bims-axbals 2025-12-28 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2025–12–28
twenty-one papers selected by
TJ Krzystek

Preclinical Evaluation of the Assembly Modulator PAV-615 in a Mouse Model of C9orf72-Associated ALS/FTD.
Maintenance and Decline of Neuronal Lysosomal Function in Aging.
Protrudin acts at ER-endosome contacts to promote KIF5-mediated endosomal tubule fission.
Human midbrain organoids reveal the characteristics of axonal mitochondria specific to dopaminergic neurons.
Basic Science and Pathogenesis.
Reduced cortico-muscular output is associated with intrinsic hypoexcitability and reduced persistent inward currents in motor cortex neurons of TDP-43Q331K ALS mice.
Basic Science and Pathogenesis.
Neuronal mitochondrial disaggregase CLPB ameliorates Huntington's disease pathology in mice.
Developing Topics.
A role for Fis1 in dendritic development.
Basic Science and Pathogenesis.
Developing Topics.
Developing Topics.
A shared DNA-repeat toxicity threshold, reached somatically at cell-type-specific rates, unites cortical and striatal neurodegeneration in Huntington's disease.
Basic Science and Pathogenesis.
Axonal growth of cortical neurons does not require paxillin.
Lysosomal swelling triggers LRRK2 activity.
A new subset of mitochondrial-derived vesicles perform inter-mitochondrial communications.
Landscape Expansion Microscopy Reveals Interactions between Membrane and Phase-Separated Organelles.
Getting to the right place at the right time - membrane trafficking and maturation in the endolysosomal system.
A neuron type-specific microexon in Ank3 /ankyrin-G modulates calcium activity and neuronal excitability.

Cells. 2025 Dec 17. pii: 2012. [Epub ahead of print]14(24):

Preclinical Evaluation of the Assembly Modulator PAV-615 in a Mouse Model of C9orf72-Associated ALS/FTD.

Jingfen Su, Jorge Alaiz Noya, Anuradha F Lingappa, Dennis Solas, Jimei Tong, Lillian Daughrity, Monica Castanedes-Casey, Aishe Kurti, Dennis W Dickson, Vishwanath R Lingappa, Leonard Petrucelli, Yongjie Zhang.

  Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative diseases that share clinical and pathological features, as well as genetic causes. A G4C2 repeat expansion in chromosome 9 open reading frame 72 (C9orf72) is the most common genetic cause of ALS and FTD, collectively referred to as c9ALS/FTD. Assembly modulation is a new therapeutic approach which appears to target allosteric sites on aberrant forms of multi-protein complexes and restore them to the healthy state. Recent findings demonstrate that tetrahydroisoquinolone (THIQ)-based protein assembly modulators can ameliorate ALS/FTD-associated phenotypes in cellular and animal models. In the present study, we investigated the effects of PAV-615, a novel and advanced THIQ-based modulator, in a c9ALS/FTD mouse model expressing 149 G4C2 repeat expansions (referred to as 149R mouse model). Specifically, PAV-615 was administered to 5-month-old 149R mice via intraperitoneal injection for one month. Motor function was evaluated using the hang wire test, while anxiety-like behavior and hyperactivity were assessed using the open-field test. Pathological markers, including dipeptide repeat (DPR) proteins, phosphorylated TAR DNA-binding protein 43 (pTDP-43) and ataxin 2-positive stress granules, were quantified by Meso Scale Discovery and immunohistochemistry assays. Compared with vehicle-treated controls, PAV-615 significantly improved motor performance and modestly reduced anxiety-like behavior and hyperactivity in 149R mice. Moreover, PAV-615 treatment significantly decreased cortical DPR, pTDP-43 and ataxin 2-positive stress granule burdens. These results support assembly modulation as a promising therapeutic approach treatment of ALS/FTD.

Keywords:  C9orf72; G4C2 repeat expansion; PAV-615; amyotrophic lateral sclerosis; anxiety-like behavior; assembly modulation; ataxin 2-positive stress granule; dipeptide repeat proteins; frontotemporal dementia; hyperactivity; motor function; phosphorylated TDP-43

DOI:  https://doi.org/10.3390/cells14242012
Cells. 2025 Dec 12. pii: 1976. [Epub ahead of print]14(24):

Maintenance and Decline of Neuronal Lysosomal Function in Aging.

Ruiling Zhong, Claire E Richardson.

  Lysosomes are central effectors of cellular maintenance, integrating the degradation of damaged organelles and protein aggregates with macromolecule recycling and metabolic signaling. In neurons, lysosomes are particularly crucial due to the cells' long lifespan, polarized architecture, and high metabolic demands. Proper regulation of lysosomal function is essential to sustain proteostasis, membrane turnover, and synaptic integrity. Although lysosomal dysfunction has been extensively studied in neurodegenerative diseases, far less is known about how lysosomal capacity and function are maintained-or fail to be maintained-with age in non-diseased neurons. In this review, we summarize current understanding of neuronal lysosomal dynamics, discuss methodological challenges in assessing lysosomal capacity and function, and highlight recent advances that reveal age-associated decline in neuronal lysosomal competence.

Keywords:  TFEB; aging; autolysosome; autophagy; endolysosome; lysosome; neuron

DOI:  https://doi.org/10.3390/cells14241976
Neurobiol Dis. 2025 Dec 20. pii: S0969-9961(25)00448-6. [Epub ahead of print] 107231

Protrudin acts at ER-endosome contacts to promote KIF5-mediated endosomal tubule fission.

Julia Kleniuk, Aishwarya G Nadadhur, Catherine Rodger, Emily Wolfenden, Isabelle A Hall, Samuel R Cheers, Eliska Zlamalova, Evan Reid.

  Defective endosomal sorting and trafficking are increasingly recognised as key drivers of neurodegeneration, including hereditary spastic paraplegia (HSP) and other motor neuron disorders. Early endosomal tubule fission (ETF) is essential for sorting cargoes for recycling and retrograde transport, yet the mechanisms coordinating this process are incompletely defined. Here, we identify the endoplasmic reticulum (ER)-resident protein protrudin-previously shown to promote axonal regeneration after injury-as a key regulator of ETF. Using CRISPR interference in human cells, we show that loss of protrudin causes marked accumulation of elongated endosomal tubules, caused by defective fission. Protrudin-mediated ETF required its ability to interact with ER-localised VAP proteins, endosomal phosphoinositides, and the kinesin motor KIF5, indicating a function at ER-endosome contact sites. The endosomal tubulation phenotype depended on dynamic microtubules and dynein and was phenocopied by KIF5 depletion, suggesting that protrudin coordinates opposing microtubule motor forces to drive fission. Beyond this direct role, protrudin connects multiple ETF machineries implicated in lipid transfer, actin regulation, and ER shaping, positioning it as a central scaffold for ETF. Importantly, depletion of protrudin or the HSP-associated kinesin KIF5A produced similar endosomal tubulation defects in human cortical neurons, underscoring the neurophysiological and disease relevance of this pathway. These findings identify protrudin as a key molecular link between ER-endosome communication, neuronal membrane trafficking, and axonal maintenance-processes whose disruption underlies neurodegenerative disease.

Keywords:  Endoplasmic reticulum; Endosomal sorting; Endosomal tubule fission; Organelle contacts

DOI:  https://doi.org/10.1016/j.nbd.2025.107231
Mol Brain. 2025 Dec 25.

Human midbrain organoids reveal the characteristics of axonal mitochondria specific to dopaminergic neurons.

Akihiko Nishijima, Mutsumi Yokota, Soichiro Kakuta, Akihiro Yamaguchi, Kei-Ichi Ishikawa, Hideyuki Okano, Wado Akamatsu, Nobutaka Hattori, Masato Koike.

  Mitochondrial dysfunction and abnormalities in mitochondrial quality control contribute to the development of neurodegenerative diseases. Parkinson's disease is a neurodegenerative disease that causes motor problems mainly due to the loss of dopaminergic neurons in the substantia nigra pars compacta. Axonal mitochondria in neurons reportedly differ in properties and morphologies from mitochondria in somata or dendrites. However, the function and morphology of axonal mitochondria in human dopaminergic neurons remain poorly understood. To define the function and morphology of axonal mitochondria in human dopaminergic neurons, we newly generated tyrosine hydroxylase (TH) reporter (TH-GFP) induced pluripotent stem cell (iPSC) lines from one control and one PRKN-mutant patient iPSC lines and differentiated these iPSC lines into dopaminergic neurons in two-dimensional monolayer cultures or three-dimensional midbrain organoids. Immunostainings with antibodies against axonal and dendritic markers showed that axons could be better distinguished from dendrites of dopaminergic neurons in the peripheral area of three-dimensional midbrain organoids than in two-dimensional monolayers. Live-cell imaging and correlative light-electron microscopy in peripheral areas of midbrain organoids derived from control TH-GFP iPSCs demonstrated that axonal mitochondria in dopaminergic neurons had lower membrane potential and were shorter in length than those in non-dopaminergic neurons. Although the mitochondrial membrane potential did not significantly differ between dopaminergic and non-dopaminergic neurons derived from PRKN-mutant patient lines, these differences tended to be similar to those in control lines. These results were also largely consistent with those of our previous study on somatic mitochondria. The findings of the present study indicate that midbrain organoids are an effective tool to distinguish axonal from dendritic mitochondria in dopaminergic neurons. This may facilitate the analysis of axonal mitochondria to provide further insights into the mechanisms of dopaminergic neuron degeneration in patients with Parkinson's disease.

Keywords:  Axonal mitochondria; Dopaminergic neurons; Electron microscopy; Live-cell imaging; Midbrain organoids

DOI:  https://doi.org/10.1186/s13041-025-01268-w
Alzheimers Dement. 2025 Dec;21 Suppl 1 e099957

Basic Science and Pathogenesis.

Bryan Kartono, Liliana Attisano, Janice Robertson.

BACKGROUND: Hexanucleotide repeat expansions in C9orf72 are the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The repeat expansions cause C9orf72 haploinsufficiency and reduced C9orf72 protein expression. C9orf72 haploinsufficiency disrupts normal synaptic function, causing alterations in neuronal morphology, glutamatergic imbalances, and dysregulation of pre- and post-synaptic proteins, linked to aberrant actin dynamics. These disruptions are central to the pathogenesis of C9orf72-related FTD. Lorlatinib, an ALK inhibitor used in cancer therapy, has known effects on regulating actin dynamics, acting through the PI3K-LIMK-cofilin pathway. Here, we explored lorlatinib as a potential therapeutic by testing its effects on rescuing neuronal phenotypes caused by C9orf72 haploinsufficiency.
METHODS: To assess lorlatinib's potential in mitigating synaptic dysfunction associated with C9orf72 haploinsufficiency, we used primary cortical neurons from C9orf72+/- mouse embryos (E13-16). We focused on dendritic arborization and vulnerability to glutamate excitotoxicity. Analyses were performed at DIV12-15 using immunocytochemistry and immunoblots to evaluate dendritic complexity and protein expression.
RESULTS: Primary cortical neurons with C9orf72 haploinsufficiency exhibit alterations in dendritic branching, indicating disrupted neuronal connectivity. Additionally, C9orf72 haploinsufficiency increases neuronal vulnerability to excitotoxicity and cell death under stress, evidenced by reduced viability following high glutamate exposure. Glutamate excitotoxicity is a common pathomechanism in neurodegenerative diseases, including FTD/ALS. This can be explained by elevated calcium-permeable AMPAR subunit GluA1 levels at post-synaptic sites, increasing vulnerability. These synaptic dysfunction phenotypes arise from aberrant activity of interrelated PI3K/Akt and LIMK1/cofilin pathways. Importantly, inhibition of ALK by lorlatinib rescues dendritic complexity and enhances neuronal resilience to glutamate-induced damage by normalizing PI3K/Akt and LIMK1/cofilin activity. Lorlatinib may mitigate synaptic dysfunction and preserve neuronal connectivity, addressing key pathomechanisms underlying C9orf72-FTD.
CONCLUSION: Our findings highlight the therapeutic potential of lorlatinib, an ALK inhibitor, in mitigating synaptic deficits caused by C9orf72 haploinsufficiency. We emphasize the role of synaptic pathology in C9orf72-FTD pathogenesis and suggest that targeting ALK with drugs like lorlatinib could offer promising therapeutic strategies for treating synaptic dysfunction caused by C9orf72 haploinsufficiency.

DOI: https://doi.org/10.1002/alz70855_099957
Neurobiol Dis. 2025 Dec 24. pii: S0969-9961(25)00464-4. [Epub ahead of print] 107247

Reduced cortico-muscular output is associated with intrinsic hypoexcitability and reduced persistent inward currents in motor cortex neurons of TDP-43Q331K ALS mice.

Jose A Viteri, Nathan R Kerr, Charles D Brennan, Priyanka Paradkar, Leena A Suleiman, Charles D Wendt, Chunhui Xu, Hong An, Meifang Wang, Hiroshi Nishimune, Joseph M Santin, W David Arnold.

  Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by spinal and cortical motor neuron loss and progressive neuromuscular decline. When ALS pathology involves the primary motor cortex (PMC), cortical excitability is often disrupted, yet how these alterations map onto motor deficits during symptomatic ALS remains unclear. To investigate this, we examined the neuromuscular function, cortico-muscular output, and neuronal excitability of symptomatic 4-month-old TDP-43Q331K mice. TDP-43 mice exhibited reduced neuromuscular excitability and impaired strength compared to WT mice. Cranial motor evoked potentials were significantly reduced in TDP-43 mice, indicating decreased cortical output to muscle. Compared to WT mice, whole-cell patch-clamp recordings from TDP-43 PMC layer V pyramidal neurons revealed intrinsic hypoexcitability, diminished persistent inward currents (PICs), and decreased excitatory synaptic activity. Corroborating PIC findings, immunohistochemical analysis showed that PMC layer V neurons exhibited reduced signal intensity of the PIC-associated proteins Nav1.6 and 5-HT2C. Bulk RNA-seq of the cortex showed distinct transcriptional profiles in TDP-43 mice, with enrichment analysis indicating altered pathways relating to ion transport, synaptic signaling, and neuronal excitability. These results suggest that cortex-wide transcriptional changes may reflect broader and additional molecular mechanisms underlying cortical hypoexcitability in ALS. Together, our results demonstrate that symptomatic TDP-43Q331K mice exhibit a reduction in cortico-muscular output and PMC neuron excitability, accompanied by reduced PICs and PIC-associated proteins within these neurons. These findings identify cortical hypoexcitability as a defining feature of the TDP-43Q331k ALS mouse model and establish multi-level associations between cortical cellular-level dysfunction and impaired motor systems output.

Keywords:  ALS; Aging; Hypoexcitability; Motor cortex; Neuromuscular

DOI:  https://doi.org/10.1016/j.nbd.2025.107247
Alzheimers Dement. 2025 Dec;21 Suppl 1 e107448

Basic Science and Pathogenesis.

Jose Torrellas, Aravindraja Chairmandurai, Adamantios Mamais, Matthew J LaVoie, Paramita Chakrabarty.

BACKGROUND: Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene, particularly the G2019S and R1441C variants, are the most prevalent genetic determinants of Parkinson's disease (PD). LRRK2 is increasingly recognized for its roles in immune signaling, with studies suggesting that interferon-gamma (IFNγ), a master immune regulator, may modulate its activity. Based on these findings, we hypothesize a synergistic interaction between LRRK2 mutations and IFNγ.
METHOD: To explore this hypothesis, we utilized neurons derived from human induced pluripotent stem cells (iPSCs) harboring homozygous G2019S or R1441C LRRK2 mutations, with isogenic wildtype (WT) controls. After differentiation into neurons, cells were treated with recombinant human IFNγ or control media on day 17 of culture and harvested after 6 hours of treatment. Six experimental conditions were analyzed: (WT + IFNγ, WT + Control, G2019S + IFNγ, G2019S + Control, R1441C + IFNγ, R1441C + Control) We analyzed these experiments using immunoblotting and immunofluorescence methods to assess interferon-pathways, tau phosphorylation, and LRRK2 substrate engagement.
RESULT: Using a dose-dependent paradigm, we optimized the minimum IFNγ dose to achieve maximal target engagement (STAT1 and phosphorylated-STAT1, pSTAT1). All three genotypes responded to IFNγ, producing equivalent levels of pSTAT1, confirming activation of the JAK-STAT pathway. We found that at baseline, levels of pSer202 tau (CP13) levels were moderately increased in both G2019S- and R1441C-LRRK2 relative to WT-LRRK2. IFNγ treatment increased CP13 levels in both G2019S- and R1441C-LRRK2, with the CP13 induction higher in G2019S-LRRK2 while WT-LRRK2 neurons did not show elevated CP13 following IFNγ treatment. IFNγ treatment phosphorylated Threonine73 on Rab10 (a LRRK2 substrate) more efficiently in G2019S-LRRK2 neurons compared to R1441C-LRRK2. We did not observe a genotype-dependent effect of IFNγ on phosphorylation of Ser106 on Rab12, another LRRK2 substrate.
CONCLUSION: Our data indicates a LRRK2 genotype related response of LRRK2 neurons to IFNγ stimulus. This data lays the groundwork for future studies to dissect mechanisms of neuronal vulnerability in PD and the interplay between LRRK2 mutations and disease pathways.

DOI: https://doi.org/10.1002/alz70855_107448
Theranostics. 2026 ;16(5): 2388-2404

Neuronal mitochondrial disaggregase CLPB ameliorates Huntington's disease pathology in mice.

Hyeonho Kim, Gaeun Hyun, Seunghye Kim, Changmo Yu, Young-Gi Hong, Jihyeon Yu, Sangsu Bae, Hyun-Woo Rhee, Jaewon Ko, Ji Won Um.

  Background: Huntington's disease (HD) is a devastating neurodegenerative disorder caused by CAG repeat expansion in the HTT gene, resulting in a polyglutamine-expanded huntingtin (HTT) protein that forms toxic aggregates. Although heat-shock proteins are known to facilitate the refolding or clearance of misfolded proteins, their precise role in modulating protein aggregation in HD remains unclear. Here, we explore the function of caseinolytic peptidase B (ClpB), a mitochondrial AAA+ ATPase and heat-shock protein, in maintaining proteostasis and synaptic integrity in HD. Methods: We examined how CLPB loss or overexpression in human embryonic kidney 293T (HEK293T) cells impacted the aggregation of wild-type HTT (HTT-Q23) and mutant HTT (HTT-Q79). In parallel, AAV-mediated ClpB knockdown or overexpression was applied to the striatum of HD model mice. and HTT aggregation and inhibitory synaptic alterations were assessed. Aggregate burden was quantified via immunostaining, and inhibitory synapse density was evaluated using VGAT immunohistochemistry and electrophysiological recordings. Results: In HEK293T cells, CLPB knockout led to abnormal aggregation of HTT-Q23 while CLPB overexpression reduced the size of HTT-Q79 aggregates. In the mouse striatum, ClpB knockdown increased HTT-Q23 aggregate numbers and altered HTT-Q79 aggregation morphology, whereas CLPB overexpression restored the density and size of VGAT-positive inhibitory synapses and improved inhibitory synaptic transmission in HD model mice. These effects of CLPB overexpression were associated with a reduced mitochondrial aggregation burden, suggesting that ClpB contributes to mitochondrial protein quality control. Conclusions: These results demonstrate that ClpB regulates both physiological and pathological HTT aggregation and contributes to maintaining inhibitory synaptic integrity. By modulating mitochondrial proteostasis, ClpB acts as a protective factor in HD pathology, highlighting its potential as a therapeutic target for neurodegenerative disorders characterized by protein misfolding.

Keywords:  ClpB; Huntington's disease; disaggregase; inhibitory synapse; mitochondria; striatum

DOI:  https://doi.org/10.7150/thno.122651
Alzheimers Dement. 2025 Dec;21 Suppl 7 e108382

Developing Topics.

Farzane S Mirfakhar, Jacob Marsh, Miguel Minaya, Stephen Pak, Gary Silverman, David Perlmutter, Shannon L Macauley, Celeste M Karch.

BACKGROUND: Tau degradation is disrupted in neurodegenerative tauopathies, such as frontotemporal dementia (FTD), which may contribute to Tau aggregation. The prevailing hypothesis has been that Tau degradation is stymied due to an imbalance in proteostasis that occurs with age.
METHOD: Here, we used Airyscan super resolution imaging to illustrate that a pathogenic FTD mutation in the MAPT gene, which encodes Tau, is sufficient to alter multiple steps of the autophagy lysosomal pathway and impair Tau degradation in stem cell derived neurons.
RESULT: We discovered lysosomes clogged with both Tau and phosphorylated Tau, stalled lysosome motility, disrupted molecular motors, enhanced autophagic flux, and slowed cargo degradation in mutant Tau neurons. Treatment of mutant Tau neurons with a small molecule autophagy enhancer drug increases autophagic flux and cargo degradation, reduces phospho-Tau levels, and reduces Tau accumulation in lysosomes without restoring defects in lysosomal motility.
CONCLUSION: This study reveals novel effects of mutant Tau and provides a window through which therapeutic treatments targeting autophagy may promote Tau homeostasis.

DOI: https://doi.org/10.1002/alz70861_108382
Sci Rep. 2025 Dec 23.

A role for Fis1 in dendritic development.

Klaudia Strucinska, Parker Kneis, Travis Pennington, Katarzyna Cizio, Patrycja Szybowska, Abigail Morgan, Joshua Weertman, Tommy L Lewis.

Mitochondrial ATP production and calcium handling are critical for metabolic regulation and neurotransmission. Thus, the formation and maintenance of the mitochondrial network is a critical component of neuronal health. Cortical pyramidal neurons contain compartment-specific mitochondrial morphologies that result from distinct axonal and dendritic mitochondrial fission and fusion profiles. We previously revealed that axonal mitochondria are maintained at a small size as a result of high axonal mitochondrial fission factor (Mff) activity. However, loss of Mff activity had little effect on cortical dendritic mitochondria, raising the question of how fission/fusion balance is controlled in the dendrites. Therefore, we sought to investigate the role of another fission factor, fission 1 (Fis1), on mitochondrial morphology, dynamics and function in cortical neurons. We knocked down Fis1 in cortical neurons both in primary culture and in vivo, and unexpectedly found that Fis1 depletion decreased mitochondrial length in the dendrites, without affecting mitochondrial size in the axon. Further, loss of Fis1 activity resulted in both increased mitochondrial motility and dynamics in the dendrites. These results argue Fis1 exhibits dendrite selectivity and plays a more complex role in neuronal mitochondrial dynamics than previously reported. Functionally, Fis1 loss resulted in reduced mitochondrial membrane potential, increased sensitivity to complex III blockade, and decreased mitochondrial calcium uptake during neuronal activity. The altered mitochondrial network culminated in elevated resting calcium levels that increased dendritic branching but reduced spine density. We conclude that Fis1 activity regulates mitochondrial morphological and functional features that influence dendritic tree arborization and connectivity.

DOI: https://doi.org/10.1038/s41598-025-33557-8
Alzheimers Dement. 2025 Dec;21 Suppl 1 e100362

Basic Science and Pathogenesis.

Maria Clara Zanellati.

BACKGROUND: Dysfunctional organelle communication networks are a common hallmark of various neurodegenerative diseases, including Alzheimer's disease (AD). Imaging organelles and their interactions in iPSC-derived neurons that harbor disease-associated mutations holds promise for understanding the mechanistic basis of neurodegeneration.
METHOD: We developed a method for multispectral imaging of eight organelles simultaneously in live cells and used this method to visualize organelle morphology and dynamics (morphodynamics) along neuronal differentiation. We transfected induced pluripotent stem cells (iPSCs) and iPSC-derived cortical neurons (iNeurons) with genetically encoded organelle markers and collected multispectral z-stack and timelapse images at five-time points throughout neuronal differentiation and maturation: iPSCs, and iNeurons at day 7, day 14, day 21, and day 28. Raw images were then subjected to linear unmixing and run through a Napari-InferSubC image analysis pipeline for segmentation and analysis of approximately 1400 morpho-metrics including organelle volume, size, shape, and number, as well as number and volume of the contacts between organelles (2- to 6-way).
RESULT: We observed dramatic remodeling of organelles during differentiation of iPSCs into iNeurons. For example, endoplasmic reticulum (ER) and mitochondria volumes increased as iPSCs differentiated into cortical neurons. We observed an increase in the overall number of the contacts throughout iNeuron maturation, accompanied by an increase in higher order contacts (3- and 4-way contacts). Examples of organelle contacts that increased during iNeuron differentiation and maturation include ER-mitochondria, known to be dysregulated in AD and Parkinson disease; mitochondria-lysosome, previously reported defective in Charcot-Marie-Tooth; and ER-peroxisome. We found that expression of VAPB, which mediates ER-peroxisome contacts and is mutated in ALS, increases as iPSCs differentiate into iNeurons. This contact is implicated in the production of plasmalogens, which are essential for the growth and maintenance of synapses in the nervous system. Knockdown of VAPB reduced plasmalogen levels and prevented the formation of synapses during iNeuron differentiation.
CONCLUSION: We uncovered a novel role for VAPB-mediated ER-peroxisome contacts in neuronal differentiation, suggesting that multi-spectral imaging can be used to interrogate organelle morphology and contacts during neuronal differentiation and neurodegeneration. As a future direction, we will use this method to reveal defects in organelle communication networks in iNeurons with AD-associated mutations.

DOI: https://doi.org/10.1002/alz70855_100362
Alzheimers Dement. 2025 Dec;21 Suppl 7 e108384

Developing Topics.

Aimee W Kao.

BACKGROUND: The lysosome is critical for maintaining normal protein homeostasis in cells and has been genetically implicated in the pathogenesis of neurodegenerative diseases including Alzheimer's Disease and frontotemporal dementia. Lysosomes are normally highly acidic organelles and acidic lysosomal pH is critical for efficient macromolecular breakdown and cellular homeostasis. Lysosomal pH becomes progressively more alkaline with age in model organisms and these changes may contribute to neurodegenerative diseases. Despite this, the genetic and molecular regulation of lysosomal pH setpoint remain incompletely understood. It is further unknown if preserving lysosomal acidity can conteract the accumulation of neurodenerative disease proteins such as tau and TDP-43. To answer these questions, we have developed a best-in-class reporter for lysosomal pH known as FIRE-pHLy (for Fluorescent Indicator Reporting on pH in the Lysosome.) METHOD: iPSC-derived neurons were generated that express FIRE-pHLy as well as CRISPRi machinery. Cells were differentiated into induced neurons and used for a whole-genome CRISPRi screen for modifiers of lysosomal pH. In addition, C. elegans were generated that express both FIRE-pHLy and an optogenetic acidifier of lysosomal pH, Acido-pHLy.
RESULT: First we demonstrate that even small changes in lysosomal pH can dramatically alter the ability of iNeuron-derived lysosomes to degrade neurodegenerative proteins such as tau. The whole-genome screen for modifiers of lysosomal pH identifed genes whose knock down either acidified or alkalnized lysosomal pH. Many disease genes were included in these groups. Amino acid metabolism was shown to be a key regulator of lysosomal pH. Using transgenic C. elegans, we show that neuronal lysosomal pH becomes more alkaline with age and chemogenetic manipulations that preserve lysosomal pH acidity extend lifespan and detoxify tau.
CONCLUSION: These findings highlight lysosomal pH as a potential therapeutic target for Alzheimer's Disease and related disorders.

DOI: https://doi.org/10.1002/alz70861_108384
Alzheimers Dement. 2025 Dec;21 Suppl 7 e108268

Developing Topics.

Yongjie Zhang, Yanwei Wu, Wei Shao, Tania F Gendron, Jorge Alaiz Noya, Caroline J Jones, Karen Jansen-West, Lillian Daughrity, Monica Castanedes-Casey, Björn Oskarsson, Dennis W Dickson, Leonard Petrucelli.

BACKGROUND: G4C2 repeat expansions in C9orf72 gene are the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Mounting evidence indicates that dipeptide repeat (DPR) proteins translated from the expanded repeats, in particular poly(GR) and poly(PR), play a significant role. We and others have shown that these positively-charged arginine-rich DPR proteins induce the assembly of stress granules (SGs) and impair their disassembly. While distinct from SGs, processing bodies (PBs) are also cytoplasmic messenger ribonucleoprotein condensates that assemble upon stress. However, the impacts of arginine-rich DPR proteins on PB assembly and dynamics have yet to be investigated.
METHOD: We conducted immunofluorescence staining for arginine-rich DPR proteins, SG- and PB-associated markers in cultured cells and mouse brain. Additionally, we performed in vitro liquid-liquid phase separation (LLPS) using recombinant proteins and assessed molecular dynamics of SG and PB through fluorescence recovery (FRAP) after photobleaching in living cells.
RESULT: Here, we observed that both GFP-(GR)100 and GFP-(PR)100 formed cytosolic inclusions immunopositive PB proteins. Unlike GFP-(GR)100 and GFP-(PR)100 inclusions, cellular cytoplasmic GFP-(GA)100 inclusions did not contain PB proteins. We subsequently examined whether the phenomena observed in cultured cells occur in vivo. We observed that PB proteins were recruited to poly(GR) inclusions in cells with aggregated poly(GR). Next, we examined the dynamics of poly(GR)-induced PBs in cultured cells using the FRAP technique. We observed that the recovery of stress-induced PBs was more rapid than that of PBs in HEK293T cells expressing GFP-(GR)100. Lastly, mechanistic studies revealed that poly(GR) interacts with PB proteins to form condensates in vitro via LLPS.
CONCLUSION: Our results provide both in vitro and in vivo evidence that arginine-rich DPRs induce the spontaneous formation of PBs via LLPS. Our results also indicate that poly(GR)-induced PBs are more stable than the transient and dynamic conventional PBs. Given that PBs uniquely enriched with mRNA degradation and decay factors, the consequences of impairing these processes are expected to contribute to abnormal RNA metabolism in c9FTD/ALS.

DOI: https://doi.org/10.1002/alz70861_108268
bioRxiv. 2025 Dec 11. pii: 2025.12.09.688862. [Epub ahead of print]

A shared DNA-repeat toxicity threshold, reached somatically at cell-type-specific rates, unites cortical and striatal neurodegeneration in Huntington's disease.

Seva Kashin, Won-Seok Lee, Tara M McDonald, Kiely Morris, Robert E Handsaker, Curtis Mello, Liv Spina, Nora M Reed, Heather de Rivera, Sri Havya Jana, Marina Hogan, Sabina Berretta, Steven A McCarroll.

Huntington's disease (HD) affects two major brain areas - the striatum and cerebral cortex - in ways that differ in timing, severity, and gene-expression changes. For these reasons, and because many cortical neurons project axons to the affected striatal neurons, striatal and cortical atrophy have long been proposed to have distinct mechanisms, with one potentially a secondary consequence of the other. In the striatum, we recently found that neurons degenerate asynchronously as their own huntingtin ( HTT) gene CAG-repeat tracts, typically inherited at 40-50 CAGs, expand somatically beyond 150 CAGs. To ask whether a similar or different dynamic affects the cerebral cortex, we analyzed HTT CAG repeats and genome-wide RNA expression together in more than 130,000 nuclei from 12 cortical areas of brain donors with HD. The resulting data revealed that cortical and striatal neurodegeneration in fact result from analogous sequences of cell-autonomous events, each instructed by somatic expansion of a neuron's own HTT CAG repeat. Analyses revealed that somatic expansion beyond a high toxicity threshold (of about 150 CAGs) is necessary and sufficient to initiate pathological changes; that this pathogenicity length threshold is shared by striatal and cortical projection neurons of all types; and that cortical area, cortical layer, and axonal projections play only incidental roles, as proxies for the true driver: profound (up to 50-fold) variation among types and subtypes of pyramidal neurons in the likelihood of reaching the 150-CAG toxicity threshold in a human lifetime. These results also suggest that containing somatic DNA-repeat expansion below this high toxicity threshold would protect both brain areas in HD.

DOI: https://doi.org/10.64898/2025.12.09.688862
Alzheimers Dement. 2025 Dec;21 Suppl 1 e096500

Basic Science and Pathogenesis.

Thomas Westergard.

BACKGROUND: Reduced levels of progranulin (PGRN) protein are associated with frontotemporal dementia (FTD), Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS). Sortilin (SORT1) is a scavenger receptor responsible for the uptake of PGRN into the cell targeting its degradation. GSK5862611 (S60-11) is an anti-Sortilin (SORT1) human IgG1 antibody developed to inhibit binding of PGRN to sortilin receptors and deplete sortilin receptors from the cell surface, resulting in an increase in extracellular PGRN levels. Similar anti-sortilin antibodies are currently in phase 2 and phase 3 clinical trials for AD and FTD-GRN, respectively. Given that PGRN plays an important role in maintaining lysosomal functions and neuronal survivability we evaluated whether enhancing PGRN levels could be effective in reversing phenotypes associated with TDP43 G298S risk variant in complex hiPSC-derived cellular models.
METHOD: hiPSC-derived bi- and tricultures comprising spinal motor neurons and astrocytes carrying TDP43 risk variant G298S mutation and wild-type or GRN edited microglia were established and treated with GSK5862611, isotype control hIgG1, or recombinant PGRN. Impacts on neurite length and TDP43 mislocalization in motor neurons as well as levels of PGRN, neurofilament light chain (NfL), and glial fibrillary acidic protein (GFAP) in the culture media were assessed.
RESULTS: Recombinant PGRN rescued the loss of neuritic length and TDP-43 mislocalization in hiPSC-derived bicultures from TDP43 G298S risk variant. Like PGRN, GSK5861611 also rescued loss of neuritic length and TDP43 mislocalization compared to untreated bicultures, however the effect was not significant when compared to isotype control hIgG1. In hiPSC-derived tricultures including GRN edited microglia, GSK5862611 increased extracellular PGRN levels and reduced TDP43 mislocalization in a dose-dependent manner. Additionally, GSK5862611 reduced NfL and GFAP levels in TDP43 tricultures with PGRN Het microglia (HumIgG1 had similar reduction in NfL).
CONCLUSION: Taken together, these results indicate that blocking Sortilin receptors increases extracellular PGRN levels while reducing TDP43 mislocalization in motor neurons, and reducing NfL and GFAP levels in complex hiPSC cellular models carrying a TDP43 G298S risk variant. These data support the hypothesis that increasing PGRN levels with an anti-sortilin antibody may be a promising therapeutic strategy in ALS.
ACKNOWLEDGEMENTS: S60-11 anti-sortilin mAb was provided by Alector.

DOI: https://doi.org/10.1002/alz70855_096500
bioRxiv. 2025 Dec 15. pii: 2025.12.11.693761. [Epub ahead of print]

Axonal growth of cortical neurons does not require paxillin.

Katelyn Rygel, Kasey Gillespie, Kristy Welshhans.

Axon growth is an essential cellular process during neural development, and its dysregulation contributes to numerous neurodevelopmental disorders. During axon growth, extracellular signals direct neurons to extend projections that connect with their synaptic targets. Paxillin is a key member of adhesion sites that control motility by linking the intracellular actin cytoskeleton to the extracellular matrix. Paxillin also binds to the cytoskeletal protein, tubulin. However, little is known about the role of adhesion proteins in neurons. Here, we use conditional paxillin knockout mice to investigate how loss of paxillin in pyramidal cortical neurons affects developing neuron morphology. Surprisingly, loss of paxillin in pyramidal cortical neurons caused no change in axon length or soma area between control ( Pxn F/F ) and conditional paxillin knockout ( Pxn F/F; Emx1-Cre ) mice at basal conditions. Following brain-derived neurotrophic factor stimulation, the loss of paxillin resulted in no change in soma area or axonal β-tubulin levels, but did result in a significant increase in axon length, as compared to control. Finally, the corpus callosum size was not significantly different between Pxn F/F and Pxn F/F; Emx1-Cre animals. In summary, these data suggest that paxillin is not required for axonal growth during neural development.

DOI: https://doi.org/10.64898/2025.12.11.693761
bioRxiv. 2025 Dec 12. pii: 2025.12.09.693280. [Epub ahead of print]

Lysosomal swelling triggers LRRK2 activity.

Tuyana Malankhanova, Zhiyong Liu, Samuel Strader, Weiping Wang, Kiwoon Sung, Zhong Li, Shawn M Ferguson, Andrew B West.

LRRK2 is implicated in lysosomal functions, but the physiological upstream cues that engage endogenous LRRK2 activity are incompletely defined. Here we show that lysosomal swelling serves as a selective and reversible trigger for LRRK2-mediated Rab phosphorylation, without requiring membrane damage. Acute inhibition of PIKfyve, but not the general disruption of phosphoinositide signaling, induces the robust accumulation of phosphorylated Rabs across endolysosomal membranes. Rescue of swelling through pharmacological restoration of lysosomal ionic imbalances from PIKfyve inhibition suppresses LRRK2 activation without restoring lysosomal function. Mechanical lysosomal swelling from indigestible osmolyte uptake causes a dose-dependent increase in LRRK2-mediated Rab phosphorylation on both swollen and non-swollen lysosomes. Together, these findings identify LRRK2 as a sensor of lysosomal volume and mechanical stress, not specifically membrane damage or PIKfyve inhibition. As lysosomal swelling is a shared pathological feature across LRRK2 -linked diseases, these results reframe LRRK2 as part of an endolysosomal surveillance system responsive to lysosomal distension.

DOI: https://doi.org/10.64898/2025.12.09.693280
Mol Biol Cell. 2025 Dec 24. mbcE25040188

A new subset of mitochondrial-derived vesicles perform inter-mitochondrial communications.

Priyanka Adla, Vani Bshivakumar, Dheeraj Pathak, Ushodaya Mattam, Prasad Tammineni, Thanuja Krishnamoorthy, Naresh B V Sepuri.

Mitochondria have a fascinating array of tools in their armory for maintaining cellular homeostasis, of which the formation of Mitochondrial-Derived Vesicles (MDVs) is the least energy-intensive. MDVs have become the 'go-to' vesicles for mitochondria to perform functions such as ferrying damaged mitochondrial proteins to lysosomes and regulating peroxisomal morphology. In a corollary to the increasing number of MDV functions, the discovery of MDV subsets has also increased. However, all the known MDV communications have been from mitochondria to other organelles. Using purified mitochondria from rat liver, we show that MDVs can be generated in vitro, and proteomic analyses reveal that liver MDVs are enriched in metabolic proteins mirroring the liver's metabolic hub status. Intriguingly, live cell imaging studies in HepG2 cells reveal a new subset of MDVs that are TOMM70+ve but TOMM20-ve. This subset of MDVs harbors metabolic enzymes, such as ALDH7A1, an aldehyde dehydrogenase. Remarkably, this class of MDVs facilitates communication between mitochondria, revealing a previously unknown communication channel. [Media: see text] [Media: see text] [Media: see text] [Media: see text].

DOI: https://doi.org/10.1091/mbc.E25-04-0188
bioRxiv. 2025 Dec 13. pii: 2025.12.10.693600. [Epub ahead of print]

Landscape Expansion Microscopy Reveals Interactions between Membrane and Phase-Separated Organelles.

Yinyin Zhuang, Zhao Zhang, Zhipeng Dai, Xiaoyu Shi.

Landscape Expansion Microscopy (land-ExM) is a light microscopy technique that visualizes both lipid and protein ultrastructural context of cells. Achieving this level of detail requires both superresolution and a high signal-to-noise ratio. Although expansion microscopy (ExM) provides superresolution, obtaining high signal-to-noise images of both proteins and lipids remains challenging. Land-ExM overcomes this limitation by using self-retention trifunctional anchors to significantly enhance protein and lipid signals in expanded samples. This improvement enables the accurate visualization of diverse membrane organelles and phase separations, as well as the three-dimensional visualization of their contact sites. As a demonstration, we revealed triple-organellar contact sites among the stress granule, the nuclear tunnel, and the nucleolus. Overall, land-ExM offers a high-contrast superresolution platform that advances our understanding of how cells spatially coordinate interactions between membrane organelles and phase separations.
eTOC Summary: Zhuang et al. introduce land-ExM, a super-resolution approach that simultaneously maps protein and lipid ultrastructure in cells with high contrast. This method visualizes 3D interactions between membrane-bound organelles and phase-separated condensates, uncovering organelle contact sites such as stress granules at nuclear tunnels adjacent to nucleoli.

DOI: https://doi.org/10.64898/2025.12.10.693600
Biol Chem. 2025 Dec 29.

Getting to the right place at the right time - membrane trafficking and maturation in the endolysosomal system.

Alexander Stockhammer, Christian Ungermann.

  The endolysosomal system connects Golgi and plasma membrane to the degradative pathway towards the lysosome and therefore presents a crossroads for endocytic recycling, secretory transport and degradation. This complexity makes protein sorting and trafficking within the endolysosomal system challenging, and it requires tight regulation so that all proteins localize correctly. Proteins are sorted by distinct sorting adaptors, which recognize sorting signals and subsequently facilitate formation of transport carriers, which deliver content to other organelles. Alternatively, organelle maturation allows passive protein transport along different trafficking routes including endosomal and autophagosomal maturation. In this review, we will provide a bird's eye overview of the divers routes along which proteins are transported within the endolysosomal system and highlight open questions in the field.

Keywords:  Arf; Rab; adapter complexes; membrane trafficking; organelle maturation; small GTPases

DOI:  https://doi.org/10.1515/hsz-2025-0187
bioRxiv. 2025 Dec 16. pii: 2025.12.12.693948. [Epub ahead of print]

A neuron type-specific microexon in Ank3 /ankyrin-G modulates calcium activity and neuronal excitability.

Shah Alam, Georgia Dermentzaki, David Cabrera-Garcia, Miao Li, Ruizhi Wang, Melissa Campbell, Ilaria Balbo, Brittany L Phillips, Min Li, Jessica Estrada, Marianna Zazhytska, Yow-Tyng Yeh, Lia Min, Elizabeth Rafikian, Elizabeth Valenzuela, Brian Joseph, Tulsi Patel, Dmytro Ustienenko, Helene Lovett, Huijuan Feng, Xiaojian Wang, Susan Brenner-Morton, Chyuan-Sheng Lin, Clarissa L Waites, Hynek Wichterle, Lizhen Chen, Mu Yang, Edmund Au, Marko Jovanovic, Stavros Lomvardas, Paul M Jenkins, Rui Yang, Sheng-Han Kuo, Yueqing Peng, Guang Yang, Neil L Harrison, Chaolin Zhang.

Recent studies have revealed many alternative exons differentially spliced across diverse neuron types in the mammalian brain, but their links to neuronal physiology remain unclear. Here we characterize a deeply conserved microexon E35a in Ank3 encoding ankyrin-G (AnkG), a multifaceted adaptor protein best known as a master organizer of the axon initial segment (AIS) and as a leading genetic risk factor for bipolar disorder. E35a is predominantly skipped in cortical glutamatergic neurons but included in cortical GABAergic neurons and cerebellar neurons, which is dictated by multiple neuronal splicing factors. In E35a-deletion mice we generated, interneurons show increased excitability and somatic Ca 2+ activity, without disruption in AIS. Biochemical analyses suggest that E35a inclusion facilitates AnkG interaction with a protein complex involving inositol trisphosphate receptors (InsP3Rs) important for intracellular Ca 2+ signaling. Alternative splicing therefore allows AnkG to modulate neuron type-specific excitability in addition to its ubiquitous pan-neuronal role in organizing the AIS.

DOI: https://doi.org/10.64898/2025.12.12.693948