The frontotemporal dementia-linked S320F mutation in the microtubule-associated protein tau promotes spontaneous aggregation, yet the structural basis of its amyloidogenesis remains unclear. Using cryo-electron microscopy, we determined the structure of an S320F295-330 tau fibril composed of parallel chains stabilized by the 306VQIVYK311 amyloid motif, with S320F buried in the fibril core and a C322-C322 disulfide linking two protofilaments. Although cysteines are dispensable for fibril formation by isolated peptide fragments in vitro, tau repeat domain constructs containing both C291 and C322 generate more potent seeds in cellular assays. In contrast, the C322S mutation suppresses spontaneous aggregation of S320F tau in cells, and combined C291S and C322S mutations inhibit seeded aggregation in both wild-type and S320F contexts. Systematic alanine mutagenesis coupled with seeding by tauopathy-derived material identifies cysteine residues as critical determinants of tau seeding, comparable in importance to core amyloid motifs. Together, these findings establish cysteines as central chemical regulators of tau aggregation and propagation. ### Competing Interest Statement J.V.A. and M.I.D. are co-founders of Handshake Bio. L.S. is a co-founder of AmyGo Solutions. Handshake Bio or Amygo did not directly fund or influence the design, execution, or interpretation of the experiments presented in this manuscript. The remaining authors declare no competing interests. United States Department of Defense, https://ror.org/0447fe631, HT9425-24-1-0641 National Institutes of Health, RF1AG065407
At the Wisconsin National Primate Research Center, we have identified a family of rhesus carrying the microtubule-associated protein tau ( MAPT ) R406W mutation linked to frontotemporal dementia (FTD). Rhesus induced pluripotent stem cells (RhiPSCs) derived from these monkeys present a unique opportunity for in vitro modeling and comparison with cells derived from MAPT R406W human carriers. Here, we report the development of a reproducible method to generate RhiPSCs compliant with the standards of the International Society for Stem Cell Research (ISSCR) to support in vitro modeling of FTD -MAPT R406W. Our stepwise approach identified efficient methods for fibroblast derivation, fibroblast reprogramming to RhiPSC, and RhiPSC maintenance over continued culture. To derive fibroblasts from MAPT wild type (WT) and R406W monkeys, a combination of manual processing and overnight enzymatic digestion was required to maximize the number of low passage fibroblasts available for reprogramming. Fibroblast reprogramming to RhiPSC using Sendai viral vectors versus oriP/EBNA1 episomal plasmids revealed the latter as most efficient. Electroporation conditions for oriP/EBNA1 reprogramming were optimized to maximize plasmid uptake and cell survival. Ultimately, eight RhiPSC lines were derived from 4 donor rhesus monkeys (n=2 WT, n=2 R406W; two clonal lines per donor) and fully characterized according to ISSCR standards. RhiPSC stemness and genetic stability was best maintained on mouse embryonic fibroblast feeders in Universal Primate Pluripotency Stem Cell medium, as opposed to Essential 12 medium supplemented with IWR1, which produced cytogenetic abnormalities. Rhesus neural progenitor cells were generated using a monolayer protocol and expressed PAX6 and NESTIN after 21 days of differentiation. Our reliable method will be useful to labs seeking to derive RhiPSCs for preclinical studies. Overall, the RhiPSCs generated from MAPT R406W carriers will be a critical resource for evaluating the molecular underpinnings of tau-related neurodegeneration across primate species. ### Competing Interest Statement The authors have declared no competing interest. NIH, R33NS115102, F31AG084303, R01NS124857, P51OD011106, R24 OD034055
Cilia are critical sensory organelles that project from the cell surface into the tissue environment, where they are surrounded by extracellular matrix (ECM). Abnormal ECM and fibrosis are two hallmarks of ciliopathies, yet the relationship between cilia and ECM is not well understood. Using the sense organs of C. elegans as a model, we found that a neomorphic mutation in the ECM gene mec-9 impacts sensory cilia function, ciliary protein localization, microtubule ultrastructure, and shedding of ciliary extracellular vesicles (EVs). We show that mec-9 is not expressed in EV releasing neurons, but rather by companion neurons in the sense organs, and may act cell non-autonomously. Our studies reveal pleiotropic roles for mec-9 in the C. elegans ciliated nervous system and provide an in vivo model to study the relationship between cilia and ECM. ### Competing Interest Statement The authors have declared no competing interest. National Institutes of Health, https://ror.org/01cwqze88, DK059418, DK116606, NS120745, F31DK103550, K12GM093854, R24OD010943 National Institutes of Health, https://ror.org/01cwqze88, R35NS105092, P40OD010440 Polycystic Kidney Disease Foundation, https://ror.org/05g0xkn45, 959686
Tau protein, the primary component in neurofibrillary tangles characteristic of Alzheimer’s Disease and related dementia disorders, normally regulates microtubule growth and stability. While tau dysfunction contributes to the progression of tauopathies, the role of microtubules in disease has remained unclear. Through forward genetic screening in Caenorhabditis elegans tauopathy models, we found multiple tubulin gene mutations that rescue tau-mediated neurodegeneration. Whole animal behavioral and in vitro biochemical assays were employed to characterize mutation-driven effects on neuron function, neurodegeneration, and effects on tubulin and tau proteins as well as microtubule function. Mutant tubulin genes were found to confer different levels of suppression correlating with the level of mutant gene expression. Mutant tubulins did not drastically alter total tau protein levels, tau phosphorylation or aggregation, however tau-induced neurodegeneration was rescued. The suppression of tau toxicity by tubulin gene mutations cannot be explained by changes in tau or tubulin expression, tau phosphorylation, or tau aggregation state. Rather the tubulin mutations appear to act by influencing global microtubule properties. In vitro experiments using C. elegans tubulin in semi-isolated and isolated contexts have indicated changes to microtubule properties without observable changes to tau-tubulin affinity. This work suggests that manipulation of microtubules can rescue tauopathy even when pathological tau species persist, supporting the importance of understanding microtubule contributions to disease progression and investigation into microtubule targeted gene therapy or small molecule approaches for tauopathy intervention. ### Competing Interest Statement The authors have declared no competing interest. * FTDP-17 : Frontotemporal Lobar Dementia with Parkinsonism chromosome 17 type AD : Alzheimer’s Disease MT(s) : microtubule(s) MAPs : Microtubule Associated Proteins TDP-43 : TAR DNA-binding protein 43 sut : suppressor of tau Pathology CeNGEN : C. elegans Gene Expression Network Aβ : Amyloid-β His : Histidine WT : wildtype Tg : transgenic
Background The centrosome is a critical regulator of cortical development, orchestrating microtubule dynamics, cell cycle progression, and neuronal migration. Disruptions in centrosome-associated proteins have been associated with a range of neurodevelopmental disorders. CEP170, a microtubule-binding protein localized to the subdistal appendages (SDA) of centrioles, has been implicated in centrosome function, yet its role in corticogenesis remains poorly defined. Methods We analyzed CEP170 expression in mouse cortex using western blotting, qPCR, scRNA-seq, and spatial transcriptomics, and examined its transcript expression in the developing human cortex using scRNA-seq. Loss-of-function phenotypes were assessed via in utero electroporation of Cep170-targeting shRNAs in embryonic mouse cortex. Cell proliferation and microtubule dynamics were analyzed using CRISPR/Cas9-generated CEP170-knockout cells, flow cytometry, microtubule regrowth assays, and immunofluorescence microscopy. Protein interactions were examined via co-immunoprecipitation and subcellular localization studies. Results We show that CEP170 is expressed in both neural progenitors and postmitotic neurons during cortical development. Cep170 knockdown in embryonic mouse cortex resulted in profound neuronal migration deficits, altered laminar fate, and abnormal dendritic morphology. CEP170 depletion also impaired progenitor cell proliferation both in vitro and in vivo. Mechanistically, C-terminal truncations disrupted CEP170 centrosomal and microtubule localization, mediated via impaired interactions with CCDC120. These truncations impaired microtubule regrowth and organization. Strikingly, partial deletion of CEP170’s centrosomal targeting and microtubule-binding domains led to severe migration deficits in the developing cortex. Conclusion Our findings identify CEP170 as a critical regulator of neural progenitor proliferation, neuronal migration, and cortical architecture via centrosome-microtubule interactions, providing new insights into centrosome-linked neurodevelopmental disorders. Graphical Abstract
Rxiv mechanobio
Rxiv cytoskeleton