Retinoic acid modulation of granule cell activity and spatial discrimination in the adult hippocampus.

Retinoic acid (RA), derived from vitamin A (retinol), plays a crucial role in modulating neuroplasticity within the adult brain. Perturbations in RA signaling have been associated with memory impairments, underscoring the necessity to elucidate RA's influence on neuronal activity, particularly within the hippocampus. In this study, we investigated the cell type and sub-regional distribution of RA-responsive granule cells (GCs) in the mouse hippocampus and delineated their properties. We discovered that RA-responsive GCs tend to exhibit a muted response to environmental novelty, typically remaining inactive. Interestingly, chronic dietary depletion of RA leads to an abnormal increase in GC activation evoked by a novel environment, an effect that is replicated by the localized application of an RA receptor beta (RARß) antagonist. Furthermore, our study shows that prolonged RA deficiency impairs spatial discrimination-a cognitive function reliant on the hippocampus-with such impairments being reversible with RA replenishment. In summary, our findings significantly contribute to a better understanding of RA's role in regulating adult hippocampal neuroplasticity and cognitive functions.

Comparative Anatomy of the Dentate Mossy Cells in Nonhuman Primates: Their Spatial Distributions and Axonal Projections Compared With Mouse Mossy Cells.

Glutamatergic mossy cells (MCs) mediate associational and commissural connectivity, exhibiting significant heterogeneity along the septotemporal axis of the mouse dentate gyrus (DG). However, it remains unclear whether the neuronal features of MCs are conserved across mammals. This study compares the neuroanatomy of MCs in the DG of mice and monkeys. The MC marker, calretinin, distinguishes two subpopulations: septal and temporal. Dual-colored fluorescence labeling is utilized to compare the axonal projection patterns of these subpopulations. In both mice and monkeys, septal and temporal MCs project axons across the longitudinal axis of the ipsilateral DG, indicating conserved associational projections. However, unlike in mice, no MC subpopulations in monkeys make commissural projections to the contralateral DG. In monkeys, temporal MCs send associational fibers exclusively to the inner molecular layer, while septal MCs give rise to wide axonal projections spanning multiple molecular layers, akin to equivalent MC subpopulations in mice. Despite conserved septotemporal heterogeneity, interspecies differences are observed in the topological organization of septal MCs, particularly in the relative axonal density in each molecular layer along the septotemporal axis of the DG. In summary, this comparative analysis sheds light on both conserved and divergent features of MCs in the DG of mice and monkeys. These findings have implications for understanding functional differentiation along the septotemporal axis of the DG and contribute to our knowledge of the anatomical evolution of the DG circuit in mammals.

Multi-proteomic analyses of 5xFAD mice reveal new molecular signatures of early-stage Alzheimer's disease.

An early diagnosis of Alzheimer's disease is crucial as treatment efficacy is limited to the early stages. However, the current diagnostic methods are limited to mid or later stages of disease development owing to the limitations of clinical examinations and amyloid plaque imaging. Therefore, this study aimed to identify molecular signatures including blood plasma extracellular vesicle biomarker proteins associated with Alzheimer's disease to aid early-stage diagnosis. The hippocampus, cortex, and blood plasma extracellular vesicles of 3- and 6-month-old 5xFAD mice were analyzed using quantitative proteomics. Subsequent bioinformatics and biochemical analyses were performed to compare the molecular signatures between wild type and 5xFAD mice across different brain regions and age groups to elucidate disease pathology. There was a unique signature of significantly altered proteins in the hippocampal and cortical proteomes of 3- and 6-month-old mice. The plasma extracellular vesicle proteomes exhibited distinct informatic features compared with the other proteomes. Furthermore, the regulation of several canonical pathways (including phosphatidylinositol 3-kinase/protein kinase B signaling) differed between the hippocampus and cortex. Twelve potential biomarkers for the detection of early-stage Alzheimer's disease were identified and validated using plasma extracellular vesicles from stage-divided patients. Finally, integrin α-IIb, creatine kinase M-type, filamin C, glutamine γ-glutamyltransferase 2, and lysosomal α-mannosidase were selected as distinguishing biomarkers for healthy individuals and early-stage Alzheimer's disease patients using machine learning modeling with approximately 79% accuracy. Our study identified novel early-stage molecular signatures associated with the progression of Alzheimer's disease, thereby providing novel insights into its pathogenesis.

Maladaptation of dentate gyrus mossy cells mediates contextual discrimination deficit after traumatic stress.

Fear overgeneralization is a maladaptive response to traumatic stress that is associated with the inability to discriminate between threat and safety contexts, a hallmark feature of post-traumatic stress disorder (PTSD). However, the neural mechanisms underlying this deficit remain unclear. Here, we show that traumatic stress exposure impairs contextual discrimination between threat and safety contexts in the learned helplessness (LH) model. Mossy cells (MCs) in the dorsal hippocampus are suppressed in response to traumatic stress. Bidirectional manipulation of MC activity in the LH model reveals that MC inhibition is causally linked to impaired contextual discrimination. Mechanistically, MC inhibition increases the number of active granule cells in a given context, significantly overlapping context-specific ensembles. Our study demonstrates that maladaptive inhibition of MCs after traumatic stress is a substantial mechanism underlying fear overgeneralization with contextual discrimination deficit, suggesting a potential therapeutic target for cognitive symptoms of PTSD.

Continuous long-range measurement of tonic dopamine with advanced FSCV for pharmacodynamic analysis of levodopa-induced dyskinesia in Parkinson's disease.

Levodopa, a dopamine prodrug, alleviates the motor symptoms of Parkinson's disease (PD), but its chronic use gives rise to levodopa-induced dyskinesia (LID). However, it remains unclear whether levodopa pharmacodynamics is altered during the progressive onset of LID. Using in vivo fast-scan cyclic voltammetry and second-derivative-based background drift removal, we continuously measured tonic dopamine levels using high temporal resolution recording over 1-h. Increases to tonic dopamine levels following acute levodopa administration were slow and marginal within the naïve PD model. However, these levels increased faster and higher in the LID model. Furthermore, we identified a strong positive correlation of dyskinetic behavior with the rate of dopamine increase, but much less with its cumulative level, at each time point. Here, we identified the altered signature of striatal DA dynamics underlying LID in PD using an advanced FSCV technique that demonstrates the long-range dynamics of tonic dopamine following drug administration.

A Neurospheroid-Based Microrobot for Targeted Neural Connections in a Hippocampal Slice.

Functional restoration by the re-establishment of cellular or neural connections remains a major challenge in targeted cell therapy and regenerative medicine. Recent advances in magnetically powered microrobots have shown potential for use in controlled and targeted cell therapy. In this study, a magnetic neurospheroid (Mag-Neurobot) that can form both structural and functional connections with an organotypic hippocampal slice (OHS) is assessed using an ex vivo model as a bridge toward in vivo application. The Mag-Neurobot consists of hippocampal neurons and superparamagnetic nanoparticles (SPIONs); it is precisely and skillfully manipulated by an external magnetic field. Furthermore, the results of patch-clamp recordings of hippocampal neurons indicate that neither the neuronal excitabilities nor the synaptic functions of SPION-loaded cells are significantly affected. Analysis of neural activity propagation using high-density multi-electrode arrays shows that the delivered Mag-Neurobot is functionally connected with the OHS. The applications of this study include functional verification for targeted cell delivery through the characterization of novel synaptic connections and the functionalities of transported and transplanted cells. The success of the Mag-Neurobot opens up new avenues of research and application; it offers a test platform for functional neural connections and neural regenerative processes through cell transplantation.

S100A10 and its binding partners in depression and antidepressant actions.

S100A10 (p11) is an emerging player in the neurobiology of depression and antidepressant actions. p11 was initially thought to be a modulator of serotonin receptor (5-HTR) trafficking and serotonergic transmission, though newly identified binding partners of p11 and neurobiological studies of these proteins have shed light on multifunctional roles for p11 in the regulation of glutamatergic transmission, calcium signaling and nuclear events related to chromatin remodeling, histone modification, and gene transcription. This review article focuses on direct binding partners of p11 in the brain including 5-HTRs, mGluR5, annexin A2, Ahnak, Smarca3, and Supt6h, as well as their roles in neuronal function, particularly in the context of depressive-like behavior as well as behavioral effects of antidepressant drug treatments in mice. In addition, we discuss neurobiological insights from recently uncovered p11 pathways in multiple types of neurons and non-neuronal cells and cast major remaining questions for future studies.

Second-Derivative-Based Background Drift Removal for a Tonic Dopamine Measurement in Fast-Scan Cyclic Voltammetry.

The dysregulation of dopamine, a neuromodulator, is associated with a broad spectrum of brain disorders, including Parkinson's disease, addiction, and schizophrenia. Quantitative measurements of dopamine are essential for understanding dopamine functional dynamics. Fast-scan cyclic voltammetry (FSCV) is the most popular electrochemical technique for measuring real-time in vivo dopamine level changes. Standard FSCV has only analyzed "phasic dopamine" (changes in seconds) because the gradual generation of background charging current is inevitable and is the primary noise source in the low-frequency band. Although "tonic dopamine" (changes in minutes to hours) is critical for understanding the dopamine system, an electrochemical technique capable of simultaneously measuring phasic and tonic dopamine in an in vivo environment has not been established. Several modified voltammetric techniques have been developed for measuring tonic dopamine; however, the sampling rates (0.1-0.05 Hz) are too low to be useful. Further investigation of the in vivo applicability of previously developed background drift removal methods for measuring tonic dopamine levels is required. We developed a second-derivative-based background removal (SDBR) method for simultaneously measuring phasic and tonic neurotransmitter levels in real-time. The performance of this technique was tested via in silico and in vitro tonic dopamine experiments. Furthermore, its applicability was tested in vivo. SDBR is a simple, robust, postprocessing technique that can extract tonic neurotransmitter levels from all FSCV data. As SDBR is calculated in individual-scan voltammogram units, it can be applied to any real-time closed-loop system that uses a neurotransmitter as a biomarker.

Comparative Phosphoproteomics of Neuro-2a Cells under Insulin Resistance Reveals New Molecular Signatures of Alzheimer's Disease.

Insulin in the brain is a well-known critical factor in neuro-development and regulation of adult neurogenesis in the hippocampus. The abnormality of brain insulin signaling is associated with the aging process and altered brain plasticity, and could promote neurodegeneration in the late stage of Alzheimer's disease (AD). The precise molecular mechanism of the relationship between insulin resistance and AD remains unclear. The development of phosphoproteomics has advanced our knowledge of phosphorylation-mediated signaling networks and could elucidate the molecular mechanisms of certain pathological conditions. Here, we applied a reliable phosphoproteomic approach to Neuro2a (N2a) cells to identify their molecular features under two different insulin-resistant conditions with clinical relevance: inflammation and dyslipidemia. Despite significant difference in overall phosphoproteome profiles, we found molecular signatures and biological pathways in common between two insulin-resistant conditions. These include the integrin and adenosine monophosphate-activated protein kinase pathways, and we further verified these molecular targets by subsequent biochemical analysis. Among them, the phosphorylation levels of acetyl-CoA carboxylase and Src were reduced in the brain from rodent AD model 5xFAD mice. This study provides new molecular signatures for insulin resistance in N2a cells and possible links between the molecular features of insulin resistance and AD.

Control of hippocampal prothrombin kringle-2 (pKr-2) expression reduces neurotoxic symptoms in five familial Alzheimer's disease mice.

BACKGROUND AND PURPOSE: There is a scarcity of information regarding the role of prothrombin kringle-2 (pKr-2), which can be generated by active thrombin, in hippocampal neurodegeneration and Alzheimer's disease (AD). EXPERIMENTAL APPROACH: To assess the role of pKr-2 in association with the neurotoxic symptoms of AD, we determined pKr-2 protein levels in post-mortem hippocampal tissues of patients with AD and the hippocampi of five familial AD (5XFAD) mice compared with those of age-matched controls and wild-type (WT) mice, respectively. In addition, we investigated whether the hippocampal neurodegeneration and object memory impairments shown in 5XFAD mice were mediated by changes to pKr-2 up-regulation. KEY RESULTS: Our results demonstrated that pKr-2 was up-regulated in the hippocampi of patients with AD and 5XFAD mice, but was not associated with amyloid-ß aggregation in 5XFAD mice. The up-regulation of pKr-2 expression was inhibited by preservation of the blood-brain barrier (BBB) via addition of caffeine to their water supply or by treatment with rivaroxaban, an inhibitor of factor Xa that is associated with thrombin production. Moreover, the prevention of up-regulation of pKr-2 expression reduced neurotoxic symptoms, such as hippocampal neurodegeneration and object recognition decline due to neurotoxic inflammatory responses in 5XFAD mice. CONCLUSION AND IMPLICATIONS: We identified a novel pathological mechanism of AD mediated by abnormal accumulation of pKr-2, which functions as an important pathogenic factor in the adult brain via blood brain barrier (BBB) breakdown. Thus, pKr-2 represents a novel target for AD therapeutic strategies and those for related conditions.

Hippocampal mossy cell involvement in behavioral and neurogenic responses to chronic antidepressant treatment.

Most antidepressants, including selective serotonin reuptake inhibitors (SSRIs), initiate their drug actions by rapid elevation of serotonin, but they take several weeks to achieve therapeutic onset. This therapeutic delay suggests slow adaptive changes in multiple neuronal subtypes and their neural circuits over prolonged periods of drug treatment. Mossy cells are excitatory neurons in the dentate hilus that regulate dentate gyrus activity and function. Here we show that neuronal activity of hippocampal mossy cells is enhanced by chronic, but not acute, SSRI administration. Behavioral and neurogenic effects of chronic treatment with the SSRI, fluoxetine, are abolished by mossy cell-specific knockout of p11 or Smarca3 or by an inhibition of the p11/AnxA2/SMARCA3 heterohexamer, an SSRI-inducible protein complex. Furthermore, simple chemogenetic activation of mossy cells using Gq-DREADD is sufficient to elevate the proliferation and survival of the neural stem cells. Conversely, acute chemogenetic inhibition of mossy cells using Gi-DREADD impairs behavioral and neurogenic responses to chronic administration of SSRI. The present data establish that mossy cells play a crucial role in mediating the effects of chronic antidepressant medication. Our results indicate that compounds that target mossy cell activity would be attractive candidates for the development of new antidepressant medications.

Obligatory roles of dopamine D1 receptors in the dentate gyrus in antidepressant actions of a selective serotonin reuptake inhibitor, fluoxetine.

Depression is a leading cause of disability. Current pharmacological treatment of depression is insufficient, and development of improved treatments especially for treatment-resistant depression is desired. Understanding the neurobiology of antidepressant actions may lead to development of improved therapeutic approaches. Here, we demonstrate that dopamine D1 receptors in the dentate gyrus act as a pivotal mediator of antidepressant actions in mice. Chronic administration of a selective serotonin reuptake inhibitor (SSRI), fluoxetine, increases D1 receptor expression in mature granule cells in the dentate gyrus. The increased D1 receptor signaling, in turn, contributes to the actions of chronic fluoxetine treatment, such as suppression of acute stress-evoked serotonin release, stimulation of adult neurogenesis and behavioral improvement. Importantly, under severely stressed conditions, chronic administration of a D1 receptor agonist in conjunction with fluoxetine restores the efficacy of fluoxetine actions on D1 receptor expression and behavioral responses. Thus, our results suggest that stimulation of D1 receptors in the dentate gyrus is a potential adjunctive approach to improve therapeutic efficacy of SSRI antidepressants.

An adiponectin receptor agonist antibody stimulates glucose uptake and fatty-acid oxidation by activating AMP-activated protein kinase.

Adiponectin (Ad) is a representative adipocytokine that regulates energy homeostasis including glucose transport and lipid oxidation through activation of AMP-activated protein kinase (AMPK) pathways. Plasma levels of Ad are reduced in obesity, which contributes to type 2 diabetes. Therefore, agents that activate the Ad signaling pathway could ameliorate metabolic diseases such as type 2 diabetes. Here, we report the identification of a high-affinitive agonist antibody against Ad receptors. The antibody was selected by using phage display of human combinatorial antibody libraries. The selected antibody induced phosphorylation of the acetyl-CoA carboxylase (ACC) and AMPK in skeletal muscle cells and stimulated glucose uptake and fatty-acid oxidation (FAO) in myotubes. In addition, the antibody significantly lowered blood glucose levels during a glucose challenge in normal mice as well as basal blood glucose levels in a type 2 diabetic mouse model. Taken together, these results suggest that the agonist antibody could be a promising therapeutic agent for the treatment of metabolic syndrome such as type 2 diabetes.

Neurotrophic interactions between neurons and astrocytes following AAV1-Rheb(S16H) transduction in the hippocampus in vivo.

BACKGROUND AND PURPOSE: We recently reported that AAV1-Rheb(S16H) transduction could protect hippocampal neurons through the induction of brain-derived neurotrophic factor (BDNF) in the rat hippocampus in vivo. It is still unclear how neuronal BDNF produced by AAV1-Rheb(S16H) transduction induces neuroprotective effects in the hippocampus and whether its up-regulation contributes to the enhance of a neuroprotective system in the adult brain. EXPERIMENTAL APPROACH: To determine the presence of a neuroprotective system in the hippocampus of patients with Alzheimer's disease (AD), we examined the levels of glial fibrillary acidic protein, BDNF and ciliary neurotrophic factor (CNTF) and their receptors, tropomyocin receptor kinase B (TrkB) and CNTF receptor α(CNTFRα), in the hippocampus of AD patients. We also determined whether AAV1-Rheb(S16H) transduction stimulates astroglial activation and whether reactive astrocytes contribute to neuroprotection in models of hippocampal neurotoxicity in vivo and in vitro. KEY RESULTS: AD patients may have a potential neuroprotective system, demonstrated by increased levels of full-length TrkB and CNTFRα in the hippocampus. Further AAV1-Rheb(S16H) transduction induced sustained increases in the levels of full-length TrkB and CNTFRα in reactive astrocytes and hippocampal neurons. Moreover, neuronal BDNF produced by Rheb(S16H) transduction of hippocampal neurons induced reactive astrocytes, resulting in CNTF production through the activation of astrocytic TrkB and the up-regulation of neuronal BDNF and astrocytic CNTF which had synergistic effects on the survival of hippocampal neurons in vivo. CONCLUSIONS AND IMPLICATIONS: The results demonstrated that Rheb(S16H) transduction of hippocampal neurons could strengthen the neuroprotective system and this intensified system may have a therapeutic value against neurodegeneration in the adult brain.

Humanized Anti-hepatocyte Growth Factor Monoclonal Antibody (YYB-101) Inhibits Ovarian Cancer Progression.

Current chemotherapy regimens have certain limitations in improving the survival rates of patients with advanced ovarian cancer. Hepatocyte growth factor (HGF) is important in ovarian cancer cell migration and invasion. This study assessed the effects of YYB-101, a humanized monoclonal anti-HGF antibody, on the growth and metastasis of ovarian cancer cells. YYB-101 suppressed the phosphorylation of the HGF receptor c-MET and inhibited the migration and invasion of SKOV3 and A2780 ovarian cancer cells. Moreover, the combination of YYB-101 and paclitaxel synergistically inhibited tumor growth in an in vivo ovarian cancer mouse xenograft model and significantly increased the overall survival (OS) rate compared with either paclitaxel or YYB-101 alone. Taken together, these findings suggest that YYB-101 has therapeutic potential in ovarian cancer when combined with conventional chemotherapy agents.

An agonist antibody prefers relapsed AML for induction of cells that kill each other.

Previously, we reported an agonist antibody to a cytokine receptor, Thrombopoietin receptor (TPOR) that effectively induces cytotoxic killer cells from precursor tumor cells isolated from newly diagnosed AML patients. Here, we show that the TPOR agonist antibody can induce even relapsed AML cells into killer cells more potently than newly diagnosed AML cells. After stimulation by the agonist antibody, these relapsed leukemic cells enter into a differentiation process of killer cells. The antibody-induced killer cells express, Granzyme B and Perforin that assault and kill other members of the AML cell population. Particularly, the agonist antibody showed potent efficacy on the AML xenograft model in mice using the NOD/LtSz-scid IL2Rγc null (NSG) mice. These results show that the TPOR agonist antibody that induces AML cells to kill each other is effective on both relapsed AML cells and in vivo. Therefore, this study suggests a new strategy for the treatment of cancer relapse after chemotherapy.

Effects of Silibinin Against Prothrombin Kringle-2-Induced Neurotoxicity in the Nigrostriatal Dopaminergic System In Vivo.

Parkinson's disease (PD) and Alzheimer's disease exhibit common features of neurodegenerative diseases and can be caused by numerous factors. A common feature of these diseases is neurotoxic inflammation by activated microglia, indicating that regulation of microglial activation is a potential mechanism for preserving neurons in the adult brain. Recently, we reported that upregulation of prothrombin kringle-2 (pKr-2), one of the domains that make up prothrombin and which is cleaved and generated by active thrombin, induces nigral dopaminergic (DA) neuronal death through neurotoxic microglial activation in the adult brain. In this study, we show that silibinin, a flavonoid found in milk thistle, can suppress the production of inducible nitric oxide synthase and neurotoxic inflammatory cytokines, such as interleukin-1ß and tumor necrosis factor-α, after pKr-2 treatment by downregulating the extracellular signal-regulated kinase signaling pathway in the mouse substantia nigra. Moreover, as demonstrated by immunohistochemical staining, measurements of the dopamine and metabolite levels, and open-field behavioral tests, silibinin treatment protected the nigrostriatal DA system resulting from the occurrence of pKr-2-triggered neurotoxic inflammation in vivo. Thus, we conclude that silibinin may be beneficial as a natural compound with anti-inflammatory effects against pKr-2-triggered neurotoxicity to protect the nigrostriatal DA pathway and its properties, and thus, may be applicable for PD therapy.

Understanding the Role of the BAI Subfamily of Adhesion G Protein-Coupled Receptors (GPCRs) in Pathological and Physiological Conditions.

Brain-specific angiogenesis inhibitors (BAIs) 1, 2, and 3 are members of the adhesion G protein-coupled receptors, subfamily B, which share a conserved seven-transmembrane structure and an N-terminal extracellular domain. In cell- and animal-based studies, these receptors have been shown to play diverse roles under physiological and pathological conditions. BAI1 is an engulfment receptor and performs major functions in apoptotic-cell clearance and interacts (as a pattern recognition receptor) with pathogen components. BAI1 and -3 also participate in myoblast fusion. Furthermore, BAI1⁻3 have been linked to tumor progression and neurological diseases. In this review, we summarize the current understanding of the functions of BAI1⁻3 in pathological and physiological conditions and discuss future directions in terms of the importance of BAIs as pharmacological targets in diseases.

Interleukin-5 suppresses Vascular Endothelial Growth Factor-induced angiogenesis through STAT5 signaling.

Interleukin-5 (IL-5) is best known as key regulator in eosinophil-associated diseases such as asthma. While a connection to vascular changes in eosinophil-associated lung diseases is still elusive, recent evidence suggests that IL-5 may have an atheroprotective role. Here, we report an unexpected anti-angiogenic potential of IL-5 on vascular endothelial cells in vitro. IL-5 significantly inhibited fundamental functions of human lung microvascular endothelial cells (HMVEC-L) in vessel formation including VEGF-induced endothelial cell proliferation, migration and tube formation. Knockdown (KD) of STAT5 abolished the direct anti-angiogenic effect of IL-5 on VEGF-induced endothelial cell proliferation, migration and tube formation.

Upregulation of neuronal astrocyte elevated gene-1 protects nigral dopaminergic neurons in vivo.

The role of astrocyte elevated gene-1 (AEG-1) in nigral dopaminergic (DA) neurons has not been studied. Here we report that the expression of AEG-1 was significantly lower in DA neurons in the postmortem substantia nigra of patients with Parkinson's disease (PD) compared to age-matched controls. Similarly, decreased AEG-1 levels were found in the 6-hydroxydopamine (6-OHDA) mouse model of PD. An adeno-associated virus-induced increase in the expression of AEG-1 attenuated the 6-OHDA-triggered apoptotic death of nigral DA neurons. Moreover, the neuroprotection conferred by the AEG-1 upregulation significantly intensified the neurorestorative effects of the constitutively active ras homolog enriched in the brain [Rheb(S16H)]. Collectively, these results demonstrated that the sustained level of AEG-1 as an important anti-apoptotic factor in nigral DA neurons might potentiate the therapeutic effects of treatments, such as Rheb(S16H) administration, on the degeneration of the DA pathway that characterizes PD.