Brain Protein Expression Research Articles

Environmental Temperature Variation Affects Brain Lipid Composition in Adult Zebrafish (Danio rerio).

This study delves deeper into the impact of environmental temperature variations on the nervous system in teleost fish. Previous research has demonstrated that exposing adult zebrafish (Danio rerio) to 18 °C and 34 °C for 4 or 21 days induces behavioural changes compared to fish kept at a control temperature of 26 °C, suggesting alterations in the nervous system. Subsequent studies revealed that these temperature conditions also modify brain protein expression, indicating potential neurotoxic effects. The primary aim of this work was to investigate the effects of prolonged exposure (21 days) to 18 °C or 34 °C on the brain lipidomes of adult zebrafish compared to a control temperature. Analysis of the brain lipidome highlighted significant alteration in the relative abundances of specific lipid molecules at 18 °C and 34 °C, confirming distinct effects induced by both tested temperatures. Exposure to 18 °C resulted in an increase in levels of phospholipids, such as phosphatidylethanolamine, alongside a general reduction in levels of sphingolipids, including sphingomyelin. Conversely, exposure to 34 °C produced more pronounced effects, with increases in levels of phosphatidylethanolamine and those of various sphingolipids such as ceramide, gangliosides, and sphingomyelin, alongside a reduction in levels of ether phospholipids, including lysophosphatidylethanolamine ether, phosphatidylethanolamine ether, and phosphatidylglycerol ether, as well as levels of glycolipids like monogalactosyldiacylglycerol. These results, when integrated with existing proteomic and behavioural data, offer new insights into the effects of thermal variations on the nervous system in teleost fish. Specifically, our proteomic and lipidomic findings suggest that elevated temperatures may disrupt mitochondrial function, increase neuronal susceptibility to oxidative stress and cytotoxicity, alter axonal myelination, impair nerve impulse transmission, hinder synapse function and neurotransmitter release, and potentially lead to increased neuronal death. These findings are particularly relevant in the fields of cell biology, neurobiology, and ecotoxicology, especially in the context of global warming.

A Combination of Astragaloside IV and Hydroxysafflor Yellow A Attenuates Cerebral Ischemia-Reperfusion Injury via NF-κB/NLRP3/Caspase-1/GSDMD Pathway.

Cerebral ischemia-reperfusion injury (IRI), occurring after blood supply restoration, contributes significantly to stroke-related deaths. This study explored the combined impact and mechanisms of astragaloside IV (AS-IV), hydroxysafflor yellow A (HSYA), and their combination in mitigating IRI. Male Sprague-Dawley (SD) rats were randomized to the Sham, MCAO, MCAO+AS-IV, MCAO+HSYA, and MCAO+AS-IV+HSYA groups. Neurological deficits and cerebral infarction were examined after restoring the blood supply to the brain. Pathomorphological changes in the cerebral cortex were observed via HE staining. IL-1β and IL-18 were quantified using ELISA. The expression of NF-κB and GSDMD in the ischemic cerebrum was analyzed using immunohistochemistry. The expression levels of NLRP3, ASC, IL-1β, Caspase-1, and GSDMD in the ischemic cerebrum were evaluated using Western blot. The MCAO+AS-IV, MCAO+HSYA, and MCAO+AS-IV+HSYA groups exhibited notably better neurological function and cerebral infarction compared with the MCAO group. The combined treatment demonstrated superior brain tissue injury alleviation. Reductions in NF-κB, GSDMD positive cells, and NLRP3/ASC/IL-1β/Caspase-1/GSDMD protein expression in the ischemic brain were significantly more pronounced with the combined therapy, indicating a synergistic effect in countering cerebral IRI via the NF-κB/NLRP3/Caspase-1/GSDMD pathway inhibition of cell pyroptosis-induced injury.

Comparative Brain Proteomic Analysis between Sham and Cerebral Ischemia Experimental Groups.

Sham control groups are essential in experimental animal studies to reduce the influence of surgical intervention. The intraluminal filament procedure is one of the most common models of middle cerebral artery occlusion (MCAO) used in the study of brain ischemia. However, a sham group is usually not included in the experimental design of these studies. In this study, we aimed to evaluate the relevance of the sham group by analyzing and comparing the brain protein profiles of the sham and MCAO groups. In the sham group, 98 dysregulated proteins were detected, compared to 171 in the ischemic group. Moreover, a comparative study of protein profiles revealed the existence of a pool of 57 proteins that appeared to be dysregulated in both sham and ischemic animals. These results indicated that the surgical procedure required for the intraluminal occlusion of the middle cerebral artery (MCA) induces changes in brain protein expression that are not associated with ischemic lesions. This study highlights the importance of including sham control groups in the experimental design, to ensure that surgical intervention does not affect the therapeutic target under study.

Regulator of G Protein Signaling 14 protein expression profile in the adult mouse brain.

Regulator of G protein signaling 14 (RGS14) is a multifunctional signaling protein that serves as a natural suppressor of synaptic plasticity in the mouse brain. Our previous studies showed that RGS14 is highly expressed in postsynaptic dendrites and spines of pyramidal neurons in hippocampal area CA2 of the developing mouse brain. However, our more recent work with adult rhesus macaque brain shows that RGS14 is found in multiple neuron populations throughout hippocampal area CA1 and CA2, caudate nucleus, putamen, globus pallidus, substantia nigra, and amygdala in the adult rhesus monkey brain. In the mouse brain, we also have observed RGS14 protein in discrete limbic regions linked to reward behavior and addiction, including the central amygdala and the nucleus accumbens, but a comprehensive mapping of RGS14 protein expression in the adult mouse brain is lacking. Here, we report that RGS14 is more broadly expressed in mouse brain than previously known. Intense RGS14 staining is observed in specific neuron populations of the hippocampal formation, amygdala, septum, bed nucleus of stria terminalis and ventral striatum/nucleus accumbens. RGS14 is also observed in axon fiber tracts including the dorsal fornix, fimbria, stria terminalis, and the ventrohippocampal commissure. Moderate RGS14 staining is observed in various other adjacent regions not previously reported. These findings show that RGS14 is expressed in brain regions that govern aspects of core cognitive functions such as sensory perception, emotion, memory, motivation, and execution of actions, and suggests that RGS14 may serve to suppress plasticity and filter inputs in these brain regions to set the overall tone on experience-to-action processes.

Identifying Novel Proteins for Chronic Pain: Integration of Human Brain Proteomes and Genome-wide Association Data

Numerous genome-wide association studies have identified risk genes for chronic pain, yet the mechanisms by which genetic variants modify susceptibility have remained elusive. We sought to identify key genes modulating chronic pain risk by regulating brain protein expression. We integrated brain proteomic data with the largest genome-wide dataset for multisite chronic pain (N = 387,649) in a proteome-wide association study (PWAS) using discovery and confirmatory proteomic datasets (N = 376 and 152) from the dorsolateral prefrontal cortex. Leveraging summary data-based Mendelian randomization and Bayesian colocalization analysis, we pinpointed potential causal genes, while a transcriptome-wide association study integrating 452 human brain transcriptomes investigated whether cis-effects on protein abundance extended to the transcriptome. Single-cell RNA-sequencing data and single-nucleus transcriptomic data revealed cell-type-specific expression patterns for identified causal genes in the dorsolateral prefrontal cortex and dorsal root ganglia (DRG), complemented by RNA microarray analysis of expression profiles in other pain-related brain regions. Of the 22 genes cis-regulating protein abundance identified by the discovery PWAS, 18 (82%) were deemed causal by summary data-based Mendelian randomization or Bayesian colocalization analysis analyses, with 7 of these 18 genes (39%) replicating in the confirmatory PWAS, including guanosine diphosphate-mannose pyrophosphorylase B, which also associated at the transcriptome level. Several causal genes exhibited selective expression in excitatory and inhibitory neurons, oligodendrocytes, and astrocytes, while most identified genes were expressed across additional pain-related brain regions. This integrative proteogenomic approach identified 18 high-confidence causal genes for chronic pain, regulated by cis-effects on brain protein levels, suggesting promising avenues for treatment research and indicating a contributory role for the DRG. PerspectiveThe current post genome-wide association study analyses identified 18 high-confidence causal genes regulating chronic pain risk via cis-modulation of brain protein abundance, suggesting promising avenues for future chronic pain therapies. Additionally, the significant expression of these genes in the DRG indicated a potential contributory role, warranting further investigation.

Lower activity of cholesteryl ester transfer protein (CETP) and the risk of dementia: a Mendelian randomization analysis

Abstract Funding Acknowledgements Type of funding sources: Private company. Main funding source(s): New Amterdam Pharma Background Elevated levels of low-density lipoprotein cholesterol (LDL-C) are linked to dementia risk, and conversely, increased plasma concentrations of high-density lipoprotein cholesterol (HDL-C) and apolipoprotein-A1 (Apo-A1) associate with decreased dementia risk. Inhibition of cholesteryl ester transfer protein (CETP) meaningfully affects the concentrations of these blood lipids and may therefore provide an opportunity to treat dementia. Methods Drug target Mendelian randomization (MR) was employed to anticipate the on-target effects of lower CETP concentration (µg/mL) on plasma lipids, cardiovascular disease, Lewy body dementia (LBD), as well as Parkinson’s dementia. Replication was sought by performed Apo-A1 and Apo-B weighted MR analyses of CETP variants. Genetic associations with protein expression in cerebrospinal fluid (CSF) and brain were consulted to identify potential associations of plasma CETP with dementia implicated proteins. Results MR analysis of lower CETP concentration (per µg/mL) recapitulated the blood lipid effects observed in clinical trials of CETP-inhibitors, as well as protective effects on CHD (odds ratio (OR) 0.92, 95% confidence interval (CI) 0.89; 0.96), heart failure, abdominal aortic aneurysm any stroke, ischemic stroke, and small vessel stroke (0.90, 95%CI 0.85; 0.96); Figure. Consideration of dementia related traits indicated that lower CETP concentrations were associated higher total brain volume (0.04 per standard deviation, 95%CI 0.02; 0.06), lower risk of LBD (OR 0.81, 95%CI 0.74; 0.89) and Parkinson’s dementia risk (OR 0.26, 95%CI 0.14; 0.48). APOE4 stratified analyses suggested the LBD effect was most pronounced in APOE-ε4+ participants (OR 0.61 95%CI 0.51; 0.73), compared to APOE-ε4- (OR 0.89 95%CI 0.79; 1.01); interaction p-value 5.81×10-4. The aforementioned associations were replicated using Apo-B and Apo-A1 weighted analyses; Figure. Additionally, MR was employed to link plasma CETP concentration to the levels of CSF and brain proteins such as EPHA2 and CSF-1, which are in development as dementia drug targets. Conclusion These results suggest that inhibition of CETP may be a viable strategy to treat dementia.

Effects of xylo-oligosaccharide on gut microbiota, brain protein expression, and lipid profile induced by high-fat diet

Midlife overweight and obesity are risk factors of cognitive decline and Alzheimer' s disease (AD) in late life. In addition to increasing risk of obesity and cognitive dysfunction, diets rich in fats also contributes to an imbalance of gut microbiota. Xylo-oligosaccharides (XOS) are a kind of prebiotic with several biological advantages, and can selectively promote the growth of beneficial microorganisms in the gut. To explore whether XOS can alleviate cognitive decline induced by high-fat diet (HFD) through improving gut microbiota composition, mice were fed with normal control or 60% HFD for 9 weeks to induce obesity. After that, mice were supplemented with XOS (30 g or 60 g/kg-diet) or without, respectively, for 12 weeks. The results showed that XOS inhibited weight gain, decreased epidydimal fat weight, and improved fasting blood sugar and blood lipids in mice. Additionally, XOS elevated spatial learning and memory function, decreased amyloid plaques accumulation, increased brain-derived neurotrophic factor levels, and improved neuroinflammation status in hippocampus. Changes in glycerolipids metabolism-associated lipid compounds caused by HFD in hippocampus were reversed after XOS intervention. On the other hand, after XOS intervention, increase in immune-mediated bacteria, Faecalibacterium was observed. In conclusion, XOS improved gut dysbiosis and ameliorated spatial learning and memory dysfunction caused by HFD by decreasing cognitive decline-associated biomarkers and changing lipid composition in hippocampus.

Response of CRH system in brain and gill of marine medaka to seawater acidification.

Corticotropin-releasing hormone (CRH) is mainly secreted by the hypothalamus to regulate stress when environmental factors change. Gills contact with water directly and may also secrete CRH to maintain local homeostasis. Ocean acidification changes water chemical parameters and is becoming an important environmental stressor for marine fish. The response of brain and gill CRH systems to ocean acidification remains unclear. In this study, marine medaka were exposed to CO2-acidified seawater (440ppm, 1000ppm, and 1800ppm CO2) for 2h, 4h, 24h, and 7 d, respectively. At 2h and 4h, the expression of crh mRNA in gills increased with increasing CO2 concentration. Crh protein is expressed mainly in the lamellae cells. crhbp and crhr1 expression also increased significantly. However, at 2h and 4h, acidification caused little changes in these genes and Crh protein expression in the brain. At 7 d, Crh-positive cells were detected in the hypothalamus; moreover, Crh protein expression in the whole brain increased. It is suggested that CRH autocrine secretion in gills is responsible for local acid-base regulation rather than systemic mobilization after short-term acidification stress, which may help the rapid regulation of body damage caused by environmental stress.

Tanshinone IIA Regulates Synaptic Plasticity in Mg2+-Free-Induced Epileptic Hippocampal Neurons via the PI3K/Akt Signaling Pathway.

Tanshinone IIA (TSIIA) is an element of the effective ingredients of Salvia miltiorrhiza Bunge (Labiatae), exhibits a significant therapeutic effect in brain neuroprotection. The focus of this study was the examination of synaptic plasticity of in Mg2+-free-induced epileptic hippocampus neurons and how TSIIA protects against it. The purity of the primary hippocampal neurons extracted from Sprague Dawley rats was assessed within 24 hours by microtubule-associated protein (MAP2) immunofluorescence staining. A hippocampal neuron model for Mg2+-free-induced spontaneous recurrent epileptiform discharge was developed, five experimental groups were then randomized: blank (Blank), model (Model), TSIIA (TSIIA, 20 µM), LY294002 (LY294002, 25 µM), and TSIIA+LY294002 (TSIIA+LY294002, 20 µM+25 µM). FIJI software was used to examine variations of neurite complexity, total length of hippocampal neurons, number of primary dendrites and density of dendritic spines. Developmental regulation brain protein (Drebrin) and brain-derived neurotrophic factor (BDNF) expression was evaluated using immunofluorescence staining and the relative expression of phospho-protein kinase B (p-Akt)/Akt, BDNF, synaptophysin (SYN) and postsynaptic density 95 (PSD-95) determined by Western blot. In contrast to the model group, TSIIA drastically reduced damage to synaptic plasticity of hippocampal neurons caused by epilepsy (p < 0.05). The TSIIA group showed a significant increase in the relative expression of PSD-95, SYN, BDNF, and p-Akt/Akt (p < 0.01). TSIIA was effective in reducing harm to the synaptic plasticity of hippocampal neurons induced by persistent status epilepticus, with the possible mechanism being regulation of the phosphatidylinositol 3-kinase 56 (PI3K)/Akt signaling pathway.

Effect of high-fat diet on the fatty acid profiles of brain in offspring mice exposed to maternal gestational diabetes mellitus.

Fatty acids play a critical role in the proper functioning of the brain. This study investigated the effects of a high-fat (HF) diet on brain fatty acid profiles of offspring exposed to maternal gestational diabetes mellitus (GDM). Insulin receptor antagonist (S961) and HF diet were used to establish the GDM animal model. Brain fatty acid profiles of the offspring mice were measured by gas chromatography at weaning and adulthood. Protein expressions of the fatty acid transport pathway Wnt3/β-catenin and the target protein major facilitator superfamily domain-containing 2a (MFSD2a) were measured in the offspring brain by Western blot. Maternal GDM increased the body weight of male offspring (P < 0.05). In weaning offspring, factorial analysis showed that maternal GDM increased the monounsaturated fatty acid (MUFA) percentage of the weaning offspring's brain (P < 0.05). Maternal GDM decreased offspring brain arachidonic acid (AA), but HF diet increased brain linoleic acid (LA) (P < 0.05). Maternal GDM and HF diet reduced offspring brain docosahexaenoic acid (DHA), and the male offspring had higher DHA than the female offspring (P < 0.05). In adult offspring, factorial analysis showed that HF diet increased brain MUFA in offspring, and male offspring had higher brain MUFA than female offspring (P < 0.05). The HF diet increased brain LA in the offspring. Male offspring had higher level of AA than female offspring (P < 0.05). HF diet reduced DHA in the brains of female offspring. The brain protein expression of β-catenin and MFSD2a in both weaning and adult female offspring was lower in the HF + GDM group than in the CON group (P < 0.05). Maternal GDM increased the susceptibility of male offspring to HF diet-induced obesity. HF diet-induced adverse brain fatty acid profiles in both male and female offspring exposed to GDM.

Caffeine-induced protein kinase A activation restores cognitive deficits induced by sleep deprivation by regulating O-GlcNAc cycling in adult zebrafish.

Sleep deprivation (SD) is widely acknowledged as a significant risk factor for cognitive impairment. In this study, intraperitoneal caffeine administration significantly ameliorated the learning and memory (L/M) deficits induced by SD and reduced aggressive behaviors in adult zebrafish. SD led to a reduction in protein kinase A (PKA) phosphorylation, phosphorylated-cAMP response element-binding protein (p-CREB), and c-Fos expression in zebrafish brain. Notably, these alterations were effectively reversed by caffeine. In addition, caffeine mitigated neuroinflammation induced by SD, as evident from suppression of the SD-mediated increase in glial fibrillary acidic protein (GFAP) and nuclear factor-κB (NF-κB) activation. Caffeine restored normal O-GlcNAcylation and O-GlcNAc transferase (OGT) levels while reversing the increased expression of O-GlcNAcase (OGA) in zebrafish brain after SD. Intriguingly, rolipram, a selective phosphodiesterase 4 (PDE4) inhibitor, effectively mitigated cognitive deficits, restored p-CREB and c-Fos levels, and attenuated the increase in GFAP in brain induced by SD. In addition, rolipram reversed the decrease in O-GlcNAcylation and OGT expression as well as elevation of OGA expression following SD. Treatment with H89, a PKA inhibitor, significantly impaired the L/M functions of zebrafish compared with the control group, inducing a decrease in O-GlcNAcylation and OGT expression and, conversely, an increase in OGA expression. The H89-induced changes in O-GlcNAc cycling and L/M dysfunction were effectively reversed by glucosamine treatment. H89 suppressed, whereas caffeine and rolipram promoted O-GlcNAc cycling in Neuro2a cells. Our collective findings underscore the interplay between PKA signaling and O-GlcNAc cycling in the regulation of cognitive function in the brain, offering potential therapeutic targets for cognitive deficits associated with SD.NEW & NOTEWORTHY Our observation highlights the intricate interplay between cAMP/PKA signaling and O-GlcNAc cycling, unveiling a novel mechanism that potentially governs the regulation of learning and memory functions. The dynamic interplay between these two pathways provides a novel and nuanced perspective on the molecular foundation of learning and memory regulation. These insights open avenues for the development of targeted interventions to treat conditions that impact cognitive function, including SD.

Faecal Microbiota Transplantation Alleviates Ferroptosis after Ischaemic Stroke

Ischaemic stroke can induce changes in the abundance of gut microbiota constituents, and the outcome of stroke may also be influenced by the gut microbiota. This study aimed to determine whether gut microbiota transplantation could rescue changes in the gut microbiota and reduce ferroptosis after stroke in rats. Male Sprague-Dawley rats (6 weeks of age) were subjected to ischaemic stroke by middle cerebral artery occlusion (MCAO). Fecal samples were collected for 16S ribosomal RNA (rRNA) sequencing to analyze the effects of FMT on the gut microbiota. Neurological deficits were evaluated using the Longa score. triphenyl tetrazolium chloride (TTC) staining was performed in the brain, and kits were used to measure malondialdehyde (MDA), iron, and glutathione (GSH) levels in the ipsilateral brain of rats. Western blotting was used to detect the protein expression levels of glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), and the transferrin receptor 2 (TFR2) in the ipsilateral brain of rats. Stroke induced significant changes in the gut microbiota, and FMT ameliorated these changes. TTC staining results showed that FMT reduced cerebral infarct volume. In addition, FMT diminished MDA and iron levels and elevated GSH levels in the ipsilateral brain. Western blot analysis showed that FMT increased GPX4 and SLC7A11 protein expression and decreased TFR2 protein expression in the ipsilateral brain after stroke. FMT can reverse gut microbiota dysbiosis, reduce cerebellar infarct volume, and decrease ferroptosis after stroke.

Resveratrol alleviates hyperglycemia-induced cardiomyocyte hypertrophy by maintaining mitochondrial homeostasis via enhancing SIRT1 expression

To investigate whether resveratrol alleviates hyperglycemia-induced cardiomyocyte hypertrophy by enhancing the expression of silent information regulation 2 homolog 1 (SIRT1) to maintain mitochondrial homeostasis. Rat cardiomyocytes H9c2 cells with or without lentivirus-mediated mRNA interference of SIRT1 were cultured in high glucose (HG) and treated with resveratrol for 72 h. The changes in superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, reactive oxygen species (ROS) level, and relative surface of the cells were examined, and the mRNA expressions of atrial natriuretic factor (ANF) and brain natriuretic peptide (BNP) and protein expressions of SIRT1, mitochondrial fusion related proteins optic atrophy protein 1 (OPA1) and mitofusin 2, mitochondrial division related proteins dynamin-related protein 1 (DRP1) and fission protein 1 (FIS1), and mitophagy-related proteins BNIP3L and LC3 were detected using RT-qPCR and Western blotting. HG exposure significantly decreased SOD activity, increased MDA content, ROS production, relative cell surface, and the mRNA expressions of ANF and BNP in the cardiomyocytes; the protein expressions of SIRT1, OPA1, mitofusin 2 and BNIP3L and LC3-Ⅱ/LC3-Ⅰ ratio were all decreased and the protein expressions of DRP1 and FIS1 increased in HG-exposed cells (P<0.01). All these changes in HG-exposed cardiomyocytes were significantly alleviated by treatment with resveratrol (P<0.05). The protective effects of resveratrol against HG exposure in the cardiomyocytes were obviously attenuated by transfection of the cells with si-SIRT1 (P<0.05). Resveratrol inhibits hyperglycemia-induced cardiomyocyte hypertrophy by reducing oxidative stress, the mechanisms of which involve enhancement of SIRT1 protein expression, regulation of mitochondrial fusion and division balance, and promoting BNIP3L-mediated mitophagy to maintain mitochondrial homeostasis in the cells.

Effect of a single psilocybin treatment on Fos protein expression in male rat brain

Psilocybin has received attention as a treatment for depression, stress disorders and drug and alcohol addiction. To help determine the mechanisms underlying its therapeutic effects, here we examined acute effects of a range of behaviourally relevant psilocybin doses (0.1–3 mg/kg SC) on regional expression of Fos, the protein product of the immediate early gene, c-fos in brain areas involved in stress, reward and motivation in male rats. We also determined the cellular phenotypes activated by psilocybin, in a co-labeling analysis with NeuN, a marker of mature neurons, or Olig1, a marker of oligodendrocytes. In adult male Sprague-Dawley rats, psilocybin increased Fos expression dose dependently in several brain regions, including the frontal cortex, nucleus accumbens, central and basolateral amygdala and locus coeruleus. These effects were most marked in the central amygdala. Double labeling experiments showed that Fos was expressed in both neurons and oligodendrocytes. These results extend previous research by determining Fos expression in multiple brain areas at a wider psilocybin dose range, and the cellular phenotypes expressing Fos. The data also highlight the amygdala, especially the central nucleus, a key brain region involved in emotional processing and learning and interconnected with other brain areas involved in stress, reward and addiction, as a potentially important locus for the therapeutic effects of psilocybin. Overall, the present findings suggest that the central amygdala may be an important site through which the initial brain activation induced by psilocybin is translated into neuroplastic changes, locally and in other regions that underlie its extended therapeutic effects.

Effects of temperature acclimation on the upper thermal tolerance of two Arctic fishes.

The thermally dynamic nearshore Beaufort Sea, Alaska, is experiencing climate change-driven temperature increases. Measuring thermal tolerance of broad whitefish (Coregonus nasus) and saffron cod (Eleginus gracilis), both important species in the Arctic ecosystem, will enhance understanding of species-specific thermal tolerances. The objectives of this study were to determine the extent that acclimating broad whitefish and saffron cod to 5°C and 15°C changed their critical thermal maximum (CTmax) and HSP70 protein and mRNA expression in brain, muscle and liver tissues. After acclimation to 5°C and 15°C, the species were exposed to a thermal ramping rate of 3.4°C · h-1 before quantifying the CTmax and HSP70 protein and transcript concentrations. Broad whitefish and saffron cod acclimated to 15°C had a significantly higher mean CTmax (27.3°C and 25.9°C, respectively) than 5°C-acclimated fish (23.7°C and 23.2°C, respectively), which is consistent with trends in CTmax between higher and lower acclimation temperatures. There were species-specific differences in thermal tolerance with 15°C-acclimated broad whitefish having higher CTmax and HSP70 protein concentrations in liver and muscle tissues than saffron cod at both acclimation temperatures. Tissue-specific differences were quantified, with brain and muscle tissues having the highest and lowest HSP70 protein concentrations, respectively, for both species and acclimation temperatures. The differences in broad whitefish CTmax between the two acclimation temperatures could be explained with brain and liver tissues from 15°C acclimation having higher HSP70a-201 and HSP70b-201 transcript concentrations than control fish that remained in lab-acclimation conditions of 8°C. The shift in CTmax and HSP70 protein and paralogous transcripts demonstrate the physiological plasticity that both species possess in responding to two different acclimation temperatures. This response is imperative to understand as aquatic temperatures continue to elevate.

Proteomic Analyses of the Mouse Brain.

Proteomics is a scientific field that aims to identify and characterize all proteins within a biological system, including their posttranslational modifications (PTMs), quantitative changes, and protein-protein interactions. Over the last two decades, proteomic approaches have been widely used in neuroscience research, providing multidimensional insights into the biology and pathology of the brain.Here, we present a basic protocol for profiling protein expression in the mouse brain, which involves total protein extraction, fractionation, digestion, and identification through liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). This method is compatible with many prevalent techniques used for protein quantitation, PTM analysis, and protein-protein interaction mapping.

Altered brain connectivity, neuroinflammation and AD‐related markers following 7 days after repetitive mild traumatic brain injury in young wild‐type mice

AbstractBackgroundTraumatic brain injuries (TBI) substantially increase the risk for neurodegenerative disorders including tauopathies and synucleinopathies. Repetitive mild TBI (rmTBI) exacerbate damage in brain impacting cognitive and emotional processes through lifetime. Therefore, there is a critical need to identify the molecular mechanisms underlying TBI‐mediated brain dysfunction. In doing so, we can develop effective therapies as the etiology for dementia remain poorly understood. Our goal is to identify early, unbiased, and novel biomarkers of brain dysfunction following rmTBI. These include defects in the brain connectome and protein expression in the areas most functionally susceptible to damage following rmTBI.MethodTo do this, we exposed young male and female C57BL/6J wild‐type mice to two rmTBI (2 × 0.6 J impacts/24 h apart) using the closed head injury model of engineered rotational acceleration (CHIMERA) or sham procedures. We measured changes in brain functional connectivity (FC) using resting state functional MRI (rsfMRI) and graph theory, examined microstructural differences in white and grey matter regions through diffusion tensor imaging (DTI) analyses, surveyed neuropathological proteins using NanoString‐GeoMx Digital spatial protein profiling spatial profiling (DSP).ResultThe rsfMRI data revealed that rmTBI induces aberrant changes in the graph metrics such as node clustering coefficient, global and local efficiency, participation coefficient, eigenvector centrality, and betweenness centrality in brain regions processing visual, auditory, and somatosensory information. The optic tract and thalamus were the most affected regions showing significant differences in phospho‐tau (S199), neuroinflammatory markers (GFAP, Iba‐1, GPNMB), and other proteolytic (cathepsin‐D) and proliferation (ki‐67) cell markers. We identified extensive white matter damage, defects in brain FC, and exacerbated anatomically distinct protein profiles.ConclusionThese findings align with results reported in individuals with TBI such as diffuse axonal damage, glial activation, and functional network disruption in the visual tract and specific thalamic regions. Upcoming studies will evaluate the potential use of using brain imaging along with molecular markers to predict longitudinal damage after rmTBI.

Integration of Alzheimer’s Disease GWAS with Molecular QTLs Reveals Prioritized Risk Genes and Disease‐associated Molecular Alterations

AbstractBackgroundDespite the success of genome‐wide association studies (GWAS) to identify genetic variants associated with complex diseases, as the majority of these variants reside in non‐coding genome, one important task is to link these variants to disease‐associated molecular alterations in risk genes. To this end, we developed a systematic gene prioritization methodology that integrated our recent Alzheimer’s disease (AD) GWAS meta‐analysis with molecular quantitative trait loci (QTL) in disease‐relevant tissues and identified highly likely candidate risk genes and molecular alterations within the majority of 42 novel loci identified. In this subsequent study, we updated our pipeline with new molecular QTL catalogues and analyzed the complete landscape of AD risk.MethodWe performed GWAS‐QTL integration in AD‐relevant tissues and cells by querying the lead variants in each locus in the molecular QTL catalogues, (ii) investigating genetic colocalization between GWAS and molecular QTL signals using coloc, and (iii) conducting transcriptome, methylome, and proteome‐wide association studies (TWAS/MWAS/PWAS) using the EADB stage I meta‐analysis GWAS on 85,934 cases and 401,577 controls. The updated gene prioritization pipeline included new analyses based on new brain protein expression QTL (pQTL), cell‐type‐specific expression QTL (ct‐eQTL), expression QTL (eQTL), histone acetylation QTL (haQTL), and methylation QTL (mQTL) catalogues. Moreover, we used Nanopore RNA sequencing to further investigate risk‐associated splicing events revealed by splicing QTL (sQTL) integration.ResultOur brain ct‐eQTL coloc results showed that while the most risk‐associated gene expression alterations are specific to microglia, the proportion of astrocyte‐specific eQTL colocalizations were doubled compared to the previous studies. These included EGFR, a previously prioritized gene due to bulk brain eQTL coloc hits, whose downregulation was regulated by the protective signal specifically in astrocytes. Importantly, in a previously unresolved locus we could prioritize HS3ST5 through astrocyte‐specific eQTLs as well. Furthermore, pQTL coloc and PWAS prioritized SNX1, a previously prioritized gene at a lower confidence together with neighboring FAM96A gene, by implicating its genetic downregulation with increased AD risk.ConclusionOur updated pipeline systematically prioritized AD risk genes and disease‐associated molecular alterations in the majority of risk loci, and contributed to better interpretation of AD GWAS and understanding of genetically‐associated molecular mechanisms underlying AD risk.

Investigating biological age as a predictor of symptom progression in Alzheimer’s disease

AbstractBackgroundAge is the strongest risk factor for dementia, including Alzheimer’s disease (AD). However, across individuals of the same chronological age there is considerable heterogeneity in rates of cognitive decline. These differences have been attributed to individual differences in age‐related biological processes, including brain protein expression. To capture these differences, we developed a measure of protein brain age from cerebrospinal fluid (CSF)‐proteins, and validated its predictive utility.MethodsIn older adults across the dementia continuum enrolled in DELCODE (training cohort; N = 488, 5‐year follow‐up) and patients with subjective cognitive decline and mild cognitive impairment (MCI) from the memory‐clinic based Fundació ACE (FACE) cohort (test cohort; N = 509, 3‐year follow‐up), 1063 CSF‐proteins were measured using OLINK‐technology. Proteins optimally predicting chronological age in A/T/N‐negative individuals without dementia from DELCODE (N = 196) were selected based on a LASSO‐regression and summarized into a BrainProteinAge‐score for all participants. A harmonized, age‐sensitive cognitive composite was created and the association between BrainProteinAge and cognitive decline assessed using structural equation modeling. Further, the predictive utility of ProteinBrainAge for incident all‐cause‐dementia was examined using time‐dependent ROC analysis in MCI patients from the test cohort.Results118 CSF‐proteins were selected and used to calculate BrainProteinAge. In the training (Est[SE] = 0.06[0.02],p&lt;0.001) and test (Est[SE] = 0.04[0.02],p = 0.013) cohorts, BrainProteinAge predicted cognitive decline beyond demographic characteristics (chronological age, sex, education) and A/T/N profiles. ProteinBrainAge captured 40% of the processes linking chronological age and cognitive decline, and improved prediction to all‐cause dementia beyond demographic characteristics and A/T/N profiles in MCI‐patients (1‐year AUC: 0.78 versus 0.82, p(difference) = 0.006). Specifically, within the AD‐continuum, BrainProteinAge (HR[CI] = 1.056[1.004‐1.110]), compared to chronological age (HR[CI] = 1.022[0.990‐1.054]), has greater predictive utility. Whilst an 8.6‐year ProteinBrainAge difference corresponded to the same risk increase observed between A+/T+ and A+/T‐ MCI patients, a difference of 24.6‐years was required for chronological age. All results remained substantively unchanged when using an optimized BrainProteinAge based on 10 proteins most strongly linked to cognitive decline.ConclusionsA new measure of biological age improves individualized prediction of cognitive decline and conversion to dementia in memory clinic patients. This provides an opportunity for precision medicine and a better understanding of age‐related biological processes contributing to dementia.

Mitragynine inhibits hippocampus neuroplasticity and its molecular mechanism

BackgroundMitragynine (MIT), the primary indole alkaloid of kratom (Mitragyna speciosa), has been associated with addictive and cognitive decline potentials. In acute studies, MIT decreases spatial memory and inhibits hippocampal synaptic transmission in long-term potentiation (LTP). This study investigated the impacts of 14-day MIT treatment on hippocampus synaptic transmission and its possible underlying mechanisms.MethodsUnder urethane anesthesia, field excitatory post-synaptic potentials (fEPSP) of the hippocampal CA1 region were recorded in the Sprague Dawley (SD) rats that received MIT (1, 5, and 10 mg/kg), morphine (MOR) 5 mg/kg, or vehicle (ip). The effects of the treatments on basal synaptic transmission, paired-pulse facilitation (PPF), and LTP were assessed in the CA1 region. Analysis of the brain's protein expression linked to neuroplasticity was then performed using a western blot.ResultsThe baseline synaptic transmission's amplitude was drastically decreased by MIT at 5 and 10 mg/kg doses, although the PPF ratio before TBS remained unchanged, the PPF ratio after TBS was significantly reduced by MIT (10 mg/kg). Strong and persistent inhibition of LTP was generated in the CA1 region by MIT (5 and 10 mg/kg) doses; this effect was not seen in MIT (1 mg/kg) treated rats. In contrast to MIT (1 mg/kg), MIT (5 and 10 mg/kg) significantly raised the extracellular glutamate levels. After exposure to MIT, GluR-1 receptor expression remained unaltered. However, NMDAε2 receptor expression was markedly downregulated. The expression of pCaMKII, pERK, pCREB, BDNF, synaptophysin, PSD-95, Delta fosB, and CDK-5 was significantly downregulated in response to MIT (5 and 10 mg/kg) exposure, while MOR (5 mg/kg) significantly raised synaptophysin and Delta fosB expression.ConclusionFindings from this work reveal that a smaller dose of MIT (1 mg/kg) poses no risk to hippocampal synaptic transmission. Alteration in neuroplasticity-associated proteins may be a molecular mechanism for MIT (5 and 10 mg/kg)-induced LTP disruption and cognitive impairments. Data from this work posit that MIT acted differently from MOR on neuroplasticity and its underlying mechanisms.Graphical abstract