Mouse Brain Tissue Research Articles

Therapeutic effect of dihydroartemisinin on Alzheimer’s disease model mice with senile macular degeneration

ObjectivesThis study focuses on the preventive and therapeutic effects of Dihydroartemisinin (DHA) on Alzheimer’s disease (AD) model mice and the effects of DHA and donepezil on amyloid β-protein deposition and autophagy in nerve cells.MethodsSix autophagy related targets were selected for molecular docking with DHA to predict the affinity between DHA and the target. The AD mouse model was established and treated with donepezil and DHA, respectively. Morris water maze was used to detect the spatial learning and memory ability of AD mice. Hematoxylin eosin (he) staining was used to observe the structural changes of cerebral cortical neurons and retina, and transmission electron microscope was used to observe the structural changes of mitochondria and synapses. Immunohistochemistry (IHC) and immunofluorescence staining were used to detect the deposition of amyloid beta protein. Western blot was used to detect the expression of apoptosis and autophagy related proteins in the brain tissue of mice in each group.ResultsThe results of molecular docking showed that the selected active compounds had good binding activity with the target. The binding energy between DHA and Aβ, Bcl-2, ATG5, LC3, Caspase3, LAMP1 is −5.7, −7.0, −5.8, −7.2, −6.9 kcal/mol. The water maze test showed that compared with the wild type (WT) group, the spatial memory ability of AD model group mice (5× FAD) was significantly decreased, and the search time (27.62 ± 6.51 s vs. 282.80 ± 17.15 s) and average path (106.30 ± 29.65 cm vs. 993.20 ± 135.80 cm) were significantly prolonged. The application of donepezil and DHA significantly shortened the exploration time and average path (donepezil: 116.10 ± 10.58 s, 529.40 ± 106.00 cm; DHA: 99.71 ± 14.22 s, 373.30 ± 60.97 cm). The path to find the platform in DHA treatment group was shorter than donepezil treatment group (P < 0.05). HE staining showed that the arrangement of nerve cells in 5× FAD mice was disordered, and IHC showed that amyloid β-protein deposition was obvious. DHA and donepezil could improve the damage of cerebral cortex structure and reduce the deposition of extracellular amyloid β-protein in AD mice. Transmission electron microscopy showed that DHA and donepezil could reduce mitochondrial vacuolation and synaptic edema. The above results showed that DHA treatment effect was better than donepezil. Compared with the conventional feeding group, autophagy and apoptosis related proteins B-cell lymphoma-2 (BCL2) and anti-thymocyte globulin (ATG) were significantly down regulated in the 5× FAD group, and the expressions of BCL2 and ATG were increased after treatment with DHA and donepezil.ConclusionsDHA combined with BCL2 and ATG protein, through promoting autophagy protein, can reduce the damage of cerebral cortex structure in AD mice, reduce the deposition of extracellular β-amyloid protein, and then improve the memory ability of AD model mice. DHA treatment is superior to donepezil monotherapy.

In-Depth Investigation on Potential Mechanism of Forest-Grown Ginseng Alleviating Alzheimer’s Disease via UHPLC-MS-Based Metabolomics

Background: Alzheimer’s disease is a central nervous system degenerative disease closely related to age with a complex pathogenesis. As a natural medicinal plant, forest-grown ginseng (GSF) contains abundant ginsenosides and offers significant neuroprotective effects. Methods: In this study, we comprehensively investigated the effect of GSF on the cell viability of PC12 cells in an AD model alongside metabolic changes in the serum and brains of mice, combined with an efficacy evaluation of PC12 cells in vitro and UHPLC-MS-based metabolomics in vivo. The goal of this study is to clarify the potential mechanism of GSF in treating AD. Results: The PC12 cell results showed that GSF can promote the proliferation of PC12 cells, reduce the content of IL-8, increase the activity of SOD, and alleviate the inflammation and oxidative stress induced by Aβ25~35. The immunohistochemical results for the mouse brain tissue also showed that GSF could reduce the inflammatory response of mouse brain tissue by reducing the overexpression of IBa1. AD was alleviated by reducing Aβ protein deposition in the mouse brain tissue. An untargeted metabolomics analysis was performed using UHPLC-Q-Exactive MS and principal component analysis (PCA) to identify the differentially expressed metabolites in the serum and brain tissue of AD mice after treatment. Twenty and seventeen different metabolites were identified in the serum and brain tissue, respectively. The pathway enrichment analysis of differential metabolites showed that GSF could treat AD by up-regulating succinic acid semialdehyde, carbamoyl phosphate, Sphingosine 1-phosphate, L-cystathionine, 2-ketobutyric acid, Vanillylmandelic acid, and D-Ribose to regulate sphingomyelin metabolism, the synthesis and metabolism of neurotransmitters and precursors, and energy metabolism. Conclusions: GSF can reduce neuroinflammation and alleviate Alzheimer’s disease by regulating the metabolic disorders of amino acids, sphingolipids, unsaturated fatty acids, and arachidonic acid in mice serum and brain tissue metabolites. These results suggest a link between metabolite imbalance and AD, and reveal the basis for the mechanism of ginsenosides in AD treatment.

HO-1 represses NF-κB signaling pathway to mediate microglia polarization and phagocytosis in intracerebral hemorrhage.

Microglia polarization plays a crucial role in inflammatory injury of brain following intracerebral hemorrhage (ICH). Heme oxygenase-1 (HO-1) has demonstrated protective properties against inflammation and promote hematoma clearance after ICH. The objective of this study was to explore impacts of HO-1 on microglia polarization and phagocytosis after ICH, along with the underlying mechanism. ICH model was constructed in C57BL/6 mice. Neurological deficit of ICH mice was evaluated. HE detected pathological changes of mouse brain tissue. Immunofluorescence staining tested co-localization between HO-1 or NF-κB p65 and IBA1. The expressions of gene and proteins were detected by RT-qPCR and Western blot, respectively. Flow cytometry determined microglial polarization phenotype and neuron apoptosis. Cell viability of neuron was assessed by CCK-8. Red blood cells labeled by PKH-26 and co-cultured with microglia for examining microglial erythrophagocytosis. Both HO-1 and NF-κB p65 phosphorylation were elevated in brain tissues of ICH mice. ZnPP, a HO-1 inhibitor, could exacerbate microglial M1 polarization and nerve injury, as well as repress microglial erythrophagocytosis in vitro and hematoma clearance in vivo. On the contrary, Tat-NBD, a NF-κB inhibitor, greatly suppressed microglial M1 polarization, and induced M2 polarization and microglial erythrophagocytosis, thus improving nerve injury and hematoma clearance after ICH. Notably, it was observed that NF-κB p65 could be activated by ZnPP treatment, and the regulatory roles of ZnPP on microglial polarization and erythrophagocytosis after ICH in vivo and in vitro were all diminished by Tat-NBD. Therefore, our data demonstrated that HO-1 alleviated nerve injury and induced M2 polarization and phagocytosis of microglia after ICH via inhibiting NF-κB signaling pathway, which could provide deepen the pathological understanding of ICH and provide potential intervention targets and drug candidate for ICH.

P-1836. SARS-CoV-2 induces blood-brain barrier and choroid plexus barrier impairments and vascular inflammation in mice

Abstract Background The coronavirus disease of 2019 (COVID-19) pandemic has led to more than 700 million confirmed cases and nearly 7 million deaths. Although Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) virus mainly infects the respiratory system, neurological complications are widely reported in both acute infection and long-COVID cases. Despite the success of vaccines and antiviral treatments, neuroinvasiveness of SARS-CoV-2 remains an important question, which is also centered on the mystery whether the virus is capable of breaching the barriers into the central nervous system.Figure 1:SARS-CoV-2 infection in the K18-hACE2 model.(A) Experimental diagram: Eight weeks-old K18-hACE2 mice were intranasally infected with SARS-CoV-2, monitored daily for symptoms, or euthanized at 5 days post-infection (DPI) to collect tissue. (B) Daily weight measurements in K18-hACE2 mice intranasally infected with SARS-CoV-2 isolate USA-WA1/2020. (C)The survival curve showing the probability of survival over 7 days after SARS-CoV-2 infection. (D) Representative mouse brain hemisphere image showing the presence of SARS-CoV-2 nucleocapsid protein by immunohistochemical staining. Boxed regions are shown on the right. Bar: 500 µm. Methods Here using the K18-hACE2 model, we investigated the impact of acute SARS-CoV-2 infection on major parts of neurovascular systems, and found increased incidence of microhemorrhage and significant disruption of both BBB and BCSFB in K18-hACE2 mice after SARS-CoV-2 infection. Cerebral microvascular injury was accompanied by substantial pericyte damage, tight junction loss, astrogliosis, and neuroinflammation in the brain parenchyma. In addition, endothelial activation and vascular inflammation occurred at both BBB and BCSFB, as shown by upregulation of VCAM-1 and COX2 markers.Figure 2:Vascular damage and BBB breakdown in SARS-CoV-2 infected K18-hACE2 model.(A) Representative images showing IgG immunohistochemical staining in K18-hACE2 mouse brain tissues with or without SARS-CoV-2 infection. Boxed regions are shown at the bottom. (B) Representative images showing immunofluorescent staining with IgG, showing the microhemorrhage site in the cortex of K18-hACE2 mice with SARS-CoV-2 infection. Bar: 50 µm. (C) Quantification of the number of microhemorrhages per field of view in the cortex, thalamus, and hippocampus (Hipp). n= 3; ***p&amp;lt; 0.001; two-tailed Student’s t-test. (D) Quantification of the diameters of the microhemorrhages in the cortex, thalamus, and hippocampus. (E) Representative images showing immunofluorescent staining for IgG at the capillary level in the cortex. K18-hACE2 mice with SARS-CoV-2 infection exhibited a significant accumulation of IgG surrounding the microvessels. Bar: 50 µm. (F) Quantification of the number of leaky small blood vessels per field of view in cortex, thalamus, and hippocampus. n= 3; ***p&amp;lt; 0.001; two-tailed Student’s t-test. (G) Quantification of the percentage of leakage vascular area to the total area of blood vessels in cortex, thalamus, and hippocampus. n= 3. Results By studying the K18-hACE2 infection model, we observed clear evidence of microvascular damage and breakdown of the blood-brain barrier (BBB). Mechanistically, SARS-CoV-2 infection caused pericyte damage, tight junction loss, endothelial activation and vascular inflammation, which together drive microvascular injury and BBB impairment. BBB tight junction loss in SARS-CoV-2 infected K18-hACE2 model. (A) The K18-hACE2 mice were used to test the BBB function after SARS-CoV-2 infection. (B) Representative images showing immunofluorescent staining for IgG, ZO-1, and Lectin in the cortex of K18-hACE2 mice. Bar: 50 µm. (C-D) Representative images showing immunofluorescent staining for ZO-1 and Lectin in the thalamus(C) and hippocampus(D) areas of K18-hACE2 mice. Bar: 50 µm. (E) Length of ZO-1-positive profiles in the cortex, thalamus, and Hipp. n= 3; ***p&amp;lt; 0.001; two-tailed Student’s t-test. (F-H) Representative images showing immunofluorescent staining for Claudin5 and Lectin in the cortex(F), thalamus (G), and hippocampus (H) areas of K18-hACE2 mice. Bar: 50 µm. (I) Claudin5 length in the cortex, thalamus and, Hipp. n= 3; ***p&amp;lt; 0.001; two-tailed Student’s t-test. Conclusion The impact of such changes, together with astrogliosis and neuroinflammation, may drive or at least contribute to the neurological complications seen in COVID-19 patients. As SARS-CoV-2 will likely remain a major health issue for years to come, our findings provide a needed understanding of its impact on the major CNS barriers and brain homeostasis at both molecular and cellular levels. Vascular inflammation in SARS-CoV-2 infected K18-hACE2 model (A-C) Representative images showing immunofluorescent staining for VCAM1 and Lectin in the cortex, thalamus, and hippocampus of K18-hACE2 mice. Bar: 50 µm. (D) Quantification of VCAM1 and lectin signal overlap in the cortex, thalamus, and hippocampus. n= 3; ***p&amp;lt; 0.001; two-tailed Student’s t-test. (E-G) Representative images showing immunostaining of COX2 and Lectin in the cortex, thalamus, and hippocampus of K18-hACE2 mice. Bar: 50 µm. (H) Quantification of COX2 and Lectin signal overlap in the cortex, thalamus, and hippocampus areas of K18-hACE2 mice. n= 3; ***p&amp;lt; 0.001; ****p&amp;lt; 0.0001; two-tailed Student’s t-test. (I) Quantification of COX2 relative fluorescence intensity in the cortex, thalamus, and hippocampus of K18-hACE2 mice. n= 3; ***p&amp;lt; 0.001; ****p&amp;lt; 0.0001; two-tailed Student’s t-test. Disclosures All Authors: No reported disclosures

Microenvironment actuated CAR T cells improve solid tumor efficacy without toxicity.

A major limiting factor in the success of chimeric antigen receptor (CAR) T cell therapy for the treatment of solid tumors is targeting tumor antigens also found on normal tissues. CAR T cells against GD2 induced rapid, fatal neurotoxicity because of CAR recognition of GD2+ normal mouse brain tissue. To improve the selectivity of the CAR T cell, we engineered a synthetic Notch receptor that selectively expresses the CAR upon binding to P-selectin, a cell adhesion protein overexpressed in tumor neovasculature. These tumor microenvironment actuated T (MEAT) cells ameliorated T cell infiltration in the brain, preventing fatal neurotoxicity while maintaining antitumor efficacy. We found that conditional CAR expression improved the persistence of tumor-infiltrating lymphocytes because of enhanced metabolic fitness of MEAT cells and the infusion of a less differentiated product. This approach increases the repertoire of targetable solid tumor antigens by restricting CAR expression and subsequent killing to cancer cells only and provides a proof-of-concept model for other targets.

H4K12 lactylation-regulated NLRP3 is involved in cigarette smoke-accelerated Alzheimer-like pathology through mTOR-regulated autophagy and activation of microglia.

Cigarette smoke (CS), an indoor environmental pollution, is an environmental risk factor for diverse neurological disorders. However, the neurotoxicological effects and mechanisms of CS on Alzheimer's disease (AD) progression remain unclear. We found that CS accelerated the progression of AD, including increasing β-amyloid (Aβ) plaque deposition and exacerbating cognitive decline. Mechanistically, CS exposure increased the levels of NOD-like receptor protein 3 (NLRP3), which impaired autophagic flux in microglia by activating the mammalian target of rapamycin (mTOR) signal. Metabolomics analysis revealed an upregulation of lactate levels and an increase in global protein lysine lactylation in the brain tissue of CS-exposed AD-transgenic mice. Immunoprecipitation-Mass Spectrometry and chromatin immunoprecipitation assays demonstrated that CS elevates H4K12 lactylation (H4K12la) levels, which accumulate at the promoter region of NLRP3, leading to the activation of its transcription. Via inhibiting lactate or NLRP3 activation, oxamate and MCC950 alleviates these CS-induced effects. Therefore, our data suggest that the CS-induced increase in lactate levels triggers NLRP3 transcriptional activation through H4K12la, which subsequently leads to mTOR-mediated autophagy dysfunction in microglia, promoting microglial activation and resulting in Aβ plaque accumulation in AD-transgenic mice. This provides a new mechanism and potential therapeutic target for AD associated with environmental factors.

Flyscan terahertz multi-plane lensless imaging with suppressed coherent noise.

Coherent lensless imaging usually suffers from coherent noise and twin-image artifacts. In the terahertz (THz) range, where wavelengths are 2 to 4 orders of magnitude longer than those in the visible spectrum, the coherent noise manifests primarily as parasitic interference fringes and edge diffraction, rather than speckle noise. In this work, to suppress the Fabry-Pérot (F-P) interference fringes, we propose a novel method, which involves the averaging over multiple diffraction patterns that are acquired at equal intervals within a sample's half-wavelength axial shift. To address edge diffraction, as well as non-uniform illumination, a normalization operation is applied. As the twin-image disturbances when dealing with a single diffraction pattern, multi-plane configuration is employed. With all these strategies combined, we propose a flyscan THz multi-plane lensless imaging technique that enables subwavelength resolution, and high-quality, full-field, and rapid complex-valued THz imaging. Furthermore, we refine two algorithms for image reconstruction: one based on the regular multi-plane alternating projection and the other based on an optimization model with total variation regularization. We experimentally verify the proposed methods, achieving a lateral resolution of 88 µm (0.74λ) at 2.52 THz, and showcase its potential for biomedical applications by imaging a section of mouse brain tissue.

CaMKIIα hub ligands are unable to reverse known phenotypes in Angelman syndrome mice.

Angelman Syndrome (AS) is a neurodevelopmental disorder caused by the loss of function of ubiquitin-protein ligase E3A (UBE3A), resulting in marked changes in synaptic plasticity. In AS mice, a dysregulation of Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIα) was previously described. This has been convincingly validated through genetic rescue of prominent phenotypes in mouse cross-breeding experiments. Selective ligands that specifically stabilize the CaMKIIα central association (hub) domain and affect different conformational states in vitro are now available. Two of these ligands, 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) and (E)-2-(5-hydroxy-2-phenyl-5,7,8,9-tetrahydro-6H-benzo[7]annulen-6-ylidene)acetic acid (Ph-HTBA), confer neuroprotection after ischemic stroke in mice where CaMKIIα is known to be dysregulated. Here, we sought to investigate whether pharmacological modulation with these prototypical CaMKIIα hub ligands presents a viable approach to alleviate AS symptoms. We performed an in vivo functional evaluation of AS mice treated for a total of 14 days with either HOCPCA or Ph-HTBA (7days pre-treatment and 7days of behavioural assessment). Both compounds were well-tolerated but unable to revert robust phenotypes of motor performance, anxiety, repetitive behaviour or seizures in AS mice. Biochemical experiments subsequently assessed CaMKIIα autophosphorylation in AS mouse brain tissue. Taken together our results indicate that pharmacological modulation of CaMKIIα via the selective hub ligands used here is not a viable treatment strategy in AS.

Mouse-derived Synaptosomes Trypsin Cleavage Assay to Characterize Synaptic Protein Sub-localization.

Neurons communicate through neurotransmission at highly specialized junctions called synapses. Each neuron forms numerous synaptic connections, consisting of presynaptic and postsynaptic terminals. Upon the arrival of an action potential, neurotransmitters are released from the presynaptic site and diffuse across the synaptic cleft to bind specialized receptors at the postsynaptic terminal. This process is tightly regulated by several proteins at both presynaptic and postsynaptic sites. The localization, abundance, and function of these proteins are essential for productive neurotransmission and are often affected in neurological and neurodegenerative disorders. Here, we outline a method for purifying mouse synaptosomes and using limited tryptic digestion to assess the subcellular localization of synaptic proteins. During synaptosomes purification, presynaptic terminals reseal and are protected from proteolysis, while postsynaptic proteins remain susceptible to tryptic cleavage. These changes can easily be evaluated by western blot analysis. This approach offers a straightforward and reliable method to evaluate the subcellular localization of synaptic proteins based on their proteolytic sensitivity, providing valuable insights into synaptic physiology and pathology. Key features • Builds upon the method developed by Boyken et al. [1] and introduces the use of isolated mouse synaptosomes to assess synaptic protein sub-localization. • Limited tryptic digestion differentiates between presynaptic and postsynaptic proteins based on proteolytic sensitivity. • Requires standard biochemical reagents and western blotting equipment and can be completed in two/three days, including synaptosome purification and western blot analysis. Graphical overview Overview of the synaptosomes trypsin cleavage assay. This protocol describes the isolation of synaptosomes from mouse brain tissue, followed by limited trypsin digestion to assess the compartmental localization of synaptic proteins. Synaptosomes, which are isolated via differential centrifugation, consist of resealed presynaptic terminals that are protected from proteolysis, while the exposed postsynaptic compartments are accessible to trypsin and undergo proteolytic cleavage. Following digestion, samples are analyzed using SDS-PAGE and western blotting. Proteins of interest are probed using specific antibodies to determine whether they are presynaptic or postsynaptic. Presynaptic proteins (e.g., synaptophysin, SNAP-25) remain intact, while postsynaptic proteins (e.g., GluA1, GluN2A) are cleaved.

CircLOC375190 promotes autophagy through modulation of the mTORC1/TFEB axis in acute ischemic stroke-induced neurological injury.

The authors explored differentially expressed circRNAs in Acute Ischemic Stroke (AIS) and revealed the role and potential downstream molecular mechanisms of circLOC375190. circLOC375190 expression was modulated by lentiviral injection in the brain of transient Middle Cerebral Artery Occlusion (tMCAO) mice. Neurological dysfunction was assessed, as well as infarction size, histopathological changes, and neuronal apoptosis in tMCAO mice. An in vitro Oxygen-Glucose Deprivation/Reoxygenation (OGD/R) PC-12 cell model was established. PC-12 cells were transfected and evaluated for viability, cytotoxicity, apoptosis, and autophagy. Inflammatory factors in mouse brain tissues and PC-12 cells were examined via enzyme-linked immunosorbent assay, and related genes were measured via real-time reverse transcriptase-polymerase chain reaction and Western blot. The ring structure of circLOC375190 was assessed by actinomycin-D and RNase-R assays. circRNA targeting to downstream factors was assessed by Fluorescence in situ hybridization assay, dual luciferase reporter assay, and RNA immunoprecipitation assay. circLOC375190 level was increased in tMCAO mice. Knocking down circLOC375190 reduced infarct size, attenuated cerebral pathological injury and neuronal apoptosis, and inhibited inflammatory damage and autophagy in tMCAO mice. circLOC375190 knockdown enhanced neuronal viability and reduced cytotoxicity, apoptosis, and autophagy in OGD/R-treated PC12 cells. Mechanistically, circLOC375190 acted as a sponge for miR-93-5p to upregulate MAP kinase interacting serine/threonine kinase 2 expression and activate the mechanistic target of rapamycin complex 1/transcription factor EB pathway. circLOC375190 exacerbates tMCAO-mediated neurological injury by regulating neuronal autophagy.

Chalcogen dihydrobenzofuran compounds as potential neuroprotective agents: an in vitro and in silico biological investigation.

Oxidative stress arises from an imbalance between reactive species (RS) production and the antioxidant defense, increasing the brain susceptibility to neurodegenerative and psychiatric diseases. Besides, changes in the expression or activity of neurotransmitter metabolism enzymes, such as monoamine oxidases (MAO), are also associated with mental disorders, including depression. Considering this, antioxidant and MAO-A activity inhibitory potential of six 2,3-chalcogenodihydrobenzofurans (2,3-DHBF) was investigated through in vitro and in silico tests. Compounds 1 to 5 incorporate sulfur (S) as chalcogen, whereas compound 6 integrates tellurium (Te). A screening (compounds 1-6) of cerebral MAO-A activity showed inhibitory activity for the compounds 2, 4, 5, and 6. Among sulfur compounds, compound 2 demonstrated superior scores in docking studies, yielding a value of - 9.9 kcal/mol. Selected for concentration-response curves, compounds 2 (with S) and 6 (with Te) inhibited MAO-A at concentrations equal to or higher than 25 μM. In a redox screening test, only compound 6 showed antioxidant effects. Concentration-response curves indicated that compound 6 reduced lipid peroxidation and protein carbonylation levels in mouse brain tissue (≥ 0.5 μM), as well as reduced RS levels (≥ 1 μM). Furthermore, the compound 6 (≥ 5 μM) was effective in reducing the ferric ion (FRAP). In radical scavenging tests such as 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), compound 6 showed significant results in concentrations from 50 μM and mimicked the enzyme glutathione S-transferase (GST) at 100 μM. In summary, this study demonstrated the cerebral antioxidant and/or MAO-A inhibition properties of 2,3-DHBF, presenting potential as neuroprotective candidates.

S100A4 Exerts Neuroprotective Effects by Attenuating Blood-Brain Barrier Disruption and Oxidative Stress via the PI3K/Akt/Nrf2 Axis in Ischemic Stroke

Ischemic stroke is a major cause of disability and mortality worldwide, with oxidative stress and blood-brain barrier (BBB) injury playing crucial roles in its pathogenesis. Our RNA sequencing results revealed that S100 calcium-binding protein A4 (S100A4) is highly expressed in the middle cerebral artery occlusion (MCAO) mouse model. We analyzed S100A4 expression in ischemic stroke patients and in mice subjected to the MCAO model. Moreover, using adeno-associated virus (AAV)-mediated knockdown of S100A4 in mice, we evaluated its effects on neurological deficits, BBB integrity, and oxidative stress in MCAO mice. Bioinformatic analyses explored the potential downstream pathways of S100A4.S100A4 expression was significantly elevated in the serum of ischemic stroke patients and brain tissues of MCAO mice. AAV-mediated knockdown of S100A4 exacerbated neurological deficits, BBB disruption, and oxidative stress in MCAO mice. The upregulation of S100A4 mitigated these outcomes, which were facilitated through the stimulation of the PI3K/Akt/Nrf2 signaling cascade.Our results illustrate that S100A4 plays a protective role in preventing neuronal damage during ischemic stroke by reducing oxidative stress and preserving BBB integrity through the PI3K/Akt/Nrf2 pathway. This highlights its promise as a potential therapeutic approach for ischemic stroke.

Electroacupuncture Pretreatment Reduces Ischemic Brain Injury by Inhibiting the Lactate Production and Its Derived Protein Lactylation Formation.

Given that electroacupuncture (EA) pretreatment inhibits lactate production and lactate-derived lysine lactation (Kla) aggravates ischemic brain injury, we aimed to investigate whether the formation of Kla protein is involved in EA pretreatment to alleviate ischemic brain injury. EA was performed on the Baihui acupoint (GV20) of male C57BL/6J mice before receiving the permanent middle cerebral artery occlusion (pMCAO) surgery. Western blot and immunofluorescent staining were used to observe neuronal survival, astrocyte activation, and protein Kla levels, and the lactate levels in ischemic brains were assayed with a commercial kit. TTCstaining and neurological function scores are performed to evaluate the brain damage in mice. We found that the increased lactate content and protein Kla levels were significantly decreased in ischemic brain tissue of mice after receiving EA pretreatment, and accompanied by the reduction of astrocyte activation and neuronal injury and death. Meantime, we found that EA pretreatment was effective in reversing the worsening of ischemic brain injury caused by lactate supplementation. However, EA pretreatment did not further reduce the lactate content and protein Kla levels and ameliorate brain injury in ischemic stroke mice after inhibition of glycolysis. Our study reveals that EA pretreatment reduced ischemic brain damage by inhibiting lactate production and its derived protein Kla formation in mice with ischemic stroke.

Radiosynthesis and evaluation of novel 18F labeled PET ligands for imaging monoacylglycerol lipase.

Monoacylglycerol lipase (MAGL) is a 33kDa cytosolic serine hydrolase that is widely distributed in the central nervous system and peripheral tissues. MAGL hydrolyzes monoacylglycerols into fatty acids and glycerol, playing a crucial role in endocannabinoid degradation. Inhibition of MAGL in the brain elevates levels of 2-arachidonoylglycerol and leads to decreased pro-inflammatory prostaglandin and thromboxane production. As such, MAGL is considered a potential target for treating neuropsychiatric disorders, metabolic syndromes, and cancer. Based on a novel spirocyclic system, we synthesized two fluorinated carbamate scaffolds as reversible MAGL inhibitors (epimers: (R)-6, IC50=18.6nM and (S)-6, IC50=1.6nM). In vitro autoradiography studies of [18F](R)-6 (codenamed [18F]MAGL-2304) and [18F](S)-6 (codenamed [18F]MAGL-2305) demonstrated heterogeneous distribution and specific binding affinity to MAGL-rich brain regions. Autoradiography with MAGL knockout mouse brain tissues confirmed the binding specificity of [18F](S)-6. Dynamic PET imaging studies revealed that [18F](S)-6 exhibited limited brain uptake and homogenous distribution in rat brains. In vivo P-gp inhibition enhanced [18F](S)-6 uptake in the brain, suggesting that [18F](S)-6 constitutes a P-gp efflux substrate. This research could provide new directions in the design of MAGL PET ligands that are based on spirocyclic scaffolds.

Comparing the effects of swimming and running wheel on clinical symptoms and myelin basic protein in mice with experimental autoimmune encephalomyelitis

Background and aims: The present investigation examines the impact of aerobic exercises, especially swimming and running wheel exercises, on the clinical manifestations and myelin basic protein (MBP) levels in mice with experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS). MBP is critical for the formation of myelin sheaths within the nervous system, and its deterioration is linked to the pathogenesis of MS. Methods: The sample of the study consisted of 96 female C57BL/6 mice. After the EAE induction, the mice were divided into 8 groups. The animals performed two exercise protocols. Then, the brain tissue was isolated, and levels of the mentioned variables were measured via the ELISA method using specific kits. The data were analyzed statistically using one-way ANOVA. Results: Results showed that lesion scores in both exercise protocols were lower in EAE mice than those in the control groups (P=0.001); in other words, swimming and receiving interferon-beta-1 reduced MBP degradation, but the decrease was not significant (P=0.09). Additionally, running wheels reduced MBP degradation, which was statistically significant (P=0.001). Conclusion: Results suggested that using a voluntary running wheel might be a more effective exercise program than swimming in preventing MBP degradation in the brain tissues of mice with EAE.

Safe and Orally Bioavailable Inhibitor of Serine Palmitoyltransferase Improves Age-Related Sarcopenia.

The accumulation of ceramides and related metabolites has emerged as a pivotal mechanism contributing to the onset of age-related diseases. However, small molecule inhibitors targeting the ceramide de novo synthesis pathway for clinical use are currently unavailable. We synthesized a safe and orally bioavailable inhibitor, termed ALT-007, targeting the rate-limiting enzyme of ceramide de novo synthesis, serine palmitoyltransferase (SPT). In a mouse model of age-related sarcopenia, ALT-007, administered through the diet, effectively restored muscle mass and function compromised by aging. Mechanistic studies revealed that ALT-007 enhances protein homeostasis in Caenorhabditis elegans and mouse models of aging and age-related diseases, such as sarcopenia and inclusion body myositis (IBM); this effect is mediated by a specific reduction in very-long chain 1-deoxy-sphingolipid species, which accumulate in both muscle and brain tissues of aged mice and in muscle cells from IBM patients. These findings unveil a promising therapeutic avenue for developing safe ceramide inhibitors to address age-related neuromuscular diseases.

Arginine depletion-induced autophagy and metabolic dysregulation are involved in the disease severity of hand, foot, and mouth disease

ABSTRACT Amino acid metabolism provides significant insight into the development and prevention of many viral diseases. Therefore, the present study aimed to compare the amino acid profiles of hand, foot, and mouth disease (HFMD) patients with those of healthy individuals and to further reveal the molecular mechanisms of HFMD severity. Using UPLC-MS/MS, we determined the plasma amino acid expression profiles of pediatric patients with HFMD (mild, n = 42; severe, n = 43) and healthy controls (n = 25). Brain tissues from CVA6-infected mice were examined using untargeted metabolomics. Several amino acids were significantly different between the three groups. Pathway analysis revealed that arginine, proline, and tryptophan metabolism are implicated in the pathogenesis of HFMD. A similar arginine depletion was observed in the brain tissues of CVA6-infected mice. Importantly, L-arginine supplementation improved the survival rate of CVA6-infected mice, inhibited virus multiplication, and reduced pathological autophagy associated with mTOR-autophagy pathway in the brain. Collectively, arginine, as the hub amino acid metabolite of the mammalian target of rapamycin (mTOR) signaling pathway affecting autophagy, plays an important role in the pathogenesis of severe HFMD. L-arginine supplementation may serve as a potential therapeutic option for critical patients with HFMD.

High-throughput gene expression analysis with TempO-LINC sensitively resolves complex brain, lung and kidney heterogeneity at single-cell resolution

We report the development and performance of a novel genomics platform, TempO-LINC, for conducting high-throughput transcriptomic analysis on single cells and nuclei. TempO-LINC works by adding cell-identifying molecular barcodes onto highly selective and high-sensitivity gene expression probes within fixed cells, without having to first generate cDNA. Using an instrument-free combinatorial indexing approach, all probes within the same fixed cell receive an identical barcode, enabling the reconstruction of single-cell gene expression profiles across as few as several hundred cells and up to 100,000 + cells per sample. The TempO-LINC approach is easily scalable based on the number of barcodes and rounds of barcoding performed; however, for the experiments reported in this study, the assay utilized over 5.3 million unique barcodes. TempO-LINC offers a robust protocol for fixing and banking cells and displays high-sensitivity gene detection from multiple diverse sample types. We show that TempO-LINC has a multiplet rate of less than 1.1% and a cell capture rate of ~ 50%. Although the assay can accurately profile the whole transcriptome (19,683 human, 21,400 mouse and 21,119 rat genes), it can be targeted to measure only actionable/informative genes and molecular pathways of interest – thereby reducing sequencing requirements. In this study, we applied TempO-LINC to profile the transcriptomes of more than 90,000 cells across multiple species and sample types, including nuclei from mouse lung, kidney and brain tissues. The data demonstrated the ability to identify and annotate more than 50 unique cell populations and positively correlate expression of cell type-specific molecular markers within them. TempO-LINC is a robust new single-cell technology that is ideal for large-scale applications/studies with high data quality.

Three-dimensional single-cell transcriptome imaging of thick tissues.

Multiplexed error-robust fluorescence in situ hybridization (MERFISH) allows genome-scale imaging of RNAs in individual cells in intact tissues. To date, MERFISH has been applied to image thin-tissue samples of ~10 µm thickness. Here, we present a thick-tissue three-dimensional (3D) MERFISH imaging method, which uses confocal microscopy for optical sectioning, deep learning for increasing imaging speed and quality, as well as sample preparation and imaging protocol optimized for thick samples. We demonstrated 3D MERFISH on mouse brain tissue sections of up to 200 µm thickness with high detection efficiency and accuracy. We anticipate that 3D thick-tissue MERFISH imaging will broaden the scope of questions that can be addressed by spatial genomics.

Deciphering the endogenous SUMO-1 landscape: a novel combinatorial peptide enrichment strategy for global profiling and disease association.

Small ubiquitin-like modifier (SUMO) plays a pivotal role in diverse cellular processes and is implicated in diseases such as cancer and neurodegenerative disorders. However, large-scale identification of endogenous SUMO-1 faces challenges due to limited enrichment methods and its lower abundance compared to SUMO-2/3. Here we propose a novel combinatorial peptide strategy, combined with anti-adhesive polymer development, to enrich endogenous SUMO-1 modified peptides, revealing a comprehensive SUMOylation landscape. Utilizing phage display, we successfully identified a linear 12-mer and a cystine-linked cyclic 7-mer peptide ligand, specifically designed to target the C-terminal regions of SUMO-1 remnants. Building upon their high affinities and satisfactory complementarity, we developed the first artificial SUMO-1 enrichment materials, ultimately establishing a combinatorial peptide strategy that facilitates a comprehensive analysis of the endogenous SUMO-1 modified proteome in both cellular and tissue contexts. We successfully mapped 1312 SUMOylation sites in HeLa cells and 1365 along with 991 endogenous SUMOylation proteins in Alzheimer's disease (AD) mouse brain tissues. Notably, our method uncovered a significant upregulation of SUMO-1 in AD mouse brain tissue, providing new insights into SUMOylation's role in disease. Overall, this work represents the most thorough exploration of SUMO-1 modified proteomics and offers robust tools for elucidating the roles of SUMO-1's biological significance.