bims-axbals Biomed News
on Axonal Biology and ALS
Issue of 2024‒04‒21
forty-five papers selected by
TJ Krzystek, ALS Therapy Development Institute
  1. Toxic gain-of-function mechanisms in C9orf72 ALS-FTD neurons drive autophagy and lysosome dysfunction.
  2. Abundant transcriptomic alterations in the human cerebellum of patients with a C9orf72 repeat expansion.
  3. Generation and characterization of monoclonal antibodies against pathologically phosphorylated TDP-43.
  4. Deep learning modeling of rare noncoding genetic variants in human motor neurons defines CCDC146 as a therapeutic target for ALS.
  5. 14-3-3θ, a novel player in TDP-43 pathophysiology: Implications for ALS/FTD.
  6. Reversal of C9orf72 mutation-induced transcriptional dysregulation and pathology in cultured human neurons by allele-specific excision.
  7. A guide to selecting high-performing antibodies for RNA-binding protein TIA1 for use in Western Blot, immunoprecipitation and immunofluorescence.
  8. Long-timescale atomistic simulations uncover loss-of-function mechanisms of uncharacterized Angiogenin mutants associated with ALS.
  9. Genetic diagnosis and detection rates using C9orf72 repeat expansion and a multi-gene panel in amyotrophic lateral sclerosis.
  10. Protocol to identify DNA-binding proteins recognizing nucleotide repeat dsDNAs.
  11. Phase-Amplitude Coupling Detection and Analysis of Human 2-Dimensional Neural Cultures in Multi-well Microelectrode Array in Vitro.
  12. Dissecting the effect of ALS mutation S375G on the conformational properties and aggregation dynamics of TDP-43370-375 fragment.
  13. Leucine-Rich Repeat Kinases.
  14. Novel Isoxazolidines as RIPK1 Inhibitors for Treating Alzheimer's Disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis.
  15. Optineurin-facilitated axonal mitochondria delivery promotes neuroprotection and axon regeneration.
  16. Directed Differentiation of Neurons from Human iPSCs for Modeling Neurological Disorders.
  17. Seeding competent TDP-43 persists in human patient and mouse muscle.
  18. AAV-mediated upregulation of VDAC1 rescues the mitochondrial respiration and sirtuins expression in a SOD1 mouse model of inherited ALS.
  19. In Vitro Patch-Clamp.
  20. Generating Homogeneous Brain Organoids from Human iPSCs.
  21. Human pluripotent stem cells as a translational toolkit in psychedelic research in vitro.
  22. Exploring the role of Prx II in mitigating endoplasmic reticulum stress and mitochondrial dysfunction in neurodegeneration.
  23. iPSC-based modeling of preeclampsia identifies epigenetic defects in extravillous trophoblast differentiation.
  24. Primary Culture of Dissociated Neurons from the Embryonic Cerebral Cortex.
  25. Current trends in basic research on Parkinson's disease: from mitochondria, lysosome to α-synuclein.
  26. Generation of Anterior Segment of the Eye Cells from hiPSCs in Microfluidic Platforms.
  27. Reprogramming fibroblast into human iBlastoids.
  28. Overview: Research on the Genetic Architecture of the Developing Cerebral Cortex in Norms and Diseases.
  29. RAB32 Ser71Arg in autosomal dominant Parkinson's disease: linkage, association, and functional analyses.
  30. Selective autophagy of macromolecular complexes: what does it take to be taken?
  31. Neuronal activity drives glymphatic waste clearance.
  32. WAV E3 ubiquitin ligases mediate degradation of IAA32/34 in the TMK1-mediated auxin signaling pathway during apical hook development.
  33. Generation of iPSCs Using Sendai Virus Vectors.
  34. Axon morphology and intrinsic cellular properties determine repetitive transcranial magnetic stimulation threshold for plasticity.
  35. PGC-1a mediated mitochondrial biogenesis promotes recovery and survival of neuronal cells from cellular degeneration.
  36. In vitro human cell culture models in a bench-to-bedside approach to epilepsy.
  37. Teaghrelin protected dopaminergic neurons in MPTP-induced Parkinson's disease animal model by promoting PINK1/Parkin-mediated mitophagy and AMPK/SIRT1/PGC1-α-mediated mitochondrial biogenesis.
  38. Delineating the contribution of ageing and physical activity to changes in mitochondrial characteristics across the lifespan.
  39. Optical recordings of organellar membrane potentials and the components of membrane conductance in lysosomes.
  40. Combining LIANA and Tensor-cell2cell to decipher cell-cell communication across multiple samples.
  41. A mechanism that transduces lysosomal damage signals to stress granule formation for cell survival.
  42. Unveiling clinical applications of bacterial extracellular vesicles as natural nanomaterials in disease diagnosis and therapeutics.
  43. Small extracellular vesicles from young plasma reverse age-related functional declines by improving mitochondrial energy metabolism.
  44. Plasminogen degrades α-synuclein, Tau and TDP-43 and decreases dopaminergic neurodegeneration in mouse models of Parkinson's disease.
  45. Immunocytochemistry of Primary Cultured Cerebral Cortical Neurons.
  1. Autophagy. 2024 Apr 18. 1-3
    Toxic gain-of-function mechanisms in C9orf72 ALS-FTD neurons drive autophagy and lysosome dysfunction.
    Jimmy Beckers, Philip Van Damme.
      Hexanucleotide repeat expansions in the C9orf72 gene are the primary genetic cause for both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two related neurodegenerative diseases. Significant advances in the elucidation of the disease mechanisms responsible for C9orf72 ALS-FTD have revealed both a toxic gain-of-function and a loss-of-function mechanism as possible underlying disease cause. As the differential contribution of both gain and loss of function in C9orf72 ALS-FTD pathogenesis remains debated, we investigated disease mechanisms in motor neurons derived from both authentic human patient C9orf72 ALS-FTD iPSCs as well as a C9orf72 knockout iPSC line. We found that patient neurons presented with less motile and enlarged lysosomes, a decrease in autophagic flux and an increase in SQSTM1/p62 puncta and insoluble TARDBP/TDP-43 species. Importantly, we found that C9orf72 knockout barely has any influence on these phenotypes and mainly results in impaired endosomal maturation. Together, our data suggest that toxic gain-of-function, rather than loss-of-function, mechanisms in C9orf72 ALS-FTD impair the autophagy-lysosome system in neurons.
    Keywords:  Autophagy; C9orf72; amyotrophic lateral sclerosis; endosome; frontotemporal dementia; lysosome
    DOI:  https://doi.org/10.1080/15548627.2024.2340415
  2. Acta Neuropathol. 2024 Apr 19. 147(1): 73
    Abundant transcriptomic alterations in the human cerebellum of patients with a C9orf72 repeat expansion.
    Evan Udine, Mariely DeJesus-Hernandez, Shulan Tian, Sofia Pereira das Neves, Richard Crook, NiCole A Finch, Matthew C Baker, Cyril Pottier, Neill R Graff-Radford, Bradley F Boeve, Ronald C Petersen, David S Knopman, Keith A Josephs, Björn Oskarsson, Sandro Da Mesquita, Leonard Petrucelli, Tania F Gendron, Dennis W Dickson, Rosa Rademakers, Marka van Blitterswijk.
      The most prominent genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) is a repeat expansion in the gene C9orf72. Importantly, the transcriptomic consequences of the C9orf72 repeat expansion remain largely unclear. Here, we used short-read RNA sequencing (RNAseq) to profile the cerebellar transcriptome, detecting alterations in patients with a C9orf72 repeat expansion. We focused on the cerebellum, since key C9orf72-related pathologies are abundant in this neuroanatomical region, yet TDP-43 pathology and neuronal loss are minimal. Consistent with previous work, we showed a reduction in the expression of the C9orf72 gene and an elevation in homeobox genes, when comparing patients with the expansion to both patients without the C9orf72 repeat expansion and control subjects. Interestingly, we identified more than 1000 alternative splicing events, including 4 in genes previously associated with ALS and/or FTLD. We also found an increase of cryptic splicing in C9orf72 patients compared to patients without the expansion and controls. Furthermore, we demonstrated that the expression level of select RNA-binding proteins is associated with cryptic splice junction inclusion. Overall, this study explores the presence of widespread transcriptomic changes in the cerebellum, a region not confounded by severe neurodegeneration, in post-mortem tissue from C9orf72 patients.
    Keywords:  Amyotrophic lateral sclerosis; C9orf72; Cryptic exons; Frontotemporal lobar degeneration; Transcriptomics
    DOI:  https://doi.org/10.1007/s00401-024-02720-2
  3. PLoS One. 2024 ;19(4): e0298080
    Generation and characterization of monoclonal antibodies against pathologically phosphorylated TDP-43.
    Paula Castellanos Otero, Tiffany W Todd, Wei Shao, Caroline J Jones, Kexin Huang, Lillian M Daughrity, Mei Yue, Udit Sheth, Tania F Gendron, Mercedes Prudencio, Björn Oskarsson, Dennis W Dickson, Leonard Petrucelli, Yong-Jie Zhang.
      Inclusions containing TAR DNA binding protein 43 (TDP-43) are a pathological hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). One of the disease-specific features of TDP-43 inclusions is the aberrant phosphorylation of TDP-43 at serines 409/410 (pS409/410). Here, we developed rabbit monoclonal antibodies (mAbs) that specifically detect pS409/410-TDP-43 in multiple model systems and FTD/ALS patient samples. Specifically, we identified three mAbs (26H10, 2E9 and 23A1) from spleen B cell clones that exhibit high specificity and sensitivity to pS409/410-TDP-43 peptides in an ELISA assay. Biochemical analyses revealed that pS409/410 of recombinant TDP-43 and of exogenous 25 kDa TDP-43 C-terminal fragments in cultured HEK293T cells are detected by all three mAbs. Moreover, the mAbs detect pS409/410-positive TDP-43 inclusions in the brains of FTD/ALS patients and mouse models of TDP-43 proteinopathy by immunohistochemistry. Our findings indicate that these mAbs are a valuable resource for investigating TDP-43 pathology both in vitro and in vivo.
    DOI:  https://doi.org/10.1371/journal.pone.0298080
  4. medRxiv. 2024 Apr 01. pii: 2024.03.30.24305115. [Epub ahead of print]
    Deep learning modeling of rare noncoding genetic variants in human motor neurons defines CCDC146 as a therapeutic target for ALS.
    Sai Zhang, Tobias Moll, Jasper Rubin-Sigler, Sharon Tu, Shuya Li, Enming Yuan, Menghui Liu, Afreen Butt, Calum Harvey, Sarah Gornall, Elham Alhalthli, Allan Shaw, Cleide Dos Santos Souza, Laura Ferraiuolo, Eran Hornstein, Tatyana Shelkovnikova, Charlotte H van Dijk, Ilia S Timpanaro, Kevin P Kenna, Jianyang Zeng, Philip S Tsao, Pamela J Shaw, Justin K Ichida, Johnathan Cooper-Knock, Michael P Snyder.
      Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disease caused by the selective and progressive death of motor neurons (MNs). Understanding the genetic and molecular factors influencing ALS survival is crucial for disease management and therapeutics. In this study, we introduce a deep learning-powered genetic analysis framework to link rare noncoding genetic variants to ALS survival. Using data from human induced pluripotent stem cell (iPSC)-derived MNs, this method prioritizes functional noncoding variants using deep learning, links cis-regulatory elements (CREs) to target genes using epigenomics data, and integrates these data through gene-level burden tests to identify survival-modifying variants, CREs, and genes. We apply this approach to analyze 6,715 ALS genomes, and pinpoint four novel rare noncoding variants associated with survival, including chr7:76,009,472:C>T linked to CCDC146 . CRISPR-Cas9 editing of this variant increases CCDC146 expression in iPSC-derived MNs and exacerbates ALS-specific phenotypes, including TDP-43 mislocalization. Suppressing CCDC146 with an antisense oligonucleotide (ASO), showing no toxicity, completely rescues ALS-associated survival defects in neurons derived from sporadic ALS patients and from carriers of the ALS-associated G4C2-repeat expansion within C9ORF72 . ASO targeting of CCDC146 may be a broadly effective therapeutic approach for ALS. Our framework provides a generic and powerful approach for studying noncoding genetics of complex human diseases.
    DOI:  https://doi.org/10.1101/2024.03.30.24305115
  5. Neuron. 2024 Apr 17. pii: S0896-6273(24)00203-4. [Epub ahead of print]112(8): 1197-1199
    14-3-3θ, a novel player in TDP-43 pathophysiology: Implications for ALS/FTD.
    Bilal Khalil, Sandrine Da Cruz.
      In this issue of Neuron, Ke et al.1 report a novel non-canonical interaction between 14-3-3θ and TDP-43 that impacts loss-of-function and gain-of-toxic pathology in TDP-43 proteinopathies. The authors further provide proof of principle for a 14-3-3θ-targeted gene therapy to reduce TDP-43-induced deficits in transgenic TDP-43 mutant mice.
    DOI:  https://doi.org/10.1016/j.neuron.2024.03.025
  6. Proc Natl Acad Sci U S A. 2024 Apr 23. 121(17): e2307814121
    Reversal of C9orf72 mutation-induced transcriptional dysregulation and pathology in cultured human neurons by allele-specific excision.
    Aradhana Sachdev, Kamaljot Gill, Maria Sckaff, Alisha M Birk, Olubankole Aladesuyi Arogundade, Katherine A Brown, Runvir S Chouhan, Patrick Oliver Issagholian-Lewin, Esha Patel, Hannah L Watry, Mylinh T Bernardi, Kathleen C Keough, Yu-Chih Tsai, Alec Simon Tulloch Smith, Bruce R Conklin, Claire Dudley Clelland.
      Efforts to genetically reverse C9orf72 pathology have been hampered by our incomplete understanding of the regulation of this complex locus. We generated five different genomic excisions at the C9orf72 locus in a patient-derived induced pluripotent stem cell (iPSC) line and a non-diseased wild-type (WT) line (11 total isogenic lines), and examined gene expression and pathological hallmarks of C9 frontotemporal dementia/amyotrophic lateral sclerosis in motor neurons differentiated from these lines. Comparing the excisions in these isogenic series removed the confounding effects of different genomic backgrounds and allowed us to probe the effects of specific genomic changes. A coding single nucleotide polymorphism in the patient cell line allowed us to distinguish transcripts from the normal vs. mutant allele. Using digital droplet PCR (ddPCR), we determined that transcription from the mutant allele is upregulated at least 10-fold, and that sense transcription is independently regulated from each allele. Surprisingly, excision of the WT allele increased pathologic dipeptide repeat poly-GP expression from the mutant allele. Importantly, a single allele was sufficient to supply a normal amount of protein, suggesting that the C9orf72 gene is haplo-sufficient in induced motor neurons. Excision of the mutant repeat expansion reverted all pathology (RNA abnormalities, dipeptide repeat production, and TDP-43 pathology) and improved electrophysiological function, whereas silencing sense expression did not eliminate all dipeptide repeat proteins, presumably because of the antisense expression. These data increase our understanding of C9orf72 gene regulation and inform gene therapy approaches, including antisense oligonucleotides (ASOs) and CRISPR gene editing.
    Keywords:  ALS; C9orf72; CRISPR; FTD; neurodegeneration
    DOI:  https://doi.org/10.1073/pnas.2307814121
  7. F1000Res. 2023 ;12 745
    A guide to selecting high-performing antibodies for RNA-binding protein TIA1 for use in Western Blot, immunoprecipitation and immunofluorescence.
    NeuroSGC/YCharOS/EDDU collaborative group
      A member of the RNA-binding protein family, T-cell intracellular antigen-1 (TIA1) regulates mRNA translation and splicing as well as cellular stress by promoting stress granule formation. Variants of the TIA1 gene have implications in neurogenerative disorders including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Reproducible research on TIA1 would be enhanced with the availability of high-quality anti-TIA1 antibodies. In this study, we characterized twelve TIA1 commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.
    Keywords:  RNA-binding protein TIA1; TIA1; Uniprot ID P31483; Western Blot; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation
    DOI:  https://doi.org/10.12688/f1000research.133645.2
  8. Arch Biochem Biophys. 2024 Apr 15. pii: S0003-9861(24)00119-X. [Epub ahead of print]756 110000
    Long-timescale atomistic simulations uncover loss-of-function mechanisms of uncharacterized Angiogenin mutants associated with ALS.
    Deeksha Dewangan, Aryaman Joshi, Aditya K Padhi.
      Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease characterized by progressive degeneration of motor neurons, resulting in respiratory failure and mortality within 3-5 years. Mutations in the Angiogenin (ANG) cause loss of ribonucleolytic and nuclear translocation activities, contributing to ALS pathogenesis. This study focused on investigating two uncharacterized ANG mutations, T11S and R122H, newly identified in the Project Mine consortium. Using extensive computational analysis, including structural modeling and microsecond-timescale molecular dynamics (MD) simulations, we observed conformational changes in the catalytic residue His114 of ANG induced by T11S and R122H mutations. These alterations impaired ribonucleolytic activity, as inferred through molecular docking and binding free energy calculations. Gibbs free energy landscape and residue-residue interaction network analysis further supported our findings, revealing the energetic states and allosteric pathway from the mutated site to His114. Additionally, we assessed the binding of NCI-65828, an inhibitor of ribonucleolytic activity of ANG, and found reduced effectiveness in binding to T11S and R122H mutants when His114 assumed a non-native conformation. This highlights the crucial role of His114 and its association with ALS. Elucidating the relationship between physical structure and functional dynamics of frequently mutated ANG mutants is essential for understanding ALS pathogenesis and developing more effective therapeutic interventions.
    Keywords:  Amyotrophic lateral sclerosis; Angiogenin; Binding free energy calculations; Loss-of-functions; Missense mutations; Molecular dynamics simulations
    DOI:  https://doi.org/10.1016/j.abb.2024.110000
  9. J Neurol. 2024 Apr 16.
    Genetic diagnosis and detection rates using C9orf72 repeat expansion and a multi-gene panel in amyotrophic lateral sclerosis.
    Dalit Barel, Daphna Marom, Penina Ponger, Alina Kurolap, Anat Bar-Shira, Idit Kaplan-Ber, Adi Mory, Beatrice Abramovich, Yuval Yaron, Vivian Drory, Hagit Baris Feldman.
      Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder. It is mostly sporadic, with the C9orf72 repeat expansion being the most common genetic cause. While the prevalence of C9orf72-ALS in patients from different populations has been studied, data regarding the yield of C9orf72 compared to an ALS gene panel testing is limited.We aimed to explore the application of C9orf72 versus a gene panel in the general Israeli population. A total of 140 ALS patients attended our Neurogenetics Clinic throughout 2018-2023. Disease onset was between ages 60 and 69 years for most patients (34%); however, a quarter had an early-onset disease (< 50 years). Overall, 119 patients (85%) were genetically evaluated: 116 (97%) were tested for the C9orf72 repeat expansion and 64 (54%) underwent gene panel testing. The C9orf72 repeat expansion had a prevalence of 21% among Ashkenazi Jewish patients compared to 5.7% in non-Ashkenazi patients, while the gene panel had a higher yield in non-Ashkenazi patients with 14% disease-causing variants compared to 5.7% in Ashkenazi Jews. Among early-onset ALS patients, panel testing was positive in 12% compared to 2.9% for C9orf72.We suggest a testing strategy for the Israeli ALS patients: C9orf72 should be the first-tier test in Ashkenazi Jewish patients, while a gene panel should be considered as the first step in non-Ashkenazi and early-onset patients. Tiered testing has important implications for patient management, including prognosis, ongoing clinical trials, and prevention in future generations. Similar studies should be implemented worldwide to uncover the diverse ALS genetic architecture and facilitate tailored care.
    Keywords:  ALS; ALS gene panel; ALS genetics; Amyotrophic lateral sclerosis; C9orf72 repeat expansion
    DOI:  https://doi.org/10.1007/s00415-024-12368-3
  10. STAR Protoc. 2024 Apr 11. pii: S2666-1667(24)00178-3. [Epub ahead of print]5(2): 103013
    Protocol to identify DNA-binding proteins recognizing nucleotide repeat dsDNAs.
    Zhiyuan Huang, Yixin Zhou, Yang Liu, Jiou Wang.
      DNA-binding proteins perform diverse functions, including regulating cellular growth and orchestrating chromatin architecture. Here, we present a protocol to discover proteins specifically interacting with a hexanucleotide repeat DNA, the expansion of which is known as the most frequent genetic cause of familial C9orf72 amyotrophic lateral sclerosis and frontotemporal dementia. We describe steps to fish out DNA-binding proteins recognizing double-stranded repeat DNAs using a SILAC (stable isotope labelling by amino acids in cell culture)-based approach and validate the results using electrophoretic mobility shift assay. For complete details on the use and execution of this protocol, please refer to Liu et al.1.
    Keywords:  Cell Biology; Cell culture; Cell separation/fractionation; Cell-based Assays; Molecular Biology; Molecular/Chemical Probes; Protein Biochemistry; Protein expression and purification
    DOI:  https://doi.org/10.1016/j.xpro.2024.103013
  11. J Neurosci Methods. 2024 Apr 12. pii: S0165-0270(24)00072-4. [Epub ahead of print] 110127
    Phase-Amplitude Coupling Detection and Analysis of Human 2-Dimensional Neural Cultures in Multi-well Microelectrode Array in Vitro.
    Yousef Salimpour, William S Anderson, Raha Dastyeb, Shiyu Liu, Guo-Li Ming, Hongjun Song, Nicholas J Maragakis, Christa W Habela.
      BACKGROUND: Human induced pluripotent stem cell (hiPSC)- derived neurons offer the possibility of studying human-specific neuronal behaviors in physiologic and pathologic states in vitro. It is unclear whether cultured neurons can achieve the fundamental network behaviors required to process information in the brain. Investigating neuronal oscillations and their interactions, as occurs in cross-frequency coupling (CFC), addresses this question.NEW METHODS: We examined whether networks of two-dimensional (2D) cultured hiPSC-derived cortical neurons grown with hiPSC-derived astrocytes on microelectrode array plates recapitulate the CFC that is present in vivo. We employed the modulation index method for detecting phase-amplitude coupling (PAC) and used offline spike sorting to analyze the contribution of single neuron spiking to network behavior.
    RESULTS: We found that PAC is present, the degree of PAC is specific to network structure, and it is modulated by external stimulation with bicuculline administration. Modulation of PAC is not driven by single neurons, but by network-level interactions.
    COMPARISON WITH EXISTING METHODS: PAC has been demonstrated in multiple regions of the human cortex as well as in organoids. This is the first report of analysis demonstrating the presence of coupling in 2D cultures.
    CONCLUSION: CFC in the form of PAC analysis explores communication and integration between groups of neurons and dynamical changes across networks. In vitro PAC analysis has the potential to elucidate the underlying mechanisms as well as capture the effects of chemical, electrical, or ultrasound stimulation; providing insight into modulation of neural networks to treat nervous system disorders in vivo.
    Keywords:  Cross-frequency coupling; Multielectrode Array; phase-amplitude coupling
    DOI:  https://doi.org/10.1016/j.jneumeth.2024.110127
  12. Biophys Chem. 2024 Mar 29. pii: S0301-4622(24)00059-0. [Epub ahead of print]310 107230
    Dissecting the effect of ALS mutation S375G on the conformational properties and aggregation dynamics of TDP-43370-375 fragment.
    Zhengdong Xu, Jianxin Zhang, Jiaxing Tang, Yehong Gong, Yu Zou, Qingwen Zhang.
      The aggregation of transactive response deoxyribonucleic acid (DNA) binding protein of 43 kDa (TDP-43) into ubiquitin-positive inclusions is closely associated with amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration, and chronic traumatic encephalopathy. The 370-375 fragment of TDP-43 (370GNNSYS375, TDP-43370-375), the amyloidogenic hexapeptides, can be prone to forming pathogenic amyloid fibrils with the characteristic of steric zippers. Previous experiments reported the ALS-associated mutation, serine 375 substituted by glycine (S375G) is linked to early onset disease and protein aggregation of TDP-43. Based on this, it is necessary to explore the underlying molecular mechanisms. By utilizing all-atom molecular dynamics (MD) simulations of 102 μs in total, we investigated the impact of S375G mutation on the conformational ensembles and oligomerization dynamics of TDP-43370-375 peptides. Our replica exchange MD simulations show that S375G mutation could promote the unstructured conformation formation and induce peptides to form a loose packed oligomer, thus inhibiting the aggregation of TDP-43370-375. Further analyses suggest that S375G mutation displays a reduction effect on the number of total hydrogen bonds and contacts among TDP-43370-375 peptides. Hydrogen bonding and polar interactions among TDP-43370-375 peptides, as well as Y374-Y374 π-π stacking interaction, are attenuated by S375G mutation. Additional microsecond MD simulations demonstrate that S375G mutation could prohibit the conformational conversion to β-structure-rich aggregates and possess an inhibitory effect on the oligomerization dynamics of TDP-43370-375. This study offers for the first time of molecular insights into the S375G mutation affecting the aggregation of TDP-43370-375 at the atomic level, and may open new avenues in the development of future site-specific mutation therapeutics.
    Keywords:  Aggregation; Amyotrophic lateral sclerosis; Molecular dynamics simulation; Molecular mechanism; S375G mutation; TDP-43
    DOI:  https://doi.org/10.1016/j.bpc.2024.107230
  13. Annu Rev Biochem. 2024 Apr 15.
    Leucine-Rich Repeat Kinases.
    Dario R Alessi, Suzanne R Pfeffer.
      Activating mutations in leucine-rich repeat kinase 2 (LRRK2) represent the most common cause of monogenic Parkinson's disease. LRRK2 is a large multidomain protein kinase that phosphorylates a specific subset of the ∼65 human Rab GTPases, which are master regulators of the secretory and endocytic pathways. After phosphorylation by LRRK2, Rabs lose the capacity to bind cognate effector proteins and guanine nucleotide exchange factors. Moreover, the phosphorylated Rabs cannot interact with their cognate prenyl-binding retrieval proteins (also known as guanine nucleotide dissociation inhibitors) and, thus, they become trapped on membrane surfaces. Instead, they gain the capacity to bind phospho-Rab-specific effector proteins, such as RILPL1, with resulting pathological consequences. Rab proteins also act upstream of LRRK2 by controlling its activation and recruitment onto membranes. LRRK2 signaling is counteracted by the phosphoprotein phosphatase PPM1H, which selectively dephosphorylates phospho-Rab proteins. We present here our current understanding of the structure, biochemical properties, and cell biology of LRRK2 and its related paralog LRRK1 and discuss how this information guides the generation of LRRK2 inhibitors for the potential benefit of patients.
    DOI:  https://doi.org/10.1146/annurev-biochem-030122-051144
  14. ACS Med Chem Lett. 2024 Apr 11. 15(4): 447-448
    Novel Isoxazolidines as RIPK1 Inhibitors for Treating Alzheimer's Disease, Multiple Sclerosis, and Amyotrophic Lateral Sclerosis.
    Ram W Sabnis.
      Provided herein are novel isoxazolidines as RIPK1 inhibitors, pharmaceutical compositions, use of such compounds in treating Alzheimer's disease, multiple sclerosis, and amyotrophic lateral sclerosis (ALS), and processes for preparing such compounds.
    DOI:  https://doi.org/10.1021/acsmedchemlett.4c00119
  15. bioRxiv. 2024 Apr 03. pii: 2024.04.02.587832. [Epub ahead of print]
    Optineurin-facilitated axonal mitochondria delivery promotes neuroprotection and axon regeneration.
    Dong Liu, Hannah C Webber, Fuyun Bian, Yangfan Xu, Manjari Prakash, Xue Feng, Ming Yang, Hang Yang, In-Jee You, Liang Li, Liping Liu, Pingting Liu, Haoliang Huang, Chien-Yi Chang, Liang Liu, Sahil H Shah, Anna La Torre, Derek S Welsbie, Yang Sun, Xin Duan, Jeffrey Louis Goldberg, Marcus Braun, Zdenek Lansky, Yang Hu.
      Optineurin (OPTN) mutations are linked to amyotrophic lateral sclerosis (ALS) and normal tension glaucoma (NTG), but a relevant animal model is lacking, and the molecular mechanisms underlying neurodegeneration are unknown. We found that OPTN C-terminus truncation (OPTNΔC) causes late-onset neurodegeneration of retinal ganglion cells (RGCs), optic nerve (ON), and spinal cord motor neurons, preceded by a striking decrease of axonal mitochondria. Surprisingly, we discover that OPTN directly interacts with both microtubules and the mitochondrial transport complex TRAK1/KIF5B, stabilizing them for proper anterograde axonal mitochondrial transport, in a C- terminus dependent manner. Encouragingly, overexpressing OPTN/TRAK1/KIF5B reverses not only OPTN truncation-induced, but also ocular hypertension-induced neurodegeneration, and promotes striking ON regeneration. Therefore, in addition to generating new animal models for NTG and ALS, our results establish OPTN as a novel facilitator of the microtubule-dependent mitochondrial transport necessary for adequate axonal mitochondria delivery, and its loss as the likely molecular mechanism of neurodegeneration.
    DOI:  https://doi.org/10.1101/2024.04.02.587832
  16. Methods Mol Biol. 2024 ;2794 141-155
    Directed Differentiation of Neurons from Human iPSCs for Modeling Neurological Disorders.
    Cheng Wang, Jonas Cerneckis, Yanhong Shi.
      Human-induced pluripotent stem cell (hiPSC) technology has enabled comprehensive human cell-based disease modeling in vitro. Due to limited accessibility of primary human neurons as well as species-specific divergence between human and rodent brain tissues, hiPSC-derived neurons have become a popular tool for studying neuronal biology in a dish. Here, we provide methods for transcription factor-driven directed differentiation of neurons from hiPSCs via a neural progenitor cell (NPC) intermediate. Doxycycline-inducible expression of neuron fate-determining transcription factors neurogenin 2 (NGN2) and achaete-scute homolog 1 (ASCL1) enables rapid and controllable differentiation of human neurons for disease modeling applications. The provided method is also designed to improve the reproducibility of human neuron differentiation by reducing the batch-to-batch variation of NPC differentiation and lentiviral transduction.
    Keywords:  Alzheimer’s disease; Directed differentiation; Disease modeling; Neurological disorders; Neurons; Stem cells; iPSCs
    DOI:  https://doi.org/10.1007/978-1-0716-3810-1_12
  17. bioRxiv. 2024 Apr 04. pii: 2024.04.03.587918. [Epub ahead of print]
    Seeding competent TDP-43 persists in human patient and mouse muscle.
    Eileen M Lynch, Sara Pittman, Jil Daw, Chiseko Ikenaga, Sheng Chen, Dhruva D Dhavale, Meredith E Jackrel, Yuna M Ayala, Paul Kotzbauer, Cindy V Ly, Alan Pestronk, Thomas E Lloyd, Conrad C Weihl.
      TAR DNA-binding protein 43 (TDP-43) is an RNA binding protein that accumulates as aggregates in the central nervous system of some neurodegenerative diseases. However, TDP-43 aggregation is also a sensitive and specific pathologic feature found in a family of degenerative muscle diseases termed inclusion body myopathy (IBM). TDP-43 aggregates from ALS and FTD brain lysates may serve as self-templating aggregate seeds in vitro and in vivo, supporting a prion-like spread from cell to cell. Whether a similar process occurs in IBM patient muscle is not clear. We developed a mouse model of inducible, muscle-specific cytoplasmic localized TDP-43. These mice develop muscle weakness with robust accumulation of insoluble and phosphorylated sarcoplasmic TDP-43, leading to eosinophilic inclusions, altered proteostasis and changes in TDP-43-related RNA processing that resolve with the removal of doxycycline. Skeletal muscle lysates from these mice also have seeding competent TDP-43, as determined by a FRET-based biosensor, that persists for weeks upon resolution of TDP-43 aggregate pathology. Human muscle biopsies with TDP-43 pathology also contain TDP-43 aggregate seeds. Using lysates from muscle biopsies of patients with IBM, IMNM and ALS we found that TDP-43 seeding capacity was specific to IBM. Surprisingly, TDP-43 seeding capacity anti-correlated with TDP-43 aggregate and vacuole abundance. These data support that TDP-43 aggregate seeds are present in IBM skeletal muscle and represent a unique TDP-43 pathogenic species not previously appreciated in human muscle disease.Summary: TDP-43 aggregate seeds persist in mouse and human skeletal muscle independent of large TDP-43 inclusions.
    DOI:  https://doi.org/10.1101/2024.04.03.587918
  18. Cell Death Discov. 2024 Apr 16. 10(1): 178
    AAV-mediated upregulation of VDAC1 rescues the mitochondrial respiration and sirtuins expression in a SOD1 mouse model of inherited ALS.
    Andrea Magrì, Cristiana Lucia Rita Lipari, Antonella Caccamo, Giuseppe Battiato, Stefano Conti Nibali, Vito De Pinto, Francesca Guarino, Angela Messina.
      Mitochondrial dysfunction represents one of the most common molecular hallmarks of both sporadic and familial forms of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder caused by the selective degeneration and death of motor neurons. The accumulation of misfolded proteins on and within mitochondria, as observed for SOD1 G93A mutant, correlates with a drastic reduction of mitochondrial respiration and the inhibition of metabolites exchanges, including ADP/ATP and NAD+/NADH, across the Voltage-Dependent Anion-selective Channel 1 (VDAC1), the most abundant channel protein of the outer mitochondrial membrane. Here, we show that the AAV-mediated upregulation of VDAC1 in the spinal cord of transgenic mice expressing SOD1 G93A completely rescues the mitochondrial respiratory profile. This correlates with the increased activity and levels of key regulators of mitochondrial functions and maintenance, namely the respiratory chain Complex I and the sirtuins (Sirt), especially Sirt3. Furthermore, the selective increase of these mitochondrial proteins is associated with an increase in Tom20 levels, the receptor subunit of the TOM complex. Overall, our results indicate that the overexpression of VDAC1 has beneficial effects on ALS-affected tissue by stabilizing the Complex I-Sirt3 axis.
    DOI:  https://doi.org/10.1038/s41420-024-01949-w
  19. Methods Mol Biol. 2024 ;2794 221-244
    In Vitro Patch-Clamp.
    Toshihiro Nomura.
      The patch-clamp technique is one of the most useful tools to analyze the function of electrically active cells such as neurons. This technique allows for the analysis of proteins (ion channels and receptors), cells (neurons), and synapses that are the building blocks of neuronal networks. Cortical development involves coordinated changes in functional measures at each of these levels of analysis that reflect both cellular and circuit maturation. This chapter explains the technical and theoretical basis of patch-clamp methodology and introduces several examples of how this technique can be applied in the context of cortical development.
    Keywords:  Action potential; Critical period; Development; Electrophysiology; Ion channel; Patch-clamp; Synapse
    DOI:  https://doi.org/10.1007/978-1-0716-3810-1_19
  20. Methods Mol Biol. 2024 ;2794 157-167
    Generating Homogeneous Brain Organoids from Human iPSCs.
    Xianwei Chen, Yanhong Shi.
      There is a high demand for the development of in vitro models for human brain development and diseases due to the inaccessibility of human brain tissues. The human iPSC-derived brain organoids provide a promising in vitro model for studying human brain development and disorders. However, it is challenging to generate a large number of brain organoids with high consistency for modeling human neurological diseases. Here, we describe a method for generating high-yield brain organoids with high consistency by combining large-scale embryoid body (EB) generation and incorporating a quality control screening step during differentiation. The method described in this chapter provides a robust way to generate brain organoids for studying human brain development and modeling neurological diseases.
    Keywords:  Alzheimer’s disease; Brain organoids; Differentiation; Disease modeling; Embryoid bodies (EBs); Induced pluripotent stem cells (iPSCs); Neurological diseases; Pluripotent stem cells (PSCs)
    DOI:  https://doi.org/10.1007/978-1-0716-3810-1_13
  21. iScience. 2024 May 17. 27(5): 109631
    Human pluripotent stem cells as a translational toolkit in psychedelic research in vitro.
    José Alexandre Salerno, Stevens Rehen.
      Psychedelics, recognized for their impact on perception, are resurging as promising treatments with rapid onset for mood and substance use disorders. Despite increasing evidence from clinical trials, questions persist about the cellular and molecular mechanisms and their precise correlation with treatment outcomes. Murine neurons and immortalized non-neural cell lines harboring overexpressed constructs have shed light on neuroplastic changes mediated by the serotonin 2A receptor (5-HT2AR) as the primary mechanism. However, limitations exist in capturing human- and disease-specific traits. Here, we discuss current accomplishments and prospects for incorporating human pluripotent stem cells (PSCs) to complement these models. PSCs can differentiate into various brain cell types, mirroring endogenous expression patterns and cell identities to recreate disease phenotypes. Brain organoids derived from PSCs resemble cell diversity and patterning, while region-specific organoids simulate circuit-level phenotypes. PSC-based models hold significant promise to illuminate the cellular and molecular substrates of psychedelic-induced phenotypic recovery in neuropsychiatric disorders.
    Keywords:  Biological sciences; Cell biology; Cellular neuroscience; Natural sciences; Neuroscience; Pharmacology; Stem cells research
    DOI:  https://doi.org/10.1016/j.isci.2024.109631
  22. Cell Commun Signal. 2024 Apr 18. 22(1): 231
    Exploring the role of Prx II in mitigating endoplasmic reticulum stress and mitochondrial dysfunction in neurodegeneration.
    Mei-Hua Jin, Lin Feng, Hong-Yi Xiang, Hu-Nan Sun, Ying-Hao Han, Taeho Kwon.
      BACKGROUND: Neurodegenerative diseases are increasingly recognized for their association with oxidative stress, which leads to progressive dysfunction and loss of neurons, manifesting in cognitive and motor impairments. This study aimed to elucidate the neuroprotective role of peroxiredoxin II (Prx II) in counteracting oxidative stress-induced mitochondrial damage, a key pathological feature of neurodegeneration.METHODS: We investigated the impact of Prx II deficiency on endoplasmic reticulum stress and mitochondrial dysfunction using HT22 cell models with knocked down and overexpressed Prx II. We observed alcohol-treated HT22 cells using transmission electron microscopy and monitored changes in the length of mitochondria-associated endoplasmic reticulum membranes and their contact with endoplasmic reticulum mitochondria contact sites (EMCSs). Additionally, RNA sequencing and bioinformatic analysis were conducted to identify the role of Prx II in regulating mitochondrial transport and the formation of EMCSs.
    RESULTS: Our results indicated that Prx II preserves mitochondrial integrity by facilitating the formation of EMCSs, which are essential for maintaining mitochondrial Ca2+ homeostasis and preventing mitochondria-dependent apoptosis. Further, we identified a novel regulatory axis involving Prx II, the transcription factor ATF3, and miR-181b-5p, which collectively modulate the expression of Armcx3, a protein implicated in mitochondrial transport. Our findings underscore the significance of Prx II in protecting neuronal cells from alcohol-induced oxidative damage and suggest that modulating the Prx II-ATF3-miR-181b-5p pathway may offer a promising therapeutic strategy against neurodegenerative diseases.
    CONCLUSIONS: This study not only expands our understanding of the cytoprotective mechanisms of Prx II but also offers necessary data for developing targeted interventions to bolster mitochondrial resilience in neurodegenerative conditions.
    Keywords:  Endoplasmic reticulum-mitochondrial interactions; Mitochondrial damage; Neurodegenerative diseases; Peroxiredoxin II; Reactive oxygen species
    DOI:  https://doi.org/10.1186/s12964-024-01613-x
  23. iScience. 2024 Apr 19. 27(4): 109569
    iPSC-based modeling of preeclampsia identifies epigenetic defects in extravillous trophoblast differentiation.
    Robert Morey, Tony Bui, Virginia Chu Cheung, Chen Dong, Joseph E Zemke, Daniela Requena, Harneet Arora, Madeline G Jackson, Donald Pizzo, Thorold W Theunissen, Mariko Horii.
      Preeclampsia (PE) is a hypertensive pregnancy disorder with increased risk of maternal and fetal morbidity and mortality. Abnormal extravillous trophoblast (EVT) development and function is considered to be the underlying cause of PE, but has not been previously modeled in vitro. We previously derived induced pluripotent stem cells (iPSCs) from placentas of PE patients and characterized abnormalities in formation of syncytiotrophoblast and responses to changes in oxygen tension. In this study, we converted these primed iPSC to naïve iPSC, and then derived trophoblast stem cells (TSCs) and EVT to evaluate molecular mechanisms underlying PE. We found that primed (but not naïve) iPSC-derived PE-EVT have reduced surface HLA-G, blunted invasive capacity, and altered EVT-specific gene expression. These abnormalities correlated with promoter hypermethylation of genes associated with the epithelial-mesenchymal transition pathway, specifically in primed-iPSC derived PE-EVT. Our findings indicate that abnormal epigenetic regulation might play a role in PE pathogenesis.
    Keywords:  Cell biology; Molecular biology; Omics; Transcriptomics
    DOI:  https://doi.org/10.1016/j.isci.2024.109569
  24. Methods Mol Biol. 2024 ;2794 169-175
    Primary Culture of Dissociated Neurons from the Embryonic Cerebral Cortex.
    Shiho Ito, Megumi Kawasaki, Takeshi Kawauchi.
      Primary neuronal culture is a valuable in vitro model for analyzing the molecular mechanisms underlying the development and function of neural circuits. In contrast to neurons in vivo, primary cultured neurons can easily be transfected with genes of interest or treated with chemicals such as agonists and inhibitors of a specific target molecule. Furthermore, time-dependent morphological changes, such as the acquisition of neuronal polarity, axon elongation, and dendrite branch formation, can be analyzed by using primary neuronal cultures. Here, we describe a method for preparing a primary culture of neurons from the developing cerebral cortex, together with a method for gene transfer to primary cultured cortical neurons.
    Keywords:  Cerebral cortex; Dissociated cell culture; Embryonic brain; Mouse; Neuron; Primary culture
    DOI:  https://doi.org/10.1007/978-1-0716-3810-1_14
  25. J Neural Transm (Vienna). 2024 Apr 13.
    Current trends in basic research on Parkinson's disease: from mitochondria, lysosome to α-synuclein.
    Hideaki Matsui, Ryosuke Takahashi.
      Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra and other brain regions. A key pathological feature of PD is the abnormal accumulation of α-synuclein protein within affected neurons, manifesting as Lewy bodies and Lewy neurites. Despite extensive research efforts spanning several decades, the underlying mechanisms of PD and disease-modifying therapies remain elusive. This review provides an overview of current trends in basic research on PD. Initially, it discusses the involvement of mitochondrial dysfunction in the pathogenesis of PD, followed by insights into the role of lysosomal dysfunction and disruptions in the vesicular transport system. Additionally, it delves into the pathological and physiological roles of α-synuclein, a crucial protein associated with PD pathophysiology. Overall, the purpose of this review is to comprehend the current state of elucidating the intricate mechanisms underlying PD and to outline future directions in understanding this disease.
    Keywords:  Lysosome; Mitochondria; Parkinson’s disease; Vesicular transport; α-synuclein
    DOI:  https://doi.org/10.1007/s00702-024-02774-2
  26. Adv Biol (Weinh). 2024 Apr 19. e2400018
    Generation of Anterior Segment of the Eye Cells from hiPSCs in Microfluidic Platforms.
    Gamze Koçak, Sude Uyulgan, Elifsu Polatlı, Vedat Sarı, Burak Kahveci, Ahmet Bursali, Leman Binokay, Tuba Reçber, Emirhan Nemutlu, Adil Mardinoğlu, Gökhan Karakülah, Canan Aslı Utine, Sinan Güven.
      Ophthalmic diseases affect many people, causing partial or total loss of vision and a reduced quality of life. The anterior segment of the eye accounts for nearly half of all visual impairment that can lead to blindness. Therefore, there is a growing demand for ocular research and regenerative medicine that specifically targets the anterior segment to improve vision quality. This study aims to generate a microfluidic platform for investigating the formation of the anterior segment of the eye derived from human induced pluripotent stem cells (hiPSC) under various spatial-mechanoresponsive conditions. Microfluidic platforms are developed to examine the effects of dynamic conditions on the generation of hiPSCs-derived ocular organoids. The differentiation protocol is validated, and mechanoresponsive genes are identified through transcriptomic analysis. Several culture strategies is implemented for the anterior segment of eye cells in a microfluidic chip. hiPSC-derived cells showed anterior eye cell characteristics in mRNA and protein expression levels under dynamic culture conditions. The expression levels of yes-associated protein and transcriptional coactivator PDZ binding motif (YAP/TAZ) and PIEZO1, varied depending on the differentiation and growth conditions of the cells, as well as the metabolomic profiles under dynamic culture conditions.
    Keywords:  hiPSC; mechanotransduction; metabolomics; ocular cell; on‐chip system; organoid; transcriptomics
    DOI:  https://doi.org/10.1002/adbi.202400018
  27. Nat Protoc. 2024 Apr 17.
    Reprogramming fibroblast into human iBlastoids.
    Jia Ping Tan, Xiaodong Liu, Jose M Polo.
      The study of early human embryogenesis has relied on the use of blastocysts donated to research or simple stem cell culture systems such as pluripotent and trophoblast stem cells, which have been seminal in shedding light on many key developmental processes. However, simple culture systems lack the necessary complexity to adequately model the spatiotemporal, cellular and molecular dynamics occurring during the early phases of embryonic development. As such, an in vitro model of the human blastocyst is advantageous in many aspects to decipher human embryogenesis. Here we describe a step-by-step protocol for the generation of induced blastoids (iBlastoids), an in vitro integrated model of the human blastocyst derived via somatic reprogramming. This protocol details the workflow for reprogramming of human dermal fibroblasts and subsequent generation of iBlastoids using the reprogramming intermediates, which together takes ~27 days (21 days for reprogramming and 6 days for iBlastoid generation). We also discuss several characterization/functional assays that can be used on the iBlastoids. We believe that a person trained in cell culture with ~1 year of experience with human somatic cell and reprogramming/cell differentiation assays would be able to perform this protocol. In short, the iBlastoids present an alternative tool as a model to the blastocyst to facilitate the scientific community in the exploration of early human development.
    DOI:  https://doi.org/10.1038/s41596-024-00984-2
  28. Methods Mol Biol. 2024 ;2794 1-12
    Overview: Research on the Genetic Architecture of the Developing Cerebral Cortex in Norms and Diseases.
    Mariko Y Momoi.
      The human brain is characterized by high cell numbers, diverse cell types with diverse functions, and intricate connectivity with an exceedingly broad surface of the cortex. Human-specific brain development was accomplished by a long timeline for maturation from the prenatal period to the third decade of life. The long timeline makes complicated architecture and circuits of human cerebral cortex possible, and it makes human brain vulnerable to intrinsic and extrinsic insults resulting in the development of variety of neuropsychiatric disorders. Unraveling the molecular and cellular processes underlying human brain development under the elaborate regulation of gene expression in a spatiotemporally specific manner, especially that of the cortex will provide a biological understanding of human cognition and behavior in health and diseases. Global research consortia and the advancing technologies in brain science including functional genomics equipped with emergent neuroinformatics such as single-cell multiomics, novel human models, and high-volume databases with high-throughput computation facilitate the biological understanding of the development of the human brain cortex. Knowing the process of interplay of the genome and the environment in cortex development will lead us to understand the human-specific cognitive function and its individual diversity. Thus, it is worthwhile to overview the recent progress in neurotechnology to foresee further understanding of the human brain and norms and diseases.
    Keywords:  Development of human cerebral cortex; Integrated functional genomics; Interplay of the genome and the environment; Pathophysiology of neuropsychiatric disorders
    DOI:  https://doi.org/10.1007/978-1-0716-3810-1_1
  29. Lancet Neurol. 2024 Apr 10. pii: S1474-4422(24)00121-2. [Epub ahead of print]
    RAB32 Ser71Arg in autosomal dominant Parkinson's disease: linkage, association, and functional analyses.
    Global Parkinson's Genetics Program (GP2)
      BACKGROUND: Parkinson's disease is a progressive neurodegenerative disorder with multifactorial causes, among which genetic risk factors play a part. The RAB GTPases are regulators and substrates of LRRK2, and variants in the LRRK2 gene are important risk factors for Parkinson's disease. We aimed to explore genetic variability in RAB GTPases within cases of familial Parkinson's disease.METHODS: We did whole-exome sequencing in probands from families in Canada and Tunisia with Parkinson's disease without a genetic cause, who were recruited from the Centre for Applied Neurogenetics (Vancouver, BC, Canada), an international consortium that includes people with Parkinson's disease from 36 sites in 24 countries. 61 RAB GTPases were genetically screened, and candidate variants were genotyped in relatives of the probands to assess disease segregation by linkage analysis. Genotyping was also done to assess variant frequencies in individuals with idiopathic Parkinson's disease and controls, matched for age and sex, who were also from the Centre for Applied Neurogenetics but unrelated to the probands or each other. All participants were aged 18 years or older. The sequencing and genotyping findings were validated by case-control association analyses using bioinformatic data obtained from publicly available clinicogenomic databases (AMP-PD, GP2, and 100 000 Genomes Project) and a private German clinical diagnostic database (University of Tübingen). Clinical and pathological findings were summarised and haplotypes were determined. In-vitro studies were done to investigate protein interactions and enzyme activities.
    FINDINGS: Between June 1, 2010, and May 31, 2017, 130 probands from Canada and Tunisia (47 [36%] female and 83 [64%] male; mean age 72·7 years [SD 11·7; range 38-96]; 109 White European ancestry, 18 north African, two east Asian, and one Hispanic] underwent whole-exome sequencing. 15 variants in RAB GTPase genes were identified, of which the RAB32 variant c.213C>G (Ser71Arg) cosegregated with autosomal dominant Parkinson's disease in three families (nine affected individuals; non-parametric linkage Z score=1·95; p=0·03). 2604 unrelated individuals with Parkinson's disease and 344 matched controls were additionally genotyped, and five more people originating from five countries (Canada, Italy, Poland, Turkey, and Tunisia) were identified with the RAB32 variant. From the database searches, in which 6043 individuals with Parkinson's disease and 62 549 controls were included, another eight individuals were identified with the RAB32 variant from four countries (Canada, Germany, UK, and USA). Overall, the association of RAB32 c.213C>G (Ser71Arg) with Parkinson's disease was significant (odds ratio [OR] 13·17, 95% CI 2·15-87·23; p=0·0055; I2=99·96%). In the people who had the variant, Parkinson's disease presented at age 54·6 years (SD 12·75, range 31-81, n=16), and two-thirds had a family history of parkinsonism. RAB32 Ser71Arg heterozygotes shared a common haplotype, although penetrance was incomplete. Findings in one individual at autopsy showed sparse neurofibrillary tangle pathology in the midbrain and thalamus, without Lewy body pathology. In functional studies, RAB32 Arg71 activated LRRK2 kinase to a level greater than RAB32 Ser71.
    INTERPRETATION: RAB32 Ser71Arg is a novel genetic risk factor for Parkinson's disease, with reduced penetrance. The variant was found in individuals with Parkinson's disease from multiple ethnic groups, with the same haplotype. In-vitro assays show that RAB32 Arg71 activates LRRK2 kinase, which indicates that genetically distinct causes of familial parkinsonism share the same mechanism. The discovery of RAB32 Ser71Arg also suggests several genetically inherited causes of Parkinson's disease originated to control intracellular immunity. This shared aetiology should be considered in future translational research, while the global epidemiology of RAB32 Ser71Arg needs to be assessed to inform genetic counselling.
    FUNDING: National Institutes of Health, the Canada Excellence Research Chairs program, Aligning Science Across Parkinson's, the Michael J Fox Foundation for Parkinson's Research, and the UK Medical Research Council.
    DOI:  https://doi.org/10.1016/S1474-4422(24)00121-2
  30. J Mol Biol. 2024 Apr 16. pii: S0022-2836(24)00169-4. [Epub ahead of print] 168574
    Selective autophagy of macromolecular complexes: what does it take to be taken?
    Javier Lizarrondo, Florian Wilfling.
      Proteins are known to perform an astonishing array of functions thanks to their ability to cooperate and modulate each other's properties. Inside cells, proteins can assemble into large multi-subunit complexes to carry out complex cellular functions. The correct assembly and maintenance of the functional state of macromolecular protein complexes is crucial for human health. Failure to do so leads to loss of function and potential accumulation of harmful materials, which is associated with a variety of human diseases such as neurodegeneration and cancer. Autophagy engulfs cytosolic material in autophagosomes, and therefore is best suited to eliminate intact macromolecular complexes without disassembling them, which could interfere with de novo assembly. In this review, we discuss the role of autophagy in the selective degradation of macromolecular complexes. We highlight the current state of knowledge for different macromolecular complexes and their selective autophagic degradation. We emphasize the gaps in our understanding of what it takes for these large macromolecular complexes to be degraded and point to future work that may shed light on the regulation of the selective degradation of macromolecular complexes by autophagy.
    Keywords:  LLPS; Selective; autophagy; cargo-recognition; phase separation; protein complexes; proteostasis
    DOI:  https://doi.org/10.1016/j.jmb.2024.168574
  31. Nat Rev Neurol. 2024 Apr 15.
    Neuronal activity drives glymphatic waste clearance.
    Lisa Kiani.
      
    DOI:  https://doi.org/10.1038/s41582-024-00963-x
  32. Proc Natl Acad Sci U S A. 2024 Apr 23. 121(17): e2314353121
    WAV E3 ubiquitin ligases mediate degradation of IAA32/34 in the TMK1-mediated auxin signaling pathway during apical hook development.
    Jun-Li Wang, Ming Wang, Li Zhang, You-Xia Li, Jing-Jing Li, Yu-Yang Li, Zuo-Xian Pu, Dan-Yang Li, Xing-Nan Liu, Wang Guo, Dong-Wei Di, Xiao-Feng Li, Guang-Qin Guo, Lei Wu.
      Auxin regulates plant growth and development through downstream signaling pathways, including the best-known SCFTIR1/AFB-Aux/IAA-ARF pathway and several other less characterized "noncanonical" pathways. Recently, one SCFTIR1/AFB-independent noncanonical pathway, mediated by Transmembrane Kinase 1 (TMK1), was discovered through the analyses of its functions in Arabidopsis apical hook development. Asymmetric accumulation of auxin on the concave side of the apical hook triggers DAR1-catalyzed release of the C-terminal of TMK1, which migrates into the nucleus, where it phosphorylates and stabilizes IAA32/34 to inhibit cell elongation, which is essential for full apical hook formation. However, the molecular factors mediating IAA32/34 degradation have not been identified. Here, we show that proteins in the CYTOKININ INDUCED ROOT WAVING 1 (CKRW1)/WAVY GROWTH 3 (WAV3) subfamily act as E3 ubiquitin ligases to target IAA32/34 for ubiquitination and degradation, which is inhibited by TMK1c-mediated phosphorylation. This antagonistic interaction between TMK1c and CKRW1/WAV3 subfamily E3 ubiquitin ligases regulates IAA32/34 levels to control differential cell elongation along opposite sides of the apical hook.
    Keywords:  Aux/IAA proteins; RING-finger E3 ubiquitin ligase; Transmembrane Kinase 1; Wavy Growth 3; noncanonical auxin signaling
    DOI:  https://doi.org/10.1073/pnas.2314353121
  33. Methods Mol Biol. 2024 ;2794 121-140
    Generation of iPSCs Using Sendai Virus Vectors.
    Hitomi Aoki.
      Induced pluripotent stem cells (iPSCs) are in vitro-derived cells capable of giving rise to several different cell types. The generation of iPSCs holds great promise for regenerative medicine and drug discovery research because it allows mature cells to be reprogrammed into a state of pluripotency. These highly versatile cells can then be induced to produce a variety of cell lineages and tissues by activating specific regulatory genes that drive their differentiation along distinct lineages. The great potential of these cells was recognized by Shinya Yamanaka who was awarded the 2012 Nobel Prize for the discovery of iPSCs. Following their discovery, various methods have now been developed for generating iPSCs. Here, we describe a method for deriving iPSCs from human dental pulp using Sendai virus vectors.
    Keywords:  Dental pulp; Induced pluripotent stem cells; Klf4; Oct3/4; Sendai virus; Sox2; c-Myc
    DOI:  https://doi.org/10.1007/978-1-0716-3810-1_11
  34. Front Cell Neurosci. 2024 ;18 1374555
    Axon morphology and intrinsic cellular properties determine repetitive transcranial magnetic stimulation threshold for plasticity.
    Christos Galanis, Lena Neuhaus, Nicholas Hananeia, Zsolt Turi, Peter Jedlicka, Andreas Vlachos.
      Introduction: Repetitive transcranial magnetic stimulation (rTMS) is a widely used therapeutic tool in neurology and psychiatry, but its cellular and molecular mechanisms are not fully understood. Standardizing stimulus parameters, specifically electric field strength, is crucial in experimental and clinical settings. It enables meaningful comparisons across studies and facilitates the translation of findings into clinical practice. However, the impact of biophysical properties inherent to the stimulated neurons and networks on the outcome of rTMS protocols remains not well understood. Consequently, achieving standardization of biological effects across different brain regions and subjects poses a significant challenge.Methods: This study compared the effects of 10 Hz repetitive magnetic stimulation (rMS) in entorhino-hippocampal tissue cultures from mice and rats, providing insights into the impact of the same stimulation protocol on similar neuronal networks under standardized conditions.
    Results: We observed the previously described plastic changes in excitatory and inhibitory synaptic strength of CA1 pyramidal neurons in both mouse and rat tissue cultures, but a higher stimulation intensity was required for the induction of rMS-induced synaptic plasticity in rat tissue cultures. Through systematic comparison of neuronal structural and functional properties and computational modeling, we found that morphological parameters of CA1 pyramidal neurons alone are insufficient to explain the observed differences between the groups. Although morphologies of mouse and rat CA1 neurons showed no significant differences, simulations confirmed that axon morphologies significantly influence individual cell activation thresholds. Notably, differences in intrinsic cellular properties were sufficient to account for the 10% higher intensity required for the induction of synaptic plasticity in the rat tissue cultures.
    Conclusion: These findings demonstrate the critical importance of axon morphology and intrinsic cellular properties in predicting the plasticity effects of rTMS, carrying valuable implications for the development of computer models aimed at predicting and standardizing the biological effects of rTMS.
    Keywords:  axons; excitation; inhibition; morphology; organotypic tissue cultures; synaptic plasticity; whole-cell patch-clamp recordings
    DOI:  https://doi.org/10.3389/fncel.2024.1374555
  35. Cell Death Discov. 2024 Apr 17. 10(1): 180
    PGC-1a mediated mitochondrial biogenesis promotes recovery and survival of neuronal cells from cellular degeneration.
    Wenting You, Kèvin Knoops, Tos T J M Berendschot, Birke J Benedikter, Carroll A B Webers, Chris P M Reutelingsperger, Theo G M F Gorgels.
      Neurodegenerative disorders are characterized by the progressive loss of structure and function of neurons, often including the death of the neuron. Previously, we reported that, by removing the cell death stimulus, dying/injured neurons could survive and recover from the process of regulated cell death, even if the cells already displayed various signs of cellular damage. Now we investigated the role of mitochondrial dynamics (fission/fusion, biogenesis, mitophagy) in both degeneration and in recovery of neuronal cells. In neuronal PC12 cells, exposure to ethanol (EtOH) induced massive neurite loss along with widespread mitochondrial fragmentation, mitochondrial membrane potential loss, reduced ATP production, and decreased total mitochondrial volume. By removing EtOH timely all these mitochondrial parameters recovered to normal levels. Meanwhile, cells regrew neurites and survived. Study of the mitochondrial dynamics showed that autophagy was activated only during the cellular degeneration phase (EtOH treatment) but not in the recovery phase (EtOH removed), and it was not dependent on the Parkin/PINK1 mediated mitophagy pathway. Protein expression of key regulators of mitochondrial fission, phospho-Drp1Ser616 and S-OPA1, increased during EtOH treatment and recovered to normal levels after removing EtOH. In addition, the critical role of PGC-1α mediated mitochondrial biogenesis in cellular recovery was revealed: inhibition of PGC-1α using SR-18292 after EtOH removal significantly impeded recovery of mitochondrial damage, regeneration of neurites, and cell survival in a concentration-dependent manner. Taken together, our study showed reversibility of mitochondrial morphological and functional damage in stressed neuronal cells and revealed that PGC-1α mediated mitochondrial biogenesis played a critical role in the cellular recovery. This molecular mechanism could be a target for neuroprotection and neurorescue in neurodegenerative diseases.
    DOI:  https://doi.org/10.1038/s41420-024-01953-0
  36. Epilepsia Open. 2024 Apr 18.
    In vitro human cell culture models in a bench-to-bedside approach to epilepsy.
    Šárka Danačíková, Barbora Straka, Jan Daněk, Vladimír Kořínek, Jakub Otáhal.
      Epilepsy is the most common chronic neurological disease, affecting nearly 1%-2% of the world's population. Current pharmacological treatment and regimen adjustments are aimed at controlling seizures; however, they are ineffective in one-third of the patients. Although neuronal hyperexcitability was previously thought to be mainly due to ion channel alterations, current research has revealed other contributing molecular pathways, including processes involved in cellular signaling, energy metabolism, protein synthesis, axon guidance, inflammation, and others. Some forms of drug-resistant epilepsy are caused by genetic defects that constitute potential targets for precision therapy. Although such approaches are increasingly important, they are still in the early stages of development. This review aims to provide a summary of practical aspects of the employment of in vitro human cell culture models in epilepsy diagnosis, treatment, and research. First, we briefly summarize the genetic testing that may result in the detection of candidate pathogenic variants in genes involved in epilepsy pathogenesis. Consequently, we review existing in vitro cell models, including induced pluripotent stem cells and differentiated neuronal cells, providing their specific properties, validity, and employment in research pipelines. We cover two methodological approaches. The first approach involves the utilization of somatic cells directly obtained from individual patients, while the second approach entails the utilization of characterized cell lines. The models are evaluated in terms of their research and clinical benefits, relevance to the in vivo conditions, legal and ethical aspects, time and cost demands, and available published data. Despite the methodological, temporal, and financial demands of the reviewed models they possess high potential to be used as robust systems in routine testing of pathogenicity of detected variants in the near future and provide a solid experimental background for personalized therapy of genetic epilepsies. PLAIN LANGUAGE SUMMARY: Epilepsy affects millions worldwide, but current treatments fail for many patients. Beyond traditional ion channel alterations, various genetic factors contribute to the disorder's complexity. This review explores how in vitro human cell models, either from patients or from cell lines, can aid in understanding epilepsy's genetic roots and developing personalized therapies. While these models require further investigation, they offer hope for improved diagnosis and treatment of genetic forms of epilepsy.
    Keywords:  drug‐resistant epilepsy; genetic testing; in vitro human cell culture; legal and ethical aspects; precision medicine
    DOI:  https://doi.org/10.1002/epi4.12941
  37. Environ Toxicol. 2024 Apr 15.
    Teaghrelin protected dopaminergic neurons in MPTP-induced Parkinson's disease animal model by promoting PINK1/Parkin-mediated mitophagy and AMPK/SIRT1/PGC1-α-mediated mitochondrial biogenesis.
    Cian-Fen Jhuo, Chun-Jung Chen, Jason T C Tzen, Wen-Ying Chen.
      Mitochondrial dysfunction, a common cellular hallmark in both familial and sporadic forms of Parkinson's disease (PD), is assumed to play a significant role in pathologic development and progression of the disease. Teaghrelin, a unique bioactive compound in some oolong tea varieties, has been demonstrated to protect SH-SY5Y cells against 1-methyl-4-phenylpyridinium induced neurotoxicity by binding to the ghrelin receptor to activate the AMPK/SIRT1/PGC-1α pathway. In this study, an animal model was established using a neurotoxin, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP), a byproduct of a prohibited drug, to evaluate the oral efficacy of teaghrelin on PD by monitoring motor dysfunction of mice in open field, pole, and bean walking tests. The results showed that MPTP-induced motor dysfunction of mice was significantly attenuated by teaghrelin supplementation. Tyrosine hydroxylase and dopamine transporter protein were found reduced in the striatum and midbrain of MPTP-treated mice, and significantly mitigated by teaghrelin supplementation. Furthermore, teaghrelin administration enhanced mitophagy and mitochondria biogenesis, which maintained cell homeostasis and prevented the accumulation of αSyn and apoptosis-related proteins. It seemed that teaghrelin protected dopaminergic neurons in MPTP-treated mice by increasing PINK1/Parkin-mediated mitophagy and AMPK/SIRT1/PGC-1α-mediated mitochondria biogenesis, highlighting its potential therapeutic role in maintaining dopaminergic neurons function in PD. Mitochondrial dysfunction, a common cellular hallmark in both familial and sporadic forms of Parkinson's disease (PD), is assumed to play a significant role in pathologic development and progression of the disease. Teaghrelin, a unique bioactive compound in some oolong tea varieties, has been demonstrated to protect SH-SY5Y cells against 1-methyl-4-phenylpyridinium induced neurotoxicity by binding to the ghrelin receptor to activate the AMPK/SIRT1/PGC-1α pathway. In this study, an animal model was established using a neurotoxin, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP), a byproduct of a prohibited drug, to evaluate the oral efficacy of teaghrelin on PD by monitoring motor dysfunction of mice in open field, pole, and bean walking tests. The results showed that MPTP-induced motor dysfunction of mice was significantly attenuated by teaghrelin supplementation. Tyrosine hydroxylase and dopamine transporter protein were found reduced in the striatum and midbrain of MPTP-treated mice, and significantly mitigated by teaghrelin supplementation. Furthermore, teaghrelin administration enhanced mitophagy and mitochondria biogenesis, which maintained cell homeostasis and prevented the accumulation of αSyn and apoptosis-related proteins. It seemed that teaghrelin protected dopaminergic neurons in MPTP-treated mice by increasing PINK1/Parkin-mediated mitophagy and AMPK/SIRT1/PGC-1α-mediated mitochondria biogenesis, highlighting its potential therapeutic role in maintaining dopaminergic neurons function in PD.
    Keywords:  Parkinson's disease; bioactive compounds; mitochondria biogenesis; mitophagy; neurotoxicity; teaghrelin
    DOI:  https://doi.org/10.1002/tox.24275
  38. Mol Aspects Med. 2024 Apr 15. pii: S0098-2997(24)00031-1. [Epub ahead of print]97 101272
    Delineating the contribution of ageing and physical activity to changes in mitochondrial characteristics across the lifespan.
    Matthew J-C Lee, Nicholas J Saner, Alessandra Ferri, Esther García-Domínguez, James R Broatch, David J Bishop.
      Ageing is associated with widespread physiological changes prominent within all tissues, including skeletal muscle and the brain, which lead to a decline in physical function. To tackle the growing health and economic burdens associated with an ageing population, the concept of healthy ageing has become a major research priority. Changes in skeletal muscle mitochondrial characteristics have been suggested to make an important contribution to the reductions in skeletal muscle function with age, and age-related changes in mitochondrial content, respiratory function, morphology, and mitochondrial DNA have previously been reported. However, not all studies report changes in mitochondrial characteristics with ageing, and there is increasing evidence to suggest that physical activity (or inactivity) throughout life is a confounding factor when interpreting age-associated changes. Given that physical activity is a potent stimulus for inducing beneficial adaptations to mitochondrial characteristics, delineating the influence of physical activity on the changes in skeletal muscle that occur with age is complicated. This review aims to summarise our current understanding and knowledge gaps regarding age-related changes to mitochondrial characteristics within skeletal muscle, as well as to provide some novel insights into brain mitochondria, and to propose avenues of future research and targeted interventions. Furthermore, where possible, we incorporate discussions of the modifying effects of physical activity, exercise, and training status, to purported age-related changes in mitochondrial characteristics.
    DOI:  https://doi.org/10.1016/j.mam.2024.101272
  39. J Physiol. 2024 Apr;602(8): 1637-1654
    Optical recordings of organellar membrane potentials and the components of membrane conductance in lysosomes.
    Cristian Castillo-Velasquez, Ella Matamala, Diego Becerra, Patricio Orio, Sebastian E Brauchi.
      The eukaryotic cell is highly compartmentalized with organelles. Owing to their function in transporting metabolites, metabolic intermediates and byproducts of metabolic activity, organelles are important players in the orchestration of cellular function. Recent advances in optical methods for interrogating the different aspects of organellar activity promise to revolutionize our ability to dissect cellular processes with unprecedented detail. The transport activity of organelles is usually coupled to the transport of charged species; therefore, it is not only associated with the metabolic landscape but also entangled with membrane potentials. In this context, the targeted expression of fluorescent probes for interrogating organellar membrane potential (Ψorg) emerges as a powerful approach, offering less-invasive conditions and technical simplicity to interrogate cellular signalling and metabolism. Different research groups have made remarkable progress in adapting a variety of optical methods for measuring and monitoring Ψorg. These approaches include using potentiometric dyes, genetically encoded voltage indicators, hybrid fluorescence resonance energy transfer sensors and photoinduced electron transfer systems. These studies have provided consistent values for the resting potential of single-membrane organelles, such as lysosomes, the Golgi and the endoplasmic reticulum. We can foresee the use of dynamic measurements of Ψorg to study fundamental problems in organellar physiology that are linked to serious cellular disorders. Here, we present an overview of the available techniques, a survey of the resting membrane potential of internal membranes and, finally, an open-source mathematical model useful to interpret and interrogate membrane-bound structures of small volume by using the lysosome as an example.
    Keywords:  Golgi; hybrid voltage sensor; lysosome; model; optical; organelle; reticulum; voltage
    DOI:  https://doi.org/10.1113/JP283825
  40. Cell Rep Methods. 2024 Apr 10. pii: S2667-2375(24)00089-4. [Epub ahead of print] 100758
    Combining LIANA and Tensor-cell2cell to decipher cell-cell communication across multiple samples.
    Hratch M Baghdassarian, Daniel Dimitrov, Erick Armingol, Julio Saez-Rodriguez, Nathan E Lewis.
      In recent years, data-driven inference of cell-cell communication has helped reveal coordinated biological processes across cell types. Here, we integrate two tools, LIANA and Tensor-cell2cell, which, when combined, can deploy multiple existing methods and resources to enable the robust and flexible identification of cell-cell communication programs across multiple samples. In this work, we show how the integration of our tools facilitates the choice of method to infer cell-cell communication and subsequently perform an unsupervised deconvolution to obtain and summarize biological insights. We explain how to perform the analysis step by step in both Python and R and provide online tutorials with detailed instructions available at https://ccc-protocols.readthedocs.io/. This workflow typically takes ∼1.5 h to complete from installation to downstream visualizations on a graphics processing unit-enabled computer for a dataset of ∼63,000 cells, 10 cell types, and 12 samples.
    Keywords:  CP: Cell biology; CP: Systems biology; cell-cell communication; context dependent; ligand-receptor interactions; multiple conditions; single-cell RNA sequencing; tensor decomposition
    DOI:  https://doi.org/10.1016/j.crmeth.2024.100758
  41. bioRxiv. 2024 Apr 02. pii: 2024.03.29.587368. [Epub ahead of print]
    A mechanism that transduces lysosomal damage signals to stress granule formation for cell survival.
    Jacob Duran, Suttinee Poolsup, Lee Allers, Monica Rosas Lemus, Qiuying Cheng, Jing Pu, Michelle Salemi, Brett Phinney, Jingyue Jia.
      Lysosomal damage poses a significant threat to cell survival. Our previous work has reported that lysosomal damage induces stress granule (SG) formation. However, the importance of SG formation in determining cell fate and the precise mechanisms through which lysosomal damage triggers SG formation remains unclear. Here, we show that SG formation is initiated via a novel calcium-dependent pathway and plays a protective role in promoting cell survival in response to lysosomal damage. Mechanistically, we demonstrate that during lysosomal damage, ALIX, a calcium-activated protein, transduces lysosomal damage signals by sensing calcium leakage to induce SG formation by controlling the phosphorylation of eIF2α. ALIX modulates eIF2α phosphorylation by regulating the association between PKR and its activator PACT, with galectin-3 exerting a negative effect on this process. We also found this regulatory event of SG formation occur on damaged lysosomes. Collectively, these investigations reveal novel insights into the precise regulation of SG formation triggered by lysosomal damage, and shed light on the interaction between damaged lysosomes and SGs. Importantly, SG formation is significant for promoting cell survival in the physiological context of lysosomal damage inflicted by SARS-CoV-2 ORF3a, adenovirus infection, Malaria hemozoin, proteopathic tau as well as environmental hazard silica.
    DOI:  https://doi.org/10.1101/2024.03.29.587368
  42. Acta Biomater. 2024 Apr 17. pii: S1742-7061(24)00196-X. [Epub ahead of print]
    Unveiling clinical applications of bacterial extracellular vesicles as natural nanomaterials in disease diagnosis and therapeutics.
    Chun Liu, Negar Yazdani, Corey S Moran, Carlos Salomon, Chaminda Jayampath Seneviratne, Sašo Ivanovski, Pingping Han.
      Bacterial extracellular vesicles (BEVs) are naturally occurring bioactive membrane-bound nanoparticles released by both gram-negative and positive bacterial species, exhibiting a multifaceted role in mediating host-microbe interactions across various physiological conditions. Increasing evidence supports BEVs as essential mediators of intercellular exchange, influencing bacterial pathogenicity, disease mechanisms, and modulating the host immune response. However, the extent to which these BEV-mediated actions can be leveraged to predict disease onset, guide treatment strategies, and determine clinical outcomes remains uncertain, particularly in terms of their clinical translation potentials. This review briefly describes BEV biogenesis and their internalisation by recipient cells and summarises methods for isolation and characterization, essential for understanding their composition and cargo. Further, it discusses the potential of biofluid-associated BEVs as biomarkers for various diseases, spanning both cancer and non-cancerous conditions. Following this, we also outline the ongoing human clinical trials of using BEVs for vaccine development. In addition to disease diagnostics, this review explores the emerging research of using natural or engineered BEVs as smart nanomaterials for applications in anti-cancer therapy and bone regeneration. This discussion extends to key factors for unlocking the clinical potential of BEVs, such as standardization of BEVs isolation and characterisation, as well as other hurdles in translating these findings to the clinical setting. We propose that addressing these hurdles through collaborative research efforts and well-designed clinical trials holds the key to fully harnessing the clinical potential of BEVs. As this field advances, this review suggests that BEV-based nanomedicine has the potential to revolutionize disease management, paving the way for innovative diagnosis, therapeutics, and personalized medicine approaches. STATEMENT OF SIGNIFICANCE: Extracellular vesicles (EVs) from both host cells and bacteria serve as multifunctional biomaterials and are emerging in the fields of biomedicine, bioengineering, and biomaterials. However, most of the current studies focus on host-derived EVs, leaving a gap in comprehensive research on bacteria-derived EVs (BEVs). Although BEVs offer an attractive option as nanomaterials for drug delivery systems, their unique nanostructure and easy-to-modify functions make them a potential method for disease diagnosis and treatment as well as vaccine development. Our work among the pioneering studies investigating the potential of BEVs as natural nanobiomaterials, plays a crucial role in both understanding the development of diseases and therapeutic interventions.
    Keywords:  bacterial extracellular vesicles; biomarker; clinical applications; diagnosis and treatment; nanobiomaterials; vaccine development
    DOI:  https://doi.org/10.1016/j.actbio.2024.04.022
  43. Nat Aging. 2024 Apr 16.
    Small extracellular vesicles from young plasma reverse age-related functional declines by improving mitochondrial energy metabolism.
    Xiaorui Chen, Yang Luo, Qing Zhu, Jingzi Zhang, Huan Huang, Yansheng Kan, Dian Li, Ming Xu, Shuohan Liu, Jianxiao Li, Jinmeng Pan, Li Zhang, Yan Guo, Binghao Wang, Guantong Qi, Zhen Zhou, Chen-Yu Zhang, Lei Fang, Yanbo Wang, Xi Chen.
      Recent investigations into heterochronic parabiosis have unveiled robust rejuvenating effects of young blood on aged tissues. However, the specific rejuvenating mechanisms remain incompletely elucidated. Here we demonstrate that small extracellular vesicles (sEVs) from the plasma of young mice counteract pre-existing aging at molecular, mitochondrial, cellular and physiological levels. Intravenous injection of young sEVs into aged mice extends their lifespan, mitigates senescent phenotypes and ameliorates age-associated functional declines in multiple tissues. Quantitative proteomic analyses identified substantial alterations in the proteomes of aged tissues after young sEV treatment, and these changes are closely associated with metabolic processes. Mechanistic investigations reveal that young sEVs stimulate PGC-1α expression in vitro and in vivo through their miRNA cargoes, thereby improving mitochondrial functions and mitigating mitochondrial deficits in aged tissues. Overall, this study demonstrates that young sEVs reverse degenerative changes and age-related dysfunction, at least in part, by stimulating PGC-1α expression and enhancing mitochondrial energy metabolism.
    DOI:  https://doi.org/10.1038/s43587-024-00612-4
  44. Sci Rep. 2024 04 13. 14(1): 8581
    Plasminogen degrades α-synuclein, Tau and TDP-43 and decreases dopaminergic neurodegeneration in mouse models of Parkinson's disease.
    Chunying Guo, Ting Wang, Haiyan Huang, Xiaolu Wang, Yugui Jiang, Jinan Li.
      Parkinson's disease (PD) is the second most frequently diagnosed neurodegenerative disease, and it is characterized by the intracellular and extracellular accumulation of α-synuclein (α-syn) and Tau, which are major components of cytosolic protein inclusions called Lewy bodies, in the brain. Currently, there is a lack of effective methods that preventing PD progression. It has been suggested that the plasminogen activation system, which is a major extracellular proteolysis system, is involved in PD pathogenesis. We investigated the functional roles of plasminogen in vitro in an okadaic acid-induced Tau hyperphosphorylation NSC34 cell model, ex vivo using brains from normal controls and methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, and in vivo in a widely used MPTP-induced PD mouse model and an α-syn overexpression mouse model. The in vitro, ex vivo and in vivo results showed that the administered plasminogen crossed the blood‒brain barrier (BBB), entered cells, and migrated to the nucleus, increased plasmin activity intracellularly, bound to α-syn through lysine binding sites, significantly promoted α-syn, Tau and TDP-43 clearance intracellularly and even intranuclearly in the brain, decreased dopaminergic neurodegeneration and increased the tyrosine hydroxylase levels in the substantia nigra and striatum, and improved motor function in PD mouse models. These findings indicate that plasminogen plays a wide range of pivotal protective roles in PD and therefore may be a promising drug candidate for PD treatment.
    Keywords:  Dopaminergic neuron; PD; Plasminogen; TDP-43; Tau; α-syn
    DOI:  https://doi.org/10.1038/s41598-024-59090-8
  45. Methods Mol Biol. 2024 ;2794 177-186
    Immunocytochemistry of Primary Cultured Cerebral Cortical Neurons.
    Shiho Ito, Takeshi Kawauchi.
      Immunocytochemistry combined with confocal or superresolution microscopy allows us to observe molecular localization and intracellular structures. However, it is challenging to analyze individual neurons in brain tissue, where neurons are densely packed. In contrast, we can easily observe structures such as the axonal growth cone and dendritic spines in dissociated individual neurons. Thus, the immunocytochemistry of primary cultured neurons is often used because it reflects the in vivo condition at least in part. Here, we describe a method for indirect fluorescence immunocytochemistry of primary cultured neurons from the embryonic cerebral cortex. This involves multiple steps including fixation, permeabilization, and antibody reaction, and in particular, we introduce an optimized protocol for permeabilization to enable the precise localization of target molecules.
    Keywords:  Brain; Cerebral cortex; Cytosolic soluble protein; Digitonin; Immunocytochemistry; Membrane-associated protein; Neuron; Primary culture; Triton X-100
    DOI:  https://doi.org/10.1007/978-1-0716-3810-1_15
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