Brain Protein Research Articles

Region specific reduction of neurogranin immunostaining in the hippocampus of Alzheimer’s disease brains

AbstractBackgroundNeurogranin (Ng) is a post‐synaptic protein involved in formation of long‐term potentials, and thus plays an important role in learning and memory. The concentration of Ng is increased in the cerebrospinal fluid (CSF) of people with Alzheimer’s disease (AD), and CSF Ng is considered to be a biomarker for synaptic dysfunction in AD. The expression of Ng protein in the human brain and its association with AD pathology, however, are less well studied.MethodUsing immunohistochemical detection methods we investigated the protein expression of Ng, beta‐amyloid (Aβ) and hyperphosphorylated paired helical filament tau (phospho‐tau; ptau) in the post‐mortem hippocampus from 12 AD patients and 12 cognitively unaffected controls (NC). We compared the number of positively stained cells (Ng, ptau) or plaques (Aβ) in the hippocampal sub‐regions (dentate gyrus, Cornu Ammonis (CA) fields 1‐3) from the 2 groups. Donor samples with intermediate AD (i.e., Braak stage III‐IV) or NC (Braak 0‐I/II) were analyzed using Image Pro Premier automated image analysis software (version 10.01). The data were normalized to the number of cells or clusters/mm2 and analyzed using a one‐way ANOVA. Using a test of circularity (Cir = (4*A)/Pi*MaxFeret2), we further investigated morphological differences in the Ng positive neurons by measuring the Ng expression in the apical dendrite of pyramidal neurons.ResultThere was a 40% reduction of Ng immunopositive neurons in AD compared to NC in the CA3(N = 24;p = 0.0003) and in the CA1(N = 24;p = 0.014) hippocampal sub‐regions. Ng positive cells did not change significantly in AD compared to NC in the other hippocampal regions. Additionally, we showed that CA1 region, Ng was localized mainly in the neuronal cell soma of AD donors, while its expression extended into the apical dendrites of NC donors (N = 24;p = 0.006). In AD, Ng immunostaining negatively correlated with ptau immunopositive neurofibrillary tangles (r = ‐0.70;p = 0.04)ConclusionSelective loss of Ng immunostaining in certain subregions of the hippocampus suggests that synaptic damage in AD maybe a region‐specific event. The mechanism underlying such regional synaptic damage and the changes in Ng expression will be key in understanding the selective vulnerability of brain regions to AD.

Germinated brown rice extract reduces brain lipid peroxidation and Aβ levels via regulations of BACE1, RAGE, IDE and LRP1 expressions in high fat/cholesterol diet-fed rats

A strong association presents between excessive amyloid-β (Aβ) buildup and oxidative stress with Alzheimer’s disease (AD). In view of the multifaceted neuroprotective effects of some dietary elements, the beneficial effect of germinated brown rice was investigated using a high fat/cholesterol diet (HFCD)-induced sporadic rat model of AD. Seven groups, including normal control, HFCD, and HFCD treated with Donepezil, Simvastatin, Probucol, and germinated brown rice ethyl acetate extract (GBR-EA; 100 and 200 mg/kg bw) were included in the present study. Biochemical assays, brain lipid peroxidation, mRNA and protein levels involved in Aβ processing and metabolism pathways were analyzed. HFCD-fed rats exhibited increased brain lipid peroxidation and Aβ levels, as well as altered expressions of proteins involved in Aβ generation, degradation and clearance. GBR-EA treatment groups showed significant reduction in lipid peroxidation and Aβ levels when compared to HFCD group. The effects of GBR-EA on Aβ levels are likely through the modulation of BACE1 and Presenilins, as well as induction of Aβ clearance and degradation. The findings revealed the potential use of germinated brown rice to attenuate HFCD diet-induced neurodegenerative alterations, likely due to reduced brain inflammation, as well as modulation of Aβ processing and metabolism pathways.

Rosavin protects the blood-brain barrier against ischemia/reperfusion-induced cerebral injury by regulating MAPK-mediated MMPs pathway.

Ischemia-reperfusion (I/R) injury is a common pathophysiological condition in ischemic stroke, involving various pathophysiological events, such as inflammation, cytotoxicity, neuronal loss and disruption of the blood-brain barrier (BBB). Rosavin is the major bioactive ingredient of Rhodiola Rosea L. with multiple therapeutic effects. The purpose of this was to investigate the role of rosavin in I/R-induced cerebral injury. A cell oxygen-glucose deprivation and reoxygenation (OGD/R) model and a mouse middle cerebral artery occlusion (MCAO) model were established to induce I/R injury in vitro and in vivo, respectively. MCAO-treated mice and OGD/R-challenged human brain microvascular endothelial cells (HBMVECs) were administrated with or without rosavin at various concentrations. Rosavin-treated mice showed reduced infarct volume, neuronal loss and neuronal cytotoxicity in I/R-injured brains. Rosavin treatment downregulated the expression of pro-inflammatory cytokines, reduced apoptosis and inhibited the activation of nuclear factor κ B in I/R-injured mice and HBMVECs. Administration with rosavin also alleviated mouse brain oedema and upregulated tight junction proteins in mouse brains after I/R injury, suggesting that rosavin protected mice against I/R-induced BBB disruption. Further analysis revealed that rosavin reduced the BBB permeability in I/R-injured mice and HBMVECs by inhibiting autophagy. Moreover, rosavin intervention inhibited I/R injury-induced activation of the mitogen-activated protein kinase (MAPK) pathway and upregulation of matrix metalloproteinases in both mouse and cell models. In conclusion, rosavin protects the BBB against I/R injury possibly by regulating the MAPK pathway. The above results provide a rationale for further exploration of rosavin as a therapeutic candidate for cerebral I/R injury.

In vivo evaluation of a novel 18F-labeled PET radioligand for translocator protein 18 kDa (TSPO) in monkey brain

Purpose[18F]SF51 was previously found to have high binding affinity and selectivity for 18 kDa translocator protein (TSPO) in mouse brain. This study sought to assess the ability of [18F]SF51 to quantify TSPO in rhesus monkey brain.MethodsPositron emission tomography (PET) imaging was performed in monkey brain (n = 3) at baseline and after pre-blockade with the TSPO ligands PK11195 and PBR28. TSPO binding was calculated as total distribution volume corrected for free parent fraction in plasma (VT/fP) using a two-tissue compartment model. Receptor occupancy and nondisplaceable uptake were determined via Lassen plot. Binding potential (BPND) was calculated as the ratio of specific binding to nondisplaceable uptake. Time stability of VT was used as an indirect probe to detect radiometabolite accumulation in the brain. In vivo and ex vivo experiments were performed in mice to determine the distribution of the radioligand.ResultsAfter [18F]SF51 injection, the concentration of brain radioactivity peaked at 2.0 standardized uptake value (SUV) at ~ 10 min and declined to 30% of the peak at 180 min. VT/fP at baseline was generally high (203 ± 15 mL· cm−3) and decreased by ~ 90% after blockade with PK11195. BPND of the whole brain was 7.6 ± 4.3. VT values reached levels similar to terminal 180-min values by 100 min and remained relatively stable thereafter with excellent identifiability (standard errors < 5%), suggesting that no significant radiometabolites accumulated in the brain. Ex vivo experiments in mouse brain showed that 96% of radioactivity was parent. No significant uptake was observed in the skull, suggesting a lack of defluorination in vivo.ConclusionThe results demonstrate that [18F]SF51 is an excellent radioligand that can quantify TSPO with a good ratio of specific to nondisplaceable uptake and has minimal radiometabolite accumulation in brain. Collectively, the results suggest that [18F]SF51 warrants further evaluation in humans.

Bone Marrow-Derived Mesenchymal Stem Cells and γ-Secretase Inhibitor Treatments Suppress Amyloid-β25-35-Induced Cognitive Impairment in Rat Dams and Cortical Degeneration in Offspring.

Alzheimer's disease (AD) is the most frequent cause of age-related neurodegeneration and ensuing cognitive impairment. Progressive deposition of extracellular amyloid beta (Aβ) aggregates (plaques) and intracellular hyperphosphorylated Tau protein (p-Tau) are the core pathological markers of AD but may precede clinical symptoms by many years, presenting a therapeutic window of opportunity. Females are more frequently afflicted by AD than males, necessitating evaluation of novel treatments for the female population. The current study examined the protective efficacies of intravenous bone marrow-derived mesenchymal stem cells (BM-MSCs) and oral gamma-secretase inhibitor-953 (GSI-953) during pregnancy on cognitive impairment in rat dams and neurodegeneration in offspring induced by intracerebroventricular injection of Aβ25-35 prior to pregnancy. The Aβ25-35 (AD) group exhibited significant (P < 0.001) impairments in the Y-maze and novel object recognition test performance prior to conception. Histological analysis of the offspring cortex revealed substantial dendritic shrinkage and activation of microglial cells, while neurochemical analysis demonstrated significant increases in the proinflammatory cytokine interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α). In contrast, BM-MSC or GSI-953 treatment of dams following Aβ25-35 injection significantly (P < 0.001) reduced the number and size of activated microglial cells, markedly increased dendrite length, and reversed proinflammatory cytokine elevations in offspring. Moreover, BM-MSC or GSI-953 treatment reversed the Aβ25-35-induced amyloid precursor protein and p-Tau elevations in the offspring brain; these changes were accompanied by upregulation of the brain-derived neurotrophic factor and downregulation of glycogen synthase kinase-3β in the serum and brain. Treatment with BM-MSCs or GSI-953 also reversed Aβ25-35-induced elevations in different gene expressions in the neonatal cortex. Finally, treatment of dams with BM-MSCs or GSI-953 prevented the Aβ25-35-induced disruption of newborn brain development. Thus, BM-MSC and GSI-953 treatments have broad-spectrum effects against Aβ25-35-induced brain pathology, including the suppression of neural inflammation, restoration of developmental plasticity, and promotion of neurotrophic signaling.

Intestine and brain TLR-4 modulation following N-acetyl-cysteine treatment in NEC rodent model

Necrotizing enterocolitis (NEC) brain injury is mediated through Toll-like receptor 4 (TLR4) on the intestinal epithelium and brain microglia. Our aim was to determine whether postnatal and/or prenatal NAC can modify NEC associated intestinal and brain TLR4 expression and brain glutathione levels in a rat model of NEC. Newborn Sprague–Dawley rats were randomized into three groups: Control (n = 33); NEC (n = 32)—hypoxia and formula feeding; and NEC-NAC (n = 34)—received NAC (300 mg/kg IP) in addition to NEC conditions. Two additional groups included pups of dams treated once daily with NAC (300 mg/kg IV) for the last 3 days of pregnancy: NAC-NEC (n = 33) or NAC-NEC-NAC (n = 36) with additional postnatal NAC. Pups were sacrificed on the fifth day, and ileum and brains harvested for TLR-4 and glutathione protein levels. Brain and ileum TLR-4 protein levels were significantly increased in NEC offspring as compared to control (brain 2.5 ± 0.6 vs. 0.88 ± 0.12 U and ileum 0.24 ± 0.04 vs. 0.09 ± 0.01, p < 0.05). When NAC was administered only to dams (NAC-NEC) a significant decrease in TLR-4 levels was demonstrated in both offspring brain (1.53 ± 0.41 vs. 2.5 ± 0.6 U, p < 0.05) and ileum (0.12 ± 0.03 vs. 0.24 ± 0.04 U, p < 0.05) as compared to NEC. The same pattern was demonstrated when NAC was administered only or postnatally. The decrease in brain and ileum glutathione levels observed in NEC offspring was reversed with all NAC treatment groups. NAC reverses the increase in ileum and brain TLR-4 levels and the decrease in brain and ileum glutathione levels associated with NEC in a rat model, and thus may protect from NEC associated brain injury.

Integrative systems biology characterizes immune-mediated neurodevelopmental changes in murine Zika virus microcephaly

Characterizing perturbation of molecular pathways in congenital Zika virus (ZIKV) infection is critical for improved therapeutic approaches. Leveraging integrative systems biology, proteomics, and RNA-seq, we analyzed embryonic brain tissues from an immunocompetent, wild-type congenital ZIKV infection mouse model. ZIKV induced a robust immune response accompanied by the downregulation of critical neurodevelopmental gene programs. We identified a negative correlation between ZIKV polyprotein abundance and host cell cycle-inducing proteins. We further captured the downregulation of genes/proteins, many of which are known to be causative for human microcephaly, including Eomesodermin/T-box Brain Protein 2 (EOMES/TBR2) and Neuronal Differentiation 2 (NEUROD2). Disturbances of distinct molecular pathways in neural progenitors and post-mitotic neurons may contribute to complex brain phenotype of congenital ZIKV infection. Overall, this report on protein- and transcript-level dynamics enhances understanding of the ZIKV immunopathological landscape through characterization of fetal immune response in the developing brain.

Potential Role of Protein Kinase FAM20C on the Brain in Raine Syndrome, an In Silico Analysis.

FAM20C (family with sequence similarity 20, member C) is a serine/threonine-specific protein kinase that is ubiquitously expressed and mainly associated with biomineralization and phosphatemia regulation. It is mostly known due to pathogenic variants causing its deficiency, which results in Raine syndrome (RNS), a sclerosing bone dysplasia with hypophosphatemia. The phenotype is recognized by the skeletal features, which are related to hypophosphorylation of different FAM20C bone-target proteins. However, FAM20C has many targets, including brain proteins and the cerebrospinal fluid phosphoproteome. Individuals with RNS can have developmental delay, intellectual disability, seizures, and structural brain defects, but little is known about FAM20C brain-target-protein dysregulation or about a potential pathogenesis associated with neurologic features. In order to identify the potential FAM20C actions on the brain, an in silico analysis was conducted. Structural and functional defects reported in RNS were described; FAM20C targets and interactors were identified, including their brain expression. Gene ontology of molecular processes, function, and components was completed for these targets, as well as for potential involved signaling pathways and diseases. The BioGRID and Human Protein Atlas databases, the Gorilla tool, and the PANTHER and DisGeNET databases were used. Results show that genes with high expression in the brain are involved in cholesterol and lipoprotein processes, plus axo-dendritic transport and the neuron part. These results could highlight some proteins involved in the neurologic pathogenesis of RNS.

Wuzang Wenyang Huayu Tang promoting learning-memory ability in vascular dementia rats via brain-gut-microbiome axis

IntroductionLots of research has revealed that vascular dementia (VD), which is closely related to the metabolic disorders of intestinal microbiota, can be treated through traditional Chinese drug by regulating the brain-gut-microbiome axis. However, the specific interaction between VD and intestinal microflora, as well as the mechanism of Wuzang Wenyang Huayu Tang (WWHT) in treating VD by regulating the brain-gut-microbiome axi remains unknown on the whole and needs to be further studied. ObjectivesTo explore the effect of WWHT on the learning and memory ability of rats with VD based on brain-gut-microbiome axis. MethodsIn this study, taking the specific pathogen-free (SPF) SD rats as the subjects, a vascular dementia rat model was created by using the two-vessel occlusion model (2-VO). The subjects were divided into six groups, namely control group, sham group, VD model group, the piracetam group, the WWHT group, and the WWHT combined with piracetam (WCP) group. After 22 days of the gavage administration method, the Morris water maze experiment was adopted to determine the learning and memory abilities of each group through the water maze, observe the effects of hematoxylin and eosin (HE) staining and transmission electron microscopy in the hippocampal area of each group, and analyze the relevant proteins in the whole brain by Western Blot (WB). In addition, the feces of each group were collected and analyzed by fecal metabolomics combined with 16S rRNA gene sequencing to explore the interaction between VD and intestinal microbiota. ResultsThrough Morris water maze experiment, it was found that the learning and memory ability of VD rats in WWHT, piracetam and WCP groups was improved. The morphology of hippocampal neuron cells were improved in the WWHT group and the number of hippocampal neuron cells were increased in the VD rats by HE. By observing the ultrastructure of hippocampal cells, it can be found that the VD model group showed cell membrane edema and large organelle lysis. The ultrastructure of the hippocampus of all the administered groups was improved. In terms of the effect on the expression levels of related proteins in the whole brain of rats, compared with the VD model group, the expression levels of Caspase-3 protein decreased in all groups after drug administration. The protein expression levels of APG5L/ATG5, Beclin-1 and LC3A/B were upregulated, indicating that the drug administration group could regulate cellular autophagy in the whole brain to further protect brain tissue. Fecal metabolite profiles revealed 27 different metabolites related to VD. These metabolites are mainly involved in related metabolic pathways, such as vitamin B metabolism, purine metabolism, pyrimidine metabolism, and histidine metabolism. Further analysis of the gut microbiota by 16S rRNA gene sequencing data showed that WWHT-treated VD rats were different in gut microbiota composition. DiscussionThrough the effect of brain-gut-microbiome axis, WWHT regulates the metabolism of vitamin B6 and tryptophan in VD rats, which can improve the intestinal microbiota of VD rats and regulate autophagy in the brain to improve cell morphology in the hippocampus, thus increasing the number of hippocampal cells to protect hippocampal neuron cells2510100, and finally improving the learning and memory ability of VD rats.

Endothelial TREM-1 receptor regulates the blood-brain barrier integrity after intracerebral hemorrhage in mice via SYK/β-catenin signaling.

Intracerebral hemorrhage (ICH) is a high mortality and disability stroke subtype. Destruction of the blood-brain barrier (BBB) is a crucial contributor to brain edema and neurological deficit after ICH. Triggering receptor expressed on myeloid cells 1 (TREM-1) has been reported to be expressed in endothelial cells, but its role in ICH remains unclear. This study aims to evaluate the role of TREM-1 on BBB permeability after ICH in mice. Two hundred and forty-two CD1 mice were used in this study. The ICH model was established by collagenase injection. LP17 was administered intranasally at 2 or 8 h after ICH to inhibit TREM-1. To explore the underlying mechanism, SYK activation CRISPR was administered intracerebroventricularly with LP17, and Anti-mouse TREM-1 rat IgG2a (a specific TREM-1 agonist) was injected intracerebroventricularly with R406 (a specific SYK inhibitor) intraperitoneally. Neurobehavioral outcome, brain water content, BBB permeability, and protein expression were evaluated. The expression level of the TREM-1 receptor increased rapidly as early as 6 h after ICH, and it was mainly expressed on the endotheliocytes in the neurovascular unit. Early and delayed administration of LP17 significantly decreased brain edema and improved neurobehavioral outcomes at 24 h after ICH. LP17 reduced the BBB permeability by increasing β-catenin, claudin-5 and ZO-1 expression. Furthermore, SYK activation CRISPR abolished the beneficial effect of LP17 on the expression of the above junction molecules. Meanwhile, R406 reversed the impact of the TREM-1 activator on the downregulation of β-catenin, claudin-5 and ZO-1 expression. This study demonstrated that TREM-1 deteriorated BBB permeability via modulating the expression of interendothelial junction molecules after ICH, and this regulation is partly mediated by the SYK/β-catenin signaling pathway.

Prioritization of Drug Targets for Neurodegenerative Diseases by Integrating Genetic and Proteomic Data From Brain and Blood

BackgroundNeurodegenerative diseases are among the most prevalent and devastating neurological disorders, with few effective prevention and treatment strategies. We aimed to integrate genetic and proteomic data to prioritize drug targets for neurodegenerative diseases. MethodsWe screened human proteomes through Mendelian randomization to identify causal mediators of Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis, frontotemporal dementia, and Lewy body dementia. For instruments, we used brain and blood protein quantitative trait loci identified from one genome-wide association study with 376 participants and another with 3301 participants, respectively. Causal associations were subsequently validated by sensitivity analyses and colocalization. The safety and druggability of identified targets were also evaluated. ResultsOur analyses showed targeting BIN1, GRN, and RET levels in blood as well as ACE, ICA1L, MAP1S, SLC20A2, and TOM1L2 levels in brain might reduce Alzheimer’s disease risk, while ICA1L, SLC20A2, and TOM1L2 were not recommended as prioritized drugs due to the identified potential side effects. Brain CD38, DGKQ, GPNMB, and SEC23IP were candidate targets for Parkinson’s disease. Among them, GPNMB was the most promising target for Parkinson’s disease with their causal relationship evidenced by studies on both brain and blood tissues. Interventions targeting FCRL3, LMAN2, and MAPK3 in blood and DHRS11, FAM120B, SHMT1, and TSFM in brain might affect multiple sclerosis risk. The risk of amyotrophic lateral sclerosis might be reduced by medications targeting DHRS11, PSMB3, SARM1, and SCFD1 in brain. ConclusionsOur study prioritized 22 proteins as targets for neurodegenerative diseases and provided preliminary evidence for drug development. Further studies are warranted to validate these targets.

Glymphatic clearance estimated using diffusion tensor imaging along perivascular spaces is reduced after traumatic brain injury and correlates with plasma neurofilament light, a biomarker of injury severity.

The glymphatic system is a perivascular fluid clearance system, most active during sleep, considered important for clearing the brain of waste products and toxins. Glymphatic failure is hypothesized to underlie brain protein deposition in neurodegenerative disorders like Alzheimer's disease. Preclinical evidence suggests that a functioning glymphatic system is also essential for recovery from traumatic brain injury, which involves release of debris and toxic proteins that need to be cleared from the brain. In a cross-sectional observational study, we estimated glymphatic clearance using diffusion tensor imaging along perivascular spaces, an MRI-derived measure of water diffusivity surrounding veins in the periventricular region, in 13 non-injured controls and 37 subjects who had experienced traumatic brain injury ∼5 months previously. We additionally measured the volume of the perivascular space using T2-weighted MRI. We measured plasma concentrations of neurofilament light chain, a biomarker of injury severity, in a subset of subjects. Diffusion tensor imaging along perivascular spaces index was modestly though significantly lower in subjects with traumatic brain injury compared with controls when covarying for age. Diffusion tensor imaging along perivascular spaces index was significantly, negatively correlated with blood levels of neurofilament light chain. Perivascular space volume did not differ in subjects with traumatic brain injury as compared with controls and did not correlate with blood levels of neurofilament light chain, suggesting it may be a less sensitive measure for injury-related perivascular clearance changes. Glymphatic impairment after traumatic brain injury could be due to mechanisms such as mislocalization of glymphatic water channels, inflammation, proteinopathy and/or sleep disruption. Diffusion tensor imaging along perivascular spaces is a promising method for estimating glymphatic clearance, though additional work is needed to confirm results and assess associations with outcome. Understanding changes in glymphatic functioning following traumatic brain injury could inform novel therapies to improve short-term recovery and reduce later risk of neurodegeneration.

A Soluble Platelet-Derived Growth Factor Receptor-β Originates via Pre-mRNA Splicing in the Healthy Brain and Is Upregulated during Hypoxia and Aging.

The platelet-derived growth factor-BB (PDGF-BB) pathway provides critical regulation of cerebrovascular pericytes, orchestrating their investment and retention within the brain microcirculation. Dysregulated PDGF Receptor-beta (PDGFRβ) signaling can lead to pericyte defects that compromise blood-brain barrier (BBB) integrity and cerebral perfusion, impairing neuronal activity and viability, which fuels cognitive and memory deficits. Receptor tyrosine kinases such as PDGF-BB and vascular endothelial growth factor-A (VEGF-A) are often modulated by soluble isoforms of cognate receptors that establish signaling activity within a physiological range. Soluble PDGFRβ (sPDGFRβ) isoforms have been reported to form by enzymatic cleavage from cerebrovascular mural cells, and pericytes in particular, largely under pathological conditions. However, pre-mRNA alternative splicing has not been widely explored as a possible mechanism for generating sPDGFRβ variants, and specifically during tissue homeostasis. Here, we found sPDGFRβ protein in the murine brain and other tissues under normal, physiological conditions. Utilizing brain samples for follow-on analysis, we identified mRNA sequences corresponding to sPDGFRβ isoforms, which facilitated construction of predicted protein structures and related amino acid sequences. Human cell lines yielded comparable sequences and protein model predictions. Retention of ligand binding capacity was confirmed for sPDGFRβ by co-immunoprecipitation. Visualizing fluorescently labeled sPDGFRβ transcripts revealed a spatial distribution corresponding to murine brain pericytes alongside cerebrovascular endothelium. Soluble PDGFRβ protein was detected throughout the brain parenchyma in distinct regions, such as along the lateral ventricles, with signals also found more broadly adjacent to cerebral microvessels consistent with pericyte labeling. To better understand how sPDGFRβ variants might be regulated, we found elevated transcript and protein levels in the murine brain with age, and acute hypoxia increased sPDGFRβ variant transcripts in a cell-based model of intact vessels. Our findings indicate that soluble isoforms of PDGFRβ likely arise from pre-mRNA alternative splicing, in addition to enzymatic cleavage mechanisms, and these variants exist under normal physiological conditions. Follow-on studies will be needed to establish potential roles for sPDGFRβ in regulating PDGF-BB signaling to maintain pericyte quiescence, BBB integrity, and cerebral perfusion-critical processes underlying neuronal health and function, and in turn, memory and cognition.

Proteomic Profiling of Mouse Brain Pyruvate Kinase Binding Proteins: A Hint for Moonlighting Functions of PKM1?

Affinity-based proteomic profiling is widely used for the identification of proteins involved in the formation of various interactomes. Since protein-protein interactions (PPIs) reflect the role of particular proteins in the cell, identification of interaction partners for a protein of interest can reveal its function. The latter is especially important for the characterization of multifunctional proteins, which can play different roles in the cell. Pyruvate kinase (PK), a classical glycolytic enzyme catalyzing the last step of glycolysis, exists in four isoforms: PKM1, PKM2, PKL, and PKR. The enzyme isoform expressed in actively dividing cells, PKM2, exhibits many moonlighting (noncanonical) functions. In contrast to PKM2, PKM1, predominantly expressed in adult differentiated tissues, lacks well-documented moonlighting functions. However, certain evidence exists that it can also perform some functions unrelated to glycolysis. In order to evaluate protein partners, bound to PKM1, in this study we have combined affinity-based separation of mouse brain proteins with mass spectrometry identification. The highly purified PKM1 and a 32-mer synthetic peptide (PK peptide), sharing high sequence homology with the interface contact region of all PK isoforms, were used as the affinity ligands. This proteomic profiling resulted in the identification of specific and common proteins bound to both affinity ligands. Quantitative affinity binding to the affinity ligands of selected identified proteins was validated using a surface plasmon resonance (SPR) biosensor. Bioinformatic analysis has shown that the identified proteins, bound to both full-length PKM1 and the PK peptide, form a protein network (interactome). Some of these interactions are relevant for the moonlighting functions of PKM1. The proteomic dataset is available via ProteomeXchange with the identifier PXD041321.

Effect of Hypoxia-Ischemia on the Expression of Iron-Related Proteins in Neonatal Rat Brains.

Hypoxic-ischemic white matter injury (WMI) pathogenesis in preterm infants is not well established, and iron-related proteins in the brain may play an important role in imbalanced iron metabolism. We aimed to investigate the iron-related protein changes in neonatal rats after hypoxia-ischemia (HI), clarify the role of iron-related proteins in hypoxic-ischemic WMI, and potentially provide a new target for the clinical treatment of hypoxic-ischemic WMI in preterm infants. We adopted a WMI animal model of bilateral common carotid artery electrocoagulation combined with hypoxia in neonatal 3-day-old Sprague-Dawley rats. We observed basic myelin protein (MBP) and iron-related protein expression in the brain (ferritin, transferrin receptor [TfR], and membrane iron transporter 1 [FPN1]) via Western blot and double immunofluorescence staining. The expression of MBP in the WMI group was significantly downregulated on postoperative days (PODs) 14, 28, and 56. Ferritin levels were significantly increased on PODs 3, 7, 14, and 28 and were most significant on POD 28, returning to the sham group level on POD 56. FPN1 levels were significantly increased on PODs 7, 28, and 56 and were still higher than those in the sham group on POD 56. TfR expression was significantly upregulated on PODs 1, 7, and 28 and returned to the sham group level on POD 56. Immunofluorescence staining showed that ferritin, TfR, and FPN1 were expressed in neurons, blood vessels, and oligodendrocytes in the cortex and corpus callosum on POD 28. Compared with the sham group, the immune-positive markers of three proteins in the WMI group were significantly increased. The expression of iron-related proteins in the brain (ferritin, FPN1, and TfR) showed spatiotemporal dynamic changes and may play an important role in hypoxic-ischemic WMI.

Contribution of the dihydropyrimidinase-like proteins family in synaptic physiology and in neurodevelopmental disorders.

The dihydropyrimidinase-like (DPYSL) proteins, also designated as the collapsin response mediators (CRMP) proteins, constitute a family of five cytosolic phosphoproteins abundantly expressed in the developing nervous system but down-regulated in the adult mouse brain. The DPYSL proteins were initially identified as effectors of semaphorin 3A (Sema3A) signaling and consequently involved in regulation of growth cone collapse in young developing neurons. To date, it has been established that DPYSL proteins mediate signals for numerous intracellular/extracellular pathways and play major roles in variety of cellular process including cell migration, neurite extension, axonal guidance, dendritic spine development and synaptic plasticity through their phosphorylation status. The roles of DPYSL proteins at early stages of brain development have been described in the past years, particularly for DPYSL2 and DPYSL5 proteins. The recent characterization of pathogenic genetic variants in DPYSL2 and in DPYSL5 human genes associated with intellectual disability and brain malformations, such as agenesis of the corpus callosum and cerebellar dysplasia, highlighted the pivotal role of these actors in the fundamental processes of brain formation and organization. In this review, we sought to establish a detailed update on the knowledge regarding the functions of DPYSL genes and proteins in brain and to highlight their involvement in synaptic processing in later stages of neurodevelopment, as well as their particular contribution in human neurodevelopmental disorders (NDDs), such as autism spectrum disorders (ASD) and intellectual disability (ID).

Programmed ageing: decline of stem cell renewal, immunosenescence, and Alzheimer's disease.

The characteristic maximum lifespan varies enormously across animal species from a few hours to hundreds of years. This argues that maximum lifespan, and the ageing process that itself dictates lifespan, are to a large extent genetically determined. Although controversial, this is supported by firm evidence that semelparous species display evolutionarily programmed ageing in response to reproductive and environmental cues. Parabiosis experiments reveal that ageing is orchestrated systemically through the circulation, accompanied by programmed changes in hormone levels across a lifetime. This implies that, like the circadian and circannual clocks, there is a master 'clock of age' (circavital clock) located in the limbic brain of mammals that modulates systemic changes in growth factor and hormone secretion over the lifespan, as well as systemic alterations in gene expression as revealed by genomic methylation analysis. Studies on accelerated ageing in mice, as well as human longevity genes, converge on evolutionarily conserved fibroblast growth factors (FGFs) and their receptors, including KLOTHO, as well as insulin-like growth factors (IGFs) and steroid hormones, as key players mediating the systemic effects of ageing. Age-related changes in these and multiple other factors are inferred to cause a progressive decline in tissue maintenance through failure of stem cell replenishment. This most severely affects the immune system, which requires constant renewal from bone marrow stem cells. Age-related immune decline increases risk of infection whereas lifespan can be extended in germfree animals. This and other evidence suggests that infection is the major cause of death in higher organisms. Immune decline is also associated with age-related diseases. Taking the example of Alzheimer's disease (AD), we assess the evidence that AD is caused by immunosenescence and infection. The signature protein of AD brain, Aβ, is now known to be an antimicrobial peptide, and Aβ deposits in AD brain may be a response to infection rather than a cause of disease. Because some cognitively normal elderly individuals show extensive neuropathology, we argue that the location of the pathology is crucial - specifically, lesions to limbic brain are likely to accentuate immunosenescence, and could thus underlie a vicious cycle of accelerated immune decline and microbial proliferation that culminates in AD. This general model may extend to other age-related diseases, and we propose a general paradigm of organismal senescence in which declining stem cell proliferation leads to programmed immunosenescence and mortality.

Effect of the obese phenotype on brain composition in congenic lean and obese LA/Ntul//-cp rats

To determine if the obese phenotype impacted on brain development and composition, groups of congenic male lean and obese littermates of the LA/Ntul//-cp rat strain were fed on a standardized nutritionally adequate diet in littermate pairs from weaning until adulthood. The obese phenotype of his strain develops early onset chronic hyperinsulinemia associated with hypertrophic-hyperplastic obesity during early postweaning growth. At 10.5 months of age, animals were sacrificed by cervical dislocation, and brain tissues excised in its entirety, weighed to the nearest mg, and measures of protein, DNA and lipid determined. Body weights (BW) of obese were significantly greater than lean. Brain mass (BrM) of lean &gt; obese, and BrM:BW of lean was greater obese. Brain protein content (BPC) and Brain DNA (BDNA) of lean was greater than obese. Brain lipid as a percent was similar in both phenotypes and net brain lipid content was proportional to brain mass. Total body fat mass of obese was significantly greater than occurred in lean littermates. Inflammatory cytokines residing in adipose tissue have been reported to contribute to DNA damage in neuronal and other tissues, impede cell replication, and accelerate cell senescence. These results indicate that brain growth and cellular development is impaired in the hyperinsulinemia-prone obese phenotype of this strain, and are likely associated with development of a chronic inflammatory syndrome and cytokine expression common to excessive fat accretion and obesity

Plasma neurodegeneration biomarker concentrations associate with glymphatic and meningeal lymphatic measures in neurological disorders

Clearance of neurotoxic brain proteins via cerebrospinal fluid (CSF) to blood has recently emerged to be crucial, and plasma biomarkers of neurodegeneration were newly introduced to predict neurological disease. This study examines in 106 individuals with neurological disorders associations between plasma biomarkers [40 and 42 amino acid-long amyloid-β (Aβ40 and Aβ42), total-tau, glial fibrillary acidic protein (GFAP), and neurofilament light (NfL)] and magnetic resonance imaging measures of CSF-mediated clearance from brain via extra-vascular pathways (proxy of glymphatic function) and CSF-to-blood clearance variables from pharmacokinetic modeling (proxy of meningeal lymphatic egress). We also examine how biomarkers vary during daytime and associate with subjective sleep quality. Plasma concentrations of neurodegeneration markers associate with indices of glymphatic and meningeal lymphatic functions in individual- and disease-specific manners, vary during daytime, but are unaffected by sleep quality. The results suggest that plasma concentrations of neurodegeneration biomarkers associate with measures of glymphatic and meningeal lymphatic function.

Secreted Amyloid Precursor Protein Alpha (sAPPα) Regulates the Cellular Proteome and Secretome of Mouse Primary Astrocytes.

Secreted amyloid precursor protein alpha (sAPPα), processed from a parent mammalian brain protein, amyloid precursor protein, can modulate learning and memory. Recently it has been shown to modulate the transcriptome and proteome of human neurons, including proteins with neurological functions. Here, we analysed whether the acute administration of sAPPα facilitated changes in the proteome and secretome of mouse primary astrocytes in culture. Astrocytes contribute to the neuronal processes of neurogenesis, synaptogenesis and synaptic plasticity. Cortical mouse astrocytes in culture were exposed to 1 nM sAPPα, and changes in both the whole-cell proteome (2 h) and the secretome (6 h) were identified with Sequential Window Acquisition of All Theoretical Fragment Ion Spectra-Mass Spectrometry (SWATH-MS). Differentially regulated proteins were identified in both the cellular proteome and secretome that are involved with neurologically related functions of the normal physiology of the brain and central nervous system. Groups of proteins have a relationship to APP and have roles in the modulation of cell morphology, vesicle dynamics and the myelin sheath. Some are related to pathways containing proteins whose genes have been previously implicated in Alzheimer's disease (AD). The secretome is also enriched in proteins related to Insulin Growth Factor 2 (IGF2) signaling and the extracellular matrix (ECM). There is the promise that a more specific investigation of these proteins will help to understand the mechanisms of how sAPPα signaling affects memory formation.