bims-axbals Biomed News
on Axonal Biology and ALS
Issue of 2024‒10‒06
twenty papers selected by
TJ Krzystek, ALS Therapy Development Institute
  1. A guide to selecting high-performing antibodies for amyloid-beta precursor protein for use in Western Blot, immunoprecipitation and immunofluorescence.
  2. Structural basis for the rescue of hyperexcitable cells by the amyotrophic lateral sclerosis drug Riluzole.
  3. RAB12-LRRK2 complex suppresses primary ciliogenesis and regulates centrosome homeostasis in astrocytes.
  4. Creation of de novo cryptic splicing for ALS and FTD precision medicine.
  5. Cell and gene therapy for amyotrophic lateral sclerosis.
  6. Reduced Levels of Neurosteroids in Cerebrospinal Fluid of Amyotrophic Lateral Sclerosis Patients.
  7. Exploiting the role of CSF NfL, CHIT1, and miR-181b as potential diagnostic and prognostic biomarkers for ALS.
  8. Understanding activity of butyrate at a cellular level.
  9. Targeting the TDP-43 low complexity domain blocks spreading of pathology in a mouse model of ALS/FTD.
  10. Establishment of human pluripotent stem cell-derived cortical neurosphere model to study pathomechanisms and chemical toxicity in Kleefstra syndrome.
  11. Therapeutic delivery of recombinant glucocerebrosidase enzyme-containing extracellular vesicles to human cells from Gaucher disease patients.
  12. RNA-binding tunes the conformational plasticity and intradomain stability of TDP-43 tandem RNA recognition motifs.
  13. Migratory autolysosome disposal mitigates lysosome damage.
  14. Regulators, mount up.
  15. Impact of perfusion on neuronal development in human derived neuronal networks.
  16. Bcl-2 Orthologues, Buffy and Debcl, Can Suppress Drp1-Dependent Age-Related Phenotypes in Drosophila.
  17. Axon guidance genes are regulated by TDP-43 and RGNEF through long-intron removal.
  18. RNA in axons, dendrites, synapses and beyond.
  19. Mitochondrial Dynamics and mRNA Translation: A Local Synaptic Tale.
  20. Adjacent Neuronal Fascicle Guides Motoneuron 24 Dendritic Branching and Axonal Routing Decisions through Dscam1 Signaling.
  1. F1000Res. 2023 ;12 956
    A guide to selecting high-performing antibodies for amyloid-beta precursor protein for use in Western Blot, immunoprecipitation and immunofluorescence.
    NeuroSGC/YCharOS/EDDU collaborative group
      The amyloid-beta precursor protein is a transmembrane protein expressed in many tissues and highly concentrated in the brain. The protein is of significant interest due to its involvement in the generation of amyloidogenic β-amyloid peptides, prone to plaque formation that is characteristic of Alzheimer's Disease. The scientific community would benefit from the availability of high-quality anti-amyloid-beta precursor protein antibodies to enhance reproducible research on this target. In this study, we characterized eleven amyloid-beta precursor protein commercial antibodies for Western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.
    Keywords:  APP; Amyloid-beta precursor protein; Uniprot ID P05067; Western Blot; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation
    DOI:  https://doi.org/10.12688/f1000research.139867.2
  2. Nat Commun. 2024 Sep 28. 15(1): 8426
    Structural basis for the rescue of hyperexcitable cells by the amyotrophic lateral sclerosis drug Riluzole.
    David Hollingworth, Frances Thomas, Dana A Page, Mohamed A Fouda, Raquel Lopez-Rios De Castro, Altin Sula, Vitaliy B Mykhaylyk, Geoff Kelly, Martin B Ulmschneider, Peter C Ruben, B A Wallace.
      Neuronal hyperexcitability is a key element of many neurodegenerative disorders including the motor neuron disease Amyotrophic Lateral Sclerosis (ALS), where it occurs associated with elevated late sodium current (INaL). INaL results from incomplete inactivation of voltage-gated sodium channels (VGSCs) after their opening and shapes physiological membrane excitability. However, dysfunctional increases can cause hyperexcitability-associated diseases. Here we reveal the atypical binding mechanism which explains how the neuroprotective ALS-treatment drug riluzole stabilises VGSCs in their inactivated state to cause the suppression of INaL that leads to reversed cellular overexcitability. Riluzole accumulates in the membrane and enters VGSCs through openings to their membrane-accessible fenestrations. Riluzole binds within these fenestrations to stabilise the inactivated channel state, allowing for the selective allosteric inhibition of INaL without the physical block of Na+ conduction associated with traditional channel pore binding VGSC drugs. We further demonstrate that riluzole can reproduce these effects on a disease variant of the non-neuronal VGSC isoform Nav1.4, where pathologically increased INaL is caused directly by mutation. Overall, we identify a model for VGSC inhibition that produces effects consistent with the inhibitory action of riluzole observed in models of ALS. Our findings will aid future drug design and supports research directed towards riluzole repurposing.
    DOI:  https://doi.org/10.1038/s41467-024-52539-4
  3. Nat Commun. 2024 Sep 29. 15(1): 8434
    RAB12-LRRK2 complex suppresses primary ciliogenesis and regulates centrosome homeostasis in astrocytes.
    Xingjian Li, Hanwen Zhu, Bik Tzu Huang, Xianting Li, Heesoo Kim, Haiyan Tan, Yuanxi Zhang, Insup Choi, Junmin Peng, Pingyi Xu, Ji Sun, Zhenyu Yue.
      The leucine-rich repeat kinase 2 (LRRK2) phosphorylates a subset of RAB GTPases, and their phosphorylation levels are elevated by Parkinson's disease (PD)-linked mutations of LRRK2. However, the precise function of the LRRK2-regulated RAB GTPase in the brain remains to be elucidated. Here, we identify RAB12 as a robust LRRK2 substrate in the mouse brain through phosphoproteomics profiling and solve the structure of RAB12-LRRK2 protein complex through Cryo-EM analysis. Mechanistically, RAB12 cooperates with LRRK2 to inhibit primary ciliogenesis and regulate centrosome homeostasis in astrocytes through enhancing the phosphorylation of RAB10 and recruiting RILPL1, while the functions of RAB12 require a direct interaction with LRRK2 and LRRK2 activity. Furthermore, the ciliary and centrosome defects caused by the PD-linked LRRK2-G2019S mutation are prevented by Rab12 deletion in astrocytes. Thus, our study reveals a physiological function of the RAB12-LRRK2 complex in regulating ciliogenesis and centrosome homeostasis. The RAB12-LRRK2 structure offers a guidance in the therapeutic development of PD by targeting the RAB12-LRRK2 interaction.
    DOI:  https://doi.org/10.1038/s41467-024-52723-6
  4. Science. 2024 Oct 04. 386(6717): 61-69
    Creation of de novo cryptic splicing for ALS and FTD precision medicine.
    Oscar G Wilkins, Max Z Y J Chien, Josette J Wlaschin, Simone Barattucci, Peter Harley, Francesca Mattedi, Puja R Mehta, Maria Pisliakova, Eugeni Ryadnov, Matthew J Keuss, David Thompson, Holly Digby, Lea Knez, Rebecca L Simkin, Juan Antinao Diaz, Matteo Zanovello, Anna-Leigh Brown, Annalucia Darbey, Rajvinder Karda, Elizabeth M C Fisher, Thomas J Cunningham, Claire E Le Pichon, Jernej Ule, Pietro Fratta.
      Loss of function of the RNA-binding protein TDP-43 (TDP-LOF) is a hallmark of amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. Here we describe TDP-REG, which exploits the specificity of cryptic splicing induced by TDP-LOF to drive protein expression when and where the disease process occurs. The SpliceNouveau algorithm combines deep learning with rational design to generate customizable cryptic splicing events within protein-coding sequences. We demonstrate that expression of TDP-REG reporters is tightly coupled to TDP-LOF in vitro and in vivo. TDP-REG enables genomic prime editing to ablate the UNC13A cryptic donor splice site specifically upon TDP-LOF. Finally, we design TDP-REG vectors encoding a TDP-43/Raver1 fusion protein that rescues key pathological cryptic splicing events, paving the way for the development of precision therapies for TDP43-related disorders.
    DOI:  https://doi.org/10.1126/science.adk2539
  5. Handb Clin Neurol. 2024 ;pii: B978-0-323-90120-8.00017-4. [Epub ahead of print]205 217-241
    Cell and gene therapy for amyotrophic lateral sclerosis.
    Alvar Paris, András Lakatos.
      Amyotrophic lateral sclerosis (ALS) is a fatal and incurable neurodegenerative disorder with rapidly progressive skeletal muscle weakness, which can also cause a variable cognitive deficit. Genetic causes are only identified in approximately 10% of all cases, with complex genotype-phenotype associations, making it challenging to identify treatment targets. What further hampers therapeutic development is a broad heterogeneity in mechanisms, possible targets, and disturbances across various cell types, aside from the cortical and spinal motor neurons that lie at the heart of the pathology of ALS. Over the last decade, significant progress in biotechnologic techniques, cell and ribonucleic acid (RNA) engineering, animal models, and patient-specific human stem cell and organoid models have accelerated both mechanistic and therapeutic discoveries. The growing number of clinical trials mirrors this. This chapter reviews the current state of human preclinical models supporting trial strategies as well as recent clinical cell and gene therapy approaches.
    Keywords:  Amyotrophic lateral sclerosis; Cell transplant; Clinical trial; Disease modeling; Human organoid; Oligonucleotide; iPSC
    DOI:  https://doi.org/10.1016/B978-0-323-90120-8.00017-4
  6. Biomolecules. 2024 Aug 28. pii: 1076. [Epub ahead of print]14(9):
    Reduced Levels of Neurosteroids in Cerebrospinal Fluid of Amyotrophic Lateral Sclerosis Patients.
    Chiara Lucchi, Cecilia Simonini, Cecilia Rustichelli, Rossella Avallone, Elisabetta Zucchi, Ilaria Martinelli, Giuseppe Biagini, Jessica Mandrioli.
      Produced by the mitochondria and endoplasmic reticulum, neurosteroids such as allopregnanolone are neuroprotective molecules that influence various neuronal functions and regulate neuroinflammation. They are reduced in neurodegenerative diseases, while in the Wobbler mouse model, allopregnanolone and its precursor progesterone showed protective effects on motor neuron degeneration. This single-center case-control study included 37 patients with amyotrophic lateral sclerosis (ALS) and 28 healthy controls. Cerebrospinal fluid (CSF) neurosteroid levels were quantified using liquid chromatography-electrospray tandem mass spectrometry and compared between the two cohorts. Neurosteroid concentrations have been correlated with neuroinflammation and neurodegeneration biomarkers detected through an automated immunoassay, along with disease features and progression. Pregnenolone, progesterone, allopregnanolone, pregnanolone, and testosterone levels were significantly lower in ALS patients' CSF compared to healthy controls. A significant inverse correlation was found between neurofilament and neurosteroid levels. Neurosteroid concentrations did not correlate with disease progression, phenotype, genotype, or survival prediction. Our study suggests the independence of the disease features and its progression, from the dysregulation of neurosteroids in ALS patients' CSF. This neurosteroid reduction may relate to disease pathogenesis or be a consequence of disease-related processes, warranting further research. The inverse correlation between neurosteroids and neurofilament levels may indicate a failure of compensatory neuroprotective mechanisms against neurodegeneration.
    Keywords:  amyotrophic lateral sclerosis; cerebrospinal fluid; neurosteroids
    DOI:  https://doi.org/10.3390/biom14091076
  7. J Neurol. 2024 Sep 28.
    Exploiting the role of CSF NfL, CHIT1, and miR-181b as potential diagnostic and prognostic biomarkers for ALS.
    Delia Gagliardi, Mafalda Rizzuti, Pegah Masrori, Domenica Saccomanno, Roberto Del Bo, Luca Sali, Megi Meneri, Simone Scarcella, Ilaria Milone, Nicole Hersmus, Antonia Ratti, Nicola Ticozzi, Vincenzo Silani, Koen Poesen, Philip Van Damme, Giacomo Pietro Comi, Stefania Corti, Federico Verde.
      Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disorder characterized by relentless and progressive loss of motor neurons. A molecular diagnosis, supported by the identification of specific biomarkers, might promote the definition of multiple biological subtypes of ALS, improving patient stratification and providing prognostic information. Here, we investigated the levels of neurofilament light chain (NfL), chitotriosidase (CHIT1) and microRNA-181b (miR-181b) in the cerebrospinal fluid (CSF) of ALS subjects (N = 210) as well as neurologically healthy and neurological disease controls (N = 218, including N = 74 with other neurodegenerative diseases) from a large European multicentric cohort, evaluating their specific or combined utility as diagnostic and prognostic biomarkers. NfL, CHIT1 and miR-181b all showed significantly higher levels in ALS subjects compared to controls, with NfL showing the most effective diagnostic performance. Importantly, all three biomarkers were increased compared to neurodegenerative disease controls and, specifically, to patients with Alzheimer's disease (AD; N = 44), with NfL and CHIT1 being also higher in ALS than in alpha-synucleinopathies (N = 22). Notably, ALS patients displayed increased CHIT1 levels despite having, compared to controls, a higher prevalence of a polymorphism lowering CHIT1 expression. While no relationship was found between CSF miR-181b and clinical measures in ALS (disease duration, functional disability, and disease progression rate), CSF NfL was the best independent predictor of disease progression and survival. This study deepens our knowledge of ALS biomarkers, highlighting the relative specificity of CHIT1 for ALS among neurodegenerative diseases and appraising the potential diagnostic utility of CSF miR-181b.
    Keywords:  ALS; Biomarker; CHIT1; CSF; MiR-181b; NfL
    DOI:  https://doi.org/10.1007/s00415-024-12699-1
  8. Neural Regen Res. 2025 Aug 01. 20(8): 2323-2324
    Understanding activity of butyrate at a cellular level.
    Prapti Chakraborty, Angela S Laird.
      
    DOI:  https://doi.org/10.4103/NRR.NRR-D-24-00468
  9. Acta Neuropathol Commun. 2024 Oct 03. 12(1): 156
    Targeting the TDP-43 low complexity domain blocks spreading of pathology in a mouse model of ALS/FTD.
    Elodie Chevalier, Mickael Audrain, Monisha Ratnam, Romain Ollier, Aline Fuchs, Kasia Piorkowska, Andrea Pfeifer, Marie Kosco-Vilbois, Tamara Seredenina, Tariq Afroz.
      Abnormal cytoplasmic localization and accumulation of pathological transactive response DNA binding protein of 43 kDa (TDP-43) underlies several devastating diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP). A key element is the correlation between disease progression and spatio-temporal propagation of TDP-43-mediated pathology in the central nervous system. Several lines of evidence support the concept of templated aggregation and cell to cell spreading of pathological TDP-43. To further investigate this mechanism in vivo, we explored the efficacy of capturing and masking the seeding-competent region of extracellular TDP-43 species. For this, we generated a novel monoclonal antibody (mAb), ACI-6677, that targets the pathogenic protease-resistant amyloid core of TDP-43. ACI-6677 has a picomolar binding affinity for TDP-43 and is capable of binding to all C-terminal TDP-43 fragments. In vitro, ACI-6677 inhibited TDP-43 aggregation and boosted removal of pathological TDP-43 aggregates by phagocytosis. When injecting FTLD-TDP brain extracts unilaterally in the CamKIIa-hTDP-43NLSm mouse model, ACI-6677 significantly limited the induction of phosphorylated TDP-43 (pTDP-43) inclusions. Strikingly, on the contralateral side, the mAb significantly prevented pTDP-43 inclusion appearance exemplifying blocking of the spreading process. Taken together, these data demonstrate for the first time that an immunotherapy targeting the protease-resistant amyloid core of TDP-43 has the potential to restrict spreading, substantially slowing or stopping progression of disease.
    Keywords:  ALS; FTD; Immunotherapy; Neuropathology; Pathomechanism; Spreading; TDP-43
    DOI:  https://doi.org/10.1186/s40478-024-01867-z
  10. Sci Rep. 2024 09 29. 14(1): 22572
    Establishment of human pluripotent stem cell-derived cortical neurosphere model to study pathomechanisms and chemical toxicity in Kleefstra syndrome.
    Andrea Balogh, Mária Bódi-Jakus, Vivien Réka Karl, Tamás Bellák, Balázs Széky, János Farkas, Federica Lamberto, David Novak, Anita Fehér, Melinda Zana, András Dinnyés.
      In the present study, we aimed to establish and characterize a mature cortical spheroid model system for Kleefstra syndrome (KS) using patient-derived iPSC. We identified key differences in the growth behavior of KS spheroids determined by reduced proliferation marked by low Ki67 and high E-cadherin expression. Conversely, in the spheroid-based neurite outgrowth assay KS outperformed the control neurite outgrowth due to higher BDNF expression. KS spheroids were highly enriched in VGLUT1/2-expressing glutamatergic and ChAT-expressing cholinergic neurons, while TH-positive catecholamine neurons were significantly underrepresented. Furthermore, high NMDAR1 expression was also detected in the KS spheroid, similarly to other patients-derived neuronal cultures, denoting high NMDAR1 expression as a general, KS-specific marker. Control and KS neuronal progenitors and neurospheres were exposed to different toxicants (paraquat, rotenone, bardoxolone, and doxorubicin), and dose-response curves were assessed after acute exposure. Differentiation stage and compound-specific differences were detected with KS neurospheres being the most sensitive to paraquat. Altogether this study describes a robust 3D model system expressing the disease-specific markers and recapitulating the characteristic pathophysiological traits. This platform is suitable for testing developing brain-adverse environmental effects interactions, drug development, and screening towards individual therapeutic strategies.
    Keywords:   In vitro toxicology; EHMT1; Human induced pluripotent stem cells; Kleefstra syndrome; Neurospheroid
    DOI:  https://doi.org/10.1038/s41598-024-72791-4
  11. Orphanet J Rare Dis. 2024 Oct 02. 19(1): 363
    Therapeutic delivery of recombinant glucocerebrosidase enzyme-containing extracellular vesicles to human cells from Gaucher disease patients.
    Keatdamrong Janpipatkul, Nareerat Sutjarit, Amornrat Tangprasittipap, Tai Chaiamarit, Pawarit Innachai, Kanoknetr Suksen, Tanida Chokpanuwat, Thipwimol Tim-Aroon, Usanarat Anurathapan, Natee Jearawiriyapaisarn, Alisa Tubsuwan, Supareak Bowornpinyo, Nithi Asavapanumas, Arthit Chairoungdua, Kanit Bhukhai, Suradej Hongeng.
      BACKGROUND: Gaucher disease (GD) is one of the most common types of lysosomal storage diseases (LSDs) caused by pathogenic variants of lysosomal β-glucocerebrosidase gene (GBA1), resulting in the impairment of Glucocerebrosidase (GCase) enzyme function and the accumulation of a glycolipid substrate, glucosylceramide (GlcCer) within lysosomes. Current therapeutic approaches such as enzyme replacement therapy and substrate reduction therapy cannot fully rescue GD pathologies, especially neurological symptoms. Meanwhile, delivery of lysosomal enzymes to the endocytic compartment of affected human cells is a promising strategy for treating neuropathic LSDs.RESULT: Here, we describe a novel approach to restore GCase enzyme in cells from neuropathic GD patients by producing extracellular vesicle (EVs)-containing GCase from cells overexpressing GBA1 gene. Lentiviral vectors containing modified GBA1 were introduced into HEK293T cells to produce a stable cell line that provides a sustainable source of functional GCase enzyme. The GBA1-overexpressing cells released EV-containing GCase enzyme, that is capable of entering into and localizing in the endocytic compartment of recipient cells, including THP-1 macrophage, SH-SY5Y neuroblastoma, and macrophages and neurons derived from induced pluripotent stem cells (iPSCs) of neuropathic GD patients. Importantly, the recipient cells exhibit higher GCase enzyme activity.
    CONCLUSION: This study presents a promising therapeutic strategy to treat severe types of LSDs. It involves delivering lysosomal enzymes to the endocytic compartment of human cells affected by conditions such as GDs with neurological symptoms, as well as potentially other neurological disorders impacting lysosomes.
    Keywords:  Extracellular vesicles; Gaucher disease; Glucocerebrosidase; HEK293T cell; Lentiviral vector; Macrophages; Neurons
    DOI:  https://doi.org/10.1186/s13023-024-03376-7
  12. Biophys J. 2024 Sep 30. pii: S0006-3495(24)00658-1. [Epub ahead of print]
    RNA-binding tunes the conformational plasticity and intradomain stability of TDP-43 tandem RNA recognition motifs.
    Busra Ozguney, Priyesh Mohanty, Jeetain Mittal.
      TAR DNA binding protein 43 (TDP-43) is a nuclear RNA/DNA-binding protein with pivotal roles in RNA-related processes such as splicing, transcription, transport, and stability. The high binding affinity and specificity of TDP-43 towards its cognate RNA sequences (GU-rich) is mediated by highly conserved residues in its tandem RNA recognition motif (RRM) domains (aa:104-263). Importantly, the loss of RNA-binding to the tandem RRMs caused by physiological stressors and chemical modifications promotes cytoplasmic mislocalization and pathological aggregation of TDP-43. Despite the substantial implications of RNA-binding in TDP-43 function and pathology, its precise effects on the intradomain stability and conformational dynamics of the tandem RRMs is not properly understood. Here, we employed all-atom molecular dynamics (MD) simulations to assess the effect of RNA-binding on the conformational landscape and intradomain stability of TDP-43 tandem RRMs. RNA limits the overall conformational space of the tandem RRMs and promotes intradomain stability through a combination of specific base stacking interactions and transient electrostatic interactions. In contrast, tandem RRMs exhibit a high intrinsic conformational plasticity in the absence of RNA which surprisingly, is accompanied by a tendency of RRM1 to adopt partially-unfolded conformations. Overall, our simulations reveal how RNA-binding dynamically tunes the structural and conformational landscape of TDP-43 tandem RRMs, contributing to physiological function and mitigating pathological aggregation.
    DOI:  https://doi.org/10.1016/j.bpj.2024.09.031
  13. J Cell Biol. 2024 Dec 02. pii: e202403195. [Epub ahead of print]223(12):
    Migratory autolysosome disposal mitigates lysosome damage.
    Takami Sho, Ying Li, Haifeng Jiao, Li Yu.
      Lysosomes, essential for intracellular degradation and recycling, employ damage-control strategies such as lysophagy and membrane repair mechanisms to maintain functionality and cellular homeostasis. Our study unveils migratory autolysosome disposal (MAD), a response to lysosomal damage where cells expel LAMP1-LC3 positive structures via autolysosome exocytosis, requiring autophagy machinery, SNARE proteins, and cell migration. This mechanism, crucial for mitigating lysosomal damage, underscores the role of cell migration in lysosome damage control and facilitates the release of small extracellular vesicles, highlighting the intricate relationship between cell migration, organelle quality control, and extracellular vesicle release.
    DOI:  https://doi.org/10.1083/jcb.202403195
  14. Science. 2024 Oct 04. 386(6717): 24-25
    Regulators, mount up.
    Yi Zeng, Aaron D Gitler.
      Cryptic exons enable delivery of therapies only to sick neurons in a motor neuron disease.
    DOI:  https://doi.org/10.1126/science.ads5951
  15. APL Bioeng. 2024 Dec;8(4): 046102
    Impact of perfusion on neuronal development in human derived neuronal networks.
    Donatella Di Lisa, Andrea Andolfi, Giacomo Masi, Giuseppe Uras, Pier Francesco Ferrari, Sergio Martinoia, Laura Pastorino.
      Advanced in vitro models of the brain have evolved in recent years from traditional two-dimensional (2D) ones, based on rodent derived cells, to three-dimensional (3D) ones, based on human neurons derived from induced pluripotent stem cells. To address the dynamic changes of the tissue microenvironment, bioreactors are used to control the in vitro microenvironment for viability, repeatability, and standardization. However, in neuronal tissue engineering, bioreactors have primarily been used for cell expansion purposes, while microfluidic systems have mainly been employed for culturing organoids. In this study, we explored the use of a commercial perfusion bioreactor to control the culture microenvironment of neuronal cells in both 2D and 3D cultures. Namely, neurons differentiated from human induced pluripotent stem cells (iNeurons) were cultured in 2D under different constant flow rates for 72 h. The impact of different flow rates on early-stage neuronal development and synaptogenesis was assessed by morphometric characterization and synaptic analysis. Based on these results, two involving variable flow rates were developed and applied again in 2D culture. The most effective protocol, in terms of positive impact on neuronal development, was then used for a preliminary study on the application of dynamic culturing conditions to neuronal cells in 3D. To this purpose, both iNeurons, co-cultured with astrocytes, and the human neuroblastoma cells SH-SY5Y were embedded into a hydrogel and maintained under perfusion for up to 28 days. A qualitative evaluation by immunocytochemistry and confocal microscopy was carried out to assess cell morphology and the formation of a 3D neuronal network.
    DOI:  https://doi.org/10.1063/5.0221911
  16. Biomolecules. 2024 Aug 30. pii: 1089. [Epub ahead of print]14(9):
    Bcl-2 Orthologues, Buffy and Debcl, Can Suppress Drp1-Dependent Age-Related Phenotypes in Drosophila.
    Azra Hasan, Brian E Staveley.
      The relationship of Amyotrophic Lateral Sclerosis, Parkinson's disease, and other age-related neurodegenerative diseases with mitochondrial dysfunction has led to our study of the mitochondrial fission gene Drp1 in Drosophila melanogaster and aspects of aging. Previously, the Drp1 protein has been demonstrated to interact with the Drosophila Bcl-2 mitochondrial proteins, and Drp1 mutations can lead to mitochondrial dysfunction and neuronal loss. In this study, the Dopa decarboxylase-Gal4 (Ddc-Gal4) transgene was exploited to direct the expression of Drp1 and Drp1-RNAi transgenes in select neurons. Here, the knockdown of Drp1 seems to compromise locomotor function throughout life but does not alter longevity. The co-expression of Buffy suppresses the poor climbing induced by the knockdown of the Drp1 function. The consequences of Drp1 overexpression, which specifically reduced median lifespan and diminished climbing abilities over time, can be suppressed through the directed co-overexpression of pro-survival Bcl-2 gene Buffy or by the co-knockdown of the pro-cell death Bcl-2 homologue Debcl. Alteration of the expression of Drp1 acts to phenocopy neurodegenerative disease phenotypes in Drosophila, while overexpression of Buffy can counteract or rescue these phenotypes to improve overall health. The diminished healthy aging due to either the overexpression of Drp1 or the RNA interference of Drp1 has produced novel Drosophila models for investigating mechanisms underlying neurodegenerative disease.
    Keywords:  Amyotrophic Lateral Sclerosis; Buffy; Debcl; Drosophila melanogaster; Drp1; Parkinson’s disease; mitochondrial dysfunction; mitochondrial fission
    DOI:  https://doi.org/10.3390/biom14091089
  17. FASEB J. 2024 Oct 15. 38(19): e70081
    Axon guidance genes are regulated by TDP-43 and RGNEF through long-intron removal.
    Yasmine Abbassi, Sara Cappelli, Eugenio Spagnolo, Alice Gennari, Giulia Visani, Simone Barattucci, Francesca Paron, Cristiana Stuani, Cristian A Droppelmann, Michael J Strong, Emanuele Buratti.
      Rho guanine nucleotide exchange factor (RGNEF) is a guanine nucleotide exchange factor (GEF) mainly involved in regulating the activity of Rho-family GTPases. It is a bi-functional protein, acting both as a guanine exchange factor and as an RNA-binding protein. RGNEF is known to act as a destabilizing factor of neurofilament light chain RNA (NEFL) and it could potentially contribute to their sequestration in nuclear cytoplasmic inclusions. Most importantly, RGNEF inclusions in the spinal motor neurons of ALS patients have been shown to co-localize with inclusions of TDP-43, the major well-known RNA-binding protein aggregating in the brain and spinal cord of human patients. Therefore, it can be hypothesized that loss-of-function of both proteins following aggregation may contribute to motor neuron death/survival in ALS patients. To further characterize their relationship, we have compared the transcriptomic profiles of neuronal cells depleted of TDP-43 and RGNEF and show that these two factors predominantly act in an antagonistic manner when regulating the expression of axon guidance genes. From a mechanistic point of view, our experiments show that the effect of these genes on the processivity of long introns can explain their mode of action. Taken together, our results show that loss-of-function of factors co-aggregating with TDP-43 can potentially affect the expression of commonly regulated neuronal genes in a very significant manner, potentially acting as disease modifiers. This finding further highlights that neurodegenerative processes at the RNA level are the result of combinatorial interactions between different RNA-binding factors that can be co-aggregated in neuronal cells. A deeper understanding of these complex scenarios may lead to a better understanding of pathogenic mechanisms occurring in patients, where more than one specific protein may be aggregating in their neurons.
    Keywords:  ALS; RGNEF; TDP‐43; hnRNPs; long‐intron processing
    DOI:  https://doi.org/10.1096/fj.202400743RR
  18. Front Mol Neurosci. 2024 ;17 1397378
    RNA in axons, dendrites, synapses and beyond.
    Richard Taylor, Nikolas Nikolaou.
      In neurons, a diverse range of coding and non-coding RNAs localize to axons, dendrites, and synapses, where they facilitate rapid responses to local needs, such as axon and dendrite extension and branching, synapse formation, and synaptic plasticity. Here, we review the extent of our current understanding of RNA subclass diversity in these functionally demanding subcellular compartments. We discuss the similarities and differences identified between axonal, dendritic and synaptic local transcriptomes, and discuss the reported and hypothesized fates and functions of localized RNAs. Furthermore, we outline the RNA composition of exosomes that bud off from neurites, and their implications for the biology of neighboring cells. Finally, we highlight recent advances in third-generation sequencing technologies that will likely provide transformative insights into splice isoform and RNA modification diversity in local transcriptomes.
    Keywords:  circular RNA (circRNA); cleavage and polyadenylation; intron retaining RNA (IR RNA); local mRNA translation; long non coding RNA (lncRNA); messenger RNA (mRNA); microRNA (miRNA); stability and degradation
    DOI:  https://doi.org/10.3389/fnmol.2024.1397378
  19. Biology (Basel). 2024 Sep 23. pii: 746. [Epub ahead of print]13(9):
    Mitochondrial Dynamics and mRNA Translation: A Local Synaptic Tale.
    Marta Zaninello, Pedro Baptista, Filipe V Duarte.
      Mitochondria are dynamic organelles that can adjust and respond to different stimuli within a cell. This plastic ability allows them to effectively coordinate several cellular functions in cells and becomes particularly relevant in highly complex cells such as neurons. An imbalance in mitochondrial dynamics can disrupt mitochondrial function, leading to abnormal cellular function and ultimately to a range of diseases, including neurodegenerative disorders. Regulation of mRNA transport and local translation inside neurons is crucial for maintaining the proteome of distal mitochondria, which is vital for energy production and synaptic function. A significant portion of the axonal transcriptome is dedicated to mRNAs for mitochondrial proteins, emphasizing the importance of local translation in sustaining mitochondrial function in areas far from the cell body. In neurons, local translation and the regulation of mRNAs encoding mitochondrial-shaping proteins could be essential for synaptic plasticity and neuronal health. The dynamics of these mRNAs, including their transport and local translation, may influence the morphology and function of mitochondria, thereby affecting the overall energy status and responsiveness of synapses. Comprehending the mitochondria-related mRNA regulation and local translation, as well as its influence on mitochondrial morphology near the synapses will help to better understand neuronal physiology and neurological diseases where mitochondrial dysfunction and impaired synaptic plasticity play a central role.
    Keywords:  mRNA; mRNA trafficking; mitochondria; mitochondrial morphology; neuron; synapse
    DOI:  https://doi.org/10.3390/biology13090746
  20. eNeuro. 2024 Sep 30. pii: ENEURO.0130-24.2024. [Epub ahead of print]
    Adjacent Neuronal Fascicle Guides Motoneuron 24 Dendritic Branching and Axonal Routing Decisions through Dscam1 Signaling.
    Kathy Clara Bui, Daichi Kamiyama.
      The formation and precise positioning of axons and dendrites are crucial for the development of neural circuits. Although juxtracrine signaling via cell-cell contact is known to influence these processes, the specific structures and mechanisms regulating neuronal process positioning within the central nervous system (CNS) remain to be fully identified. Our study investigates motoneuron 24 (MN24) in the Drosophila embryonic CNS, which is characterized by a complex yet stereotyped axon projection pattern, known as 'axonal routing.' In this motoneuron, the primary dendritic branches project laterally toward the midline, specifically emerging at the sites where axons turn. We observed that Scp2-positive neurons contribute to the lateral fascicle structure in the ventral nerve cord (VNC) near MN24 dendrites. Notably, the knockout of the Down syndrome cell adhesion molecule (Dscam1) results in the loss of dendrites and disruption of proper axonal routing in MN24, while not affecting the formation of the fascicle structure. Through cell-type specific knockdown and rescue experiments of Dscam1, we have determined that the interaction between MN24 and Scp2-positive fascicle, mediated by Dscam1, promotes the development of both dendrites and axonal routing. Our findings demonstrate that the holistic configuration of neuronal structures, such as axons and dendrites, within single motoneurons can be governed by local contact with the adjacent neuron fascicle, a novel reference structure for neural circuitry wiring.Significance Summary We uncover a key neuronal structure serving as a guiding reference for neural circuitry within the Drosophila embryonic CNS, highlighting the essential role of an adjacent axonal fascicle in precisely coordinating axon and dendrite positioning in motoneuron 24 (MN24). Our investigation of cell-cell interactions between motoneurons and adjacent axonal fascicles-crucial for initiating dendrite formation, soma mislocation, and axonal pathfinding in MN24-emphasizes the neuronal fascicle's significance in neural circuit formation through Dscam1-mediated inter-neuronal communication. This enhances our understanding of the molecular underpinnings of motoneuron morphogenesis in Drosophila Given the occurrence of analogous axon fascicle formations within the vertebrate spinal cord, such structures may play a conserved role in the morphogenesis of motoneurons via Dscam1 across phyla.
    DOI:  https://doi.org/10.1523/ENEURO.0130-24.2024
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