bims-axbals Biomed News
on Axonal biology and ALS
Issue of 2025–05–25
27 papers selected by
TJ Krzystek



  1. Autophagy Rep. 2024 ;3(1): 2291250
      The pathological accumulation of the nuclear protein TDP-43 (TAR DNA-binding protein 43 kDa) in the cytoplasm is characteristic of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP), and its spread through the brain and spinal cord is closely associated with the progression of these two diseases. However, the mechanisms through which the TDP-43 pathology propagates throughout the central nervous system remain unclear. We recently reported the role of (macro)autophagy in the secretion of TDP-43 via extracellular vesicles (EVs). We found that among the autophagy modulators, bafilomycin A1 (Baf) and GRN (granulin precursor) deficiency impair the formation of autolysosomes and promote the secretion of TDP-43 by EVs. TDP-43 loading on EVs involves autophagy-related proteins and the knockdown of TDP-43 augmented Baf-induced EV release. Thus, our results suggest that the loss-of-function of TDP-43 accelerates release of EVs possibly derived from autophagosomes, which may mediate cell-to-cell spread of the TDP-43 pathology.
    Keywords:  ALS; FTLD-TDP; TDP-43; autophagy; extracellular vesicle; lysosome; prion; progranulin
    DOI:  https://doi.org/10.1080/27694127.2023.2291250
  2. Nat Commun. 2025 May 19. 16(1): 4633
      Mutations in FUS and TARDBP cause amyotrophic lateral sclerosis (ALS), but the precise mechanisms of selective motor neuron degeneration remain unresolved. To address if pathomechanisms are shared across mutations and related to either gain- or loss-of-function, we performed single-cell RNA sequencing across isogenic induced pluripotent stem cell-derived neuron types, harbouring FUS P525L, FUS R495X, TARDBP M337V mutations or FUS knockout. Transcriptional changes were far more pronounced in motor neurons than interneurons. About 20% of uniquely dysregulated motor neuron transcripts were shared across FUS mutations, half from gain-of-function. Most indicated mitochondrial impairments, with attenuated pathways shared with mutant TARDBP M337V as well as C9orf72-ALS patient motor neurons. Mitochondrial motility was impaired in ALS motor axons, even with nuclear localized FUS mutants, demonstrating shared toxic gain-of-function mechanisms across FUS- and TARDBP-ALS, uncoupled from protein mislocalization. These early mitochondrial dysfunctions unique to motor neurons may affect survival and represent therapeutic targets in ALS.
    DOI:  https://doi.org/10.1038/s41467-025-59679-1
  3. Biochim Biophys Acta Mol Basis Dis. 2025 May 14. pii: S0925-4439(25)00254-6. [Epub ahead of print] 167906
      Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder affecting motor neurons. TAR DNA-binding protein 43 (TDP-43) mis-localisation from the nucleus to the cytoplasm is the major pathological characteristic of ALS. Telomeres are repetitive DNA sequences found in complex with proteins at chromosomal ends. The shelterin protein complex protects telomeres from DNA damage by producing characteristic t-loop structures, and telomere repeat binding factor 2 (TRF2) has an essential role in this process. Telomere dysregulation is reported in ALS, but conflicting findings have been obtained. Here we examined if telomere dysregulation is present in cortical neurons in a mouse model with pathological mis-localisation of TDP-43 to the cytoplasm - TDP-43 rNLS - compared to controls, and in cortical primary neurons expressing TDP-43 ALS associated mutations (A315T, A90V). We demonstrate that telomeres are significantly longer and of more variable in length in this model compared to controls. This was proceeded by downregulation of TRF2 in early disease stages with subsequent upregulation of TRF2 at advanced disease in TDP43 rNLS mice. A trend towards TRF2 upregulation was also present in human ALS. We detected dysregulation of catalytic subunit of telomerase, TERT and trend towards upregulation of telomere interacting protein, Rif 1 in these mice and human ALS spinal cord lysates. The longer telomeres were independent of the alternative lengthening of telomeres (ALT). Similarly, no DNA damage at telomere sites was detected. Our findings imply that telomere protection is compromised, leading to longer telomeres in cortical neurons in ALS associated with TDP-43 pathology.
    Keywords:  ALS; DNA damage; Neurodegeneration; TDP-43 pathology; Telomere dysfunction; Telomeres
    DOI:  https://doi.org/10.1016/j.bbadis.2025.167906
  4. Proc Natl Acad Sci U S A. 2025 May 27. 122(21): e2502294122
      Stathmin-2 (also known as SCG10) is encoded by the STMN2 gene, whose mRNA is one of the most abundantly expressed in human motor neurons. In almost all instances of ALS and other TDP-43 proteinopathies, stathmin-2 encoding mRNAs are cryptically spliced and polyadenylated in motor neurons, a pathogenic consequence of nuclear loss of function of the RNA binding protein TDP-43. While stathmin-2 has been shown to enhance regeneration after axonal injury to axons of cultured motor neurons, here, we show that after crush injury within the adult murine nervous system of wild-type or stathmin-2-null mice, the presence of stathmin-2 reduces axonal and neuromuscular junction degeneration and stimulates reinnervation and functional recovery. Mechanistically, although stathmin-2 has been proposed to function through direct binding to α/β tubulin heterodimers and correspondingly to affect microtubule assembly and dynamics, stathmin-2's role in axon regeneration after axotomy is shown to be independent of its tubulin binding abilities.
    Keywords:  NMNAT2; SCG10; STMN2; axon regeneration; microtubules
    DOI:  https://doi.org/10.1073/pnas.2502294122
  5. Acta Neuropathol Commun. 2025 May 19. 13(1): 108
      Alzheimer's disease (AD) is characterized by the accumulation and spread of Tau intraneuronal inclusions throughout most of the telencephalon, leaving hindbrain regions like the cerebellum and spinal cord largely spared. These neuropathological observations, along with the identification of specific vulnerable sub-populations from AD brain-derived single nuclei transcriptomics, suggest that a subset of brain regions and neuronal subtypes possess a selective vulnerability to Tau pathology. Given the inability to culture neurons from patient brains, a disease-relevant in vitro model which recapitulates these features would serve as a critical tool to validate modulators of vulnerability and resilience. Using our recently established platform for inducing endogenous Tau aggregation in human induced pluripotent stem cell (hiPSC)-derived cortical excitatory neurons via application of AD brain-derived exogenous Tau aggregates, we explored whether Tau aggregates preferentially induce aggregation in specific neuronal subtypes. We compared Tau seeding in hiPSC-derived neuron subtypes representing regional identities across the forebrain, midbrain, and hindbrain. Higher susceptibility (i.e. more Tau aggregation) was consistently observed among cortical neuron subtypes, with CTIP2-positive, somatostatin (SST)-positive cortical inhibitory neurons showing the greatest aggregation levels across hiPSC lines from multiple donors. hiPSC-neurons also delineated between the disease-specific vulnerabilities of different protein aggregates, as α-synuclein preformed fibrils showed an increased propensity to induce aggregates in midbrain dopaminergic (mDA)-like neurons, mimicking Parkinson's disease (PD)-specific susceptibility. Aggregate uptake and degradation rates were insufficient to explain differential susceptibility. The absence of a consistent transcriptional response following aggregate seeding further indicated that intrinsic neuronal subtype-specific properties could drive susceptibility. The present data provides evidence that hiPSC-neurons exhibit features of selective neuronal vulnerability which manifest in a cell autonomous manner, suggesting that mining intrinsic (or basal) transcriptomic signatures of more vulnerable compared to more resilient hiPSC-neurons could uncover the molecular underpinnings of differential susceptibility to protein aggregation found in a variety of neurodegenerative diseases.
    Keywords:  Alzheimer’s disease; Parkinson’s disease; Selective vulnerability; Tau; hiPSC-derived neurons
    DOI:  https://doi.org/10.1186/s40478-025-02000-4
  6. Autophagy Rep. 2025 ;4(1): 2474796
      Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) represent two extremes of a neurodegenerative disease spectrum characterised by overlapping genetic, clinical, and neuropathological features. This review covers the intricate relationship between both ALS and FTD and defects in the autophagy and endolysosomal pathway as recent evidence has pointed towards alterations in these pathways as being a root cause of disease pathogenesis. Here, we review the current knowledge on the interplay between ALS/FTD and lysosomebased proteostasis pathways and carefully asses the steps of the autophagy and endolysosomal pathways that are impaired by ALS or FTDcausing variants. Finally, we present a comprehensive overview of therapeutic strategies aimed at restoring autophagic and lysosomal function as potential avenues for mitigating the impact of these devastating diseases. Through this review, we aim to enhance the understanding of the pathophysiological mechanisms involving autophagy and/or the endolysosomal system that underlie the ALS-FTD spectrum and underscore the necessity for specific therapeutic approaches that target these shared vulnerabilities.
    Keywords:  Amyotrophic lateral sclerosis (ALS); autophagosome; autophagy; endolysosome; endosome; frontotemporal dementia (FTD); lysosome; neurodegeneration
    DOI:  https://doi.org/10.1080/27694127.2025.2474796
  7. Sci Rep. 2025 May 23. 15(1): 17879
      Transactive response DNA-binding Protein 43 (TDP-43) aggregation is a key pathological feature in Amyotrophic Lateral Sclerosis and related neurodegenerative diseases. This study investigates the inhibitory effects of Epigallocatechin-3-gallate (EGCG), a polyphenol found in green tea, on TDP-43 aggregation. Using a combination of fluorescence assays, NMR spectroscopy, and computational modeling, we demonstrate that Epigallocatechin-3-gallate significantly delays the nucleation phase of TDP-43 aggregation process, thus inhibiting the formation of TDP-43 aggregates in vitro. Additionally, we proved a direct interaction of the compound with the RNA recognition motifs of TDP-43 and modeled the mechanism of interaction. Our findings reveal that EGCG stabilizes the RRM domains, counteracting aggregation by interfering with the early stages of the amyloidogenic pathway. Furthermore, EGCG's stability under experimental conditions was ensured using reducing agents, highlighting the importance of maintaining its reduced form for reproducible results. These insights underscore the therapeutic potential of EGCG in TDP-43 proteinopathies and provide a foundation for developing targeted treatments for ALS and related disorders.
    Keywords:  ALS; EGCG stability; Protein aggregation; Protein-ligand interaction; RNA-binding proteins; TDP-43
    DOI:  https://doi.org/10.1038/s41598-025-02035-6
  8. Glia. 2025 May 22.
      Amyotrophic lateral sclerosis (ALS) is defined by motor neuron death. However, recent research has identified non-cell-autonomous mechanisms, with significant involvement of glia in disease progression. We link previous observations of intracellular protein aggregates in glia to the autophagy pathway, the primary mediator of intracellular degradation of large protein aggregates. While dysfunctional autophagy is reported in ALS motor neurons, pre-clinical and clinical outcomes of autophagy modulators have been inconsistent, indicating the need for a nuanced understanding of autophagy dynamics across CNS cell types and ALS-affected regions. We hypothesized that glial autophagy is defective in ALS, with glial-type-specific dysfunction. To investigate in vivo autophagy dynamics, we intercrossed SOD1G93A mice with transgenic RFP-EGFP-LC3 autophagy reporter mice, enabling the quantification of autophagy degradation, termed flux. Investigation of autophagy dynamics in SOD1 oligodendrocytes, microglia, and astrocytes at key disease stages uncovered useful insights. While oligodendrocytes seemed to mount effective compensatory autophagic responses to combat mutant SOD1, significantly increased autophagy flux was observed in symptomatic spinal microglia and astrocytes in comparison to controls. Symptomatic SOD1 astrocytes displayed greater autophagy dysfunction compared to microglia, with subcellular analysis revealing cell compartment-specific, transient autophagy defects that returned to control levels by end stage. Interestingly, spinal glia showed more pronounced and earlier autophagy dysfunction compared to motor cortex glia, where autophagy dysfunction emerged later in disease end stage, aligning with greater spinal cord pathology reported in this model. Our results suggest that cell-type- and time-specific targeting might be essential when developing autophagy therapeutics for ALS, with prioritization of astrocytic autophagy modulation.
    Keywords:  ALS; SOD1; SOD1G93A; astrocytes; autophagy; microglia; oligodendrocytes
    DOI:  https://doi.org/10.1002/glia.70045
  9. Mol Neurodegener. 2025 May 20. 20(1): 59
       BACKGROUND: Neuronal primary cilia, vital for signaling and cell-cycle regulation, have been implicated in maintaining neuronal identity. While a link between primary ciliary defects and neurodegenerative diseases is emerging, the precise pathological mechanisms remain unclear.
    METHODS: We studied the genetic contribution of NEK1 to ALS pathogenesis by analyzing the exome sequences of 920 Korean patients with ALS. To understand the disease contribution of NEK1 variants in ALS, we performed a series of functional studies using patient fibroblasts focusing on primary cilia and microtubule-related phenotypes. In addition, these findings were validated in iPSC-derived motor neurons (iPSC-MNs).
    RESULTS: NIMA-related kinase 1 (NEK1), a gene encoding a serine/threonine kinase involved in cell cycle regulation, has been identified as a risk gene for amyotrophic lateral sclerosis (ALS). Here, we report that mutations in NEK1 cause primary ciliary abnormality, cell cycle re-entry, and disrupted tubulin acetylation in ALS. We analyzed the whole-exome sequences of 920 Korean patients with sporadic ALS and identified 16 NEK1 variants in 23 patients. We found that two novel variants, p.E853Rfs*9 and p.M1?, reduced NEK1 expression, resulting in loss-of-function (LOF) and one synonymous splicing variant (p.Q132=) exhibited an aberrant isoform lacking exon 5. All three NEK1 variants exhibited abnormal primary ciliary structure, impaired sonic hedgehog signaling, and altered cell-cycle progression. Furthermore, the ALS-linked variants induced intracellular calcium overload followed by Aurora kinase A (AurA)-histone deacetylase (HDAC)6 activation, resulting in ciliary disassembly. These defects were restored by treatment with the intracellular Ca2+ chelator, BAPTA. We also found that NEK1 variants cause decreased α-tubulin acetylation, mitochondrial alteration, and impaired DNA damage response (DDR). Notably, drug treatment to inhibit HDAC6 restored the NEK1-dependent deficits in patient fibroblasts. And, we confirmed that data found in patient fibroblasts were reproduced in iPSC-MNs model.
    CONCLUSIONS: Our results suggest that NEK1 contributes to ALS pathogenesis through the LOF mechanism, and HDAC6 inhibition provides an attractive therapeutic strategy for NEK1 variants associated ALS treatment.
    Keywords:   NEK1 ; Amyotrophic lateral sclerosis; Cell cycle; DNA damage response; Microtubule; Mitochondria; Primary cilia
    DOI:  https://doi.org/10.1186/s13024-025-00848-7
  10. Sci Rep. 2025 May 21. 15(1): 17646
      A slow decline in the autophagy-lysosomal pathway is a hallmark of the normal aging brain. Yet, an acceleration of this cellular function may propel neurodegenerative events. In fact, mutations in genes associated with the autophagy-lysosomal pathway can lead to Parkinson's disease. Also, amyloidogenic protein deposition is observed in lysosomal storage disorders, which are caused by genetic mutations representing risk factors for Parkinson's disease. For example, Gaucher's disease GBA1 mutations leading to defects in lysosomal sphingolipid metabolism cause α-synuclein accumulation. We observed that increased lysosomal Tau accumulation is found in human dermal fibroblasts engineered for inducible Tau expression. Inhibition of the GBA1 product GCase augmented Tau-dependent lysosomal stress and Tau accumulation. Here, we show increased Tau seed-induced Tau accumulation in Gaucher's fibroblasts carrying GBA1 mutations when compared to normal fibroblasts. Pharmacological enhancement of GCase reversed this effect, notably, also in normal fibroblasts. This suggests that boosting GCase activity may represent a therapeutic strategy to slow down aging-dependent lysosomal deficits and brain protein deposition.
    DOI:  https://doi.org/10.1038/s41598-025-02346-8
  11. Pharmacol Ther. 2025 May 14. pii: S0163-7258(25)00088-9. [Epub ahead of print]272 108876
      The inherent technical difficulties, ethical/regulatory issues and costs of experimental studies in animal models is prompting investigators to replace as much as possible living organisms with in vitro physiological models named organoids and assembloids. Generated from induced pluripotent stem cells, these three-dimensional structures approximate the complexity of tissues and their interactions, enabling personalized disease modelling and drug testing. The integration of multiple components in assembloids further enhances their predictive value for multi-system interactions and toxicities. This review describes how neuromuscular organoids, incorporating functional neuromuscular junctions and contractile muscle tissue, have been used to replicate, in vitro, complex neuromuscular morpho-functional structures, offering very valuable platforms to study molecular mechanisms and drug effects in models of incurable diseases such as spinal muscular atrophy and amyotrophic lateral sclerosis. In the cardiological field, cardiac organoids and assembloids are proving reliable models for testing drug effects at molecular, morphological, electrophysiological and mechanical level. Recently, the integration of neuronal components into cardiac organoids has provided a potential approach to investigate autonomic function, a fundamental aspect of many neurological, neuromuscular and cardiac diseases. Challenges and limitations still remain, including the non-uniform differentiation protocols across studies, the incomplete maturation of cell phenotypes, and the lack of integrated pharmacokinetic modelling. We discussed some future developments aimed at overcoming such hurdles. Despite their current limitations, organoids and assembloids clearly hold great promises and will help advancing many fields of biomedicine.
    Keywords:  Assembloids; Cardiac diseases; Drug testing; Neuromuscular diseases; Organoids
    DOI:  https://doi.org/10.1016/j.pharmthera.2025.108876
  12. Mol Aspects Med. 2025 May 20. pii: S0098-2997(25)00033-0. [Epub ahead of print]103 101369
      A wide range of human diseases are associated with protein misfolding and amyloid aggregates. Recent studies suggest that in certain neurological disorders, including Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Dementia (FTD) and various tauopathies, protein aggregation may be promoted by virus-like particles (VLPs) formed by endogenous retroviruses (ERVs). The molecular mechanisms by which these VLPs contribute to protein aggregation, however, remain enigmatic. Here, we discuss possible molecular mechanisms of ERV-derived VLPs in the formation and spread of protein aggregates. An intriguing possibility is that liquid-like condensates may facilitate the formation of both protein aggregates and ERV-derived VLPs. We also describe how RNA chaperoning, and the encapsulation and trafficking of misfolded proteins, may contribute to protein homeostasis through the elimination of protein aggregates from cells. Based on these insights, we discuss future potential therapeutic opportunities.
    DOI:  https://doi.org/10.1016/j.mam.2025.101369
  13. Sci Adv. 2025 May 23. 11(21): eadv1281
      Action potentials are initiated and modulated at the axon initial segment (AIS) by highly clustered ion channels. Voltage-gated Kv1 potassium channels underlie most outward AIS K+ current. AIS Kv1 channels exist in a large protein complex including ADAM22, Caspr2, and LGI1. However, their clustering mechanisms remain unknown. Because Kv1 channels have a highly conserved PDZ-binding motif, we used CRISPR-based genome editing to screen 18 PDZ domain-containing proteins identified in our previous AIS proximity proteome for their AIS localization. Among these, we found that the scaffolding proteins SCRIB and PSD93 are highly enriched at the AIS. Using CRISPR-mediated knockout, cell surface clustering assays, and coimmunoprecipitation, we show that SCRIB and PSD93 bind to and are required for AIS Kv1 channel clustering, whereas SCRIB links the AIS Kv1 channel protein complex to the master AIS scaffolding protein AnkyrinG. These results define a hierarchy of scaffolding proteins that combine to cluster AIS Kv1 channels.
    DOI:  https://doi.org/10.1126/sciadv.adv1281
  14. J Mol Biol. 2025 May 16. pii: S0022-2836(25)00285-2. [Epub ahead of print] 169219
      An infamous hallmark of neurodegenerative diseases is the accumulation of misfolded or unfolded proteins forming inclusions in the brain. The accumulation of these abnormal structures is a mysterious one, given that cells devote significant resources to integrate complementary pathways to ensure proteome integrity and proper protein folding. Aberrantly folded protein species are rapidly targeted for disposal by the ubiquitin-proteasome system (UPS), and even if this should fail, and the species accumulates, the cell can also rely on the lysosome-mediated degradation pathways of autophagy. Despite the many safeguards in place, failure to maintain protein homeostasis commonly occurs during, or preceding, the onset of disease. Over the last decade and a half, studies suggest that the failure of autophagy may explain the disruption in protein homeostasis observed in disease. In this review, we will examine how the highly complex cells of the brain can become vulnerable to failure of aggregate clearance at specific points during the processive pathway of autophagy, contributing to aggregate accumulation in brains with neurodegenerative disease.
    Keywords:  Neurodegeneration; Protein aggregation; glia; neurons; protein homeostasis
    DOI:  https://doi.org/10.1016/j.jmb.2025.169219
  15. Neuromolecular Med. 2025 May 21. 27(1): 42
      Parkinson's disease (PD) is a chronic and progressive neurodegenerative disorder for which there are currently no curative therapies. Therefore, the need for innovative treatments for this illness is critical. The glucosylceramidase beta 1 (GBA1) and leucine-rich repeated kinase 2 (LRRK2) genes have been postulated as potential genetically defined drug targets. We report for the first time that the LRRK2 inhibitor PF-06447475 (PF-475) not only restores GCase enzyme activity, but also increases mitochondrial membrane potential, significantly decreases DJ-1 Cys106-SO3, reduces lysosome accumulation, and diminishes cleaved caspase-3 (CC3) in GBA1 K198E fibroblasts. Furthermore, in addition to a significant reduction in p-Ser935 LRRK2 kinase, we found that PF-475 reduced p-Thr73 RAB 10 and p-Ser129 α-Syn in mutant skin fibroblasts. In addition, we found that the GCase activator GCA (NCGC00188758) increased GCase activity and decreased lysosomal accumulation, but did not affect p-Ser935 LRRK2, ∆Ψm, p-Ser129 α-Syn, DJ-1 Cys106-SO3, or CC3 in K198E GBA1 fibroblasts. The GCase inhibitor conduritol-β-epoxide (CBE), used as an internal control, significantly reduced GCase and left the other pathological markers largely unaltered in GBA1 K198E, but reduced GCase and increased the accumulation of lysosomes only in WT GBA1 fibroblasts. Taken together, these results suggest that LRRK2 is a critical signaling kinase in the pathogenic mechanism associated with the lysosomal GBA1/GCase K198E variant. Our findings suggest that the use of LRRK2 inhibitors in PD patients with GBA1 mutations, such as K198E, may be effective in reversing GBA1/GCase deficiency, autophagy impairment, oxidative stress, and neuronal death.
    Keywords:  Fibroblasts; GBA; LRRK2; PF-06447475; Parkinson’s disease; RAB10
    DOI:  https://doi.org/10.1007/s12017-025-08864-y
  16. Science. 2025 May 22. eadr3498
      Mitochondria fulfill central functions in metabolism and energy supply. They express their own genome, which encodes key subunits of the oxidative phosphorylation system. However, central mechanisms underlying mitochondrial gene expression remain enigmatic. A lack of suitable technologies to target mitochondrial protein synthesis in cells has limited experimental access. Here, we silenced the translation of specific mitochondrial mRNAs in living human cells by delivering synthetic peptide-morpholino chimeras. This approach allowed us to perform a comprehensive temporal monitoring of cellular responses. Our study provides insights into mitochondrial translation, its integration into cellular physiology, and provides a strategy to address mitochondrial gene expression in living cells. The approach can potentially be used to analyze mechanisms and pathophysiology of mitochondrial gene expression in a range of cellular model systems.
    DOI:  https://doi.org/10.1126/science.adr3498
  17. Proc Natl Acad Sci U S A. 2025 May 27. 122(21): e2422752122
      Many neurodegenerative disorders (NDDs) preferentially affect neurons with long or complex axonal arbors but the cellular and molecular bases for neurite length-dependent vulnerability of neurons to degeneration is largely unknown. Using Drosophila sensory neurons as a model system we show that neuronal activation of the integrated stress response triggers expression of the Interleukin-6 homolog unpaired 3 (upd3), which is both necessary and sufficient for axon length-dependent degeneration of presynapses. Upd3 activates phagocytic glia, triggering phagocytic removal of presynapses preferentially on neurons with long axons, thus revealing an intrinsic axon length-dependent vulnerability to glial insult. Finally, we found that axon length-dependent presynapse loss in fly models of human NDDs utilized this pathway, requiring upd3 and glial expression of the phagocytic receptor draper. Our studies identify inflammatory cytokine signaling and glial phagocytosis as key determinants of axon length-dependent vulnerability, thus mechanistically linking these hallmarks of NDDs.
    Keywords:  axon; cytokine; glia; neurodegeneration; neuron
    DOI:  https://doi.org/10.1073/pnas.2422752122
  18. Autophagy Rep. 2025 ;4(1): 2438563
      LC3-interacting region (LIR) motifs are essential for recruiting proteins onto autophagosomes, the hallmark of autophagy. We recently explored the relevance of the specific position of the LIRs in RavZ and ATG4B (autophagy-related 4B). RavZ's N-terminal LIRs drive substrate recognition and enzymatic activity, while its C-terminal LIR aids membrane localization. In contrast, ATG4B's C-terminal LIR is indispensable for LC3B (microtubule-associated protein 1 light chain 3B)-phosphatidylethanolamine (PE) delipidation on autophagosomes but not required for cytosolic LC3B priming, which is mediated solely by its catalytic domain (CAD). These findings underscore the structural adaptation of LIRs for context-specific functions. This novel nuanced understanding provides a framework for developing therapeutic tools to modulate autophagy by precisely targeting LIRs or their associated processes, offering potential treatment for diseases like neurodegenerative disorders and infections characterized by autophagy dysregulation.
    Keywords:  ATG4B; Autophagy; LC3/GABARAP; LIR; RavZ; delipidation
    DOI:  https://doi.org/10.1080/27694127.2024.2438563
  19. Alzheimers Dement. 2025 May;21(5): e14621
       INTRODUCTION: Recent advancements in immunological methods accurately quantify biofluid biomarkers for Alzheimer's disease (AD) pathology. Despite progress, more biomarkers, ideally in blood, are needed for effective disease monitoring for AD and other neurodegenerative proteinopathies.
    METHODS: We used the Nucleic Acid Linked Immuno-Sandwich Assay (NULISA) central nervous system panel for biomarker quantification in plasma, serum, and cerebrospinal fluid of patients with AD, mild cognitive impairment, Lewy body dementia, progranulin (GRN) mutation carriers.
    RESULTS: NULISA identified phosphorylated tau217 and neurofilament light chain as the most deregulated biomarkers in the AD continuum and GRN mutation carriers, respectively. Importantly, numerous novel proteomic changes were observed in each disease endophenotype, which included synaptic processing, inflammation, microglial reactivity, TAR DNA-binding protein 43, and α-synuclein pathology.
    DISCUSSION: We underline the potential of next-generation biomarker identification tools to detect novel proteomic features that also incorporate established biomarkers. These findings highlight the importance of continued biomarker discovery to improve treatment decisions and help us better understand the complexities of neurodegenerative disorders.
    HIGHLIGHTS: The, direct, or indirect, measures in blood that complement phosphorylated tau (p-tau)217 for other proteinopathies or disease progression are urgently needed. Significant novel proteomic changes were observed in each disease endophenotype in plasma, serum, and cerebrospinal fluid, which included proteins involved in synaptic processing, inflammation, microglial reactivity, TAR DNA-binding protein 43, and α-synuclein pathology. Nucleic Acid Linked Immuno-Sandwich Assay continued to unbiasely highlight p-tau217 and neurofilament light chain as the most significantly deregulated blood biomarkers in the Alzheimer's disease continuum and progranulin mutation carriers, respectively.
    Keywords:  Lewy body disease; Nucleic Acid Linked Immuno‐Sandwich Assay; discovery; frontotemporal dementia; plasma biomarkers; proteomics
    DOI:  https://doi.org/10.1002/alz.14621
  20. Chembiochem. 2025 May 21. e202500156
      Small GTPases comprise a diverse class of signaling proteins in mammalian cells and regulate a variety of cellular processes such as cell growth, cell movement, vesicle formation, and nuclear transport. Due to their involvement in critical cellular pathways, changes in the activation state of small GTPases due to genetic mutations or alterations in gene expression can lead to human disease. As such, the ability to control the activity of small GTPases is paramount in understanding the precise role these proteins play in human biology and in reducing their impacts on related diseases. Herein, we present important advances made in the development of small molecule- and protein engineering-based strategies to control the activity of small GTPases. Current approaches within each area are discussed within their historical contexts along with commentary on the importance that each technology has had on improving our ability to regulate small GTPase activity. Given this ever-evolving toolbox for controlling small GTPase signaling, we anticipate continued growth in the study of this protein class.
    Keywords:  Inhibitors; protein engineering; signal transduction; small GTPases; split-proteins
    DOI:  https://doi.org/10.1002/cbic.202500156
  21. Redox Biol. 2025 May 13. pii: S2213-2317(25)00191-0. [Epub ahead of print]84 103678
      Cytosolic thioredoxin (Trx) is a critical redox protein that converts protein disulfides to thiols via catalytic activity of thioredoxin reductase-1 (TrxR1) and NADPH. Thioredoxin-2 (Trx2) is a mitochondria-localized isoform. It is generally believed that Trx and Trx2 perform similar functions within the cytosol and mitochondria respectively. Here, we demonstrate that cytosolic Trx shuttles into mitochondria in the presence of normal levels of Trx2 in physiological state and higher levels of Trx translocate to mitochondria in oxidative stress conditions such as exposure to high concentrations of oxygen. This shuttle is required to maintain mitochondrial structure and function during physiological and oxidative stress conditions. Further, reduced Trx (Trx-SH) shuttle into mitochondria to protect against the downregulation of several mitochondrially coded genes and proteins of respiratory chain complexes in oxidative stress. Translocation of Trx occurs only in the reduced state as oxidized or cysteine mutant Trx is unable to translocate to the mitochondria. Accumulation of mitochondrial DNA damage product 8-Oxo-dG in hyperoxia is decreased in the presence of higher levels of cytosolic Trx within the mitochondrion. Collectively, our data demonstrate that shuttling of reduced cytosolic Trx into mitochondria protects against mitochondrial DNA damage, decreased gene and protein expression of respiratory chain complexes and mitochondrial dysfunction resulting in restoration of their native function and cell survival in physiological and oxidative stress conditions.
    DOI:  https://doi.org/10.1016/j.redox.2025.103678
  22. Autophagy Rep. 2024 ;3(1): 2432848
      OPTN (optineurin), an amyotrophic lateral sclerosis (ALS)-associated modifier, plays vital roles in autophagy and cellular vesicular transport in mammals. OPTN can associate with RAB8A and the GTPase-activating protein TBC1D17, and facilitate the negative regulation of RAB8A by TBC1D17 (TBC domain family member 17). Recently, we reported the biochemical and structural characterizations of the interactions between OPTN, RAB8A and TBC1D17. We determined the crystal structure of the leucine-zipper domain (LZD) of OPTN with the GTP-bound active RAB8A and uncovered the molecular mechanism underpinning the specific interaction of OPTN with RAB8A. Moreover, we revealed that OPTN LZD and the TBC (Tre-2/Bub2/Cdc16) domain of TBC1D17 competitively bind to active RAB8A, while the central coiled-coil domain of OPTN and the active RAB8A can simultaneously interact with TBC1D17 TBC. In summary, our study provided mechanistic insights into the interaction of OPTN with RAB8A, and revealed the interaction relationship among OPTN, RAB8A and TBC1D17.
    Keywords:  RAB8A; TBC1D17; Vesicular transport; optineurin
    DOI:  https://doi.org/10.1080/27694127.2024.2432848
  23. Front Cell Dev Biol. 2025 ;13 1538377
      Neurodegenerative diseases (NDs) such as Alzheimer's, Parkinson's and Huntington's diseases as well as ataxias and fronto-temporal disorders are all characterized by the progressive accumulation of protein aggregates (amyloids) into inclusions bodies. In addition, recent experimental evidence is challenging the conventional view of the disease by revealing the ability of some of these disease-relevant proteins to be transferred between cells by means of extracellular vesicles (EVs), allowing the mutant protein to seed oligomers involving both the mutant and wild type forms of the protein. Abnormal secretion and levels of EVs are closely related to the pathogenesis of neurodegenerative diseases and contribute to disease progression. Numerous studies have proposed EVs as therapeutic targets or biomarkers for neurodegenerative diseases. In this review, we summarize and discuss the role of small heat shock proteins (sHSPs) and autophagy in cellular quality control and turn-over of the major aggregation-prone proteins associated to neurodegenerative disorders. We also highlight the advanced research progress on mechanisms regulating unconventional secretion, secretory autophagy and EVs biogenesis and their contribution in the pathological processes underlining these diseases. Finally, we outline the latest research on the roles of EVs in neurodegenerative diseases and their potential diagnostic and therapeutic significance for the treatment of these clinically relevant conditions.
    Keywords:  autophagy; extracellular vesicles and exosomes; neurodegenerative diseases; protein misfolding; protein oligomerization and aggregation; small heat shock proteins; unconventional protein secretion
    DOI:  https://doi.org/10.3389/fcell.2025.1538377
  24. Autophagy Rep. 2024 ;3(1): 2346064
      Neurons are highly differentiated and compartmentalized cells that conduct cellular processes in a spatiotemporally regulated manner. Autophagy in neurons occurs locally under stimulation and contributes to synaptic plasticity. Little is known about the initial steps leading to autophagy upon neuronal stimulation and the role of autophagic compartments at the postsynaptic part of the synapse. Here, we summarize our recent manuscript on Rab11 role in autophagy initiation in the dendritic spines. We showed that Rab11 maintains in the dendritic spines Atg9A and is necessary for LC3+ vesicles to emerge at the postsynapse. We hypothesize that autophagosomes arise due to an interplay between NMDA receptor stimulation and local mTOR kinase activity. We suggest that autophagosomes are not, in fact, responsible for dendritic spine pruning.
    Keywords:  Atg9A; Rab11; autophagy; dendritic spines; mTOR; neurons; synaptic plasticity
    DOI:  https://doi.org/10.1080/27694127.2024.2346064
  25. Eur J Med Chem. 2025 May 17. pii: S0223-5234(25)00539-2. [Epub ahead of print]294 117774
      Targeted protein degradation using proteolysis-targeting chimeras (PROTACs) has emerged as a powerful strategy for modulating protein function. In this study, we developed mTOR-targeting PROTACs by conjugating the mTOR agonist MHY-1485 to the Cereblon (CRBN) ligand pomalidomide, demonstrating that even activators can serve as effective warheads for targeted protein degradation. Through systematic screening, we identified PD-M6 as a potent bifunctional molecule capable of degrading mTOR (DC50 = 4.8 μM), reversing the proliferative effects of MHY-1485, and inhibiting cell proliferation (IC50 = 11.3 μM) while inducing autophagy, akin to the mTOR known inhibitor rapamycin. Proteomic analysis further revealed that PD-M6 downregulated key proteins in the mTOR signaling pathway, including LAMTOR1, MAPKAP1, and CASTOR1, which are involved in proteasome-mediated degradation, cell division, apoptosis, and lysosomal signaling. Notably, PD-M6 specifically induced the degradation of LAMTOR1. These findings highlight a novel approach for designing PROTACs from agonists, broadening the scope of targeted protein degradation strategies for therapeutic applications.
    Keywords:  Autophagy; Degradation; PROTACs; Proteomics; mTOR
    DOI:  https://doi.org/10.1016/j.ejmech.2025.117774
  26. Autophagy Rep. 2025 ;4(1): 2501365
      Autophagy has been implicated in various cellular processes, including non-conventional secretion. Our previous findings suggest that ATP is loaded into amphisomes and secreted upon autophagy stimulation at focal adhesion sites in a VAMP7-dependent manner. Here, we demonstrate that the knockout (KO) of VAMP7, along with its partners RAB21 and its guanine nucleotide exchange factor (GEF) VARP, inhibits ATP release, indicating a key role for this pathway in amphisome secretion. Constitutively inactive RAB21 also inhibited ATP secretion. RAB21 overexpression rescued starvation-induced ATP secretion in RAB21 KO, but not in VAMP7 or VARP KO cells. RAB21-LC3-positive vesicles redistributed to the cell periphery upon starvation. KO cells and overexpression experiments showed that RAB21 plays a positive role in autophagosome biogenesis, particularly in controlling the number of LC3-II- and DFCP1-positive structures upon starvation, suggesting a role in the early steps of autophagosome formation. Accordingly, VARP partially colocalized with LC3 upon starvation. Together, these findings identify a novel role for RAB21 in regulating autophagic ATP secretion likely in amphisome biogenesis and their localization in the cell periphery.
    Keywords:  ATP release; LC3; RAB proteins; VAMP7; macroautophagy; secretory autophagy
    DOI:  https://doi.org/10.1080/27694127.2025.2501365
  27. Bio Protoc. 2025 Mar 20. 15(6): e5251
      The growth cone is a highly motile tip structure that guides axonal elongation and directionality in differentiating neurons. Migrating immature neurons also exhibit a growth cone-like structure (GCLS) at the tip of the leading process. However, it remains unknown whether the GCLS in migrating immature neurons shares the morphological and molecular features of axonal growth cones and can thus be considered equivalent to them. Here, we describe a detailed method for time-lapse imaging and optical manipulation of growth cones using a super-resolution laser-scanning microscope. To observe growth cones in elongating axons and migrating neurons, embryonic cortical neurons and neonatal ventricular-subventricular zone (V-SVZ)-derived neurons, respectively, were transfected with plasmids encoding fluorescent protein-conjugated cytoskeletal probes and three-dimensionally cultured in Matrigel, which mimics the in vivo background. At 2-5 days in vitro, the morphology and dynamics of these growth cones and their associated cytoskeletal molecules were assessed by time-lapse super-resolution imaging. The use of photoswitchable cytoskeletal inhibitors, which can be reversibly and precisely controlled by laser illumination at two different wavelengths, revealed the spatiotemporal regulatory machinery and functional significance of growth cones in neuronal migration. Furthermore, machine learning-based methods enabled us to automatically segment growth cone morphology from elongating axons and the leading process. This protocol provides a cutting-edge methodology for studying the growth cone in developmental and regenerative neuroscience, being adaptable for various cell biology and imaging applications. Key features • Three-dimensional primary culture of migrating and differentiating neurons in Matrigel. • Visualization of fine morphology and dynamics of growth cones using super-resolution imaging. • Optical manipulation of cytoskeletal molecules in growth cones using photoswitchable inhibitors. • Machine learning-based extraction of growth cone morphology.
    Keywords:  Elongating axons; F-actin; Growth cone; Microtubules; Migrating neurons; Optical manipulation; Photoswitchable inhibitor; Postnatal neurogenesis; Super-resolution imaging; Ventricular–subventricular zone
    DOI:  https://doi.org/10.21769/BioProtoc.5251