bims-axbals 2024-09-15 papers

bims-axbals

Biomed News

on Axonal Biology and ALS

Issue of 2024‒09‒15
twenty-six papers selected by
TJ Krzystek, ALS Therapy Development Institute

A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence.
A robust evaluation of TDP-43, poly GP, cellular pathology and behavior in a AAV-C9ORF72 (G4C2)66 mouse model.
An Essential Role for Calnexin in ER-Phagy and the Unfolded Protein Response.
Modeling Lewy body disease with SNCA triplication iPSC-derived cortical organoids and identifying therapeutic drugs.
Fully defined NGN2 neuron protocol reveals diverse signatures of neuronal maturation.
CHCHD10P80L knock-in zebrafish display a mild ALS-like phenotype.
Targeting EGLN2/PHD1 protects motor neurons and normalizes the astrocytic interferon response.
Downregulation of Lnc-ABCA12-3 modulates UBQLN1 expression and protein homeostasis pathways in amyotrophic lateral sclerosis.
The Six-Transmembrane Enzyme GDE2 Is Required for the Release of Molecularly Distinct Small Extracellular Vesicles from Neurons.
Novel imaging tools to study mitochondrial morphology in Caenorhabditis elegans.
Microtubules, Membranes, and Movement: New Roles for Stathmin-2 in Axon Integrity.
Ptbp2 re-expression rescues axon growth defects in Smn-deficient motoneurons.
Loss-of-function variants in RNA binding motif protein X-linked induce neuronal defects contributing to amyotrophic lateral sclerosis pathogenesis.
Combination AAV therapy with galectin-1 and SOD1 downregulation demonstrates superior therapeutic effect in a severe ALS mouse model.
A miR-383-5p signalling hub coordinates the axon regeneration response to inflammation.
Emerging targets in amyotrophic lateral sclerosis (ALS): The promise of ATP-binding cassette (ABC) transporter modulation.
Generation and characterization of cortical organoids from iPSC-derived dental pulp stem cells using traditional and innovative approaches.
Astrocytic LRRK2 Controls Synaptic Connectivity via Regulation of ERM Phosphorylation.
Quantification of serum TDP-43 and neurofilament light chain in patients with amyotrophic lateral sclerosis stratified by UNC13A genotype.
Engineered NLS-chimera downregulates expression of aggregation-prone endogenous FUS.
Induce Pluripotent Stem Cells (iPSC) Technology in Depression.
V-ATPase Dysfunction in the Brain: Genetic Insights and Therapeutic Opportunities.
Proteome dynamics in iPSC-derived human dopaminergic neurons.
Experimental Cell Models for Investigating Neurodegenerative Diseases.
SUMOylation modulates mitochondrial dynamics in an in vitro rotenone model of Parkinson's disease.
Brain organoids: A new tool for modelling of neurodevelopmental disorders.

F1000Res. 2024 ;13 922

A guide to selecting high-performing antibodies for Huntingtin (UniProt ID: P42858) for use in western blot, immunoprecipitation, and immunofluorescence.

NeuroSGC/YCharOS/EDDU collaborative group

  Huntingtin encodes a 3144 amino acid protein, with a polyglutamine repeat tract at the N-terminus. Expansion of this repeat tract above a pathogenic threshold of 36 repeats is the causative mutation of Huntington's disease, a neurodegenerative disorder characterized by loss of striatal neurons. Here we have characterized twenty Huntingtin commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Keywords:  HTT; Huntingtin; UniProt ID P42858; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation; western blot

DOI:  https://doi.org/10.12688/f1000research.153670.1
bioRxiv. 2024 Aug 27. pii: 2024.08.27.607409. [Epub ahead of print]

A robust evaluation of TDP-43, poly GP, cellular pathology and behavior in a AAV-C9ORF72 (G4C2)66 mouse model.

Emily G Thompson, Olivia Spead, S Can Akerman, Carrie Curcio, Benjamin L Zaepfel, Erica R Kent, Thomas Philips, Balaji G Vijayakumar, Anna Zacco, Weibo Zhou, Guhan Nagappan, Jeffrey D Rothstein.

  The G4C2 hexanucleotide repeat expansion in C9ORF72 is the major genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9-ALS/FTD). Despite considerable efforts, the development of mouse models of C9-ALS/FTD useful for therapeutic development has proven challenging due to the intricate interplay of genetic and molecular factors underlying this neurodegenerative disorder, in addition to species differences. This study presents a robust investigation of the cellular pathophysiology and behavioral outcomes in a previously described AAV mouse model of C9-ALS expressing 66 G4C2 hexanucleotide repeats. Despite displaying key molecular ALS pathological markers including RNA foci, dipeptide repeat (DPR) protein aggregation, p62 positive stress granule formation as well as mild gliosis, the AAV-(G4C2)66 mouse model in this study exhibits negligible neuronal loss, no motor deficits, and functionally unimpaired TAR DNA-binding protein-43 (TDP-43). While our findings indicate and support that this is a robust and pharmacologically tractable model for investigating the molecular mechanisms and cellular consequences of (G4C2) repeat driven DPR pathology, it is not suitable for investigating the development of disease associated neurodegeneration, TDP-43 dysfunction, gliosis, and motor performance. Our findings underscore the complexity of ALS pathogenesis involving genetic mutations and protein dysregulation and highlight the need for more comprehensive model systems that reliably replicate the multifaceted cellular and behavioral aspects of C9-ALS.

Keywords:  Amyotrophic Lateral Sclerosis; C9orf72 repeat expansion; Dipeptide Repeats; GFAP; NfL; p62

DOI:  https://doi.org/10.1101/2024.08.27.607409
Cells. 2024 Sep 06. pii: 1498. [Epub ahead of print]13(17):

An Essential Role for Calnexin in ER-Phagy and the Unfolded Protein Response.

Daniel Wolf, Chiara Röder, Michael Sendtner, Patrick Lüningschrör.

  ER-phagy is a specialized form of autophagy, defined by the lysosomal degradation of ER subdomains. ER-phagy has been implicated in relieving the ER from misfolded proteins during ER stress upon activation of the unfolded protein response (UPR). Here, we identified an essential role for the ER chaperone calnexin in regulating ER-phagy and the UPR in neurons. We showed that chemical induction of ER stress triggers ER-phagy in the somata and axons of primary cultured motoneurons. Under basal conditions, the depletion of calnexin leads to an enhanced ER-phagy in axons. However, upon ER stress induction, ER-phagy did not further increase in calnexin-deficient motoneurons. In addition to increased ER-phagy under basal conditions, we also detected an elevated proteasomal turnover of insoluble proteins, suggesting enhanced protein degradation by default. Surprisingly, we detected a diminished UPR in calnexin-deficient early cortical neurons under ER stress conditions. In summary, our data suggest a central role for calnexin in orchestrating both ER-phagy and the UPR to maintain protein homeostasis within the ER.

Keywords:  ER stress; ER-phagy; UPR; calnexin; unfolded protein response

DOI:  https://doi.org/10.3390/cells13171498
Sci Adv. 2024 Sep 13. 10(37): eadk3700

Modeling Lewy body disease with SNCA triplication iPSC-derived cortical organoids and identifying therapeutic drugs.

Yunjung Jin, Fuyao Li, Zonghua Li, Tadafumi C Ikezu, Justin O'Leary, Manikandan Selvaraj, Yiyang Zhu, Yuka A Martens, Shunsuke Koga, Hannah Santhakumar, Yonghe Li, Wenyan Lu, Yang You, Kiara Lolo, Michael DeTure, Alexandra I Beasley, Mary D Davis, Pamela J McLean, Owen A Ross, Takahisa Kanekiyo, Tsuneya Ikezu, Thomas Caulfield, Jonathan Carr, Zbigniew K Wszolek, Guojun Bu, Dennis W Dickson, Na Zhao.

Aggregated α-synuclein (α-SYN) proteins, encoded by the SNCA gene, are hallmarks of Lewy body disease (LBD), affecting multiple brain regions. However, the specific mechanisms underlying α-SYN pathology in cortical neurons, crucial for LBD-associated dementia, remain unclear. Here, we recapitulated α-SYN pathologies in human induced pluripotent stem cells (iPSCs)-derived cortical organoids generated from patients with LBD with SNCA gene triplication. Single-cell RNA sequencing, combined with functional and molecular validation, identified synaptic and mitochondrial dysfunction in excitatory neurons exhibiting high expression of the SNCA gene, aligning with observations in the cortex of autopsy-confirmed LBD human brains. Furthermore, we screened 1280 Food and Drug Administration-approved drugs and identified four candidates (entacapone, tolcapone, phenazopyridine hydrochloride, and zalcitabine) that inhibited α-SYN seeding activity in real-time quaking-induced conversion assays with human brains, reduced α-SYN aggregation, and alleviated mitochondrial dysfunction in SNCA triplication organoids and excitatory neurons. Our findings establish human cortical LBD models and suggest potential therapeutic drugs targeting α-SYN aggregation for LBD.

DOI: https://doi.org/10.1126/sciadv.adk3700
Cell Rep Methods. 2024 Aug 30. pii: S2667-2375(24)00236-4. [Epub ahead of print] 100858

Fully defined NGN2 neuron protocol reveals diverse signatures of neuronal maturation.

Xiwei Shan, Ai Zhang, Mitchell G Rezzonico, Ming-Chi Tsai, Carlos Sanchez-Priego, Yingjie Zhang, Michelle B Chen, Meena Choi, José Miguel Andrade López, Lilian Phu, Amber L Cramer, Qiao Zhang, Jillian M Pattison, Christopher M Rose, Casper C Hoogenraad, Claire G Jeong.

  NGN2-driven induced pluripotent stem cell (iPSC)-to-neuron conversion is a popular method for human neurological disease modeling. In this study, we present a standardized approach for generating neurons utilizing clonal, targeted-engineered iPSC lines with defined reagents. We demonstrate consistent production of excitatory neurons at scale and long-term maintenance for at least 150 days. Temporal omics, electrophysiological, and morphological profiling indicate continued maturation to postnatal-like neurons. Quantitative characterizations through transcriptomic, imaging, and functional assays reveal coordinated actions of multiple pathways that drive neuronal maturation. We also show the expression of disease-related genes in these neurons to demonstrate the relevance of our protocol for modeling neurological disorders. Finally, we demonstrate efficient generation of NGN2-integrated iPSC lines. These workflows, profiling data, and functional characterizations enable the development of reproducible human in vitro models of neurological disorders.

Keywords:  CP: Neuroscience; CP: Stem cell; NGN2; disease modeling; iPSC; multi-omics profiling; neuron maturation; neuronal differentiation

DOI:  https://doi.org/10.1016/j.crmeth.2024.100858
Exp Neurol. 2024 Sep 09. pii: S0014-4886(24)00271-1. [Epub ahead of print] 114945

CHCHD10P80L knock-in zebrafish display a mild ALS-like phenotype.

Virginie Petel Légaré, Ziyaan A Harji, Christian J Rampal, Hana Antonicka, Tyler J N Gurberg, Olivia Persia, Esteban C Rodríguez, E A Shoubridge, Gary A B Armstrong.

  Mutations in the nuclear-encoded mitochondrial gene CHCHD10 have been observed in patients with a spectrum of diseases that include amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To investigate the pathogenic nature of disease-associated variants of CHCHD10 we generated a zebrafish knock-in (KI) model expressing the orthologous ALS-associated CHCHD10P80L variant (zebrafish: Chchd10P83L). Larval chchd10P83L/P83L fish displayed reduced Chchd10 protein expression levels, motor impairment, reduced survival and abnormal neuromuscular junctions (NMJ). These deficits were not accompanied by changes in transcripts involved in the integrated stress response (ISR), phenocopying previous findings in our knockout (chchd10-/-). Adult, 11-month old chchd10P83L/P83L zebrafish, displayed smaller slow- and fast-twitch muscle cell cross-sectional areas compared to wild type zebrafish muscle cells. Motoneurons in the spinal cord of chchd10P83L/P83L zebrafish displayed similar cross-sectional areas to that of wild type motor neurons and significantly fewer motor neurons were observed when compared to chchd2-/- adult spinal cords. Bulk RNA sequencing using whole spinal cords of 7-month old fish revealed transcriptional changes associated with neuroinflammation, apoptosis, amino acid metabolism and mt-DNA inflammatory response in our chchd10P83L/P83L model. The findings presented here, suggest that the CHCHD10P80L variant confers an ALS-like phenotype when expressed in zebrafish.

Keywords:  Als; CHCHD10; Knock-in mutation; Mitochondria; Motor neuron; Neurodegeneration; Zebrafish

DOI:  https://doi.org/10.1016/j.expneurol.2024.114945
Cell Rep. 2024 Sep 09. pii: S2211-1247(24)01070-2. [Epub ahead of print]43(9): 114719

Targeting EGLN2/PHD1 protects motor neurons and normalizes the astrocytic interferon response.

Christine Germeys, Tijs Vandoorne, Kristofer Davie, Suresh Poovathingal, Kara Heeren, Wendy Vermeire, FatemehArefeh Nami, Matthieu Moisse, Annelies Quaegebeur, Annerieke Sierksma, Laura Rué, Adrià Sicart, Caroline Eykens, Lenja De Cock, Bart De Strooper, Peter Carmeliet, Philip Van Damme, Katrien De Bock, Ludo Van Den Bosch.

  Neuroinflammation and dysregulated energy metabolism are linked to motor neuron degeneration in amyotrophic lateral sclerosis (ALS). The egl-9 family hypoxia-inducible factor (EGLN) enzymes, also known as prolyl hydroxylase domain (PHD) enzymes, are metabolic sensors regulating cellular inflammation and metabolism. Using an oligonucleotide-based and a genetic approach, we showed that the downregulation of Egln2 protected motor neurons and mitigated the ALS phenotype in two zebrafish models and a mouse model of ALS. Single-nucleus RNA sequencing of the murine spinal cord revealed that the loss of EGLN2 induced an astrocyte-specific downregulation of interferon-stimulated genes, mediated via the stimulator of interferon genes (STING) protein. In addition, we found that the genetic deletion of EGLN2 restored this interferon response in patient induced pluripotent stem cell (iPSC)-derived astrocytes, confirming the link between EGLN2 and astrocytic interferon signaling. In conclusion, we identified EGLN2 as a motor neuron protective target normalizing the astrocytic interferon-dependent inflammatory axis in vivo, as well as in patient-derived cells.

Keywords:  ALS; C9orf72; CP: Neuroscience; EGLN2; PHD1; SOD1; astrocyte; cGAS/STING; interferon; motor neuron; single-nucleus RNA sequencing

DOI:  https://doi.org/10.1016/j.celrep.2024.114719
Sci Rep. 2024 Sep 13. 14(1): 21383

Downregulation of Lnc-ABCA12-3 modulates UBQLN1 expression and protein homeostasis pathways in amyotrophic lateral sclerosis.

Yujiao Yu, Dejiang Pang, Jingxuan Huang, Chunyu Li, Yiyuan Cui, Huifang Shang.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron degeneration. Dysregulation of long non-coding RNAs (lncRNAs) has been implicated in ALS pathogenesis but their roles remain unclear. Previous studies found lnc-ABCA12-3 was downregulated in ALS patients. We aim to characterize the expression and function of lnc-ABCA12-3 in ALS and explore its mechanisms of action. Lnc-ABCA12-3 expression was analyzed in PBMCs from ALS patients and correlated with clinical outcomes. Effect of modulating lnc-ABCA12-3 expression was assessed in cell models using assays of apoptosis, protein homeostasis and pathway analysis. RNA pull-down and interaction studies were performed to identify lnc-ABCA12-3 binding partners. Lnc-ABCA12-3 was downregulated in ALS patients, correlating with faster progression and shorter survival. Overexpression of lnc-ABAC12-3 conferred protection against oxidative stress-induced apoptosis, while knockdown lnc-ABCA12-3 enhanced cell death. Lnc-ABCA12-3 maintained protein quality control pathways, including ubiquitination, autophagy and stress granule formation, by regulating the ubiquitin shuttle protein UBQLN1. This study identified lnc-ABCA12-3 as a novel regulatory lncRNA implicated in ALS pathogenesis by modulating cellular survival and stress responses through interactions with UBQLN1, influencing disease progression. Lnc-ABCA12-3 may influence ALS through regulating protein homeostasis pathways.

DOI: https://doi.org/10.1038/s41598-024-72666-8
Cells. 2024 Aug 24. pii: 1414. [Epub ahead of print]13(17):

The Six-Transmembrane Enzyme GDE2 Is Required for the Release of Molecularly Distinct Small Extracellular Vesicles from Neurons.

Kyle T Shuler, Josue Llamas-Rodriguez, Reuben Levy-Myers, Shanthini Sockanathan.

  Extracellular vesicles (EVs) are implicated in a multitude of physiological and pathophysiological processes in the nervous system; however, their biogenesis and cargoes are not well defined. Glycerophosphodiester Phosphodiesterase 2 (GDE2 or GDPD5) is a six-transmembrane protein that cleaves the Glycosylphosphatidylinositol (GPI)-anchor that tethers some proteins to the membrane and has important roles in neurodevelopment and disease-relevant pathways of neuronal survival. We show here that GDE2 regulates the number of small EVs (sEVs) released from the cell surface of neurons via its GPI-anchor cleavage activity and contributes to the loading of protein cargo through enzymatic and non-enzymatic mechanisms. Proteomic profiling reveals that GDE2 releases at least two distinct EV populations, one containing GDE2 itself and the other harboring the putative ectosomal markers CD9 and BSG. sEVs released by GDE2 are enriched in cytoskeletal and actin-remodeling proteins, suggesting a potential mechanism for GDE2-dependent EV release. Further, sEV populations released by GDE2 are enriched in proteins responsible for modulating synaptic activity and proteins that are critical for cellular redox homeostasis. These studies identify GDE2 as a novel regulator of molecularly distinct sEV populations from neurons with potential roles in the synaptic and redox pathways required for neuronal function and survival.

Keywords:  GDE2; cytoskeleton; ectosomes; extracellular vesicles; neurons; redox; synapse

DOI:  https://doi.org/10.3390/cells13171414
Life Sci Alliance. 2024 Nov;pii: e202402918. [Epub ahead of print]7(11):

Novel imaging tools to study mitochondrial morphology in Caenorhabditis elegans.

Miriam Valera-Alberni, Pallas Yao, Silvia Romero-Sanz, Anne Lanjuin, William B Mair.

Mitochondria exhibit a close interplay between their structure and function. Understanding this intricate relationship requires advanced imaging techniques that can capture the dynamic nature of mitochondria and their impact on cellular processes. However, much of the work on mitochondrial dynamics has been performed in single celled organisms or in vitro cell culture. Here, we introduce novel genetic tools for live imaging of mitochondrial morphology in the nematode Caenorhabditis elegans, addressing a pressing need for advanced techniques in studying organelle dynamics within live intact multicellular organisms. Through a comprehensive analysis, we directly compare our tools with existing methods, demonstrating their advantages for visualizing mitochondrial morphology and contrasting their impact on organismal physiology. We reveal limitations of conventional techniques, whereas showcasing the utility and versatility of our approaches, including endogenous CRISPR tags and ectopic labeling. By providing a guide for selecting the most suitable tools based on experimental goals, our work advances mitochondrial research in C. elegans and enhances the strategic integration of diverse imaging modalities for a holistic understanding of organelle dynamics in living organisms.

DOI: https://doi.org/10.26508/lsa.202402918
J Neurosci Res. 2024 Sep;102(9): e25382

Microtubules, Membranes, and Movement: New Roles for Stathmin-2 in Axon Integrity.

Emma J C Thornburg-Suresh, Daniel W Summers.

  Neurons establish functional connections responsible for how we perceive and react to the world around us. Communication from a neuron to its target cell occurs through a long projection called an axon. Axon distances can exceed 1 m in length in humans and require a dynamic microtubule cytoskeleton for growth during development and maintenance in adulthood. Stathmins are microtubule-associated proteins that function as relays between kinase signaling and microtubule polymerization. In this review, we describe the prolific role of Stathmins in microtubule homeostasis with an emphasis on emerging roles for Stathmin-2 (Stmn2) in axon integrity and neurodegeneration. Stmn2 levels are altered in Amyotrophic Lateral Sclerosis and loss of Stmn2 provokes motor and sensory neuropathies. There is growing potential for employing Stmn2 as a disease biomarker or even a therapeutic target. Meeting this potential requires a mechanistic understanding of emerging complexity in Stmn2 function. In particular, Stmn2 palmitoylation has a surprising contribution to axon maintenance through undefined mechanisms linking membrane association, tubulin interaction, and axon transport. Exploring these connections will reveal new insight on neuronal cell biology and novel opportunities for disease intervention.

Keywords:  ALS; Stmn2; axon degeneration; cytoskeleton; neurodegeneration; palmitoylation

DOI:  https://doi.org/10.1002/jnr.25382
Front Mol Neurosci. 2024 ;17 1393779

Ptbp2 re-expression rescues axon growth defects in Smn-deficient motoneurons.

Saeede Salehi, Abdolhossein Zare, Gayatri Gandhi, Michael Sendtner, Michael Briese.

  Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by mutations or deletions in the survival motoneuron 1 (SMN1) gene, resulting in deficiency of the SMN protein that is essential for motoneuron function. Smn depletion in mice disturbs axonal RNA transport and translation, thereby contributing to axon growth impairment, muscle denervation, and motoneuron degeneration. However, the mechanisms whereby Smn loss causes axonal defects remain unclear. RNA localization and translation in axons are controlled by RNA-binding proteins (RBP) and we recently observed that the neuronal RBP Ptbp2 modulates axon growth in motoneurons. Here, we identify Smn as an interactor of Ptbp2 in the cytosolic compartments of motoneurons. We show that the expression level of Ptbp2 is reduced in axons but not in the somata of Smn-depleted motoneurons. This is accompanied by reduced synthesis of the RBP hnRNP R in axons. Re-expression of Ptbp2 in axons compensates for the deficiency of Smn and rescues the defects in axon elongation and growth cone maturation observed in Smn-deficient motoneurons. Our data suggest that Ptbp2 and Smn are components of cytosolic mRNP particles, contributing to the precise spatial and temporal control of protein synthesis within axons and axon terminals.

Keywords:  Ptbp2; SMN; axon growth; axonal RNA transport; axonal translation; spinal muscular atrophy

DOI:  https://doi.org/10.3389/fnmol.2024.1393779
MedComm (2020). 2024 Sep;5(9): e712

Loss-of-function variants in RNA binding motif protein X-linked induce neuronal defects contributing to amyotrophic lateral sclerosis pathogenesis.

Di He, Xinyi He, Dongchao Shen, Liyang Liu, Xunzhe Yang, Meng Hao, Yi Wang, Yi Li, Qing Liu, Mingsheng Liu, Jiucun Wang, Xue Zhang, Liying Cui.

  Despite being one of the most prevalent RNA modifications, the role of N6-methyladenosine (m6A) in amyotrophic lateral sclerosis (ALS) remains ambiguous. In this investigation, we explore the contribution of genetic defects of m6A-related genes to ALS pathogenesis. We scrutinized the mutation landscape of m6A genes through a comprehensive analysis of whole-exome sequencing cohorts, encompassing 508 ALS patients and 1660 population-matched controls. Our findings reveal a noteworthy enrichment of RNA binding motif protein X-linked (RBMX) variants among ALS patients, with a significant correlation between pathogenic m6A variants and adverse clinical outcomes. Furthermore, Rbmx knockdown in NSC-34 cells overexpressing mutant TDP43Q331K results in cell death mediated by an augmented p53 response. Similarly, RBMX knockdown in ALS motor neurons derived from induced pluripotent stem cells (iPSCs) manifests morphological defects and activation of the p53 pathway. Transcriptional analysis using publicly available single-cell sequencing data from the primary motor cortex indicates that RBMX-regulated genes selectively influence excitatory neurons and exhibit enrichment in ALS-implicated pathways. Through integrated analyses, our study underscores the emerging roles played by RBMX in ALS, suggesting a potential nexus between the disease and dysregulated m6A-mediated mRNA metabolism.

Keywords:  ALS; RBMX; m6A modification; single‐cell sequencing; whole‐exome sequencing

DOI:  https://doi.org/10.1002/mco2.712
Mol Ther Methods Clin Dev. 2024 Sep 12. 32(3): 101312

Combination AAV therapy with galectin-1 and SOD1 downregulation demonstrates superior therapeutic effect in a severe ALS mouse model.

Megan C Baird, Shibi B Likhite, Tatyana A Vetter, Joseph R Caporale, Holly B Girard, Florence S Roussel, Abigail E Howard, Maura K Schwartz, Addison R Reed, Abuzar Kaleem, Xiaojin Zhang, Kathrin C Meyer.

  Neuroinflammation is a miscreant in accelerating progression of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). However, treatments targeting neuroinflammation alone have led to disappointing results in clinical trials. Both neuronal and non-neuronal cell types have been implicated in the pathogenesis of ALS, and multiple studies have shown correction of each cell type has beneficial effects on disease outcome. Previously, we shown that AAV9-mediated superoxide dismutase 1 (SOD1) suppression in motor neurons and astrocytes significantly improves motor function and extends survival in ALS mouse models. Despite neuron and astrocyte correction, ALS mice still succumb to death with microgliosis observed in endpoint tissue. Therefore, we hypothesized that the optimal therapeutic approach will target and simultaneously correct motor neurons, astrocytes, and microglia. Here, we developed a novel approach to indirectly target microglia with galectin-1 (Gal1) and combined this with our previously established AAV9.SOD1.short hairpin RNA treatment. We show Gal1 conditioning of SOD1 G93A microglia decreases inflammatory markers and rescues motor neuron death in vitro. When paired with SOD1 downregulation, we found a synergistic effect of combination treatment in vivo and show a significant extension of survival of SOD1 G93A mice over SOD1 suppression alone. These results highlight the importance of targeting inflammatory microglia as a critical component in future therapeutic development.

Keywords:  AAV; SOD1G93A mouse; amyotrophic lateral sclerosis; combination therapy; galectin; gene therapy; microglia; motor neuron disease; neuroinflammation; non-cell autonomous toxicity

DOI:  https://doi.org/10.1016/j.omtm.2024.101312
J Neurosci. 2024 Sep 12. pii: e1822232024. [Epub ahead of print]

A miR-383-5p signalling hub coordinates the axon regeneration response to inflammation.

Matthew A Hintermayer, Camille A Juźwik, Barbara Morquette, Elizabeth Hua, Julia Zhang, Sienna Drake, Shan Shan Shi, Isabel Rambaldi, Vamshi Vangoor, Jeroen Pasterkamp, Craig Moore, Alyson E Fournier.

Neuroinflammation can positively influence axon regeneration following injury in the central nervous system (CNS). Inflammation promotes the release of neurotrophic molecules and stimulates intrinsic pro-regenerative molecular machinery in neurons, but the detailed mechanisms driving this effect are not fully understood. We evaluated how microRNAs are regulated in retinal neurons in response to intraocular inflammation to identify their potential role in axon regeneration. We found that miR-383-5p is downregulated in retinal ganglion cells in response to zymosan-induced intraocular inflammation. MiR-383-5p downregulation in neurons is sufficient to promote axon growth in vitro, and the intravitreal injection of a miR-383-5p inhibitor into the eye promotes axon regeneration following optic nerve crush. MiR-383-5p directly targets ciliary neurotrophic factor (CNTF) receptor components and miR-383-5p inhibition sensitizes adult retinal neurons to the outgrowth-promoting effects of CNTF. Interestingly, we also demonstrate that CNTF treatment is sufficient to reduce miR-383-5p levels in neurons, constituting a positive-feedback module whereby initial CNTF treatment reduces miR-383-5p levels, which then disinhibits CNTF receptor components to sensitize neurons to the ligand. Additionally, miR-383-5p inhibition de-represses the mitochondrial antioxidant protein peroxiredoxin-3 (PRDX3) which was required for the pro-regenerative effects associated with miR-383-5p loss of function in vitro. We have thus identified a positive feedback mechanism that facilitates neuronal CNTF sensitivity in neurons, and a new molecular signalling module that promotes inflammation-induced axon regeneration.Significance statement Inflammation can both positively and negatively influence the neuronal response to injury. Identifying molecular signalling pathways that mimic pro-regenerative effects of inflammation while bypassing cytotoxic effects is important for our understanding of the precise functions of inflammation in CNS injury and repair. We demonstrate that miR-383-5p is suppressed in neurons in response to inflammatory stimuli and regulates members of the ciliary neurotrophic factor (CNTF) receptor complex, as well as the expression of an antioxidant protein to improve axon regeneration in an optic nerve crush model. These findings identify a new molecular signalling module that promotes axon regeneration and that may bypass detrimental effects of inflammation.

DOI: https://doi.org/10.1523/JNEUROSCI.1822-23.2024
Behav Brain Res. 2024 Sep 05. pii: S0166-4328(24)00398-X. [Epub ahead of print]476 115242

Emerging targets in amyotrophic lateral sclerosis (ALS): The promise of ATP-binding cassette (ABC) transporter modulation.

Maneesh Mohan, Ashi Mannan, Aayush Nauriyal, Thakur Gurjeet Singh.

  Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative primarily affecting motor neurons, leading to disability and neuronal death, and ATP-Binding Cassette (ABC) transporter due to their role in drug efflux and modulation of various cellular pathways contributes to the pathogenesis of ALS. In this article, we extensively investigated various molecular and mechanistic pathways linking ALS transporter to the pathogenesis of ALS; this involves inflammatory pathways such as Mitogen-Activated Protein Kinase (MAPK), Phosphatidylinositol-3-Kinase/Protein Kinase B (PI3K/Akt), Toll-Like Receptor (TLR), Glycogen Synthase Kinase 3β (GSK-3β), Nuclear Factor Kappa-B (NF-κB), and Cyclooxygenase (COX). Oxidative pathways such as Astrocytes, Glutamate, Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), Sirtuin 1 (SIRT-1), Forkhead box protein O (FOXO), Extracellular signal-regulated kinase (ERK). Additionally, we delve into the role of autophagic pathways like TAR DNA-binding protein 43 (TDP-43), AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR), and lastly, the apoptotic pathways. Furthermore, by understanding these intricate interactions, we aim to develop novel therapeutic strategies targeting ABC transporters, improving drug delivery, and ultimately offering a promising avenue for treating ALS.

Keywords:  ATP-binding cassette (ABC) transporters; Apoptosis; Inflammation; Molecular pathways; Motor neurons; Neurodegeneration; Oxidative stress

DOI:  https://doi.org/10.1016/j.bbr.2024.115242
Neurochem Int. 2024 Sep 04. pii: S0197-0186(24)00181-5. [Epub ahead of print] 105854

Generation and characterization of cortical organoids from iPSC-derived dental pulp stem cells using traditional and innovative approaches.

André Luíz Teles E Silva, Bruno Yukio Yokota-Moreno, Mariana Silva Branquinho, Geisa Rodrigues Salles, Thiago Cattuzo de Souza, Ronald Almeida de Carvalho, Gabriel Batista, Elisa Varella Branco, Karina Griesi-Oliveira, Maria Rita Passos Bueno, Marimélia Aparecida Porcionatto, Roberto Hirochi Herai, Lionel Fernel Gamarra, Andrea Laurato Sertié.

  Cortical organoids derived from human induced pluripotent stem cells (hiPSCs) represent a powerful in vitro experimental system to investigate human brain development and disease, often inaccessible to direct experimentation. However, despite steady progress in organoid technology, several limitations remain, including high cost and variability, use of hiPSCs derived from tissues harvested invasively, unexplored three-dimensional (3D) structural features and neuronal connectivity. Here, using a cost-effective and reproducible protocol as well as conventional two-dimensional (2D) immunostaining, we show that cortical organoids generated from hiPSCs obtained by reprogramming stem cells from human exfoliated deciduous teeth (SHED) recapitulate key aspects of human corticogenesis, such as polarized organization of neural progenitor zones with the presence of outer radial glial stem cells, and differentiation of superficial- and deep-layer cortical neurons and glial cells. We also show that 3D bioprinting and magnetic resonance imaging of intact cortical organoids are alternative and complementary approaches to unravel critical features of the 3D architecture of organoids. Finally, extracellular electrical recordings in whole organoids showed functional neuronal networks. Together, our findings suggest that SHED-derived cortical organoids constitute an attractive model of human neurodevelopment, and support the notion that a combination of 2D and 3D techniques to analyze organoid structure and function may help improve this promising technology.

Keywords:  2D and 3D structural analysis; SHED-derived cortical organoids; Stem cells from human exfoliated deciduous teeth (SHED); extracellular measurements of neuronal connectivity

DOI:  https://doi.org/10.1016/j.neuint.2024.105854
bioRxiv. 2024 Aug 28. pii: 2023.04.09.536178. [Epub ahead of print]

Astrocytic LRRK2 Controls Synaptic Connectivity via Regulation of ERM Phosphorylation.

Shiyi Wang, Ryan Baumert, Gabrielle Séjourné, Dhanesh Sivadasan Bindu, Kylie Dimond, Kristina Sakers, Leslie Vazquez, Jessica Moore, Christabel Xin Tan, Tetsuya Takano, Maria Pia Rodriguez, Scott H Soderling, Albert R La Spada, Cagla Eroglu.

Astrocytes, a major glial cell type of the brain, regulate synapse numbers and function. However, whether astrocyte dysfunction can cause synaptic pathologies in neurological disorders such as Parkinson's Disease (PD) is unknown. Here, we investigated the impact of the most common PD-linked mutation in the leucine-rich repeat kinase 2 ( LRRK2 ) gene (G2019S) on the synaptic functions of astrocytes. We found that both in human and mouse cortex, the LRRK2 G2019S mutation causes astrocyte morphology deficits and enhances the phosphorylation of the ERM proteins (Ezrin, Radixin, and Moesin), which are important components of perisynaptic astrocyte processes. Reducing ERM phosphorylation in LRRK2 G2019S mouse astrocytes restored astrocyte morphology and corrected excitatory synaptic deficits. Using an in vivo BioID proteomic approach, we found Ezrin, the most abundant astrocytic ERM protein, interacts with the Autophagy-Related 7 (Atg7), a master regulator of catabolic processes. The Ezrin/Atg7 interaction is inhibited by Ezrin phosphorylation, thus diminished in the LRRK2 G2019S astrocytes. Importantly, Atg7 function is required to maintain proper astrocyte morphology. These studies reveal an astrocytic molecular mechanism that could serve as a therapeutic target in PD.

DOI: https://doi.org/10.1101/2023.04.09.536178
J Neurol Sci. 2024 Sep 02. pii: S0022-510X(24)00345-9. [Epub ahead of print]466 123210

Quantification of serum TDP-43 and neurofilament light chain in patients with amyotrophic lateral sclerosis stratified by UNC13A genotype.

Valeria Casiraghi, Ilaria Milone, Alberto Brusati, Silvia Peverelli, Alberto Doretti, Barbara Poletti, Luca Maderna, Claudia Morelli, Nicola Ticozzi, Vincenzo Silani, Federico Verde, Antonia Ratti.

  Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative condition affecting upper and/or lower motor neurons and characterized neuropathologically by TDP-43 proteinopathy. Given its role in ALS pathobiology, it is currently under debate whether TDP-43 might represent a suitable ALS biomarker to be measured in patients' biofluids. The rs12608932 A > C single nucleotide polymorphism in the UNC13A gene is a risk factor for ALS and patients homozygous for the high-risk C allele display a higher burden of TDP-43 neuropathology than homozygotes for the low-risk A allele, although the association with TDP-43 levels in biofluids has never been evaluated. In this study, we measured serum levels of TDP-43 and neurofilament light chain (NFL) by Simoa technology in a cohort of 69 ALS patients stratified according to the UNC13A rs12608932 genotype compared to 43 neurologically healthy controls. By multiple linear regression analysis, serum TDP-43 was significantly elevated in ALS patients compared to controls, with UNC13A AA and AC, but not CC, ALS patients showing higher serum TDP-43 levels than controls. We also confirmed that serum NFL concentration was increased in ALS patients, without any correlation with the UNC13A genotype. Our results indicate that serum TDP-43 is higher in ALS patients compared to controls and that, in contrast to NFL, this increase is specifically associated with the UNC13A rs12608932 AA and AC genotypes, but not with the high-risk CC genotype. Studies in larger cohorts will be needed to confirm these findings and to elucidate the biological link between serum TDP-43 levels and UNC13A genotype.

Keywords:  ALS; NFL; TDP-43; UNC13A

DOI:  https://doi.org/10.1016/j.jns.2024.123210
Nat Commun. 2024 Sep 10. 15(1): 7887

Engineered NLS-chimera downregulates expression of aggregation-prone endogenous FUS.

Miyuki Hayashi, Amandeep Girdhar, Ying-Hui Ko, Kevin M Kim, Jacquelyn A DePierro, Joseph R Buchler, Nikhita Arunprakash, Aditya Bajaj, Gino Cingolani, Lin Guo.

Importin β-superfamily nuclear import receptors (NIRs) mitigate mislocalization and aggregation of RNA-binding proteins (RBPs), like FUS and TDP-43, which are implicated in neurodegenerative diseases. NIRs potently disaggregate RBPs by recognizing their nuclear localization signal (NLS). However, disease-causing mutations in NLS compromise NIR binding and activity. Here, we define features that characterize the anti-aggregation activity of NIR and NLS. We find that high binding affinity between NIR and NLS, and optimal NLS location relative to the aggregating domain plays a role in determining NIR disaggregation activity. A designed FUS chimera (FUSIBB), carrying the importin β binding (IBB) domain, is solubilized by importin β in vitro, translocated to the nucleus in cultured cells, and downregulates the expression of endogenous FUS. In this study, we posit that guiding the mutual recognition of NLSs and NIRs will aid the development of therapeutics, illustrated by the highly soluble FUSIBB replacing the aggregation-prone endogenous FUS.

DOI: https://doi.org/10.1038/s41467-024-52151-6
Adv Exp Med Biol. 2024 ;1456 85-91

Induce Pluripotent Stem Cells (iPSC) Technology in Depression.

Apurva Kumar, Laura Stertz, Antonio L Teixeira.

  Induced pluripotent stem cells (iPSCs) are a promising in vitro model for drug-screening and precision-based psychiatry for the treatment of major depressive disorders (MDD). In this chapter, we explore different uses for iPSC technology, three-dimensional (3D) organoids models, and mesenchymal stem cells therapy in MDD, as well their potential and limitations.

Keywords:  Depression; Neurogenesis; Organoids; Stem cells; iPSC

DOI:  https://doi.org/10.1007/978-981-97-4402-2_5
Cells. 2024 Aug 28. pii: 1441. [Epub ahead of print]13(17):

V-ATPase Dysfunction in the Brain: Genetic Insights and Therapeutic Opportunities.

Antonio Falace, Greta Volpedo, Marcello Scala, Federico Zara, Pasquale Striano, Anna Fassio.

  Vacuolar-type ATPase (v-ATPase) is a multimeric protein complex that regulates H+ transport across membranes and intra-cellular organelle acidification. Catabolic processes, such as endocytic degradation and autophagy, strictly rely on v-ATPase-dependent luminal acidification in lysosomes. The v-ATPase complex is expressed at high levels in the brain and its impairment triggers neuronal dysfunction and neurodegeneration. Due to their post-mitotic nature and highly specialized function and morphology, neurons display a unique vulnerability to lysosomal dyshomeostasis. Alterations in genes encoding subunits composing v-ATPase or v-ATPase-related proteins impair brain development and synaptic function in animal models and underlie genetic diseases in humans, such as encephalopathies, epilepsy, as well as neurodevelopmental, and degenerative disorders. This review presents the genetic and functional evidence linking v-ATPase subunits and accessory proteins to various brain disorders, from early-onset developmental epileptic encephalopathy to neurodegenerative diseases. We highlight the latest emerging therapeutic strategies aimed at mitigating lysosomal defects associated with v-ATPase dysfunction.

Keywords:  lysosomal dysfunction; neurodegeneration; neurodevelopmental disorders; v-ATPse

DOI:  https://doi.org/10.3390/cells13171441
Mol Cell Proteomics. 2024 Sep 07. pii: S1535-9476(24)00128-2. [Epub ahead of print] 100838

Proteome dynamics in iPSC-derived human dopaminergic neurons.

Claudia Cavarischia-Rega, Karan Sharma, Julia C Fitzgerald, Boris Macek.

  Dopaminergic neurons participate in fundamental physiological processes and are the cell type primarily affected in Parkinson's disease. Their analysis is challenging due to the intricate nature of their function, involvement in diverse neurological processes, heterogeneity and localization in deep brain regions. Consequently, most of the research on the protein dynamics of dopaminergic neurons has been performed in animal cells ex vivo. Here we use iPSC-derived human mid-brain specific dopaminergic neurons to study general features of their proteome biology and provide datasets for protein turnover and dynamics, including a human axonal translatome. We cover the proteome to a depth of 9,409 proteins and use dynamic SILAC to measure the half-life of more than 4,300 proteins. We report uniform turnover rates of conserved cytosolic protein complexes such as the proteasome and map the variable rates of turnover of the respiratory chain complexes in these cells. We use differential dynamic SILAC labeling in combination with microfluidic devices to analyze local protein synthesis and transport between axons and soma. We report 105 potentially novel axonal markers and detect translocation of 269 proteins between axons and the soma in the time frame of our analysis (120 hours). Importantly, we provide evidence for local synthesis of 154 proteins in the axon and their retrograde transport to the soma, among them several proteins involved in RNA editing such as ADAR1 and the RNA helicase DHX30, involved in the assembly of mitochondrial ribosomes. Our study provides a workflow and resource for future applications of quantitative proteomics in iPSC-derived human neurons.

Keywords:  axon; dopaminergic neurons; iPSC; protein turnover; proteomics

DOI:  https://doi.org/10.1016/j.mcpro.2024.100838
Int J Mol Sci. 2024 Sep 09. pii: 9747. [Epub ahead of print]25(17):

Experimental Cell Models for Investigating Neurodegenerative Diseases.

Cecilia Evangelisti, Sherin Ramadan, Antonio Orlacchio, Emanuele Panza.

  Experimental models play a pivotal role in biomedical research, facilitating the understanding of disease mechanisms and the development of novel therapeutics. This is particularly true for neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, and motor neuron disease, which present complex challenges for research and therapy development. In this work, we review the recent literature about experimental models and motor neuron disease. We identified three main categories of models that are highly studied by scientists. In fact, experimental models for investigating these diseases encompass a variety of approaches, including modeling the patient's cell culture, patient-derived induced pluripotent stem cells, and organoids. Each model offers unique advantages and limitations, providing researchers with a range of tools to address complex biological questions. Here, we discuss the characteristics, applications, and recent advancements in terms of each model system, highlighting their contributions to advancing biomedical knowledge and translational research.

Keywords:  gene editing; iPSCs; model organisms; organoid; primary cells

DOI:  https://doi.org/10.3390/ijms25179747
Mol Cell Neurosci. 2024 Sep 10. pii: S1044-7431(24)00054-X. [Epub ahead of print]131 103969

SUMOylation modulates mitochondrial dynamics in an in vitro rotenone model of Parkinson's disease.

Ericks Sousa Soares, Letícia Yoshitome Queiroz, Ellen Gerhardt, Rui Daniel S Prediger, Tiago Fleming Outeiro, Helena Iturvides Cimarosti.

  SUMOylation is a post-translational modification essential for various biological processes. SUMO proteins bind to target substrates in a three-step enzymatic pathway, which is rapidly reversible by the action of specific proteases, known as SENPs. Studies have shown that SUMOylation is dysregulated in several human disorders, including neurodegenerative diseases that are characterized by the progressive loss of neurons, mitochondrial dysfunction, deficits in autophagy, and oxidative stress. Considering the potential neuroprotective roles of SUMOylation, the aim of this study was to investigate the effects of SENP3 knockdown in H4 neuroglioma cells exposed to rotenone, an in vitro model of cytotoxicity that mimics dopaminergic loss in Parkinson's disease (PD). The current data show that SENP3 knockdown increases SUMO-2/3 conjugates, which is accompanied by reduced levels of the mitochondrial fission protein Drp1 and increased levels of the mitochondrial fusion protein OPA1. Of high interest, SENP3 knockdown prevented rotenone-induced superoxide production and cellular death. Taken together, these findings highlight the importance of SUMOylation in maintaining mitochondrial homeostasis and the neuroprotective potential of this modification in PD.

Keywords:  Mitochondria; Parkinson's disease; SENP3 knockdown; SUMOylation

DOI:  https://doi.org/10.1016/j.mcn.2024.103969
J Cell Mol Med. 2024 Sep;28(17): e18560

Brain organoids: A new tool for modelling of neurodevelopmental disorders.

Yirizhati Aili, Nuersimanguli Maimaitiming, Zengliang Wang, Yongxin Wang.

  Neurodevelopmental disorders are mostly studied using mice as models. However, the mouse brain lacks similar cell types and structures as those of the human brain. In recent years, emergence of three-dimensional brain organoids derived from human embryonic stem cells or induced pluripotent stem cells allows for controlled monitoring and evaluation of early neurodevelopmental processes and has opened a window for studying various aspects of human brain development. However, such organoids lack original anatomical structure of the brain during maturation, and neurodevelopmental maturation processes that rely on unique cellular interactions and neural network connections are limited. Consequently, organoids are difficult to be used extensively and effectively while modelling later stages of human brain development and disease progression. To address this problem, several methods and technologies have emerged that aim to enhance the sophisticated regulation of brain organoids developmental processes through bioengineering approaches, which may alleviate some of the current limitations. This review discusses recent advances and application areas of human brain organoid culture methods, aiming to generalize optimization strategies for organoid systems, improve the ability to mimic human brain development, and enhance the application value of organoids.

Keywords:  brain organoids; induced pluripotent stem cells; neurodevelopmental diseases; preclinical models

DOI:  https://doi.org/10.1111/jcmm.18560