bims-axbals 2024-06-30 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2024–06–30
23 papers selected by
TJ Krzystek, ALS Therapy Development Institute

The exocyst subunit EXOC2 regulates the toxicity of expanded GGGGCC repeats in C9ORF72-ALS/FTD.
Defective mitochondria-lysosomal axis enhances the release of extracellular vesicles containing mitochondrial DNA and proteins in Huntington's disease.
Stress-induced TDP-43 nuclear condensation causes splicing loss of function and STMN2 depletion.
Physiological regulation of neuronal Wnt activity is essential for TDP-43 localization and function.
Extracellular Vesicles from NSC-34 MN-like Cells Transfected with Mutant SOD1 Modulate Inflammatory Status of Raw 264.7 Macrophages.
The Neuromuscular Disorder Mediated by Extracellular Vesicles in Amyotrophic Lateral Sclerosis.
Shotgun Proteomics Links Proteoglycan-4+ Extracellular Vesicles to Cognitive Protection in Amyotrophic Lateral Sclerosis.
The Spatiotemporal Expression of SOCS3 in the Brainstem and Spinal Cord of Amyotrophic Lateral Sclerosis Mice.
Treating amyotrophic lateral sclerosis with allogeneic Schwann cell-derived exosomal vesicles: a case report.
Downregulation of Protease Cathepsin D and Upregulation of Pathologic α-Synuclein Mediate Paucity of DNAJC6-Induced Degeneration of Dopaminergic Neurons.
Assessing MicroRNA Profiles from Low Concentration Extracellular Vesicle RNA Utilizing NanoString nCounter Technology.
Xenografted human iPSC-derived neurons with the familial Alzheimer's disease APPV717I mutation reveal dysregulated transcriptome signatures linked to synaptic function and implicate LINGO2 as a disease signaling mediator.
Altered expression of human myxovirus resistance protein a in amyotrophic lateral sclerosis.
Neuroinflammation and Lysosomal Abnormalities Characterise the Essential Role for Oxidation Resistance 1 in the Developing and Adult Cerebellum.
Downregulation of the silent potassium channel Kv8.1 increases motor neuron vulnerability in amyotrophic lateral sclerosis.
Design of a covalent protein-protein interaction inhibitor of SRPKs to suppress angiogenesis and invasion of cancer cells.
Comment to: "SCA4 Unravelled After More than 25 Years Using Advanced Genomic Technologies".
Molecular motor tug-of-war regulates elongasome cell wall synthesis dynamics in Bacillus subtilis.
Pharmacologic Activation of Integrated Stress Response Kinases Inhibits Pathologic Mitochondrial Fragmentation.
A hydrophobic core stabilizes the residual structure in the RRM2 intermediate state of the ALS-linked protein TDP-43.
Haloperidol, Olanzapine, and Risperidone Induce Morphological Changes in an In Vitro Model of Human Hippocampal Neurogenesis.
Sunitinib-mediated inhibition of STAT3 in skeletal muscle and spinal cord does not affect the disease in a mouse model of ALS.
PTPRS is a novel marker for early Tau pathology and synaptic integrity in Alzheimer's disease.

Cell Rep. 2024 Jun 26. pii: S2211-1247(24)00703-4. [Epub ahead of print]43(7): 114375

The exocyst subunit EXOC2 regulates the toxicity of expanded GGGGCC repeats in C9ORF72-ALS/FTD.

Dilara O Halim, Gopinath Krishnan, Evan P Hass, Soojin Lee, Mamta Verma, Sandra Almeida, Yuanzheng Gu, Deborah Y Kwon, Thomas G Fazzio, Fen-Biao Gao.

  GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this genetic mutation leads to neurodegeneration remains largely unknown. Using CRISPR-Cas9 technology, we deleted EXOC2, which encodes an essential exocyst subunit, in induced pluripotent stem cells (iPSCs) derived from C9ORF72-ALS/FTD patients. These cells are viable owing to the presence of truncated EXOC2, suggesting that exocyst function is partially maintained. Several disease-relevant cellular phenotypes in C9ORF72 iPSC-derived motor neurons are rescued due to, surprisingly, the decreased levels of dipeptide repeat (DPR) proteins and expanded G4C2 repeats-containing RNA. The treatment of fully differentiated C9ORF72 neurons with EXOC2 antisense oligonucleotides also decreases expanded G4C2 repeats-containing RNA and partially rescued disease phenotypes. These results indicate that EXOC2 directly or indirectly regulates the level of G4C2 repeats-containing RNA, making it a potential therapeutic target in C9ORF72-ALS/FTD.

Keywords:  ALS; ASO; C9ORF72; CP: Cell biology; CP: Neuroscience; DPR; FTD; exocyst; iPSC; neurodegeneration; neuron; poly(GR)

DOI:  https://doi.org/10.1016/j.celrep.2024.114375
J Extracell Biol. 2022 Oct;1(10): e65

Defective mitochondria-lysosomal axis enhances the release of extracellular vesicles containing mitochondrial DNA and proteins in Huntington's disease.

Margarida Beatriz, Rita Vilaça, Sandra I Anjo, Bruno Manadas, Cristina Januário, A Cristina Rego, Carla Lopes.

  Mitochondrial and autophagy dysfunction are mechanisms proposed to be involved in the pathogenesis of several neurodegenerative diseases. Huntington's disease (HD) is a progressive neurodegenerative disorder associated with mutant Huntingtin-induced abnormalities in neuronal mitochondrial dynamics and quality control. Former studies suggest that the removal of defective mitochondria may be compromised in HD. Mitochondrial quality control (MQC) is a complex, well-orchestrated pathway that can be compromised through mitophagy dysregulation or impairment in the mitochondria-lysosomal axis. Another mitochondrial stress response is the generation of mitochondrial-derived vesicles that fuse with the endolysosomal system and form multivesicular bodies that are extruded from cells as extracellular vesicles (EVs). In this work, we aimed to study the presence of mitochondrial components in human EVs and the relation to the dysfunction of both mitochondria and the autophagy pathway. We comprehensively characterized the mitochondrial and autophagy alterations in premanifest and manifest HD carriers and performed a proteomic and genomic EVs profile. We observed that manifest HD patients exhibit mitochondrial and autophagy impairment associated with enhanced EVs release. Furthermore, we detected mitochondrial DNA and proteins in EVs released by HD cells and in neuronal-derived EVs including VDAC-1 and alpha and beta subunits of ATP synthase F1. HD-extracellular vesicles transport higher levels of mitochondrial genetic material in manifest HD patients, suggesting an alternative pathway for the secretion of reactive mitochondrial components. This study provides a novel framework connecting EVs enhanced release of mitochondrial components to mitochondrial and lysosomal dysfunction in HD.

Keywords:  Huntington's disease; autophagy; extracellular vesicles; mitochondrial DNA; mitochondrial dysfunction; neuronal‐derived extracellular vesicles

DOI:  https://doi.org/10.1002/jex2.65
Cell Rep. 2024 Jun 27. pii: S2211-1247(24)00750-2. [Epub ahead of print]43(7): 114421

Stress-induced TDP-43 nuclear condensation causes splicing loss of function and STMN2 depletion.

Wan-Ping Huang, Brittany C S Ellis, Rachel E Hodgson, Anna Sanchez Avila, Vedanth Kumar, Jessica Rayment, Tobias Moll, Tatyana A Shelkovnikova.

  TDP-43 protein is dysregulated in several neurodegenerative diseases, which often have a multifactorial nature and may have extrinsic stressors as a "second hit." TDP-43 undergoes reversible nuclear condensation in stressed cells including neurons. Here, we demonstrate that stress-inducible nuclear TDP-43 condensates are RNA-depleted, non-liquid assemblies distinct from the known nuclear bodies. Their formation requires TDP-43 oligomerization and ATP and is inhibited by RNA. Using a confocal nanoscanning assay, we find that amyotrophic lateral sclerosis (ALS)-linked mutations alter stress-induced TDP-43 condensation by changing its affinity to liquid-like ribonucleoprotein assemblies. Stress-induced nuclear condensation transiently inactivates TDP-43, leading to loss of interaction with its protein binding partners and loss of function in splicing. Splicing changes are especially prominent and persisting for STMN2 RNA, and STMN2 protein becomes rapidly depleted early during stress. Our results point to early pathological changes to TDP-43 in the nucleus and support therapeutic modulation of stress response in ALS.

Keywords:  ALS; CP: Molecular biology; STMN2; TDP-43; condensate; nuclear body; splicing; stathmin-2; stress

DOI:  https://doi.org/10.1016/j.celrep.2024.114421
EMBO J. 2024 Jun 25.

Physiological regulation of neuronal Wnt activity is essential for TDP-43 localization and function.

Nan Zhang, Anna Westerhaus, Macey Wilson, Ethan Wang, Loyal Goff, Shanthini Sockanathan.

  Nuclear exclusion of the RNA- and DNA-binding protein TDP-43 can induce neurodegeneration in different diseases. Diverse processes have been implicated to influence TDP-43 mislocalization, including disrupted nucleocytoplasmic transport (NCT); however, the physiological pathways that normally ensure TDP-43 nuclear localization are unclear. The six-transmembrane enzyme glycerophosphodiester phosphodiesterase 2 (GDE2 or GDPD5) cleaves the glycosylphosphatidylinositol (GPI) anchor that tethers some proteins to the membrane. Here we show that GDE2 maintains TDP-43 nuclear localization by regulating the dynamics of canonical Wnt signaling. Ablation of GDE2 causes aberrantly sustained Wnt activation in adult neurons, which is sufficient to cause NCT deficits, nuclear pore abnormalities, and TDP-43 nuclear exclusion. Disruption of GDE2 coincides with TDP-43 abnormalities in postmortem tissue from patients with amyotrophic lateral sclerosis (ALS). Further, GDE2 deficits are evident in human neural cell models of ALS, which display erroneous Wnt activation that, when inhibited, increases mRNA levels of genes regulated by TDP-43. Our study identifies GDE2 as a critical physiological regulator of Wnt signaling in adult neurons and highlights Wnt pathway activation as an unappreciated mechanism contributing to nucleocytoplasmic transport and TDP-43 abnormalities in disease.

Keywords:  GDE2; Neurodegeneration; Nucleocytoplasmic Transport; TDP-43; WNT

DOI:  https://doi.org/10.1038/s44318-024-00156-8
Genes (Basel). 2024 Jun 03. pii: 735. [Epub ahead of print]15(6):

Extracellular Vesicles from NSC-34 MN-like Cells Transfected with Mutant SOD1 Modulate Inflammatory Status of Raw 264.7 Macrophages.

Elisabetta Carata, Marco Muci, Stefania Mariano, Simona Di Giulio, Annamaria Nigro, Alessandro Romano, Elisa Panzarini.

  Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease targeting the brain and spinal cord. Non-neuronal cells, including macrophages, may contribute to the disruption of motor neurons (MNs), neuromuscular junction dismantling and clinical signs of ALS. Understanding the modality and the effect of MNs-macrophage communication is pivotal. Here, we focus on extracellular vesicle (EVS)-mediated communication and, in particular, we analyze the response of macrophages. NSC-34 cells transfected with mutant SOD1 (G93A, A4V, G85R, G37R) and differentiated towards MN-like cells, and Raw 264.7 macrophages are the cellular models of the study. mSOD1 NSC-34 cells release a high number of vesicles, both large-lEVs (300 nm diameter) and small-sEVs (90 nm diameter), containing inflammation-modulating molecules, and are efficiently taken up by macrophages. RT-PCR analysis of inflammation mediators demonstrated that the conditioned medium of mSOD1 NSC-34 cells polarizes Raw 264.7 macrophages towards both pro-inflammatory and anti-inflammatory phenotypes. sEVs act on macrophages in a time-dependent manner: an anti-inflammatory response mediated by TGFβ firstly starts (12 h); successively, the response shifts towards a pro-inflammation IL-1β-mediated (48 h). The response of macrophages is strictly dependent on the SOD1 mutation type. The results suggest that EVs impact physiological and behavioral macrophage processes and are of potential relevance to MN degeneration.

Keywords:  NSC-34 cells; Raw 264.7 macrophages; amyotrophic lateral sclerosis; anti-inflammatory macrophages; extracellular vesicles; mSOD1; pro-inflammatory macrophages

DOI:  https://doi.org/10.3390/genes15060735
Curr Issues Mol Biol. 2024 Jun 14. 46(6): 5999-6017

The Neuromuscular Disorder Mediated by Extracellular Vesicles in Amyotrophic Lateral Sclerosis.

Elisabetta Carata, Marco Muci, Simona Di Giulio, Tiziano Di Giulio, Stefania Mariano, Elisa Panzarini.

  Amyotrophic lateral sclerosis (ALS) represents a neurodegenerative disorder characterized by the progressive loss of both upper and lower motor neurons, resulting in muscular atrophy and eventual paralysis. While much research has concentrated on investigating the impact of major mutations associated with ALS on motor neurons and central nervous system (CNS) cells, recent studies have unveiled that ALS pathogenesis extends beyond CNS imbalances, encompassing dysregulation in other tissues such as skeletal muscle. Evidence from animal models and patients supports this broader perspective. Skeletal muscle, once considered solely as an effector organ, is now recognized as possessing significant secretory activity capable of influencing motor neuron survival. However, the precise cellular and molecular mechanisms underlying the detrimental effects observed in muscle and its associated structures in ALS remain poorly understood. Additionally, emerging data suggest that extracellular vesicles (EVs) may play a role in the establishment and function of the neuromuscular junction (NMJ) under both physiological and pathological conditions and in wasting and regeneration of skeletal muscles, particularly in neurodegenerative diseases like ALS. This review aims to explore the key findings about skeletal muscle involvement in ALS, shedding light on the potential underlying mechanisms and contributions of EVs and their possible application for the design of biosensors.

Keywords:  amyotrophic lateral sclerosis; biosensors; extracellular vesicles; miRNA; skeletal muscle

DOI:  https://doi.org/10.3390/cimb46060358
Biomolecules. 2024 Jun 19. pii: 727. [Epub ahead of print]14(6):

Shotgun Proteomics Links Proteoglycan-4+ Extracellular Vesicles to Cognitive Protection in Amyotrophic Lateral Sclerosis.

Beatrice Vilardo, Fabiola De Marchi, Davide Raineri, Marcello Manfredi, Veronica De Giorgis, Alen Bebeti, Lorenza Scotti, Natasa Kustrimovic, Giuseppe Cappellano, Letizia Mazzini, Annalisa Chiocchetti.

  Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder lacking reliable biomarkers for early diagnosis and disease progression monitoring. This study aimed to identify the novel biomarkers in plasmatic extracellular vesicles (EVs) isolated from ALS patients and healthy controls (HCs). A total of 61 ALS patients and 30 age-matched HCs were enrolled in the study and the protein content of circulating EVs was analyzed by shotgun proteomics. The study was divided into a discovery phase (involving 12 ALS and 12 HC patients) and a validation one (involving 49 ALS and 20 HC patients). In the discovery phase, more than 300 proteins were identified, with 32 proteins showing differential regulation in ALS patients compared to HCs. In the validation phase, over 400 proteins were identified, with 20 demonstrating differential regulation in ALS patients compared to HCs. Notably, seven proteins were found to be common to both phases, all of which were significantly upregulated in EVs from ALS patients. Most of them have previously been linked to ALS since they have been detected in the serum or cerebrospinal fluid of ALS patients. Among them, proteoglycan (PRG)-4, also known as lubricin, was of particular interest since it was significantly increased in ALS patients with normal cognitive and motor functions. This study highlights the significance of EVs as a promising avenue for biomarker discovery in ALS. Moreover, it sheds light on the unexpected role of PRG-4 in relation to cognitive status in ALS patients.

Keywords:  amyotrophic lateral sclerosis (ALS); cognition; cognitive impairment; extracellular vesicles (EVs); proteoglycan 4 (PRG-4); proteomics

DOI:  https://doi.org/10.3390/biom14060727
Brain Sci. 2024 May 31. pii: 564. [Epub ahead of print]14(6):

The Spatiotemporal Expression of SOCS3 in the Brainstem and Spinal Cord of Amyotrophic Lateral Sclerosis Mice.

Ching-Yi Lin, Veronica Vanoverbeke, David Trent, Kathryn Willey, Yu-Shang Lee.

  Amyotrophic lateral sclerosis (ALS) is characterized by the progressive loss of motor neurons from the brain and spinal cord. The excessive neuroinflammation is thought to be a common determinant of ALS. Suppressor of cytokine signaling-3 (SOCS3) is pathologically upregulated after injury/diseases to negatively regulate a broad range of cytokines/chemokines that mediate inflammation; however, the role that SOCS3 plays in ALS pathogenesis has not been explored. Here, we found that SOCS3 protein levels were significantly increased in the brainstem of the superoxide dismutase 1 (SOD1)-G93A ALS mice, which is negatively related to a progressive decline in motor function from the pre-symptomatic to the early symptomatic stage. Moreover, SOCS3 levels in both cervical and lumbar spinal cords of ALS mice were also significantly upregulated at the pre-symptomatic stage and became exacerbated at the early symptomatic stage. Concomitantly, astrocytes and microglia/macrophages were progressively increased and reactivated over time. In contrast, neurons were simultaneously lost in the brainstem and spinal cord examined over the course of disease progression. Collectively, SOCS3 was first found to be upregulated during ALS progression to directly relate to both increased astrogliosis and increased neuronal loss, indicating that SOCS3 could be explored to be as a potential therapeutic target of ALS.

Keywords:  amyotrophic lateral sclerosis (ALS); neuroinflammation; pre-Bötzinger Complex (preBötC); suppressor of cytokine signaling-3 (SOCS3)

DOI:  https://doi.org/10.3390/brainsci14060564
Neural Regen Res. 2024 Apr 03.

Treating amyotrophic lateral sclerosis with allogeneic Schwann cell-derived exosomal vesicles: a case report.

Pascal J Goldschmidt-Clermont, Aisha Khan, George Jimsheleishvili, Patricia Graham, Adriana Brooks, Risset Silvera, Alexander J P Goldschmidt, Damien D Pearse, W Dalton Dietrich, Allan D Levi, James D Guest.

Schwann cells are essential for the maintenance and function of motor neurons, axonal networks, and the neuromuscular junction. In amyotrophic lateral sclerosis, where motor neuron function is progressively lost, Schwann cell function may also be impaired. Recently, important signaling and potential trophic activities of Schwann cell-derived exosomal vesicles have been reported. This case report describes the treatment of a patient with advanced amyotrophic lateral sclerosis using serial intravenous infusions of allogeneic Schwann cell-derived exosomal vesicles, marking, to our knowledge, the first instance of such treatment. An 81-year-old male patient presented with a 1.5-year history of rapidly progressive amyotrophic lateral sclerosis. After initial diagnosis, the patient underwent a combination of generic riluzole, sodium phenylbutyrate for the treatment of amyotrophic lateral sclerosis, and taurursodiol. The patient volunteered to participate in an FDA-approved single-patient expanded access treatment and received weekly intravenous infusions of allogeneic Schwann cell-derived exosomal vesicles to potentially restore impaired Schwann cell and motor neuron function. We confirmed that cultured Schwann cells obtained from the amyotrophic lateral sclerosis patient via sural nerve biopsy appeared impaired (senescent) and that exposure of the patient's Schwann cells to allogeneic Schwann cell-derived exosomal vesicles, cultured expanded from a cadaver donor improved their growth capacity in vitro. After a period of observation lasting 10 weeks, during which amyotrophic lateral sclerosis Functional Rating Scale-Revised and pulmonary function were regularly monitored, the patient received weekly consecutive infusions of 1.54 × 1012 (×2), and then consecutive infusions of 7.5 × 1012 (×6) allogeneic Schwann cell-derived exosomal vesicles diluted in 40 mL of Dulbecco's phosphate-buffered saline. None of the infusions were associated with adverse events such as infusion reactions (allergic or otherwise) or changes in vital signs. Clinical lab serum neurofilament and cytokine levels measured prior to each infusion varied somewhat without a clear trend. A more sensitive in-house assay suggested possible inflammasome activation during the disease course. A trend for clinical stabilization was observed during the infusion period. Our study provides a novel approach to address impaired Schwann cells and possibly motor neuron function in patients with amyotrophic lateral sclerosis using allogeneic Schwann cell-derived exosomal vesicles. Initial findings suggest that this approach is safe.

DOI: https://doi.org/10.4103/NRR.NRR-D-23-01815
Int J Mol Sci. 2024 Jun 18. pii: 6711. [Epub ahead of print]25(12):

Downregulation of Protease Cathepsin D and Upregulation of Pathologic α-Synuclein Mediate Paucity of DNAJC6-Induced Degeneration of Dopaminergic Neurons.

Ching-Chi Chiu, Ying-Ling Chen, Yi-Hsin Weng, Shu-Yu Liu, Hon-Lun Li, Tu-Hsueh Yeh, Hung-Li Wang.

  A homozygous mutation of the DNAJC6 gene causes autosomal recessive familial type 19 of Parkinson's disease (PARK19). To test the hypothesis that PARK19 DNAJC6 mutations induce the neurodegeneration of dopaminergic cells by reducing the protein expression of functional DNAJC6 and causing DNAJC6 paucity, an in vitro PARK19 model was constructed by using shRNA-mediated gene silencing of endogenous DANJC6 in differentiated human SH-SY5Y dopaminergic neurons. shRNA targeting DNAJC6 induced the neurodegeneration of dopaminergic cells. DNAJC6 paucity reduced the level of cytosolic clathrin heavy chain and the number of lysosomes in dopaminergic neurons. A DNAJC6 paucity-induced reduction in the lysosomal number downregulated the protein level of lysosomal protease cathepsin D and impaired macroautophagy, resulting in the upregulation of pathologic α-synuclein or phospho-α-synucleinSer129 in the endoplasmic reticulum (ER) and mitochondria. The expression of α-synuclein shRNA or cathepsin D blocked the DNAJC6 deficiency-evoked degeneration of dopaminergic cells. An increase in ER α-synuclein or phospho-α-synucleinSer129 caused by DNAJC6 paucity activated ER stress, the unfolded protein response and ER stress-triggered apoptotic signaling. The lack of DNAJC6-induced upregulation of mitochondrial α-synuclein depolarized the mitochondrial membrane potential and elevated the mitochondrial level of superoxide. The DNAJC6 paucity-evoked ER stress-related apoptotic cascade, mitochondrial malfunction and oxidative stress induced the degeneration of dopaminergic neurons via activating mitochondrial pro-apoptotic signaling. In contrast with the neuroprotective function of WT DNAJC6, the PARK19 DNAJC6 mutants (Q789X or R927G) failed to attenuate the tunicamycin- or rotenone-induced upregulation of pathologic α-synuclein and stimulation of apoptotic signaling. Our data suggest that PARK19 mutation-induced DNAJC6 paucity causes the degeneration of dopaminergic neurons via downregulating protease cathepsin D and upregulating neurotoxic α-synuclein. Our results also indicate that PARK19 mutation (Q789X or R927G) impairs the DNAJC6-mediated neuroprotective function.

Keywords:  DNAJC6; PARK19; cathepsin D; dopaminergic neurons; α-synuclein

DOI:  https://doi.org/10.3390/ijms25126711
Methods Mol Biol. 2024 ;2822 101-123

Assessing MicroRNA Profiles from Low Concentration Extracellular Vesicle RNA Utilizing NanoString nCounter Technology.

Rachel E Crossland, Jamie Macdonald, Xiao-Nong Wang.

  Extracellular vesicles (EV) are rich in small RNA; however, a frequent caveat can be low abundance of EV RNA content, especially in clinical studies. NanoString MicroRNA Assays allow for multiplexed profiling of n = 800 mature microRNAs and can be applied to assess EV microRNA cargo. Here, we describe a method to adapt NanoString nCounter microRNA profiling to assess mature microRNA expression in low-concentration RNA samples, including concentrating the RNA, quantifying the RNA, and performing the NanoString protocol. Twelve samples can be assessed at one time using this method.

Keywords:  Extracellular vesicle; Molecular profiling; NanoString; microRNA

DOI:  https://doi.org/10.1007/978-1-0716-3918-4_9
Acta Neuropathol. 2024 Jun 25. 147(1): 107

Xenografted human iPSC-derived neurons with the familial Alzheimer's disease APPV717I mutation reveal dysregulated transcriptome signatures linked to synaptic function and implicate LINGO2 as a disease signaling mediator.

Wenhui Qu, Matti Lam, Julie J McInvale, Jason A Mares, Sam Kwon, Nelson Humala, Aayushi Mahajan, Trang Nguyen, Kelly A Jakubiak, Jeong-Yeon Mun, Thomas G Tedesco, Osama Al-Dalahmah, Syed A Hussaini, Andrew A Sproul, Markus D Siegelin, Philip L De Jager, Peter Canoll, Vilas Menon, Gunnar Hargus.

  Alzheimer's disease (AD) is the most common cause of dementia, and disease mechanisms are still not fully understood. Here, we explored pathological changes in human induced pluripotent stem cell (iPSC)-derived neurons carrying the familial AD APPV717I mutation after cell injection into the mouse forebrain. APPV717I mutant iPSCs and isogenic controls were differentiated into neurons revealing enhanced Aβ42 production, elevated phospho-tau, and impaired neurite outgrowth in APPV717I neurons. Two months after transplantation, APPV717I and control neural cells showed robust engraftment but at 12 months post-injection, APPV717I grafts were smaller and demonstrated impaired neurite outgrowth compared to controls, while plaque and tangle pathology were not seen. Single-nucleus RNA-sequencing of micro-dissected grafts, performed 2 months after cell injection, identified significantly altered transcriptome signatures in APPV717I iPSC-derived neurons pointing towards dysregulated synaptic function and axon guidance. Interestingly, APPV717I neurons showed an increased expression of genes, many of which are also upregulated in postmortem neurons of AD patients including the transmembrane protein LINGO2. Downregulation of LINGO2 in cultured APPV717I neurons rescued neurite outgrowth deficits and reversed key AD-associated transcriptional changes related but not limited to synaptic function, apoptosis and cellular senescence. These results provide important insights into transcriptional dysregulation in xenografted APPV717I neurons linked to synaptic function, and they indicate that LINGO2 may represent a potential therapeutic target in AD.

Keywords:  Alzheimer’s disease; Disease modeling; Grafts; Induced pluripotent stem cells; Single-nucleus RNA sequencing; Transplantation; iPSCs; snRNA-seq

DOI:  https://doi.org/10.1007/s00401-024-02755-5
J Neuropathol Exp Neurol. 2024 Jun 25. pii: nlae052. [Epub ahead of print]

Altered expression of human myxovirus resistance protein a in amyotrophic lateral sclerosis.

Hiroyuki Honda, Shoko Sadashima, Motoi Yoshimura, Naonori Sakurada, Sachiko Koyama, Kaoru Yagita, Hideomi Hamasaki, Hideko Noguchi, Hajime Arahata, Naokazu Sasagasako.

  Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder. The etiology of sporadic ALS (sALS) has not yet been clarified. An increasing body of evidence suggests the involvement of viral infections and interferons (IFNs). Human myxovirus resistance protein A (MxA) is an IFN-induced dynamin-like GTPase that acts as a potent antiviral factor. This study examined MxA expression in ALS patient spinal cords using immunohistochemistry. Thirty-two cases of sALS (pathologically proven ALS-TDP), 10 non-ALS, other neurological disease control cases were examined. In most ALS cases, MxA cytoplasmic condensates were observed in the remaining spinal anterior horn neurons. The ALS group had a significantly higher rate of MxA-highly expressing neurons than the non-ALS group. Colocalization of MxA cytoplasmic condensate and transactive response DNA-binding protein 43 kDa (TDP-43)-positive inclusions was rarely observed. Because MxA has antiviral activity induced by IFNs, our results suggest that IFNs are involved in the pathogenesis of ALS in spinal cord anterior horn neurons. Our study also suggests that monitoring viral infections and IFN activation in patients with ALS may be critically important.

Keywords:  ALS; Interferon; MxA; Myxovirus resistance protein; Virus

DOI:  https://doi.org/10.1093/jnen/nlae052
Antioxidants (Basel). 2024 Jun 03. pii: 685. [Epub ahead of print]13(6):

Neuroinflammation and Lysosomal Abnormalities Characterise the Essential Role for Oxidation Resistance 1 in the Developing and Adult Cerebellum.

Eboni M V Bucknor, Errin Johnson, Stephanie Efthymiou, Javeria R Alvi, Tipu Sultan, Henry Houlden, Reza Maroofian, Ehsan G Karimiani, Mattéa J Finelli, Peter L Oliver.

  Loss-of-function mutations in the TLDc family of proteins cause a range of severe childhood-onset neurological disorders with common clinical features that include cerebellar neurodegeneration, ataxia and epilepsy. Of these proteins, oxidation resistance 1 (OXR1) has been implicated in multiple cellular pathways related to antioxidant function, transcriptional regulation and cellular survival; yet how this relates to the specific neuropathological features in disease remains unclear. Here, we investigate a range of loss-of-function mouse model systems and reveal that constitutive deletion of Oxr1 leads to a rapid and striking neuroinflammatory response prior to neurodegeneration that is associated with lysosomal pathology. We go on to show that neuroinflammation and cell death in Oxr1 knockouts can be completely rescued by the neuronal expression of Oxr1, suggesting that the phenotype is driven by the cell-intrinsic defects of neuronal cells lacking the gene. Next, we generate a ubiquitous, adult inducible knockout of Oxr1 that surprisingly displays rapid-onset ataxia and cerebellar neurodegeneration, establishing for the first time that the distinctive pathology associated with the loss of Oxr1 occurs irrespective of developmental stage. Finally, we describe two new homozygous human pathogenic variants in OXR1 that cause neurodevelopmental delay, including a novel stop-gain mutation. We also compare functionally two missense human pathogenic mutations in OXR1, including one newly described here, that cause different clinical phenotypes but demonstrate partially retained neuroprotective activity against oxidative stress. Together, these data highlight the essential role of Oxr1 in modulating neuroinflammatory and lysosomal pathways in the mammalian brain and support the hypothesis that OXR1 protein dosage may be critical for pathological outcomes in disease.

Keywords:  ataxia; cerebellum; lysosome; neuroinflammation; oxidative stress

DOI:  https://doi.org/10.3390/antiox13060685
Brain Commun. 2024 ;6(3): fcae202

Downregulation of the silent potassium channel Kv8.1 increases motor neuron vulnerability in amyotrophic lateral sclerosis.

Xuan Huang, Seungkyu Lee, Kuchuan Chen, Riki Kawaguchi, Ole Wiskow, Sulagna Ghosh, Devlin Frost, Laura Perrault, Roshan Pandey, Joseph R Klim, Bruno Boivin, Crystal Hermawan, Kenneth J Livak, Daniel H Geschwind, Brian J Wainger, Kevin C Eggan, Bruce P Bean, Clifford J Woolf.

  While voltage-gated potassium channels have critical roles in controlling neuronal excitability, they also have non-ion-conducting functions. Kv8.1, encoded by the KCNV1 gene, is a 'silent' ion channel subunit whose biological role is complex since Kv8.1 subunits do not form functional homotetramers but assemble with Kv2 to modify its ion channel properties. We profiled changes in ion channel expression in amyotrophic lateral sclerosis patient-derived motor neurons carrying a superoxide dismutase 1(A4V) mutation to identify what drives their hyperexcitability. A major change identified was a substantial reduction of KCNV1/Kv8.1 expression, which was also observed in patient-derived neurons with C9orf72 expansion. We then studied the effect of reducing KCNV1/Kv8.1 expression in healthy motor neurons and found it did not change neuronal firing but increased vulnerability to cell death. A transcriptomic analysis revealed dysregulated metabolism and lipid/protein transport pathways in KCNV1/Kv8.1-deficient motor neurons. The increased neuronal vulnerability produced by the loss of KCNV1/Kv8.1 was rescued by knocking down Kv2.2, suggesting a potential Kv2.2-dependent downstream mechanism in cell death. Our study reveals, therefore, unsuspected and distinct roles of Kv8.1 and Kv2.2 in amyotrophic lateral sclerosis-related neurodegeneration.

Keywords:  ALS; ion channels; motor neuron; neurodegeneration

DOI:  https://doi.org/10.1093/braincomms/fcae202
Commun Chem. 2024 Jun 27. 7(1): 144

Design of a covalent protein-protein interaction inhibitor of SRPKs to suppress angiogenesis and invasion of cancer cells.

Gongli Cai, Yishu Bao, Qingyun Li, Pang-Hung Hsu, Jiang Xia, Jacky Chi Ki Ngo.

Serine-arginine (SR) proteins are splicing factors that play essential roles in both constitutive and alternative pre-mRNA splicing. Phosphorylation of their C-terminal RS domains by SR protein kinases (SRPKs) regulates their localization and diverse cellular activities. Dysregulation of phosphorylation has been implicated in many human diseases, including cancers. Here, we report the development of a covalent protein-protein interaction inhibitor, C-DBS, that targets a lysine residue within the SRPK-specific docking groove to block the interaction and phosphorylation of the prototypic SR protein SRSF1. C-DBS exhibits high specificity and conjugation efficiency both in vitro and in cellulo. This self-cell-penetrating inhibitor attenuates the phosphorylation of endogenous SR proteins and subsequently inhibits the angiogenesis, migration, and invasion of cancer cells. These findings provide a new foundation for the development of covalent SRPK inhibitors for combatting diseases such as cancer and viral infections and overcoming the resistance encountered by ATP-competitive inhibitors.

DOI: https://doi.org/10.1038/s42004-024-01230-2
Mov Disord. 2024 Jun;39(6): 1077

Comment to: "SCA4 Unravelled After More than 25 Years Using Advanced Genomic Technologies".

Martin Paucar, Daniel Nilsson, Martin Engvall, José Laffita-Mesa, Anna Wedell, Per Svenningsson.



Keywords:   ZFHX3; spinocerebellar ataxia type 4 (SCA4)

DOI:  https://doi.org/10.1002/mds.29827
Nat Commun. 2024 Jun 26. 15(1): 5411

Molecular motor tug-of-war regulates elongasome cell wall synthesis dynamics in Bacillus subtilis.

Stuart Middlemiss, Matthieu Blandenet, David M Roberts, Andrew McMahon, James Grimshaw, Joshua M Edwards, Zikai Sun, Kevin D Whitley, Thierry Blu, Henrik Strahl, Séamus Holden.

Most rod-shaped bacteria elongate by inserting new cell wall material into the inner surface of the cell sidewall. This is performed by class A penicillin binding proteins (PBPs) and a highly conserved protein complex, the elongasome, which moves processively around the cell circumference and inserts long glycan strands that act as barrel-hoop-like reinforcing structures, thereby giving rise to a rod-shaped cell. However, it remains unclear how elongasome synthesis dynamics and termination events are regulated to determine the length of these critical cell-reinforcing structures. To address this, we developed a method to track individual elongasome complexes around the entire circumference of Bacillus subtilis cells for minutes-long periods using single-molecule fluorescence microscopy. We found that the B. subtilis elongasome is highly processive and that processive synthesis events are frequently terminated by rapid reversal or extended pauses. We found that cellular levels of RodA regulate elongasome processivity, reversal and pausing. Our single-molecule data, together with stochastic simulations, show that elongasome dynamics and processivity are regulated by molecular motor tug-of-war competition between several, likely two, oppositely oriented peptidoglycan synthesis complexes associated with the MreB filament. Altogether these results demonstrate that molecular motor tug-of-war is a key regulator of elongasome dynamics in B. subtilis, which likely also regulates the cell shape via modulation of elongasome processivity.

DOI: https://doi.org/10.1038/s41467-024-49785-x
bioRxiv. 2024 Jun 10. pii: 2024.06.10.598126. [Epub ahead of print]

Pharmacologic Activation of Integrated Stress Response Kinases Inhibits Pathologic Mitochondrial Fragmentation.

Kelsey R Baron, Samantha Oviedo, Sophia Krasny, Mashiat Zaman, Rama Aldakhlallah, Prakhyat Mathur, Gerald Pfeffer, Michael J Bollong, Timothy Shutt, Danielle A Grotjahn, R Luke Wiseman.

Excessive mitochondrial fragmentation is associated with the pathologic mitochondrial dysfunction implicated in the pathogenesis of etiologically-diverse diseases, including many neurodegenerative disorders. The integrated stress response (ISR) - comprising the four eIF2α kinases PERK, GCN2, PKR, and HRI - is a prominent stress-responsive signaling pathway that regulates mitochondrial morphology and function in response to diverse types of pathologic insult. This suggests that pharmacologic, stress-independent activation of the ISR represents a potential strategy to mitigate pathologic mitochondrial fragmentation associated with human disease. Here, we show that pharmacologic, stress-independent activation of the ISR kinases HRI or GCN2 promotes adaptive mitochondrial elongation and prevents mitochondrial fragmentation induced by the calcium ionophore ionomycin. Further, we show that stress-independent activation of these ISR kinases reduces mitochondrial fragmentation and restores basal mitochondrial morphology in patient fibroblasts expressing the pathogenic D414V variant of the pro-fusion mitochondrial GTPase MFN2 associated with neurological dysfunctions including ataxia, optic atrophy, and sensorineural hearing loss. These results identify pharmacologic, stress-independent activation of ISR kinases as a potential strategy to prevent pathologic mitochondrial fragmentation induced by disease-relevant chemical and genetic insults, further motivating the pursuit of highly selective ISR kinase-activating compounds as a therapeutic strategy to mitigate mitochondrial dysfunction implicated in diverse human diseases.

DOI: https://doi.org/10.1101/2024.06.10.598126
bioRxiv. 2024 Jun 12. pii: 2024.06.12.598648. [Epub ahead of print]

A hydrophobic core stabilizes the residual structure in the RRM2 intermediate state of the ALS-linked protein TDP-43.

Brian C Mackness, Brittany R Morgan, Laura M Deveau, Sagar V Kathuria, Jill A Zitzewitz, Francesca Massi.

Folding intermediates mediate both protein folding and the misfolding and aggregation observed in human diseases, including amyotrophic lateral sclerosis (ALS), and are prime targets for therapeutic interventions. In this study, we identified the core nucleus of structure for a folding intermediate in the second RNA recognition motif (RRM2) of the ALS-linked RNA-binding protein, TDP-43, using a combination of experimental and computational approaches. Urea equilibrium unfolding studies revealed that the RRM2 intermediate state consists of collapsed residual secondary structure localized to the N-terminal half of RRM2, while the C-terminus is largely disordered. Steered molecular dynamics simulations and mutagenesis studies yielded key stabilizing hydrophobic contacts that, when mutated to alanine, severely disrupt the overall fold of RRM2. In combination, these findings suggest a role for this RRM intermediate in normal TDP-43 function as well as serving as a template for misfolding and aggregation through the low stability and non-native secondary structure.

DOI: https://doi.org/10.1101/2024.06.12.598648
Biomolecules. 2024 Jun 13. pii: 688. [Epub ahead of print]14(6):

Haloperidol, Olanzapine, and Risperidone Induce Morphological Changes in an In Vitro Model of Human Hippocampal Neurogenesis.

Bálint Jezsó, Sára Kálmán, Kiara Gitta Farkas, Edit Hathy, Katalin Vincze, Dzsenifer Kovács-Schoblocher, Julianna Lilienberg, Csongor Tordai, Zsófia Nemoda, László Homolya, Ágota Apáti, János M Réthelyi.

   BACKGROUND: Induced pluripotent stem cell (iPSC) based neuronal differentiation is valuable for studying neuropsychiatric disorders and pharmacological mechanisms at the cellular level. We aimed to examine the effects of typical and atypical antipsychotics on human iPSC-derived neural progenitor cells (NPCs).
METHODS: Proliferation and neurite outgrowth were measured by live cell imaging, and gene expression levels related to neuronal identity were analyzed by RT-QPCR and immunocytochemistry during differentiation into hippocampal dentate gyrus granule cells following treatment of low- and high-dose antipsychotics (haloperidol, olanzapine, and risperidone).
RESULTS: Antipsychotics did not modify the growth properties of NPCs after 3 days of treatment. However, the characteristics of neurite outgrowth changed significantly in response to haloperidol and olanzapine. After three weeks of differentiation, mRNA expression levels of the selected neuronal markers increased (except for MAP2), while antipsychotics caused only subtle changes. Additionally, we found no changes in MAP2 or GFAP protein expression levels as a result of antipsychotic treatment.
CONCLUSIONS: Altogether, antipsychotic medications promoted neurogenesis in vitro by influencing neurite outgrowth rather than changing cell survival or gene expression. This study provides insights into the effects of antipsychotics on neuronal differentiation and highlights the importance of considering neurite outgrowth as a potential target of action.

Keywords:  antipsychotics; hippocampal neurogenesis; human induced pluripotent stem cells; neural differentiation; neurite outgrowth

DOI:  https://doi.org/10.3390/biom14060688
Neurobiol Dis. 2024 Jun 22. pii: S0969-9961(24)00176-1. [Epub ahead of print]199 106576

Sunitinib-mediated inhibition of STAT3 in skeletal muscle and spinal cord does not affect the disease in a mouse model of ALS.

Massimo Tortarolo, Andrea David Re Cecconi, Laura Camporeale, Cassandra Margotta, Giovanni Nardo, Laura Pasetto, Valentina Bonetto, Mariarita Galbiati, Valeria Crippa, Angelo Poletti, Rosanna Piccirillo, Caterina Bendotti.

  Variability in disease onset and progression is a hallmark of amyotrophic lateral sclerosis (ALS), both in sporadic and genetic forms. Recently, we found that SOD1-G93A transgenic mice expressing the same amount of mutant SOD1 but with different genetic backgrounds, C57BL/6JOlaHsd and 129S2/SvHsd, show slow and rapid muscle wasting and disease progression, respectively. Here, we investigated the different molecular mechanisms underlying muscle atrophy. Although both strains showed similar denervation-induced degradation of muscle proteins, only the rapidly progressing mice exhibited early and sustained STAT3 activation that preceded atrophy in gastrocnemius muscle. We therefore investigated the therapeutic potential of sunitinib, a tyrosine kinase inhibitor known to inhibit STAT3 and prevent cancer-induced muscle wasting. Although sunitinib treatment reduced STAT3 activation in the gastrocnemius muscle and lumbar spinal cord, it did not preserve spinal motor neurons, improve neuromuscular impairment, muscle atrophy and disease progression in the rapidly progressing SOD1-G93A mice. Thus, the effect of sunitinib is not equally positive in different diseases associated with muscle wasting. Moreover, given the complex role of STAT3 in the peripheral and central compartments of the neuromuscular system, the present study suggests that its broad inhibition may lead to opposing effects, ultimately preventing a potential positive therapeutic action in ALS.

Keywords:  Amyotrophic lateral sclerosis; Atrogenes; Disease progression; Genetic background; SOD1; STAT3, muscle atrophy; Spinal cord; Sunitinib

DOI:  https://doi.org/10.1016/j.nbd.2024.106576
Sci Rep. 2024 06 26. 14(1): 14718

PTPRS is a novel marker for early Tau pathology and synaptic integrity in Alzheimer's disease.

PREVENT-AD research group

  We examined the role of protein tyrosine phosphatase receptor sigma (PTPRS) in the context of Alzheimer's disease and synaptic integrity. Publicly available datasets (BRAINEAC, ROSMAP, ADC1) and a cohort of asymptomatic but "at risk" individuals (PREVENT-AD) were used to explore the relationship between PTPRS and various Alzheimer's disease biomarkers. We identified that PTPRS rs10415488 variant C shows features of neuroprotection against early Tau pathology and synaptic degeneration in Alzheimer's disease. This single nucleotide polymorphism correlated with higher PTPRS transcript abundance and lower p(181)Tau and GAP-43 levels in the CSF. In the brain, PTPRS protein abundance was significantly correlated with the quantity of two markers of synaptic integrity: SNAP25 and SYT-1. We also found the presence of sexual dimorphism for PTPRS, with higher CSF concentrations in males than females. Male carriers for variant C were found to have a 10-month delay in the onset of AD. We thus conclude that PTPRS acts as a neuroprotective receptor in Alzheimer's disease. Its protective effect is most important in males, in whom it postpones the age of onset of the disease.

Keywords:  Alzheimer’s disease; Autophagy; Cerebrospinal fluid; Protein-tyrosine phosphatase receptors; Synaptic markers; Tau pathology

DOI:  https://doi.org/10.1038/s41598-024-65104-2