bims-axbals Biomed News
on Axonal biology and ALS
Issue of 2025–02–09
27 papers selected by
TJ Krzystek, ALS Therapy Development Institute
  1. A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence.
  2. Unlocking Disease-Modifying Treatments for TDP-43-Mediated Neurodegeneration.
  3. Muscle-specific Bet1L knockdown induces neuromuscular denervation, motor neuron degeneration, and motor dysfunction in a rat model of familial ALS.
  4. UBE3A controls axon initial segment in the cortical pyramidal neurons.
  5. Kaempferol enhances ER-mitochondria coupling and protects motor neurons from mitochondrial dysfunction and ER stress in C9ORF72-ALS.
  6. Somatic CAG-repeat expansion drives neuronal loss in Huntington's disease.
  7. Chaperone dysfunction in motor neuron disease: new insights from studies of the SMN complex.
  8. Development and validation of a sensitive sandwich ELISA against human PINK1.
  9. Proteomic analysis reveals distinct cerebrospinal fluid signatures across genetic frontotemporal dementia subtypes.
  10. Super-Enhancer Protects Cells From Toxicity of C9orf72 Poly(proline-arginine) by Inducing the Expression of KPNA2/KPNB1.
  11. ALS plasma biomarkers reveal neurofilament and pTau correlate with disease onset and progression.
  12. The Huntingtin Transport Complex.
  13. Mec1-mediated Atg9 phosphorylation regulates the PAS recruitment of Atg9 vesicles upon energy stress.
  14. Amyloid-β can activate JNK signalling via WNT5A-ROR2 to reduce synapse formation in Alzheimer's disease.
  15. Loss of Fanconi anemia proteins causes a reliance on lysosomal exocytosis.
  16. VPS41 recruits biosynthetic LAMP-positive vesicles through interaction with Arl8b.
  17. The autophagy component LC3 regulates lymphocyte adhesion via LFA1 transport in response to outside-in signaling.
  18. Cognitive impairment in epilepsy progression: Adult neurogenesis loss at critical window.
  19. Hsp70 chaperones, Ssa1 and Ssa2, limit poly(A) binding protein aggregation.
  20. Oxidative Stress Promotes Axonal Atrophy through Alterations in Microtubules and EB1 Function.
  21. Parkinson's disease mutant Miro1 causes mitochondrial dysfunction and dopaminergic neuron loss.
  22. Recent advances in small molecule inhibitors of deubiquitinating enzymes.
  23. Coordination between ESCRT function and Rab conversion during endosome maturation.
  24. Glycogen synthase is required for heat shock-mediated autophagy induction in neuronal cells.
  25. High-throughput discovery of inhibitory protein fragments with AlphaFold.
  26. Aspirin inhibits proteasomal degradation and promotes α-synuclein aggregate clearance through K63 ubiquitination.
  27. Harmine promotes axon regeneration through enhancing glucose metabolism.
  1. F1000Res. 2024 ;13 1559
    A guide to selecting high-performing antibodies for VAPB (UniProt ID: O95292) for use in western blot, immunoprecipitation, and immunofluorescence.
    NeuroSGC/YCharOS/EDDU collaborative group
      VAPB is an adaptor protein known for its role as an anchor for other proteins at the endoplasmic reticulum. A mutant form of VAPB has been linked to amyotrophic lateral sclerosis and the underlying mechanisms resulting from this defect are studied by researchers in this area to uncover its implication in the disease. Here we have characterized six VAPB 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:  O95292; VAMP-associated protein B/C; VAPB; Vesicle-associated membrane protein-associated protein B/C; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation; western blot
    DOI:  https://doi.org/10.12688/f1000research.160226.1
  2. Bioessays. 2025 Feb 03. e202400257
    Unlocking Disease-Modifying Treatments for TDP-43-Mediated Neurodegeneration.
    Rebecca San Gil, Adam K Walker.
      Neurons degenerate in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), causing progressive and inevitably fatal neurological decline. The best therapeutic strategies target underlying disease mediators, but after decades of intensive research, the causes of these neurodegenerative diseases remain elusive. Recently, coordinated activities of large consortia, increasing open access to large datasets, new methods such as cryo-transmission electron microscopy, and advancements in high-resolution omics technologies have offered new insights into the biology of disease that bring us closer to understanding mechanisms of neurodegeneration. In particular, improved understanding of the roles of the key pathological protein TAR DNA binding protein 43 (TDP-43) in disease has revealed intriguing new opportunities that provide hope for better diagnostic tools and effective treatments for ALS and FTD.
    Keywords:  TARDBP; chaperones; frontotemporal dementia; motor neuron disease; neurodegenerative disease; proteogenomics
    DOI:  https://doi.org/10.1002/bies.202400257
  3. Front Neurosci. 2025 ;19 1527181
    Muscle-specific Bet1L knockdown induces neuromuscular denervation, motor neuron degeneration, and motor dysfunction in a rat model of familial ALS.
    Adam Eckardt, Charles Marble, Bradley Fern, Henry Moritz, Charles Kotula, Jiayi Ke, Clarisse Rebancos, Samantha Robertson, Hiroshi Nishimune, Masatoshi Suzuki.
      Amyotrophic lateral sclerosis (ALS) is a neuromuscular disease characterized by specific loss of motor neurons in the spinal cord and brain stem. Although ALS has historically been characterized as a motor neuron disease, there is evidence that motor neurons degenerate in a retrograde manner, beginning in the periphery at the neuromuscular junctions (NMJs) and skeletal muscle. We recently reported a vesicle trafficking protein Bet1L (Bet1 Golgi Vesicular Membrane Trafficking Protein Like) as a new molecule possibly linked to NMJ degeneration in ALS. In this study, we tested the hypothesis that Bet1L gene silencing in skeletal muscle could influence NMJ integrity, motor neuron function, and survival in a rat model of familial ALS (SOD1G93A transgenic). Small interfering RNA (siRNA) targeting the Bet1L gene was injected on a weekly basis into the hindlimb muscle of pre-symptomatic ALS and wild-type (WT) rats. After 3 weeks, intramuscular Bet1L siRNA injection significantly increased the number of denervated NMJs in the injected muscle. Bet1L knockdown decreased motor neuron size in the lumbar spinal cord, which innervated the siRNA-injected hindlimb. Impaired motor function was identified in the hindlimbs of Bet1L siRNA-injected rats. Notably, the effects of Bet1L knockdown on NMJ and motor neuron degeneration were more significant in ALS rats when compared to WT rats. Together, Bet1L knockdown induces denervation of NMJs, but also this knockdown accelerates the disease progression in ALS. Our results provide new evidence to support the potential roles of Bet1L as a key molecule in NMJ maintenance and ALS pathogenesis.
    Keywords:  Bet1L; SOD1G93A rats; amyotrophic lateral sclerosis (ALS); distal axonopathy; motor neuron; neuromuscular junction; skeletal muscle; small interfering RNA (siRNA)
    DOI:  https://doi.org/10.3389/fnins.2025.1527181
  4. Biochem Biophys Res Commun. 2025 Feb 01. pii: S0006-291X(25)00143-3. [Epub ahead of print]751 151429
    UBE3A controls axon initial segment in the cortical pyramidal neurons.
    Xinlang Liu, Zhuqian Jiang, Yoshinori Otani, Xiaowei Zhu, Yanyan Yu, Abu Md Mamun Tarif, Raihana Nasrin Ferdousy, Tatsuya Kishino, Masashi Fujitani.
      The axon initial segment (AIS) is a critical regulator of neuronal excitability and the initiation site of action potentials. Alterations in the structural features of AIS, such as length and position, have been shown to influence neuronal function, a phenomenon known as activity-dependent AIS plasticity. In addition to their physiological functions, abnormalities in the AIS have been implicated in various neurological disorders. UBE3A is an E3 ubiquitin ligase crucial for protein degradation in neurons. In mature neurons, only the maternal allele of the UBE3A gene is active, and the paternal allele is silenced. However, the role of UBE3A in controlling AIS in the cortical pyramidal neurons has not yet been fully elucidated. In this study, we compared wild-type mice with three different Ube3a-deficient mice and observed specific elongation of the AIS in the prelimbic cortex of the medial prefrontal cortex but not in the somatosensory cortex or motor cortex, as previously reported. Interestingly, we also showed that UBE3A controls AIS length in a cell-autonomous manner using cultured cortical neurons derived from Ube3a-floxed (Ube3aflox/flox) mice. This study indicates that UBE3A controls AIS length through a cell-autonomous mechanism in vitro. However, non-cell-autonomous mechanisms, including feedback from inhibitory neurons or connections with the hippocampus, may also influence adult AIS length in vivo, thereby helping to maintain the excitability homeostasis of cortical pyramidal neurons in a region-specific manner.
    Keywords:  Angelman syndrome; Axon initial segment; Prelimbic cortex; Pyramidal neurons; Ubiquitin-protein ligase E3A
    DOI:  https://doi.org/10.1016/j.bbrc.2025.151429
  5. Acta Neuropathol Commun. 2025 Feb 01. 13(1): 21
    Kaempferol enhances ER-mitochondria coupling and protects motor neurons from mitochondrial dysfunction and ER stress in C9ORF72-ALS.
    Federica Pilotto, Paulien Hermine Smeele, Olivier Scheidegger, Rim Diab, Martina Schobesberger, Julieth Andrea Sierra-Delgado, Smita Saxena.
      Repeat expansions in the C9ORF72 gene are a frequent cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Considerable progress has been made in identifying C9ORF72-mediated disease and resolving its underlying etiopathogenesis. The contributions of intrinsic mitochondrial deficits as well as chronic endoplasmic reticulum stress to the development of the C9ORF72-linked pathology are well established. Nevertheless, to date, no cure or effective therapy is available, and thus attempts to find a potential drug target, have received increasing attention. Here, we investigated the mode of action and therapeutic effect of a naturally occurring dietary flavanol, kaempferol in preclinical rodent and human models of C9ORF72-ALS. Notably, kaempferol treatment of C9ORF72-ALS human patient-derived motor neurons/neurons, resolved mitochondrial deficits, promoted resiliency against severe ER stress, and conferred neuroprotection. Treatment of symptomatic C9ORF72 mice with kaempferol, normalized mitochondrial calcium uptake, restored mitochondria function, and diminished ER stress. Importantly, in vivo, chronic kaempferol administration ameliorated pathological motor dysfunction and inhibited motor neuron degeneration, highlighting the translational potential of kaempferol. Lastly, in silico modelling identified a novel kaempferol target and mechanistically the neuroprotective mechanism of kaempferol is through the iP3R-VDAC1 pathway via the modulation of GRP75 expression. Thus, kaempferol holds great promise for treating neurodegenerative diseases where both mitochondrial and ER dysfunction are causally linked to the pathophysiology.
    DOI:  https://doi.org/10.1186/s40478-025-01927-y
  6. Neuron. 2025 Feb 05. pii: S0896-6273(25)00012-1. [Epub ahead of print]113(3): 342-344
    Somatic CAG-repeat expansion drives neuronal loss in Huntington's disease.
    Gillian P Bates.
      Using single-cell technologies on postmortem brains, Handsaker et al.1 have demonstrated that substantial somatic expansion of the CAG repeat that causes Huntington's disease results in progressive transcriptional dysregulation and drives the loss of spiny projection neurons in the caudate.
    DOI:  https://doi.org/10.1016/j.neuron.2025.01.008
  7. Genetics. 2025 Feb 05. pii: iyae223. [Epub ahead of print]
    Chaperone dysfunction in motor neuron disease: new insights from studies of the SMN complex.
    A Gregory Matera.
      Spinal muscular atrophy and amyotrophic lateral sclerosis are devastating neurodegenerative diseases characterized by motor neuron loss. Although these 2 disorders have distinct genetic origins, recent studies suggest that they share common etiological mechanisms rooted in proteostatic dysfunction. At the heart of this emerging understanding is the survival motor neuron (SMN) complex.
    Keywords:  ALS; SMA; SMN; TDP-43; amyotrophic lateral sclerosis; genetic suppressors; heat shock proteins; hspA; spinal muscular atrophy; survival motor neuron
    DOI:  https://doi.org/10.1093/genetics/iyae223
  8. Autophagy. 2025 Feb 06. 1-16
    Development and validation of a sensitive sandwich ELISA against human PINK1.
    Zahra Baninameh, Jens O Watzlawik, Bernardo A Bustillos, Gabriella Fiorino, Tingxiang Yan, Szymon L Lewicki, Haonan Zhang, Dennis W Dickson, Joanna Siuda, Zbigniew K Wszolek, Wolfdieter Springer, Fabienne C Fiesel.
      The ubiquitin kinase and ligase PINK1 and PRKN together label damaged mitochondria for their elimination in lysosomes by selective autophagy (mitophagy). This cytoprotective quality control pathway is genetically linked to familial Parkinson disease but is also altered during aging and in other neurodegenerative disorders. However, the molecular mechanisms of these mitophagy changes remain uncertain. In healthy mitochondria, PINK1 protein is continuously imported, cleaved, and degraded, but swiftly accumulates on damaged mitochondria, where it triggers the activation of the mitophagy pathway by phosphorylating its substrates ubiquitin and PRKN. Levels of PINK1 protein can therefore be used as a proxy for mitochondrial damage and mitophagy initiation. However, validated methodologies to sensitively detect and quantify PINK1 protein are currently not available. Here, we describe the development and thorough validation of a novel immunoassay to measure human PINK1 on the Meso Scale Discovery platform. The final assay showed excellent linearity, parallelism, and sensitivity. Even in the absence of mitochondrial stress (i.e. at basal conditions), when PINK1 protein is usually not detectable by immunoblotting, significant differences were obtained when comparing samples from patient fibroblasts or differentiated neurons with and without PINK1 expression. Of note, PINK1 protein levels were found increased in human postmortem brain with normal aging, but not in brains with Alzheimer disease, suggesting that indeed different molecular mechanisms are at play. In summary, we have developed a novel sensitive PINK1 immunoassay that will complement other efforts to decipher the roles and biomarker potential of the PINK1-PRKN mitophagy pathway in the physiological and pathological context. Abbreviations: AD: Alzheimer disease; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; ECL: electrochemiluminescence; ELISA: enzyme-linked immunosorbent assay; iPSC: induced pluripotent stem cell; KO: knockout; LLOQ: lower limit of quantification; MSD: Meso Scale Discovery; PD: Parkinson disease; p-S65-Ub: serine-65 phosphorylated ubiquitin; Ub: ubiquitin; ULOQ: upper limit of quantification; WT: wild-type.
    Keywords:  Autophagy; P-S65-Ub; PINK1; Parkin; mitophagy; ubiquitin
    DOI:  https://doi.org/10.1080/15548627.2025.2457915
  9. Sci Transl Med. 2025 Feb 05. 17(784): eadm9654
    Proteomic analysis reveals distinct cerebrospinal fluid signatures across genetic frontotemporal dementia subtypes.
    GENFI
      We used an untargeted mass spectrometric approach, tandem mass tag proteomics, for the identification of proteomic signatures in genetic frontotemporal dementia (FTD). A total of 238 cerebrospinal fluid (CSF) samples from the Genetic FTD Initiative were analyzed, including samples from 107 presymptomatic (44 C9orf72, 38 GRN, and 25 MAPT) and 55 symptomatic (27 C9orf72, 17 GRN, and 11 MAPT) mutation carriers as well as 76 mutation-negative controls ("noncarriers"). We found shared and distinct proteomic alterations in each genetic form of FTD. Among the proteins significantly altered in symptomatic mutation carriers compared with noncarriers, we found that a set of proteins including neuronal pentraxin 2 and fatty acid binding protein 3 changed across all three genetic forms of FTD and patients with Alzheimer's disease from previously published datasets. We observed differential changes in lysosomal proteins among symptomatic mutation carriers with marked abundance decreases in MAPT carriers but not other carriers. Further, we identified mutation-associated proteomic changes already evident in presymptomatic mutation carriers. Weighted gene coexpression network analysis combined with gene ontology annotation revealed clusters of proteins enriched in neurodegeneration and glial responses as well as synapse- or lysosome-related proteins indicating that these are the central biological processes affected in genetic FTD. These clusters correlated with measures of disease severity and were associated with cognitive decline. This study revealed distinct proteomic changes in the CSF of patients with genetic FTD, providing insights into the pathological processes involved in the disease. In addition, we identified proteins that warrant further exploration as diagnostic and prognostic biomarker candidates.
    DOI:  https://doi.org/10.1126/scitranslmed.adm9654
  10. Cell Biochem Funct. 2025 Feb;43(2): e70053
    Super-Enhancer Protects Cells From Toxicity of C9orf72 Poly(proline-arginine) by Inducing the Expression of KPNA2/KPNB1.
    Miaomiao Chen, Henglu Cui, Xiaoyu Zhang, Shuyan Ma, Jinjing Guo, Zhaoxiu Liu, Donghua Gu, Yihui Fan.
      Hexanucleotide repeat expansions in C9orf72 are the most common genetic mutation associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9-ALS/FTD). Dipeptide repeat (DPR) proteins, such as poly(proline-arginine) (polyPR) generated from G4C2 repeat expansions, have been shown to be highly toxic. In this study, PR20 was labeled with fluorescein isothiocyanate (FITC) to track its cellular localization. Several cell lines demonstrated survival under PR20 treatment by sequestering PR20 in the cytoplasm. Treatment with JQ-1 or Ivermectin (Iver) translocated PR20 into the nucleus, leading to cell death. Mechanistically, KPNA2/KPNB1 interacted with PR20 in the cytoplasm and hindered PR20 from entering the cell nucleus. Genetic silencing of KPNA2/KPNB1 converted PR20-resistant cells into PR20-sensitive cells. Treatment with JQ1 significantly reduced the protein levels of KPNA2/KPNB1, allowing PR20 to enter the nucleus. Overexpression of KPNA2 or KPNB1 effectively blocked cell death induced by co-treatment with JQ-1 and PR20. Our results indicate that super-enhancers shield cells from PR20 toxicity by upregulating the expression of KPNA2/KPNB1.
    Keywords:  C9orf72; KPNA2; KPNB1; amyotrophic lateral sclerosis (ALS); dipeptide repeat (DPR); frontotemporal dementia (FTD); super‐enhancer
    DOI:  https://doi.org/10.1002/cbf.70053
  11. Ann Clin Transl Neurol. 2025 Feb 06.
    ALS plasma biomarkers reveal neurofilament and pTau correlate with disease onset and progression.
    Eleanor V Thomas, Changee Han, Woo Jae Kim, Seneshaw Asress, Yingjie Li, Jennifer A Taylor, Marla Gearing, Christina N Fournier, Zachary T McEachin, Nicholas T Seyfried, Jonathan D Glass.
       OBJECTIVE: We performed a pilot screen to assess the utility of the NULISA™ (Nucleic-acid-Linked Immuno-Sandwich Assay) platform in the identification of amyotrophic lateral sclerosis (ALS) biomarkers.
    METHODS: Plasma from 86 individuals (48 ALS, 18 asymptomatic C9orf72 repeat expansion carriers (AsymC9), and 20 healthy controls) was analyzed via a multiplexed NULISA™ assay that includes 120 neurodegeneration-associated proteins. Statistical analysis of NULISA™ results was performed to identify proteins differentially expressed in plasma and their correlation with disease-associated parameters.
    RESULTS: ALS plasma showed elevation of the established biomarkers, neurofilament light chain (NEFL) and neurofilament heavy chain (NEFH). Compared to controls and AsymC9, microtubule-associated protein tau (MAPT), phosphorylated tau 181 (pTau181), phosphorylated tau 217 (pTau217), phosphorylated tau 231 (pTau231), and phosphorylated TDP-43 (pTDP-43) were elevated in ALS. NEFL levels positively correlated with pTau181, pTau217, pTau231, and pTDP-43. MAPT and pTDP-43 were also correlated with pTau181, pTau217 and pTau231. Elevated pTau was negatively correlated with survival and ALSFRS-R. Spinal onset ALS was associated with higher pTau181, pTau217, and pTau231.
    INTERPRETATION: We confirm previous reports showing elevated pTau181 in ALS plasma and show elevation of other phosphorylated tau forms, pTau217 and pTau231, typically observed in Alzheimer's disease. We provide preliminary data showing the detection and elevation of pTDP-43-409/410 in a subset of ALS samples compared to healthy controls. Neurofilament and tau levels are highly correlated suggesting their elevation may reflect a common pathology and disease state. Total and phosphorylated tau are correlated with multiple disease measures, such as ALS duration, ALSFRS-R, and site of onset.
    DOI:  https://doi.org/10.1002/acn3.70001
  12. Biochemistry. 2025 Feb 05.
    The Huntingtin Transport Complex.
    Emily N P Prowse, Brooke A Turkalj, Lale Gursu, Adam G Hendricks.
      A dynamic network of scaffolding molecules, adaptor proteins, and motor proteins work together to orchestrate the movement of proteins, mRNA, and vesicular cargoes. Defects in intracellular transport can often lead to neurodegeneration. Huntingtin (HTT) is a ubiquitously expressed scaffolding protein with a multitude of cellular roles, including regulating the transport of various organelles. HTT is remarkable in its ability to regulate the transport of a wide range of cargoes, including BDNF vesicles, APP vesicles, early endosomes, autophagosomes, lysosomes, and mitochondria. This interaction network allows huntingtin to control microtubule-based transport by kinesin and dynein, as well as actin-based transport by myosin VI. By forming complexes with multiple motor adaptors, huntingtin regulates a variety of cargoes and guides cargoes through the different stages of biosynthesis, signaling, and degradation. Accordingly, pathogenic polyglutamine expansions seen in Huntington's Disease (HD) dysregulate huntingtin transport complexes, resulting in defects in transport and neurodegeneration.
    Keywords:  HAP1; HAP40; dynein; huntingtin; kinesin; optineurin
    DOI:  https://doi.org/10.1021/acs.biochem.4c00811
  13. Proc Natl Acad Sci U S A. 2025 Feb 11. 122(6): e2422582122
    Mec1-mediated Atg9 phosphorylation regulates the PAS recruitment of Atg9 vesicles upon energy stress.
    Siyu Fan, Shuling Dong, Weijing Yao, Yi Zhang, Mingzhu Fan, Shan Feng, Choufei Wu, Liqin Zhang, Cong Yi.
      Mec1 plays an essential role in both the DNA damage response and glucose starvation-induced autophagy. We recently reported that Mec1 regulates glucose starvation-induced autophagy through its direct binding to Atg13. However, the role of Mec1's kinase activity in autophagy remains unclear. In this study, we demonstrate that the kinase activity of Mec1 is required for glucose starvation-induced autophagy by regulating the phagophore assembly site (PAS) recruitment of Atg9 vesicles. Mechanistic and functional analyses identified Atg9 as a direct phosphorylation substrate of Mec1, with phosphorylation occurring at the S35, T203, and T243 sites. Mutations at these sites reduce the association of Atg9 with Atg17, Atg23, and Atg27, thereby impairing the PAS recruitment of Atg9 vesicles. Notably, we found that the Mec1-Atg13 binding is a prerequisite for the phosphorylation of Atg9 by Mec1. Furthermore, Mec1-mediated phosphorylation of Atg9 is also crucial for the PAS recruitment of Atg9 vesicles in response to DNA damage. We thus propose that Mec1's kinase activity regulates the PAS recruitment of Atg9 vesicles by phosphorylating Atg9 in response to energy stress and DNA damage.
    Keywords:  Atg9; DNA damage–induced autophagy; Mec1; Saccharomyces cerevisiae; glucose starvation–induced autophagy
    DOI:  https://doi.org/10.1073/pnas.2422582122
  14. J Cell Sci. 2025 Feb 01. pii: JCS263526. [Epub ahead of print]138(3):
    Amyloid-β can activate JNK signalling via WNT5A-ROR2 to reduce synapse formation in Alzheimer's disease.
    Kevin Fang, Ehsan Pishva, Thomas Piers, Steffen Scholpp.
      Wnt signalling is an essential signalling system in neurogenesis, with a crucial role in synaptic plasticity and neuronal survival, processes that are disrupted in Alzheimer's disease (AD). Within this network, the Wnt/β-catenin pathway has been studied for its neuroprotective role, and this is suppressed in AD. However, the involvement of the non-canonical Wnt-planar cell polarity (Wnt/PCP) pathway in AD remains to be determined. This study investigates the role of ROR2, a Wnt/PCP co-receptor, in synaptogenesis. We demonstrate that WNT5A-ROR2 signalling activates the JNK pathway, leading to synapse loss in mature neurons. This effect mirrors the synaptotoxic actions of Aβ1-42 and DKK1, which are elevated in AD. Notably, blocking ROR2 and JNK mitigates Aβ1-42 and DKK1-induced synapse loss, suggesting their dependence on ROR2. In induced pluripotent stem cell (iPSC)-derived cortical neurons carrying a PSEN1 mutation, known to increase the Aβ42/40 ratio, we observed increased WNT5A-ROR2 clustering and reduced numbers of synapses. Inhibiting ROR2 or JNK partially rescued synaptogenesis in these neurons. These findings suggest that, unlike the Wnt/β-catenin pathway, the Wnt/PCP-ROR2 signalling pathway can operate in a feedback loop with Aβ1-42 to enhance JNK signalling and contribute to synapse loss in AD.
    Keywords:  Alzheimer's disease; JNK signalling; KOLF2.1J; Presenilin1; Synaptogenesis; Wnt/PCP
    DOI:  https://doi.org/10.1242/jcs.263526
  15. bioRxiv. 2025 Jan 24. pii: 2025.01.23.634631. [Epub ahead of print]
    Loss of Fanconi anemia proteins causes a reliance on lysosomal exocytosis.
    Becky Xu Hua Fu, Albert Xu, Hua Li, Daniel E Johnson, Jennifer R Grandis, Luke A Gilbert.
      Mutations in the FA pathway lead to a rare genetic disease that increases risk of bone marrow failure, acute myeloid leukemia, and solid tumors. FA patients have a 500 to 800-fold increase in head and neck squamous cell carcinoma compared to the general population and the treatment for these malignancies are ineffective and limited due to the deficiency in DNA damage repair. Using unbiased CRISPR-interference screening, we found the loss of FA function renders cells dependent on key exocytosis genes such as SNAP23. Further investigation revealed that loss of FA pathway function induced deficiencies in lysosomal health, dysregulation of autophagy and increased lysosomal exocytosis. The compromised cellular state caused by the loss of FA genes is accompanied with decreased lysosome abundance and increased lysosomal membrane permeabilization in cells. We found these signatures in vitro across multiple cell types and cell lines and in clinically relevant FA patient cancers. Our findings are the first to connect the FA pathway to lysosomal exocytosis and thus expands our understanding of FA as a disease and of induced dependencies in FA mutant cancers.
    DOI:  https://doi.org/10.1101/2025.01.23.634631
  16. J Cell Biol. 2025 Apr 03. pii: e202405002. [Epub ahead of print]224(4):
    VPS41 recruits biosynthetic LAMP-positive vesicles through interaction with Arl8b.
    Paolo Sanzà, Jan van der Beek, Derk Draper, Cecilia de Heus, Tineke Veenendaal, Corlinda Ten Brink, Ginny G Farías, Nalan Liv, Judith Klumperman.
      Vacuolar protein sorting 41 (VPS41), a component of the homotypic fusion and protein sorting (HOPS) complex for lysosomal fusion, is essential for the trafficking of lysosomal membrane proteins via lysosome-associated membrane protein (LAMP) carriers from the trans-Golgi network (TGN) to endo/lysosomes. However, the molecular mechanisms underlying this pathway and VPS41's role herein remain poorly understood. Here, we investigated the effects of ectopically localizing VPS41 to mitochondria on LAMP distribution. Using electron microscopy, we identified that mitochondrial-localized VPS41 recruited LAMP1- and LAMP2A-positive vesicles resembling LAMP carriers. The retention using selective hooks (RUSH) system further revealed that newly synthesized LAMPs were specifically recruited by mitochondrial VPS41, a function not shared by other HOPS subunits. Notably, we identified the small GTPase Arl8b as a critical factor for LAMP carrier trafficking. Arl8b was present on LAMP carriers and bound to the WD40 domain of VPS41, enabling their recruitment. These findings reveal a unique role of VPS41 in recruiting TGN-derived LAMP carriers and expand our understanding of VPS41-Arl8b interactions beyond endosome-lysosome fusion, providing new insights into lysosomal trafficking mechanisms.
    DOI:  https://doi.org/10.1083/jcb.202405002
  17. Nat Commun. 2025 Feb 04. 16(1): 1343
    The autophagy component LC3 regulates lymphocyte adhesion via LFA1 transport in response to outside-in signaling.
    Naoyuki Kondo, Yuko Mimori-Kiyosue, Keizo Tokuhiro, Giuseppe Pezzotti, Tatsuo Kinashi.
      The leukocyte integrin LFA1 is indispensable for immune responses, orchestrating lymphocyte trafficking and adhesion. While LFA1 activation induces LFA1 clustering at the cell contact surface via outside-in signaling, the regulatory mechanisms remain unclear. Here, we uncovered a previously hidden function of the autophagosome component LC3 beyond its role in autophagy by bridging two seemingly unrelated pathways: LFA1 transport and autophagosome transport. LFA1 clusters co-trafficked with LC3, facilitating LFA1 accumulation at the contact surface. LC3b knockout decreased lymphocyte adhesiveness. LFA1 activation did not induce autophagy, whereas it increased mTOR and AMPK activity. LFA1-dependent AMPK activation enhances LFA1 and LC3 clustering and adhesion. Inhibiting Mst1 kinase-mediated LC3 phosphorylation promoted LC3-mediated LFA1 recruitment to the contact surface through direct interaction with RAPL, uncovering an unprecedented integrin recruitment route. These findings uncover a function of LC3 and expand our understanding of lymphocyte regulation via LFA1.
    DOI:  https://doi.org/10.1038/s41467-025-56631-1
  18. Cell Stem Cell. 2025 Feb 06. pii: S1934-5909(25)00004-9. [Epub ahead of print]32(2): 173-175
    Cognitive impairment in epilepsy progression: Adult neurogenesis loss at critical window.
    Yi Wang, Zhong Chen.
      The mechanisms underlying cognitive impairment in epilepsy remain poorly understood. Ammothumkandy et al.1 reveal that the loss of adult immature neurons correlates with auditory verbal learning deficits in human mesial temporal lobe epilepsy during a critical disease period, underscoring the pivotal role of adult neurogenesis in specific cognitive domain.
    DOI:  https://doi.org/10.1016/j.stem.2025.01.004
  19. bioRxiv. 2025 Jan 20. pii: 2025.01.17.633617. [Epub ahead of print]
    Hsp70 chaperones, Ssa1 and Ssa2, limit poly(A) binding protein aggregation.
    Hannah E Buchholz, Sean A Martin, Jane E Dorweiler, Claire M Radtke, Adam S Knier, Natalia B Beans, Anita L Manogaran.
      Molecular chaperones play a central role in maintaining protein homeostasis. The highly conserved Hsp70 family of chaperones have major functions in folding of nascent peptides, protein refolding, and protein aggregate disassembly. In yeast, loss of two Hsp70 proteins, Ssa1 and Ssa2, is associated with decreased cellular growth and shortened lifespan. While heterologous or mutant temperature sensitive proteins form anomalous large cytoplasmic inclusions in ssa1Δssa2Δ strains, it is unclear how endogenous wildtype proteins behave and are regulated in the presence of limiting Hsp70s. Using the wildtype yeast Poly A binding protein (Pab1), which is involved in mRNA binding and forms stress granules (SGs) upon heat shock, Pab1 forms large inclusions in approximately half of ssa1Δssa2Δ cells in the absence of stress. Overexpression of Ssa1, Hsp104, and Sis1 almost completely limits the formation of these large inclusions in ssa1Δssa2Δ , suggesting that excess Ssa1, Hsp104 and Sis1 can each compensate for the lower levels of Ssa proteins. Upon heat shock, SGs also form in cells whether large Pab1 inclusions are present or not. Surprisingly, cells containing only SGs disassemble faster than wildtype, whereas cells with both large inclusions disassemble slower albeit completely. We suspect that disassembly of these large inclusions is linked to the elevated heat shock response and elevated Hsp104 and Sis1 levels in ssa1Δssa2Δ strains. We also observed that wildtype cultures grown to saturation also form large Pab1-GFP inclusions. These inclusions can be partially rescued by overexpression of Ssa1. Taken together, our data suggests that Hsp70 not only plays a role in limiting unwanted protein aggregation in normal cells, but as cells age, the depletion of active Hsp70 possibly underlies the age-related aggregation of endogenous proteins.
    DOI:  https://doi.org/10.1101/2025.01.17.633617
  20. Aging Dis. 2025 Jan 16.
    Oxidative Stress Promotes Axonal Atrophy through Alterations in Microtubules and EB1 Function.
    Samuel Shields, Emilia Gregory, Oliver Wilkes, IIlana Gozes, Natalia Sanchez-Soriano.
      Axons are crucial for transmitting neurochemical signals. As organisms age, the ability of neurons to maintain their axons declines; hence, aged axons are more susceptible to damage or dysfunction. Understanding how aging causes axonal vulnerability is crucial for developing strategies to enhance overall resilience of neurons and prevent neuronal deterioration during aging and in age-related neurodegenerative diseases. Increasing levels of reactive oxygen species (ROS) causes oxidative stress - a hallmark of aging and age-related diseases. Despite this association, a causal relationship between oxidative stress and neuronal aging remains unclear, particularly in how subcellular physiology may be affected by ROS. By using Drosophila-derived primary neuronal cultures and a recently developed in vivo neuronal model of aging, which involves the visualisation of Drosophila medulla neurons, we investigated the interplay between oxidative stress, neuronal aging and the microtubule cytoskeleton. Our results showed that oxidative stress is a key driver of axonal and synaptic decay, as shown by an enhanced appearance of axonal swellings, microtubule alterations (in both axons and synapses) and morphological transformation of axonal terminals during aging. We demonstrated that increasing the levels of ROS sensitises microtubule plus end-binding protein 1 (EB1), leading to microtubule defects that effect neuronal integrity. Furthermore, manipulating EB1 proved to be a valuable therapeutic strategy to prevent aging hallmarks enhanced in conditions of elevated ROS. In summary, we demonstrate a mechanistic pathway linking cellular oxidative stress with changes in the microtubule cytoskeleton leading to axonal deterioration during aging and provide evidence of the therapeutic potential of enhancing microtubule plus-end physiology to improve the resilience of axons.
    DOI:  https://doi.org/10.14336/AD.2024.0839
  21. Brain. 2025 Feb 06. pii: awaf051. [Epub ahead of print]
    Parkinson's disease mutant Miro1 causes mitochondrial dysfunction and dopaminergic neuron loss.
    Axel Chemla, Giuseppe Arena, Ginevra Sacripanti, Kyriaki Barmpa, Alise Zagare, Pierre Garcia, Vyron Gorgogietas, Paul Antony, Jochen Ohnmacht, Alexandre Baron, Jaqueline Jung, Frida Lind-Holm Mogensen, Alessandro Michelucci, Anne-Marie Marzesco, Manuel Buttini, Thorsten Schmidt, Anne Grünewald, Jens C Schwamborn, Rejko Krüger, Cláudia Saraiva.
      The complex and heterogeneous nature of Parkinson's disease (PD) is still not fully understood, however, increasing evidence supports mitochondrial impairment as a major driver of neurodegeneration. Miro1, a mitochondrial GTPase encoded by the RHOT1 gene, is involved in mitochondrial transport, mitophagy and mitochondrial calcium buffering, and is therefore essential for maintaining mitochondrial homeostasis. Recently, Miro1 has been linked genetically and pathophysiologically to PD, further supported by the identification of heterozygous variants of Miro1 in patients. Herein, we used patient-derived cellular models alongside knock-in mice to investigate Miro1-dependent pathophysiological processes and molecular mechanisms underlying neurodegeneration in PD. Experimental work performed in induced pluripotent stem cells (iPSC)-derived models, including midbrain organoids and dopaminergic neuronal cell cultures from a PD patient carrying the p.R272Q Miro1 mutation as well as healthy and isogenic controls, indicated that the p.R272Q Miro1 mutation leads to increased oxidative stress, disrupted mitochondrial bioenergetics and altered cellular metabolism. This was accompanied by increased α-synuclein levels and a significant reduction of dopaminergic neurons. Moreover, the p.R272Q Miro1 mutation - located in the calcium-binding domain of the GTPase - disrupted calcium homeostasis. This resulted in the calcium-dependent activation of calpain proteases and the subsequent cleavage of α-synuclein. Knock-in mice expressing p.R285Q Miro1 (the orthologue of the human p.R272Q mutation) displayed accumulation of phosphorylated α-synuclein in the striatum and a significant loss of dopaminergic neurons in the substantia nigra, accompanied by behavioral alterations. These findings demonstrate that mutant Miro1 is sufficient to comprehensively model PD-relevant phenotypes in vitro and in vivo, reinforcing its pivotal role in PD pathogenesis.
    Keywords:  calcium homeostasis; knock-in mice; neurodegeneration; p.R272Q Miro1; patient-specific iPSC-derived models; α-synuclein
    DOI:  https://doi.org/10.1093/brain/awaf051
  22. Eur J Med Chem. 2025 Jan 28. pii: S0223-5234(25)00089-3. [Epub ahead of print]287 117324
    Recent advances in small molecule inhibitors of deubiquitinating enzymes.
    Pengwei Liu, Zhengyang Chen, Yiting Guo, Qiaojun He, Chenghao Pan.
      Proteins play a pivotal role in maintaining cellular homeostasis. Their degradation primarily orchestrated through the ubiquitin-proteasome system (UPS) and cellular autophagy. Dysfunction of the UPS is associated with various human diseases, including cancer, autoimmune disorders, and neurodegenerative conditions. Consequently, the UPS has emerged as a promising therapeutic target. Deubiquitinases (DUBs) have garnered significant attention as potential targets for therapeutic intervention due to their role in modulating protein stability and function. This review focuses on recent advancements of DUBs, particularly their relevance in the UPS and their potential as drug targets. Notably, inhibitors targeting specific DUBs, such as USP1, USP7, USP14, and USP30 have shown promise in preclinical and clinical studies for cancer therapy. Additionally, DUB inhibitors have been involved in novel therapeutic approaches lately, including as targets for proteolysis-targeting chimeras (PROTACs) or as tools in deubiquitinase-targeting chimeras (DUBTACs).
    Keywords:  Deubiquitinating enzymes; Drug development; Protein homeostasis; Small-molecule inhibitors
    DOI:  https://doi.org/10.1016/j.ejmech.2025.117324
  23. EMBO J. 2025 Feb 05.
    Coordination between ESCRT function and Rab conversion during endosome maturation.
    Daniel P Ott, Samit Desai, Jachen A Solinger, Andres Kaech, Anne Spang.
      The endosomal pathway is essential for regulating cell signaling and cellular homeostasis. Rab5 positive early endosomes receive proteins from the plasma membrane. Dependent on a ubiquitin mark on the protein, they will be either recycled or sorted into intraluminal vesicles (ILVs) by endosomal sorting complex required for transport (ESCRT) proteins. During endosome maturation Rab5 is replaced by Rab7 on endosomes that are able to fuse with lysosomes to form endolysosomes. However, whether ESCRT-driven ILV formation and Rab5-to-Rab7 conversion are coordinated remains unknown. Here we show that loss of early ESCRTs led to enlarged Rab5 positive endosomes and prohibited Rab conversion. Reduction of ubiquitinated cargo alleviated this phenotype. Moreover, ubiquitinated proteins on the endosomal limiting membrane prevented the displacement of the Rab5 guanine nucleotide exchange factor (GEF) RABX-5 by the GEF for Rab7, SAND-1/CCZ-1. Overexpression of Rab7 could partially overcome this block, even in the absence of SAND-1 or CCZ1, suggesting the presence of a second Rab7 GEF. Our data reveal a hierarchy of events in which cargo corralling by ESCRTs is upstream of Rab conversion, suggesting that ESCRT-0 and ubiquitinated cargo could act as timers that determine the onset of Rab conversion.
    Keywords:   C. elegans ; Endosomes; Live Cell Imaging; Mammalian Cells; Rab GTPases
    DOI:  https://doi.org/10.1038/s44318-025-00367-7
  24. Biol Open. 2025 Feb 06. pii: bio.061605. [Epub ahead of print]
    Glycogen synthase is required for heat shock-mediated autophagy induction in neuronal cells.
    Pratibha Bhadauriya, Akanksha Onkar, Kamali Nagarajan, Kavikumar Angamuthu Karuppusamy, Subramaniam Ganesh, Saloni Agarwal.
      Autophagy is an essential cellular process that facilitates the degradation of aggregated proteins and damaged organelles to maintain cellular homeostasis and promote cell survival. Recent studies have indicated a direct role for glycogen synthase (GS) in activating neuronal autophagy and in conferring protection against cytotoxic misfolded proteins. Since heat shock induces protein misfolding and autophagy is an essential component of the heat shock response that clears the misfolded proteins, we looked at the possible role of GS in heat shock response pathways in neuronal cells. We demonstrate an increase in the activity and level of GS and a concomitant increase in the glycogen level during the heat shock and post-heat shock recovery period. These changes had a direct correlation with autophagy induction. We further demonstrate that heat shock transcription factor 1 regulates the level and activation of GS during heat shock and that GS is essential for the induction of autophagy during heat stress in neuronal cells. Intriguingly, the partial knock-down of GS led to increased death due to heat shock in neuronal cells and Drosophila. Our study offers a novel insight into the role of GS and glycogen metabolic pathways in heat shock response in neuronal cells.
    Keywords:  Metabolism; Misfolded proteins; Neuronal cells; Proteolysis; Stress response
    DOI:  https://doi.org/10.1242/bio.061605
  25. Proc Natl Acad Sci U S A. 2025 Feb 11. 122(6): e2322412122
    High-throughput discovery of inhibitory protein fragments with AlphaFold.
    Andrew Savinov, Sebastian Swanson, Amy E Keating, Gene-Wei Li.
      Peptides can bind to specific sites on larger proteins and thereby function as inhibitors and regulatory elements. Peptide fragments of larger proteins are particularly attractive for achieving these functions due to their inherent potential to form native-like binding interactions. Recently developed experimental approaches allow for high-throughput measurement of protein fragment inhibitory activity in living cells. However, it has thus far not been possible to predict de novo which of the many possible protein fragments bind to protein targets, let alone act as inhibitors. We have developed a computational method, FragFold, that employs AlphaFold to predict protein fragment binding to full-length proteins in a high-throughput manner. Applying FragFold to thousands of fragments tiling across diverse proteins revealed peaks of predicted binding along each protein sequence. Comparisons with experimental measurements establish that our approach is a sensitive predictor of fragment function: Evaluating inhibitory fragments from known protein-protein interaction interfaces, we find 87% are predicted by FragFold to bind in a native-like mode. Across full protein sequences, 68% of FragFold-predicted binding peaks match experimentally measured inhibitory peaks. Deep mutational scanning experiments support the predicted binding modes and uncover superior inhibitory peptides in high throughput. Further, FragFold is able to predict previously unknown protein binding modes, explaining prior genetic and biochemical data. The success rate of FragFold demonstrates that this computational approach should be broadly applicable for discovering inhibitory protein fragments across proteomes.
    Keywords:  FragFold; deep mutational scanning; inhibitory peptides; massively parallel; protein interactions
    DOI:  https://doi.org/10.1073/pnas.2322412122
  26. Nat Commun. 2025 Feb 07. 16(1): 1438
    Aspirin inhibits proteasomal degradation and promotes α-synuclein aggregate clearance through K63 ubiquitination.
    Jing Gao, Yang Liu, Chenfang Si, Rui Guo, Shouqiao Hou, Xiaosong Liu, Houfang Long, Di Liu, Daichao Xu, Zai-Rong Zhang, Cong Liu, Bing Shan, Christoph W Turck, Kaiwen He, Yaoyang Zhang.
      Aspirin is a potent lysine acetylation inducer, but its impact on lysine ubiquitination and ubiquitination-directed protein degradation is unclear. Herein, we develop the reversed-pulsed-SILAC strategy to systematically profile protein degradome in response to aspirin. By integrating degradome, acetylome, and ubiquitinome analyses, we show that aspirin impairs proteasome activity to inhibit proteasomal degradation, rather than directly suppressing lysine ubiquitination. Interestingly, aspirin increases lysosomal degradation-implicated K63-linked ubiquitination. Accordingly, using the major pathological protein of Parkinson's disease (PD), α-synuclein (α-syn), as an example of protein aggregates, we find that aspirin is able to reduce α-syn in cultured cells, neurons, and PD model mice with rescued locomotor ability. We further reveal that the α-syn aggregate clearance induced by aspirin is K63-ubiquitination dependent in both cells and PD mice. These findings suggest two complementary mechanisms by which aspirin regulates the degradation of soluble and insoluble proteins, providing insights into its diverse pharmacological effects that can aid in future drug development efforts.
    DOI:  https://doi.org/10.1038/s41467-025-56737-6
  27. J Biol Chem. 2025 Feb 02. pii: S0021-9258(25)00101-2. [Epub ahead of print] 108254
    Harmine promotes axon regeneration through enhancing glucose metabolism.
    Ruixuan Liu, Bing Zhou.
      Axon regeneration requires a substantial mitochondrial energy supply. However, injured mature neurons often fail to regenerate due to their inability to meet these elevated energy demands. Our findings indicate that harmine compensates for the energy deficit following axonal injury by enhancing the coupling between glucose metabolism and mitochondrial homeostasis, thereby promoting axon regeneration. Notably, harmine facilitates mitochondrial biogenesis and enhances mitophagy, ensuring efficient mitochondrial turnover and energy supply. Thus, harmine plays a crucial role in enhancing glucose metabolism to maintain mitochondrial function, demonstrating significant potential in treating mature neuronal axon injuries and sciatic nerve injuries.
    Keywords:  axon regeneration; energy supply; glucose metabolism; harmine; metabolic coupling; mitochondrial function; neuron
    DOI:  https://doi.org/10.1016/j.jbc.2025.108254
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