bims-axbals 2024-10-13 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2024–10–13
34 papers selected by
TJ Krzystek, ALS Therapy Development Institute

Hyperexcitability precedes CA3 hippocampal neurodegeneration in a dox-regulatable TDP-43 mouse model of ALS-FTD.
Therapeutic potential of oleanolic acid in modulation of PI3K/Akt/mTOR/STAT-3/GSK-3β signaling pathways and neuroprotection against methylmercury-induced neurodegeneration.
A pathogenic mutation in the ALS/FTD gene VCP induces mitochondrial hypermetabolism by modulating the permeability transition pore.
KIF1C facilitates retrograde transport of lysosomes through Hook3 and dynein.
Loss of mitochondrial Ca2+ response and CaMKII/ERK activation by LRRK2R1441G mutation correlate with impaired depolarization-induced mitophagy.
Conformational Enigma of TDP-43 Misfolding in Neurodegenerative Disorders.
Apache is a neuronal player in autophagy required for retrograde axonal transport of autophagosomes.
Absence of pretaporter restrains features of the parkin phenotype in Drosophila.
Endo-IP and Lyso-IP Toolkit for Endolysosomal Profiling of Human Induced Neurons.
Vulnerability of neurofilament-expressing neurons in frontotemporal dementia.
Synaptic Mitochondria: a crucial factor in the aged hippocampus.
C9orf72 poly-PR forms anisotropic condensates causative of nuclear TDP-43 pathology.
Secretion of endoplasmic reticulum protein VAPB/ALS8 requires topological inversion.
The Human Neural Cell Atlas of Zika Infection in developing human brain tissue: viral pathogenesis, innate immunity, and lineage reprogramming.
CRISPR/Cas9 gene editing in induced pluripotent stem cells to investigate the feline hypertrophic cardiomyopathy causing MYBPC3/R820W mutation.
α-Synuclein pathology disrupts mitochondrial function in dopaminergic and cholinergic neurons at-risk in Parkinson's disease.
The membrane curvature-inducing REEP1-4 proteins generate an ER-derived vesicular compartment.
Modeling Parkinson's disease pathology in human dopaminergic neurons by sequential exposure to α-synuclein fibrils and proinflammatory cytokines.
Neuroprotective effects of ellorarxine in neuronal models of degeneration.
Small RNAs in plasma extracellular vesicles define biomarkers of premanifest changes in Huntington's disease.
Dopey-dependent regulation of extracellular vesicles maintains neuronal morphology.
Isolation of small extracellular vesicles from regenerating muscle tissue using tangential flow filtration and size exclusion chromatography.
Impaired axon initial segment structure and function in a model of ARHGEF9 developmental and epileptic encephalopathy.
Autophagy controls neuronal differentiation by regulating the WNT-DVL signaling pathway.
Deep learning pipeline for automated cell profiling from cyclic imaging.
A Siah3 of relief: A role for mitophagy as a fail-safe during developmental axon pruning.
Proteomic characterization of ubiquitin carboxyl-terminal hydrolase 19 deficient cells reveals a role for USP19 in secretion of lysosomal proteins.
Pathogenic Huntingtin aggregates alter actin organization and cellular stiffness resulting in stalled clathrin mediated endocytosis.
Drp1 acetylation mediated by CDK5-AMPK-GCN5L1 axis promotes cerebral ischemic injury via facilitating mitochondrial fission.
One-step nanoscale expansion microscopy reveals individual protein shapes.
Cell type specification and diversity in subpallial organoids.
Stick-slip unfolding favors self-association of expanded HTT mRNA.
Delivery Aspects for Implementing siRNA Therapeutics for Blood Diseases.
TBC1D15-regulated mitochondria-lysosome membrane contact exerts neuroprotective effects by alleviating mitochondrial calcium overload in seizure.

bioRxiv. 2024 Sep 24. pii: 2024.09.24.612703. [Epub ahead of print]

Hyperexcitability precedes CA3 hippocampal neurodegeneration in a dox-regulatable TDP-43 mouse model of ALS-FTD.

William Rodemer, Irene Ra, Elizabeth Jia, Jaskeerat Gujral, Bin Zhang, Kevt'her Hoxha, Bo Xing, Sanya Mehta, Madona Farag, Silvia Porta, Frances E Jensen, Delia M Talos, Virginia M-Y Lee.

Neuronal hyperexcitability is a hallmark of amyotrophic lateral sclerosis (ALS) but its relationship with the TDP-43 aggregates that comprise the predominant pathology in over 90% of ALS cases remains unclear. Emerging evidence in tissue and slice culture models indicate that TDP-43 pathology induces neuronal hyperexcitability suggesting it may be responsible for the excitotoxicity long believed to be a major driver of ALS neuron death. Here, we characterized hyperexcitability and neurodegeneration in the hippocampus of doxycycline-regulatable rNLS8 mice (NEFH-tTA x tetO-hTDP-43ΔNLS), followed by treatment with AAV encoded DREADDs and anti-seizure medications to measure the effect on behavioral function and neurodegeneration. We found that approximately half of the CA3 neurons in the dorsal hippocampus are lost between 4 and 6 weeks after TDP-43ΔNLS induction. Neurodegeneration was preceded by selective hyperexcitability in the mossy fiber - CA3 circuit, leading us to hypothesize that glutamate excitotoxicity may be a significant contributor to neurodegeneration in this model. Interestingly, hippocampal injection of AAV encoded inhibitory DREADDs (hM4Di) and daily activation with CNO ligand rescued anxiety deficits on elevated zero maze (EZM) but did not reduce neurodegeneration. Therapeutic doses of the anti-seizure medications, valproic acid and levetiracetam, did not improve behavior or prevent neurodegeneration. These results highlight the complexity of TDP-43 - induced alterations to neuronal excitability and suggest that whereas targeting hyperexcitability can meliorate some behavioral deficits, it may not be sufficient to halt or slow neurodegeneration in TDP-43-related proteinopathies.
Significance Statement: Cytoplasmic aggregates of TAR DNA Binding Protein 43 (TDP-43) are the predominant pathology in over 90% of Amyotrophic lateral sclerosis (ALS) and the majority of frontotemporal lobar degeneration (FTLD-TDP) cases. Understanding how TDP-43 pathology promotes neurodegeneration may lead to therapeutic strategies to slow disease progression in humans. Recent reports in mouse and cell culture models suggest loss-of-normal TDP-43 function may drive neuronal hyperexcitability, a key physiological hallmark of ALS and possible contributor to neurodegeneration. In this study, we identified region-specific hyperexcitability that precedes neurodegeneration in the inducible rNLS8 TDP-43 mouse model. Suppressing hyperexcitability with chemogenetics improved behavioral function but did not reduce hippocampal neuron loss. Anti-seizure medications had no beneficial effects suggesting directly targeting hyperexcitability may not be therapeutically effective.

DOI: https://doi.org/10.1101/2024.09.24.612703
Neurochem Int. 2024 Oct 03. pii: S0197-0186(24)00203-1. [Epub ahead of print]180 105876

Therapeutic potential of oleanolic acid in modulation of PI3K/Akt/mTOR/STAT-3/GSK-3β signaling pathways and neuroprotection against methylmercury-induced neurodegeneration.

Ramaish Sharma, Sidharth Mehan, Zuber Khan, Ghanshyam Das Gupta, Acharan S Narula.

  Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder that gradually deteriorates motor neurons, leading to demyelination, muscle weakness, and eventually respiratory failure. The disease involves several pathological processes, such as increased glutamate levels, mitochondrial dysfunction, and persistent neuroinflammation, often exacerbated by environmental toxins like mercury. This study explores the therapeutic potential of Olea europaea active phytoconstituents oleanolic acid (OLA) against ALS by targeting the overactivated PI3K/Akt/mTOR/STAT-3/GSK-3β signalling pathways. Methods involved in-silico studies, in vitro and in vivo experiments in which varying doses of methylmercury 5 mg/kg, p.o. and OLA (100 and 200 mg/kg, i.p.) were administered to rats for 42 days. Behavioural assessments, gross morphological, histopathological, and neurochemical parameters were measured in cerebrospinal fluid (CSF), blood plasma, and brain homogenates (cerebral cortex, hippocampus, striatum, midbrain, cerebellum) along with complete blood count (CBC) analysis. Results revealed OLA's significant neuroprotective properties. OLA effectively modulated targeted pathways, reducing pro-inflammatory cytokines, restoring normal levels of myelin basic protein (MBP) and neurofilament light chain (NEFL), and reducing histopathological changes. Gross pathological studies indicated less tissue damage, while CBC analysis showed improved hematology parameters. Additionally, the combination of OLA and edaravone (10 mg/kg, i.p.) demonstrated enhanced efficacy, improving motor functions and extending survival in ALS model rats. In conclusion, OLA exhibits significant therapeutic potential for ALS, acting as a potent modulator of key pathological signaling pathways. The findings suggest the feasibility of integrating OLA into existing treatment regimens, potentially improving clinical outcomes for ALS patients. However, further research must validate these findings in human clinical trials.

Keywords:  Amyotrophic lateral sclerosis; Demyelination; Methylmercury; Neurodegeneration; Neuroprotection; PI3K/Akt/mTOR/STAT-3/GSK-3β

DOI:  https://doi.org/10.1016/j.neuint.2024.105876
Acta Neuropathol Commun. 2024 Oct 10. 12(1): 161

A pathogenic mutation in the ALS/FTD gene VCP induces mitochondrial hypermetabolism by modulating the permeability transition pore.

Silke Vanderhaeghe, Jovan Prerad, Arun Kumar Tharkeshwar, Elien Goethals, Katlijn Vints, Jimmy Beckers, Wendy Scheveneels, Eveline Debroux, Katrien Princen, Philip Van Damme, Marc Fivaz, Gerard Griffioen, Ludo Van Den Bosch.

  Valosin-containing protein (VCP) is a ubiquitously expressed type II AAA+ ATPase protein, implicated in both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This study aimed to explore the impact of the disease-causing VCPR191Q/wt mutation on mitochondrial function using a CRISPR/Cas9-engineered neuroblastoma cell line. Mitochondria in these cells are enlarged, with a depolarized mitochondrial membrane potential associated with increased respiration and electron transport chain activity. Our results indicate that mitochondrial hypermetabolism could be caused, at least partially, by increased calcium-induced opening of the permeability transition pore (mPTP), leading to mild mitochondrial uncoupling. In conclusion, our findings reveal a central role of the ALS/FTD gene VCP in maintaining mitochondrial homeostasis and suggest a model of pathogenesis based on progressive alterations in mPTP physiology and mitochondrial energetics.

Keywords:  Amyotrophic lateral sclerosis; Frontotemporal dementia; Mitochondria; Mitochondrial dysfunction; Mitochondrial permeability transition pore; VCP

DOI:  https://doi.org/10.1186/s40478-024-01866-0
Commun Biol. 2024 Oct 11. 7(1): 1305

KIF1C facilitates retrograde transport of lysosomes through Hook3 and dynein.

Takeshi Saji, Mitsuharu Endo, Yasushi Okada, Yasuhiro Minami, Michiru Nishita.

Lysosomes, crucial cellular organelles, undergo bidirectional transport along microtubules, mediated by motor proteins such as cytoplasmic dynein-1 (dynein) and various kinesins. While the kinesin-3 family member KIF1C is established in mediating anterograde vesicle transport, its role in lysosomal transport remains unclear. Our study reveals that KIF1C unexpectedly supports the retrograde transport of lysosomes, driven by dynein, and contributes to their perinuclear localization. Notably, while KIF1C facilitates this perinuclear positioning, its motor activity is not required and, instead, exerts an inhibitory effect on this process. Mechanistically, KIF1C facilitates this process by interacting with the dynein-activating adaptor Hook3, which associates with the lysosome-anchored protein RUFY3. This regulatory mechanism is critical for the efficient degradation of cargo in autophagic and endocytic pathways. Our findings identify an unconventional, non-motor role for KIF1C in activating dynein-driven lysosomal transport, expanding our understanding of its functional diversity in cellular trafficking.

DOI: https://doi.org/10.1038/s42003-024-07023-6
Cell Commun Signal. 2024 Oct 10. 22(1): 485

Loss of mitochondrial Ca2+ response and CaMKII/ERK activation by LRRK2R1441G mutation correlate with impaired depolarization-induced mitophagy.

Eunice Eun-Seo Chang, Huifang Liu, Zoe Yuen-Kiu Choi, Yasine Malki, Steffi Xi-Yue Zhang, Shirley Yin-Yu Pang, Michelle Hiu-Wai Kung, David B Ramsden, Shu-Leong Ho, Philip Wing-Lok Ho.

   BACKGROUND: Stress-induced activation of ERK/Drp1 serves as a checkpoint in the segregation of damaged mitochondria for autophagic clearance (mitophagy). Elevated cytosolic calcium (Ca2+) activates ERK, which is pivotal to mitophagy initiation. This process is altered in Parkinson's disease (PD) with mutations in leucine-rich repeat kinase 2 (LRRK2), potentially contributing to mitochondrial dysfunction. Pathogenic LRRK2 mutation is linked to dysregulated cellular Ca2+ signaling but the mechanism involved remains unclear.
METHODS: Mitochondrial damages lead to membrane depolarization. To investigate how LRRK2 mutation impairs cellular response to mitochondrial damages, mitochondrial depolarization was induced by artificial uncoupler (FCCP) in wild-type (WT) and LRRK2R1441G mutant knockin (KI) mouse embryonic fibroblasts (MEFs). The resultant cytosolic Ca2+ flux was assessed using live-cell Ca2+ imaging. The role of mitochondria in FCCP-induced cytosolic Ca2+ surge was confirmed by co-treatment with the mitochondrial sodium-calcium exchanger (NCLX) inhibitor. Cellular mitochondrial quality and function were evaluated by Seahorse™ real-time cell metabolic analysis, flow cytometry, and confocal imaging. Mitochondrial morphology was visualized using transmission electron microscopy (TEM). Activation (phosphorylation) of stress response pathways were assessed by immunoblotting.
RESULTS: Acute mitochondrial depolarization induced by FCCP resulted in an immediate cytosolic Ca2+ surge in WT MEFs, mediated predominantly via mitochondrial NCLX. However, such cytosolic Ca2+ response was abolished in LRRK2 KI MEFs. This loss of response in KI was associated with impaired activation of Ca2+/calmodulin-dependent kinase II (CaMKII) and MEK, the two upstream kinases of ERK. Treatment of LRRK2 inhibitor did not rescue this phenotype indicating that it was not caused by mutant LRRK2 kinase hyperactivity. KI MEFs exhibited swollen mitochondria with distorted cristae, depolarized mitochondrial membrane potential, and reduced mitochondrial Ca2+ store and mitochondrial calcium uniporter (MCU) expression. These mutant cells also exhibited lower cellular ATP: ADP ratio albeit higher basal respiration than WT, indicating compensation for mitochondrial dysfunction. These defects may hinder cellular stress response and signals to Drp1-mediated mitophagy, as evident by impaired mitochondrial clearance in the mutant.
CONCLUSIONS: Pathogenic LRRK2R1441G mutation abolished mitochondrial depolarization-induced Ca2+ response and impaired the basal mitochondrial clearance. Inherent defects from LRRK2 mutation have weakened the cellular ability to scavenge damaged mitochondria, which may further aggravate mitochondrial dysfunction and neurodegeneration in PD.

Keywords:  Calcium-dependent pathways; Cellular stress response; LRRK2 mutation; Mitochondrial dysfunction; Mitophagy; NCLX; Parkinson disease

DOI:  https://doi.org/10.1186/s12964-024-01844-y
ACS Omega. 2024 Oct 01. 9(39): 40286-40297

Conformational Enigma of TDP-43 Misfolding in Neurodegenerative Disorders.

Meenakshi Pillai, Santosh Kumar Jha.

Misfolding and aggregation of the protein remain some of the most common phenomena observed in neurodegeneration. While there exist multiple neurodegenerative disorders characterized by accumulation of distinct proteins, what remains particularly interesting is the ability of these proteins to undergo a conformational change to form aggregates. TDP-43 is one such nucleic acid binding protein whose misfolding is associated with many neurogenerative diseases including amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar degeneration (FTLD). TDP-43 protein assumes several different conformations and oligomeric states under the diseased condition. In this review, we explore the intrinsic relationship between the conformational variability of TDP-43 protein, with a particular focus on the RRM domains, and its propensity to undergo aggregation. We further emphasize the probable mechanism behind the formation of these conformations and suggest a potential diagnostic and therapeutic strategy in the context of these conformational states of the protein.

DOI: https://doi.org/10.1021/acsomega.4c04119
Cell Mol Life Sci. 2024 Oct 05. 81(1): 416

Apache is a neuronal player in autophagy required for retrograde axonal transport of autophagosomes.

Barbara Parisi, Alessandro Esposito, Enrico Castroflorio, Mattia Bramini, Sara Pepe, Antonella Marte, Fabrizia C Guarnieri, Flavia Valtorta, Pietro Baldelli, Fabio Benfenati, Anna Fassio, Silvia Giovedì.

  Neurons are dependent on efficient quality control mechanisms to maintain cellular homeostasis and function due to their polarization and long-life span. Autophagy is a lysosomal degradative pathway that provides nutrients during starvation and recycles damaged and/or aged proteins and organelles. In neurons, autophagosomes constitutively form in distal axons and at synapses and are trafficked retrogradely to the cell soma to fuse with lysosomes for cargo degradation. How the neuronal autophagy pathway is organized and controlled remains poorly understood. Several presynaptic endocytic proteins have been shown to regulate both synaptic vesicle recycling and autophagy. Here, by combining electron, fluorescence, and live imaging microscopy with biochemical analysis, we show that the neuron-specific protein APache, a presynaptic AP-2 interactor, functions in neurons as an important player in the autophagy process, regulating the retrograde transport of autophagosomes. We found that APache colocalizes and co-traffics with autophagosomes in primary cortical neurons and that induction of autophagy by mTOR inhibition increases LC3 and APache protein levels at synaptic boutons. APache silencing causes a blockade of autophagic flux preventing the clearance of p62/SQSTM1, leading to a severe accumulation of autophagosomes and amphisomes at synaptic terminals and along neurites due to defective retrograde transport of TrkB-containing signaling amphisomes along the axons. Together, our data identify APache as a regulator of the autophagic cycle, potentially in cooperation with AP-2, and hypothesize that its dysfunctions contribute to the early synaptic impairments in neurodegenerative conditions associated with impaired autophagy.

Keywords:  AP-2; Amphisome; LC3; Retrograde trafficking; Synapse; Torin1; TrkB; mTOR

DOI:  https://doi.org/10.1007/s00018-024-05441-7
Exp Neurol. 2024 Oct 09. pii: S0014-4886(24)00323-6. [Epub ahead of print] 114997

Absence of pretaporter restrains features of the parkin phenotype in Drosophila.

María Constanza Silvera, Rafael Cantera, María José Ferreiro.

   BACKGROUND: Scientific research based on model organisms can help to understand the biology of Parkinson's Disease, the second most prevalent neurodegenerative disease. Drosophila melanogaster mutant for the gene parkin, homologous to human's PARK2, exhibit well-characterized phenotypes including loss of dopaminergic neurons, lower survival and motor defects. Through the transcriptomic analysis of an exceptional case of reversible neurodegeneration in Drosophila, our group identified that the gene pretaporter, homologous to TXNDC5 of humans, was downregulated in the reversal phase. Here, we explore the hypothesis that the lack of expression of pretaporter will restrain phenotypes observed in Drosophila parkin mutants.
METHODS: After establishing by immunochemistry that Pretaporter is expressed in PPL1 dopaminergic neurons, we constructed pretaporter-parkin double mutants flies to investigate the hypothesis through immunohistochemistry, survival and climbing assays.
CONCLUSIONS: It was found that the loss-of-function mutation in pretaporter significatively restrains the phenotype caused by the loss-of-function mutation in parkin in several key aspects: it abolished the loss of PPL1 neurons normally seen in parkin mutant flies, promoted their survival in both sexes and reduced the decay in motor ability in parkin female flies. We propose that the absence of Pretaporter in parkin mutant flies prevents the death of dopaminergic neurons by rendering them resistant to Draper-mediated-phagocytosis.

Keywords:  Drosophila; Parkin; Parkinson's disease; Pretaporter

DOI:  https://doi.org/10.1016/j.expneurol.2024.114997
bioRxiv. 2024 Sep 26. pii: 2024.09.24.614704. [Epub ahead of print]

Endo-IP and Lyso-IP Toolkit for Endolysosomal Profiling of Human Induced Neurons.

Frances V Hundley, Miguel A Gonzalez-Lozano, Lena M Gottschalk, Aslan N K Cook, Jiuchun Zhang, Joao A Paulo, J Wade Harper.

Plasma membrane protein degradation and recycling is regulated by the endolysosomal system, wherein endosomes bud from the plasma membrane into the cytosol and mature into degradative lysosomes. As such, the endolysosomal system plays a critical role in determining the abundance of proteins on the cell surface, influencing cellular identity and function. Highly polarized cells, like neurons, rely on the endolysosomal system for axonal and dendritic specialization and synaptic compartmentalization. The importance of this system to neuronal function is reflected by the prevalence of risk variants in components of the system in several neurodegenerative diseases, ranging from Parkinson's to Alzheimer's disease. Nevertheless, our understanding of endocytic cargo and core endolysosomal machinery in neurons is limited, in part due to technical limitations. Here, we developed a toolkit for capturing EEA1-postive endosomes (Endo-IP) and TMEM192-positive lysosomes (Lyso-IP) in stem cell-derived induced neurons (iNeurons). We demonstrated its utility by revealing the endolysosomal protein landscapes for cortical-like iNeurons and stem cells. This allowed us to globally profile endocytic cargo, identifying hundreds of transmembrane proteins, including neurogenesis and synaptic proteins, as well as endocytic cargo with predicted SNX17 or SNX27 recognition motifs. By contrast, parallel lysosome profiling reveals a simpler protein repertoire, reflecting in part temporally controlled recycling or degradation for many endocytic targets. This system will facilitate mechanistic interrogation of endolysosomal components found as risk factors in neurodegenerative disease.

DOI: https://doi.org/10.1101/2024.09.24.614704
Mol Cell Neurosci. 2024 Oct 04. pii: S1044-7431(24)00059-9. [Epub ahead of print] 103974

Vulnerability of neurofilament-expressing neurons in frontotemporal dementia.

Nina Daniels, Aidan D Bindoff, James C Vickers, Anna E King, Jessica M Collins.

  Frontotemporal dementia (FTD) is an umbrella term for several early onset dementias, that are caused by frontotemporal lobar degeneration (FTLD), which involves the atrophy of the frontal and temporal lobes of the brain. Neuron loss in the frontal and temporal lobes is a characteristic feature of FTLD, however the selective vulnerability of different neuronal populations in this group of diseases is not fully understood. Neurofilament-expressing neurons have been shown to be selectively vulnerable in other neurodegenerative diseases, including Alzheimer's disease and amyotrophic lateral sclerosis, therefore we sought to investigate whether this neuronal population is vulnerable in FTLD. We also examined whether neuronal sub-type vulnerability differed between FTLD with TDP-43 inclusions (FTLD-TDP) and FTLD with tau inclusions (FTLD-Tau). Post-mortem human tissue from the superior frontal gyrus (SFG) of FTLD-TDP (n = 15), FTLD-Tau (n = 8) and aged Control cases (n = 6) was immunolabelled using antibodies against non-phosphorylated neurofilaments (SMI32 antibody), calretinin and NeuN, to explore neuronal cell loss. The presence of non-phosphorylated neurofilament immunolabelling in axons of the SFG white matter was also quantified as a measure of axon pathology, as axonal neurofilaments are normally phosphorylated. We demonstrate the selective loss of neurofilament-expressing neurons in both FTLD-TDP and FTLD-Tau cases compared to aged Controls. We also show that non-phosphorylated neurofilament axonal pathology in the SFG white matter was associated with increasing age, but not FTLD. This data suggests neurofilament-expressing neurons are vulnerable in both FTLD-TDP and FTLD-Tau.

Keywords:  Axon; Frontotemporal dementia; Neurodegeneration; Neurofilaments; Pathology; SMI32

DOI:  https://doi.org/10.1016/j.mcn.2024.103974
Ageing Res Rev. 2024 Oct 04. pii: S1568-1637(24)00342-8. [Epub ahead of print] 102524

Synaptic Mitochondria: a crucial factor in the aged hippocampus.

Karina A Cicali, Cheril Tapia-Rojas.

  Aging is a multifaceted biological process characterized by progressive molecular and cellular damage accumulation. The brain hippocampus undergoes functional deterioration with age, caused by cellular deficits, decreased synaptic communication, and neuronal death, ultimately leading to memory impairment. One of the factors contributing to this dysfunction is the loss of mitochondrial function. In neurons, mitochondria are categorized into synaptic and non-synaptic pools based on their location. Synaptic mitochondria, situated at the synapses, play a crucial role in maintaining neuronal function and synaptic plasticity, whereas non-synaptic mitochondria are distributed throughout other neuronal compartments, supporting overall cellular metabolism and energy supply. The proper function of synaptic mitochondria is essential for synaptic transmission as they provide the energy required and regulate calcium homeostasis at the communication sites between neurons. Maintaining the structure and functionality of synaptic mitochondria involves intricate processes, including mitochondrial dynamics such as fission, fusion, transport, and quality control mechanisms. These processes ensure that mitochondria remain functional, replace damaged organelles, and sustain cellular homeostasis at synapses. Notably, deficiencies in these mechanisms have been increasingly associated with aging and the onset of age-related neurodegenerative diseases. Synaptic mitochondria from the hippocampus are particularly vulnerable to age-related changes, including alterations in morphology and a decline in functionality, which significantly contribute to decreased synaptic activity during aging. This review comprehensively explores the critical roles that mitochondrial dynamics and quality control mechanisms play in preserving synaptic activity and neuronal function. It emphasizes the emerging evidence linking the deterioration of synaptic mitochondria to the aging process and the development of neurodegenerative diseases, highlighting the importance of these organelles from hippocampal neurons as potential therapeutic targets for mitigating cognitive decline and synaptic degeneration associated with aging. The novelty of this review lies in its focus on the unique vulnerability of hippocampal synaptic mitochondria to aging, underscoring their importance in maintaining brain function across the lifespan.

Keywords:  Aging; Cognitive Decline; Mitochondria; Mitochondrial Dysfunction; Non-synaptic; Synaptic

DOI:  https://doi.org/10.1016/j.arr.2024.102524
iScience. 2024 Oct 18. 27(10): 110937

C9orf72 poly-PR forms anisotropic condensates causative of nuclear TDP-43 pathology.

Rachel E Hodgson, Jessica A Rayment, Wan-Ping Huang, Anna Sanchez Avila, Brittany C S Ellis, Ya-Hui Lin, Nikita Soni, Guillaume M Hautbergue, Tatyana A Shelkovnikova.

  Proteinaceous inclusions formed by C9orf72-derived dipeptide-repeat (DPR) proteins are a histopathological hallmark in ∼50% of familial amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) cases. However, DPR aggregation/inclusion formation could not be efficiently recapitulated in cell models for four out of five DPRs. In this study, using optogenetics, we achieved chemical-free poly-PR condensation/aggregation in cultured cells including human motor neurons, with spatial and temporal control. Strikingly, nuclear poly-PR condensates had anisotropic, hollow-center appearance, resembling TDP-43 anisosomes, and their growth was limited by RNA. These condensates induced abnormal TDP-43 granulation in the nucleus without stress response activation. Cytoplasmic poly-PR aggregates forming under prolonged opto-stimulation were more persistent than its nuclear condensates, selectively sequestered TDP-43 in a demixed state and surrounded spontaneous stress granules. Thus, poly-PR condensation accompanied by nuclear TDP-43 dysfunction may constitute an early pathological event in C9-ALS/FTD. Anisosome-type condensates of disease-linked proteins may represent a common molecular species in neurodegenerative disease.

Keywords:  Biochemistry; Cell biology; Molecular biology

DOI:  https://doi.org/10.1016/j.isci.2024.110937
Nat Commun. 2024 Oct 10. 15(1): 8777

Secretion of endoplasmic reticulum protein VAPB/ALS8 requires topological inversion.

Kosuke Kamemura, Rio Kozono, Mizuki Tando, Misako Okumura, Daisuke Koga, Satoshi Kusumi, Kanako Tamai, Aoi Okumura, Sayaka Sekine, Daichi Kamiyama, Takahiro Chihara.

VAMP-associated protein (VAP) is a type IV integral transmembrane protein at the endoplasmic reticulum (ER). Mutations in human VAPB/ALS8 are associated with amyotrophic lateral sclerosis (ALS). The N-terminal major sperm protein (MSP) domain of VAPB (Drosophila Vap33) is cleaved, secreted, and acts as a signaling ligand for several cell-surface receptors. Although extracellular functions of VAPB are beginning to be understood, it is unknown how the VAPB/Vap33 MSP domain facing the cytosol is secreted to the extracellular space. Here we show that Vap33 is transported to the plasma membrane, where the MSP domain is exposed extracellularly by topological inversion. The externalized MSP domain is cleaved by Matrix metalloproteinase 1/2 (Mmp1/2). Overexpression of Mmp1 restores decreased levels of extracellular MSP domain derived from ALS8-associated Vap33 mutants. We propose an unprecedented secretion mechanism for an ER-resident membrane protein, which may contribute to ALS8 pathogenesis.

DOI: https://doi.org/10.1038/s41467-024-53097-5
bioRxiv. 2024 Sep 29. pii: 2024.09.27.615512. [Epub ahead of print]

The Human Neural Cell Atlas of Zika Infection in developing human brain tissue: viral pathogenesis, innate immunity, and lineage reprogramming.

Birth Defects Research Laboratory (BDRL)

Zika virus (ZIKV) infection during pregnancy can lead to fetal brain infection and developmental anomalies collectively known as congenital Zika syndrome (CZS). To define the molecular features underlying CZS in a relevant human cell model, we evaluated ZIKV infection and neurodevelopment in primary fetal brain explants and induced pluripotent stem cell-derived mixed neural cultures at single cell resolution. We identified astrocytes as key innate immune sentinel cells detecting ZIKV and producing IFN-β. In contrast, neural progenitor cells displayed impaired innate immunity and supported high levels of viral replication. ZIKV infection of neurons suppressed differentiation and synaptic signaling networks and programmed a molecular switch from neurogenesis to astrogliogenesis. We identified a universal ZIKV-driven cellular stress response linked to intrinsic apoptosis and regulated by IFN-β. These findings reveal how innate immune signaling intersects with ZIKV-driven perturbations in cellular function to influence CZS outcomes including neuron developmental dysfunction and apoptotic cell death.

DOI: https://doi.org/10.1101/2024.09.27.615512
PLoS One. 2024 ;19(10): e0311761

CRISPR/Cas9 gene editing in induced pluripotent stem cells to investigate the feline hypertrophic cardiomyopathy causing MYBPC3/R820W mutation.

Luke C Dutton, Jayesh Dudhia, Deborah J Guest, David J Connolly.

Hypertrophic cardiomyopathy (HCM) is the most common heart disease in domestic cats, often leading to congestive heart failure and death, with current treatment strategies unable to reverse or prevent progression of the disease. The underlying pathological processes driving HCM remain unclear, which hinders novel drug discovery. The aim of this study was to generate a cellular model of the feline HCM-causing MYBPC3 mutation R820W. Using CRISPR/Cas9 gene editing we introduced the R820W mutation into a human induced pluripotent stem cell (iPSC) line. We differentiated both homozygous mutant clones and isogenic control clones to cardiomyocytes (iPSC-CMs). Protein quantification indicated that haploinsufficiency is not the disease mechanism of the mutation. Homozygous mutant iPSC-CMs had a larger cell area than isogenic controls, with the sarcomere structure and incorporation of cMyBP-C appearing similar between mutant and control iPSC-CMs. Contraction kinetic analysis indicated that homozygous iPSC-CMs have impaired relaxation and are hypocontractile compared to isogenic control iPSC-CMs. In summary, we demonstrate successful generation of an iPSC model of a feline MYBPC3 mutation, with the cellular model recapitulating aspects of HCM including cellular hypertrophy and impaired relaxation kinetics. We anticipate that further study of this model will lead to improved understanding of the disease-causing molecular mechanism, ultimately leading to novel drug discovery.

DOI: https://doi.org/10.1371/journal.pone.0311761
Mol Neurodegener. 2024 Oct 08. 19(1): 69

α-Synuclein pathology disrupts mitochondrial function in dopaminergic and cholinergic neurons at-risk in Parkinson's disease.

Fanni F Geibl, Martin T Henrich, Zhong Xie, Enrico Zampese, Jun Ueda, Tatiana Tkatch, David L Wokosin, Elena Nasiri, Constantin A Grotmann, Valina L Dawson, Ted M Dawson, Navdeep S Chandel, Wolfgang H Oertel, D James Surmeier.

   BACKGROUND: Pathological accumulation of aggregated α-synuclein (aSYN) is a common feature of Parkinson's disease (PD). However, the mechanisms by which intracellular aSYN pathology contributes to dysfunction and degeneration of neurons in the brain are still unclear. A potentially relevant target of aSYN is the mitochondrion. To test this hypothesis, genetic and physiological methods were used to monitor mitochondrial function in substantia nigra pars compacta (SNc) dopaminergic and pedunculopontine nucleus (PPN) cholinergic neurons after stereotaxic injection of aSYN pre-formed fibrils (PFFs) into the mouse brain.
METHODS: aSYN PFFs were stereotaxically injected into the SNc or PPN of mice. Twelve weeks later, mice were studied using a combination of approaches, including immunocytochemical analysis, cell-type specific transcriptomic profiling, electron microscopy, electrophysiology and two-photon-laser-scanning microscopy of genetically encoded sensors for bioenergetic and redox status.
RESULTS: In addition to inducing a significant neuronal loss, SNc injection of PFFs induced the formation of intracellular, phosphorylated aSYN aggregates selectively in dopaminergic neurons. In these neurons, PFF-exposure decreased mitochondrial gene expression, reduced the number of mitochondria, increased oxidant stress, and profoundly disrupted mitochondrial adenosine triphosphate production. Consistent with an aSYN-induced bioenergetic deficit, the autonomous spiking of dopaminergic neurons slowed or stopped. PFFs also up-regulated lysosomal gene expression and increased lysosomal abundance, leading to the formation of Lewy-like inclusions. Similar changes were observed in PPN cholinergic neurons following aSYN PFF exposure.
CONCLUSIONS: Taken together, our findings suggest that disruption of mitochondrial function, and the subsequent bioenergetic deficit, is a proximal step in the cascade of events induced by aSYN pathology leading to dysfunction and degeneration of neurons at-risk in PD.

Keywords:  Alpha-synuclein; Bioenergetics; Dopaminergic; Electrophysiology; Lewy pathology; Mitochondria; Parkinson’s disease; Pedunculopontine nucleus; Substantia Nigra; Transcriptome

DOI:  https://doi.org/10.1186/s13024-024-00756-2
Nat Commun. 2024 Oct 05. 15(1): 8655

The membrane curvature-inducing REEP1-4 proteins generate an ER-derived vesicular compartment.

Yoko Shibata, Emily E Mazur, Buyan Pan, Joao A Paulo, Steven P Gygi, Suyog Chavan, L Sebastian Alexis Valerio, Jiuchun Zhang, Tom A Rapoport.

The endoplasmic reticulum (ER) is shaped by abundant membrane curvature-generating proteins that include the REEP family member REEP5. The REEP1 subfamily, consisting of four proteins in mammals (REEP1-4), is less abundant and lack a N-terminal region. Mutations in REEP1 and REEP2 cause Hereditary Spastic Paraplegia, but the function of these four REEP proteins remains enigmatic. Here we show that REEP1-4 reside in a unique vesicular compartment and identify features that determine their localization. Mutations in REEP1-4 that compromise curvature generation, including those causing disease, relocalize the proteins to the bulk ER. These mutants interact with wild-type proteins to retain them in the ER, consistent with their autosomal-dominant disease inheritance. REEP1 vesicles contain the membrane fusogen atlastin-1, but not general ER proteins. We propose that REEP1-4 generate these vesicles themselves by budding from the ER, and that they cycle back to the ER by atlastin-mediated fusion. The vesicles may serve to regulate ER tubule dynamics.

DOI: https://doi.org/10.1038/s41467-024-52901-6
Nat Neurosci. 2024 Oct 08.

Modeling Parkinson's disease pathology in human dopaminergic neurons by sequential exposure to α-synuclein fibrils and proinflammatory cytokines.

Armin Bayati, Riham Ayoubi, Adriana Aguila, Cornelia E Zorca, Ghislaine Deyab, Chanshuai Han, Sherilyn Junelle Recinto, Emmanuelle Nguyen-Renou, Cecilia Rocha, Gilles Maussion, Wen Luo, Irina Shlaifer, Emily Banks, Ian McDowell, Esther Del Cid Pellitero, Xue Er Ding, Behrang Sharif, Philippe Séguéla, Moein Yaqubi, Carol X-Q Chen, Zhipeng You, Narges Abdian, Heidi M McBride, Edward A Fon, Jo Anne Stratton, Thomas M Durcan, Patrick C Nahirney, Peter S McPherson.

Lewy bodies (LBs), α-synuclein-enriched intracellular inclusions, are a hallmark of Parkinson's disease (PD) pathology, yet a cellular model for LB formation remains elusive. Recent evidence indicates that immune dysfunction may contribute to the development of PD. In this study, we found that induced pluripotent stem cell (iPSC)-derived human dopaminergic (DA) neurons form LB-like inclusions after treatment with α-synuclein preformed fibrils (PFFs) but only when coupled to a model of immune challenge (interferon-γ or interleukin-1β treatment) or when co-cultured with activated microglia-like cells. Exposure to interferon-γ impairs lysosome function in DA neurons, contributing to LB formation. The knockdown of LAMP2 or the knockout of GBA in conjunction with PFF administration is sufficient for inclusion formation. Finally, we observed that the LB-like inclusions in iPSC-derived DA neurons are membrane bound, suggesting that they are not limited to the cytoplasmic compartment but may be formed due to dysfunctions in autophagy. Together, these data indicate that immune-triggered lysosomal dysfunction may contribute to the development of PD pathology.

DOI: https://doi.org/10.1038/s41593-024-01775-4
Front Neurosci. 2024 ;18 1422294

Neuroprotective effects of ellorarxine in neuronal models of degeneration.

Azita Kouchmeshky, Andrew Whiting, Peter McCaffery.

   Introduction: Retinoic acid (RA) was first recognised to be important for the central nervous system (CNS) in its developmental regulatory role and, given this action, it has been proposed in the adult CNS to regulate plasticity and promote regeneration. These types of roles have included support of neurogenesis, induction of neurite outgrowth, and protection from neuronal death. These functions are predominantly mediated by the retinoic acid receptor (RAR) transcription factor, and hence agonists for the RARs have been tested in a variety of models of neurodegeneration. This present study employs several in vitro models less explored for the action of RAR agonists to reverse neurodegeneration.
Methods: A series of assays are used in which neuronal cells are placed under the types of stress that have been linked to neurodegeneration, in particular amyotrophic lateral sclerosis (ALS), and the neuroprotective influence of a new potent agonist for RAR, ellorarxine, is tested out. In these assays, neuronal cells were subjected to excitotoxic stress induced by glutamate, proteostasis disruption caused by epoxomicin, and oxidative stress leading to stress granule formation triggered by sodium arsenite.
Results: Ellorarxine effectively reversed neuronal death in excitotoxic and proteostasis disruption assays and mitigated stress granule formation induced by sodium arsenite. This study also highlights for the first time the novel observation of RAR modulation of stress granules, although it is unknown whether this change in stress granules will be neuroprotective or potentially regenerative. Furthermore, the distribution of RAR agonists following intraperitoneal injection was assessed in mice, revealing preferential accumulation in the central nervous system, particularly in the spinal cord, compared to the liver. Gene expression studies in the spinal cord demonstrated that ellorarxine induces transcriptional changes at a low dose (0.01 mg/kg).
Discussion: These findings underscore the therapeutic potential of RAR agonists, such as ellorarxine, for ALS and potentially other neurodegenerative diseases.

Keywords:  amyotrophic lateral sclerosis; excitotoxicity; proteostasis; retinoic acid; stress granules

DOI:  https://doi.org/10.3389/fnins.2024.1422294
J Extracell Vesicles. 2024 Oct;13(10): e12522

Small RNAs in plasma extracellular vesicles define biomarkers of premanifest changes in Huntington's disease.

Marina Herrero-Lorenzo, Jesús Pérez-Pérez, Georgia Escaramís, Saül Martínez-Horta, Rocío Pérez-González, Elisa Rivas-Asensio, Jaime Kulisevsky, Ana Gámez-Valero, Eulàlia Martí.

  Despite the advances in the understanding of Huntington's disease (HD), there is a need for molecular biomarkers to categorize mutation carriers during the preclinical stage of the disease preceding functional decline. Small RNAs (sRNAs) are a promising source of biomarkers since their expression levels are highly sensitive to pathobiological processes. Here, using an optimized method for plasma extracellular vesicles (EVs) purification and an exhaustive analysis pipeline of sRNA sequencing data, we show that EV-sRNAs are downregulated early in mutation carriers and that this deregulation is associated with premanifest cognitive performance. Seven candidate sRNAs (tRF-Glu-CTC, tRF-Gly-GCC, miR-451a, miR-21-5p, miR-26a-5p, miR-27a-3p and let7a-5p) were validated in additional subjects, showing a significant diagnostic accuracy at premanifest stages. Of these, miR-21-5p was significantly decreased over time in a longitudinal study; and miR-21-5p and miR-26a-5p levels correlated with cognitive changes in the premanifest cohort. In summary, the present results suggest that deregulated plasma EV-sRNAs define an early biosignature in mutation carriers with specific species highlighting the progression and cognitive changes occurring at the premanifest stage.

Keywords:  Huntington's disease; biomarker; extracellular vesicles; miRNA; premanifest; small RNA; tRF

DOI:  https://doi.org/10.1002/jev2.12522
Curr Biol. 2024 Oct 01. pii: S0960-9822(24)01229-6. [Epub ahead of print]

Dopey-dependent regulation of extracellular vesicles maintains neuronal morphology.

Seungmee Park, Nathaniel Noblett, Lauren Pitts, Antonio Colavita, Ann M Wehman, Yishi Jin, Andrew D Chisholm.

  Mature neurons maintain their distinctive morphology for extended periods in adult life. Compared to developmental neurite outgrowth, axon guidance, and target selection, relatively little is known of mechanisms that maintain the morphology of mature neurons. Loss of function in C. elegans dip-2, a member of the conserved lipid metabolic regulator Dip2 family, results in progressive overgrowth of neurites in adults. We find that dip-2 mutants display specific genetic interactions with sax-2, the C. elegans ortholog of Drosophila Furry and mammalian FRY. Combined loss of dip-2 and sax-2 results in failure to maintain neuronal morphology and elevated release of neuronal extracellular vesicles (EVs). By screening for suppressors of dip-2(0) sax-2(0) double mutant defects, we identified gain-of-function (gf) mutations in the conserved Dopey family protein PAD-1 and its associated phospholipid flippase TAT-5/ATP9A that restore normal neuronal morphology and normal levels of EV release to dip-2(0) sax-2(0) double mutants. Neuron-specific knockdown suggests that PAD-1(gf) can act cell autonomously in neurons. PAD-1(gf) displays increased association with the plasma membrane in oocytes and inhibits EV release in multiple cell types. Our findings uncover a novel functional network of DIP-2, SAX-2, PAD-1, and TAT-5 that maintains neuronal morphology and modulates EV release.

Keywords:  DIP-2 lipid regulator; PAD-1/Dopey; SAX-2/Fry; TAT-5 flippase; endosomal trafficking; neuronal maintenance

DOI:  https://doi.org/10.1016/j.cub.2024.09.018
Skelet Muscle. 2024 Oct 11. 14(1): 22

Isolation of small extracellular vesicles from regenerating muscle tissue using tangential flow filtration and size exclusion chromatography.

Uxia Gurriaran-Rodriguez, Yves De Repentigny, Rashmi Kothary, Michael A Rudnicki.

  We have recently made the strikingly discovery that upon a muscle injury, Wnt7a is upregulated and secreted from new regenerating myofibers on the surface of exosomes to elicit its myogenerative response distally. Despite recent advances in extracellular vesicle (EVs) isolation from diverse tissues, there is still a lack of specific methodology to purify EVs from muscle tissue. To eliminate contamination with non-EV secreted proteins and cytoplasmic fragments, which are typically found when using classical methodology, such as ultracentrifugation, we adapted a protocol combining Tangential Flow Filtration (TFF) and Size Exclusion Chromatography (SEC). We found that this approach allows simultaneous purification of Wnt7a, bound to EVs (retentate fraction) and free non-EV Wnt7a (permeate fraction). Here we described this optimized protocol designed to specifically isolate EVs from hind limb muscle explants, without cross-contamination with other sources of non-EV bounded proteins. The first step of the protocol is to remove large EVs with sequential centrifugation. Extracellular vesicles are then concentrated and washed in exchange buffer by TFF. Lastly, SEC is performed to remove any soluble protein traces remaining after TFF. Overall, this procedure can be used to isolate EVs from conditioned media or biofluid that contains EVs derived from any cell type or tissue, improving reproducibility, efficiency, and purity of EVs preparations. Our purification protocol results in high purity EVs that maintain structural integrity and thus fully compatible with in vitro and in vivo bioactivity and analytic assays.

Keywords:  Myoregeneration; Skeletal muscle; Tangential flow filtration; Tissue derived extracellular vesicles

DOI:  https://doi.org/10.1186/s13395-024-00355-1
Proc Natl Acad Sci U S A. 2024 Oct 15. 121(42): e2400709121

Impaired axon initial segment structure and function in a model of ARHGEF9 developmental and epileptic encephalopathy.

Wanqi Wang, Damian J Williams, Jia Jie Teoh, Divyalakshmi Soundararajan, Aamir Zuberi, Cathleen M Lutz, Wayne N Frankel, Christopher D Makinson.

  Developmental and epileptic encephalopathies (DEE) are rare but devastating and largely intractable childhood epilepsies. Genetic variants in ARHGEF9, encoding a scaffolding protein important for the organization of the postsynaptic density of inhibitory synapses, are associated with DEE accompanied by complex neurological phenotypes. In a mouse model carrying a patient-derived ARHGEF9 variant associated with severe disease, we observed aggregation of postsynaptic proteins and loss of functional inhibitory synapses at the axon initial segment (AIS), altered axo-axonic synaptic inhibition, disrupted action potential generation, and complex seizure phenotypes consistent with clinical observations. These results illustrate diverse roles of ARHGEF9 that converge on regulation of the structure and function of the AIS, thus revealing a pathological mechanism for ARHGEF9-associated DEE. This unique example of a neuropathological condition associated with multiple AIS dysfunctions may inform strategies for treating neurodevelopmental diseases.

Keywords:  axon initial segment; epilepsy; hippocampus; mouse model

DOI:  https://doi.org/10.1073/pnas.2400709121
Autophagy. 2024 Oct 09. 1-18

Autophagy controls neuronal differentiation by regulating the WNT-DVL signaling pathway.

Vincencius Vidyawan, Lesly Puspita, Virginia Blessy Juwono, Magdalena Deline, Kelvin Pieknell, Mi-Yoon Chang, Sang-Hun Lee, Jae-Won Shim.

  Macroautophagy/autophagy dysregulation is associated with various neurological diseases, including Vici syndrome. We aimed to determine the role of autophagy in early brain development. We generated neurons from human embryonic stem cells and developed a Vici syndrome model by introducing a loss-of-function mutation in the EPG5 gene. Autophagy-related genes were upregulated at the neuronal progenitor cell stage. Inhibition of autolysosome formation with bafilomycin A1 treatment at the neuronal progenitor cell stage delayed neuronal differentiation. Notably, WNT (Wnt family member) signaling may be part of the underlying mechanism, which is negatively regulated by autophagy-mediated DVL2 (disheveled segment polarity protein 2) degradation. Disruption of autolysosome formation may lead to failure in the downregulation of WNT signaling, delaying neuronal differentiation. EPG5 mutations disturbed autolysosome formation, subsequently inducing defects in progenitor cell differentiation and cortical layer generation in organoids. Disrupted autophagy leads to smaller organoids, recapitulating Vici syndrome-associated microcephaly, and validating the disease relevance of our study.Abbreviations: BafA1: bafilomycin A1; co-IP: co-immunoprecipitation; DVL2: dishevelled segment polarity protein 2; EPG5: ectopic P-granules 5 autophagy tethering factor; gRNA, guide RNA; hESC: human embryonic stem cells; KO: knockout; mDA, midbrain dopamine; NIM: neural induction media; NPC: neuronal progenitor cell; qPCR: quantitative polymerase chain reaction; UPS: ubiquitin-proteasome system; WNT: Wnt family member; WT: wild type.

Keywords:  EPG5; Vici syndrome; WNT signal pathway; human embryonic stem cells; macroautophagy; neuronal differentiation

DOI:  https://doi.org/10.1080/15548627.2024.2407707
Sci Rep. 2024 10 09. 14(1): 23600

Deep learning pipeline for automated cell profiling from cyclic imaging.

Christian Landeros, Juhyun Oh, Ralph Weissleder, Hakho Lee.

  Cyclic fluorescence microscopy enables multiple targets to be detected simultaneously. This, in turn, has deepened our understanding of tissue composition, cell-to-cell interactions, and cell signaling. Unfortunately, analysis of these datasets can be time-prohibitive due to the sheer volume of data. In this paper, we present CycloNET, a computational pipeline tailored for analyzing raw fluorescent images obtained through cyclic immunofluorescence. The automated pipeline pre-processes raw image files, quickly corrects for translation errors between imaging cycles, and leverages a pre-trained neural network to segment individual cells and generate single-cell molecular profiles. We applied CycloNET to a dataset of 22 human samples from head and neck squamous cell carcinoma patients and trained a neural network to segment immune cells. CycloNET efficiently processed a large-scale dataset (17 fields of view per cycle and 13 staining cycles per specimen) in 10 min, delivering insights at the single-cell resolution and facilitating the identification of rare immune cell clusters. We expect that this rapid pipeline will serve as a powerful tool to understand complex biological systems at the cellular level, with the potential to facilitate breakthroughs in areas such as developmental biology, disease pathology, and personalized medicine.

Keywords:  Cell segmentation; Cyclic microscopy; Imaging analysis; Machine learning; Software

DOI:  https://doi.org/10.1038/s41598-024-74597-w
Sci Signal. 2024 10 08. 17(857): eads1228

A Siah3 of relief: A role for mitophagy as a fail-safe during developmental axon pruning.

Rina L Davidson, David J Simon.

Developmental axon pruning is controlled by a careful balance of pro- and anti-apoptotic signals, which are activated in response to external cues to sculpt mature neuronal circuitry. In this issue of Science Signaling, Abraham et al. define a safeguard against apoptotic axon pruning and illustrate that Siah3 represses Parkin-mediated mitophagy to control the availability of axonal mitochondria that activate the pruning process.

DOI: https://doi.org/10.1126/scisignal.ads1228
Mol Cell Proteomics. 2024 Oct 08. pii: S1535-9476(24)00144-0. [Epub ahead of print] 100854

Proteomic characterization of ubiquitin carboxyl-terminal hydrolase 19 deficient cells reveals a role for USP19 in secretion of lysosomal proteins.

Simone Bonelli, Margot Lo Pinto, Yihong Ye, Stephan A Mueller, Stefan F Lichtenthaler, Simone D Scilabra.

  Ubiquitin carboxyl-terminal hydrolase 19 (USP19) is a unique deubiquitinase (DUB), characterized by multiple variants generated by alternative splicing. Several variants bear a C-terminal transmembrane domain that anchors them to the endoplasmic reticulum (ER). Other than regulating protein stability by preventing proteasome degradation, USP19 has been reported to rescue substrates from ER-associated protein degradation (ERAD) in a catalytic-independent manner, promote autophagy and address proteins to lysosomal degradation via endosomal microautophagy. USP19 has recently emerged as the protein responsible for the unconventional secretion of misfolded proteins including Parkinson's disease-associated protein α-synuclein. Despite mounting evidence that USP19 plays crucial roles in several biological processes, the underlying mechanisms are unclear due to lack of information on the physiological substrates of USP19. Herein, we used high-resolution quantitative proteomics to analyze changes in the secretome and cell proteome induced by loss of USP19 to identify proteins whose secretion or turnover is regulated by USP19. We found that ablation of USP19 induced significant proteomic alterations both in and out of the cell. Loss of USP19 impaired the release of several lysosomal proteins, including legumain (LGMN) and several cathepsins. In order to understand the underlaying mechanism, we dissected the USP19-regulated secretion of LGMN in several cell types. We found that LGMN was not a DUB substrate of USP19 and that its USP19-dependent release did not require their direct interaction. LGMN secretion occurred by a mechanism that involved the Golgi apparatus, autophagosome formation and lysosome function. This mechanism resembled the recently described "lysosomal exocytosis", by which lysosomal hydrolases are secreted, when ubiquitination of p62 is increased in cells lacking deubiquitinases such as USP15 and USP17. In conclusion, our proteomic characterization of USP19 has identified a collection of proteins in the secretome and within the cell that are regulated by USP19, which link USP19 to secretion of lysosomal proteins, including LGMN.

Keywords:  legumain; lysosomal exocytosis; proteomics; secretory autophagy; ubiquitin carboxyl-terminal hydrolase 19; unconventional secretion

DOI:  https://doi.org/10.1016/j.mcpro.2024.100854
Elife. 2024 Oct 09. pii: e98363. [Epub ahead of print]13

Pathogenic Huntingtin aggregates alter actin organization and cellular stiffness resulting in stalled clathrin mediated endocytosis.

Surya Bansi Singh, Shatruhan Singh Rajput, Aditya Sharma, Sujal Kataria, Priyanka Dutta, Vaishnavi Ananthanarayanan, Amitabha Nandi, Shivaprasad Patil, Amitabha Majumdar, Deepa Subramanyam.

  Aggregation of mutant forms of Huntingtin is the underlying feature of neurodegeneration observed in Huntington's disorder. In addition to neurons, cellular processes in non-neuronal cell types are also shown to be affected. Cells expressing neurodegeneration-associated mutant proteins show altered uptake of ligands, suggestive of impaired endocytosis, in a manner as yet unknown. Using live cell imaging, we show that clathrin-mediated endocytosis (CME) is affected in Drosophila hemocytes and mammalian cells containing Huntingtin aggregates. This is also accompanied by alterations in the organization of the actin cytoskeleton resulting in increased cellular stiffness. Further, we find that Huntingtin aggregates sequester actin and actin-modifying proteins. Overexpression of Hip1 or Arp3 (actin-interacting proteins) could restore CME and cellular stiffness in cells containing Huntingtin aggregates. Neurodegeneration driven by pathogenic Huntingtin was also rescued upon overexpression of either Hip1 or Arp3 in Drosophila. Examination of other pathogenic aggregates revealed that TDP-43 also displayed defective CME, altered actin organization and increased stiffness, similar to pathogenic Huntingtin. Together, our results point to an intimate connection between dysfunctional CME, actin misorganization and increased cellular stiffness caused by alteration in the local intracellular environment by pathogenic aggregates.

Keywords:  D. melanogaster; cell biology

DOI:  https://doi.org/10.7554/eLife.98363
Mol Med. 2024 Oct 10. 30(1): 173

Drp1 acetylation mediated by CDK5-AMPK-GCN5L1 axis promotes cerebral ischemic injury via facilitating mitochondrial fission.

Jiejie Zhang, Shan Wang, Haitao Zhang, Xiaotong Yang, Xin Ren, Lei Wang, Yihan Yang, Yi Yang, Ya Wen.

  The aberrant acetylation of mitochondrial proteins is involved in the pathogenesis of multiple diseases including neurodegenerative diseases and cerebral ischemic injury. Previous studies have shown that depletion of mitochondrial NAD+, which is necessary for mitochondrial deacetylase activity, leads to decreased activity of mitochondrial deacetylase and thus causes hyperacetylation of mitochondrial proteins in ischemic brain tissues, which results in altered mitochondrial dynamics. However, it remains largely unknown about how mitochondrial dynamics-related protein Drp1 is acetylated in ischemic neuronal cells and brain tissues. Here, we showed that Drp1 and GCN5L1 expression was up-regulated in OGD-treated neuronal cells and ischemic brain tissues induced by dMCAO, accompanied by the increased mitochondrial fission, mtROS accumulation, and cell apoptosis. Further, we confirmed that ischemia/hypoxia promoted Drp1 interaction with GCN5L1 in neuronal cells and brain tissues. GCN5L1 knockdown attenuated, while its overexpression enhanced Drp1 acetylation and mitochondrial fission, indicating that GCN5L1 plays a crucial role in ischemia/hypoxia-induced mitochondrial fission by acetylating Drp1. Mechanistically, ischemia/hypoxia induced Drp1 phosphorylation by CDK5 upregulation-mediated activation of AMPK in neuronal cells, which in turn facilitated the interaction of GCN5L1 with Drp1, thus enhancing Drp1 acetylation and mitochondrial fission. Accordingly, inhibition of AMPK alleviated ischemia/hypoxia- induced Drp1 acetylation and mitochondrial fission and protected brain tissues from ischemic damage. These findings provide a novel insight into the functional roles of GCN5L1 in regulating Drp1 acetylation and identify a previously unrecognized CDK5-AMPK-GCN5L1 pathway that mediates the acetylation of Drp1 in ischemic brain tissues.

Keywords:  AMPK; Acetylation; CDK5; Drp1; GCN5L1; Ischemic stroke; Mitochondrial fission; Neuronal cells

DOI:  https://doi.org/10.1186/s10020-024-00948-y
Nat Biotechnol. 2024 Oct 09.

One-step nanoscale expansion microscopy reveals individual protein shapes.

Ali H Shaib, Abed Alrahman Chouaib, Rajdeep Chowdhury, Jonas Altendorf, Daniel Mihaylov, Chi Zhang, Donatus Krah, Vanessa Imani, Russell K W Spencer, Svilen Veselinov Georgiev, Nikolaos Mougios, Mehar Monga, Sofiia Reshetniak, Tiago Mimoso, Han Chen, Parisa Fatehbasharzad, Dagmar Crzan, Kim-Ann Saal, Mohamad Mahdi Alawieh, Nadia Alawar, Janna Eilts, Jinyoung Kang, Alireza Soleimani, Marcus Müller, Constantin Pape, Luis Alvarez, Claudia Trenkwalder, Brit Mollenhauer, Tiago F Outeiro, Sarah Köster, Julia Preobraschenski, Ute Becherer, Tobias Moser, Edward S Boyden, A Radu Aricescu, Markus Sauer, Felipe Opazo, Silvio O Rizzoli.

The attainable resolution of fluorescence microscopy has reached the subnanometer range, but this technique still fails to image the morphology of single proteins or small molecular complexes. Here, we expand the specimens at least tenfold, label them with conventional fluorophores and image them with conventional light microscopes, acquiring videos in which we analyze fluorescence fluctuations. One-step nanoscale expansion (ONE) microscopy enables the visualization of the shapes of individual membrane and soluble proteins, achieving around 1-nm resolution. We show that conformational changes are readily observable, such as those undergone by the ~17-kDa protein calmodulin upon Ca2+ binding. ONE is also applied to clinical samples, analyzing the morphology of protein aggregates in cerebrospinal fluid from persons with Parkinson disease, potentially aiding disease diagnosis. This technology bridges the gap between high-resolution structural biology techniques and light microscopy, providing new avenues for discoveries in biology and medicine.

DOI: https://doi.org/10.1038/s41587-024-02431-9
Front Genet. 2024 ;15 1440583

Cell type specification and diversity in subpallial organoids.

Narciso Pavon, Yubing Sun, ChangHui Pak.

  Neural organoids have emerged as valuable tools for studying the developing brain, sparking enthusiasm and driving their adoption in disease modeling, drug screening, and investigating fetal neural development. The increasing popularity of neural organoids as models has led to a wide range of methodologies aimed at continuous improvement and refinement. Consequently, research groups often improve and reconfigure protocols to create region-specific organoids, resulting in diverse phenotypes, including variations in morphology, gene expression, and cell populations. While these improvements are exciting, routine adoptions of such modifications and protocols in the research laboratories are often challenging due to the reiterative empirical testing necessary to validate the cell types generated. To address this challenge, we systematically compare the similarities and differences that exist across published protocols that generates subpallial-specific organoids to date. In this review, we focus specifically on exploring the production of major GABAergic neuronal subtypes, especially Medium Spiny Neurons (MSNs) and Interneurons (INs), from multiple subpallial organoid protocols. Importantly, we look to evaluate the cell type diversity and the molecular pathways manipulated to generate them, thus broadening our understanding of the existing subpallial organoids as well as assessing the in vitro applicability of specific patterning factors. Lastly, we discuss the current challenges and outlook on the improved patterning of region-specific neural organoids. Given the critical roles MSN and IN dysfunction play in neurological disorders, comprehending the GABAergic neurons generated by neural organoids will undoubtedly facilitate clinical translation.

Keywords:  MSNs; brain region-specific organoids; fgf; iPSCs; neural organoids; patterning; shh; subpallium

DOI:  https://doi.org/10.3389/fgene.2024.1440583
Nat Commun. 2024 Oct 09. 15(1): 8738

Stick-slip unfolding favors self-association of expanded HTT mRNA.

Brett M O'Brien, Roumita Moulick, Gabriel Jiménez-Avalos, Nandakumar Rajasekaran, Christian M Kaiser, Sarah A Woodson.

In Huntington's Disease (HD) and related disorders, expansion of CAG trinucleotide repeats produces a toxic gain of function in affected neurons. Expanded huntingtin (expHTT) mRNA forms aggregates that sequester essential RNA binding proteins, dysregulating mRNA processing and translation. The physical basis of RNA aggregation has been difficult to disentangle owing to the heterogeneous structure of the CAG repeats. Here, we probe the folding and unfolding pathways of expHTT mRNA using single-molecule force spectroscopy. Whereas normal HTT mRNAs unfold reversibly and cooperatively, expHTT mRNAs with 20 or 40 CAG repeats slip and unravel non-cooperatively at low tension. Slippage of CAG base pairs is punctuated by concerted rearrangement of adjacent CCG trinucleotides, trapping partially folded structures that readily base pair with another RNA strand. We suggest that the conformational entropy of the CAG repeats, combined with stable CCG base pairs, creates a stick-slip behavior that explains the aggregation propensity of expHTT mRNA.

DOI: https://doi.org/10.1038/s41467-024-52764-x
Biochemistry. 2024 Oct 10.

Delivery Aspects for Implementing siRNA Therapeutics for Blood Diseases.

Saba Abbasi Dezfouli, Marcel E Michailides, Hasan Uludag.

  Hematological disorders result in significant health consequences, and traditional therapies frequently entail adverse reactions without addressing the root cause. A potential solution for hematological disorders characterized by gain-of-function mutations lies in the emergence of small interfering RNA (siRNA) molecules as a therapeutic option. siRNAs are a class of RNA molecules composed of double-stranded RNAs that can degrade specific mRNAs, thereby inhibiting the synthesis of underlying disease proteins. Therapeutic interventions utilizing siRNA can be tailored to selectively target genes implicated in diverse hematological disorders, including sickle cell anemia, β-thalassemia, and malignancies such as lymphoma, myeloma, and leukemia. The development of efficient siRNA silencers necessitates meticulous contemplation of variables such as the RNA backbone, stability, and specificity. Transportation of siRNA to specific cells poses a significant hurdle, prompting investigations of diverse delivery approaches, including chemically modified forms of siRNA and nanoparticle formulations with various biocompatible carriers. This review delves into the crucial role of siRNA technology in targeting and treating hematological malignancies and disorders. It sheds light on the latest research, development, and clinical trials, detailing how various pharmaceutical approaches leverage siRNA against blood disorders, mainly concentrating on cancers. It outlines the preferred molecular targets and physiological barriers to delivery while emphasizing the growing potential of various therapeutic delivery methods. The need for further research is articulated in the context of overcoming the shortcomings of siRNA in order to enrich discussions around siRNA's role in managing blood disorders and aiding the scientific community in advancing more targeted and effective treatments.

Keywords:  Hematological malignancies; RNAi; delivery systems; leukemia; siRNA

DOI:  https://doi.org/10.1021/acs.biochem.4c00327
Sci Rep. 2024 10 10. 14(1): 23782

TBC1D15-regulated mitochondria-lysosome membrane contact exerts neuroprotective effects by alleviating mitochondrial calcium overload in seizure.

Yinyin Xie, Wanwan Zhang, Tingting Peng, Xiaoyi Wang, Xiaolei Lian, Jiao He, Cui Wang, Nanchang Xie.

  Mitochondrial calcium overload plays an important role in the neurological insults in seizure. The Rab7 GTPase-activating protein, Tre-2/Bub2/Cdc16 domain family member 15 (TBC1D15), is involved in the regulation of mitochondrial calcium dynamics by mediating mitochondria-lysosome membrane contact. However, whether TBC1D15-regulated mitochondria-lysosome membrane contact and mitochondrial calcium participate in neuronal injury in seizure is unclear. We aimed to investigate the effect of TBC1D15-regulated mitochondria-lysosome membrane contact on epileptiform discharge-induced neuronal damage and further explore the underlying mechanism. Lentiviral vectors (Lv) infection and stereotaxic adeno-associated virus (AAV) injection were used to regulate TBC1D15 expression before establishing in vitro epileptiform discharge and in vivo status epilepticus (SE) models. TBC1D15's effect on inter-organellar interactions, mitochondrial calcium levels and neuronal injury in seizure was evaluated. The results showed that abnormalities in mitochondria-lysosome membrane contact, mitochondrial calcium overload, mitochondrial dysfunction, increased levels of reactive oxygen species, and prominent neuronal damage were partly relieved by TBC1D15 overexpression, whereas TBC1D15 knockdown markedly deteriorated these phenomena. Further examination revealed that epileptiform discharge-induced mitochondrial calcium overload in primary hippocampal neurons was closely associated with abnormal mitochondria-lysosome membrane contact. This study highlights the crucial role played by TBC1D15-regulated mitochondria-lysosome membrane contact in epileptiform discharge-induced neuronal injury by alleviating mitochondrial calcium overload.

Keywords:  Mitochondrial calcium overload; Mitochondria–lysosome membrane contact; Seizure; TBC1D15

DOI:  https://doi.org/10.1038/s41598-024-74388-3