Biology subjects

Nonaka, T.

Publications and source records attributed to Nonaka, T..

8 recordsLinked to original sources

bioRxiv2026

The 329HHK331 motif is essential for Alzheimer's disease tau filament fold

Cryo-electron microscopy (cryo-EM) has revealed disease-specific tau filament folds, yet the local sequence elements that determine them remain poorly understood. Here we focused on the 329HHK331 motif near an inter-protofilament interface in Alzheimers disease (AD)-type tau filaments, and analyzed recombinant dGAE filaments of wild-type (WT) and mutants in this motif. All mutants formed amyloid-like filaments in vitro, but their morphologies differed. In cultured cells, WT filaments efficiently seeded WT tau aggregation. Filaments with three-residue changes (deletion or Ala substitution) showed almost no seeding activity, whereas two-residue deletions retained partial activity. Cryo-EM showed that WT dGAE filaments form a quadruple helical filament of two protofilament dimers. Each dimer comprises two C-shaped protofilaments, centered on a 333GGG335-mediated inter-protofilament interaction and supported by flanking 329HHK331-336QVE338 contacts. Three-residue alterations abolished interactions with the QVE motif at the protofilament interface, thereby displacing 333GGG335 and forming non-C-shaped protofilament structures that are intrinsically poor templates for tau seeding. By contrast, two-residue deletions maintained the C-shaped protofilament structure because the remaining residue formed alternative inter-protofilament interactions. These findings suggest that the 329HHK331 region is a key determinant of the AD-like C-shaped protofilament fold and link this motif to tau seeding, providing insight into disease-specific tau filament formation.

neuroscience↗

bioRxiv2025

α-Synuclein aggregates induce mitochondrial damage and trigger innate immunity to drive neuron-microglia communication

Tunneling nanotubes (TNTs) enable direct intercellular transfer of macromolecules, organelles, and pathogenic protein aggregates. While -synuclein (-Syn) aggregates are known to promote TNT formation, the underlying mechanisms remain poorly defined. Here, using human neuronal and microglial cell lines, as well as iPSC-derived dopaminergic neurons and microglia, we show that -Syn aggregates induce severe mitochondrial damage, leading to cytosolic release of mitochondrial DNA (mtDNA) and activation of the cGAS-STING-NF- {kappa}B-IRF3 pathway. This innate immune response drives actin cytoskeleton remodeling and the formation of TNT-like structures, promoting intercellular transfer of -Syn from neurons to microglia. Additionally, neuronal cells transfer damaged mitochondria to microglia, where they undergo lysosome-mediated degradation. Neuron-to-microglia communication under -Syn- induced stress also triggers a bystander inflammatory response in microglia, suggesting a neuroimmune activation. Our findings identify mitochondrial damage and STING-mediated inflammation as key drivers of TNT formation and -Syn propagation, highlighting new potential targets to modulate disease progression in Synucleinopathies.

cell biology↗

bioRxiv2024

Heteromeric amyloid filaments of ANXA11 and TDP-43 in FTLD-TDP Type C

Neurodegenerative diseases are characterised by the abnormal filamentous assembly of specific proteins in the central nervous system1. Human genetic studies established a causal role for protein assembly in neurodegeneration2. However, the underlying molecular mechanisms remain largely unknown, which is limiting progress in developing clinical tools for these diseases. Recent advances in electron cryo-microscopy (cryo-EM) have enabled the structures of the protein filaments to be determined from patient brains1. All diseases studied to date have been characterised by the self-assembly of a single intracellular protein in homomeric amyloid filaments, including that of TAR DNA-binding protein 43 (TDP-43) in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) Types A and B3,4. Here, we used cryo-EM to determine filament structures from the brains of individuals with FTLD-TDP Type C, one of the most common forms of sporadic FTLD-TDP. Unexpectedly, the structures revealed that a second protein, annexin A11 (ANXA11), co-assembles with TDP-43 in heteromeric amyloid filaments. The ordered filament fold is formed by TDP-43 residues G282/284-N345 and ANXA11 residues L39-L74 from their respective low-complexity domains (LCDs). Regions of TDP-43 and ANXA11 previously implicated in protein-protein interactions form an extensive hydrophobic interface at the centre of the filament fold. Immunoblots of the filaments revealed that the majority of ANXA11 exists as a [~]22 kDa N-terminal fragment (NTF) lacking the annexin core domain. Immunohistochemistry of brain sections confirmed the co-localisation of ANXA11 and TDP-43 in inclusions, redefining the histopathology of FTLD-TDP Type C. This work establishes a central role for ANXA11 in FTLD-TDP Type C. The unprecedented formation of heteromeric amyloid filaments in human brain revises our understanding of amyloid assembly and may be of significance for the pathogenesis of neurodegenerative diseases.

neuroscience↗

bioRxiv2024

Differential alpha-Synuclein-induced Autophagy Dysfunction in Neuronal and Microglial Cells drives Tunneling Nanotubes and Aggregate Spread to Microglia

Tunneling nanotubes (TNTs) play a crucial role in intercellular communication, enabling a dynamic network for the transfer of molecular cargo over long distances between connected cells. Previous studies have demonstrated efficient, directional transfer of -Synuclein (-Syn) aggregates from neurons to microglia, with endosomal trafficking and lysosomal processing identified as the primary events following -Syn internalization. Using human neuronal and microglial cell lines, we found that microglia exhibit higher lysosomal turnover, particularly through lysophagy, whereas neuronal lysosomes display compromised degradative capacity and impaired autophagic flux. This deficiency results in less efficient degradation of aggregates in neurons. Moreover, perturbation of autophagy enhances TNT-mediated transfer of aggregate from neuronal cells to microglia. In contrast, microglia co-cultured with -Syn-containing neurons upregulate autophagy flux, enabling efficient degradation of the transferred aggregates. These findings were further validated using human induced pluripotent stem cells (hiPSC)-derived neurons and microglia. Overall, our study highlights the distinct responses of neurons and microglia to -Syn aggregates and identifies dysfunctional autophagy in neurons as a key driver of the preferential and directional transfer of aggregates to microglia.

cell biology↗

bioRxiv2022

Structure and Function of Gli123 Involved in Mycoplasma mobile Gliding

Mycoplasma mobile is a fish pathogen that glides on solid surfaces by means of a unique mechanism. The gliding machinery of M. mobile is composed of internal and surface structures. In the present study, we focused on the function and structure of Gli123, a surface protein that is essential for the localization of other surface proteins. The amino acid sequence of Gli123, which is 1128 amino acids long, contains lipoprotein-specific repeats. We isolated the native Gli123 protein from M. mobile cells and a recombinant protein, rGli123, from Escherichia coli. The isolated rGli123 complemented a non-binding and non-gliding mutant of M. mobile that lacked Gli123. Circular dichroism and rotary-shadowing electron microscopy (EM) showed that rGli123 has a structure that is not significantly different from that of the native protein. Rotary-shadowing EM suggested that the molecules changed their shape between globular and rod-like structures, depending on the ionic strength of the solution. Negative-staining EM coupled with single-particle analysis revealed that Gli123 forms a globular structure featuring a small protrusion with dimensions of 20.0, 14.5, and 16.0 nm. Small-angle X-ray scattering analyses indicated a rod-like structure composed of several tandem globular domains with total dimensions of approximately 34 nm length and 4 nm width. Both molecular structures were suggested to be dimers based on the predicted molecular size and structure. Gli123 may have evolved by multiplication of repeating lipoprotein units and acquired clumping role of surface proteins. IMPORTANCEMycoplasmas are pathogenic bacteria that are widespread in animals. They are characterized by small cell and genome sizes but are equipped with unique abilities to escape host immunity, such as surface variation and gliding. Here, we focused on a surface-localizing protein that is essential for Mycoplasma mobile gliding. The findings of this study suggested that the protein undergoes drastic conformational changes between its rod-like and globular structures. These changes may be caused by a repetitive structure common in the surface proteins that is responsible for the modulation of the cell surface structure and related to the assembly process for the surface gliding machinery. An evolutionary process for this unique mycoplasma gliding mechanism has also been suggested in the present study.

microbiology↗

bioRxiv2021

AIM2 inflammasome activation in astrocytes occurs during the late phase of EAE

Inflammasomes are a class of innate immune signaling platforms that activate in response to an array of cellular damage and pathogens. Inflammasomes promote inflammation under many circumstances to enhance immunity against pathogens and inflammatory responses through their effector cytokines, IL-1{beta} and IL-18. Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are such autoimmune conditions influenced by inflammasomes. Despite work investigating inflammasomes during EAE, little remains known concerning the role of inflammasomes in the central nervous system (CNS) during the disease. Here we use multiple genetically modified mouse models to monitor activated inflammasomes in situ based on ASC oligomerization in the spinal cord. Using inflammasome reporter mice, we found heightened inflammasome activation in astrocytes after the disease peak. In contrast, microglia and CNS-infiltrated myeloid cells had few activated inflammasomes in the CNS during EAE. Astrocyte inflammasome activation was dependent on AIM2, but low IL-1{beta} expression and no significant signs of cell death were found in astrocytes during EAE. Thus, the AIM2 inflammasome activation in astrocytes may have a distinct role from traditional inflammasome-mediated inflammation. SIGNIFICANCE STATEMENTInflammasome activation in the peripheral immune system is pathogenic in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). However, inflammasome activity in the central nervous system (CNS) is largely unexplored. Here, we used genetically modified mice to determine inflammasome activation in the CNS during EAE. Our data indicated heightened AIM2 inflammasome activation in astrocytes after the disease peak. Unexpectedly, neither CNS-infiltrated myeloid cells nor microglia were the primary cells with activated inflammasomes in SC during EAE. Despite AIM2 inflammasome activation, astrocytes did not undergo apparent cell death and produced little of the proinflammatory cytokine, IL-1{beta}, during EAE. This study showed that CNS inflammasome activation occurs during EAE without associating with IL-1{beta}-mediated inflammation.

immunology↗

bioRxiv2020

Dectin-1 limits central nervous system autoimmunity through a non-canonical pathway

Pathologic roles for innate immunity in neurologic disorders are well-described, but protective aspects of the immune response are less understood. Dectin-1, a C-type lectin receptor (CLR), is largely known to induce inflammation. However, we found that Dectin-1 is protective in experimental autoimmune encephalomyelitis (EAE), while its canonical signaling mediator, Card9, promotes the disease. Notably, Dectin-1 does not respond to heat-killed Mycobacteria, an adjuvant to induce EAE. Myeloid cells mediate the protective function of Dectin-1 in EAE and upregulate gene expression of neuroprotective molecules, including Oncostatin M (Osm) through a non-canonical Card9-independent pathway, mediated by NFAT. Furthermore, we found that the Osm receptor (OsmR) functions specifically in astrocytes to reduce EAE severity. Our study revealed a new mechanism of protective myeloid-astrocyte crosstalk regulated by a non-canonical Dectin-1 pathway and identifies novel therapeutic targets for CNS autoimmunity. Graphical AbstractO_LIDectin-1 is a protective C-type lectin receptor (CLR) in experimental autoimmune encephalomyelitis (EAE) C_LIO_LIDectin-1 promotes expression of Osm, a neuroprotective IL-6 family cytokine, in myeloid cells C_LIO_LIOsmR signaling in astrocytes limits EAE progression and promotes remission C_LIO_LINon-canonical Card9-independent signaling drives a distinct Dectin-1-mediated transcriptional program to induce expression of Osm and other factors with protective or anti-inflammatory functions C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/080481v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@885aa2org.highwire.dtl.DTLVardef@1f68087org.highwire.dtl.DTLVardef@70263aorg.highwire.dtl.DTLVardef@ddc3b4_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2020

α-Synuclein strains that cause distinct pathologies differentially inhibit proteasome

Abnormal -synuclein aggregation has been implicated in several diseases and is known to spread in a prion-like manner. There is a relationship between protein aggregate structure (strain) and clinical phenotype in prion diseases, however, whether differences in the strains of -synuclein aggregates account for the different pathologies remained unclear. Here, we generated two types of -synuclein fibrils from identical monomer and investigated their seeding and propagation ability in mice and primary-cultured neurons. One -synuclein fibril induced marked accumulation of phosphorylated -synuclein and ubiquitinated protein aggregates, while the other did not, indicating the formation of -synuclein two strains. Notably, the former -synuclein strain inhibited proteasome activity and co-precipitated with 26S proteasome complex. Further examination indicated that structural differences in the C-terminal region of -synuclein strains lead to different effects on proteasome activity. These results provide a possible molecular mechanism to account for the different pathologies induced by different -synuclein strains.

neuroscience↗