bims-axbals 2025-01-12 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2025–01–12
thirty-one papers selected by
TJ Krzystek, ALS Therapy Development Institute

Dual-targeting CRISPR-CasRx reduces C9orf72 ALS/FTD sense and antisense repeat RNAs in vitro and in vivo.
Patient-derived Induced Pluripotent Stem Cells as a Model to Study Frontotemporal Dementia Pathologies.
Human iPSC-derived microglial cells protect neurons from neurodegeneration in long-term cultured adhesion brain organoids.
Chronic Oxidative Stress and Stress Granule Formation in UBQLN2 ALS Neurons: Insights into Neuronal Degeneration and Potential Therapeutic Targets.
A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD.
Gain-of-function ANXA11 mutation cause late-onset ALS with aberrant protein aggregation, neuroinflammation and autophagy impairment.
TDP-43 Cryptic RNAs in Perry Syndrome: Differences across Brain Regions and TDP-43 Proteinopathies.
Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells.
Aberrant splicing in Huntington's disease accompanies disrupted TDP-43 activity and altered m6A RNA modification.
Schwann Cells in Neuromuscular Disorders: A Spotlight on Amyotrophic Lateral Sclerosis.
The MIR-NAT MAPT-AS1 does not regulate Tau expression in human neurons.
Generation of Retinal Ganglion Cells from Reprogrammed Keratocytes of Non-Glaucoma and Glaucoma Donors.
Axodendritic targeting of TAU and MAP2 and microtubule polarization in iPSC-derived versus SH-SY5Y-derived human neurons.
Regulation of physiological and pathological condensates by molecular chaperones.
A Proteomic Examination of Plasma Extracellular Vesicles Across Colorectal Cancer Stages Uncovers Biological Insights That Potentially Improve Prognosis.
Exploring the Role of Axons in ALS from Multiple Perspectives.
Neuronal Imaging at 8-Bit Depth to Combine High Spatial and High Temporal Resolution With Acquisition Rates Up To 40 kHz.
Alpha-Synuclein Effects on Mitochondrial Quality Control in Parkinson's Disease.
Synaptic vesicle characterization of iPSC-derived dopaminergic neurons provides insight into distinct secretory vesicle pools.
Widespread Distribution of α-Synuclein Oligomers in LRRK2 -related Parkinson's Disease.
Poly-ubiquitylated transmembrane proteins outcompete other cargo for limited space inside clathrin-coated vesicles.
Computational Generation of Long-range Axonal Morphologies.
TBC1D24 interacts with the v-ATPase and regulates intraorganellar pH in neurons.
DNA damage and its links to neuronal aging and degeneration.
Curcumin Improves Hippocampal Cell Bioenergetics, Redox and Inflammatory Markers, and Synaptic Proteins, Regulating Mitochondrial Calcium Homeostasis.
Novel high-content and open-source image analysis tools for profiling mitochondrial morphology in neurological cell models.
MPicker: visualizing and picking membrane proteins for cryo-electron tomography.
Genetic Ablation of Sarm1 Mitigates Disease Acceleration after Traumatic Brain Injury in the SOD1G93A Transgenic Mouse Model of Amyotrophic Lateral Sclerosis.
A synapse-specific refractory period for plasticity at individual dendritic spines.
Huntingtin interactome reveals huntingtin role in regulation of double strand break DNA damage response (DSB/DDR), chromatin remodeling and RNA processing pathways.
Mitochondria- and ER-associated actin are required for mitochondrial fusion.

Nat Commun. 2025 Jan 08. 16(1): 459

Dual-targeting CRISPR-CasRx reduces C9orf72 ALS/FTD sense and antisense repeat RNAs in vitro and in vivo.

Liam Kempthorne, Deniz Vaizoglu, Alexander J Cammack, Mireia Carcolé, Martha J Roberts, Alla Mikheenko, Alessia Fisher, Pacharaporn Suklai, Bhavana Muralidharan, François Kroll, Thomas G Moens, Lidia Yshii, Stijn Verschoren, Benedikt V Hölbling, Francisco C Moreira, Eszter Katona, Rachel Coneys, Paula de Oliveira, Yong-Jie Zhang, Karen Jansen, Lillian M Daughrity, Alexander McGown, Tennore M Ramesh, Ludo Van Den Bosch, Gabriele Lignani, Ahad A Rahim, Alyssa N Coyne, Leonard Petrucelli, Jason Rihel, Adrian M Isaacs.

The most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) is an intronic G4C2 repeat expansion in C9orf72. The repeats undergo bidirectional transcription to produce sense and antisense repeat RNA species, which are translated into dipeptide repeat proteins (DPRs). As toxicity has been associated with both sense and antisense repeat-derived RNA and DPRs, targeting both strands may provide the most effective therapeutic strategy. CRISPR-Cas13 systems mature their own guide arrays, allowing targeting of multiple RNA species from a single construct. We show CRISPR-Cas13d variant CasRx effectively reduces overexpressed C9orf72 sense and antisense repeat transcripts and DPRs in HEK cells. In C9orf72 patient-derived iPSC-neuron lines, CRISPR-CasRx reduces endogenous sense and antisense repeat RNAs and DPRs and protects against glutamate-induced excitotoxicity. AAV delivery of CRISPR-CasRx to two distinct C9orf72 repeat mouse models significantly reduced both sense and antisense repeat-containing transcripts. This highlights the potential of RNA-targeting CRISPR systems as therapeutics for C9orf72 ALS/FTD.

DOI: https://doi.org/10.1038/s41467-024-55550-x
bioRxiv. 2024 Dec 17. pii: 2024.12.11.628042. [Epub ahead of print]

Patient-derived Induced Pluripotent Stem Cells as a Model to Study Frontotemporal Dementia Pathologies.

Sonia Infante-Tadeo, Diane L Barber.

The neurodegenerative disorder Frontotemporal Dementia (FTD) can be caused by a repeat expansion (GGGGCC; G4C2) in C9orf72. The function of wild-type C9orf72 and the mechanism by which the C9orf72-G4C2 mutation causes FTD, however, remain unresolved. Diverse disease models including human brain samples and differentiated neurons from patient-derived induced pluripotent stem cells (iPSCs) identified some hallmarks associated with FTD, but these models have limitations, including biopsies capturing only a static snapshot of dynamic processes and differentiated neurons being labor-intensive, costly, and post-mitotic. We find that patient-derived iPSCs, without being differentiated into neurons, exhibit established FTD hallmarks, including increased lysosome pH, decreased lysosomal cathepsin activity, cytosolic TDP-43 proteinopathy, and increased nuclear TFEB. Moreover, lowering lysosome pH in FTD iPSCs mitigates TDP-43 proteinopathy, suggesting a key role for lysosome dysfunction. RNA-seq reveals dysregulated transcripts in FTD iPSCs affecting calcium signaling, cell death, synaptic function, and neuronal development. We confirm differences in protein expression for some dysregulated genes not previously linked to FTD, including CNTFR (neuronal survival), Annexin A2 (anti-apoptotic), NANOG (neuronal development), and moesin (cytoskeletal dynamics). Our findings underscore the potential of FTD iPSCs as a model for studying FTD cellular pathology and for drug screening to identify therapeutics.
SIGNIFICANCE STATEMENT: Understanding the cellular pathology of Frontotemporal Dementia linked to a GGGGCC expansion in the C9orf72 gene remains a challenge.This study shows that undifferentiated patient-derived iPSCs exhibit hallmark FTD characteristics, including lysosome dysfunction and TDP-43 proteinopathy, and identifies dysregulated genes related to neurodegeneration.These findings highlight patient-derived iPSCs as a valuable model for studying FTD pathology and for drug screening, potentially guiding future research in therapeutic development.

DOI: https://doi.org/10.1101/2024.12.11.628042
Commun Biol. 2025 Jan 09. 8(1): 30

Human iPSC-derived microglial cells protect neurons from neurodegeneration in long-term cultured adhesion brain organoids.

Xianwei Chen, Guoqiang Sun, Lizhao Feng, E Tian, Yanhong Shi.

Brain organoid models have greatly facilitated our understanding of human brain development and disease. However, key brain cell types, such as microglia, are lacking in most brain organoid models. Because microglia have been shown to play important roles in brain development and pathologies, attempts have been made to add microglia to brain organoids through co-culture. However, only short-term microglia-organoid co-cultures can be established, and it remains challenging to have long-lasting survival of microglia in organoids to mimic long-term residency of microglia in the brain. In this study, we developed an adhesion brain organoid (ABO) platform that allows prolonged culture of brain organoids (greater than a year). Moreover, the long-term (LT)-ABO system contains abundant astrocytes and can support prolonged survival and ramification of microglia. Furthermore, we showed that microglia in the LT-ABO could protect neurons from neurodegeneration by increasing synaptic density and reducing p-Tau level and cell death in the LT-ABO. Therefore, the microglia-containing LT-ABO platform generated in this study provides a promising human cellular model for studying neuron-glia and glia-glia interactions in brain development and the pathogenesis of neurodegenerative diseases such as Alzheimer's disease.

DOI: https://doi.org/10.1038/s42003-024-07401-0
Int J Mol Sci. 2024 Dec 15. pii: 13448. [Epub ahead of print]25(24):

Chronic Oxidative Stress and Stress Granule Formation in UBQLN2 ALS Neurons: Insights into Neuronal Degeneration and Potential Therapeutic Targets.

Ao Gu, Yiti Zhang, Jianfeng He, Mingri Zhao, Lingjie Ding, Wanxi Liu, Jianing Xiao, Jiali Huang, Mujun Liu, Xionghao Liu.

  The pathogenesis of neurodegenerative diseases results from the interplay between genetic and environmental factors. Aging and chronic oxidative stress are critical contributors to neurodegeneration. UBQLN2, a ubiquitin-related protein, aids in protein degradation and protects against oxidative stress. In ALS neurons harboring UBQLN2 mutations, oxidative stress accelerates pathological changes, yet the precise mechanisms remain unclear. Using induced motor neurons (iMNs) derived from UBQLN2 P497H iPSCs, we observed ALS-like phenotypes, including TDP-43 mislocalization, increased cell death, and reduced viability. Sodium arsenite (SA)-induced oxidative stress triggered stress granule formation, while autophagy dysfunction exacerbated neuronal degeneration. CHX and bosutinib treatments reduced ubiquitinated protein accumulation and alleviated degeneration, highlighting potential therapeutic pathways. These findings emphasize the role of chronic oxidative stress and stress granule formation in UBQLN2 ALS, offering insights into novel therapeutic targets.

Keywords:  ALS; UBQLN2; motor neurons; neurodegenerative diseases; oxidative stress; stress granule

DOI:  https://doi.org/10.3390/ijms252413448
Nat Commun. 2025 Jan 08. 16(1): 460

A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD.

Tristan X McCallister, Colin K W Lim, Mayuri Singh, Sijia Zhang, Najah S Ahsan, William M Terpstra, Alisha Y Xiong, M Alejandra Zeballos C, Jackson E Powell, Jenny Drnevich, Yifei Kang, Thomas Gaj.

An abnormal expansion of a GGGGCC (G4C2) hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we develop here a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform curbed the expression of the G4C2 repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci, reduced the production of a dipeptide repeat protein, and reversed transcriptional deficits. This high-fidelity system possessed improved transcriptome-wide specificity compared to its native form and mediated targeting in motor neuron-like cells derived from a patient with ALS. These results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.

DOI: https://doi.org/10.1038/s41467-024-55548-5
Acta Neuropathol Commun. 2025 Jan 04. 13(1): 2

Gain-of-function ANXA11 mutation cause late-onset ALS with aberrant protein aggregation, neuroinflammation and autophagy impairment.

Qing Liu, Ye Sun, Baodong He, Haodong Chen, Lijing Wang, Gaojie Wang, Kang Zhang, Ximeng Zhao, Xinzhe Zhang, Dongchao Shen, Xue Zhang, Liying Cui.

  Mutations in the ANXA11 gene, encoding an RNA-binding protein, have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), but the underlying in vivo mechanisms remain unclear. This study examines the clinical features of ALS patients harboring the ANXA11 hotspot mutation p.P36R, characterized by late-onset motor neuron disease and occasional multi-system involvement. To elucidate the pathogenesis, we developed a knock-in mouse model carrying the p.P36R mutation. In both heterozygous and homozygous mutant mice, ANXA11 protein levels were comparable to those in wild-type. Both groups exhibited late-onset motor dysfunction at approximately 10 months of age, with similar survival rates to wild-type (> 24 months) and no signs of dementia. Pathological analysis revealed early abnormal aggregates in spinal cord motor neurons, cortical neurons, and muscle cells of homozygous mice. From 2 months of age, we observed mislocalized ANXA11 aggregates, SQSTM1/p62-positive inclusions, and cytoplasmic TDP-43 mislocalization, which intensified with disease progression. Importantly, mutant ANXA11 co-aggregated with TDP-43 and SQSTM1/p62-positive inclusions. Electron microscopy of the gastrocnemius muscle uncovered myofibrillar abnormalities, including sarcomeric disorganization, Z-disc dissolution, and subsarcolemmal electron-dense structures within autophagic vacuoles. Autophagic flux, initially intact at 2 months, was impaired by 9 months, as evidenced by decreased Beclin-1 and LC3BII/I levels and increased SQSTM1/p62 expression, coinciding with mTORC1 hyperactivation. Significant motor neuron loss and neuroinflammation were detected by 9 months, with marked muscle dystrophy apparent by 12 months compared to wild-type controls. These findings implicate the gain-of-function ANXA11 mutation drives late-onset motor neuron disease by early presymptomatic proteinopathy, progressive neuronal degeneration, neuroinflammation, and autophagic dysfunction.

Keywords:  ANXA11; Amyotrophic lateral sclerosis; Autophagy; Mouse model; Neuroinflammation; TDP-43 proteinopathy

DOI:  https://doi.org/10.1186/s40478-024-01919-4
Mov Disord. 2025 Jan 09.

TDP-43 Cryptic RNAs in Perry Syndrome: Differences across Brain Regions and TDP-43 Proteinopathies.

Sarah R Pickles, Jesus Gonzalez Bejarano, Anand Narayan, Lillian Daughrity, Candela Maroto Cidfuentes, Madison M Reeves, Mei Yue, Paula Castellanos Otero, Virginia Estades Ayuso, Judy Dunmore, Yuping Song, Jimei Tong, Michael DeTure, Bailey Rawlinson, Monica Castanedes-Casey, Jaroslaw Dulski, Catalina Cerquera-Cleves, Yongjie Zhang, Keith A Josephs, Dennis W Dickson, Leonard Petrucelli, Zbigniew K Wszolek, Mercedes Prudencio.

   BACKGROUND: Perry syndrome (PS) is a rare and fatal hereditary autosomal dominant neurodegenerative disorder caused by mutations in dynactin (DCTN1). PS brains accumulate inclusions positive for ubiquitin, transactive-response DNA-binding protein of 43 kDa (TDP-43), and to a lesser extent dynactin.
OBJECTIVES: Little is known regarding the contributions of TDP-43, an RNA binding protein that represses cryptic exon inclusion, in PS. Therefore, we sought to identify the degree of TDP-43 dysfunction in two regions of PS brains.
METHODS: We evaluated the levels of insoluble pTDP-43 and TDP-43-regulated cryptic RNAs and protein in the caudate nucleus and substantia nigra of 7 PS cases, 12 cases of frontotemporal lobar degeneration (FTLD) with TDP-43 pathology, and 11 cognitively healthy controls without TDP-43 pathology.
RESULTS: Insoluble pTDP-43 protein levels were detected in PS brains to a similar extent in the caudate nucleus and substantia nigra but lower than those in FTLD brains. The caudate nucleus of PS showed accumulation of eight TDP-43-regulated cryptic RNAs (ACTL6B, CAMK2B, STMN2, UNC13A, KCNQ2, ATG4B, GPSM2, and HDGFL2) and cryptic protein (HDGFL2) characteristic of FTLD. Conversely, only one cryptic target, UNC13A, reached significance in the substantia nigra despite similar pTDP-43 levels.
CONCLUSION: We detected TDP-43 cryptic RNAs and protein in PS caudate nucleus. Given the importance of cryptic exon biology in the development of biomarkers, and the identification of novel targets for therapeutic intervention, it is imperative we understand the consequences of TDP-43 dysfunction across different brain regions and determine the targets that are specific and common to TDP-43 proteinopathies. © 2025 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Keywords:  Perry syndrome; cryptic; frontotemporal dementia; frontotemporal lobar degeneration; transactive‐response DNA‐binding protein of 43 kDa (TDP‐43)

DOI:  https://doi.org/10.1002/mds.30104
J Vis Exp. 2024 Dec 13.

Vascular Organoid Generation from Human-Induced Pluripotent Stem Cells.

Cong Xu, Siyu He, Yuefei Zhu, Gazelle Crasto, Caroline Chen, Morgan L Clay, Yeh-Hsing Lao, Kam W Leong.

Vascular organoids derived from human induced pluripotent stem cells (hiPSCs) recapitulate the cell type diversity and complex architecture of human vascular networks. This three-dimensional (3D) model holds substantial potential for vascular pathology modeling and in vitro drug screening. Despite recent advances, a key technical challenge remains in reproducibly generating organoids with consistent quality, which is crucial for downstream assays and applications. Here, a modified protocol is presented that improves both the homogeneity and reproducibility of vascular organoid generation. The modified protocol incorporates the use of microwells and the CEPT cocktail (chroman 1, emricasan, polyamines, and the integrated stress response inhibitor, trans-ISRIB) to improve embryoid body formation and cell survival. Differentiated, mature vascular organoids generated using this protocol are characterized by whole-mount 3D immunofluorescence microscopy to analyze their morphology and complex vasculature. This protocol enables the production of high-quality vascular organoids in a scalable manner, potentially facilitating their use in disease modeling and drug screening applications.

DOI: https://doi.org/10.3791/67125
Nat Neurosci. 2025 Jan 06.

Aberrant splicing in Huntington's disease accompanies disrupted TDP-43 activity and altered m6A RNA modification.

Thai B Nguyen, Ricardo Miramontes, Carlos Chillon-Marinas, Roy Maimon, Sonia Vazquez-Sanchez, Alice L Lau, Nicolette R McClure, Zhuoxing Wu, Keona Q Wang, Whitney E England, Monika Singha, Jennifer T Stocksdale, Marie Heath, Ki-Hong Jang, Sunhee Jung, Karen Ling, Paymann Jafar-Nejad, Jharrayne I McKnight, Leanne N Ho, Osama Al Dalahmah, Richard L M Faull, Joan S Steffan, Jack C Reidling, Cholsoon Jang, Gina Lee, Don W Cleveland, Clotilde Lagier-Tourenne, Robert C Spitale, Leslie M Thompson.

Huntington's disease (HD) is caused by a CAG repeat expansion in the HTT gene, leading to altered gene expression. However, the mechanisms leading to disrupted RNA processing in HD remain unclear. Here we identify TDP-43 and the N6-methyladenosine (m6A) writer protein METTL3 to be upstream regulators of exon skipping in multiple HD systems. Disrupted nuclear localization of TDP-43 and cytoplasmic accumulation of phosphorylated TDP-43 occurs in HD mouse and human brains, with TDP-43 also co-localizing with HTT nuclear aggregate-like bodies distinct from mutant HTT inclusions. The binding of TDP-43 onto RNAs encoding HD-associated differentially expressed and aberrantly spliced genes is decreased. Finally, m6A RNA modification is reduced on RNAs abnormally expressed in the striatum of HD R6/2 mouse brain, including at clustered sites adjacent to TDP-43 binding sites. Our evidence supports TDP-43 loss of function coupled with altered m6A modification as a mechanism underlying alternative splicing in HD.

DOI: https://doi.org/10.1038/s41593-024-01850-w
Cells. 2025 Jan 03. pii: 47. [Epub ahead of print]14(1):

Schwann Cells in Neuromuscular Disorders: A Spotlight on Amyotrophic Lateral Sclerosis.

Kathryn R Moss, Smita Saxena.

  Amyotrophic Lateral Sclerosis (ALS) is a complex neurodegenerative disease primarily affecting motor neurons, leading to progressive muscle atrophy and paralysis. This review explores the role of Schwann cells in ALS pathogenesis, highlighting their influence on disease progression through mechanisms involving demyelination, neuroinflammation, and impaired synaptic function. While Schwann cells have been traditionally viewed as peripheral supportive cells, especially in motor neuron disease, recent evidence indicates that they play a significant role in ALS by impacting motor neuron survival and plasticity, influencing inflammatory responses, and altering myelination processes. Furthermore, advancements in understanding Schwann cell pathology in ALS combined with lessons learned from studying Charcot-Marie-Tooth disease Type 1 (CMT1) suggest potential therapeutic strategies targeting these cells may support nerve repair and slow disease progression. Overall, this review aims to provide comprehensive insights into Schwann cell classification, physiology, and function, underscoring the critical pathological contributions of Schwann cells in ALS and suggests new avenues for targeted therapeutic interventions aimed at modulating Schwann cell function in ALS.

Keywords:  ALS; CMT1; NMJ; Schwann cell; myelin; satellite glial cells; terminal Schwann cells

DOI:  https://doi.org/10.3390/cells14010047
PLoS One. 2025 ;20(1): e0314973

The MIR-NAT MAPT-AS1 does not regulate Tau expression in human neurons.

Rafaela Policarpo, Leen Wolfs, Saul Martínez-Montero, Lina Vandermeulen, Ines Royaux, Gert Van Peer, Pieter Mestdagh, Bart De Strooper, Annerieke Sierksma, Constantin d'Ydewalle.

The MAPT gene encodes Tau protein, a member of the large family of microtubule-associated proteins. Tau forms large insoluble aggregates that are toxic to neurons in several neurological disorders, and neurofibrillary Tau tangles represent a key pathological hallmark of Alzheimer's disease (AD) and other tauopathies. Lowering Tau expression levels constitutes a potential treatment for AD but the mechanisms that regulate Tau expression at the transcriptional or translational level are not well understood. Natural antisense transcripts (NATs) are a particular class of long non-coding RNAs (lncRNAs) that can regulate expression of their overlapping protein-coding genes at the epigenetic, transcriptional, or translational level. We and others identified a long non-coding RNA associated with the MAPT gene, named MAPT antisense 1 (MAPT-AS1). We confirmed that MAPT-AS1 is expressed in neurons in human post mortem brain tissue. To study the role of MAPT-AS1 on MAPT expression regulation, we modulated the expression of this lncRNA in human neuroblastoma cell lines and in human induced pluripotent stem cell (iPSC) derived neurons. In contrast to previous reports, we observed no changes on MAPT mRNA or Tau protein levels upon modulation of MAPT-AS1 levels in these cellular models. Our data suggest that MAPT-AS1 does not regulate Tau expression levels in human neurons in vitro. Thus, MAPT-AS1 does not represent a valuable therapeutic target to lower Tau expression in patients affected by tauopathies including AD.

DOI: https://doi.org/10.1371/journal.pone.0314973
Curr Protoc. 2025 Jan;5(1): e70091

Generation of Retinal Ganglion Cells from Reprogrammed Keratocytes of Non-Glaucoma and Glaucoma Donors.

Shahna S Hameed, Tasneem P Sharma.

  Human induced pluripotent stem cell (hiPSC)-based disease modeling can be successfully recapitulated to mimic disease characteristics across various human pathologies. Glaucoma, a progressive optic neuropathy, primarily affects the retinal ganglion cells (RGCs). While multiple groups have successfully generated RGCs from non-diseased hiPSCs, producing RGCs from glaucomatous human samples holds significant promise for understanding disease pathology by revealing patient-specific disease signatures. Given that keratocytes originate from the neural crest and previous reports suggest that ocular fibroblasts from glaucomatous donors carry pathogenic signatures, it is highly plausible that these signatures imprinted within the keratocytes will also be present in the derived RGCs. Thus, we aimed to generate RGCs from both glaucomatous and non-glaucomatous donor keratocytes and validate disease-specific signatures in 3D retinal organoids and in isolated RGCs. Our protocol describes the generation of iPSCs from keratocytes of both glaucomatous and non-glaucomatous donors, followed by their differentiation into retinal organoids. Subsequent isolation and culturing of RGCs were performed. Disease signatures in the RGCs were validated in both 3D retinal organoids (ROs) and 2D RGC cultures, and glaucomatous RGCs in 3D and 2D cultures demonstrated increased cleaved CASP3 and significant RGC loss, indicating disease imprints in the hiPSC-derived RGCs. This model offers a venue and high throughput platform for studying glaucomatous disease pathology and holds significant potential for drug discovery using RGCs derived from human donors. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Culturing of keratocytes from human cadaveric donors Basic Protocol 2: Reprogramming donor keratocytes into iPSCs Basic Protocol 3: Evaluation of chromosomal loss during reprogramming in iPSCs by karyotyping Basic Protocol 4: Generation of 3D ROs Basic Protocol 5: Dissociation and culturing of RGCs from 3D ROs Support Protocol 1: Immunostaining for phenotypic characterization of cells Support Protocol 2: Sectioning of 3D ROs and immunostaining Support Protocol 3: Western blotting for cleaved CASP3 and THY1.

Keywords:  glaucoma; induced pluripotent stem cells; keratocytes; retinal ganglion cells; retinal organoids

DOI:  https://doi.org/10.1002/cpz1.70091
Open Life Sci. 2024 ;19(1): 20221010

Axodendritic targeting of TAU and MAP2 and microtubule polarization in iPSC-derived versus SH-SY5Y-derived human neurons.

Helen Breuer, Michael Bell-Simons, Hans Zempel.

  Cell polarity is crucial in neurons, characterized by distinct axonal and dendritic structures. Neurons generally have one long axon and multiple shorter dendrites, marked by specific microtubule (MT)-associated proteins, e.g., MAP2 for dendrites and TAU for axons, while the scaffolding proteins AnkG and TRIM46 mark the axon-initial-segment. In tauopathies, such as Alzheimer's disease (AD), TAU sorting, and neuronal polarity are disrupted, leading to MT loss. However, modeling and studying MTs in human neuronal cells relevant to the study of AD and TAU-related neurodegenerative diseases (NDD) is challenging. To study MT dynamics in human neurons, we compared two cell culture systems: SH-SY5Y-derived neurons (SHN) and induced pluripotent stem cell-derived neurons (iN). Using immunostaining and EB3-tdTomato time-lapse imaging, we found AnkG absent in SHN but present in iN, while TRIM46 was present in both. TAU and MAP2 showed axonal and dendritic enrichment, respectively, similar to mouse primary neurons. Both neuron types exhibited polarized MT structures, with unidirectional MTs in axons and bidirectional MTs in dendrites. Polymerization speeds were similar; however, iNs had more retrograde MT growth events, while SHN showed a higher overall number of growth events. Thus, SHN and iN are both suitable for studying neuronal cell polarity, with SHN being particularly suitable if the focus is not the AIS.

Keywords:  AnkG/TRIM46; EB3-trafficking; axon-initial-segment; dendrite; neuronal cell polarity

DOI:  https://doi.org/10.1515/biol-2022-1010
FEBS J. 2025 Jan 05.

Regulation of physiological and pathological condensates by molecular chaperones.

Nadeen Akaree, Valentina Secco, Flonia Levy-Adam, Amal Younis, Serena Carra, Reut Shalgi.

  Biomolecular condensates are dynamic membraneless compartments that regulate a myriad of cellular functions. A particular type of physiological condensate called stress granules (SGs) has gained increasing interest due to its role in the cellular stress response and various diseases. SGs, composed of several hundred RNA-binding proteins, form transiently in response to stress to protect mRNAs from translation and disassemble when the stress subsides. Interestingly, SGs contain several aggregation-prone proteins, such as TDP-43, FUS, hnRNPA1, and others, which are typically found in pathological inclusions seen in autopsy tissues from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. Moreover, mutations in these genes lead to the familial form of ALS and FTD. This has led researchers to propose that pathological aggregation is seeded by aberrant SGs: SGs that fail to properly disassemble, lose their dynamic properties, and become pathological condensates which finally 'mature' into aggregates. Here, we discuss the evidence supporting this model for various ALS/FTD-associated proteins. We further continue to focus on molecular chaperone-mediated regulation of ALS/FTD-associated physiological condensates on one hand, and pathological condensates on the other. In addition to SGs, we review ALS/FTD-relevant nuclear condensates, namely paraspeckles, anisosomes, and nucleolar amyloid bodies, and discuss their emerging regulation by chaperones. As the majority of chaperoning mechanisms regulate physiological condensate disassembly, we highlight parallel themes of physiological and pathological condensation regulation across different chaperone families, underscoring the potential for early disease intervention.

Keywords:  ALS; FTD; FUS; LLPS; TDP‐43; aggregation; chaperones; condensates; proteostasis; stress granules

DOI:  https://doi.org/10.1111/febs.17390
Cancers (Basel). 2024 Dec 21. pii: 4259. [Epub ahead of print]16(24):

A Proteomic Examination of Plasma Extracellular Vesicles Across Colorectal Cancer Stages Uncovers Biological Insights That Potentially Improve Prognosis.

Abidali Mohamedali, Benjamin Heng, Ardeshir Amirkhani, Shivani Krishnamurthy, David Cantor, Peter Jun Myung Lee, Joo-Shik Shin, Michael Solomon, Gilles J Guillemin, Mark S Baker, Seong Beom Ahn.

   BACKGROUND: Recent advancements in understanding plasma extracellular vesicles (EVs) and their role in disease biology have provided additional unique insights into the study of Colorectal Cancer (CRC).
METHODS: This study aimed to gain biological insights into disease progression from plasma-derived extracellular vesicle proteomic profiles of 80 patients (20 from each CRC stage I-IV) against 20 healthy age- and sex-matched controls using a high-resolution SWATH-MS proteomics with a reproducible centrifugation method to isolate plasma EVs.
RESULTS: We applied the High-Stringency Human Proteome Project (HPP) guidelines for SWATH-MS analysis, which refined our initial EV protein identification from 1362 proteins (10,993 peptides) to a more reliable and confident subset of 853 proteins (6231 peptides). In early-stage CRC, we identified 11 plasma EV proteins with differential expression between patients and healthy controls (three up-regulated and eight down-regulated), many of which are involved in key cancer hallmarks. Additionally, within the same cohort, we analysed EV proteins associated with tumour recurrence to identify potential prognostic indicators for CRC. A subset of up-regulated proteins associated with extracellular vesicle formation (GDI1, NSF, and TMED9) and the down-regulation of TSG101 suggest that micro-metastasis may have occurred earlier than previously anticipated.
DISCUSSION: By employing stringent proteomic analysis and a robust SWATH-MS approach, we identified dysregulated EV proteins that potentially indicate early-stage CRC and predict recurrence risk, including proteins involved in metabolism, cytoskeletal remodelling, and immune response. While our findings underline discrepancies with other studies due to differing isolation and stringency parameters, they provide valuable insights into the complexity of the EV proteome, emphasising the need for standardised protocols and larger, well-controlled studies to validate potential biomarkers.

Keywords:  SWATH-MS; colorectal cancer; exosomes; extracellular vesicles; plasma microvesicle proteins; protein biomarkers

DOI:  https://doi.org/10.3390/cancers16244259
Cells. 2024 Dec 17. pii: 2076. [Epub ahead of print]13(24):

Exploring the Role of Axons in ALS from Multiple Perspectives.

Xiaosu Chen, Shuchang Lv, Jinmeng Liu, Yingjun Guan, Chunjie Xu, Xiaonan Ma, Mu Li, Xue Bai, Kexin Liu, Haoyun Zhang, Qiupeng Yan, Fenghua Zhou, Yanchun Chen.

  Amyotrophic lateral sclerosis (ALS), commonly known as motor neuron disease, is a neurodegenerative disorder characterized by the progressive degeneration of both upper and lower motor neurons. This pathological process results in muscle weakness and can culminate in paralysis. To date, the precise etiology of ALS remains unclear. However, a burgeoning body of research indicates that axonal dysfunction is a pivotal element in the pathogenesis of ALS and significantly influences the progression of disease. Dysfunction of axons in ALS can result in impediments to nerve impulse transmission, leading to motor impairment, muscle atrophy, and other associated complications that severely compromise patients' quality of life and survival prognosis. In this review, we concentrate on several key areas: the ultrastructure of axons, the mechanisms of axonal degeneration in ALS, the impact of impaired axonal transport on disease progression in ALS, and the potential for axonal regeneration within the central nervous system (CNS). Our objective is to achieve a more holistic and profound understanding of the multifaceted role that axons play in ALS, thereby offering a more intricate and refined perspective on targeted axonal therapeutic interventions.

Keywords:  amyotrophic lateral sclerosis; axon degeneration; axon regeneration; axon transport; therapy

DOI:  https://doi.org/10.3390/cells13242076
J Biophotonics. 2025 Jan 10. e202400513

Neuronal Imaging at 8-Bit Depth to Combine High Spatial and High Temporal Resolution With Acquisition Rates Up To 40 kHz.

Fatima Abbas, Ömer Yusuf İpek, Philippe Moreau, Marco Canepari.

  A challenge in neuroimaging is acquiring frame sequences at high temporal resolution from the largest possible number of pixels. Measuring 1%-10% fluorescence changes normally requires 12-bit or higher bit depth, constraining the frame size allowing imaging in the kHz range. We resolved Ca2+ or membrane potential signals from cell populations or single neurons in brain slices by acquiring fluorescence at 8-bit depth and by binning pixels offline, achieving unprecedented frame sizes at kHz rates. In hippocampal slices stained with the Ca2+ indicator Fluo-4 AM, we resolved transients at 2 kHz from large frames. Along the apical dendrite of a layer-5 pyramidal neuron, we measured Ca2+ signals associated with a back-propagating action potential at 10 kHz. Finally, in the axon initial segment of the same cell type, we recorded an action potential at 40 kHz by voltage-sensitive dye imaging. This approach unlocks the potential for a range of imaging measurements.

Keywords:  brain slices; calcium imaging; neurons; voltage imaging

DOI:  https://doi.org/10.1002/jbio.202400513
Biomolecules. 2024 Dec 22. pii: 1649. [Epub ahead of print]14(12):

Alpha-Synuclein Effects on Mitochondrial Quality Control in Parkinson's Disease.

Lydia Shen, Ulf Dettmer.

  The maintenance of healthy mitochondria is essential for neuronal survival and relies upon mitochondrial quality control pathways involved in mitochondrial biogenesis, mitochondrial dynamics, and mitochondrial autophagy (mitophagy). Mitochondrial dysfunction is critically implicated in Parkinson's disease (PD), a brain disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra. Consequently, impaired mitochondrial quality control may play a key role in PD pathology. This is affirmed by work indicating that genes such as PRKN and PINK1, which participate in multiple mitochondrial processes, harbor PD-associated mutations. Furthermore, mitochondrial complex-I-inhibiting toxins like MPTP and rotenone are known to cause Parkinson-like symptoms. At the heart of PD is alpha-synuclein (αS), a small synaptic protein that misfolds and aggregates to form the disease's hallmark Lewy bodies. The specific mechanisms through which aggregated αS exerts its neurotoxicity are still unknown; however, given the vital role of both αS and mitochondria to PD, an understanding of how αS influences mitochondrial maintenance may be essential to elucidating PD pathogenesis and discovering future therapeutic targets. Here, the current knowledge of the relationship between αS and mitochondrial quality control pathways in PD is reviewed, highlighting recent findings regarding αS effects on mitochondrial biogenesis, dynamics, and autophagy.

Keywords:  PGC-1α; PINK1/Parkin; Parkinson’s disease; mitochondrial dysfunction; mitochondrial fragmentation; mitophagy; α-synuclein

DOI:  https://doi.org/10.3390/biom14121649
NPJ Parkinsons Dis. 2025 Jan 09. 11(1): 16

Synaptic vesicle characterization of iPSC-derived dopaminergic neurons provides insight into distinct secretory vesicle pools.

Kenshiro Fujise, Jaya Mishra, Martin Shaun Rosenfeld, Nisha Mohd Rafiq.

The dysfunction of dopaminergic (DA) neurons is central to Parkinson's disease. Distinct synaptic vesicle (SV) populations, differing in neurotransmitter content (dopamine vs. glutamate), may vary due to differences in trafficking and exocytosis. However, the structural organization of these vesicles remains unclear. In this study, we examined axonal varicosities in human iPSC-derived DA and glutamatergic neurons (i3Neurons). i3Neurons primarily contained small, clear SVs (40-50 nm), whereas DA neurons contained larger, pleiomorphic vesicles including dense core and empty vesicles, in addition to the classical SVs. VMAT2-positive vesicles in DA neurons, which load dopamine, were spatially segregated from VGLUT1/2-positive vesicles in an SV-like reconstitution system. These vesicles also colocalized with SV markers (e.g., VAMP2, SV2C), and can be clustered by synapsin. Moreover, DA axonal terminals in mouse striata showed similar vesicle pool diversity. These findings reveal structural differences in DA neurons' vesicles, highlighting iPSC-derived neurons as effective models for studying presynaptic structures.

DOI: https://doi.org/10.1038/s41531-024-00862-4
bioRxiv. 2024 Dec 20. pii: 2024.12.18.629265. [Epub ahead of print]

Widespread Distribution of α-Synuclein Oligomers in LRRK2 -related Parkinson's Disease.

Hiroaki Sekiya, Lukas Franke, Yuki Hashimoto, Mariko Takata, Katsuya Nishida, Naonobu Futamura, Kazuko Hasegawa, Hisatomo Kowa, Owen A Ross, Pamela J McLean, Tatsushi Toda, Zbigniew K Wszolek, Dennis W Dickson.

Mutations in leucine-rich repeat kinase 2 ( LRRK2 ) are the most common cause of familial and sporadic Parkinson's disease (PD). While the clinical features of LRRK2 -PD patients resemble those of typical PD, there are significant differences in the pathological findings. The pathological hallmark of definite PD is the presence of α-synuclein (αSYN)-positive Lewy-related pathology; however, approximately half of LRRK2 -PD cases do not have Lewy-related pathology. Lewy-related pathology is a late-stage αSYN aggregation that can be visualized with hematoxylin and eosin stains or conventional immunohistochemistry (IHC). Increasing evidence has indicated that αSYN oligomers, which represent the early-stage of αSYN aggregation, may have neurotoxicity. Visualization of αSYN oligomers requires specialized staining techniques, such as αSYN-proximity ligation assay (PLA). The distribution and severity of αSYN oligomers in the human brain of LRRK2 -PD patients remain unknown. In this study, we performed phosphorylated αSYN-IHC and αSYN-PLA staining on postmortem brain sections of patients with three pathogenic LRRK2 mutants: p.G2019S (n=5), p.I2020T (n=5), and p.R1441C (n=4). The severity of Lewy-related pathology and αSYN oligomers were assessed semi-quantitatively in the brainstem, limbic lobe, basal ganglia, and cerebral cortex. αSYN oligomers were detected in LRRK2 -PD cases even in cases without Lewy-related pathology; a negative correlation was observed between Lewy-related pathology and αSYN oligomers (r=-0.26 [-0.39, -0.12]; P<0.0001). Our findings suggest that αSYN oligomers may represent a common pathological feature of LRRK2 -PD. Notably, patients harboring p.G2019S and p.I2020T had significantly higher levels of αSYN oligomers in those without Lewy-related pathology compared to those with Lewy-related pathology. These cases also had a trend toward shorter disease duration. These results imply that in LRRK2 -PD, αSYN oligomers may initially accumulate in the brain but do not progress to form Lewy-related pathology. The present study suggests that targeting αSYN oligomers may be a therapeutic strategy for LRRK2 -PD even if there is no Lewy-related pathology.

DOI: https://doi.org/10.1101/2024.12.18.629265
bioRxiv. 2024 Dec 18. pii: 2024.12.17.628947. [Epub ahead of print]

Poly-ubiquitylated transmembrane proteins outcompete other cargo for limited space inside clathrin-coated vesicles.

Hao-Yang Liu, Grant Ashby, Feng Yuan, Susovan Sarkar, Carl C Hayden, Jon M Huibregtse, Jeanne C Stachowiak.

Endocytic recycling of transmembrane proteins is essential to cell signaling, ligand uptake, protein traffic and degradation. The intracellular domains of many transmembrane proteins are ubiquitylated, which promotes their internalization by clathrin-mediated endocytosis. How might this enhanced internalization impact endocytic uptake of transmembrane proteins that lack ubiquitylation? Recent work demonstrates that diverse transmembrane proteins compete for space within highly crowded endocytic structures, suggesting that enhanced internalization of one group of transmembrane proteins may come at the expense of other groups. Here we show that preferential internalization of poly-ubiquitylated transmembrane proteins results in reduced endocytosis of mono-ubiquitylated and non-ubiquitylated proteins. Using a combination of live-cell imaging and ligand uptake assays, we confirmed that increased ubiquitylation correlates with increased internalization by clathrin-coated vesicles. Further, poly-ubiquitylated receptors significantly outcompeted their mono-ubiquitylated and non-ubiquitylated counterparts for localization to endocytic sites and uptake of extracellular ligands. These findings demonstrate the inherent interdependence of transmembrane protein recycling, suggesting that clathrin-coated vesicles act as selective filters, prioritizing highly ubiquitylated transmembrane proteins for uptake while leaving proteins with little or no ubiquitylation behind. Given that poly-ubiquitylation is thought to signal protein aging and damage, our findings suggest a mechanism for selective internalization of high priority cargo proteins, with simultaneously exclusion and protection of functional proteins that lack poly-ubiquitylation.
Significance: Ubiquitylation is essential for maintaining cellular homeostasis by regulating transmembrane protein trafficking, degradation, and signaling. Traditionally, poly-ubiquitylation has been viewed primarily as a signal for protein degradation, while mono-ubiquitylation is considered sufficient to trigger endocytosis. However, our work reveals a previously unrecognized role for poly-ubiquitylation in clathrin-mediated endocytosis, demonstrating that poly-ubiquitylated transmembrane proteins outcompete their non-ubiquitylated counterparts for incorporation into clathrin-coated vesicles, thereby establishing a competitive framework for endocytic cargo sorting. This mechanism reveals a selective sorting mechanism driven by the extent of ubiquitylation, which could regulate the removal of damaged proteins while protecting functional proteins at the plasma membrane.

DOI: https://doi.org/10.1101/2024.12.17.628947
Neuroinformatics. 2025 Jan 10. 23(1): 3

Computational Generation of Long-range Axonal Morphologies.

Adrien Berchet, Remy Petkantchin, Henry Markram, Lida Kanari.

  Long-range axons are fundamental to brain connectivity and functional organization, enabling communication between different brain regions. Recent advances in experimental techniques have yielded a substantial number of whole-brain axonal reconstructions. While previous computational generative models of neurons have predominantly focused on dendrites, generating realistic axonal morphologies is more challenging due to their distinct targeting. In this study, we present a novel algorithm for axon synthesis that combines algebraic topology with the Steiner tree algorithm, an extension of the minimum spanning tree, to generate both the local and long-range compartments of axons. We demonstrate that our computationally generated axons closely replicate experimental data in terms of their morphological properties. This approach enables the generation of biologically accurate long-range axons that span large distances and connect multiple brain regions, advancing the digital reconstruction of the brain. Ultimately, our approach opens up new possibilities for large-scale in-silico simulations, advancing research into brain function and disorders.

Keywords:  Algebraic topology; Axon synthesis; Brain connectivity; Neuronal morphology; Steiner tree

DOI:  https://doi.org/10.1007/s12021-024-09696-0
iScience. 2025 Jan 17. 28(1): 111515

TBC1D24 interacts with the v-ATPase and regulates intraorganellar pH in neurons.

Sara Pepe, Davide Aprile, Enrico Castroflorio, Antonella Marte, Simone Giubbolini, Samir Hopestone, Anna Parsons, Tânia Soares, Fabio Benfenati, Peter L Oliver, Anna Fassio.

  The vacuolar ATPase (v-ATPase) is essential for acidification of intracellular organelles, including synaptic vesicles. Its activity is controlled by cycles of association and dissociation of the ATP hydrolysis (V1) and proton transport (V0) multi-protein subunits. Mutations in genes coding for both v-ATPase subunits and TBC1D24 cause neurodevelopmental disorders with overlapping syndromes; therefore, it is important to investigate their potentially interrelated functions. Here, we reveal that TBC1D24 interacts with the v-ATPase in the brain. Using a constitutive Tbc1d24 knockout mouse model, we observed accumulation of lysosomes and non-degraded lipid materials in neuronal tissue. In Tbc1d24 knockout neurons, we detected V1 mis-localization with increased pH at endo-lysosomal compartments and autophagy impairment. Furthermore, synaptic vesicles endocytosis and reacidification were impaired. Thus, we demonstrate that TBC1D24 is a positive regulator of v-ATPase activity in neurons suggesting that alteration of pH homeostasis could underlie disorders associated with TBC1D24 and the v-ATPase.

Keywords:  cellular neuroscience; molecular neuroscience

DOI:  https://doi.org/10.1016/j.isci.2024.111515
Neuron. 2025 Jan 08. pii: S0896-6273(24)00879-1. [Epub ahead of print]113(1): 7-28

DNA damage and its links to neuronal aging and degeneration.

Ilse Delint-Ramirez, Ram Madabhushi.

  DNA damage is a major risk factor for the decline of neuronal functions with age and in neurodegenerative diseases. While how DNA damage causes neurodegeneration is still being investigated, innovations over the past decade have provided significant insights into this issue. Breakthroughs in next-generation sequencing methods have begun to reveal the characteristics of neuronal DNA damage hotspots and the causes of DNA damage. Chromosome conformation capture-based approaches have shown that, while DNA damage and the ensuing cellular response alter chromatin topology, chromatin organization at damage sites also affects DNA repair outcomes in neurons. Additionally, neuronal activity results in the formation of programmed DNA breaks, which could burden DNA repair mechanisms and promote neuronal dysfunction. Finally, emerging evidence implicates DNA damage-induced inflammation as an important contributor to the age-related decline in neuronal functions. Together, these discoveries have ushered in a new understanding of the significance of genome maintenance for neuronal function.

Keywords:  DNA damage; active DNA demethylation; mutations; neurodegeneration; neuroinflammation; topoisomerase

DOI:  https://doi.org/10.1016/j.neuron.2024.12.001
Neurotox Res. 2025 Jan 08. 43(1): 3

Curcumin Improves Hippocampal Cell Bioenergetics, Redox and Inflammatory Markers, and Synaptic Proteins, Regulating Mitochondrial Calcium Homeostasis.

Claudia Jara, Angie K Torres, Han S Park-Kang, Lisette Sandoval, Claudio Retamal, Alfonso Gonzalez, Micaela Ricca, Sebastián Valenzuela, Michael P Murphy, Nibaldo C Inestrosa, Cheril Tapia-Rojas.

  Mitochondria produces energy through oxidative phosphorylation (OXPHOS), maintaining calcium homeostasis, survival/death cell signaling mechanisms, and redox balance. These mitochondrial functions are especially critical for neurons. The hippocampus is crucial for memory formation in the brain, which is a process with high mitochondrial function demand. Loss of hippocampal function in aging is related to neuronal damage, where mitochondrial impairment is critical. Synaptic and mitochondrial dysfunction are early events in aging; both are regulated reciprocally and contribute to age-associated memory loss together. We previously showed that prolonged treatment with Curcumin or Mitoquinone (MitoQ) improves mitochondrial functions in aged mice, exerting similar neuroprotective effects. Curcumin has been described as an anti-inflammatory and antioxidant compound, and MitoQ is a potent antioxidant directly targeting mitochondria; however, whether Curcumin exerts a direct impact on the mitochondria is unclear. In this work, we study whether Curcumin could have a mechanism similar to MitoQ targeting the mitochondria. We utilized hippocampal slices of 4-6-month-old C57BL6 mice to assess the cellular changes induced by acute Curcumin treatment ex-vivo compared to MitoQ. Our results strongly suggest that both compounds improve the synaptic structure, oxidative state, and energy production in the hippocampus. Nevertheless, Curcumin and MitoQ modify mitochondrial function differently; MitoQ improves the mitochondrial bioenergetics state, reducing ROS production and increasing ATP generation. In contrast, Curcumin reduces mitochondrial calcium levels and prevents calcium overload related to mitochondrial swelling. Thus, Curcumin is described as a new regulator of mitochondrial calcium homeostasis and could be used in pathological events involving calcium deregulation and excitotoxicity, such as aging and neurodegenerative diseases.

Keywords:  Curcumin; Hippocampus; MitoQ; Mitochondria

DOI:  https://doi.org/10.1007/s12640-024-00726-y
SLAS Discov. 2025 Jan 06. pii: S2472-5552(25)00001-2. [Epub ahead of print] 100208

Novel high-content and open-source image analysis tools for profiling mitochondrial morphology in neurological cell models.

Marcus Y Chin, David A Joy, Madhuja Samaddar, Anil Rana, Johann Chow, Takashi Miyamoto, Meredith Calvert.

  Mitochondria undergo dynamic morphological changes depending on cellular cues, stress, genetic factors, or disease. The structural complexity and disease-relevance of mitochondria have stimulated efforts to generate image analysis tools for describing mitochondrial morphology for therapeutic development. Using high-content analysis, we measured multiple morphological parameters and employed unbiased feature clustering to identify the most robust pair of texture metrics that described mitochondrial state. Here, we introduce a novel image analysis pipeline to enable rapid and accurate profiling of mitochondrial morphology in various cell types and pharmacological perturbations. We applied a high-content adapted implementation of our tool, MitoProfilerHC, to quantify mitochondrial morphology changes in i) a mammalian cell dose response study and ii) compartment-specific drug effects in primary neurons. Next, we expanded the usability of our pipeline by using napari, a Python-powered image analysis tool, to build an open-source version of MitoProfiler and validated its performance and applicability. In conclusion, we introduce MitoProfiler as both a high-content-based and an open-source method to accurately quantify mitochondrial morphology in cells, which we anticipate to greatly facilitate mechanistic discoveries in mitochondrial biology and disease.

Keywords:  Mitochondria; high-content imaging; high-throughput screening; image analysis; mitochondrial morphology; napari plugin; neurons; open-source

DOI:  https://doi.org/10.1016/j.slasd.2025.100208
Nat Commun. 2025 Jan 08. 16(1): 472

MPicker: visualizing and picking membrane proteins for cryo-electron tomography.

Xiaofeng Yan, Shudong Li, Weilin Huang, Hao Wang, Tianfang Zhao, Mingtao Huang, Niyun Zhou, Yuan Shen, Xueming Li.

Advancements in cryo-electron tomography (cryoET) allow the structure of macromolecules to be determined in situ, which is crucial for studying membrane protein structures and their interactions in the cellular environment. However, membranes are often highly curved and have a strong contrast in cryoET tomograms, which masks the signals from membrane proteins. These factors pose difficulties in observing and revealing the structures of membrane proteins in situ. Here, we report a membrane-flattening method and the corresponding software, MPicker, designed for the visualization, localization, and orientation determination of membrane proteins in cryoET tomograms. This method improves the visualization of proteins on and around membranes by generating a flattened tomogram that eliminates membrane curvature and reduces the spatial complexity of membrane protein analysis. In MPicker, we integrated approaches for automated particle picking and coarse alignment of membrane proteins for sub-tomogram averaging. MPicker was tested on tomograms of various cells to evaluate the method for visualizing, picking, and analyzing membrane proteins.

DOI: https://doi.org/10.1038/s41467-024-55767-w
Ann Neurol. 2025 Jan 10.

Genetic Ablation of Sarm1 Mitigates Disease Acceleration after Traumatic Brain Injury in the SOD1G93A Transgenic Mouse Model of Amyotrophic Lateral Sclerosis.

Elif O Dogan, Sean R Simonini, James Bouley, Alexandra Weiss, Robert H Brown, Nils Henninger.

OBJECTIVE: Approximately 20% of familial cases of amyotrophic lateral sclerosis (ALS) are caused by mutations in the gene encoding superoxide dismutase 1 (SOD1). Epidemiological data have identified traumatic brain injury (TBI) as an exogenous risk factor for ALS; however, the mechanisms by which TBI may worsen SOD1 ALS remain largely undefined.
METHODS: We sought to determine whether repetitive TBI (rTBI) accelerates disease onset and progression in the transgenic SOD1G93A mouse ALS model, and whether loss of the primary regulator of axonal degeneration sterile alpha and TIR motif containing 1 (Sarm1) mitigates the histological and behavioral pathophysiology. We subjected wild-type (n = 23), Sarm1 knockout (KO; n = 17), SOD1G93A (n = 19), and SOD1G93AxSarm1KO (n = 26) mice of both sexes to rTBI or sham surgery at age 64 days (62-68 days). Body weight and ALS-deficit score were serially assessed up to 17 weeks after surgery and histopathology assessed in layer V of the primary motor cortex at the study end point.
RESULTS: In sham injured SOD1G93A mice, genetic ablation of Sarm1 did not attenuate axonal loss, improve neurological deficits, or survival. The rTBI accelerated onset of G93A-SOD1 ALS, as indicated by accentuated body weight loss, earlier onset of hindlimb tremor, and shortened survival. The rTBI also triggered TDP-43 mislocalization, enhanced axonal and neuronal loss, microgliosis, and astrocytosis. Loss of Sarm1 significantly diminished the impact of rTBI on disease progression and rescued rTBI-associated neuropathology.
INTERPRETATION: SARM1-mediated axonal death pathway promotes pathogenesis after TBI in SOD1G93A mice suggesting that anti-SARM1 therapeutics are a viable approach to preserve neurological function in injury-accelerated G93A-SOD1 ALS. ANN NEUROL 2025.

DOI: https://doi.org/10.1002/ana.27174
Proc Natl Acad Sci U S A. 2025 Jan 14. 122(2): e2410433122

A synapse-specific refractory period for plasticity at individual dendritic spines.

Juan C Flores, Dipannita Sarkar, Karen Zito.

  How newly formed memories are preserved while brain plasticity is ongoing has been a source of debate. One idea is that synapses which experienced recent plasticity become resistant to further plasticity, a type of metaplasticity often referred to as saturation. Here, we probe the local dendritic mechanisms that limit plasticity at recently potentiated synapses. We show that recently potentiated individual synapses exhibit a synapse-specific refractory period for further potentiation. We further found that the refractory period is associated with reduced postsynaptic CaMKII signaling; however, stronger synaptic activation fully restored CaMKII signaling but only partially restored the ability for further plasticity. Importantly, the refractory period is released after one hour, a timing that coincides with the enrichment of several postsynaptic proteins to preplasticity levels. Notably, increasing the level of the postsynaptic scaffolding protein, PSD95, but not of PSD93, overcomes the refractory period. Our results support a model in which potentiation at a single synapse is sufficient to initiate a synapse-specific refractory period that persists until key postsynaptic proteins regain their steady-state synaptic levels.

Keywords:  PSD-95; dendritic spine; metaplasticity; synaptic plasticity; two-photon imaging

DOI:  https://doi.org/10.1073/pnas.2410433122
bioRxiv. 2024 Dec 27. pii: 2024.12.27.630542. [Epub ahead of print]

Huntingtin interactome reveals huntingtin role in regulation of double strand break DNA damage response (DSB/DDR), chromatin remodeling and RNA processing pathways.

Tamara Ratovitski, Chloe D Holland, Robert N O'Meally, Alexey V Shevelkin, Tianze Shi, Robert N Cole, Mali Jiang, Christopher A Ross.

  Huntington's Disease (HD), a progressive neurodegenerative disorder with no disease-modifying therapies, is caused by a CAG repeat expansion in the HD gene encoding polyglutamine-expanded huntingtin (HTT) protein. Mechanisms of HD cellular pathogenesis and cellular functions of the normal and mutant HTT proteins are still not completely understood. HTT protein has numerous interaction partners, and it likely provides a scaffold for assembly of multiprotein complexes many of which may be altered in HD. Previous studies have implicated DNA damage response in HD pathogenesis. Gene transcription and RNA processing has also emerged as molecular mechanisms associated with HD. Here we used multiple approaches to identify HTT interactors in the context of DNA damage stress. Our results indicate that HTT interacts with many proteins involved in the regulation of interconnected DNA repair/remodeling and RNA processing pathways. We present evidence for a role for HTT in double strand break repair mechanism. We demonstrate HTT functional interaction with a major DNA damage response kinase DNA-PKcs and association of both proteins with nuclear speckles. We show that S1181 phosphorylation of HTT is regulated by DSB, and can be carried out (at least in vitro) by DNA-PK. Furthermore, we show HTT interactions with RNA binding proteins associated with nuclear speckles, including two proteins encoded by genes at HD modifier loci, TCERG1 and MED15, and with chromatin remodeling complex BAF. These interactions of HTT may position it as an important scaffolding intermediary providing integrated regulation of gene expression and RNA processing in the context of DNA repair mechanisms.

Keywords:  DNA damage; Huntington’s Disease; RNA processing; huntingtin; neurodegeneration; post-translational modifications; protein interactions

DOI:  https://doi.org/10.1101/2024.12.27.630542
Nat Commun. 2025 Jan 07. 16(1): 451

Mitochondria- and ER-associated actin are required for mitochondrial fusion.

Priya Gatti, Cara Schiavon, Julien Cicero, Uri Manor, Marc Germain.

Mitochondria are crucial for cellular metabolism and signalling. Mitochondrial activity is modulated by mitochondrial fission and fusion, which are required to properly balance metabolic functions, transfer material between mitochondria, and remove defective mitochondria. Mitochondrial fission occurs at mitochondria-endoplasmic reticulum (ER) contact sites, and requires the formation of actin filaments that drive mitochondrial constriction and the recruitment of the fission protein DRP1. The role of actin in mitochondrial fusion remains entirely unexplored. Here we show that preventing actin polymerisation on either mitochondria or the ER disrupts both fission and fusion. We show that fusion but not fission is dependent on Arp2/3, whereas both fission and fusion require INF2 formin-dependent actin polymerization. We also show that mitochondria-associated actin marks fusion sites prior to the fusion protein MFN2. Together, our work introduces a method for perturbing organelle-associated actin and demonstrates a previously unknown role for actin in mitochondrial fusion.

DOI: https://doi.org/10.1038/s41467-024-55758-x