bims-axbals 2025-08-10 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2025–08–10
38 papers selected by
TJ Krzystek

Sporadic ALS induced pluripotent stem cell derived neurons reveal hallmarks of TDP-43 loss of function.
Convergent activation of the integrated stress response and ER-mitochondria uncoupling in VAPB-associated ALS.
Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device.
Cytoplasmic TDP-43 leads to early functional impairments without neurodegeneration in a Serotonergic Neuron-Specific C. elegans Model.
Dysregulated Expression of Inflammasome and Extracellular Matrix Genes in C9orf72-ALS/FTD Microglia.
Hide-and-Seek genome editing reveals that Gephyrin is required for axo-axonic synapse assembly.
Evaluating TRIM46's Role in Axon Initial Segment Formation.
14-3-3 binding maintains the Parkinson's associated kinase LRRK2 in an inactive state.
Neuron-derived extracellular vesicles in plasma present a potential non-invasive biomarker for Huntingtin protein and RNA assessment in Huntington disease.
LRRK2 kinase activity regulates Parkinson's disease-relevant lipids at the lysosome.
The role of astrocytes in human Huntington's disease pathology.
Machine learning-based proteomics profiling of ALS identifies downregulation of RPS29 that maintains protein homeostasis and STMN2 level.
Effects of natural Lithium and Lithium isotopes on voltage gated sodium channel activity in SH-SY5Y and IPSC derived cortical neurons.
Cryptic intronic transcriptional initiation generates efficient endogenous mRNA templates for C9orf72-associated RAN translation.
SOD1 is delivered to lysosomes via autophagy to maintain lysosomal function and integrity.
Transcriptome-based screening in TARDBP/TDP-43 knock-in motor neurons identifies the NEDD8-activating enzyme inhibitor MLN4924.
The complex web of membrane contact sites in brain aging and neurodegeneration.
G2019S LRRK2 mutation causing Parkinson's disease without Lewy bodies.
Expression of amyotrophic lateral sclerosis associated protein disulfide isomerase A3 D217N variant recapitulates early morphological alterations at the neuromuscular junction.
Dominant-negative isoform of TDP-43 is regulated by ALS-linked RNA-binding proteins.
Restoring DSCAM expression rescues neuronal morphology and axon guidance deficits in Down syndrome.
ATP Synthase Abundance in Neuronal Extracellular Vesicles Reflects Changes in the Mitochondria of Parent Neurons.
Protocol for the induction of human spinal motor neurons from human induced pluripotent stem cells for studying amyotrophic lateral sclerosis.
Treatment of age-related decreases in GTP levels restores endocytosis and autophagy.
PDZD8 links organelle crosstalk to synaptic remodeling via autophagy.
Specialized axon initial segment enables low firing threshold and rapid action potential output in fast-spiking interneurons of the human neocortex.
OPTN deficiency through CRISPR/Cas9 downregulates autophagy and mitophagy in a SOD1-G93A-expressing transgenic cell line.
Generation of human iPSC-derived pancreatic organoids to study pancreas development and disease.
Drug-Induced Reversible Lysosomal Changes Tracked in Live Cells by Holo-Tomographic Flow Cytometry.
A 3D iPSC retina model reveals non-cell-autonomous and non-neuronal mechanism of photoreceptor degeneration in a lysosomal storage disorder.
Accumulation of TDP-43 causes karyopherin-α4 pathology that characterises amyotrophic lateral sclerosis.
Lysosomal membrane homeostasis and its importance in physiology and disease.
Amyotrophic lateral sclerosis in Mainland China: clinical translational challenges and opportunities.
Application of brain organoids in neurodevelopmental disorders.
Knock-out of Tpm4.2/Actin Filaments Alters Neuronal Signaling, Neurite Outgrowth, and Behavioral Phenotypes in Mice.
Inhibition of Sirtuin 2 enhances autophagy and restores neuronal function in aged hippocampal neurons.
The membrane skeleton is constitutively remodeled in neurons by calcium signaling.
Autophagy activator AA-20 improves proteostasis and extends Caenorhabditis elegans lifespan.

Nat Commun. 2025 Aug 02. 16(1): 7092

Sporadic ALS induced pluripotent stem cell derived neurons reveal hallmarks of TDP-43 loss of function.

Jeffrey D Rothstein, Olivia Keeley, Caroline Warlick, Timothy M Miller, Cindy V Ly, Jonathan D Glass, Alyssa N Coyne.

Nuclear loss and cytoplasmic buildup of the RNA-binding protein TDP-43 is a hallmark of ALS and related disorders. While studies using artificial TDP-43 depletion in neurons have revealed changes in gene expression and splicing, their relevance to actual patients remained unclear. Induced pluripotent stem cell (iPSC)-derived neurons (iPSNs) from 180 individuals, including controls, C9orf72 ALS/FTD, and sporadic ALS (sALS) patients were used to generate and analyze ~32,500 qRT-PCR data points across 20 genes which identified variable, time-dependent signatures of TDP-43 loss of function in individual lines. Notably, the same changes were also seen in postmortem brain tissue from the same patients, confirming that iPSNs accurately model disease. Inducing damage to the nuclear pore complex, specifically by reducing the nucleoporin POM121 in healthy iPSNs, was enough to replicate the molecular changes associated with ALS/FTD TDP-43 dysfunction. This directly links nuclear pore integrity to TDP-43-related pathology. Encouragingly, repairing nuclear pore injury in sALS iPSNs restored normal gene processing disrupted by TDP-43 loss. This study (1) provides a valuable population-scale resource for studying TDP-43 dysfunction in ALS, (2) confirms that patient-derived iPSNs closely reflect disease processes seen in the brain, and (3) demonstrates that targeting nuclear pore injury may offer a promising therapeutic strategy in ALS.

DOI: https://doi.org/10.1038/s41467-025-62482-7
EMBO Mol Med. 2025 Aug 05.

Convergent activation of the integrated stress response and ER-mitochondria uncoupling in VAPB-associated ALS.

Curran Landry, James P Costanzo, Miguel Mitne-Neto, Mayana Zatz, Ashleigh E Schaffer, Maria Hatzoglou, Alysson R Muotri, Helen C Miranda.

  Vesicle-associated membrane protein-associated protein-B (VAPB) is an endoplasmic reticulum (ER) membrane-bound protein. The P56S mutation in VAPB causes a dominant, familial form of amyotrophic lateral sclerosis (ALS). However, the mechanism by which this mutation leads to motor neuron (MN) degeneration remains unclear. Utilizing inducible pluripotent stem cell (iPSC)-derived MNs expressing either wild-type (WT) or P56S VAPB, we demonstrate that the mutant protein reduces neuronal firing and disrupts ER-mitochondria-associated membranes (ER MAMs), with a time-dependent decline in mitochondrial membrane potential (MMP), hallmarks of MN pathology. These findings were validated in patient-derived iPSC-MNs. Additionally, VAPB P56S MNs show increased susceptibility to ER stress, elevated expression of the Integrated Stress Response (ISR) regulator ATF4 under stress, and reduced global protein synthesis. Notably, pharmacological ISR inhibition using ISRIB rescued ALS-associated phenotypes in both VAPB P56S and patient-derived iPSC-MNs. We present the first evidence that the VAPB P56S mutation activates ISR signaling via mitochondrial dysfunction in human MNs. These findings support ISR modulation as a strategy for ALS intervention and highlight the need for patient stratification in clinical trials.

Keywords:  ALS (Amyotrophic Lateral Sclerosis); ER-MAM (Endoplasmic Reticulum Mitochondria Associated Membrane); ISR (Integrated Stress Response); Neurodegeneration; VAPB ((Vesicle Associated Membrane Protein Associated Protein B)

DOI:  https://doi.org/10.1038/s44321-025-00279-3
Front Cell Neurosci. 2025 ;19 1590732

Investigation of early axonal phenotypes in an iPSC-derived ALS cellular model using a microfluidic device.

Asako Otomo, Keiko Nishijima, Yuta Murakami, Mitsuru Ishikawa, Haruka Yudahira, Kento Shimakura, Hideyuki Okano, Masashi Aoki, Hiroshi Kimura, Shinji Hadano.

   Introduction: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease caused by the loss of upper and lower motor neurons. Mutations in the FUS/TLS gene have been reported as the second most common mutation in Japanese patients with familial ALS. In recent years, lower motor neurons (LMNs) differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients have been widely used to analyze the mechanisms of neuronal cell death and degeneration.
Methods: In this study, we developed a microfluidic device designed to observe axonal growth, morphology, and trafficking at high resolution in neurons derived from induced pluripotent stem cells (iPSCs) and tested whether our microfluidic device effectively evaluates neurodegenerative phenotypes. We used iPSCs carrying homozygous FUS/TLS mutations (FUS_H517D) to induce LMNs by expressing NEUROG2, ISL1, and LHX3 under the control of the tetracycline regulation system.
Results and discussions: After seven days of in vitro differentiation (DIV7), we confirmed that over 95% of iPSCs differentiated into HB9-positive LMNs. Notably, the cell viability of FUS_H517D LMNs was comparable to that of LMNs differentiated from iPSCs without the FUS/TLS mutation at DIV7. However, by DIV14 and DIV21, the viability of FUS_H517D LMNs was notably lower than that of control LMNs, indicating degeneration of FUS_H517D LMNs after differentiation. Using our microfluidic device, we assessed axonal phenotypes in FUS_H517D LMNs. Under oxidative stress conditions, we observed that the axonal length of FUS_H517D LMNs was significantly shorter than that of control cells as early as DIV7, with this axonal growth restriction becoming more pronounced by DIV11. This suggests that axonal growth restriction is an early detectable phenotype in degenerating neurons. Additionally, we examined mitochondrial trafficking within axons in our device, which is often disrupted in degenerative neurons. Our results showed a significant increase in the number of motile mitochondria in FUS_H517D LMNs, with retrograde transport accounting for a large portion of trafficking. Our microfluidic device-based culture and evaluation system using FUS_H517D LMNs offers a valuable ALS cellular model focused on early axonal phenotypes. This approach contributes to the study of molecular mechanisms underlying axonal degeneration in ALS.

Keywords:  FUS/TLS; amyotrophic lateral sclerosis (ALS); iPSCs; lower motor neurons; microfluidic device

DOI:  https://doi.org/10.3389/fncel.2025.1590732
bioRxiv. 2025 Jul 31. pii: 2025.07.30.667669. [Epub ahead of print]

Cytoplasmic TDP-43 leads to early functional impairments without neurodegeneration in a Serotonergic Neuron-Specific C. elegans Model.

Ailín Lacour, Florencia Vassallu, Diego Rayes, Lionel Muller Igaz.

TDP-43 proteinopathies, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), are marked by the pathological cytoplasmic accumulation of TAR DNA-binding protein 43 (TDP-43), leading to progressive neuronal dysfunction and degeneration. To investigate the early functional consequences of TDP-43 mislocalization, we generated Caenorhabditis elegans models expressing either wild-type human TDP-43 or a variant with a mutated nuclear localization signal (ΔNLS), specifically in serotonergic neurons. These neurons were chosen because i) serotonin deficits are a feature of ALS/FTD and ii) in C. elegans , they regulate well-characterized behaviors, providing a straightforward readout of neuronal function. We found that expression of either TDP-43 variant impaired serotonin-dependent behaviors-including pharyngeal pumping, egg-laying, and locomotion slowing upon food encounter-with the cytoplasmic ΔNLS form causing more severe deficits. Serotonergic neurons remained i) morphologically intact, indicating that neuronal dysfunction precedes overt neurodegeneration; and ii) partially responsive to the selective serotonin reuptake inhibitor fluoxetine, suggesting that neurotransmitter release is still partially functional. Altogether, our findings demonstrate that cytoplasmic TDP-43 disrupts neuronal signaling and behavior early in disease progression. This C. elegans model provides a genetically tractable system to dissect early mechanisms of TDP-43-mediated dysfunction and to identify therapeutic strategies targeting predegenerative stages of ALS/FTD.

DOI: https://doi.org/10.1101/2025.07.30.667669
ASN Neuro. 2025 ;17(1): 2542998

Dysregulated Expression of Inflammasome and Extracellular Matrix Genes in C9orf72-ALS/FTD Microglia.

Louise Thiry, Nisha S Pulimood, Ye Man Tang, Stefano Stifani.

  Hexanucleotide repeat expansion (HRE) in the non-coding region of the gene C9orf72 is the most prevalent mutation in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The C9orf72 HRE contributes to neuron degeneration in ALS/FTD through both cell-autonomous mechanisms and non-cell autonomous disease processes involving glial cells such as microglia. The molecular mechanisms underlying the contribution of C9orf72-HRE microglia to neuron death in ALS/FTD remain to be fully elucidated. In this study, we generated microglia from human C9orf72-HRE and isogenic iPSCs using three different microglia derivation methods. RNA sequencing analysis reveals a cell-autonomous dysregulation of extracellular matrix (ECM) genes and genes involved in pathways underlying inflammasome activation in C9orf72-HRE microglia. In agreement with elevated expression of inflammasome components, conditioned media from C9orf72-HRE microglia enhance the death of C9orf72-HRE motor neurons implicating microglia-secreted molecules in non-cell autonomous mechanisms of C9orf72 HRE pathology. These findings suggest that aberrant activation of inflammasome-mediated mechanisms in C9orf72-HRE microglia results in a pro-inflammatory phenotype that contributes to non-cell autonomous mechanisms of motor neuron degeneration in ALS/FTD.

Keywords:  Amyotrophic lateral sclerosis; C9orf72; RNA sequencing; extracellular matrix; inflammasome; microglia

DOI:  https://doi.org/10.1080/17590914.2025.2542998
Proc Natl Acad Sci U S A. 2025 Aug 12. 122(32): e2500726122

Hide-and-Seek genome editing reveals that Gephyrin is required for axo-axonic synapse assembly.

Yuki Ogawa, Duc V M Nguyen, Ayano Ogawa, Matthew N Rasband.

  The visualization and manipulation of proteins in neurons is widely used to deduce their functions. While every experimental approach has limitations, the concurrent knock-in and knockout of two different proteins can be especially challenging. To this end, we developed Hide-and-Seek genome editing, which allows the simultaneous visualization and knockout of proteins in neurons using Adeno-associated viral vectors and the CRISPR/Cas9 system. We demonstrate the efficacy and flexibility of this method for rapid, efficient, and simultaneous knock-in and knockout of proteins in vitro and in vivo, at the synapse, axon initial segment (AIS), nucleus, and mitochondria. Using Hide-and-Seek, we show that the scaffolding protein Gephyrin is required for the proper assembly of axo-axonic synapses at the AIS.

Keywords:  AAV; CRISPR; axon; axon initial segment; inhibitory synapse

DOI:  https://doi.org/10.1073/pnas.2500726122
J Neurosci. 2025 Aug 06. pii: e0337252025. [Epub ahead of print]45(32):

Evaluating TRIM46's Role in Axon Initial Segment Formation.

Sophie Belmonte.

DOI: https://doi.org/10.1523/JNEUROSCI.0337-25.2025
Nat Commun. 2025 Aug 05. 16(1): 7226

14-3-3 binding maintains the Parkinson's associated kinase LRRK2 in an inactive state.

Juliana A Martinez Fiesco, Alexandra Beilina, Astrid Alvarez de la Cruz, Ning Li, Riley D Metcalfe, Mark R Cookson, Ping Zhang.

Leucine-rich repeat kinase 2 (LRRK2) is an essential regulator in cellular signaling and a major contributor to Parkinson's disease (PD) pathogenesis. 14-3-3 proteins are critical modulators of LRRK2 activity, yet the structural basis of their interaction has remained unclear. Here, we present the cryo-electron microscopy structure of the LRRK2:14-3-32 autoinhibitory complex, revealing how a 14-3-3 dimer stabilizes an autoinhibited LRRK2 monomer through dual-site anchoring. The dimer engages both phosphorylated S910/S935 sites and the COR-A/B subdomains within the Roc-COR GTPase region. This spatial configuration constrains LRR domain mobility, reinforces the inactive conformation, and likely impedes LRRK2 dimerization and oligomer formation. Structure-guided mutagenesis studies show that PD-associated mutations at the COR:14-3-32 interface and within the GTPase domain weaken 14-3-3 binding and impair its inhibitory effect on LRRK2 kinase activity. Furthermore, we demonstrate that type I LRRK2 kinase inhibitor, which stabilizes the kinase domain in its active conformation, reduces 14-3-3 binding and promotes dephosphorylation at pS910 and pS935. Together, these findings provide a structural basis for understanding how LRRK2 is maintained in an inactive state, elucidate the mechanistic role of 14-3-3 in LRRK2 regulation, inform the interpretation of PD biomarkers, and suggest therapeutic strategies aimed at enhancing LRRK2-14-3-3 interactions to treat PD and related disorders.

DOI: https://doi.org/10.1038/s41467-025-62337-1
bioRxiv. 2025 Jul 21. pii: 2025.07.17.665403. [Epub ahead of print]

Neuron-derived extracellular vesicles in plasma present a potential non-invasive biomarker for Huntingtin protein and RNA assessment in Huntington disease.

Inês Caldeira Brás, Yuanyun Xie, Amber Lee Southwell.

Huntington disease (HD) is a neurodegenerative disease caused by a trinucleotide repeat expansion in the HTT gene encoding an elongated polyglutamine tract in the huntingtin (HTT) protein. The use of biomarkers has become a major component in preclinical studies focusing on HTT lowering strategies. Quantification of soluble mutant HTT (mHTT) in cerebrospinal fluid (CSF) has served as a pharmacodynamic readout and as potential disease progression biomarker. However, development of future assays for HTT measurement from other biofluids, such as blood, will facilitate the access to human samples since CSF collection is an invasive outpatient procedure. Brain cells, in particular neurons, secrete extracellular vesicles (EVs) that cross the blood-brain barrier and circulate in blood. Importantly, EVs have been identified to be involved in HTT export from cells to the extracellular space. However, it is unknow which vesicle subtype correlates better with HD progression. Our work investigates the potential of EVs as non-invasive sources of clinical biomarkers in liquid biopsies. We developed an optimized ultracentrifugation protocol for the purification of ectosomes and exosomes from human samples and plasma of humanized HD mouse models. Ectosomes are larger vesicles that bud from the plasma membrane of cells, whereas exosomes originate from multivesicular bodies and are afterwards released to the extracellular space. Consistent with previous published data in other model systems, ectosomes isolated from plasma of the Hu97/18 mouse model contain both wild-type (wt) and mHTT in higher levels than in exosomes. Similar results were observed in media from HD induced pluripotent stem cells (iPSCs)-differentiated neurons and in Hu97/18 primary neuronal cultures. Interestingly, we also found higher levels of HTT transcripts in this EV subtype. We further demonstrate that initial storage of the samples using a slow freezing protocol preserves HTT and EV protein marker levels, highlighting the importance of sample preparation for EV isolation and analysis. Our results also show that plasma contains vesicles originated from neuronal cells that can be isolated using neuron-specific markers, such as ATPase Na+/K+ transporting subunit alpha 3 (ATP1A3), allowing the evaluation of HTT levels in the brain through vesicles circulating in the blood. Overall, our results demonstrate that HTT protein measurement from EVs isolated from blood can be a potential less-invasive disease biomarker. We also demonstrate that EVs subtypes contain different HTT protein and RNA levels, important for the development of consistent and reliable biomarkers. Further characterization of neuron-specific EVs content from patient-derived biofluids will lead to the development of novel clinical biomarkers and for evaluation of therapeutic strategies.

DOI: https://doi.org/10.1101/2025.07.17.665403
Mol Neurodegener. 2025 Aug 06. 20(1): 89

LRRK2 kinase activity regulates Parkinson's disease-relevant lipids at the lysosome.

Michael T Maloney, Xiang Wang, Rajarshi Ghosh, Shan V Andrews, Romeo Maciuca, Shababa T Masoud, Maayan Agam, Richard M Caprioli, Giuseppe Astarita, Vitaliy V Bondar, John Chen, Chi-Lu Chiu, Sonnet S Davis, Audrey Cheuk-Nga Ho, Hoang N Nguyen, Nicholas E Propson, Michelle L Reyzer, Oliver B Davis, Matthew C Deen, Sha Zhu, Gilbert Di Paolo, David J Vocadlo, Anthony A Estrada, Javier de Vicente, Joseph W Lewcock, Annie Arguello, Jung H Suh, Sarah Huntwork-Rodriguez, Anastasia G Henry.

   BACKGROUND: Pathogenic variants in LRRK2 lead to increased kinase activity, and LRRK2 kinase inhibition is being explored in clinical studies as a therapeutic approach for Parkinson's Disease (PD). LRRK2 inhibitors reduce urine levels of bis(monoacylglycerol)phosphate (BMP), a key endolysosomal lipid involved in glycosphingolipid (GSL) catabolism, in preclinical models and clinical subjects. However, how LRRK2 regulates BMP and its significance with respect to lysosomal dysfunction in PD are poorly defined.
METHODS: Using a combination of genetic and pharmacological approaches to modulate LRRK2 kinase activity, we explored the mechanisms by which LRRK2 can regulate the levels of BMP and PD-relevant GSLs across cellular models, including iPSC-derived microglia, and in tissues and biofluids from mice using mass spectrometry. The impact of LRRK2 activity on various aspects of lysosomal function, including endolysosomal GCase activity, was assessed using live-cell imaging and lysosomal immunoprecipitation. We employed imaging mass-spectrometry and FACS-based methods to specifically examine how LRRK2 modulates BMP and GSL levels across different cell types and regions of the brain. To confirm the relevance of our findings to disease, we measured lysosomal biomarkers in urine and cerebrospinal fluid (CSF) from human subjects carrying variants in LRRK2 associated with PD risk and from subjects dosed with a LRRK2 kinase inhibitor.
RESULTS: Our data demonstrate that LRRK2 can employ distinct mechanisms to control intracellular BMP levels and modulate lysosomal homeostasis depending on the tissue examined. We show that LRRK2 deletion or inhibition lowers urine BMP levels by reducing the secretion of BMP-containing vesicles from kidney into urine. In other cell types such as microglia, LRRK2-mediated inhibition of β-glucocerebrosidase (GCase), a PD-linked enzyme involved in GSL catabolism, leads to lysosomal GSL accumulation and increases BMP levels as a compensatory response to restore lysosomal homeostasis. LRRK2 inhibition normalizes lysosomal function and reduces GSL levels in preclinical models and CSF from LRRK2-PD patients.
CONCLUSIONS: Our study highlights the therapeutic potential of LRRK2 kinase inhibition to improve PD-associated lysosomal dysfunction and supports the utility of GSLs as CSF-based biomarkers of LRRK2 activity.
TRIAL REGISTRATION: This work includes results from the following phase 1b study in PD patients: ClinicalTrials.gov ID: NCT03710707; https://clinicaltrials.gov/study/NCT03710707?intr=dnl201&rank=2 . The date of registration was 10/18/2018.

Keywords:  BMP and glycosphingolipids; LRRK2; Lysosome; Parkinson’s disease

DOI:  https://doi.org/10.1186/s13024-025-00880-7
J Huntingtons Dis. 2025 Aug;14(3): 214-228

The role of astrocytes in human Huntington's disease pathology.

Osama Al-Dalahmah, James E Goldman.

  Huntington's disease (HD) is a neurodegenerative disorder caused by a repeat expansion in the HTT gene. The disease is well known for severe and progressive loss of neurons in the caudate and putamen, although other areas are also involved. Much of the attention on understanding the mechanisms underlying HD has focused on the neurons. The brain also contains large numbers of glial cells, such as astrocytes, oligodendrocytes, and microglia, which also become dysfunctional in HD. Astrocytes are one of the most abundant cell types in the central nervous system and are critical for regulating the brain environment and supporting neurons in many ways. In this review, we discuss the changes in astrocytes during the evolution of HD in the human brain. We detail the key phenotypes of astrocytes in human HD, which encompass reactive astrogliosis, loss of homeostatic function, gain of a neuroprotective function, changes in lipid metabolism, huntingtin protein aggregation, and limited somatic repeat expansion. We briefly discuss the conservation of these phenotypes in mouse models and propose a model of how astrocyte states change in human HD. Finally, we present open questions for astrocyte researchers in the HD field. Together, this review represents a valuable resource for readers interested in astrocytic changes in human HD.

Keywords:  Huntington's disease; astrocytes; glutamate transporters; mHTT; metallothionines; transcriptomics

DOI:  https://doi.org/10.1177/18796397251344173
Commun Biol. 2025 Aug 07. 8(1): 1177

Machine learning-based proteomics profiling of ALS identifies downregulation of RPS29 that maintains protein homeostasis and STMN2 level.

Wei Xu, Zhipeng Guo, Yian Guan, Shihui Lv, Xue Gao, Wenchen Luo, Tianlin Cheng, Zhicheng Shao, Bangbao Tao, Tao Wang, Zhixin Qiu.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease. The molecular understanding of ALS is hampered by the lack of experimental models recapitulating disease heterogeneity and analytical framework integrating multi-omics datasets. Here, we developed a pipeline integrating machine learning and consensus clustering to analyze a large-scale dataset of patient-derived motor neuron models from Answer ALS. Compared to the transcriptome, proteomic profiling closely correlates with ALS pathology, which is interrogated to identify 110 proteomics-based biomarkers (Proteomics Markers for ALS 110, PMA110). Functional enrichment highlights dysregulation of ALS pathways, including protein translation and neuronal function. By integrating ALS subtype-specific proteins with patient postmortem proteomics, we found that RPS29 was consistently downregulated in ALS models and patient motor neurons. RPS29 is required for neuronal viability by maintaining ribosome profiling and accurate translation, and suppressing pathological translation. RPS29 downregulation suppresses translation of STMN2, an essential protein for motor neurons, in iPSC-derived motor neurons. Taken together, this study provides a robust framework for ALS proteomics, identifies RPS29 as a quality controller of protein translation, and presents a translational mechanism for STMN2 maintenance in ALS.

DOI: https://doi.org/10.1038/s42003-025-08578-8
Sci Rep. 2025 Aug 07. 15(1): 28901

Effects of natural Lithium and Lithium isotopes on voltage gated sodium channel activity in SH-SY5Y and IPSC derived cortical neurons.

Irina Bukhteeva, James D Livingstone, Kartar Singh, Evgeny V Pavlov, Michael A Beazely, Michel J P Gingras, Zoya Leonenko.

  Although lithium (Li) is a widely used treatment for bipolar disorder, its exact mechanisms of action remain elusive. Research has shown that the two stable Li isotopes, which differ in their mass and nuclear spin, can induce distinct effects in both in vivo and in vitro studies. Since sodium (Na+) channels are the primary pathway for Li+ entry into cells, we examined how Li+ affects the current of Na+ channels using whole-cell patch-clamp techniques on SH-SY5Y neuroblastoma cells and human iPSC-derived cortical neurons. Our findings indicate that mammalian Na+ channels in both neuronal models studied here display no selectivity between Na+ and Li+, unlike previously reported bacterial Na+ channels. We observed differences between the two neuronal models in three measured parameters ([Formula: see text]). We saw no statistically significant differences between any ions in SH-SY5Y cells, but small differences in the half-maximum activation potential ([Formula: see text]) between Na+ and 6Li+ and between 7Li+ and 6Li+ were found in iPSC-derived cortical neurons. Although Na+ channels are widely expressed and important in neuronal function, the very small differences observed in this work suggest that Li+ regulation through Na+ channels is likely not the primary mechanism underlying Li+ isotope differentiation.

Keywords:  IPSC-derived cortical neurons; Lithium isotopes; Mammalian sodium channels; Patch-clamp; SH-SY5Y

DOI:  https://doi.org/10.1038/s41598-025-12893-9
Proc Natl Acad Sci U S A. 2025 Aug 12. 122(32): e2507334122

Cryptic intronic transcriptional initiation generates efficient endogenous mRNA templates for C9orf72-associated RAN translation.

Shannon L Miller, Katelyn M Green, Bradley Crone, Jessica A Switzenberg, Elizabeth M H Tank, Amy Krans, Karen Jansen-West, Clare M Wieland, Eric W Ji, Leonard Petrucelli, Sami J Barmada, Alan P Boyle, Peter K Todd.

  Intronic GGGGCC hexanucleotide repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Despite its intronic location, this repeat avidly supports synthesis of pathogenic dipeptide repeat (DPR) proteins via repeat-associated non-AUG (RAN) translation. However, the template RNA species that undergoes RAN translation endogenously remains unclear. Using long-read based 5' RNA ligase-mediated rapid amplification of cDNA ends (5' Repeat-RLM-RACE), we identified C9orf72 transcripts initiating within intron 1 in a C9BAC mouse model, patient-derived iNeurons, and iNeuron-derived polysomes. These cryptic m7G-capped mRNAs are at least partially polyadenylated and are more abundant than transcripts derived from intron retention or circular intron lariats. In RAN translation reporter assays, intronic template transcripts-even those with short (32 nucleotide) leaders-exhibited robust expression compared to exon-intron and repeat-containing lariat reporters. To assess endogenous repeat-containing lariat RNA contributions to RAN translation, we enhanced endogenous lariat stability by knocking down the lariat debranching enzyme Dbr1. However, this modulation did not impact DPR production in patient-derived iNeurons. These findings identify cryptic, linear, m7G-capped intron-initiating C9orf72 mRNAs as an endogenous template for RAN translation and DPR production, with implications for disease pathogenesis and therapeutic development.

Keywords:  ALS; neurodegeneration; repeat expansion disease; transcription; translation

DOI:  https://doi.org/10.1073/pnas.2507334122
J Cell Biol. 2025 Oct 06. pii: e202501007. [Epub ahead of print]224(10):

SOD1 is delivered to lysosomes via autophagy to maintain lysosomal function and integrity.

Yanzhe Zheng, Meng Li, Xuelin Chen, Ze Zheng, Zixuan Chen, Ruilin Tian, Yan G Zhao.

The gene encoding superoxide dismutase 1 (SOD1) is often mutated in familial amyotrophic lateral sclerosis (ALS), affecting motor neurons. Compared with ALS-associated mutant SOD1, the function of WT SOD1 is less explored. We demonstrate that during starvation, WT and mutant SOD1 are transported into lysosomes. Genome-wide CRISPR interference (CRISPRi) screening identified autophagy-related proteins and the autophagic receptor TP53INP1 as key mediators. TP53INP1 binds ATG8 family proteins, preferentially LC3C, and directly interacts with SOD1. Within lysosomes, SOD1 retains its enzymatic activity. Starvation induces elevated levels of lysosomal reactive oxygen species (ROS), which are further increased by knocking down SOD1 or TP53INP1. Lysosomal degradation activities and membrane integrity are also compromised in the absence of SOD1 or TP53INP1. We reveal a novel function of SOD1 in maintaining lysosomal activity and integrity, and a previously unrecognized role of autophagy in delivering cytosolic enzymes into lysosomes for catalytic purposes, rather than for degradation.

DOI: https://doi.org/10.1083/jcb.202501007
Sci Rep. 2025 Aug 05. 15(1): 28555

Transcriptome-based screening in TARDBP/TDP-43 knock-in motor neurons identifies the NEDD8-activating enzyme inhibitor MLN4924.

Sarah Lépine, Gilles Maussion, Alexandria Schneider, Angela Nauleau-Javaudin, María José Castellanos-Montiel, Georgina Jiménez Ambriz, Dan Spiegelman, Narges Abdian, Anna Krystina Franco-Flores, Ghazal Haghi, Lale Gursu, Michael R Fiorini, Allison A Dilliott, Sali M K Farhan, Mathilde Chaineau, Thomas M Durcan.

A growing body of knowledge implicates perturbed RNA homeostasis in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that currently has no cure and few available treatments. Dysregulation of the multifunctional RNA-binding protein TDP-43 is increasingly regarded as a convergent feature of this disease, evidenced at the neuropathological level by the detection of TDP-43 pathology in most patient tissues, and at the genetic level by the identification of disease-associated mutations in its coding gene TARDBP. To characterize the transcriptional landscape induced by TARDBP mutations, we performed whole-transcriptome profiling of motor neurons (MNs) differentiated from two knock-in iPSC lines expressing the ALS-linked TDP-43 variants p.A382T or p.G348C. Our results show that the TARDBP mutations significantly altered the expression profiles of mRNAs and microRNAs of the 14q32 cluster in MNs. Using mutation-induced gene signatures and the Connectivity Map database, we identified compounds predicted to restore gene expression toward wild-type levels. Among top-scoring compounds selected for further investigation, the NEDD8-activating enzyme inhibitor MLN4924 effectively improved cell viability and neuronal activity, highlighting a possible role for protein post-translational modification via NEDDylation in the pathobiology of TDP-43 in ALS.

DOI: https://doi.org/10.1038/s41598-025-12147-8
Cell Mol Life Sci. 2025 Aug 08. 82(1): 301

The complex web of membrane contact sites in brain aging and neurodegeneration.

Domenico Azarnia Tehran, Paola Pizzo.

  To sustain the essential biological functions required for life, eukaryotic cells rely on complex interactions between different intracellular compartments. Membrane contact sites (MCS), regions where organelles come into close proximity, have recently emerged as major hubs for cellular communication, mediating a broad range of physiological processes, including calcium signalling, lipid synthesis and bioenergetics. MCS are particularly abundant and indispensable in polarized and long-lived cells, such as neurons, where they support both structural and functional integrity. In this review, we explore the functional diversity, molecular composition, and dynamic regulation of key mammalian MCS: endoplasmic reticulum (ER)-plasma membrane, ER-mitochondria and contact sites involving lipid droplets. We highlight their central role in neuronal health and discuss how MCS dysfunction has increasingly been recognized as a hallmark of brain aging and various neurodegenerative diseases, most notably Alzheimer's disease, where altered MCS dynamics contribute to pathogenesis. Finally, we emphasize the therapeutic potential of targeting MCS and outline key unanswered questions to guide future research.

Keywords:  Inter-organelle crosstalk; Neuronal homeostasis; Organelle contacts; Synaptic dysfunction; Therapeutic targets

DOI:  https://doi.org/10.1007/s00018-025-05830-6
BMJ Case Rep. 2025 Aug 08. pii: bcr0820080632. [Epub ahead of print]2009

G2019S LRRK2 mutation causing Parkinson's disease without Lewy bodies.

Carles Gaig, María José Martí, Mario Ezquerra, Adriana Cardozo, Maria Jesus Rey, Eduardo Tolosa.

The G2019S leucine-rich repeat kinase 2 gene (LRRK2) mutation has been identified in a significant proportion of familial and sporadic cases of Parkinson's disease (PD). Until now, information on the neuropathological changes associated with the G2019S LRRK2 mutation has been sparse. We report a 77-year-old patient who presented with a 14 year history of PD but, unexpectedly, histopathological examination disclosed mild neuronal loss in the substantia nigra without α-synuclein, tau or ubiquitin cytoplasmic inclusions. A G2019S LRRK2 mutation was eventually detected. The present case confirms that clinical PD caused by G2019S mutations can be associated with non-specific nigral degeneration without Lewy.

DOI: https://doi.org/10.1136/bcr.08.2008.0632
Neurobiol Dis. 2025 Aug 01. pii: S0969-9961(25)00261-X. [Epub ahead of print] 107045

Expression of amyotrophic lateral sclerosis associated protein disulfide isomerase A3 D217N variant recapitulates early morphological alterations at the neuromuscular junction.

Martin Sepulveda, Francisca MartinezTraub, Patricia Ojeda, Jessica Mella, Jorge Ojeda, Cristina Pinto, Rodrigo Diaz, Pablo Rozas, Claudia Sepulveda, Bredford Kerr, Vania Morales, Mirva Saaranen, Lloyd Ruddock, Danilo B Medinas, Juan Pablo Henriquez, Claudio Hetz.

  Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by neuromuscular connectivity decline followed by motoneuron loss. Altered proteostasis is suggested as a transversal pathogenic mechanism, notably involving dysfunction at the level of the endoplasmic reticulum (ER). Protein disulfide isomerases (PDIs) are key enzymes that catalyze protein folding and disulfide bond formation in the ER. Importantly, PDIs function is disrupted in ALS. We previously identified mutations in the gene encoding PDIA3 (also known as Grp58 or ERp57) as risk factors for ALS, which were associated with altered neuromuscular junction (NMJ) organization when expressed in zebrafish, a phenotype recapitulated in PDIA3-null mice. Here, we generated a transgenic mouse line overexpressing the ALS-linked PDIA3 variant D217N and performed a comprehensive characterization of ALS-like features. The transgenic line exhibited moderate overexpression of mutant PDIA3D217N, which led to morphological alterations at the NMJ resembling those observed in ALS models and patients, along with abnormal distribution of oxidative and glycolytic muscle fibers. However, mutant PDIA3D217N expression did not result in motor impairment, coordination deficits, or motoneuron loss. At the molecular level, we observed reduced expression of SV2 in the spinal cord, an important synaptic protein involved in NMJ function. Our findings further support the involvement of PDIA3 dysfunction as a risk factor in the emergence of early features of ALS.

Keywords:  Amyotrophic lateral sclerosis; Endoplasmic reticulum; Neuromuscular junction; Protein disulfide isomerase; Proteostasis

DOI:  https://doi.org/10.1016/j.nbd.2025.107045
J Cell Biol. 2025 Oct 06. pii: e202406097. [Epub ahead of print]224(10):

Dominant-negative isoform of TDP-43 is regulated by ALS-linked RNA-binding proteins.

Minami Hasegawa-Ogawa, Asako Onda-Ohto, Takumasa Nakajo, Arisa Funabashi, Ayane Ohya, Ryota Yazaki, Hirotaka James Okano.

TDP-43, an RNA-binding protein (RBP) encoded by the TARDBP gene, is crucial for understanding the pathogenesis of neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. Dysregulated TDP-43 causes motor neuron loss, highlighting the need for proper expression levels. Here, we identify a dominant-negative isoform among the multiple TARDBP splicing variants and validate its endogenous expression using a developed antibody against its translated product. Furthermore, we revealed that ALS-associated RBPs regulate its expression: hnRNP K promotes its splicing and expression, while hnRNP A1 and FUS suppress these processes through distinct mechanisms. hnRNP A1 inhibits hnRNP K-mediated splicing, and FUS represses the dominant-negative isoform through both its translational inhibition and hnRNP K suppression. Notably, ALS-mutant FUS weakens this regulatory mechanism, leading to impaired repression of hnRNP K and the dominant-negative isoform. Our findings suggest a regulatory network involving ALS-linked RBPs that govern TDP-43 isoform expression and provide new insights into how disruptions in this network contribute to ALS pathogenesis.

DOI: https://doi.org/10.1083/jcb.202406097
bioRxiv. 2025 Jul 31. pii: 2025.07.25.666804. [Epub ahead of print]

Restoring DSCAM expression rescues neuronal morphology and axon guidance deficits in Down syndrome.

Manasi Agrawal, Nikita Kirkise, Katelyn Rygel, Pabitra K Sahoo, Carly J Vincent, Daniel Joyner, Ricardo Aguilar-Alvarez, Trey R Philip, Parker S Dhillon, Juhi Patel, Jeffery Twiss, Aaron M Jasnow, Kristy Welshhans.

Down syndrome (DS) results from the triplication of human chromosome 21 (HSA21) and is the leading cause of intellectual disability. Down syndrome cell adhesion molecule ( DSCAM ) is located on HSA21 and is overproduced in DS. DSCAM is a receptor for netrin-1 and important for neural wiring in the developing brain. Using a Dscam gain-of-function mouse model and human induced pluripotent stem cell (hiPSC)-derived cortical neurons, in combination with cellular, molecular, and behavioral approaches, this study aims to understand how DSCAM triplication and its subsequent excessive production contribute to changes in neural development and intellectual disability in DS. Analysis of morphological parameters revealed impaired neuronal development and loss of netrin-1-mediated axon guidance in mouse hippocampal pyramidal neurons overexpressing DSCAM. Furthermore, DSCAM overexpression reduces interhemispheric connectivity in vivo , and hippocampal- dependent learning in adult mice. DS hiPSC-derived excitatory pyramidal neurons exhibit a similar phenotype: impaired morphological development and loss of netrin-1-mediated axon guidance. Remarkably, normalization of DSCAM in DS hiPSC-derived neurons rescues many of these neuronal phenotypes, including reduced axon length and deficits in axon guidance. These results suggest that DSCAM plays an essential role in the development of neurons and neuronal networks, and its overproduction contributes to intellectual disability in DS.

DOI: https://doi.org/10.1101/2025.07.25.666804
J Extracell Vesicles. 2025 Aug;14(8): e70140

ATP Synthase Abundance in Neuronal Extracellular Vesicles Reflects Changes in the Mitochondria of Parent Neurons.

Pamela J Yao, Carlos Nogueras-Ortiz, Krishna Ananthu Pucha, Dimitrios Kapogiannis.

  Mitochondrial proteins are found in extracellular vesicles (EVs) such as neuron-derived EVs (NEVs). Yet whether and how NEV-borne mitochondrial proteins relate to the state of mitochondria in the parent neurons is unclear. Studying the mitochondrial ATP synthase in primary hippocampal neurons and their released EVs, we discovered that the abundance of ATP synthase in NEVs echoes the catalytic activity level of ATP synthase in neurons. We also observed, unexpectedly, that within the neuron, the quantity of ATP synthase remains constant irrespective of the level of its activity. Using non-canonical amino acid tagging coupled with proximity ligation assay, we found that the amount of nascent ATP synthase is linearly correlated to its activity, which may contribute to maintaining the overall quantity of ATP synthase in the neuron stable. Furthermore, we identified a sub-population of mitochondria-derived vesicles (MDVs) that carry ATP synthase and are not targeted to lysosomal degradation. Our findings suggest a strategy used by neurons in regulating and fine-tuning mitochondrial ATP synthase through MDV and NEV generation. Further studies are needed to elucidate the relationship between ATP synthase-containing-NEVs and -MDVs.

Keywords:  ATP synthase; extracellular vesicles; mitochondria; mitochondrial‐derived vesicles; mitovesicles; neurons

DOI:  https://doi.org/10.1002/jev2.70140
STAR Protoc. 2025 Aug 05. pii: S2666-1667(25)00422-8. [Epub ahead of print]6(3): 104016

Protocol for the induction of human spinal motor neurons from human induced pluripotent stem cells for studying amyotrophic lateral sclerosis.

Selena Setsu, Satoru Morimoto, Shiho Nakamura, Fumiko Ozawa, Hideyuki Okano.

  Here, we present a protocol for inducing spinal lower motor neurons (LMNs) from human induced pluripotent stem cells (iPSCs). We describe steps for preparation of a chemically induced transitional state (CTraS), transduction with Sendai virus, and LMN differentiation and maintenance. We then detail procedures for live imaging for single-cell-based survival analysis and neurite length of LMNs using BioStation and immunocytochemistry for induction efficiency check. This protocol is optimized for amyotrophic lateral sclerosis (ALS) research and large-scale screening. For complete details on the use and execution of this protocol, please refer to Setsu et al.1.

Keywords:  Antibody; Cell Biology; Cell Differentiation; Cell culture; Cell-based Assays; Gene Expression; Microscopy; Molecular Biology; Neuroscience; Single Cell; Stem Cells

DOI:  https://doi.org/10.1016/j.xpro.2025.104016
Geroscience. 2025 Aug 02.

Treatment of age-related decreases in GTP levels restores endocytosis and autophagy.

R A Santana, J M McWhirt, G J Brewer.

  Age-related declines in neuronal bioenergetic levels may limit vesicular trafficking and autophagic clearance of damaged organelles and proteins. Age-related ATP depletion would impact cognition dependent on ionic homeostasis, but limits on proteostasis powered by GTP are less clear. We used neurons isolated from aged 3xTg-AD Alzheimer's model mice and a novel genetically encoded fluorescent GTP sensor (GEVAL) to evaluate live GTP levels in situ. We report an age-dependent reduction in ratiometric measurements of free/bound GTP levels in living hippocampal neurons. Free GTP colocalized in the mitochondria decreased with age accompanied by the accumulation of free GTP-labeled vesicular structures. The energy dependence of autophagy was demonstrated by depletion of GTP with rapamycin stimulation, while bafilomycin inhibition of autophagy raised GTP levels. Twenty-four-hour supplementation of aged neurons with the NAD precursor nicotinamide and the Nrf2 redox modulator EGCG restored GTP levels to youthful levels and mobilized endocytosis and lysosomal consumption for autophagy via the respective GTPases Rab7 and Arl8b. This vesicular mobilization promoted the clearance of intraneuronal Aβ aggregates, improved viability, and lowered protein oxidative nitration in AD model neurons. Our results reveal age- and AD-related neuronal GTP energy deficits that impair autophagy and endocytosis. GTP deficits were remediated by an external NAD precursor together with a Nrf2 redox modulator which suggests a translational path.

Keywords:  Aging; Alzheimer; EGCG; Energetics; Neuron; Nicotinamide; Redox

DOI:  https://doi.org/10.1007/s11357-025-01786-4
Autophagy. 2025 Aug 03. 1-2

PDZD8 links organelle crosstalk to synaptic remodeling via autophagy.

Rajan S Thakur, Kate M O'Connor-Giles.

  Synapse formation and plasticity require coordinating cellular processes from signaling to protein turnover over long distances, placing high demands on intracellular communication. Membrane contact sites (MCSs) between organelles are specialized compartments for coordinating cellular processes, yet their functions in the developing nervous system remain poorly understood. Through an in vivo CRISPR screen in Drosophila, we identified the conserved endoplasmic reticulum (ER) MCS tethering protein Pdzd8 as a regulator of activity-dependent synapse development. Our in vivo studies demonstrate that Pdzd8 functions at ER-late endosome/lysosome MCSs to promote lysosomal maturation and increase autophagic flux during periods of high demand such as prolonged neuronal activity.

Keywords:  Autophagy; PDZD8; lipid transfer; lysosomes; membrane contact sites; synapse

DOI:  https://doi.org/10.1080/15548627.2025.2537983
bioRxiv. 2025 Jul 23. pii: 2024.10.28.620622. [Epub ahead of print]

Specialized axon initial segment enables low firing threshold and rapid action potential output in fast-spiking interneurons of the human neocortex.

Emoke Bakos, Ádám Tiszlavicz, Viktor Szegedi, Abdennour Douida, Szabina Furdan, Daphne K Welter, Jonathan M Landry, Balazs Bende, Gabor Hutoczki, Pal Barzo, Gabor Tamas, Vladimir Benes, Attila Szucs, Karri Lamsa.

The mammalian brain exhibits notable interspecies variation. Microanatomical and molecular differences in homologous neurons, those with similar locations and developmental origins across species, are best characterized in the neocortical mantle, the center of complex brain functions; however, the purpose of these differences remains unclear. We performed whole-cell microelectrode recordings along with microanatomical and molecular analyses of human fast-spiking parvalbumin (pvalb)-expressing interneurons in neocortical tissue resected during brain surgery, comparing them with similar data obtained from the mouse neocortex. The action potential (AP) firing threshold was lower in human neurons than in mouse neurons. This was due to a deficiency in low-voltage-activated inhibitory Kv1.1 and Kv1.2 potassium channels in the axon initial segment (AIS), a specialized axonal region that determines AP threshold and initiation, in human cells. In contrast, Kv1 ion channels were prominent in mouse neurons. The AIS was also elongated in humans. Computational simulations of fast-spiking interneurons revealed that the human-type AIS lowers the AP threshold and shortens the time lag for AP initiation. We found that the low membrane AP firing threshold in pvalb neurons is closely linked to slow membrane potential kinetics in the soma. Thus, the human AIS supports fast in-fast out circuit function in human pvalb neurons, compensating for electrically slow somatic membrane responses. When formulating therapeutic strategies that involve fast- spiking neurons, it is crucial to take into account the molecular and functional species differences. Short blurb: Fast-spiking neurons in the human neocortex feature structural and molecular specializations in the axon initial segment that lower firing thresholds and minimize input- output delay.

DOI: https://doi.org/10.1101/2024.10.28.620622
IBRO Neurosci Rep. 2025 Dec;19 307-316

OPTN deficiency through CRISPR/Cas9 downregulates autophagy and mitophagy in a SOD1-G93A-expressing transgenic cell line.

Di Wen, Qiusheng Li, Yuanyuan Li, Wenyu Yan, Yanyan Wang, Yakun Liu.

  Amyotrophic lateral sclerosis (ALS) is characterized by the loss of upper and lower motor neurons (MNs) and is the most common adult paralysis neurodegenerative disease. Dysregulated autophagy, which has been reported in the pathogenesis of familial ALS, has been found in superoxide dismutase 1 (SOD1) transgenic mice and cell lines. Optineurin (OPTN) is a signal regulator that coordinates many crucial cellular processes, including autophagy, mitophagy and aggrephagy. Recent studies have shown that OPTN gene mutations are correlated with ALS, glaucoma and Paget's disease of the bone. Indeed, defects in autophagosome-lysosome fusion have been reported in patients with ALS-associated OPTN mutations. However, the exact function of OPTN in the pathology of ALS remains unknown. To determine the function of OPTN, we generated OPTN-knockdown cell lines from SOD1-G93A-expressing NSC34 cells with the clustered regularly interspaced short palindromic repeats/associated system 9 (CRISPR/Cas9) approach. In our research, we observed that the loss of OPTN resulted in the impairment of autophagy and mitophagy pathways. Moreover, the mitochondrial transmembrane potential was depolarized by LV-sgRNA-OPTN. On the basis of observations of live cells, the production of reactive oxygen species (ROS) was increased, the autophagic flux decreased, and the autophagic flux merged with that of mitochondria according to confocal live-cell imaging. A decreased LC3-II and an increased p62 levels indicated that autophagy pathway activation was decreased. The protein levels of VDAC1 and TBK1 decreased after OPTN knockdown, suggesting that mitophagy was blocked. Our results suggest that OPTN plays a pivotal role in regulating autophagy and mitophagy.

Keywords:  Amyotrophic lateral sclerosis; Autophagy; CRISPR/Cas9; Mitophagy; OPTN; SOD1-G93A transgenic cell line

DOI:  https://doi.org/10.1016/j.ibneur.2025.07.011
F1000Res. 2025 ;14 575

Generation of human iPSC-derived pancreatic organoids to study pancreas development and disease.

Jean-Francois Darrigrand, Abigail Isaacson, Francesca Maria Spagnoli.

  The pancreas has vital endocrine and exocrine functions that can be affected by life-threatening diseases such as diabetes and pancreatic cancer. Although animal models are essential for understanding pancreatic development and disease, they are limited by their low throughput and major species-specific molecular and physiological differences. Generating 3D in vitro models, such as organoids, that are physiologically relevant is essential for investigating pancreatic development and disease in the human context. However, the production of human stem cell-derived pancreatic organoids with a proper branched architecture and correct patterning of cell domains remains challenging. Here, we successfully developed a protocol that efficiently and reproducibly generated organoids from human induced pluripotent stem cells (hiPSCs) by optimizing organoid culture format and media. Our differentiation protocol promotes acinar cell differentiation and generates organoids with branches patterned into the central trunk and peripheral tip domains without relying on animal-derived matrices for organoid culture. This platform opens the door to high-throughput investigations of human pancreatic development in a system that recapitulates the most important aspects of pancreatic tissue architecture. Lastly, we anticipate that this system will contribute to the replacement of animal models used to investigate diseases, such as pancreatic cancer.

Keywords:  3D pancreatic model; acinar fate differentiation; branching morphogenesis; human induced pluripotent stem cells (hiPSCs); lineage specification; organoid differentiation; pancreatic diseases.; replacement

DOI:  https://doi.org/10.12688/f1000research.162496.1
ACS Nano. 2025 Aug 06.

Drug-Induced Reversible Lysosomal Changes Tracked in Live Cells by Holo-Tomographic Flow Cytometry.

Daniele Pirone, Michela Schiavo, Giusy Giugliano, Sandro Montefusco, Lisa Miccio, Pasquale Memmolo, Diego Luis Medina, Pietro Ferraro.

  Lysosomal storage diseases (LSDs) are genetic disorders caused by enzyme deficiencies that lead to lysosomal dysfunction and progressive cell damage. Accurate visualization and quantification of lysosomal morphology and subcellular localization are essential parameters for understanding the pathology and disease progression of different LSDs, as well as for developing effective therapies. Here, we successfully identified and characterized lysosomes using a holo-tomographic flow cytometry (HTFC) technique, which allows for label-free, high-content, and high-throughput 3D imaging of lysosomal compartments in single live cells. This study could complement traditional gold-standard methods to overcome the actual limitations. Leveraging this technology, we propose quantitative biomarkers of lysosomal accumulation in LSD-affected cells. In fact, by generating refractive index tomograms, we achieved accurate measurement and comprehensive 3D visualization of cytoplasmic lysosomal aggregation in suspended single cells. Through experimental validation and advanced computational analyses, we identified a quantitative correlation between the 3D lysosomal architecture and the efficacy of various therapeutic strategies, including genetic and pharmacological interventions. This work represents a significant advance in lysosomal research and may support future efforts to improve diagnostics and develop targeted therapies for LSDs.

Keywords:  digital holography; drug testing; high-content imaging; imaging flow cytometry; label-free biomarkers; lysosomal storage diseases; quantitative phase imaging

DOI:  https://doi.org/10.1021/acsnano.5c08530
bioRxiv. 2025 Jul 15. pii: 2025.07.10.664233. [Epub ahead of print]

A 3D iPSC retina model reveals non-cell-autonomous and non-neuronal mechanism of photoreceptor degeneration in a lysosomal storage disorder.

Jimin Han, Nathaniel Foley, Sonal Dalvi, Janet A H Tang, Amit Chatterjee, Lal Krishan Kumar, Chad A Galloway, Kumar Singh, Yashoda Subedi, Kevin Ling, Alison Heffer, Richard T Libby, Danielle S W Benoit, Anthony L Cook, Vera Bonilha, Edward Schuchman, Jennifer J Hunter, Ruchira Singh.

Disruption of photoreceptor-retinal pigment epithelium (RPE) interface with loss of photoreceptor outer segments (POSs) in the retina is a pathological hallmark of several neurodegenerative and retinal diseases including lysosomal storage disorder's like CLN3 disease. However, the retina is a functional composite in vivo; and in vitro stem cell models of retina that enable investigation of the photoreceptor-RPE interface in healthy and diseased retina are lacking. Here, we developed a 3D human pluripotent stem cell (hPSC)-derived retina model to investigate the photoreceptor-RPE interface in healthy and disease tissue. Using this 3D hPSC retina model, we demonstrated that the most common disease causing CLN3 mutation ( CLN3 Δ ex7-8 ) leads to reduced levels of acid ceramidase (AC) and consequently altered sphingolipid metabolism and signaling and POS loss in CLN3 disease. Consistent with the 3D hPSC retina model, altered sphingolipid metabolism and signaling coincided with POS loss in a large animal model of CLN3 disease, CLN3 miniswine. Therapeutically, recombinant human acid ceramidase (rhAC) targeted both altered sphingolipid metabolism and retina degeneration in the CLN3 hPSC retina model and the CLN3 miniswine eye. These findings demonstrate a proof-of-concept that rhAC can rescue disease phenotype in a large animal model of CLN3 disease and suggest that rhAC could be a therapeutic approach for CLN3 disease.
One Sentence Summary: Acid ceramidase deficiency and consequently altered sphingolipid signaling promotes disease phenotype(s) in a lysosomal storage disorder, CLN3 disease.

DOI: https://doi.org/10.1101/2025.07.10.664233
Front Neurosci. 2025 ;19 1558227

Accumulation of TDP-43 causes karyopherin-α4 pathology that characterises amyotrophic lateral sclerosis.

Manpreet Singh Atwal, Jerneja Nimac, Urša Čerček, Sarah Ricarda Goesch, Hannah Rebecca Goesch, Paraskevi Tziortzouda, Tiziana Ercolani, Anna Zatorska, Terouz Pasha, Ivo Carre, Jacqueline Mitchell, Claire Troakes, Bart Tummers, Vera Župunski, Boris Rogelj, Tibor Hortobágyi, Frank Hirth.

  Cytoplasmic mislocalisation and nuclear depletion of TDP-43 are pathological hallmarks of amyotrophic lateral sclerosis (ALS), including mutations in the C9ORF72 gene that characterise the most common genetic form of ALS (C9ALS). Studies in human cells and animal models have associated cytoplasmic mislocalisation of TDP-43 with abnormalities in nuclear transport receptors, referred to as karyopherins, that mediate the nucleocytoplasmic shuttling of TDP-43. Yet the relationship between karyopherin abnormalities and TDP-43 pathology are unclear. Here we report karyopherin-α4 (KPNA4) pathology in the spinal cord of TDP-43-positive sporadic ALS and C9ALS patients. Structural analyses revealed the selective interaction between KPNA subtypes, especially KPNA4, with the nuclear localisation signal (NLS) of TDP-43. Targeted cytoplasmic mislocalisation and nuclear depletion of TDP-43 caused KPNA4 pathology in human cells. Similar phenotypes were observed in Drosophila whereby cytoplasmic accumulation of the TDP-43 homolog, TBPH, caused the nuclear decrease and cytosolic mislocalisation of the KPNA4 homolog, Importin-α3 (Impα3). In contrast, induced accumulation of Impα3 was not sufficient to cause TBPH mislocalisation. Instead, targeted gain of Impα3 in the presence of accumulating cytosolic TBPH, restored Impα3 localisation and partially rescued nuclear TBPH. These results demonstrate that cytoplasmic accumulation of TDP-43 causes karyopherin pathology that characterises ALS spinal cord. Together with earlier reports, our findings establish KPNA4 abnormalities as a molecular signature of TDP-43 proteinopathies and identify it as a potential therapeutic target to sustain nuclear TDP-43 essential for cellular homeostasis affected in ALS and frontotemporal dementia.

Keywords:  C9ORF72; KPNA4; TDP-43; amyotrophic lateral sclerosis; karyopherin; nuclear import

DOI:  https://doi.org/10.3389/fnins.2025.1558227
Nat Rev Mol Cell Biol. 2025 Aug 04.

Lysosomal membrane homeostasis and its importance in physiology and disease.

Maja Radulovic, Chonglin Yang, Harald Stenmark.

Lysosomes are membranous organelles that are crucial for cell function and organ physiology. Serving as the terminal stations of the endocytic pathway, lysosomes have fundamental roles in the degradation of endogenous and exogenous macromolecules and particles as well as damaged or superfluous organelles. Moreover, the lysosomal membrane is a docking and activation platform for several signalling components, including mTOR complex 1 (mTORC1), which orchestrates metabolic signalling in the cell. The integrity of their membrane is crucial for lysosomes to function as hubs for the regulation of cell metabolism. Various agents, including pathogens, nanoparticles and drugs, can compromise lysosomal membrane integrity. Membrane permeabilization causes leakage of proteases and cations into the cytosol, which can induce cell death pathways and innate immunity signalling. Multiple pathways repair damaged lysosomes, and severely damaged lysosomes are degraded by an autophagic process, lysophagy. Moreover, lysosome damage activates transcriptional programmes that orchestrate lysosome biogenesis to replenish the cellular lysosome pool. In this Review, we discuss recent insights into the mechanisms that ensure the maintenance of lysosomal membrane homeostasis, including novel mechanisms of lysosomal membrane repair and the interplay between lysosome damage, repair, lysophagy and lysosome biogenesis. We highlight the importance of lysosomal membrane homeostasis in cell function, physiology, disease and ageing, and discuss the potential for therapeutic exploitation of lysosomal membrane permeabilization.

DOI: https://doi.org/10.1038/s41580-025-00873-w
Curr Opin Neurol. 2025 Aug 08.

Amyotrophic lateral sclerosis in Mainland China: clinical translational challenges and opportunities.

Ji He, Dongsheng Fan.

   PURPOSE OF REVIEW: Amyotrophic lateral sclerosis (ALS) imposes a growing medical and socioeconomic burden in China. This review synthesizes recent advances in understanding ALS epidemiology, biomarker discovery, therapeutic innovations, and policy frameworks in China. It highlights the urgency of addressing challenges, including fragmented healthcare resources, translational medicine gaps, and regional inequities, while emphasizing China's unique contributions to global ALS research.
RECENT FINDINGS: Chinese ALS cohorts exhibit distinct epidemiological profiles, including a younger mean age of onset and prolonged median survival. Policy initiatives, such as ALS inclusion in rare disease registries and insurance reforms, aim to reduce financial burdens of patients. Multimodal biomarker exploration has advanced integrated diagnostic models combining neurofilament light chain (NfL) and clinical data platforms. Neuroimaging and electrophysiological studies reveal glymphatic dysfunction, white matter degeneration, and neuromuscular junction abnormalities, with novel links to hepatic metabolism. Genomic analyses identify population-specific variants. Therapeutic innovations in China include not only biopharmaceuticals, but also integrative traditional Chinese medicine (TCM) approaches.
SUMMARY: China's ALS landscape is transitioning towards precision medicine through biomarker-guided diagnostics and multidisciplinary care models. Key priorities include establishing a national ALS registry, standardizing biomarker validation, and expanding clinical trials to bridge translational medicine gaps.

Keywords:  Chinese population; amyotrophic lateral sclerosis; biomarkers; clinical trails

DOI:  https://doi.org/10.1097/WCO.0000000000001421
Biochem Biophys Res Commun. 2025 Jul 23. pii: S0006-291X(25)01120-9. [Epub ahead of print]779 152405

Application of brain organoids in neurodevelopmental disorders.

Chao Zhao, Qi Yong, Lu Xia, Tengfei Zhu, Kun Xia.

  Understanding the etiology of human neurological and psychiatric disorders remains challenging due to species-specific genomic architectures, prolonged developmental timelines, and the unique cytoarchitectural features of the human brain, which are inadequately replicated in conventional animal models. To address these limitations, human pluripotent stem cell (hPSC)-derived brain organoids have emerged as an ethically accessible experimental system that recapitulates critical aspects of early human neurodevelopment in vitro. These three-dimensional (3D) models mimic the generation of neural progenitors, their proliferation and differentiation into neurons and glial cells, as well as the dynamic cell-cell interactions that govern cortical lamination and circuit assembly. Recent advancements in organoid culture protocols-including the fusion of region-specific brain organoids, vascularization strategies, and the incorporation of microglia-have yielded more physiologically relevant models. Such innovations enable the development of powerful bioassays to investigate disease mechanisms underlying neurodevelopmental disorders (NDDs). Despite persistent challenges related to functional synaptic maturation and modeling the blood-brain barrier, the integration of single-cell multiomics,organoid-on-a-chip systems,and xenotransplantation approaches holds promise for unraveling human-specific pathophysiology and accelerating therapeutic discovery.This review synthesizes the progress in brain organoid technologies and highlights their potential to deepen mechanistic insights into brain disorders while advancing translational research.

Keywords:  ASD; Brain organoid; NDDs; iPSC

DOI:  https://doi.org/10.1016/j.bbrc.2025.152405
Mol Neurobiol. 2025 Aug 02.

Knock-out of Tpm4.2/Actin Filaments Alters Neuronal Signaling, Neurite Outgrowth, and Behavioral Phenotypes in Mice.

Sian Genoud, Chanchanok Chaichim, Rossana Rosa Porto, Tamara Tomanic, Holly Stefen, Esmeralda Paric, Soumalya Sarkar, Dasol Yoo, Wendi Gao, Edna C Hardeman, Peter W Gunning, Tim Karl, John Power, Thomas Fath.

  Tropomyosins (Tpm) are master regulators of actin dynamics through forming co-polymers with filamentous actin. Despite the well-understood function of muscle Tpms in the contractile apparatus of muscle cells, much less is known about the diverse physiological function of cytoplasmic Tpms in eukaryotic cells. Here, we investigated the role of the Tpm4.2 isoform in neuronal processes including signaling, neurite outgrowth, and receptor recycling using primary neurons from Tpm4.2 knock-out mice. Live imaging of calcium and electrophysiology data demonstrated increased frequency, yet reduced strength of single neuron spikes. Calcium imaging further showed an increase in neuronal networks. In vitro assays of Tpm4.2 knock-out neurons displayed impaired recycling of the AMPA neurotransmitter receptor subunit GluA1. Morphometric analysis of neurite growth showed increased dendritic complexity and altered dendritic spine morphology in Tpm4.2 knock-out primary neurons. Behavioral analysis of Tpm4.2 knock-out mice displayed heightened anxiety in the open field test, while the elevated plus maze displayed heightened anxiety only in females. Our study depicts the multi-faceted role of the Tpm4.2 isoform and its co-polymer F-actin population in neurons, with potential implications for better understanding diseases of the nervous system which involve actin cytoskeleton dysfunction.

Keywords:  Actin cytoskeleton; Neuronal signaling; Neurons; Tyopomyosin

DOI:  https://doi.org/10.1007/s12035-025-05259-9
Brain Res Bull. 2025 Aug 07. pii: S0361-9230(25)00313-2. [Epub ahead of print]230 111501

Inhibition of Sirtuin 2 enhances autophagy and restores neuronal function in aged hippocampal neurons.

Zhenyuan Zhang, Lan Zhang, Yidan Zhang, Yuan Zhao, Ya Gao, Cong Zhang, Dongxiao Li, Xiangjian Zhang, Guofeng Yang, Jian Zhang.

  Age-related cognitive decline is linked to impaired autophagy and hippocampal dysfunction. This study investigates the role of Sirtuin 2 (SIRT2) in age-related cognitive decline, focusing on its impact on autophagy and hippocampal function. Quantitative proteomic analysis revealed 67 significantly dysregulated proteins in the hippocampus of naturally aged male mice, including upregulated SIRT2 and impaired autophagy. To explore the role of SIRT2 in brain aging and its association with autophagy, naturally aged male mice received AK7, a SIRT2-specific inhibitor, for four consecutive weeks, followed by behavioral assessment using the Morris water maze. Western blot and immunofluorescence analyses were applied to assess mTOR phosphorylation, LC3B-II turnover, and SQSTM1/p62 degradation, complemented by in vitro validation in a D-galactose-induced HT-22 cellular senescence model. The results indicated that AK7 administration improved cognitive performance in aged mice, while simultaneously reducing mTOR phosphorylation and enhancing autophagy markers. In vitro, SIRT2 genetic knockdown restored the mTOR phosphorylation, LC3B-II/LC3I ratio, and SQSTM1/p62 accumulation, while also reducing senescence markers (including TNF-α, P21,Trp53) in D-galactose-induced HT-22 cells. These effects were abolished by mTOR activation, confirming mTOR as a downstream mediator of SIRT2. Our findings highlight SIRT2 inhibition as a promising therapeutic strategy to counteract age-related cognitive decline through the modulation of mTOR-dependent autophagy in the hippocampus.

Keywords:  Autophagy; Brain aging; Proteomic; Sirtuin 2 (SIRT2)

DOI:  https://doi.org/10.1016/j.brainresbull.2025.111501
Science. 2025 Aug 07. 389(6760): eadn6712

The membrane skeleton is constitutively remodeled in neurons by calcium signaling.

Evan Heller, Naina Kurup, Xiaowei Zhuang.

The membrane skeleton in neurons adopts a periodic lattice structure in which actin filaments, capped by adducin and tropomodulin, form ring-shaped structures connected by spectrin tetramers along neurites. This membrane-associated periodic skeleton (MPS) is important for many neuronal functions. Using live-cell super-resolution imaging, we found that the MPS is surprisingly dynamic, undergoing local disassembly and reformation constitutively in axons. MPS remodeling is driven by calcium signaling, leading to actin-ring destabilization through protein kinase C-mediated adducin phosphorylation and to spectrin degradation by calpain. Formin, an actin-nucleating and -polymerizing enzyme, plays a dual role in MPS remodeling and maintenance. MPS remodeling is enhanced by neuronal activity and functionally facilitates endocytosis. Our results highlight the importance of a dynamic membrane skeletal structure in neuronal function.

DOI: https://doi.org/10.1126/science.adn6712
Proc Natl Acad Sci U S A. 2025 Aug 12. 122(32): e2423455122

Autophagy activator AA-20 improves proteostasis and extends Caenorhabditis elegans lifespan.

Ee Phie Tan, Nora Lyang, Saam Doroodian, Pablo Sanz-Martinez, Jin Xu, Sviatlana Zaretski, José L Nieto-Torres, Hiroshi Ebata, Shaun H Y Lim, William C Hou, Khalyd J Clay, Leonard Yoon, Lynée A Massey, Derek Rhoades, Danny Garza, Kristen A Johnson, Alan To, Lydia Ambaye, Emily P Bentley, Michael Petrascheck, Alexandra Stolz, Jeffery W Kelly, Malene Hansen.

  The degradation of cellular components through autophagy is essential for longevity and healthy aging. However, autophagy function decreases with aging, contributing to age-related diseases. In this study, we characterized a small-molecule activator of autophagy called AA-20 that enhances autophagy and lipid droplet clearance in human cells and in the nematode Caenorhabditis elegans. AA-20 reduces polyglutamine aggregation in an autophagy-dependent manner in both human cells and C. elegans, where it also promotes fitness. Consistently, we found that AA-20 extends lifespan in WT C. elegans, but not in autophagy-deficient mutants. Interestingly, our findings suggest that AA-20 acts, at least in part, through a mechanism involving the transcription factor EB, but without inhibiting the protein kinase mammalian target of rapamycin complex 1. Collectively, our results identify an autophagy activator AA-20, which may have potential therapeutic implications for aging-related proteinopathies and lipid storage disorders.

Keywords:  C. elegans; autophagy activator; healthspan; lifespan; lipophagy

DOI:  https://doi.org/10.1073/pnas.2423455122