bims-axbals 2023-11-12 papers

bims-axbals

Biomed News

on Axonal Biology and ALS

Issue of 2023‒11‒12
27 papers selected by
TJ Krzystek, ALS Therapy Development Institute

Synaptic remodeling follows upper motor neuron hyperexcitability in a rodent model of TDP-43.
Efficacy and safety of a SOD1-targeting artificial miRNA delivered by AAV9 in mice are impacted by miRNA scaffold selection.
FUS gene mutation in amyotrophic lateral sclerosis: a new case report and systematic review.
Immunological drivers of amyotrophic lateral sclerosis.
Analysis of damage-associated molecular patterns in amyotrophic lateral sclerosis based on ScRNA-seq and bulk RNA-seq data.
Mitochondrial Inherited Disorders and their Correlation with Neurodegenerative Diseases.
Biomarkers in amyotrophic lateral sclerosis: current status and future prospects.
Mitochondrial regulation in human pluripotent stem cells during reprogramming and β cell differentiation.
Unveiling the SOD1-mediated ALS phenotype: insights from a comprehensive meta-analysis.
Artificial Intelligence Applications in the Diagnosis of Neuromuscular Diseases: A Narrative Review.
Limitations of the human iPSC-derived neuron model for early-onset Alzheimer's disease.
LRRK2 Attenuates Antioxidant Response in Familial Parkinson's Disease Derived Neural Stem Cells.
Long-termin vitroculture of 3D brain tissue model based on chitosan thermogel.
CLIP-Seq analysis enables the design of protective ribosomal RNA bait oligonucleotides against C9ORF72 ALS/FTD poly-GR pathophysiology.
Establishment of human induced pluripotent stem cell line (SDQLCHi060-A) from a patient with AUTS2 syndrome carrying a heterozygous mutation in AUTS2 gene.
Long-term live cell imaging during differentiation of human iPSC-derived neurons.
Novel Strategies for Angiogenesis in Tissue Injury: Therapeutic Effects of iPSCs-Derived Exosomes.
Novel single-nucleus transcriptomics unravels developmental and functionally controlled principles of mammalian neuromuscular junctions.
Induced pluripotent stem cells derived from patients carrying mitochondrial mutations exhibit altered bioenergetics and aberrant differentiation potential.
Regulation of axon pathfinding by astroglia across genetic model organisms.
Mechanism and therapeutic potential of targeting cGAS-STING signaling in neurological disorders.
Sphingolipids: From structural components to signaling hubs.
Differentiation of Neurons, Astrocytes, Oligodendrocytes and Microglia From Human Induced Pluripotent Stem Cells to Form Neural Tissue-On-Chip: A Neuroinflammation Model to Evaluate the Therapeutic Potential of Extracellular Vesicles Derived from Mesenchymal Stem Cells.
A conserved requirement for RME-8/DNAJC13 in neuronal autophagic lysosome reformation.
Mitochondrial phosphagen kinases support the volatile power demands of motor nerve terminals.
Combined 3D bioprinting and tissue-specific ECM system reveals the influence of brain matrix on stem cell differentiation.
CBAP regulates the function of Akt-associated TSC protein complexes to modulate mTORC1 signaling.

Front Cell Neurosci. 2023 ;17 1274979

Synaptic remodeling follows upper motor neuron hyperexcitability in a rodent model of TDP-43.

Marcus S Dyer, G Lorenzo Odierna, Rosemary M Clark, Adele Woodhouse, Catherine A Blizzard.

  Amyotrophic Lateral Sclerosis (ALS) is an incurable disease characterized by relentlessly progressive degeneration of the corticomotor system. Cortical hyperexcitability has been identified as an early pre-symptomatic biomarker of ALS. This suggests that hyperexcitability occurs upstream in the ALS pathological cascade and may even be part of the mechanism that drives development of symptoms or loss of motor neurons in the spinal cord. However, many studies also indicate a loss to the synaptic machinery that mediates synaptic input which raises the question of which is the driver of disease, and which is a homeostatic response. Herein, we used an inducible mouse model of TDP-43 mediated ALS that permits for the construction of detailed phenotypic timelines. Our work comprehensively describes the relationship between intrinsic hyperexcitability and altered synaptic input onto motor cortical layer 5 pyramidal neurons over time. As a result, we have constructed the most complete timeline of electrophysiological changes following induction of TDP-43 dysfunction in the motor cortex. We report that intrinsic hyperexcitability of layer 5 pyramidal neurons precedes changes to excitatory synaptic connections, which manifest as an overall loss of inputs onto layer 5 pyramidal neurons. This finding highlights the importance of hyperexcitability as a primary mechanism of ALS and re-contextualizes synaptic changes as possibly representing secondary adaptive responses. Recognition of the relationship between intrinsic hyperexcitability and reduced excitatory synaptic input has important implications for the development of useful therapies against ALS. Novel strategies will need to be developed that target neuronal output by managing excitability against synapses separately.

Keywords:  TDP-43; amyotrophic lateral sclerosis; excitatory postsynaptic currents; hyperexcitability; motor neuron disease; synapse; timeline

DOI:  https://doi.org/10.3389/fncel.2023.1274979
Mol Ther Nucleic Acids. 2023 Dec 12. 34 102057

Efficacy and safety of a SOD1-targeting artificial miRNA delivered by AAV9 in mice are impacted by miRNA scaffold selection.

Shukkwan K Chen, Zachary C E Hawley, Maria I Zavodszky, Sam Hana, Daniel Ferretti, Branka Grubor, Michael Hawes, Shanqin Xu, Stefan Hamann, Galina Marsh, Patrick Cullen, Ravi Challa, Thomas M Carlile, Hang Zhang, Wan-Hung Lee, Andrea Peralta, Pete Clarner, Cong Wei, Kathryn Koszka, Feng Gao, Shih-Ching Lo.

  Toxic gain-of-function mutations in superoxide dismutase 1 (SOD1) contribute to approximately 2%-3% of all amyotrophic lateral sclerosis (ALS) cases. Artificial microRNAs (amiRs) delivered by adeno-associated virus (AAV) have been proposed as a potential treatment option to silence SOD1 expression and mitigate disease progression. Primary microRNA (pri-miRNA) scaffolds are used in amiRs to shuttle a hairpin RNA into the endogenous miRNA pathway, but it is unclear whether different primary miRNA (pri-miRNA) scaffolds impact the potency and safety profile of the expressed amiR in vivo. In our process to develop an AAV amiR targeting SOD1, we performed a preclinical characterization of two pri-miRNA scaffolds, miR155 and miR30a, sharing the same guide strand sequence. We report that, while the miR155-based vector, compared with the miR30a-based vector, leads to a higher level of the amiR and more robust suppression of SOD1 in vitro and in vivo, it also presents significantly greater risks for CNS-related toxicities in vivo. Despite miR30a-based vector showing relatively lower potency, it can significantly delay the development of ALS-like phenotypes in SOD1-G93A mice and increase survival in a dose-dependent manner. These data highlight the importance of scaffold selection in the pursuit of highly efficacious and safe amiRs for RNA interference gene therapy.

Keywords:  ALS; MT: oligonucleotides: therapies; RNAi; SOD1; adeno-associated virus; gene therapy; preclinical; primary miRNA scaffold

DOI:  https://doi.org/10.1016/j.omtn.2023.102057
Amyotroph Lateral Scler Frontotemporal Degener. 2023 Nov 05. 1-15

FUS gene mutation in amyotrophic lateral sclerosis: a new case report and systematic review.

Xin Xiao, Min Li, Zhi Ye, Xiaoyan He, Jun Wei, Yunhong Zha.

  OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease associated with upper and lower motor neuron degeneration and necrosis, characterized by progressive muscle weakness, atrophy, and paralysis. The FUS mutation-associated ALS has been classified as ALS6. We reported a case of ALS6 with de novo mutation and investigated retrospectively the characteristics of cases with FUS mutation.METHODS: We reported a male patient with a new heterozygous variant of the FUS gene and comprehensively reviewed 173 ALS cases with FUS mutation. The literature was reviewed from the PubMed MEDLINE electronic database (https://www.ncbi.nlm.nih.gov/pubmed) using "Amyotrophic Lateral Sclerosis and Fus mutation" or "Fus mutation" as key words from 1 January 2009 to 1 January 2022.
RESULTS: We report a case of ALS6 with a new mutation point (c.1225-1227delGGA) and comprehensively review 173 ALS cases with FUS mutation. Though ALS6 is all with FUS mutation, it is still a highly heterogenous subtype. The average onset age of ALS6 is 35.2 ± 1.3 years, which is much lower than the average onset age of ALS (60 years old). Juvenile FUS mutations have an aggressive progression of disease, with an average time from onset to death or tracheostomy of 18.2 ± 0.5 months. FUS gene has the characteristics of early onset, faster progress, and shorter survival, especially in deletion mutation p.G504Wfs *12 and missense mutation of p.P525L.
CONCLUSIONS: ALS6 is a highly heterogenous subtype. Our study could allow clinicians to better understand the non-ALS typical symptoms, phenotypes, and pathophysiology of ALS6.

Keywords:  Amyotrophic lateral sclerosis; FUS; de novo mutation; genetics; phenotype

DOI:  https://doi.org/10.1080/21678421.2023.2272170
Sci Transl Med. 2023 Nov 08. 15(721): eadj9332

Immunological drivers of amyotrophic lateral sclerosis.

Tania F Gendron, Leonard Petrucelli.

Amyotrophic lateral sclerosis (ALS), a devastating motor neuron disease involving complex genetic and environmental factors, is associated with neuroinflammation. Preclinical and clinical studies support immune system involvement in ALS pathogenesis, thereby spurring investigations into potential pathogenic mechanisms, immune response biomarkers, and ALS therapeutics.

DOI: https://doi.org/10.1126/scitranslmed.adj9332
Front Neurosci. 2023 ;17 1259742

Analysis of damage-associated molecular patterns in amyotrophic lateral sclerosis based on ScRNA-seq and bulk RNA-seq data.

Yue Shi, Ruixia Zhu.

  Background: Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disorder characterized by the progressive loss of motor neurons. Despite extensive research, the exact etiology of ALS remains elusive. Emerging evidence highlights the critical role of the immune system in ALS pathogenesis and progression. Damage-Associated Molecular Patterns (DAMPs) are endogenous molecules released by stressed or damaged cells, acting as danger signals and activating immune responses. However, their specific involvement in ALS remains unclear.Methods: We obtained single-cell RNA sequencing (scRNA-seq) data of ALS from the primary motor cortex in the Gene Expression Omnibus (GEO) database. To better understand genes associated with DAMPs, we performed analyses on cell-cell communication and trajectory. The abundance of immune-infiltrating cells was assessed using the single-sample Gene Set Enrichment Analysis (ssGSEA) method. We performed univariate Cox analysis to construct the risk model and utilized the least absolute shrinkage and selection operator (LASSO) analysis. Finally, we identified potential small molecule drugs targeting ALS by screening the Connectivity Map database (CMap) and confirmed their potential through molecular docking analysis.
Results: Our study annotated 10 cell types, with the expression of genes related to DAMPs predominantly observed in microglia. Analysis of intercellular communication revealed 12 ligand-receptor pairs in the pathways associated with DAMPs, where microglial cells acted as ligands. Among these pairs, the SPP1-CD44 pair demonstrated the greatest contribution. Furthermore, trajectory analysis demonstrated distinct differentiation fates of different microglial states. Additionally, we constructed a risk model incorporating four genes (TRPM2, ROCK1, HSP90AA1, and HSPA4). The validity of the risk model was supported by multivariate analysis. Moreover, external validation from dataset GSE112681 confirmed the predictive power of the model, which yielded consistent results with datasets GSE112676 and GSE112680. Lastly, the molecular docking analysis suggested that five compounds, namely mead-acid, nifedipine, nifekalant, androstenol, and hydrastine, hold promise as potential candidates for the treatment of ALS.
Conclusion: Taken together, our study demonstrated that DAMP entities were predominantly observed in microglial cells within the context of ALS. The utilization of a prognostic risk model can accurately predict ALS patient survival. Additionally, genes related to DAMPs may present viable drug targets for ALS therapy.

Keywords:  ALS; DAMPs; immune infiltration; molecular docking; single-cell sequencing

DOI:  https://doi.org/10.3389/fnins.2023.1259742
Endocr Metab Immune Disord Drug Targets. 2023 Nov 01.

Mitochondrial Inherited Disorders and their Correlation with Neurodegenerative Diseases.

Sofjana Gushi, Dr Vasileios Balis PhD Mrsb.

  Mitochondria are essential organelles for the survival of a cell because they produce energy. The cells that need more mitochondria are neurons because they perform a variety of tasks that are necessary to support brain homeostasis. The build-up of abnormal proteins in neurons, as well as their interactions with mitochondrial proteins, or MAM proteins, cause serious health issues. As a result, mitochondrial functions, such as mitophagy, are impaired, resulting in the disorders described in this review. They are also due to mtDNA mutations, which alter the heritability of diseases. The topic of disease prevention, as well as the diagnosis, requires further explanation and exploration. Finally, there are treatments that are quite promising, but more detailed research is needed.

Keywords:  Mitochondria; diagnosis; heritability.; mitochondrial DNA; mitophagy; neurodegeneration; prevention; treatment

DOI:  https://doi.org/10.2174/0118715303250271231018103202
Nat Rev Neurol. 2023 Nov 10.

Biomarkers in amyotrophic lateral sclerosis: current status and future prospects.

Roisin McMackin, Peter Bede, Caroline Ingre, Andrea Malaspina, Orla Hardiman.

Disease heterogeneity in amyotrophic lateral sclerosis poses a substantial challenge in drug development. Categorization based on clinical features alone can help us predict the disease course and survival, but quantitative measures are also needed that can enhance the sensitivity of the clinical categorization. In this Review, we describe the emerging landscape of diagnostic, categorical and pharmacodynamic biomarkers in amyotrophic lateral sclerosis and their place in the rapidly evolving landscape of new therapeutics. Fluid-based markers from cerebrospinal fluid, blood and urine are emerging as useful diagnostic, pharmacodynamic and predictive biomarkers. Combinations of imaging measures have the potential to provide important diagnostic and prognostic information, and neurophysiological methods, including various electromyography-based measures and quantitative EEG-magnetoencephalography-evoked responses and corticomuscular coherence, are generating useful diagnostic, categorical and prognostic markers. Although none of these biomarker technologies has been fully incorporated into clinical practice or clinical trials as a primary outcome measure, strong evidence is accumulating to support their clinical utility.

DOI: https://doi.org/10.1038/s41582-023-00891-2
Front Endocrinol (Lausanne). 2023 ;14 1236472

Mitochondrial regulation in human pluripotent stem cells during reprogramming and β cell differentiation.

Ila Tewari Jasra, Nerea Cuesta-Gomez, Kevin Verhoeff, Braulio A Marfil-Garza, Nidheesh Dadheech, A M James Shapiro.

  Mitochondria are the powerhouse of the cell and dynamically control fundamental biological processes including cell reprogramming, pluripotency, and lineage specification. Although remarkable progress in induced pluripotent stem cell (iPSC)-derived cell therapies has been made, very little is known about the role of mitochondria and the mechanisms involved in somatic cell reprogramming into iPSC and directed reprogramming of iPSCs in terminally differentiated cells. Reprogramming requires changes in cellular characteristics, genomic and epigenetic regulation, as well as major mitochondrial metabolic changes to sustain iPSC self-renewal, pluripotency, and proliferation. Differentiation of autologous iPSC into terminally differentiated β-like cells requires further metabolic adaptation. Many studies have characterized these alterations in signaling pathways required for the generation and differentiation of iPSC; however, very little is known regarding the metabolic shifts that govern pluripotency transition to tissue-specific lineage differentiation. Understanding such metabolic transitions and how to modulate them is essential for the optimization of differentiation processes to ensure safe iPSC-derived cell therapies. In this review, we summarize the current understanding of mitochondrial metabolism during somatic cell reprogramming to iPSCs and the metabolic shift that occurs during directed differentiation into pancreatic β-like cells.

Keywords:  Diabetes Mellitus; beta cells (β Cells); induced pluripotent stem (iPS) cells; islet transplantation; stem cells

DOI:  https://doi.org/10.3389/fendo.2023.1236472
J Neurol. 2023 Nov 06.

Unveiling the SOD1-mediated ALS phenotype: insights from a comprehensive meta-analysis.

Teuta Domi, Paride Schito, Giacomo Sferruzza, Tommaso Russo, Laura Pozzi, Federica Agosta, Paola Carrera, Nilo Riva, Massimo Filippi, Angelo Quattrini, Yuri Matteo Falzone.

  BACKGROUND AND OBJECTIVES: Amyotrophic lateral sclerosis associated with mutations in SOD1 (SOD1-ALS) might be susceptible to specific treatment. The aim of the study is to outline the clinical features of SOD1-ALS patients by comparing them to patients without ALS major gene variants and patients with variants in other major ALS genes. Defining SOD1-ALS phenotype may assist clinicians in identifying patients who should be prioritized for genetic testing.METHODS: We performed an extensive literature research including original studies which reported the clinical features of SOD1-ALS and at least one of the following patient groups: C9ORF72 hexanucleotide repeat expansion (C9-ALS), TARDBP (TARDBP-ALS), FUS (FUS-ALS) or patients without a positive test for a major-ALS gene (N-ALS). A random effects meta-analytic model was applied to clinical data extracted encompassing sex, site and age of onset. To reconstruct individual patient survival data, the published Kaplan-Meier curves were digitized. Data were measured as odds ratio (OR) or standardized mean difference (SMD) as appropriate. Median survival was compared between groups.
RESULTS: Twenty studies met the inclusion criteria. We identified 721 SOD1-ALS, 470 C9-ALS, 183 TARDBP-ALS, 113 FUS-ALS and 2824 N-ALS. SOD1-ALS showed a higher rate of spinal onset compared with N-ALS and C9-ALS (OR = 4.85, 95% CI = 3.04-7.76; OR = 10.47, 95% CI = 4.32-27.87) and an earlier onset compared with N-ALS (SMD = - 0.45, 95% CI = - 0.72 to - 0.18). SOD1-ALS had a similar survival compared with N-ALS (p = 0.14), a longer survival compared with C9-ALS (p < 0.01) and FUS-ALS (p = 0.019) and a shorter survival compared with TARDBP-ALS (p < 0.01).
DISCUSSION: This study indicates the presence of a specific SOD1-ALS phenotype. Insights in SOD1-ALS clinical features are important in genetic counseling, disease prognosis and support patients' stratification in clinical trials.

Keywords:  ALS; ALS genetics; Metanalysis; Motor neuron disease; SOD1

DOI:  https://doi.org/10.1007/s00415-023-12074-6
Cureus. 2023 Nov;15(11): e48458

Artificial Intelligence Applications in the Diagnosis of Neuromuscular Diseases: A Narrative Review.

Martha C Piñeros-Fernández.

  The accurate diagnosis of neuromuscular diseases (NMD) is in many cases difficult; the starting point is the clinical approach based on the course of the disease and a careful physical examination of the patient. Electrodiagnostic tests, imaging, muscle biopsy, and genetics are fundamental complementary studies for the diagnosis of NMD. The large volume of data obtained from such studies makes it necessary to look for efficient solutions, such as artificial intelligence (AI) applications, which can help classify, synthesize, and organize the information of patients with NMD to facilitate their accurate and timely diagnosis. The objective of this study was to describe the usefulness of AI applications in the diagnosis of patients with neuromuscular diseases. A narrative review was done, including publications on artificial intelligence applied to the diagnostic methods of NMD currently existing. Twelve studies were included. Two of the studies focused on muscle ultrasound, five of the studies on muscle MRI, two studies on electromyography, two studies on amyotrophic lateral sclerosis (ALS) biomarkers, and one study on genes related to myopathies. The accuracy of classification using different classification algorithms used in each of the studies included in this narrative review was already 90% in most studies. In conclusion, the future design of more accurate algorithms applied to NMD with greater precision will have an impact on the earlier diagnosis of this group of diseases.

Keywords:  applications of medical informatics; artificial intelligence; diagnostic accuracy; machine learning; neuromuscular diseases

DOI:  https://doi.org/10.7759/cureus.48458
Mol Brain. 2023 Nov 03. 16(1): 75

Limitations of the human iPSC-derived neuron model for early-onset Alzheimer's disease.

Phoebe Valdes, Kenneth W Henry, Michael Q Fitzgerald, Koushik Muralidharan, Andrew B Caldwell, Srinivasan Ramachandran, Lawrence S B Goldstein, William C Mobley, Douglas R Galasko, Shankar Subramaniam.

  Non-familial Alzheimer's disease (AD) occurring before 65 years of age is commonly referred to as early-onset Alzheimer's disease (EOAD) and constitutes ~ 5-6% of all AD cases (Mendez et al. in Continuum 25:34-51, 2019). While EOAD exhibits the same clinicopathological changes such as amyloid plaques, neurofibrillary tangles (NFTs), brain atrophy, and cognitive decline (Sirkis et al. in Mol Psychiatry 27:2674-88, 2022; Caldwell et al. in Mol Brain 15:83, 2022) as observed in the more prevalent late-onset AD (LOAD), EOAD patients tend to have more severe cognitive deficits, including visuospatial, language, and executive dysfunction (Sirkis et al. in Mol Psychiatry 27:2674-88, 2022). Patient-derived induced pluripotent stem cells (iPSCs) have been used to model and study penetrative, familial AD (FAD) mutations in APP, PSEN1, and PSEN2 (Valdes et al. in Research Square 1-30, 2022; Caldwell et al. in Sci Adv 6:1-16, 2020) but have been seldom used for sporadic forms of AD that display more heterogeneous disease mechanisms. In this study, we sought to characterize iPSC-derived neurons from EOAD patients via RNA sequencing. A modest difference in expression profiles between EOAD patients and non-demented control (NDC) subjects resulted in a limited number of differentially expressed genes (DEGs). Based on this analysis, we provide evidence that iPSC-derived neuron model systems, likely due to the loss of EOAD-associated epigenetic signatures arising from iPSC reprogramming, may not be ideal models for studying sporadic AD.

Keywords:  Early-onset Alzheimer’s disease; RNA-seq; Systems biology; iPSC neurons

DOI:  https://doi.org/10.1186/s13041-023-01063-5
Cells. 2023 Oct 31. pii: 2550. [Epub ahead of print]12(21):

LRRK2 Attenuates Antioxidant Response in Familial Parkinson's Disease Derived Neural Stem Cells.

Jeffrey Kim, Etienne W Daadi, Elyas Sebastien Daadi, Thomas Oh, Michela Deleidi, Marcel M Daadi.

  Parkinson's disease (PD) is the second most prevalent neurodegenerative disease, characterized by the loss of midbrain dopaminergic neurons which leads to impaired motor and cognitive functions. PD is predominantly an idiopathic disease; however, about 5% of cases are linked to hereditary mutations. The most common mutation in both familial and sporadic PD is the G2019S mutation of leucine-rich repeat kinase 2 (LRRK2). Currently, it is not fully understood how this mutation leads to PD pathology. In this study, we isolated self-renewable, multipotent neural stem cells (NSCs) from induced pluripotent stem cells (iPSCs) harboring the G2019S LRRK2 mutation and compared them with their isogenic gene corrected counterparts using single-cell RNA-sequencing. Unbiased single-cell transcriptomic analysis revealed perturbations in many canonical pathways, specifically NRF2-mediated oxidative stress response, and glutathione redox reactions. Through various functional assays, we observed that G2019S iPSCs and NSCs exhibit increased basal levels of reactive oxygen species (ROS). We demonstrated that mutant cells show significant increase in the expression for KEAP1 and decrease in NRF2 associated with a reduced antioxidant response. The decreased viability of mutant NSCs in the H2O2-induced oxidative stress assay was rescued by two potent antioxidant drugs, PrC-210 at concentrations of 500 µM and 1 mM and Edaravone at concentrations 50 µM and 100 µM. Our data suggest that the hyperactive LRRK2 G2019S kinase activity leads to increase in KEAP1, which binds NRF2 and leads to its degradation, reduction in the antioxidant response, increased ROS, mitochondria dysfunction and cell death observed in the PD phenotype.

Keywords:  Parkinson’s disease; induced pluripotent stem cells; neural stem cells; single cell transcriptomics; target identification

DOI:  https://doi.org/10.3390/cells12212550
Biofabrication. 2023 Nov 10. 16(1):

Long-termin vitroculture of 3D brain tissue model based on chitosan thermogel.

Donatella Di Lisa, Lorenzo Muzzi, Alberto Lagazzo, Andrea Andolfi, Sergio Martinoia, Laura Pastorino.

  Methods for studying brain function and disease heavily rely onin vivoanimal models,ex-vivotissue slices, and 2D cell culture platforms. These methods all have limitations that significantly impact the clinical translatability of results. Consequently, models able to better recapitulate some aspects ofin vivohuman brain are needed as additional preclinical tools. In this context, 3D hydrogel-basedin vitromodels of the brain are considered promising tools. To create a 3D brain-on-a-chip model, a hydrogel capable of sustaining neuronal maturation over extended culture periods is required. Among biopolymeric hydrogels, chitosan-β-glycerophosphate (CHITO-β-GP) thermogels have demonstrated their versatility and applicability in the biomedical field over the years. In this study, we investigated the ability of this thermogel to encapsulate neuronal cells and support the functional maturation of a 3D neuronal network in long-term cultures. To the best of our knowledge, we demonstrated for the first time that CHITO-β-GP thermogel possesses optimal characteristics for promoting neuronal growth and the development of an electrophysiologically functional neuronal network derived from both primary rat neurons and neurons differentiated from human induced pluripotent stem cells (h-iPSCs) co-cultured with astrocytes. Specifically, two different formulations were firstly characterized by rheological, mechanical and injectability tests. Primary nervous cells and neurons differentiated from h-iPSCs were embedded into the two thermogel formulations. The 3D cultures were then deeply characterized by immunocytochemistry, confocal microscopy, and electrophysiological recordings, employing both 2D and 3D micro-electrode arrays. The thermogels supported the long-term culture of neuronal networks for up to 100 d. In conclusion, CHITO-β-GP thermogels exhibit excellent mechanical properties, stability over time under culture conditions, and bioactivity toward nervous cells. Therefore, they are excellent candidates as artificial extracellular matrices in brain-on-a-chip models, with applications in neurodegenerative disease modeling, drug screening, and neurotoxicity evaluation.

Keywords:  3D neuronal cultures; brain-on-a-chip; chitosan-β-glycerophosphate thermogel; injectability; soft hydrogels

DOI:  https://doi.org/10.1088/1758-5090/ad0979
Sci Adv. 2023 Nov 10. 9(45): eadf7997

CLIP-Seq analysis enables the design of protective ribosomal RNA bait oligonucleotides against C9ORF72 ALS/FTD poly-GR pathophysiology.

Juan A Ortega, Ivan R Sasselli, Marco Boccitto, Andrew C Fleming, Tyler R Fortuna, Yichen Li, Kohei Sato, Tristan D Clemons, Elizabeth D Mckenna, Thao P Nguyen, Eric N Anderson, Jesus Asin, Justin K Ichida, Udai B Pandey, Sandra L Wolin, Samuel I Stupp, Evangelos Kiskinis.

Amyotrophic lateral sclerosis and frontotemporal dementia patients with a hexanucleotide repeat expansion in C9ORF72 (C9-HRE) accumulate poly-GR and poly-PR aggregates. The pathogenicity of these arginine-rich dipeptide repeats (R-DPRs) is thought to be driven by their propensity to bind low-complexity domains of multivalent proteins. However, the ability of R-DPRs to bind native RNA and the significance of this interaction remain unclear. Here, we used computational and experimental approaches to characterize the physicochemical properties of R-DPRs and their interaction with RNA. We find that poly-GR predominantly binds ribosomal RNA (rRNA) in cells and exhibits an interaction that is predicted to be energetically stronger than that for associated ribosomal proteins. Critically, modified rRNA "bait" oligonucleotides restore poly-GR-associated ribosomal deficits and ameliorate poly-GR toxicity in patient neurons and Drosophila models. Our work strengthens the hypothesis that ribosomal function is impaired by R-DPRs, highlights a role for direct rRNA binding in mediating ribosomal dysfunction, and presents a strategy for protecting against C9-HRE pathophysiological mechanisms.

DOI: https://doi.org/10.1126/sciadv.adf7997
Stem Cell Res. 2023 Nov 03. pii: S1873-5061(23)00228-3. [Epub ahead of print]73 103242

Establishment of human induced pluripotent stem cell line (SDQLCHi060-A) from a patient with AUTS2 syndrome carrying a heterozygous mutation in AUTS2 gene.

Chunlai Gao, Yi Liu, Ning Liu, Zilong Li, Xiaomeng Yang, Yanan Yang, Yue Xi, Jianjun Tian, Zhongtao Gai.

AUTS2 syndrome is a neurodevelopmental disorder caused by pathogenic variants and deletions of the AUTS2 gene, resulting in intellectual disability, microcephaly, and other phenotypes. Here, we generated a human induced pluripotent stem cell (iPSC) line from a 21-month-old boy with AUTS2 syndrome caused by a heterozygous mutation (c.1486C > T, p.Q496X) in the AUTS2 gene. The iPSCs had normal morphology and karyotype, expressed pluripotency markers, showed differentiation potential in vitro, and carried the AUTS2 gene mutation.

DOI: https://doi.org/10.1016/j.scr.2023.103242
STAR Protoc. 2023 Nov 07. pii: S2666-1667(23)00666-4. [Epub ahead of print]4(4): 102699

Long-term live cell imaging during differentiation of human iPSC-derived neurons.

Jingqi Wang, Paul A Gleeson, Lou Fourriere.

  Live-cell imaging is crucial to appreciate the dynamics and the complexity of cellular interaction processes. However, live-cell imaging of human neurons is challenging due to neuronal sensitivity. Here, we describe a long-term live-cell imaging protocol for neurons derived from human induced pluripotent stem cells. By using an IncuCyte live-cell imaging system, we have obtained information on neuronal dynamics during the different stages of neurogenesis. The protocol has also been developed to monitor the dynamics of the neuronal intracellular organelles. For complete details on the use and execution of this protocol, please refer to Wang et al.1.

Keywords:  Cell Biology; Cell Culture; Cell Differentiation; Microscopy; Neuroscience; Stem Cells

DOI:  https://doi.org/10.1016/j.xpro.2023.102699
Angiology. 2023 Nov 07. 33197231213192

Novel Strategies for Angiogenesis in Tissue Injury: Therapeutic Effects of iPSCs-Derived Exosomes.

Jiaxin Zhang, Maoning Shi, Jing Wang, Fei Li, Chenxu Du, Gang Su, Xiaodong Xie, Shiweng Li.

  Regeneration after tissue injury is a dynamic and complex process, and angiogenesis is necessary for normal physiological activities and tissue repair. Induced pluripotent stem cells are a new approach in regenerative medicine, which provides good model for the study of difficult-to-obtain human tissues, patient-specific therapy, and tissue repair. As an innovative cell-free therapeutic strategy, the main advantages of the treatment of induced pluripotent stem cells (iPSCs)-derived exosomes are low in tumorigenicity and immunogenicity, which become an important pathway for tissue injury. This review focuses on the mechanism of the angiogenic effect of iPSCs-derived exosomes on wound repair in tissue injury and their potential therapeutic targets, with a view to providing a theoretical basis for the use of iPSCs-derived exosomes in clinical therapy.

Keywords:  angiogenesis; exosomes (exos); induced pluripotent stem cells (iPSCs); regenerative medicine; wound healing

DOI:  https://doi.org/10.1177/00033197231213192
J Neurochem. 2023 Nov 09.

Novel single-nucleus transcriptomics unravels developmental and functionally controlled principles of mammalian neuromuscular junctions.

Hermona Soreq.

  Prof Ohno's team (Ohkawara et al. 2023, current issue) underscored the dynamic and functional features that co-shape the embryonic and early post-natal development of mammalian neuromuscular junctions (NMJs) using single-nucleus transcriptomics which provides specific insights into the activities of individually studied nuclei and their functional characteristics. Unlike other single-nucleus transcriptomics studies, which tend to be limited to single developmental time points, this article provides novel views of the complex developmental and regulatory dynamics and embryonic cell type origins underscoring the formation of functioning mammalian NMJs by combining this transcriptomic approach with interference tests in cultured C2C12 myotubes. This reveals intriguing novel links between the particular nicotinic acetylcholine receptor genes (nAChR) and regulator transcripts thereof and enables outlining the sequential development of functioning NMJs along embryogenesis and soon after delivery. Specifically, the timewise and cell type origins of the studied nuclei emerged as essential for NMJ neurogenesis and inter-cellular transfer of specific regulators has been indicated. Breaking the barriers between distinct research subdisciplines, this study opens new neurochemistry research directions that recombine developmental, regulatory, and functional transcriptomics in NMJ-including tissues. Moreover, these findings may facilitate tests of diverse pharmaceutical and therapeutic modulators of neuromuscular functioning in health and disease, assisting the translational research progress in treating devastating neuromuscular states such as in amyotrophic lateral sclerosis, myasthenia gravis or individuals poisoned occupationally or otherwise with anticholinesterase inhibitors.

Keywords:  Editorial Highlight

DOI:  https://doi.org/10.1111/jnc.15986
Stem Cell Res Ther. 2023 Nov 07. 14(1): 320

Induced pluripotent stem cells derived from patients carrying mitochondrial mutations exhibit altered bioenergetics and aberrant differentiation potential.

Fibi Meshrkey, Kelly M Scheulin, Christopher M Littlejohn, Joshua Stabach, Bibhuti Saikia, Vedant Thorat, Yimin Huang, Thomas LaFramboise, Edward J Lesnefsky, Raj R Rao, Franklin D West, Shilpa Iyer.

  BACKGROUND: Human mitochondrial DNA mutations are associated with common to rare mitochondrial disorders, which are multisystemic with complex clinical pathologies. The pathologies of these diseases are poorly understood and have no FDA-approved treatments leading to symptom management. Leigh syndrome (LS) is a pediatric mitochondrial disorder that affects the central nervous system during early development and causes death in infancy. Since there are no adequate models for understanding the rapid fatality associated with LS, human-induced pluripotent stem cell (hiPSC) technology has been recognized as a useful approach to generate patient-specific stem cells for disease modeling and understanding the origins of the phenotype.METHODS: hiPSCs were generated from control BJ and four disease fibroblast lines using a cocktail of non-modified reprogramming and immune evasion mRNAs and microRNAs. Expression of hiPSC-associated intracellular and cell surface markers was identified by immunofluorescence and flow cytometry. Karyotyping of hiPSCs was performed with cytogenetic analysis. Sanger and next-generation sequencing were used to detect and quantify the mutation in all hiPSCs. The mitochondrial respiration ability and glycolytic function were measured by the Seahorse Bioscience XFe96 extracellular flux analyzer.
RESULTS: Reprogrammed hiPSCs expressed pluripotent stem cell markers including transcription factors POU5F1, NANOG and SOX2 and cell surface markers SSEA4, TRA-1-60 and TRA-1-81 at the protein level. Sanger sequencing analysis confirmed the presence of mutations in all reprogrammed hiPSCs. Next-generation sequencing demonstrated the variable presence of mutant mtDNA in reprogrammed hiPSCs. Cytogenetic analyses confirmed the presence of normal karyotype in all reprogrammed hiPSCs. Patient-derived hiPSCs demonstrated decreased maximal mitochondrial respiration, while mitochondrial ATP production was not significantly different between the control and disease hiPSCs. In line with low maximal respiration, the spare respiratory capacity was lower in all the disease hiPSCs. The hiPSCs also demonstrated neural and cardiac differentiation potential.
CONCLUSION: Overall, the hiPSCs exhibited variable mitochondrial dysfunction that may alter their differentiation potential and provide key insights into clinically relevant developmental perturbations.

Keywords:  Bioenergetics; Differentiation; Mitochondrial disease; Pluripotent stem cell; Reprogramming; Respiration; hiPSC

DOI:  https://doi.org/10.1186/s13287-023-03546-7
Front Cell Neurosci. 2023 ;17 1241957

Regulation of axon pathfinding by astroglia across genetic model organisms.

Georgia Rapti.

  Glia and neurons are intimately associated throughout bilaterian nervous systems, and were early proposed to interact for patterning circuit assembly. The investigations of circuit formation progressed from early hypotheses of intermediate guideposts and a "glia blueprint", to recent genetic and cell manipulations, and visualizations in vivo. An array of molecular factors are implicated in axon pathfinding but their number appears small relatively to circuit complexity. Comprehending this circuit complexity requires to identify unknown factors and dissect molecular topographies. Glia contribute to both aspects and certain studies provide molecular and functional insights into these contributions. Here, I survey glial roles in guiding axon navigation in vivo, emphasizing analogies, differences and open questions across major genetic models. I highlight studies pioneering the topic, and dissect recent findings that further advance our current molecular understanding. Circuits of the vertebrate forebrain, visual system and neural tube in zebrafish, mouse and chick, the Drosophila ventral cord and the C. elegans brain-like neuropil emerge as major contexts to study glial cell functions in axon navigation. I present astroglial cell types in these models, and their molecular and cellular interactions that drive axon guidance. I underline shared principles across models, conceptual or technical complications, and open questions that await investigation. Glia of the radial-astrocyte lineage, emerge as regulators of axon pathfinding, often employing common molecular factors across models. Yet this survey also highlights different involvements of glia in embryonic navigation or pioneer axon pathfinding, and unknowns in the molecular underpinnings of glial cell functions. Future cellular and molecular investigations should complete the comprehensive view of glial roles in circuit assembly.

Keywords:  axon guidance; development; embryo; glia-neuron interactions; model organisms

DOI:  https://doi.org/10.3389/fncel.2023.1241957
Mol Neurodegener. 2023 Nov 08. 18(1): 79

Mechanism and therapeutic potential of targeting cGAS-STING signaling in neurological disorders.

Yige Huang, Bangyan Liu, Subhash C Sinha, Sadaf Amin, Li Gan.

  DNA sensing is a pivotal component of the innate immune system that is responsible for detecting mislocalized DNA and triggering downstream inflammatory pathways. Among the DNA sensors, cyclic GMP-AMP synthase (cGAS) is a primary player in detecting cytosolic DNA, including foreign DNA from pathogens and self-DNA released during cellular damage, culminating in a type I interferon (IFN-I) response through stimulator of interferon genes (STING) activation. IFN-I cytokines are essential in mediating neuroinflammation, which is widely observed in CNS injury, neurodegeneration, and aging, suggesting an upstream role for the cGAS DNA sensing pathway. In this review, we summarize the latest developments on the cGAS-STING DNA-driven immune response in various neurological diseases and conditions. Our review covers the current understanding of the molecular mechanisms of cGAS activation and highlights cGAS-STING signaling in various cell types of central and peripheral nervous systems, such as resident brain immune cells, neurons, and glial cells. We then discuss the role of cGAS-STING signaling in different neurodegenerative conditions, including tauopathies, Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, as well as aging and senescence. Finally, we lay out the current advancements in research and development of cGAS inhibitors and assess the prospects of targeting cGAS and STING as therapeutic strategies for a wide spectrum of neurological diseases.

Keywords:  ALS; Alzheimer disease; Antiviral; Cytosolic dsDNA; Interferon response; Neuroinflammation; Parkinson disease; STING; cGAS

DOI:  https://doi.org/10.1186/s13024-023-00672-x
Enzymes. 2023 ;pii: S1874-6047(23)00009-4. [Epub ahead of print]54 171-201

Sphingolipids: From structural components to signaling hubs.

Batoul M Issleny, Rama Jamjoum, Saurav Majumder, Johnny Stiban.

  In late November 2019, Prof. Lina M. Obeid passed away from cancer, a disease she spent her life researching and studying its intricate molecular underpinnings. Along with her husband, Prof. Yusuf A. Hannun, Obeid laid down the foundations of sphingolipid biochemistry and oversaw its remarkable evolution over the years. Lipids are a class of macromolecules that are primarily associated with cellular architecture. In fact, lipids constitute the perimeter of the cell in such a way that without them, there cannot be cells. Hence, much of the early research on lipids identified the function of this class of biological molecules as merely structural. Nevertheless, unlike proteins, carbohydrates, and nucleic acids, lipids are elaborately diverse as they are not made up of monomers in polymeric forms. This diversity in structure is clearly mirrored by functional pleiotropy. In this chapter, we focus on a major subset of lipids, sphingolipids, and explore their historic rise from merely inert structural components of plasma membranes to lively and necessary signaling molecules that transmit various signals and control many cellular processes. We will emphasize the works of Lina Obeid since she was an integral pillar of the sphingolipid research world.

Keywords:  Apoptosis; Autophagy; Cellular signaling; Gene expression; Lina M. Obeid; Lipid signaling; Sphingolipid functions; Structural components; Unorthodox structures

DOI:  https://doi.org/10.1016/bs.enz.2023.07.003
Stem Cell Rev Rep. 2023 Nov 08.

Differentiation of Neurons, Astrocytes, Oligodendrocytes and Microglia From Human Induced Pluripotent Stem Cells to Form Neural Tissue-On-Chip: A Neuroinflammation Model to Evaluate the Therapeutic Potential of Extracellular Vesicles Derived from Mesenchymal Stem Cells.

Pelin Saglam-Metiner, Elif Duran, Leila Sabour-Takanlou, Cigir Biray-Avci, Ozlem Yesil-Celiktas.

  Advances in stem cell (SC) technology allow the generation of cellular models that recapitulate the histological, molecular and physiological properties of humanized in vitro three dimensional (3D) models, as well as production of cell-derived therapeutics such as extracellular vesicles (EVs). Improvements in organ-on-chip platforms and human induced pluripotent stem cells (hiPSCs) derived neural/glial cells provide unprecedented systems for studying 3D personalized neural tissue modeling with easy setup and fast output. Here, we highlight the key points in differentiation procedures for neurons, astrocytes, oligodendrocytes and microglia from single origin hiPSCs. Additionally, we present a well-defined humanized neural tissue-on-chip model composed of differentiated cells with the same genetic backgrounds, as well as the therapeutic potential of bone marrow mesenchymal stem cells (BMSCs)-derived extracellular vesicles to propose a novel treatment for neuroinflammation derived diseases. Around 100 nm CD9 + EVs promote a more anti-inflammatory and pro-remodeling of cell-cell interaction cytokine responses on tumor necrosis factor-α (TNF-α) induced neuroinflammation in neural tissue-on-chip model which is ideal for modeling authentic neural-glial patho-physiology.

Keywords:  Differentiation; Extracellular vesicles; Induced pluripotent stem cells; Neuroinflammation; Organ-on-a-chip

DOI:  https://doi.org/10.1007/s12015-023-10645-8
Autophagy. 2023 Nov 09. 1-17

A conserved requirement for RME-8/DNAJC13 in neuronal autophagic lysosome reformation.

Sierra B Swords, Nuo Jia, Anne Norris, Jil Modi, Qian Cai, Barth D Grant.

  Autophagosomes fuse with lysosomes, forming autolysosomes that degrade engulfed cargo. To maintain lysosomal capacity, autophagic lysosome reformation (ALR) must regenerate lysosomes from autolysosomes using a membrane tubule-based process. Maintaining lysosomal capacity is required to maintain cellular health, especially in neurons where lysosomal dysfunction has been repeatedly implicated in neurodegenerative disease. The DNA-J domain HSC70 co-chaperone RME-8/DNAJC13 has been linked to endosomal coat protein regulation and to neurological disease. We report new analysis of the requirements for the RME-8/DNAJC13 protein in neurons, focusing on intact C. elegans mechanosensory neurons, and primary mouse cortical neurons in culture. Loss of RME-8/DNAJC13 in both systems results in accumulation of grossly elongated autolysosomal tubules. Further C. elegans analysis revealed a similar autolysosome tubule accumulation defect in mutants known to be required for ALR in mammals, including mutants lacking bec-1/BECN1/Beclin1 and vps-15/PIK3R4/p150 that regulate the class III phosphatidylinositol 3-kinase (PtdIns3K) VPS-34, and dyn-1/dynamin that severs ALR tubules. Clathrin is also an important ALR regulator implicated in autolysosome tubule formation and release. In C. elegans we found that loss of RME-8 causes severe depletion of clathrin from neuronal autolysosomes, a phenotype shared with bec-1 and vps-15 mutants. We conclude that RME-8/DNAJC13 plays a previously unrecognized role in ALR, likely affecting autolysosome tubule severing. Additionally, in both systems, loss of RME-8/DNAJC13 reduced macroautophagic/autophagic flux, suggesting feedback regulation from ALR to autophagy. Our results connecting RME-8/DNAJC13 to ALR and autophagy provide a potential mechanism by which RME-8/DNAJC13 could influence neuronal health and the progression of neurodegenerative disease.Abbreviation: ALR, autophagic lysosome reformation; ATG-13/EPG-1, AuTophaGy (yeast Atg homolog)-13; ATG-18, AuTophaGy (yeast Atg homolog)-18; AV, autophagic vacuole; CLIC-1, Clathrin Light Chain-1; EPG-3, Ectopic P Granules-3; EPG-6, Ectopic P Granules-6; LGG-1, LC3, GABARAP and GATE-16 family-1; MAP1LC3/LC3, microtubule-associated protein 1 light chain 3; PD, Parkinson disease; PtdIns3P, phosphatidylinositol-3-phosphate; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate; RME-8, Receptor Mediated Endocytosis-8; SNX-1, Sorting NeXin-1; VPS-34, related to yeast Vacuolar Protein Sorting factor-34.

Keywords:  Autophagy; clathrin; endocytosis; lysosomes; neurodegeneration; trafficking

DOI:  https://doi.org/10.1080/15548627.2023.2269028
J Physiol. 2023 Nov 09.

Mitochondrial phosphagen kinases support the volatile power demands of motor nerve terminals.

Karlis A Justs, Sergio Sempertegui, Danielle V Riboul, Carlos D Oliva, Ryan J Durbin, Sarah Crill, Michal Stawarski, Chenchen Su, Robert B Renden, Yaouen Fily, Gregory T Macleod.

  Motor neurons are the longest neurons in the body, with axon terminals separated from the soma by as much as a meter. These terminals are largely autonomous with regard to their bioenergetic metabolism and must burn energy at a high rate to sustain muscle contraction. Here, through computer simulation and drawing on previously published empirical data, we determined that motor neuron terminals in Drosophila larvae experience highly volatile power demands. It might not be surprising then, that we discovered the mitochondria in the motor neuron terminals of both Drosophila and mice to be heavily decorated with phosphagen kinases - a key element in an energy storage and buffering system well-characterized in fast-twitch muscle fibres. Knockdown of arginine kinase 1 (ArgK1) in Drosophila larval motor neurons led to several bioenergetic deficits, including mitochondrial matrix acidification and a faster decline in the cytosol ATP to ADP ratio during axon burst firing. KEY POINTS: Neurons commonly fire in bursts imposing highly volatile demands on the bioenergetic machinery that generates ATP. Using a computational approach, we built profiles of presynaptic power demand at the level of single action potentials, as well as the transition from rest to sustained activity. Phosphagen systems are known to buffer ATP levels in muscles and we demonstrate that phosphagen kinases, which support such phosphagen systems, also localize to mitochondria in motor nerve terminals of fruit flies and mice. By knocking down phosphagen kinases in fruit fly motor nerve terminals, and using fluorescent reporters of the ATP:ADP ratio, lactate, pH and Ca2+ , we demonstrate a role for phosphagen kinases in stabilizing presynaptic ATP levels. These data indicate that the maintenance of phosphagen systems in motor neurons, and not just muscle, could be a beneficial initiative in sustaining musculoskeletal health and performance.

Keywords:  ATP; energy; mitochondria; presynaptic

DOI:  https://doi.org/10.1113/JP284872
Front Cell Dev Biol. 2023 ;11 1258993

Combined 3D bioprinting and tissue-specific ECM system reveals the influence of brain matrix on stem cell differentiation.

Martina Zamponi, Peter A Mollica, Yara Khodour, Julie S Bjerring, Robert D Bruno, Patrick C Sachs.

  We have previously shown that human and murine breast extracellular matrix (ECM) can significantly impact cellular behavior, including stem cell fate determination. It has been established that tissue-specific extracellular matrix from the central nervous system has the capacity to support neuronal survival. However, the characterization of its influence on stem cell differentiation and its adaptation to robust 3D culture models is underdeveloped. To address these issues, we combined our 3D bioprinter with hydrogels containing porcine brain extracellular matrix (BMX) to test the influence of the extracellular matrix on stem cell differentiation. Our 3D bioprinting system generated reproducible 3D neural structures derived from mouse embryonic stem cells (mESCs). We demonstrate that the addition of BMX preferentially influences 3D bioprinted mESCs towards neural lineages compared to standard basement membrane (Geltrex/Matrigel) hydrogels alone. Furthermore, we demonstrate that we can transplant these 3D bioprinted neural cellular structures into a mouse's cleared mammary fat pad, where they continue to grow into larger neural outgrowths. Finally, we demonstrate that direct injection of human induced pluripotent stem cells (hiPSCS) and neural stem cells (NSCs) suspended in pure BMX formed neural structures in vivo. Combined, these findings describe a unique system for studying brain ECM/stem cell interactions and demonstrate that BMX can direct pluripotent stem cells to differentiate down a neural cellular lineage without any additional specific differentiation stimuli.

Keywords:  3D bioprinting; brain; cellular microenvironment; extracellular matrix; fate determination; neural differentiation; stem cells

DOI:  https://doi.org/10.3389/fcell.2023.1258993
J Biol Chem. 2023 Nov 08. pii: S0021-9258(23)02483-3. [Epub ahead of print] 105455

CBAP regulates the function of Akt-associated TSC protein complexes to modulate mTORC1 signaling.

Wei-Ting Liao, Yun-Jung Chiang, Hsin-Fang Yang-Yen, Li-Chung Hsu, Zee-Fen Chang, Jeffrey Jy Yen.

  The Akt-Rheb-mTORC1 pathway plays a crucial role in regulating cell growth, but the mechanisms underlying the activation of Rheb-mTORC1 by Akt remain unclear. In our previous study, we found that CBAP was highly expressed in human T-ALL cells and primary tumors, and its deficiency led to reduced phosphorylation of TSC2/S6K1 signaling proteins, as well as impaired cell proliferation and leukemogenicity. We also demonstrated that CBAP was required for Akt-mediated TSC2 phosphorylation in vitro. In response to insulin, CBAP was also necessary for phosphorylation of TSC2/S6K1 and the dissociation of TSC2 from the lysosomal membrane. Here we report that CBAP interacts with AKT and TSC2, and knockout of CBAP or serum starvation leads to an increase in TSC1 in the Akt/TSC2 immunoprecipitation complexes. Lysosomal-anchored CBAP was found to override serum starvation and promote S6K1 and 4EBP1 phosphorylation and c-Myc expression in a TSC2-dependent manner. Additionally, recombinant CBAP inhibited the GAP activity of TSC2 complexes in vitro, leading to increased Rheb-GTP loading, likely due to the competition between TSC1 and CBAP for binding to the HBD domain of TSC2. Overexpression of the N26 region of CBAP, which is crucial for binding to TSC2, resulted in a decrease in mTORC1 signaling and an increase in TSC1 association with the TSC2/AKT complex, ultimately leading to increased GAP activity toward Rheb and impaired cell proliferation. Thus, we propose that CBAP can modulate the stability of TSC1-TSC2 as well as promote translocation of TSC1/TSC2 complexes away from lysosomes to regulate Rheb-mTORC1 signaling.

Keywords:  Akt; Rheb; cell growth; mTORC1 activation; small GTPase; tumor cell biology

DOI:  https://doi.org/10.1016/j.jbc.2023.105455