Publications by authors named "Ludo Van Den Bosch"

153 Publications

Generation of Human Motor Units with Functional Neuromuscular Junctions in Microfluidic Devices.

J Vis Exp 2021 09 7(175). Epub 2021 Sep 7.

Department of Neurosciences, Experimental Neurology, and Leuven Brain Institute, KU Leuven - University of Leuven; VIB Center for Brain & Disease Research, Laboratory of Neurobiology, ;

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle contraction. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), NMJs degenerate, resulting in muscle atrophy and progressive paralysis. The underlying mechanism of NMJ degeneration is unknown, largely due to the lack of translatable research models. This study aimed to create a versatile and reproducible in vitro model of a human motor unit with functional NMJs. Therefore, human induced pluripotent stem cell (hiPSC)-derived motor neurons and human primary mesoangioblast (MAB)-derived myotubes were co-cultured in commercially available microfluidic devices. The use of fluidically isolated micro-compartments allows for the maintenance of cell-specific microenvironments while permitting cell-to-cell contact through microgrooves. By applying a chemotactic and volumetric gradient, the growth of motor neuron-neurites through the microgrooves promoting myotube interaction and the formation of NMJs were stimulated. These NMJs were identified immunocytochemically through co-localization of motor neuron presynaptic marker synaptophysin (SYP) and postsynaptic acetylcholine receptor (AChR) marker α-bungarotoxin (Btx) on myotubes and characterized morphologically using scanning electron microscopy (SEM). The functionality of the NMJs was confirmed by measuring calcium responses in myotubes upon depolarization of the motor neurons. The motor unit generated using standard microfluidic devices and stem cell technology can aid future research focusing on NMJs in health and disease.
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http://dx.doi.org/10.3791/62959DOI Listing
September 2021

Association of Variants in the SPTLC1 Gene With Juvenile Amyotrophic Lateral Sclerosis.

JAMA Neurol 2021 Oct;78(10):1236-1248

Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, United Kingdom.

Importance: Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of ALS characterized by age of symptom onset less than 25 years and a variable presentation.

Objective: To identify the genetic variants associated with juvenile ALS.

Design, Setting, And Participants: In this multicenter family-based genetic study, trio whole-exome sequencing was performed to identify the disease-associated gene in a case series of unrelated patients diagnosed with juvenile ALS and severe growth retardation. The patients and their family members were enrolled at academic hospitals and a government research facility between March 1, 2016, and March 13, 2020, and were observed until October 1, 2020. Whole-exome sequencing was also performed in a series of patients with juvenile ALS. A total of 66 patients with juvenile ALS and 6258 adult patients with ALS participated in the study. Patients were selected for the study based on their diagnosis, and all eligible participants were enrolled in the study. None of the participants had a family history of neurological disorders, suggesting de novo variants as the underlying genetic mechanism.

Main Outcomes And Measures: De novo variants present only in the index case and not in unaffected family members.

Results: Trio whole-exome sequencing was performed in 3 patients diagnosed with juvenile ALS and their parents. An additional 63 patients with juvenile ALS and 6258 adult patients with ALS were subsequently screened for variants in the SPTLC1 gene. De novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient) were identified in 3 unrelated patients diagnosed with juvenile ALS and failure to thrive. A fourth variant (p.Leu39del) was identified in a patient with juvenile ALS where parental DNA was unavailable. Variants in this gene have been previously shown to be associated with autosomal-dominant hereditary sensory autonomic neuropathy, type 1A, by disrupting an essential enzyme complex in the sphingolipid synthesis pathway.

Conclusions And Relevance: These data broaden the phenotype associated with SPTLC1 and suggest that patients presenting with juvenile ALS should be screened for variants in this gene.
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http://dx.doi.org/10.1001/jamaneurol.2021.2598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8406220PMC
October 2021

Potential Therapeutic Role of HDAC Inhibitors in FUS-ALS.

Front Mol Neurosci 2021 9;14:686995. Epub 2021 Aug 9.

Vlaams Instituut voor Biotechnologie (VIB), Center for Brain and Disease Research, Laboratory of Neurobiology, Leuven, Belgium.

Mutations in the gene cause amyotrophic lateral sclerosis (ALS-FUS). However, the exact pathogenic mechanism of mutant fused in sarcoma (FUS) protein is not completely understood. FUS is an RNA binding protein (RBP) localized predominantly in the nucleus, but ALS-linked FUS mutations can affect its nuclear localization signal impairing its import into the nucleus. This mislocalization to the cytoplasm facilitates FUS aggregation in cytoplasmic inclusions. Therapies targeting post translational modifications are rising as new treatments for ALS, in particular acetylation which could have a role in the dynamics of RBPs. Research using histone deacetylase (HDAC) inhibitors in FUS-ALS models showed that HDACs can influence cytoplasmic FUS localization. Inhibition of HDACs could promote acetylation of the FUS RNA binding domain (RRM) and altering its RNA interactions resulting in FUS maintenance in the nucleus. In addition, acetylation of FUS RRMs might also favor or disfavor its incorporation into pathological inclusions. In this review, we summarize and discuss the evidence for the potential role of HDACs in the context of FUS-ALS and we propose a new hypothesis based on this overview.
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http://dx.doi.org/10.3389/fnmol.2021.686995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8380926PMC
August 2021

Induced pluripotent stem cell-derived motor neurons of CMT type 2 patients reveal progressive mitochondrial dysfunction.

Brain 2021 Sep;144(8):2471-2485

Peripheral Neuropathy Research Group, Department of Biomedical Sciences, University of Antwerp, Antwerp, 2610, Belgium.

Axonal Charcot-Marie-Tooth neuropathies (CMT type 2) are caused by inherited mutations in various genes functioning in different pathways. The types of genes and multiplicity of mutations reflect the clinical and genetic heterogeneity in CMT2 disease, which complicates its diagnosis and has inhibited the development of therapies. Here, we used CMT2 patient-derived pluripotent stem cells (iPSCs) to identify common hallmarks of axonal degeneration shared by different CMT2 subtypes. We compared the cellular phenotypes of neurons differentiated from CMT2 patient iPSCs with those from healthy controls and a CRISPR/Cas9-corrected isogenic line. Our results demonstrated neurite network alterations along with extracellular electrophysiological abnormalities in the differentiated motor neurons. Progressive deficits in mitochondrial and lysosomal trafficking, as well as in mitochondrial morphology, were observed in all CMT2 patient lines. Differentiation of the same CMT2 iPSC lines into peripheral sensory neurons only gave rise to cellular phenotypes in subtypes with sensory involvement, supporting the notion that some gene mutations predominantly affect motor neurons. We revealed a common mitochondrial dysfunction in CMT2-derived motor neurons, supported by alterations in the expression pattern and oxidative phosphorylation, which could be recapitulated in the sciatic nerve tissue of a symptomatic mouse model. Inhibition of a dual leucine zipper kinase could partially ameliorate the mitochondrial disease phenotypes in CMT2 subtypes. Altogether, our data reveal shared cellular phenotypes across different CMT2 subtypes and suggests that targeting such common pathomechanisms could allow the development of a uniform treatment for CMT2.
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http://dx.doi.org/10.1093/brain/awab226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8418338PMC
September 2021

Exploring the alternative: Fish, flies and worms as preclinical models for ALS.

Neurosci Lett 2021 08 10;759:136041. Epub 2021 Jun 10.

KU Leuven - University of Leuven, Department of Neurosciences, Experimental Neurology, and Leuven Brain Institute (LBI), Leuven, Belgium; VIB, Center for Brain & Disease Research, Laboratory of Neurobiology, Leuven, Belgium. Electronic address:

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disorder characterized by the loss of upper and lower motor neurons. In general, patients succumb to respiratory insufficiency due to respiratory muscle weakness. Despite many promising therapeutic strategies primarily identified in rodent models, patient trials remain rather unsuccessful. There is a clear need for alternative approaches, which could provide directions towards the justified use of rodents and which increase the likelihood to identify new promising clinical candidates. In the last decades, the use of fast genetic approaches and the development of high-throughput screening platforms in the nematode Caenorhabditis elegans, in the fruit fly (Drosophila melanogaster) and in zebrafish (Danio rerio) have contributed to new insights into ALS pathomechanisms, disease modifiers and therapeutic targets. In this mini-review, we provide an overview of these alternative small animal studies, modeling the most common ALS genes and discuss the most recent preclinical discoveries. We conclude that small animal models will not replace rodent models, yet they clearly represent an important asset for preclinical studies.
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http://dx.doi.org/10.1016/j.neulet.2021.136041DOI Listing
August 2021

Reply to 'TDP43 aggregates: the 'Schrödinger's cat' in amyotrophic lateral sclerosis'.

Nat Rev Neurosci 2021 08;22(8):515

Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, UK.

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http://dx.doi.org/10.1038/s41583-021-00478-0DOI Listing
August 2021

In-silico prediction of in-vitro protein liquid-liquid phase separation experiments outcomes with multi-head neural attention.

Bioinformatics 2021 May 13. Epub 2021 May 13.

SWITCH Lab, Leuven, KU, Belgium.

Motivation: Proteins able to undergo Liquid-Liquid Phase Separation (LLPS) in-vivo and in-vitro are drawing a lot of interest, due to their functional relevance for cell life. Nevertheless, the proteome-scale experimental screening of these proteins seems unfeasible, because besides being expensive and time consuming, LLPS is heavily influenced by multiple environmental conditions such as concentration, pH and temperature, thus requiring a combinatorial number of experiments for each protein.

Results: To overcome this problem, we propose an Neural Network model able to predict the LLPS behavior of proteins given specified experimental conditions, effectively predicting the outcome of in-vitro experiments. Our model can be used to rapidly screen proteins and experimental conditions searching for LLPS, thus reducing the search space that needs to be covered experimentally. We experimentally validate Droppler's prediction on the the TAR DNA-binding protein in different experimental conditions, showing the consistency of its predictions.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btab350DOI Listing
May 2021

Liquid-Liquid Phase Separation Enhances TDP-43 LCD Aggregation but Delays Seeded Aggregation.

Biomolecules 2021 04 8;11(4). Epub 2021 Apr 8.

Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI), KU Leuven-University of Leuven, 3000 Leuven, Belgium.

Aggregates of TAR DNA-binding protein (TDP-43) are a hallmark of several neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Although TDP-43 aggregates are an undisputed pathological species at the end stage of these diseases, the molecular changes underlying the initiation of aggregation are not fully understood. The aim of this study was to investigate how phase separation affects self-aggregation and aggregation seeded by pre-formed aggregates of either the low-complexity domain (LCD) or its short aggregation-promoting regions (APRs). By systematically varying the physicochemical conditions, we observed that liquid-liquid phase separation (LLPS) promotes spontaneous aggregation. However, we noticed less efficient seeded aggregation in phase separating conditions. By analyzing a broad range of conditions using the Hofmeister series of buffers, we confirmed that stabilizing hydrophobic interactions prevail over destabilizing electrostatic forces. RNA affected the cooperativity between LLPS and aggregation in a "reentrant" fashion, having the strongest positive effect at intermediate concentrations. Altogether, we conclude that conditions which favor LLPS enhance the subsequent aggregation of the TDP-43 LCD with complex dependence, but also negatively affect seeding kinetics.
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http://dx.doi.org/10.3390/biom11040548DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8068378PMC
April 2021

Human motor units in microfluidic devices are impaired by FUS mutations and improved by HDAC6 inhibition.

Stem Cell Reports 2021 Sep 22;16(9):2213-2227. Epub 2021 Apr 22.

KU Leuven - University of Leuven, Department of Neurosciences, Experimental Neurology, and Leuven Brain Institute, Leuven, Belgium; VIB, Center for Brain & Disease Research, Laboratory of Neurobiology, Leuven, Belgium. Electronic address:

Neuromuscular junctions (NMJs) ensure communication between motor neurons (MNs) and muscle; however, in MN disorders, such as amyotrophic lateral sclerosis (ALS), NMJs degenerate resulting in muscle atrophy. The aim of this study was to establish a versatile and reproducible in vitro model of a human motor unit to investigate the effects of ALS-causing mutations. Therefore, we generated a co-culture of human induced pluripotent stem cell (iPSC)-derived MNs and human primary mesoangioblast-derived myotubes in microfluidic devices. A chemotactic and volumetric gradient facilitated the growth of MN neurites through microgrooves resulting in the interaction with myotubes and the formation of NMJs. We observed that ALS-causing FUS mutations resulted in reduced neurite outgrowth as well as an impaired neurite regrowth upon axotomy. NMJ numbers were likewise reduced in the FUS-ALS model. Interestingly, the selective HDAC6 inhibitor, Tubastatin A, improved the neurite outgrowth, regrowth, and NMJ morphology, prompting HDAC6 inhibition as a potential therapeutic strategy for ALS.
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http://dx.doi.org/10.1016/j.stemcr.2021.03.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8452598PMC
September 2021

-derived arginine-containing dipeptide repeats associate with axonal transport machinery and impede microtubule-based motility.

Sci Adv 2021 Apr 9;7(15). Epub 2021 Apr 9.

KU Leuven-University of Leuven, Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI), Leuven, Belgium.

A hexanucleotide repeat expansion in the gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How this mutation leads to these neurodegenerative diseases remains unclear. Here, we show using patient stem cell-derived motor neurons that the repeat expansion impairs microtubule-based transport, a process critical for neuronal survival. Cargo transport defects are recapitulated by treating neurons from healthy individuals with proline-arginine and glycine-arginine dipeptide repeats (DPRs) produced from the repeat expansion. Both arginine-rich DPRs similarly inhibit axonal trafficking in adult neurons in vivo. Physical interaction studies demonstrate that arginine-rich DPRs associate with motor complexes and the unstructured tubulin tails of microtubules. Single-molecule imaging reveals that microtubule-bound arginine-rich DPRs directly impede translocation of purified dynein and kinesin-1 motor complexes. Collectively, our study implicates inhibitory interactions of arginine-rich DPRs with axonal transport machinery in -associated ALS/FTD and thereby points to potential therapeutic strategies.
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http://dx.doi.org/10.1126/sciadv.abg3013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034861PMC
April 2021

Tetrahydroquinoline-Capped Histone Deacetylase 6 Inhibitor SW-101 Ameliorates Pathological Phenotypes in a Charcot-Marie-Tooth Type 2A Mouse Model.

J Med Chem 2021 04 8;64(8):4810-4840. Epub 2021 Apr 8.

Bright Minds Biosciences, Toronto, ON M5H 3V9, Canada.

Histone deacetylase 6 (HDAC6) is a promising therapeutic target for the treatment of neurodegenerative disorders. SW-100 (), a phenylhydroxamate-based HDAC6 inhibitor (HDAC6i) bearing a tetrahydroquinoline (THQ) capping group, is a highly potent and selective HDAC6i that was shown to be effective in mouse models of Fragile X syndrome and Charcot-Marie-Tooth disease type 2A (CMT2A). In this study, we report the discovery of a new THQ-capped HDAC6i, termed SW-101 (), that possesses excellent HDAC6 potency and selectivity, together with markedly improved metabolic stability and druglike properties compared to SW-100 (). X-ray crystallography data reveal the molecular basis of HDAC6 inhibition by SW-101 (). Importantly, we demonstrate that SW-101 () treatment elevates the impaired level of acetylated α-tubulin in the distal sciatic nerve, counteracts progressive motor dysfunction, and ameliorates neuropathic symptoms in a CMT2A mouse model bearing mutant 2. Taken together, these results bode well for the further development of SW-101 () as a disease-modifying HDAC6i.
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http://dx.doi.org/10.1021/acs.jmedchem.0c02210DOI Listing
April 2021

Lighting Up the Plasma Membrane: Development and Applications of Fluorescent Ligands for Transmembrane Proteins.

Chemistry 2021 Jun 1;27(34):8605-8641. Epub 2021 Jun 1.

Department of Chemistry, Molecular Design and Synthesis, KU Leuven Campus Arenberg Celestijnenlaan 200F -, box 2404, 3001, Leuven, Belgium.

Despite the fact that transmembrane proteins represent the main therapeutic targets for decades, complete and in-depth knowledge about their biochemical and pharmacological profiling is not fully available. In this regard, target-tailored small-molecule fluorescent ligands are a viable approach to fill in the missing pieces of the puzzle. Such tools, coupled with the ability of high-precision optical techniques to image with an unprecedented resolution at a single-molecule level, helped unraveling many of the conundrums related to plasma proteins' life-cycle and druggability. Herein, we review the recent progress made during the last two decades in fluorescent ligand design and potential applications in fluorescence microscopy of voltage-gated ion channels, ligand-gated ion channels and G-coupled protein receptors.
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http://dx.doi.org/10.1002/chem.202100296DOI Listing
June 2021

HDAC6 inhibition restores TDP-43 pathology and axonal transport defects in human motor neurons with TARDBP mutations.

EMBO J 2021 04 10;40(7):e106177. Epub 2021 Mar 10.

Department of Neurosciences, Experimental Neurology, Leuven Brain Institute (LBI), KU Leuven - University of Leuven, Leuven, Belgium.

TDP-43 is the major component of pathological inclusions in most ALS patients and in up to 50% of patients with frontotemporal dementia (FTD). Heterozygous missense mutations in TARDBP, the gene encoding TDP-43, are one of the common causes of familial ALS. In this study, we investigate TDP-43 protein behavior in induced pluripotent stem cell (iPSC)-derived motor neurons from three ALS patients with different TARDBP mutations, three healthy controls and an isogenic control. TARDPB mutations induce several TDP-43 changes in spinal motor neurons, including cytoplasmic mislocalization and accumulation of insoluble TDP-43, C-terminal fragments, and phospho-TDP-43. By generating iPSC lines with allele-specific tagging of TDP-43, we find that mutant TDP-43 initiates the observed disease phenotypes and has an altered interactome as indicated by mass spectrometry. Our findings also indicate that TDP-43 proteinopathy results in a defect in mitochondrial transport. Lastly, we show that pharmacological inhibition of histone deacetylase 6 (HDAC6) restores the observed TDP-43 pathologies and the axonal mitochondrial motility, suggesting that HDAC6 inhibition may be an interesting therapeutic target for neurodegenerative disorders linked to TDP-43 pathology.
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http://dx.doi.org/10.15252/embj.2020106177DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8013789PMC
April 2021

Quantitative Nucleocytoplasmic Transport Assays in Cellular Models of Neurodegeneration.

Bio Protoc 2020 Jun 20;10(12):e3659. Epub 2020 Jun 20.

Department of Microbiology, Immunology and Transplantation - Laboratory of Virology and Chemotherapy, KU Leuven - Rega Institute for Medical Research, Leuven, Belgium.

Nucleocytoplasmic transport deficits are suggested to play a role in neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). Given the importance and complexity of this process, understanding when these aberrations occur and which pathways are involved is of great importance. Here, we make use of CRISPR-Cas9 technology to design cell lines stably expressing fluorophore proteins shuttling between the nucleus and cytoplasm by karyopherins of choice. To validate this protocol, we measured an ALS-associated nucleocytoplasmic transport pathway in the presence of the disease-associated peptide poly-PR. This technique allows measuring a particular active nucleocytoplasmic transport pathway in intact cells in a neurodegenerative disease-associated context. Moreover, these experiments can be performed without the need for expensive equipment and have the potential to be upscaled for high-throughput screening purposes.
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http://dx.doi.org/10.21769/BioProtoc.3659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842572PMC
June 2020

Triad of TDP43 control in neurodegeneration: autoregulation, localization and aggregation.

Nat Rev Neurosci 2021 04 2;22(4):197-208. Epub 2021 Mar 2.

Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical Neuroscience Institute, Institute of Psychiatry, Psychology and Neuroscience, King's College London, London, UK.

Cytoplasmic aggregation of TAR DNA-binding protein 43 (TDP43; also known as TARDBP or TDP-43) is a key pathological feature of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). TDP43 typically resides in the nucleus but can shuttle between the nucleus and the cytoplasm to exert its multiple functions, which include regulation of the splicing, trafficking and stabilization of RNA. Cytoplasmic mislocalization and nuclear loss of TDP43 have both been associated with ALS and FTD, suggesting that calibrated levels and correct localization of TDP43 - achieved through an autoregulatory loop and tightly controlled nucleocytoplasmic transport - safeguard its normal function. Furthermore, TDP43 can undergo phase transitions, including its dispersion into liquid droplets and its accumulation into irreversible cytoplasmic aggregates. Thus, autoregulation, nucleocytoplasmic transport and phase transition are all part of an intrinsic control system regulating the physiological levels and localization of TDP43, and together are essential for the cellular homeostasis that is affected in neurodegenerative disease.
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http://dx.doi.org/10.1038/s41583-021-00431-1DOI Listing
April 2021

AAV9-mediated gene delivery of MCT1 to oligodendrocytes does not provide a therapeutic benefit in a mouse model of ALS.

Mol Ther Methods Clin Dev 2021 Mar 20;20:508-519. Epub 2021 Jan 20.

Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI), KU Leuven - University of Leuven, Leuven, Belgium.

Oligodendrocyte dysfunction has been implicated in the pathophysiology of amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder characterized by progressive motor neuron loss. The failure of trophic support provided by oligodendrocytes is associated with a concomitant reduction in oligodendroglial monocarboxylate transporter 1 (MCT1) expression and is detrimental for the long-term survival of motor neuron axons. Therefore, we established an adeno-associated virus 9 (AAV9)-based platform by which MCT1 was targeted mostly to white matter oligodendrocytes to investigate whether this approach could provide a therapeutic benefit in the SOD1 mouse model of ALS. Despite good oligodendrocyte transduction and AAV-mediated MCT1 transgene expression, the disease outcome of SOD1 mice was not altered. Our study further increases our current understanding about the complex nature of oligodendrocyte pathology in ALS and provides valuable insights into the future development of therapeutic strategies to efficiently modulate these cells.
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http://dx.doi.org/10.1016/j.omtm.2021.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878966PMC
March 2021

A generic approach to study the kinetics of liquid-liquid phase separation under near-native conditions.

Commun Biol 2021 01 19;4(1):77. Epub 2021 Jan 19.

VIB-VUB Center for Structural Biology, VIB, Brussels, Belgium.

Understanding the kinetics, thermodynamics, and molecular mechanisms of liquid-liquid phase separation (LLPS) is of paramount importance in cell biology, requiring reproducible methods for studying often severely aggregation-prone proteins. Frequently applied approaches for inducing LLPS, such as dilution of the protein from an urea-containing solution or cleavage of its fused solubility tag, often lead to very different kinetic behaviors. Here we demonstrate that at carefully selected pH values proteins such as the low-complexity domain of hnRNPA2, TDP-43, and NUP98, or the stress protein ERD14, can be kept in solution and their LLPS can then be induced by a jump to native pH. This approach represents a generic method for studying the full kinetic trajectory of LLPS under near native conditions that can be easily controlled, providing a platform for the characterization of physiologically relevant phase-separation behavior of diverse proteins.
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http://dx.doi.org/10.1038/s42003-020-01596-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815728PMC
January 2021

Impact of prolonged sepsis on neural and muscular components of muscle contractions in a mouse model.

J Cachexia Sarcopenia Muscle 2021 04 19;12(2):443-455. Epub 2021 Jan 19.

Clinical Division and Laboratory of Intensive Care Medicine, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium.

Background: Prolonged critically ill patients frequently develop debilitating muscle weakness that can affect both peripheral nerves and skeletal muscle. In-depth knowledge on the temporal contribution of neural and muscular components to muscle weakness is currently incomplete.

Methods: We used a fluid-resuscitated, antibiotic-treated, parenterally fed murine model of prolonged (5 days) sepsis-induced muscle weakness (caecal ligation and puncture; n = 148). Electromyography (EMG) measurements were performed in two nerve-muscle complexes, combined with histological analysis of neuromuscular junction denervation, axonal degeneration, and demyelination. In situ muscle force measurements distinguished neural from muscular contribution to reduced muscle force generation. In myofibres, imaging and biomechanics were combined to evaluate myofibrillar contractile calcium sensitivity, sarcomere organization, and fibre structural properties. Myosin and actin protein content and titin gene expression were measured on the whole muscle.

Results: Five days of sepsis resulted in increased EMG latency (P = 0.006) and decreased EMG amplitude (P < 0.0001) in the dorsal caudal tail nerve-tail complex, whereas only EMG amplitude was affected in the sciatic nerve-gastrocnemius muscle complex (P < 0.0001). Myelin sheath abnormalities (P = 0.2), axonal degeneration (number of axons; P = 0.4), and neuromuscular junction denervation (P = 0.09) were largely absent in response to sepsis, but signs of axonal swelling [higher axon area (P < 0.0001) and g-ratio (P = 0.03)] were observed. A reduction in maximal muscle force was present after indirect nerve stimulation (P = 0.007) and after direct muscle stimulation (P = 0.03). The degree of force reduction was similar with both stimulations (P = 0.2), identifying skeletal muscle, but not peripheral nerves, as the main contributor to muscle weakness. Myofibrillar calcium sensitivity of the contractile apparatus was unaffected by sepsis (P ≥ 0.6), whereas septic myofibres displayed disorganized sarcomeres (P < 0.0001) and altered myofibre axial elasticity (P < 0.0001). Septic myofibres suffered from increased rupturing in a passive stretching protocol (25% more than control myofibres; P = 0.04), which was associated with impaired myofibre active force generation (P = 0.04), linking altered myofibre integrity to function. Sepsis also caused a reduction in muscle titin gene expression (P = 0.04) and myosin and actin protein content (P = 0.05), but not the myosin-to-actin ratio (P = 0.7).

Conclusions: Prolonged sepsis-induced muscle weakness may predominantly be related to a disruption in myofibrillar cytoarchitectural structure, rather than to neural abnormalities.
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http://dx.doi.org/10.1002/jcsm.12668DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8061378PMC
April 2021

Spatiotemporal Proteomic Analysis of Stress Granule Disassembly Using APEX Reveals Regulation by SUMOylation and Links to ALS Pathogenesis.

Mol Cell 2020 12 19;80(5):876-891.e6. Epub 2020 Nov 19.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel. Electronic address:

Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics. We discover 109 novel SG proteins and characterize distinct SG substructures. We reveal dozens of disassembly-engaged proteins (DEPs), some of which play functional roles in SG disassembly, including small ubiquitin-like modifier (SUMO) conjugating enzymes. We further demonstrate that SUMOylation regulates SG disassembly and SG formation. Parallel proteomics with amyotrophic lateral sclerosis (ALS)-associated C9ORF72 dipeptides uncovered attenuated DEP recruitment during SG disassembly and impaired SUMOylation. Accordingly, SUMO activity ameliorated C9ORF72-ALS-related neurodegeneration in Drosophila. By dissecting the SG spatiotemporal proteomic landscape, we provide an in-depth resource for future work on SG function and reveal basic and disease-relevant mechanisms of SG disassembly.
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http://dx.doi.org/10.1016/j.molcel.2020.10.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816607PMC
December 2020

TDP-43 proteinopathies: a new wave of neurodegenerative diseases.

J Neurol Neurosurg Psychiatry 2020 11 11. Epub 2020 Nov 11.

Westmead Clinical School, University of Sydney, Sydney, New South Wales, Australia

Inclusions of pathogenic deposits containing TAR DNA-binding protein 43 (TDP-43) are evident in the brain and spinal cord of patients that present across a spectrum of neurodegenerative diseases. For instance, the majority of patients with sporadic amyotrophic lateral sclerosis (up to 97%) and a substantial proportion of patients with frontotemporal lobar degeneration (~45%) exhibit TDP-43 positive neuronal inclusions, suggesting a role for this protein in disease pathogenesis. In addition, TDP-43 inclusions are evident in familial ALS phenotypes linked to multiple gene mutations including the TDP-43 gene coding () and unrelated genes (eg, ). While TDP-43 is an essential RNA/DNA binding protein critical for RNA-related metabolism, determining the pathophysiological mechanisms through which TDP-43 mediates neurodegeneration appears complex, and unravelling these molecular processes seems critical for the development of effective therapies. This review highlights the key physiological functions of the TDP-43 protein, while considering an expanding spectrum of neurodegenerative diseases associated with pathogenic TDP-43 deposition, and dissecting key molecular pathways through which TDP-43 may mediate neurodegeneration.
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http://dx.doi.org/10.1136/jnnp-2020-322983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803890PMC
November 2020

Role and therapeutic potential of liquid-liquid phase separation in amyotrophic lateral sclerosis.

J Mol Cell Biol 2021 04;13(1):15-28

Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI), KU Leuven-University of Leuven, 3000 Leuven, Belgium.

Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disease selectively affecting motor neurons, leading to progressive paralysis. Although most cases are sporadic, ∼10% are familial. Similar proteins are found in aggregates in sporadic and familial ALS, and over the last decade, research has been focused on the underlying nature of this common pathology. Notably, TDP-43 inclusions are found in almost all ALS patients, while FUS inclusions have been reported in some familial ALS patients. Both TDP-43 and FUS possess 'low-complexity domains' (LCDs) and are considered as 'intrinsically disordered proteins', which form liquid droplets in vitro due to the weak interactions caused by the LCDs. Dysfunctional 'liquid-liquid phase separation' (LLPS) emerged as a new mechanism linking ALS-related proteins to pathogenesis. Here, we review the current state of knowledge on ALS-related gene products associated with a proteinopathy and discuss their status as LLPS proteins. In addition, we highlight the therapeutic potential of targeting LLPS for treating ALS.
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http://dx.doi.org/10.1093/jmcb/mjaa049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8036000PMC
April 2021

repeat expansions confer risk for amyotrophic lateral sclerosis and contribute to TDP-43 mislocalization.

Brain Commun 2020 19;2(2):fcaa064. Epub 2020 May 19.

Department of Basic and Clinical Neuroscience, Maurice Wohl Clinical Neuroscience Institute and United Kingdom Dementia Research Institute, King's College London, London SE5 9NU, UK.

Increasingly, repeat expansions are being identified as part of the complex genetic architecture of amyotrophic lateral sclerosis. To date, several repeat expansions have been genetically associated with the disease: intronic repeat expansions in , polyglutamine expansions in and polyalanine expansions in . Together with previously published data, the identification of an amyotrophic lateral sclerosis patient with a family history of spinocerebellar ataxia type 1, caused by polyglutamine expansions in , suggested a similar disease association for the repeat expansion in . We, therefore, performed a large-scale international study in 11 700 individuals, in which we showed a significant association between intermediate repeat expansions and amyotrophic lateral sclerosis (=3.33 × 10). Subsequent functional experiments have shown that ATXN1 reduces the nucleocytoplasmic ratio of TDP-43 and enhances amyotrophic lateral sclerosis phenotypes in , further emphasizing the role of polyglutamine repeat expansions in the pathophysiology of amyotrophic lateral sclerosis.
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http://dx.doi.org/10.1093/braincomms/fcaa064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7425293PMC
May 2020

C9orf72 loss-of-function: a trivial, stand-alone or additive mechanism in C9 ALS/FTD?

Acta Neuropathol 2020 11 2;140(5):625-643. Epub 2020 Sep 2.

Department of Neurosciences, Experimental Neurology, and Leuven Brain Institute (LBI), KU Leuven-University of Leuven, 3000, Leuven, Belgium.

A repeat expansion in C9orf72 is responsible for the characteristic neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in a still unresolved manner. Proposed mechanisms involve gain-of-functions, comprising RNA and protein toxicity, and loss-of-function of the C9orf72 gene. Their exact contribution is still inconclusive and reports regarding loss-of-function are rather inconsistent. Here, we review the function of the C9orf72 protein and its relevance in disease. We explore the potential link between reduced C9orf72 levels and disease phenotypes in postmortem, in vitro, and in vivo models. Moreover, the significance of loss-of-function in other non-coding repeat expansion diseases is used to clarify its contribution in C9orf72 ALS/FTD. In conclusion, with evidence pointing to a multiple-hit model, loss-of-function on itself seems to be insufficient to cause neurodegeneration in C9orf72 ALS/FTD.
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http://dx.doi.org/10.1007/s00401-020-02214-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547039PMC
November 2020

Opportunities for histone deacetylase inhibition in amyotrophic lateral sclerosis.

Br J Pharmacol 2021 03 26;178(6):1353-1372. Epub 2020 Aug 26.

Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI), KU Leuven-University of Leuven, Leuven, Belgium.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. ALS patients suffer from a progressive loss of motor neurons, leading to respiratory failure within 3 to 5 years after diagnosis. Available therapies only slow down the disease progression moderately or extend the lifespan by a few months. Epigenetic hallmarks have been linked to the disease, creating an avenue for potential therapeutic approaches. Interference with one class of epigenetic enzymes, histone deacetylases, has been shown to affect neurodegeneration in many preclinical models. Consequently, it is crucial to improve our understanding about histone deacetylases and their inhibitors in (pre)clinical models of ALS. We conclude that selective inhibitors with high tolerability and safety and sufficient blood-brain barrier permeability will be needed to interfere with both epigenetic and non-epigenetic targets of these enzymes. LINKED ARTICLES: This article is part of a themed issue on Neurochemistry in Japan. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.6/issuetoc.
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http://dx.doi.org/10.1111/bph.15217DOI Listing
March 2021

Focus on the heterogeneity of amyotrophic lateral sclerosis.

Amyotroph Lateral Scler Frontotemporal Degener 2020 11 25;21(7-8):485-495. Epub 2020 Jun 25.

Mario Negri-ALS Study Group, Istituto di Ricerche Farmacologiche Mario Negri IRCCS, Milano, Italy.

The clinical manifestations of amyotrophic lateral sclerosis (ALS) are variable in terms of age at disease onset, site of onset, progression of symptoms, motor neuron involvement, and the occurrence of cognitive and behavioral changes. Genetic background is a key determinant of the ALS phenotype. The mortality of the disease also varies with the ancestral origin of the affected population and environmental factors are likely to be associated with ALS at least within some cohorts. Disease heterogeneity is likely underpinned by the presence of different pathogenic mechanisms. A variety of ALS animal models can be informative about the heterogeneity of the neuropathological or genetic aspects of the disease and can support the development of new therapeutic intervention. Evolving biomarkers can contribute to the identification of differing genotypes and phenotypes, and can be used to explore whether genotypic and phenotypic differences in animal models might help to provide a better definition of the heterogeneity of ALS in humans. These include neurofilaments, peripheral blood mononuclear cells, extracellular vesicles, microRNA and imaging findings. These biomarkers might predict not only the development of the disease, but also the variability in progression, although robust validation is required. A promising area of progress in modeling the heterogeneity of human ALS is represented by the use of human induced pluripotent stem cell (iPSCs)-derived motor neurons. Although the translational value of iPSCs remains unclear, this model is attractive in the perspective of replicating the heterogeneity of sporadic ALS as a first step toward a personalized medicine strategy.
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http://dx.doi.org/10.1080/21678421.2020.1779298DOI Listing
November 2020

The multifaceted role of kinases in amyotrophic lateral sclerosis: genetic, pathological and therapeutic implications.

Brain 2020 06;143(6):1651-1673

KU Leuven-University of Leuven, Department of Neurosciences, Experimental Neurology, and Leuven Brain Institute (LBI), Leuven, Belgium.

Amyotrophic lateral sclerosis is the most common degenerative disorder of motor neurons in adults. As there is no cure, thousands of individuals who are alive at present will succumb to the disease. In recent years, numerous causative genes and risk factors for amyotrophic lateral sclerosis have been identified. Several of the recently identified genes encode kinases. In addition, the hypothesis that (de)phosphorylation processes drive the disease process resulting in selective motor neuron degeneration in different disease variants has been postulated. We re-evaluate the evidence for this hypothesis based on recent findings and discuss the multiple roles of kinases in amyotrophic lateral sclerosis pathogenesis. We propose that kinases could represent promising therapeutic targets. Mainly due to the comprehensive regulation of kinases, however, a better understanding of the disturbances in the kinome network in amyotrophic lateral sclerosis is needed to properly target specific kinases in the clinic.
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http://dx.doi.org/10.1093/brain/awaa022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296858PMC
June 2020

CMT2Q-causing mutation in the Dhtkd1 gene lead to sensory defects, mitochondrial accumulation and altered metabolism in a knock-in mouse model.

Acta Neuropathol Commun 2020 03 13;8(1):32. Epub 2020 Mar 13.

Department of Medical Genetics, E-Institutes of Shanghai Universities, Shanghai Jiao Tong University School of Medicine (SJTUSM), 280 South Chongqing Road, Shanghai, 200025, People's Republic of China.

Charcot-Marie-Tooth disease (CMT) is a group of inherited neurological disorders of the peripheral nervous system. CMT is subdivided into two main types: a demyelinating form, known as CMT1, and an axonal form, known as CMT2. Nearly 30 genes have been identified as a cause of CMT2. One of these is the 'dehydrogenase E1 and transketolase domain containing 1' (DHTKD1) gene. We previously demonstrated that a nonsense mutation [c.1455 T > G (p.Y485*)] in exon 8 of DHTKD1 is one of the disease-causing mutations in CMT2Q (MIM 615025). The aim of the current study was to investigate whether human disease-causing mutations in the Dhtkd1 gene cause CMT2Q phenotypes in a mouse model in order to investigate the physiological function and pathogenic mechanisms associated with mutations in the Dhtkd1 gene in vivo. Therefore, we generated a knock-in mouse model with the Dhtkd1 point mutation. We observed that the Dhtkd1 expression level in sciatic nerve of knock-in mice was significantly lower than in wild-type mice. Moreover, a histopathological phenotype was observed, reminiscent of a peripheral neuropathy, including reduced large axon diameter and abnormal myelination in peripheral nerves. The knock-in mice also displayed clear sensory defects, while no abnormalities in the motor performance were observed. In addition, accumulation of mitochondria and an elevated energy metabolic state was observed in the knock-in mice. Taken together, our study indicates that the Dhtkd1 knock-in mice partially recapitulate the clinical phenotypes of CMT2Q patients and we hypothesize that there might be a compensatory effect from the elevated metabolic state in the knock-in mice that enables them to maintain their normal locomotor function.
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http://dx.doi.org/10.1186/s40478-020-00901-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7071680PMC
March 2020

HDAC6 inhibitors: Translating genetic and molecular insights into a therapy for axonal CMT.

Brain Res 2020 04 30;1733:146692. Epub 2020 Jan 30.

KU Leuven - University of Leuven, Department of Neurosciences, Experimental Neurology and Leuven Brain Institute (LBI), Leuven, Belgium; VIB - Center for Brain & Disease Research, Laboratory of Neurobiology, Leuven, Belgium. Electronic address:

Histone deacetylase 6 (HDAC6) plays a central role in various processes that are key for neuronal survival. In this review, we summarize the current evidence related to disease pathways in the axonal form of Charcot-Marie-Tooth disease (CMT) and highlight the role of HDAC6 in these pathways. We hypothesize that HDAC6 might in fact actively contribute to the pathogenesis of certain forms of axonal CMT. HDAC6 plays a deacetylase activity-dependent, negative role in axonal transport and axonal regeneration, which are both processes implicated in axonal CMT. On the other hand, HDAC6 coordinates a protective response during elimination of toxic misfolded proteins, but this is mostly mediated independent of its deacetylase activity. The current mechanistic insights on these functions of HDAC6 in axonal CMT, along with the selective druggability against its deacetylase activity, make the targeting of HDAC6 particularly attractive. We elaborate on the preclinical studies that demonstrated beneficial effects of HDAC6 inhibitors in axonal CMT models and outline possible modes of action. Overall, this overview ultimately provides a rationale for the use of small-molecule HDAC6 inhibitors as a therapeutic strategy for this devastating disease.
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http://dx.doi.org/10.1016/j.brainres.2020.146692DOI Listing
April 2020

EphA4 loss improves social memory performance and alters dendritic spine morphology without changes in amyloid pathology in a mouse model of Alzheimer's disease.

Alzheimers Res Ther 2019 12 12;11(1):102. Epub 2019 Dec 12.

Department of Neurosciences, Experimental Neurology, and Leuven Brain Institute (LBI), KU Leuven - University of Leuven, Leuven, Belgium.

Background: EphA4 is a receptor of the ephrin system regulating spine morphology and plasticity in the brain. These processes are pivotal in the pathophysiology of Alzheimer's disease (AD), characterized by synapse dysfunction and loss, and the progressive loss of memory and other cognitive functions. Reduced EphA4 signaling has been shown to rescue beta-amyloid-induced dendritic spine loss and long-term potentiation (LTP) deficits in cultured hippocampal slices and primary hippocampal cultures. In this study, we investigated whether EphA4 ablation might preserve synapse function and ameliorate cognitive performance in the APPPS1 transgenic mouse model of AD.

Methods: A postnatal genetic ablation of EphA4 in the forebrain was established in the APPPS1 mouse model of AD, followed by a battery of cognitive tests at 9 months of age to investigate cognitive function upon EphA4 loss. A Golgi-Cox staining was used to explore alterations in dendritic spine density and morphology in the CA1 region of the hippocampus.

Results: Upon EphA4 loss in APPPS1 mice, we observed improved social memory in the preference for social novelty test without affecting other cognitive functions. Dendritic spine analysis revealed altered synapse morphology as characterized by increased dendritic spine length and head width. These modifications were independent of hippocampal plaque load and beta-amyloid peptide levels since these were similar in mice with normal versus reduced levels of EphA4.

Conclusion: Loss of EphA4 improved social memory in a mouse model of Alzheimer's disease in association with alterations in spine morphology.
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http://dx.doi.org/10.1186/s13195-019-0554-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6909519PMC
December 2019
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