Astrocyte Gene Research Articles

Vector-Mediated Delivery of Transgenes and RNA Interference-Based Gene Silencing Sequences to Astrocytes for Disease Management: Advances and Prospectives.

Astrocytes are a type of important glial cell in the brain that serve crucial functions in regulating neuronal activity, facilitating communication between neurons, and keeping everything in balance. In this abstract, we explore current methods and future approaches for using vectors to precisely target astrocytes in the fight against various illnesses. In order to deliver therapeutic cargo selectively to astrocytes, researchers have made tremendous progress by using viral vectors such as adeno-associated viruses (AAVs) and lentiviruses. It has been established that engineered viral vectors are capable of either crossing the blood-brain barrier (BBB) or being delivered intranasally, which facilitates their entrance into the brain parenchyma. These vectors are able to contain transgenes that code for neuroprotective factors, synaptic modulators, or anti-inflammatory medicines, which pave the way for multiple approaches to disease intervention. Strategies based on RNA interference (RNAi) make vector-mediated astrocyte targeting much more likely to work. Small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) are two types of RNA that can be made to silence disease-related genes in astrocytes. Vector-mediated delivery in conjunction with RNAi techniques provides a powerful toolkit for investigating the complex biological pathways that contribute to disease development. However, there are still a number of obstacles to overcome in order to perfect the specificity, safety, and duration of vector-mediated astrocyte targeting. In order to successfully translate research findings into clinical practise, it is essential to minimise off-target effects and the risk of immunogenicity. To demonstrate the therapeutic effectiveness of these strategies, rigorous preclinical investigation and validation are required.

Hemorrhagic stroke-induced subtype of inflammatory reactive astrocytes disrupts blood-brain barrier.

Astrocytes undergo disease-specific transcriptomic changes upon brain injury. However, phenotypic changes of astrocytes and their functions remain unclear after hemorrhagic stroke. Here we reported hemorrhagic stroke induced a group of inflammatory reactive astrocytes with high expression of Gfap and Vimentin, as well as inflammation-related genes lipocalin-2 (Lcn2), Complement component 3 (C3), and Serpina3n. In addition, we demonstrated that depletion of microglia but not macrophages inhibited the expression of inflammation-related genes in inflammatory reactive astrocytes. RNA sequencing showed that blood-brain barrier (BBB) disruption-related gene matrix metalloproteinase-3 (MMP3) was highly upregulated in inflammatory reactive astrocytes. Pharmacological inhibition of MMP3 in astrocytes or specific deletion of astrocytic MMP3 reduced BBB disruption and improved neurological outcomes of hemorrhagic stroke mice. Our study demonstrated that hemorrhagic stroke induced a group of inflammatory reactive astrocytes that were actively involved in disrupting BBB through MMP3, highlighting a specific group of inflammatory reactive astrocytes as a critical driver for BBB disruption in neurological diseases.

A phenotypic screening platform for identifying chemical modulators of astrocyte reactivity.

Disease, injury and aging induce pathological reactive astrocyte states that contribute to neurodegeneration. Modulating reactive astrocytes therefore represent an attractive therapeutic strategy. Here we describe the development of an astrocyte phenotypic screening platform for identifying chemical modulators of astrocyte reactivity. Leveraging this platform for chemical screening, we identify histone deacetylase 3 (HDAC3) inhibitors as effective suppressors of pathological astrocyte reactivity. We demonstrate that HDAC3 inhibition reduces molecular and functional characteristics of reactive astrocytes in vitro. Transcriptional and chromatin mapping studies show that HDAC3 inhibition disarms pathological astrocyte gene expression and function while promoting the expression of genes associated with beneficial astrocytes. Administration of RGFP966, a small molecule HDAC3 inhibitor, blocks reactive astrocyte formation and promotes neuroprotection in vivo in mice. Collectively, these results establish a platform for discovering modulators of reactive astrocyte states, inform the mechanisms that control astrocyte reactivity and demonstrate the therapeutic benefits of modulating astrocyte reactivity for neurodegenerative diseases.

Disease-Associated Neurotoxic Astrocyte Markers in Alzheimer Disease Based on Integrative Single-Nucleus RNA Sequencing

Alzheimer disease (AD) is an irreversible neurodegenerative disease, and astrocytes play a key role in its onset and progression. The aim of this study is to analyze the characteristics of neurotoxic astrocytes and identify novel molecular targets for slowing down the progression of AD. Single-nucleus RNA sequencing (snRNA-seq) data were analyzed from various AD cohorts comprising about 210,654 cells from 53 brain tissue. By integrating snRNA-seq data with bulk RNA-seq data, crucial astrocyte types and genes associated with the prognosis of patients with AD were identified. The expression of neurotoxic astrocyte markers was validated using 5 × FAD and wild-type (WT) mouse models, combined with experiments such as western blot, quantitative real-time PCR (qRT-PCR), and immunofluorescence. A group of neurotoxic astrocytes closely related to AD pathology was identified, which were involved in inflammatory responses and pathways related to neuron survival. Combining snRNA and bulk tissue data, ZEP36L, AEBP1, WWTR1, PHYHD1, DST and RASL12 were identified as toxic astrocyte markers closely related to disease severity, significantly elevated in brain tissues of 5 × FAD mice and primary astrocytes treated with Aβ. Among them, WWTR1 was significantly increased in astrocytes of 5 × FAD mice, driving astrocyte inflammatory responses, and has been identified as an important marker of neurotoxic astrocytes. snRNA-seq analysis reveals the biological functions of neurotoxic astrocytes. Six genes related to AD pathology were identified and validated, among which WWTR1 may be a novel marker of neurotoxic astrocytes.

Repetitive head trauma and apoE4 induce chronic cerebrovascular alterations that impair tau elimination from the brain

Repetitive mild traumatic brain injuries (r-mTBI) sustained in the military or contact sports have been associated with the accumulation of extracellular tau in the brain, which may contribute to the pathogenesis of neurodegenerative tauopathies. The expression of the apolipoprotein E4 (apoE4) isoform has been associated with higher levels of tau in the brain, and worse clinical outcomes after r-mTBI, though the influence of apoE genotype on extracellular tau dynamics in the brain is poorly understood. We recently demonstrated that extracellular tau can be eliminated across blood-brain barrier (BBB), which is progressively impaired following r-mTBI. The current studies investigated the influence of repetitive mild TBI (r-mTBI) and apoE genotype on the elimination of extracellular solutes from the brain. Following intracortical injection of biotin-labeled tau into humanized apoE-Tr mice, the levels of exogenous tau residing in the brain of apoE4 mice were elevated compared to other isoforms, indicating reduced tau elimination. Additionally, we found exposure to r-mTBI increased tau residence in apoE2 mice, similar to our observations in E2FAD animals. Each of these findings may be the result of diminished tau efflux via LRP1 at the BBB, as LRP1 inhibition significantly reduced tau uptake in endothelial cells and decreased tau transit across an in vitro model of the BBB (basolateral-to-apical). Notably, we showed that injury and apoE status, (particularly apoE4) resulted in chronic alterations in BBB integrity, pericyte coverage, and AQP4 polarization. These aberrations coincided with an atypical reactive astrocytic gene signature indicative of diminished CSF-ISF exchange. Our work found that CSF movement was reduced in the chronic phase following r-mTBI (>18 months post injury) across all apoE genotypes. In summary, we show that apoE genotype strongly influences cerebrovascular homeostasis, which can lead to age-dependent deficiencies in the elimination of toxic proteins from the brain, like tau, particularly in the aftermath of head trauma.

Comparative Transcriptomic Analysis of Cerebellar Astrocytes across Developmental Stages and Brain Regions.

Astrocytes are the most abundant glial cell type in the central nervous system, and they play a crucial role in normal brain function. While gliogenesis and glial differentiation occur during perinatal cerebellar development, the processes that occur during early postnatal development remain obscure. In this study, we conducted transcriptomic profiling of postnatal cerebellar astrocytes at postnatal days 1, 7, 14, and 28 (P1, P7, P14, and P28), identifying temporal-specific gene signatures at each specific time point. Comparing these profiles with region-specific astrocyte differentially expressed genes (DEGs) published for the cortex, hippocampus, and olfactory bulb revealed cerebellar-specific gene signature across these developmental timepoints. Moreover, we conducted a comparative analysis of cerebellar astrocyte gene signatures with gene lists from pediatric brain tumors of cerebellar origin, including ependymoma and medulloblastoma. Notably, genes downregulated at P14, such as Kif11 and HMGB2, exhibited significant enrichment across all pediatric brain tumor groups, suggesting the importance of astrocytic gene repression during cerebellar development to these tumor subtypes. Collectively, our studies describe gene expression patterns during cerebellar astrocyte development, with potential implications for pediatric tumors originating in the cerebellum.

Anti-inflammatory Effects of Umbilical Cord Mesenchymal Stem Cell and Autologous Conditioned Serum on Oligodendrocyte, Astrocyte, and Microglial Specific Gene in Cuprizone Animal Model.

Inflammation, myelin loss, astrocytosis, and microgliosis are pathological signs of the autoimmune and demyelinating disease known as multiple sclerosis (MS). Axonal and neuronal degenerations have basic molecular pathways. The remyelination process can be influenced by the secretome of mesenchymal stem cells due to their capacity for immunomodulation, differentiation, and neuroprotection. Microglial cells are divided into two subgroups: M1 and M2 phenotypes. A crucial component of the microglial function is the colony stimulating factor 1 receptor (CSF1R). We aimed to evaluate the immunomodulating effects of secretome and conditioned serum on the microglial phenotypes and improvement of demyelination in a cuprizone model of MS. The study used 48 male C57BL/6 mice, which were randomly distributed into 6 subgroups (n = 8), i.e., control, cuprizone, MSC (confluency 40% and 80%) secretome group, and blood derived conditioned serum (autologous and humanized). The animals were fed with 0.2% cuprizone diet for 12 weeks. Supplements were injected into the lateral tail vein using a 27-gauge needle every 3 days 500 μl per injection. At 14 days after transplantation, animals from each group were sacrificed and analyzed by Real time PCR. The results showed that the administration of MSC secretome can efficiently reduce expression of pro-inflammatory cytokines (IL-1, IL6 and TNF-α) in the corpus callosum; also, conditioned serum downregulated IL-1. Moreover, the oligodendrocyte-specific gene was upregulated by secretome and conditioned serum treatment. Also, the expression of microglial- specific gene was reduced after treatment. These findings demonstrated that the secretome isolated from MSCs used as a therapy decreased and increased the M1 and M2 levels, respectively, to control neuroinflammation in CPZ mice. In conclusion, the current study showed the viability of devising a method to prepare suitable MSCs and secreted factor to cure neurodegenerative diseases, as well as the capability of regulating MSC secretome patterns by manipulating the cell density.

Single Cell RNASeq Analysis of AD and Control Post Mortem Brain Samples

Alzheimer’s disease (AD) is a devastative late life disease, molecularly characterized by biochemical lesions of amyloid β plaques and Tau tangles. the 2nd most frequent one worldwide with no early detection methods/cure. I have applied single cell sequencing and analysed post mortem brain samples (using a Chromium machine) from 2 AD and 2 control frontal cortex samples. All the samples had ethics approval, and the RNA was tested for quality. The samples were obtained from the Edenborough MRC brain bank. Very few genes have been linked to far to AD (mainly through GWAS studies [1]). Overall, I quantified about 24,000 nuclei. Using statistical, bioinformatics and computational analyses I have detected significant differential expression changes in astrocyte, neuronal and microglial marker genes. I also conducted network analyses. The percentage of reads passing filter was slightly low (68%). Overall output was a bit lower than optimal. There was still over 1.6Bn clusters generated though, which is within the specification for an S1 flow-cell, and base call quality (quality control) was excellent: 97% >Q30. The read counts mapping to genome occupancy rate was also fairy low (63-81%). The number of barcodes is as follows: sample 1 (AD, Braak I-II) - 6,223 sample 2 (control) 6,262 sample 3 (AD Braak I-II) 5,332 sample 4 (AD, Braak I-II) 5,332 as well. The data is available for download under the GEO (accession number GSE175814). to assess global and cell-type specific gene expression impacts in Alzheimer’s versus control brain samples, we performed a holistic comparison of matching cell populations in AD and controls with the software cell Harmony. To identify possible gene regulatory network underlying transcriptional regulation in these distinct populations, we further explored prior evidenced transcription factor targets relationships from cell Harmony. Importantly, we observe both common and cell-type specific core transcription factors regulators, with both enriched downstream targets and regulated transcription factors. Upregulated networks in Astrocytes were associated with broad regulation by HIF1A, SOX2, NRF1, and RB1. the proportion of endothelial cells was higher in the Alzheimer’s disease samples than the neurological control samples. results reveal that the cell type-specific transcriptomic changes in Alzheimer’s disease are associated with 4 molecular pathways: angiogenesis in endothelial cells, immune response in endothelial cells and microglia, myelination in oligodendrocytes, and synaptic signalling in astrocytes and neurons. I also compared the results with microarray data from large cohort studies that examined samples from the prefrontal cortex (Alzheimer’s disease: n = 310; Neurological controls: n = 157) or temporal cortex (Alzheimer’s disease: n = 106; Neurological controls: n = 135). Among the DEGs identified in our snRNA-seq analysis, 1,113 and 764 genes were significantly differentially expressed in the microarray data from the prefrontal cortex and temporal cortex, respectively. subcluster analysis of microglia identified 13 subpopulations; VGF signalling was also found as changed. In the future, imaging of post mortem Alzheimer’s patients may verify brain cellular morphology/cell volume of vascular changes in the Alzheimer’s compared to control samples. My study findings [2] may open new venues to our understanding of the disease underlying molecular processes and cellular signaling. Regarding the glial cell changes, our main conclusion is that the glial cells may be reactive in the disease and not causal.

Brain-wide circuit-specific targeting of astrocytes

Astrocytes are integral components of brain circuitry. They enwrap synapses, react to neuronal activity, and regulate synaptic transmission. Astrocytes are heterogeneous and exhibit distinct features and functions in different circuits. Selectively targeting the astrocytes associated with a given neuronal circuit would enable elucidation of their circuit-specific functions but has been technically challenging to date. Recently, we constructed anterograde transneuronal viral vectors based on yellow fever vaccine YFV-17D. Among them, the replication-incompetent YFVΔNS1-Cre can selectively turn on reporter genes in postsynaptic neurons if the viral gene NS1 is expressed in postsynaptic neurons. Here we show that without exogenous expression of NS1 at the postsynaptic sites, locally injected YFVΔNS1-Cre selectively turns on reporter genes in astrocytes in downstream brain regions. The targeting of astrocytes can occur across the whole brain but is specific for the neuronal circuits traced. Therefore, YFVΔNS1-Cre provides a tool for selective genetic targeting of astrocytes to reveal their circuit-specific roles.

GABA transporters gene expression associates with [18F]FDG‐PET signal in regions typically hypometabolic in Alzheimer’s disease

AbstractBackgroundIt is suggested that the [18F]fluorodeoxyglucose(FDG)‐PET signal reflects astrocyte and neuronal activity, with the excitatory glutamate uptake by astrocytes as the trigger for brain glucose metabolism. The inhibitory neurotransmitter γ‐aminobutyric acid (GABA) is taken up by neurons and astrocytes through the GABA transporters (GAT) 1 and 3. However, its role in the [18F]FDG‐PET signal in healthy aging and Alzheimer’s disease (AD) remains poorly investigated. Thus, we aimed to explore associations between neuronal and astrocytic GABA transporters gene expression with [18F]FDG‐PET in cognitively unimpaired (CU) and AD individuals.MethodWe obtained [18F]FDG‐PET imaging data of CU and AD from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) (Table 1). GAT1 and GAT3 gene expression in the brain was obtained from the Allen Brain Atlas (Table 1, Figure 1). We extracted SUVr (pons as reference region) and mRNA values using the RMINC package and performed Pearson’s correlation to test the association between [18F]FDG‐PET and GABA transporters mRNA (p‐value < 0.05).ResultIn brain regions typically hypometabolic in AD, we observed a strong positive association of [18F]FDG‐PET SUVr with GAT1 and GAT3 gene expression in CU (GAT1: r = 0.93; p < 0.0001; Figure 2A, GAT3: r = 0.94; p < 0.0001; Figure 2B) and AD individuals (GAT1: r = 0.9; p < 0.0001; Figure 2A, GAT3: r = 0.92; p < 0.0001; Figure 2B). By contrast, in brain regions typically resilient, we found a weak correlation with CU individuals (GAT1: r = 0.32; p = 0.016; Figure 2C, GAT3: r = 0.26; p = 0.04; Figure 2D) and no significant correlation in AD patients (GAT1: r = 0.24; p = 0.07; Figure 2C, GAT3: r = 0.24; p = 0.059; Figure 2D).ConclusionOur results demonstrate a significant strong positive association between [18F]FDG‐PET SUVr and GABA transporters gene expression in CU and AD in hypometabolic regions. These findings suggest that GABA transporters are involved with brain glucose metabolism in regions classically vulnerable to AD pathology.

GABA transporters gene expression associates with [18F]FDG‐PET signal in regions typically hypometabolic in Alzheimer’s disease

AbstractBackgroundIt is suggested that the [18F]fluorodeoxyglucose(FDG)‐PET signal reflects astrocyte and neuronal activity, with the excitatory glutamate uptake by astrocytes as the trigger for brain glucose metabolism. The inhibitory neurotransmitter γ‐aminobutyric acid (GABA) is taken up by neurons and astrocytes through the GABA transporters (GAT) 1 and 3. However, its role in the [18F]FDG‐PET signal in healthy aging and Alzheimer’s disease (AD) remains poorly investigated. Thus, we aimed to explore associations between neuronal and astrocytic GABA transporters gene expression with [18F]FDG‐PET in cognitively unimpaired (CU) and AD individuals.MethodWe obtained [18F]FDG‐PET imaging data of CU and AD from the Alzheimer’s Disease Neuroimaging Initiative (ADNI) (Table 1). GAT1 and GAT3 gene expression in the brain was obtained from the Allen Brain Atlas (Table 1, Figure 1). We extracted SUVr (pons as reference region) and mRNA values using the RMINC package and performed Pearson’s correlation to test the association between [18F]FDG‐PET and GABA transporters mRNA (p‐value < 0.05).ResultIn brain regions typically hypometabolic in AD, we observed a strong positive association of [18F]FDG‐PET SUVr with GAT1 and GAT3 gene expression in CU (GAT1: r = 0.93; p < 0.0001; Figure 2A, GAT3: r = 0.94; p < 0.0001; Figure 2B) and AD individuals (GAT1: r = 0.9; p < 0.0001; Figure 2A, GAT3: r = 0.92; p < 0.0001; Figure 2B). By contrast, in brain regions typically resilient, we found a weak correlation with CU individuals (GAT1: r = 0.32; p = 0.016; Figure 2C, GAT3: r = 0.26; p = 0.04; Figure 2D) and no significant correlation in AD patients (GAT1: r = 0.24; p = 0.07; Figure 2C, GAT3: r = 0.24; p = 0.059; Figure 2D).ConclusionOur results demonstrate a significant strong positive association between [18F]FDG‐PET SUVr and GABA transporters gene expression in CU and AD in hypometabolic regions. These findings suggest that GABA transporters are involved with brain glucose metabolism in regions classically vulnerable to AD pathology. ”

Association between brain [18F]FDG‐PET signal and genetic cell markers in the brain tissue

AbstractBackgroundBrain glucose hypometabolism, indexed by [18F]FDG‐PET, is considered a biomarker of neurodegeneration in Alzheimer’s disease (AD). However, other brain cells, such as astrocytes and microglia, also consume considerable amounts of glucose and may contribute to the [18F]FDG‐PET signal in the brain. Thus, the cellular source of the [18F]FDG‐PET signal remains controversial. Here, we aimed to evaluate whether canonical markers of different brain cell types (neuron, oligodendrocyte, astrocyte, and microglia) associate with brain [18F]FDG‐PET signal in humans and a rat model of human amyloidosis.Method[18F]FDG‐PET images were acquired in ten‐month‐old APP/PS1 (TgF344‐AD, n = 8) and wild‐type (WT, n = 6) rats. Next, we evaluated the gene expression of NeuN, Olig2, GFAP, S100B, and IBA1 in the frontal and temporoparietal cortices and cerebellum. Association maps integrating mRNA with brain [18F]FDG‐PET signal were conducted at the voxel level using RMINC. [18F]FDG‐PET and brain tissue mRNA (GFAP, ACSA2, S100B, GLAST, IBA1, CSF1R, TREM2, Olig2, Cldn11, NeuN) of cognitively unimpaired patients were obtained from ADNI and Allen Brain Atlas database, respectively. In humans, mean regional [18F]FDG‐PET SUVr (reference region: pons) and mRNA expression were used to evaluate their association using a gaussian model. Differences were considered statistically significant at p<0.05 (t>2).ResultGFAP and S100B mRNA levels in the temporoparietal and frontal cortices positively correlated with brain [18F]FDG‐PET signal (local maxima, GFAPt(13) = 9.4, S100Bt(13) = 6.6; Fig1B‐C). No associations were found with TREM2, Olig2 and NeuN (t(13)<2; Fig1D‐F). We expanded our analysis by comparing gene astrocyte markers and astrocyte gene cell markers in humans. At the regional level, we found that FDG‐PET SUVr and astrocyte mRNA were positively associated: S100B (R‐squared = 0.34), GFAP (R‐squared = 0.33), GLAST (R‐squared = 0.31) and ACSA‐2 (R‐squared = 0.31; Fig1G).ConclusionOur findings suggest astrocyte mRNA markers are more closely associated with brain [18F]FDG‐PET signal than neuronal, oligodendrocyte, and microglial mRNA markers.

Astrocyte Gpc5 overexpression prevents hippocampal synaptic hyperexcitability in APP/PS1 mouse

AbstractBackgroundAstrocytes are essential regulators of synaptic function throughout life. They regulate the formation, maturation and elimination of new synapses, as well as modulate synaptic transmission, and provide nutritional support to neurons. Our lab and others have shown that with healthy aging astrocytes change their properties altering the expression of genes important for synaptic maintenance in brain regions vulnerable to Alzheimer’s disease (AD). We now investigated whether astrocytes present the same age‐associated changes in mouse models of AD. We also assess whether these astrocyte changes are contributing to the synaptic dysfunction and cognitive alterations observed in AD mouse models.MethodWe use BacTRAP technology to isolate hippocampal astrocyte RNA from two different mouse models of Alzheimer’s disease: a model of Tau pathology (MAPTP301S) and amyloidosis (APPswe/PS1dE9). We analyze astrocyte transcriptome at 3 different timepoints: 4, 6 and 12 months, and in male and female mice independently. To assess the contribution of astrocyte alterations to synaptic dysfunction, we manipulate astrocyte gene expression using a viral approach in APP/PS1 mice, and characterized synaptic function using electrophysiology path‐clamp recordings, and spatial learning with Barnes maze test.ResultBoth APP/PS1 and MAPTP301S models showed an upregulation of genes involved in astrocyte reactivity and neuroinflammation and a downregulation of genes important for synaptic support. Among the genes that were downregulated was Glypican 5 (Gpc5), an astrocyte factor important for synaptic maturation and stabilization. Interestingly, Gpc5 downregulation has also been reported in several other human Alzheimer’s disease transcriptional studies. When we overexpress Gpc5 in astrocytes in APP/PS1 mice, we found that APP/PS1 mice injected with the control virus showed an increase postsynaptic frequency in CA1 pyramidal neurons, and this hyperexcitability was no present in APP/PS1 mice overexpressing Gpc5. In addition, preliminary cognitive results from the Barnes maze test suggest that astrocyte Gpc5 overexpression also improves spatial learning in APP/PS1 and WT mice.ConclusionThis data show that astrocyte Gpc5 downregulation might contribute to synaptic dysfunction in AD. Overexpressing Gpc5 in astrocytes prevents synaptic hyperexcitability in APP/PS1 mice. These results open exciting new venues for using astrocytic factors as novel targets in AD.

Association between brain [18F]FDG‐PET signal and genetic cell markers in the brain tissue

AbstractBackgroundBrain glucose hypometabolism, indexed by [18F]FDG‐PET, is considered a biomarker of neurodegeneration in Alzheimer’s disease (AD). However, other brain cells, such as astrocytes and microglia, also consume considerable amounts of glucose and may contribute to the [18F]FDG‐PET signal in the brain. Thus, the cellular source of the [18F]FDG‐PET signal remains controversial. Here, we aimed to evaluate whether canonical markers of different brain cell types (neuron, oligodendrocyte, astrocyte, and microglia) associate with brain [18F]FDG‐PET signal in humans and a rat model of human amyloidosis.Method[18F]FDG‐PET images were acquired in ten‐month‐old APP/PS1 (TgF344‐AD, n = 8) and wild‐type (WT, n = 6) rats. Next, we evaluated the gene expression of NeuN, Olig2, GFAP, S100B, and IBA1 in the frontal and temporoparietal cortices and cerebellum. Association maps integrating mRNA with brain [18F]FDG‐PET signal were conducted at the voxel level using RMINC. [18F]FDG‐PET and brain tissue mRNA (GFAP, ACSA2, S100B, GLAST, IBA1, CSF1R, TREM2, Olig2, Cldn11, NeuN) of cognitively unimpaired patients were obtained from ADNI and Allen Brain Atlas database, respectively. In humans, mean regional [18F]FDG‐PET SUVr (reference region: pons) and mRNA expression were used to evaluate their association using a gaussian model. Differences were considered statistically significant at p<0.05 (t>2).ResultGFAP and S100B mRNA levels in the temporoparietal and frontal cortices positively correlated with brain [18F]FDG‐PET signal (local maxima, GFAPt(13) = 9.4, S100Bt(13) = 6.6; Fig1B‐C). No associations were found with TREM2, Olig2 and NeuN (t(13)<2; Fig1D‐F). We expanded our analysis by comparing gene astrocyte markers and astrocyte gene cell markers in humans. At the regional level, we found that FDG‐PET SUVr and astrocyte mRNA were positively associated: S100B (R‐squared = 0.34), GFAP (R‐squared = 0.33), GLAST (R‐squared = 0.31) and ACSA‐2 (R‐squared = 0.31; Fig1G).ConclusionOur findings suggest astrocyte mRNA markers are more closely associated with brain [18F]FDG‐PET signal than neuronal, oligodendrocyte, and microglial mRNA markers.

Restoring periodontal tissue homoeostasis prevents cognitive decline by reducing the number of Serpina3nhigh astrocytes in the hippocampus

Cognitive decline has been linked to periodontitis through an undetermined pathophysiological mechanism. This study aimed to explore the mechanism underlying periodontitis-related cognitive decline and identify therapeutic strategies for this condition. Using single-nucleus RNA sequencing we found that changes in astrocyte number, gene expression, and cell‒cell communication were associated with cognitive decline in mice with periodontitis. In addition, activation of the NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome was observed to decrease the phagocytic capability of macrophages and reprogram macrophages to a more proinflammatory state in the gingiva, thus aggravating periodontitis. To further investigate this finding, lipid-based nanoparticles carrying NLRP3 siRNA (NPsiNLRP3) were used to inhibit overactivation of the NLRP3 inflammasome in gingival macrophages, restoring the oral microbiome and reducing periodontal inflammation. Furthermore, gingival injection of NPsiNLRP3 reduced the number of Serpina3nhigh astrocytes in the hippocampus and prevented cognitive decline. This study provides a functional basis for the mechanism by which the destruction of periodontal tissues can worsen cognitive decline and identifies nanoparticle-mediated restoration of gingival macrophage function as a novel treatment for periodontitis-related cognitive decline.

Nicotine, THC, and Dolutegravir Modulate E-Cigarette-Induced Changes in Addiction- and Inflammation-Associated Genes in Rat Brains and Astrocytes.

E-cigarette use has been marketed as a safer alternative to traditional cigarettes, as a means of smoking cessation, and are used at a higher rate than the general population in people with HIV (PWH). Early growth receptor 2 (EGR2) and Activity-Regulated Cytoskeleton-Associated Protein (ARC) have a role in addiction, synaptic plasticity, inflammation, and neurodegeneration. This study showed that 10 days of exposure to e-cigarette vapor altered gene expression in the brains of 6-month-old, male, Sprague Dawley rats. Specifically, the e-cigarette solvent vapor propylene glycol (PG) downregulated EGR2 and ARC mRNA expression in frontal cortex, an effect which was reversed by nicotine (NIC) and THC, suggesting that PG could have a protective role against NIC and cannabis dependence. However, in vitro, PG upregulated EGR2 and ARC mRNA expression at 18 h in cultured C6 rat astrocytes suggesting that PG may have neuroinflammatory effects. PG-induced upregulation of EGR2 and ARC mRNA was reversed by NIC but not THC. The HIV antiretroviral DTG reversed the effect NIC had on decreasing PG-induced upregulation of EGR2, which is concerning because EGR2 has been implicated in HIV latency reversal, T-cell apoptosis, and neuroinflammation, a process that underlies the development of HIV-associated neurocognitive disorders.

A human in vitro neuronal model for studying homeostatic plasticity at the network level

Mechanisms that underlie homeostatic plasticity have been extensively investigated at single-cell levels in animal models, but are less well understood at the network level. Here, we used microelectrode arrays to characterize neuronal networks following induction of homeostatic plasticity in human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons co-cultured with rat astrocytes. Chronic suppression of neuronal activity through tetrodotoxin (TTX) elicited a time-dependent network re-arrangement. Increased expression of AMPA receptors and the elongation of axon initial segments were associated with increased network excitability following TTX treatment. Transcriptomic profiling of TTX-treated neurons revealed up-regulated genes related to extracellular matrix organization, while down-regulated genes related to cell communication; also astrocytic gene expression was found altered. Overall, our study shows that hiPSC-derived neuronal networks provide a reliable invitro platform to measure and characterize homeostatic plasticity at network and single-cell levels; this platform can be extended to investigate altered homeostatic plasticity in brain disorders.

Transcriptomic and morphological maturation of human astrocytes in cerebral organoids.

Cerebral organoids (CerOrgs) derived from human induced pluripotent stem cells (iPSCs) are a valuable tool to study human astrocytes and their interaction with neurons and microglia. The timeline of astrocyte development and maturation in this model is currently unknown and this limits the value and applicability of the model. Therefore, we generated CerOrgs from three healthy individuals and assessed astrocyte maturation after 5, 11, 19, and 37 weeks in culture. At these four time points, the astrocyte lineage was isolated based on the expression of integrin subunit alpha 6 (ITGA6). Based on the transcriptome of the isolated ITGA6-positive cells, astrocyte development started between 5 and 11 weeks in culture and astrocyte maturation commenced after 11 weeks in culture. After 19 weeks in culture, the ITGA6-positive astrocytes had the highest expression of human mature astrocyte genes, and the predicted functional properties were related to brain homeostasis. After 37 weeks in culture, a subpopulation of ITGA6-negative astrocytes appeared, highlighting the heterogeneity within the astrocytes. The morphology shifted from an elongated progenitor-like morphology to the typical bushy astrocyte morphology. Based on the morphological properties, predicted functional properties, and the similarities with the human mature astrocyte transcriptome, we concluded that ITGA6-positive astrocytes have developed optimally in 19-week-old CerOrgs.

An 840 kb distant upstream enhancer is a crucial regulator of catecholamine-dependent expression of the Bdnf gene in astrocytes.

Brain-derived neurotrophic factor (BDNF) plays a fundamental role in the developing and adult nervous system, contributing to neuronal survival, differentiation, and synaptic plasticity. Dysregulation of BDNF synthesis, secretion or signaling has been associated with many neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. Although the transcriptional regulation of the Bdnf gene has been extensively studied in neurons, less is known about the regulation and function of BDNF in non-neuronal cells. The most abundant type of non-neuronal cells in the brain, astrocytes, express BDNF in response to catecholamines. However, genetic elements responsible for this regulation have not been identified. Here,we investigated four potential Bdnf enhancer regions and based on reporter gene assays, CRISPR/Cas9 engineering and CAPTURE-3C-sequencing we conclude that a region 840 kb upstream of the Bdnf gene regulates catecholamine-dependent expression of Bdnf in rodent astrocytes. We also provide evidence that this regulation is mediated by CREB and AP1 family transcription factors. This is the first report of an enhancer coordinating the transcription of Bdnf gene in non-neuronal cells.

Selective neuroimmune modulation by type I interferon drives neuropathology and neurologic dysfunction following traumatic brain injury.

Accumulating evidence suggests that type I interferon (IFN-I) signaling is a key contributor to immune cell-mediated neuropathology in neurodegenerative diseases. Recently, we demonstrated a robust upregulation of type I interferon-stimulated genes in microglia and astrocytes following experimental traumatic brain injury (TBI). The specific molecular and cellular mechanisms by which IFN-I signaling impacts the neuroimmune response and neuropathology following TBI remains unknown. Using the lateral fluid percussion injury model (FPI) in adult male mice, we demonstrated that IFN α/β receptor (IFNAR) deficiency resulted in selective and sustained blockade of type I interferon-stimulated genes following TBI as well as decreased microgliosis and monocyte infiltration. Molecular alteration of reactive microglia also occurred with diminished expression of genes needed for MHC class I antigen processing and presentation following TBI. This was associated with decreased accumulation of cytotoxic T cells in the brain. The IFNAR-dependent modulation of the neuroimmune response was accompanied by protection from secondary neuronal death, white matter disruption, and neurobehavioral dysfunction. These data support further efforts to leverage the IFN-I pathway for novel, targeted therapy of TBI.