Background Dementia is a progressive disorder characterized by memory impairment and cognitive decline; however, the systemic molecular alterations associated with aging and dementia remain incompletely understood. In this study, we performed mass spectrometry-based plasma proteomic profiling in two independent cohorts to identify molecular signatures linked to cognitive function. Methods In the healthy aging cohort, 2,411 proteins were identified from 119 successfully analyzed plasma samples collected from individuals aged 20–79 years. In the cognitive decline cohort comprising non-demented controls (NC; n = 20), patients with mild cognitive impairment (MCI; n = 20), and those with Alzheimer’s disease (AD; n = 20) diagnosed according to National Institute on Aging–Alzheimer’s Association criteria, 1,494 proteins were identified. Differential protein expression was assessed using permutation-based false discovery rate correction (FDR < 0.05). Results In the healthy aging cohort, plasma proteomic changes occurred gradually with age and were enriched in immune and inflammatory pathways. In contrast, the cognitive decline cohort showed distinct alterations enriched in actin- and platelet-related proteins. Cluster analysis revealed stage-specific molecular signatures emerging during the transition from normal cognition to mild cognitive impairment and AD. Integrated comparison of the two cohorts suggested that aging- and AD-associated proteomic changes followed divergent trajectories. Notably, among the proteins significantly altered in AD (FDR < 0.05), 80 have not previously been reported in plasma dementia studies. Conclusions Plasma alterations associated with cognitive decline are not merely an extension of normal aging but may reflect distinct systemic processes involving immune regulation, actin, and platelet function. These findings from a discovery cohort provide insight into systemic molecular mechanisms underlying cognitive decline and identify candidate blood-based biomarkers and potential therapeutic targets for dementia. Further validation in larger longitudinal studies with balanced cohort designs, appropriate statistical adjustments, and standardized experimental procedures are warranted.

Age-related declines in cognitive and motor function are influenced not only by intrinsic brain changes but also by systemic factors, including circulating blood components. Recent studies have suggested that rejuvenation of the hematopoietic system may influence brain ageing; however, the effects of bone marrow transplantation (BMT) during middle age remain poorly understood. In this study, we examined behavioral outcomes following heterochronic BMT in 8-month-old mice conditioned with busulfan and transplanted with bone marrow cells from either young (2-month-old) or age-matched donors. Three months post-transplantation, mice receiving young bone marrow showed changes in wire-hang performance and showed differences in open-field parameters, while recognition memory measured by the novel object recognition task was unchanged. Immunohistochemical analysis showed a tendency toward increased doublecortin- and EdU-positive cells in the dentate gyrus of young-donor BMT mice; however, this difference did not reach statistical significance. Together, these findings suggest that heterochronic BMT may be associated with changes in selected behavioral measures in middle-aged mice and warrant further studies to clarify the relationship between systemic factors and age-related brain changes.

Focal cortical dysplasia type II (FCDII) is a malformation of cortical development caused by somatic mutations in the mTOR signaling pathway. Two hallmark pathological cell types in FCDII, dysmorphic neurons (DNs) and balloon cells (BCs), arise as a result of somatic mutations in the mTOR signaling pathway and are implicated in the pathophysiology of drug-resistant epilepsy. However, how these somatic mutations reshape cell states within the human cortex remains poorly understood. Here, we integrate imaging-based spatial transcriptomics (iST), single-nucleus RNA sequencing, and proteomics of surgically resected FCDIIb tissue to define the transcriptional and proteomic profiles of DNs and BCs. Spatial mapping of iST data resolved transcriptional signatures in histologically validated DNs and BCs within FCDIIb sections. Integrative omics analysis further revealed that DNs show upregulation of PI3K-AKT-mTOR and p53-CROT metabolic programs accompanied by suppression of synaptic signaling, whereas BCs exhibit transcriptional signatures of reactive astrocytes with increased phagocytic and immune-like activity. These data delineate cell-type-specific consequences of somatic mTOR pathway mutations at single-cell resolution and reveal previously unrecognized metabolic and immunoregulatory mechanisms contributing to epileptogenesis in drug-resistant epilepsy. Our study establishes a spatial multi-omics framework for dissecting human cortical malformations and highlights potential therapeutic targets for drug-resistant epilepsy. ### Competing Interest Statement The authors have declared no competing interest. Japan Agency for Medical Research and Development, JP20ek0109374, JP24wm0425005h0004, 25ek0109764h0001, 25wm0625508h0001, 21wm0425019, 25wm0625126 Japan Society for the Promotion of Science, JP22K15134, JP20K15919, JP23K14295, JP22K09273, JP22H02730 Japan Health Research Promotion Bureau, 2024-D-01 Intramural Research Grant of NCNP, 3-9, 4-5, 4-6, 7-8, 3-8 Tokumori Yasumoto Memorial Trust Takeda Science Foundation

With Japan's aging population, the number of individuals diagnosed with dementia has been steadily rising, creating significant social and economic challenges. Dementia is caused by various underlying conditions that lead to acquired brain injury. It is characterized by a progressive decline in cognitive function, which can impair activities of daily living (ADLs) and social interactions. However, current medical interventions for neurodegenerative dementias remain insufficient to achieve a complete cure.

Hippocampal pyramidal neurons exhibit diverse spike patterns and gene expression profiles. However, their relationships with single neurons are not fully understood. In this study, we designed an electrophysiology-based experimental procedure to identify gene expression profiles using RNA sequencing of single hippocampal pyramidal neurons whose spike patterns were recorded in living mice. This technique involves a sequence of experiments consisting of in vivo juxtacellular recording and labeling, brain slicing, cell collection, and transcriptome analysis. We demonstrated that the expression levels of a subset of genes in individual hippocampal pyramidal neurons were significantly correlated with their spike burstiness, submillisecond-level spike rise times or spike rates, directly measured by in vivo electrophysiological recordings. Because this methodological approach can be applied across a wide range of brain regions, it is expected to contribute to studies on various neuronal heterogeneities to understand how physiological spike patterns are associated with gene expression profiles.

In the central nervous system, astrocytes enable appropriate synapse function through glutamate clearance from the synaptic cleft; however, it remains unclear how astrocytic glutamate transporters function at peri-synaptic contact. Here, we report that Down syndrome cell adhesion molecule (DSCAM) in Purkinje cells controls synapse formation and function in the developing cerebellum. Dscam-mutant mice show defects in CF synapse translocation as is observed in loss of function mutations in the astrocytic glutamate transporter GLAST expressed in Bergmann glia. These mice show impaired glutamate clearance and the delocalization of GLAST away from the cleft of parallel fibre (PF) synapse. GLAST complexes with the extracellular domain of DSCAM. Riluzole, as an activator of GLAST-mediated uptake, rescues the proximal impairment in CF synapse formation in Purkinje cell-selective Dscam-deficient mice. DSCAM is required for motor learning, but not gross motor coordination. In conclusion, the intercellular association of synaptic and astrocyte proteins is important for synapse formation and function in neural transmission.

DSCAM (Down syndrome cell adhesion molecule) is a unique neuronal adhesion protein with extensively documented multifaceted functionalities. DSCAM also has interesting properties in vertebrates and invertebrates, respectively. In Drosophila species, particularly, Dscam exhibits remarkable genetic diversity, with tens of thousands of splicing isoforms that modulate the specificity of neuronal wiring. Interestingly, this splice variant diversity of Dscam is absent in vertebrates. DSCAM plays a pivotal role in mitigating excessive adhesion between identical cell types, thereby maintaining the structural and functional coherence of neural networks. DSCAM contributes to the oversight of selective intercellular interactions such as synaptogenesis; however, the precise regulatory mechanisms underlying the promotion and inhibition of cell adhesion involved remain unclear. In this review, we aim to delineate the distinct molecules that interact with DSCAM and their specific roles within the biological landscapes of Drosophila and vertebrates. By integrating these comparative insights, we aim to elucidate the multifunctional nature of DSCAM, particularly its capacity to facilitate or deter intercellular adhesion.

Down Syndrome, a genetic disorder resulting from trisomy of chromosome 21, is frequently associated with neurodevelopmental anomalies and vascular and hematologic pathologies. Moreover, the predisposition of Down Syndrome patients to develop early onset Alzheimer's disease post 50 years of age has been postulated to be linked with the trisomy state of the amyloid precursor protein gene located on chromosome 21. However, the pathogenesis of Alzheimer's disease remains elusive since the accumulation of amyloid beta does not necessarily cause Alzheimer's disease, and antibody therapy aimed at removing amyloid beta does not achieve remission. Recent literature has provided intriguing evidence indicating the amelioration of cognitive function in geriatric mice upon administration of plasma from younger cohorts (Castellano et al., Nature, 2017), implicating a potential connection between cerebrovascular and hematologic conditions and insinuating that blood components may exert influence on cerebral function. Consequently, this study is designed to investigate whether the transplantation of bone marrow cells from Down Syndrome model mice leads to alterations in brain functionality. In this presentation, we will present data obtained to date and discuss potential mechanisms that regulate brain function by the hemopoietic stem cell.

Dementia, characterized by cognitive and memory deficits, leads to brain atrophy, cell death, and insoluble substance accumulation. Alzheimer's disease (AD), linked to the APP gene, is the most common dementia type. Amyloid-beta's production and aggregation can forecast AD progression 20 years before onset, though the exact triggers remain unclear. Treatments focus on symptoms, with no cure available. Juvenile Alzheimer's in Down syndrome adults is prevalent and resembles isolated Alzheimer's, making it a preventive treatment target (Fortea et al., Lancet, 2020). However, its initial pathogenesis is still unknown (Flores Aguilar et al., Brain, 2020). Our study found that wild type mice transplanted with hematopoietic stem cells from Down syndrome models had impaired short-term memory, indicating bone marrow-derived cells' influence on brain memory. Analyses of hippocampal neurons also showed changed neuronal properties. We are currently examining the mechanisms behind brain function changes induced by bone marrow transplantation, and further investigation is vital for understanding dementia's pathogenesis and opening paths for therapeutic development.

Parkinson's disease (PD) is characterized by dopaminergic (DAergic) neuronal loss in the substantia nigra pars compacta (SNpc), resulting from α-synuclein (αSyn) toxicity. We previously reported that αSyn oligomerization and toxicity are regulated by the fatty-acid binding protein 3 (FABP3), and the therapeutic effects of the FABP3 ligand, MF1, was successfully demonstrated in PD models. Here, we developed a novel and potent ligand, HY-11-9, which has a higher affinity for FABP3 (Kd = 11.7 ± 8.8) than MF1 (Kd = 302.8 ± 130.3). We also investigated whether the FABP3 ligand can ameliorate neuropathological deterioration after the onset of disease in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinsonism. Motor deficits were observed two weeks after MPTP treatment. Notably, oral administration of HY-11-9 (0.03 mg/kg) improved motor deficits in both beam-walking and rotarod tasks, whereas MF1 failed to improve the motor deficits in both tasks. Consistent with the behavioral tasks, HY-11-9 recovered dopamine neurons from MPTP toxicity in the substantia nigra and ventral tegmental areas. Furthermore, HY-11-9 reduced the accumulation of phosphorylated-serine129-α-synuclein (pS129-αSyn) and colocalization with FABP3 in tyrosine hydroxylase (TH)-positive DA neurons in the PD mouse model. Overall, HY-11-9 significantly improved MPTP-induced behavioral and neuropathological deterioration, suggesting that it may be a potential candidate for PD therapy.

Astrocytes are morphologically complex, with a myriad of processes which allow contact with other astrocytes, blood vessels, and neurons. Adhesion molecules expressed by these cells regulate this connectivity. Adhesion molecules are required to form and maintain functional neural circuits, but their importance and mechanisms of action, particularly in astrocyte-neuron contact, remain unresolved. Several studies of neuron-astrocyte connections have demonstrated the vital functions of adhesion molecules, including neuron-glia cell adhesion molecules, astrotactins, and protocadherins. In this review, we provide an overview and perspective of astrocyte-neuron contacts mediated by adhesion molecules in developing neural circuits and synapse formation, especially in the cerebellum. We also outline a novel mechanism of interaction between neurons and astrocytes in the tripartite synapses that has been recently found by our group.

Autism susceptibility candidate 2 (AUTS2), a risk gene for autism spectrum disorders (ASDs), is implicated in telencephalon development. Because AUTS2 is also expressed in the cerebellum where defects have been linked to ASDs, we investigated AUTS2 functions in the cerebellum. AUTS2 is specifically localized in Purkinje cells (PCs) and Golgi cells during postnatal development. Auts2 conditional knockout (cKO) mice exhibited smaller and deformed cerebella containing immature-shaped PCs with reduced expression of Cacna1a. Auts2 cKO and knock-down experiments implicated AUTS2 participation in elimination and translocation of climbing fiber synapses and restriction of parallel fiber synapse numbers. Auts2 cKO mice exhibited behavioral impairments in motor learning and vocal communications. Because Cacna1a is known to regulate synapse development in PCs, it suggests that AUTS2 is required for PC maturation to elicit normal development of PC synapses and thus the impairment of AUTS2 may cause cerebellar dysfunction related to psychiatric illnesses such as ASDs.

For normal neurogenesis and circuit formation, delamination of differentiating neurons from the proliferative zone must be precisely controlled; however, the regulatory mechanisms underlying cell attachment are poorly understood. Here, we show that Down syndrome cell adhesion molecule (DSCAM) controls neuronal delamination by local suppression of the RapGEF2-Rap1-N-cadherin cascade at the apical endfeet in the dorsal midbrain. Dscam transcripts were expressed in differentiating neurons, and DSCAM protein accumulated at the distal part of the apical endfeet. Cre-loxP-based neuronal labeling revealed that Dscam knockdown impaired endfeet detachment from ventricles. DSCAM associated with RapGEF2 to inactivate Rap1, whose activity is required for membrane localization of N-cadherin. Correspondingly, Dscam knockdown increased N-cadherin localization and ventricular attachment area at the endfeet. Furthermore, excessive endfeet attachment by Dscam knockdown was restored by co-knockdown of RapGEF2 or N-cadherin Our findings shed light on the molecular mechanism that regulates a critical step in early neuronal development.

The layer structure has been intensively characterized in the developing neocortex and cerebellum based on the various molecular markers. However, as to the developing dorsal midbrain, comprehensive analyses have not been intensely carried out, and thus, the name as well as the definition of each layer is not commonly shared. Here, we redefined the three layers, such as the ventricular zone, intermediate zone and marginal zone, based on various markers for proliferation and differentiation in embryonic dorsal midbrain. Biphasic Ki67 expression defines the classical VZ, in which there is clear separation of the mitotic and interphase zones. Next, we mapped the distribution of immature neurons to the defined layers, based on markers for glutamatergic and GABAergic lineage. Interestingly, Tbr2 and Neurog2 were expressed in the postmitotic neurons. We also report that active (phosphorylated) JNK is a useful marker to demarcate layers during the embryonic stage. Finally, we validated the final arrival layers of the migratory glutamatergic and GABAergic neurons. These results form a foundation for analyses of brain development, especially in the proliferation and migration of excitatory and inhibitory neurons in the dorsal midbrain.

The cerebellum represents a good model system to investigate the molecular machinery underlying the synapse development, including its initiation, maturation and elimination depending on the neuronal activity. Multiple climbing fibers (CFs, axons of Inferior olivary neurons) innervate single purkinje cell soma and make synaptic-contact in early developmental stage. As advance the stage, single CF (Winner-CF) strengthened depend on neural activity. This

Microtubules (MTs) play critical roles in various cellular events, including cell migration. End-binding proteins (EBs) accumulate at the ends of growing MTs and regulate MT end dynamics by recruiting other plus end-tracking proteins (+TIPs). However, how EBs contribute to MT dynamics through +TIPs remains elusive. We focused on tau-tubulin kinase 2 (TTBK2) as an EB1/3-binding kinase and confirmed that TTBK2 acted as a +TIP. We identified MT-depolymerizing kinesin KIF2A as a novel substrate of TTBK2. TTBK2 phosphorylated KIF2A at S135 in intact cells in an EB1/3-dependent fashion and inactivated its MT-depolymerizing activity in vitro. TTBK2 depletion reduced MT lifetime (facilitated shrinkage and suppressed rescue) and impaired HeLa cell migration, and these phenotypes were partially restored by KIF2A co-depletion. Expression of nonphosphorylatable KIF2A, but not wild-type KIF2A, reduced MT lifetime and slowed down the cell migration. These findings indicate that TTBK2 with EB1/3 phosphorylates KIF2A and antagonizes KIF2A-induced depolymerization at MT plus ends for cell migration.