Brains Of 5xFAD Mice Research Articles

CD8+ T cells exacerbate AD-like symptoms in mouse model of amyloidosis

Alzheimer’s disease (AD) is linked to toxic Aβ plaques in the brain and activation of innate responses. Recent findings however suggest that the disease may also depend on the adaptive immunity, as B cells exacerbate and CD8+ T cells limit AD-like pathology in mouse models of amyloidosis. Here, by artificially blocking or augmenting CD8+ T cells in the brain of 5xFAD mice, we provide evidence that AD-like pathology is promoted by pathogenic, proinflammatory cytokines and exhaustion markers expressing CXCR6+ CD39+CD73+/- CD8+ TRM-like cells. The CD8+ T cells appear to act by targeting disease associated microglia (DAM), as we find them in tight complexes with microglia around Aβ plaques in the brain of mice and humans with AD. We also report that these CD8+ T cells are induced by B cells in the periphery, further underscoring the pathogenic importance of the adaptive immunity in AD. We propose that CD8+ T cells and B cells should be considered as therapeutic targets for control of AD, as their ablation at the onset of AD is sufficient to decrease CD8+ T cells in the brain and block the amyloidosis-linked neurodegeneration.

Engineered exosomes with a photoinducible protein delivery system enable CRISPR-Cas-based epigenome editing in Alzheimer's disease.

Effective intracellular delivery of therapeutic proteins can potentially treat a wide array of diseases. However, efficient delivery of functional proteins across the cell membrane remains challenging. Exosomes are nanosized vesicles naturally secreted by various types of cells and may serve as promising nanocarriers for therapeutic biomolecules. Here, we engineered exosomes equipped with a photoinducible cargo protein release system, termed mMaple3-mediated protein loading into and release from exosome (MAPLEX), in which cargo proteins can be loaded into the exosomes by fusing them with photocleavable protein (mMaple3)-conjugated exosomal membrane markers and subsequently released from the exosomal membrane by inducing photocleavage with blue light illumination. Using this system, we first induced transcriptional regulation by delivering octamer-binding transcription factor 4 and SRY-box transcription factor 2 to fibroblasts in vitro. Second, we induced in vivo gene recombination in Cre reporter mice by delivering Cre recombinase. Last, we achieved targeted epigenome editing in the brains of 5xFAD and 3xTg-AD mice, two models of Alzheimer's disease. Administration of MAPLEXs loaded with β-site amyloid precursor protein cleaving enzyme 1 (Bace1)-targeting single guide RNA-incorporated dCas9 ribonucleoprotein complexes, coupled with the catalytic domain of DNA methyltransferase 3A, resulted in successful methylation of the targeted CpG sites within the Bace1 promoter. This approach led to a significant reduction in Bace1 expression, improved recognition memory impairment, and reduced amyloid pathology in 5xFAD and 3xTg-AD mice. These results suggest that MAPLEX is an efficient intracellular protein delivery system that can deliver diverse therapeutic proteins for multiple diseases.

Herpes simplex virus 1 accelerates the progression of Alzheimer’s disease by modulating microglial phagocytosis and activating NLRP3 pathway

Accumulating evidence implicates that herpes simplex virus type 1 (HSV-1) has been linked to the development and progression of Alzheimer’s disease (AD). HSV-1 infection induces β-amyloid (Aβ) deposition in vitro and in vivo, but the effect and precise mechanism remain elusive. Here, we show that HSV-1 infection of the brains of transgenic 5xFAD mice resulted in accelerated Aβ deposition, gliosis, and cognitive dysfunction. We demonstrate that HSV-1 infection induced the recruitment of microglia to the viral core to trigger microglial phagocytosis of HSV-GFP-positive neuronal cells. In addition, we reveal that the NLRP3 inflammasome pathway induced by HSV-1 infection played a crucial role in Aβ deposition and the progression of AD caused by HSV-1 infection. Blockade of the NLRP3 inflammasome signaling reduces Aβ deposition and alleviates cognitive decline in 5xFAD mice after HSV-1 infection. Our findings support the notion that HSV-1 infection is a key factor in the etiology of AD, demonstrating that NLRP3 inflammasome activation functions in the interface of HSV-1 infection and Aβ deposition in AD.

Targeting soluble amyloid-beta oligomers with a novel nanobody

The classical amyloid cascade hypothesis postulates that the aggregation of amyloid plaques and the accumulation of intracellular hyperphosphorylated Tau tangles, together, lead to profound neuronal death. However, emerging research has demonstrated that soluble amyloid-β oligomers (SAβOs) accumulate early, prior to amyloid plaque formation. SAβOs induce memory impairment and disrupt cognitive function independent of amyloid-β plaques, and even in the absence of plaque formation. This work describes the development and characterization of a novel anti-SAβO (E3) nanobody generated from an alpaca immunized with SAβO. In-vitro assays and in-vivo studies using 5XFAD mice indicate that the fluorescein (FAM)-labeled E3 nanobody recognizes both SAβOs and amyloid-β plaques. The E3 nanobody traverses across the blood–brain barrier and binds to amyloid species in the brain of 5XFAD mice. Imaging of mouse brains reveals that SAβO and amyloid-β plaques are not only different in size, shape, and morphology, but also have a distinct spatial distribution in the brain. SAβOs are associated with neurons, while amyloid plaques reside in the extracellular matrix. The results of this study demonstrate that the SAβO nanobody can serve as a diagnostic agent with potential theragnostic applications in Alzheimer’s disease.

Chemically Driven Clearance of Amyloid Aggregates by Polyfunctionalized Furo[2,3-b:4,5-b']dipyridine-Chalcone Hybrids to Ameliorate Memory in an Alzheimer Mouse Model.

The aberrant assembly of amyloid-β (Aβ) is implicated in Alzheimer's disease (AD). Recent clinical outcomes of Aβ-targeted immunotherapy reinforce the notion that clearing Aβ burden is a potential therapeutic approach for AD. Herein, to develop drug candidates for chemically driven clearance of Aβ aggregates, we synthesized 51 novel polyfunctionalized furo[2,3-b:4,5-b']dipyridine-chalcone hybrid compounds. After conducting two types of cell-free anti-Aβ functional assays, Aβ aggregation prevention and Aβ aggregate clearance, we selected YIAD-0336, (E)-8-((1H-pyrrol-2-yl)methylene)-10-(4-chlorophenyl)-2,4-dimethyl-7,8-dihydropyrido[3',2':4,5]furo[3,2-b]quinolin-9(6H)-one, for further in vivo investigations. As YIAD-0336 exhibited a low blood-brain barrier penetration profile, it was injected along with aggregated Aβ directly into the intracerebroventricular region of ICR mice and ameliorated spatial memory in Y-maze tests. Next, YIAD-0336 was orally administered to 5XFAD transgenic mice with intravenous injections of mannitol, and YIAD-0336 significantly removed Aβ plaques from the brains of 5XFAD mice. Collectively, YIAD-0336 dissociated toxic aggregates in the mouse brain and hence alleviated cognitive deterioration. Our findings indicate that chemically driven clearance of Aβ aggregates is a promising therapeutic approach for AD.

Caffeoylquinic Acid Mitigates Neuronal Loss and Cognitive Decline in 5XFAD Mice Without Reducing the Amyloid-β Plaque Burden.

Caffeoylquinic acid (CQA), which is abundant in coffee beans and Centella asiatica, reportedly improves cognitive function in Alzheimer's disease (AD) model mice, but its effects on neuroinflammation, neuronal loss, and the amyloid-β (Aβ) plaque burden have remained unclear. To assess the effects of a 16-week treatment with CQA on recognition memory, working memory, Aβ levels, neuronal loss, neuroinflammation, and gene expression in the brains of 5XFAD mice, a commonly used mouse model of familial AD. 5XFAD mice at 7 weeks of age were fed a 0.8% CQA-containing diet for 4 months and then underwent novel object recognition (NOR) and Y-maze tests. The Aβ levels and plaque burden were analyzed by enzyme-linked immunosorbent assay and immunofluorescent staining, respectively. Immunostaining of markers of mature neurons, synapses, and glial cells was analyzed. AmpliSeq transcriptome analysis and quantitative reverse-transcription-polymerase chain reaction were performed to assess the effect of CQA on gene expression levels in the cerebral cortex of the 5XFAD mice. CQA treatment for 4 months improved recognition memory and ameliorated the reduction of mature neurons and synaptic function-related gene mRNAs. The Aβ levels, plaque burden, and glial markers of neuroinflammation seemed unaffected. These findings suggest that CQA treatment mitigates neuronal loss and improves cognitive function without reducing Aβ levels or neuroinflammation. Thus, CQA is a potential therapeutic compound for AD, improving cognitive function via as-yet unknown mechanisms independent of reductions in Aβ or neuroinflammation.

Identification of Cinnamein, a Component of Balsam of Tolu/Peru, as a New Ligand of PPARα for Plaque Reduction and Memory Protection in a Mouse Model of Alzheimer's Disease.

Despite intense investigations, no effective treatment is yet available to reduce plaques and protect memory and learning in patients with Alzheimer's disease (AD), the most common neurodegenerative disorder. Therefore, it is important to identify a non-toxic, but effective, treatment option for AD. Cinnamein, a nontoxic compound, is naturally available in Balsam of Peru and Tolu Balsam. We examined whether cinnamein treatment could decrease plaques and improve cognitive functions in 5XFAD mouse model of AD. We employed in silico analysis, time-resolved fluorescence energy transfer assay, thermal shift assay, primary neuron isolation, western blot, immunostaining, immunohistochemistry, Barnes maze, T maze, and open field behavior. Oral administration of cinnamein led to significant reduction in amyloid-β plaque deposits in the brain and protection of spatial learning and memory in 5XFAD mice. Peroxisome proliferator-activated receptor alpha (PPARα), a nuclear hormone receptor, is involved in plaque lowering and increase in hippocampal plasticity. While investigating underlying mechanisms, we found that cinnamein served as a ligand of PPARα. Accordingly, oral cinnamein upregulated the level of PPARα, but not PPARβ, in the hippocampus, and remained unable to decrease plaques from the hippocampus and improve memory and learning in 5XFAD mice lacking PPARα. While A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) is one of the drivers of nonamyloidogenic pathway, transcription factor EB (TFEB) is considered as the master regulator of autophagy. Cinnamein treatment was found to upregulate both ADAM10 and TFEB in the brain of 5XFAD mice via PPARα. Our results suggest that this balsam component may have therapeutic importance in AD.

Liver-specific adiponectin gene therapy suppresses microglial NLRP3-inflammasome activation for treating Alzheimer’s disease

Adiponectin (APN) is an adipokine which predominantly expresses in adipocytes with neuroprotective and anti-inflammatory effects. We have recently indicated that circulatory trimeric APN can enter the brain by crossing the blood–brain barrier (BBB) and modulate microglia-mediated neuroinflammation. Here, we found that the microglial NLR family pyrin domain containing 3 (NLRP3)-inflammasome activation was exacerbated in APN−/−5xFAD mice in age-dependent manner. The focus of this study was to develop a new and tractable therapeutic approach for treating Alzheimer’s disease (AD)-related pathology in 5xFAD mice using peripheral APN gene therapy. We have generated and transduced adeno-associated virus (AAV2/8) expressing the mouse mutated APN gene (APNC39S) into the liver of 5xFAD mice that generated only low-molecular-weight trimeric APN (APNTri). Single dose of AAV2/8-APNC39S in the liver increased circulatory and cerebral APN levels indicating the overexpressed APNTri was able to cross the BBB. Overexpression of APNTri decreased both the soluble and fibrillar Aβ in the brains of 5xFAD mice. AAV2/8-APNTri treatment reduced Aβ-induced IL-1β and IL-18 secretion by suppressing microglial NLRP3-inflammasome activation. The memory functions improved significantly in AAV-APNTri-treated 5xFAD mice with reduction of dystrophic neurites. These findings demonstrate that peripheral gene delivery to overexpress trimeric APN can be a potential therapy for AD.Graphical

Galectin-9 and Tim-3 are upregulated in response to microglial activation induced by the peptide Amyloid-β (25–35)

Galectins are a group of β-galactoside-binding lectins associated with regulating immunological response. In the brains of AD patients and 5xFAD (familial AD) mice, galectin-3 (Gal-3) was highly upregulated and found to be expressed in microglia associated with Aβ plaques. However, the participation of other galectins, specifically galectin-9 (Gal-9) and T-cell immunoglobulin and mucin domain 3 (Tim-3) receptors, are unknown in the inflammatory response. The experimental model of the Aβ25–35 peptide will allow us to study the mechanisms of neuroinflammation and describe the changes in the expression of the Gal-9 and Tim-3 receptor. This study aimed to evaluate whether Aβ25–35 peptide administration into the lateral ventricles of rats upregulated Gal-9 and Tim-3 implicated in the modulation of neuroinflammation. The vehicle or Aβ25–35 peptide (1 μg/μL) was bilaterally administered into the lateral ventricles of the rat, and control group. After the administration of the Aβ25–35 peptide, animals were tested for learning (day 29) and spatial memory (day 30) in the novel object recognition test (NOR). On day 31, hippocampus was examined for morphological changes by Nilss stain, biochemical changes by NO2 and MDA, immunohistochemical analysis by astrocytes (GFAP), microglia (Iba1), Gal-9 and Tim-3, and western blot. Our results show the administration of the Aβ25–35 peptide into the lateral ventricles of rats induce memory impairment in the NOR by increases the oxidative stress and inflammatory response. This result is associated with an upregulation of Gal-9 and Tim-3 predominantly detected in the microglia cells of Aβ25–35-treated rats with respect to the control group. Gal-9 and Tim-3 are upregulated in activated microglia that could modulate the inflammatory response and damage in neurodegenerative processes induced by the Aβ25–35 peptide. Therefore, we suggest that Gal-9 and Tim-3 participate in the inflammatory process induced by the administration of the Aβ25–35 peptide.

Autosomal Dominant Alzheimer's Disease Mutations in Human Microglia Are Not Sufficient to Trigger Amyloid Pathology in WT Mice but Might Affect Pathology in 5XFAD Mice.

Several genetic variants that affect microglia function have been identified as risk factors for Alzheimer's Disease (AD), supporting the importance of this cell type in disease progression. However, the effect of autosomal dominant mutations in the amyloid precursor protein (APP) or the presenilin (PSEN1/2) genes has not been addressed in microglia in vivo. We xenotransplanted human microglia derived from non-carriers and carriers of autosomal dominant AD (ADAD)-causing mutations in the brain of hCSF1 WT or 5XFAD mice. We observed that ADAD mutations in microglia are not sufficient to trigger amyloid pathology in WT mice. In 5XFAD mice, we observed a non-statistically significant increase in amyloid plaque volume and number of dystrophic neurites, coupled with a reduction in plaque-associated microglia in the brain of mice xenotransplanted with ADAD human microglia compared to mice xenotransplanted with non-ADAD microglia. In addition, we observed a non-statistically significant impairment in working and contextual memory in 5XFAD mice xenotransplanted with ADAD microglia compared to those xenotransplanted with non-ADAD-carrier microglia. We conclude that, although not sufficient to initiate amyloid pathology in the healthy brain, mutations in APP and PSEN1 in human microglia might cause mild changes in pathological and cognitive outcomes in 5XFAD mice in a manner consistent with increased AD risk.

Photostimulation of lymphatic clearance of β-amyloid from mouse brain: a new strategy for the therapy of Alzheimer’s disease

Alzheimer’s disease (AD) is an age-related neurodegenerative disorder that poses a significant burden on socio-economic and healthcare systems worldwide. However, the currently available therapy of AD is limited, and new strategies are needed to enhance the clearance of β-amyloid (Aβ) protein and improve cognitive function. Photobiomodulation (PBM) is a non-invasive and effective therapeutic method that has shown promise in treating various brain diseases. Here, we demonstrate that 1267-nm PBM significantly alleviates cognitive decline in the 5xFAD mouse model of AD and is safe as it does not induce a significant increase in cortical temperature. Moreover, with the combination of 3D tissue optical clearing imaging and automatic brain region segmentation, we show that PBM-mediated reductions of Aβ plaques in different subregions of prefrontal cortex and the hippocampus are different. The PBM-induced lymphatic clearance of Aβ from the brain is associated with improvement of memory and cognitive functions in 5xFAD mice. Our results suggest that the modulation of meningeal lymphatic vessels (MLVs) should play an important role in promoting Aβ clearance. Collectively, this pilot study demonstrates that PBM can safely accelerate lymphatic clearance of Aβ from the brain of 5xFAD mice, promoting improvement of neurocognitive status of AD animals suggesting that PBM can be an effective and bedside therapy for AD.Graphical

A brain penetrant small‐molecule SYK inhibitor for the treatment of Alzheimer’s and neuroinflammatory diseases

AbstractBackgroundSpleen tyrosine kinase (Syk) is a cytosolic pro‐inflammatory signalling kinase that is associated with several neuroinflammatory conditions, including Alzheimer’s disease. We have developed structurally novel QED‐701, a brain‐penetrant, small molecule inhibitor of Syk that possesses excellent pharmacokinetic properties and displays anti‐inflammatory efficacy in vivo.MethodQED‐701 was administered to 5xFAD mice for 4‐months, beginning at 8‐weeks of age. At termination, the pro‐inflammatory profile in the brain was assessed by qPCR and histology was performed for amyloid, neuron health and microglial activation.ResultQED‐701 significantly reduced inflammation in the brain of 5xFAD mice, in a dose‐dependent manner that correlates to Syk inhibition in the CNS.ConclusionQED‐701 is a preclinical candidate for the treatment of Alzheimer’s disease and other neuroinflammatory conditions. It is a potent and selective inhibitor of Syk that readily enters the brain and displays anti‐inflammatory efficacy in the 5xFAD model of Alzheimer’s disease.

Modulating amyloid‐beta proteostasis in the brain of 5xFAD mice by selective expression of angiotensin converting enzyme in microglia

AbstractBackgroundAlzheimer’s Disease (AD) is a common and progressive neurodegenerative disease characterized by abnormalities in amyloid‐beta (Ab) proteostasis in the brain. Abnormal cleavage of Ab and aggregation into extracellular amyloid‐containing plaques is widely believed to contribute to neuroinflammation, synapse loss and neurotoxicity in AD. Microglia are brain‐resident myeloid‐lineage cells that mediate synapse homeostasis and the immune response to Ab deposition in the brain, and many Genome‐Wide Association Studies (GWAS) have identified late‐onset AD (LOAD) risk‐associated genes that are primarily expressed in microglia, suggesting their important role in disease pathogenesis (Kunkle et al., 2019, Wightman et al. 2021). Identifying signaling pathways in microglia that modulate Ab proteostasis may provide important targets for disease therapy.MethodThe gene encoding for Angiotensin Converting Enzyme (ACE) has been identified as an AD risk‐associated gene (Kunkle et al., 2019, Wightman et al, 2021), and ACE modulates the function of myeloid‐lineage cells, such as neutrophils and macrophages. ACE expression in macrophages appears to modulate their ability to decrease Ab plaque load in the brain and reverse cognitive abnormalities in AD model mice. Here, we developed a novel mouse model to express ACE specifically in microglia (RACE). We mated RACE mice to 5xFAD mice and examined the role of ACE expression in microglia using molecular, physiologic and behavioral techniques. Single nucleus RNA sequencing (10x Genomics) and digital spatial profiling on tissues sections (Nanostring GeoMx) was used to identify gene expression modulated by ACE in microglia.ResultWe find that ACE expression in microglia in 5xFAD mice increases amyloid phagocytosis, reduces plaque burden, rescues synapse and neuron loss, enhances microglial recruitment to plaques, rescues learning and memory deficits, and modulates gene expression associated with injury and autophagy in plaque‐associated microglia. Single‐nucleus RNA sequencing, proteomic profiling, synaptic plasticity, and pharmacological studies are in process to interrogate the molecular basis of ACE function in microglia.ConclusionACE expression in microglia has the remarkable capacity to restore Ab proteostasis, rescue synapse and neuron loss, and learning and memory abnormalities in 5xFAD mice. Signaling pathways and gene expression modulated by ACE in microglia may be targets for novel disease therapy.

Long noncoding RNA MALAT1; A novel and potential therapeutic target for Alzheimer’s disease

AbstractBackgroundAlzheimer disease (AD) is the most common form of dementia, characterized pathologically by amyloid plaques and neurofibrillary tangles. Despite considerable advances in the knowledge of AD pathogenesis, we know little about preventing or delaying the ongoing neurodegenerative process, indicating that there remain critical gaps in our understanding of disease mechanisms and opportunities for new treatment approaches. Long noncoding RNAs (lncRNAs) are larger than 200 nucleotides, structurally resemble mRNAs but do not encode proteins. LncRNAs participate in regulation of several physiological and pathophysiological processes by modulating gene expression and have been documented in several neurodegenerative diseases. Among them, metastasis associated lung adenocarcinoma transcript 1 (MALAT1) is a highly abundant and evolutionary conserved lncRNA and regulates a subset of genes involved in synaptic plasticity, cognitive function and memory.MethodTo test the hypothesis that lncRNA MALAT1 plays an important role in AD pathology, we quantified expression levels of lncRNA MALAT1 in the brains of age‐matched AD patients and cognitively normal controls (Non‐AD) by QRT‐PCR and RNAScope in situ hybridization assays, respectively. We further examined the lncRNA MALAT1 expressions and AD‐related neuropathology in the brains of 5XFAD transgenic mouse model of AD and their WT littermates using dual RNAScope/Immunofluorescence technique.ResultWe observed reduced expressions of lncRNA MALAT1 in the hippocampus of AD brains compared to the non‐AD control brains. Next, we found marked reduced expression of lncRNA MALAT1 in the brain of 5xFAD mice compared to non‐transgenic mice. Interestingly, decreased expression levels of lncRNA MALAT1 are correlated with AD‐related neuropathology and memory deficits in 5xFAD mice.ConclusionThese findings provided compelling evidence that AD is associated with altered expression of lncRNA MALAT1 which may influence several pathways related to neural damage and memory deficits in AD. Because expression patterns, interacting proteins, pathways, and biological functions of lncRNA MALAT1 are remain largely unexplored in AD, understanding how lncRNA MALAT1 regulates AD‐related neuropathology may allow development of effective therapies for this incurable disease.

Dysregulated bioenergetics pathways in the Alzheimer’s mouse brain

AbstractBackgroundAlzheimer’s disease (AD) is the most common cause of dementia in the aging population. The pathological characteristics include extracellular senile plaques and intracellular neurofibrillary tangles. In addition, mitochondrial dysfunction, oxidative stress, and neuroinflammation contribute to AD pathogenesis.MethodRNA‐seq analysis was done to compare the transcriptome in the brain of 5XFAD mice to the WT brain.ResultThere was significant changes in the expression of genes related to pathways such as, oxidation‐reduction, oxidative phosphorylation, innate immune response, ribosomal protein synthesis, and ubiquitin proteosome system. The most striking feature was the alteration of the oxidation‐reduction process with changes in the expression of a large number of mitochondrial genes. We also observed an upregulation of several immune response genes. Gene interaction network of oxidation‐reduction related genes further confirmed a tight “Gordian knot” of mitochondrial genes. Furthermore, gene interaction analysis of all the 1288 genes showed at least three distinct interaction clusters, with the predominant one relating to cellular energeticsConclusionWe identified 1288 genes distinctly different in the 5XFAD brain compared to the WT brain and found cellular energetics to be the most distinct gene cluster in the AD mouse brain compared to the WT.

Interaction of amisulpride with GLUT1 at the blood-brain barrier. Relevance to Alzheimer's disease.

Blood-brain barrier (BBB) dysfunction may be involved in the increased sensitivity of Alzheimer's disease (AD) patients to antipsychotics, including amisulpride. Studies indicate that antipsychotics interact with facilitated glucose transporters (GLUT), including GLUT1, and that GLUT1 BBB expression decreases in AD. We tested the hypotheses that amisulpride (charge: +1) interacts with GLUT1, and that BBB transport of amisulpride is compromised in AD. GLUT1 substrates, GLUT1 inhibitors and GLUT-interacting antipsychotics were identified by literature review and their physicochemical characteristics summarised. Interactions between amisulpride and GLUT1 were studied using in silico approaches and the human cerebral endothelial cell line, hCMEC/D3. Brain distribution of [3H]amisulpride was determined using in situ perfusion in wild type (WT) and 5xFamilial AD (5xFAD) mice. With transmission electron microscopy (TEM) we investigated brain capillary degeneration in WT mice, 5xFAD mice and human samples. Western blots determined BBB transporter expression in mouse and human. Literature review revealed that, although D-glucose has no charge, charged molecules can interact with GLUT1. GLUT1 substrates are smaller (184.95±6.45g/mol) than inhibitors (325.50±14.40g/mol) and GLUT-interacting antipsychotics (369.38±16.04). Molecular docking showed beta-D-glucose (free energy binding: -15.39kcal/mol) and amisulpride (-29.04kcal/mol) interact with GLUT1. Amisulpride did not affect [14C]D-glucose hCMEC/D3 accumulation. [3H]amisulpride uptake into the brain (except supernatant) of 5xFAD mice compared to WT remained unchanged. TEM revealed brain capillary degeneration in human AD. There was no difference in GLUT1 or P-glycoprotein BBB expression between WT and 5xFAD mice. In contrast, caudate P-glycoprotein, but not GLUT1, expression was decreased in human AD capillaries versus controls. This study provides new details about the BBB transport of amisulpride, evidence that amisulpride interacts with GLUT1 and that BBB transporter expression is altered in AD. This suggests that antipsychotics could potentially exacerbate the cerebral hypometabolism in AD. Further research into the mechanism of amisulpride transport by GLUT1 is important for improving antipsychotics safety.

Truncated Dyrk1A aggravates neuronal apoptosis by inhibiting ASF-mediated Bcl-x exon 2b inclusion.

Aggravated neuronal loss, caused mainly by neuronal apoptosis, is observed in the brain of patients with Alzheimer's disease (AD) and animal models of AD. A truncated form of Dual-specific and tyrosine phosphorylation-regulated protein kinase 1A (Dyrk1A) plays a vital role in AD pathogenesis. Downregulation of anti-apoptotic Bcl-xL is tightly correlated with neuronal loss in AD. However, the molecular regulation of neuronal apoptosis and Bcl-x expression by Dyrk1A in AD remains largely elusive. Here, we aimed to explore the role and molecular mechanism of Dyrk1A in apoptosis. Cell Counting Kit-8 (CCK8), flow cytometry, and TdT-mediated dUTP Nick-End Labeling (TUNEL) were used to check apoptosis. The cells, transfected with Dyrk1A or/and ASF with Bcl-x minigene, were used to assay Bcl-x expression by RT-PCR and Western blots. Co-immunoprecipitation, autoradiography, and immunofluorescence were conducted to check the interaction of ASF and Dyrk1A. Gene set enrichment analysis (GSEA) of apoptosis-related genes was performed in mice overexpressing Dyrk1A (TgDyrk1A) and AD model 5xFAD mice. Dyrk1A promoted Bcl-xS expression and apoptosis. Splicing factor ASF promoted Bcl-x exon 2b inclusion, leading to increased Bcl-xL expression. Dyrk1A suppressed ASF-mediated Bcl-x exon 2b inclusion via phosphorylation. The C-terminus deletion of Dyrk1A facilitated its binding and kinase activity to ASF. Moreover, Dyrk1a1-483 further suppressed the ASF-mediated Bcl-x exon 2b inclusion and aggravated apoptosis. The truncated Dyrk1A, increased Bcl-xS, and enrichment of apoptosis-related genes was observed in the brain of 5xFAD mice. We speculate that increased Dyrk1A and truncated Dyrk1A may aggravate neuronal apoptosis by decreasing the ratio of Bcl-xL/Bcl-xS via phosphorylating ASF in AD.

Effects of Chronic, Low-Dose Cannabinoids, Cannabidiol, Delta-9-Tetrahydrocannabinol and a Combination of Both, on Amyloid Pathology in the 5xFAD Mouse Model of Alzheimer's Disease.

Background: There is an urgent need for novel therapies to treat Alzheimer's disease. Among others, the use of cannabinoids such as delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) has been proposed as a putative approach based on their anti-inflammatory effects. Methods: The present work was designed to explore the effects of chronic (28 days) treatment with low doses of cannabinoids: CBD (0.273 mg/kg), THC (0.205 mg/kg) or a combination of both (CBD:THC; 0.273 mg/kg:0.205 mg/kg) in the 5xFAD mouse model of AD. Results: Our data revealed that THC-treated 5xFAD mice (but not other treatment groups) exhibited anxiogenic and depressant-like behavior. A significant improvement in spatial memory was observed only in the CBD:THC-treated group. Interestingly, all cannabinoid-treated groups showed significantly increased cortical levels of the insoluble form of beta amyloid 1-42. These effects were not accompanied by changes in molecular parameters of inflammation at the mRNA or protein level. Conclusions: These data reveal differential effects of chronic, low-dose cannabinoids and point to a role of these cannabinoids in the processing of amyloid peptides in the brains of 5xFAD mice.

Alzheimer's disease-induced phagocytic microglia express a specific profile of coding and non-coding RNAs.

Alzheimer's disease (AD) is a neurodegenerative disease and the main cause of dementia in the elderly. AD pathology is characterized by accumulation of microglia around the beta-amyloid (Aβ) plaques which assumes disease-specific transcriptional signatures, as for the disease-associated microglia (DAM). However, the regulators of microglial phagocytosis are still unknown. We isolated Aβ-laden microglia from the brain of 5xFAD mice for RNA sequencing to characterize the transcriptional signature in phagocytic microglia and to identify the key non-coding RNAs capable of regulating microglial phagocytosis. Through spatial sequencing, we show the transcriptional changes of microglia in the AD mouse brain in relation to Aβ proximity. Finally, we show that phagocytic messenger RNAs are regulated by miR-7a-5p, miR-29a-3p and miR-146a-5p microRNAs and segregate the DAM population into phagocytic and non-phagocytic states. Our study pinpoints key regulators of microglial Aβ clearing capacity suggesting new targets for future therapeutic approaches.

Interactions of Cellular Energetic Gene Clusters in the Alzheimer's Mouse Brain.

Alzheimer's disease (AD) is the most common cause of dementia in the aging population. The pathological characteristics include extracellular senile plaques and intracellular neurofibrillary tangles. In addition, mitochondrial dysfunction, oxidative stress, and neuroinflammation contribute to AD pathogenesis. In this study, we sought to determine the crosstalk between different pathways in the brain of 5XFAD mice, a mouse model for amyloid pathology, by RNA-seq analysis. We observed significant changes in the expression of genes (1288 genes; adj p value < 0.05; log2-fold > 1 and < 1) related to pathways including oxidation-reduction, oxidative phosphorylation, innate immune response, ribosomal protein synthesis, and ubiquitin proteosome system. The most striking feature was the downregulation of genes related to oxidation-reduction process with changes in the expression of a large number of mitochondrial genes. We also observed an upregulation of several immune response genes. Gene interaction network of oxidation-reduction related genes further confirmed a tight cluster of mitochondrial genes. Furthermore, gene interaction analysis of all the 1288 genes showed at least three distinct interaction clusters, with the predominant one relating to cellular energetics. In summary, we identified 1288 genes distinctly different in the 5XFAD brain compared to the WT brain and found cellular energetics to be the most distinct gene cluster in the AD mouse brain.