis as the predominant death route in Parkinson's disease.
  14. CryoVesNet: A dedicated framework for synaptic vesicle segmentation in cryo-electron tomograms.
  15. Hemozoin induces malaria via activation of DNA damage, p38 MAPK and neurodegenerative pathways in a human iPSC-derived neuronal model of cerebral malaria.
  16. NMNAT1 Is Essential for Human iPS Cell Differentiation to the Retinal Lineage.
  17. Nanomagnetic Guidance Shapes the Structure-Function Relationship of Developing Cortical Networks.
  18. Cellular Mechanisms of RET Receptor Dysfunction in Multiple Endocrine Neoplasia 2.
  19. Mitochondria break free: Mitochondria-derived vesicles in aging and associated conditions.
  20. Inhibiting mtDNA transcript translation alters Alzheimer's disease-associated biology.
  21. Neuronal Vulnerability of the Entorhinal Cortex to Tau Pathology in Alzheimer's Disease.
  22. A protein phosphatase 1 specific phosphatase targeting peptide (PhosTAP) to identify the PP1 phosphatome.
  23. Comparison of Gene-Editing Approaches for Severe Congenital Neutropenia-Causing Mutations in the ELANE Gene.
  24. ALS-like pathology diminishes swelling of spinal astrocytes in the SOD1 animal model.
  25. Insights into the structure of NLR family member X1: Paving the way for innovative drug discovery.
  26. An Effective DNA Methylation Biomarker Screening Mechanism for Amyotrophic Lateral Sclerosis (ALS) Based on Comorbidities and Gene Function Analysis.
  27. Lysosomal Ca2+ release-facilitated TFEB nuclear translocation alleviates ischemic brain injury by attenuating autophagic/lysosomal dysfunction in neurons.
  28. Effects of microgravity on human iPSC-derived neural organoids on the International Space Station.
  29. Formation of Human Thymus Organoids in Three-Dimensional Fibrin Hydrogels.
  30. Complement C5a Implication in Axonal Growth After Injury.
  31. Thioflavin-T: application as a neuronal body and nucleolar stain and the blue light photo enhancement effect.
  32. A click-based electrocorticographic brain-computer interface enables long-term high-performance switch scan spelling.
  33. Using human disease mutations to understand de novo DNA methyltransferase function.
  34. Anti-NMDAR1 antibody impairs dendritic branching in immature cultured neurons.
  35. Fluorescent Reporters, Imaging, and Artificial Intelligence Toolkits to Monitor and Quantify Autophagy, Heterophagy, and Lysosomal Trafficking Fluxes.
  36. Actin Depolymerizing Factor Destrin Regulates Cilia Development and Function during Vertebrate Embryogenesis.
  37. miRNA packaging into small extracellular vesicles and implications in pain.
  38. PGC-1α regulation by FBXW7 through a novel mechanism linking chaperone-mediated autophagy and the ubiquitin-proteasome system.
  39. Elevated peripheral inflammation is associated with choroid plexus enlargement in independent sporadic amyotrophic lateral sclerosis cohorts.
  40. Loss of CLN3 in microglia leads to impaired lipid metabolism and myelin turnover.
  41. High-throughput capture and in situ protein analysis of extracellular vesicles by chemical probe-based array.
  42. Melatonin modulates the Notch1 signaling pathway and Sirt3 in the hippocampus of hypoxic-ischemic neonatal rats.
  1. Biochim Biophys Acta Mol Basis Dis. 2024 Oct 18. pii: S0925-4439(24)00534-9. [Epub ahead of print]1871(1): 167540
    Severe dynein dysfunction in cholinergic neurons exacerbates ALS-like phenotypes in a new mouse model.
    Fabio A Simoes, Eleni Christoforidou, Raphaelle Cassel, Luc Dupuis, Majid Hafezparast.
      Cytoplasmic dynein 1, a motor protein essential for retrograde axonal transport, is increasingly implicated in the pathogenesis of neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). In this study, we developed a novel mouse model that combines the Legs at odd angles (Loa, F580Y) point mutation in the dynein heavy chain with a cholinergic neuron-specific knockout of the dynein heavy chain. This model, for the first time, allows us to investigate the impact of Loa allele exclusivity in these neurons into adulthood. Our findings reveal that this selective increase in dynein dysfunction exacerbated the phenotypes observed in heterozygous Loa mice including pre-wean survival, reduced body weight and grip strength. Additionally, it induced ALS-like pathology in neuromuscular junctions (NMJs) not seen in heterozygous Loa mice. Notably, we also found a previously unobserved significant increase in neurons displaying TDP-43 puncta in both Loa mutants, suggesting early TDP-43 mislocalisation - a hallmark of ALS. The novel model also exhibited a concurrent rise in p62 puncta that did not co-localise with TDP-43, indicating broader impairments in autophagic clearance mechanisms. Overall, this new model underscores the fact that dynein impairment alone can induce ALS-like pathology and provides a valuable platform to further explore the role of dynein in ALS.
    Keywords:  ALS; Amyotrophic lateral sclerosis; Dynein; MND; Motor neurons; Neuromuscular junctions; TDP-43
    DOI:  https://doi.org/10.1016/j.bbadis.2024.167540
  2. Brain Commun. 2024 ;6(5): fcae350
    Dynactin-1 mediates rescue of impaired axonal transport due to reduced mitochondrial bioenergetics in amyotrophic lateral sclerosis motor neurons.
    Ruxandra Dafinca, Carlota Tosat-Bitrian, Emily Carroll, Björn F Vahsen, Javier Gilbert-Jaramillo, Jakub Scaber, Emily Feneberg, Errin Johnson, Kevin Talbot.
      Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the motor system with complex determinants, including genetic and non-genetic factors. A key pathological signature of ALS is the cytoplasmic mislocalization and aggregation of TDP-43 in affected motor neurons, which is found in 97% of cases. Recent reports have shown that mitochondrial dysfunction plays a significant role in motor neuron degeneration in ALS, and TDP-43 modulates several mitochondrial transcripts. In this study, we used induced pluripotent stem cell-derived motor neurons from ALS patients with TDP-43 mutations and a transgenic TDP-43M337V mouse model to determine how TDP-43 mutations alter mitochondrial function and axonal transport. We detected significantly reduced mitochondrial respiration and ATP production in patient induced pluripotent stem cell-derived motor neurons, linked to an interaction between TDP-43M337V with ATPB and COX5A. A downstream reduction in speed of retrograde axonal transport in patient induced pluripotent stem cell-derived motor neurons was detected, which correlated with downregulation of the motor protein complex, DCTN1/dynein. Overexpression of DCTN1 in patient induced pluripotent stem cell-derived motor neurons significantly increased the percentage of retrograde travelling mitochondria and reduced the percentage of stationary mitochondria. This study shows that ALS induced pluripotent stem cell-derived motor neurons with mutations in TDP-43 have deficiencies in essential mitochondrial functions with downstream effects on retrograde axonal transport, which can be partially rescued by DCTN1 overexpression.
    Keywords:  amyotrophic lateral sclerosis; axonal transport; induced pluripotent stem cells; mitochondrial dysfunction; motor proteins
    DOI:  https://doi.org/10.1093/braincomms/fcae350
  3. F1000Res. 2024 ;13 77
    A guide to selecting high-performing antibodies for PLC-gamma-2 for use in Western Blot, immunoprecipitation and immunofluorescence.
    NeuroSGC/YCharOS/EDDU collaborative group
      Phosphatidylinositol-specific phospholipase C gamma 2 (PLC-gamma-2) is an enzyme that regulates the function of immune cells. PLC-gamma-2 has been implicated in neurodegenerative and autoimmune disorders, yet investigation of this protein has been limited by a lack of independently characterized antibodies. Here we have characterized eleven PLC-gamma-2 commercial antibodies for use in Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.
    Keywords:  PLC-gamma-2; PLC-γ2; PLCG2; Phosphatidylinositol-specific phospholipase C gamma 2; Uniprot ID P16885; Western Blot; antibody characterization; antibody validation; immunofluorescence ; immunoprecipitation
    DOI:  https://doi.org/10.12688/f1000research.146156.1
  4. Neuron. 2024 Oct 23. pii: S0896-6273(24)00695-0. [Epub ahead of print]112(20): 3375-3377
    Interference of nuclear speckles: A nexus of RNA foci, dipeptide repeats, and mis-splicing in C9ORF72 ALS/FTD.
    Haining Zhu.
      In this issue of Neuron, Wu et al.1 show that nuclear speckle proteins are sequestered by both nuclear RNA foci and cytoplasmic dipeptide repeat aggregates in C9ORF72-ALS/FTD. Consequently, dysregulation of splicing induces widespread splicing alterations and contributes to neurodegeneration.
    DOI:  https://doi.org/10.1016/j.neuron.2024.10.001
  5. Mol Brain. 2024 Oct 23. 17(1): 77
    NDRG1 upregulation by ubiquitin proteasome system dysfunction aggravates neurodegeneration.
    Tomonori Hoshino, Atsushi Mukai, Hirofumi Yamashita, Hidemi Misawa, Makoto Urushitani, Yoshitaka Tashiro, Shu-Ichi Matsuzawa, Ryosuke Takahashi.
      Protein turnover is crucial for cell survival, and the impairment of proteostasis leads to cell death. Aging is associated with a decline in proteostasis, as the progressive accumulation of damaged proteins is a hallmark of age-related disorders such as neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). We previously discovered that the declining function of the ubiquitin-proteasome system (UPS) in motor neurons contributes to sporadic ALS pathologies, such as progressive motor neuron loss, protein accumulation, and glial activation. However, the mechanisms of UPS dysfunction-induced cell damage, such as cell death and aggregation, are not fully understood. This study used transcriptome analysis of motor neurons with UPS dysfunction and found that the expression of N-myc downstream regulated 1 (NDRG1) gets upregulated by UPS dysfunction. Additionally, the upregulation of NDRG1 induces cell death in the Neuro2a mouse neuroblastoma cell line. These results suggest that NDRG1 is a potential marker for UPS dysfunction and may play a role in neurodegeneration, such as that seen in ALS.
    Keywords:  Amyotrophic lateral sclerosis; Cell death; NDRG1; Neurodegeneration; Proteasome; Psmc4 (Rpt3)
    DOI:  https://doi.org/10.1186/s13041-024-01150-1
  6. J Mol Biol. 2024 Oct 17. pii: S0022-2836(24)00445-5. [Epub ahead of print] 168823
    A hydrophobic core stabilizes the residual structure in the RRM2 intermediate state of the ALS-linked protein TDP-43.
    Brian C Mackness, Brittany R Morgan, Laura M Deveau, Sagar V Kathuria, Jill A Zitzewitz, Francesca Massi.
      Folding intermediates mediate both protein folding and the misfolding and aggregation observed in human diseases, including amyotrophic lateral sclerosis (ALS), and are prime targets for therapeutic interventions. In this study, we identified the core nucleus of structure for a folding intermediate in the second RNA recognition motif (RRM2) of the ALS-linked RNA-binding protein, TDP-43, using a combination of experimental and computational approaches. Urea equilibrium unfolding studies revealed that the RRM2 intermediate state consists of collapsed residual secondary structure localized to the N-terminal half of RRM2, while the C-terminus is largely disordered. Steered molecular dynamics simulations and mutagenesis studies yielded key stabilizing hydrophobic contacts that, when mutated to alanine, severely disrupt the overall fold of RRM2. In combination, these findings suggest a role for this RRM intermediate in normal TDP-43 function as well as serving as a template for misfolding and aggregation through the low stability and non-native secondary structure.
    Keywords:  neurodegenerative diseases; protein conformation; protein misfolding; protein stability; protein structure
    DOI:  https://doi.org/10.1016/j.jmb.2024.168823
  7. Brain Sci. 2024 Sep 27. pii: 978. [Epub ahead of print]14(10):
    Sleep Apnea and Amyotrophic Lateral Sclerosis: Cause, Correlation, Any Relation?
    P Hande Ozdinler.
      Amyotrophic lateral sclerosis (ALS) is a motor neuron disease with progressive neurodegeneration, affecting both the cortical and the spinal component of the motor neuron circuitry in patients. The cellular and molecular basis of selective neuronal vulnerability is beginning to emerge. Yet, there are no effective cures for ALS, which affects more than 200,000 people worldwide each year. Recent studies highlight the importance of the glymphatic system and its proper function for the clearance of the cerebral spinal fluid, which is achieved mostly during the sleep period. Therefore, a potential link between problems with sleep and neurodegenerative diseases has been postulated. This paper discusses the present understanding of this potential correlation.
    Keywords:  CSF; glymphatic system; neurodegeneration; spine loss
    DOI:  https://doi.org/10.3390/brainsci14100978
  8. FASEB J. 2024 Oct;38(20): e70121
    The role of polo-like kinases 2 in the proteasomal and lysosomal degradation of alpha-synuclein in neurons.
    Chun Chau Sung, Wai Yun Lam, Kenny K K Chung.
      Parkinson's disease (PD) is a neurodegenerative disorder caused by the degeneration of dopaminergic neurons in the brain stem. PD is mostly sporadic, but familial PD (FPD) cases are recorded in different studies. The first gene mutation that is linked to FPD is α-synuclein (α-syn). It was then found that α-syn is also accumulated in Lewy body (LB), a classical pathological hallmark in PD patients. Different studies have shown that α-syn accumulation and aggregation can be a crucial factor contributing to the degeneration of dopaminergic neurons in PD. α-syn has been found to be degraded by the ubiquitin proteasomal system (UPS) and autophagy-lysosomal pathway (ALP). In this study, we initially explored how α-syn phosphorylation by GRK6, PLK2 and CK2α would facilitate its degradation in relation to the UPS or ALP. Unexpectedly, we found that the degradation of α-syn through PLK2 phosphorylation could be modulated by UPS and ALP in a novel mechanism. Specially, attenuation of UPS could increase the amount of PLK2 and then could facilitate the phosphorylation and degradation of α-syn through ALP. To test this further in vivo, we attenuate the proteasomal activity in a well-established A53T α-syn transgenic PD mouse model. We found that attenuation of proteasomal activity in the A53T α-syn transgenic mice could reduce the accumulation of α-syn in the striatum and midbrain. Based on our results, this study provides a new insight into how α-syn is degraded through the UPS and ALP.
    Keywords:  autophagy–lysosomal pathway; phosphorylation; protein degradation; ubiquitin‐proteasomal system; α‐synuclein
    DOI:  https://doi.org/10.1096/fj.202401035R
  9. J Transl Med. 2024 Oct 21. 22(1): 953
    CSF and blood levels of Neurofilaments, T-Tau, P-Tau, and Abeta-42 in amyotrophic lateral sclerosis: a systematic review and meta-analysis.
    Elmira Agah, Helia Mojtabavi, Atefeh Behkar, Arash Heidari, Atra Ajdari, Zoha Shaka, Seyed Vahid Mousavi, Negar Firoozeh, Abbas Tafakhori, Nima Rezaei.
      Recent literature suggests that markers of neuroaxonal damage, such as neurofilaments and tau protein, might serve as potential biomarkers for ALS. We conducted this systematic review and meta-analysis study to compare cerebrospinal fluid (CSF) and blood levels of total tau (t-tau), phosphorylated tau (p-tau), amyloid beta peptide 42 (Abeta-42), and neurofilaments in ALS patients and controls. A systematic search of Cochrane Library, PubMed, Embase, and ISI Web of Science was conducted on March 18, 2022, and updated on January 26, 2023. Observational studies that compared the concentrations of neurofilament light chain (NfL), neurofilament heavy chain (NFH), t-tau, p-tau, or Abeta-42 in CSF or peripheral blood of ALS patients and controls were included. Data from relevant studies were independently extracted and screened for quality using a standard tool, by at least two authors. Meta-analysis was conducted when a minimum of 3 studies reported the same biomarker within the same biofluid. A total of 100 studies were eligible for at least one meta-analysis. CSF and blood levels of NfL (standardized mean difference (SMD) [95% CI]; CSF: 1.46 [1.25-1.68]; blood: 1.35 [1.09-1.60]) and NFH (CSF: 1.32 [1.13-1.50], blood: 0.90 [0.58-1.22]) were significantly higher in ALS patients compared with controls. The pooled differences between ALS patients and controls were not significant for CSF t-tau, blood t-tau, and CSF Abeta-42, but CSF p-tau was lower in ALS patients (-0.27 [-0.47- -0.07]). Significantly decreased p-tau/t-tau ratios were found in ALS patients compared with controls (-0.84 [-1.16- -0.53]). Heterogeneity was considerable in most of our meta-analyses. CSF and blood neurofilament levels, as well as the CSF p-tau/t-tau ratio, might be potential candidates for improving ALS diagnosis. Further research is warranted to better understand the underlying mechanisms and the clinical implications of these biomarker alterations.
    Keywords:  ALS; Abeta-42; Alzheimer’s disease biomarkers; Amyotrophic lateral sclerosis; Biomarkers; Meta-analysis; Neurofilaments; Phosphorylated-tau; Systematic review; Total-tau
    DOI:  https://doi.org/10.1186/s12967-024-05767-7
  10. J Fungi (Basel). 2024 Oct 19. pii: 727. [Epub ahead of print]10(10):
    Recent Advances in Diagnostic Approaches for Mucormycosis.
    Jawad Safiia, Marco Aurelio Díaz, Hassan Alshaker, Christine J Atallah, Paul Sakr, Dimitrios G Moshovitis, Ahmad Nawlo, Andres E Franceschi, Alexis Liakos, Sophia Koo.
      Mucormycosis, an invasive fungal infection caused by members of the order Mucorales, often progresses fulminantly if not recognized in a timely manner. This comprehensive review discusses the latest developments in diagnostic approaches for mucormycosis, from traditional histopathology and culture-based methods to advanced and emerging techniques such as molecular assays, imaging, serology, and metabolomics. We discuss challenges in the diagnosis of mucormycosis and emphasize the importance of rapid and accurate identification of this life-threatening infection.
    Keywords:  Mucorales; fungal biomarkers; medical mycology; metabolomics; mucormycosis; volatile organic compounds
    DOI:  https://doi.org/10.3390/jof10100727
  11. Sci Rep. 2024 10 22. 14(1): 24875
    Validation of a functional human AD model with four AD therapeutics utilizing patterned ipsc-derived cortical neurons integrated with microelectrode arrays.
    Julbert Caneus, Kaveena Autar, Nesar Akanda, Marcella Grillo, Christopher J Long, Max Jackson, Sarah Lindquist, Xiufang Guo, Dave Morgan, James J Hickman.
      Preclinical methods are needed for screening potential Alzheimer's disease (AD) therapeutics that recapitulate phenotypes found in the Mild Cognitive Impairment (MCI) stage or even before this stage of the disease. This would require a phenotypic system that reproduces cognitive deficits without significant neuronal cell death to mimic the clinical manifestations of AD during these stages. Long-term potentiation (LTP), which is a correlate of learning and memory, was induced in mature human iPSC-derived cortical neurons cultured on microelectrode arrays utilizing circuit patterns connecting two adjacent electrodes. We demonstrated an LTP system that modeled the MCI and pre-MCI stages of Alzheimer's and validated this functional system utilizing four AD therapeutics, which was also verified utilizing patch-clamp electrophysiology. LTP was induced by tetanic electrical stimulation, and LTP maintenance was significantly reduced in the presence of Amyloid-Beta 42 (Aβ42) oligomers compared to the controls, however, co-treatment with AD therapeutics (Donepezil, Memantine, Rolipram and Saracatinib) corrected Aβ42-induced LTP impairment. The results illustrate the utility of the system as a validated platform to model MCI AD pathology, and potentially for the pre-MCI phase before significant neuronal death. This system also has the potential to become an ideal platform for high-content therapeutic screening for other neurodegenerative diseases.
    Keywords:  Alzheimer’s disease; Amyloid-beta42; Drug efficacy; Human-on-a-Chip; Long-term potentiation; Microelectrode arrays
    DOI:  https://doi.org/10.1038/s41598-024-73869-9
  12. Front Cell Dev Biol. 2024 ;12 1477173
    Molecular mechanisms of polarized transport to the apical plasma membrane.
    Masataka Kunii, Akihiro Harada.
      Cell polarity is essential for cellular function. Directional transport within a cell is called polarized transport, and it plays an important role in cell polarity. In this review, we will introduce the molecular mechanisms of polarized transport, particularly apical transport, and its physiological importance.
    Keywords:  Rab8; apical transport; cell polarity; polarized transport; unconventional transport
    DOI:  https://doi.org/10.3389/fcell.2024.1477173
  13. Mol Biomed. 2024 Oct 24. 5(1): 44
    Identification of nitric oxide-mediated necroptosis as the predominant death route in Parkinson's disease.
    Ting Zhang, Wenjing Rui, Yue Sun, Yunyun Tian, Qiaoyan Li, Qian Zhang, Yanchun Zhao, Zongzhi Liu, Tiepeng Wang.
      Parkinson's disease (PD) involves multiple forms of neuronal cell death, but the dominant pathway involved in disease progression remains unclear. This study employed RNA sequencing (RNA-seq) of brain tissue to explore gene expression patterns across different stages of PD. Using the Scaden deep learning algorithm, we predicted neurocyte subtypes and modelled dynamic interactions for five classic cell death pathways to identify the predominant routes of neuronal death during PD progression. Our cell type-specific analysis revealed an increasing shift towards necroptosis, which was strongly correlated with nitric oxide synthase (NOS) expression across most neuronal subtypes. In vitro experiments confirmed that nitric oxide (NO) is a key mediator of necroptosis, leading to nuclear shrinkage and decreased mitochondrial membrane potential via phosphorylation of the PIP1/PIP3/MLKL signalling cascade. Importantly, specific necroptosis inhibitors significantly mitigated neuronal damage in both in vitro and in vivo PD models. Further analysis revealed that NO-mediated necroptosis is prevalent in early-onset Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS) and across multiple brain regions but not in brain tumours. Our findings suggest that NO-mediated necroptosis is a critical pathway in PD and other neurodegenerative disorders, providing potential targets for therapeutic intervention.
    Keywords:  Necroptosis; Neurodegenerative disease; Nitric oxide; Parkinson's disease
    DOI:  https://doi.org/10.1186/s43556-024-00213-y
  14. J Cell Biol. 2025 Jan 06. pii: e202402169. [Epub ahead of print]224(1):
    CryoVesNet: A dedicated framework for synaptic vesicle segmentation in cryo-electron tomograms.
    Amin Khosrozadeh, Raphaela Seeger, Guillaume Witz, Julika Radecke, Jakob B Sørensen, Benoît Zuber.
      Cryo-electron tomography (cryo-ET) has the potential to reveal cell structure down to atomic resolution. Nevertheless, cellular cryo-ET data is highly complex, requiring image segmentation for visualization and quantification of subcellular structures. Due to noise and anisotropic resolution in cryo-ET data, automatic segmentation based on classical computer vision approaches usually does not perform satisfactorily. Communication between neurons relies on neurotransmitter-filled synaptic vesicle (SV) exocytosis. Cryo-ET study of the spatial organization of SVs and their interconnections allows a better understanding of the mechanisms of exocytosis regulation. Accurate SV segmentation is a prerequisite to obtaining a faithful connectivity representation. Hundreds of SVs are present in a synapse, and their manual segmentation is a bottleneck. We addressed this by designing a workflow consisting of a convolutional network followed by post-processing steps. Alongside, we provide an interactive tool for accurately segmenting spherical vesicles. Our pipeline can in principle segment spherical vesicles in any cell type as well as extracellular and in vitro spherical vesicles.
    DOI:  https://doi.org/10.1083/jcb.202402169
  15. Sci Rep. 2024 10 23. 14(1): 24959
    Hemozoin induces malaria via activation of DNA damage, p38 MAPK and neurodegenerative pathways in a human iPSC-derived neuronal model of cerebral malaria.
    Abida Lslam Pranty, Leon-Phillip Szepanowski, Wasco Wruck, Akua Afriyie Karikari, James Adjaye.
      Malaria caused by Plasmodium falciparum infection results in severe complications including cerebral malaria (CM), in which approximately 30% of patients end up with neurological sequelae. Sparse in vitro cell culture-based experimental models which recapitulate the molecular basis of CM in humans has impeded progress in our understanding of its etiology. This study employed healthy human induced pluripotent stem cells (iPSCs)-derived neuronal cultures stimulated with hemozoin (HMZ) - the malarial toxin as a model for CM. Secretome, qRT-PCR, Metascape, and KEGG pathway analyses were conducted to assess elevated proteins, genes, and pathways. Neuronal cultures treated with HMZ showed enhanced secretion of interferon-gamma (IFN-γ), interleukin (IL)1-beta (IL-1β), IL-8 and IL-16. Enrichment analysis revealed malaria, positive regulation of cytokine production and positive regulation of mitogen-activated protein kinase (MAPK) cascade which confirm inflammatory response to HMZ exposure. KEGG assessment revealed up-regulation of malaria, MAPK and neurodegenerative diseases-associated pathways which corroborates findings from previous studies. Additionally, HMZ induced DNA damage in neurons. This study has unveiled that exposure of neuronal cultures to HMZ, activates molecules and pathways similar to those observed in CM and neurodegenerative diseases. Furthermore, our model is an alternative to rodent experimental models of CM.
    Keywords:  Cerebral malaria; DNA damage; Hemozoin; Neuro-inflammation; Neurons; iPSCs
    DOI:  https://doi.org/10.1038/s41598-024-76259-3
  16. Invest Ophthalmol Vis Sci. 2024 Oct 01. 65(12): 37
    NMNAT1 Is Essential for Human iPS Cell Differentiation to the Retinal Lineage.
    Hiroshi Kuribayashi, Toshiro Iwagawa, Akira Murakami, Takeshi Kawamura, Yutaka Suzuki, Sumiko Watanabe.
      Purpose: The gene encoding nicotinamide mononucleotide adenylyltransferase 1 (NMNAT1), a nicotinamide adenine dinucleotide synthetase localized in the cell nucleus, is a causative factor in Leber's congenital amaurosis, which is the earliest onset type of inherited retinal degeneration. We sought to investigate the roles of NMNAT1 in early retinal development.Methods: We used human induced pluripotent stem cells (hiPSCs) and established NMNAT1-knockout (KO) hiPSCs using CRISPR/cas9 technology to reveal the roles of NMNAT1 in human retinal development.
    Results: NMNAT1 was not essential for the survival and proliferation of immature hiPSCs; therefore, we subjected NMNAT1-KO hiPSCs to retinal organoid (RO) differentiation culture. The expression levels of immature hiPSC-specific genes decreased in a similar manner after organoid culture initiation up to 2 weeks in the control and NMNAT1-KO. Neuroectoderm-specific genes were induced in the control and NMNAT1-KO organoids within a few days after starting the organoid culture; PAX6 and TUBB3 were higher in NMNAT1-KO organoids up to 7 days than in the control organoids. However, the induction of genes involving retinal early development, such as RAX, which was induced at around day 10 in this culture, was considerably reduced in NMNAT1-KO organoids. Morphological examination also showed failure of retinal primordial structure formation, which became visible at around 2 weeks of the control culture, in the NMNAT1-KO organoids. Decreased intracellular NAD levels and poly(ADP-ribosyl)ation were observed in NMNAT1-KO organoids at 7 to 10 days of the culture. Mass spectrometry analysis of inhibited proteins in the poly(ADP-ribosyl)ation pathway identified poly(ADP-ribosyl)ation of poly(ADP-ribose) polymerase 1 (PARP1) as a major protein.
    Conclusions: These results indicate that NMNAT1 was dispensable for neural lineage differentiation but essential for the commitment of retinal fate differentiation in hiPSCs. The NMNAT1-NAD-PARP1 axis may play a critical role in the appropriate development of human retinal lineage differentiation.
    DOI:  https://doi.org/10.1167/iovs.65.12.37
  17. Nano Lett. 2024 Oct 21.
    Nanomagnetic Guidance Shapes the Structure-Function Relationship of Developing Cortical Networks.
    Connor L Beck, Conner T Killeen, Sara C Johnson, Anja Kunze.
      In this study, we implement large-scale nanomagnetic guidance on cortical neurons to guide dissociated neuronal networks during development. Cortical networks cultured over microelectrode arrays were exposed to functionalized magnetic nanoparticles, followed by magnetic field exposure to guide neurites over 14 days in vitro. Immunofluorescence of the axonal protein Tau revealed a greater number of neurites that were longer and aligned with the nanomagnetic force relative to nonguided networks. This was further confirmed through brightfield imaging on the microelectrode arrays during development. Spontaneous electrophysiological recordings revealed that the guided networks exhibited increased firing rates and frequency in force-aligned connectivity identified through Granger Causality. Applying this methodology across networks with nonuniform force directions increased local activity in target regions, identified as regions in the direction of the nanomagnetic force. Altogether, these results demonstrate that nanomagnetic forces guide the structure and function of dissociated cortical neuron networks at the millimeter scale.
    Keywords:  Axon guidance; Electrophysiology; Magnetic nanoparticles; Microelectrode arrays; Neural circuit guidance; Neural networks
    DOI:  https://doi.org/10.1021/acs.nanolett.4c03156
  18. Endocr Relat Cancer. 2024 Oct 01. pii: ERC-24-0187. [Epub ahead of print]
    Cellular Mechanisms of RET Receptor Dysfunction in Multiple Endocrine Neoplasia 2.
    Timothy J Walker, Lois M Mulligan.
      REarranged during Transfection (RET) is a developmentally important receptor tyrosine kinase that has been identified as an oncogenic driver in a number of cancers. Activating RET point-mutations give rise to the inherited cancer syndrome Multiple Endocrine Neoplasia type 2 (MEN2), characterized by medullary thyroid carcinoma. There are two MEN2 subtypes, MEN2A and MEN2B, that differ in tumour aggressiveness and the associated constellation of other disease features, which are caused by distinct patterns of RET amino acid substitution mutations. MEN2A-RET mutations affecting extracellular cysteine residues promote ligand independent dimerization and constitutive RET activity, while MEN2B is caused by a single amino acid change in the tyrosine kinase domain of RET, releasing autoinhibition and producing a more active MEN2B-RET kinase that can promote signalling as monomers or dimers in the absence of ligand. These mutations cause intrinsic biochemical changes in RET structure and activation but also trigger extrinsic effects that alter RET cellular location, interactions and mechanisms of downregulation that can prolong or mislocate RET activity, changing or enhancing functional outcomes. Together, changes in specific combinations of RET-mediated effects associated with different mutations give rise to the distinct MEN2 disease phenotypes. Here, we discuss the current understanding of the intrinsic and extrinsic characteristics of RET MEN2A cysteine and MEN2B mutants and how these contribute to transforming cellular processes and to differences in tumour progression and disease aggressiveness.
    DOI:  https://doi.org/10.1530/ERC-24-0187
  19. Ageing Res Rev. 2024 Oct 19. pii: S1568-1637(24)00367-2. [Epub ahead of print]102 102549
    Mitochondria break free: Mitochondria-derived vesicles in aging and associated conditions.
    Luigi Ferrucci, Flora Guerra, Cecilia Bucci, Emanuele Marzetti, Anna Picca.
      Mitophagy is the intracellular recycling system that disposes damaged/inefficient mitochondria and allows biogenesis of new organelles to ensure mitochondrial quality is optimized. Dysfunctional mitophagy has been implicated in human aging and diseases. Multiple evolutionarily selected, redundant mechanisms of mitophagy have been identified, but their specific roles in human health and their potential exploitation as therapeutic targets are unclear. Recently, the characterization of the endosomal-lysosomal system has revealed additional mechanisms of mitophagy and mitochondrial quality control that operate via the production of mitochondria-derived vesicles (MDVs). Circulating MDVs can be isolated and characterized to provide an unprecedented opportunity to study this type of mitochondrial recycling in vivo and to relate it to human physiology and pathology. Defining the role of MDVs in human physiology, pathology, and aging is hampered by the lack of standardized methods to isolate, validate, and characterize these vesicles. Hence, some basic questions about MDVs remain unanswered. While MDVs are generated directly through the extrusion of mitochondrial membranes within the cell, a set of circulating extracellular vesicles leaking from the endosomal-lysosomal system and containing mitochondrial portions have also been identified and warrant investigation. Preliminary research indicates that MDV generation serves multiple biological roles and contributes to restoring cell homeostasis. However, studies have shown that MDVs may also be involved in pathological conditions. Therefore, further research is warranted to establish when/whether MDVs are supporting disease progression and/or are extracting damaged mitochondrial components to alleviate cellular oxidative burden and restore redox homeoastasis. This information will be relevant for exploiting these vesicles for therapeutic purpose. Herein, we provide an overview of preclinical and clinical studies on MDVs in aging and associated conditions and discuss the interplay between MDVs and some of the hallmarks of aging (mitophagy, inflammation, and proteostasis). We also outline open questions on MDV research that should be prioritized by future investigations.
    Keywords:  Exosomes; Extracellular vesicles; Inflammaging; Mitochondrial DNA; Mitochondrial quality control; Mitophagy
    DOI:  https://doi.org/10.1016/j.arr.2024.102549
  20. Alzheimers Dement. 2024 Oct 23.
    Inhibiting mtDNA transcript translation alters Alzheimer's disease-associated biology.
    Alexander P Gabrielli, Lesya Novikova, Amol Ranjan, Xiaowan Wang, Nicholas J Ernst, Dhanushki Abeykoon, Anysja Roberts, Annie Kopp, Clayton Mansel, Linlan Qiao, Colton R Lysaker, Ian W Wiedling, Heather M Wilkins, Russell H Swerdlow.
      INTRODUCTION: Alzheimer's disease (AD) features changes in mitochondrial structure and function. Investigators debate where to position mitochondrial pathology within the chronology and context of other AD features.METHODS: To address whether mitochondrial dysfunction alters AD-implicated genes and proteins, we treated SH-SY5Y cells and induced pluripotent stem cell (iPSC)-derived neurons with chloramphenicol, an antibiotic that inhibits mtDNA-generated transcript translation. We characterized adaptive, AD-associated gene, and AD-associated protein responses.
    RESULTS: SH-SY5Y cells and iPSC neurons responded to mtDNA transcript translation inhibition by increasing mtDNA copy number and transcription. Nuclear-expressed respiratory chain mRNA and protein levels also changed. There were AD-consistent concordant and model-specific changes in amyloid precursor protein, beta amyloid, apolipoprotein E, tau, and α-synuclein biology.
    DISCUSSION: Primary mitochondrial dysfunction induces compensatory organelle responses, changes nuclear gene expression, and alters the biology of AD-associated genes and proteins in ways that may recapitulate brain aging and AD molecular phenomena.
    HIGHLIGHTS: In AD, mitochondrial dysfunction could represent a disease cause or consequence. We inhibited mitochondrial translation in human neuronal cells and neurons. Mitochondrial and nuclear gene expression shifted in adaptive-consistent patterns. APP, Aβ, APOE, tau, and α-synuclein biology changed in AD-consistent patterns. Mitochondrial stress creates an environment that promotes AD pathology.
    Keywords:  Alzheimer's disease; amyloid; mitochondria; neurons; translation
    DOI:  https://doi.org/10.1002/alz.14275
  21. Br J Biomed Sci. 2024 ;81 13169
    Neuronal Vulnerability of the Entorhinal Cortex to Tau Pathology in Alzheimer's Disease.
    Simi Zhang, Chelsea Ann Crossley, Qi Yuan.
      This review delves into the entorhinal cortex (EC) as a central player in the pathogenesis of Alzheimer's Disease (AD), emphasizing its role in the accumulation and propagation of tau pathology. It elucidates the multifaceted functions of the EC, encompassing memory formation, spatial navigation, and olfactory processing, while exploring how disruptions in these processes contribute to cognitive decline in AD. The review discusses the intricate interplay between tau pathology and EC vulnerability, highlighting how alterations in neuronal firing patterns and synaptic function within the EC exacerbate cognitive impairments. Furthermore, it elucidates how specific neuronal subtypes within the EC exhibit differential susceptibility to tau-induced damage, contributing to disease progression. Early detection methods, such as imaging techniques and assessments of EC blood flow, are examined as potential tools for identifying tau pathology in the preclinical stages of AD. These approaches offer promise for improving diagnostic accuracy and enabling timely intervention. Therapeutic strategies targeting tau pathology within the EC are explored, including the clearance of pathological tau aggregates and the inhibition of tau aggregation processes. By understanding the molecular and cellular mechanisms underlying EC vulnerability, researchers can develop more targeted and effective interventions to slow disease progression. The review underscores the importance of reliable biomarkers to assess disease progression and therapeutic efficacy in clinical trials targeting the EC. Ultimately, it aims to contribute to the development of more effective management strategies for AD, emphasizing the translation of research findings into clinical practice to address the growing societal burden of the disease.
    Keywords:  Alzheimer’s disease; early detection; entorhinal cortex; memory; tau pathology
    DOI:  https://doi.org/10.3389/bjbs.2024.13169
  22. Proc Natl Acad Sci U S A. 2024 Oct 29. 121(44): e2415383121
    A protein phosphatase 1 specific phosphatase targeting peptide (PhosTAP) to identify the PP1 phosphatome.
    Meng S Choy, Hieu T Nguyen, Ganesan S Kumar, Wolfgang Peti, Arminja N Kettenbach, Rebecca Page.
      Phosphoprotein phosphatases (PPPs) are the key serine/threonine phosphatases that regulate all essential signaling cascades. In particular, Protein Phosphatase 1 (PP1) dephosphorylates ~80% of all ser/thr phosphorylation sites. Here, we developed a phosphatase targeting peptide (PhosTAP) that binds all PP1 isoforms and does so with a stronger affinity than any other known PP1 regulator. This PhosTAP can be used as a PP1 recruitment tool for Phosphorylation Targeting Chimera (PhosTAC)-type recruitment in in vitro and cellular experiments, as well as in phosphoproteomics experiments to identify PP1-specific substrates and phosphosites. The latter is especially important to further our understanding of cellular signaling, as the identification of substrates and especially phosphosites that are targeted by specific phosphatases lags behind that of their kinase counterparts. Using PhosTAP-based proteomics, we show that, counter to our current understanding, many PP1 regulators are also substrates, that the number of residues between regulator PP1-binding and phosphosites vary significantly, and that PP1 counteracts the activities of mitotic kinases. Finally, we also found that Haspin kinase is a direct substrate of PP1 and that its PP1-dependent dephosphorylation modulates its activity during anaphase. Together, we show that PP1-specific PhosTAPs are a powerful tool for +studying PP1 activity in vitro and in cells.
    Keywords:  phosphatase targeting peptide (PhosTAP); phosphoprotein phosphatases (PPP); protein engineering; protein phosphatase 1 (PP1); protein–protein interactions
    DOI:  https://doi.org/10.1073/pnas.2415383121
  23. CRISPR J. 2024 Oct;7(5): 258-271
    Comparison of Gene-Editing Approaches for Severe Congenital Neutropenia-Causing Mutations in the ELANE Gene.
    Malte Ulrich Ritter, Masoud Nasri, Benjamin Dannenmann, Perihan Mir, Benjamin Secker, Diana Amend, Maksim Klimiankou, Karl Welte, Julia Skokowa.
      Safety considerations for gene therapies of inherited preleukemia syndromes, including severe congenital neutropenia (CN), are paramount. We compared several strategies for CRISPR/Cas9 gene editing of autosomal-dominant ELANE mutations in CD34+ cells from two CN patients head-to-head. We tested universal and allele-specific ELANE knockout, ELANE mutation correction by homology-directed repair (HDR) with AAV6, and allele-specific HDR with ssODN. All strategies were not toxic, had at least 30% editing, and rescued granulopoiesis in vitro. In contrast to published data, allele-specific indels in the last exon of ELANE also restored granulopoiesis. Moreover, by implementing patient-derived induced pluripotent stem cells for GUIDE-Seq off-target analysis, we established a clinically relevant "personalized" assessment of off-target activity of gene editing on the background of the patient's genome. We found that allele-specific approaches had the most favorable off-target profiles. Taken together, a well-defined head-to-head comparison pipeline for selecting the appropriate gene therapy is essential for diseases, with several gene editing strategies available.
    DOI:  https://doi.org/10.1089/crispr.2024.0006
  24. Front Cell Neurosci. 2024 ;18 1472374
    ALS-like pathology diminishes swelling of spinal astrocytes in the SOD1 animal model.
    Tereza Filipi, Jana Tureckova, Ondrej Vanatko, Martina Chmelova, Monika Kubiskova, Natalia Sirotova, Stanislava Matejkova, Lydia Vargova, Miroslava Anderova.
      Astrocytes are crucial for the functioning of the nervous system as they maintain the ion homeostasis via volume regulation. Pathological states, such as amyotrophic lateral sclerosis (ALS), affect astrocytes and might even cause a loss of such functions. In this study, we examined astrocytic swelling/volume recovery in both the brain and spinal cord of the SOD1 animal model to determine the level of their impairment caused by the ALS-like pathology. Astrocyte volume changes were measured in acute brain or spinal cord slices during and after exposure to hyperkalemia. We then compared the results with alterations of extracellular space (ECS) diffusion parameters, morphological changes, expression of the Kir4.1 channel and the potassium concentration measured in the cerebrospinal fluid, to further disclose the link between potassium and astrocytes in the ALS-like pathology. Morphological analysis revealed astrogliosis in both the motor cortex and the ventral horns of the SOD1 spinal cord. The activated morphology of SOD1 spinal astrocytes was associated with the results from volume measurements, which showed decreased swelling of these cells during hyperkalemia. Furthermore, we observed lower shrinkage of ECS in the SOD1 spinal ventral horns. Immunohistochemical analysis then confirmed decreased expression of the Kir4.1 channel in the SOD1 spinal cord, which corresponded with the diminished volume regulation. Despite astrogliosis, cortical astrocytes in SOD1 mice did not show alterations in swelling nor changes in Kir4.1 expression, and we did not identify significant changes in ECS parameters. Moreover, the potassium level in the cerebrospinal fluid did not deviate from the physiological concentration. The results we obtained thus suggest that ALS-like pathology causes impaired potassium uptake associated with Kir4.1 downregulation in the spinal astrocytes, but based on our data from the cortex, the functional impairment seems to be independent of the morphological state.
    Keywords:  SOD1; amyotrophic lateral sclerosis; astrocytes; extracellular space; potassium uptake; volume regulation
    DOI:  https://doi.org/10.3389/fncel.2024.1472374
  25. Comput Struct Biotechnol J. 2024 Dec;23 3506-3513
    Insights into the structure of NLR family member X1: Paving the way for innovative drug discovery.
    Shannon Jewell, Thanh Binh Nguyen, David B Ascher, Avril A B Robertson.
      Nucleotide-binding oligomerization domain, leucine rich repeat containing X1 (NLRX1) is a negative regulator of the nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) pathway, with a significant role in the context of inflammation. Altered expression of NLRX1 is prevalent in inflammatory diseases leading to interest in NLRX1 as a drug target. There is a lack of structural information available for NLRX1 as only the leucine-rich repeat domain of NLRX1 has been crystallised. This lack of structural data limits progress in understanding function and potential druggability of NLRX1. We have modelled full-length NLRX1 by combining experimental, homology modelled and AlphaFold2 structures. The full-length model of NLRX1 was used to explore protein dynamics, mutational tolerance and potential functions. We identified a new RNA binding site in the previously uncharacterized N-terminus, which served as a basis to model protein-RNA complexes. The structure of the adenosine triphosphate (ATP) binding domain revealed a potential catalytic functionality for the protein as a member of the ATPase Associated with Diverse Cellular Activity family of proteins. Finally, we investigated the interactions of NLRX1 with small molecule activators in development, revealing a binding site that has not previously been discussed in literature. The model generated here will help to catalyse efforts towards creating new drug molecules to target NLRX1 and may be used to inform further studies on functionality of NLRX1.
    Keywords:  ATPase; Inflammasome; Inflammation; NLRX1
    DOI:  https://doi.org/10.1016/j.csbj.2024.09.013
  26. Bioengineering (Basel). 2024 Oct 12. pii: 1020. [Epub ahead of print]11(10):
    An Effective DNA Methylation Biomarker Screening Mechanism for Amyotrophic Lateral Sclerosis (ALS) Based on Comorbidities and Gene Function Analysis.
    Cing-Han Yang, Jhen-Li Huang, Li-Kai Tsai, David Taniar, Tun-Wen Pai.
      This study used epigenomic methylation differential expression analysis to identify primary biomarkers in patients with amyotrophic lateral sclerosis (ALS). We combined electronic medical record datasets from MIMIC-IV (United States) and NHIRD (Taiwan) to explore ALS comorbidities in depth and discover any comorbidity-related biomarkers. We also applied word2vec to these two clinical diagnostic medical databases to measure similarities between ALS and other similar diseases and evaluated the statistical assessment of the odds ratio to discover significant comorbidities for ALS subjects. Important and representative DNA methylation biomarker candidates could be effectively selected by cross-comparing similar diseases to ALS, comorbidity-related genes, and differentially expressed methylation loci for ALS subjects. The screened epigenomic and comorbidity-related biomarkers were clustered based on their genetic functions. The candidate DNA methylation biomarkers associated with ALS were comprehensively discovered. Gene ontology annotations were then applied to analyze and cluster the candidate biomarkers into three different groups based on gene function annotations. The results showed that a potential testing kit for ALS detection can be composed of SOD3, CACNA1H, and ERBB4 for effective early screening of ALS using blood samples. By developing an effective DNA methylation biomarker screening mechanism, early detection and prophylactic treatment of high-risk ALS patients can be achieved.
    Keywords:  DNA methylation; amyotrophic lateral sclerosis (ALS); bioinformatics; biomarker screening platform; comorbidity
    DOI:  https://doi.org/10.3390/bioengineering11101020
  27. Sci Rep. 2024 10 22. 14(1): 24836
    Lysosomal Ca2+ release-facilitated TFEB nuclear translocation alleviates ischemic brain injury by attenuating autophagic/lysosomal dysfunction in neurons.
    Qian Lei, Xuemei Chen, Yajie Xiong, Shangdan Li, Jiaqian Wang, Hongyun He, Yihao Deng.
      Neuronal death was frequently driven by autophagic/lysosomal dysfunction after ischemic stroke, whereas how to restore the impaired autophagic flux remained elusive. Autophagic/lysosomal signaling could be augmented after transcription factor EB (TFEB) nuclear translocation, which was facilitated by its dephosphorylation. A key TFEB dephosphorylase was calcineurin (CaN), whose activity was drastically regulated by cytosolic calcium ion concentration ([Ca2+]) controlled by lysosomal Ca2+ channel-like protein of TRPML1. Our research shows that ML-SA1, an agonist of the TRPML1 channel, significantly enhanced the lysosomal Ca2+ release and the CaN expression in penumbric neurons, subsequently promoted TFEB nuclear translocation, and greatly reversed autophagy/lysosome dysfunction. Moreover, ML-SA1 treatment significantly reduced neuronal loss, infarct size, and neurological deficits. By contrast, ML-SI3, an inhibitor of TRPML1, inhibited the lysosomal Ca2+ release conversely, aggravated the impairment of autophagic flux and consequentially exacerbated brain stroke lesion. These studies suggest that TRPML1 elevation alleviates ischemic brain injury by restoring autophagic/lysosomal dysfunction via Lysosomal Ca2+ release-facilitated TFEB nuclear translocation in neurons.
    Keywords:  Autophagic/lysosomal dysfunction; Ischemic stroke; Lysosomal Ca2+ ; Neuroprotection; TFEB; TRPML1
    DOI:  https://doi.org/10.1038/s41598-024-75802-6
  28. Stem Cells Transl Med. 2024 Oct 23. pii: szae070. [Epub ahead of print]
    Effects of microgravity on human iPSC-derived neural organoids on the International Space Station.
    Davide Marotta, Laraib Ijaz, Lilianne Barbar, Madhura Nijsure, Jason Stein, Nicolette Pirjanian, Ilya Kruglikov, Twyman Clements, Jana Stoudemire, Paula Grisanti, Scott A Noggle, Jeanne F Loring, Valentina Fossati.
      Research conducted on the International Space Station (ISS) in low-Earth orbit (LEO) has shown the effects of microgravity on multiple organs. To investigate the effects of microgravity on the central nervous system, we developed a unique organoid strategy for modeling specific regions of the brain that are affected by neurodegenerative diseases. We generated 3-dimensional human neural organoids from induced pluripotent stem cells (iPSCs) derived from individuals affected by primary progressive multiple sclerosis (PPMS) or Parkinson's disease (PD) and non-symptomatic controls, by differentiating them toward cortical and dopaminergic fates, respectively, and combined them with isogenic microglia. The organoids were cultured for a month using a novel sealed cryovial culture method on the International Space Station (ISS) and a parallel set that remained on Earth. Live samples were returned to Earth for analysis by RNA expression and histology and were attached to culture dishes to enable neurite outgrowth. Our results show that both cortical and dopaminergic organoids cultured in LEO had lower levels of genes associated with cell proliferation and higher levels of maturation-associated genes, suggesting that the cells matured more quickly in LEO. This study is continuing with several more missions in order to understand the mechanisms underlying accelerated maturation and to investigate other neurological diseases. Our goal is to make use of the opportunity to study neural cells in LEO to better understand and treat neurodegenerative disease on Earth and to help ameliorate potentially adverse neurological effects of space travel.
    Keywords:  Parkinson’s disease; induced pluripotent stem cells; microglia; microgravity; multiple sclerosis; neurons; organoids
    DOI:  https://doi.org/10.1093/stcltm/szae070
  29. J Vis Exp. 2024 Oct 04.
    Formation of Human Thymus Organoids in Three-Dimensional Fibrin Hydrogels.
    Manon d'Arco, Nathan Provin, Pierre Maminirina, Olivier Baron, Carole Guillonneau, Laurent David, Matthieu Giraud.
      Generation of a functional and self-tolerant T cell repertoire is a complex process dependent on the thymic microenvironment and, primarily, on the properties of its extracellular matrix (ECM). Thymic epithelial cells (TECs) are crucial in thymopoiesis, nurturing and selecting developing T cells by filtering self-reactive clones. TECs have been empirically demonstrated to be particularly sensitive to physical and chemical clues supplied by the ECM and classical monolayer cell culture leads to a quick loss of functionality until their death. Because of this delicate maintenance combined with relative rarity, and despite the high stakes in modeling thymus biology in vitro, models able to faithfully mimic the TEC niche at scale and over time are still lacking. Here, we describe the formation of a multicellular human thymic organoid model, in which the TEC compartment is derived from human induced pluripotent stem cells (iPSC) and reaggregated with primary early thymocyte progenitors in a three-dimensional (3D) fibrin-based hydrogel. This model answers current needs for a scalable culture system that reproduces the thymic microenvironment ex vivo and demonstrates functionality, i.e., the ability to produce T cells and to support thymus organoid growth over several weeks. Thus, we propose a practical in vitro model of thymus functionality through iPSC-derived organoids that would benefit research on TEC biology and T cell generation ex vivo.
    DOI:  https://doi.org/10.3791/66795
  30. Cells. 2024 Oct 18. pii: 1729. [Epub ahead of print]13(20):
    Complement C5a Implication in Axonal Growth After Injury.
    Aurélie Cotten, Charlotte Jeanneau, Patrick Decherchi, Imad About.
      Complement C5a protein has been shown to play a major role in tissue regeneration through interaction with its receptor (C5aR) on target cells. Expression of this receptor has been reported in the nervous system which, upon injury, has no treatment to restore the lost functions. This work aimed at investigating the Complement C5a effect on axonal growth after axotomy in vitro. Primary hippocampal neurons were isolated from embryonic Wistar rats. Cell expression of C5aR mRNA was verified by RT-PCR while its membrane expression, localization, and phosphorylation were investigated by immunofluorescence. Then, the effects of C5a on injured axonal growth were investigated using a 3D-printed microfluidic device. Immunofluorescence demonstrated that the primary cultures contained only mature neurons (93%) and astrocytes (7%), but no oligodendrocytes or immature neurons. Immunofluorescence revealed a co-localization of NF-L and C5aR only in the mature neurons where C5a induced the phosphorylation of its receptor. C5a application on injured axons in the microfluidic devices significantly increased both the axonal growth speed and length. Our findings highlight a new role of C5a in regeneration demonstrating an enhancement of axonal growth after axotomy. This may provide a future therapeutic tool in the treatment of central nervous system injury.
    Keywords:  Complement C5a; axon regeneration; axotomy; neuron injury
    DOI:  https://doi.org/10.3390/cells13201729
  31. Sci Rep. 2024 10 22. 14(1): 24846
    Thioflavin-T: application as a neuronal body and nucleolar stain and the blue light photo enhancement effect.
    Jin-Hong Min, Heela Sarlus, Sho Oasa, Robert A Harris.
      Thioflavin-T (THT) is a common and indispensable tool for the study of amyloid pathologies and protein aggregation, both in vitro and for histological samples. In this study we expand the use of THT beyond its canonical usage for staining amyloid plaques and demonstrate its novel use as an easy and rapid stain comparable to fluorescent Nissl staining, allowing for clear discernment of neuronal cell bodies and also nucleoli in fixed tissue and live cells. We believe that this is of value for any lab that studies central nervous system (CNS) tissues. Furthermore, we show that THT could potentially be used as a an alternative to the use of fluorescent reporters or other more costly RNA binding compounds in the study of nucleolar dynamics owing to its ability to clearly stain nucleoli in live cells. We also discovered the previously unreported effect of blue light exposure on the photo enhancement of THT excited by a 488 nm laser in stained tissue sample and how to avoid complications arising from this effect. Finally, we provide a simple protocol that can be easily adjusted either for using THT as a neuronal cell body and nucleoli stain, compatible with antibody based staining methods tested up to 4 fluorophores, or alternatively by using an additional washing step the protocol may be used for amyloid plaque detection in fixed brain tissue.
    Keywords:  Alzheimer’s disease; Amyloid Beta; Brain; Neuron; Nissl; Photo Enhancement; Thioflavin-T
    DOI:  https://doi.org/10.1038/s41598-024-74359-8
  32. Commun Med (Lond). 2024 Oct 22. 4(1): 207
    A click-based electrocorticographic brain-computer interface enables long-term high-performance switch scan spelling.
    Daniel N Candrea, Samyak Shah, Shiyu Luo, Miguel Angrick, Qinwan Rabbani, Christopher Coogan, Griffin W Milsap, Kevin C Nathan, Brock A Wester, William S Anderson, Kathryn R Rosenblatt, Alpa Uchil, Lora Clawson, Nicholas J Maragakis, Mariska J Vansteensel, Francesco V Tenore, Nicolas F Ramsey, Matthew S Fifer, Nathan E Crone.
      BACKGROUND: Brain-computer interfaces (BCIs) can restore communication for movement- and/or speech-impaired individuals by enabling neural control of computer typing applications. Single command click detectors provide a basic yet highly functional capability.METHODS: We sought to test the performance and long-term stability of click decoding using a chronically implanted high density electrocorticographic (ECoG) BCI with coverage of the sensorimotor cortex in a human clinical trial participant (ClinicalTrials.gov, NCT03567213) with amyotrophic lateral sclerosis. We trained the participant's click detector using a small amount of training data (<44 min across 4 days) collected up to 21 days prior to BCI use, and then tested it over a period of 90 days without any retraining or updating.
    RESULTS: Using a click detector to navigate a switch scanning speller interface, the study participant can maintain a median spelling rate of 10.2 characters per min. Though a transient reduction in signal power modulation can interrupt usage of a fixed model, a new click detector can achieve comparable performance despite being trained with even less data (<15 min, within 1 day).
    CONCLUSIONS: These results demonstrate that a click detector can be trained with a small ECoG dataset while retaining robust performance for extended periods, providing functional text-based communication to BCI users.
    DOI:  https://doi.org/10.1038/s43856-024-00635-3
  33. Biochem Soc Trans. 2024 Oct 24. pii: BST20231017. [Epub ahead of print]
    Using human disease mutations to understand de novo DNA methyltransferase function.
    Willow Rolls, Marcus D Wilson, Duncan Sproul.
      DNA methylation is a repressive epigenetic mark that is pervasive in mammalian genomes. It is deposited by DNA methyltransferase enzymes (DNMTs) that are canonically classified as having de novo (DNMT3A and DNMT3B) or maintenance (DNMT1) function. Mutations in DNMT3A and DNMT3B cause rare Mendelian diseases in humans and are cancer drivers. Mammalian DNMT3 methyltransferase activity is regulated by the non-catalytic region of the proteins which contain multiple chromatin reading domains responsible for DNMT3A and DNMT3B recruitment to the genome. Characterising disease-causing missense mutations has been central in dissecting the function and regulation of DNMT3A and DNMT3B. These observations have also motivated biochemical studies that provide the molecular details as to how human DNMT3A and DNMT3B mutations drive disorders. Here, we review progress in this area highlighting recent work that has begun dissecting the function of the disordered N-terminal regions of DNMT3A and DNMT3B. These studies have elucidated that the N-terminal regions of both proteins mediate novel chromatin recruitment pathways that are central in our understanding of human disease mechanisms. We also discuss how disease mutations affect DNMT3A and DNMT3B oligomerisation, a process that is poorly understood in the context of whole proteins in cells. This dissection of de novo DNMT function using disease-causing mutations provides a paradigm of how genetics and biochemistry can synergise to drive our understanding of the mechanisms through which chromatin misregulation causes human disease.
    Keywords:  cancer; epigenetics; genetic disease; methylation
    DOI:  https://doi.org/10.1042/BST20231017
  34. J Appl Biomed. 2024 Sep;22(3): 136-140
    Anti-NMDAR1 antibody impairs dendritic branching in immature cultured neurons.
    Pascal Jorratt, Aneta Petruskova.
      Anti-N-methyl D-aspartate receptor (anti-NMDAR) encephalitis is an autoimmune disorder characterized by IgG antibodies targeting NMDAR. The prevalence is remarkably higher in women and some develop the condition during pregnancy. While immunotherapies have shown good outcomes for pregnant mothers and their infants, the impact on early neurodevelopment remains elusive. This study investigates the effects of anti-NMDAR antibody on the development of primary cortical cultures. Anti-NMDAR antibody was administered to the cultures at day in vitro 5 for the following 5 days to assess dendritic branching and arbor complexity, and at day in vitro 14 for measuring the expression of brain-derived neurotrophic factor (BDNF) and synaptic proteins. Immature cultured neurons treated with anti-NMDAR antibody exhibited impaired dendritic branching and arbor complexity. Interestingly, BDNF expression was unaffected in mature neurons. Additionally, GluN1 expression, a mandatory NMDAR subunit, was significantly reduced, while no significant alterations were observed in PSD-95, gephyrin and synaptophysin expression. These findings shed light on the structural and synaptic impacts of anti-NMDAR antibody on immature neurons, providing evidence for their consequences in early neuronal development.
    Keywords:  Anti-NMDAR encephalitis; BDNF; Dendritic branching; Neuronal development; Synaptic proteins
    DOI:  https://doi.org/10.32725/jab.2024.019
  35. Traffic. 2024 Oct;25(10): e12957
    Fluorescent Reporters, Imaging, and Artificial Intelligence Toolkits to Monitor and Quantify Autophagy, Heterophagy, and Lysosomal Trafficking Fluxes.
    Mikhail Rudinskiy, Diego Morone, Maurizio Molinari.
      Lysosomal compartments control the clearance of cell-own material (autophagy) or of material that cells endocytose from the external environment (heterophagy) to warrant supply of nutrients, to eliminate macromolecules or parts of organelles present in excess, aged, or containing toxic material. Inherited or sporadic mutations in lysosomal proteins and enzymes may hamper their folding in the endoplasmic reticulum (ER) and their lysosomal transport via the Golgi compartment, resulting in lysosomal dysfunction and storage disorders. Defective cargo delivery to lysosomal compartments is harmful to cells and organs since it causes accumulation of toxic compounds and defective organellar homeostasis. Assessment of resident proteins and cargo fluxes to the lysosomal compartments is crucial for the mechanistic dissection of intracellular transport and catabolic events. It might be combined with high-throughput screenings to identify cellular, chemical, or pharmacological modulators of these events that may find therapeutic use for autophagy-related and lysosomal storage disorders. Here, discuss qualitative, quantitative and chronologic monitoring of autophagic, heterophagic and lysosomal protein trafficking in fixed and live cells, which relies on fluorescent single and tandem reporters used in combination with biochemical, flow cytometry, light and electron microscopy approaches implemented by artificial intelligence-based technology.
    Keywords:  ER‐phagy; ER‐to‐lysosome‐associated degradation (ERLAD); artificial intelligence; autophagy; autophagy flux; endolysosomes (EL); heterophagy; lysosomal storage disorders (LSD); lysosomes; tandem fluorescent reporters
    DOI:  https://doi.org/10.1111/tra.12957
  36. Dev Reprod. 2024 Sep;28(3): 109-119
    Actin Depolymerizing Factor Destrin Regulates Cilia Development and Function during Vertebrate Embryogenesis.
    Youni Kim, Hyun-Kyung Lee, Kyeong-Yeon Park, Tayaba Ismail, Hongchan Lee, Hyun-Shik Lee.
      The actin cytoskeleton plays fundamental roles in ciliogenesis and the actin depolymerizing factor destrin regulates actin dynamics by treadmilling actin filaments and increasing globular actin pools. However, the specific developmental roles of destrin in ciliogenesis have not been fully elucidated. Here, we investigated the function of destrin in ciliogenesis using Xenopus laevis and human retinal pigmented epithelial (hRPE1) cells. We discovered the loss of destrin increased the number of multiciliated cells in the Xenopus epithelium and impeded cilia motility. Additionally, destrin depletion remarkably reduced the length of primary cilia in the Xenopus neural tube and hRPE1 cells by affecting actin dynamics. Immunofluorescence using markers of ciliary components indicated that destrin controls the directionality and polarity of basal bodies and axonemal elongation by modulating actin dynamics, independent of basal body docking. In conclusion, destrin plays a significant role during vertebrate ciliogenesis regulating both primary and multicilia development. Our data suggest new insights for understanding the roles of actin dynamics in cilia development.
    Keywords:  Ciliogenesis; Destrin; F-actin; Multiciliated cells; Primary cilia; Xenopus laevis
    DOI:  https://doi.org/10.12717/DR.2024.28.3.109
  37. Pain Rep. 2024 Dec;9(6): e1198
    miRNA packaging into small extracellular vesicles and implications in pain.
    Jason T DaCunza, Jason R Wickman, Seena K Ajit.
      Extracellular vesicles (EVs) are a heterogenous group of lipid bilayer bound particles naturally released by cells. These vesicles are classified based on their biogenesis pathway and diameter. The overlap in size of exosomes generated from the exosomal pathway and macrovesicles that are pinched off from the surface of the plasma membrane makes it challenging to isolate pure populations. Hence, isolated vesicles that are less than 200 nm are called small extracellular vesicles (sEVs). Extracellular vesicles transport a variety of cargo molecules, and multiple mechanisms govern the packaging of cargo into sEVs. Here, we discuss the current understanding of how miRNAs are targeted into sEVs, including the role of RNA binding proteins and EXOmotif sequences present in miRNAs in sEV loading. Several studies in human pain disorders and rodent models of pain have reported alterations in sEV cargo, including miRNAs. The sorting mechanisms and target regulation of miR-939, a miRNA altered in individuals with complex regional pain syndrome, is discussed in the context of inflammation. We also provide a broad overview of the therapeutic strategies being pursued to utilize sEVs in the clinic and the work needed to further our understanding of EVs to successfully deploy sEVs as a pain therapeutic.
    Keywords:  Exosomes; Pain; Small extracellular vesicles; miR-939; miRNA; sEV biogenesis
    DOI:  https://doi.org/10.1097/PR9.0000000000001198
  38. FEBS J. 2024 Oct 21.
    PGC-1α regulation by FBXW7 through a novel mechanism linking chaperone-mediated autophagy and the ubiquitin-proteasome system.
    Simona Eleuteri, Bao Wang, Gianni Cutillo, Tracy Shi Zhang Fang, Kai Tao, Yan Qu, Qian Yang, Wenyi Wei, David K Simon.
      Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a key regulator of mitochondrial biogenesis and antioxidative defenses, and it may play a critical role in Parkinson's disease (PD). F-box/WD repeat domain-containing protein (FBXW7), an E3 protein ligase, promotes the degradation of substrate proteins through the ubiquitin-proteasome system (UPS) and leads to the clearance of PGC-1α. Here, we elucidate a novel post-translational mechanism for regulating PGC-1α levels in neurons. We show that enhancing chaperone-mediated autophagy (CMA) activity promotes the CMA-mediated degradation of FBXW7 and consequently increases PGC-1α. We confirm the relevance of this pathway in vivo by showing decreased FBXW7 and increased PGC-1α as a result of boosting CMA selectively in dopaminergic (DA) neurons by overexpressing lysosomal-associated membrane protein 2A (LAMP2A) in TH-Cre-LAMP2-loxp conditional mice. We further demonstrate that these mice are protected against MPTP-induced oxidative stress and neurodegeneration. These results highlight a novel regulatory pathway for PGC-1α in DA neurons and suggest targeted increasing of CMA or decreasing FBXW7 in DA neurons as potential neuroprotective strategies in PD.
    Keywords:  CMA; Fbxw7; PGC‐1α; Parkinson's disease
    DOI:  https://doi.org/10.1111/febs.17276
  39. Fluids Barriers CNS. 2024 Oct 21. 21(1): 83
    Elevated peripheral inflammation is associated with choroid plexus enlargement in independent sporadic amyotrophic lateral sclerosis cohorts.
    Sujuan Sun, Yujing Chen, Yan Yun, Bing Zhao, Qingguo Ren, Xiaohan Sun, Xiangshui Meng, Chuanzhu Yan, Pengfei Lin, Shuangwu Liu.
      BACKGROUND: Using neuroimaging techniques, growing evidence has suggested that the choroid plexus (CP) volume is enlarged in multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). Notably, the CP has been suggested to play an important role in inflammation-induced CNS damage under disease conditions. However, to our knowledge, no study has investigated the relationships between peripheral inflammation and CP volume in sporadic ALS patients. Thus, in this study, we aimed to verify CP enlargement and explore its association with peripheral inflammation in vivo in independent ALS cohorts.METHODS: Based on structural MRI data, CP volume was measured using Gaussian mixture models and further manually corrected in two independent cohorts of sporadic ALS patients and healthy controls (HCs). Serum inflammatory protein levels were measured using a novel high-sensitivity Olink proximity extension assay (PEA) technique. Xtreme gradient boosting (XGBoost) was used to explore the contribution of peripheral inflammatory factors to CP enlargement. Then, partial correlation analyses were performed.
    RESULTS: CP volumes were significantly higher in ALS patients than in HCs in the independent cohorts. Compared with HCs, serum levels of CRP, IL-6, CXCL10, and 35 other inflammatory factors were significantly increased in ALS patients. Using the XGBoost approach, we established a model-based importance of features, and the top three predictors of CP volume in ALS patients were CRP, IL-6, and CXCL10 (with gains of 0.24, 0.18, and 0.15, respectively). Correlation analyses revealed that CRP, IL-6, and CXCL10 were significantly associated with CP volume in ALS patients (r = 0.462 ∼ 0.636, p < 0.001).
    CONCLUSION: Our study is the first to reveal a consistent and replicable contribution of peripheral inflammation to CP enlargement in vivo in sporadic ALS patients. Given that CP enlargement has been recently detected in other brain diseases, these findings should consider extending to other disease conditions with a peripheral inflammatory component.
    Keywords:  ALS; Choroid plexus; Inflammation; MRI
    DOI:  https://doi.org/10.1186/s12987-024-00586-w
  40. Commun Biol. 2024 Oct 22. 7(1): 1373
    Loss of CLN3 in microglia leads to impaired lipid metabolism and myelin turnover.
    Seda Yasa, Elisabeth S Butz, Alessio Colombo, Uma Chandrachud, Luca Montore, Sarah Tschirner, Matthias Prestel, Steven D Sheridan, Stephan A Müller, Janos Groh, Stefan F Lichtenthaler, Sabina Tahirovic, Susan L Cotman.
      Loss-of-function mutations in CLN3 cause juvenile Batten disease, featuring neurodegeneration and early-stage neuroinflammation. How loss of CLN3 function leads to early neuroinflammation is not yet understood. Here, we have comprehensively studied microglia from Cln3∆ex7/8 mice, a genetically accurate disease model. Loss of CLN3 function in microglia leads to lysosomal storage material accumulation and abnormal morphology of subcellular organelles. Moreover, pathological proteomic signatures are indicative of defects in lysosomal function and abnormal lipid metabolism. Consistent with these findings, CLN3-deficient microglia are unable to efficiently turnover myelin and metabolize the associated lipids, showing defects in lipid droplet formation and cholesterol accumulation. Accordingly, we also observe impaired myelin integrity in aged Cln3∆ex7/8 mouse brain. Autophagy inducers and cholesterol-lowering drugs correct the observed microglial phenotypes. Taken together, these data implicate a cell-autonomous defect in CLN3-deficient microglia that impacts their ability to support neuronal cell health, suggesting microglial targeted therapies should be considered for CLN3 disease.
    DOI:  https://doi.org/10.1038/s42003-024-07057-w
  41. Nat Protoc. 2024 Oct 22.
    High-throughput capture and in situ protein analysis of extracellular vesicles by chemical probe-based array.
    Xin Feng, Ao Shen, Wei Zhang, Shengnan Jia, Anton Iliuk, Yuling Wang, Wenke Zhang, Ying Zhang, W Andy Tao, Lianghai Hu.
      Extracellular vesicles (EVs) are small particles with phospholipid bilayers that carry a diverse range of cargoes including nucleic acids, proteins and metabolites. EVs have important roles in various cellular processes and are increasingly recognized for their ubiquitous role in cell-cell communications and potential applications in therapeutics and diagnostics. Although many methods have been developed for the characterization and measurement of EVs, analyzing them from biofluids remains a challenge with regard to throughput and sensitivity. Recently, we introduced an approach to facilitate high-throughput analysis of EVs from trace amounts of sample. In this method, an amphiphile-dendrimer supramolecular probe (ADSP) is coated onto a nitrocellulose membrane for array-based capture and to enable an in situ immunoblotting assay. Here, we describe the protocol for our array-based method of EV profiling. We describe an enhanced version of the method that incorporates an automated printing workstation, ensuring high throughput and reproducibility. We further demonstrate the use of our array to profile specific glycosylations on the EV surface using click chemistry of an azide group introduced by metabolic labeling. In this protocol, the synthesis of ADSP and the fabrication of ADSP nitrocellulose membrane array can be completed on the same day. EVs are efficiently captured from biological or clinical samples through a 30-min incubation, followed by an immunoblotting assay within a 3-h window, thus providing a high-throughput platform for EV isolation and in situ targeted analysis of EV proteins and their modifications.
    DOI:  https://doi.org/10.1038/s41596-024-01082-z
  42. Sci Rep. 2024 10 23. 14(1): 25069
    Melatonin modulates the Notch1 signaling pathway and Sirt3 in the hippocampus of hypoxic-ischemic neonatal rats.
    Atefeh Mohammadi, Water Balduini, Silvia Carloni.
      The Notch1 signaling pathway plays a crucial role in the development of the central nervous system, governing pivotal functional activities in the brain, such as neurogenesis. Sirt3 is instrumental in managing mitochondrial homeostasis and is essential to cell survival. Dysregulation of these signaling pathways is implicated in the pathogenesis of a wide range of diseases, including neurodegenerative disorders such as stroke. We have previously shown that melatonin significantly improved the perinatal brain damage caused by hypoxia-ischemia (HI) through the activation of several protective mechanisms such as restoring mitochondria status and increasing the hippocampal cell proliferation. This study assessed whether melatonin affects the Notch1 signaling pathway and Sirt3 after neonatal HI. Results show that HI significantly increased Notch1 expression both in hippocampal neurons and glial cells as well as the expression of the key proteins of the pathway NICD, HES1, and c-Myc. Melatonin significantly prevented the Notch1 signaling pathway activation induced by HI, maintaining NICD and HES1 expression to control levels. In the same neurons, melatonin also prevents the Sirt3 depletion caused by HI. In summary, this study provides new insights into the effects of melatonin on the Notch1 signaling pathway and Sirt3 in in vivo neonatal brain ischemia. We suggest that the rapid modulation of the Notch1 signaling pathway and Sirt3 induced by melatonin may support neuronal survival during ischemia.
    Keywords:  Neonatal brain ischemia; Neuroprotection; Notch; Sirtuin 3
    DOI:  https://doi.org/10.1038/s41598-024-76307-y
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