bims-axbals 2024-03-10 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2024–03–10
twelve papers selected by
TJ Krzystek, ALS Therapy Development Institute

Homozygous ALS-linked mutations in TARDBP/TDP-43 lead to hypoactivity and synaptic abnormalities in human iPSC-derived motor neurons.
Neuromuscular organoids model spinal neuromuscular pathologies in C9orf72 amyotrophic lateral sclerosis.
RNA aptamer reveals nuclear TDP-43 pathology is an early aggregation event that coincides with STMN-2 cryptic splicing and precedes clinical manifestation in ALS.
Loss of WIPI4 in neurodegeneration causes autophagy-independent ferroptosis.
Identification of molecular signatures defines the differential proteostasis response in induced spinal and cranial motor neurons.
α-Synuclein seeds in amyotrophic lateral sclerosis.
Location of the axon initial segment assembly can be predicted from neuronal shape.
Recent advances in nanoparticulate RNA delivery systems.
AI-organoid integrated systems for biomedical studies and applications.
Cortical neurons obtained from patient-derived iPSCs with GNAO1 p.G203R variant show altered differentiation and functional properties.
LRRK2 kinase inhibition reverses G2019S mutation-dependent effects on tau pathology progression.
RANBP17 overexpression restores nucleocytoplasmic transport and ameliorates neurodevelopment in induced DYT1 dystonia motor neurons.

iScience. 2024 Mar 15. 27(3): 109166

Homozygous ALS-linked mutations in TARDBP/TDP-43 lead to hypoactivity and synaptic abnormalities in human iPSC-derived motor neurons.

Sarah Lépine, Angela Nauleau-Javaudin, Eric Deneault, Carol X-Q Chen, Narges Abdian, Anna Krystina Franco-Flores, Ghazal Haghi, María José Castellanos-Montiel, Gilles Maussion, Mathilde Chaineau, Thomas Martin Durcan.

  Cytoplasmic mislocalization and aggregation of the RNA-binding protein TDP-43 is a pathological hallmark of the motor neuron (MN) disease amyotrophic lateral sclerosis (ALS). Furthermore, while mutations in TARDBP (encoding TDP-43) have been associated with ALS, the pathogenic consequences of these mutations remain poorly understood. Using CRISPR-Cas9, we engineered two homozygous knock-in induced pluripotent stem cell lines carrying mutations in TARDBP encoding TDP-43A382T and TDP-43G348C, two common yet understudied ALS TDP-43 variants. Motor neurons (MNs) differentiated from knock-in iPSCs had normal viability and displayed no significant changes in TDP-43 subcellular localization, phosphorylation, solubility, or aggregation compared with isogenic control MNs. However, our results highlight synaptic impairments in both TDP-43A382T and TDP-43G348C MN cultures, as reflected in synapse abnormalities and alterations in spontaneous neuronal activity. Collectively, our findings suggest that MN dysfunction may precede the occurrence of TDP-43 pathology and neurodegeneration in ALS and further implicate synaptic and excitability defects in the pathobiology of this disease.

Keywords:  Cellular neuroscience; Molecular neuroscience

DOI:  https://doi.org/10.1016/j.isci.2024.109166
Cell Rep. 2024 Mar 01. pii: S2211-1247(24)00220-1. [Epub ahead of print]43(3): 113892

Neuromuscular organoids model spinal neuromuscular pathologies in C9orf72 amyotrophic lateral sclerosis.

Chong Gao, Qinghua Shi, Xue Pan, Jiajia Chen, Yuhong Zhang, Jiali Lang, Shan Wen, Xiaodong Liu, Tian-Lin Cheng, Kai Lei.

  Hexanucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Due to the lack of trunk neuromuscular organoids (NMOs) from ALS patients' induced pluripotent stem cells (iPSCs), an organoid system was missing to model the trunk spinal neuromuscular neurodegeneration. With the C9orf72 ALS patient-derived iPSCs and isogenic controls, we used an NMO system containing trunk spinal cord neural and peripheral muscular tissues to show that the ALS NMOs could model peripheral defects in ALS, including contraction weakness, neural denervation, and loss of Schwann cells. The neurons and astrocytes in ALS NMOs manifested the RNA foci and dipeptide repeat proteins. Acute treatment with the unfolded protein response inhibitor GSK2606414 increased the glutamatergic muscular contraction 2-fold and reduced the dipeptide repeat protein aggregation and autophagy. This study provides an organoid system for spinal neuromuscular pathologies in ALS and its application for drug testing.

Keywords:  ALS; C9orf72; CP: Neuroscience; GSK2606414; NMO; autophagy; dipeptide repeat proteins; muscle denervation; neural degeneration

DOI:  https://doi.org/10.1016/j.celrep.2024.113892
Acta Neuropathol. 2024 Mar 05. 147(1): 50

RNA aptamer reveals nuclear TDP-43 pathology is an early aggregation event that coincides with STMN-2 cryptic splicing and precedes clinical manifestation in ALS.

Holly Spence, Fergal M Waldron, Rebecca S Saleeb, Anna-Leigh Brown, Olivia M Rifai, Martina Gilodi, Fiona Read, Kristine Roberts, Gillian Milne, Debbie Wilkinson, Judi O'Shaughnessy, Annalisa Pastore, Pietro Fratta, Neil Shneider, Gian Gaetano Tartaglia, Elsa Zacco, Mathew H Horrocks, Jenna M Gregory.

  TDP-43 is an aggregation-prone protein which accumulates in the hallmark pathological inclusions of amyotrophic lateral sclerosis (ALS). However, the analysis of deeply phenotyped human post-mortem samples has shown that TDP-43 aggregation, revealed by standard antibody methods, correlates poorly with symptom manifestation. Recent identification of cryptic-splicing events, such as the detection of Stathmin-2 (STMN-2) cryptic exons, are providing evidence implicating TDP-43 loss-of-function as a potential driving pathomechanism but the temporal nature of TDP-43 loss and its relation to the disease process and clinical phenotype is not known. To address these outstanding questions, we used a novel RNA aptamer, TDP-43APT, to detect TDP-43 pathology and used single molecule in situ hybridization to sensitively reveal TDP-43 loss-of-function and applied these in a deeply phenotyped human post-mortem tissue cohort. We demonstrate that TDP-43APT identifies pathological TDP-43, detecting aggregation events that cannot be detected by classical antibody stains. We show that nuclear TDP-43 pathology is an early event, occurring prior to cytoplasmic accumulation and is associated with loss-of-function measured by coincident STMN-2 cryptic splicing pathology. Crucially, we show that these pathological features of TDP-43 loss-of-function precede the clinical inflection point and are not required for region specific clinical manifestation. Furthermore, we demonstrate that gain-of-function in the form of extensive cytoplasmic accumulation, but not loss-of-function, is the primary molecular correlate of clinical manifestation. Taken together, our findings demonstrate implications for early diagnostics as the presence of STMN-2 cryptic exons and early TDP-43 aggregation events could be detected prior to symptom onset, holding promise for early intervention in ALS.

Keywords:   Stathmin-2 ; Amyotrophic lateral sclerosis; Cognition; Cryptic splicing; Loss-of-function; Neuropathology; RNA aptamer; TDP-43

DOI:  https://doi.org/10.1007/s00401-024-02705-1
Nat Cell Biol. 2024 Mar 07.

Loss of WIPI4 in neurodegeneration causes autophagy-independent ferroptosis.

Ye Zhu, Motoki Fujimaki, Louisa Snape, Ana Lopez, Angeleen Fleming, David C Rubinsztein.

β-Propeller protein-associated neurodegeneration (BPAN) is a rare X-linked dominant disease, one of several conditions that manifest with neurodegeneration and brain iron accumulation. Mutations in the WD repeat domain 45 (WDR45) gene encoding WIPI4 lead to loss of function in BPAN but the cellular mechanisms of how these trigger pathology are unclear. The prevailing view in the literature is that BPAN is simply the consequence of autophagy deficiency given that WIPI4 functions in this degradation pathway. However, our data indicate that WIPI4 depletion causes ferroptosis-a type of cell death induced by lipid peroxidation-via an autophagy-independent mechanism, as demonstrated both in cell culture and in zebrafish. WIPI4 depletion increases ATG2A localization at endoplasmic reticulum-mitochondrial contact sites, which enhances phosphatidylserine import into mitochondria. This results in increased mitochondrial synthesis of phosphatidylethanolamine, a major lipid prone to peroxidation, thus enabling ferroptosis. This mechanism has minimal overlap with classical ferroptosis stimuli but provides insights into the causes of neurodegeneration in BPAN and may provide clues for therapeutic strategies.

DOI: https://doi.org/10.1038/s41556-024-01373-3
Cell Rep. 2024 Mar 07. pii: S2211-1247(24)00213-4. [Epub ahead of print]43(3): 113885

Identification of molecular signatures defines the differential proteostasis response in induced spinal and cranial motor neurons.

Ana Paula Zen Petisco Fiore, Shuvadeep Maity, Lauren Jeffery, Disi An, Justin Rendleman, Dylan Iannitelli, Hyungwon Choi, Esteban Mazzoni, Christine Vogel.

  Amyotrophic lateral sclerosis damages proteostasis, affecting spinal and upper motor neurons earlier than a subset of cranial motor neurons. To aid disease understanding, we exposed induced cranial and spinal motor neurons (iCrMNs and iSpMNs) to proteotoxic stress, under which iCrMNs showed superior survival, quantifying the transcriptome and proteome for >8,200 genes at 0, 12, and 36 h. Two-thirds of the proteome showed cell-type differences. iSpMN-enriched proteins related to DNA/RNA metabolism, and iCrMN-enriched proteins acted in the endoplasmic reticulum (ER)/ER chaperone complex, tRNA aminoacylation, mitochondria, and the plasma/synaptic membrane, suggesting that iCrMNs expressed higher levels of proteins supporting proteostasis and neuronal function. When investigating the increased proteasome levels in iCrMNs, we showed that the activity of the 26S proteasome, but not of the 20S proteasome, was higher in iCrMNs than in iSpMNs, even after a stress-induced decrease. We identified Ublcp1 as an iCrMN-specific regulator of the nuclear 26S activity.

Keywords:  CP: Cell biology; CP: Neuroscience; Ublcp1; amyotrophic lateral sclerosis; motor neurons; proteasome; unfolded protein response

DOI:  https://doi.org/10.1016/j.celrep.2024.113885
Nat Rev Neurol. 2024 Mar 05.

α-Synuclein seeds in amyotrophic lateral sclerosis.

Ian Fyfe.

DOI: https://doi.org/10.1038/s41582-024-00949-9
iScience. 2024 Mar 15. 27(3): 109264

Location of the axon initial segment assembly can be predicted from neuronal shape.

Zhuang Xu, Christopher N Angstmann, Yuhuang Wu, Holly Stefen, Esmeralda Parić, Thomas Fath, Paul M G Curmi.

  The axon initial segment (AIS) is located at the proximal axon demarcating the boundary between axonal and somatodendritic compartments. The AIS facilitates the generation of action potentials and maintenance of neuronal polarity. In this study, we show that the location of AIS assembly, as marked by Ankyrin G, corresponds to the nodal plane of the lowest-order harmonic of the Laplace-Beltrami operator solved over the neuronal shape. This correlation establishes a coupling between location of AIS assembly and neuronal cell morphology. We validate this correlation for neurons with atypical morphology and neurons containing multiple AnkG clusters on distinct neurites, where the nodal plane selects the appropriate axon showing enriched Tau. Based on our findings, we propose that Turing patterning systems are candidates for dynamically governing AIS location. Overall, this study highlights the importance of neuronal cell morphology in determining the precise localization of the AIS within the proximal axon.

Keywords:  Cellular neuroscience; Classification Description: Molecular neuroscience

DOI:  https://doi.org/10.1016/j.isci.2024.109264
Proc Natl Acad Sci U S A. 2024 Mar 12. 121(11): e2307798120

Recent advances in nanoparticulate RNA delivery systems.

Jacob Witten, Yizong Hu, Robert Langer, Daniel G Anderson.

Nanoparticle-based RNA delivery has shown great progress in recent years with the approval of two mRNA vaccines for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and a liver-targeted siRNA therapy. Here, we discuss the preclinical and clinical advancement of new generations of RNA delivery therapies along multiple axes. Improvements in cargo design such as RNA circularization and data-driven untranslated region optimization can drive better mRNA expression. New materials discovery research has driven improved delivery to extrahepatic targets such as the lung and splenic immune cells, which could lead to pulmonary gene therapy and better cancer vaccines, respectively. Other organs and even specific cell types can be targeted for delivery via conjugation of small molecule ligands, antibodies, or peptides to RNA delivery nanoparticles. Moreover, the immune response to any RNA delivery nanoparticle plays a crucial role in determining efficacy. Targeting increased immunogenicity without induction of reactogenic side effects is crucial for vaccines, while minimization of immune response is important for gene therapies. New developments have addressed each of these priorities. Last, we discuss the range of RNA delivery clinical trials targeting diverse organs, cell types, and diseases and suggest some key advances that may play a role in the next wave of therapies.

DOI: https://doi.org/10.1073/pnas.2307798120
Bioeng Transl Med. 2024 Mar;9(2): e10641

AI-organoid integrated systems for biomedical studies and applications.

Sudhiksha Maramraju, Andrew Kowalczewski, Anirudh Kaza, Xiyuan Liu, Jathin Pranav Singaraju, Mark V Albert, Zhen Ma, Huaxiao Yang.

  In this review, we explore the growing role of artificial intelligence (AI) in advancing the biomedical applications of human pluripotent stem cell (hPSC)-derived organoids. Stem cell-derived organoids, these miniature organ replicas, have become essential tools for disease modeling, drug discovery, and regenerative medicine. However, analyzing the vast and intricate datasets generated from these organoids can be inefficient and error-prone. AI techniques offer a promising solution to efficiently extract insights and make predictions from diverse data types generated from microscopy images, transcriptomics, metabolomics, and proteomics. This review offers a brief overview of organoid characterization and fundamental concepts in AI while focusing on a comprehensive exploration of AI applications in organoid-based disease modeling and drug evaluation. It provides insights into the future possibilities of AI in enhancing the quality control of organoid fabrication, label-free organoid recognition, and three-dimensional image reconstruction of complex organoid structures. This review presents the challenges and potential solutions in AI-organoid integration, focusing on the establishment of reliable AI model decision-making processes and the standardization of organoid research.

Keywords:  artificial intelligence; deep learning; disease modeling; drug evaluation; human pluripotent stem cells (hPSCs); machine learning; organoid; regenerative medicine

DOI:  https://doi.org/10.1002/btm2.10641
Heliyon. 2024 Mar 15. 10(5): e26656

Cortical neurons obtained from patient-derived iPSCs with GNAO1 p.G203R variant show altered differentiation and functional properties.

Maria Cristina Benedetti, Tiziano D'andrea, Alessio Colantoni, Denis Silachev, Valeria de Turris, Zaira Boussadia, Valentina A Babenko, Egor A Volovikov, Lilia Belikova, Alexandra N Bogomazova, Rita Pepponi, Dosh Whye, Elizabeth D Buttermore, Gian Gaetano Tartaglia, Maria A Lagarkova, Vladimir L Katanaev, Ilya Musayev, Simone Martinelli, Sergio Fucile, Alessandro Rosa.

  Pathogenic variants in the GNAO1 gene, encoding the alpha subunit of an inhibitory heterotrimeric guanine nucleotide-binding protein (Go) highly expressed in the mammalian brain, have been linked to encephalopathy characterized by different combinations of neurological symptoms, including developmental delay, hypotonia, epilepsy and hyperkinetic movement disorder with life-threatening paroxysmal exacerbations. Currently, there are only symptomatic treatments, and little is known about the pathophysiology of GNAO1-related disorders. Here, we report the characterization of a new in vitro model system based on patient-derived induced pluripotent stem cells (hiPSCs) carrying the recurrent p.G203R amino acid substitution in Gαo, and a CRISPR-Cas9-genetically corrected isogenic control line. RNA-Seq analysis highlighted aberrant cell fate commitment in neuronal progenitor cells carrying the p.G203R pathogenic variant. Upon differentiation into cortical neurons, patients' cells showed reduced expression of early neural genes and increased expression of astrocyte markers, as well as premature and defective differentiation processes leading to aberrant formation of neuronal rosettes. Of note, comparable defects in gene expression and in the morphology of neural rosettes were observed in hiPSCs from an unrelated individual harboring the same GNAO1 variant. Functional characterization showed lower basal intracellular free calcium concentration ([Ca2+]i), reduced frequency of spontaneous activity, and a smaller response to several neurotransmitters in 40- and 50-days differentiated p.G203R neurons compared to control cells. These findings suggest that the GNAO1 pathogenic variant causes a neurodevelopmental phenotype characterized by aberrant differentiation of both neuronal and glial populations leading to a significant alteration of neuronal communication and signal transduction.

Keywords:  Encephalopathy; GNAO1; Induced pluripotent stem cell; Movement disorder; neural rosette; wnt

DOI:  https://doi.org/10.1016/j.heliyon.2024.e26656
Transl Neurodegener. 2024 Mar 04. 13(1): 13

LRRK2 kinase inhibition reverses G2019S mutation-dependent effects on tau pathology progression.

Noah Lubben, Julia K Brynildsen, Connor M Webb, Howard L Li, Cheryl E G Leyns, Lakshmi Changolkar, Bin Zhang, Emily S Meymand, Mia O'Reilly, Zach Madaj, Daniella DeWeerd, Matthew J Fell, Virginia M Y Lee, Dani S Bassett, Michael X Henderson.

   BACKGROUND: Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease (PD). These mutations elevate the LRRK2 kinase activity, making LRRK2 kinase inhibitors an attractive therapeutic. LRRK2 kinase activity has been consistently linked to specific cell signaling pathways, mostly related to organelle trafficking and homeostasis, but its relationship to PD pathogenesis has been more difficult to define. LRRK2-PD patients consistently present with loss of dopaminergic neurons in the substantia nigra but show variable development of Lewy body or tau tangle pathology. Animal models carrying LRRK2 mutations do not develop robust PD-related phenotypes spontaneously, hampering the assessment of the efficacy of LRRK2 inhibitors against disease processes. We hypothesized that mutations in LRRK2 may not be directly related to a single disease pathway, but instead may elevate the susceptibility to multiple disease processes, depending on the disease trigger. To test this hypothesis, we have previously evaluated progression of α-synuclein and tau pathologies following injection of proteopathic seeds. We demonstrated that transgenic mice overexpressing mutant LRRK2 show alterations in the brain-wide progression of pathology, especially at older ages.
METHODS: Here, we assess tau pathology progression in relation to long-term LRRK2 kinase inhibition. Wild-type or LRRK2G2019S knock-in mice were injected with tau fibrils and treated with control diet or diet containing LRRK2 kinase inhibitor MLi-2 targeting the IC50 or IC90 of LRRK2 for 3-6 months. Mice were evaluated for tau pathology by brain-wide quantitative pathology in 844 brain regions and subsequent linear diffusion modeling of progression.
RESULTS: Consistent with our previous work, we found systemic alterations in the progression of tau pathology in LRRK2G2019S mice, which were most pronounced at 6 months. Importantly, LRRK2 kinase inhibition reversed these effects in LRRK2G2019S mice, but had minimal effect in wild-type mice, suggesting that LRRK2 kinase inhibition is likely to reverse specific disease processes in G2019S mutation carriers. Additional work may be necessary to determine the potential effect in non-carriers.
CONCLUSIONS: This work supports a protective role of LRRK2 kinase inhibition in G2019S carriers and provides a rational workflow for systematic evaluation of brain-wide phenotypes in therapeutic development.

Keywords:  Cell-to-cell spread; G2019S; Genetic risk; MLi-2; Mapt; Transmission

DOI:  https://doi.org/10.1186/s40035-024-00403-2
J Neurosci. 2024 Mar 04. pii: e1728232024. [Epub ahead of print]

RANBP17 overexpression restores nucleocytoplasmic transport and ameliorates neurodevelopment in induced DYT1 dystonia motor neurons.

Masuma Akter, Haochen Cui, Md Abir Hosain, Jinmei Liu, Yuntian Duan, Baojin Ding.

DYT1 dystonia is a debilitating neurological movement disorder, and it represents the most frequent and severe form of hereditary primary dystonia. There is currently no cure for this disease due to its unclear pathogenesis. In our previous study utilizing patient-specific motor neurons (MNs), we identified distinct cellular deficits associated with the disease, including deformed nucleus, disrupted neurodevelopment, and the compromised nucleocytoplasmic transport (NCT) functions. However, the precise molecular mechanisms underlying these cellular impairments have remained elusive. In this study, we revealed the genome-wide changes of gene expression in DYT1 MNs through transcriptomic analysis. We found that those dysregulated genes are intricately involved in neurodevelopment and various biological processes. Interestingly, we identified that the expression level of RANBP17, a RAN binding protein crucial for NCT regulation, exhibited a significant reduction in DYT1 MNs. By manipulating RANBP17 expression, we further demonstrated that RANBP17 plays an important role in facilitating nuclear transport of both protein and transcript cargos in induced human neurons. Excitingly, the overexpression of RANBP17 emerged as a substantial mitigating factor, effectively restoring impaired NCT activity and rescuing neurodevelopmental deficits observed in DYT1 MNs. These findings shed light on the intricate molecular underpinnings of impaired NCT in DYT1 neurons and provide novel insights into the pathophysiology of DYT1 dystonia, potentially leading to the development of innovative treatment strategies.Significance Statement DYT1 dystonia is a debilitating neurological movement disorder, and currently, there is no cure available due to its unclear pathogenesis. However, the inaccessibility of patient neurons greatly hinders the progress of research on this disease. In this study, we generated DYT1 patient-specific neurons from induced pluripotent stem cells (iPSCs) and examined genome-wide changes in gene expression. We have identified that RANBP17, a nuclear transport regulator, plays a substantial mitigating role, effectively rescuing cellular deficits observed in DYT1 neurons. These findings shed light on the intricate molecular underpinnings in DYT1 dystonia and have the potential to lead to the development of innovative treatment strategies.

DOI: https://doi.org/10.1523/JNEUROSCI.1728-23.2024