bims-axbals 2025-04-06 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2025–04–06
25 papers selected by
TJ Krzystek

Amyotrophic Lateral Sclerosis: Focus on Cytoplasmic Trafficking and Proteostasis.
A guide to selecting high-performing antibodies for TAF15 (UniProt ID: Q92804) for use in western blot, immunoprecipitation, and immunofluorescence.
TDP-43 seeding induces cytoplasmic aggregation heterogeneity and nuclear loss of function of TDP-43.
Klotho overexpression protects human cortical neurons from β-amyloid induced neuronal toxicity.
Seeded aggregation of TDP-43 induces its loss of function and reveals early pathological signatures.
Restoration of Miro1's N-terminal GTPase function alleviates prenatal stress-induced mitochondrial fission via Drp1 modulation.
PPM1M, a LRRK2-counteracting, phosphoRab12-preferring phosphatase with potential link to Parkinson's disease.
Defective anterograde protein-trafficking contributes to endoplasmic reticulum-stress in a CLN1 disease model.
LRRK2 Mediates α-Synuclein-Induced Neuroinflammation and Ferroptosis through the p62-Keap1-Nrf2 Pathway in Parkinson's Disease.
Zebrafish TDP43 positively regulates p65-mediated apoptotic pathway.
Generation of an induced pluripotent stem cell (iPSC) line (INNDSUi009-A) from a patient with amyotrophic lateral sclerosis due to SOD1 mutation.
A non-fluorescent immunohistochemistry method for measuring autophagy flux using MAP1LC3/LC3 and SQSTM1 as core markers.
The Endo-GeneScreen Platform Identifies Drug-Like Probes that Regulate Endogenous Protein Levels within Physiological Contexts.
A human iPSC-derived midbrain neural stem cell model of prenatal opioid exposure and withdrawal: A proof of concept study.
The interneuron hypothesis of amyotrophic lateral sclerosis.
Multi-omics study reveals differential expression and phosphorylation of autophagy-related proteins in autism spectrum disorder.
Retromer promotes the lysosomal turnover of mtDNA.
Stathmin 1 regulates mitophagy and cellular function in hematopoietic stem cells.
Inhibiting acute, axonal DLK palmitoylation is neuroprotective and avoids deleterious effects of cell-wide DLK inhibition.
The life and times of brain autophagic vesicles.
PDZD8 promotes autophagy at ER-lysosome membrane contact sites to regulate activity-dependent synaptic growth.
Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9.
RAF1 kinase contributes to autophagic lysosome reformation.
Seizure and redox rescue in a model of glucose transport deficiency.
Three's company: Membrane waltz among organelles.

Mol Neurobiol. 2025 Apr 03.

Amyotrophic Lateral Sclerosis: Focus on Cytoplasmic Trafficking and Proteostasis.

Shrilaxmi Ms, Saradindu Banerjee, Santosh R D'Mello, Somasish Ghosh Dastidar.

  Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease characterized by the pathological loss of upper and lower motor neurons. Whereas most ALS cases are caused by a combination of environmental factors and genetic susceptibility, in a relatively small proportion of cases, the disorder results from mutations in genes that are inherited. Defects in several different cellular mechanisms and processes contribute to the selective loss of motor neurons (MNs) in ALS. Prominent among these is the accumulation of aggregates of misfolded proteins or peptides which are toxic to motor neurons. These accumulating aggregates stress the ability of the endoplasmic reticulum (ER) to function normally, cause defects in the transport of proteins between the ER and Golgi, and impair the transport of RNA, proteins, and organelles, such as mitochondria, within axons and dendrites, all of which contribute to the degeneration of MNs. Although dysfunction of a variety of cellular processes combines towards the pathogenesis of ALS, in this review, we focus on recent advances concerning the involvement of defective ER stress, vesicular transport between the ER and Golgi, and axonal transport.

Keywords:  Amyotrophic lateral sclerosis; Axonal transport; Endoplasmic reticulum stress; Neurodegenerative diseases; Unfolded protein response; Vesicular transport

DOI:  https://doi.org/10.1007/s12035-025-04831-7
F1000Res. 2025 ;14 37

A guide to selecting high-performing antibodies for TAF15 (UniProt ID: Q92804) for use in western blot, immunoprecipitation, and immunofluorescence.

NeuroSGC/YCharOS/EDDU collaborative group

  TAF15 (TATA-box binding protein-associated factor 15) is a member of the FET protein family, known for their roles in transcriptional regulation and RNA metabolism. Here we have characterized five TAF15 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:  Q92804; TAF15; TATA-binding protein-associated factor 2N; TATA-box binding protein associated factor 15; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation; western blot

DOI:  https://doi.org/10.12688/f1000research.160371.1
Neuron. 2025 Mar 26. pii: S0896-6273(25)00176-X. [Epub ahead of print]

TDP-43 seeding induces cytoplasmic aggregation heterogeneity and nuclear loss of function of TDP-43.

Jens Rummens, Bilal Khalil, Günseli Yıldırım, Pedro Silva, Valentina Zorzini, Nicolas Peredo, Marta Wojno, Meine Ramakers, Ludo Van Den Bosch, Philip Van Damme, Kristofer Davie, Jelle Hendrix, Frederic Rousseau, Joost Schymkowitz, Sandrine Da Cruz.

  Cytoplasmic aggregation and nuclear depletion of TAR DNA-binding protein 43 (TDP-43) are hallmarks of several neurodegenerative disorders. Yet, recapitulating both features in cellular systems has been challenging. Here, we produced amyloid-like fibrils from recombinant TDP-43 low-complexity domain and demonstrate that sonicated fibrils trigger TDP-43 pathology in human cells, including induced pluripotent stem cell (iPSC)-derived neurons. Fibril-induced cytoplasmic TDP-43 inclusions acquire distinct biophysical properties, recapitulate pathological hallmarks such as phosphorylation, ubiquitin, and p62 accumulation, and recruit nuclear endogenous TDP-43, leading to its loss of function. A transcriptomic signature linked to both aggregation and nuclear loss of TDP-43, including disease-specific cryptic splicing, is identified. Cytoplasmic TDP-43 aggregates exhibit time-dependent heterogeneous morphologies as observed in patients-including compacted, filamentous, or fragmented-which involve upregulation/recruitment of protein clearance pathways. Ultimately, cell-specific progressive toxicity is provoked by seeded TDP-43 pathology in human neurons. These findings identify TDP-43-templated aggregation as a key mechanism driving both cytoplasmic gain of function and nuclear loss of function, offering a valuable approach to identify modifiers of sporadic TDP-43 proteinopathies.

Keywords:  ALS; FTD; LLPS; RNA metabolism; TDP-43; pathology; prion-like seeding; protein aggregation

DOI:  https://doi.org/10.1016/j.neuron.2025.03.004
Mol Brain. 2025 Mar 28. 18(1): 27

Klotho overexpression protects human cortical neurons from β-amyloid induced neuronal toxicity.

Mohammed R Shaker, Salam Salloum-Asfar, Rowaida Z Taha, Ibrahim Javed, Ernst J Wolvetang.

  Klotho, a well-known aging suppressor protein, has been implicated in neuroprotection and the regulation of neuronal senescence. While previous studies have demonstrated its anti-aging properties in human brain organoids, its potential to mitigate neurodegenerative processes triggered by β-amyloid remains underexplored. In this study, we utilised human induced pluripotent stem cells (iPSCs) engineered with a doxycycline-inducible system to overexpress KLOTHO and generated 2D cortical neuron cultures from these cells. These neurons were next exposed to pre-aggregated β-amyloid 1-42 oligomers to model the neurotoxicity associated with Alzheimer's disease. Our data reveal that upregulation of KLOTHO significantly reduced β-amyloid-induced neuronal degeneration and apoptosis, as evidenced by decreased cleaved caspase-3 expression and preservation of axonal integrity. Additionally, KLOTHO overexpression prevented the loss of dendritic branching and mitigated reductions in axonal diameter, hallmark features of neurodegenerative pathology. These results highlight Klotho's protective role against β-amyloid-induced neurotoxicity in human cortical neurons and suggest that its age-related decline may contribute to neurodegenerative diseases such as Alzheimer's disease. Our findings underscore the therapeutic potential of Klotho-based interventions in mitigating age-associated neurodegenerative processes.

Keywords:  Alzheimer’s disease; Cortical neurons; Klotho; Neurodegeneration; Neuroprotection; iPSCs; β-amyloid

DOI:  https://doi.org/10.1186/s13041-025-01199-6
Neuron. 2025 Mar 26. pii: S0896-6273(25)00180-1. [Epub ahead of print]

Seeded aggregation of TDP-43 induces its loss of function and reveals early pathological signatures.

Carlo Scialò, Weijia Zhong, Somanath Jagannath, Oscar Wilkins, Davide Caredio, Marian Hruska-Plochan, Flavio Lurati, Martina Peter, Elena De Cecco, Luigi Celauro, Adriano Aguzzi, Giuseppe Legname, Pietro Fratta, Magdalini Polymenidou.

  Neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) results from both gain of toxicity and loss of normal function of the RNA-binding protein TDP-43, but their mechanistic connection remains unclear. Increasing evidence suggests that TDP-43 aggregates act as self-templating seeds, propagating pathology through the central nervous system via a prion-like cascade. We developed a robust TDP-43-seeding platform for quantitative assessment of TDP-43 aggregate uptake, cell-to-cell spreading, and loss of function within living cells, while they progress toward pathology. We show that both patient-derived and recombinant TDP-43 pathological aggregates were abundantly internalized by human neuron-like cells, efficiently recruited endogenous TDP-43, and formed cytoplasmic inclusions reminiscent of ALS/FTD pathology. Combining a fluorescent reporter of TDP-43 function with RNA sequencing and proteomics, we demonstrated aberrant cryptic splicing and a loss-of-function profile resulting from TDP-43-templated aggregation. Our data highlight known and novel pathological signatures in the context of seed-induced TDP-43 loss of function.

Keywords:  ALS/FTD; RNA-binding proteins; TDP-43; aggregation; cryptic splicing; loss of function; low-complexity domain; neurodegeneration; seeding; spreading

DOI:  https://doi.org/10.1016/j.neuron.2025.03.008
Cell Commun Signal. 2025 Apr 02. 23(1): 166

Restoration of Miro1's N-terminal GTPase function alleviates prenatal stress-induced mitochondrial fission via Drp1 modulation.

Gee Euhn Choi, Ji Yong Park, Mo Ran Park, Chang Woo Chae, Young Hyun Jung, Jae Ryong Lim, Jee Hyeon Yoon, Ji Hyeon Cho, Ho Jae Han.

   BACKGROUND: Prenatal stress exposure irreversibly impairs mitochondrial dynamics, including mitochondrial trafficking and morphology in offspring, leading to neurodevelopmental and neuropsychiatric disorders in adulthood. Thus, understanding the molecular mechanism controlling mitochondrial dynamics in differentiating neurons is crucial to prevent the prenatal stress-induced impairments in behavior. We investigated the interplay between mitochondrial transport and fusion/fission in differentiating neurons exposed to prenatal stress, leading to ensuing behavior impairments, and then tried to identify the primary regulator that modulates both phenomena.
METHODS: We used primary hippocampal neurons of mice exposed to prenatal stress and human induced-pluripotent stem cell (hiPSC)-derived neurons, for investigating the impact of glucocorticoid on mitochondrial dynamics during differentiation. For constructing mouse models, we used AAV vectors into mouse pups exposed to prenatal stress to regulate protein expressions in hippocampal regions.
RESULTS: We first observed that prenatal exposure to glucocorticoids induced motility arrest and fragmentation of mitochondria in differentiating neurons derived from mouse fetuses (E18) and human induced pluripotent stem cells (hiPSCs). Further, glucocorticoid exposure during neurogenesis selectively downregulated Miro1 and increased Drp1 phosphorylation (Ser616). MIRO1 overexpression restored mitochondrial motility and increased intramitochondrial calcium influx through ER-mitochondria contact (ERMC) formation. Furthermore, we determined that the N-terminal GTPase domain of Miro1 plays a critical role in ERMC formation, which then decreased Drp1 phosphorylation (Ser616). Similarly, prenatal corticosterone exposure led to impaired neuropsychiatric and cognitive function in the offspring by affecting mitochondrial distribution and synaptogenesis, rescued by Miro1WT, but not N-terminal GTPase active form Miro1P26V, expression.
CONCLUSION: Prenatal glucocorticoid-mediated Miro1 downregulation contributes to dysfunction in mitochondrial dynamics through Drp1 phosphorylation (Ser616) in differentiating neurons.

Keywords:  ER-mitochondria contacts; Miro; Mitochondrial dynamics; Neurodegeneration; Prenatal glucocorticoid

DOI:  https://doi.org/10.1186/s12964-025-02172-5
bioRxiv. 2025 Mar 19. pii: 2025.03.19.644182. [Epub ahead of print]

PPM1M, a LRRK2-counteracting, phosphoRab12-preferring phosphatase with potential link to Parkinson's disease.

Claire Y Chiang, Neringa Pratuseviciute, Yu-En Lin, Ayan Adhikari, Wondwossen M Yeshaw, Chloe Flitton, Pemba L Sherpa, Francesca Tonelli, Irena Rektorova, Timothy Lynch, Joanna Siuda, Monika Rudzińska-Bar, Oleksandr Pulyk, Peter Bauer, Christian Beetz, Dennis W Dickson, Owen A Ross, Zbigniew K Wszolek, Christine Klein, Alexander Zimprich, Dario R Alessi, Esther M Sammler, Suzanne R Pfeffer.

Leucine-rich repeat kinase 2 (LRRK2) phosphorylates a subset of Rab GTPases that regulate receptor trafficking; activating mutations in LRRK2 are linked to Parkinson's disease. Rab phosphorylation is a transient event that can be reversed by phosphatases, including PPM1H, that acts on phosphoRab8A and phosphoRab10. Here we report a phosphatome-wide siRNA screen that identified PPM1M as a phosphoRab12-preferring phosphatase that also acts on phosphoRab8A and phosphoRab10. Upon knockout from cells or mice, PPM1M displays selectivity for phosphoRab12. As shown previously for mice harboring LRRK2 pathway mutations, knockout of Ppm1m leads to primary cilia loss in striatal cholinergic interneurons. We have also identified a rare PPM1M mutation in patients with Parkinson's disease that is catalytically inactive when tested in vitro and in cells. These findings identify PPM1M as a key player in the LRRK2 signaling pathway and provide a new therapeutic target for the possible benefit of patients with Parkinson's disease.
Teaser: Parkinson's linked Rab phosphorylation is reversed by PPM1M; the inactive D440N variant is implicated in rare patient cases.

DOI: https://doi.org/10.1101/2025.03.19.644182
Neurobiol Dis. 2025 Mar 28. pii: S0969-9961(25)00106-8. [Epub ahead of print] 106890

Defective anterograde protein-trafficking contributes to endoplasmic reticulum-stress in a CLN1 disease model.

Nisha Plavelil, Abhilash P Appu, K C Gopal, Avisek Mondal, Neil Perkins, Anil B Mukherjee.

  Lysosomal storage disorders (LSDs) represent 70 inherited metabolic diseases, in most of which neurodegeneration is a devastating manifestation. The CLN1 disease is a fatal neurodegenerative LSD, caused by inactivating mutations in the CLN1 gene encoding palmitoyl-protein thioesterase-1 (PPT1). S-palmitoylation, a reversable posttranslational modification by saturated fatty acids (generally palmitate) facilitates endosomal trafficking of many proteins, especially in the brain. While palmitoyl-acyltransferases (called ZDHHCs) catalyze S-palmitoylation, depalmitoylation is mediated by palmitoyl-protein thioesterases (PPTs). We previously reported that in Cln1-/- mice, which mimic human CLN1-disease, endoplasmic reticulum (ER)-stress leads to unfolded protein response (UPR) contributing to neurodegeneration. However, the mechanism underlying ER-stress has remained elusive. The anterograde (ER to Golgi) protein-trafficking is mediated via COPII (coat protein complex II) vesicles, whereas the retrograde transport (Golgi to ER) is mediated by COPI vesicles. We hypothesized that dysregulated anterograde protein-trafficking causing stagnation of proteins in the ER leads to ER-stress in Cln1-/- mice. We found that the levels of five COPII vesicle-associated proteins (i.e. Sar1, Sec23, Sec24, Sec13 and Sec31) are significantly higher in the ER-fractions of cortical tissues from Cln1-/- mice compared with those from their WT littermates. Remarkably, all COPII proteins, except Sec13, undergo S-palmitoylation. Moreover, CLN8, a Batten disease-protein, requires dynamic S-palmitoylation (palmitoylation-depalmitoylation) for ER-Golgi trafficking. Intriguingly, Ppt1-deficiency in Cln1-/- mice impairs ER-Golgi trafficking of Cln8-protein along with several other COPII-associated proteins. We propose that impaired anterograde trafficking causes excessive accumulation of proteins in the ER causing ER-stress and UPR contributing to neurodegeneration in CLN1 disease.

Keywords:  CLN1 disease; ER-stress; Lysosomal storage disease; Neurodegeneration; Palmitoyl-protein thioesterase-1; S-palmitoylation; Unfolded protein response

DOI:  https://doi.org/10.1016/j.nbd.2025.106890
Inflammation. 2025 Apr 02.

LRRK2 Mediates α-Synuclein-Induced Neuroinflammation and Ferroptosis through the p62-Keap1-Nrf2 Pathway in Parkinson's Disease.

Xinjie Liu, Zijian Zheng, Cheng Xue, Xiangrong Wang, Jianwei Li, Zheng Liu, Wenqiang Xin, Xinping Xu, Dongwei Zhou, Longping Yao, Guohui Lu.

  Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder worldwide, characterized by the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta and the abnormal aggregation of α-synuclein (α-syn). Despite extensive research, the mechanisms underlying microglial-mediated neuroinflammation and ferroptosis in PD remain inadequately understood. In particular, the role of leucine-rich repeat kinase 2 (LRRK2) in microglial cells and its modulation of the p62-Keap1-Nrf2 signaling pathway warrant further investigation.In this study, we present novel findings demonstrating that LRRK2 regulates microglial neuroinflammation and ferroptosis through the p62-Keap1-Nrf2 signaling axis in the context of PD. Using α-syn-stimulated BV2 microglial cells, we found that LRRK2 inhibition significantly reduced the production of pro-inflammatory cytokines and enhanced the activation of the p62-Keap1-Nrf2 pathway, thereby mitigating ferroptosis and oxidative stress. Furthermore, conditioned medium from LRRK2-inhibited microglia conferred neuroprotective effects on cultured neurons, highlighting the therapeutic potential of targeting LRRK2 in microglia.Importantly, these in vitro findings were corroborated in the MPTP-induced PD mouse model, where LRRK2 inhibition led to diminished microglial activation, decreased apoptosis of midbrain dopaminergic neurons, and upregulation of the p62-Keap1-Nrf2 pathway.Our study fills a critical gap in understanding the microglial mechanisms mediated by LRRK2 and provides novel insights into the pathogenesis of PD. These findings suggest that targeting LRRK2 in microglia may represent a promising therapeutic strategy for PD.

Keywords:  Ferroptosis; LRRK2; Neuroinflammation; Parkinson’s disease; p62-Keap1-Nrf2 pathway

DOI:  https://doi.org/10.1007/s10753-025-02291-8
Int J Biol Macromol. 2025 Mar 27. pii: S0141-8130(25)03151-4. [Epub ahead of print]308(Pt 3): 142599

Zebrafish TDP43 positively regulates p65-mediated apoptotic pathway.

Tingting Yu, Miaomiao Li, Meifeng Li, Quanling Zhang, Hongying Zhang, Zeyin Jiang, Shanghong Wang, Huiling Mao, Dongming Li, Lihua Fan, Chengyu Hu, Xiaowen Xu.

  TAR DNA-binding protein 43 (TDP43) is a multifunctional RNA/DNA binding protein that serves as a hallmark of neurodegeneration in amyotrophic lateral sclerosis (ALS) and is associated with the inflammatory response related to nuclear factor κB (NF-κB) pathway. However, the relationship between TDP43 and NF-κB is not well known. In this study, zebrafish TDP43 (DrTDP43) can be induced by grass carp reovirus (GCRV) or spring viremia of carp virus (SVCV). DrTDP43 enhances the nuclear factor-kappaB (NF-κB) activity and the expression of p65 and TNFα, as well as promotes the phosphorylation of p65 in response to stimulation of GCRV and SVCV. Further assays indicate that DrTDP43 primarily resides in the nucleus and interacts with p65 via its RRM1. DrTDP43 is required for p65 to induce pro-inflammatory cytokine production (IL-6, IL-10, TNFα, IL-1β). It disrupts mitochondrial membrane potential and exacerbates apoptosis via downregulating Bcl2 and upregulating Bax, caspase3, and eIF2α. Moreover, knockdown of TDP43 decreases the content of reactive oxygen species (ROS) and the number of apoptotic cells in zebrafish larvae, which is attributed to the lower lever of p65 phosphorylation and expression of TNFα, Bax and cleaved-caspase3. In a word, these results establish TDP43 as a critical activator of the NF-κB-mediated apoptotic pathway during antiviral responses, which reveals a previously unrecognized host defense mechanism.

Keywords:  Apoptosis; Nuclear factor-kappaB; TAR DNA-binding protein 43; Zebrafish; p65

DOI:  https://doi.org/10.1016/j.ijbiomac.2025.142599
Stem Cell Res. 2025 Mar 31. pii: S1873-5061(25)00054-6. [Epub ahead of print]85 103704

Generation of an induced pluripotent stem cell (iPSC) line (INNDSUi009-A) from a patient with amyotrophic lateral sclerosis due to SOD1 mutation.

Bo Li, Haoran Mu, Didi Shan, Yitong Yang, Yingxin Wang, Jianing Li, Hongxu Wang, Xiaohan Sun, Xinbo Ji, Zexin Zhan, Yichang Jiao, Yao Tang, Bo Kong, Bo Gao, Yu Wang, Ping Sun, Fuchen Liu.

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive degeneration of nerve cells in the spinal cord and brain. We generated and characterized a human induced pluripotent stem cell (iPSC) line from skin fibroblasts of a patient with ALS due to SOD1 Mutation. The pluripotency of these iPSCs was verified by the expression of several pluripotency markers at both RNA and protein levels, as well as their capability to differentiate into all three germ layers.

DOI: https://doi.org/10.1016/j.scr.2025.103704
FEBS Open Bio. 2025 Apr 03.

A non-fluorescent immunohistochemistry method for measuring autophagy flux using MAP1LC3/LC3 and SQSTM1 as core markers.

Shahla Shojaei, Amir Barzegar Behrooz, Marco Cordani, Mahmoud Aghaei, Negar Azarpira, Daniel J Klionsky, Saeid Ghavami.

  Macroautophagy/autophagy is a crucial cellular process for degrading and recycling damaged proteins and organelles, playing a significant role in diseases such as cancer and neurodegeneration. Evaluating autophagy flux, which tracks autophagosome formation, maturation, and degradation, is essential for understanding disease mechanisms. Current fluorescence-based methods are resource-intensive, requiring advanced equipment and expertise, limiting their use in clinical laboratories. Here, we introduce a non-fluorescent immunohistochemistry (IHC) method using MAP1LC3/LC3 and SQSTM1 as core markers for autophagy flux assessment. LC3 levels reflect autophagosome formation, whereas SQSTM1 degradation and a decrease in the number of its puncta indicate active flux (i.e., lysosomal turnover). We optimized chromogenic detection using diaminobenzidine (DAB) staining and developed a scoring system based on puncta number and the percentage of stained cells. This accessible, cost-effective method enables reliable autophagy quantification using a standard light microscope, bridging the gap between experimental research and clinical diagnostics. Our protocol allows accurate autophagy evaluation in fixed tissues, offering practical applications in biomedical research and clinical pathology assessment.

Keywords:  autophagometer; autophagy flux measurement; cellular homeostasis analysis; chromogenic detection; cost‐effective autophagy assay; non‐fluorescent immunohistochemistry

DOI:  https://doi.org/10.1002/2211-5463.70014
bioRxiv. 2025 Mar 13. pii: 2025.03.13.643156. [Epub ahead of print]

The Endo-GeneScreen Platform Identifies Drug-Like Probes that Regulate Endogenous Protein Levels within Physiological Contexts.

Preston Samowitz, Laszlo Radnai, Thomas Vaissiere, Sheldon D Michaelson, Camilo Rojas, Ryan Mitchell, Murat Kilinc, Austin Edwards, Justin Shumate, Richard Hawkins, Virneliz Fernandez-Vega, Timothy P Spicer, Louis Scampavia, Theodore Kamenecka, Courtney A Miller, Gavin Rumbaugh.

Traditional phenotypic drug discovery platforms have suffered from poor scalability and a lack of mechanistic understanding of newly discovered phenotypic probes. To address this, we created Endo- GeneScreen (EGS), a high-throughput enabled screening platform that identifies bioactive small molecules capable of regulating endogenous protein expression encoded by any preselected target gene within a biologically appropriate context. As a proof-of-concept, EGS successfully identified drug candidates that up-regulate endogenous expression of neuronal Syngap1, a gene that causes a neurodevelopmental disorder when haploinsufficient. For example, SR-1815, a previously unknown and undescribed kinase inhibitor, alleviated major cellular consequences of Syngap1 loss-of-function by restoring normal SynGAP protein levels and dampening neuronal hyperactivity within haploinsufficient neurons. Moreover, we demonstrate that EGS assays accelerate preclinical development of identified drug candidates and facilitate mode-of-action deconvolution studies. Thus, EGS identifies first-in-class bioactive small molecule probes that promote biological discovery and precision therapeutic development.

DOI: https://doi.org/10.1101/2025.03.13.643156
PLoS One. 2025 ;20(4): e0319418

A human iPSC-derived midbrain neural stem cell model of prenatal opioid exposure and withdrawal: A proof of concept study.

Rhea Sullivan, Quinn Ahrens, Sara L Mills-Huffnagle, Irina A Elcheva, Steven D Hicks.

A growing body of clinical literature has described neurodevelopmental delays in infants with chronic prenatal opioid exposure and withdrawal. Despite this, the mechanism of how opioids impact the developing brain remains unknown. Here, we developed an in vitro model of prenatal morphine exposure and withdrawal using healthy human induced pluripotent stem cell (iPSC)-derived midbrain neural progenitors in monolayer. To optimize our model, we identified that a longer neural induction and regional patterning period increases expression of canonical opioid receptors mu and kappa in midbrain neural progenitors compared to a shorter protocol (OPRM1, two-tailed t-test, p = 0.004; OPRK1, p = 0.0003). Next, we showed that the midbrain neural progenitors derived from a longer iPSC neural induction also have scant toll-like receptor 4 (TLR4) expression, a key player in neonatal opioid withdrawal syndrome pathophysiology. During morphine withdrawal, differentiating neural progenitors experience cyclic adenosine monophosphate overshoot compared to cell exposed to vehicle (p = 0.0496) and morphine exposure conditions (p, = 0.0136, 1-way ANOVA). Finally, we showed that morphine exposure and withdrawal alters proportions of differentiated progenitor cell fates (2-way ANOVA, F = 16.05, p < 0.0001). Chronic morphine exposure increased proportions of nestin positive progenitors (p = 0.0094), and decreased proportions of neuronal nuclear antigen positive neurons (NEUN) (p = 0.0047) compared to those exposed to vehicle. Morphine withdrawal decreased proportions of glial fibrillary acidic protein positive cells of astrocytic lineage (p = 0.044), and increased proportions of NEUN-positive neurons (p < 0.0001) compared to those exposed to morphine only. Applications of this paradigm include mechanistic studies underscoring neural progenitor cell fate commitments in early neurodevelopment during morphine exposure and withdrawal.

DOI: https://doi.org/10.1371/journal.pone.0319418
Brain. 2025 Apr 03. 148(4): 1045-1046

The interneuron hypothesis of amyotrophic lateral sclerosis.

Bryan J Traynor.

DOI: https://doi.org/10.1093/brain/awaf104
Sci Rep. 2025 Mar 29. 15(1): 10878

Multi-omics study reveals differential expression and phosphorylation of autophagy-related proteins in autism spectrum disorder.

Eden Deri, Shashank Kumar Ojha, Maryam Kartawy, Igor Khaliulin, Haitham Amal.

Our multi-omics study investigated the molecular mechanisms underlying autism spectrum disorder (ASD) using Shank3Δ4-22 and Cntnap2-/- mouse models. Through global- and phospho- proteomics of the mouse cortex, we focused on shared molecular changes and found that autophagy was particularly affected in both models. Global proteomics identified a small number of differentially expressed proteins that significantly impact postsynaptic components and synaptic function, including key pathways such as mTOR signaling. Phosphoproteomics revealed unique phosphorylation sites in autophagy-related proteins such as ULK2, RB1CC1, ATG16L1, and ATG9, suggesting that altered phosphorylation patterns contribute to impaired autophagic flux in ASD. SH-SY5Y cells with SHANK3 gene deletion showed elevated LC3-II and p62 levels, indicating autophagosome accumulation and autophagy initiation, while the reduced level of the lysosomal activity marker LAMP1 suggested impaired autophagosome-lysosome fusion. The study highlights the involvement of reactive nitrogen species and nitric oxide (NO) on autophagy disruption. Importantly, inhibition of neuronal NO synthase (nNOS) by 7-NI normalized autophagy markers levels in the SH-SY5Y cells and primary cultured neurons. We have previously shown that nNOS inhibition improved synaptic and behavioral phenotypes in Shank3Δ4-22 and Cntnap2-/- mouse models. Our multi-omics study reveals differential expression and phosphorylation of autophagy-related proteins in ASD but further investigation is needed to prove the full involvement of autophagy in ASD. Our study underscores the need for further examination into the functional consequences of the identified phosphorylation sites, which may offer potential novel therapeutic autophagy-related targets for ASD treatment.

DOI: https://doi.org/10.1038/s41598-025-95860-8
Sci Adv. 2025 Apr 04. 11(14): eadr6415

Retromer promotes the lysosomal turnover of mtDNA.

Parisa Kakanj, Mari Bonse, Arya Kshirsagar, Aylin Gökmen, Felix Gaedke, Ayesha Sen, Belén Mollá, Elisabeth Vogelsang, Astrid Schauss, Andreas Wodarz, David Pla-Martín.

Mitochondrial DNA (mtDNA) is exposed to multiple insults produced by normal cellular function. Upon mtDNA replication stress, the mitochondrial genome transfers to endosomes for degradation. Using proximity biotinylation, we found that mtDNA stress leads to the rewiring of the mitochondrial proximity proteome, increasing mitochondria's association with lysosomal and vesicle-related proteins. Among these, the retromer complex, particularly VPS35, plays a pivotal role by extracting mitochondrial components. The retromer promotes the formation of mitochondrial-derived vesicles shuttled to lysosomes. The mtDNA, however, directly shuttles to a recycling organelle in a BAX-dependent manner. Moreover, using a Drosophila model carrying a long deletion on the mtDNA (ΔmtDNA), we found that ΔmtDNA activates a specific transcriptome profile to counteract mitochondrial damage. Here, Vps35 expression restores mtDNA homoplasmy and alleviates associated defects. Hence, we demonstrate the existence of a previously unknown quality control mechanism for the mitochondrial matrix and the essential role of lysosomes in mtDNA turnover to relieve mtDNA damage.

DOI: https://doi.org/10.1126/sciadv.adr6415
bioRxiv. 2025 Mar 13. pii: 2025.03.10.642434. [Epub ahead of print]

Stathmin 1 regulates mitophagy and cellular function in hematopoietic stem cells.

Luana Chiquetto, Meg Schuetz, Qian Dong, Mia Warmka, Liana Valin, Ashley Jones, Patrick Hunt, Copper Petermeier, Jie Wang, Nate Roundy, Zev J Greenberg, Wei Yang, Christine R Zhang, Grant A Challen, Cliff J Luke, Robert A J Signer, Wandy L Beatty, Stephen Sykes, Weikai Li, David J Kast, Laura G Schuettpelz.

Stathmin 1 is a cytoplasmic phosphoprotein that regulates microtubule dynamics via promotion of microtubule catastrophe and sequestration of free tubulin heterodimers. Stathmin 1 is highly expressed in hematopoietic stem cells (HSCs), and overexpressed in leukemic cells, however its role in HSCs is not known. Herein, we found that loss of Stathmin 1 is associated with altered microtubule architecture in HSCs, and markedly impaired HSC function. Transcriptomic studies suggested alterations in oxidative phosphorylation in Stmn1 -/- HSCs, and further mechanistic studies revealed defective mitochondrial structure and function in the absence of Stathmin 1 with increased ROS production. Microtubules associate with mitochondria and lysosomes to facilitate autophagosome formation and mitophagy, and indeed we found that this critical mitochondrial quality control process is impaired in Stathmin 1-deficient HSCs. Finally, stimulation of autophagy improved the colony forming ability of Stmn1 -/- hematopoietic stem and progenitor cells. Together, our data identify Stathmin 1 as a novel regulator of mitophagy and mitochondrial health in HSCs.
Key Points: The microtubule regulating protein Stathmin 1 is highly expressed in HSPCs and promotes normal microtubule architecture.Loss of Stathmin 1 in HSPCs leads to impaired autophagy with abnormal mitochondrial morphology, decreased respiratory capacity, and impaired cellular function.

DOI: https://doi.org/10.1101/2025.03.10.642434
Nat Commun. 2025 Apr 03. 16(1): 3031

Inhibiting acute, axonal DLK palmitoylation is neuroprotective and avoids deleterious effects of cell-wide DLK inhibition.

Xiaotian Zhang, Heykyeong Jeong, Jingwen Niu, Sabrina M Holland, Brittany N Rotanz, John Gordon, Margret B Einarson, Wayne E Childers, Gareth M Thomas.

Inhibiting dual leucine-zipper kinase (DLK) could potentially ameliorate diverse neuropathological conditions, but a direct inhibitor of DLK's kinase domain caused unintended side effects in human patients, indicative of neuronal cytoskeletal disruption. We sought a more precise intervention and show here that axon-to-soma pro-degenerative signaling requires acute, axonal palmitoylation of DLK. To identify potential modulators of this modification, we screened >28,000 compounds using a high-content imaging readout of DLK's palmitoylation-dependent subcellular localization. Several hits alter DLK localization in non-neuronal cells, reduce DLK retrograde signaling and protect cultured dorsal root ganglion neurons from neurodegeneration. Mechanistically, the two most neuroprotective compounds selectively prevent DLK's stimulus-dependent palmitoylation and subsequent recruitment to axonal vesicles, but do not affect palmitoylation of other axonal proteins assessed and avoid the cytoskeletal disruption associated with direct DLK inhibition. Our hit compounds also reduce pro-degenerative retrograde signaling in vivo, revealing a previously unrecognized neuroprotective strategy.

DOI: https://doi.org/10.1038/s41467-025-58036-6
J Mol Biol. 2025 Mar 26. pii: S0022-2836(25)00171-8. [Epub ahead of print] 169105

The life and times of brain autophagic vesicles.

Lisa Gambarotto, Erin Wosnitzka, Vassiliki Nikoletopoulou.

  Most of the knowledge on the mechanisms and functions of autophagy originates from studies in yeast and other cellular models. How this valuable information is translated to the brain, one of the most complex and evolving organs, has been intensely investigated. Fueled by the tight dependence of the mammalian brain on autophagy, and the strong links of human brain diseases with autophagy impairment, the field has revealed adaptations of the autophagic machinery to the physiology of neurons and glia, the highly specialized cell types of the brain. Here, we first provide a detailed account of the tools available for studying brain autophagy; we then focus on the recent advancements in understanding how autophagy is regulated in brain cells, and how it contributes to their homeostasis and integrated functions. Finally, we discuss novel insights and open questions that the new knowledge has raised in the field.

Keywords:  autophagic vesicles; brain; glia; homeostasis; neurons; selective autophagy; synapse

DOI:  https://doi.org/10.1016/j.jmb.2025.169105
Cell Rep. 2025 Mar 28. pii: S2211-1247(25)00254-2. [Epub ahead of print]44(4): 115483

PDZD8 promotes autophagy at ER-lysosome membrane contact sites to regulate activity-dependent synaptic growth.

Rajan S Thakur, Kate M O'Connor-Giles.

  Building synaptic connections requires coordinating a host of cellular activities from cell signaling to protein turnover, placing a high demand on intracellular communication. Membrane contact sites (MCSs) formed between organelles have emerged as key signaling hubs for coordinating diverse cellular activities, yet their roles in the developing nervous system remain obscure. We investigate the in vivo function of the endoplasmic reticulum (ER) MCS tethering and lipid-transfer protein PDZD8, which was recently linked to intellectual disability, in the nervous system. We find that PDZD8 is required for activity-dependent synaptic bouton formation in multiple paradigms. PDZD8 is sufficient to drive excess synaptic bouton formation through an autophagy-dependent mechanism and required for synapse development when autophagy is limited. PDZD8 accelerates autophagic flux by promoting lysosome maturation at ER-late endosome/lysosome MCSs. We propose that PDZD8 functions in the nervous system to increase autophagy during periods of high demand, including activity-dependent synaptic growth.

Keywords:  CP: Cell biology; CP: Neuroscience; Drosophila; autophagy; lipid transfer protein; lysosomes; membrane contact sites; neurodevelopment; synapse

DOI:  https://doi.org/10.1016/j.celrep.2025.115483
Sci Rep. 2025 Apr 01. 15(1): 11116

Genetic editing of primary human dorsal root ganglion neurons using CRISPR-Cas9.

Seph M Palomino, Katherin A Gabriel, Juliet M Mwirigi, Anna Cervantes, Peter Horton, Geoffrey Funk, Aubin Moutal, Laurent F Martin, Rajesh Khanna, Theodore J Price, Amol Patwardhan.

CRISPR-Cas9 is now the leading method for genome editing and is advancing for the treatment of human disease. CRIPSR has promise in treating neurological diseases, but traditional viral-vector-delivery approaches have neurotoxicity limiting their use. Here we describe a simple method for non-viral transfection of primary human DRG (hDRG) neurons for CRISPR-Cas9 editing. We edited TRPV1, NTSR2, and CACNA1E using a lipofection method with CRISPR-Cas9 plasmids containing reporter tags (GFP or mCherry). Transfection was successfully demonstrated by the expression of the reporters two days post-administration. CRISPR-Cas9 editing was confirmed at the genome level with a T7-endonuclease-I assay; protein level with immunocytochemistry and Western blot; and functional level through capsaicin-induced Ca2+ accumulation in a high-throughput compatible fluorescent imaging plate reader (FLIPR) system. This work establishes a reliable, target specific, non-viral CRISPR-Cas9-mediated genetic editing in primary human neurons with potential for future clinical application for sensory diseases.

DOI: https://doi.org/10.1038/s41598-025-91153-2
Cell Rep. 2025 Apr 03. pii: S2211-1247(25)00261-X. [Epub ahead of print]44(4): 115490

RAF1 kinase contributes to autophagic lysosome reformation.

Stefanie Toifl, Sebastian Didusch, Karin Ehrenreiter, Enrico Desideri, Coralie Dorard, Manuela Baccarini.

  Autophagic lysosome reformation (ALR) is crucial for lysosomal homeostasis and therefore for different autophagic processes. Despite recent advances, the signaling mechanisms regulating ALR are incompletely understood. We show that RAF1, a member of the RAS/RAF/MEK/ERK pathway initiated by growth factors, has an essential, kinase-dependent role in lysosomal biology. RAF1 ablation impairs autophagy, and a proxisome screen identifies several proteins involved in autophagic and lysosomal pathways in the RAF1 molecular space. Two of these, SPG11 and the lipid phosphatase MTMR4, are RAF1 substrates. RAF1 ablation causes the appearance of enlarged autolysosomes and alters the phosphoinositide composition of autolysosomes. RAF1 and MTMR4 colocalize on autolysosomes, and overexpression of a MTMR4 mutant mimicking phosphorylation of the RAF1-dependent site rescues the lysosomal phenotypes induced by RAF1 ablation. Our data identify an RAF1 function in lysosomal homeostasis and a substrate through which the kinase regulates phospholipid metabolism at the lysosome, ALR, and autophagy.

Keywords:  CP: Cell biology; RAF1 interactome; RAF1 substrates; autophagic lysosome reformation; autophagy; lysosomal homeostasis

DOI:  https://doi.org/10.1016/j.celrep.2025.115490
PLoS Comput Biol. 2025 Apr 04. 21(4): e1012959

Seizure and redox rescue in a model of glucose transport deficiency.

Jay S Coggan, Polina Shichkova, Henry Markram, Daniel Keller.

Disruptions of energy supply to the brain are associated with many neurodegenerative pathologies and are difficult to study due to numerous interlinked metabolic pathways. We explored the effects of diminished energy supply on brain metabolism using a computational model of the neuro-glia-vasculature ensemble, in the form of a neuron, an astrocyte and local blood supply. As a case study, we investigated the glucose transporter type-1 deficiency syndrome (GLUT1-DS), a childhood affliction characterized by impaired glucose utilization and associated with phenotypes including seizures. Compared to neurons, astrocytes exhibited markedly higher metabolite concentration variabilities for all but a few redox species. This effect could signal a role for astrocytes in absorbing the shock of blood nutrient fluctuations. Redox balances were disrupted in GLUT1-DS with lower levels of reducing equivalent carriers NADH and ATP. The best non-glucose nutrient or pharmacotherapies for re-establishing redox normalcy involved lactate, the keto-diet (β-hydroxybutyrate), NAD and Q10 supplementation, suggesting a possible glucose sparing mechanism. GLUT1-DS seizures resulted from after-discharge neuronal firing caused by post-stimulus ATP reductions and impaired Na+/K+-ATPase, which can be rescued by restoring either normal glucose or by relatively small increases in neuronal ATP.

DOI: https://doi.org/10.1371/journal.pcbi.1012959
Biochim Biophys Acta Bioenerg. 2025 Apr 01. pii: S0005-2728(25)00021-0. [Epub ahead of print] 149555

Three's company: Membrane waltz among organelles.

Valentin Guyard, Francesca Giordano.

  The study of membrane contact sites (MCS) has profoundly transformed our understanding of inter-organelle communication. These sites, where the membranes of two organelles are closely apposed, facilitate the transfer of small molecules such as lipids and ions. They are especially crucial for the maintenance of the structure and function of organelles like mitochondria and lipid droplets, which are largely excluded from vesicular trafficking. The significant advancements in imaging techniques, and molecular and cell biology research have shown that MCS are more complex than what originally thought and can involve more than two organelles. This has revealed the intricate nature and critical importance of these subcellular connections. Here, we provide an overview of newly described three-way inter-organelles associations, and the proteins involved in these MCS. We highlight the roles these contacts play in key cellular processes such as lipid droplet biogenesis and mitochondrial division. Additionally, we discuss the latest advances in super-resolution imaging that enable the study of these complex three-way interactions. Ongoing research, driven by technological innovations, promises to uncover further insights into their roles in fundamental cellular processes and their implications for health and disease.

Keywords:  Lipid droplet biogenesis; Membrane contact sites; Mitochondria; Mitochondria dynamics

DOI:  https://doi.org/10.1016/j.bbabio.2025.149555