bims-axbals 2024-03-03 papers

bims-axbals

Biomed News

on Axonal Biology and ALS

Issue of 2024–03–03
37 papers selected by
TJ Krzystek, ALS Therapy Development Institute

Understanding age-related pathologic changes in TDP-43 functions and their consequence on RNA splicing and signalling in health and disease.
Generation of human iPSC-derived phrenic-like motor neurons to model respiratory motor neuron degeneration in ALS.
Ribosomal quality control factors inhibit repeat-associated non-AUG translation from GC-rich repeats.
Human Motor Neurons Elicit Pathological Hallmarks of ALS and Reveal Potential Biomarkers of the Disease in Response to Prolonged IFNγ Exposure.
Molecular hallmarks of ageing in amyotrophic lateral sclerosis.
PolyGR and polyPR knock-in mice reveal a conserved neuroprotective extracellular matrix signature in C9orf72 ALS/FTD neurons.
Generation of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Astrocytes for Amyotrophic Lateral Sclerosis and Other Neurodegenerative Disease Studies.
Cerebrospinal fluid and blood exosomes as biomarkers for amyotrophic lateral sclerosis; a systematic review.
Lessons from inducible pluripotent stem cell models on neuronal senescence in aging and neurodegeneration.
hnRNP R regulates mitochondrial movement and membrane potential in axons of motoneurons.
The Application of Brain Organoid for Drug Discovery in Mitochondrial Diseases.
Multiomics Evaluation of Human iPSCs and iPSC-Derived Neurons.
Gene editing improves endoplasmic reticulum-mitochondrial contacts and unfolded protein response in Friedreich's ataxia iPSC-derived neurons.
Effects of motor cortical and peripheral axonal hyperexcitability on survival in amyotrophic lateral sclerosis.
Quantifying differentiation of progenitor populations using cerebral organoid models for neurodevelopmental disorders.
Mutual interaction of neurons and astrocytes derived from iPSCs with APP V717L mutation developed the astrocytic phenotypes of Alzheimer's disease.
FBXL4: safeguarding against mitochondrial depletion through suppression of mitophagy.
α-Synuclein: Multiple pathogenic roles in trafficking and proteostasis pathways in Parkinson's disease.
RNA-mediated ribonucleoprotein assembly controls TDP-43 nuclear retention.
Restoring functional TDP-43 oligomers in ALS and laminopathic cellular models through baicalein-induced reconfiguration of TDP-43 aggregates.
PTCH1-mutant human cerebellar organoids exhibit altered neural development and recapitulate early medulloblastoma tumorigenesis.
Tom20 gates PINK1 activity and mediates its tethering of the TOM and TIM23 translocases upon mitochondrial stress.
Neuronal exosomal miRNAs modulate mitochondrial functions and cell death in bystander neuronal cells under Parkinson's disease stress conditions.
A Neglected Gene: The Role of the ANG Gene in the Pathogenesis of Amyotrophic Lateral Sclerosis.
Cellular reprogramming as a tool to model human aging in a dish.
Loss of GTF2I promotes neuronal apoptosis and synaptic reduction in human cellular models of neurodevelopment.
Nur77 increases mitophagy and decreases aggregation of α-synuclein by modulating the p-c-Abl/p-PHB2 Y121 in α-synuclein PFF SH-SY5Y cells and mice.
Spreading of motor neuron degeneration in ALS is not so random.
Isolation of Extracellular Vesicles Using Titanium Dioxide Microspheres.
Most axonal mitochondria in cortical pyramidal neurons lack mitochondrial DNA and consume ATP.
G2019S selective LRRK2 kinase inhibitor abrogates mitochondrial DNA damage.
Primary cilia promote the differentiation of human neurons through the WNT signaling pathway.
Combinatorial selective ER-phagy remodels the ER during neurogenesis.
mitoTALEN reduces the mutant mtDNA load in neurons.
Drosophila melanogaster Neuromuscular Junction as a Model to Study Synaptopathies and Neuronal Autophagy.
Myosin F controls actin organization and dynamics in Toxoplasma gondii.
A ratiometric ER calcium sensor for quantitative comparisons across cell types and subcellular regions.

Ageing Res Rev. 2024 Feb 22. pii: S1568-1637(24)00064-3. [Epub ahead of print] 102246

Understanding age-related pathologic changes in TDP-43 functions and their consequence on RNA splicing and signalling in health and disease.

Flora Cheng, Tyler Chapman, Selina Zhang, Marco Morsch, Roger Chung, Albert Lee, Stephanie L Rayner.

  TAR DNA binding protein-43 (TDP-43) is a key component in RNA splicing which plays a crucial role in the aging process. In neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) and Limbic-predominant age-related TDP-43 Encephalopathy (LATE), TDP-43 can be mutated, mislocalised out of the nucleus of neurons and glial cells and form cytoplasmic inclusions. These TDP-43 alterations can lead to its RNA splicing dysregulation and contribute to mis-splicing of various types of RNA, such as mRNA, microRNA, and circRNA. These changes can result in the generation of an altered transcriptome and proteome within cells, ultimately changing the diversity and quantity of gene products. In this review, we summarise the findings of novel atypical RNAs resulting from TDP-43 dysfunction and their potential biomarkers or targets for therapeutic development.

Keywords:  Circular RNA; Cryptic exon; MicroRNA; Neurodegeneration; Spliceosome; TDP-43

DOI:  https://doi.org/10.1016/j.arr.2024.102246
Commun Biol. 2024 Feb 28. 7(1): 238

Generation of human iPSC-derived phrenic-like motor neurons to model respiratory motor neuron degeneration in ALS.

Louise Thiry, Julien Sirois, Thomas M Durcan, Stefano Stifani.

The fatal motor neuron (MN) disease Amyotrophic Lateral Sclerosis (ALS) is characterized by progressive MN degeneration. Phrenic MNs (phMNs) controlling the activity of the diaphragm are prone to degeneration in ALS, leading to death by respiratory failure. Understanding of the mechanisms of phMN degeneration in ALS is limited, mainly because human experimental models to study phMNs are lacking. Here we describe a method enabling the derivation of phrenic-like MNs from human iPSCs (hiPSC-phMNs) within 30 days. This protocol uses an optimized combination of small molecules followed by cell-sorting based on a cell-surface protein enriched in hiPSC-phMNs, and is highly reproducible using several hiPSC lines. We show further that hiPSC-phMNs harbouring ALS-associated amplification of the C9orf72 gene progressively lose their electrophysiological activity and undergo increased death compared to isogenic controls. These studies establish a previously unavailable protocol to generate human phMNs offering a disease-relevant system to study mechanisms of respiratory MN dysfunction.

DOI: https://doi.org/10.1038/s42003-024-05925-z
Nucleic Acids Res. 2024 Feb 27. pii: gkae137. [Epub ahead of print]

Ribosomal quality control factors inhibit repeat-associated non-AUG translation from GC-rich repeats.

Yi-Ju Tseng, Amy Krans, Indranil Malik, Xiexiong Deng, Evrim Yildirim, Sinem Ovunc, Elizabeth M H Tank, Karen Jansen-West, Ross Kaufhold, Nicolas B Gomez, Roger Sher, Leonard Petrucelli, Sami J Barmada, Peter K Todd.

A GGGGCC (G4C2) hexanucleotide repeat expansion in C9ORF72 causes amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), while a CGG trinucleotide repeat expansion in FMR1 leads to the neurodegenerative disorder Fragile X-associated tremor/ataxia syndrome (FXTAS). These GC-rich repeats form RNA secondary structures that support repeat-associated non-AUG (RAN) translation of toxic proteins that contribute to disease pathogenesis. Here we assessed whether these same repeats might trigger stalling and interfere with translational elongation. We find that depletion of ribosome-associated quality control (RQC) factors NEMF, LTN1 and ANKZF1 markedly boost RAN translation product accumulation from both G4C2 and CGG repeats while overexpression of these factors reduces RAN production in both reporter assays and C9ALS/FTD patient iPSC-derived neurons. We also detected partially made products from both G4C2 and CGG repeats whose abundance increased with RQC factor depletion. Repeat RNA sequence, rather than amino acid content, is central to the impact of RQC factor depletion on RAN translation-suggesting a role for RNA secondary structure in these processes. Together, these findings suggest that ribosomal stalling and RQC pathway activation during RAN translation inhibits the generation of toxic RAN products. We propose augmenting RQC activity as a therapeutic strategy in GC-rich repeat expansion disorders.

DOI: https://doi.org/10.1093/nar/gkae137
J Neurosci. 2024 Feb 27. pii: e1787232024. [Epub ahead of print]

Human Motor Neurons Elicit Pathological Hallmarks of ALS and Reveal Potential Biomarkers of the Disease in Response to Prolonged IFNγ Exposure.

Changho Chun, Jung Hyun Lee, Mark Bothwell, Paul Nghiem, Alec S T Smith, David L Mack.

Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative disorder marked by progressive motor neuron degeneration and muscle denervation. A recent transcriptomic study integrating a wide range of human ALS samples revealed that upregulation of p53, a downstream target of inflammatory stress, is commonly detected in familial and sporadic ALS cases by a mechanism linked to TDP-43 dysfunction. In this study, we show that prolonged IFNγ treatment of human iPSC-derived spinal motor neurons results in severe cytoplasmic aggregation of TDP-43. Either TDP-43 dysfunction resulting from IFNγ exposure or from an ALS-associated TDP-43 mutation was associated with activation of the p53 pathway. This was accompanied by hyperactivation of neuronal firing, followed by the complete loss of their electrophysiological function. Through comparative single-cell transcriptome analysis, we have identified significant alterations in ALS-associated genes in motor neurons exposed to IFNγ, implicating their direct involvement in ALS pathology. Interestingly, IFNγ was found to induce significant levels of PD-L1 expression in motor neurons without affecting the levels of any other immune checkpoint proteins. This finding suggests a potential role for excessive PD-L1 expression in ALS development, given that PD-L1 was recently reported to impair neuronal firing ability in mice. Our findings suggest that exposing motor neurons to IFNγ could directly derive ALS pathogenesis, even without the presence of inherent genetic mutation or functional glia component. Furthermore, this study provides a comprehensive list of potential candidate genes for future immunotherapeutic targets with which to treat sporadic forms of ALS, which account for 90% of all reported cases.Significance statement ALS is currently an incurable neurodegenerative disease that primarily damages motor function. 90% of the reported cases have unknown reasons, but their progression is extremely fast once triggered. A pathologic hallmark of ALS is an aggregation of RNA/DNA binding protein, TDP-43, but its pathologic link to the disease is yet to be elucidated. In this study, we found the Interferon-gamma (IFNγ), an immune-derived cytokine activates p53 pathway which appears in common in post-mortem ALS tissue. Importantly, IFNγ triggered a significant cytoplasmic TDP-43 aggregation and impaired electrophysiological function of human motor neurons. Furthermore, we found that ALS risk genes related to the mitochondrial dysfunction are aberrantly expressed under the IFNγ exposure, which constitutes potential therapeutic targets of immune-dysregulated neurodegeneration in ALS.

DOI: https://doi.org/10.1523/JNEUROSCI.1787-23.2024
Cell Mol Life Sci. 2024 Mar 02. 81(1): 111

Molecular hallmarks of ageing in amyotrophic lateral sclerosis.

Cyril Jones Jagaraj, Sina Shadfar, Sara Assar Kashani, Sayanthooran Saravanabavan, Fabiha Farzana, Julie D Atkin.

  Amyotrophic lateral sclerosis (ALS) is a fatal, severely debilitating and rapidly progressing disorder affecting motor neurons in the brain, brainstem, and spinal cord. Unfortunately, there are few effective treatments, thus there remains a critical need to find novel interventions that can mitigate against its effects. Whilst the aetiology of ALS remains unclear, ageing is the major risk factor. Ageing is a slowly progressive process marked by functional decline of an organism over its lifespan. However, it remains unclear how ageing promotes the risk of ALS. At the molecular and cellular level there are specific hallmarks characteristic of normal ageing. These hallmarks are highly inter-related and overlap significantly with each other. Moreover, whilst ageing is a normal process, there are striking similarities at the molecular level between these factors and neurodegeneration in ALS. Nine ageing hallmarks were originally proposed: genomic instability, loss of telomeres, senescence, epigenetic modifications, dysregulated nutrient sensing, loss of proteostasis, mitochondrial dysfunction, stem cell exhaustion, and altered inter-cellular communication. However, these were recently (2023) expanded to include dysregulation of autophagy, inflammation and dysbiosis. Hence, given the latest updates to these hallmarks, and their close association to disease processes in ALS, a new examination of their relationship to pathophysiology is warranted. In this review, we describe possible mechanisms by which normal ageing impacts on neurodegenerative mechanisms implicated in ALS, and new therapeutic interventions that may arise from this.

Keywords:  ALS; Ageing; Molecular hallmarks; Neurodegenerative diseases

DOI:  https://doi.org/10.1007/s00018-024-05164-9
Nat Neurosci. 2024 Feb 29.

PolyGR and polyPR knock-in mice reveal a conserved neuroprotective extracellular matrix signature in C9orf72 ALS/FTD neurons.

Carmelo Milioto, Mireia Carcolé, Ashling Giblin, Rachel Coneys, Olivia Attrebi, Mhoriam Ahmed, Samuel S Harris, Byung Il Lee, Mengke Yang, Robert A Ellingford, Raja S Nirujogi, Daniel Biggs, Sally Salomonsson, Matteo Zanovello, Paula de Oliveira, Eszter Katona, Idoia Glaria, Alla Mikheenko, Bethany Geary, Evan Udine, Deniz Vaizoglu, Sharifah Anoar, Khrisha Jotangiya, Gerard Crowley, Demelza M Smeeth, Mirjam L Adams, Teresa Niccoli, Rosa Rademakers, Marka van Blitterswijk, Anny Devoy, Soyon Hong, Linda Partridge, Alyssa N Coyne, Pietro Fratta, Dario R Alessi, Ben Davies, Marc Aurel Busche, Linda Greensmith, Elizabeth M C Fisher, Adrian M Isaacs.

Dipeptide repeat proteins are a major pathogenic feature of C9orf72 amyotrophic lateral sclerosis (C9ALS)/frontotemporal dementia (FTD) pathology, but their physiological impact has yet to be fully determined. Here we generated C9orf72 dipeptide repeat knock-in mouse models characterized by expression of 400 codon-optimized polyGR or polyPR repeats, and heterozygous C9orf72 reduction. (GR)400 and (PR)400 knock-in mice recapitulate key features of C9ALS/FTD, including cortical neuronal hyperexcitability, age-dependent spinal motor neuron loss and progressive motor dysfunction. Quantitative proteomics revealed an increase in extracellular matrix (ECM) proteins in (GR)400 and (PR)400 spinal cord, with the collagen COL6A1 the most increased protein. TGF-β1 was one of the top predicted regulators of this ECM signature and polyGR expression in human induced pluripotent stem cell neurons was sufficient to induce TGF-β1 followed by COL6A1. Knockdown of TGF-β1 or COL6A1 orthologues in polyGR model Drosophila exacerbated neurodegeneration, while expression of TGF-β1 or COL6A1 in induced pluripotent stem cell-derived motor neurons of patients with C9ALS/FTD protected against glutamate-induced cell death. Altogether, our findings reveal a neuroprotective and conserved ECM signature in C9ALS/FTD.

DOI: https://doi.org/10.1038/s41593-024-01589-4
Bio Protoc. 2024 Feb 20. 14(4): e4936

Generation of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Astrocytes for Amyotrophic Lateral Sclerosis and Other Neurodegenerative Disease Studies.

Katarina Stoklund Dittlau, Abinaya Chandrasekaran, Kristine Freude, Ludo Van Den Bosch.

  Astrocytes are increasingly recognized for their important role in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS). In ALS, astrocytes shift from their primary function of providing neuronal homeostatic support towards a reactive and toxic role, which overall contributes to neuronal toxicity and cell death. Currently, our knowledge on these processes is incomplete, and time-efficient and reproducible model systems in a human context are therefore required to understand and therapeutically modulate the toxic astrocytic response for future treatment options. Here, we present an efficient and straightforward protocol to generate human induced pluripotent stem cell (hiPSC)-derived astrocytes implementing a differentiation scheme based on small molecules. Through an initial 25 days, hiPSCs are differentiated into astrocytes, which are matured for 4+ weeks. The hiPSC-derived astrocytes can be cryopreserved at every passage during differentiation and maturation. This provides convenient pauses in the protocol as well as cell banking opportunities, thereby limiting the need to continuously start from hiPSCs. The protocol has already proven valuable in ALS research but can be adapted to any desired research field where astrocytes are of interest. Key features • This protocol requires preexisting experience in hiPSC culturing for a successful outcome. • The protocol relies on a small molecule differentiation scheme and an easy-to-follow methodology, which can be paused at several time points. • The protocol generates >50 × 106 astrocytes per differentiation, which can be cryopreserved at every passage, ensuring a large-scale experimental output.

Keywords:  Amyotrophic lateral sclerosis; Astrocyte; Human induced pluripotent stem cell; Neurodegeneration; Small-molecule differentiation

DOI:  https://doi.org/10.21769/BioProtoc.4936
Diagn Pathol. 2024 Mar 01. 19(1): 47

Cerebrospinal fluid and blood exosomes as biomarkers for amyotrophic lateral sclerosis; a systematic review.

Shahram Darabi, Armin Ariaei, Auob Rustamzadeh, Dariush Afshari, Enam Alhagh Charkhat Gorgich, Leila Darabi.

   BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive and fatal motor neuron disease. Due to the limited knowledge about potential biomarkers that help in early diagnosis and monitoring disease progression, today's diagnoses are based on ruling out other diseases, neurography, and electromyography examination, which takes a time-consuming procedure.
METHODS: PubMed, ScienceDirect, and Web of Science were explored to extract articles published from January 2015 to June 2023. In the searching strategy following keywords were included; amyotrophic lateral sclerosis, biomarkers, cerebrospinal fluid, serum, and plama.
RESULTS: A total number of 6 studies describing fluid-based exosomal biomarkers were included in this study. Aggregated proteins including SOD1, TDP-43, pTDP-43, and FUS could be detected in the microvesicles (MVs). Moreover, TDP-43 and NFL extracted from plasma exosomes could be used as prognostic biomarkers. Also, downregulated miR-27a-3p detected through exoEasy Maxi and exoQuick Kit in the plasma could be measured as a diagnostic biomarker. Eventually, the upregulated level of CORO1A could be used to monitor disease progression.
CONCLUSION: Based on the results, each biomarker alone is insufficient to evaluate ALS. CNS-derived exosomes contain multiple ALS-related biomarkers (SOD1, TDP-43, pTDP-43, FUS, and miRNAs) that are detectable in cerebrospinal fluid and blood is a proper alternation. Exosome detecting kits listed as exoEasy, ExoQuick, Exo-spin, ME kit, ExoQuick Plus, and Exo-Flow, are helpful to reach this purpose.

Keywords:  Amyotrophic lateral sclerosis; Biomarkers; Exosomes; Extracellular vesicles

DOI:  https://doi.org/10.1186/s13000-024-01473-6
Nat Aging. 2024 Mar 01.

Lessons from inducible pluripotent stem cell models on neuronal senescence in aging and neurodegeneration.

Isabelle R de Luzy, Michael K Lee, William C Mobley, Lorenz Studer.

Age remains the central risk factor for many neurodegenerative diseases including Parkinson's disease, Alzheimer's disease and amyotrophic lateral sclerosis. Although the mechanisms of aging are complex, the age-related accumulation of senescent cells in neurodegeneration is well documented and their clearance can alleviate disease-related features in preclinical models. Senescence-like characteristics are observed in both neuronal and glial lineages, but their relative contribution to aging and neurodegeneration remains unclear. Human pluripotent stem cell-derived neurons provide an experimental model system to induce neuronal senescence. However, the extensive heterogeneity in the profile of senescent neurons and the methods to assess senescence remain major challenges. Here, we review the evidence of cellular senescence in neuronal aging and disease, discuss human pluripotent stem cell-based model systems used to investigate neuronal senescence and propose a panel of cellular and molecular hallmarks to characterize senescent neurons. Understanding the role of neuronal senescence may yield novel therapeutic opportunities in neurodegenerative disease.

DOI: https://doi.org/10.1038/s43587-024-00586-3
Neurobiol Dis. 2024 Feb 24. pii: S0969-9961(24)00053-6. [Epub ahead of print]193 106454

hnRNP R regulates mitochondrial movement and membrane potential in axons of motoneurons.

Sophia Dithmar, Abdolhossein Zare, Saeede Salehi, Michael Briese, Michael Sendtner.

  Axonal mitochondria defects are early events in the pathogenesis of motoneuron disorders such as spinal muscular atrophy and amyotrophic lateral sclerosis. The RNA-binding protein hnRNP R interacts with different motoneuron disease-related proteins such as SMN and TDP-43 and has important roles in axons of motoneurons, including axonal mRNA transport. However, whether hnRNP R also modulates axonal mitochondria is currently unknown. Here, we show that axonal mitochondria exhibit altered function and motility in hnRNP R-deficient motoneurons. Motoneurons lacking hnRNP R show decreased anterograde and increased retrograde transport of mitochondria in axons. Furthermore, hnRNP R-deficiency leads to mitochondrial hyperpolarization, caused by decreased complex I and reversed complex V activity within the respiratory chain. Taken together, our data indicate a role for hnRNP R in regulating transport and maintaining functionality of axonal mitochondria in motoneurons.

Keywords:  Axon; Mitochondria; Motoneuron; hnRNP R

DOI:  https://doi.org/10.1016/j.nbd.2024.106454
Int J Biochem Cell Biol. 2024 Feb 27. pii: S1357-2725(24)00047-5. [Epub ahead of print] 106556

The Application of Brain Organoid for Drug Discovery in Mitochondrial Diseases.

Kristina Xiao Liang.

  Mitochondrial diseases are difficult to treat due to the complexity and multifaceted nature of mitochondrial dysfunction. Brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells designed to mimic brain-like development and function. Brain organoids have received a lot of attention in recent years as powerful tools for modeling human diseases, brain development, and drug screening. Screening compounds for mitochondrial diseases using brain organoids could provide a more physiologically relevant platform for drug discovery. Brain organoids offer the possibility of personalized medicine because they can be derived from patient-specific cells, allowing testing of drugs tailored to specific genetic mutations. In this article, we highlight how brain organoids offer a promising avenue for screening compounds for mitochondrial diseases and address the challenges and limitations associated with their use. We hope this review will provide new insights into the further progress of brain organoids for mitochondrial screening studies.

Keywords:  Brain organoid; drug discovery; drug screening; iPSCs; mitochondrial diseases

DOI:  https://doi.org/10.1016/j.biocel.2024.106556
J Proteome Res. 2024 Feb 28.

Multiomics Evaluation of Human iPSCs and iPSC-Derived Neurons.

Gwang Bin Lee, Wan Nur Atiqah Binti Mazli, Ling Hao.

  Human induced pluripotent stem cells (iPSCs) can be differentiated into neurons, providing living human neurons to model brain diseases. However, it is unclear how different types of molecules work together to regulate stem cell and neuron biology in healthy and disease states. In this study, we conducted integrated proteomics, lipidomics, and metabolomics analyses with confident identification, accurate quantification, and reproducible measurements to compare the molecular profiles of human iPSCs and iPSC-derived neurons. Proteins, lipids, and metabolites related to mitosis, DNA replication, pluripotency, glycosphingolipids, and energy metabolism were highly enriched in iPSCs, whereas synaptic proteins, neurotransmitters, polyunsaturated fatty acids, cardiolipins, and axon guidance pathways were highly enriched in neurons. Mutations in the GRN gene lead to the deficiency of the progranulin (PGRN) protein, which has been associated with various neurodegenerative diseases. Using this multiomics platform, we evaluated the impact of PGRN deficiency on iPSCs and neurons at the whole-cell level. Proteomics, lipidomics, and metabolomics analyses implicated PGRN's roles in neuroinflammation, purine metabolism, and neurite outgrowth, revealing commonly altered pathways related to neuron projection, synaptic dysfunction, and brain metabolism. Multiomics data sets also pointed toward the same hypothesis that neurons seem to be more susceptible to PGRN loss compared to iPSCs, consistent with the neurological symptoms and cognitive impairment from patients carrying inherited GRN mutations.

Keywords:  GRN; iPSC; iPSC-derived neuron; lipidomics; metabolomics; multiomics; neuron; neuron differentiation; progranulin; proteomics

DOI:  https://doi.org/10.1021/acs.jproteome.3c00790
Front Pharmacol. 2024 ;15 1323491

Gene editing improves endoplasmic reticulum-mitochondrial contacts and unfolded protein response in Friedreich's ataxia iPSC-derived neurons.

Priyanka Mishra, Anusha Sivakumar, Avalon Johnson, Carla Pernaci, Anna S Warden, Lilas Rony El-Hachem, Emily Hansen, Rafael A Badell-Grau, Veenita Khare, Gabriela Ramirez, Sydney Gillette, Angelyn B Solis, Peng Guo, Nicole Coufal, Stephanie Cherqui.

  Friedreich ataxia (FRDA) is a multisystemic, autosomal recessive disorder caused by homozygous GAA expansion mutation in the first intron of frataxin (FXN) gene. FXN is a mitochondrial protein critical for iron-sulfur cluster biosynthesis and deficiency impairs mitochondrial electron transport chain functions and iron homeostasis within the organelle. Currently, there is no effective treatment for FRDA. We have previously demonstrated that single infusion of wild-type hematopoietic stem and progenitor cells (HSPCs) resulted in prevention of neurologic and cardiac complications of FRDA in YG8R mice, and rescue was mediated by FXN transfer from tissue engrafted, HSPC-derived microglia/macrophages to diseased neurons/myocytes. For a future clinical translation, we developed an autologous stem cell transplantation approach using CRISPR/Cas9 for the excision of the GAA repeats in FRDA patients' CD34+ HSPCs; this strategy leading to increased FXN expression and improved mitochondrial functions. The aim of the current study is to validate the efficiency and safety of our gene editing approach in a disease-relevant model. We generated a cohort of FRDA patient-derived iPSCs and isogenic lines that were gene edited with our CRISPR/Cas9 approach. iPSC derived FRDA neurons displayed characteristic apoptotic and mitochondrial phenotype of the disease, such as non-homogenous microtubule staining in neurites, increased caspase-3 expression, mitochondrial superoxide levels, mitochondrial fragmentation, and partial degradation of the cristae compared to healthy controls. These defects were fully prevented in the gene edited neurons. RNASeq analysis of FRDA and gene edited neurons demonstrated striking improvement in gene clusters associated with endoplasmic reticulum (ER) stress in the isogenic lines. Gene edited neurons demonstrated improved ER-calcium release, normalization of ER stress response gene, XBP-1, and significantly increased ER-mitochondrial contacts that are critical for functional homeostasis of both organelles, as compared to FRDA neurons. Ultrastructural analysis for these contact sites displayed severe ER structural damage in FRDA neurons, that was undetected in gene edited neurons. Taken together, these results represent a novel finding for disease pathogenesis showing dramatic ER structural damage in FRDA, validate the efficacy profile of our FXN gene editing approach in a disease relevant model, and support our approach as an effective strategy for therapeutic intervention for Friedreich's ataxia.

Keywords:  Friedreich’s ataxia; calcium homeostasis; endoplasmic reticulum-mitochondrial contacts; gene editing; induced pluripotent stem cells; neuronal apoptosis; neurons; unfolded protein response

DOI:  https://doi.org/10.3389/fphar.2024.1323491
J Neurol Neurosurg Psychiatry. 2024 Feb 28. pii: jnnp-2023-333039. [Epub ahead of print]

Effects of motor cortical and peripheral axonal hyperexcitability on survival in amyotrophic lateral sclerosis.

Ryo Otani, Kazumoto Shibuya, Yo-Ichi Suzuki, Tomoki Suichi, Marie Morooka, Yuya Aotsuka, Moeko Ogushi, Satoshi Kuwabara.

   BACKGROUND: Increased 'cortical' and 'peripheral' excitability are reportedly associated with shorter survival in amyotrophic lateral sclerosis (ALS) patients, suggesting that hyperexcitability contributes to motor neuron death. However, whether upper or lower motor function has a greater impact on survival is unclear. We aimed to investigate the component that strongly impacts the prognosis of ALS.
METHODS: A total of 103 consecutive patients with ALS who underwent cortical (threshold tracking transcranial magnetic stimulation (TMS)) and motor nerve excitability tests were included. Motor cortical excitability was evaluated using short-interval intracortical inhibition (SICI) during TMS. Motor axonal excitability was assessed using the strength-duration time constant (SDTC). Survival time was defined as the time from examination to death or tracheostomy.
RESULTS: Compared with healthy subjects, patients with ALS had lower SICI and longer SDTC (p<0.05), indicating increased excitability of cortical motor neurons and motor axons. According to the SICI and SDTC findings, patients were divided into the following four groups: 'cortical high and peripheral high (high-high)', 'high-low', 'low-high' and 'low-low' groups. In Kaplan-Meier curves, the 'high-high' and 'low-high' groups showed significantly shorter survival than the other groups. Multivariate analysis revealed that increased cortical (HR=5.3, p<0.05) and peripheral (HR=20.0, p<0.001) excitability were significantly associated with shorter survival.
CONCLUSIONS: In patients with ALS, both motor cortical and peripheral hyperexcitability independently affected survival time, with peripheral hyperexcitability having a greater impact on shorter survival. The modulation of neuronal/axonal excitability is a potential therapeutic target for ALS.

Keywords:  ALS; MAGNETIC STIMULATION; MOTOR NEURON DISEASE; NEUROPHYSIOLOGY

DOI:  https://doi.org/10.1136/jnnp-2023-333039
STAR Protoc. 2024 Feb 29. pii: S2666-1667(24)00069-8. [Epub ahead of print]5(1): 102904

Quantifying differentiation of progenitor populations using cerebral organoid models for neurodevelopmental disorders.

Annika L Schroder, Martin Fairbanks-Santana, Jennifer Rakotomamonjy, Alicia Guemez-Gamboa.

  Neurodevelopmental disorders are characterized by complex phenotypes that often result from concomitant dysregulation of cell proliferation, differentiation, or other crucial developmental processes. Here, we present a protocol to quantify differentiation of progenitor populations during early stages of neurogenesis in induced pluripotent stem cell (iPSC)-derived cerebral organoids. We describe steps for organoid differentiation and maturation, sample preparation, immunofluorescence, and imaging and analysis using epifluorescence microscopy. This protocol can be used to compare cerebral organoids from control and patient-derived iPSCs. For complete details on the use and execution of this protocol, please refer to Rakotomamonjy et al. (2023).1.

Keywords:  Cell Differentiation; Microscopy; Neuroscience; Organoids; Stem Cells

DOI:  https://doi.org/10.1016/j.xpro.2024.102904
Inflamm Regen. 2024 Feb 28. 44(1): 8

Mutual interaction of neurons and astrocytes derived from iPSCs with APP V717L mutation developed the astrocytic phenotypes of Alzheimer's disease.

Sopak Supakul, Rei Murakami, Chisato Oyama, Tomoko Shindo, Yuki Hatakeyama, Maika Itsuno, Hiroko Bannai, Shinsuke Shibata, Sumihiro Maeda, Hideyuki Okano.

   BACKGROUND: The development of induced pluripotent stem cells (iPSCs) technology has enabled human cellular disease modeling for inaccessible cell types, such as neural cells in the brain. However, many of the iPSC-derived disease models established to date typically involve only a single cell type. These monoculture models are inadequate for accurately simulating the brain environment, where multiple cell types interact. The limited cell type diversity in monoculture models hinders the accurate recapitulation of disease phenotypes resulting from interactions between different cell types. Therefore, our goal was to create cell models that include multiple interacting cell types to better recapitulate disease phenotypes.
METHODS: To establish a co-culture model of neurons and astrocytes, we individually induced neurons and astrocytes from the same iPSCs using our novel differentiation methods, and then co-cultured them. We evaluated the effects of co-culture on neurons and astrocytes using immunocytochemistry, immuno-electron microscopy, and Ca2+ imaging. We also developed a co-culture model using iPSCs from a patient with familial Alzheimer's disease (AD) patient (APP V717L mutation) to investigate whether this model would manifest disease phenotypes not seen in the monoculture models.
RESULTS: The co-culture of the neurons and astrocytes increased the branching of astrocyte processes, the number of GFAP-positive cells, neuronal activities, the number of synapses, and the density of presynaptic vesicles. In addition, immuno-electron microscopy confirmed the formation of a tripartite synaptic structure in the co-culture model, and inhibition of glutamate transporters increased neuronal activity. Compared to the co-culture model of the control iPSCs, the co-culture model of familial AD developed astrogliosis-like phenotype, which was not observed in the monoculture model of astrocytes.
CONCLUSIONS: Co-culture of iPSC-derived neurons and astrocytes enhanced the morphological changes mimicking the in vivo condition of both cell types. The formation of the functional tripartite synaptic structures in the co-culture model suggested the mutual interaction between the cells. Furthermore, the co-culture model with the APP V717L mutation expressed in neurons exhibited an astrocytic phenotype reminiscent of AD brain pathology. These results suggest that our co-culture model is a valuable tool for disease modeling of neurodegenerative diseases.

Keywords:  Alzheimer’s disease; Astrocytes; Co-culture model; Induced pluripotent stem cells (iPSCs); Neurons; Tripartite synapse

DOI:  https://doi.org/10.1186/s41232-023-00310-5
Autophagy. 2024 Feb 29. 1-3

FBXL4: safeguarding against mitochondrial depletion through suppression of mitophagy.

Prajakta Kulkarni, Giang Thanh Nguyen-Dien, Keri-Lyn Kozul, Julia K Pagan.

  Mitophagy is a critical mitochondrial quality control process that selectively removes dysfunctional or excess mitochondria through the autophagy-lysosome system. The process is tightly controlled to ensure cellular and physiological homeostasis. Insufficient mitophagy can result in failure to remove damaged mitochondria and consequent cellular degeneration, but it is equally important to appropriately restrain mitophagy to prevent excessive mitochondrial depletion. Here, we discuss our recent discovery that the SKP1-CUL1-F-box (SCF)-FBXL4 (F-box and leucine-rich repeat protein 4) E3 ubiquitin ligase localizes to the mitochondrial outer membrane, where it constitutively mediates the ubiquitination and degradation of BNIP3L/NIX and BNIP3 mitophagy receptors to suppress mitophagy. The post-translational regulation of BNIP3L and BNIP3 is disrupted in mitochondrial DNA depletion syndrome 13 (MTDPS13), a multi-systemic disorder caused by mutations in the FBXL4 gene and characterized by elevated mitophagy and mitochondrial DNA/mtDNA depletion in patient fibroblasts. Our results demonstrate that mitophagy is not solely stimulated in response to specific conditions but is instead also actively suppressed through the continuous degradation of BNIP3L and BNIP3 mediated by the SCF-FBXL4 ubiquitin ligase. Thus, cellular conditions or signaling events that prevent the FBXL4-mediated turnover of BNIP3L and BNIP3 on specific mitochondria are expected to facilitate their selective removal.

Keywords:  BNIP3; BNIP3L/NIX; FBXL4; MTDPS13; mitophagy; ubiquitin ligase

DOI:  https://doi.org/10.1080/15548627.2024.2318077
Neuroscientist. 2024 Feb 29. 10738584241232963

α-Synuclein: Multiple pathogenic roles in trafficking and proteostasis pathways in Parkinson's disease.

Annie J Zalon, Drew J Quiriconi, Caleb Pitcairn, Joseph R Mazzulli.

  Parkinson's disease (PD) is a common age-related neurodegenerative disorder characterized by the loss of dopaminergic neurons in the midbrain. A hallmark of both familial and sporadic PD is the presence of Lewy body inclusions composed mainly of aggregated α-synuclein (α-syn), a presynaptic protein encoded by the SNCA gene. The mechanisms driving the relationship between α-syn accumulation and neurodegeneration are not completely understood, although recent evidence indicates that multiple branches of the proteostasis pathway are simultaneously perturbed when α-syn aberrantly accumulates within neurons. Studies from patient-derived midbrain cultures that develop α-syn pathology through the endogenous expression of PD-causing mutations show that proteostasis disruption occurs at the level of synthesis/folding in the endoplasmic reticulum (ER), downstream ER-Golgi trafficking, and autophagic-lysosomal clearance. Here, we review the fundamentals of protein transport, highlighting the specific steps where α-syn accumulation may intervene and the downstream effects on proteostasis. Current therapeutic efforts are focused on targeting single pathways or proteins, but the multifaceted pathogenic role of α-syn throughout the proteostasis pathway suggests that manipulating several targets simultaneously will provide more effective disease-modifying therapies for PD and other synucleinopathies.

Keywords:  Parkinson’s disease; autophagy; protein trafficking; synuclein

DOI:  https://doi.org/10.1177/10738584241232963
PLoS Biol. 2024 Feb 29. 22(2): e3002527

RNA-mediated ribonucleoprotein assembly controls TDP-43 nuclear retention.

Patricia M Dos Passos, Erandika H Hemamali, Lohany D Mamede, Lindsey R Hayes, Yuna M Ayala.

TDP-43 is an essential RNA-binding protein strongly implicated in the pathogenesis of neurodegenerative disorders characterized by cytoplasmic aggregates and loss of nuclear TDP-43. The protein shuttles between nucleus and cytoplasm, yet maintaining predominantly nuclear TDP-43 localization is important for TDP-43 function and for inhibiting cytoplasmic aggregation. We previously demonstrated that specific RNA binding mediates TDP-43 self-assembly and biomolecular condensation, requiring multivalent interactions via N- and C-terminal domains. Here, we show that these complexes play a key role in TDP-43 nuclear retention. TDP-43 forms macromolecular complexes with a wide range of size distribution in cells and we find that defects in RNA binding or inter-domain interactions, including phase separation, impair the assembly of the largest species. Our findings suggest that recruitment into these macromolecular complexes prevents cytoplasmic egress of TDP-43 in a size-dependent manner. Our observations uncover fundamental mechanisms controlling TDP-43 cellular homeostasis, whereby regulation of RNA-mediated self-assembly modulates TDP-43 nucleocytoplasmic distribution. Moreover, these findings highlight pathways that may be implicated in TDP-43 proteinopathies and identify potential therapeutic targets.

DOI: https://doi.org/10.1371/journal.pbio.3002527
Sci Rep. 2024 02 26. 14(1): 4620

Restoring functional TDP-43 oligomers in ALS and laminopathic cellular models through baicalein-induced reconfiguration of TDP-43 aggregates.

Hsiang-Yu Chang, I-Fan Wang.

  A group of misfolded prone-to-aggregate domains in disease-causing proteins has recently been shown to adopt unique conformations that play a role in fundamental biological processes. These processes include the formation of membrane-less sub-organelles, alternative splicing, and gene activation and silencing. The cellular responses are regulated by the conformational switching of prone-to-aggregate domains, independently of changes in RNA or protein expression levels. Given this, targeting the misfolded states of disease-causing proteins to redirect them towards their physiological conformations is emerging as an effective therapeutic strategy for diseases caused by protein misfolding. In our study, we successfully identified baicalein as a potent structure-correcting agent. Our findings demonstrate that baicalein can reconfigure existing TDP-43 aggregates into an oligomeric state both in vitro and in disease cells. This transformation effectively restores the bioactivity of misfolded TDP-43 proteins in cellular models of ALS and premature aging in progeria. Impressively, in progeria cells where defective lamin A interferes with TDP-43-mediated exon skipping, the formation of pathological TDP-43 aggregates is promoted. Baicalein, however, restores the functionality of TDP-43 and mitigates nuclear shape defects in these laminopathic cells. This establishes a connection between lamin A and TDP-43 in the context of aging. Our findings suggest that targeting physiological TDP-43 oligomers could offer a promising therapeutic avenue for treating aging-associated disorders.

Keywords:  ALS; Aggregates; Baicalein; FTLD-U; Hutchinson-Gilford progeria syndrome (HGPS); Low complexity (LC) domain; Physiological oligomers; TDP-43

DOI:  https://doi.org/10.1038/s41598-024-55229-9
Dis Model Mech. 2024 Feb 01. pii: dmm050323. [Epub ahead of print]17(2):

PTCH1-mutant human cerebellar organoids exhibit altered neural development and recapitulate early medulloblastoma tumorigenesis.

Max J van Essen, Elizabeth J Apsley, Joey Riepsaame, Ruijie Xu, Paul A Northcott, Sally A Cowley, John Jacob, Esther B E Becker.

  Patched 1 (PTCH1) is the primary receptor for the sonic hedgehog (SHH) ligand and negatively regulates SHH signalling, an essential pathway in human embryogenesis. Loss-of-function mutations in PTCH1 are associated with altered neuronal development and the malignant brain tumour medulloblastoma. As a result of differences between murine and human development, molecular and cellular perturbations that arise from human PTCH1 mutations remain poorly understood. Here, we used cerebellar organoids differentiated from human induced pluripotent stem cells combined with CRISPR/Cas9 gene editing to investigate the earliest molecular and cellular consequences of PTCH1 mutations on human cerebellar development. Our findings demonstrate that developmental mechanisms in cerebellar organoids reflect in vivo processes of regionalisation and SHH signalling, and offer new insights into early pathophysiological events of medulloblastoma tumorigenesis without the use of animal models.

Keywords:  CRISPR; Cerebellum; Development; Medulloblastoma; Patched 1; Sonic hedgehog; iPSCs

DOI:  https://doi.org/10.1242/dmm.050323
Proc Natl Acad Sci U S A. 2024 Mar 05. 121(10): e2313540121

Tom20 gates PINK1 activity and mediates its tethering of the TOM and TIM23 translocases upon mitochondrial stress.

Mohamed A Eldeeb, Andrew N Bayne, Armaan Fallahi, Thomas Goiran, Emma J MacDougall, Andrea Soumbasis, Cornelia E Zorca, Jace-Jones Tabah, Rhalena A Thomas, Nathan Karpilovsky, Meghna Mathur, Thomas M Durcan, Jean-François Trempe, Edward A Fon.

  Mutations in PTEN-induced putative kinase 1 (PINK1) cause autosomal recessive early-onset Parkinson's disease (PD). PINK1 is a Ser/Thr kinase that regulates mitochondrial quality control by triggering mitophagy mediated by the ubiquitin (Ub) ligase Parkin. Upon mitochondrial damage, PINK1 accumulates on the outer mitochondrial membrane forming a high-molecular-weight complex with the translocase of the outer membrane (TOM). PINK1 then phosphorylates Ub, which enables recruitment and activation of Parkin followed by autophagic clearance of the damaged mitochondrion. Thus, Parkin-dependent mitophagy hinges on the stable accumulation of PINK1 on the TOM complex. Yet, the mechanism linking mitochondrial stressors to PINK1 accumulation and whether the translocases of the inner membrane (TIMs) are also involved remain unclear. Herein, we demonstrate that mitochondrial stress induces the formation of a PINK1-TOM-TIM23 supercomplex in human cultured cell lines, dopamine neurons, and midbrain organoids. Moreover, we show that PINK1 is required to stably tether the TOM to TIM23 complexes in response to stress such that the supercomplex fails to accumulate in cells lacking PINK1. This tethering is dependent on an interaction between the PINK1 N-terminal-C-terminal extension module and the cytosolic domain of the Tom20 subunit of the TOM complex, the disruption of which, by either designer or PD-associated PINK1 mutations, inhibits downstream mitophagy. Together, the findings provide key insight into how PINK1 interfaces with the mitochondrial import machinery, with important implications for the mechanisms of mitochondrial quality control and PD pathogenesis.

Keywords:  PINK1; mitochondrial import; mitochondrial quality control; mitophagy; proteolysis

DOI:  https://doi.org/10.1073/pnas.2313540121
Neurotoxicology. 2024 Feb 22. pii: S0161-813X(24)00019-6. [Epub ahead of print]

Neuronal exosomal miRNAs modulate mitochondrial functions and cell death in bystander neuronal cells under Parkinson's disease stress conditions.

Fatema Currim, Shatakshi Shukla, Jyoti Singh, Dhruv Gohel, Minal Mane, Anjali Shinde, Milton Roy, Shani Goyani, Hitesh Vasiyani, Aswathy Chandran, Jean-Christophe Rochet, Jason Cannon, Rajesh Singh.

  Parkinson's Disease (PD) is a chronic neurodegenerative disorder characterized by progressive loss of midbrain dopaminergic neurons in the substantia nigra part of the brain. Pathology spread to numerous brain regions and cell types suggests that intercellular communication is essential to PD progression. Exosomes mediate intercellular communication between neurons, glia, and other cell types throughout PD-relevant brain regions. However, the mechanism remains unclear, and its implication in PD pathology, is not well understood. In the current study, we explored the role of exosomes in modulating the response to PD-relevant toxicants. In cellular models of PD, neuronal cell-derived exosomes are readily internalized by recipient neuronal cells as intact vesicles. Internalized exosomes in bystander neuronal cells localize to mitochondria and dysregulate mitochondrial functions, leading to cell death under PD stress conditions. NGS analysis of exosomes released by neuronal cells subjected to PD stress conditions showed that levels of specific miRNAs were altered in exosomes under PD stress conditions. Bioinformatic analysis of the miRNA targets revealed enriched pathways related to neuronal processes and morphogenesis, apoptosis, and ageing. Levels of two miRNAs, hsa-miR-30a-5p and hsa-miR-181c-5p, were downregulated in exosomes under PD stress conditions. Expression of the identified miRNAs in neuronal cells led to their enrichment in exosomes, and exosome uptake in neuronal cells ameliorated mitochondrial dysfunction induced by PD stress conditions and rescued cell death. In conclusion, loss of enrichment of specific miRNAs, including miR-30a-5p and miR-181c-5p, under PD stress conditions causes mitochondrial dysfunction and neuronal death, and hence may lead to progression of PD.

Keywords:  Parkinson’s Disease; exosomal miRNAs; intercellular communication; neuron-neuron communication

DOI:  https://doi.org/10.1016/j.neuro.2024.02.005
Aging Dis. 2024 Feb 22.

A Neglected Gene: The Role of the ANG Gene in the Pathogenesis of Amyotrophic Lateral Sclerosis.

Yu Zhang, Yanan Li, Shen Bin, Xi Cheng, Qi Niu.

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive neurodegenerative disease with a poor prognosis. To date, more than 40 ALS-related genes have been identified. However, there is still a lack of targeted therapeutic drugs for the treatment of ALS, especially for patients with acute onset and severe disease. A series of studies reported missense heterozygous mutations with loss of function in the coding region of the ANG gene in ALS patients. ANG deficiency is related to the pathogenesis of ALS, but the underlying mechanism has not been determined. This article aimed to synthesize and consolidate the knowledge of the pathological mechanism of ALS induced by ANG mutation and provide a theoretical basis for ALS diagnosis and targeted therapy. This article further delves into the mechanisms underlying the current understanding of the structure and function of the ANG gene, the association between ANG and ALS, and its pathogenesis. Mutations in ANG may lead to the development of ALS through the loss of neuroprotective function, induction of oxidative stress, or inhibition of rRNA synthesis. ANG mutations and genetic and environmental factors may cause disease heterogeneity and more severe disease than in ALS patients with the wild-type gene. Exploring this mechanism is expected to provide a new approach for ALS treatment through increasing ANG expression or angiogenin activity. However, the related study is still in its infancy; therefore, this article also highlights the need for further exploration of the application of ANG gene mutations in clinical trials and animal experiments is needed to achieve improved early diagnosis and treatment of ALS.

DOI: https://doi.org/10.14336/AD.2024.0107
Nat Commun. 2024 Feb 28. 15(1): 1816

Cellular reprogramming as a tool to model human aging in a dish.

Patricia R Pitrez, Luis M Monteiro, Oliver Borgogno, Xavier Nissan, Jerome Mertens, Lino Ferreira.

The design of human model systems is highly relevant to unveil the underlying mechanisms of aging and to provide insights on potential interventions to extend human health and life span. In this perspective, we explore the potential of 2D or 3D culture models comprising human induced pluripotent stem cells and transdifferentiated cells obtained from aged or age-related disorder-affected donors to enhance our understanding of human aging and to catalyze the discovery of anti-aging interventions.

DOI: https://doi.org/10.1038/s41467-024-46004-5
Cell Rep. 2024 Feb 27. pii: S2211-1247(24)00195-5. [Epub ahead of print]43(3): 113867

Loss of GTF2I promotes neuronal apoptosis and synaptic reduction in human cellular models of neurodevelopment.

Jason W Adams, Annabelle Vinokur, Janaína S de Souza, Charles Austria, Bruno S Guerra, Roberto H Herai, Karl J Wahlin, Alysson R Muotri.

  Individuals with Williams syndrome (WS), a neurodevelopmental disorder caused by hemizygous loss of 26-28 genes at 7q11.23, characteristically portray a hypersocial phenotype. Copy-number variations and mutations in one of these genes, GTF2I, are associated with altered sociality and are proposed to underlie hypersociality in WS. However, the contribution of GTF2I to human neurodevelopment remains poorly understood. Here, human cellular models of neurodevelopment, including neural progenitors, neurons, and three-dimensional cortical organoids, are differentiated from CRISPR-Cas9-edited GTF2I-knockout (GTF2I-KO) pluripotent stem cells to investigate the role of GTF2I in human neurodevelopment. GTF2I-KO progenitors exhibit increased proliferation and cell-cycle alterations. Cortical organoids and neurons demonstrate increased cell death and synaptic dysregulation, including synaptic structural dysfunction and decreased electrophysiological activity on a multielectrode array. Our findings suggest that changes in synaptic circuit integrity may be a prominent mediator of the link between alterations in GTF2I and variation in the phenotypic expression of human sociality.

Keywords:  CP: Developmental biology; CP: Neuroscience; GTF2I; Williams syndrome; brain organoid; cortical organoid; neurodevelopment; stem cells

DOI:  https://doi.org/10.1016/j.celrep.2024.113867
Eur J Med Chem. 2024 Feb 23. pii: S0223-5234(24)00131-4. [Epub ahead of print]268 116251

Nur77 increases mitophagy and decreases aggregation of α-synuclein by modulating the p-c-Abl/p-PHB2 Y121 in α-synuclein PFF SH-SY5Y cells and mice.

Shiyi Yin, Mengmeng Shen, Yongjiang Zhang, Jiannan Wu, Run Song, Xiaoyi Lai, Zhenzhen Tian, Tingting Wang, Weina Jin, Junqiang Yan.

  Parkinson's disease (PD) is characterized by the progressive death of dopamine (DA) neurons and the pathological accumulation of α-synuclein (α-syn) fibrils. In our previous study, simulated PHB2 phosphorylation was utilized to clarify the regulatory role of c-Abl in PHB2-mediated mitophagy in PD models. In this investigation, we employed an independently patented PHB2Y121 phosphorylated antibody in the PD model to further verify that the c-Abl inhibitor STI571 can impede PHB2Y121 phosphorylation, decrease the formation of α-Syn polymers, and improve autophagic levels. The specific involvement of Nur77 in PD pathology has remained elusive. We also investigate the contribution of Nur77, a nuclear transcription factor, to α-syn and mitophagy in PD. Our findings demonstrate that under α-syn, Nur77 translocates from the cytoplasm to the mitochondria, improving PHB-mediated mitophagy by regulating c-Abl phosphorylation. Moreover, Nur77 overexpression alleviates the expression level of pS129-α-syn and the loss of DA neurons in α-syn PFF mice, potentially associated with the p-c-Abl/p-PHB2 Y121 axis. This study provides initial in vivo and in vitro evidence that Nur77 protects PD DA neurons by modulating the p-c-Abl/p-PHB2 Y121 axis, and STI571 holds promise as a treatment for PD.

Keywords:  Mitophagy; Nur77; PHB2; Parkinson's disease; STI571; c-Abl; α-synuclein

DOI:  https://doi.org/10.1016/j.ejmech.2024.116251
Rev Neurol (Paris). 2024 Feb 29. pii: S0035-3787(24)00415-6. [Epub ahead of print]

Spreading of motor neuron degeneration in ALS is not so random.

P Corcia, P Couratier.

DOI: https://doi.org/10.1016/j.neurol.2024.02.384
Adv Exp Med Biol. 2024 ;1443 1-22

Isolation of Extracellular Vesicles Using Titanium Dioxide Microspheres.

Veronica Feijoli Santiago, Livia Rosa-Fernandes, Janaina Macedo-da-Silva, Claudia B Angeli, Simon Ngao Mule, Claudio R F Marinho, Ana Claudia Torrecilhas, Suely N K Marie, Giuseppe Palmisano.

  Extracellular vesicles (EVs) are bilayer membrane particles released from several cell types to the extracellular environment. EVs have a crucial role in cell-cell communication, involving different biological processes in health and diseases. Due to the potential of biomarkers for several diseases as diagnostic and therapeutic tools, it is relevant to understand the biology of the EVs and their content. One of the current challenges involving EVs is regarding the purification method, which is a critical step for EV's functional and characterization studies. Ultracentrifugation is the most used method for EV isolation, where the nanoparticles are separated in sequential centrifugation to isolate the EVs based on their size. However, for viscous biofluids such as plasma, there is a co-isolation of the most abundant proteins, which can impair the EV's protein identification due to the low abundance of these proteins and signal suppression by the most abundant plasma proteins. Emerging techniques have gained attention in recent years. Titanium dioxide (TiO2) is one of the most promising techniques due to its property for selective isolation based on the interaction with phospholipids in the EV membrane. Using a small amount of TiO2 beads and a low volume of plasma, it is possible to isolate EVs with reduced plasma protein co-isolation. This study describes a comprehensive workflow for the isolation and characterization of plasma extracellular vesicles (EVs) using mass spectrometry-based proteomics techniques. The aim of this chapter is describe the EV isolation using TiO2 beads enrichment and high-throughput mass spectrometry techniques to efficiently identify the protein composition of EVs in a fast and straightforward manner.

Keywords:  Extracellular vesicles; MALDI-TOF MS; Mass spectrometry; Proteomics; Titanium dioxide beads

DOI:  https://doi.org/10.1007/978-3-031-50624-6_1
bioRxiv. 2024 Feb 13. pii: 2024.02.12.579972. [Epub ahead of print]

Most axonal mitochondria in cortical pyramidal neurons lack mitochondrial DNA and consume ATP.

Yusuke Hirabayashi, Tommy L Lewis, Yudan Du, Daniel M Virga, Aubrianna M Decker, Giovanna Coceano, Jonatan Alvelid, Maëla A Paul, Stevie Hamilton, Parker Kneis, Yasufumi Takahashi, Jellert T Gaublomme, Ilaria Testa, Franck Polleux.

In neurons of the mammalian central nervous system (CNS), axonal mitochondria are thought to be indispensable for supplying ATP during energy-consuming processes such as neurotransmitter release. Here, we demonstrate using multiple, independent, in vitro and in vivo approaches that the majority (~80-90%) of axonal mitochondria in cortical pyramidal neurons (CPNs), lack mitochondrial DNA (mtDNA). Using dynamic, optical imaging analysis of genetically encoded sensors for mitochondrial matrix ATP and pH, we demonstrate that in axons of CPNs, but not in their dendrites, mitochondrial complex V (ATP synthase) functions in a reverse way, consuming ATP and protruding H+ out of the matrix to maintain mitochondrial membrane potential. Our results demonstrate that in mammalian CPNs, axonal mitochondria do not play a major role in ATP supply, despite playing other functions critical to regulating neurotransmission such as Ca2+ buffering.

DOI: https://doi.org/10.1101/2024.02.12.579972
NPJ Parkinsons Dis. 2024 Mar 01. 10(1): 49

G2019S selective LRRK2 kinase inhibitor abrogates mitochondrial DNA damage.

Nicholas Pena, Tara Richbourg, Claudia P Gonzalez-Hunt, Rui Qi, Paul Wren, Carrolee Barlow, Natalie F Shanks, Holly J Carlisle, Laurie H Sanders.

Pathogenic mutations in LRRK2 cause Parkinson's disease (PD). The G2019S variant is the most common, which results in abnormally high kinase activity. Compounds that target LRRK2 kinase activity are currently being developed and tested in clinical trials. We recently found that G2019S LRRK2 causes mitochondrial DNA (mtDNA) damage and treatment with multiple classes of LRRK2 kinase inhibitors at concentrations associated with dephosphorylation of LRRK2 reversed mtDNA damage to healthy control levels. Because maintaining the normal function of LRRK2 in heterozygous G2019S LRRK2 carriers while specifically targeting the G2019S LRRK2 activity could have an advantageous safety profile, we explored the efficacy of a G2019S mutant selective LRRK2 inhibitor to reverse mtDNA damage in G2019S LRRK2 models and patient cells relative to non-selective LRRK2 inhibitors. Potency of LRRK2 kinase inhibition by EB-42168, a G2019S mutant LRRK2 kinase inhibitor, and MLi-2, a non-selective inhibitor, was determined by measuring phosphorylation of LRRK2 at Ser935 and/or Ser1292 using quantitative western immunoblot analysis. The Mito DNADX assay, which allows for the accurate real-time quantification of mtDNA damage in a 96-well platform, was performed in parallel. We confirmed that EB-42168 selectively inhibits LRRK2 phosphorylation on G2019S LRRK2 relative to wild-type LRRK2. On the other hand, MLi-2 was equipotent for wild-type and G2019S LRRK2. Acute treatment with EB-42168 inhibited LRRK2 phosphorylation and also restored mtDNA damage to healthy control levels. We further investigated the relationship between LRRK2 kinase activity, mtDNA damage and mitophagy. Levels of mtDNA damage caused by G2019S LRRK2 were fully re-established within 2 h of a LRRK2 inhibitor wash out and recovery experiment, indicating the mtDNA damage phenotype is highly dynamic. G2019S LRRK2 mitophagy defects were not alleviated with LRRK2 kinase inhibition, suggesting that mitophagy is not mechanistically regulating LRRK2 kinase-mediated reversal of mtDNA damage in this acute timeframe. Abrogation of mtDNA damage with the mutant selective tool inhibitor EB-42168 demonstrates the potential of a precision medicine approach for LRRK2 G2019S PD. Levels of mtDNA damage may serve as a potential pharmacodynamic biomarker of altered kinase activity that could be useful for small molecule development and clinical trials.

DOI: https://doi.org/10.1038/s41531-024-00660-y
BMC Biol. 2024 Feb 27. 22(1): 48

Primary cilia promote the differentiation of human neurons through the WNT signaling pathway.

Andrea Coschiera, Masahito Yoshihara, Gilbert Lauter, Sini Ezer, Mariangela Pucci, Haonan Li, Alan Kavšek, Christian G Riedel, Juha Kere, Peter Swoboda.

   BACKGROUND: Primary cilia emanate from most human cell types, including neurons. Cilia are important for communicating with the cell's immediate environment: signal reception and transduction to/from the ciliated cell. Deregulation of ciliary signaling can lead to ciliopathies and certain neurodevelopmental disorders. In the developing brain cilia play well-documented roles for the expansion of the neural progenitor cell pool, while information about the roles of cilia during post-mitotic neuron differentiation and maturation is scarce.
RESULTS: We employed ciliated Lund Human Mesencephalic (LUHMES) cells in time course experiments to assess the impact of ciliary signaling on neuron differentiation. By comparing ciliated and non-ciliated neuronal precursor cells and neurons in wild type and in RFX2 -/- mutant neurons with altered cilia, we discovered an early-differentiation "ciliary time window" during which transient cilia promote axon outgrowth, branching and arborization. Experiments in neurons with IFT88 and IFT172 ciliary gene knockdowns, leading to shorter cilia, confirm these results. Cilia promote neuron differentiation by tipping WNT signaling toward the non-canonical pathway, in turn activating WNT pathway output genes implicated in cyto-architectural changes.
CONCLUSIONS: We provide a mechanistic entry point into when and how ciliary signaling coordinates, promotes and translates into anatomical changes. We hypothesize that ciliary alterations causing neuron differentiation defects may result in "mild" impairments of brain development, possibly underpinning certain aspects of neurodevelopmental disorders.

Keywords:  Axon branching; Neuron differentiation; Primary cilia; Transcriptomics time-course; WNT signaling

DOI:  https://doi.org/10.1186/s12915-024-01845-w
Nat Cell Biol. 2024 Mar 01.

Combinatorial selective ER-phagy remodels the ER during neurogenesis.

Melissa J Hoyer, Cristina Capitanio, Ian R Smith, Julia C Paoli, Anna Bieber, Yizhi Jiang, Joao A Paulo, Miguel A Gonzalez-Lozano, Wolfgang Baumeister, Florian Wilfling, Brenda A Schulman, J Wade Harper.

The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions, ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis, where a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodelling, as autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic boutons, and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, our understanding of the mechanisms underlying selective removal of the ER and the role of individual ER-phagy receptors is limited. Here we combine a genetically tractable induced neuron (iNeuron) system for monitoring ER remodelling during in vitro differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodelling via selective autophagy. Through analysis of single and combinatorial ER-phagy receptor mutants, we delineate the extent to which each receptor contributes to both the magnitude and selectivity of ER protein clearance. We define specific subsets of ER membrane or lumenal proteins as preferred clients for distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, and directly visualize tubular ER membranes within autophagosomes in neuronal projections by cryo-electron tomography. This molecular inventory of ER proteome remodelling and versatile genetic toolkit provide a quantitative framework for understanding the contributions of individual ER-phagy receptors for reshaping ER during cell state transitions.

DOI: https://doi.org/10.1038/s41556-024-01356-4
Mol Ther Nucleic Acids. 2024 Mar 12. 35(1): 102132

mitoTALEN reduces the mutant mtDNA load in neurons.

Sandra R Bacman, Jose Domingo Barrera-Paez, Milena Pinto, Derek Van Booven, James B Stewart, Anthony J Griswold, Carlos T Moraes.

  Mutations within mtDNA frequently give rise to severe encephalopathies. Given that a majority of these mtDNA defects exist in a heteroplasmic state, we harnessed the precision of mitochondrial-targeted TALEN (mitoTALEN) to selectively eliminate mutant mtDNA within the CNS of a murine model harboring a heteroplasmic mutation in the mitochondrial tRNA alanine gene (m.5024C>T). This targeted approach was accomplished by the use of AAV-PHP.eB and a neuron-specific synapsin promoter for effective neuronal delivery and expression of mitoTALEN. We found that most CNS regions were effectively transduced and showed a significant reduction in mutant mtDNA. This reduction was accompanied by an increase in mitochondrial tRNA alanine levels, which are drastically reduced by the m.5024C>T mutation. These results showed that mitochondrial-targeted gene editing can be effective in reducing CNS-mutant mtDNA in vivo, paving the way for clinical trials in patients with mitochondrial encephalopathies.

Keywords:  AAV-PHPeB; CNS; MT: RNA/DNA editing; TALEN; gene therapy; heteroplasmy; mitochondria

DOI:  https://doi.org/10.1016/j.omtn.2024.102132
Methods Mol Biol. 2024 ;2761 97-120

Drosophila melanogaster Neuromuscular Junction as a Model to Study Synaptopathies and Neuronal Autophagy.

Anushka Chakravorty, Vasu Sheeba, Ravi Manjithaya.

  Neuronal synapse dysfunction is a key characteristic of several neurodegenerative disorders, such as Alzheimer's disease, spinocerebellar ataxias, and Huntington's disease. Modeling these disorders to study synaptic dysfunction requires a robust and reproducible method for assaying the subtle changes associated with synaptopathies in terms of structure and function of the synapses. Drosophila melanogaster neuromuscular junctions (NMJs) serve as good models to study such alterations. Further, modifications in the microenvironment of synapses can sometimes reflect in the behavior of the animal, which can also be assayed in a high-throughput manner. The methods outlined in this chapter highlight assays to study the behavioral changes associated with synaptic dysfunction in a spinocerebellar ataxia type 3 (SCA3) model. Further, molecular assessment of alterations in NMJ structure and function is also summarized, followed by effects of autophagy pathway upregulation in providing neuroprotection. These methods can be further extended and modified to study the therapeutic effects of drugs or small molecules in providing neuroprotection for any synaptopathy models.

Keywords:  Autophagy; Drosophila; Locomotion; Neurodegeneration; Neuromuscular junction; Synaptopathy

DOI:  https://doi.org/10.1007/978-1-0716-3662-6_9
Mol Biol Cell. 2024 Feb 28. mbcE23120510

Myosin F controls actin organization and dynamics in Toxoplasma gondii.

Jacob A Kellermeier, Aoife T Heaslip.

Intracellular cargo transport is a ubiquitous cellular process in all eukaryotes. In many cell types, membrane bound cargo is associated with molecular motors which transport cargo along microtubule and actin tracks. In Toxoplasma gondii (T. gondii), an obligate intracellular parasite in the phylum Apicomplexa, organization of the endomembrane pathway depends on actin and an unconventional myosin motor, myosin F (MyoF). Loss of MyoF and actin disrupts vesicle transport, organelle positioning, and division of the apicoplast, a non-photosynthetic plastid organelle. How this actomyosin system contributes to these cellular functions is still unclear. Using live-cell imaging, we observed that MyoF-EmeraldFP (MyoF-EmFP) displayed a dynamic and filamentous-like organization in the parasite cytosol, reminiscent of cytosolic actin filament dynamics. MyoF was not associated with the Golgi, apicoplast or dense granule surfaces, suggesting that it does not function using the canonical cargo transport mechanism. Instead, we found that loss of MyoF resulted in a dramatic rearrangement of the actin cytoskeleton in interphase parasites accompanied by significantly reduced actin dynamics. However, actin organization during parasite replication and motility was unaffected by the loss of MyoF. These findings revealed that MyoF is an actin organizing protein in Toxoplasma and facilitates cargo movement using an unconventional transport mechanism. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

DOI: https://doi.org/10.1091/mbc.E23-12-0510
bioRxiv. 2024 Feb 16. pii: 2024.02.15.580492. [Epub ahead of print]

A ratiometric ER calcium sensor for quantitative comparisons across cell types and subcellular regions.

Ryan J Farrell, Kirsten G Bredvik, Michael B Hoppa, S Thomas Hennigan, Timothy A Brown, Timothy A Ryan.

The endoplasmic reticulum (ER) is an important regulator of Ca2+ in cells and dysregulation of ER calcium homeostasis can lead to numerous pathologies. Understanding how various pharmacological and genetic perturbations of ER Ca2+ homeostasis impacts cellular physiology would likely be facilitated by more quantitative measurements of ER Ca2+ levels that allow easier comparisons across conditions. Here, we developed a ratiometric version of our original ER-GCaMP probe that allows for more quantitative comparisons of the concentration of Ca2+ in the ER across cell types and sub-cellular compartments. Using this approach we show that the resting concentration of ER Ca2+ in primary dissociated neurons is substantially lower than that in measured in embryonic fibroblasts.

DOI: https://doi.org/10.1101/2024.02.15.580492