bims-axbals 2024-11-10 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2024–11–10
25 papers selected by
TJ Krzystek, ALS Therapy Development Institute

Human iPSC-derived MSCs induce neurotrophic effects and improve metabolic activity in acute neuronal injury models.
Possible changes in motor neuron discharge characteristics in presymptomatic amyotrophic lateral sclerosis.
Inhibition of RNA splicing triggers CHMP7 nuclear entry, impacting TDP-43 function and leading to the onset of ALS cellular phenotypes.
The Basis of Cognitive and Behavioral Dysfunction in Amyotrophic Lateral Sclerosis.
The R1441C-Lrrk2 mutation induces myeloid immune cell exhaustion in an age- and sex-dependent manner in mice.
Unraveling the multifaceted insights into amyotrophic lateral sclerosis: Genetic underpinnings, pathogenesis, and therapeutic horizons.
ALS-associated FUS mutation reshapes the RNA and protein composition of stress granules.
Disruption of neuronal actin barrier promotes the entry of disease-implicated proteins to exacerbate amyotrophic lateral sclerosis pathology.
Dilated cardiomyopathy-associated skeletal muscle actin (ACTA1) mutation R256H disrupts actin structure and function and causes cardiomyocyte hypocontractility.
Axon demyelination and degeneration in a zebrafish spastizin model of hereditary spastic paraplegia.
Unveiling the cell biology of hippocampal neurons with dendritic axon origin.
Hyaluronic Acid-Based 3D Bioprinted Hydrogel Structure for Directed Axonal Guidance and Modeling Innervation In Vitro.
Evaluation of LC3-II Release via Extracellular Vesicles in Relation to the Accumulation of Intracellular LC3-positive Vesicles.
Inducible Pluripotent Stem Cell Models to Study Bone Marrow Failure and MDS Predisposition Syndromes.
A diarylheptanoid derivative mediates glycogen synthase kinase 3β to promote the porcine muscle satellite cell proliferation: Implications for cultured meat production.
Network medicine informed multiomics integration identifies drug targets and repurposable medicines for Amyotrophic Lateral Sclerosis.
A novel multiplexed immunoassay for surface-exposed proteins in plasma extracellular vesicles.
Impairment of the trans-Golgi-Lysosomal Pathway Accelerates Dopaminergic Neuronal Senescence in LRRK2R1627P Rats.
Analysis of mitochondrial protein aggregation and disaggregation.
USP14 inhibition enhances Parkin-independent mitophagy in iNeurons.
Isolation of Small Extracellular Vesicles (sEVs) from the Apoplastic Wash Fluid of Nicotiana benthamiana Leaves.
Mitochondrial dysfunction and inflammasome activation in neurodegenerative diseases: Mechanisms and therapeutic implications.
Organoids and organoid extracellular vesicles-based disease treatment strategies.
Shikonin Induces Autophagy and Apoptosis in Esophageal Cancer EC9706 Cells by Regulating the AMPK/mTOR/ULK Axis.
A switch from lysosomal degradation to secretory autophagy initiates osteogenic bone metastasis in prostate cancer.

J Neurosci. 2024 Nov 04. pii: e0606242024. [Epub ahead of print]

Human iPSC-derived MSCs induce neurotrophic effects and improve metabolic activity in acute neuronal injury models.

Keiji Kawatani, Genesis Omana Suarez, Ralph B Perkerson, Ephraim E Parent, Toshihiko Nambara, Joshua A Knight, Tammee M Parsons, Kshama Gupta, Francis Shue, Alla Alnobani, Prasanna Vibhute, Hancheng Cai, Hugo Guerrero-Cázares, John A Copland, Alfredo Quiñones-Hinojosa, Takahisa Kanekiyo.

Mesenchymal stromal cell (MSC) therapy has regenerative potentials to treat various pathological conditions including neurological diseases. MSCs isolated from various organs can differentiate into specific cell types to repair organ damages. However, their paracrine mechanisms are predicted to predominantly mediate their immunomodulatory, pro-angiogenic, and regenerative properties. While preclinical studies highlight the significant potential of MSC therapy in mitigating neurological damage from stroke and traumatic brain injury, the variability in clinical trial outcomes may stem from the inherent heterogeneity of somatic MSCs. Accumulating evidence has demonstrated that induced pluripotent stem cells (iPSCs) are an ideal alternative resource for the unlimited expansion and biomanufacturing of MSCs. Thus, we investigated how iPSC-derived MSCs (iMSCs) influence properties of iPSC-derived neurons. Our findings demonstrate that the secretome from iMSCs possesses neurotrophic effects, improving neuronal survival and promoting neuronal outgrowth and synaptic activity in vitro Additionally, the iMSCs enhance metabolic activity via mitochondrial respiration in neurons, both in vitro and in mouse models. Glycolytic pathways also increased following the administration of iMSC secretome to iPSC-derived neurons. Consistently, in vivo experiments showed that intravenous administration of iMSCs compensated for the elevated energetic demand in male mice with irradiation-induced brain injury by restoring synaptic metabolic activity during acute brain damage. 18F-FDG PET imaging also detected an increase in brain glucose uptake following iMSC administration. Together, our results highlight the potential of iMSC-based therapy in treating neuronal damage in various neurological disorders, while paving the way for future research and potential clinical applications of iMSCs in regenerative medicine.Significance Statement Regenerative biotherapeutics using MSCs have emerged as a promising intervention for treating various neurological diseases. Our study explored the potential beneficial effects of human iPSC-derived MSCs (iMSCs) on neurons. We demonstrated that molecules secreted into the culture medium by iMSCs enhance regenerative capabilities by improving neuronal survival, growth, and metabolic activity, as well as synaptic functions, in human iPSC-derived neurons. Mouse experiments also suggested the potential of iMSC therapy to mitigate synaptic mitochondrial dysfunction and enhance brain glucose uptake during acute radiation-induced brain injury, steps that contribute to restoring normal neuronal function. Our results highlight that iMSCs may be a promising alternative cell product for treating neuronal damage, overcoming the inconsistent efficacy of somatic MSCs due to cell variability.

DOI: https://doi.org/10.1523/JNEUROSCI.0606-24.2024
J Physiol. 2024 Nov 05.

Possible changes in motor neuron discharge characteristics in presymptomatic amyotrophic lateral sclerosis.

Maria Piotrkiewicz.



Keywords:  afterhyperpolarization duration; amyotrophic lateral sclerosis; biomarkers; early diagnosis; hypothesis; prodromic period; spinal motoneurones

DOI:  https://doi.org/10.1113/JP287788
Neuron. 2024 Oct 31. pii: S0896-6273(24)00728-1. [Epub ahead of print]

Inhibition of RNA splicing triggers CHMP7 nuclear entry, impacting TDP-43 function and leading to the onset of ALS cellular phenotypes.

Norah Al-Azzam, Jenny H To, Vaishali Gautam, Lena A Street, Chloe B Nguyen, Jack T Naritomi, Dylan C Lam, Assael A Madrigal, Benjamin Lee, Wenhao Jin, Anthony Avina, Orel Mizrahi, Jasmine R Mueller, Willard Ford, Cara R Schiavon, Elena Rebollo, Anthony Q Vu, Steven M Blue, Yashwin L Madakamutil, Uri Manor, Jeffrey D Rothstein, Alyssa N Coyne, Marko Jovanovic, Gene W Yeo.

  Amyotrophic lateral sclerosis (ALS) is linked to the reduction of certain nucleoporins in neurons. Increased nuclear localization of charged multivesicular body protein 7 (CHMP7), a protein involved in nuclear pore surveillance, has been identified as a key factor damaging nuclear pores and disrupting transport. Using CRISPR-based microRaft, followed by gRNA identification (CRaft-ID), we discovered 55 RNA-binding proteins (RBPs) that influence CHMP7 localization, including SmD1, a survival of motor neuron (SMN) complex component. Immunoprecipitation-mass spectrometry (IP-MS) and enhanced crosslinking and immunoprecipitation (CLIP) analyses revealed CHMP7's interactions with SmD1, small nuclear RNAs, and splicing factor mRNAs in motor neurons (MNs). ALS induced pluripotent stem cell (iPSC)-MNs show reduced SmD1 expression, and inhibiting SmD1/SMN complex increased CHMP7 nuclear localization. Crucially, overexpressing SmD1 in ALS iPSC-MNs restored CHMP7's cytoplasmic localization and corrected STMN2 splicing. Our findings suggest that early ALS pathogenesis is driven by SMN complex dysregulation.

Keywords:  ALS; CHMP7; CRISPR screen; RNA splicing; SMN complex; SmD1; TDP-43; amyotrophic lateral sclerosis; neurodegeneration

DOI:  https://doi.org/10.1016/j.neuron.2024.10.007
Brain Behav. 2024 Nov;14(11): e70115

The Basis of Cognitive and Behavioral Dysfunction in Amyotrophic Lateral Sclerosis.

Alexander Bampton, Caroline McHutchison, Kevin Talbot, Michael Benatar, Alexander G Thompson, Martin R Turner.

   OBJECTIVE: To summarize and evaluate evidence pertaining to the clinical, genetic, histopathological, and neuroimaging correlates of cognitive and behavioral dysfunction in amyotrophic lateral sclerosis (ALS).
METHODOLOGY: We comprehensively reviewed the literature on cognitive and behavioral manifestations of ALS, narrating findings from both cross-sectional and longitudinal studies. We discussed knowledge gaps in the evidence base and key limitations affecting studies to date, before formulating a framework for future research paradigms aimed at investigating clinicopathological correlates of neuropsychological dysfunction in ALS.
RESULTS: Studies have demonstrated clinical associations with cognitive dysfunction in ALS e.g., bulbar-onset of symptoms, pathological associations (extramotor TDP-43 deposition), and imaging associations (frontotemporal involvement). The most common behavioral deficit, apathy, is highly associated with verbal fluency, but longitudinal studies assessing behavioral dysfunction in ALS are comparatively lacking.
CONCLUSION: Longitudinal studies have been helpful in identifying several potential correlates of cognitive and behavioral dysfunction but have frequently been confounded by selection bias and inappropriate testing platforms. This review provides a framework for more robust assessment of clinicopathological associations of neuropsychological abnormalities in ALS in the future, advocating for greater utilization of pre-symptomatic C9orf72 repeat expansion-carrying cohorts.

Keywords:  C9orf72; TDP‐43; amyotrophic lateral sclerosis; behavioral; cognitive; frontotemporal dementia; motor neurone disease

DOI:  https://doi.org/10.1002/brb3.70115
Sci Transl Med. 2024 Nov 06. 16(772): eadl1535

The R1441C-Lrrk2 mutation induces myeloid immune cell exhaustion in an age- and sex-dependent manner in mice.

Rebecca L Wallings, Karen McFarland, Hannah A Staley, Noelle Neighbarger, Susen Schaake, Norbert Brüggemann, Simone Zittel, Tatiana Usnich, Christine Klein, Esther M Sammler, Malú Gámez Tansey.

Age is the greatest risk factor for many neurodegenerative diseases, yet immune system aging, a contributor to neurodegeneration, is understudied. Genetic variation in the LRRK2 gene affects risk for both familial and sporadic Parkinson's disease (PD). The leucine-rich repeat kinase 2 (LRRK2) protein is implicated in peripheral immune cell signaling, but the effects of an aging immune system on LRRK2 function remain unclear. We analyzed peritoneal macrophages from R1441C-Lrrk2 knock-in mice and observed a biphasic, age-dependent effect of the R1441C-Lrrk2 mutation on peritoneal macrophage function. We report increases in antigen presentation, anti-inflammatory cytokine production, lysosomal activity, and pathogen uptake in peritoneal macrophages from young (2- to 3-month-old) female R1441C-Lrrk2 mice. Conversely, macrophages from aged (18- to 21-month-old) female R1441C-Lrrk2 mice exhibited decreased antigen presentation after inflammatory insult, decreased lysosomal function, and pathogen uptake, with a concomitant increase in DNA fragmentation in the presence of pathogens. This immune cell exhaustion phenotype was not observed in male R1441C-Lrrk2 mice and was driven by increased LRRK2 protein kinase activity. This phenotype was also observed in human peripheral myeloid cells, with monocyte-derived macrophages from patients with PD who had either the R1441C- or Y1699C-LRRK2 mutation exhibiting decreased pathogen uptake and increased PDL1 expression, consistent with immune cell exhaustion. Our findings that LRRK2 mutations conferred an immunological advantage at a young age but could predispose the carrier to age-acquired immune cell exhaustion have implications for the therapeutic development of LRRK2 inhibitors.

DOI: https://doi.org/10.1126/scitranslmed.adl1535
Mutat Res Rev Mutat Res. 2024 Nov 02. pii: S1383-5742(24)00031-0. [Epub ahead of print]794 108518

Unraveling the multifaceted insights into amyotrophic lateral sclerosis: Genetic underpinnings, pathogenesis, and therapeutic horizons.

Ramaish Sharma, Zuber Khan, Sidharth Mehan, Ghanshyam Das Gupta, Acharan S Narula.

  Amyotrophic Lateral Sclerosis (ALS), a progressive neurodegenerative disease, primarily impairs upper and lower motor neurons, leading to debilitating motor dysfunction and eventually respiratory failure, widely known as Lou Gehrig's disease. ALS presents with diverse symptomatology, including dysarthria, dysphagia, muscle atrophy, and hyperreflexia. The prevalence of ALS varies globally, with incidence rates ranging from 1.5 to 3.8 per 100,000 individuals, significantly affecting populations aged 45-80. A complex interplay of genetic and environmental factors underpins ALS pathogenesis. Key genetic contributors include mutations in chromosome 9 open reading frame 72 (C9ORF72), superoxide dismutase type 1 (SOD1), Fusedin sarcoma (FUS), and TAR DNA-binding protein (TARDBP) genes, accounting for a considerable fraction of both familial (fALS) and sporadic (sALS) cases. The disease mechanism encompasses aberrant protein folding, mitochondrial dysfunction, oxidative stress, excitotoxicity, and neuroinflammation, contributing to neuronal death. This review consolidates current insights into ALS's multifaceted etiology, highlighting the roles of environmental exposures (e.g., toxins, heavy metals) and their interaction with genetic predispositions. We emphasize the polygenic nature of ALS, where multiple genetic variations cumulatively influence disease susceptibility and progression. This aspect underscores the challenges in ALS diagnosis, which currently lacks specific biomarkers and relies on symptomatology and familial history. Therapeutic strategies for ALS, still in nascent stages, involve symptomatic management and experimental approaches targeting molecular pathways implicated in ALS pathology. Gene therapy, focusing on specific ALS mutations, and stem cell therapy emerge as promising avenues. However, effective treatments remain elusive, necessitating a deeper understanding of ALS's genetic architecture and the development of targeted therapies based on personalized medicine principles. This review aims to provide a comprehensive understanding of ALS, encouraging further research into its complex genetic underpinnings and the development of innovative, effective treatment modalities.

Keywords:  Amyotrophic lateral sclerosis; Gene therapy; Genetic mutations (C9ORF72, SOD1, FUS, TDP-43); Motor neuron degeneration; Neuroinflammation; Polygenic etiology

DOI:  https://doi.org/10.1016/j.mrrev.2024.108518
Nucleic Acids Res. 2024 Nov 04. pii: gkae942. [Epub ahead of print]

ALS-associated FUS mutation reshapes the RNA and protein composition of stress granules.

Davide Mariani, Adriano Setti, Francesco Castagnetti, Erika Vitiello, Lorenzo Stufera Mecarelli, Gaia Di Timoteo, Andrea Giuliani, Angelo D'Angelo, Tiziana Santini, Eleonora Perego, Sabrina Zappone, Nara Liessi, Andrea Armirotti, Giuseppe Vicidomini, Irene Bozzoni.

Stress granules (SG) are part of a cellular protection mechanism where untranslated messenger RNAs and RNA-binding proteins are stored upon conditions of cellular stress. Compositional variations due to qualitative or quantitative protein changes can disrupt their functionality and alter their structure. This is the case of different forms of amyotrophic lateral sclerosis (ALS) where a causative link has been proposed between the cytoplasmic de-localization of mutant proteins, such as FUS (Fused in Sarcoma), and the formation of cytotoxic inclusions. Here, we describe the SG transcriptome in neuroblastoma cells and define several features for RNA recruitment in these condensates. We demonstrate that SG dynamics and RNA content are strongly modified by the incorporation of mutant FUS, switching to a more unstructured, AU-rich SG transcriptome. Moreover, we show that mutant FUS, together with its protein interactors and their target RNAs, are responsible for the reshaping of the mutant SG transcriptome with alterations that can be linked to neurodegeneration. Our data describe the molecular differences between physiological and pathological SG in ALS-FUS conditions, showing how FUS mutations impact the RNA and protein composition of these condensates.

DOI: https://doi.org/10.1093/nar/gkae942
Neural Regen Res. 2025 Sep 01. 20(9): 2589-2590

Disruption of neuronal actin barrier promotes the entry of disease-implicated proteins to exacerbate amyotrophic lateral sclerosis pathology.

Mikito Shimizu, Tatsusada Okuno.

DOI: https://doi.org/10.4103/NRR.NRR-D-24-00661
Proc Natl Acad Sci U S A. 2024 Nov 12. 121(46): e2405020121

Dilated cardiomyopathy-associated skeletal muscle actin (ACTA1) mutation R256H disrupts actin structure and function and causes cardiomyocyte hypocontractility.

Ankit Garg, Silvia Jansen, Lina Greenberg, Rui Zhang, Kory J Lavine, Michael J Greenberg.

  Skeletal muscle actin (ACTA1) mutations are a prevalent cause of skeletal myopathies consistent with ACTA1's high expression in skeletal muscle. Rare de novo mutations in ACTA1 associated with combined cardiac and skeletal myopathies have been reported, but ACTA1 represents only ~20% of the total actin pool in cardiomyocytes, making its role in cardiomyopathy controversial. Here we demonstrate how a mutation in an actin isoform expressed at low levels in cardiomyocytes can cause cardiomyopathy by focusing on a unique ACTA1 variant, R256H. We previously identified this variant in a family with dilated cardiomyopathy, who had reduced systolic function without clinical skeletal myopathy. Using a battery of multiscale biophysical tools, we show that R256H has potent effects on ACTA1 function at the molecular scale and in human cardiomyocytes. Importantly, we demonstrate that R256H acts in a dominant manner, where the incorporation of small amounts of mutant protein into thin filaments is sufficient to disrupt molecular contractility, and that this effect is dependent on the presence of troponin and tropomyosin. To understand the structural basis of this change in regulation, we resolved a structure of R256H filaments using cryoelectron microscopy, and we see alterations in actin's structure that have the potential to disrupt interactions with tropomyosin. Finally, we show that ACTA1R256H/+ human-induced pluripotent stem cell cardiomyocytes demonstrate reduced contractility and sarcomeric organization. Taken together, we demonstrate that R256H has multiple effects on ACTA1 function that are sufficient to cause reduced contractility and establish a likely causative relationship between ACTA1 R256H and clinical cardiomyopathy.

Keywords:  actin; cardiomyopathy; contractility; muscle

DOI:  https://doi.org/10.1073/pnas.2405020121
Open Biol. 2024 Nov;14(11): 240100

Axon demyelination and degeneration in a zebrafish spastizin model of hereditary spastic paraplegia.

Vranda Garg, Selina André, Luisa Heyer, Gudrun Kracht, Torben Ruhwedel, Patricia Scholz, Till Ischebeck, Hauke B Werner, Christian Dullin, Jacob Engelmann, Wiebke Möbius, Martin C Göpfert, Roland Dosch, Bart R H Geurten.

  Hereditary spastic paraplegias (HSPs) are a diverse set of neurological disorders characterized by progressive spasticity and weakness in the lower limbs caused by damage to the axons of the corticospinal tract. More than 88 genetic mutations have been associated with HSP, yet the mechanisms underlying these disorders are not well understood. We replicated the pathophysiology of one form of HSP known as spastic paraplegia 15 (SPG15) in zebrafish. This disorder is caused in humans by mutations in the ZFYVE26 gene, which codes for a protein called SPASTIZIN. We show that, in zebrafish, the significant reduction of Spastizin caused degeneration of large motor neurons. Motor neuron degeneration is associated with axon demyelination in the spinal cord and impaired locomotion in the spastizin mutants. Our findings reveal that the reduction in Spastizin compromises axonal integrity and affects the myelin sheath, ultimately recapitulating the pathophysiology of HSPs.

Keywords:  C-start escape response; Hereditary spastic paraplegia (HSP); lower limb spasticity; mauthner neurons; spastizin; zebrafish

DOI:  https://doi.org/10.1098/rsob.240100
J Cell Biol. 2025 Jan 06. pii: e202403141. [Epub ahead of print]224(1):

Unveiling the cell biology of hippocampal neurons with dendritic axon origin.

Yuhao Han, Daniela Hacker, Bronte Catharina Donders, Christopher Parperis, Roland Thuenauer, Christophe Leterrier, Kay Grünewald, Marina Mikhaylova.

In mammalian axon-carrying-dendrite (AcD) neurons, the axon emanates from a basal dendrite, instead of the soma, to create a privileged route for action potential generation at the axon initial segment (AIS). However, it is unclear how such unusual morphology is established and whether the structure and function of the AIS in AcD neurons are preserved. By using dissociated hippocampal cultures as a model, we show that the development of AcD morphology can occur prior to synaptogenesis and independently of the in vivo environment. A single precursor neurite first gives rise to the axon and then to the AcD. The AIS possesses a similar cytoskeletal architecture as the soma-derived AIS and similarly functions as a trafficking barrier to retain axon-specific molecular composition. However, it does not undergo homeostatic plasticity, contains lesser cisternal organelles, and receives fewer inhibitory inputs. Our findings reveal insights into AcD neuron biology and underscore AIS structural differences based on axon onset.

DOI: https://doi.org/10.1083/jcb.202403141
Adv Healthc Mater. 2024 Nov 06. e2402504

Hyaluronic Acid-Based 3D Bioprinted Hydrogel Structure for Directed Axonal Guidance and Modeling Innervation In Vitro.

Laura Honkamäki, Oskari Kulta, Paula Puistola, Karoliina Hopia, Promise Emeh, Lotta Isosaari, Anni Mörö, Susanna Narkilahti.

  Neurons form predefined connections and innervate target tissues through elongating axons, which are crucial for the development, maturation, and function of these tissues. However, innervation is often overlooked in tissue engineering (TE) applications. Here, multimaterial 3D bioprinting is used to develop a novel 3D axonal guidance structure in vitro. The approach uses the stiffness difference of acellular hyaluronic acid-based bioink printed as two alternating, parallel-aligned filaments. The structure has soft passages incorporated with guidance cues for axonal elongation while the stiff bioink acts as a structural support and contact guidance. The mechanical properties and viscosity differences of the bioinks are confirmed. Additionally, human pluripotent stem cell (hPSC) -derived neurons form a 3D neuronal network in the softer bioink supplemented with guidance cues whereas the stiffer restricts the network formation. Successful 3D multimaterial bioprinting of the axonal structure enables complete innervation by peripheral neurons via soft passages within 14 days of culture. This model provides a novel, stable, and long-term platform for studies of 3D innervation and axonal dynamics in health and disease.

Keywords:  axon orientation; human pluripotent stem cell‐derived neurons; hyaluronic acid; multi‐material 3D bioprinting

DOI:  https://doi.org/10.1002/adhm.202402504
J Vis Exp. 2024 Oct 18.

Evaluation of LC3-II Release via Extracellular Vesicles in Relation to the Accumulation of Intracellular LC3-positive Vesicles.

Shun-Ichi Ito, Yoshinori Tanaka.

(Macro)autophagy represents a fundamental cellular degradation pathway. In this process, double-membraned vesicles known as autophagosomes engulf cytoplasmic contents, subsequently fusing with lysosomes for degradation. Beyond the canonical role, autophagy-related genes also modulate a secretory pathway involving the release of inflammatory molecules, tissue repair factors, and extracellular vesicles (EVs). Notably, the process of disseminating pathological proteins between cells, particularly in neurodegenerative diseases affecting the brain and spinal cord, underscores the significance of understanding this phenomenon. Recent research suggests that the transactive response DNA-binding protein 43 kDa (TDP-43), a key player in amyotrophic lateral sclerosis and frontotemporal lobar degeneration, is released in an autophagy-dependent manner via EVs enriched with the autophagosome marker microtubule-associated proteins 1A/1B light chain 3B-II (LC3-II), especially when autophagosome-lysosome fusion is inhibited. To elucidate the mechanism underlying the formation and release of LC3-II-positive EVs, it is imperative to establish an accessible and reproducible method for evaluating both intracellular and extracellular LC3-II-positive vesicles. This study presents a detailed protocol for assessing LC3-II levels via immunoblotting in cellular and EV fractions obtained through differential centrifugation. Bafilomycin A1 (Baf), an inhibitor of autophagosome-lysosome fusion, serves as a positive control to enhance the levels of intracellular and extracellular LC3-II-positive vesicles. Tumor susceptibility gene 101 (TSG101) is used as a marker for multivesicular bodies. Applying this protocol, it is demonstrated that siRNA-mediated knockdown of syntaxin-6 (STX6), a genetic risk factor for sporadic Creutzfeldt-Jakob disease, augments LC3-II levels in the EV fraction of cells treated with Baf while showing no significant effect on TSG101 levels. These findings suggest that STX6 may negatively regulate the extracellular release of LC3-II via EVs, particularly under conditions where autophagosome-lysosome fusion is impaired. Combined with established methods for evaluating autophagy, this protocol provides valuable insights into the role of specific molecules in the formation and release of LC3-II-positive EVs.

DOI: https://doi.org/10.3791/67385
Exp Hematol. 2024 Nov 02. pii: S0301-472X(24)00534-4. [Epub ahead of print] 104669

Inducible Pluripotent Stem Cell Models to Study Bone Marrow Failure and MDS Predisposition Syndromes.

Sushree S Sahoo, Majd Khiami, Marcin W Wlodarski.

Induced pluripotent stem cells (iPSCs) have emerged as powerful tools for in vitro modeling of bone marrow failure (BMF) syndromes and hereditary conditions predisposing to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). This review synthesizes recent advances in iPSC-based disease modeling for various inherited BMF/MDS disorders, including Fanconi anemia, dyskeratosis congenita, Diamond Blackfan anemia syndrome, Shwachman-Diamond syndrome, severe congenital neutropenia, and GATA2, RUNX1, ETV6, ANKRD26, SAMD9, SAMD9L and ADH5/ALDH2 syndromes. While the majority of these iPSC lines are derived from patient cells, some are generated by introducing patient-specific mutations into healthy iPSC backgrounds, offering complementary approaches to disease modeling. The review highlights the ability of iPSCs to recapitulate key disease phenotypes, such as impaired hematopoietic differentiation, telomere dysfunction, and defects in DNA repair or ribosome biogenesis. We discuss how these models have enhanced our understanding of disease pathomechanisms, hematopoietic defects, and potential therapeutic approaches. Challenges in generating and maintaining disease-specific iPSCs are examined, particularly for disorders involving DNA repair. We emphasize the necessity of creating isogenic controls to elucidate genotype-phenotype relationships. Furthermore, we address limitations of current iPSC models, including genetic variability among iPSC clones derived from the same patient, and difficulties in achieving robust engraftment of iPSC-derived hematopoietic progenitor cells in mouse transplantation models. The review also explores future directions, including the potential of iPSC models for drug discovery and personalized medicine approaches. This review underscores the significance of iPSC technology in advancing our understanding of inherited hematopoietic disorders and its potential to inform novel therapeutic strategies.

DOI: https://doi.org/10.1016/j.exphem.2024.104669
Biochem Biophys Res Commun. 2024 Dec 03. pii: S0006-291X(24)01386-X. [Epub ahead of print]736 150850

A diarylheptanoid derivative mediates glycogen synthase kinase 3β to promote the porcine muscle satellite cell proliferation: Implications for cultured meat production.

Utid Suriya, Ratchakrit Srikuea, Tanida Chokpanuwat, Kanoknetr Suksen, Wasina Watcharanapapan, Palida Saleepimol, Dusit Laohasinnarong, Apichart Suksamrarn, Kyaw Zwar Myint, Tavan Janvilisri, Arthit Chairoungdua, Kanit Bhukhai.

  Skeletal muscle stem cells, or satellite cells, are vital for cultured meat production, driving proliferation and differentiation to form muscle fibers in vitro. However, these abilities are often compromised after long-term in vitro culturing due to a loss of their stemness characteristics. Therefore, effective pharmacological agents that enhance satellite cell proliferation and maintain stemness ability are needed for optimal cell growth for cultured meat production. In this study, the effects of the identified glycogen synthase kinase 3β (GSK3β) inhibitors, ASPP 049, a diarylheptanoid isolated from Curcuma comosa rhizomes, and CHIR 99021 on porcine muscle satellite cell (PMSC) proliferation and Wnt/β-catenin signaling pathway were investigated. We found that both compounds enhanced cell viability and proliferation while preserving the stemness marker, as evidenced by increased expression of the skeletal muscle stem cell marker, Pax7 protein. Molecular dynamics simulations showed that ASPP 049 and CHIR 99021 exhibited differing binding affinities, primarily through hydrophobic interactions, suggesting potential for the design of more potent inhibitors in the future. Despite its weaker binding, ASPP 049 still showed significant effects on the regulation of the Wnt/β-catenin signaling pathway via increased phosphorylation of GSK3β at Ser9 and decreased the phosphorylation of β-catenin at Ser33, Ser37, and Thr41, thereby subsequently activating Wnt transcriptional activity. This study highlights the potential of ASPP 049 and CHIR 99021 to enhance PMSC proliferation and maintain stemness ability, offering a promising avenue for improving cultured meat production.

Keywords:  Cell proliferation; Cultured meat; GSK3β inhibitor; Molecular docking; Pax7; Porcine satellite cells; Wnt/β-catenin signaling

DOI:  https://doi.org/10.1016/j.bbrc.2024.150850
NPJ Syst Biol Appl. 2024 Nov 05. 10(1): 128

Network medicine informed multiomics integration identifies drug targets and repurposable medicines for Amyotrophic Lateral Sclerosis.

Mucen Yu, Jielin Xu, Ranjan Dutta, Bruce Trapp, Andrew A Pieper, Feixiong Cheng.

Amyotrophic Lateral Sclerosis (ALS) is a devastating, immensely complex neurodegenerative disease by lack of effective treatments. We developed a network medicine methodology via integrating human brain multi-omics data to prioritize drug targets and repurposable treatments for ALS. We leveraged non-coding ALS loci effects from genome-wide associated studies (GWAS) on human brain expression quantitative trait loci (QTL) (eQTL), protein QTL (pQTL), splicing QTL (sQTL), methylation QTL (meQTL), and histone acetylation QTL (haQTL). Using a network-based deep learning framework, we identified 105 putative ALS-associated genes enriched in known ALS pathobiological pathways. Applying network proximity analysis of predicted ALS-associated genes and drug-target networks under the human protein-protein interactome (PPI) model, we identified potential repurposable drugs (i.e., Diazoxide and Gefitinib) for ALS. Subsequent validation established preclinical evidence for top-prioritized drugs. In summary, we presented a network-based multi-omics framework to identify drug targets and repurposable treatments for ALS and other neurodegenerative disease if broadly applied.

DOI: https://doi.org/10.1038/s41540-024-00449-y
J Extracell Vesicles. 2024 Nov;13(11): e70007

A novel multiplexed immunoassay for surface-exposed proteins in plasma extracellular vesicles.

Emma Tordoff, Jillian Allen, Katya Elgart, Ahmed Elsherbini, Vrinda Kalia, Haotian Wu, Erden Eren, Dimitrios Kapogiannis, Olesia Gololobova, Kenneth Witwer, Olga Volpert, Erez Eitan.

  Small membranous extracellular vesicles (EV) incorporate proteins and nucleic acids from the parent cell. Proteins exposed on EV surface are dictated by cellular origin and biogenesis pathway. To better understand the EV origin and function, it is important to develop methods that reveal surface protein composition of heterogeneous EV populations in culture supernatants and in biofluids. Tetraspanins CD9, CD63, and CD81 are common and abundant EV markers. However, their relative enrichment (profile) on EVs of specific cellular origins is not fully elucidated. We introduce LuminEV, a novel version of the Luminex assay for the multiplexed analysis of EV surface proteins. Optimized LuminEV reagents enable direct, specific, and sensitive measurements of EV markers in biofluids and in culture supernatants, bypassing EV isolation step. LuminEV assay for CD9, CD63, and CD81 was validated by comparing simplex and multiplex measurements, establishing linearity, spike-in recovery, inter- and intra-assay precision, and reproducibility between operators. LuminEV measurements of CD9, CD63, and CD81 in conditioned media from 15 cell lines revealed strong variations between cell types and showed high sensitivity, which enabled EV detection without prior concentration. Using tetraspanin levels as a readout, we noted suppression and induction of EV release from the cultured cells by GW6869 and monensin. Measurement of EV CD9, CD63, and CD81 in blood plasma from 70 disease-free donors showed respective abundance of 72, 16, and 12%. CD63 displayed weak, albeit significant, negative correlation with age and was slightly lower in female samples. The assay was then used to detect cell type-specific EV surface markers, including CD235a (erythrocytes), GAP43 (neurons), and CD68 (macrophages), and to detect differences in tetraspanin profiles between healthy and diseased donors. In summary, LuminEV offers robust and sensitive approach for multiplexed assessment of EV surface proteins, to facilitate the research into EV biology, biomarker, and therapeutic applications.

Keywords:  assay validation; extracellular vesicles; surface marker analysis; tetraspanin composition

DOI:  https://doi.org/10.1002/jev2.70007
Aging Dis. 2024 Nov 01.

Impairment of the trans-Golgi-Lysosomal Pathway Accelerates Dopaminergic Neuronal Senescence in LRRK2R1627P Rats.

Qiumei Yang, Shimin Pang, Chunsong Zhao, Yanyan Wang, Jing Lu, Zhenyu Yue, Piu Chan.

Leucine-rich repeat kinase 2 (LRRK2)-R1628P mutation has been shown to be one of the common risk factors for Parkinson's disease (PD) in Asian populations, but the mechanism by which R1628P mutations cause neuronal dysfunction remains unknown. We used LRRK2R1627P knock-in rats (human LRRK2-R1628P corresponds to rat LRRK2-R1627P) to investigate the R1627P mutation on function of dopaminergic neurons (DANs) and their susceptibility to the environmental toxin Lipopolysaccharide (LPS) during aging. LRRK2R1627P rats showed no significant loss of DANs, dopamine and its metabolites, or motor dysfunction; however, spontaneous exploration and olfactory discrimination reduced, and dendritic spines of DANs showed degeneration. We found decreased pThr73-Rab10 located on the trans-Golgi, disrupted Golgi structure and lipofuscin accumulation in aged LRRK2R1627P rat DANs, and the protein related to trans-Golgi complex and regulating lysosome function were significantly reduced. Although the neuroinflammation of brain was not obvious in the aging process, we confirmed a decrease in the ratio of CD4+/CD8+ and B cells, an increase in inflammatory factors (TLR4, NFKB, TNF-α) in the periphery. Furthermore, we demonstrated that the R1627P mutation caused the abnormal accumulation of α-Syn in the aged rat intestine. LPS exacerbated pathological α-Syn aggregation in the small intestine of LRRK2 transgenic rats and spread to the brain via the gut-brain axis. This led to microgliosis in the substantia nigra, creating a pro-inflammatory environment and inducing DANs degeneration. Gut-brain axis disruption may be a key determinant of progression to R1628P-PD in R1628P carriers. This insight has important clinical implications and highlights the importance of monitoring and addressing gut-brain axis integrity in individuals with LRRK2 mutations.

DOI: https://doi.org/10.14336/AD.2024.0883
Methods Enzymol. 2024 ;pii: S0076-6879(24)00388-4. [Epub ahead of print]707 475-498

Analysis of mitochondrial protein aggregation and disaggregation.

Wolfgang Voos, Anne Wilkening, Robin Ostermann, Michael Bruderek, Witold Jaworek, Laura Ruland.

  Deficits of mitochondrial functions have been identified in many human pathologies, in particular in age-related human neurodegenerative diseases. Hence, the molecular causes for mitochondrial dysfunction and potential protection mechanisms have become a major topic in modern cell biology. Apart from defects in their structural integrity, problems in mitochondrial protein biogenesis, including polypeptide transport, folding and assembly to active enzymes, all may result in some degree of functional defects of the organelle. An accumulation of misfolded polypeptides inside mitochondria, confounded by the dual source of mitochondrial polypeptides, will result in the formation of protein aggregates. Such aggregate accumulation bears a cell-toxic potential, resulting in mitochondrial and correlated cellular damages, summarized in the term "aggregate proteotoxicity". Here, we discuss methods to analyze protein aggregation in the mitochondrial matrix compartment. We also address techniques to characterize the biochemical mechanisms that reduce aggregate proteotoxicity, the disaggregation or resolubilization of aggregated polypeptides and the sequestration and neutralization of mitochondrial aggregates at specific sites inside a cell.

Keywords:  ATP-dependent proteases; Human cells; Mitochondria; Molecular chaperones; Protein aggregation; Proteotoxicity; Yeast

DOI:  https://doi.org/10.1016/bs.mie.2024.07.048
Pharmacol Res. 2024 Oct 30. pii: S1043-6618(24)00429-8. [Epub ahead of print] 107484

USP14 inhibition enhances Parkin-independent mitophagy in iNeurons.

Bernardo Greta, A Prado Miguel, Dashtmian Anna Roshani, Mariavittoria Favaro, Sofia Mauri, Alice Borsetto, Elena Marchesan, Joao A Paulo, Steve P Gygi, Daniel J Finley, Elena Ziviani.

  Loss of proteostasis is well documented during physiological aging and depends on the progressive decline in the activity of two major degradative mechanisms: the ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway. This decline in proteostasis is exacerbated in age-associated neurodegenerative diseases, such as Parkinson's Disease (PD). In PD, patients develop an accumulation of aggregated proteins and dysfunctional mitochondria, which leads to ROS production, neuroinflammation and neurodegeneration. We recently reported that inhibition of the deubiquitinating enzyme USP14, which is known to enhance both the UPS and autophagy, increases lifespan and rescues the pathological phenotype of two Drosophila models of PD. Studies on the effects of USP14 inhibition in mammalian neurons have not yet been conducted. To close this gap, we exploited iNeurons differentiated from human embryonic stem cells (hESCs), and investigated the effect of inhibiting USP14 in these cultured neurons. Quantitative global proteomics analysis performed following genetic ablation or pharmacological inhibition of USP14 demonstrated that USP14 loss of function specifically promotes mitochondrial autophagy in iNeurons. Biochemical and imaging data also showed that USP14 inhibition enhances mitophagy. The mitophagic effect of USP14 inhibition proved to be PINK1/Parkin- independent, instead relying on expression of the mitochondrial E3 Ubiquitin Ligase MITOL/MARCH5. Notably, USP14 inhibition normalized the mitochondrial defects of Parkin KO human neurons.

Keywords:  Autophagy; MARCH5/MITOL; Mitophagy; PINK1; Parkin; UPS; USP14

DOI:  https://doi.org/10.1016/j.phrs.2024.107484
Curr Protoc. 2024 Nov;4(11): e70026

Isolation of Small Extracellular Vesicles (sEVs) from the Apoplastic Wash Fluid of Nicotiana benthamiana Leaves.

Mahmoud K Eldahshoury, Konstantina Katsarou, Joshua T Farley, Kriton Kalantidis, Carine de Marcos Lousa.

  Extracellular vesicles (EVs) are small membranous vesicles secreted by cells into their surrounding extracellular environment. Similar to mammalian EVs, plant EVs have emerged as essential mediators of intercellular communication in plants that facilitate the transfer of biological material between cells. They also play essential roles in diverse physiological processes including stress responses, developmental regulation, and defense mechanisms against pathogens. In addition, plant EVs have demonstrated promising health benefits as well as potential therapeutic effects in mammalian health. Despite the plethora of potential applications using plant EVs, their isolation and characterization remains challenging. In contrast to mammalian EVs, which benefit from more standardized isolation protocols, methods for isolating plant EVs can vary depending on the starting material used, resulting in diverse levels of purity and composition. Additionally, the field suffers from the lack of plant EV markers. Nevertheless, three main EV subclasses have been described from leaf apoplasts: tetraspanin 8 positive (TET8), penetration-1-positive (PEN1), and EXPO vesicles derived from exocyst-positive organelles (EXPO). Here, we present an optimized protocol for the isolation and enrichment of small EVs (sEVs; <200 nm) from the apoplastic fluid from Nicotiana benthamiana leaves by ultracentrifugation. We analyze the preparation through transmitted electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. We believe this method will establish a basic protocol for the isolation of EVs from N. benthamiana leaves, and we discuss technical considerations to be evaluated by each researcher working towards improving their plant sEV preparations. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Isolation and enrichment of small extracellular vesicles (sEVs) from the apoplastic fluid of Nicotiana benthamiana leaves.

Keywords:  EV; Nicotiana benthamiana; PEN1; TET8; apoplastic fluid; plant; small extracellular vesicles (sEVs)

DOI:  https://doi.org/10.1002/cpz1.70026
Mitochondrion. 2023 Oct 28. pii: S1567-7249(23)00087-9. [Epub ahead of print]

Mitochondrial dysfunction and inflammasome activation in neurodegenerative diseases: Mechanisms and therapeutic implications.

Olia Hamzeh, Fatemeh Rabiei, Mahdi Shakeri, Hadi Parsian, Payam Saadat, Sahar Rostami-Mansoor.

  Impaired mitochondrial function is crucial to the pathogenesis of several neurodegenerative diseases. It causes the release of mitochondrial DNA (mtDNA), mitochondrial reactive oxygen species (mtROS), ATP, and cardiolipin, which activate the nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome. NLRP3 inflammasome is an important innate immune system element contributing to neuroinflammation and neurodegeneration. Therefore, targeting the NLRP3 inflammasome has become an interesting therapeutic approach for treating neurodegenerative diseases. This review describes the role of mitochondrial abnormalities and over-activated inflammasomes in the progression of neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), Multiple sclerosis (MS), Amyotrophic lateral sclerosis (ALS), and Friedrich ataxia (FRDA). We also discuss the therapeutic strategies focusing on signaling pathways associated with inflammasome activation, which potentially alleviate neurodegenerative symptoms and impede disease progression.

Keywords:  Inflammasome; Mitochondrial dysfunction; NLRP3; Neurodegenerative disease

DOI:  https://doi.org/10.1016/j.mito.2023.10.003
J Nanobiotechnology. 2024 Nov 06. 22(1): 679

Organoids and organoid extracellular vesicles-based disease treatment strategies.

Guangyin Zhou, Ruiyang Li, Shihao Sheng, Jingtao Huang, Fengjin Zhou, Yan Wei, Han Liu, Jiacan Su.

  Organoids are "mini-organs" that self-organize and differentiate from stem cells under in vitro 3D culture conditions, mimicking the spatial structure and function of tissues in vivo. Extracellular vesicles (EVs) are nanoscale phospholipid bilayer vesicles secreted by living cells, rich in bioactive molecules, with excellent biocompatibility and low immunogenicity. Compared to EVs, organoid-derived EVs (OEVs) exhibit higher yield and enhanced biological functions. Organoids possess stem cell characteristics, and OEVs are capable of delivering active substances, making both highly promising for medical applications. In this review, we provide an overview of the fundamental biological principles of organoids and OEVs, and discuss their current applications in disease treatment. We then focus on the differences between OEVs and traditional EVs. Subsequently, we present methods for the engineering modification of OEVs. Finally, we critically summarize the advantages and challenges of organoids and OEVs. In conclusion, we believe that a deeper understanding of organoids and OEVs will provide innovative solutions to complex diseases.

Keywords:  Disease treatment; Engineering modification; Extracellular vesicles; Organoids; Organoids extracellular vesicles

DOI:  https://doi.org/10.1186/s12951-024-02917-3
Anal Cell Pathol (Amst). 2024 ;2024 7752299

Shikonin Induces Autophagy and Apoptosis in Esophageal Cancer EC9706 Cells by Regulating the AMPK/mTOR/ULK Axis.

Junli Zhang, Jiayi Guo, Biao Gu, Fen Wang, Yi Li, Ling Shang, Wendi Jiang, Junrao Ma, Wenjuan Wu.

  Shikonin is a plant medicine extracted from Lithospermum, which dominate influential antioxidant and antitumor effect. Here, we report that shikonin was capable of inducing human esophageal cancer EC9706 cell apoptosis and autophagy, in a time- and dose-dependent manner. Shikonin exposure repressed cell viability and migration and invasion capabilities and caused EC9706 cell autophagy and apoptosis by activating the AMPK/mTOR/ULK axis. Autophagy inhibition secured EC9706 cells against shikonin-induced autophagy and apoptosis and reversed the upregulation of AMPK and ULK phosphorylation and downregulation of mTOR phosphorylation provoked by shikonin. In summary, shikonin instigates EC9706 cell apoptosis and autophagy using the target AMPK/mTOR/ULK signal pathway axis, which provides a potential new target to treat human esophageal cancer.

Keywords:  apoptosis; autophagy; esophageal cancer; shikonin; the AMPK/mTOR/ULK axis

DOI:  https://doi.org/10.1155/2024/7752299
J Extracell Vesicles. 2024 Nov;13(11): e70002

A switch from lysosomal degradation to secretory autophagy initiates osteogenic bone metastasis in prostate cancer.

Xiaoyu Wei, Mengmeng Liang, Min Deng, Ji Zheng, Fei Luo, Qinyu Ma.

  The identification of both autophagy-related material degradation and unconventional secretion has paved the way for significant breakthroughs linking autophagy to a plethora of physiological processes and disease conditions. However, the mechanisms that coordinate these two pathways remain elusive. Here, we demonstrate that a switch from the lysosomal degradation to a secretory autophagy pathway is governed by protein tyrosine phosphatase 1B (PTP1B, encoded by PTPN1). Dephosphorylation at two tyrosine residues of syntaxin17 (STX17) by PTP1B reduces autophagosome-lysosome fusion while switching the cells to a secretory autophagy pathway. Both PTP1B overexpression and tumour-derived extracellular vesicles (EVs) can activate the secretory autophagy pathway in osteoblasts. Moreover, we demonstrate that osteoblastic LC3+ EVs, generated via the secretory autophagy pathway, are the primary contributor to tumour-associated bone remodelling in prostate cancer. Depletion of tumour-derived EVs secretion or genetic ablation of osteoblastic PTP1B rescues aberrant bone remodelling and lesions, highlighting the relevance between LC3+ EVs and the formation of bone metastatic niche. Our results reveal the significance of tumour-regulated PTP1B in the fate decision of autophagosomes, and propose a role ofLC3+ EVs in shaping the bone metastatic niche.

Keywords:  autophagosome‐lysosome fusion; bone metastatic niche; extracellular vesicles; secretory autophagy; tumour‐associated bone remodelling

DOI:  https://doi.org/10.1002/jev2.70002