bims-axbals 2024-10-20 papers

bims-axbals

Biomed News

on Axonal Biology and ALS

Issue of 2024‒10‒20
24 papers selected by
TJ Krzystek, ALS Therapy Development Institute

A guide to selecting high-performing antibodies for CSNK2A1 (UniProt ID: P68400) for use in western blot, immunoprecipitation and immunofluorescence.
TDP43 aggregation at ER-exit sites impairs ER-to-Golgi transport.
Ataxin-2 polyglutamine expansions aberrantly sequester TDP-43 ribonucleoprotein condensates disrupting mRNA transport and local translation in neurons.
Multiple System Atrophy: Pathology, Pathogenesis, and Path Forward.
How is Excitotoxicity Being Modelled in iPSC-Derived Neurons?
A Hybrid 2D-to-3D in vitro Differentiation Platform Improves Outcomes of Cerebral Cortical Organoid Generation in hiPSCs.
Three-Dimensional iPSC-Based In Vitro Cardiac Models for Biomedical and Pharmaceutical Research Applications.
Plasma membrane repair defect in Alzheimer's disease neurons is driven by the reduced dysferlin expression.
Autophagy, aging, and age-related neurodegeneration.
Neuronal lineage tracing from progenitors in human cortical organoids reveals mechanisms of neuronal production, diversity, and disease.
Altered muscle cholesterol transport in ALS.
A novel ER stress regulator ARL6IP5 induces reticulophagy to ameliorate the prion burden.
Mitophagy as a guardian against cellular aging.
In vivo imaging in mouse spinal cord reveals that microglia prevent degeneration of injured axons.
Subtype-specific neurons from patient iPSCs display distinct neuropathological features of Alzheimer's disease.
Autophagy adaptors mediate Parkin-dependent mitophagy by forming sheet-like liquid condensates.
RNA Binding Protein Dysfunction Links Smoldering/Slowly Expanding Lesions to Neurodegeneration in Multiple Sclerosis.
Unraveling autophagic imbalances and therapeutic insights in Mecp2-deficient models.
The Role of Sirtuin 1 in Ageing and Neurodegenerative Disease: A Molecular Perspective.
An update on autophagy disorders.
Alterations in the axon initial segment plasticity is involved in early pathogenesis in Alzheimer's disease.
Potential Use of Organoids in Regenerative Medicine.
Live-Cell Calcium Imaging in 3D Intestinal Organoids.
Molecular Imaging of Human Brain Organoids Using Mass Spectrometry.

F1000Res. 2024 ;13 781

A guide to selecting high-performing antibodies for CSNK2A1 (UniProt ID: P68400) for use in western blot, immunoprecipitation and immunofluorescence.

NeuroSGC/YCharOS/EDDU collaborative group

  Casein kinase II subunit alpha (CSNK2A1), a serine/threonine kinase, phosphorylates multiple protein substrates and is involved in diverse cellular and biological processes. Implicated in various human diseases, high-performing antibodies would help evaluate its potential as a therapeutic target and benefit the scientific community. In this study, we have characterized ten CSNK2A1 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:  CSNK2A1; Casein kinase II subunit alpha; UniProt ID P68400; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation; western blot

DOI:  https://doi.org/10.12688/f1000research.153243.2
Nat Commun. 2024 Oct 19. 15(1): 9026

TDP43 aggregation at ER-exit sites impairs ER-to-Golgi transport.

Hongyi Wu, Loo Chien Wang, Belle M Sow, Damien Leow, Jin Zhu, Kathryn M Gallo, Kathleen Wilsbach, Roshni Gupta, Lyle W Ostrow, Crystal J J Yeo, Radoslaw M Sobota, Rong Li.

Protein aggregation plays key roles in age-related degenerative diseases, but how different proteins coalesce to form inclusions that vary in composition, morphology, molecular dynamics and confer physiological consequences is poorly understood. Here we employ a general reporter based on mutant Hsp104 to identify proteins forming aggregates in human cells under common proteotoxic stress. We identify over 300 proteins that form different inclusions containing subsets of aggregating proteins. In particular, TDP43, implicated in Amyotrophic Lateral Sclerosis (ALS), partitions dynamically between two distinct types of aggregates: stress granule and a previously unknown non-dynamic (solid-like) inclusion at the ER exit sites (ERES). TDP43-ERES co-aggregation is induced by diverse proteotoxic stresses and observed in the motor neurons of ALS patients. Such aggregation causes retention of secretory cargos at ERES and therefore delays ER-to-Golgi transport, providing a link between TDP43 aggregation and compromised cellular function in ALS patients.

DOI: https://doi.org/10.1038/s41467-024-52706-7
Dev Cell. 2024 Oct 12. pii: S1534-5807(24)00572-0. [Epub ahead of print]

Ataxin-2 polyglutamine expansions aberrantly sequester TDP-43 ribonucleoprotein condensates disrupting mRNA transport and local translation in neurons.

Denethi Wijegunawardana, Asima Nayak, Sonali S Vishal, Neha Venkatesh, Pallavi P Gopal.

  Altered RNA metabolism and misregulation of transactive response DNA-binding protein of 43 kDa (TDP-43), an essential RNA-binding protein (RBP), define amyotrophic lateral sclerosis (ALS). Intermediate-length polyglutamine (polyQ) expansions of Ataxin-2, a like-Sm (LSm) RBP, are associated with increased risk for ALS, but the underlying biological mechanisms remain unknown. Here, we studied the spatiotemporal dynamics and mRNA regulatory functions of TDP-43 and Ataxin-2 ribonucleoprotein (RNP) condensates in rodent (rat) primary cortical neurons and mouse motor neuron axons in vivo. We report that Ataxin-2 polyQ expansions aberrantly sequester TDP-43 within RNP condensates and disrupt both its motility along the axon and liquid-like properties. We provide evidence that Ataxin-2 governs motility and translation of neuronal RNP condensates and that Ataxin-2 polyQ expansions fundamentally perturb spatial localization of mRNA and suppress local translation. Overall, our results support a model in which Ataxin-2 polyQ expansions disrupt stability, localization, and/or translation of critical axonal and cytoskeletal mRNAs, particularly important for motor neuron integrity.

Keywords:  Ataxin-2; TDP-43; amyotrophic lateral sclerosis; axonal transport; liquid-liquid phase separation; local translation; mRNA localization; neuronal cell biology; neuronal transport granules; ribonucleoprotein condensates

DOI:  https://doi.org/10.1016/j.devcel.2024.09.023
Annu Rev Pathol. 2024 Oct 15.

Multiple System Atrophy: Pathology, Pathogenesis, and Path Forward.

Alain Ndayisaba, Glenda M Halliday, Vikram Khurana.

Multiple system atrophy (MSA) is a fatal neurodegenerative disease characterized by autonomic failure and motor impairment. The hallmark pathologic finding in MSA is widespread oligodendroglial cytoplasmic inclusions rich in aggregated α-synuclein (αSyn). MSA is widely held to be an oligodendroglial synucleinopathy, and we outline lines of evidence to support this assertion, including the presence of early myelin loss. We consider emerging data that support the possibility of neuronal or immune dysfunction as primary drivers of MSA. These hypotheses are placed in the context of a major recent discovery that αSyn is conformationally distinct in MSA versus other synucleinopathies such as Parkinson's disease. We outline emerging techniques in epidemiology, genetics, and molecular pathology that will shed more light on this mysterious disease. We anticipate a future in which cutting-edge developments in personalized disease modeling, including with pluripotent stem cells, bridge mechanistic developments at the bench and real benefits at the bedside.

DOI: https://doi.org/10.1146/annurev-pathmechdis-051122-104528
Neurotox Res. 2024 Oct 15. 42(5): 43

How is Excitotoxicity Being Modelled in iPSC-Derived Neurons?

Jan L Cheng, Anthony L Cook, Jana Talbot, Sharn Perry.

  Excitotoxicity linked either to environmental causes (pesticide and cyanotoxin exposure), excitatory neurotransmitter imbalance, or to intrinsic neuronal hyperexcitability, is a pathological mechanism central to neurodegeneration in amyotrophic lateral sclerosis (ALS). Investigation of excitotoxic mechanisms using in vitro and in vivo animal models has been central to understanding ALS mechanisms of disease. In particular, advances in induced pluripotent stem cell (iPSC) technologies now provide human cell-based models that are readily amenable to environmental and network-based excitotoxic manipulations. The cell-type specific differentiation of iPSC, combined with approaches to modelling excitotoxicity that include editing of disease-associated gene variants, chemogenetics, and environmental risk-associated exposures make iPSC primed to examine gene-environment interactions and disease-associated excitotoxic mechanisms. Critical to this is knowledge of which neurotransmitter receptor subunits are expressed by iPSC-derived neuronal cultures being studied, how their activity responds to antagonists and agonists of these receptors, and how to interpret data derived from multi-parameter electrophysiological recordings. This review explores how iPSC-based studies have contributed to our understanding of ALS-linked excitotoxicity and highlights novel approaches to inducing excitotoxicity in iPSC-derived neurons to further our understanding of its pathological pathways.

Keywords:  AMPAR; Calcium; DREADDs; Glutamate; Kainic acid; NMDAR

DOI:  https://doi.org/10.1007/s12640-024-00721-3
Curr Protoc. 2024 Oct;4(10): e70022

A Hybrid 2D-to-3D in vitro Differentiation Platform Improves Outcomes of Cerebral Cortical Organoid Generation in hiPSCs.

Dosh Whye, Erika M Norabuena, Gayathri Rajaram Srinivasan, Delaney Wood, Taryn J Polanco, Nina R Makhortova, Mustafa Sahin, Elizabeth D Buttermore.

  Three-dimensional (3D) cerebral cortical organoids are popular in vitro cellular model systems widely used to study human brain development and disease, compared to traditional stem cell-derived methods that use two-dimensional (2D) monolayer cultures. Despite the advancements made in protocol development for cerebral cortical organoid derivation over the past decade, limitations due to biological, mechanistic, and technical variables remain in generating these complex 3D cellular systems. Building from our previously established differentiation system, we have made modifications to our existing 3D cerebral cortical organoid protocol that resolve several of these technical and biological challenges when working with diverse groups of human induced pluripotent stem cell (hiPSC) lines. This improved protocol blends a 2D monolayer culture format for the specification of neural stem cells and expansion of neuroepithelial progenitor cells with a 3D system for improved self-aggregation and subsequent organoid development. Furthermore, this "hybrid" approach is amenable to both an accelerated cerebral cortical organoid protocol as well as an alternative long-term differentiation protocol. In addition to establishing a hybrid technical format, this protocol also offers phenotypic and morphological characterization of stage-specific cellular profiles using antibodies and fluorescent-based dyes for live cell imaging. © 2024 Wiley Periodicals LLC. Basic Protocol 1: hiPSC-based 2D monolayer specification into neural stem cells (NSCs) Basic Protocol 2: Serial passaging and 2D monolayer expansion of neuroepithelial progenitor cells (NPCs) Support Protocol 1: Direct cryopreservation and rapid thawing of NSCs and NPCs Basic Protocol 3: Bulk aggregation of 3D neurospheres and accelerated cerebral cortical organoid differentiation Alternate Protocol 1: Bulk aggregation of 3D neurospheres and long-term cerebral cortical organoid differentiation Support Protocol 2: High-throughput 3D neurosphere formation and 2D neurosphere migration assay Support Protocol 3: LIVE/DEAD stain cell imaging assay of 3D neurospheres Support Protocol 4: NeuroFluor NeuO live cell dye for 3D cerebral cortical organoids.

Keywords:  cerebral organoids; cortical organoids; human induced pluripotent stem cells; neural stem cells; neurospheres

DOI:  https://doi.org/10.1002/cpz1.70022
Int J Mol Sci. 2024 Oct 04. pii: 10690. [Epub ahead of print]25(19):

Three-Dimensional iPSC-Based In Vitro Cardiac Models for Biomedical and Pharmaceutical Research Applications.

Simona Bufi, Rosaria Santoro.

  Cardiovascular diseases are a major cause of death worldwide. Advanced in vitro models can be the key stone for a better understanding of the mechanisms at the basis of the different pathologies, supporting the development of novel therapeutic protocols. In particular, the implementation of induced pluripotent stem cell (iPSC) technology allows for the generation of a patient-specific pluripotent cell line that is able to differentiate in several organ-specific cell subsets while retaining the patient genetic background, thus putting the basis for personalized in vitro modeling toward personalized medicine. The design of iPSC-based models able to recapitulate the complexity of the cardiac environment is a critical goal. Here, we review some of the published efforts to exploit three dimensional (3D) iPSC-based methods to recapitulate the relevant cardiomyopathies, including genetically and non-genetically determined cardiomyopathies and cardiotoxicity studies. Finally, we discuss the actual method limitations and the future perspectives in the field.

Keywords:  cardiac; cardiomyopathy; engineered heart tissue; in vitro modeling; microtissue; organoid

DOI:  https://doi.org/10.3390/ijms251910690
FASEB J. 2024 Oct 31. 38(20): e70099

Plasma membrane repair defect in Alzheimer's disease neurons is driven by the reduced dysferlin expression.

Hannah R Bulgart, Miguel A Lopez Perez, Alexis Tucker, Gianni N Giarrano, Kassidy Banford, Olivia Miller, Sidney W G Bonser, Loren E Wold, Douglas Scharre, Noah Weisleder.

  Alzheimer's disease (AD) is the most common neurodegenerative disease, and a defect in neuronal plasma membrane repair could exacerbate neurotoxicity, neuronal death, and disease progression. In this study, application of AD patient cerebrospinal fluid (CSF) and recombinant human Aβ to otherwise healthy neurons induces defective neuronal plasma membrane repair in vitro and ex vivo. We identified Aβ as the biochemical component in patient CSF leading to compromised repair capacity and depleting Aβ rescued repair capacity. These elevated Aβ levels reduced expression of dysferlin, a protein that facilitates membrane repair, by altering autophagy and reducing dysferlin trafficking to sites of membrane injury. Overexpression of dysferlin and autophagy inhibition rescued membrane repair. Overall, these findings indicate an AD pathogenic mechanism where Aβ impairs neuronal membrane repair capacity and increases susceptibility to cell death. This suggests that membrane repair could be therapeutically targeted in AD to restore membrane integrity and reduce neurotoxicity and neuronal death.

Keywords:  Alzheimer's disease; autophagy; dysferlin; membrane repair; neurodegeneration

DOI:  https://doi.org/10.1096/fj.202401731RR
Neuron. 2024 Oct 08. pii: S0896-6273(24)00663-9. [Epub ahead of print]

Autophagy, aging, and age-related neurodegeneration.

Jennifer E Palmer, Niall Wilson, Sung Min Son, Pawel Obrocki, Lidia Wrobel, Matea Rob, Michael Takla, Viktor I Korolchuk, David C Rubinsztein.

  Autophagy is a conserved mechanism that degrades damaged or superfluous cellular contents and enables nutrient recycling under starvation conditions. Many neurodegeneration-associated proteins are autophagy substrates, and autophagy upregulation ameliorates disease in many animal models of neurodegeneration by enhancing the clearance of toxic proteins, proinflammatory molecules, and dysfunctional organelles. Autophagy inhibition also induces neuronal and glial senescence, a phenomenon that occurs with increasing age in non-diseased brains as well as in response to neurodegeneration-associated stresses. However, aging and many neurodegeneration-associated proteins and mutations impair autophagy. This creates a potentially detrimental feedback loop whereby the accumulation of these disease-associated proteins impairs their autophagic clearance, facilitating their further accumulation and aggregation. Thus, understanding how autophagy interacts with aging, senescence, and neurodegenerative diseases in a temporal, cellular, and genetic context is important for the future clinical application of autophagy-modulating therapies in aging and neurodegeneration.

Keywords:  Alzheimer’s disease; Huntington’s disease; Parkinson’s disease; aging; autophagy; frontotemporal dementia; motor neuron disease; neurodegeneration; senescence

DOI:  https://doi.org/10.1016/j.neuron.2024.09.015
Cell Rep. 2024 Oct 11. pii: S2211-1247(24)01213-0. [Epub ahead of print]43(10): 114862

Neuronal lineage tracing from progenitors in human cortical organoids reveals mechanisms of neuronal production, diversity, and disease.

Luke A D Bury, Shuai Fu, Anthony Wynshaw-Boris.

  The contribution of progenitor subtypes to generating the billions of neurons produced during human cortical neurogenesis is not well understood. We developed the cortical organoid lineage-tracing (COR-LT) system for human cortical organoids. Differential fluorescent reporter activation in distinct progenitor cells leads to permanent reporter expression, enabling the progenitor cell lineage of neurons to be determined. Surprisingly, nearly all excitatory neurons produced in cortical organoids were generated indirectly from intermediate progenitor cells. Additionally, neurons of different progenitor lineages were transcriptionally distinct. Isogenic lines made from an autistic individual with and without a likely pathogenic CTNNB1 variant demonstrated that the variant substantially altered the proportion of neurons derived from specific progenitor cell lineages, as well as the lineage-specific transcriptional profiles of these neurons, suggesting a pathogenic mechanism for this mutation. These results suggest individual progenitor subtypes play roles in generating the diverse neurons of the human cerebral cortex.

Keywords:  CP: Stem cell research; PTEN; autism spectrum disorder; beta-catenin; genetic lineage tracing; human neurogenesis; induced pluripotent stem cells; organoids

DOI:  https://doi.org/10.1016/j.celrep.2024.114862
Nat Rev Neurol. 2024 Oct 14.

Altered muscle cholesterol transport in ALS.

Heather Wood.

DOI: https://doi.org/10.1038/s41582-024-01029-8
Autophagy. 2024 Oct 12.

A novel ER stress regulator ARL6IP5 induces reticulophagy to ameliorate the prion burden.

Kajal Kamble, Ujjwal Kumar, Harsh Aahra, Mohit Yadav, Sumnil Bhola, Sarika Gupta.

  Prion disease is a fatal and infectious neurodegenerative disorder caused by the trans-conformation conversion of PRNP/PrPC to PRNP/PrPSc. Accumulated PRNP/PrPSc-induced ER stress causes chronic unfolded protein response (UPR) activation, which is one of the fundamental steps in prion disease progression. However, the role of various ER-resident proteins in prion-induced ER stress is elusive. This study demonstrated that ARL6IP5 is compensatory upregulated in response to chronically activated UPR in the cellular prion disease model (RML-ScN2a). Furthermore, overexpression of ARL6IP5 overcomes ER stress by lowering the expression of chronically activated UPR pathway proteins. We discovered that ARL6IP5 induces reticulophagy to reduce the PRNP/PrPSc burden by releasing ER stress. Conversely, the knockdown of ARL6IP5 leads to inefficient macroautophagic/autophagic flux and elevated PRNP/PrPSc burden. Our study also uncovered that ARL6IP5-induced reticulophagy depends on Ca2+-mediated AMPK activation and can induce 3 MA-inhibited autophagic flux. The detailed mechanistic study revealed that ARL6IP5-induced reticulophagy involves interaction with soluble reticulophagy receptor CALCOCO1 and lysosomal marker LAMP1, leading to degradation in lysosomes. Here, we delineate the role of ARL6IP5 as a novel ER stress regulator and reticulophagy inducer that can effectively reduce the misfolded PRNP/PrPSc burden. Our research opens up a new avenue of selective autophagy in prion disease and represents a potential therapeutic target.

Keywords:  Autophagy; ER stress; Reticulophagy/er-phagy; prion burden/PrPSc burden; prion disease

DOI:  https://doi.org/10.1080/15548627.2024.2410670
Autophagy. 2024 Oct 14. 1-3

Mitophagy as a guardian against cellular aging.

Tetsushi Kataura, Niall Wilson, Gailing Ma, Viktor I Korolchuk.

  Mitophagy, the selective autophagic clearance of damaged mitochondria, is considered vital for maintaining mitochondrial quality and cellular homeostasis; however, its molecular mechanisms, particularly under basal conditions, and its role in cellular physiology remain poorly characterized. We recently demonstrated that basal mitophagy is a key feature of primary human cells and is downregulated by immortalization, suggesting its dependence on the primary cell state. Mechanistically, we demonstrated that the PINK1-PRKN-SQSTM1 pathway regulates basal mitophagy, with SQSTM1 sensing superoxide-enriched mitochondria through its redox-sensitive cysteine residues, which mediate SQSTM1 oligomerization and mitophagy activation. We developed STOCK1N-57534, a small molecule that targets and promotes this SQSTM1 activation mechanism. Treatment with STOCK1N-57534 reactivates mitophagy downregulated in senescent and naturally aged donor-derived primary cells, improving cellular senescence(-like) phenotypes. Our findings highlight that basal mitophagy is protective against cellular senescence and aging, positioning its pharmacological reactivation as a promising anti-aging strategy.Abbreviation: IR: ionizing radiation; ROS: reactive oxygen species; SARs: selective autophagy receptors.

Keywords:  Aging; SQSTM1/p62; autophagy; mitochondria; mitophagy; senescence

DOI:  https://doi.org/10.1080/15548627.2024.2414461
Nat Commun. 2024 Oct 13. 15(1): 8837

In vivo imaging in mouse spinal cord reveals that microglia prevent degeneration of injured axons.

Wanjie Wu, Yingzhu He, Yujun Chen, Yiming Fu, Sicong He, Kai Liu, Jianan Y Qu.

Microglia, the primary immune cells in the central nervous system, play a critical role in regulating neuronal function and fate through their interaction with neurons. Despite extensive research, the specific functions and mechanisms of microglia-neuron interactions remain incompletely understood. In this study, we demonstrate that microglia establish direct contact with myelinated axons at Nodes of Ranvier in the spinal cord of mice. The contact associated with neuronal activity occurs in a random scanning pattern. In response to axonal injury, microglia rapidly transform their contact into a robust wrapping form, preventing acute axonal degeneration from extending beyond the nodes. This wrapping behavior is dependent on the function of microglial P2Y12 receptors, which may be activated by ATP released through axonal volume-activated anion channels at the nodes. Additionally, voltage-gated sodium channels (NaV) and two-pore-domain potassium (K2P) channels contribute to the interaction between nodes and glial cells following injury, and inhibition of NaV delays axonal degeneration. Through in vivo imaging, our findings reveal a neuroprotective role of microglia during the acute phase of single spinal cord axon injury, achieved through neuron-glia interaction.

DOI: https://doi.org/10.1038/s41467-024-53218-0
Cell Regen. 2024 Oct 10. 13(1): 21

Subtype-specific neurons from patient iPSCs display distinct neuropathological features of Alzheimer's disease.

Ran Tao, Chunmei Yue, Zhijie Guo, Wenke Guo, Yao Yao, Xianfa Yang, Zhen Shao, Chao Gao, Jianqing Ding, Lu Shen, Shengdi Chen, Naihe Jing.

  Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by massive neuronal loss in the brain. Both cortical glutamatergic neurons and basal forebrain cholinergic neurons (BFCNs) in the AD brain are selectively vulnerable. The degeneration and dysfunction of these two subtypes of neurons are closely associated with the cognitive decline of AD patients. The determination of cellular and molecular mechanisms involved in AD pathogenesis, especially in the early stage, will largely facilitate the understanding of this disease and the development of proper intervention strategies. However, due to the inaccessibility of living neurons in the brains of patients, it remains unclear how cortical glutamatergic neurons and BFCNs respond to pathological stress in the early stage of AD. In this study, we established in vitro differentiation systems that can efficiently differentiate patient-derived iPSCs into BFCNs. We found that AD-BFCNs secreted less Aβ peptide than cortical glutamatergic neurons did, even though the Aβ42/Aβ40 ratio was comparable to that of cortical glutamatergic neurons. To further mimic the neurotoxic niche in AD brain, we treated iPSC-derived neurons with Aβ42 oligomer (AβO). BFCNs are less sensitive to AβO induced tau phosphorylation and expression than cortical glutamatergic neurons. However, AβO could trigger apoptosis in both AD-cortical glutamatergic neurons and AD-BFCNs. In addition, AD iPSC-derived BFCNs and cortical glutamatergic neurons exhibited distinct electrophysiological firing patterns and elicited different responses to AβO treatment. These observations revealed that subtype-specific neurons display distinct neuropathological changes during the progression of AD, which might help to understand AD pathogenesis at the cellular level.

Keywords:  Alzheimer’s disease (AD); Basal forebrain cholinergic neuron (BFCN); Cellular model; Cortical glutamatergic neuron; iPSC

DOI:  https://doi.org/10.1186/s13619-024-00204-y
EMBO J. 2024 Oct 17.

Autophagy adaptors mediate Parkin-dependent mitophagy by forming sheet-like liquid condensates.

Zi Yang, Saori R Yoshii, Yuji Sakai, Jing Zhang, Haruka Chino, Roland L Knorr, Noboru Mizushima.

  During PINK1- and Parkin-mediated mitophagy, autophagy adaptors are recruited to damaged mitochondria to promote their selective degradation. Autophagy adaptors such as optineurin (OPTN) and NDP52 facilitate mitophagy by recruiting the autophagy-initiation machinery, and assisting engulfment of damaged mitochondria through binding to ubiquitinated mitochondrial proteins and autophagosomal ATG8 family proteins. Here, we demonstrate that OPTN and NDP52 form sheet-like phase-separated condensates with liquid-like properties on the surface of ubiquitinated mitochondria. The dynamic and liquid-like nature of OPTN condensates is important for mitophagy activity, because reducing the fluidity of OPTN-ubiquitin condensates suppresses the recruitment of ATG9 vesicles and impairs mitophagy. Based on these results, we propose a dynamic liquid-like, rather than a stoichiometric, model of autophagy adaptors to explain the interactions between autophagic membranes (i.e., ATG9 vesicles and isolation membranes) and mitochondrial membranes during Parkin-mediated mitophagy. This model underscores the importance of liquid-liquid phase separation in facilitating membrane-membrane contacts, likely through the generation of capillary forces.

Keywords:  Autophagy; Liquid–Liquid Phase Separation; Mitophagy; Optineurin; Wetting

DOI:  https://doi.org/10.1038/s44318-024-00272-5
Ann Neurol. 2024 Oct 18.

RNA Binding Protein Dysfunction Links Smoldering/Slowly Expanding Lesions to Neurodegeneration in Multiple Sclerosis.

Miranda L Messmer, Hannah E Salapa, Bogdan F Popescu, Michael C Levin.

OBJECTIVE: Despite the advances in treatments for multiple sclerosis (MS), unremitting neurodegeneration continues to drive disability and disease progression. Smoldering/slowly expanding lesions (SELs) and dysfunction of the RNA binding protein (RBP) heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) are pathologic hallmarks of MS cortex and intricately tied to disability and neurodegeneration, respectively. We hypothesized that neuronal hnRNP A1 dysfunction contributes to neurodegeneration and is exacerbated by smoldering/SELs in progressive MS.METHODS: Neuronal hnRNP A1 pathology (nucleocytoplasmic mislocalization of hnRNP A1) was examined in healthy control and MS brains using immunohistochemistry. MS cases were stratified by severity of hnRNP A1 pathology to examine the link between RBP dysfunction, demyelination, and neurodegeneration.
RESULTS: We found that smoldering/SELs were only present within a subset of MS tissues characterized by elevated neuronal hnRNP A1 pathology (MS-A1high) in adjacent cortical gray matter. In contrast to healthy controls and MS with low hnRNP A1 pathology (MS-A1low), MS-A1high showed elevated markers of neurodegeneration, including neuronal loss and injury, brain atrophy, axonal loss, and axon degeneration. Additionally, we discovered a subpopulation of morphologically intact neurons lacking expression of NeuN, a neuron-specific RBP, in cortical projection neurons in MS-A1high cases.
INTERPRETATION: hnRNP A1 dysfunction contributes to neurodegeneration and may be exacerbated by smoldering/SELs in progressive MS. The discovery of NeuN-negative neurons suggests that some cortical neurons may only be injured and not lost. By characterizing RBP pathology in MS cortex, this study has important implications for understanding the pathogenic mechanisms driving neurodegeneration, the substrate of disability and disease progression. ANN NEUROL 2024.

DOI: https://doi.org/10.1002/ana.27114
EMBO Mol Med. 2024 Oct 14.

Unraveling autophagic imbalances and therapeutic insights in Mecp2-deficient models.

Alessandro Esposito, Tommaso Seri, Martina Breccia, Marzia Indrigo, Giuseppina De Rocco, Francesca Nuzzolillo, Vanna Denti, Francesca Pappacena, Gaia Tartaglione, Simone Serrao, Giuseppe Paglia, Luca Murru, Stefano de Pretis, Jean-Michel Cioni, Nicoletta Landsberger, Fabrizia Claudia Guarnieri, Michela Palmieri.

  Loss-of-function mutations in MECP2 are associated to Rett syndrome (RTT), a severe neurodevelopmental disease. Mainly working as a transcriptional regulator, MeCP2 absence leads to gene expression perturbations resulting in deficits of synaptic function and neuronal activity. In addition, RTT patients and mouse models suffer from a complex metabolic syndrome, suggesting that related cellular pathways might contribute to neuropathogenesis. Along this line, autophagy is critical in sustaining developing neuron homeostasis by breaking down dysfunctional proteins, lipids, and organelles.Here, we investigated the autophagic pathway in RTT and found reduced content of autophagic vacuoles in Mecp2 knock-out neurons. This correlates with defective lipidation of LC3B, probably caused by a deficiency of the autophagic membrane lipid phosphatidylethanolamine. The administration of the autophagy inducer trehalose recovers LC3B lipidation, autophagosomes content in knock-out neurons, and ameliorates their morphology, neuronal activity and synaptic ultrastructure. Moreover, we provide evidence for attenuation of motor and exploratory impairment in Mecp2 knock-out mice upon trehalose administration. Overall, our findings open new perspectives for neurodevelopmental disorders therapies based on the concept of autophagy modulation.

Keywords:  Autophagy; MeCP2; Metabolism; Neurons; Rett Syndrome

DOI:  https://doi.org/10.1038/s44321-024-00151-w
Ageing Res Rev. 2024 Oct 16. pii: S1568-1637(24)00363-5. [Epub ahead of print] 102545

The Role of Sirtuin 1 in Ageing and Neurodegenerative Disease: A Molecular Perspective.

Riya Thapa, Ehssan Moglad, Muhammad Afzal, Gaurav Kumar, Asif Ahmad Bhat, Waleed Hassan Almalki, Imran Kazmi, Sami I Alzarea, Kumud Pant, Thakur Gurjeet Singh, Sachin Kumar Singh, Haider Ali.

  Sirtuin 1 (SIRT1), an NAD+-dependent deacetylase, has emerged as a key regulator of cellular processes linked to ageing and neurodegeneration. SIRT1 modulates various signalling pathways, including those involved in autophagy, oxidative stress, and mitochondrial function, which are critical in the pathogenesis of neurodegenerative diseases. This review explores the therapeutic potential of SIRT1 in several neurodegenerative disorders, including Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and Amyotrophic Lateral Sclerosis (ALS). Preclinical studies have demonstrated that SIRT1 activators, such as resveratrol, SRT1720, and SRT2104, can alleviate disease symptoms by reducing oxidative stress, enhancing autophagic flux, and promoting neuronal survival. Ongoing clinical trials are evaluating the efficacy of these SIRT1 activators, providing hope for future therapeutic strategies targeting SIRT1 in neurodegenerative diseases. This review explores the role of SIRT1 in ageing and neurodegenerative diseases, with a particular focus on its molecular mechanisms, therapeutic potential, and clinical applications.

Keywords:  Alzheimer's Disease; Cellular Homeostasis; Huntington's Disease; Neurodegenerative Diseases; Parkinson's Disease; Sirtuin 1

DOI:  https://doi.org/10.1016/j.arr.2024.102545
J Inherit Metab Dis. 2024 Oct 17.

An update on autophagy disorders.

Hormos Salimi Dafsari, Diego Martinelli, Afshin Saffari, Darius Ebrahimi-Fakhari, Manolis Fanto, Carlo Dionisi-Vici, Heinz Jungbluth.

  Macroautophagy is a highly conserved cellular pathway for the degradation and recycling of defective cargo including proteins, organelles, and macromolecular complexes. As autophagy is particularly relevant for cellular homeostasis in post-mitotic tissues, congenital disorders of autophagy, due to monogenic defects in key autophagy genes, share a common "clinical signature" including neurodevelopmental, neurodegenerative, and neuromuscular features, as well as variable abnormalities of the eyes, skin, heart, bones, immune cells, and other organ systems, depending on the expression pattern and the specific function of the defective proteins. Since the clinical and genetic resolution of EPG5-related Vici syndrome, the paradigmatic congenital disorder of autophagy, the widespread use of massively parallel sequencing has resulted in the identification of a growing number of autophagy-associated disease genes, encoding members of the core autophagy machinery as well as related proteins. Recently identified monogenic disorders linking selective autophagy, vesicular trafficking, and other pathways have further expanded the molecular and phenotypical spectrum of congenital disorders of autophagy as a clinical disease spectrum. Moreover, significant advances in basic research have enhanced the understanding of the underlying pathophysiology as a basis for therapy development. Here, we review (i) autophagy in the context of other intracellular trafficking pathways; (ii) the main congenital disorders of autophagy and their typical clinico-pathological signatures; and (iii) the recommended primary health surveillance in monogenic disorders of autophagy based on available evidence. We further discuss recently identified molecular mechanisms that inform the current understanding of autophagy in health and disease, as well as perspectives on future therapeutic approaches.

Keywords:  autophagy; cellular trafficking; congenital disorders; neurodegeneration; neurodevelopment

DOI:  https://doi.org/10.1002/jimd.12798
MedComm (2020). 2024 Nov;5(11): e768

Alterations in the axon initial segment plasticity is involved in early pathogenesis in Alzheimer's disease.

Yu Li, Han Wang, Yiming Wang, Zhiya Chen, Yiqiong Liu, Wu Tian, Xinrui Kang, Abolghasem Pashang, Don Kulasiri, Xiaoli Yang, Hung Wing Li, Yan Zhang.

  Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder, characterized by the early presence of amyloid-β (Aβ) and hyperphosphorylated tau. Identifying the neuropathological changes preceding cognitive decline is crucial for early intervention. Axon initial segment (AIS) maintains the orderly structure of the axon and is responsible for initiating action potentials (APs). To investigate the role of AIS in early stages of AD pathogenesis, we focused on alterations in the AIS of neurons from APP/PS1 mouse models harboring familial AD mutations. AIS length and electrophysiological properties were assessed in neurons using immunostaining and patch-clamp techniques. The expression and function of ankyrin G (AnkG) isoforms were evaluated by western blot and rescue experiments. We observed a significant shortening of AIS in APP/PS1 mice, which correlated with impaired action potential propagation. Furthermore, a decrease in the 480 kDa isoform of AnkG was observed. Rescue of this isoform restored AIS plasticity and improved long-term potentiation in APP/PS1 neurons. Our study implicates AIS plasticity alterations and AnkG dysregulation as early events in AD. The restoration of AIS integrity by the 480 kDa AnkG isoform presents a potential therapeutic strategy for AD, underscoring the importance of targeting AIS stability in neurodegenerative diseases.

Keywords:  Alzheimer's disease; ankyrin G; axon initial segment; plasticity

DOI:  https://doi.org/10.1002/mco2.768
Tissue Eng Regen Med. 2024 Oct 16.

Potential Use of Organoids in Regenerative Medicine.

Wahyunia L Septiana, Jeanne A Pawitan.

  BACKGROUND: In vitro cell culture is crucial for studying human diseases and development. Compared to traditional monolayer cultures, 3D culturing with organoids offers significant advantages by more accurately replicating natural tissues' structural and functional features. This advancement enhances disease modeling, drug testing, and regenerative medicine applications. Organoids, derived from stem cells, mimic tissue physiology in a more relevant manner. Despite their promise, the clinical use of regenerative medicine currently needs to be improved by reproducibility, scalability, and maturation issues.METHODS: This article overviews recent organoid research, focusing on their types, sources, 3D culturing methods, and applications in regenerative medicine. A literature review of "organoid" and "regenerative medicine" in PubMed/MEDLINE highlighted relevant studies published over the past decade, emphasizing human-sourced organoids and their regenerative benefits, as well as the availability of free full-text articles. The review uses descriptive data, including tables and text, to illustrate the challenges and potential of organoids in regenerative medicine.
RESULTS: The transition from 2D to 3D models, particularly organoids, has significantly advanced in vitro research. This review covers a decade of progress in various organoid types-such as liver, cholangiocyte, intestinal, pancreatic, cardiac, brain, thymus, and mammary organoids-and their 3D culture methods and applications. It addresses critical issues of maturity, scalability, and reproducibility and underscores the need for standardization and improved production techniques to facilitate broader clinical applications in regenerative medicine.
CONCLUSIONS: Successful therapy requires increased scalability and standardization. Organoids have enormous potential in biological research, notwithstanding obstacles.

Keywords:  3D cell culture; In vitro models; Organoids; Regenerative medicine; Stem cells; Tissue engineering

DOI:  https://doi.org/10.1007/s13770-024-00672-y
Methods Mol Biol. 2025 ;2861 213-221

Live-Cell Calcium Imaging in 3D Intestinal Organoids.

Eva Rath, Tamara Zietek.

  Live-cell Ca2+ imaging is an important tool to detect activation of receptors by a putative ligand/drug and complements studies on transport processes, as intracellular Ca2+ changes provide direct evidence for substrate fluxes. Organoid-based systems offer numerous advantages over other in vitro systems such as cell lines, primary cells, or tissue explants, and in particular, intestinal organoid culture has revolutionized research on functional gastrointestinal processes. Calcium imaging using the fluorescent Ca2+ indicator Fura-2-AM can be applied to 3D intestinal organoids, which show an excellent dye-loading efficiency. Here we describe live-cell Ca2+ imaging in intestinal organoids, an important technique to improve research on malabsorption syndromes, secretory diarrhea, and metabolic disorders.

Keywords:  3D-organoid culture; Calcium imaging; Drug transport; Incretin secretion; Intestinal organoids; Live-cell imaging; Nutrient absorption

DOI:  https://doi.org/10.1007/978-1-0716-4164-4_16
J Vis Exp. 2024 Sep 27.

Molecular Imaging of Human Brain Organoids Using Mass Spectrometry.

Saleh M Khalil, Gerarda Cappuccio, Feng Li, Mirjana Maletic-Savatic.

Brain organoid models serve as a powerful tool for studying human brain development and function. Mass spectrometry imaging (MSI), a cutting-edge technology, allows us to map the spatial distribution of diverse molecules such as lipids, sugars, amino acids, drugs, and their metabolites within these organoids, all without the need for specific molecular probes. High-quality MSI data hinge on meticulous sample preparation. Fixatives play a pivotal role, but conventional options such as glutaraldehyde, paraformaldehyde, and cryopreserving such as sucrose may inadvertently impact tissue metabolites. Optimal fixation entails flash freezing in liquid nitrogen. However, for small organoids, a more suitable approach involves transitioning the organoids directly from the incubator into a warmed embedding solution, followed by freezing in dry ice-cooled ethanol. Another critical step is the embedding prior to cryosectioning, which also requires materials compatible with MSI, as traditional options can interfere with matrix deposition and ionization. Here, an optimized protocol for high resolution-MALDI-MSI of human brain organoids is presented, encompassing sample preparation, sectioning, and imaging using mass spectrometry. This method showcases the molecular distribution of small metabolites, such as amino acids, with high mass accuracy and sensitivity. As such, coupled with complementary studies of brain organoids, it can assist in illuminating complex processes governing early brain development, metabolic cell fate trajectories, and distinctive metabolite signatures. Furthermore, it provides insights into the precise locations of molecules within the organoid, enriching our understanding of the spatial organization of 3D brain organoid models. As the field continues to advance, a growing number of studies leveraging MSI to delve into brain organoids and complex biological systems is anticipated, thereby deepening the understanding of the metabolic aspects of human brain function and development.

DOI: https://doi.org/10.3791/66997