bims-axbals 2025-06-22 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2025–06–22
twenty-two papers selected by
TJ Krzystek

A guide to selecting high-performing antibodies for Rab10 (UniProt ID: P61026) for use in western blot, immunoprecipitation, and immunofluorescence.
A guide to selecting high-performing antibodies for STING1 (Uniprot ID: Q86WV6) for use in western blot, immunoprecipitation, and immunofluorescence.
Telmisartan is neuroprotective in a hiPSC-derived spinal microtissue model for C9orf72 ALS via inhibition of neuroinflammation.
Exploring NEK1 genetic variability in Italian amyotrophic lateral sclerosis patients.
Enhancing neuronal viability: The protective role of 10% human cerebrospinal fluid in primary neuronal cultures.
ALS-associated TDP-43 aggregates drive innate and adaptive immune cell activation.
Dopamine and cortical neurons with different Parkinsonian mutations show variation in lysosomal and mitochondrial dysfunction.
YAP maintains the dynamics of TDP-43 condensates and antagonizes TDP-43 pathological aggregates.
Cell autonomous microglia defects in a stem cell model of frontotemporal dementia tau.
Synaptic mitochondria in aging and neurodegenerative diseases: unraveling their functional decline and vulnerability.
Regulation of LRRK2 activity by metabolic stress and heavy metal exposure.
ATG9 not just an Autophagy Related Protein.
One gene, many phenotypes: the role of KIF5A in neurodegenerative and neurodevelopmental diseases.
Change of spiny neuron structure in the basal ganglia song circuit and its regulation by miR-9 during song development.
Fasting the mitochondria to prevent neurodegeneration: the role of ceramides.
Non-canonical roles of mitotic proteins in cortical neurons.
Development of a Microfluidics-Based Approach for Investigating Microtubule Polymer Mechanics.
Lysosomal targeting of liposomes with acidic pH and Cathepsin B induces protein aggregate clearance.
A Novel Method for Culturing Telencephalic Neurons in Axolotls.
An Efficient Organoid Cutting Method for Long-Term Culture and High-Throughput Analyses.
Formation of seeding-competent α-synuclein aggregates in parkin-deficient iPSC-derived human neurons.
Scale-down optimization of a robust, parallelizable human induced pluripotent stem cell bioprocess for high-throughput research.

F1000Res. 2024 ;13 1061

A guide to selecting high-performing antibodies for Rab10 (UniProt ID: P61026) for use in western blot, immunoprecipitation, and immunofluorescence.

NeuroSGC/YCharOS/EDDU collaborative group

  Rab10 is a small GTPase involved in cargo transport from the trans-Golgi network to the plasma membrane and endocytic recycling back to the cell membrane. It has garnered significant interest in neurodegenerative disease research, particularly due to its phosphorylation by the LRRK2 kinase. This relationship underscores the importance of Rab10 in cellular processes related to disease pathology, specifically Parkinson's disease. The accessibility of renewable and selective antibodies against Rab10 would advance research efforts, enabling further understanding of its function and implications in disease. Here, we have characterized eight Rab10 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Keywords:  RAB10; Rab10; UniProt ID: P61026; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation; western blot

DOI:  https://doi.org/10.12688/f1000research.156209.1
F1000Res. 2024 ;13 1049

A guide to selecting high-performing antibodies for STING1 (Uniprot ID: Q86WV6) for use in western blot, immunoprecipitation, and immunofluorescence.

NeuroSGC/YCharOS/EDDU collaborative group

  Stimulator of interferon genes protein (STING1) is an immune adaptor protein which promotes innate immune defense mechanisms against pathogens. To enhance our understanding of STING1-associated disease, it is essential to make high-performing antibodies accessible to the scientific community. This study aims to improve reliability of STING1 research as we have characterized sixteen STING1 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Keywords:  MITA; MPYS; STING1; Uniprot ID: Q86WV6; antibody characterization; antibody validation; hSTING; immunofluorescence; immunoprecipitation; mediator of IRF3 activation; stimulator of interferon genes protein; transmembrane protein 173; western blot

DOI:  https://doi.org/10.12688/f1000research.155929.2
Stem Cell Reports. 2025 Jun 16. pii: S2213-6711(25)00139-0. [Epub ahead of print] 102535

Telmisartan is neuroprotective in a hiPSC-derived spinal microtissue model for C9orf72 ALS via inhibition of neuroinflammation.

Berkiye Sonustun, Björn F Vahsen, Mario Ledesma-Terrón, Zhuoning Li, Laura Tuffery, Nan Xu, Elizabeth L Calder, Johannes Jungverdorben, Leslie Weber, Aaron Zhong, David G Miguez, Mara Monetti, Ting Zhou, Elisa Giacomelli, Lorenz Studer.

  Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive motor neuron (MN) loss. The most common genetic cause, a hexanucleotide repeat expansion in C9orf72 (C9-ALS), disrupts microglial function, contributing to neuroinflammation, a key disease driver. To investigate this, we developed a three-dimensional spinal microtissue (SM) model incorporating human induced pluripotent stem cell (hiPSC)-derived MNs, astrocytes, and microglia. Screening 190 Food and Drug Administration (FDA)-approved compounds, we identified sartans-angiotensin II receptor I blockers (ARBs)-as potent inhibitors of neuroinflammation. Telmisartan, a highly brain-penetrant ARB, significantly reduced the levels of pro-inflammatory cytokines interleukin (IL)-6 and IL-8 and rescued MN loss in C9-ALS SMs. Our findings suggest that C9-ALS microglia drive MN toxicity and that telmisartan can effectively mitigate inflammation and preserve MN viability. This work lays the groundwork for modeling disease-related neuroinflammation and points to telmisartan as a therapeutic candidate worth further exploration for treating C9-ALS.

Keywords:  3D microtissue; C9orf72; amyotrophic lateral sclerosis; astrocytes; drug screen; microglia; motor neurons; neuroinflammation; sartans; triculture

DOI:  https://doi.org/10.1016/j.stemcr.2025.102535
J Neurol. 2025 Jun 19. 272(7): 469

Exploring NEK1 genetic variability in Italian amyotrophic lateral sclerosis patients.

Viviana Pensato, Silvia Peverelli, Cinzia Tiloca, Stefania Magri, Alberto Brusati, Monica Pingue, Claudia Morelli, Eleonora Dalla Bella, Arianna Manini, Pierpaola Tannorella, Alberto Doretti, Jessica Mandrioli, Fabrizia Terenghi, Alessandro Prelle, Nilo Riva, Federico Verde, Roberto Eleopra, Franco Taroni, Giuseppe Lauria Pinter, Vincenzo Silani, Nicola Ticozzi, Cinzia Gellera, Antonia Ratti.

   BACKGROUND: Mutations in NEK1, encoding for a serine/threonine kinase which regulates several biological processes, are associated with amyotrophic lateral sclerosis (ALS).
METHODS: NEK1 was analysed by amplicon deep sequencing in a cohort of 1016 Italian sporadic and familial ALS patients previously screened for C9orf72, SOD1, TARDBP and FUS mutations.
RESULTS: We identified 28 rare NEK1 variants in 29 patients (2.85%) of whom 20/782 were sporadic (2.5%), 6/107 familial (5%) and 3/127 of unknown aetiology (2.3%). Variants were classified as pathogenic (P; n = 1), likely pathogenic (LP; n = 6 in 7 patients) and of unknown significance (VUS; n = 21) according the American College of Medical Genetics and Genomics criteria. Notably, 64% of the identified variants (18/28, including 4 LP and 14 VUS) were novel. Among the 29 patients with rare NEK1 variants, 7 (of whom 5 were familial cases) had additional variants in one of the four main ALS causative genes. Moreover, 23 patients carried the already reported NEK1 p.Arg261His risk variant (VUS) alone or in addition to SOD1 mutations (n = 1) or C9orf72 repeat expansion (n = 2) and to the NEK1 p.Asp128Val variant (n = 1). Genotype-phenotype correlation analysis showed no significant differences in age at onset or survival in NEK1 variant carriers, independently on the variant type. No flail arm phenotype, but atypical features, including sensory symptoms, were present in NEK1 carriers.
CONCLUSION: Our study further expands NEK1 genetic variability by identifying novel rare variants and confirming ALS oligogenic nature since 19.6% of NEK1 patients also carried mutations in one of the four main ALS-associated genes.

Keywords:   NEK1 ; ALS; Genetic screening; IPSC; Oligogenicity

DOI:  https://doi.org/10.1007/s00415-025-13153-6
Brain Res. 2025 Jun 13. pii: S0006-8993(25)00343-9. [Epub ahead of print]1864 149782

Enhancing neuronal viability: The protective role of 10% human cerebrospinal fluid in primary neuronal cultures.

Vineet Arora, Alicia Bernhardt, Alessandro Napoli, Mijail Serruya.

  Human cerebrospinal fluid (hCSF) is a physiologically rich medium containing neurotrophic factors, signaling molecules, and essential metabolites that support neuronal development, survival, and function. While its neuroprotective properties have been demonstrated in organotypic brain slices and human iPSC-derived models, its application in primary rodent cortical neuron cultures-a foundational system for studying synaptic development and neurodegeneration-remains underexplored. In this study, we systematically evaluated the effects of hCSF supplementation on neuronal viability in primary cortical cultures derived from embryonic day 18 (E18) rat embryos. To determine the optimal concentration, we tested a range of media:hCSF ratios and identified 90:10 (i.e., 10% hCSF) as the most effective for enhancing neuronal survival. Cell viability was assessed using two complementary assays: SYTOX Green for detecting dead cells and Calcein AM/Ethidium Homodimer-2 (EthD2) dual-staining for quantifying live/dead cell populations. Our results show that 10% hCSF supplementation significantly reduces cell death and improves overall neuronal health under standard in vitro conditions. This optimized approach offers a reproducible and physiologically relevant strategy for improving dissociated cortical neuron cultures and has important implications for in vitro modeling of neurodegenerative diseases, neurotoxicity screening, and regenerative neuroscience research.

Keywords:  Cell death reduction; E18 rat embryos; Human cerebrospinal fluid (hCSF); Neuronal survival; Neuronal viability; Neuroprotection; Neurotrophic factors; Primary neuronal cultures

DOI:  https://doi.org/10.1016/j.brainres.2025.149782
iScience. 2025 Jun 20. 28(6): 112648

ALS-associated TDP-43 aggregates drive innate and adaptive immune cell activation.

Baggio A Evangelista, Joey V Ragusa, Kyle Pellegrino, Yijia Wu, Ivana Yoseli Quiroga-Barber, Shannon R Cahalan, Omeed K Arooji, Jillann A Madren, Sally Schroeter, Joe Cozzarin, Ling Xie, Xian Chen, Kristen K White, J Ashley Ezzell, Marie A Iannone, Sarah Cohen, Douglas H Phanstiel, Rick B Meeker, Todd J Cohen.

  Amyotrophic lateral sclerosis (ALS) is the most common and fatal motor neuron disease. Approximately 90% of ALS patients exhibit pathology of the master RNA regulator, transactive response DNA binding protein (TDP-43). Despite the prevalence TDP-43 pathology in ALS motor neurons, recent findings suggest immune dysfunction is a determinant of disease progression in patients. Whether TDP-43 aggregates elicit immune responses remains underexplored. In this study, we demonstrate that TDP-43 aggregates are internalized by antigen-presenting cell populations, cause vesicle rupture, and drive innate and adaptive immune cell activation by way of antigen presentation. Using a multiplex imaging platform, we observed enrichment of activated microglia/macrophages in ALS white matter that correlated with phosphorylated TDP-43 accumulation, CD8 T cell infiltration, and major histocompatibility complex expression. Taken together, this study sheds light on a novel cellular response to TDP-43 aggregates through an immunological lens.

Keywords:  Immunity; Neuroscience; Omics

DOI:  https://doi.org/10.1016/j.isci.2025.112648
NPJ Parkinsons Dis. 2025 Jun 20. 11(1): 177

Dopamine and cortical neurons with different Parkinsonian mutations show variation in lysosomal and mitochondrial dysfunction.

Jessica Chedid, Yan Li, Adahir Labrador-Garrido, Dad Abu-Bonsrah, Chiara Pavan, Tyra Fraser, Dmitry Ovchinnikov, Melanie Zhong, Ryan Davis, Dario Strbenac, Jennifer A Johnston, Lachlan H Thompson, Deniz Kirik, Clare L Parish, Glenda M Halliday, Carolyn M Sue, Gautam Wali, Nicolas Dzamko.

Mutations causing Parkinson's disease (PD) give diverse pathological phenotypes whose cellular correlates remain to be determined. Those with PRKN mutations have significantly earlier selective vulnerability of dopamine neurons, those with SNCA mutations have increased alpha-synuclein deposition, while those with LRRK2 mutations have additional deposition of tau. Yet all three mutation types are implicated in mitochondrial and/or lysosomal dysfunction. To compare cellular dysfunctions associated with these different pathological phenotypes, an unbiased high-content imaging platform was developed to assess both lysosomal and mitochondrial dysfunction, along with alpha-synuclein and tau protein deposition using induced pluripotent stem cell (iPSC) derived cortical and ventral midbrain neurons. Different PD mutations caused cell type specific dysfunctions, likely to impact on both selective neuronal vulnerability and the pathologies observed in PD. Comparison of dopamine neurons identified that both lysosomal and mitochondrial dysfunction were predominant with PRKN lof mutations, whereas SNCA A53T and LRRK2 R1441G mutations had increased tau deposition. In contrast, cortical neurons with SNCA and LRRK2 mutations both had mitochondrial and autophagy impairments without protein deposition, with LRRK2 cells additionally showing decreased glucocerebrosidase activity and increased alpha-synuclein phosphorylation.

DOI: https://doi.org/10.1038/s41531-025-01048-2
Nat Cell Biol. 2025 Jun 20.

YAP maintains the dynamics of TDP-43 condensates and antagonizes TDP-43 pathological aggregates.

Jiaqi Zhang, Jiaojiao Hu, Ruogu Liu, Tian Zhou, Xuewei Luo, Peigang Liang, Zaichao Xie, Qinyue Zhao, Yan Chen, Dan Du, Cong Liu, Yiming Zheng, Dan Li, Bo Wang.

Recent studies exploring the underlying pathomechanisms of amyotrophic lateral sclerosis (ALS), a fatal motor neuron disorder, have focused on biomolecular condensates. Here we reveal an unexpected function for YAP, a central component of the Hippo pathway, in regulating the dynamic behaviour of stress granules and TDP-43 condensates, a role that is independent of its transcriptional activity in the Hippo pathway. YAP directly binds to TDP-43. This interaction directly promotes the homotypic multimerization and phase separation of TDP-43 while inhibiting its hyperphosphorylation and solidification under stress conditions. Remarkably, YAP, whose messenger RNA levels are reduced in patients with ALS, is found to co-localize with pathological hyperphosphorylated TDP-43 aggregates in the brains of patients with ALS. In addition, elevation of YAP/Yorkie (a fly homologue of mammalian YAP) expression substantially reduces TDP-43 toxicity in primary neuron and transgenic fly models of ALS. Our findings highlight an unexpected role of YAP in managing ALS-associated biomolecular condensates, presenting important implications for potential ALS treatments.

DOI: https://doi.org/10.1038/s41556-025-01685-y
Mol Psychiatry. 2025 Jun 17.

Cell autonomous microglia defects in a stem cell model of frontotemporal dementia tau.

Abhirami K Iyer, Lisa Vermunt, Farzaneh S Mirfakhar, Miguel Minaya, Mariana Acquarone, Rama Krishna Koppisetti, Arun Renganathan, Shih-Feng You, Emma P Danhash, Kylie J Schache, Anthony Verbeck, Grant Galasso, Scott M Lee, Guangming Huang, Katherine J Miller, Jacob Marsh, Alissa L Nana, Salvatore Spina, William W Seeley, Lea T Grinberg, Sally Temple, Charlotte E Teunissen, Chihiro Sato, Celeste M Karch.

Neuronal dysfunction has been extensively studied as a central feature of neurodegenerative tauopathies. However, across neurodegenerative diseases, there is strong evidence for active involvement of immune cells like microglia in driving disease pathophysiology. Here, we demonstrate that MAPT mRNA and tau protein are expressed in microglia in human brains and in human induced pluripotent stem cell (iPSC)-derived microglia like cells (iMGLs). Using iMGLs harboring the MAPT IVS10 + 16 mutation and isogenic controls, we demonstrate that a tau mutation is sufficient to alter microglial transcriptional states. We discovered that MAPT IVS10 + 16 microglia exhibit cytoskeletal abnormalities, stalled phagocytosis, disrupted TREM2/TYROBP networks, and altered metabolism. Additionally, we found that secretory factors from MAPT IVS10 + 16 iMGLs impact neuronal health, reducing synaptic density in neurons. Key features observed in vitro were recapitulated in human brain tissue and cerebrospinal fluid from MAPT mutations carriers. Together, our findings that MAPT IVS10 + 16 drives cell-intrinsic dysfunction in microglia that impacts neuronal health has major implications for development of therapeutics for tauopathies.

DOI: https://doi.org/10.1038/s41380-025-03073-2
Neural Regen Res. 2025 Jun 19.

Synaptic mitochondria in aging and neurodegenerative diseases: unraveling their functional decline and vulnerability.

Karina A Cicali, Angie K Torres, Cheril Tapia-Rojas.

   ABSTRACT: Aging is a physiological and complex process produced by accumulative age-dependent cellular damage, which significantly impacts brain regions like the hippocampus, an essential region involved in memory and learning. A crucial factor contributing to this decline is the dysfunction of mitochondria, particularly those located at synapses. Synaptic mitochondria are specialized organelles that produce the energy required for synaptic transmission but are also important for calcium homeostasis at these sites. In contrast, non-synaptic mitochondria primarily involve cellular metabolism and long-term energy supply. Both pools of mitochondria differ in their form, proteome, functionality, and cellular role. The proper functioning of synaptic mitochondria depends on processes such as mitochondrial dynamics, transport, and quality control. However, synaptic mitochondria are particularly vulnerable to age-associated damage, characterized by oxidative stress, impaired energy production, and calcium dysregulation. These changes compromise synaptic transmission, reducing synaptic activity and cognitive decline during aging. In the context of neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's, the decline of synaptic mitochondrial function is even more pronounced. These diseases are marked by pathological protein accumulation, disrupted mitochondrial dynamics, and heightened oxidative stress, accelerating synaptic dysfunction and neuronal loss. Due to their specialized role and location, synaptic mitochondria are among the first organelles to exhibit dysfunction, underscoring their critical role in disease progression. This review delves into the main differences at structural and functional levels between synaptic and nonsynaptic mitochondria, emphasizing the vulnerability of synaptic mitochondria to the aging process and neurodegeneration. These approaches highlight the potential of targeting synaptic mitochondria to mitigate age-associated cognitive impairment and synaptic degeneration. This review emphasizes the distinct vulnerabilities of hippocampal synaptic mitochondria, highlighting their essential role in sustaining brain function throughout life and their promise as therapeutic targets for safeguarding the cognitive capacities of people of advanced age.

Keywords:  aging; hippocampus; memory; mitochondria; synaptic mitochondria

DOI:  https://doi.org/10.4103/NRR.NRR-D-24-01571
Brain Res. 2025 Jun 14. pii: S0006-8993(25)00346-4. [Epub ahead of print]1863 149785

Regulation of LRRK2 activity by metabolic stress and heavy metal exposure.

Michalis Kentros, Jordan Follett, Nitya Subrahmanian, Aravindraja Chairmandurai, Katerina Melachroinou, Diane B Re, Rafael de Cabo, Ruth Chia, Jillian H Kluss, Alexandra Beilina, Heather Mortiboys, Matthew J LaVoie, Hardy J Rideout, Mark R Cookson, Adamantios Mamais.

  Genetic variability in the gene encoding leucine-rich repeat kinase 2 (LRRK2) is associated with both familial and sporadic Parkinson's disease (PD). While LRRK2 is known to modulate vesicular trafficking and stress signaling through its phosphorylation and kinase activity, how it responds to metabolic and environmental stressors remains poorly understood. Here, we show that acute inhibition of glycolysis and oxidative phosphorylation triggers rapid, reversible dephosphorylation of LRRK2 at constitutive sites in cells, ex vivo brain slices, and primary astrocytes. In contrast, glucose deprivation modestly increases LRRK2 kinase activity and Rab substrate phosphorylation. In vivo, chronic 2-deoxyglucose treatment reduces S935 phosphorylation in kidney tissue, linking energy stress to LRRK2 modulation in peripheral organs. Strikingly, manganese (Mn), a PD-relevant environmental toxicant, robustly activates LRRK2, inducing pS1292 autophosphorylation and phosphorylation of Rab8a, Rab10 and Rab12, while suppressing S935 phosphorylation after a 24 hrs exposure. Time-resolved analysis revealed distinct temporal substrate regulation, with rapid Rab12 phosphorylation and pRab10 levels gradually increasing and peaking only after 24 h. Phosphorylated Rab10 remains closely associated with both lysosomal and centrosomal membranes under Mn stress. Mn impaired mitochondrial respiration and increased ROS, and antioxidant treatment rescued Rab10 phosphorylation, establishing a redox-dependent mechanism of LRRK2 activation. Together, these findings reveal stressor-specific modes of LRRK2 regulation and suggest that LRRK2 integrates metabolic and environmental signals via redox-sensitive pathways relevant to PD pathogenesis.

Keywords:  LRRK2; Manganese; Metabolic stress

DOI:  https://doi.org/10.1016/j.brainres.2025.149785
J Mol Biol. 2025 Jun 11. pii: S0022-2836(25)00354-7. [Epub ahead of print] 169288

ATG9 not just an Autophagy Related Protein.

Emily Millard, Sharon A Tooze.

  Autophagy proteins coordinate the biogenesis of a phagophore, the formation and maturation of an autophagosome. Genetic mutations of these proteins can result in dysregulated autophagy, stalled autophagosome biogenesis, and lead to cell death. ATG9, the sole transmembrane ATG (autophagy related) protein governs the nucleation of the phagophore. At a molecular level ATG9 has been shown to be a lipid scramblase capable of redistributing lipids across the lipid bilayer. ATG9-positive vesicles can also deliver lipid-modifying enzymes to alter the lipid composition of membranes. Both functions are required for autophagy. However, ATG proteins, including ATG9, play key molecular roles in pathways unrelated to autophagy. ATG9 has been shown to function in multiple pathways at the Golgi, plasma membrane, and lysosomes. ATG9 can also play an important role in immune signalling. The trafficking of ATG9 in ATG9-positive vesicles is essential to many of these pathways. In this review we highlight the functions of ATG9 in autophagy and autophagy-unrelated pathways, here referred to as "non-canonical functions", and summarise the broader role of ATG9A in cell homeostasis.

Keywords:  ATG9A; Atg9; autophagy; membrane trafficking

DOI:  https://doi.org/10.1016/j.jmb.2025.169288
Cell Commun Signal. 2025 Jun 16. 23(1): 287

One gene, many phenotypes: the role of KIF5A in neurodegenerative and neurodevelopmental diseases.

Marta Cozzi, Barbara Tedesco, Veronica Ferrari, Marta Chierichetti, Paola Pramaggiore, Laura Cornaggia, Rocio Magdalena, Maria Brodnanova, Ali Mohamed, Carmelo Milioto, Margherita Piccolella, Mariarita Galbiati, Paola Rusmini, Valeria Crippa, Cinzia Gellera, Stefania Magri, Franco Taroni, Riccardo Cristofani, Angelo Poletti.



Keywords:  Amyotrophic lateral sclerosis; Charcot-Marie-Tooth disease; Hereditary spastic paraplegia; KIF5A; Neonatal intractable myoclonus

DOI:  https://doi.org/10.1186/s12964-025-02277-x
J Neurosci. 2025 Jun 13. pii: e2276232025. [Epub ahead of print]

Change of spiny neuron structure in the basal ganglia song circuit and its regulation by miR-9 during song development.

Hannah Jarrell, Ansab Akhtar, Max Horowitz, Zhi Huang, Zhimin Shi, ZhiDe Fang, XiaoChing Li.

Juvenile zebra finches learn to sing by imitating conspecific songs of adults during a sensitive period early in life. Area X is a basal ganglia nucleus of the song control circuit specialized for song-related sensory-motor learning during song development. The structural plasticity and the molecular mechanisms regulating neuronal structure in Area X during song development and maturation are unclear. In this study, we examined the structure of spiny neurons, the main neuron type in Area X, at key stages of song development in male zebra finches. We report that dendritic arbor of spiny neurons expands during the sensitive period for song learning, and this initial growth is followed by pruning of dendrites and spines accompanied by changes in spine morphology as the song circuit matures. Previously, we showed that overexpression of miR-9 in Area X impairs song learning and performance and alters the expression of many genes that have important roles in neuronal structure and function (Shi et al., 2018). As an extension of that study, we report here that overexpression of miR-9 in spiny neurons in juvenile zebra finches reduces dendritic arbor complexity and spine density in a developmental stage-specific manner. We also show that miR-9 regulates structural maintenance of spiny neurons in adulthood. Together, these findings reveal dynamic microstructural changes in the song circuit during the sensitive period of song development and provide evidence that miR-9 regulates neuronal structure during song development and maintenance.Significance Statement Song development in juvenile zebra finches provides a model to study sensitive period plasticity for language development and related neural developmental disorders in humans. Area X is a basal ganglia nucleus essential for song-related sensory-motor learning in the zebra finch. We show that dendritic arbor of spiny neurons in Area X undergoes an initial growth and expansion followed by pruning of dendrites and spines during song development, and that this process is regulated by miR-9 in a developmental stage specific manner. These findings reveal the temporal profiles of structural development of key neurons in the basal ganglia song circuit and reveal a possible molecular mechanism for restricting sensitive period plasticity during vocal development.

DOI: https://doi.org/10.1523/JNEUROSCI.2276-23.2025
Front Neurosci. 2025 ;19 1602149

Fasting the mitochondria to prevent neurodegeneration: the role of ceramides.

Luis Armando Valenzuela-Ahumada, Octavio Fabián Mercado-Gómez, Rubi Viveros-Contreras, Rosalinda Guevara-Guzmán, Alberto Camacho-Morales.

  Neurodegenerative diseases affect up to 349.2 million individuals worldwide. Preclinical and clinical advances have documented that altered energy homeostasis and mitochondria dysfunction is a hallmark of neurological disorders. Diet-derived ceramides species might target and disrupt mitochondria function leading to defective energy balance and neurodegeneration. Ceramides as bioactive lipid species affect mitochondria function by several mechanism including changes in membrane chemical composition, inhibition of the respiratory chain, ROS overproduction and oxidative stress, and also by activating mitophagy. Promising avenues of intervention has documented that intermittent fasting (IF) is able to benefit and set proper energy metabolism. IF is an eating protocol that involves alternating periods of fasting with periods of eating which modulate ceramide metabolism and mitochondria function in neurons. This review will address the detrimental effect of ceramides on mitochondria membrane composition, respiratory chain, ROS dynamics and mitophagy in brain contributing to neurodegeneration. We will focus on effect of IF on ceramide metabolism as a potential avenue to improve mitochondria function and prevention of neurodegeneration.

Keywords:  ceramides; intermittent fasting; microglia; mitophagy; neurodegeneration

DOI:  https://doi.org/10.3389/fnins.2025.1602149
Trends Neurosci. 2025 Jun 14. pii: S0166-2236(25)00105-5. [Epub ahead of print]

Non-canonical roles of mitotic proteins in cortical neurons.

Joana Cavaco, Sara Carvalhal.

  Mitotic proteins are traditionally studied for their role in chromosome segregation during cell division. However, research increasingly highlights the important non-canonical roles of mitotic proteins beyond mitosis, particularly in the mammalian cerebral cortex. Alterations in the expression levels or mutations of mitotic proteins are increasingly linked to brain disorders such as primary microcephaly and Alzheimer's disease. A central, unresolved question remains: how do mitotic proteins contribute to neuronal pathogenesis? Here, we review emerging literature on the non-canonical roles of mitotic proteins in mature neurons. Additionally, we discuss how these contribute to the complex mechanisms underlying neurodevelopmental and neurodegenerative disorders. We also discuss their potential for identifying therapeutic strategies and as biomarkers in brain pathologies.

Keywords:  brain; mitosis; neurodegenerative disorders; neuronal migration; synapsis, neurodevelopmental disorders

DOI:  https://doi.org/10.1016/j.tins.2025.05.010
J Vis Exp. 2025 May 30.

Development of a Microfluidics-Based Approach for Investigating Microtubule Polymer Mechanics.

Matthew Rogers, Laura Richardson, Marija Zanic.

In this protocol, we describe the design and fabrication of a microfluidic device developed for the investigation of microtubule polymer mechanics. The design utilizes the intrinsic benefits of Polydimethylsiloxane (PDMS)-based microfluidic devices and introduces several features to enable a robust and customizable high-throughput experimental approach. The developed device incorporates redundant bubble-trapping capabilities to prevent the occurrence of detrimental air bubbles. Furthermore, the device interfaces with an automated flow control system to reduce manual intervention and enable high-throughput analyses. Commercial simulation software is utilized to better develop and understand the fluid transport using this system. Finally, we demonstrate the capability to conduct multiple experiments simultaneously within a single device by growing microtubule extensions with distinct fluorescent labels in different sections of the device. Overall, this microfluidic flow system can be used to probe microtubule polymer mechanics and provides improvements in experimental design for broader microtubule in vitro studies. The synthesis of microfabrication, automated flow control, and computational modeling approaches enables a flexible system ideally suited for probing the cellular cytoskeleton in vitro.

DOI: https://doi.org/10.3791/68185
Cell Commun Signal. 2025 Jun 19. 23(1): 296

Lysosomal targeting of liposomes with acidic pH and Cathepsin B induces protein aggregate clearance.

Minsol Jeon, Da-Eun Kim, So Young Choi, Seoyoung Kim, Seongchan Kim, Hyojin Lee, Hyunkyung Kim.

  The autophagy-lysosomal pathway is a cellular degradation mechanism that regulates protein quality by eliminating aggregates and maintaining normal protein function. It has been reported that aging itself reduces lysosomal proteolytic activity in age-related neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease. Reduction in lysosomal function may underlie the accumulation of protein aggregates such as amyloid beta (Aβ), tau, and α-synuclein. Some of these protein aggregates may cause additional lysosomal dysfunction and create a vicious cycle leading to a gradual increase in protein aggregation. In this study, liposome-based lysosomal pH-modulating particles (LPPs), containing a liquid solution to adjust lysosomal pH, have been developed to restore lysosomal function. The results demonstrate that acidic LPPs effectively restore lysosomal function by recovering lysosomal pH and facilitating the removal of protein aggregates. These findings demonstrated that acidic LPPs could effectively recover the abnormal lysosomal function via restoration of lysosomal pH and enhance the clearance of protein aggregates. Furthermore, the simultaneous introduction of Cathepsin B (CTSB) proteins and acidic LPP revealed a synergistic effect, promoting lysosomal pH recovery and enhancing aggregates removal. These findings suggest a novel strategy for improving lysosomal clearance activity in proteinopathies.

Keywords:  Aggregate clearance; Autophagy; Cathepsin; Lysosome; Proteinopathy

DOI:  https://doi.org/10.1186/s12964-025-02310-z
J Comp Neurol. 2025 Jun;533(6): e70066

A Novel Method for Culturing Telencephalic Neurons in Axolotls.

Sevginur Bostan, Safiye Serdengeçti, F Kemal Bayat, Sadık Bay, AyşeServer Sezer, Neşe Ayşit, Gürkan Öztürk.

  The axolotl (Ambystoma mexicanum), a neotenic salamander with remarkable regenerative capabilities, serves as a key model for studying nervous system regeneration. Despite its potential, the cellular and molecular mechanisms underlying this regenerative capacity remain poorly understood, partly due to the lack of reliable in vitro models for axolotl neural cells. In this study, we developed a novel protocol for primary cultures of adult axolotl telencephalon/pallium, enabling the maintenance of viable and functionally active neural cells. Using calcium imaging and immunocytochemistry, we demonstrated the presence of neuronal and glial markers, synaptic connections, and spontaneous calcium activity, highlighting the functional integrity of the cultured cells. Our findings reveal that these cultures can be maintained in both serum and serum-free conditions, with neurons exhibiting robust neurite outgrowth and responsiveness to injury. This protocol addresses a critical gap in axolotl research by providing a controlled in vitro system to study neurogenesis and regeneration. By offering insights into the regenerative mechanisms of axolotl neurons, this work lays the foundation for comparative studies with mammalian systems, potentially informing therapeutic strategies for neurodegenerative diseases and CNS injuries in humans.

Keywords:  axolotl; primary neuron culture; regeneration; telencephalon

DOI:  https://doi.org/10.1002/cne.70066
Tissue Eng Regen Med. 2025 Jun 16.

An Efficient Organoid Cutting Method for Long-Term Culture and High-Throughput Analyses.

Nicholas A Chartrain, Marina V Pryzhkova, Juliana I Candelaria, Kristin H Gilchrist, Philip W Jordan.

   BACKGROUND: Human organoid models are invaluable for developmental studies, disease modeling, and personalized medicine research. However, long-term maintenance is challenging due to hypoxia and nutrient limitations as organoids grow. Cutting organoids improves viability, but current methods have low throughput and are prone to causing culture contamination. This study introduces an efficient organoid cutting method to enhance long-term culture and enable high-throughput analyses.
METHODS: We employed three-dimensional (3D) printing to fabricate four classes of organoid cutting jigs with blade guides that were compared and optimized for consistent sectioning of human pluripotent stem cell (hPSC)-derived organoids. Organoids were cultured in mini-spin bioreactors and cut every three weeks, beginning on day 35. Organoid health and growth were evaluated by size increase and proliferative marker expression. Additionally, we utilized 3D printed molds to create GelMA or Geltrex-embedded organoid arrays and silicone molds for optimal cutting temperature compound (OCT)-embedding of organoid arrays.
RESULTS: All 3D printed jigs enabled rapid and uniform organoid cutting under sterile conditions. We determined that a flat-bottom cutting jig design had superior cutting efficiency. Cutting improved nutrient diffusion, increased cell proliferation, and enhanced organoid growth during long-term culture. The mold-based approaches enabled the creation of densely packed organoid arrays and cryosections with evenly distributed organoids.
CONCLUSION: This novel organoid cutting and arraying method overcomes limitations in long-term organoid culture and high-throughput processing. The simplicity of the cutter design and handling make it a versatile tool for diverse types of organoids. By enhancing organoid viability and enabling consistent sample preparation, this approach facilitates improved organ development and disease modeling, drug screening, and high-throughput analyses, including single-cell spatial transcriptomics applications.

Keywords:  Bioengineering; High-throughput; Human pluripotent stem cells; Long-term culture; Organoid

DOI:  https://doi.org/10.1007/s13770-025-00731-y
NPJ Parkinsons Dis. 2025 Jun 21. 11(1): 180

Formation of seeding-competent α-synuclein aggregates in parkin-deficient iPSC-derived human neurons.

Sissel Ida Schmidt, Justyna Okarmus, Daniel Aghaie Madsen, Julie Schmidt Hansen, Emil Gregersen, Hjalte Gram, Lucas S Winkelmann, Anderson Souza Oliveira, Rachel Heon-Roberts, Brent J Ryan, Kristine Freude, Morten Blaabjerg, Poul Henning Jensen, Morten Meyer.

Loss-of-function mutations in PARK2 (parkin) cause early-onset familial Parkinson's disease (PD) and may also contribute to sporadic PD. While Lewy bodies, enriched in aggregated phosphorylated α-synuclein (α-Syn), are typical in PD, their presence in PARK2-mediated PD remains debated. Using human isogenic PARK2-/- induced pluripotent stem cell-derived neurons, we investigated α-Syn pathology under parkin deficiency. PARK2-/- neurons showed elevated intracellular aggregated and total α-Syn levels, increased α-Syn release, and higher levels of aggregation-inducing α-Syn seeds. These neurons also displayed more pSer129 α-Syn+ inclusions, which were further enhanced by α-Syn preformed fibril (PFF) exposure. Moreover, we identified synaptic loss in the PARK2-/- neurons, exacerbated by PFF treatment, and dysregulated Ca2+ homeostasis consistent with enhanced activity of the smooth endoplasmic reticulum Ca2+-ATPase (SERCA). Our data provide an important contribution to the debate on the role of α-Syn in the pathology of PARK2-related PD and challenge the view of PARK2-related PD as a non-synucleinopathy.

DOI: https://doi.org/10.1038/s41531-025-01038-4
Biotechnol Rep (Amst). 2025 Sep;47 e00900

Scale-down optimization of a robust, parallelizable human induced pluripotent stem cell bioprocess for high-throughput research.

James Colter, Tiffany Dang, Julia Malinovska, Jessica May Corpuz, Dora Modrcin, Roman Krawetz, Kartikeya Murari, Michael Scott Kallos.

  Human induced pluripotent stem cell (hiPSC) derived therapeutics require clinically relevant quantities of high-quality cell populations for applications in regenerative medicine. The lack of efficacy exhibited across clinical trials suggests deeper understanding of the networks governing phenotype is needed. Further, costs limit study throughput in characterizing the artificial niche relative to outcomes. We present herein an optimized strategy to enable high-throughput hiPSC expansion at <20 mL research scale. We assessed viability of single cell inoculation and aggregate preformation to facilitate proliferation. We modeled aggregate characteristics against agitation rate. Our results demonstrate tunable control with fold expansion comparable to commercial systems. Marker quantification and teratoma assay confirm functional pluripotency. This approach constitutes a scalable protocol to accelerate hiPSC research, and a significant step in advancing the rate of progress in elucidating links to derivative functionality. This work will enable statistically rigorous studies targeting hiPSC and downstream phenotype for clinical manufacturing.

Keywords:  Human induced pluripotent stem cells; Optimization; Stem cell bioprocessing; Stirred Tank Bioreactors

DOI:  https://doi.org/10.1016/j.btre.2025.e00900