bims-axbals 2024-11-24 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2024–11–24
twenty papers selected by
TJ Krzystek, ALS Therapy Development Institute

Regulation of TAR DNA binding protein 43 (TDP-43) homeostasis by cytosolic DNA accumulation.
Autophagic stress activates distinct compensatory secretory pathways in neurons.
CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.
Type-II kinase inhibitors that target Parkinson's Disease-associated LRRK2.
PINK1 controls RTN3L-mediated ER autophagy by regulating peripheral tubule junctions.
A Novel Human TBCK- Neuronal Cell Model Results in Severe Neurodegeneration and Partial Rescue with Mitochondrial Fission Inhibition.
Elevated Cerebrospinal Fluid Ubiquitin Carboxyl-Terminal Hydrolase Isozyme L1 in Asymptomatic C9orf72 Hexanucleotide Repeat Expansion Carriers.
Genipin rescues developmental and degenerative defects in familial dysautonomia models and accelerates axon regeneration.
Neuromuscular Organoids to Study Spinal Cord Development and Disease.
Editorial: Human brain organoids to model neurodegenerative diseases at the BOSS23 Brain Organoid Summer School.
Development and validation a novel FEZF2 based fluorescent reporter for corticospinal motor neurons.
Synchronized motion of interface residues for evaluating protein-RNA complex binding affinity: Application to aptamer-mediated inhibition of TDP-43 aggregates.
Cerebrospinal fluid levels of NfM in relation to NfL and pNfH as prognostic markers in amyotrophic lateral sclerosis.
Overexpression of autophagy enhancer PACER/RUBCNL in neurons accelerates disease in the SOD1G93A ALS mouse model.
Reprogrammed human lateral ganglionic eminence precursors generate striatal neurons and restore motor function in a rat model of Huntington's disease.
Impact of Ageing and Disuse on Neuromuscular Junction and Mitochondrial Function and Morphology: Current Evidence and Controversies.
In vitro electrophysiological drug testing on neuronal networks derived from human induced pluripotent stem cells.
Optimized simple culture protocol for inducing mature myotubes from MYOD1-overexpressed human iPS cells.
Salidroside facilitates neuroprotective effects in ischemic stroke by promoting axonal sprouting through promoting autophagy.
Human iPSC-derived microglia sense and dampen hyperexcitability of cortical neurons carrying the epilepsy-associated SCN2A-L1342P mutation.

J Biol Chem. 2024 Nov 15. pii: S0021-9258(24)02501-8. [Epub ahead of print] 107999

Regulation of TAR DNA binding protein 43 (TDP-43) homeostasis by cytosolic DNA accumulation.

Cha Yang, Cynthia Leifer, Jan Lammerding, Fenghua Hu.

  TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA binding protein predominantly localized in the nucleus under physiological conditions. TDP-43 proteinopathy, characterized by cytoplasmic aggregation and nuclear loss, is associated with many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Thus it is crucial to understand the molecular mechanism regulating TDP-43 homeostasis. Here, we show that the uptake of oligodeoxynucleotides (ODNs) induces reversible TDP-43 cytoplasmic puncta formation in both neurons and glia and ODNs facilitate the liquid-liquid phase separation of TDP-43 in vitro. Importantly, persistent accumulation of DNA in the cytoplasm leads to nuclear depletion of TDP-43 and enhanced production of a short isoform of TDP-43 (sTDP-43). In addition, in response to ODN uptake, the nuclear import receptor karyopherin subunit β1 (KPNB1) is sequestered in the cytosolic TDP-43 puncta. ALS-linked Q331K mutation decreases the dynamics of cytoplasmic TDP-43 puncta and increases the levels of sTDP-43. Moreover, the TDP-43 cytoplasmic puncta are induced by DNA damage and by impaired nuclear envelope integrity due to Lamin A/C deficiency. In summary, our data support that abnormal DNA accumulation in the cytoplasm may be one of the key mechanisms leading to TDP-43 proteinopathy and provides novel insights into molecular mechanisms of ALS caused by TDP-43 mutations.

Keywords:  DNA molecules; Lamin; TDP-43; phase separation; proteinopathy

DOI:  https://doi.org/10.1016/j.jbc.2024.107999
bioRxiv. 2024 Nov 07. pii: 2024.11.07.621551. [Epub ahead of print]

Autophagic stress activates distinct compensatory secretory pathways in neurons.

Sierra D Palumbos, Jacob Popolow, Juliet Goldsmith, Erika L F Holzbaur.

Autophagic dysfunction is a hallmark of neurodegenerative disease, leaving neurons vulnerable to the accumulation of damaged organelles and proteins. However, the late onset of diseases suggests that compensatory quality control mechanisms may be engaged to delay the deleterious effects induced by compromised autophagy. Neurons expressing common familial Parkinson's disease (PD)-associated mutations in LRRK2 kinase exhibit defective autophagy. Here, we demonstrate that both primary murine neurons and human iPSC-derived neurons harboring pathogenic LRRK2 upregulate the secretion of extracellular vesicles. We used unbiased proteomics to characterize the secretome of LRRK2 G2019S neurons and found that autophagic cargos including mitochondrial proteins were enriched. Based on these observations, we hypothesized that autophagosomes are rerouted toward secretion when cell-autonomous degradation is compromised, likely to mediate clearance of undegraded cellular waste. Immunoblotting confirmed the release of autophagic cargos and immunocytochemistry demonstrated that secretory autophagy was upregulated in LRRK2 G2019S neurons. We also found that LRRK2 G2019S neurons upregulate the release of exosomes containing miRNAs. Live-cell imaging confirmed that this upregulation of exosomal release was dependent on hyperactive LRRK2 activity, while pharmacological experiments indicate that this release staves off apoptosis. Finally, we show that markers of both vesicle populations are upregulated in plasma from mice expressing pathogenic LRRK2. In sum, we find that neurons expressing pathogenic LRRK2 upregulate the compensatory release of secreted autophagosomes and exosomes, to mediate waste disposal and transcellular communication, respectively. We propose that this increased secretion contributes to the maintenance of cellular homeostasis, delaying neurodegenerative disease progression over the short term while potentially contributing to increased neuroinflammation over the longer term.
SIGNIFICANCE: A hallmark feature of many neurodegenerative diseases is autophagy dysfunction, resulting in the accumulation of damaged proteins and organelles that is detrimental to neuronal health. The late onset of neurodegenerative diseases, however, suggests alternative quality control mechanisms may delay neuronal degeneration. Here, we demonstrate that neurons expressing a Parkinson's Disease-causing mutation upregulate the release of two extracellular vesicle populations. First, we observe the increased expulsion of secreted autophagosomes to mediate cellular waste disposal. Second, we observe the increased release of exosomes, likely to facilitate transcellular communication. Thus, we propose that increases in secretory autophagy and exosome release are a homeostatic response in neurons undergoing chronic stress.

DOI: https://doi.org/10.1101/2024.11.07.621551
Sci Adv. 2024 Nov 22. 10(47): eadn5417

CalDAG-GEFI acts as a guanine nucleotide exchange factor for LRRK2 to regulate LRRK2 function and neurodegeneration.

Qinfang Liu, Bingxu Huang, Noah Guy Lewis Guiberson, Shifan Chen, Dong Zhu, Gang Ma, Xin-Ming Ma, Jill R Crittenden, Jianzhong Yu, Ann M Graybiel, Ted M Dawson, Valina L Dawson, Yulan Xiong.

Mutations in LRRK2 are the most common genetic cause of Parkinson's disease (PD). LRRK2 protein contains two enzymatic domains: a GTPase (Roc-COR) and a kinase domain. Disease-causing mutations are found in both domains. Now, studies have focused largely on LRRK2 kinase activity, while attention to its GTPase function is limited. LRRK2 is a guanine nucleotide-binding protein, but the mechanism of direct regulation of its GTPase activity remains unclear and its physiological GEF is not known. Here, we identified CalDAG-GEFI (CDGI) as a physiological GEF for LRRK2. CDGI interacts with LRRK2 and increases its GDP to GTP exchange activity. CDGI modulates LRRK2 cellular functions and LRRK2-induced neurodegeneration in both LRRK2 Drosophila and mouse models. Together, this study identified the physiological GEF for LRRK2 and provides strong evidence that LRRK2 GTPase is regulated by GAPs and GEFs. The LRRK2 GTPase, GAP, or GEF activities have the potential to serve as therapeutic targets, which is distinct from the direct LRRK2 kinase inhibition.

DOI: https://doi.org/10.1126/sciadv.adn5417
bioRxiv. 2024 Oct 31. pii: 2024.09.17.613365. [Epub ahead of print]

Type-II kinase inhibitors that target Parkinson's Disease-associated LRRK2.

Nicolai D Raig, Katherine J Surridge, Marta Sanz-Murillo, Verena Dederer, Andreas Krämer, Martin P Schwalm, Lewis Elson, Deep Chatterjee, Sebastian Mathea, Thomas Hanke, Andres E Leschziner, Samara L Reck-Peterson, Stefan Knapp.

Aberrant increases in kinase activity of leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease (PD). Numerous LRRK2-selective type-I kinase inhibitors have been developed and some have entered clinical trials. In this study, we present the first LRRK2-selective type-II kinase inhibitors. Targeting the inactive conformation of LRRK2 is functionally distinct from targeting the active-like conformation using type-I inhibitors. We designed these inhibitors using a combinatorial chemistry approach fusing selective LRRK2 type-I and promiscuous type-II inhibitors by iterative cycles of synthesis supported by structural biology and activity testing. Our current lead structures are selective and potent LRRK2 inhibitors. Through cellular assays, cryo-electron microscopy structural analysis, and in vitro motility assays, we show that our inhibitors stabilize the open, inactive kinase conformation. These new conformation-specific compounds will be invaluable as tools to study LRRK2's function and regulation, and expand the potential therapeutic options for PD.

DOI: https://doi.org/10.1101/2024.09.17.613365
J Cell Biol. 2024 Dec 02. pii: e202407193. [Epub ahead of print]223(12):

PINK1 controls RTN3L-mediated ER autophagy by regulating peripheral tubule junctions.

Ravi Chidambaram, Kamal Kumar, Smriti Parashar, Gowsalya Ramachandran, Shuliang Chen, Susan Ferro-Novick.

Here, we report that the RTN3L-SEC24C endoplasmic reticulum autophagy (ER-phagy) receptor complex, the CUL3KLHL12 E3 ligase that ubiquitinates RTN3L, and the FIP200 autophagy initiating protein, target mutant proinsulin (Akita) condensates for lysosomal delivery at ER tubule junctions. When delivery was blocked, Akita condensates accumulated in the ER. In exploring the role of tubulation in these events, we unexpectedly found that loss of the Parkinson's disease protein, PINK1, reduced peripheral tubule junctions and blocked ER-phagy. Overexpression of the PINK1 kinase substrate, DRP1, increased junctions, reduced Akita condensate accumulation, and restored lysosomal delivery in PINK1-depleted cells. DRP1 is a dual-functioning protein that promotes ER tubulation and severs mitochondria at ER-mitochondria contact sites. DRP1-dependent ER tubulating activity was sufficient for suppression. Supporting these findings, we observed PINK1 associating with ER tubules. Our findings show that PINK1 shapes the ER to target misfolded proinsulin for RTN3L-SEC24C-mediated macro-ER-phagy at defined ER sites called peripheral junctions. These observations may have important implications for understanding Parkinson's disease.

DOI: https://doi.org/10.1083/jcb.202407193
bioRxiv. 2024 Oct 31. pii: 2024.10.30.621078. [Epub ahead of print]

A Novel Human TBCK- Neuronal Cell Model Results in Severe Neurodegeneration and Partial Rescue with Mitochondrial Fission Inhibition.

Rajesh Angireddy, Bhanu Chandra Karisetty, Kaitlin A Katsura, Abdias Díaz, Svathi Murali, Sarina Smith, Laura Ohl, Kelly Clark, Andrew V Kossenkov, Elizabeth J K Bhoj.

Background and Objectives: TBCK syndrome is a rare fatal pediatric neurodegenerative disease caused by biallelic loss-of-function mutations in the TBCK gene. Previous studies by our lab and others have implicated mTOR, autophagy, lysosomes, and intracellular mRNA transport, however the exact primary pathologic mechanism is unknown. This gap has prevented the development of targeted therapies.
Methods: We employed a human neural progenitor cell line (NPC), ReNcell VM, which can differentiate into neurons and astrocytes, to understand the role of TBCK in mTORC1 activity and neuronal autophagy and cellular mechanisms of pathology. We used shRNA technology to knockdown TBCK in ReNcells.
Results: These data showed that loss of TBCK did not inhibit mTORC1 activity in neither NPC nor neurons. Additionally, analysis of eight patient-derived cells and TBCK knock down HeLa cells showed that mTORC1 inhibition is inconsistent across different patients and cell types. We showed that TBCK knockdown in ReNcells affected NPC differentiation to neurons and astrocytes. Specifically, differentiation defects are coupled to cell cycle defects in NPC and increased cell death during differentiation. RNAseq analysis indicated the downregulation of several different neurodevelopmental and differentiation pathways. We observed a higher number of LC3-positive vesicles in the soma and neurites of TBCK knockdown cells. Further, TBCK knockdown altered mitochondrial dynamics and membrane potential in NPC, neurons and astrocytes. We found partial mitochondrial rescue with the mitochondrial fission inhibitor mdivi- 1.
Discussion: This work outlines a new Human Cell Model for TBCK-related neurodegeneration and the essential role of mitochondrial health and partial rescue with mitochondrial fission inhibitor. This data, along with human neurons and astrocytes, illuminate mechanisms of neurodegeneration and provide a possible novel therapeutic avenue for affected patients.

DOI: https://doi.org/10.1101/2024.10.30.621078
Ann Neurol. 2024 Nov 16.

Elevated Cerebrospinal Fluid Ubiquitin Carboxyl-Terminal Hydrolase Isozyme L1 in Asymptomatic C9orf72 Hexanucleotide Repeat Expansion Carriers.

Elizabeth R Dellar, Iolanda Vendrell, Benazir Amein, David G Lester, Evan C Edmond, Katie Yoganathan, Thanuja Dharmadasa, Aitana Sogorb-Esteve, Roman Fischer, Kevin Talbot, Jonathan D Rohrer, Martin R Turner, Alexander G Thompson.

OBJECTIVE: To identify biochemical changes in individuals at higher risk of developing amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD) via C9orf72 hexanucleotide repeat expansion (HRE) heterozygosity.
METHODS: Cross-sectional observational study of 48 asymptomatic C9orf72 HRE carriers, 39 asymptomatic non-carrier controls, 19 people with sporadic ALS, 10 with C9orf72 ALS, 14 with sporadic FTD, and 10 with C9orf72 FTD. Relative abundance of 30 pre-defined cerebrospinal fluid biomarkers of ALS and FTD were compared in asymptomatic C9orf72 HRE carriers and age-matched non-carrier controls. Differential abundance of these proteins was quantified using data independent acquisition mass spectrometry or electro chemiluminescent assay for neurofilament light chain. Unbiased analysis of the entire cerebrospinal fluid proteome was then carried out.
RESULTS: Ubiquitin carboxyl-hydrolase isozyme L1 levels were higher in asymptomatic C9orf72 HRE carriers compared with age-matched non-carriers (log2fold change 0.20, FDR-adjusted p-value = 0.034), whereas neurofilament light chain levels did not significantly differ. Ubiquitin carboxyl-hydrolase isozyme L1 levels remained elevated after matching of groups by neurofilament levels (p = 0.011), and after adjusting for age, sex, and neurofilament levels. A significant difference was also observed when restricting analysis to younger participants (<37) matched by neurofilament level (p = 0.007).
INTERPRETATION: Elevated cerebrospinal fluid ubiquitin carboxyl-hydrolase isozyme L1 levels in C9orf72 HRE carriers can occur in the absence of increased neurofilament levels, potentially reflecting either compensatory or pathogenic mechanisms preceding rapid neuronal loss. This brings forward the window on changes associated with the C9orf72 HRE carrier state, with potential to inform understanding of penetrance and approaches to prevention. ANN NEUROL 2024.

DOI: https://doi.org/10.1002/ana.27133
Sci Transl Med. 2024 Nov 20. 16(774): eadq2418

Genipin rescues developmental and degenerative defects in familial dysautonomia models and accelerates axon regeneration.

Kenyi Saito-Diaz, Paula Dietrich, Tripti Saini, Md Mamunur Rashid, Hsueh-Fu Wu, Mohamed Ishan, Xin Sun, Sydney Bedillion, Archie Jayesh Patel, Anthony Robert Prudden, Camryn Gale Wzientek, Trinity Nora Knight, Ya-Wen Chen, Geert-Jan Boons, Shuibing Chen, Lorenz Studer, Michael Tiemeyer, Bingqian Xu, Ioannis Dragatsis, Hong-Xiang Liu, Nadja Zeltner.

The peripheral nervous system (PNS) is essential for proper body function. A high percentage of the world's population suffers from nerve degeneration or peripheral nerve damage. Despite this, there are major gaps in the knowledge of human PNS development and degeneration; therefore, there are no available treatments. Familial dysautonomia (FD) is a devastating disorder caused by a homozygous point mutation in the gene ELP1. FD specifically affects the development and causes degeneration of the PNS. We previously used patient-derived induced pluripotent stem cells (iPSCs) to show that peripheral sensory neurons (SNs) recapitulate the developmental and neurodegenerative defects observed in FD. Here, we conducted a chemical screen to identify compounds that rescue the SN differentiation inefficiency in FD. We identified that genipin restores neural crest and SN development in patient-derived iPSCs and in two mouse models of FD. Additionally, genipin prevented FD degeneration in SNs derived from patients with FD, suggesting that it could be used to ameliorate neurodegeneration. Moreover, genipin cross-linked the extracellular matrix (ECM), increased the stiffness of the ECM, reorganized the actin cytoskeleton, and promoted transcription of yes-associated protein-dependent genes. Last, genipin enhanced axon regeneration in healthy sensory and sympathetic neurons (part of the PNS) and in prefrontal cortical neurons (part of the central nervous system) in in vitro axotomy models. Our results suggest that genipin has the potential to treat FD-related neurodevelopmental and neurodegenerative phenotypes and to enhance neuronal regeneration of healthy neurons after injury. Moreover, this suggests that the ECM can be targeted to treat FD.

DOI: https://doi.org/10.1126/scitranslmed.adq2418
Methods Mol Biol. 2024 Nov 22.

Neuromuscular Organoids to Study Spinal Cord Development and Disease.

Tobias Grass, Zeynep Dokuzluoglu, Natalia Rodríguez-Muela.

  Many aspects of neurodegenerative disease pathology remain unresolved. Why do certain neuronal subpopulations acquire vulnerability to stress or mutations in ubiquitously expressed genes, while others remain resilient? Do these neurons harbor intrinsic marks that make them prone to degeneration? Do these diseases have a neurodevelopmental component? Lacking this fundamental knowledge hampers the discovery of efficacious treatments. While it is well established that human organoids enable the modeling of brain-related diseases, we still lack an organoid model that recapitulates the regionalization complexity and physiology of the spinal cord. Here, we describe an advanced experimental protocol to generate neuromuscular organoids composed of a wide rostro-caudal (RC) diversity of spinal motor neurons (spMNs) and mesodermal progenitor-derived muscle cells. This model therefore allows for the robust and reproducible study of neuromuscular unit development and disease.

Keywords:  Human induced pluripotent stem cells; Neuromesodermal progenitors; Neuromuscular spinal cord organoids; Skeletal and smooth muscle; Spinal motor neurons

DOI:  https://doi.org/10.1007/7651_2024_574
Front Cell Neurosci. 2024 ;18 1501036

Editorial: Human brain organoids to model neurodegenerative diseases at the BOSS23 Brain Organoid Summer School.

Fabio Cavaliere, Dirk M Hermann, Chiara Magliaro.



Keywords:  Alzheimer's disease; Amyotrophic lateral sclerosis; Parkinson's disease; brain organoids; neurodegenerative diseases

DOI:  https://doi.org/10.3389/fncel.2024.1501036
Metab Brain Dis. 2024 Nov 19. 40(1): 17

Development and validation a novel FEZF2 based fluorescent reporter for corticospinal motor neurons.

Ronghua Wu, Skandha Ramakrishnan, Haixu Lin, Zhangji Dong, Mei Liu, Liang Qiang.

  Corticospinal motor neurons (CSMNs), also named upper motor neurons, are the giant pyramidal neurons called Betz cells. In mammals, the majority of CSMNs reside within layer V of the primary motor cortex, where they extend long axon bundles named the pyramidal tract into the brainstem and the spinal cord to control voluntary movement. CSMN lesions are implicated in a variety of neurodegenerative disorders, such Amyotrophic Lateral Sclerosis, Primary Lateral Sclerosis and Hereditary Spastic paraplegia. Although FEZF2-CTIP2 genetic axis have been indicated as the cardinal molecular pathway underlying the development of CSMNs, these proteins are transcription factors that are mostly used to label the nuclei of CSMNs in the fixed cells and tissues. Therefore, a fluorescent reporter to mark CSMNs will be invaluable in identifying living CSMNs, including their extended processes, for time-lapse imaging and high-throughput molecular analyses with much more improved specificity. Based on the in-silico analysis, we identified a putative region within the promoter sequence of FEZF2 and assembled it with an indispensable enhancer motif at its downstream of the gene to form a complex promoter that drives the expression of reporter GFP. The plasmid and virus of FEZF2:eGFP reporter constructs were further validated for its use in specifically labeling CSMNs in primary neuronal cultures from the embryonic rat motor cortex, postnatal mouse cortex. This innovative molecular labeling tool has the potential to offer indispensable support in diverse experimental setups, enabling a comprehensive understanding of the susceptibility and specificity of CSMNs in a wide array of neurological disorders.

Keywords:  Corticospinal Motor Neuron; Fezf2; Fluorescent Reporter; Hereditary Spastic Paraplegia; Lentivirus

DOI:  https://doi.org/10.1007/s11011-024-01482-w
Protein Sci. 2024 Dec;33(12): e5201

Synchronized motion of interface residues for evaluating protein-RNA complex binding affinity: Application to aptamer-mediated inhibition of TDP-43 aggregates.

Francesco Paolo Panei, Lorenzo Di Rienzo, Elsa Zacco, Alexandros Armaos, Gian Gaetano Tartaglia, Giancarlo Ruocco, Edoardo Milanetti.

  Investigating the binding between proteins and aptamers, such as peptides or RNA molecules, is of crucial importance both for understanding the molecular mechanisms that regulate cellular activities and for therapeutic applications in several pathologies. Here, a new computational procedure, employing mainly docking, clustering analysis, and molecular dynamics simulations, was designed to estimate the binding affinities between a protein and some RNA aptamers, through the investigation of the dynamical behavior of the predicted molecular complex. Using the state-of-the-art software catRAPID, we computationally designed a set of RNA aptamers interacting with the TAR DNA-binding protein 43 (TDP-43), a protein associated with several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). We thus devised a computational protocol to predict the RNA-protein molecular complex, so that the structural and dynamical behavior of such a complex can be investigated through extensive molecular dynamics simulation. We hypothesized that the coordinated and synchronized motion of the protein-binding residues, when in contact with RNA molecule, is a critical requisite in order to have a stable binding. Indeed, we calculated the motion covariance exhibited by the interface residues during molecular dynamics simulation: we tested the results against experimental measurements of binding affinity (in this case, the dissociation constant) for six RNA molecules, resulting in a linear correlation of about 0.9. Our findings suggest that the synchronized movement of interface residues plays a pivotal role in ensuring the stability within RNA-protein complexes, moreover providing insights into the contribution of each interface residue. This promising pipeline could thus contribute to the design of RNA aptamers interacting with proteins.

Keywords:  binding affinity prediction; computational method; computational molecular biophysics; molecular dynamics simulations; protein–RNA interactions; synchronized motion

DOI:  https://doi.org/10.1002/pro.5201
Amyotroph Lateral Scler Frontotemporal Degener. 2024 Nov 22. 1-11

Cerebrospinal fluid levels of NfM in relation to NfL and pNfH as prognostic markers in amyotrophic lateral sclerosis.

Jennie Olofsson, Sofia Bergström, Sára Mravinacová, Ulf Kläppe, Linn Öijerstedt, Henrik Zetterberg, Kaj Blennow, Caroline Ingre, Peter Nilsson, Anna Månberg.

  Objective: To evaluate the prognostic potential of neurofilament medium chain (NfM) in CSF from patients with ALS and explore its relationship with the extensively studied neurofilament light chain (NfL) and phosphorylated heavy chain (pNfH). Method: CSF levels of NfL, NfM, and pNfH were analyzed in 235 samples from patients with ALS, ALS mimics, and healthy controls in a well-characterized cohort from Karolinska ALS Clinical Research Center in Stockholm, Sweden. NfM levels were analyzed using an antibody-based suspension bead-array and NfL and pNfH levels were measured using ELISA. Clinical data, including ALS Revised Functional Rating Scale (ALSFRS-R), and survival outcomes were utilized for disease progression estimations. Result: Increased NfM levels were observed in patients with ALS compared with mimics and healthy controls. Similarly, higher NfM levels were found in fast compared with slow progressing patients for baseline and longitudinal progression when evaluating both total and subscores of ALSFRS-R. These findings were consistent with the results observed for NfL and pNfH. All three proteins, used individually as well as in combination, showed comparable performance when classifying fast vs slow progressing patients (AUCs 0.78-0.85). For all neurofilaments, higher survival probability was observed for patients with low CSF levels. Conclusion: Based on this cross-sectional study, the prognostic value provided by NfM aligns with the more established markers, NfL and pNfH. Additional investigations with independent cohorts and longitudinal studies are needed to further assess the potential added value of NfM.

Keywords:  amyotrophic lateral sclerosis; cerebrospinal fluid; neurofilament medium; progression; survival

DOI:  https://doi.org/10.1080/21678421.2024.2428930
Biol Res. 2024 Nov 17. 57(1): 86

Overexpression of autophagy enhancer PACER/RUBCNL in neurons accelerates disease in the SOD1G93A ALS mouse model.

Luis Labrador, Leonardo Rodriguez, Sebastián Beltran, Fernanda Hernandez, Laura Gomez, Patricia Ojeda, Cristian Bergmann, Melissa Calegaro-Nassif, Bredford Kerr, Danilo B Medinas, Patricio Manque, Ute Woehlbier.

  Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal paralytic disorder associated with motor neuron death. Mutant superoxide dismutase 1 (SOD1) misfolding and aggregation have been linked to familial ALS, with the accumulation of abnormal wild-type SOD1 species being also observed in postmortem tissue of sporadic ALS cases. Both wild-type and mutated SOD1 are reported to contribute to motoneuron cell death. The autophagic pathway has been shown to be dysregulated in ALS. Recent evidence suggests a dual time-dependent role of autophagy in the progression of the disease. PACER, also called RUBCNL (Rubicon-like), is an enhancer of autophagy and has been found diminished in its levels during ALS pathology in mice and humans. Pacer loss of function disturbs the autophagy process and leads to the accumulation of SOD1 aggregates, as well as sensitizes neurons to death. Therefore, here we investigated if constitutive overexpression of PACER in neurons since early development is beneficial in an in vivo model of ALS. We generated a transgenic mouse model overexpressing human PACER in neurons, which then was crossbred with the mutant SOD1G93A ALS mouse model. Unexpectedly, PACER/SOD1G93A double transgenic mice exhibited an earlier disease onset and shorter lifespan than did littermate SOD1G93A mice. The overexpression of PACER in neurons in vivo and in vitro increased the accumulation of SOD1 aggregates, possibly due to impaired autophagy. These results suggest that similar to Pacer loss-of function, Pacer gain-of function is detrimental to autophagy, increases SOD1 aggregation and worsens ALS pathogenesis. In a wider context, our results indicate the requirement to maintain a fine balance of PACER protein levels to sustain proteostasis.

Keywords:  Amyotrophic lateral sclerosis; Autophagy; KIAA0226L; PACER; RUBCNL; SQSTM1; Superoxide dismutase 1; p62

DOI:  https://doi.org/10.1186/s40659-024-00567-1
Stem Cell Res Ther. 2024 Nov 22. 15(1): 448

Reprogrammed human lateral ganglionic eminence precursors generate striatal neurons and restore motor function in a rat model of Huntington's disease.

Amy McCaughey-Chapman, Anne Lieke Burgers, Catharina Combrinck, Laura Marriott, David Gordon, Bronwen Connor.

   BACKGROUND: Huntington's disease (HD) is a genetic neurological disorder predominantly characterised by the progressive loss of GABAergic medium spiny neurons in the striatum resulting in motor dysfunction. One potential strategy for the treatment of HD is the development of cell replacement therapies to restore neuronal circuitry and function by the replacement of lost neurons. We propose the generation of lineage-specific human lateral ganglionic eminence precursors (hiLGEP) using direct reprogramming technology provides a novel and clinically viable cell source for cell replacement therapy for HD.
METHODS: hiLGEPs were derived by direct reprogramming of adult human dermal fibroblasts (aHDFs) using chemically modified mRNA (cmRNA) and a defined reprogramming medium. hiLGEPs were differentiated in vitro using an optimised striatal differentiation medium. Acquisition of a striatal precursor and neural cell fate was assessed through gene expression and immunocytochemical analysis of key markers. hiLGEP-derived striatal neuron functionality in vitro was demonstrated by calcium imaging using Cal-520. To investigate the ability for hiLGEP to survive, differentiate and functionally integrate in vivo, we transplanted hiLGEPs into the striatum of quinolinic acid (QA)-lesioned rats and performed behavioural assessment using the cylinder test over the course of 14 weeks. Survival and differentiation of hiLGEPs was assessed at 8 and 14-weeks post-transplant by immunohistochemical analysis.
RESULTS: We demonstrate the capability to generate hiLGEPs from aHDFs using cmRNA encoding the pro-neural genes SOX2 and PAX6, combined with a reprogramming medium containing Gö6983, Y-27,632, N-2 and Activin A. hiLGEPs generated functional DARPP32 + neurons following 14 days of culture in BrainPhys™ media supplemented with dorsomorphin and Activin A. We investigated the ability for hiLGEPs to survive transplantation, differentiate to medium spiny-like striatal neurons and improve motor function in the QA lesion rat model of HD. Fourteen weeks after transplantation, we observed STEM121 + neurons co-expressing MAP2, DARPP32, GAD65/67, or GABA. Rats transplanted with hiLGEPs also demonstrated reduction in motor function impairment as determined by spontaneous exploratory forelimb use when compared to saline transplanted animals.
CONCLUSION: This study provides proof-of-concept and demonstrates for the first time that aHDFs can be directly reprogrammed to hiLGEPs which survive transplantation, undergo neuronal differentiation to generate medium spiny-like striatal neurons, and reduce functional impairment in the QA lesion rat model of HD.

Keywords:  Cell replacement therapy; Direct reprogramming; Huntington’s disease; Lateral ganglionic eminence precursor cells; Striatum

DOI:  https://doi.org/10.1186/s13287-024-04057-9
Ageing Res Rev. 2024 Nov 16. pii: S1568-1637(24)00404-5. [Epub ahead of print] 102586

Impact of Ageing and Disuse on Neuromuscular Junction and Mitochondrial Function and Morphology: Current Evidence and Controversies.

Evgeniia Motanova, Marco Pirazzini, Samuele Negro, Ornella Rossetto, Marco Narici.

  Inactivity and ageing can have a detrimental impact on skeletal muscle and the neuromuscular junction (NMJ). Decreased physical activity results in muscle atrophy, impaired mitochondrial function, and NMJ instability. Ageing is associated with a progressive decrease in muscle mass, deterioration of mitochondrial function in the motor axon terminals and in myofibres, NMJ instability and loss of motor units. Focusing on the impact of inactivity and ageing, this review examines the consequences on NMJ stability and the role of mitochondrial dysfunction, delving into their complex relationship with ageing and disuse. Evidence suggests that mitochondrial dysfunction can be a pathogenic driver for NMJ alterations, with studies revealing the role of mitochondrial defects in motor neuron degeneration and NMJ instability. Two perspectives behind NMJ instability are discussed: one is that mitochondrial dysfunction in skeletal muscle triggers NMJ deterioration, the other envisages dysfunction of motor terminal mitochondria as a primary contributor to NMJ instability. While evidence from these studies supports both perspectives on the relationship between NMJ dysfunction and mitochondrial impairment, gaps persist in the understanding of how mitochondrial dysfunction can cause NMJ deterioration. Further research, both in humans and in animal models, is essential for unravelling the mechanisms and potential interventions for age- and inactivity-related neuromuscular and mitochondrial alterations.

Keywords:  Ageing; Disuse; Mitochondrial Ca(2+); Mitochondrial dysfunction; Neuromuscular junction; Skeletal muscle

DOI:  https://doi.org/10.1016/j.arr.2024.102586
Stem Cell Res Ther. 2024 Nov 17. 15(1): 433

In vitro electrophysiological drug testing on neuronal networks derived from human induced pluripotent stem cells.

Giulia Parodi, Giorgia Zanini, Linda Collo, Roberta Impollonia, Chiara Cervetto, Monica Frega, Michela Chiappalone, Sergio Martinoia.

   BACKGROUND: In vitro models for drug testing constitute a valuable and simplified in-vivo-like assay to better comprehend the biological drugs effect. In particular, the combination of neuronal cultures with Micro-Electrode Arrays (MEAs) constitutes a reliable system to investigate the effect of drugs aimed at manipulating the neural activity and causing controlled changes in the electrophysiology. While chemical modulation in rodents' models has been extensively studied in the literature, electrophysiological variations caused by chemical modulation on neuronal networks derived from human induced pluripotent stem cells (hiPSCs) still lack a thorough characterization.
METHODS: In this work, we created three different configurations of hiPSCs-derived neuronal networks composed of fully glutamatergic neurons (100E), 75% of glutamatergic and 25% of GABAergic neurons (75E25I) and fully GABAergic neurons (100I). We focused on the effects caused by antagonists of three of the most relevant ionotropic receptors of the human brain, i.e., 2-amino-5-phosphonovaleric (APV, NMDA receptors antagonist), 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, AMPA receptors antagonist), and bicuculline, picrotoxin and pentylenetetrazole (BIC, PTX, and PTZ, respectively, GABAA receptors antagonists).
RESULTS: We found that APV and CNQX completely abolished the network bursting activity and caused major changes in the functional connectivity. On the other hand, the effect of BIC, PTX and PTZ mostly affected configurations in which the inhibitory component was present by increasing the firing and network bursting activity as well as the functional connectivity.
CONCLUSIONS: Our work revealed that hiPSCs-derived neuronal networks are very sensitive to pharmacological manipulation of the excitatory ionotropic glutamatergic and inhibitory ionotropic GABAergic transmission, representing a preliminary and necessary step forward in the field of drug testing that can rely on pathological networks of human origin.

Keywords:  Drug testing; Electrophysiology; Human induced pluripotent stem cells; Micro-Electrode Arrays

DOI:  https://doi.org/10.1186/s13287-024-04018-2
Sci Rep. 2024 Nov 20. 14(1): 28783

Optimized simple culture protocol for inducing mature myotubes from MYOD1-overexpressed human iPS cells.

Eiji Wada, Nao Susumu, Yuya Okuzaki, Akitsu Hotta, Hidetoshi Sakurai, Yukiko K Hayashi.

  The forced expression system of MYOD1, a master gene for myogenic differentiation, can efficiently and rapidly reproduce muscle differentiation of human induced pluripotent stem cells (hiPSCs). Despite these advantages of the MYOD1 overexpression system, developed myotubes are relatively immature and do not recapitulate several aspects of striated muscle fibers. Here, we developed a simple optimized protocol using an alternative culture medium for maximizing the advantages of the MYOD1 overexpression system, and successfully improved the formation of multinucleated mature myotubes within 10 days. In this study, we generated hiPSCs derived from healthy donors and an individual with congenial muscular dystrophy caused by LMNA mutation (laminopathy), and compared disease-associated phenotypes in differentiated myotubes generated by the conventional method and by our new optimized culture method. Using our optimized method, abnormal myonuclear shape was pronounced in the patient-derived iPSCs. In addition, abnormal accumulation of the nuclear membrane protein emerin was observed in LMNA-mutant hiPSCs. Our new culture method is expected to be widely applicable as a MYOD1 overexpression model of hiPSC-derived skeletal muscle cells for the analysis of a variety of muscle diseases.

Keywords:  Differentiation of skeletal muscle cells; Induced pluripotent stem cells; Laminopathy; Muscular dystrophy; Newly developed culture method

DOI:  https://doi.org/10.1038/s41598-024-79745-w
Phytomedicine. 2024 Nov 09. pii: S0944-7113(24)00866-3. [Epub ahead of print]135 156208

Salidroside facilitates neuroprotective effects in ischemic stroke by promoting axonal sprouting through promoting autophagy.

Wenfang Lai, Yanfeng He, Binbin Zhou, Qingqing Wu, Huiling Wu, Jingquan Chen, Xuerui Zheng, Ru Jia, Pu Lin, Guizhu Hong, Jianyu Chen.

   BACKGROUND: Ischemic stroke is a common cerebrovascular disease characterized by high incidence, disability, mortality, and recurrence. The limitations of current pharmacological treatments, which have primarily single neuroprotective action and a narrow therapeutic time window, lead to unsatisfactory therapeutic efficacy. Activation of autophagy can facilitate neural regeneration.
OBJECTIVE: To clarify whether salidroside can promote axonal sprouting through autophagy resulting in protecting neurons.
METHODS: In vivo, a Middle Cerebral Artery Occlusion/reperfusion (MCAO/IR) model was used, and in vitro, an Oxygen-Glucose Deprivation/Reoxygenation (OGD/R)-induced primary neuronal cell model was employed to evaluate the neuroprotective effects of salidroside. BDA neurotracer, immunofluorescence, and Western blot (WB) were utilized to determine its impact on axonal sprouting and the levels of related proteins (MAP2, GAP43, and PSD-95). Proteomics, transmission electron microscopy (TEM), and WB were applied to identify the effects on autophagy-related proteins (beclin1, LC3, p62, and LAMP2), autophagosomes and lysosomes. The mechanism of salidroside in promoting axonal sprouting through inducing autophagy was further confirmed by blocking with the autophagy inhibitor 3-MA.
RESULTS: Salidroside reduced neurologic deficits and infarct volume induced by MCAO/IR in vivo and protected OGD/R induced primary neuronal cells in vitro. Both in vivo and in vitro, it increased the number and length of axons and upregulated the expression of key axonal proteins (MAP2, GAP43, and PSD-95) and mediated autophagy-related proteins. Mechanistic studies showed that the promoting effects of salidroside on autophagy and axonal sprouting disappeared after the blockade by 3-MA.
CONCLUSION: This study reports for the first time that the neuroprotective effect of salidroside in ischemic stroke can be executed through mediating autophagy-related protein (beclin1, LC3, p62, and LAMP2), resulting in induced axonal sprouting or mature protein (MAP2, GAP43, and PSD-95).

Keywords:  Autophagy; Axonal Regeneration; Middle Cerebral Artery Occlusion; Neuroprotection; Salidroside

DOI:  https://doi.org/10.1016/j.phymed.2024.156208
J Neurosci. 2024 Nov 18. pii: e2027232024. [Epub ahead of print]

Human iPSC-derived microglia sense and dampen hyperexcitability of cortical neurons carrying the epilepsy-associated SCN2A-L1342P mutation.

Zhefu Que, Maria I Olivero-Acosta, Morgan Robinson, Ian Chen, Jingliang Zhang, Kyle Wettschurack, Jiaxiang Wu, Tiange Xiao, C Max Otterbacher, Vinayak Shankar, Hope Harlow, Seoyong Hong, Benjamin Zirkle, Muhan Wang, Ningren Cui, Purba Mandal, Xiaoling Chen, Brody Deming, Manasi Halurkar, Yuanrui Zhao, Jean-Christophe Rochet, Ranjie Xu, Amy L Brewster, Long-Jun Wu, Chongli Yuan, William C Skarnes, Yang Yang.

Neuronal hyperexcitability is a hallmark of epilepsy. It has been recently shown in rodent models of seizures that microglia, the brain's resident immune cells, can respond to and modulate neuronal excitability. However, how human microglia interact with human neurons to regulate hyperexcitability mediated by an epilepsy-causing genetic mutation found in patients is unknown. The SCN2A gene is responsible for encoding the voltage-gated sodium channel Nav1.2, one of the leading contributors to monogenic epilepsies. Previously, we demonstrated that the recurring Nav1.2-L1342P mutation leads to hyperexcitability in a male donor (KOLF2.1) hiPSC-derived cortical neuron model. Microglia originate from a different lineage (yolk sac) and are not naturally present in hiPSCs-derived neuronal cultures. To study how microglia respond to neurons carrying a disease-causing mutation and influence neuronal excitability, we established a co-culture model comprising hiPSC-derived neurons and microglia. We found that microglia display increased branch length and enhanced process-specific calcium signal when co-cultured with Nav1.2-L1342P neurons. Moreover, the presence of microglia significantly lowered the repetitive action potential firing and current density of sodium channels in neurons carrying the mutation. Additionally, we showed that co-culturing with microglia led to a reduction in sodium channel expression within the axon initial segment of Nav1.2-L1342P neurons. Furthermore, we demonstrated that Nav1.2-L1342P neurons release a higher amount of glutamate compared to control neurons. Our work thus reveals a critical role of human iPSCs-derived microglia in sensing and dampening hyperexcitability mediated by an epilepsy-causing mutation.Significance Statement Seizure studies in mouse models have highlighted the role of microglia in modulating neuronal activity, particularly in the promotion or suppression of seizures. However, a gap persists in comprehending the influence of human microglia on intrinsically hyperexcitable neurons carrying epilepsy-associated pathogenic mutations. This research addresses this gap by investigating human microglia and their impact on neuronal functions. Our findings demonstrate that microglia exhibit dynamic morphological alterations and calcium fluctuations in the presence of neurons carrying an epilepsy-associated SCN2A mutation. Furthermore, microglia suppressed the excitability of hyperexcitable neurons, suggesting a potential beneficial role. This study underscores the role of microglia in the regulation of abnormal neuronal activity, providing insights into therapeutic strategies for neurological conditions associated with hyperexcitability.

DOI: https://doi.org/10.1523/JNEUROSCI.2027-23.2024