bims-axbals 2024-08-11 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2024–08–11
27 papers selected by
TJ Krzystek, ALS Therapy Development Institute

Poly-GR repeats associated with ALS/FTD gene C9ORF72 impair translation elongation and induce a ribotoxic stress response in neurons.
Axonopathy Underlying Amyotrophic Lateral Sclerosis: Unraveling Complex Pathways and Therapeutic Insights.
Overexpression of Toxic Poly(Glycine-Alanine) Aggregates in Primary Neuronal Cultures Induces Time-Dependent Autophagic and Synaptic Alterations but Subtle Activity Impairments.
TDP-43 dysfunction leads to bioenergetic failure and lipid metabolic rewiring in human cells.
Generation of Human-Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells of C9ORF72-Associated Amyotrophic Lateral Sclerosis Patients.
DNAJA2 and Hero11 mediate similar conformational extension and aggregation suppression of TDP-43.
Stress granule formation helps to mitigate neurodegeneration.
Lysosomal TBK1 responds to amino acid availability to relieve Rab7-dependent mTORC1 inhibition.
Neonicotinoid Pesticides Affect Developing Neurons in Experimental Mouse Models and in Human Induced Pluripotent Stem Cell (iPSC)-Derived Neural Cultures and Organoids.
Modeling riboflavin transporter deficiency type 2: from iPSC-derived motoneurons to iPSC-derived astrocytes.
LRRK2 kinase activity is necessary for development and regeneration in Nematostella vectensis.
Wnt5a Promotes Axon Elongation in Coordination with the Wnt-Planar Cell Polarity Pathway.
Protocol for transplantation of cells derived from human midbrain organoids into a Parkinson's disease mouse model to restore motor function.
TBK1 p.Y153Qfs*9 variant may be associated with young-onset, rapidly progressive amyotrophic lateral sclerosis through a haploinsufficiency mechanism.
iPSC-Based Disease Modeling and Functional Assessment of Neurons in Patients with Metabolic Disorder.
Extracellular Vesicle Isolation and Mass Spectrometry-Based Proteomic Analysis in Arabidopsis thaliana.
From Young to Old: Mimicking Neuronal Aging in Directly Converted Neurons from Young Donors.
[Amyotrophic lateral sclerosis associated with a new pathogenic variant of the ERBB4 gene].
A decrease in Fkbp52 alters autophagosome maturation and A152T-tau clearance in vivo.
Mechanisms of autophagy-lysosome dysfunction in neurodegenerative diseases.
Identification of Neighboring Proteins of Endosomal Regulators by Using TurboID-Based Proximity Labeling.
Transcriptomics and weighted protein network analyses of the LRRK2 protein interactome reveal distinct molecular signatures for sporadic and LRRK2 Parkinson's Disease.
Nesprin-2 coordinates opposing microtubule motors during nuclear migration in neurons.
Methods for Monitoring ER-Phagy.
Multi-omic analysis of Huntington's disease reveals a compensatory astrocyte state.
Analysis of Mitophagy Reporters in Drosophila.
The Autophagy Paradox: A New Hypothesis in Neurodegenerative Disorders.

Sci Signal. 2024 Aug 06. 17(848): eadl1030

Poly-GR repeats associated with ALS/FTD gene C9ORF72 impair translation elongation and induce a ribotoxic stress response in neurons.

Daoyuan Dong, Zhe Zhang, Yini Li, Malgorzata J Latallo, Shaopeng Wang, Blake Nelson, Rong Wu, Gopinath Krishnan, Fen-Biao Gao, Bin Wu, Shuying Sun.

Hexanucleotide repeat expansion in the C9ORF72 gene is the most frequent inherited cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansion results in multiple dipeptide repeat proteins, among which arginine-rich poly-GR proteins are highly toxic to neurons and decrease the rate of protein synthesis. We investigated whether the effect on protein synthesis contributes to neuronal dysfunction and degeneration. We found that the expression of poly-GR proteins inhibited global translation by perturbing translation elongation. In iPSC-differentiated neurons, the translation of transcripts with relatively slow elongation rates was further slowed, and stalled, by poly-GR. Elongation stalling increased ribosome collisions and induced a ribotoxic stress response (RSR) mediated by ZAKα that increased the phosphorylation of the kinase p38 and promoted cell death. Knockdown of ZAKα or pharmacological inhibition of p38 ameliorated poly-GR-induced toxicity and improved the survival of iPSC-derived neurons from patients with C9ORF72-ALS/FTD. Our findings suggest that targeting the RSR may be neuroprotective in patients with ALS/FTD caused by repeat expansion in C9ORF72.

DOI: https://doi.org/10.1126/scisignal.adl1030
Neurosci Bull. 2024 Aug 04.

Axonopathy Underlying Amyotrophic Lateral Sclerosis: Unraveling Complex Pathways and Therapeutic Insights.

Tongshu Luan, Qing Li, Zhi Huang, Yu Feng, Duo Xu, Yujie Zhou, Yiqing Hu, Tong Wang.

  Amyotrophic Lateral Sclerosis (ALS) is a complex neurodegenerative disorder characterized by progressive axonopathy, jointly leading to the dying back of the motor neuron, disrupting both nerve signaling and motor control. In this review, we highlight the roles of axonopathy in ALS progression, driven by the interplay of multiple factors including defective trafficking machinery, protein aggregation, and mitochondrial dysfunction. Dysfunctional intracellular transport, caused by disruptions in microtubules, molecular motors, and adaptors, has been identified as a key contributor to disease progression. Aberrant protein aggregation involving TDP-43, FUS, SOD1, and dipeptide repeat proteins further amplifies neuronal toxicity. Mitochondrial defects lead to ATP depletion, oxidative stress, and Ca2+ imbalance, which are regarded as key factors underlying the loss of neuromuscular junctions and axonopathy. Mitigating these defects through interventions including neurotrophic treatments offers therapeutic potential. Collaborative research efforts aim to unravel ALS complexities, opening avenues for holistic interventions that target diverse pathological mechanisms.

Keywords:  Amyotrophic lateral sclerosis; Axon trafficking; Axonopathy; Mitochondrial defect; Neurotrophic factor; Protein aggregation

DOI:  https://doi.org/10.1007/s12264-024-01267-2
Cells. 2024 Aug 03. pii: 1300. [Epub ahead of print]13(15):

Overexpression of Toxic Poly(Glycine-Alanine) Aggregates in Primary Neuronal Cultures Induces Time-Dependent Autophagic and Synaptic Alterations but Subtle Activity Impairments.

Christina Steffke, Shreya Agarwal, Edor Kabashi, Alberto Catanese.

  The pathogenic expansion of the intronic GGGGCC hexanucleotide located in the non-coding region of the C9orf72 gene represents the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). This mutation leads to the accumulation of toxic RNA foci and dipeptide repeats (DPRs), as well as reduced levels of the C9orf72 protein. Thus, both gain and loss of function are coexisting pathogenic aspects linked to C9orf72-ALS/FTD. Synaptic alterations have been largely described in C9orf72 models, but it is still not clear which aspect of the pathology mostly contributes to these impairments. To address this question, we investigated the dynamic changes occurring over time at the synapse upon accumulation of poly(GA), the most abundant DPR. Overexpression of this toxic form induced a drastic loss of synaptic proteins in primary neuron cultures, anticipating autophagic defects. Surprisingly, the dramatic impairment characterizing the synaptic proteome was not fully matched by changes in network properties. In fact, high-density multi-electrode array analysis highlighted only minor reductions in the spike number and firing rate of poly(GA) neurons. Our data show that the toxic gain of function linked to C9orf72 affects the synaptic proteome but exerts only minor effects on the network activity.

Keywords:  ALS; C9orf72; MEA; autophagy; poly(GA); synapse

DOI:  https://doi.org/10.3390/cells13151300
Redox Biol. 2024 Aug 05. pii: S2213-2317(24)00279-9. [Epub ahead of print]75 103301

TDP-43 dysfunction leads to bioenergetic failure and lipid metabolic rewiring in human cells.

Miriam Ceron-Codorniu, Pascual Torres, Anna Fernàndez-Bernal, Santiago Rico-Rios, José Ce Serrano, Maria P Miralles, Maria Beltran, Ana Garcera, Rosa M Soler, Reinald Pamplona, Manuel Portero-Otín.

  The dysfunction of TAR DNA-binding protein 43 (TDP-43) is implicated in various neurodegenerative diseases, though the specific contributions of its toxic gain-of-function versus loss-of-function effects remain unclear. This study investigates the impact of TARDBP loss on cellular metabolism and viability using human-induced pluripotent stem cell-derived motor neurons and HeLa cells. TARDBP silencing led to reduced metabolic activity and cell growth, accompanied by neurite degeneration and decreased oxygen consumption rates in both cell types. Notably, TARDBP depletion induced a metabolic shift, impairing ATP production, increasing metabolic inflexibility, and elevating free radical production, indicating a critical role for TDP-43 in maintaining cellular bioenergetics. Furthermore, TARDBP loss triggered non-apoptotic cell death, increased ACSL4 expression, and reprogrammed lipid metabolism towards lipid droplet accumulation, while paradoxically enhancing resilience to ferroptosis inducers. Overall, our findings highlight those essential cellular traits such as ATP production, metabolic activity, oxygen consumption, and cell survival are highly dependent on TARDBP function.

Keywords:  ACSL4; Amyotrophic lateral sclerosis; Homeostasis; In vitro models; TDP-43

DOI:  https://doi.org/10.1016/j.redox.2024.103301
Methods Mol Biol. 2024 ;2835 135-146

Generation of Human-Induced Pluripotent Stem Cells from Peripheral Blood Mononuclear Cells of C9ORF72-Associated Amyotrophic Lateral Sclerosis Patients.

Kalina Andrysiak, Jacek Stępniewski, Magdalena Spaczyńska-Boczar, Katarzyna Łapicka-Bodzioch, Agnieszka Słowik, Józef Dulak.

  Disease modeling of neuromuscular disorders, such as amyotrophic lateral sclerosis (ALS), is hindered by limited accessibility of affected cells. This problem can be overcome by generation of human induced pluripotent stem cells (hiPSC), which can be then differentiated into required cells. Here, we describe the detailed protocol of hiPSC establishment from peripheral blood mononuclear cells (PBMC) of two ALS patients with detected expansion of G4C2 (GGGGCC) repeats in the first intron of C9ORF72 gene, known to be linked with the most common form of familial ALS.Successful PBMC reprogramming with non-integrating Sendai vectors was confirmed by expression of pluripotency markers: OCT4, NANOG, SSEA4, and TRA-1-60 in obtained hiPSC and their ability to differentiate into cells of three germ layers.The generated ALS-patient-specific hiPSC create a possibility for deciphering molecular basis of this devastating neuromuscular disease.

Keywords:  Amyotrophic lateral sclerosis; Human induced pluripotent stem cells; Sendai vectors; Somatic cell reprogramming

DOI:  https://doi.org/10.1007/978-1-0716-3995-5_12
RNA. 2024 Aug 08. pii: rna.080165.124. [Epub ahead of print]

DNAJA2 and Hero11 mediate similar conformational extension and aggregation suppression of TDP-43.

Andy Y W Lam, Kotaro Tsuboyama, Hisashi Tadakuma, Yukihide Tomari.

  Many RNA binding proteins (RBPs) contain low-complexity domains (LCDs) with prion-like compositions. These long intrinsically disordered regions regulate their solubility, contributing to their physiological roles in RNA processing and organization. However, this also makes these RBPs prone to pathological misfolding and aggregation that are characteristic of neurodegenerative diseases. For example, TAR DNA-binding protein 43 (TDP-43) forms pathological aggregates associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). While molecular chaperones are well-known suppressors of these aberrant events, we recently reported that highly disordered, hydrophilic and charged heat-resistant obscure (Hero) proteins may have similar effects. Specifically, Hero proteins can maintain the activity of other proteins from denaturing conditions in vitro, while their overexpression can suppress cellular aggregation and toxicity associated with aggregation-prone proteins. However, it is unclear how these protective effects are achieved. Here, we utilized single-molecule FRET to monitor the conformations of the aggregation-prone prion-like LCD of TDP-43. While we observed high conformational heterogeneity in wild-type LCD, the ALS-associated mutation A315T promoted collapsed conformations. In contrast, an Hsp40 chaperone, DNAJA2, and a Hero protein, Hero11 stabilized extended states of the LCD, consistent with their ability to suppress the aggregation of TDP-43. Our results link single-molecule effects on conformation to macro effects on bulk aggregation, where a Hero protein, like a chaperone, can maintain the conformational integrity of a client protein to prevent its aggregation.

Keywords:  Hero protein; TAR DNA-binding protein 43; chaperone; intrinsically disordered protein; single-molecule FRET

DOI:  https://doi.org/10.1261/rna.080165.124
Nucleic Acids Res. 2024 Aug 06. pii: gkae655. [Epub ahead of print]

Stress granule formation helps to mitigate neurodegeneration.

M Rebecca Glineburg, Evrim Yildirim, Nicolas Gomez, Genesis Rodriguez, Jaclyn Pak, Xingli Li, Christopher Altheim, Jacob Waksmacki, Gerald M McInerney, Sami J Barmada, Peter K Todd.

Cellular stress pathways that inhibit translation initiation lead to transient formation of cytoplasmic RNA/protein complexes known as stress granules. Many of the proteins found within stress granules and the dynamics of stress granule formation and dissolution are implicated in neurodegenerative disease. Whether stress granule formation is protective or harmful in neurodegenerative conditions is not known. To address this, we took advantage of the alphavirus protein nsP3, which selectively binds dimers of the central stress granule nucleator protein G3BP and markedly reduces stress granule formation without directly impacting the protein translational inhibitory pathways that trigger stress granule formation. In Drosophila and rodent neurons, reducing stress granule formation with nsP3 had modest impacts on lifespan even in the setting of serial stress pathway induction. In contrast, reducing stress granule formation in models of ataxia, amyotrophic lateral sclerosis and frontotemporal dementia largely exacerbated disease phenotypes. These data support a model whereby stress granules mitigate, rather than promote, neurodegenerative cascades.

DOI: https://doi.org/10.1093/nar/gkae655
EMBO J. 2024 Aug 05.

Lysosomal TBK1 responds to amino acid availability to relieve Rab7-dependent mTORC1 inhibition.

Gabriel Talaia, Amanda Bentley-DeSousa, Shawn M Ferguson.

  Lysosomes play a pivotal role in coordinating macromolecule degradation and regulating cell growth and metabolism. Despite substantial progress in identifying lysosomal signaling proteins, understanding the pathways that synchronize lysosome functions with changing cellular demands remains incomplete. This study uncovers a role for TANK-binding kinase 1 (TBK1), well known for its role in innate immunity and organelle quality control, in modulating lysosomal responsiveness to nutrients. Specifically, we identify a pool of TBK1 that is recruited to lysosomes in response to elevated amino acid levels. This lysosomal TBK1 phosphorylates Rab7 on serine 72. This is critical for alleviating Rab7-mediated inhibition of amino acid-dependent mTORC1 activation. Furthermore, a TBK1 mutant (E696K) associated with amyotrophic lateral sclerosis and frontotemporal dementia constitutively accumulates at lysosomes, resulting in elevated Rab7 phosphorylation and increased mTORC1 activation. This data establishes the lysosome as a site of amino acid regulated TBK1 signaling that is crucial for efficient mTORC1 activation. This lysosomal pool of TBK1 has broader implications for lysosome homeostasis, and its dysregulation could contribute to the pathogenesis of ALS-FTD.

Keywords:  ALS-FTD; Lysosome; Nutrient Sensing; TBK1; mTORC1

DOI:  https://doi.org/10.1038/s44318-024-00180-8
Cells. 2024 Jul 31. pii: 1295. [Epub ahead of print]13(15):

Neonicotinoid Pesticides Affect Developing Neurons in Experimental Mouse Models and in Human Induced Pluripotent Stem Cell (iPSC)-Derived Neural Cultures and Organoids.

Alessandro Mariani, Davide Comolli, Roberto Fanelli, Gianluigi Forloni, Massimiliano De Paola.

  Neonicotinoids are synthetic, nicotine-derived insecticides used worldwide to protect crops and domestic animals from pest insects. The reported evidence shows that they are also able to interact with mammalian nicotine receptors (nAChRs), triggering detrimental responses in cultured neurons. Exposure to high neonicotinoid levels during the fetal period induces neurotoxicity in animal models. Considering the persistent exposure to these insecticides and the key role of nAChRs in brain development, their potential neurotoxicity on mammal central nervous system (CNS) needs further investigations. We studied here the neurodevelopmental effects of different generations of neonicotinoids on CNS cells in mouse fetal brain and primary cultures and in neuronal cells and organoids obtained from human induced pluripotent stem cells (iPSC). Neonicotinoids significantly affect neuron viability, with imidacloprid (IMI) inducing relevant alterations in synaptic protein expression, neurofilament structures, and microglia activation in vitro, and in the brain of prenatally exposed mouse fetuses. IMI induces neurotoxic effects also on developing human iPSC-derived neurons and cortical organoids. Collectively, the current findings show that neonicotinoids might induce impairment during neuro/immune-development in mouse and human CNS cells and provide new insights in the characterization of risk for the exposure to this class of pesticides.

Keywords:  developmental neurotoxicity; iPSC; neonicotinoids; organoids; pesticides

DOI:  https://doi.org/10.3390/cells13151295
Front Cell Neurosci. 2024 ;18 1440555

Modeling riboflavin transporter deficiency type 2: from iPSC-derived motoneurons to iPSC-derived astrocytes.

Valentina Magliocca, Angela Lanciotti, Elena Ambrosini, Lorena Travaglini, Veronica D'Ezio, Valentina D'Oria, Stefania Petrini, Michela Catteruccia, Keith Massey, Marco Tartaglia, Enrico Bertini, Tiziana Persichini, Claudia Compagnucci.

   Introduction: Riboflavin transporter deficiency type 2 (RTD2) is a rare neurodegenerative autosomal recessive disease caused by mutations in the SLC52A2 gene encoding the riboflavin transporters, RFVT2. Riboflavin (Rf) is the precursor of FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide), which are involved in different redox reactions, including the energetic metabolism processes occurring in mitochondria. To date, human induced pluripotent stem cells (iPSCs) have given the opportunity to characterize RTD2 motoneurons, which reflect the most affected cell type. Previous works have demonstrated mitochondrial and peroxisomal altered energy metabolism as well as cytoskeletal derangement in RTD2 iPSCs and iPSC-derived motoneurons. So far, no attention has been dedicated to astrocytes.
Results and discussion: Here, we demonstrate that in vitro differentiation of astrocytes, which guarantee trophic and metabolic support to neurons, from RTD2 iPSCs is not compromised. These cells do not exhibit evident morphological differences nor significant changes in the survival rate when compared to astrocytes derived from iPSCs of healthy individuals. These findings indicate that differently from what had previously been documented for neurons, RTD2 does not compromise the morpho-functional features of astrocytes.

Keywords:  astrocytes; in vitro disease modeling; induced pluripotent stem cells; motoneurons; neurodegenerative autosomal recessive disease; redox state; riboflavin transporter deficiency

DOI:  https://doi.org/10.3389/fncel.2024.1440555
Neural Dev. 2024 Aug 08. 19(1): 16

LRRK2 kinase activity is necessary for development and regeneration in Nematostella vectensis.

Grace Holmes, Sophie R Ferguson, Patrick Alfryn Lewis, Karen Echeverri.

   BACKGROUND: The starlet sea anemone, Nematostella vectensis, is an emerging model organism with a high regenerative capacity, which was recently found to possess an orthologue to the human Leucine Rich Repeat Kinase 2 (LRRK2) gene. Mutations in this gene are the most common cause of inherited Parkinson's Disease (PD), highlighting the importance of understanding its function. Despite two decades of research, however, the function of LRRK2 is not well established.
METHODS: To investigate the function of LRRKs in Nematostella vectensis, we applied small molecule inhibitors targeting the kinase activity of LRRK2 to examine its function in development, homeostasis and regeneration in Nematostella vectensis.
RESULTS: In vivo analyses inhibiting the kinase function of this enzyme demonstrated a role of nvLRRK2 in development and regeneration of N. vectensis. These findings implicate a developmental role of LRRK2 in Nematostella, adding to the expanding knowledge of its physiological function.
CONCLUSIONS: Our work introduces a new model organism with which to study LRRK biology. We report that LRRK kinase activity is necessary for the development and regeneration of Nematostella. Given the short generation time, genetic trackability and in vivo imaging capabilities, this work introduces Nematostella vectensis as a new model in which to study genes linked to neurodegenerative diseases such as Parkinson's.

Keywords:   Nematostella vectensis ; Development; LRRK; Regeneration

DOI:  https://doi.org/10.1186/s13064-024-00193-3
Cells. 2024 Jul 28. pii: 1268. [Epub ahead of print]13(15):

Wnt5a Promotes Axon Elongation in Coordination with the Wnt-Planar Cell Polarity Pathway.

Samar Ahmad, Liliana Attisano.

  The establishment of neuronal polarity, involving axon specification and outgrowth, is critical to achieve the proper morphology of neurons, which is important for neuronal connectivity and cognitive functions. Extracellular factors, such as Wnts, modulate diverse aspects of neuronal morphology. In particular, non-canonical Wnt5a exhibits differential effects on neurite outgrowth depending upon the context. Thus, the role of Wnt5a in axon outgrowth and neuronal polarization is not completely understood. In this study, we demonstrate that Wnt5a, but not Wnt3a, promotes axon outgrowth in dissociated mouse embryonic cortical neurons and does so in coordination with the core PCP components, Prickle and Vangl. Unexpectedly, exogenous Wnt5a-induced axon outgrowth was dependent on endogenous, neuronal Wnts, as the chemical inhibition of Porcupine using the IWP2- and siRNA-mediated knockdown of either Porcupine or Wntless inhibited Wnt5a-induced elongation. Importantly, delayed treatment with IWP2 did not block Wnt5a-induced elongation, suggesting that endogenous Wnts and Wnt5a act during specific timeframes of neuronal polarization. Wnt5a in fibroblast-conditioned media can associate with small extracellular vesicles (sEVs), and we also show that these Wnt5a-containing sEVs are primarily responsible for inducing axon elongation.

Keywords:  Wnt5a; axon elongation; extracellular vesicles; neuronal polarity; planar cell polarity

DOI:  https://doi.org/10.3390/cells13151268
STAR Protoc. 2024 Aug 08. pii: S2666-1667(24)00416-7. [Epub ahead of print]5(3): 103251

Protocol for transplantation of cells derived from human midbrain organoids into a Parkinson's disease mouse model to restore motor function.

Chong-Lei Fu, Xi Jiang, Bo-Cheng Dong, Dan Li, Xin-Yu She, Jun Yao.

  Midbrain organoids provide an innovative cellular source for transplantation therapies of neurodegenerative diseases. Here, we present a protocol for midbrain organoid-derived cell transplantation into a Parkinson's disease mouse model. We describe steps for midbrain organoid generation, single-cell suspension preparation, and cell transplantation. This approach is valuable for studying the efficacy of midbrain organoids as a potential cellular source for restoring motor function. For complete details on the use and execution of this protocol, please refer to Fu et al.1.

Keywords:  Model Organisms; Neuroscience; Organoids

DOI:  https://doi.org/10.1016/j.xpro.2024.103251
J Chin Med Assoc. 2024 Aug 09.

TBK1 p.Y153Qfs*9 variant may be associated with young-onset, rapidly progressive amyotrophic lateral sclerosis through a haploinsufficiency mechanism.

Shih-Yu Fang, Pei-Chien Tsai, Kang-Yang Jih, Fang-Chi Hsu, Yi-Chu Liao, Chih-Chao Yang, Yi-Chung Lee.

BACKGROUND: TBK1 variants have been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia spectrum disorder. The current study elucidated the clinical and molecular genetic features of a novel TBK1 variant identified in a patient with young-onset, rapidly progressive ALS.
METHODS: The coding regions of TBK1, SOD1, TARDBP, and FUS were genetically analyzed using Sanger sequencing. Repeat-primed PCR was used to survey the GGGGCC repeat in C9ORF72. The study participant underwent a comprehensive clinical evaluation. The functional effects of the TBK1 variant were analyzed through in vitro transfection studies.
RESULTS: We identified a novel frameshift truncating TBK1 variant, c.456_457delGT (p.Y153Qfs*9), in a man with ALS. The disease initially manifested as right hand weakness at the age of 39 years but progressed rapidly, with the revised ALS Functional Rating Scale score declining at an average monthly rate of 1.92 points in the first year after diagnosis. The patient had no cognitive dysfunction. However, Technetium-99m single photon emission tomography indicated hypoperfusion in his bilateral superior and middle frontal cortices. In vitro studies revealed that the p.Y153Qfs*9 variant resulted in a truncated TBK1 protein product, reduced TBK1 protein expression, loss of kinase function, reduced interaction with optineurin, and impaired dimerization.
CONCLUSION: The heterozygous TBK1 p.Y153Qfs*9 variant may be associated with young-onset, rapidly progressive ALS through a haploinsufficiency mechanism.

DOI: https://doi.org/10.1097/JCMA.0000000000001147
Methods Mol Biol. 2024 ;2835 111-120

iPSC-Based Disease Modeling and Functional Assessment of Neurons in Patients with Metabolic Disorder.

Abhay Srivastava, Sanjiv Dhingra.

  The advancement in technology has allowed us to identify and accurately detect new mutations causing genetic disorders. However, their underlying physiological mechanisms of manifestation are not well understood. This chapter is a non-invasive blueprint to how iPSC-based disease modeling can be used to understand the neural activity and provide mechanistic insights for inborn disorder patients with neurological dysfunction seen more prominently with metabolic disorder patients. It has increasingly become easier to create personalized iPSCs from both specific patients and corresponding age and sex-matched controls by using their blood samples. These iPSCs can be used to generate any cell type of the body. This chapter covers how iPSCs can be generated from blood cells and their characterization followed by instructions on differentiating these iPSCs into mature neurons in a petri dish. The chapter most importantly describes how these mature neurons can be evaluated for their activity by using multi-well microelectrode array system and its analysis. This method of generating personalized iPSC derived neurons and their endpoint assessment can be applied to many clinical and preclinical studies. This iPSC-based application can be extrapolated to study any condition which can affect neuronal activity.

Keywords:  Cell differentiation; Metabolic disorder; Multi-well microelectrode array; Neuronal activity; iPSC-based disease modeling

DOI:  https://doi.org/10.1007/978-1-0716-3995-5_10
Methods Mol Biol. 2024 ;2841 75-83

Extracellular Vesicle Isolation and Mass Spectrometry-Based Proteomic Analysis in Arabidopsis thaliana.

Jiayang Gao, Jinyu Chen, Hai Zhang, Liwen Jiang, Yong Cui.

  Extracellular vesicles (EVs) can transport various biological materials, including proteins, lipids, nucleic acids, and metabolites, through the unconventional protein secretion (UPS) pathway. Plant EVs can be classified into at least three major types: tetraspanin 8 (TET8)-positive EVs, penetration 1 (PEN1)-positive EVs, and exocyst-positive organelle (EXPO)-derived EVs. However, the research progress of plant EVs has been hindered due to the limitations inherent in EV isolation techniques. Moreover, since previous research on plant EVs has primarily focused on the interaction between plants and microbes, the biogenesis, transport, and secretion of plant EVs remain unexplored. Recent advances in centrifugation methods for extraction of apoplastic wash fluids, combined with mass spectrometry-based proteomic analysis, provide approaches to identify regulators and cargoes of plant EVs and thus serve as an important step for future studies on the biogenesis and function of plant EVs. Here, we illustrate detailed methods of EV isolation and mass spectrometry-based proteomic analysis in Arabidopsis.

Keywords:  Apoplastic wash fluids; Exocyst-positive organelle (EXPO); Extracellular vesicle; Mass spectrometry

DOI:  https://doi.org/10.1007/978-1-0716-4059-3_6
Cells. 2024 Jul 26. pii: 1260. [Epub ahead of print]13(15):

From Young to Old: Mimicking Neuronal Aging in Directly Converted Neurons from Young Donors.

Nimmy Varghese, Amandine Grimm, M Zameel Cader, Anne Eckert.

  A substantial challenge in human brain aging is to find a suitable model to mimic neuronal aging in vitro as accurately as possible. Using directly converted neurons (iNs) from human fibroblasts is considered a promising tool in human aging since it retains the aging-associated mitochondrial donor signature. Still, using iNs from aged donors can pose certain restrictions due to their lower reprogramming and conversion efficacy than those from younger individuals. To overcome these limitations, our study aimed to establish an in vitro neuronal aging model mirroring features of in vivo aging by acute exposure on young iNs to either human stress hormone cortisol or the mitochondrial stressor rotenone, considering stress as a trigger of in vivo aging. The impact of rotenone was evident in mitochondrial bioenergetic properties by showing aging-associated deficits in mitochondrial respiration, cellular ATP, and MMP and a rise in glycolysis, mitochondrial superoxide, and mitochondrial ROS; meanwhile, cortisol only partially induced an aging-associated mitochondrial dysfunction. To replicate the in vivo aging-associated mitochondrial dysfunctions, using rotenone, a mitochondrial complex I inhibitor, proved to be superior to the cortisol model. This work is the first to use stress on young iNs to recreate aging-related mitochondrial impairments.

Keywords:  cortisol; directly converted neurons from human fibroblasts; mitochondria; rotenone; stress

DOI:  https://doi.org/10.3390/cells13151260
Zh Nevrol Psikhiatr Im S S Korsakova. 2024 ;124(7): 165-168

[Amyotrophic lateral sclerosis associated with a new pathogenic variant of the ERBB4 gene].

E V Pervushina, M A Kutlubaev, E V Saifullina, E V Gaisina, L A Smakova, I M Khidiyatova.

  Amyotrophic lateral sclerosis (ALS) is a sporadic disease in most of the cases; in 10-15% of cases genetic forms are recorded. A genetic form of ALS associated with the mutation in the ERBB4 gene (ALS19) has been reported in 2013. A protein encoded by the ERBB4 is probably involved in ubiquitous component of the pathogenesis of ALS. We present a case of ALS associated with a new pathogenic variant of the ERBB4 gene, with early bulbar onset and slow progression of the disease within 10 years.

Keywords:  ERBB4; amyotrophic lateral sclerosis; monogenic disorders; motor neuron disease

DOI:  https://doi.org/10.17116/jnevro2024124071165
Front Cell Neurosci. 2024 ;18 1425222

A decrease in Fkbp52 alters autophagosome maturation and A152T-tau clearance in vivo.

Emilie Lesport, Lucie Commeau, Mélanie Genet, Etienne-Emile Baulieu, Marcel Tawk, Julien Giustiniani.

  The failure of the autophagy-lysosomal pathway to clear the pathogenic forms of Tau exacerbates the pathogenesis of tauopathies. We have previously shown that the immunophilin FKBP52 interacts both physically and functionally with Tau, and that a decrease in FKBP52 protein levels is associated with Tau deposition in affected human brains. We have also shown that FKBP52 is physiologically present within the lysosomal system in healthy human neurons and that a decrease in FKBP52 expression alters perinuclear lysosomal positioning and Tau clearance during Tau-induced proteotoxic stress in vitro. In this study, we generate a zebrafish fkbp4 loss of function mutant and show that axonal retrograde trafficking of Lamp1 vesicles is altered in this mutant. Moreover, using our transgenic HuC::mCherry-EGFP-LC3 line, we demonstrate that the autophagic flux is impaired in fkbp4 mutant embryos, suggesting a role for Fkbp52 in the maturation of autophagic vesicles. Alterations in both axonal transport and autophagic flux are more evident in heterozygous rather than homozygous fkbp4 mutants. Finally, taking advantage of the previously described A152T-Tau transgenic fish, we show that the clearance of pathogenic A152T-Tau mutant proteins is slower in fkbp4 +/- mutants in comparison to fkbp4 +/+ larvae. Altogether, these results indicate that Fkbp52 is required for the normal trafficking and maturation of lysosomes and autophagic vacuoles along axons, and that its decrease is sufficient to hinder the clearance of pathogenic Tau in vivo.

Keywords:  FKBP52; autophagy; lysosomes; tau; tauopathies; zebrafish

DOI:  https://doi.org/10.3389/fncel.2024.1425222
Nat Rev Mol Cell Biol. 2024 Aug 06.

Mechanisms of autophagy-lysosome dysfunction in neurodegenerative diseases.

Ralph A Nixon, David C Rubinsztein.

Autophagy is a lysosome-based degradative process used to recycle obsolete cellular constituents and eliminate damaged organelles and aggregate-prone proteins. Their postmitotic nature and extremely polarized morphologies make neurons particularly vulnerable to disruptions caused by autophagy-lysosomal defects, especially as the brain ages. Consequently, mutations in genes regulating autophagy and lysosomal functions cause a wide range of neurodegenerative diseases. Here, we review the role of autophagy and lysosomes in neurodegenerative diseases such as Alzheimer disease, Parkinson disease and frontotemporal dementia. We also consider the strong impact of cellular ageing on lysosomes and autophagy as a tipping point for the late-age emergence of related neurodegenerative disorders. Many of these diseases have primary defects in autophagy, for example affecting autophagosome formation, and in lysosomal functions, especially pH regulation and calcium homeostasis. We have aimed to provide an integrative framework for understanding the central importance of autophagic-lysosomal function in neuronal health and disease.

DOI: https://doi.org/10.1038/s41580-024-00757-5
Methods Mol Biol. 2024 ;2841 121-130

Identification of Neighboring Proteins of Endosomal Regulators by Using TurboID-Based Proximity Labeling.

Danni Lin, Jiayi Kuang, Caiji Gao.

  In the endomembrane system, multivesicular bodies (MVBs) play a crucial role in sorting ubiquitinated membrane proteins into intraluminal vesicles for degradation upon fusion with vacuoles or lysosomes. This process involves regulations by multiprotein complexes, including endosomal sorting complex required for transport (ESCRT) I-III, and accessory proteins. Although many organellar proteomes have been identified in plant cells, the information of specific proteomes associated with regulators engaged in MVB biogenesis remains limited. Here, using the ESCRT component FREE1 as an example, we describe a method to identify neighboring proteins of endosomal regulators by using an approach of TurboID-based proximity labeling.

Keywords:  Arabidopsis; MVB; Organellar proteome; Proximity labeling technology; TurboID

DOI:  https://doi.org/10.1007/978-1-0716-4059-3_11
NPJ Parkinsons Dis. 2024 Aug 03. 10(1): 144

Transcriptomics and weighted protein network analyses of the LRRK2 protein interactome reveal distinct molecular signatures for sporadic and LRRK2 Parkinson's Disease.

Yibo Zhao, Matthew Bracher-Smith, Yuelin Li, Kirsten Harvey, Valentina Escott-Price, Patrick A Lewis, Claudia Manzoni.

Mutations in the LRRK2 gene are the most common genetic cause of familial Parkinson's Disease (LRRK2-PD) and an important risk factor for sporadic PD (sPD). Multiple clinical trials are ongoing to evaluate the benefits associated with the therapeutical reduction of LRRK2 kinase activity. In this study, we described the changes of transcriptomic profiles (whole blood mRNA levels) of LRRK2 protein interactors in sPD and LRRK2-PD cases as compared to healthy controls with the aim of comparing the two PD conditions. We went on to model the protein-protein interaction (PPI) network centred on LRRK2, which was weighted to reflect the transcriptomic changes on expression and co-expression levels of LRRK2 protein interactors. Our results showed that LRRK2 interactors present both similar and distinct alterations in expression levels and co-expression behaviours in the sPD and LRRK2-PD cases; suggesting that, albeit being classified as the same disease based on clinical features, LRRK2-PD and sPD display significant differences from a molecular perspective. Interestingly, the similar changes across the two PD conditions result in decreased connectivity within a topological cluster of the LRRK2 PPI network associated with protein metabolism/biosynthesis and ribosomal metabolism suggesting protein homoeostasis and ribosomal dynamics might be affected in both sporadic and familial PD in comparison with controls.

DOI: https://doi.org/10.1038/s41531-024-00761-8
J Cell Biol. 2024 Nov 04. pii: e202405032. [Epub ahead of print]223(11):

Nesprin-2 coordinates opposing microtubule motors during nuclear migration in neurons.

Chuying Zhou, You Kure Wu, Fumiyoshi Ishidate, Takahiro K Fujiwara, Mineko Kengaku.

Nuclear migration is critical for the proper positioning of neurons in the developing brain. It is known that bidirectional microtubule motors are required for nuclear transport, yet the mechanism of the coordination of opposing motors is still under debate. Using mouse cerebellar granule cells, we demonstrate that Nesprin-2 serves as a nucleus-motor adaptor, coordinating the interplay of kinesin-1 and dynein. Nesprin-2 recruits dynein-dynactin-BicD2 independently of the nearby kinesin-binding LEWD motif. Both motor binding sites are required to rescue nuclear migration defects caused by the loss of function of Nesprin-2. In an intracellular cargo transport assay, the Nesprin-2 fragment encompassing the motor binding sites generates persistent movements toward both microtubule minus and plus ends. Nesprin-2 drives bidirectional cargo movements over a prolonged period along perinuclear microtubules, which advance during the migration of neurons. We propose that Nesprin-2 keeps the nucleus mobile by coordinating opposing motors, enabling continuous nuclear transport along advancing microtubules in migrating cells.

DOI: https://doi.org/10.1083/jcb.202405032
Methods Mol Biol. 2024 ;2845 109-126

Methods for Monitoring ER-Phagy.

Marisa Di Monaco, Marian Peteri, Jin Rui Liang.

  The endoplasmic reticulum (ER) serves as a central hub for protein synthesis, folding, and lipid biosynthesis in eukaryotic cells. Maintaining ER homeostasis is essential for optimal cellular function, and one mechanism that has garnered attention is endoplasmic reticulum-specific autophagy, or ER-phagy. ER-phagy selectively removes specific ER portions, playing a pivotal role in cellular health and adaptation to environmental stressors. ER-phagy can be induced by diverse cellular conditions such as amino acid starvation, disruption of ER quality control mechanisms, and accumulation of misfolded ER protein, highlighting cellular adaptability and the significance of ER-phagy in stress responses. Clinically relevant mutations in ER-phagy receptors are implicated in various diseases, underlining the fundamental importance of ER-phagy in ER homeostasis. Here, we provide comprehensive protocols and general considerations while investigating ER-phagy using three fundamental techniques-Western blotting, immunofluorescence, and flow cytometry-commonly used in ER-phagy detection and quantitation.

Keywords:  Autophagy; ER-phagy; Endoplasmic reticulum; FACS; Fluorescent reporters; Immunofluorescence; Selective autophagy; Western blotting

DOI:  https://doi.org/10.1007/978-1-0716-4067-8_9
Nat Commun. 2024 Aug 08. 15(1): 6742

Multi-omic analysis of Huntington's disease reveals a compensatory astrocyte state.

Fahad Paryani, Ji-Sun Kwon, Christopher W Ng, Kelly Jakubiak, Nacoya Madden, Kenneth Ofori, Alice Tang, Hong Lu, Shengnan Xia, Juncheng Li, Aayushi Mahajan, Shawn M Davidson, Anna O Basile, Caitlin McHugh, Jean Paul Vonsattel, Richard Hickman, Michael C Zody, David E Housman, James E Goldman, Andrew S Yoo, Vilas Menon, Osama Al-Dalahmah.

The mechanisms underlying the selective regional vulnerability to neurodegeneration in Huntington's disease (HD) have not been fully defined. To explore the role of astrocytes in this phenomenon, we used single-nucleus and bulk RNAseq, lipidomics, HTT gene CAG repeat-length measurements, and multiplexed immunofluorescence on HD and control post-mortem brains. We identified genes that correlated with CAG repeat length, which were enriched in astrocyte genes, and lipidomic signatures that implicated poly-unsaturated fatty acids in sensitizing neurons to cell death. Because astrocytes play essential roles in lipid metabolism, we explored the heterogeneity of astrocytic states in both protoplasmic and fibrous-like (CD44+) astrocytes. Significantly, one protoplasmic astrocyte state showed high levels of metallothioneins and was correlated with the selective vulnerability of distinct striatal neuronal populations. When modeled in vitro, this state improved the viability of HD-patient-derived spiny projection neurons. Our findings uncover key roles of astrocytic states in protecting against neurodegeneration in HD.

DOI: https://doi.org/10.1038/s41467-024-50626-0
Methods Mol Biol. 2024 ;2845 79-93

Analysis of Mitophagy Reporters in Drosophila.

Alvaro Sanchez-Martinez, Aitor Martinez, Alexander J Whitworth.

  Mitophagy is the degradation of mitochondria via the autophagy-lysosome system, disruption of which has been linked to multiple neurodegenerative diseases. As a flux process involving the identification, tagging, and degradation of subcellular components, the analysis of mitophagy benefits from the microscopy analysis of fluorescent reporters. Studying the pathogenic mechanisms of disease also benefits from analysis in animal models in order to capture the complex interplay of molecular and cell biological phenomena. Here, we describe protocols to analyze mitophagy reporters in Drosophila by light microscopy.

Keywords:  Brain; Drosophila; Light microscopy; Mitochondria; Mitophagy; Muscle; Neurodegeneration; Reporter; mito-QC; mtx-QC

DOI:  https://doi.org/10.1007/978-1-0716-4067-8_7
Neurochem Int. 2024 Aug 05. pii: S0197-0186(24)00154-2. [Epub ahead of print] 105827

The Autophagy Paradox: A New Hypothesis in Neurodegenerative Disorders.

Haleh Barmaki, Alireza Nourazarian, Behrouz Shademan, Khaki-Khatibi Fatemeh.

  A recent study showed that while autophagy is usually tied to protein and organelle turnover, it can also play dual roles in neurodegenerative diseases. Traditionally, autophagy was seen as protective since it removes damaged proteins and organelles. but new data suggests autophagy can sometimes promote neuron death. and This review tackles autophagy's seemingly contradictory effects in neurodegeneration, or the "autophagy paradox. " It offers a framework for understanding autophagy in neurodegenerative research and the cellular processes involved. In short, our data uncovers a harmful autophagy role in certain situations, conflicting the view that it's always beneficial. We describe the distinct, disease-specific autophagy pathways functioning in various neurodegenerative diseases. Part two concerns potential therapeutic implications of manipulating autophagy and current strategies targeting the autophagic system, suggesting interesting areas for future research into tailored modulators. This could eventually enable activating or controlling specific autophagy pathways and aid in developing more effective treatments. Researchers believe more molecular-level research is needed so patient-tailored autophagy-modulating therapeutics can be developed given this dilemma. Moreover, research must translate faster into effective neurodegenerative disease treatment options. This article aims to provide a wholly new perspective on autophagy's classically described role in these severe diseases, challenging current dogma and opening new therapeutic avenue options.

Keywords:  Autophagy; Cell Death; Genetic Therapy; Neurodegenerative Diseases; Neuronal Homeostasis

DOI:  https://doi.org/10.1016/j.neuint.2024.105827