bims-axbals 2024-09-22 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2024–09–22
29 papers selected by
TJ Krzystek, ALS Therapy Development Institute

Molecular pathology, developmental changes and synaptic dysfunction in (pre-) symptomatic human C9ORF72-ALS/FTD cerebral organoids.
CRISPR-Cas13d targeting suppresses repeat-associated non-AUG translation of C9orf72 hexanucleotide repeat RNA.
Motor Cortical Neuronal Hyperexcitability Associated with α-Synuclein Aggregation.
Identification of high-performing antibodies for SPARC-related modular calcium-binding protein 1 (SMOC-1) for use in Western Blot and immunoprecipitation.
A comprehensive review of electrophysiological techniques in amyotrophic lateral sclerosis research.
Gigaxonin, mutated in Giant Axonal Neuropathy, interacts with TDP-43 and other RNA binding proteins.
Next-Gen Therapeutics: Pioneering Drug Discovery with iPSCs, Genomics, AI, and Clinical Trials in a Dish.
Epigenetics in the formation of pathological aggregates in amyotrophic lateral sclerosis.
Atomistic Insights into the Stabilization of TDP-43 Protofibrils by ATP.
Single-Molecule Sequencing of the C9orf72 Repeat Expansion in Patient iPSCs.
Dysregulated FOXO1 activity drives skeletal muscle intrinsic dysfunction in amyotrophic lateral sclerosis.
N-acetylaspartate promotes glycolytic-to-oxidative fiber-type switch and resistance to atrophic stimuli in myotubes.
Glial swip-10 controls systemic mitochondrial function, oxidative stress, and neuronal viability via copper ion homeostasis.
Location and function of TDP-43 in platelets, alterations in neurodegenerative diseases and arising considerations for current plasma biobank protocols.
Biallelic PTPMT1 variants disrupt cardiolipin metabolism and lead to a neurodevelopmental syndrome.
Generation of CRISPR/Cas9-edited human iPSC lines carrying homozygous and heterozygous SAMD9 p.I983S mutations.
PROTAC-mediated vimentin degradation promotes terminal erythroid differentiation of pluripotent stem cells.
The p53 target DRAM1 modulates calcium homeostasis and ER stress by promoting contact between lysosomes and the ER through STIM1.
Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways.
Targeting ferroptosis with the lipoxygenase inhibitor PTC-041 as a therapeutic strategy for the treatment of Parkinson's disease.
Prion protein pathology in Ubiquilin 2 models of ALS.
Viral overexpression of human alpha-synuclein in mouse substantia nigra dopamine neurons results in hyperdopaminergia but no neurodegeneration.
Human TDP43 is required for ALS‑related annexin A11 toxicity in Drosophila.
Crosstalk between ubiquitination and translation in neurodevelopmental disorders.
Quantitative proteomics of patient fibroblasts reveal biomarkers and diagnostic signatures of mitochondrial disease.
iPLA2β loss leads to age-related cognitive decline and neuroinflammation by disrupting neuronal mitophagy.
Ready-to-use iPSC-derived microglia progenitors for the treatment of CNS disease in mouse models of neuropathic mucopolysaccharidoses.
27-Hydroxycholesterol Enhances Secretion of Extracellular Vesicles by ROS-Induced Dysregulation of Lysosomes.
Characterization of fission and fusion mitochondrial dynamics in HD fibroblasts according to patient's severity status.

Acta Neuropathol Commun. 2024 Sep 18. 12(1): 152

Molecular pathology, developmental changes and synaptic dysfunction in (pre-) symptomatic human C9ORF72-ALS/FTD cerebral organoids.

Astrid T van der Geest, Channa E Jakobs, Tijana Ljubikj, Christiaan F M Huffels, Marta Cañizares Luna, Renata Vieira de Sá, Youri Adolfs, Marina de Wit, Daan H Rutten, Marthe Kaal, Maria M Zwartkruis, Mireia Carcolé, Ewout J N Groen, Elly M Hol, Onur Basak, Adrian M Isaacs, Henk-Jan Westeneng, Leonard H van den Berg, Jan H Veldink, Domino K Schlegel, R Jeroen Pasterkamp.

  A hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Human brain imaging and experimental studies indicate early changes in brain structure and connectivity in C9-ALS/FTD, even before symptom onset. Because these early disease phenotypes remain incompletely understood, we generated iPSC-derived cerebral organoid models from C9-ALS/FTD patients, presymptomatic C9ORF72-HRE (C9-HRE) carriers, and controls. Our work revealed the presence of all three C9-HRE-related molecular pathologies and developmental stage-dependent size phenotypes in cerebral organoids from C9-ALS/FTD patients. In addition, single-cell RNA sequencing identified changes in cell type abundance and distribution in C9-ALS/FTD organoids, including a reduction in the number of deep layer cortical neurons and the distribution of neural progenitors. Further, molecular and cellular analyses and patch-clamp electrophysiology detected various changes in synapse structure and function. Intriguingly, organoids from all presymptomatic C9-HRE carriers displayed C9-HRE molecular pathology, whereas the extent to which more downstream cellular defects, as found in C9-ALS/FTD models, were detected varied for the different presymptomatic C9-HRE cases. Together, these results unveil early changes in 3D human brain tissue organization and synaptic connectivity in C9-ALS/FTD that likely constitute initial pathologies crucial for understanding disease onset and the design of therapeutic strategies.

Keywords:  ALS; Brain; C9ORF72; Development; Neural organoid; Presymptomatic

DOI:  https://doi.org/10.1186/s40478-024-01857-1
J Clin Invest. 2024 Sep 17. pii: e179016. [Epub ahead of print]

CRISPR-Cas13d targeting suppresses repeat-associated non-AUG translation of C9orf72 hexanucleotide repeat RNA.

Honghe Liu, Xiao-Feng Zhao, Yu-Ning Lu, Lindsey R Hayes, Jiou Wang.

  A hexanucleotide GGGGCC repeat expansion in the non-coding region of C9orf72 gene is the most common genetic mutation identified in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The resulting repeat RNA and dipeptide repeat proteins from non-conventional repeat translation have been recognized as important markers associated with the diseases. CRISPR-Cas13d, a powerful RNA targeting tool, has faced challenges in effectively targeting RNA with stable secondary structures. Here we report that CRISPR-Cas13d can be optimized to specifically target GGGGCC repeat RNA. Our results demonstrate that the CRISPR-Cas13d system can be harnessed to significantly diminish the translation of poly-dipeptides originating from the GGGGCC repeat RNA. This efficacy has been validated in various cell types, including induced pluripotent stem cells and differentiated motor neurons originating from C9orf72-ALS patients, as well as in C9orf72 repeat transgenic mice. These findings demonstrate the application of CRISPR-Cas13d in targeting RNA with intricate higher-order structures and suggest a potential therapeutic approach for ALS and FTD.

Keywords:  Gene therapy; Genetics; Molecular biology; Neurodegeneration; Neuroscience

DOI:  https://doi.org/10.1172/JCI179016
Res Sq. 2024 Sep 03. pii: rs.3.rs-4797540. [Epub ahead of print]

Motor Cortical Neuronal Hyperexcitability Associated with α-Synuclein Aggregation.

Hong-Yuan Chu, Liqiang Chen, Hiba Douja Chehade.

Dysfunction of the cerebral cortex is thought to underlie motor and cognitive impairments in Parkinson disease (PD). While cortical function is known to be suppressed by abnormal basal ganglia output following dopaminergic degeneration, it remains to be determined how the deposition of Lewy pathology disrupts cortical circuit integrity and function. Moreover, it is also unknown whether cortical Lewy pathology and midbrain dopaminergic degeneration interact to disrupt cortical function in late-stage. To begin to address these questions, we injected α-synuclein (αSyn) preformed fibrils (PFFs) into the dorsolateral striatum of mice to seed αSyn pathology in the cortical cortex and induce degeneration of midbrain dopaminergic neurons. Using this model system, we reported that αSyn aggregates accumulate in the motor cortex in a layer- and cell-subtype-specific pattern. Particularly, intratelencephalic neurons (ITNs) showed earlier accumulation and greater extent of αSyn aggregates relative to corticospinal neurons (CSNs). Moreover, we demonstrated that the intrinsic excitability and inputs resistance of αSyn aggregates-bearing ITNs in the secondary motor cortex (M2) are increased, along with a noticeable shrinkage of cell bodies and loss of dendritic spines. Last, neither the intrinsic excitability of CSNs nor their thalamocortical input was altered by a partial striatal dopamine depletion associated with αSyn pathology. Our results documented motor cortical neuronal hyperexcitability associated with αSyn aggregation and provided a novel mechanistic understanding of cortical circuit dysfunction in PD.

DOI: https://doi.org/10.21203/rs.3.rs-4797540/v1
F1000Res. 2023 ;12 1279

Identification of high-performing antibodies for SPARC-related modular calcium-binding protein 1 (SMOC-1) for use in Western Blot and immunoprecipitation.

NeuroSGC/YCharOS/EDDU collaborative group

  SPARC-related modular calcium-binding protein 1, otherwise known as SMOC-1, is a secreted glycoprotein involved in various cell biological processes including cell-matrix interactions, osteoblast differentiation, embryonic development, and homeostasis. SMOC-1 was found to be elevated in asymptomatic Alzheimer's disease (AD) patient cortex as well as being enriched in amyloid plaques and in AD patientcerebrospinal fluid, arguing for SMOC-1 as a promising biomarker for AD. Having access to high-quality SMOC-1 antibodies is crucial for the scientific community. It can ensure the consistency and reliability of SMOC-1 research, and further the exploration of its potential as both a therapeutic target or diagnostic marker.. In this study, we characterized seven SMOC-1 commercial antibodies for Western blot and immunoprecipitation, using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified successful antibodies in the tested applications and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Keywords:  SMOC-1; SMOC1; SPARC-related modular calcium binding protein 1; Uniprot ID Q9H4F8; antibody characterization; antibody validation; immunoprecipitation; western blot

DOI:  https://doi.org/10.12688/f1000research.141800.2
Front Cell Neurosci. 2024 ;18 1435619

A comprehensive review of electrophysiological techniques in amyotrophic lateral sclerosis research.

Keyuan Ren, Qinglong Wang, Douglas Jiang, Ethan Liu, Julie Alsmaan, Rui Jiang, Seward B Rutkove, Feng Tian.

  Amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease, is characterized by progressive motor neuron degeneration, leading to widespread weakness and respiratory failure. While a variety of mechanisms have been proposed as causes of this disease, a full understanding remains elusive. Electrophysiological alterations, including increased motor axon excitability, likely play an important role in disease progression. There remains a critical need for non-animal disease models that can integrate electrophysiological tools to better understand underlying mechanisms, track disease progression, and evaluate potential therapeutic interventions. This review explores the integration of electrophysiological technologies with ALS disease models. It covers cellular and clinical electrophysiological tools and their applications in ALS research. Additionally, we examine conventional animal models and highlight advancements in humanized models and 3D organoid technologies. By bridging the gap between these models, we aim to enhance our understanding of ALS pathogenesis and facilitate the development of new therapeutic strategies.

Keywords:  amyotrophic lateral sclerosis; disease modeling; electrical impedance myography; electrophysiology; excitability; excitotoxicity; organoid

DOI:  https://doi.org/10.3389/fncel.2024.1435619
bioRxiv. 2024 Sep 05. pii: 2024.09.03.611033. [Epub ahead of print]

Gigaxonin, mutated in Giant Axonal Neuropathy, interacts with TDP-43 and other RNA binding proteins.

Cassandra L Phillips, Maryam Faridounnia, Rachel A Battaglia, Baggio A Evangelista, Todd J Cohen, Puneet Opal, Thomas W Bouldin, Diane Armao, Natasha T Snider.

Giant Axonal Neuropathy (GAN) is a neurodegenerative disease caused by loss-of-function mutations in the KLHL16 gene, encoding the cytoskeleton regulator gigaxonin. In the absence of functional gigaxonin, intermediate filament (IF) proteins accumulate in neurons and other cell types due to impaired turnover and transport. GAN neurons exhibit distended, swollen axons and distal axonal degeneration, but the mechanisms behind this selective neuronal vulnerability are unknown. Our objective was to identify novel gigaxonin interactors pertinent to GAN neurons. Unbiased proteomics revealed a statistically significant predominance of RNA-binding proteins (RBPs) within the soluble gigaxonin interactome and among differentially-expressed proteins in iPSC-neuron progenitors from a patient with classic GAN. Among the identified RBPs was TAR DNA-binding protein 43 (TDP-43), which associated with the gigaxonin protein and its mRNA transcript. TDP-43 co-localized within large axonal neurofilament IFs aggregates in iPSC-motor neurons derived from a GAN patient with the 'axonal CMT-plus' disease phenotype. Our results implicate RBP dysfunction as a potential underappreciated contributor to GAN-related neurodegeneration.
Summary: This work reveals that the neurodegeneration-associated protein and cytoskeleton regulator gigaxonin and its mRNA associate with numerous RNA binding proteins. These findings shift understanding of normal gigaxonin function and provide insights into how disease-causing mutations in the gigaxonin-encoding gene ( KLHL16 ) may ignite a pathogenic cascade in neurons.

DOI: https://doi.org/10.1101/2024.09.03.611033
Annu Rev Pharmacol Toxicol. 2024 Sep 16.

Next-Gen Therapeutics: Pioneering Drug Discovery with iPSCs, Genomics, AI, and Clinical Trials in a Dish.

Zehra Yildirim, Kyle Swanson, Xuekun Wu, James Zou, Joseph Wu.

In the high-stakes arena of drug discovery, the journey from bench to bedside is hindered by a daunting 92% failure rate, primarily due to unpredicted toxicities and inadequate therapeutic efficacy in clinical trials. The FDA Modernization Act 2.0 heralds a transformative approach, advocating for the integration of alternative methods to conventional animal testing, including cell-based assays that employ human induced pluripotent stem cell (iPSC)-derived organoids, and organ-on-a-chip technologies, in conjunction with sophisticated artificial intelligence (AI) methodologies. Our review explores the innovative capacity of iPSC-derived clinical trial in a dish models designed for cardiovascular disease research. We also highlight how integrating iPSC technology with AI can accelerate the identification of viable therapeutic candidates, streamline drug screening, and pave the way toward more personalized medicine. Through this, we provide a comprehensive overview of the current landscape and future implications of iPSC and AI applications being navigated by the research community and pharmaceutical industry.

DOI: https://doi.org/10.1146/annurev-pharmtox-022724-095035
Front Mol Neurosci. 2024 ;17 1417961

Epigenetics in the formation of pathological aggregates in amyotrophic lateral sclerosis.

Veronica Noches, Danae Campos-Melo, Cristian A Droppelmann, Michael J Strong.

  The progressive degeneration of motor neurons in amyotrophic lateral sclerosis (ALS) is accompanied by the formation of a broad array of cytoplasmic and nuclear neuronal inclusions (protein aggregates) largely containing RNA-binding proteins such as TAR DNA-binding protein 43 (TDP-43) or fused in sarcoma/translocated in liposarcoma (FUS/TLS). This process is driven by a liquid-to-solid phase separation generally from proteins in membrane-less organelles giving rise to pathological biomolecular condensates. The formation of these protein aggregates suggests a fundamental alteration in the mRNA expression or the levels of the proteins involved. Considering the role of the epigenome in gene expression, alterations in DNA methylation, histone modifications, chromatin remodeling, non-coding RNAs, and RNA modifications become highly relevant to understanding how this pathological process takes effect. In this review, we explore the evidence that links epigenetic mechanisms with the formation of protein aggregates in ALS. We propose that a greater understanding of the role of the epigenome and how this inter-relates with the formation of pathological LLPS in ALS will provide an attractive therapeutic target.

Keywords:  ALS; DNA methylation; RNA modifications; chromatin remodeling enzymes; epigenetics; histone modifications; non-coding RNAs; protein aggregates

DOI:  https://doi.org/10.3389/fnmol.2024.1417961
J Chem Inf Model. 2024 Sep 18.

Atomistic Insights into the Stabilization of TDP-43 Protofibrils by ATP.

Zhengdong Xu, Jiaxing Tang, Yehong Gong, Jianxin Zhang, Yu Zou.

The aberrant accumulation of the transactive response deoxyribonucleic acid (DNA)-binding protein of 43 kDa (TDP-43) aggregates in the cytoplasm of motor neurons is the main pathological hallmark of amyotrophic lateral sclerosis (ALS). Previous experiments reported that adenosine triphosphate (ATP), the universal energy currency for all living cells, could induce aggregation and enhance the folding of TDP-43 fibrillar aggregates. However, the significance of ATP on TDP-43 fibrillation and the mechanism behind it remain elusive. In this work, we conducted multiple atomistic molecular dynamics (MD) simulations totaling 20 μs to search the critical nucleus size of TDP-43282-360 and investigate the impact of ATP molecules on preformed protofibrils. The results reveal that the trimer is the critical nucleus for TDP-43282-360 fibril formation and the tetramer is the minimal stable nucleus. When ATP molecules bind to the TDP-43282-360 trimer and tetramer, they can consolidate the TDP-43282-360 protofibrils by increasing the content of the β-sheet structure and promoting the formation of hydrogen bonds (H-bonds). Binding site analyses show that the N-terminus of TDP-43282-360 protofibrils is the main binding site of ATP, and R293 dominates the direct binding of ATP. Further analyses reveal that the π-π, cation-π, salt bridge, and H-bonding interactions together contribute to the binding of ATP to TDP-43282-360 protofibrils. This study decoded the detailed stabilization mechanism of protofibrillar TDP-43282-360 oligomers by ATP, and may provide new avenues for the development of drug design against ALS.

DOI: https://doi.org/10.1021/acs.jcim.4c01140
Bio Protoc. 2024 Sep 05. 14(17): e5060

Single-Molecule Sequencing of the C9orf72 Repeat Expansion in Patient iPSCs.

Yu-Chih Tsai, Katherine A Brown, Mylinh T Bernardi, John Harting, Claire D Clelland.

  A hexanucleotide GGGGCC repeat expansion in the C9orf72 gene is the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). C9orf72 repeat expansions are currently identified with long-range PCR or Southern blot for clinical and research purposes, but these methods lack accuracy and sensitivity. The GC-rich and repetitive content of the region cannot be amplified by PCR, which leads traditional sequencing approaches to fail. We turned instead to PacBio single-molecule sequencing to detect and size the C9orf72 repeat expansion without amplification. We isolated high molecular weight genomic DNA from patient-derived iPSCs of varying repeat lengths and then excised the region containing the C9orf72 repeat expansion from naked DNA with a CRISPR/Cas9 system. We added adapters to the cut ends, capturing the target region for sequencing on PacBio's Sequel, Sequel II, or Sequel IIe. This approach enriches the C9orf72 repeat region without amplification and allows the repeat expansion to be consistently and accurately sized, even for repeats in the thousands. Key features • This protocol is adapted from PacBio's previous "no-amp targeted sequencing utilizing the CRISPR-Cas9 system." • Optimized for sizing C9orf72 repeat expansions in patient-derived iPSCs and applicable to DNA from any cell type, blood, or tissue. • Requires high molecular weight naked DNA. • Compatible with Sequel I and II but not Revio.

Keywords:  C9orf72; No amplification; Repeat expansion; Single-molecule sequencing; iPSCs

DOI:  https://doi.org/10.21769/BioProtoc.5060
Acta Neuropathol. 2024 Sep 16. 148(1): 43

Dysregulated FOXO1 activity drives skeletal muscle intrinsic dysfunction in amyotrophic lateral sclerosis.

Mónica Zufiría, Oihane Pikatza-Menoio, Maddi Garciandia-Arcelus, Xabier Bengoetxea, Andrés Jiménez, Amaia Elicegui, María Levchuk, Olatz Arnold-García, Jon Ondaro, Pablo Iruzubieta, Laura Rodríguez-Gómez, Uxoa Fernández-Pelayo, Mikel Muñoz-Oreja, Ana Aiastui, José Manuel García-Verdugo, Vicente Herranz-Pérez, Miren Zulaica, Juan José Poza, Rebeca Ruiz-Onandi, Roberto Fernández-Torrón, Juan Bautista Espinal, Mario Bonilla, Ana Lersundi, Gorka Fernández-Eulate, Javier Riancho, Ainara Vallejo-Illarramendi, Ian James Holt, Amets Sáenz, Edoardo Malfatti, Stéphanie Duguez, Lorea Blázquez, Adolfo López de Munain, Gorka Gerenu, Francisco Gil-Bea, Sonia Alonso-Martín.

  Amyotrophic Lateral Sclerosis (ALS) is a multisystemic neurodegenerative disorder, with accumulating evidence indicating metabolic disruptions in the skeletal muscle preceding disease symptoms, rather than them manifesting as a secondary consequence of motor neuron (MN) degeneration. Hence, energy homeostasis is deeply implicated in the complex physiopathology of ALS and skeletal muscle has emerged as a key therapeutic target. Here, we describe intrinsic abnormalities in ALS skeletal muscle, both in patient-derived muscle cells and in muscle cell lines with genetic knockdown of genes related to familial ALS, such as TARDBP (TDP-43) and FUS. We found a functional impairment of myogenesis that parallels defects of glucose oxidation in ALS muscle cells. We identified FOXO1 transcription factor as a key mediator of these metabolic and functional features in ALS muscle, via gene expression profiling and biochemical surveys in TDP-43 and FUS-silenced muscle progenitors. Strikingly, inhibition of FOXO1 mitigated the impaired myogenesis in both the genetically modified and the primary ALS myoblasts. In addition, specific in vivo conditional knockdown of TDP-43 or FUS orthologs (TBPH or caz) in Drosophila muscle precursor cells resulted in decreased innervation and profound dysfunction of motor nerve terminals and neuromuscular synapses, accompanied by motor abnormalities and reduced lifespan. Remarkably, these phenotypes were partially corrected by foxo inhibition, bolstering the potential pharmacological management of muscle intrinsic abnormalities associated with ALS. The findings demonstrate an intrinsic muscle dysfunction in ALS, which can be modulated by targeting FOXO factors, paving the way for novel therapeutic approaches that focus on the skeletal muscle as complementary target tissue.

Keywords:  Amyotrophic lateral sclerosis; FOXO1; FUS; Glycolysis; Myogenesis; TDP-43

DOI:  https://doi.org/10.1007/s00401-024-02794-y
Cell Death Dis. 2024 Sep 19. 15(9): 686

N-acetylaspartate promotes glycolytic-to-oxidative fiber-type switch and resistance to atrophic stimuli in myotubes.

Serena Castelli, Enrico Desideri, Leonardo Laureti, Federica Felice, Angela De Cristofaro, Silvia Scaricamazza, Giacomo Lazzarino, Maria Rosa Ciriolo, Fabio Ciccarone.

N-acetylaspartate (NAA) is a neuronal metabolite that can be extruded in extracellular fluids and whose blood concentration increases in several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Aspartoacylase (ASPA) is the enzyme responsible for NAA breakdown. It is abundantly expressed in skeletal muscle and most other human tissues, but the role of NAA catabolism in the periphery is largely neglected. Here we demonstrate that NAA treatment of differentiated C2C12 muscle cells increases lipid turnover, mitochondrial biogenesis and oxidative metabolism at the expense of glycolysis. These effects were ascribed to NAA catabolism, as CRISPR/Cas9 ASPA KO cells are insensitive to NAA administration. Moreover, the metabolic switch induced by NAA was associated with an augmented resistance to atrophic stimuli. Consistently with in vitro results, SOD1-G93A ALS mice show an increase in ASPA levels in those muscles undergoing the glycolytic to oxidative switch during the disease course. The impact of NAA on the metabolism and resistance capability of myotubes supports a role for this metabolite in the phenotypical adaptations of skeletal muscle in neuromuscular disorders.

DOI: https://doi.org/10.1038/s41419-024-07047-0
Proc Natl Acad Sci U S A. 2024 Sep 24. 121(39): e2320611121

Glial swip-10 controls systemic mitochondrial function, oxidative stress, and neuronal viability via copper ion homeostasis.

Peter Rodriguez, Vrinda Kalia, Cristina Fenollar-Ferrer, Chelsea L Gibson, Zayna Gichi, Andre Rajoo, Carson D Matier, Aidan T Pezacki, Tong Xiao, Lucia Carvelli, Christopher J Chang, Gary W Miller, Andy V Khamoui, Jana Boerner, Randy D Blakely.

  Cuprous copper [Cu(I)] is an essential cofactor for enzymes that support many fundamental cellular functions including mitochondrial respiration and suppression of oxidative stress. Neurons are particularly reliant on mitochondrial production of ATP, with many neurodegenerative diseases, including Parkinson's disease, associated with diminished mitochondrial function. The gene MBLAC1 encodes a ribonuclease that targets pre-mRNA of replication-dependent histones, proteins recently found in yeast to reduce Cu(II) to Cu(I), and when mutated disrupt ATP production, elevates oxidative stress, and severely impacts cell growth. Whether this process supports neuronal and/or systemic physiology in higher eukaryotes is unknown. Previously, we identified swip-10, the putative Caenorhabditis elegans ortholog of MBLAC1, establishing a role for glial swip-10 in limiting dopamine (DA) neuron excitability and sustaining DA neuron viability. Here, we provide evidence from computational modeling that SWIP-10 protein structure mirrors that of MBLAC1 and locates a loss of function coding mutation at a site expected to disrupt histone RNA hydrolysis. Moreover, we find through genetic, biochemical, and pharmacological studies that deletion of swip-10 in worms negatively impacts systemic Cu(I) levels, leading to deficits in mitochondrial respiration and ATP production, increased oxidative stress, and neurodegeneration. These phenotypes can be offset in swip-10 mutants by the Cu(I) enhancing molecule elesclomol and through glial expression of wildtype swip-10. Together, these studies reveal a glial-expressed pathway that supports systemic mitochondrial function and neuronal health via regulation of Cu(I) homeostasis, a mechanism that may lend itself to therapeutic strategies to treat devastating neurodegenerative diseases.

Keywords:  C. elegans; copper; glia; neurodegeneration; swip-10

DOI:  https://doi.org/10.1073/pnas.2320611121
Sci Rep. 2024 09 18. 14(1): 21837

Location and function of TDP-43 in platelets, alterations in neurodegenerative diseases and arising considerations for current plasma biobank protocols.

Ruth Luthi-Carter, Sara Cappelli, Morgan Le Roux-Bourdieu, Noemie Tentillier, James P Quinn, Tiziana Petrozziello, Lathika Gopalakrishnan, Purva Sethi, Himanshi Choudhary, Giorgia Bartolini, Elias Gebara, Cristiana Stuani, Laure Font, Jiyan An, Vanessa Ortega, Jessica Sage, Edina Kosa, Bianca A Trombetta, Roberto Simeone, Tamara Seredenina, Tariq Afroz, James D Berry, Steven E Arnold, Becky C Carlyle, Oskar Adolfsson, Ghazaleh Sadri-Vakili, Emanuele Buratti, Robert Bowser, Abdulbaki Agbas.

The TAR DNA Binding Protein 43 (TDP-43) has been implicated in the pathogenesis of human neurodegenerative diseases and exhibits hallmark neuropathology in amyotrophic lateral sclerosis (ALS). Here, we explore its tractability as a plasma biomarker of disease and describe its localization and possible functions in the cytosol of platelets. Novel TDP-43 immunoassays were developed on three different technical platforms and qualified for specificity, signal-to-noise ratio, detection range, variation, spike recovery and dilution linearity in human plasma samples. Surprisingly, implementation of these assays demonstrated that biobank-archived plasma samples yielded considerable heterogeneity in TDP-43 levels. Importantly, subsequent investigation attributed these differences to variable platelet recovery. Fractionations of fresh blood revealed that ≥ 95% of the TDP-43 in platelet-containing plasma was compartmentalized within the platelet cytosol. We reasoned that this highly concentrated source of TDP-43 comprised an interesting substrate for biochemical analyses. Additional characterization of platelets revealed the presence of the disease-associated phosphoserine 409/410 TDP-43 proteoform and many neuron- and astrocyte-expressed TDP-43 mRNA targets. Considering these striking similarities, we propose that TDP-43 may serve analogous functional roles in platelets and synapses, and that the study of platelet TDP-43 might provide a window into disease-related TDP-43 dyshomeostasis in the central nervous system.

DOI: https://doi.org/10.1038/s41598-024-70822-8
Brain. 2024 Aug 30. pii: awae268. [Epub ahead of print]

Biallelic PTPMT1 variants disrupt cardiolipin metabolism and lead to a neurodevelopmental syndrome.

Micol Falabella, Chiara Pizzamiglio, Luis Carlos Tabara, Benjamin Munro, Mohamed S Abdel-Hamid, Ece Sonmezler, William L Macken, Shanti Lu, Lisa Tilokani, Padraig J Flannery, Nina Patel, Simon A S Pope, Simon J R Heales, Dania B H Hammadi, Charlotte L Alston, Robert W Taylor, Hanns Lochmuller, Cathy E Woodward, Robyn Labrum, Jana Vandrovcova, Henry Houlden, Efstathia Chronopoulou, Germaine Pierre, Reza Maroofian, Michael G Hanna, Jan-Willem Taanman, Semra Hiz, Yavuz Oktay, Maha S Zaki, Rita Horvath, Julien Prudent, Robert D S Pitceathly.

  Primary mitochondrial diseases (PMDs) are among the most common inherited neurological disorders. They are caused by pathogenic variants in mitochondrial or nuclear DNA that disrupt mitochondrial structure and/or function, leading to impaired oxidative phosphorylation (OXPHOS). One emerging subcategory of PMDs involves defective phospholipid (PL) metabolism. Cardiolipin (CL), the signature PL of mitochondria, resides primarily in the inner mitochondrial membrane, where it is biosynthesised and remodelled via multiple enzymes and is fundamental to several aspects of mitochondrial biology. Genes that contribute to CL biosynthesis have recently been linked with PMD. However, the pathophysiological mechanisms that underpin human CL-related PMDs are not fully characterised. Here, we report six individuals, from three independent families, harbouring biallelic variants in PTPMT1, a mitochondrial tyrosine phosphatase required for de novo CL biosynthesis. All patients presented with a complex, neonatal/infantile onset neurological and neurodevelopmental syndrome comprising developmental delay, microcephaly, facial dysmorphism, epilepsy, spasticity, cerebellar ataxia and nystagmus, sensorineural hearing loss, optic atrophy, and bulbar dysfunction. Brain MRI revealed a variable combination of corpus callosum thinning, cerebellar atrophy, and white matter changes. Using patient-derived fibroblasts and skeletal muscle tissue, combined with cellular rescue experiments, we characterise the molecular defects associated with mutant PTPMT1 and confirm the downstream pathogenic effects that loss of PTPMT1 has on mitochondrial structure and function. To further characterise the functional role of PTPMT1 in CL homeostasis, we established a zebrafish ptpmt1 knockout model associated with abnormalities in body size, developmental alterations, decreased total CL levels, and OXPHOS deficiency. Together, these data indicate that loss of PTPMT1 function is associated with a new autosomal recessive PMD caused by impaired CL metabolism, highlight the contribution of aberrant CL metabolism towards human disease, and emphasise the importance of normal CL homeostasis during neurodevelopment.

Keywords:  cardiolipin; mitochondria; mitochondrial dynamics; neurodevelopmental syndrome; primary mitochondrial disease

DOI:  https://doi.org/10.1093/brain/awae268
Stem Cell Res. 2024 Aug 12. pii: S1873-5061(24)00230-7. [Epub ahead of print]81 103532

Generation of CRISPR/Cas9-edited human iPSC lines carrying homozygous and heterozygous SAMD9 p.I983S mutations.

Majd Khiami, Yan Ju, Lei Han, Jonathan Klein, Min-Joon Han, Shondra M Pruett-Miller, Marcin W Wlodarski.

  Induced pluripotent stem cells (iPSCs) harboring patient derived SAMD9 mutation offer a unique platform to study the multi-organ involvement observed in this rare disease, referred to as myelodysplasia, infections, restriction of growth, adrenal hypoplasia, genital phenotypes, and enteropathy (MIRAGE) syndrome. The pluripotent nature of iPSCs allows in vitro differentiation into various somatic cell types representing multiple organ systems affected in SAMD9-mutated patients. Hence, in this paper, we present a CRISPR/Cas9-engineered iPSC model carrying SAMD9 c.2948T>G, p.I983S mutation previously reported in two patients with severe MIRAGE syndrome.

Keywords:  Bone marrow failure; CRISPR/Cas9; Hematology; SAMD9; iPSC

DOI:  https://doi.org/10.1016/j.scr.2024.103532
Stem Cell Res Ther. 2024 Sep 18. 15(1): 310

PROTAC-mediated vimentin degradation promotes terminal erythroid differentiation of pluripotent stem cells.

Hao Yan, Ruge Zang, Tiantian Cui, Yiming Liu, Biao Zhang, Lingpin Zhao, Hongyu Li, Juannian Zhou, Haiyang Wang, Quan Zeng, Lei Xu, Yuqi Zhou, Xuetao Pei, Jiafei Xi, Wen Yue.

   BACKGROUND: Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), can undergo erythroid differentiation, offering a potentially invaluable resource for generating large quantities of erythroid cells. However, the majority of erythrocytes derived from hPSCs fail to enucleate compared with those derived from cord blood progenitors, with an unknown molecular basis for this difference. The expression of vimentin (VIM) is retained in erythroid cells differentiated from hPSCs but is absent in mature erythrocytes. Further exploration is required to ascertain whether VIM plays a critical role in enucleation and to elucidate the underlying mechanisms.
METHODS: In this study, we established a hESC line with reversible vimentin degradation (dTAG-VIM-H9) using the proteolysis-targeting chimera (PROTAC) platform. Various time-course studies, including erythropoiesis from CD34+ human umbilical cord blood and three-dimensional (3D) organoid culture from hESCs, morphological analysis, quantitative real-time PCR (qRT-PCR), western blotting, flow cytometry, karyotyping, cytospin, Benzidine-Giemsa staining, immunofluorescence assay, and high-speed cell imaging analysis, were conducted to examine and compare the characteristics of hESCs and those with vimentin degradation, as well as their differentiated erythroid cells.
RESULTS: Vimentin expression diminished during normal erythropoiesis in CD34+ cord blood cells, whereas it persisted in erythroid cells differentiated from hESC. Depletion of vimentin using the degradation tag (dTAG) system promotes erythroid enucleation in dTAG-VIM-H9 cells. Nuclear polarization of erythroblasts is elevated by elimination of vimentin.
CONCLUSIONS: VIM disappear during the normal maturation of erythroid cells, whereas they are retained in erythroid cells differentiated from hPSCs. We found that retention of vimentin during erythropoiesis impairs erythroid enucleation from hPSCs. Using the PROTAC platform, we validated that vimentin degradation by dTAG accelerates the enucleation rate in dTAG-VIM-H9 cells by enhancing nuclear polarization.

Keywords:  Cultured RBCs; Enucleation; Erythropoiesis; PROTAC; Polarization; Vimentin

DOI:  https://doi.org/10.1186/s13287-024-03910-1
Proc Natl Acad Sci U S A. 2024 Sep 24. 121(39): e2400531121

The p53 target DRAM1 modulates calcium homeostasis and ER stress by promoting contact between lysosomes and the ER through STIM1.

Xiying Wang, Ji Geng, Suman Rimal, Yuxiu Sui, Jie Pan, Zhenghong Qin, Bingwei Lu.

  It is well established that DNA Damage Regulated Autophagy Modulator 1 (DRAM1), a lysosomal protein and a target of p53, participates in autophagy. The cellular functions of DRAM1 beyond autophagy remain elusive. Here, we show p53-dependent upregulation of DRAM1 in mitochondrial damage-induced Parkinson's disease (PD) models and exacerbation of disease phenotypes by DRAM1. We find that the lysosomal location of DRAM1 relies on its intact structure including the cytosol-facing C-terminal domain. Excess DRAM1 disrupts endoplasmic reticulum (ER) structure, triggers ER stress, and induces protective ER-phagy. Mechanistically, DRAM1 interacts with stromal interacting molecule 1 (STIM1) to tether lysosomes to the ER and perturb STIM1 function in maintaining intracellular calcium homeostasis. STIM1 overexpression promotes cellular health by restoring calcium homeostasis, ER stress response, ER-phagy, and AMP-activated protein kinase (AMPK)-Unc-51 like autophagy activating kinase 1 (ULK1) signaling in cells with excess DRAM1. Thus, by promoting organelle contact between lysosomes and the ER, DRAM1 modulates ER structure and function and cell survival under stress. Our results suggest that DRAM1 as a lysosomal protein performs diverse roles in cellular homeostasis and stress response. These findings may have significant implications for our understanding of the role of the p53/DRAM1 axis in human diseases, from cancer to neurodegenerative diseases.

Keywords:  DRAM1; ER; ER-phagy; calcium homeostasis; lysosome

DOI:  https://doi.org/10.1073/pnas.2400531121
bioRxiv. 2024 Sep 03. pii: 2024.08.30.610524. [Epub ahead of print]

Temporal control of acute protein aggregate turnover by UBE3C and NRF1-dependent proteasomal pathways.

Kelsey L Hickey, Alexandra Panov, Enya Miguel Whelan, Tillman Schäfer, Arda Mizrak, Ron R Kopito, Wolfgang Baumeister, Rubén Fernández-Busnadiego, J Wade Harper.

  A hallmark of neurodegenerative diseases is the progressive loss of proteostasis, leading to the accumulation of misfolded proteins or protein aggregates, with subsequent cytotoxicity. To combat this toxicity, cells have evolved degradation pathways (ubiquitin-proteasome system and autophagy) that detect and degrade misfolded proteins. However, studying the underlying cellular pathways and mechanisms has remained a challenge, as formation of many types of protein aggregates is asynchronous, with individual cells displaying distinct kinetics, thereby hindering rigorous time-course studies. Here, we merge a kinetically tractable and synchronous agDD-GFP system for aggregate formation with targeted gene knockdowns, to uncover degradation mechanisms used in response to acute aggregate formation. We find that agDD-GFP forms amorphous aggregates by cryo-electron tomography at both early and late stages of aggregate formation. Aggregate turnover occurs in a proteasome-dependent mechanism in a manner that is dictated by cellular aggregate burden, with no evidence of the involvement of autophagy. Lower levels of misfolded agDD-GFP, enriched in oligomers, utilizes UBE3C-dependent proteasomal degradation in a pathway that is independent of RPN13 ubiquitylation by UBE3C. Higher aggregate burden activates the NRF1 transcription factor to increase proteasome subunit transcription, and subsequent degradation capacity of cells. Loss or gain of NRF1 function alters the turnover of agDD-GFP under conditions of high aggregate burden. Together, these results define the role of UBE3C in degradation of this class of misfolded aggregation-prone proteins and reveals a role for NRF1 in proteostasis control in response to widespread protein aggregation.

Keywords:  Biological Sciences; Cell Biology; NRF1; Protein aggregates; Protein quality control; Protein turnover; UBE3C; Ubiquitin-proteasome system

DOI:  https://doi.org/10.1101/2024.08.30.610524
PLoS One. 2024 ;19(9): e0309893

Targeting ferroptosis with the lipoxygenase inhibitor PTC-041 as a therapeutic strategy for the treatment of Parkinson's disease.

Angela Minnella, Kevin P McCusker, Akiko Amagata, Beatrice Trias, Marla Weetall, Joey C Latham, Sloane O'Neill, Richard K Wyse, Matthew B Klein, Jeffrey K Trimmer.

Parkinson's disease is the second most common neurodegenerative disorder, affecting nearly 10 million people worldwide. Ferroptosis, a recently identified form of regulated cell death characterized by 15-lipoxygenase-mediated hydroperoxidation of membrane lipids, has been implicated in neurodegenerative disorders including amyotrophic lateral sclerosis and Parkinson's disease. Pharmacological inhibition of 15 -lipoxygenase to prevent iron- and lipid peroxidation-associated ferroptotic cell death is a rational strategy for the treatment of Parkinson's disease. We report here the characterization of PTC-041 as an anti-ferroptotic reductive lipoxygenase inhibitor developed for the treatment of Parkinson's disease. In these studies, PTC-041 potently protects primary human Parkinson's disease patient-derived fibroblasts from lipid peroxidation and subsequent ferroptotic cell death and prevents ferroptosis-related neuronal loss and astrogliosis in primary rat neuronal cultures. Additionally, PTC-041 prevents ferroptotic-mediated α-synuclein protein aggregation and nitrosylation in vitro, suggesting a potential role for anti-ferroptotic lipoxygenase inhibitors in mitigating pathogenic aspects of synucleinopathies such as Parkinson's disease. We further found that PTC-041 protects against synucleinopathy in vivo, demonstrating that PTC-041 treatment of Line 61 transgenic mice protects against α-synuclein aggregation and phosphorylation as well as prevents associated neuronal and non-neuronal cell death. Finally, we show that. PTC-041 protects against 6-hydroxydopamine-induced motor deficits in a hemiparkinsonian rat model, further validating the potential therapeutic benefits of lipoxygenase inhibitors in the treatment of Parkinson's disease.

DOI: https://doi.org/10.1371/journal.pone.0309893
Neurobiol Dis. 2024 Sep 17. pii: S0969-9961(24)00274-2. [Epub ahead of print] 106674

Prion protein pathology in Ubiquilin 2 models of ALS.

Nhat T Le, Nam Chu, Gunjan Joshi, Nicole R Higgins, Ouada Nebie, Niyi Adelakun, Marie Butts, Mervyn J Monteiro.

  Mutations in UBQLN2 cause ALS and frontotemporal dementia (FTD). The pathological signature in UBQLN2 cases is deposition of highly unusual types of inclusions in the brain and spinal cord that stain positive for UBQLN2. However, what role these inclusions play in pathogenesis remains unclear. Here we show cellular prion protein (PrPC) is found in UBQLN2 inclusions in both mouse and human neuronal induced pluripotent (IPSC) models of UBQLN2 mutations, evidenced by the presence of aggregated forms of PrPC with UBQLN2 inclusions. Turnover studies indicated that the P497H UBQLN2 mutation slows PrPC protein degradation and leads to mislocalization of PrPC in the cytoplasm. Immunoprecipitation studies indicated UBQLN2 and PrPC bind together in a complex. The abnormalities in PrPC caused by UBQLN2 mutations may be relevant in disease pathogenesis.

Keywords:  Amyotrophic lateral sclerosis; Human neuronal induced pluripotent models; Mouse models; Prion protein; Ubiquilin 2

DOI:  https://doi.org/10.1016/j.nbd.2024.106674
Exp Neurol. 2024 Sep 15. pii: S0014-4886(24)00285-1. [Epub ahead of print]382 114959

Viral overexpression of human alpha-synuclein in mouse substantia nigra dopamine neurons results in hyperdopaminergia but no neurodegeneration.

Sofia Ines Garcia Moreno, Fabian Limani, Iina Ludwig, Catherine Gilbert, Christian Pifl, Thomas S Hnasko, Thomas Steinkellner.

  Loss of select neuronal populations such as midbrain dopamine (DA) neurons is a pathological hallmark of Parkinson's disease (PD). The small neuronal protein α-synuclein has been related both genetically and neuropathologically to PD, yet how and if it contributes to selective vulnerability remains elusive. Here, we describe the generation of a novel adeno-associated viral vector (AAV) for Cre-dependent overexpression of wild-type human α-synuclein. Our strategy allows us to restrict α-synuclein to select neuronal populations and hence investigate the cell-autonomous effects of elevated α-synuclein in genetically-defined cell types. Since DA neurons in the substantia nigra pars compacta (SNc) are particularly vulnerable in PD, we investigated in more detail the effects of increased α-synuclein in these cells. AAV-mediated overexpression of wildtype human α-synuclein in SNc DA neurons increased the levels of α-synuclein within these cells and augmented phosphorylation of α-synuclein at serine-129, which is considered a pathological feature of PD and other synucleinopathies. However, despite abundant α-synuclein overexpression and hyperphosphorylation we did not observe any dopaminergic neurodegeneration up to 90 days post virus infusion. In contrast, we noticed that overexpression of α-synuclein resulted in increased locomotor activity and elevated striatal DA levels suggesting that α-synuclein enhanced dopaminergic activity. We therefore conclude that cell-autonomous effects of elevated α-synuclein are not sufficient to trigger acute dopaminergic neurodegeneration.

Keywords:  Adeno-associated viral vectors; Alpha-synuclein; Animal model of Parkinson's disease; Cre recombinase; Dopamine neuron vulnerability; Parkinson's disease

DOI:  https://doi.org/10.1016/j.expneurol.2024.114959
Biomed Rep. 2024 Nov;21(5): 165

Human TDP43 is required for ALS‑related annexin A11 toxicity in Drosophila.

Jodi Barnard, Rachel Hunt, Mert Yucel, David Mazaud, Bradley N Smith, Manolis Fanto.

  Genomics allows identification of genes and mutations associated with amyotrophic lateral sclerosis (ALS). Mutations in annexin A11 (ANXA11) are responsible for ~1% of all familial ALS and fronto-temporal dementia cases. The present study used the fruit fly, Drosophila melanogaster, to assess the mechanism of toxicity of ANXA11 mutants in residues that are conserved in the fly ANXB11 protein, the closest homolog to human ANXA11. In immune fluorescence, lifespan and negative geotaxis assays ANXA11 mutants, while displaying some degree of alteration in localization and function, did not exert any relevant organism toxicity in Drosophila. However, they showed a specific interaction with human TAR DNA-binding protein (TDP43). The present study illustrated that the ANXA11 mutants interact with human TDP43, but not the fly TAR DNA-binding protein-43 homolog (TBPH) or other ALS-associated genes such as super oxide dismutase 1, to shorten lifespan and increase negative geotaxis defects. This sheds light both on the mechanisms underlying ALS, further elucidating the intricate molecular network implicated in ALS and placing ANXA11 as a key player in its pathology, and on the complexity of using Drosophila as a model organism for researching genes in ALS.

Keywords:  Drosophila; TDP43; amyotrophic lateral sclerosis; annexin A11; neurodegeneration

DOI:  https://doi.org/10.3892/br.2024.1853
Front Mol Neurosci. 2024 ;17 1398048

Crosstalk between ubiquitination and translation in neurodevelopmental disorders.

Nagore Elu, Srividya Subash, Susana R Louros.

  Ubiquitination is one of the most conserved post-translational modifications and together with mRNA translation contributes to cellular protein homeostasis (proteostasis). Temporal and spatial regulation of proteostasis is particularly important during synaptic plasticity, when translation of specific mRNAs requires tight regulation. Mutations in genes encoding regulators of mRNA translation and in ubiquitin ligases have been associated with several neurodevelopmental disorders. RNA metabolism and translation are regulated by RNA-binding proteins, critical for the spatial and temporal control of translation in neurons. Several ubiquitin ligases also regulate RNA-dependent mechanisms in neurons, with numerous ubiquitination events described in splicing factors and ribosomal proteins. Here we will explore how ubiquitination regulates translation in neurons, from RNA biogenesis to alternative splicing and how dysregulation of ubiquitin signaling can be the underlying cause of pathology in neurodevelopmental disorders, such as Fragile X syndrome. Finally we propose that targeting ubiquitin signaling is an attractive novel therapeutic strategy for neurodevelopmental disorders where mRNA translation and ubiquitin signaling are disrupted.

Keywords:  FMRP; UBE3A; neurodevelopmental disorders; ribosome; splicing; translation; ubiquitin

DOI:  https://doi.org/10.3389/fnmol.2024.1398048
JCI Insight. 2024 Sep 17. pii: e178645. [Epub ahead of print]

Quantitative proteomics of patient fibroblasts reveal biomarkers and diagnostic signatures of mitochondrial disease.

Sandrina P Correia, Marco F Moedas, Lucie S Taylor, Karin Naess, Albert Z Lim, Robert McFarland, Zuzanna Kazior, Anastasia Rumyantseva, Rolf Wibom, Martin Engvall, Helene Bruhn, Nicole Lesko, Ákos Végvári, Lukas Käll, Matthias Trost, Charlotte L Alston, Christoph Freyer, Robert W Taylor, Anna Wedell, Anna Wredenberg.

   BACKGROUND: Mitochondrial diseases belong to the group of inborn errors of metabolism (IEM), with a prevalence of 1:2,000-1:5,000. They are the most common form of IEM, but despite advances in next-generation sequencing technologies, almost half of the patients are left genetically undiagnosed.
METHODS: We investigated a cohort of 61 patients with defined mitochondrial disease to improve diagnostics, identify biomarkers, and correlate metabolic pathways to specific disease groups. Clinical presentations were structured using human phenotype ontology terms, and mass spectrometry-based proteomics was performed on primary fibroblasts. Additionally, we integrated six patients carrying variants of uncertain significance (VUS) to test proteomics as a diagnostic expansion.
RESULTS: Proteomic profiles from patient samples could be classified according to their biochemical and genetic characteristics, with the expression of five proteins (GPX4, MORF4L1, MOXD1, MSRA and TMED9) correlating with the disease cohort, and thus, acting as putative biomarkers. Pathway analysis showed a deregulation of inflammatory and mitochondrial stress responses. This included the upregulation of glycosphingolipid metabolism and mitochondrial protein import, as well as the downregulation of arachidonic acid metabolism. Furthermore, we could assign pathogenicity to a VUS in MRPS23 by demonstrating the loss of associated mitochondrial ribosome subunits.
CONCLUSION: We established mass spectrometry-based proteomics on patient fibroblasts as a viable and versatile tool for diagnosing patients with mitochondrial disease.
FUNDING: The NovoNordisk Foundation, Knut and Alice Wallenberg Foundation, Wellcome Centre for Mitochondrial Research, UK Medical Research Council, and the UK NHS Highly Specialised Service for Rare Mitochondrial Disorders of Adults and Children.

Keywords:  Metabolism; Mitochondria; Molecular diagnosis; Proteomics

DOI:  https://doi.org/10.1172/jci.insight.178645
J Neuroinflammation. 2024 Sep 18. 21(1): 228

iPLA2β loss leads to age-related cognitive decline and neuroinflammation by disrupting neuronal mitophagy.

Li Jiao, Wenxin Shao, Wenqi Quan, Longjiang Xu, Penghui Liu, Jinling Yang, Xiaozhong Peng.

BACKGROUND: During brain aging, disturbances in neuronal phospholipid metabolism result in impaired cognitive function and dysregulation of neurological processes. Mutations in iPLA2β are associated with neurodegenerative conditions that significantly impact brain phospholipids. iPLA2β deficiency exacerbates mitochondrial dysfunction and abnormal mitochondrial accumulation. We hypothesized that iPLA2β contributes to age-related cognitive decline by disrupting neuronal mitophagy.
METHODOLOGY: We used aged wild-type (WT) mice and iPLA2β-/- mice as natural aging models to assess cognitive performance, iPLA2β expression in the cortex, levels of chemokines and inflammatory cytokines, and mitochondrial dysfunction, with a specific focus on mitophagy and the mitochondrial phospholipid profile. To further elucidate the role of iPLA2β, we employed adeno-associated virus (AAV)-mediated iPLA2β overexpression in aged mice and re-evaluated these parameters.
RESULTS: Our findings revealed a significant reduction in iPLA2β levels in the prefrontal cortex of aged brains. Notably, iPLA2β-deficient mice exhibited impaired learning and memory. Loss of iPLA2β in the PFC of aged mice led to increased levels of chemokines and inflammatory cytokines. This damage was associated with altered mitochondrial morphology, reduced ATP levels due to dysregulation of the parkin-independent mitophagy pathway, and changes in the mitochondrial phospholipid profile. AAV-mediated overexpression of iPLA2β alleviated age-related parkin-independent mitophagy pathway dysregulation in primary neurons and the PFC of aged mice, reduced inflammation, and improved cognitive function.
CONCLUSIONS: Our study suggests that age-related iPLA2β loss in the PFC leads to cognitive decline through the disruption of mitophagy. These findings highlight the potential of targeting iPLA2β to ameliorate age-related neurocognitive disorders.

DOI: https://doi.org/10.1186/s12974-024-03219-z
Nat Commun. 2024 Sep 16. 15(1): 8132

Ready-to-use iPSC-derived microglia progenitors for the treatment of CNS disease in mouse models of neuropathic mucopolysaccharidoses.

Panagiotis Douvaras, Diego F Buenaventura, Bruce Sun, Ashley Lepack, Elizabeth Baker, Elizabeth Simpson, Mark Ebel, Gregory Lallos, Deven LoSchiavo, Nicholas Stitt, Nathaniel Adams, Conor McAuliffe, Ana Forton-Juarez, Brian Kosmyna, Elizabeth Pereira, Benjamin Burnett, David Dilworth, Stephanie Fisher, Jing Wang, Peter Tonge, Mark Tomishima, Carlos Paladini, Dan Wilkinson, Chew-Li Soh, Maya Srinivas, Christoph Patsch, Stefan Irion.

Mucopolysaccharidoses are inherited metabolic disorders caused by the deficiency in lysosomal enzymes required to break down glycosaminoglycans. Accumulation of glycosaminoglycans leads to progressive, systemic degenerative disease. The central nervous system is particularly affected, resulting in developmental delays, neurological regression, and early mortality. Current treatments fail to adequately address neurological defects. Here we explore the potential of human induced pluripotent stem cell (hiPSC)-derived microglia progenitors as a one-time, allogeneic off-the-shelf cell therapy for several mucopolysaccharidoses (MPS). We show that hiPSC-derived microglia progenitors, possessing normal levels of lysosomal enzymes, can deliver functional enzymes into four subtypes of MPS knockout cell lines through mannose-6-phosphate receptor-mediated endocytosis in vitro. Additionally, our findings indicate that a single administration of hiPSC-derived microglia progenitors can reduce toxic glycosaminoglycan accumulation and prevent behavioral deficits in two different animal models of MPS. Durable efficacy is observed for eight months after transplantation. These results suggest a potential avenue for treating MPS with hiPSC-derived microglia progenitors.

DOI: https://doi.org/10.1038/s41467-024-52400-8
Endocrinology. 2024 Sep 19. pii: bqae127. [Epub ahead of print]

27-Hydroxycholesterol Enhances Secretion of Extracellular Vesicles by ROS-Induced Dysregulation of Lysosomes.

Anasuya Das Gupta, Jaena Park, Janet E Sorrells, Hannah Kim, Natalia Krawczynska, Dhanya Pradeep, Yu Wang, Hashni Epa Vidana Gamage, Adam T Nelczyk, Stephen A Boppart, Marni D Boppart, Erik R Nelson.

  Extracellular vesicles (EVs) serve as crucial mediators of cell-to-cell communication in normal physiology as well as in diseased states, and have been largely studied in regard to their role in cancer progression. However, the mechanisms by which their biogenesis and secretion are regulated by metabolic or endocrine factors remain unknown. Here, we delineate a mechanism by which EV secretion is regulated by a cholesterol metabolite, 27-Hydroxycholesterol (27HC), where treatment of myeloid immune cells (RAW 264.7 and J774A.1) with 27HC impairs lysosomal homeostasis, leading to shunting of multivesicular bodies (MVBs) away from lysosomal degradation, towards secretion as EVs. This altered lysosomal function is likely caused by mitochondrial dysfunction and subsequent increase in reactive oxygen species (ROS). Interestingly, cotreatment with a mitochondria-targeted antioxidant rescued the lysosomal impairment and attenuated the 27HC-mediated increase in EV secretion. Overall, our findings establish how a cholesterol metabolite regulates EV secretion and paves the way for the development of strategies to regulate cancer progression by controlling EV secretion.

Keywords:  27-hydroxycholesterol; extracellular vesicles; lysosome; mitochondria; myeloid immune cell; reactive oxygen species

DOI:  https://doi.org/10.1210/endocr/bqae127
Neurobiol Dis. 2024 Sep 14. pii: S0969-9961(24)00267-5. [Epub ahead of print]201 106667

Characterization of fission and fusion mitochondrial dynamics in HD fibroblasts according to patient's severity status.

Saja Gharaba, Uri Sprecher, Adam Baransi, Noam Muchtar, Miguel Weil.

  Huntington's Disease (HD) is an inheritable neurodegenerative condition caused by an expanded CAG trinucleotide repeat in the HTT gene with a direct correlation between CAG repeats expansion and disease severity with earlier onset-of- disease. Previously we have shown that primary skin fibroblasts from HD patients exhibit unique phenotype disease features, including distinct nuclear morphology and perturbed actin cap linked with cell motility, that are correlated with the HD patient disease severity. Here we provide further evidence that mitochondrial fission-fusion morphology balance dynamics, classified using a custom image-based high-content analysis (HCA) machine learning tool, that improved correlation with HD severity status. This mitochondrial phenotype is supported by appropriate changes in fission-fusion biomarkers (Drp1, MFN1, MFN2, VAT1) levels in the HD patients' fibroblasts. These findings collectively point towards a dysregulation in mitochondrial dynamics, where both fission and fusion processes may be disrupted in HD cells compared to healthy controls. This study shows for the first time a methodology that enables identification of HD phenotype before patient's disease onset (Premanifest). Therefore, we believe that this tool holds a potential for improving precision in HD patient's diagnostics bearing the potential to evaluate alterations in mitochondrial dynamics throughout the progression of HD, offering valuable insights into the molecular mechanisms and drug therapy evaluation underlying biological differences in any disease stage.

Keywords:  Fission and fusion; Huntington's disease; Image-based high content analysis; Machine learning classifier; Mitochondrial morphology; Primary skin fibroblast; Single-cell analysis

DOI:  https://doi.org/10.1016/j.nbd.2024.106667