bims-axbals 2024-09-08 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2024–09–08
seventeen papers selected by
TJ Krzystek, ALS Therapy Development Institute

ALS-associated C21ORF2 variant disrupts DNA damage repair, mitochondrial metabolism, neuronal excitability and NEK1 levels in human motor neurons.
Patient mutations in DRP1 perturb synaptic maturation of cortical neurons.
Identification of selective and non-selective C9ORF72 targeting in vivo active siRNAs.
Molecular Visualization of Neuronal TDP43 Pathology In Situ.
Comparative analysis of neurofilaments and biomarkers of muscular damage in amyotrophic lateral sclerosis.
Modelling neurocardiac physiology and diseases using human pluripotent stem cells: current progress and future prospects.
Generation of Induced-Primary Retinal Pigment Epithelium from Human Retinal Organoids.
Lysosomal dysfunction in α-synuclein pathology: molecular mechanisms and therapeutic strategies.
Nuclear proteasomes buffer cytoplasmic proteins during autophagy compromise.
NEK1 haploinsufficiency worsens DNA damage, but not defective ciliogenesis, in C9ORF72 patient-derived iPSC-motoneurons.
AAV-Mediated Expression of miR-17 Enhances Neurite and Axon Regeneration In Vitro.
Young small extracellular vesicles rejuvenate replicative senescence by remodeling Drp1 translocation-mediated mitochondrial dynamics.
Generation of human region-specific brain organoids with medullary spinal trigeminal nuclei.
Reversible control of kinase signaling through chemical-induced dephosphorylation.
α-Bungarotoxin labelling of AMPA receptor-associated TARPs in living neurons.
Loss of Smek1 Induces Tauopathy and Triggers Neurodegeneration by Regulating Microtubule Stability.
A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence.

Acta Neuropathol Commun. 2024 Sep 04. 12(1): 144

ALS-associated C21ORF2 variant disrupts DNA damage repair, mitochondrial metabolism, neuronal excitability and NEK1 levels in human motor neurons.

Pavol Zelina, Anna Aster de Ruiter, Christy Kolsteeg, Ilona van Ginneken, Harmjan R Vos, Laura F Supiot, Boudewijn M T Burgering, Frank J Meye, Jan H Veldink, Leonard H van den Berg, R Jeroen Pasterkamp.

  Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease leading to motor neuron loss. Currently mutations in > 40 genes have been linked to ALS, but the contribution of many genes and genetic mutations to the ALS pathogenic process remains poorly understood. Therefore, we first performed comparative interactome analyses of five recently discovered ALS-associated proteins (C21ORF2, KIF5A, NEK1, TBK1, and TUBA4A) which highlighted many novel binding partners, and both unique and shared interactors. The analysis further identified C21ORF2 as a strongly connected protein. The role of C21ORF2 in neurons and in the nervous system, and of ALS-associated C21ORF2 variants is largely unknown. Therefore, we combined human iPSC-derived motor neurons with other models and different molecular cell biological approaches to characterize the potential pathogenic effects of C21ORF2 mutations in ALS. First, our data show C21ORF2 expression in ALS-relevant mouse and human neurons, such as spinal and cortical motor neurons. Further, the prominent ALS-associated variant C21ORF2-V58L caused increased apoptosis in mouse neurons and movement defects in zebrafish embryos. iPSC-derived motor neurons from C21ORF2-V58L-ALS patients, but not isogenic controls, show increased apoptosis, and changes in DNA damage response, mitochondria and neuronal excitability. In addition, C21ORF2-V58L induced post-transcriptional downregulation of NEK1, an ALS-associated protein implicated in apoptosis and DDR. In all, our study defines the pathogenic molecular and cellular effects of ALS-associated C21ORF2 mutations and implicates impaired post-transcriptional regulation of NEK1 downstream of mutant C21ORF72 in ALS.

Keywords:  Amyotrophic lateral sclerosis; Genetic mutation; IPSC; Interactor; Motor neuron

DOI:  https://doi.org/10.1186/s40478-024-01852-6
bioRxiv. 2024 Aug 26. pii: 2024.08.23.609462. [Epub ahead of print]

Patient mutations in DRP1 perturb synaptic maturation of cortical neurons.

T B Baum, C Bodnya, J Costanzo, V Gama.

With the advent of exome sequencing, a growing number of children are being identified with de novo loss of function mutations in the dynamin 1 like ( DNM1L) gene encoding the large GTPase essential for mitochondrial fission, dynamin-related protein 1 (DRP1); these mutations result in severe neurodevelopmental phenotypes, such as developmental delay, optic atrophy, and epileptic encephalopathies. Though it is established that mitochondrial fission is an essential precursor to the rapidly changing metabolic needs of the developing cortex, it is not understood how identified mutations in different domains of DRP1 uniquely disrupt cortical development and synaptic maturation. We leveraged the power of induced pluripotent stem cells (iPSCs) harboring DRP1 mutations in either the GTPase or stalk domains to model early stages of cortical development in vitro . High-resolution time-lapse imaging of axonal transport in mutant DRP1 cortical neurons reveals mutation-specific changes in mitochondrial motility of severely hyperfused mitochondrial structures. Transcriptional profiling of mutant DRP1 cortical neurons during maturation also implicates mutation dependent alterations in synaptic development and calcium regulation gene expression. Disruptions in calcium dynamics were confirmed using live functional recordings of 100 DIV (days in vitro) mutant DRP1 cortical neurons. These findings and deficits in pre- and post-synaptic marker colocalization using super resolution microscopy, strongly suggest that altered mitochondrial morphology of DRP1 mutant neurons leads to pathogenic dysregulation of synaptic development and activity.

DOI: https://doi.org/10.1101/2024.08.23.609462
Mol Ther Nucleic Acids. 2024 Sep 10. 35(3): 102291

Identification of selective and non-selective C9ORF72 targeting in vivo active siRNAs.

James W Gilbert, Zachary Kennedy, Bruno M D C Godinho, Ashley Summers, Alexandra Weiss, Dimas Echeverria, Brianna Bramato, Nicholas McHugh, David Cooper, Ken Yamada, Matthew Hassler, Hélène Tran, Fen Biao Gao, Robert H Brown, Anastasia Khvorova.

  A hexanucleotide (G4C2) repeat expansion (HRE) within intron one of C9ORF72 is the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). C9ORF72 haploinsufficiency, formation of RNA foci, and production of dipeptide repeat (DPR) proteins have been proposed as mechanisms of disease. Here, we report the first example of disease-modifying siRNAs for C9ORF72 driven ALS/FTD. Using a combination of reporter assay and primary cortical neurons derived from a C9-ALS/FTD mouse model, we screened a panel of more than 150 fully chemically stabilized siRNAs targeting different C9ORF72 transcriptional variants. We demonstrate the lack of correlation between siRNA efficacy in reporter assay versus native environment; repeat-containing C9ORF72 mRNA variants are found to preferentially localize to the nucleus, and thus C9ORF72 mRNA accessibility and intracellular localization have a dominant impact on functional RNAi. Using a C9-ALS/FTD mouse model, we demonstrate that divalent siRNAs targeting C9ORF72 mRNA variants specifically or non-selectively reduce the expression of C9ORF72 mRNA and significantly reduce DPR proteins. Interestingly, siRNA silencing all C9ORF72 mRNA transcripts was more effective in removing intranuclear mRNA aggregates than targeting only HRE-containing C9ORF72 mRNA transcripts. Combined, these data support RNAi-based degradation of C9ORF72 as a potential therapeutic paradigm.

Keywords:  C9ORF72 amyotrophic lateral sclerosis, ALS; MT: Oligonucleotides: Therapies and Applications; dipeptide-repeat proteins; frontotemporal dementia, FTD; mRNA localization; oligonucleotide therapeutics; siRNA

DOI:  https://doi.org/10.1016/j.omtn.2024.102291
bioRxiv. 2024 Aug 19. pii: 2024.08.19.608477. [Epub ahead of print]

Molecular Visualization of Neuronal TDP43 Pathology In Situ.

Amanda L Erwin, Matthew L Chang, Martin G Fernandez, Durga Attili, Jennifer E Russ, Renaldo Sutanto, Emile S Pinarbasi, Michael Bekier, Tyler S Brant, Terry Hahn, Megan Dykstra, Dafydd Thomas, Xingli Li, Ryan D Baldridge, Elizabeth M H Tank, Sami J Barmada, Shyamal Mosalaganti.

Nuclear exclusion and cytoplasmic accumulation of the RNA-binding protein TDP43 are characteristic of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Despite this, the origin and ultrastructure of cytosolic TDP43 deposits remain unknown. Accumulating evidence suggests that abnormal RNA homeostasis can drive pathological TDP43 mislocalization, enhancing RNA misprocessing due to loss of nuclear TDP43 and engendering a cycle that ends in cell death. Here, we show that adding small monovalent oligonucleotides successfully recapitulates pathological TDP43 mislocalization and aggregation in iPSC-derived neurons (iNeurons). By employing a multimodal in situ cryo-correlative light and electron microscopy pipeline, we examine how RNA influences the localization and aggregation of TDP43 in near-native conditions. We find that mislocalized TDP43 forms ordered fibrils within lysosomes and autophagosomes in iNeurons as well as in patient tissue, and provide the first high-resolution snapshots of TDP43 aggregates in situ . In so doing, we provide a cellular model for studying initial pathogenic events underlying ALS, FTLD, and related TDP43-proteinopathies.

DOI: https://doi.org/10.1101/2024.08.19.608477
Brain Commun. 2024 ;6(5): fcae288

Comparative analysis of neurofilaments and biomarkers of muscular damage in amyotrophic lateral sclerosis.

Maximilian Vidovic, Hanna Sophie Lapp, Constanze Weber, Lydia Plitzko, Michael Seifert, Petra Steinacker, Markus Otto, Andreas Hermann, René Günther.

  Diagnosis of the fatal neurodegenerative disease amyotrophic lateral sclerosis is challenging. Neurofilaments, indicative of neuronal damage, along with creatine kinase, creatinine, myoglobin, and troponin T, representing muscular damage, have been identified as promising fluid biomarkers. This study aims to comprehensively assess and compare their diagnostic and prognostic potential in a 'real-world' cohort of patients with amyotrophic lateral sclerosis. About 77 patients with amyotrophic lateral sclerosis and its clinical variants, and 26 age- and sex-matched controls with various neuromuscular and neurodegenerative diseases, were retrospectively included in this monocentric, cross-sectional study. Neurofilaments in cerebrospinal fluid and biomarkers of muscular damage in serum were measured and correlated with demographic features, motor function, survival time, clinical phenotypes, and the extent of upper and lower motor neuron involvement. Neurofilament, myoglobin, and troponin T concentrations were higher in patients with amyotrophic lateral sclerosis compared to disease controls. Higher neurofilament levels correlated with lower motor function and faster disease progression rate, while higher creatine kinase and creatinine concentrations were linked to preserved motor function. In contrast, troponin T elevation indicated poorer fine and gross motor functions. Increased neurofilament levels were associated with shorter survival, whereas biomarkers of muscular damage lacked survival correlation. Neurofilament concentrations were higher in classical amyotrophic lateral sclerosis than in progressive muscular atrophy, while myoglobin and troponin T levels were elevated in progressive muscular atrophy compared to primary lateral sclerosis. Neurofilaments were predominantly linked to upper motor neuron involvement. Our findings confirmed the robust diagnostic and prognostic value of neurofilaments in amyotrophic lateral sclerosis. Elevated neurofilament concentrations were associated with higher disease severity, faster disease progression, shorter survival, and predominant upper motor neuron degeneration. Biomarkers of muscular damage were inferior in distinguishing amyotrophic lateral sclerosis from other neuromuscular and neurodegenerative diseases. However, they may serve as complementary biomarkers and support in discriminating clinical variants of amyotrophic lateral sclerosis.

Keywords:  ALS; amyotrophic lateral sclerosis; biomarkers; biomarkers of muscular damage; neurofilaments

DOI:  https://doi.org/10.1093/braincomms/fcae288
J Physiol. 2024 Sep 05.

Modelling neurocardiac physiology and diseases using human pluripotent stem cells: current progress and future prospects.

Hsueh-Fu Wu, Charlotte Hamilton, Harrison Porritt, Annika Winbo, Nadja Zeltner.

  Throughout our lifetime the heart executes cycles of contraction and relaxation to meet the body's ever-changing metabolic needs. This vital function is continuously regulated by the autonomic nervous system. Cardiovascular dysfunction and autonomic dysregulation are also closely associated; however, the degrees of cause and effect are not always readily discernible. Thus, to better understand cardiovascular disorders, it is crucial to develop model systems that can be used to study the neurocardiac interaction in healthy and diseased states. Human pluripotent stem cell (hiPSC) technology offers a unique human-based modelling system that allows for studies of disease effects on the cells of the heart and autonomic neurons as well as of their interaction. In this review, we summarize current understanding of the embryonic development of the autonomic, cardiac and neurocardiac systems, their regulation, as well as recent progress of in vitro modelling systems based on hiPSCs. We further discuss the advantages and limitations of hiPSC-based models in neurocardiac research.

Keywords:  autonomic nervous system; cardiology; disease modelling; human pluripotent stem cell; neurocardiology

DOI:  https://doi.org/10.1113/JP286416
Methods Mol Biol. 2025 ;2848 197-214

Generation of Induced-Primary Retinal Pigment Epithelium from Human Retinal Organoids.

Miguel Flores-Bellver, M Valeria Canto-Soler.

  Retinal pigment epithelium (RPE) cells derived from induced pluripotent stem cells (iPSCs) serve multiple roles, including among others, modeling RPE development in normal and pathological conditions, investigating mechanisms of RPE physiology, modeling retinal diseases involving the RPE, and developing strategies for regenerative therapies. We have developed a simple and efficient protocol to generate RPE tissue from human iPSCs-derived retinal organoids. The RPE tissue present in the retinal organoids is analogous to the native human RPE in differentiation timeline, histological organization, and key features of functional maturation. Building upon this system, we established a method to generate functionally mature, polarized RPE monolayers comparable to human primary RPE. This comprehensive protocol outlines the steps for isolating and culturing RPE tissue using retinal organoids. The outcome is a pure population of cells expressing mature RPE signatures and organized in a characteristic cobblestone monolayer featuring robust ultrastructural polarization. These RPE monolayers also exhibit the functional hallmarks of bona fide mature RPE cells, providing a suitable system to mimic the biology and function of the native human RPE.

Keywords:  In vitro modeling; RPE; Retinal organoid; Stem cells; hiPSC

DOI:  https://doi.org/10.1007/978-1-0716-4087-6_13
Cell Mol Life Sci. 2024 Sep 03. 81(1): 382

Lysosomal dysfunction in α-synuclein pathology: molecular mechanisms and therapeutic strategies.

Lijun Dai, Miao Liu, Wei Ke, Liam Chen, Xin Fang, Zhentao Zhang.

  In orchestrating cell signaling, facilitating plasma membrane repair, supervising protein secretion, managing waste elimination, and regulating energy consumption, lysosomes are indispensable guardians that play a crucial role in preserving intracellular homeostasis. Neurons are terminally differentiated post-mitotic cells. Neuronal function and waste elimination depend on normal lysosomal function. Converging data suggest that lysosomal dysfunction is a critical event in the etiology of Parkinson's disease (PD). Mutations in Glucosylceramidase Beta 1 (GBA1) and leucine-rich repeat kinase 2 (LRRK2) confer an increased risk for the development of parkinsonism. Furthermore, lysosomal dysfunction has been observed in the affected neurons of sporadic PD (sPD) patients. Given that lysosomal hydrolases actively contribute to the breakdown of impaired organelles and misfolded proteins, any compromise in lysosomal integrity could incite abnormal accumulation of proteins, including α-synuclein, the major component of Lewy bodies in PD. Clinical observations have shown that lysosomal protein levels in cerebrospinal fluid may serve as potential biomarkers for PD diagnosis and as signs of lysosomal dysfunction. In this review, we summarize the current evidence regarding lysosomal dysfunction in PD and discuss the intimate relationship between lysosomal dysfunction and pathological α-synuclein. In addition, we discuss therapeutic strategies that target lysosomes to treat PD.

Keywords:  Biomarkers; Chaperone-mediated autophagy; GBA1; TMEM175; Therapeutic strategies

DOI:  https://doi.org/10.1007/s00018-024-05419-5
Nat Cell Biol. 2024 Aug 29.

Nuclear proteasomes buffer cytoplasmic proteins during autophagy compromise.

So Jung Park, Sung Min Son, Antonio Daniel Barbosa, Lidia Wrobel, Eleanna Stamatakou, Ferdinando Squitieri, Gabriel Balmus, David C Rubinsztein.

Autophagy is a conserved pathway where cytoplasmic contents are engulfed by autophagosomes, which then fuse with lysosomes enabling their degradation. Mutations in core autophagy genes cause neurological conditions, and autophagy defects are seen in neurodegenerative diseases such as Parkinson's disease and Huntington's disease. Thus, we have sought to understand the cellular pathway perturbations that autophagy-perturbed cells are vulnerable to by seeking negative genetic interactions such as synthetic lethality in autophagy-null human cells using available data from yeast screens. These revealed that loss of proteasome and nuclear pore complex components cause synergistic viability changes akin to synthetic fitness loss in autophagy-null cells. This can be attributed to the cytoplasm-to-nuclear transport of proteins during autophagy deficiency and subsequent degradation of these erstwhile cytoplasmic proteins by nuclear proteasomes. As both autophagy and cytoplasm-to-nuclear transport are defective in Huntington's disease, such cells are more vulnerable to perturbations of proteostasis due to these synthetic interactions.

DOI: https://doi.org/10.1038/s41556-024-01488-7
Hum Mol Genet. 2024 Sep 02. pii: ddae121. [Epub ahead of print]

NEK1 haploinsufficiency worsens DNA damage, but not defective ciliogenesis, in C9ORF72 patient-derived iPSC-motoneurons.

Serena Santangelo, Sabrina Invernizzi, Marta Nice Sorce, Valeria Casiraghi, Silvia Peverelli, Alberto Brusati, Claudia Colombrita, Nicola Ticozzi, Vincenzo Silani, Patrizia Bossolasco, Antonia Ratti.

  The hexanucleotide G4C2 repeat expansion (HRE) in C9ORF72 gene is the major cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leading to both loss- and gain-of-function pathomechanisms. The wide clinical heterogeneity among C9ORF72 patients suggests potential modifying genetic and epigenetic factors. Notably, C9ORF72 HRE often co-occurs with other rare variants in ALS/FTD-associated genes, such as NEK1, which encodes for a kinase involved in multiple cell pathways, including DNA damage response and ciliogenesis. In this study, we generated induced pluripotent stem cells (iPSCs) and differentiated motoneurons (iPSC-MNs) from an ALS patient carrying both C9ORF72 HRE and a NEK1 loss-of-function mutation to investigate the biological effect of NEK1 haploinsufficiency on C9ORF72 pathology in a condition of oligogenicity. Double mutant C9ORF72/NEK1 cells showed increased pathological C9ORF72 RNA foci in iPSCs and higher DNA damage levels in iPSC-MNs compared to single mutant C9ORF72 cells, but no effect on DNA damage response. When we analysed the primary cilium, we observed a defective ciliogenesis in C9ORF72 iPSC-MNs which was not worsened by NEK1 haploinsufficiency in the double mutant iPSC-MNs. Altogether, our study shows that NEK1 haploinsufficiency influences differently DNA damage and cilia length, potentially acting as a modifier at biological level in an in vitro ALS patient-derived disease model of C9ORF72 pathology.

Keywords:   C9ORF72 ; NEK1 ; ALS; iPSC-motoneurons; primary cilium

DOI:  https://doi.org/10.1093/hmg/ddae121
Int J Mol Sci. 2024 Aug 21. pii: 9057. [Epub ahead of print]25(16):

AAV-Mediated Expression of miR-17 Enhances Neurite and Axon Regeneration In Vitro.

Raquel Alves Almeida, Carolina Gomes Ferreira, Victor Ulysses Souza Matos, Julia Meireles Nogueira, Marina Pimenta Braga, Lucas Caldi Gomes, Erika Cristina Jorge, Frederico Marianetti Soriani, Uwe Michel, Vinicius Toledo Ribas.

  Neurodegenerative disorders, including traumatic injuries to the central nervous system (CNS) and neurodegenerative diseases, are characterized by early axonal damage, which does not regenerate in the adult mammalian CNS, leading to permanent neurological deficits. One of the primary causes of the loss of regenerative ability is thought to be a developmental decline in neurons' intrinsic capability for axon growth. Different molecules are involved in the developmental loss of the ability for axon regeneration, including many transcription factors. However, the function of microRNAs (miRNAs), which are also modulators of gene expression, in axon re-growth is still unclear. Among the various miRNAs recently identified with roles in the CNS, miR-17, which is highly expressed during early development, emerges as a promising target to promote axon regeneration. Here, we used adeno-associated viral (AAV) vectors to overexpress miR-17 (AAV.miR-17) in primary cortical neurons and evaluate its effects on neurite and axon regeneration in vitro. Although AAV.miR-17 had no significant effect on neurite outgrowth and arborization, it significantly enhances neurite regeneration after scratch lesion and axon regeneration after axotomy of neurons cultured in microfluidic chambers. Target prediction and functional annotation analyses suggest that miR-17 regulates gene expression associated with autophagy and cell metabolism. Our findings suggest that miR-17 promotes regenerative response and thus could mitigate neurodegenerative effects.

Keywords:  axon; damage; miR-17; regeneration

DOI:  https://doi.org/10.3390/ijms25169057
J Nanobiotechnology. 2024 Sep 05. 22(1): 543

Young small extracellular vesicles rejuvenate replicative senescence by remodeling Drp1 translocation-mediated mitochondrial dynamics.

Yingying Peng, Tingting Zhao, Shuxuan Rong, Shuqing Yang, Wei Teng, Yunyi Xie, Yan Wang.

   BACKGROUND: Human mesenchymal stem cells have attracted interest in regenerative medicine and are being tested in many clinical trials. In vitro expansion is necessary to provide clinical-grade quantities of mesenchymal stem cells; however, it has been reported to cause replicative senescence and undefined dysfunction in mesenchymal stem cells. Quality control assessments of in vitro expansion have rarely been addressed in ongoing trials. Young small extracellular vesicles from the remnant pulp of human exfoliated deciduous teeth stem cells have demonstrated therapeutic potential for diverse diseases. However, it is still unclear whether young small extracellular vesicles can reverse senescence-related declines.
RESULTS: We demonstrated that mitochondrial structural disruption precedes cellular dysfunction during bone marrow-derived mesenchymal stem cell replication, indicating mitochondrial parameters as quality assessment indicators of mesenchymal stem cells. Dynamin-related protein 1-mediated mitochondrial dynamism is an upstream regulator of replicative senescence-induced dysfunction in bone marrow-derived mesenchymal stem cells. We observed that the application of young small extracellular vesicles could rescue the pluripotency dissolution, immunoregulatory capacities, and therapeutic effects of replicative senescent bone marrow-derived mesenchymal stem cells. Mechanistically, young small extracellular vesicles could promote Dynamin-related protein 1 translocation from the cytoplasm to the mitochondria and remodel mitochondrial disruption during replication history.
CONCLUSIONS: Our findings show that Dynamin-related protein 1-mediated mitochondrial disruption is associated with the replication history of bone marrow-derived mesenchymal stem cells. Young small extracellular vesicles from human exfoliated deciduous teeth stem cells alleviate replicative senescence by promoting Dynamin-related protein 1 translocation onto the mitochondria, providing evidence for a potential rejuvenation strategy.

Keywords:  Mesenchymal stem cell; Mitochondrial dynamics; Replicative senescence; Small extracellular vesicles; Stem cells from the remnant pulp of human exfoliated deciduous teeth (SHED)

DOI:  https://doi.org/10.1186/s12951-024-02818-5
Cell Stem Cell. 2024 Aug 24. pii: S1934-5909(24)00290-X. [Epub ahead of print]

Generation of human region-specific brain organoids with medullary spinal trigeminal nuclei.

Wei Pang, Jinkui Zhu, Kexin Yang, Xiaona Zhu, Wei Zhou, Linlin Jiang, Xuran Zhuang, Yantong Liu, Jianfeng Wei, Xiaoxiang Lu, Yao Yin, Ziling Chen, Yangfei Xiang.

  Brain organoids with nucleus-specific identities provide unique platforms for studying human brain development and diseases at a finer resolution. Despite its essential role in vital body functions, the medulla of the hindbrain has seen a lack of in vitro models, let alone models resembling specific medullary nuclei, including the crucial spinal trigeminal nucleus (SpV) that relays peripheral sensory signals to the thalamus. Here, we report a method to differentiate human pluripotent stem cells into region-specific brain organoids resembling the dorsal domain of the medullary hindbrain. Importantly, organoids specifically recapitulated the development of the SpV derived from the dorsal medulla. We also developed an organoid system to create the trigeminothalamic projections between the SpV and the thalamus by fusing these organoids, namely human medullary SpV-like organoids (hmSpVOs), with organoids representing the thalamus (hThOs). Our study provides a platform for understanding SpV development, nucleus-based circuit organization, and related disorders in the human brain.

Keywords:  brain organoid; hPSCs; medulla; spinal trigeminal nucleus; trigeminothalamic projection

DOI:  https://doi.org/10.1016/j.stem.2024.08.004
Commun Biol. 2024 Aug 31. 7(1): 1073

Reversible control of kinase signaling through chemical-induced dephosphorylation.

Ying Sun, Rihong Zhou, Jin Hu, Shan Feng, Qi Hu.

The coordination between kinases and phosphatases is crucial for regulating the phosphorylation levels of essential signaling molecules. Methods enabling precise control of kinase activities are valuable for understanding the kinase functions and for developing targeted therapies. Here, we use the abscisic acid (ABA)-induced proximity system to reversibly control kinase signaling by recruiting phosphatases. Using this method, we found that the oncogenic tyrosine kinase BCR::ABL1 can be inhibited by recruiting various cytoplasmic phosphatases. We also discovered that the oncogenic serine/threonine kinase BRAF(V600E), which has been reported to bypass phosphorylation regulation, can be positively regulated by protein phosphatase 1 (PP1) and negatively regulated by PP5. Additionally, we observed that the dual-specificity kinase MEK1 can be inhibited by recruiting PP5. This suggests that bifunctional molecules capable of recruiting PP5 to MEK or RAF kinases could be promising anticancer drug candidates. Thus, the ABA-induced dephosphorylation method enables rapid screening of phosphatases to precisely control kinase signaling.

DOI: https://doi.org/10.1038/s42003-024-06771-9
Neuroscience. 2024 Aug 30. pii: S0306-4522(24)00440-8. [Epub ahead of print]

α-Bungarotoxin labelling of AMPA receptor-associated TARPs in living neurons.

Cecile Bats, Ian D Coombs, Mark Farrant, Stuart G Cull-Candy.

  In mammalian central neurons AMPARs are clustered at glutamatergic synapses where they mediate fast excitatory transmission. In addition to four pore-forming subunits (GluA1-4), AMPARs contain auxiliary transmembrane AMPAR regulatory proteins (γ2, γ3, γ4, γ5, γ7 or γ8) whose incorporation can vary between neuron types, brain regions, and stages of development. As well as modulating the functional properties of AMPARs, these auxiliary subunits play a central role in AMPAR trafficking. Directly visualizing TARPs could therefore provide a valuable insight into mechanisms underlying these processes. Although antibodies are routinely used for the detection of surface proteins, our experience suggests anti-TARP antibodies are too bulky to access their target, possibly due to close interactions between the extracellular domains of TARP and AMPAR subunits. We therefore assessed the utility of a small monovalent probe - fluorescent α-bungarotoxin (α-Btx) - for TARP labelling in living neurons. We inserted the bungarotoxin binding site (BBS) within the extracellular domain of TARPs to enable their detection in cells exposed to fluorescent α-Btx. Focusing on the prototypical TARP γ2, we demonstrate that the small size of fluorescent α-Btx allows it to bind to the BBS-tagged TARP when associated with AMPARs. Importantly, labelled γ2 enhances AMPAR function in the same way as unmodified γ2. In living neurons, fluorescent α-Btx-labelled γ2 associates with AMPAR clusters at synapses. As a proof-of-principle, we employed our method to compare the surface trafficking of γ2 and γ7 in cerebellar stellate neurons. Our approach provides a simple way to visualize TARPs within AMPARs in living cells.

Keywords:  AMPA receptor; AMPAR auxiliary subunits; Bungarotoxin; Live imaging; TARP

DOI:  https://doi.org/10.1016/j.neuroscience.2024.08.036
Adv Sci (Weinh). 2024 Aug 29. e2400584

Loss of Smek1 Induces Tauopathy and Triggers Neurodegeneration by Regulating Microtubule Stability.

Ruo-Nan Duan, Ai Liu, Yue-Qing Sun, Yun-Fang Xie, Shi-Jun Wei, Shang Gao, Yi-Ming Liu, Xi Li, Wen-Jie Sun, Jiang-Xia Li, Chuan-Zhu Yan, Qi-Ji Liu.

  Suppressor of Mek1 (Smek1) is a regulatory subunit of protein phosphatase 4. Genome-wide association studies have shown the protective effect of SMEK1 in Alzheimer's disease (AD). However, the physiological and pathological roles of Smek1 in AD and other tauopathies are largely unclear. Here, the role of Smek1 in preventing neurodegeneration is investigated in tauopathy. Smek1 is downregulated in the aged human brain. Through single-cell sequencing, a novel neuronal cluster is identified that possesses neurodegenerative characteristics in Smek1-/- mice. Smek1 deficiency caused markedly more severe motor and cognitive impairments in mice, as well as neuronal loss, gliosis, and tau hyperphosphorylation at major glycogen synthase kinase 3β (Gsk3β) sites. Protein-protein interaction analysis revealed that the Ran-binding domain (RanBD) in the N-terminus of Smek1 facilitated binding with kinesin family member 2A (Kif2a). Depletion of Smek1 resulted in cytoplasmic aggregation of Kif2a, axon outgrowth defects, and impaired mitochondrial axonal trafficking. Downregulation of Kif2a markedly attenuated tau hyperphosphorylation and axon outgrowth defects in shSmek1 cells. For the first time, this study demonstrates that Smek1 deficiency progressively induces neurodegeneration by exacerbating tau pathology and mitochondrial dysfunction in an age-dependent manner.

Keywords:  Kif2a; Smek1; neurodegeneration; tau phosphorylation; tauopathy

DOI:  https://doi.org/10.1002/advs.202400584
F1000Res. 2024 ;13 481

A guide to selecting high-performing antibodies for Protein-glutamine gamma-glutamyltransferase 2 (TGM2) for use in western blot, immunoprecipitation and immunofluorescence.

Neuro/SGC/EDDU collaborative group

  Protein-glutamine gamma-glutamyltransferase 2 (TGM2) is a Ca 2+ dependent enzyme that catalyzes transglutaminase cross-linking modifications. TGM2 is involved in various diseases, either in a protective or contributory manner, making it a crucial protein to study and determine its therapeutic potential. Identifying high-performing TGM2 antibodies would facilitate these investigations. Here we have characterized seventeen TGM2 commercial antibodies for western blot and sixteen for immunoprecipitation, and immunofluorescence. The implemented standardized experimental protocol is based on comparing read-outs in knockout cell lines against their isogenic parental controls. This study is part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Keywords:  Protein-glutamine gamma-glutamyltransferase 2; TGM; TGM2; Uniprot ID P21980; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation; western blot

DOI:  https://doi.org/10.12688/f1000research.150684.2