Nobuhiko Ohno

Neurons extend axons toward spatially defined but often distant targets to establish brain circuits. Callosal projection neurons exemplify this organization, linking corresponding cortical territories between the two hemispheres ¹ . Growing axons rely on growth cones to regulate direction and speed through densely packed tissues ^2,3 . Although growth cone morphology and dynamics have been extensively characterized in cultured neurons and invertebrate models, how growth cones navigate the three-dimensional environment of the developing mammalian brain remains poorly explored. Here, we combined a genetically encoded electron microscopy (EM)-labeling strategy and zero-shot deep-learning-based tracing with high-resolution volume EM to reconstruct growth cones of identified callosal projection neurons in the developing mouse brain. Mediolaterally extending growth cones exhibited a planar, fan-shaped morphology, preferentially expanded along the dorsoventral axis. Filopodia extended from the edges of this plane and aligned predominantly with the same axis but showed no directional bias toward the growing direction. Longitudinal in vivo imaging revealed that increased growth cone complexity associated with filopodial extension coincided with lower instantaneous velocity but preceded accelerated extension. Together, these findings reveal a dorsoventrally oriented planar growth cone architecture in which filopodial extension precedes accelerated axon growth, suggesting a structural constraint that may restrict axon navigation to the callosal plane.

Aging is associated with impairments in cognitive flexibility, a key executive function supported by the medial prefrontal cortex (mPFC), yet the biological mechanisms underlying individual variability in age‐related decline remain poorly understood. Here we investigated behavioral, ultrastructural, and proteomic correlates of cognitive inflexibility in mice across aging. Using a touchscreen‐based attentional set‐shifting task, we observed substantial individual variability in cognitive inflexibility among aged C57BL/6J mice. Volume electron microscopy of the mPFC revealed age‐related reductions in synaptic density, but these structural changes did not correlate with cognitive performance. Instead, the proportion of synapses containing presynaptic mitochondria was inversely associated with cognitive flexibility in aged mice. To identify molecular correlates, we performed proteomic profiling of mPFC whole tissue and synaptosome fractions. Proteins associated with individual variability in cognitive inflexibility were largely distinct from those associated with chronological aging. Notably, synaptosomal proteins negatively correlated with cognitive performance were strongly enriched for mitochondrial pathways, including oxidative phosphorylation, mitochondrial translation, and the tricarboxylic acid cycle. Consistent with these findings, the mitochondria‐targeted antioxidant MitoQ improved attentional set‐shifting performance in aged mice without affecting initial learning. Proteomic analyses revealed that MitoQ reduced the abundance of synaptosomal mitochondrial proteins, particularly those involved in mitochondrial apoptotic signaling. Together, these results suggest that synaptic mitochondrial oxidative stress in the mPFC contributes to individual vulnerability to cognitive inflexibility. Targeting synaptic mitochondrial oxidative stress may therefore represent a promising strategy to preserve executive function during aging.

Background Podocyte infolding glomerulopathy (PIG) is a rare glomerular disease, with case reports and cohort studies lacking clear morphological diagnostic criteria. We aimed to establish transmission electron microscopy (TEM)–based diagnostic criteria for PIG and classify its morphological subtypes. Methods This retrospective nationwide cohort study was conducted by the PIG Working Group of the Japanese Society of Nephrology. Clinical data and TEM findings were collected from patients diagnosed with or suspected of PIG at 69 institutions across Japan between 1991–2025. PIG was defined by TEM as podocyte infolding exceeding half the glomerular basement membrane (GBM) thickness at two or more sites within a single capillary loop or the presence of microspheres or microtubules within the GBM. Cases were classified into morphological subtypes, and their clinical characteristics were analyzed. Results Among 103 registered cases, 93 met diagnostic criteria. Four morphological subtypes were identified: Type A, primary podocyte infolding without prominent microspheres or microtubules (n = 21); Type B, predominant microspheres (n = 34); Type C, microtubules with or without microspheres (n = 38); and Type D, dense clusters of microspheres (n = 10). Types B and C were more common in younger females and frequently associated with systemic lupus erythematosus (58% and 71%, respectively), whereas Types A and D occurred mainly in older males and were not associated with SLE. Conclusions We established TEM-based diagnostic criteria for PIG and identified four morphological subtypes, suggesting two clinicopathological phenotypes: SLE-associated (B and C) and non–SLE-associated (A and D).

Memories leave lasting physical changes at the synaptic level. Although stable, larger spines are thought to support memory, the high turnover of dendritic spines and the drifting of neuronal representations after memory formation suggest alternative possibilities. To elucidate the structural trace underlying memory retention, we used a mouse model of artificial hibernation. During hibernation, hippocampal neurons exhibited a substantial reduction in their activity and an extensive elimination of dendritic spines and synapses. Despite these changes, their memory and associated hippocampal neuronal representations remained intact. We found that a subset of spines characterized by synaptic contacts with multisynaptic boutons is maintained during hibernation. These findings suggest that synaptic engram architecture, rather than larger spines per se, is resilient to network remodeling and associated with long-term memory retention.

The molecular mechanisms underlying metastasis still remain unclear. We previously established a suspension culture using low‐attachment culture dishes and demonstrated that cell lines adapted through 2 months suspension culture (termed FL sublines) exhibited higher metastatic potential than their parental counterparts. In this study, we identified the molecules involved in acquiring these phenotypes under low‐adhesion conditions. We showed that detached tumor cells in suspension culture formed spheroids that recapitulated tumor cells in the spread‐through‐air space (STAS), and demonstrated that the anti‐adhesion molecule mucin 21 was upregulated in detached lung cancer cells independent of driver mutations. Analyses of both cell lines and primary tumors revealed that mucin 21 is overexpressed in terminal respiratory unit‐type lung adenocarcinomas. Mucin 21‐knockout cells showed reduced viability and proliferation under adherent and low‐attachment conditions, accompanied by enhanced anoikis. Transmission electron microscopy revealed that the intercellular spaces observed in FL sublines during suspension culture were reduced in mucin 21‐knockout cells, suggesting impaired acquisition of low adhesive properties. Thus, mucin 21 appears crucial for the survival of terminal respiratory unit‐type lung adenocarcinoma cells under both adherent and low‐adhesion conditions.

CEP152 is essential for centriole function and neurodevelopment, and pathogenic recessive variants in CEP152 cause primary microcephaly. We identified new compound heterozygous CEP152 variants, c.314 G > A,p.(W105*) and c.2689 A > T,p.(K897*), in a microcephalic patient and analyzed them alongside a homozygous variant c.95 A > C,p.(Q32P) associated with severe microcephaly with marked gyral simplification. In vitro assays revealed distinct effects: p.K897* prevented centrosomal localization, p.W105* led to protein degradation, and p.Q32P retained centrosomal targeting but disrupted binding to Polo-like kinase 4, a key centriole biogenesis kinase and CEP152 partner. In vivo, both Cep152 ^W105*/K897* and Cep152 ^Q32P/Q32P knock-in mice displayed microcephaly; notably, Cep152 ^Q32P/Q32P mice also exhibited severe cortical defects during brain development. Cellular analyses revealed centrosome dysfunction, mitotic errors, and increased apoptosis, which were exacerbated in Cep152 ^Q32P/Q32P brains. Morphological examination, including electron microscopy, further demonstrated structural abnormalities of the centrosomes and centrioles in Cep152 ^Q32P/Q32P brains. Electrophysiological and gene expression analyses confirmed variant-specific neuronal impairments, which correlate with clinical severity. Collectively, these findings demonstrate that distinct CEP152 variants disrupt neurodevelopment through different mechanisms, thereby explaining the spectrum of microcephaly severity and associated phenotypes.

The memory trace at the neuronal and synaptic levels remains controversial. Stable, larger spines are thought to support memory, but the high turnover of dendritic spines and the drifting of neuronal representations following memory formation suggest alternative possibilities. To elucidate a structural trace underlying memory retention, we utilize a mouse model of artificial hibernation. During hibernation, hippocampal neurons exhibited a substantial reduction in their activity and an extensive elimination of dendritic spines and synapses. Despite these changes, their memory and associated hippocampal neuronal representations are intact after arousal. We find that a subset of spines is maintained during hibernation. These spatially clustered engram-engram synapses are exclusively protected from elimination and characterized by synaptic contacts with multi-synaptic boutons. These findings suggest that synaptic engram architecture, rather than larger spines per se, is resilient to network remodeling and underlies long-term memory retention.

Myelin formation by oligodendrocytes is essential for the regulation of the conduction velocity and proper brain function. To ensure accurate information processing in response to experiences such as sensory stimuli and learning, oligodendrocytes adjust their number and morphology. In addition, oligodendrocyte morphology changes with senescence and in the presence of neurodegenerative diseases. Thus, visualizing oligodendrocytes and analyzing their morphology is crucial for understanding how our brains change under such conditions. Herein, we describe the methods for labeling and analyzing the morphologies of individual oligodendrocytes in mouse white matter at the light microscopic level. PDGFRa-CreERT2:Tau-mGFP and PLP-CreERT2:Tau-mGFP mice enable us to visualize and analyze later-born or early-born oligodendrocyte morphology. In addition, sparse oligodendrocyte labeling with attenuated rabies virus expressing GFP enables the visualization and morphological analysis of individual oligodendrocytes in various brain white matter regions without the need for transgenic animals. Furthermore, the combination with immunostaining in thick tissues enables the identification of labeled oligodendrocytes and myelin sheaths, as well as their interactions with neuronal axons. These methods are suitable for revealing how oligodendrocytes adapt their morphologies depending on environmental stimuli or pathological conditions.

The activity of oligodendrocyte progenitor cells (OPCs) and oligodendrocytes (OLs) throughout life drives myelination, which is crucial for rapid neuronal communication. OLs in the aging brain demonstrate a reduced capacity for myelin formation and maintenance, but the underlying differentiation of individual OLs and morphological changes of their myelin in aging remain unclear. Here, we utilized Pdgfra‐CreER^T2:Tau‐mGFP double transgenic mice to selectively label and visualize newly generated OLs in aged (78‐week‐old) mice and compared them with those in young (8‐week‐old) mice. We revealed a significantly lower percentage of newly generated OLs that differentiated into mature OLs and a decreased rate of myelinating OLs accumulation in aged mice compared with young mice. Additionally, newly generated myelinating mature OLs in aged mice demonstrated significantly greater height compared with those in young mice. Furthermore, myelin internodes were significantly shorter and significantly fewer in aged mice compared with young mice. Our results indicate age‐related impairments in the differentiation efficiency of aged OPCs and age‐related morphological changes in OLs. These alterations in newly generated OLs may contribute to impaired myelination, reduced myelin turnover, and disrupted myelin maintenance in aged mice.