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.

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.

MDGA1 negatively regulates amyloid precursor protein-mediated synapse inhibition in the hippocampus.

Balanced synaptic inhibition, controlled by multiple synaptic adhesion proteins, is critical for proper brain function. MDGA1 (meprin, A-5 protein, and receptor protein-tyrosine phosphatase mu [MAM] domain-containing glycosylphosphatidylinositol anchor protein 1) suppresses synaptic inhibition in mammalian neurons, yet the molecular mechanisms underlying MDGA1-mediated negative regulation of GABAergic synapses remain unresolved. Here, we show that the MDGA1 MAM domain directly interacts with the extension domain of amyloid precursor protein (APP). Strikingly, MDGA1-mediated synaptic disinhibition requires the MDGA1 MAM domain and is prominent at distal dendrites of hippocampal CA1 pyramidal neurons. Down-regulation of APP in presynaptic GABAergic interneurons specifically suppressed GABAergic, but not glutamatergic, synaptic transmission strength and inputs onto both the somatic and dendritic compartments of hippocampal CA1 pyramidal neurons. Moreover, APP deletion manifested differential effects in somatostatin- and parvalbumin-positive interneurons in the hippocampal CA1, resulting in distinct alterations in inhibitory synapse numbers, transmission, and excitability. The infusion of MDGA1 MAM protein mimicked postsynaptic MDGA1 gain-of-function phenotypes that involve the presence of presynaptic APP. The overexpression of MDGA1 wild type or MAM, but not MAM-deleted MDGA1, in the hippocampal CA1 impaired novel object-recognition memory in mice. Thus, our results establish unique roles of APP-MDGA1 complexes in hippocampal neural circuits, providing unprecedented insight into trans-synaptic mechanisms underlying differential tuning of neuronal compartment-specific synaptic inhibition.

Stimulation of the Migration and Expansion of Adult Mouse Neural Stem Cells by the FPR2-Specific Peptide WKYMVm.

Neural stem cells (NSCs) are multipotent cells capable of self-renewal and differentiation into different nervous system cells. Mouse NSCs (mNSCs) are useful tools for studying neurogenesis and the therapeutic applications of neurodegenerative diseases in mammals. Formyl peptide receptor 2 (FPR2), expressed in the central nervous system and brain, is involved in the migration and differentiation of murine embryonic-derived NSCs. In this study, we explored the effect of FPR2 activation in adult mNSCs using the synthetic peptide Trp-Lys-Tyr-Met-Val-D-Met-NH2 (WKYMVm), an agonist of FPR2. After isolation of NSCs from the subventricular zone of the adult mouse brain, they were cultured in two culture systems-neurospheres or adherent monolayers-to demonstrate the expression of NSC markers and phenotypes. Under different conditions, mNSCs differentiated into neurons and glial cells such as astrocytes, microglia, and oligodendrocytes. Treatment with WKYMVm stimulated the chemotactic migration of mNSCs. Moreover, WKYMVm-treated mNSCs were found to promote proliferation; this result was confirmed by the expansion of mNSCs in Matrigel and the increase in the number of Ki67-positive cells. Incubation of mNSCs with WKYMVm in a supplement-free medium enhanced the survival rate of the mNSCs. Together, these results suggest that WKYMVm-induced activation of FPR2 stimulates cellular responses in adult NSCs.

Application of Proteomics in Cancer: Recent Trends and Approaches for Biomarkers Discovery.

Proteomics has become an important field in molecular sciences, as it provides valuable information on the identity, expression levels, and modification of proteins. For example, cancer proteomics unraveled key information in mechanistic studies on tumor growth and metastasis, which has contributed to the identification of clinically applicable biomarkers as well as therapeutic targets. Several cancer proteome databases have been established and are being shared worldwide. Importantly, the integration of proteomics studies with other omics is providing extensive data related to molecular mechanisms and target modulators. These data may be analyzed and processed through bioinformatic pipelines to obtain useful information. The purpose of this review is to provide an overview of cancer proteomics and recent advances in proteomic techniques. In particular, we aim to offer insights into current proteomics studies of brain cancer, in which proteomic applications are in a relatively early stage. This review covers applications of proteomics from the discovery of biomarkers to the characterization of molecular mechanisms through advances in technology. Moreover, it addresses global trends in proteomics approaches for translational research. As a core method in translational research, the continued development of this field is expected to provide valuable information at a scale beyond that previously seen.

Emodin induces collagen type I synthesis in Hs27 human dermal fibroblasts.

Fibrillar collagen and elastic fibers are the main components of the dermal extracellular matrix (ECM), which confers mechanical strength and resilience to the skin. In particular, type I collagen produced by fibroblasts is the most abundant collagen that determines the general strength of the ECM, thereby contributing to the prevesntion of the skin-aging process. Although the natural anthraquinone derivative emodin (1,3,8-trihydroxy-6-methylanthraquinone) exerts numerous beneficial effects, including antiviral, anticancer, anti-inflammatory and wound-healing effects in diverse cells, the effect of emodin on collagen expression or skin aging is not fully understood. The present study demonstrated that exposure to emodin increased type I collagen synthesis in a concentration- and time-dependent manner in Hs27 human dermal fibroblasts. Subsequent experiments showed that emodin strongly increased collagen type I levels without altering cell proliferation or cellular matrix metalloproteinase-1 (MMP-1) expression. Additionally, it was determined that increased phosphorylation of 5' AMP-activated protein kinase, following emodin treatment, was responsible for increased type I collagen synthesis. These findings clearly indicate that emodin plays an important role in collagen type I synthesis in dermal fibroblasts, thereby making it a potential drug candidate for treating skin aging and wrinkles.

LAR-RPTPs Directly Interact with Neurexins to Coordinate Bidirectional Assembly of Molecular Machineries.

Neurexins (Nrxns) and LAR-RPTPs (leukocyte common antigen-related protein tyrosine phosphatases) are presynaptic adhesion proteins responsible for organizing presynaptic machineries through interactions with nonoverlapping extracellular ligands. Here, we report that two members of the LAR-RPTP family, PTPσ and PTPδ, are required for the presynaptogenic activity of Nrxns. Intriguingly, Nrxn1 and PTPσ require distinct sets of intracellular proteins for the assembly of specific presynaptic terminals. In addition, Nrxn1α showed robust heparan sulfate (HS)-dependent, high-affinity interactions with Ig domains of PTPσ that were regulated by the splicing status of PTPσ. Furthermore, Nrxn1α WT, but not a Nrxn1α mutant lacking HS moieties (Nrxn1α ΔHS), inhibited postsynapse-inducing activity of PTPσ at excitatory, but not inhibitory, synapses. Similarly, cis expression of Nrxn1α WT, but not Nrxn1α ΔHS, suppressed the PTPσ-mediated maintenance of excitatory postsynaptic specializations in mouse cultured hippocampal neurons. Lastly, genetics analyses using male or female Drosophila Dlar and Dnrx mutant larvae identified epistatic interactions that control synapse formation and synaptic transmission at neuromuscular junctions. Our results suggest a novel synaptogenesis model whereby different presynaptic adhesion molecules combine with distinct regulatory codes to orchestrate specific synaptic adhesion pathways.SIGNIFICANCE STATEMENT We provide evidence supporting the physical interactions of neurexins with leukocyte common-antigen related receptor tyrosine phosphatases (LAR-RPTPs). The availability of heparan sulfates and alternative splicing of LAR-RPTPs regulate the binding affinity of these interactions. A set of intracellular presynaptic proteins is involved in common for Nrxn- and LAR-RPTP-mediated presynaptic assembly. PTPσ triggers glutamatergic and GABAergic postsynaptic differentiation in an alternative splicing-dependent manner, whereas Nrxn1α induces GABAergic postsynaptic differentiation in an alternative splicing-independent manner. Strikingly, Nrxn1α inhibits the glutamatergic postsynapse-inducing activity of PTPσ, suggesting that PTPσ and Nrxn1α might control recruitment of a different pool of postsynaptic machinery. Drosophila orthologs of Nrxns and LAR-RPTPs mediate epistatic interactions in controlling synapse structure and strength at neuromuscular junctions, underscoring the physiological significance in vivo.

Structural basis of SALM3 dimerization and synaptic adhesion complex formation with PTPσ.

Synaptic adhesion molecules play an important role in the formation, maintenance and refinement of neuronal connectivity. Recently, several leucine rich repeat (LRR) domain containing neuronal adhesion molecules have been characterized including netrin G-ligands, SLITRKs and the synaptic adhesion-like molecules (SALMs). Dysregulation of these adhesion molecules have been genetically and functionally linked to various neurological disorders. Here we investigated the molecular structure and mechanism of ligand interactions for the postsynaptic SALM3 adhesion protein with its presynaptic ligand, receptor protein tyrosine phosphatase σ (PTPσ). We solved the crystal structure of the dimerized LRR domain of SALM3, revealing the conserved structural features and mechanism of dimerization. Furthermore, we determined the complex structure of SALM3 with PTPσ using small angle X-ray scattering, revealing a 2:2 complex similar to that observed for SALM5. Solution studies unraveled additional flexibility for the complex structure, but validated the uniform mode of action for SALM3 and SALM5 to promote synapse formation. The relevance of the key interface residues was further confirmed by mutational analysis with cellular binding assays and artificial synapse formation assays. Collectively, our results suggest that SALM3 dimerization is a pre-requisite for the SALM3-PTPσ complex to exert synaptogenic activity.

Molecular Mechanisms of Synaptic Specificity: Spotlight on Hippocampal and Cerebellar Synapse Organizers.

Synapses and neural circuits form with exquisite specificity during brain development to allow the precise and appropriate flow of neural information. Although this property of synapses and neural circuits has been extensively investigated for more than a century, molecular mechanisms underlying this property are only recently being unveiled. Recent studies highlight several classes of cell-surface proteins as organizing hubs in building structural and functional architectures of specific synapses and neural circuits. In the present mini-review, we discuss recent findings on various synapse organizers that confer the distinct properties of specific synapse types and neural circuit architectures in mammalian brains, with a particular focus on the hippocampus and cerebellum.

Distribution of TGF-beta isoforms and signaling intermediates in corneal fibrotic wound repair.

In this study, temporal and spatial distribution of three TGF-beta isoforms and their downstream signaling pathways including pSmad2 and p38MAPK were examined during fibrotic wound repair. In normal chick corneas, TGF-beta1, -2, and -3 were weakly detected in Bowman's layer (BL). In healing corneas, TGF-beta1 was primarily deposited in the fibrin clot and the unwounded BL. TGF-beta2 was highly expressed in healing epithelial and endothelial cells, and numerous active fibroblasts/myofibroblasts. TGF-beta3 was mainly detected in the unwound region of basal epithelial cells. alpha-Smooth muscle actin (alpha-SMA) was initially appeared in the posterior region of repairing stroma at day 3, and was detected in the entire healing stroma by day 7. Notably, alpha-SMA was absent in the central region of healing stroma by day 14, and its staining pattern was similar to those of TGF-beta2 and p38MAPK. By contrast, pSmad2 was mainly detected in the fibroblasts. In normal cornea, laminin was mainly detected in both epithelial basement membrane (BM) and Descemet's membrane (DM). By contrast to reconstitution of the BM in the wound region, the DM was not repaired although endothelial layer was regenerated, indicating that high levels of TGF-beta2 were released into the posterior region of healing stroma on day 14. High levels of alpha-SMA staining, shown in cultured repair stromal cells from healing corneas on day 14 and in TGF-beta2 treated normal stromal cells, were significantly reduced by p38MAPK inhibition. Collectively, this study suggests that TGF-beta2-mediated myofibroblast transformation is mediated, at least partly, by the p38MAPK pathway in vivo.

FCCP depolarizes plasma membrane potential by activating proton and Na+ currents in bovine aortic endothelial cells.

We investigated the effects of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore and uncoupler of mitochondrial oxidative phosphorylation in mitochondria, on plasma membrane potential and ionic currents in bovine aortic endothelial cells (BAECs). The membrane potential and ionic currents of BAECs were recorded using the patch-clamp technique in current-clamp and voltage-clamp modes, respectively. FCCP activated ionic currents and depolarized the plasma membrane potential in a dose-dependent manner. Neither the removal of extracellular Ca2+ nor pretreatment with BAPTA/AM affected the FCCP-induced currents, implying that the currents are not associated with the FCCP-induced intracellular [Ca2+]i increase. FCCP-induced currents were significantly influenced by the changes in extracellular or intracellular pH; the increased proton gradient produced by lowering the extracellular pH or intracellular alkalinization augmented the changes in membrane potential and ionic currents caused by FCCP. FCCP-induced currents were significantly reduced under extracellular Na+-free conditions. The reversal potentials of FCCP-induced currents under Na+-free conditions were well fitted to the calculated equilibrium potential for protons. Interestingly, FCCP-induced Na+ transport (subtracted currents, I(control)- I(Na+-free) was closely dependent on extracellular pH, whereas FCCP-induced H+transport was not significantly affected by the absence of Na+. These results suggest that the FCCP-induced ionic currents and depolarization, which are strongly dependent on the plasmalemmal proton gradient, are likely to be mediated by both H+ and Na+ currents across the plasma membrane. The relationship between H+ and Na+ transport still needs to be determined.