Lupus autoantibodies initiate neuroinflammation sustained by continuous HMGB1:RAGE signaling and reversed by increased LAIR-1 expression.

Cognitive impairment is a frequent manifestation of neuropsychiatric systemic lupus erythematosus, present in up to 80% of patients and leading to a diminished quality of life. In the present study, we used a model of lupus-like cognitive impairment that is initiated when antibodies that crossreact with excitatory neuronal receptors penetrate the hippocampus, causing immediate, self-limited, excitotoxic death of hippocampal neurons, which is then followed by a significant loss of dendritic complexity in surviving neurons. This injury creates a maladaptive equilibrium that is sustained in mice for at least 1 year. We identified a feedforward loop of microglial activation and microglia-dependent synapse elimination dependent on neuronal secretion of high mobility group box 1 protein (HMGB1) which binds the receptor for advanced glycation end products (RAGE) and leads to microglial secretion of C1q, upregulation of interleukin-10 with consequent downregulation of leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1), an inhibitory receptor for C1q. Treatment with a centrally acting angiotensin-converting enzyme inhibitor or with an angiotensin-receptor blocker restored a healthy equilibrium, microglial quiescence and intact spatial memory.

Genetic variants of phospholipase C-γ2 alter the phenotype and function of microglia and confer differential risk for Alzheimer's disease.

Genetic association studies have demonstrated the critical involvement of the microglial immune response in Alzheimer's disease (AD) pathogenesis. Phospholipase C-gamma-2 (PLCG2) is selectively expressed by microglia and functions in many immune receptor signaling pathways. In AD, PLCG2 is induced uniquely in plaque-associated microglia. A genetic variant of PLCG2, PLCG2P522R, is a mild hypermorph that attenuates AD risk. Here, we identified a loss-of-function PLCG2 variant, PLCG2M28L, that confers an increased AD risk. PLCG2P522R attenuated disease in an amyloidogenic murine AD model, whereas PLCG2M28L exacerbated the plaque burden associated with altered phagocytosis and Aß clearance. The variants bidirectionally modulated disease pathology by inducing distinct transcriptional programs that identified microglial subpopulations associated with protective or detrimental phenotypes. These findings identify PLCG2M28L as a potential AD risk variant and demonstrate that PLCG2 variants can differentially orchestrate microglial responses in AD pathogenesis that can be therapeutically targeted.

Circulating HIV-Specific Interleukin-21(+)CD4(+) T Cells Represent Peripheral Tfh Cells with Antigen-Dependent Helper Functions.

A central effort in HIV vaccine development is to generate protective broadly neutralizing antibodies, a process dependent on T follicular helper (Tfh) cells. The feasibility of using peripheral blood counterparts of lymph node Tfh cells to assess the immune response and the influence of viral and vaccine antigens on their helper functions remain obscure. We assessed circulating HIV-specific IL-21(+)CD4(+) T cells and showed transcriptional and phenotypic similarities to lymphoid Tfh cells, and hence representing peripheral Tfh (pTfh) cells. pTfh cells were functionally active and B cell helper quality differed depending on antigen specificity. Furthermore, we found higher frequency of pTfh cells in peripheral blood mononuclear cell specimens from the ALVAC+AIDSVAX (RV144) HIV vaccine trial associated with protective antibody responses compared to the non-protective DNA+Ad5 vaccine trial. Together, we identify IL-21(+)CD4(+) T cells as pTfh cells, implicating them as key populations in the generation of vaccine-evoked antibody responses.

Incubation of palatable food craving is associated with brain-wide neuronal activation in mice.

Studies using rodent models have shown that relapse to drug or food seeking increases progressively during abstinence, a behavioral phenomenon termed "incubation of craving." Mechanistic studies of incubation of craving have focused on specific neurobiological targets within preselected brain areas. Recent methodological advances in whole-brain immunohistochemistry, clearing, and imaging now allow unbiased brain-wide cellular resolution mapping of regions and circuits engaged during learned behaviors. However, these whole-brain imaging approaches were developed for mouse brains, while incubation of drug craving has primarily been studied in rats, and incubation of food craving has not been demonstrated in mice. Here, we established a mouse model of incubation of palatable food craving and examined food reward seeking after 1, 15, and 60 abstinence days. We then used the neuronal activity marker Fos with intact-brain mapping procedures to identify corresponding patterns of brain-wide activation. Relapse to food seeking was significantly higher after 60 abstinence days than after 1 or 15 days. Using unbiased ClearMap analysis, we identified increased activation of multiple brain regions, particularly corticostriatal structures, following 60 but not 1 or 15 abstinence days. We used orthogonal SMART2 analysis to confirm these findings within corticostriatal and thalamocortical subvolumes and applied expert-guided registration to investigate subdivision and layer-specific activation patterns. Overall, we 1) identified brain-wide activity patterns during incubation of food seeking using complementary analytical approaches and 2) provide a single-cell resolution whole-brain atlas that can be used to identify functional networks and global architecture underlying the incubation of food craving.

Role of Piriform Cortex and Its Afferent Projections in Relapse to Fentanyl Seeking after Food Choice-Induced Voluntary Abstinence.

We previously demonstrated a role of piriform cortex (Pir) in relapse to fentanyl seeking after food choice-induced voluntary abstinence. Here, we used this model to further study the role of Pir and its afferent projections in fentanyl relapse. We trained male and female rats to self-administer palatable food pellets for 6 d (6 h/day) and fentanyl (2.5 µg/kg/infusion, i.v.) for 12 d (6 h/day). We assessed relapse to fentanyl seeking after 12 voluntary abstinence sessions, achieved through a discrete choice procedure between fentanyl and palatable food (20 trials/session). We determined projection-specific activation of Pir afferents during fentanyl relapse with Fos plus the retrograde tracer cholera toxin B (injected into Pir). Fentanyl relapse was associated with increased Fos expression in anterior insular cortex (AI) and prelimbic cortex (PL) neurons projecting to Pir. We next used an anatomical disconnection procedure to determine the causal role of these two projections (AI→Pir and PL→Pir) in fentanyl relapse. Contralateral but not ipsilateral disconnection of AI→Pir projections decreased fentanyl relapse but not reacquisition of fentanyl self-administration. In contrast, contralateral but not ipsilateral disconnection of PL→Pir projections modestly decreased reacquisition but not relapse. Fluorescence-activated cell sorting and quantitative PCR data showed molecular changes within Pir Fos-expressing neurons associated with fentanyl relapse. Finally, we found minimal or no sex differences in fentanyl self-administration, fentanyl versus food choice, and fentanyl relapse. Our results indicate that AI→Pir and PL→Pir projections play dissociable roles in nonreinforced relapse to fentanyl seeking versus reacquisition of fentanyl self-administration after food choice-induced voluntary abstinence.SIGNIFICANCE STATEMENT We previously showed a role of Pir in fentanyl relapse after food choice-induced voluntary abstinence in rats, a procedure mimicking human abstinence or a significant reduction in drug self-administration because of the availability of alternative nondrug rewards. Here, we aimed to further characterize the role of Pir in fentanyl relapse by investigating the role of Pir afferent projections and analyzing molecular changes in relapse-activated Pir neurons. We identified dissociable roles of two Pir afferent projections (AI→Pir and PL→Pir) in relapse to fentanyl seeking versus reacquisition of fentanyl self-administration after voluntary abstinence. We also characterized molecular changes within Pir Fos-expressing neurons associated with fentanyl relapse.

Flow Cytometry of Synaptoneurosomes (FCS) Reveals Increased Ribosomal S6 and Calcineurin Proteins in Activated Medial Prefrontal Cortex to Nucleus Accumbens Synapses.

Learning and behavior activate cue-specific patterns of sparsely distributed cells and synapses called ensembles that undergo memory-encoding engram alterations. While Fos is often used to label selectively activated cell bodies and identify neuronal ensembles, there is no comparable endogenous marker to label activated synapses and identify synaptic ensembles. For the purpose of identifying candidate synaptic activity markers, we optimized a flow cytometry of synaptoneurosome (FCS) procedure for assessing protein alterations in activated synapses from male and female rats. After injecting yellow fluorescent protein (YFP)-expressing adeno-associated virus into medial prefrontal cortex (mPFC) to label terminals in nucleus accumbens (NAc) of rats, we injected 20 mg/kg cocaine in a novel context (cocaine+novelty) to activate synapses, and prepared NAc synaptoneurosomes 0-60 min following injections. For FCS, we used commercially available antibodies to label presynaptic and postsynaptic markers synaptophysin and PSD-95 as well as candidate markers of synaptic activity [activity-regulated cytoskeleton protein (Arc), CaMKII and phospho-CaMKII, ribosomal protein S6 (S6) and phospho-S6, and calcineurin and phospho-calcineurin] in YFP-labeled synaptoneurosomes. Cocaine+novelty increased the percentage of S6-positive synaptoneurosomes at 5-60 min and calcineurin-positive synaptoneurosomes at 5-10 min. Electron microscopy verified that S6 and calcineurin levels in synaptoneurosomes were increased 10 min after cocaine+novelty. Pretreatment with the anesthetic chloral hydrate blocked cocaine+novelty-induced S6 and calcineurin increases in synaptoneurosomes, and novel context exposure alone (without cocaine) increased S6, both of which indicate that these increases were due to neural activity per se. Overall, FCS can be used to study protein alterations in activated synapses coming from specifically labeled mPFC projections to NAc.SIGNIFICANCE STATEMENT Memories are formed during learning and are stored in the brain by long-lasting molecular and cellular alterations called engrams formed within specific patterns of cue-activated neurons called neuronal ensembles. While Fos has been used to identify activated ensemble neurons and the engrams within them, we have not had a similar marker for activated synapses that can be used to identify synaptic engrams. Here we developed a procedure for high-throughput in-line analysis of flow cytometry of synaptoneurosome (FCS) and found that ribosomal S6 protein and calcineurin were increased in activated mPFC-NAc synapses. FCS can be used to study protein alterations in activated synapses within specifically labeled circuits.

Dementia exacerbates periodontal bone loss in females.

BACKGROUND: Periodontitis is a chronic inflammatory disease defined by the pathologic loss of the periodontal ligament and alveolar bone in relation to aging. Although clinical cohort studies reported that periodontitis is significantly elevated in males compared to females, emerging evidence indicates that females with dementia are at a greater risk for periodontitis and decreased alveolar bone. OBJECTIVE: This study aimed to evaluate whether dementia is a potential sex-dependent risk factor for periodontal bone loss using an experimental model of periodontitis induced in the triple transgenic (3x-Tg) dementia-like mice and clinical samples collected from senior 65 plus age patients with diagnosed dementia. MATERIALS AND METHODS: We induced periodontitis in dementia-like triple-transgenic (3x-Tg) male and female mice and age-matched wild-type (WT) control mice by ligature placement. Then, alveolar bone loss and osteoclast activity were evaluated using micro-CT and in situ imaging assays. In addition, we performed dental examinations on patients with diagnosed dementia. Finally, dementia-associated Aß42 and p-Tau (T181) and osteoclastogenic receptor activator of nuclear factor kappa-Β ligand (RANKL) in gingival crevicular fluid (GCF) collected from mice and clinical samples were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Alveolar bone loss and in situ osteoclast activity were significantly elevated in periodontal lesions of 3x-Tg females but not males, compared to wild-type control mice. In addition, we also observed that the probing pocket depth (PPD) was also significantly elevated in female patients with dementia. Using ELISA assay, we observed that females had elevated levels of osteoclastogenic RANKL and dementia-associated Aß42 and p-Tau (T181) in the GCF collected from experimental periodontitis lesions and clinical samples. CONCLUSION: Altogether, we demonstrate that females with dementia have an increased risk for periodontal bone loss compared to males.

Nexinhib20 Inhibits Neutrophil Adhesion and ß2 Integrin Activation by Antagonizing Rac-1-Guanosine 5'-Triphosphate Interaction.

Neutrophils are critical for mediating inflammatory responses. Inhibiting neutrophil recruitment is an attractive approach for preventing inflammatory injuries, including myocardial ischemia-reperfusion (I/R) injury, which exacerbates cardiomyocyte death after primary percutaneous coronary intervention in acute myocardial infarction. In this study, we found out that a neutrophil exocytosis inhibitor Nexinhib20 inhibits not only exocytosis but also neutrophil adhesion by limiting ß2 integrin activation. Using a microfluidic chamber, we found that Nexinhib20 inhibited IL-8-induced ß2 integrin-dependent human neutrophil adhesion under flow. Using a dynamic flow cytometry assay, we discovered that Nexinhib20 suppresses intracellular calcium flux and ß2 integrin activation after IL-8 stimulation. Western blots of Ras-related C3 botulinum toxin substrate 1 (Rac-1)-GTP pull-down assays confirmed that Nexinhib20 inhibited Rac-1 activation in leukocytes. An in vitro competition assay showed that Nexinhib20 antagonized the binding of Rac-1 and GTP. Using a mouse model of myocardial I/R injury, Nexinhib20 administration after ischemia and before reperfusion significantly decreased neutrophil recruitment and infarct size. Our results highlight the translational potential of Nexinhib20 as a dual-functional neutrophil inhibitory drug to prevent myocardial I/R injury.

Transgenic amyloid precursor protein mouse models of amyloidosis. Incomplete models for Alzheimer's disease but effective predictors of anti-amyloid therapies.

INTRODUCTION: Amyloid precursor protein (APP) transgenic mice are models of Alzheimer's disease (AD) amyloidosis, not all of AD. Diffuse, compacted, and vascular deposits in APP mice mimic those found in AD cases. METHODS: Most interventional studies in APP mice start treatment early in the process of amyloid deposition, consistent with a prevention treatment regimen. Most clinical trials treat patients with established amyloid deposits in a therapeutic treatment regimen. RESULTS: The first treatment to reduce amyloid and cognitive impairment in mice was immunotherapy. The APP mouse models not only predicted efficacy, but presaged the vascular leakage called ARIA. The recent immunotherapy clinical trials that removed amyloid and slowed cognitive decline confirms the utility of these early APP models when used in therapeutic designs. DISCUSSION: New mouse models of AD pathologies will add to the research armamentarium, but the early models have accurately predicted responses to amyloid therapies in humans.

Inpp5d haplodeficiency alleviates tau pathology in the PS19 mouse model of Tauopathy.

INTRODUCTION: A noncoding variant (rs35349669) within INPP5D, a lipid and protein phosphatase restricted to microglia in the brain, is linked to increased susceptibility to Alzheimer's disease (AD). While Inpp5d is well-studied in amyloid pathology, its role in tau pathology remains unclear. METHODS: PS19 Tauopathy mice were crossed with Inpp5d-haplodeficient (Inpp5d+/-) mice to examine the impact of Inpp5d in tau pathology. RESULTS: Increased INPP5D expression correlated positively with phospho-Tau AT8 in PS19 mice. Inpp5d haplodeficiency mitigated hyperphosphorylated tau levels (AT8, AT180, AT100, and PHF1) and motor deficits in PS19 mice. Transcriptomic analysis revealed an up-regulation of genes associated with immune response and cell migration. DISCUSSION: Our findings define an association between INPP5D expression and tau pathology in PS19 mice. Alleviation in hyperphosphorylated tau, motor deficits, and transcriptomics changes in haplodeficient-Inpp5d PS19 mice indicate that modulation in INPP5D expression may provide therapeutic potential for mitigating tau pathology and improving motor deficits. HIGHLIGHTS: The impact of Inpp5d in the context of tau pathology was studied in the PS19 mouse model. INPP5D expression is associated with tau pathology. Reduced Inpp5d expression in PS19 mice improved motor functions and decreased total and phospho-Tau levels. Inpp5d haplodeficiency in PS19 mice modulates gene expression patterns linked to immune response and cell migration. These data suggest that inhibition of Inpp5d may be a therapeutic approach in tauopathies.

Loss of Inpp5d has disease-relevant and sex-specific effects on glial transcriptomes.

INTRODUCTION: Inpp5d is genetically associated with Alzheimer's disease risk. Loss of Inpp5d alters amyloid pathology in models of amyloidosis. Inpp5d is expressed predominantly in microglia but its function in brain is poorly understood. METHODS: We performed single-cell RNA sequencing to study the effect of Inpp5d loss on wild-type mouse brain transcriptomes. RESULTS: Loss of Inpp5d has sex-specific effects on the brain transcriptome. Affected genes are enriched for multiple neurodegeneration terms. Network analyses reveal a gene co-expression module centered around Inpp5d in female mice. Inpp5d loss alters Pleotrophin (PTN), Prosaposin (PSAP), and Vascular Endothelial Growth Factor A (VEGFA) signaling probability between cell types. DISCUSSION: Our data suggest that the normal function of Inpp5d is entangled with mechanisms involved in neurodegeneration. We report the effect of Inpp5d loss without pathology and show that this has dramatic effects on gene expression. Our study provides a critical reference for researchers of neurodegeneration, allowing separation of disease-specific changes mediated by Inpp5d in disease from baseline effects of Inpp5d loss. HIGHLIGHTS: Loss of Inpp5d has different effects in male and female mice. Genes dysregulated by Inpp5d loss relate to neurodegeneration. Total loss of Inpp5d in female mice collapses a conserved gene co-expression module. Loss of microglial Inpp5d affects the transcriptome of other cell types.

In vivo validation of late-onset Alzheimer's disease genetic risk factors.

INTRODUCTION: Genome-wide association studies have identified over 70 genetic loci associated with late-onset Alzheimer's disease (LOAD), but few candidate polymorphisms have been functionally assessed for disease relevance and mechanism of action. METHODS: Candidate genetic risk variants were informatically prioritized and individually engineered into a LOAD-sensitized mouse model that carries the AD risk variants APOE ε4/ε4 and Trem2*R47H. The potential disease relevance of each model was assessed by comparing brain transcriptomes measured with the Nanostring Mouse AD Panel at 4 and 12 months of age with human study cohorts. RESULTS: We created new models for 11 coding and loss-of-function risk variants. Transcriptomic effects from multiple genetic variants recapitulated a variety of human gene expression patterns observed in LOAD study cohorts. Specific models matched to emerging molecular LOAD subtypes. DISCUSSION: These results provide an initial functionalization of 11 candidate risk variants and identify potential preclinical models for testing targeted therapeutics. HIGHLIGHTS: A novel approach to validate genetic risk factors for late-onset AD (LOAD) is presented. LOAD risk variants were knocked in to conserved mouse loci. Variant effects were assayed by transcriptional analysis. Risk variants in Abca7, Mthfr, Plcg2, and Sorl1 loci modeled molecular signatures of clinical disease. This approach should generate more translationally relevant animal models.

Characterizing molecular and synaptic signatures in mouse models of late-onset Alzheimer's disease independent of amyloid and tau pathology.

INTRODUCTION: MODEL-AD (Model Organism Development and Evaluation for Late-Onset Alzheimer's Disease) is creating and distributing novel mouse models with humanized, clinically relevant genetic risk factors to capture the trajectory and progression of late-onset Alzheimer's disease (LOAD) more accurately. METHODS: We created the LOAD2 model by combining apolipoprotein E4 (APOE4), Trem2*R47H, and humanized amyloid-beta (Aß). Mice were subjected to a control diet or a high-fat/high-sugar diet (LOAD2+HFD). We assessed disease-relevant outcome measures in plasma and brain including neuroinflammation, Aß, neurodegeneration, neuroimaging, and multi-omics. RESULTS: By 18 months, LOAD2+HFD mice exhibited sex-specific neuron loss, elevated insoluble brain Aß42, increased plasma neurofilament light chain (NfL), and altered gene/protein expression related to lipid metabolism and synaptic function. Imaging showed reductions in brain volume and neurovascular uncoupling. Deficits in acquiring touchscreen-based cognitive tasks were observed. DISCUSSION: The comprehensive characterization of LOAD2+HFD mice reveals that this model is important for preclinical studies seeking to understand disease trajectory and progression of LOAD prior to or independent of amyloid plaques and tau tangles. HIGHLIGHTS: By 18 months, unlike control mice (e.g., LOAD2 mice fed a control diet, CD), LOAD2+HFD mice presented subtle but significant loss of neurons in the cortex, elevated levels of insoluble Ab42 in the brain, and increased plasma neurofilament light chain (NfL). Transcriptomics and proteomics showed changes in gene/proteins relating to a variety of disease-relevant processes including lipid metabolism and synaptic function. In vivo imaging revealed an age-dependent reduction in brain region volume (MRI) and neurovascular uncoupling (PET/CT). LOAD2+HFD mice also demonstrated deficits in acquisition of touchscreen-based cognitive tasks.

Advancements in APOE and dementia research: Highlights from the 2023 AAIC Advancements: APOE conference.

INTRODUCTION: The apolipoprotein E gene (APOE) is an established central player in the pathogenesis of Alzheimer's disease (AD), with distinct apoE isoforms exerting diverse effects. apoE influences not only amyloid-beta and tau pathologies but also lipid and energy metabolism, neuroinflammation, cerebral vascular health, and sex-dependent disease manifestations. Furthermore, ancestral background may significantly impact the link between APOE and AD, underscoring the need for more inclusive research. METHODS: In 2023, the Alzheimer's Association convened multidisciplinary researchers at the "AAIC Advancements: APOE" conference to discuss various topics, including apoE isoforms and their roles in AD pathogenesis, progress in apoE-targeted therapeutic strategies, updates on disease models and interventions that modulate apoE expression and function. RESULTS: This manuscript presents highlights from the conference and provides an overview of opportunities for further research in the field. DISCUSSION: Understanding apoE's multifaceted roles in AD pathogenesis will help develop targeted interventions for AD and advance the field of AD precision medicine. HIGHLIGHTS: APOE is a central player in the pathogenesis of Alzheimer's disease. APOE exerts a numerous effects throughout the brain on amyloid-beta, tau, and other pathways. The AAIC Advancements: APOE conference encouraged discussions and collaborations on understanding the role of APOE.

The Novavax Heterologous COVID Booster Demonstrates Lower Reactogenicity Than mRNA: A Targeted Review.

COVID-19 continues to be a global health concern and booster doses are necessary for maintaining vaccine-mediated protection, limiting the spread of SARS-CoV-2. Despite multiple COVID vaccine options, global booster uptake remains low. Reactogenicity, the occurrence of adverse local/systemic side effects, plays a crucial role in vaccine uptake and acceptance, particularly for booster doses. We conducted a targeted review of the reactogenicity of authorized/approved mRNA and protein-based vaccines demonstrated by clinical trials and real-world evidence. It was found that mRNA-based boosters show a higher incidence and an increased severity of reactogenicity compared with the Novavax protein-based COVID vaccine, NVX-CoV2373. In a recent NIAID study, the incidence of pain/tenderness, swelling, erythema, fatigue/malaise, headache, muscle pain, or fever was higher in individuals boosted with BNT162b2 (0.4 to 41.6% absolute increase) or mRNA-1273 (5.5 to 55.0% absolute increase) compared with NVX-CoV2373. Evidence suggests that NVX-CoV2373, when utilized as a heterologous booster, demonstrates less reactogenicity compared with mRNA vaccines, which, if communicated to hesitant individuals, may strengthen booster uptake rates worldwide.

Control of the temporal development of Alzheimer's disease pathology by the MR1/MAIT cell axis.

BACKGROUND: Neuroinflammation is an important feature of Alzheimer's disease (AD). Understanding which aspects of the immune system are important in AD may lead to new therapeutic approaches. We study the major histocompatibility complex class I-related immune molecule, MR1, which is recognized by an innate-like T cell population called mucosal-associated invariant T (MAIT) cells. METHODS: Having found that MR1 gene expression is elevated in the brain tissue of AD patients by mining the Agora database, we sought to examine the role of the MR1/MAIT cell axis in AD pathology. Brain tissue from AD patients and the 5XFAD mouse model of AD were used to analyze MR1 expression through qPCR, immunofluorescence, and flow cytometry. Furthermore, mice deficient in MR1 and MAIT cells were crossed with the 5XFAD mice to produce a model to study how the loss of this innate immune axis alters AD progression. Moreover, 5XFAD mice were also used to study brain-resident MAIT cells over time. RESULTS: In tissue samples from AD patients and 5XFAD mice, MR1 expression was substantially elevated in the microglia surrounding plaques vs. those that are further away (human AD: P < 0.05; 5XFAD: P < 0.001). In 5XFAD mice lacking the MR1/MAIT cell axis, the development of amyloid-beta plaque pathology occurred at a significantly slower rate than in those mice with MR1 and MAIT cells. Furthermore, in brain tissue from 5XFAD mice, there was a temporal increase in MAIT cell numbers (P < 0.01) and their activation state, the latter determined by detecting an upregulation of both CD69 (P < 0.05) and the interleukin-2 receptor alpha chain (P < 0.05) via flow cytometry. CONCLUSIONS: Together, these data reveal a previously unknown role for the MR1/MAIT cell innate immune axis in AD pathology and its potential utility as a novel therapeutic target.

Transcriptomic analysis reveals novel age-independent immunomodulatory proteins as a mode of cerebroprotection in P2X4 receptor knockout mice after ischemic stroke.

Identification of new potential drug target proteins and their plausible mechanisms for stroke treatment is critically needed. We previously showed that genetic deletion and short-term pharmacological inhibition of P2X4, a purinergic receptor for adenosine triphosphate (ATP), provides acute cerebroprotection. However, potential mechanisms remain unknown. Therefore, we employed RNA-Seq technology to identify the gene expression profiles and pathway analysis followed by qPCR validation of differentially expressed genes (DEGs). This analysis identified roles of DEGs in certain biological processes responsible for P2X4R-dependent cerebroprotection after stroke. We subjected both young and aged male and female global P2X4 receptor knock out (P2X4RKO) and littermate WT (WT) mice to ischemic stroke. After three days, mice were sacrificed, and total RNA was isolated using Trizol and subjected to RNA-Seq and NanoString-mediated qPCR. DESeq2, Gene Ontology (GO), and Ingenuity Pathway Analysis (IPA) were used to identify gene expression profiles and biological pathways. We found 2246 DEGs in P2X4R KO vs. WT tissue after stroke. Out of these DEGs, 1920 genes were downregulated and 325 genes were upregulated in P2X4R KO. GO/IPA analysis of the top 300 DEGs suggests an enrichment of inflammation and extracellular matrix component genes. qPCR validation of the top 30 DEGs revealed downregulation of two common age-independent genes in P2X4R KO mice: Interleukin-6 (Il-6), an inflammatory cytokine, and Cytotoxic T Lymphocyte-Associated Protein 2 alpha (Ctla2a), an immunosuppressive factor. These data suggest that P2X4R-mediated cerebroprotection after stroke is initiated by attenuation of immune modulatory pathways in both young and aged mice of both sexes.

PLCß2 Promotes VEGF-Induced Vascular Permeability.

BACKGROUND: Regulation of vascular permeability is critical to maintaining tissue metabolic homeostasis. VEGF (vascular endothelial growth factor) is a key stimulus of vascular permeability in acute and chronic diseases including ischemia reperfusion injury, sepsis, and cancer. Identification of novel regulators of vascular permeability would allow for the development of effective targeted therapeutics for patients with unmet medical need. METHODS: In vitro and in vivo models of VEGFA-induced vascular permeability, pathological permeability, quantitation of intracellular calcium release and cell entry, and phosphatidylinositol 4,5-bisphosphate levels were evaluated with and without modulation of PLC (phospholipase C) ß2. RESULTS: Global knock-out of PLCß2 in mice resulted in blockade of VEGFA-induced vascular permeability in vivo and transendothelial permeability in primary lung endothelial cells. Further work in an immortalized human microvascular cell line modulated with stable knockdown of PLCß2 recapitulated the observations in the mouse model and primary cell assays. Additionally, loss of PLCß2 limited both intracellular release and extracellular entry of calcium following VEGF stimulation as well as reduced basal and VEGFA-stimulated levels of phosphatidylinositol 4,5-bisphosphate compared to control cells. Finally, loss of PLCß2 in both a hyperoxia-induced lung permeability model and a cardiac ischemia:reperfusion model resulted in improved animal outcomes when compared with wild-type controls. CONCLUSIONS: The results implicate PLCß2 as a key positive regulator of VEGF-induced vascular permeability through regulation of both calcium flux and phosphatidylinositol 4,5-bisphosphate levels at the cellular level. Targeting of PLCß2 in a therapeutic setting may provide a novel approach to regulating vascular permeability in patients.

INPP5D deficiency attenuates amyloid pathology in a mouse model of Alzheimer's disease.

INTRODUCTION: Inositol polyphosphate-5-phosphatase (INPP5D) is a microglia-enriched lipid phosphatase in the central nervous system. A non-coding variant (rs35349669) in INPP5D increases the risk for Alzheimer's disease (AD), and elevated INPP5D expression is associated with increased plaque deposition. INPP5D negatively regulates signaling via several microglial cell surface receptors, including triggering receptor expressed on myeloid cells 2 (TREM2); however, the impact of INPP5D inhibition on AD pathology remains unclear. METHODS: We used the 5xFAD mouse model of amyloidosis to assess how Inpp5d haplodeficiency regulates amyloid pathogenesis. RESULTS: Inpp5d haplodeficiency perturbs the microglial intracellular signaling pathways regulating the immune response, including phagocytosis and clearing of amyloid beta (Aß). It is important to note that Inpp5d haploinsufficiency leads to the preservation of cognitive function. Spatial transcriptomic analysis revealed that pathways altered by Inpp5d haploinsufficiency are related to synaptic regulation and immune cell activation. CONCLUSION: These data demonstrate that Inpp5d haplodeficiency enhances microglial functions by increasing plaque clearance and preserves cognitive abilities in 5xFAD mice. Inhibition of INPP5D is a potential therapeutic strategy for AD.

Impact of fetal exposure to mycotoxins on longissimus muscle fiber hypertrophy and miRNA profile.

BACKGROUND: Longissimus muscle samples were collected from lambs exposed in utero to mycotoxins [E-, endophyte-free tall fescue seed without ergot alkaloids (negative control) or E + , endophyte-infected tall fescue seed containing ergot alkaloids] during mid-gestation (MID; E + /E-) or late-gestation (LATE; E-/E +) harvested at two developmental stages (FETAL, gestational d133) or (MAT, near maturity, 250 d of age; n = 3/treatment/developmental stage). Muscle samples were examined to determine the impact of in utero mycotoxin exposure on skeletal muscle fiber hypertrophy and the miRNA profile at FETAL and MAT. RESULTS: Longissimus weight was greater (P < 0.05) in E + /E- lambs compared to E-/E + lambs at MAT; however, FETAL longissimus weight did not differ (P > 0.10) between fescue treatments. Type I fiber cross sectional area was larger (P < 0.10) for E + /E- than E-/E + at MAT but did not differ (P > 0.10) between fescue treatments at FETAL. Type II fiber area was larger (P < 0.05) at MAT in E + /E- compared to E-/E + but did not differ (P < 0.05) between fescue treatments at FETAL. Cross-sectional Type I and Type II longissimus muscle fiber area increased (P < 0.05) from FETAL to MAT by 6.86-fold and 10.83-fold, respectively. The ratio of Type II:Type I muscle fibers was lower (P = 0.04) at MAT compared to FETAL. There were 120 miRNA differentially expressed (q < 0.05) between FETAL and MAT. Maternal fescue treatment did not alter (q > 0.05) expression of miRNAs in the longissimus muscle. miR-133, -29a, -22-3p, and -410-3p were identified as highly significant with a log2 fold change > 4. In vitro satellite cell cultures showed that selected miRNAs (miR-22-3p, 29a, 27a, and 133a) are differentially regulated during proliferation and differentiation indicating a role of miRNA in muscle hypertrophy. CONCLUSIONS: Exposure to mycotoxins did not alter fiber type but had long-term impacts on postnatal muscle hypertrophy and cross-sectional area. The miRNA profile of the longissimus was not altered by Maternal mycotoxin exposure at FETAL or MAT. Developmental age altered the miRNA transcriptome and mRNA expression of known genes related to muscle growth. These results indicate that Maternal exposure to E + fescue seed during LATE gestation can alter postnatal muscle hypertrophy in sheep; however, these changes are not regulated by the miRNA transcriptome of the longissimus muscle.