bims-axbals 2024-11-03 papers

bims-axbals

Biomed News

on Axonal Biology and ALS

Issue of 2024‒11‒03
37 papers selected by
TJ Krzystek, ALS Therapy Development Institute

Skeletal myotubes expressing ALS mutant SOD1 induce pathogenic changes, impair mitochondrial axonal transport, and trigger motoneuron death.
HDGFL2 cryptic protein: a portal to detection and diagnosis in neurodegenerative disease.
Amyotrophic Lateral Sclerosis: Insights and New Prospects in Disease Pathophysiology, Biomarkers and Therapies.
Polyethyleneimine facilitates the growth and electrophysiological characterization of iPSC-derived motor neurons.
A variant of the Hspa8 synaptic chaperone modifies disease in a SOD1G86R mouse model of amyotrophic lateral sclerosis.
Short Repeat Ribonucleic Acid Reduces Cytotoxicity by Preventing the Aggregation of TDP-43 and Its 25 KDa Carboxy-Terminal Fragment.
Cell-Type Resolved Protein Atlas of Brain Lysosomes Identifies SLC45A1-Associated Disease as a Lysosomal Disorder.
Axon Initial Segment Structure and Function in Health and Disease.
A Review of Biomarkers of Amyotrophic Lateral Sclerosis: A Pathophysiologic Approach.
Protocol for a phase 2 study of bosutinib for amyotrophic lateral sclerosis using real-world data: induced pluripotent stem cell-based drug repurposing for amyotrophic lateral sclerosis medicine (iDReAM) study.
A de novo Missense Mutation in PPP2R5D Alters Dopamine Pathways and Morphology of iPSC-derived Midbrain Neurons.
Ortholog of autism candidate gene RBM27 regulates mitoribosomal assembly factor MALS-1 to protect against mitochondrial dysfunction and axon degeneration during neurodevelopment.
F-BAR proteins CIP4 and FBP17 function in cortical neuron radial migration and process outgrowth.
Reporter Alleles in hiPSCs: Visual Cues on Development and Disease.
Rethinking antisense oligonucleotide therapeutics for amyotrophic lateral sclerosis.
Discovery of Novel Inhibitors against ALS-Related SOD1(A4V) Aggregation through the Screening of a Chemical Library Using Differential Scanning Fluorimetry (DSF).
Accumulation of F-actin drives brain aging and limits healthspan in Drosophila.
Rab11 suppresses neuronal stress signaling by localizing dual leucine zipper kinase to axon terminals for protein turnover.
Astragaloside IV reduces mutant Ataxin-3 levels and supports mitochondrial function in Spinocerebellar Ataxia Type 3.
Design of peptide therapeutics as protein-protein interaction inhibitors to treat neurodegenerative diseases.
Transcriptomic alterations in APP/PS1 mice astrocytes lead to early postnatal axon initial segment structural changes.
CNN-Based Neurodegenerative Disease Classification Using QR-Represented Gait Data.
Netrin-1 stimulated axon growth requires the polyglutamylase TTLL1.
[Research progress on the mechanism of autophagy flow injury caused by lysosomal dysfunction after cerebral ischemia].
Targeted protein degradation via cellular trafficking of nanoparticles.
Targeting Protein Aggregation in ALS.
Overview of nomenclature and diagnosis of amyotrophic lateral sclerosis.
Mitochondrial fission controls astrocyte morphogenesis and organization in the cortex.
Measurement of α-synuclein as protein cargo in plasma extracellular vesicles.
Split-Luciferase Reassembly Assay to Measure Endoplasmic Reticulum-Mitochondria Contacts in Live Cells.
Neurexin1 level in Huntington's Disease and decreased Neurexin1 in disease progression.
Mild cognitive impairment in amyotrophic lateral sclerosis: current view.
In organello silencing of mitochondrial gene expression.
Recombinant human plasma gelsolin suppresses persistent neuroinflammation and restores hippocampal neurogenesis in murine model of decompression sickness.
New evidence of vascular defects in neurodegenerative diseases revealed by single cell RNA sequencing.
miR-124 coordinates metabolic regulators acting at early stages of human neurogenesis.
Monitoring mitochondrial protein import by live cell imaging.

Mol Med. 2024 Oct 25. 30(1): 185

Skeletal myotubes expressing ALS mutant SOD1 induce pathogenic changes, impair mitochondrial axonal transport, and trigger motoneuron death.

Pablo Martínez, Mónica Silva, Sebastián Abarzúa, María Florencia Tevy, Enrique Jaimovich, Martha Constantine-Paton, Fernando J Bustos, Brigitte van Zundert.

  Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motoneurons (MNs), and despite progress, there is no effective treatment. A large body of evidence shows that astrocytes expressing ALS-linked mutant proteins cause non-cell autonomous toxicity of MNs. Although MNs innervate muscle fibers and ALS is characterized by the early disruption of the neuromuscular junction (NMJ) and axon degeneration, there are controversies about whether muscle contributes to non-cell-autonomous toxicity to MNs. In this study, we generated primary skeletal myotubes from myoblasts derived from ALS mice expressing human mutant SOD1G93A (termed hereafter mutSOD1). Characterization revealed that mutSOD1 skeletal myotubes display intrinsic phenotypic and functional differences compared to control myotubes generated from non-transgenic (NTg) littermates. Next, we analyzed whether ALS myotubes exert non-cell-autonomous toxicity to MNs. We report that conditioned media from mutSOD1 myotubes (mutSOD1-MCM), but not from control myotubes (NTg-MCM), induced robust death of primary MNs in mixed spinal cord cultures and compartmentalized microfluidic chambers. Our study further revealed that applying mutSOD1-MCM to the MN axonal side in microfluidic devices rapidly reduces mitochondrial axonal transport while increasing Ca2 + transients and reactive oxygen species (i.e., H2O2). These results indicate that soluble factor(s) released by mutSOD1 myotubes cause MN axonopathy that leads to lethal pathogenic changes.

Keywords:  ALS; Axonopathy; Mitochondria; Motoneuron; Muscle; Myotubes; Pathology

DOI:  https://doi.org/10.1186/s10020-024-00942-4
Mol Neurodegener. 2024 Oct 25. 19(1): 79

HDGFL2 cryptic protein: a portal to detection and diagnosis in neurodegenerative disease.

Ellen A Albagli, Anna Calliari, Tania F Gendron, Yong-Jie Zhang.



Keywords:  Amyotrophic lateral sclerosis; Biomarkers; Cerebrospinal fluid; Cryptic peptide; Frontotemporal dementia; Hepatoma derived growth factor 2; Neurodegeneration; TAR DNA-binding protein 43

DOI:  https://doi.org/10.1186/s13024-024-00768-y
Pharmaceuticals (Basel). 2024 Oct 18. pii: 1391. [Epub ahead of print]17(10):

Amyotrophic Lateral Sclerosis: Insights and New Prospects in Disease Pathophysiology, Biomarkers and Therapies.

Jameel M Al-Khayri, Mamtha Ravindran, Akshatha Banadka, Chendanda Devaiah Vandana, Kushalva Priya, Praveen Nagella, Kowshik Kukkemane.

  Amyotrophic Lateral Sclerosis (ALS) is a severe neurodegenerative disorder marked by the gradual loss of motor neurons, leading to significant disability and eventual death. Despite ongoing research, there are still limited treatment options, underscoring the need for a deeper understanding of the disease's complex mechanisms and the identification of new therapeutic targets. This review provides a thorough examination of ALS, covering its epidemiology, pathology, and clinical features. It investigates the key molecular mechanisms, such as protein aggregation, neuroinflammation, oxidative stress, and excitotoxicity that contribute to motor neuron degeneration. The role of biomarkers is highlighted for their importance in early diagnosis and disease monitoring. Additionally, the review explores emerging therapeutic approaches, including inhibitors of protein aggregation, neuroinflammation modulators, antioxidant therapies, gene therapy, and stem cell-based treatments. The advantages and challenges of these strategies are discussed, with an emphasis on the potential for precision medicine to tailor treatments to individual patient needs. Overall, this review aims to provide a comprehensive overview of the current state of ALS research and suggest future directions for developing effective therapies.

Keywords:  amyotrophic lateral sclerosis; diagnosis; neuroinflammation; pathology; therapies

DOI:  https://doi.org/10.3390/ph17101391
Sci Rep. 2024 10 30. 14(1): 26106

Polyethyleneimine facilitates the growth and electrophysiological characterization of iPSC-derived motor neurons.

Meimei Yang, Daofeng You, Gang Liu, Yin Lu, Guangming Yang, Timothy O'Brien, David C Henshall, Orla Hardiman, Li Cai, Min Liu, Sanbing Shen.

  Induced pluripotent stem cell (iPSC) technology, in combination with electrophysiological characterization via multielectrode array (MEA), has facilitated the utilization of iPSC-derived motor neurons (iPSC-MNs) as highly valuable models for underpinning pathogenic mechanisms and developing novel therapeutic interventions for motor neuron diseases (MNDs). However, the challenge of MN adherence to the MEA plate and the heterogeneity presented in iPSC-derived cultures raise concerns about the reproducibility of the findings obtained from these cellular models. We discovered that one novel factor modulating the electrophysiological activity of iPSC-MNs is the extracellular matrix (ECM) used in the coating to support in vitro growth, differentiation and maturation of iPSC-MNs. The current study showed that two coating conditions, namely, Poly-L-ornithine/Matrigel (POM) and Polyethyleneimine (PEI) strongly promoted attachment of iPSC-MNs on MEA culture dishes compared to three other coating conditions, and both facilitated the maturation of iPSC-MNs as characterized by the detection of extensive electrophysiological activities from the MEA plates. POM coating accelerated the maturation of the iPSC-MNs for up to 5 weeks, which suits modeling of neurodevelopmental disorders. However, the application of PEI resulted in more even distribution of the MNs on the culture dish and reduced variability of electrophysiological signals from the iPSC-MNs in 7-week cultures, which permitted the detection of enhanced excitability in iPSC-MNs from patients with amyotrophic lateral sclerosis (ALS). This study provides a comprehensive comparison of five coating conditions and offers POM and PEI as favorable coatings for in vitro modeling of neurodevelopmental and neurodegenerative disorders, respectively.

Keywords:  Extracellular matrix; Matrigel; Multielectrode array; Poly-l-ornithine; Polyethyleneimine; iPSC-derived motor neurons

DOI:  https://doi.org/10.1038/s41598-024-77710-1
Exp Neurol. 2024 Oct 24. pii: S0014-4886(24)00350-9. [Epub ahead of print]383 115024

A variant of the Hspa8 synaptic chaperone modifies disease in a SOD1G86R mouse model of amyotrophic lateral sclerosis.

Taishi Takeda, Yoon-Ra Her, Jeong-Ki Kim, Narendra N Jha, Umrao R Monani.

  Amyotrophic lateral sclerosis (ALS) is a relatively common and invariably fatal, paralyzing motor neuron disease for which there are few treatment options. ALS is frequently associated with ubiquitin-positive motor neuronal aggregates, a pathology suggestive of perturbed proteostasis. Indeed, cellular chaperones, which are involved in protein trafficking and degradation often underlie familial ALS. Spinal muscular atrophy (SMA) is a second, common paralytic condition resulting from motor neuron loss and muscle atrophy. While SMA is now effectively treated, mechanisms underlying motor neuron degeneration in the disease remain far from clear. To address mechanistic questions about SMA, we recently identified a genetic modifier of the disease. The factor, a G470R variant in the constitutively expressed cellular chaperone, Hspa8, arrested motor neuron loss, prevented the abnormal accumulation of neurofilament aggregates at nerve terminals and suppressed disease. Hspa8 is best known for its role in autophagy. Amongst its many clients is the ALS-associated superoxide dismutase 1 (SOD1) protein. Given its suppression of the SMA phenotype, we tested potential disease-mitigating effects of Hspa8G470R in a mutant SOD1 mouse model of ALS. Unexpectedly, disease in mutant SOD1 mice expressing the G470R variant was aggravated. Motor performance of the mice deteriorated, muscle atrophy worsened, and lifespan shrunk even further. Paradoxically, SOD1 protein in spinal cord tissue of the mice was dramatically reduced. Our results suggest that Hspa8 modulates the ALS phenotype. However, rather than mitigating disease, the G470R variant exacerbates it.

Keywords:  Amyotrophic lateral sclerosis; Hspa8; Modifier; Mouse models; SOD1; Spinal muscular atrophy

DOI:  https://doi.org/10.1016/j.expneurol.2024.115024
JACS Au. 2024 Oct 28. 4(10): 3896-3909

Short Repeat Ribonucleic Acid Reduces Cytotoxicity by Preventing the Aggregation of TDP-43 and Its 25 KDa Carboxy-Terminal Fragment.

Ai Fujimoto, Masataka Kinjo, Akira Kitamura.

TAR DNA/RNA-binding protein 43 kDa (TDP-43) proteinopathy is a hallmark of neurodegenerative disorders, such as amyotrophic lateral sclerosis, in which cytoplasmic aggregates containing TDP-43 and its C-terminal fragments, such as TDP-25, are observed in degenerative neuronal cells. However, few reports have focused on small molecules that can reduce their aggregation and cytotoxicity. Here, we show that short RNA repeats of GGGGCC and AAAAUU are aggregation suppressors of TDP-43 and TDP-25. TDP-25 interacts with these RNAs, as well as TDP-43, despite the lack of major RNA-recognition motifs using fluorescence cross-correlation spectroscopy. Expression of these RNAs significantly decreases the number of cells harboring cytoplasmic aggregates of TDP-43 and TDP-25 and ameliorates cell death by TDP-25 and mislocalized TDP-43 without altering the cellular transcriptome of molecular chaperones. Consequently, short RNA repeats of GGGGCC and AAAAUU can maintain proteostasis by preventing the aggregation of TDP-43 and TDP-25.

DOI: https://doi.org/10.1021/jacsau.4c00566
bioRxiv. 2024 Oct 14. pii: 2024.10.14.618295. [Epub ahead of print]

Cell-Type Resolved Protein Atlas of Brain Lysosomes Identifies SLC45A1-Associated Disease as a Lysosomal Disorder.

Ali Ghoochani, Julia C Heiby, Eshaan S Rawat, Uche N Medoh, Domenico Di Fraia, Wentao Dong, Marc Gastou, Kwamina Nyame, Nouf N Laqtom, Natalia Gomez-Ospina, Alessandro Ori, Monther Abu-Remaileh.

Mutations in lysosomal genes cause neurodegeneration and neurological lysosomal storage disorders (LSDs). Despite their essential role in brain homeostasis, the cell-type-specific composition and function of lysosomes remain poorly understood. Here, we report a quantitative protein atlas of the lysosome from mouse neurons, astrocytes, oligodendrocytes, and microglia. We identify dozens of novel lysosomal proteins and reveal the diversity of the lysosomal composition across brain cell types. Notably, we discovered SLC45A1, mutations in which cause a monogenic neurological disease, as a neuron-specific lysosomal protein. Loss of SLC45A1 causes lysosomal dysfunction in vitro and in vivo. Mechanistically, SLC45A1 plays a dual role in lysosomal sugar transport and stabilization of V1 subunits of the V-ATPase. SLC45A1 deficiency depletes the V1 subunits, elevates lysosomal pH, and disrupts iron homeostasis causing mitochondrial dysfunction. Altogether, our work redefines SLC45A1-associated disease as a LSD and establishes a comprehensive map to study lysosome biology at cell-type resolution in the brain and its implications for neurodegeneration.

DOI: https://doi.org/10.1101/2024.10.14.618295
Physiol Rev. 2024 Oct 31.

Axon Initial Segment Structure and Function in Health and Disease.

Paul M Jenkins, Kevin J Bender.

  At the simplest level, neurons are structured to integrate synaptic input and perform computational transforms on that input, converting it into an action potential (AP) code. This process-converting synaptic input into AP output-typically occurs in a specialized region of axon termed the axon initial segment (AIS). The AIS, as its name implies, is often contained to the first section of axon abutted to the soma and is home to a dizzying array of ion channels, attendant scaffolding proteins, intracellular organelles, extracellular proteins, and, in some cases, synapses. The AIS serves multiple roles as the final arbiter for determining if inputs are sufficient to evoke APs, as a gatekeeper that physically separates the somatodendritic domain from the axon proper, and as a regulator of overall neuronal excitability, dynamically tuning its size to best suit the needs of parent neurons. These complex roles have received considerable attention from experimentalists and theoreticians alike. Here, we review recent advances in our understanding of the AIS and its role in neuronal integration and polarity in health and disease.

Keywords:  ankyrin; axon initial segment; excitability; ion channel; polarity

DOI:  https://doi.org/10.1152/physrev.00030.2024
Int J Mol Sci. 2024 Oct 10. pii: 10900. [Epub ahead of print]25(20):

A Review of Biomarkers of Amyotrophic Lateral Sclerosis: A Pathophysiologic Approach.

Rawiah S Alshehri, Ahmad R Abuzinadah, Moafaq S Alrawaili, Muteb K Alotaibi, Hadeel A Alsufyani, Rajaa M Alshanketi, Aysha A AlShareef.

  Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons. The heterogeneous nature of ALS at the clinical, genetic, and pathological levels makes it challenging to develop diagnostic and prognostic tools that fit all disease phenotypes. Limitations associated with the functional scales and the qualitative nature of mainstay electrophysiological testing prompt the investigation of more objective quantitative assessment. Biofluid biomarkers have the potential to fill that gap by providing evidence of a disease process potentially early in the disease, its progression, and its response to therapy. In contrast to other neurodegenerative diseases, no biomarker has yet been validated in clinical use for ALS. Several fluid biomarkers have been investigated in clinical studies in ALS. Biofluid biomarkers reflect the different pathophysiological processes, from protein aggregation to muscle denervation. This review takes a pathophysiologic approach to summarizing the findings of clinical studies utilizing quantitative biofluid biomarkers in ALS, discusses the utility and shortcomings of each biomarker, and highlights the superiority of neurofilaments as biomarkers of neurodegeneration over other candidate biomarkers.

Keywords:  CSF; amyotrophic lateral sclerosis; biomarker; blood; pathophysiology

DOI:  https://doi.org/10.3390/ijms252010900
BMJ Open. 2024 Oct 26. 14(10): e082142

Protocol for a phase 2 study of bosutinib for amyotrophic lateral sclerosis using real-world data: induced pluripotent stem cell-based drug repurposing for amyotrophic lateral sclerosis medicine (iDReAM) study.

Keiko Imamura, Yuishin Izumi, Naohiro Egawa, Takashi Ayaki, Makiko Nagai, Kazutoshi Nishiyama, Yasuhiro Watanabe, Takenobu Murakami, Ritsuko Hanajima, Hiroshi Kataoka, Takao Kiriyama, Hitoki Nanaura, Kazuma Sugie, Takehisa Hirayama, Osamu Kano, Masahiro Nakamori, Hirofumi Maruyama, Shotaro Haji, Koji Fujita, Naoki Atsuta, Harutsugu Tatebe, Takahiko Tokuda, Naoto Takahashi, Akiko Morinaga, Riko Tabuchi, Motoki Oe, Mihoko Kobayashi, Kasia Lobello, Satoshi Morita, Gen Sobue, Ryosuke Takahashi, Haruhisa Inoue.

  INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a progressive, severe neurodegenerative disease caused by motor neuron death. Development of a medicine for ALS is urgently needed, and induced pluripotent cell-based drug repurposing identified a Src/c-Abl inhibitor, bosutinib, as a candidate for molecular targeted therapy of ALS. A phase 1 study confirmed the safety and tolerability of bosutinib in a 12-week treatment of ALS patients. The objectives of this study are to evaluate the efficacy and longer-term safety of bosutinib in ALS patients.METHODS AND ANALYSIS: An open-label, multicentre phase 2 study was designed. The study consisted of a 12-week observation period, a 1-week transitional period, a 24-week study treatment period and a 4-week follow-up period. Following the transitional period, patients whose total Revised ALS Functional Rating Scale (ALSFRS-R) score declined by 1 to 4 points during the 12-week observation period were to receive bosutinib for 24 weeks. In this study, 25 ALS patients will be enrolled; patients will be randomly assigned to the following groups: 12 patients in the 200 mg quaque die (QD) group and 13 patients in the 300 mg QD group of bosutinib. The safety and exploratory efficacy of bosutinib in ALS patients for 24 weeks will be assessed. Efficacy using the ALSFRS-R score will be compared with the external published data from an edaravone study (MCI186-19) and registry data from a multicentre ALS cohort study, the Japanese Consortium for Amyotrophic Lateral Sclerosis Research.
ETHICS AND DISSEMINATION: This study was approved by the ethics committees of Kyoto University, Tokushima University, Kitasato University, Tottori University, Nara Medical University School of Medicine, Toho University and Hiroshima University. The findings will be disseminated in peer-reviewed journals and at scientific conferences.
TRIAL REGISTRATION NUMBER: jRCT2051220002; Pre-results, NCT04744532; Pre-results.

Keywords:  Clinical trials; NEUROLOGY; REGISTRIES

DOI:  https://doi.org/10.1136/bmjopen-2023-082142
Stem Cells. 2024 Oct 26. pii: sxae068. [Epub ahead of print]

A de novo Missense Mutation in PPP2R5D Alters Dopamine Pathways and Morphology of iPSC-derived Midbrain Neurons.

Jasmine L Carter, Julian A N M Halmai, Jennifer J Waldo, Paula A Vij, Maribel Anguiano, Isaac J Villegas, Yu Xin Du, Jan Nolta, Kyle D Fink.

  Induced pluripotent stem cell (iPSC) models of neurodevelopmental disorders (NDDs) have promoted an understanding of commonalities and differences within or across patient populations by revealing the underlying molecular and cellular mechanisms contributing to disease pathology. Here, we focus on developing a human model for PPP2R5D-related NDD, called Jordan syndrome, which has been linked to Early-Onset Parkinson's Disease (EOPD). Here we sought to understand the underlying molecular and cellular phenotypes across multiple cell states and neuronal subtypes in order to gain insight into Jordan syndrome pathology. Our work revealed that iPSC-derived midbrain neurons from Jordan syndrome patients display significant differences in dopamine-associated pathways and neuronal architecture. We then evaluated a CRISPR-based approach for editing heterozygous dominant G-to-A mutations at the transcript level in patient-derived neural stem cells. Our findings show site-directed RNA editing is influenced by sgRNA length and cell type. These studies support the potential for a CRISPR RNA editor system to selectively edit mutant transcripts harboring G-to-A mutations in neural stem cells while providing an alternative editing technology for those suffering from NDDs.

Keywords:  EOPD; Jordans Syndrome; NDD; RNA editing and CRISPR/Cas13; disease modeling; iPSC-derived neuron; midbrain neuron; stem cell

DOI:  https://doi.org/10.1093/stmcls/sxae068
PLoS Biol. 2024 Oct 31. 22(10): e3002876

Ortholog of autism candidate gene RBM27 regulates mitoribosomal assembly factor MALS-1 to protect against mitochondrial dysfunction and axon degeneration during neurodevelopment.

Tamjid A Chowdhury, David A Luy, Garrett Scapellato, Dorian Farache, Amy S Y Lee, Christopher C Quinn.

Mitochondrial dysfunction is thought to be a key component of neurodevelopmental disorders such as autism, intellectual disability, and attention-deficit hyperactivity disorder (ADHD). However, little is known about the molecular mechanisms that protect against mitochondrial dysfunction during neurodevelopment. Here, we address this question through the investigation of rbm-26, the Caenorhabditis elegans ortholog of the RBM27 autism candidate gene, which encodes an RNA-binding protein whose role in neurons is unknown. We report that RBM-26 (RBM26/27) protects against axonal defects by negatively regulating expression of the MALS-1 (MALSU1) mitoribosomal assembly factor. Autism-associated missense variants in RBM-26 cause a sharp decrease in RBM-26 protein expression along with defects in axon overlap and axon degeneration that occurs during larval development. Using a biochemical screen, we identified the mRNA for the MALS-1 mitoribosomal assembly factor as a binding partner for RBM-26. Loss of RBM-26 function causes a dramatic overexpression of mals-1 mRNA and MALS-1 protein. Moreover, genetic analysis indicates that this overexpression of MALS-1 is responsible for the mitochondrial and axon degeneration defects in rbm-26 mutants. These observations reveal a mechanism that regulates expression of a mitoribosomal assembly factor to protect against axon degeneration during neurodevelopment.

DOI: https://doi.org/10.1371/journal.pbio.3002876
bioRxiv. 2024 Oct 25. pii: 2024.10.25.620310. [Epub ahead of print]

F-BAR proteins CIP4 and FBP17 function in cortical neuron radial migration and process outgrowth.

Lauren A English, Russell J Taylor, Connor J Cameron, Emily A Broker, Erik W Dent.

Neurite initiation from newly born neurons is a critical step in neuronal differentiation and migration. Neuronal migration in the developing cortex is accompanied by dynamic extension and retraction of neurites as neurons progress through bipolar and multipolar states. However, there is a relative lack of understanding regarding how the dynamic extension and retraction of neurites is regulated during neuronal migration. In recent work we have shown that CIP4, a member of the F-BAR family of membrane bending proteins, inhibits cortical neurite formation in culture, while family member FBP17 induces premature neurite outgrowth. These results beg the question of how CIP4 and FBP17 function in radial neuron migration and differentiation in vivo , including the timing and manner of neurite extension and retraction. Indeed, the regulation of neurite outgrowth is essential for the transitions between bipolar and multipolar states during radial migration. To examine the effects of modulating expression of CIP4 and FBP17 in vivo , we used in utero electroporation, in combination with our published Double UP technique, to compare knockdown or overexpression cells with control cells within the same mouse tissue of either sex. We show that either knockdown or overexpression of CIP4 and FBP17 results in the marked disruption of radial neuron migration by modulating neuronal morphology and neurite outgrowth, consistent with our findings in culture. Our results demonstrate that the F-BAR proteins CIP4 and FBP17 are essential for proper radial migration in the developing cortex and thus play a key role in cortical development.SIGNIFICANCE STATEMENT: During embryonic development, radial migration of newly born cortical neurons is a complex process that underlies the proper formation of the neocortex, the outermost layers of neurons in the brain. Disruptions in radial migration results in profound effects on cognitive function and can lead to devastating developmental disabilities. To better understand this critical process in brain development we examined two members of the F-BAR family of membrane bending proteins, CIP4 and FBP17, which are present in the developing brain. We demonstrate that intracellular concentrations of these proteins must be tightly regulated. Increasing or decreasing levels of either protein has profound effects on neuronal morphology and proper radial migration, suggesting they are key players in cortical development.

DOI: https://doi.org/10.1101/2024.10.25.620310
Int J Mol Sci. 2024 Oct 13. pii: 11009. [Epub ahead of print]25(20):

Reporter Alleles in hiPSCs: Visual Cues on Development and Disease.

Gustavo Caldeira Cotta, Rachel Castro Teixeira Dos Santos, Guilherme Mattos Jardim Costa, Samyra Maria Dos Santos Nassif Lacerda.

  Reporter alleles are essential for advancing research with human induced pluripotent stem cells (hiPSCs), notably in developmental biology and disease modeling. This study investigates the state-of-the-art gene-editing techniques tailored for generating reporter alleles in hiPSCs, emphasizing their effectiveness in investigating cellular dynamics and disease mechanisms. Various methodologies, including the application of CRISPR/Cas9 technology, are discussed for accurately integrating reporter genes into the specific genomic loci. The synthesis of findings from the studies utilizing these reporter alleles reveals insights into developmental processes, genetic disorder modeling, and therapeutic screening, consolidating the existing knowledge. These hiPSC-derived models demonstrate remarkable versatility in replicating human diseases and evaluating drug efficacy, thereby accelerating translational research. Furthermore, this review addresses challenges and future directions in refining the reporter allele design and application to bolster their reliability and relevance in biomedical research. Overall, this investigation offers a comprehensive perspective on the methodologies, applications, and implications of reporter alleles in hiPSC-based studies, underscoring their essential role in advancing both fundamental scientific understanding and clinical practice.

Keywords:  developmental biology; disease modeling; human iPSCs; reporter alleles

DOI:  https://doi.org/10.3390/ijms252011009
Ann Clin Transl Neurol. 2024 Oct 29.

Rethinking antisense oligonucleotide therapeutics for amyotrophic lateral sclerosis.

Daisuke Ito, Kensuke Okada.

Antisense oligonucleotides, which are used to silence target genes, are gaining attention as a novel drug discovery modality for proteinopathies. However, while clinical trials for neurodegenerative diseases like amyotrophic lateral sclerosis have been conducted in recent years, the results have not always been favorable. The results from a Phase III trial of the antisense oligonucleotide, that is, tofersen, which targets SOD1 mRNA, showed decreased levels of cerebrospinal fluid SOD1 and plasma neurofilament light chain but no improvements in primary clinical endpoint. Moreover, case reports pertaining to patients with amyotrophic lateral sclerosis carrying FUS and C9orf72 mutations who received antisense oligonucleotide-based treatments have demonstrated a notable reduction in the targeted protein (thus providing the proof of mechanism) but with no discernible clinical benefits. There are several possible reasons why antisense oligonucleotides knockdown fails to achieve proof of concept, which need to be addressed: on-target adverse effects resulting from the loss of function of target gene and irreversible neuronal death cascade due to toxic protein accumulation, among other factors. This review provides an overview of the current status and discusses the prospects of antisense oligonucleotides treatment for amyotrophic lateral sclerosis.

DOI: https://doi.org/10.1002/acn3.52234
Pharmaceuticals (Basel). 2024 Sep 27. pii: 1286. [Epub ahead of print]17(10):

Discovery of Novel Inhibitors against ALS-Related SOD1(A4V) Aggregation through the Screening of a Chemical Library Using Differential Scanning Fluorimetry (DSF).

Maria Giannakou, Ifigeneia Akrani, Angeliki Tsoka, Vassilios Myrianthopoulos, Emmanuel Mikros, Constantinos Vorgias, Dimitris G Hatzinikolaou.

  BACKGROUND: Cu/Zn Superoxide Dismutase 1 (SOD1) is a 32 kDa cytosolic dimeric metalloenzyme that neutralizes superoxide anions into oxygen and hydrogen peroxide. Mutations in SOD1 are associated with ALS, a disease causing motor neuron atrophy and subsequent mortality. These mutations exert their harmful effects through a gain of function mechanism, rather than a loss of function. Despite extensive research, the mechanism causing selective motor neuron death still remains unclear. A defining feature of ALS pathogenesis is protein misfolding and aggregation, evidenced by ubiquitinated protein inclusions containing SOD1 in affected motor neurons. This work aims to identify compounds countering SOD1(A4V) misfolding and aggregation, which could potentially aid in ALS treatment.METHODS: The approach employed was in vitro screening of a library comprising 1280 pharmacologically active compounds (LOPAC®) in the context of drug repurposing. Using differential scanning fluorimetry (DSF), these compounds were tested for their impact on SOD1(A4V) thermal stability.
RESULTS AND CONCLUSIONS: Dimer stability was the parameter chosen as the criterion for screening, since the dissociation of the native SOD1 dimer is the step prior to its in vitro aggregation. The screening revealed one compound raising protein-ligand Tm by 6 °C, eleven inducing a higher second Tm, suggesting a stabilization effect, and fourteen reducing Tm from 10 up to 26 °C, suggesting possible interactions or non-specific binding.

Keywords:  ALS; DSF technique; SOD1; chemical library scanning; drug discovery; drug repurposing; protein-misfolding diseases

DOI:  https://doi.org/10.3390/ph17101286
Nat Commun. 2024 Oct 25. 15(1): 9238

Accumulation of F-actin drives brain aging and limits healthspan in Drosophila.

Edward T Schmid, Joseph M Schinaman, Naomi Liu-Abramowicz, Kylie S Williams, David W Walker.

The actin cytoskeleton is a key determinant of cell structure and homeostasis. However, possible tissue-specific changes to actin dynamics during aging, notably brain aging, are not understood. Here, we show that there is an age-related increase in filamentous actin (F-actin) in Drosophila brains, which is counteracted by prolongevity interventions. Critically, decreasing F-actin levels in aging neurons prevents age-onset cognitive decline and extends organismal healthspan. Mechanistically, we show that autophagy, a recycling process required for neuronal homeostasis, is disabled upon actin dysregulation in the aged brain. Remarkably, disrupting actin polymerization in aged animals with cytoskeletal drugs restores brain autophagy to youthful levels and reverses cellular hallmarks of brain aging. Finally, reducing F-actin levels in aging neurons slows brain aging and promotes healthspan in an autophagy-dependent manner. Our data identify excess actin polymerization as a hallmark of brain aging, which can be targeted to reverse brain aging phenotypes and prolong healthspan.

DOI: https://doi.org/10.1038/s41467-024-53389-w
Elife. 2024 Oct 30. pii: RP96592. [Epub ahead of print]13

Rab11 suppresses neuronal stress signaling by localizing dual leucine zipper kinase to axon terminals for protein turnover.

Seung Mi Kim, Yaw Quagraine, Monika Singh, Jung Hwan Kim.

  Dual leucine zipper kinase (DLK) mediates multiple neuronal stress responses, and its expression levels are constantly suppressed to prevent excessive stress signaling. We found that Wallenda (Wnd), the Drosophila ortholog of DLK, is highly enriched in the axon terminals of Drosophila sensory neurons in vivo and that this subcellular localization is necessary for Highwire-mediated Wnd protein turnover under normal conditions. Our structure-function analysis found that Wnd palmitoylation is essential for its axon terminal localization. Palmitoylation-defective Wnd accumulated in neuronal cell bodies, exhibited dramatically increased protein expression levels, and triggered excessive neuronal stress responses. Defective intracellular transport is implicated in neurodegenerative conditions. Comprehensive dominant-negative Rab protein screening identified Rab11 as an essential factor for Wnd localization in axon terminals. Consequently, Rab11 loss-of-function increased the protein levels of Wnd and induced neuronal stress responses. Inhibiting Wnd activity significantly ameliorated neuronal loss and c-Jun N-terminal kinase signaling triggered by Rab11 loss-of-function. Taken together, these suggest that DLK proteins are constantly transported to axon terminals for protein turnover and a failure of such transport can lead to neuronal loss. Our study demonstrates how subcellular protein localization is coupled to protein turnover for neuronal stress signaling.

Keywords:  D. melanogaster; DLK; Highwire; Rab11; axon; cell biology; neuronal stress; neuroscience; protein turnover

DOI:  https://doi.org/10.7554/eLife.96592
Sci Rep. 2024 10 29. 14(1): 25979

Astragaloside IV reduces mutant Ataxin-3 levels and supports mitochondrial function in Spinocerebellar Ataxia Type 3.

Yongshiou Lin, Wenling Cheng, Juichih Chang, Yuling Wu, Mingli Hsieh, Chinsan Liu.

  This study investigated the therapeutic effects of astragaloside IV (AST) on spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), a neurodegenerative disorder. Human neuroblastoma SK-N-SH cells expressing mutant ataxin-3 protein with 78 CAG repeats (MJD78) were employed as an in vitro model. Protein expression analysis demonstrated that AST treatment reduced mutant ataxin-3 protein expression and aggregation by enhancing the autophagic process in MJD78 cells. Elevated oxidative stress levels in MJD78 cells were significantly reduced following AST treatment, which also enhanced antioxidant capacity, as evidenced by flow cytometry and antioxidant enzyme activity assays. Furthermore, AST treatment ameliorated mitochondrial dysfunction in MJD78 cells, including improvements in mitochondrial membrane potential, respiration, and mitochondrial dynamics. In conclusion, AST administration increased antioxidant capacity, reduced both cellular and mitochondrial oxidative stress, and improved mitochondrial quality control processes through fusion, fission, and autophagy. These mechanisms collectively reduced intracellular mutant ataxin-3 protein aggregation, thereby achieving therapeutic efficacy in the SCA3 model.

Keywords:  Astragaloside IV; Autophagy; Mitochondrial dysfunction; Oxidative stress; Spinocerebellar ataxia type 3

DOI:  https://doi.org/10.1038/s41598-024-77763-2
RSC Adv. 2024 Oct 29. 14(47): 34637-34642

Design of peptide therapeutics as protein-protein interaction inhibitors to treat neurodegenerative diseases.

Daryl Ariawan, Kanishka P M Thananthirige, Ali El-Omar, Julia van der Hoven, Sian Genoud, Holly Stefen, Thomas Fath, Janet van Eersel, Lars M Ittner, Ole Tietz.

Peptide therapeutics are an emerging class of drugs to treat neurodegenerative diseases by inhibiting protein-protein interactions (PPIs). Nerinetide has recently emerged as a promising therapeutic for the treatment of ischemic stroke and Alzheimer's Disease (AD). The design of this potent neuroprotective agent includes a cell penetrating peptide sequence that achieves delivery into neurons and a protein-protein inhibitory sequence that achieves inhibition of protein complex formation through mimicry. In this study, we deconstruct the nerinetide sequence and study the relationship between plasma stability, intraneuronal delivery and drug efficacy to provide design guidelines for the development of next generation, peptidic PPI inhibitors to treat neurodegenerative diseases.

DOI: https://doi.org/10.1039/d4ra05040a
Cell Mol Life Sci. 2024 Nov 01. 81(1): 444

Transcriptomic alterations in APP/PS1 mice astrocytes lead to early postnatal axon initial segment structural changes.

María José Benitez, Diana Retana, Lara Ordoñez-Gutiérrez, Inés Colmena, María José Goméz, Rebeca Álvarez, María Ciorraga, Ana Dopazo, Francisco Wandosell, Juan José Garrido.

  Alzheimer´s disease (AD) is characterized by neuronal function loss and degeneration. The integrity of the axon initial segment (AIS) is essential to maintain neuronal function and output. AIS alterations are detected in human post-mortem AD brains and mice models, as well as, neurodevelopmental and mental disorders. However, the mechanisms leading to AIS deregulation in AD and the extrinsic glial origin are elusive. We studied early postnatal differences in AIS cellular/molecular mechanisms in wild-type or APP/PS1 mice and combined neuron-astrocyte co-cultures. We observed AIS integrity alterations, reduced ankyrinG expression and shortening, in APP/PS1 mice from P21 and loss of AIS integrity at 21 DIV in wild-type and APP/PS1 neurons in the presence of APP/PS1 astrocytes. AnkyrinG decrease is due to mRNAs and protein reduction of retinoic acid synthesis enzymes Rdh1 and Aldh1b1, as well as ADNP (Activity-dependent neuroprotective protein) in APP/PS1 astrocytes. This effect was mimicked by wild-type astrocytes expressing ADNP shRNA. In the presence of APP/PS1 astrocytes, wild-type neurons AIS is recovered by inhibition of retinoic acid degradation, and Adnp-derived NAP peptide (NAPVSIPQ) addition or P2X7 receptor inhibition, both regulated by retinoic acid levels. Moreover, P2X7 inhibitor treatment for 2 months impaired AIS disruption in APP/PS1 mice. Our findings extend current knowledge on AIS regulation, providing data to support the role of astrocytes in early postnatal AIS modulation. In conclusion, AD onset may be related to very early glial cell alterations that induce AIS and neuronal function changes, opening new therapeutic approaches to detect and avoid neuronal function loss.

Keywords:  ADNP; AnkyrinG; Astrocytes; Axon initial segment; Neurodegeneration; P2X7; Retinoic acid

DOI:  https://doi.org/10.1007/s00018-024-05485-9
Brain Behav. 2024 Oct;14(10): e70100

CNN-Based Neurodegenerative Disease Classification Using QR-Represented Gait Data.

Çağatay Berke Erdaş, Emre Sümer.

PURPOSE: The primary aim of this study is to develop an effective and reliable diagnostic system for neurodegenerative diseases by utilizing gait data transformed into QR codes and classified using convolutional neural networks (CNNs). The objective of this method is to enhance the precision of diagnosing neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), Parkinson's disease (PD), and Huntington's disease (HD), through the introduction of a novel approach to analyze gait patterns.METHODS: The research evaluates the CNN-based classification approach using QR-represented gait data to address the diagnostic challenges associated with neurodegenerative diseases. The gait data of subjects were converted into QR codes, which were then classified using a CNN deep learning model. The dataset includes recordings from patients with Parkinson's disease (n = 15), Huntington's disease (n = 20), and amyotrophic lateral sclerosis (n = 13), and from 16 healthy controls.
RESULTS: The accuracy rates obtained through 10-fold cross-validation were as follows: 94.86% for NDD versus control, 95.81% for PD versus control, 93.56% for HD versus control, 97.65% for ALS versus control, and 84.65% for PD versus HD versus ALS versus control. These results demonstrate the potential of the proposed system in distinguishing between different neurodegenerative diseases and control groups.
CONCLUSION: The results indicate that the designed system may serve as a complementary tool for the diagnosis of neurodegenerative diseases, particularly in individuals who already present with varying degrees of motor impairment. Further validation and research are needed to establish its wider applicability.

DOI: https://doi.org/10.1002/brb3.70100
Front Neurosci. 2024 ;18 1436312

Netrin-1 stimulated axon growth requires the polyglutamylase TTLL1.

Kyle R Northington, Jasmynn Calderon, Emily A Bates.

  Introduction: In the developing brain, neurons extend an axonal process through a complex and changing environment to form synaptic connections with the correct targets in response to extracellular cues. Microtubule and actin filaments provide mechanical support and drive axon growth in the correct direction. The axonal cytoskeleton responds to extracellular guidance cues. Netrin-1 is a multifunctional guidance cue that can induce alternate responses based on the bound receptor. The mechanism by which actin responds to Netrin-1 is well described. However, how Netrin-1 influences the microtubule cytoskeleton is less understood. Appropriate microtubule function is required for axon pathfinding, as mutations in tubulin phenocopy axon crossing defects of Netrin-1 and DCC mutants. Microtubule stabilization is required for attractive guidance cue response. The C-terminal tails of microtubules can be post-translationally modified. Post-translational modifications (PTMs) help control the microtubule cytoskeleton.Methods: We measured polyglutamylation in cultured primary mouse cortical neurons before and after Netrin-1 stimulation. We used immunohistochemistry to measure how Netrin-1 stimulation alters microtubule-associated protein localization. Next, we manipulated TTLL1 to determine if Netrin-1-induced axon growth and MAP localization depend on polyglutamylation levels.
Results: In this study, we investigated if Netrin-1 signaling alters microtubule PTMs in the axon. We found that microtubule polyglutamylation increases after Netrin-1 stimulation. This change in polyglutamylation is necessary for Netrin-1-induced axonal growth rate increases. We next determined that MAP1B and DCX localization changes in response to Netrin-1. These proteins can both stabilize the microtubule cytoskeleton and may be responsible for Netrin-1-induced growth response in neurons. The changes in DCX and MAP1B depend on TTLL1, a protein responsible for microtubule polyglutamylation.

Keywords:  DCX = doublecortin; Netrin-1; TTLL1; axon growth and guidance; microtubule polyglutamylation; microtubule-associated protein 1B; tubulin (microtubules)

DOI:  https://doi.org/10.3389/fnins.2024.1436312
Sheng Li Xue Bao. 2024 Oct 25. 76(5): 783-790

[Research progress on the mechanism of autophagy flow injury caused by lysosomal dysfunction after cerebral ischemia].

Jia-Qian Wang, Hong-Yun He, Yi-Hao Deng.

Ischemic stroke is an acute cerebrovascular disease caused by cerebral vascular obstruction, which is the third leading cause of human death and disability. Multiple studies have demonstrated that autophagy plays a positive role in neurons after ischemic stroke. Autophagy is the main intracellular mechanism that mediates the degradation and recycling of various substrates in lysosomes, so it is very important to maintain normal function of lysosomes. However, cerebral ischemia can result in significant impairment of lysosomal function, subsequently leading to disruption in autophagy flow and exacerbation of neuronal injury. This review elucidates the mechanism of autophagic flux injury resulting from lysosomal dysfunction induced by impaired fusion between autophagosomes and lysosomes, alterations in the acidic environment within lysosomes, and diminished biosynthesis of lysosomes following ischemic stroke. The lysosome is regarded as the primary focal point for investigating the mechanism of autophagic flux injury, with the aim of modulating neuronal autophagic flux to improve cerebral ischemia-induced brain injury. This approach holds potential for exerting a neuroprotective effect and providing a novel avenue for stroke treatment.
Nat Nanotechnol. 2024 Oct 28.

Targeted protein degradation via cellular trafficking of nanoparticles.

Yang Liu, Runhan Liu, Jiawei Dong, Xue Xia, Haoying Yang, Sijun Wei, Linlin Fan, Mengke Fang, Yan Zou, Meng Zheng, Kam W Leong, Bingyang Shi.

Strategies that selectively bind proteins of interest and target them to the intracellular protein recycling machinery for targeted protein degradation have recently emerged as powerful tools for undruggable targets in biomedical research and the pharmaceutical industry. However, targeting any new protein of interest with current degradation tools requires a laborious case-by-case design for different diseases and cell types, especially for extracellular targets. Here we observe that nanoparticles can mediate specific receptor-independent internalization of a bound protein and further develop a general strategy for degradation of extracellular proteins of interest by making full use of clinically approved components. This extremely flexible strategy aids in targeted protein degradation tool development and provides knowledge for targeted drug therapies and nanomedicine design.

DOI: https://doi.org/10.1038/s41565-024-01801-3
Biomolecules. 2024 Oct 18. pii: 1324. [Epub ahead of print]14(10):

Targeting Protein Aggregation in ALS.

Michele Perni, Benedetta Mannini.

  Proteinopathies involve the abnormal accumulation of specific proteins. Maintaining the balance of the proteome is a finely regulated process managed by a complex network of cellular machinery responsible for protein synthesis, folding, and degradation. However, stress and ageing can disrupt this balance, leading to widespread protein aggregation. Currently, several therapies targeting protein aggregation are in clinical trials for ALS. These approaches mainly focus on two strategies: addressing proteins that are prone to aggregation due to mutations and targeting the cellular mechanisms that maintain protein homeostasis to prevent aggregation. This review will cover these emerging drugs. Advances in ALS research not only offer hope for better outcomes for ALS patients but also provide valuable insights and methodologies that can benefit the broader field of neurodegenerative disease drug discovery.

Keywords:  amyotrophic lateral sclerosis; clinical trials; drug discovery; inflammation; neurodegeneration; protein aggregation; protein homeostasis; therapeutics

DOI:  https://doi.org/10.3390/biom14101324
Ann Med. 2024 Dec;56(1): 2422572

Overview of nomenclature and diagnosis of amyotrophic lateral sclerosis.

Renshi Xu.

  The nomenclature of amyotrophic lateral sclerosis (ALS) currently is blurred, indistinct and no accurate and haven't been properly updated since the first description, which is far from being suitable for the current implementation of clinical practise and scientific research of ALS, and urgently need an solution. Furthermore, the current diagnostic criteria need also further been improved, because the current clinical diagnosis of ALS majorly depends on the clinical manifestations yet. Up to now, no any objective clinical auxiliary examination can be helpful to diagnose ALS besides the electromyogram identifying the lower motor neuron damage, which isn't conducive to early diagnosis and prolongs the time of ALS confirmed diagnosis. In this mini review, we discussed the current doubt about the nomenclature and diagnostic criteria of ALS, and prospected in order to further improve and normalize the nomenclature and diagnosis of ALS.

Keywords:  Amyotrophic lateral sclerosis; diagnosis; doubt; insight; motor neuron disease; nomenclature

DOI:  https://doi.org/10.1080/07853890.2024.2422572
bioRxiv. 2024 Oct 22. pii: 2024.10.22.619706. [Epub ahead of print]

Mitochondrial fission controls astrocyte morphogenesis and organization in the cortex.

Maria Pia Rodriguez Salazar, Sprihaa Kolanukuduru, Valentina Ramirez, Boyu Lyu, Gabrielle Sejourne, Hiromi Sesaki, Guoqiang Yu, Cagla Eroglu.

Dysfunctional mitochondrial dynamics are a hallmark of devastating neurodevelopmental disorders such as childhood refractory epilepsy. However, the role of glial mitochondria in proper brain development is not well understood. We show that astrocyte mitochondria undergo extensive fission while populating astrocyte distal branches during postnatal cortical development. Loss of mitochondrial fission regulator, Dynamin-related protein 1 (Drp1), decreases mitochondrial localization to distal astrocyte processes, and this mitochondrial mislocalization reduces astrocyte morphological complexity. Functionally, astrocyte-specific conditional deletion of Drp1 induces astrocyte reactivity and disrupts astrocyte organization in the cortex. These morphological and organizational deficits are accompanied by loss of astrocytic gap junction protein Connexin 43. These findings uncover a crucial role for mitochondrial fission in coordinating astrocytic morphogenesis and organization, revealing the regulation of astrocytic mitochondria dynamics as a critical step in neurodevelopment.Summary: During cortical astrocyte morphogenesis, mitochondria decrease in size to populate distal astrocyte processes. Drp1-mediated mitochondrial fission is necessary for peripheral astrocyte process formation. Astrocyte-specific Drp1 loss induces astrocyte reactivity, disrupts cortical astrocyte organization, and dysregulates gap-junction protein Connexin 43 abundance.

DOI: https://doi.org/10.1101/2024.10.22.619706
Proc Natl Acad Sci U S A. 2024 Nov 05. 121(45): e2408949121

Measurement of α-synuclein as protein cargo in plasma extracellular vesicles.

Tal Gilboa, Dmitry Ter-Ovanesyan, Shih-Chin Wang, Sara Whiteman, George T Kannarkat, George M Church, Alice S Chen-Plotkin, David R Walt.

  Extracellular vesicles (EVs) are released by all cells and hold great promise as a class of biomarkers. This promise has led to increased interest in measuring EV proteins from both total EVs as well as brain-derived EVs in plasma. However, measuring cargo proteins in EVs has been challenging because EVs are present at low levels, and EV isolation methods are imperfect at separating EVs from free proteins. Thus, knowing whether a protein measured after EV isolation is truly inside EVs is difficult. In this study, we developed methods to measure whether a protein is inside EVs and quantify the ratio of a protein in EVs relative to total plasma. To achieve this, we combined a high-yield size-exclusion chromatography protocol with an optimized protease protection assay and Single Molecule Array (Simoa) digital enzyme-linked immunoassays (ELISAs) for ultrasensitive measurement of proteins inside EVs. We applied these methods to analyze α-synuclein and confirmed that a small fraction of the total plasma α-synuclein is inside EVs. Additionally, we developed a highly sensitive Simoa assay for phosphorylated α-synuclein (phosphorylated at the Ser129 residue). We found enrichment in the phosphorylated α-synuclein to total α-synuclein ratio inside EVs relative to outside EVs. Finally, we applied the methods we developed to measure total and phosphorylated α-synuclein inside EVs from Parkinson's disease and Lewy body dementia patient samples. This work provides a framework for determining the levels of proteins in EVs and represents an important step in the development of EV diagnostics for diseases of the brain, as well as other organs.

Keywords:  Parkinson’s Disease; alpha synuclein; biomarker; exosomes; extracellular vesicles

DOI:  https://doi.org/10.1073/pnas.2408949121
J Vis Exp. 2024 Oct 11.

Split-Luciferase Reassembly Assay to Measure Endoplasmic Reticulum-Mitochondria Contacts in Live Cells.

Connie Chen, Katherine A Rafael, Ginam Cho, Youngshin Lim.

Endoplasmic reticulum (ER)-mitochondria contact sites play a critical role in cell health and homeostasis, such as the regulation of Ca2+ and lipid homeostasis, mitochondrial dynamics, autophagosome and mitophagosome biogenesis, and apoptosis. Failure to maintain normal ER-mitochondrial coupling is implicated in many neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and hereditary spastic paraplegia. It is of considerable significance to explore how the dysregulation of ER-mitochondrial contacts could lead to cell death and whether repairing these contacts to the normal level could ameliorate neurodegenerative conditions. Thus, improved assays that measure the level of these contacts could help to illuminate the pathogenic mechanisms of these diseases. Ultimately, establishing simple and reliable assays will facilitate the development of new therapeutic strategies. Here we describe a split-luciferase assay to quantitatively measure the level of ER-mitochondria contacts in live cells. This assay can be used to study the pathophysiological role of these contacts as well as to identify their modulators in high-throughput screening.

DOI: https://doi.org/10.3791/66862
Neurosci Res. 2024 Oct 29. pii: S0168-0102(24)00133-0. [Epub ahead of print]

Neurexin1 level in Huntington's Disease and decreased Neurexin1 in disease progression.

Kyoungjoo Cho, Gyung Whan Kim.

  Huntington's disease (HD) is a neurodegenerative disorder characterized by the presence of abnormally expanded polyglutamine tracts in huntingtin protein (HTT). Mutant HTT disrupts synaptic transmission and plasticity, particularly in the striatum and cortex, leading to early dysfunctions, such as altered neurotransmitter release, impaired synaptic vesicle recycling, and disrupted postsynaptic receptor function. Synaptic loss precedes neuronal degeneration and contributes to disease progression. Neurexin1 (NRXN1), a synaptic cell adhesion molecule primarily located in the presynaptic membrane, plays a crucial role in maintaining synaptic integrity. The present study investigated the role of NRXN1 in HD. This study researched whether the changed level has been related to expanded polyQ stretch and disease progression. Here, we report a reduction in NRXN1 levels in post-symptomatic HD mice and in neuronal cells expressing abnormally expanded polyQ tracts. Mutant HTT was found to decrease NRXN1 levels while increasing LAMP2A levels, which promotes lysosomal degradation of NRXN1. In HD cells expressing Q111, downregulated LAMP2A restored NRXN1 levels and maintained cell proliferation compared with cells expressing Q7. These findings suggest that NRXN1 is regulated by LAMP2A-mediated way and that decreased NRXN1 levels are associated with symptomatic progression and neuronal cell loss in HD.

Keywords:  Huntington’s disease; LAMP2; Neurexin1

DOI:  https://doi.org/10.1016/j.neures.2024.10.006
J Neural Transm (Vienna). 2024 Oct 29.

Mild cognitive impairment in amyotrophic lateral sclerosis: current view.

Kurt A Jellinger.

  Amyotrophic lateral sclerosis (ALS) is a fatal multi-system neurodegenerative disorder with no effective treatment or cure. Although primarily characterized by motor degeneration, cognitive dysfunction is an important non-motor symptom that has a negative impact on patient and caregiver burden. Mild cognitive deficits are present in a subgroup of non-demented patients with ALS, often preceding motor symptoms. Detailed neuropsychological assessments reveal deficits in a variety of cognitive domains, including those of verbal fluency and retrieval, language, executive function, attention and verbal memory. Mild cognitive impairment (MCI), a risk factor for developing dementia, affects between 10% and over 50% of ALS patients. Neuroimaging revealed atrophy of frontal and temporal cortices, disordered white matter Integrity, volume reduction in amygdala and thalamus, hypometabolism in the frontal and superior temporal gyrus and anterior insula. Neuronal loss in non-motor brain areas, associated with TDP-43 deposition, one of the morphological hallmarks of ALS, is linked to functional disruption of frontostriatal and frontotemporo-limbic connectivities as markers for cognitive deficits in ALS, the pathogenesis of which is still poorly understood. Early diagnosis by increased cerebrospinal fluid or serum levels of neurofilament light/heavy chain or glial fibrillary acidic protein awaits confirmation for MCI in ALS. These fluid biomarkers and early detection of brain connectivity signatures before structural changes will be helpful not only in establishing early premature diagnosis but also in clarifying the pathophysiological mechanisms of MCI in ALS, which might serve as novel targets for prohibition/delay and future adequate treatment of this debilitating disorder.

Keywords:  ALS; Brain connectivities; Fluid biomarkers; Mild cognitive impairment; Multi-modal neuroimaging; Treatment modalities

DOI:  https://doi.org/10.1007/s00702-024-02850-7
Methods Enzymol. 2024 ;pii: S0076-6879(24)00371-9. [Epub ahead of print]706 501-518

In organello silencing of mitochondrial gene expression.

Mats Koschel, Luis Daniel Cruz-Zaragoza.

  Mitochondria contain proteins from two genetic origins. Most mitochondrial proteins are encoded in the nuclear genome, translated in the cytosol, and subsequently imported into the different mitochondrial sub-compartments. A small number is encoded in the mitochondrial DNA (mtDNA). The manipulation of the mtDNA gene expression represents a challenge. Here, we present an in vitro approach using morpholinos chemically linked to a precursor protein to silence gene expression in purified human mitochondria. The protocol is demonstrated with a Jac1-morpholino chimera specifically targeting COX1 mRNA. The chimera import and mitochondrial translation requirements are described in a step-by-step procedure, where the dose-dependent effect of reducing COX1 translation is observed. The affinity and specificity of chimera-mRNA binding also show great applicability to purify transcript-associated proteins by using the imported chimera construct as bait for immunoprecipitation. This new strategy opens up the possibility to address mechanistic questions about gene expression and physiology in mitochondria.

Keywords:  Gene expression; In vitro; Mitochondria; Morpholino; Silencing

DOI:  https://doi.org/10.1016/bs.mie.2024.07.035
J Neurophysiol. 2024 Oct 30.

Recombinant human plasma gelsolin suppresses persistent neuroinflammation and restores hippocampal neurogenesis in murine model of decompression sickness.

Abid R Bhat, Awadhesh K Arya, Veena M Bhopale, Zuha Imtiyaz, Stephen R Thom.

  Loss of plasma gelsolin (pGSN), a protein that lyses actin filaments, is implicated in the pathology of inflammatory and neurodegenerative diseases. We hypothesized that because pGSN is depleted in a murine model of decompression sickness (DCS), supplementation by administration of human recombinant (rhu-) pGSN would ameliorate inflammatory events. We observed that pGSN levels were persistently decreased in mice for at least 12 days post-exposure to 790 kPa of air for 2 hours. This decline was associated with elevated levels of inflammatory microparticles (MPs) in the blood and cervical lymph nodes, which previously were shown to cause neuroinflammation. Additionally, these mice exhibited reduced expression of synaptic proteins, impaired neurogenesis as well as impaired cognitive and motor functions. Rhu-pGSN ameliorated the inflammatory changes and resulted in restored synaptic protein expression, neurogenesis and neurological function. These findings demonstrate that neuronal dysfunction in our murine model of DCS is mediated by MPs and that rhu-pGSN can ameliorate injury even when administered in a delayed fashion.

Keywords:  Decompression stress; microparticles; neurogenesis; neuroinflammation; plasma gelsolin

DOI:  https://doi.org/10.1152/jn.00332.2024
Clin Sci (Lond). 2024 Nov 06. 138(21): 1377-1394

New evidence of vascular defects in neurodegenerative diseases revealed by single cell RNA sequencing.

Jiaying Qiu, Siwan Peng, Ruobing Qu, Liucheng Wu, Lingyan Xing, Luzhong Zhang, Junjie Sun.

  Neurodegenerative diseases (NDs) involve the progressive loss of neuronal structure or function in the brain and spinal cord. Despite their diverse etiologies, NDs manifest similar pathologies. Emerging research identifies vascular defects as a previously neglected hallmark of NDs. The development and popularization of single-cell RNA sequencing (scRNA-seq) technologies have significantly advanced our understanding of brain vascular cell types and their molecular characteristics, including gene expression changes at the single-cell level in NDs. These unprecedented insights deepen our understanding of the pathogenic mechanisms underlying NDs. However, the occurrence and role of vascular defects in disease progression remain largely unexplored. In this paper, we systematically summarize recent advances in the structure and organization of the central nervous system vasculature in mice, healthy individuals, and patients with NDs, focussing primarily on disease-specific alterations in vascular cell types or subtypes. Combining scRNA-seq with pathology evidence, we propose that vascular defects, characterized by disruptions in cell types and structural integrity, may serve as common early features of NDs. Finally, we discuss several pathways through which vascular defects in NDs lead to neuronal degeneration. A deeper understanding of the causes and contributions of vascular defects to NDs aids in elucidating the pathogenic mechanisms and developing meaningful therapeutic interventions.

Keywords:  Alzheimer's disease; Neurodegenerative disease; ScRNA-seq; Spinal muscular atrophy; Vascular defects; blood brain barrier

DOI:  https://doi.org/10.1042/CS20241658
Commun Biol. 2024 Oct 25. 7(1): 1393

miR-124 coordinates metabolic regulators acting at early stages of human neurogenesis.

Geurim Son, Yongwoo Na, Yongsung Kim, Ji-Hoon Son, Gregory D Clemenson, Simon T Schafer, Jong-Yeon Yoo, Sarah L Parylak, Apua Paquola, Hyunsu Do, Dayeon Kim, Insook Ahn, Mingyu Ju, Chanhee S Kang, Younghee Ju, Eunji Jung, Aidan H McDonald, Youngjin Park, Gilhyun Kim, Se-Bum Paik, Junho Hur, Joon Kim, Yong-Mahn Han, Seung-Hee Lee, Fred H Gage, Jong-Seo Kim, Jinju Han.

Metabolic dysregulation of neurons is associated with diverse human brain disorders. Metabolic reprogramming occurs during neuronal differentiation, but it is not fully understood which molecules regulate metabolic changes at the early stages of neurogenesis. In this study, we report that miR-124 is a driver of metabolic change at the initiating stage of human neurogenesis. Proteome analysis has shown the oxidative phosphorylation pathway to be the most significantly altered among the differentially expressed proteins (DEPs) in the immature neurons after the knockdown of miR-124. In agreement with these proteomics results, miR-124-depleted neurons display mitochondrial dysfunctions, such as decreased mitochondrial membrane potential and cellular respiration. Moreover, morphological analyses of mitochondria in early differentiated neurons after miR-124 knockdown result in smaller and less mature shapes. Lastly, we show the potential of identified DEPs as novel metabolic regulators in early neuronal development by validating the effects of GSTK1 on cellular respiration. GSTK1, which is upregulated most significantly in miR-124 knockdown neurons, reduces the oxygen consumption rate of neural cells. Collectively, our data highlight the roles of miR-124 in coordinating metabolic maturation at the early stages of neurogenesis and provide insights into potential metabolic regulators associated with human brain disorders characterized by metabolic dysfunctions.

DOI: https://doi.org/10.1038/s42003-024-07089-2
Methods Enzymol. 2024 ;pii: S0076-6879(24)00363-X. [Epub ahead of print]706 437-447

Monitoring mitochondrial protein import by live cell imaging.

F X Reymond Sutandy.

  The majority of mitochondrial proteins are synthesized in the cytosol and must be imported into mitochondria to attain their mature forms and execute their functions. Disruption of mitochondrial functions, whether caused by external or internal stress, may compromise mitochondrial protein import. Therefore, monitoring mitochondrial protein import has become a standard approach to assess mitochondrial health and gain insights into mitochondrial biology, especially during stress. This chapter describes a detailed protocol for monitoring mitochondrial import in live cells using microscopy. Co-localization between mitochondria and a genetic reporter of mitochondrially targeted enhanced GFP (eGFP) is employed to evaluate mitochondrial protein import efficiency under different physiological conditions. Overall, this technique provides a simple and robust approach to assess mitochondrial protein import efficiency within its native cellular environment.

Keywords:  MTS; mitochondria; protein import; stress response

DOI:  https://doi.org/10.1016/bs.mie.2024.07.027