Primary Human Astrocytes Research Articles

Single-cell analysis of osmoregulation reveals heterogeneity of aquaporin 4 functionality in human astrocytes.

The water channel aquaporin 4 (AQP4) contributes to water flow and waste removal across the blood-brain barrier and its levels, organization and localization are perturbed in various neurological diseases, including Alzheimer's Disease. This renders AQP4 a potentially valuable therapeutic target. However, most functional assays aimed at identifying modulators of AQP4 function are performed with primary rodent cells and do not consider inter-cellular variations in AQP4 abundance and presentation. To address this, we have established and applied a robust live cell microscopy assay that captures the contribution of AQP4 in the osmotically driven (de-)quenching of the vital dye Calcein-AM with single-cell resolution. Using human astrocytoma cells, we found that performing measurements on cellular regions instead of whole fields of view yielded a more sensitive readout of the osmotic response, which correlated with AQP4 abundance. Stable co-expression of the two major AQP4 isoforms, but not of the individual isoforms, provoked a faster adaptation to osmotic changes, while siRNA-mediated knockdown of AQP4 had the opposite effect. Post-hoc correlation with the canonical membrane marker CD44 revealed that the speed of the osmotic response scaled with AQP4 membrane enrichment. Coating the substrate with laminin promoted AQP4 membrane enrichment, while cell confinement with fixed-size micropatterns further increased the speed of osmoregulation, underscoring the influence of extracellular factors. The osmotic response of primary fetal astrocytes and human iPSC-derived astrocyte models was comparable to AQP4-deficient astrocytoma cells, in line with their low AQP4 levels and indicative of their immature state. In conclusion, a correlative single-cell approach based on the quantification of Calcein-AM quenching capacity, AQP4 abundance and AQP4 membrane enrichment, allows resolving osmoregulation in a more sensitive manner and reveals heterogeneity between and within human astrocyte (-like) cultures, which could prove crucial for future screens aimed at identifying AQP4 modulators.

3D Neurovascular Unit Tissue Model to Assess Responses to Traumatic Brain Injury.

The neurovascular unit (NVU) is a critical interface in the central nervous system that links vascular interactions with glial and neural tissue. Disruption of the NVU has been linked to the onset and progression of neurodegenerative diseases. Despite its significance the NVU remains challenging to study in a physiologically relevant manner. Here, a 3D cell triculture model of the NVU is developed that incorporates human primary brain microvascular endothelial cells, astrocytes, and pericytes into a tissue system that can be sustained invitro for several weeks. This tissue model helps recapitulate the complexity of the NVU and can be used to interrogate the mechanisms of disease and cell-cell interactions. The NVU tissue model displays elevated cell death and inflammatory responses following mechanical damage, to emulate traumatic brain injury (TBI) under controlled laboratory conditions, including lactate dehydrogenase (LDH) release, elevated inflammatory markers TNF-α and monocyte chemoattractant cytokines MCP-2 and MCP-3 and reduced expression of the tight junction marker ZO-1. This 3D tissue model serves as a tool for deciphering mechanisms of TBIs and immune responses associated with the NVU.

DEVELOPMENT OF A HUMAN-SPECIfiC 3D IN VITRO PAEDIATRIC CEREBELLAR TUMOUR MODEL USING DECELLULARISED BRAIN AUTOPSY TISSUE

Abstract AIMS Group-3 medulloblastoma (MB) is a paediatric cerebellar tumour with a dismal prognosis. Our objective was to establish a 3D in vitro model termed ‘tumoursphere matrix’ to recapitulate physiologically-relevant communication between tumour cells and reactive brain elements, aiming to discern differential gene expression in cancer cells upon co-culture with astrocytes and human brain extracellular matrix (ECM). METHOD Non-disease human autopsy brain was decellularised to generate ECM and extensively characterised to show DNA reduction, ECM ligand retention and compatibility with different cell lines. D283, D341 and D425 cells were co-cultured with primary human cerebellar astrocytes within a PEGDA hydrogel containing decellularised ECM. Following 7 days of culture, co-cultured cells were separated using fluorescence activated cell sorting to generate individual cell populations for transcriptomic analysis. RESULTS ECM characterisation showed a significant reduction in cellular material whilst retaining ECM ligands. Reduction of DNA content to 174 ng/mg was corroborated by an absence of nuclei in immunohistochemical staining. Colorimetric assays indicated collagen and glycosaminoglycan retention in decellularised ECM, and immunofluorescence confirmed retention of fibronectin, laminin and hyaluronic acid. Detection of all ECM ligands was cross-validated by Orbitrap-Secondary Ion Mass Spectrometry. Primary human cerebellar astrocytes, D283, D341 and D425 cells were cultured as spheroids within the ECM/PEGDA hydrogel for 7 days with no significant decrease in cellular viability. Cytokine and Human Matrix Metalloproteinase (MMP) Antibody Arrays showed retained cytokine and MMP expression in all three cell lines in the presence of ECM material. CONCLUSION We have developed a bespoke 3D model which can co-culture cells for over 7 days, whilst recapitulating the in vivo tumour environment. RNA sequencing on all 3 cell lines in mono and co-culture is currently in progress to identify differentially expressed genes in medulloblastoma cells upon astrocytic and brain ECM crosstalk. We hypothesise that biochemical signalling underlying this communication network will reveal putative therapeutic vulnerabilities.

A Human Brain-Chip for Modeling Brain Pathologies and Screening Blood–Brain Barrier Crossing Therapeutic Strategies

Background/Objectives: The limited translatability of preclinical experimental findings to patients remains an obstacle for successful treatment of brain diseases. Relevant models to elucidate mechanisms behind brain pathogenesis, including cell-specific contributions and cell-cell interactions, and support successful targeting and prediction of drug responses in humans are urgently needed, given the species differences in brain and blood-brain barrier (BBB) functions. Human microphysiological systems (MPS), such as Organ-Chips, are emerging as a promising approach to address these challenges. Here, we examined and advanced a Brain-Chip that recapitulates aspects of the human cortical parenchyma and the BBB in one model. Methods: We utilized human primary astrocytes and pericytes, human induced pluripotent stem cell (hiPSC)-derived cortical neurons, and hiPSC-derived brain microvascular endothelial-like cells and included for the first time on-chip hiPSC-derived microglia. Results: Using Tumor necrosis factor alpha (TNFα) to emulate neuroinflammation, we demonstrate that our model recapitulates in vivo-relevant responses. Importantly, we show microglia-derived responses, highlighting the Brain-Chip’s sensitivity to capture cell-specific contributions in human disease-associated pathology. We then tested BBB crossing of human transferrin receptor antibodies and conjugated adeno-associated viruses. We demonstrate successful in vitro/in vivo correlation in identifying crossing differences, underscoring the model’s capacity as a screening platform for BBB crossing therapeutic strategies and ability to predict in vivo responses. Conclusions: These findings highlight the potential of the Brain-Chip as a reliable and time-efficient model to support therapeutic development and provide mechanistic insights into brain diseases, adding to the growing evidence supporting the value of MPS in translational research and drug discovery.

Astrocytes initiate autophagic flux and maintain cell viability after internalizing non-active native extracellular α-synuclein

Astrocytes are tasked with regulating the synaptic environment. Early stages of various neurodegenerative diseases are characterized by synapse loss, and astrocytic atrophy and dysfunction has been proposed as a possible cause. α-Synuclein (αS) is a highly expressed neuronal protein located in the synapse that can be released in the extracellular space. Evidence points to astrocytes as being responsible for uptake and degradation of extracellular αS. Therefore, misfolded active fibrillized αS resulting in protein inclusions and aggregates could be due to astrocytic dysfunction. Despite these pathological hallmarks and lines of evidence, the autophagic function of astrocytes in response to monomeric non-active αS to model healthy conditions has not been investigated. Human primary cortical astrocytes were treated with 100 nM of extracellular monomeric non-active αS alone, and in combination with N-terminal binding monomeric γ-synuclein (γS) as a control. Western blot analysis and super resolution imaging of HiLyte-488 labeled αS confirmed successful internalization of αS at 12, 24 and 48 h after treatment, while αS dimers were only observed at 48 h. Western blot analysis also confirmed αS's ability to induce autophagic flux by 48 h. Annexin V/PI flow cytometry results revealed increased early apoptosis at 24 h, but which resolved itself by 48 h, indicating no cell death in cortical astrocytes at all time points, suggesting astrocytes can manage the protein degradation demand of monomeric αS in healthy physiological conditions. Likewise, astrocytes reduced secretion of apolipoprotein (ApoE), a protein involved in pro-inflammatory pathways, synapse regulation, and autophagy by 12 h. Similarly, total c-JUN protein levels, a transcription factor involved in pro-inflammatory pathways increased by 12 h in the nuclear fraction. Therefore, astrocytes are able to respond and degrade αS in healthy physiological conditions, and astrocyte dysfunction could precede detrimental αS accumulation.

Tuning a bioengineered hydrogel for studying astrocyte reactivity in glioblastoma

Astrocytes play many essential roles in the central nervous system (CNS) and are altered significantly in disease. These reactive astrocytes contribute to neuroinflammation and disease progression in many pathologies, including glioblastoma (GB), an aggressive form of brain cancer. Current in vitro platforms do not allow for accurate modeling of reactive astrocytes. In this study, we sought to engineer a simple bioengineered hydrogel platform that would support the growth of primary human astrocytes and allow for accurate analysis of various reactive states. After validating this platform using morphological analysis and qPCR, we then used the platform to begin investigating how astrocytes respond to GB derived extracellular vesicles (EVs) and soluble factors (SF). These studies reveal that EVs and SFs induce distinct astrocytic states. In future studies, this platform can be used to study how astrocytes transform the tumor microenvironment in GB and other diseases of the CNS. Statement of significanceRecent work has shown that astrocytes help maintain brain homeostasis and may contribute to disease progression in diseases such as glioblastoma (GB), a deadly primary brain cancer. In vitro models allow researchers to study basic mechanisms of astrocyte biology in healthy and diseased conditions, however current in vitro systems do not accurately mimic the native brain microenvironment. In this study, we show that our hydrogel system supports primary human astrocyte culture with an accurate phenotype and allows us to study how astrocytes change in response to a variety of inflammatory signals in GB. This platform could be used further investigate astrocyte behavior and possible therapeutics that target reactive astrocytes in GB and other brain diseases.

Engineered 3D human neurovascular model of Alzheimer's disease to study vascular dysfunction

The blood–brain barrier (BBB) serves as a selective filter that prevents harmful substances from entering the healthy brain. Dysfunction of this barrier is implicated in several neurological diseases. In the context of Alzheimer's disease (AD), BBB breakdown plays a significant role in both the initiation and progression of the disease. This study introduces a three-dimensional (3D) self-assembled in vitro model of the human neurovascular unit to recapitulate some of the complex interactions between the BBB and AD pathologies. It incorporates primary human brain endothelial cells, pericytes and astrocytes, and stem cell-derived neurons and astrocytes harboring Familial AD (FAD) mutations. Over an extended co-culture period, the model demonstrates increased BBB permeability, dysregulation of key endothelial and pericyte markers, and morphological alterations mirroring AD pathologies. The model enables visualization of amyloid-beta (Aβ) accumulation in both neuronal and vascular compartments. This model may serve as a versatile tool for neuroscience research and drug development to provide insights into the dynamic relationship between vascular dysfunction and AD pathogenesis.

Assessment and Evaluation of Contemporary Approaches for Astrocyte Differentiation from hiPSCs: A Modeling Paradigm for Alzheimer's Disease

BackgroundAstrocytes have recently gained attention as key players in the pathogenesis of neurodegenerative diseases, including Alzheimer's disease. Numerous differentiation protocols have been developed to study human astrocytes in vitro. However, the properties of the resulting glia are inconsistent, making it difficult to select an appropriate method for a given research question. Therefore, we compared three approaches for the generation of iPSC-derived astrocytes. We performed a detailed analysis using a widely used long serum-free (LSFP) and short serum-free (SSFP) protocol, as well as a TUSP protocol using serum for a limited time of differentiation.ResultsWe used RNA sequencing and immunochemistry to characterize the cultures. Astrocytes generated by the LSFP and SSFP methods differed significantly in their characteristics from those generated by the TUSP method using serum. The TUSP astrocytes had a less neuronal pattern, showed a higher degree of extracellular matrix formation, and were more mature. The short-term presence of FBS in the medium facilitated the induction of astroglia characteristics but did not result in reactive astrocytes. Data from cell-type deconvolution analysis applied to bulk transcriptomes from the cultures assessed their similarity to primary and fetal human astrocytes.ConclusionsOverall, our analyses highlight the need to consider the advantages and disadvantages of a given differentiation protocol for solving specific research tasks or drug discovery studies with iPSC-derived astrocytes.

Human Immunodeficiency Virus (HIV-1) Targets Astrocytes via Cell-Free and Cell-Associated Infection.

Infection of astrocytes by Human Immunodeficiency Virus (HIV-1) remains a topic of debate, with conflicting data, yet instances of astrocytes containing viral DNA have been observed in vivo. In this study, we aimed to elucidate potential routes through which astrocytes could be infected and their ability to produce infectious particles using primary human astrocytes. We infected primary astrocytes derived from either neuroprogenitor cells (NPCs) or induced pluripotent stem cells (iPSCs) that express both C-X-C chemokine receptor type 4 (CXCR4) and the C-C chemokine receptor type 5 (CCR5) coreceptors, using either cell-free HIV-1 virus directly or cell-associated virus indirectly through infected macrophages and microglia. Low-level infectivity by cell-free viruses was primarily attributed to a defect in the entry process. Bypassing HIV-specific receptor-mediated entry using pseudotyped viruses resulted in productive infection and the release of infectious particles. These findings suggest that astrocytes may be one of the potential sources of neurotoxicity in HIV-associated neurocognitive disorders (HAND) and could possibly act as reservoirs for HIV in the central nervous system (CNS).

Electrostimulation via a 3D-printed, biomimetic, neurotrophic, electroconductive scaffold for the promotion of axonal regrowth after spinal cord injury

Spinal cord injury (SCI) is a devastating neurotrauma, affecting 250,000 to 500,000 people annually, and typically results in paralysis. Electrostimulation can promote neuronal growth, but the formation of a lesion cavity post-SCI inhibits regrowth, limiting its efficacy. Bridging the lesion with a structured, electroactive substrate to direct electrostimulation to growing neurites could support and drive neuronal regrowth through the lesion to enable functional recovery but to date, no such platform exists. This study describes the development of an electroconductive (15 ± 5 S/m), 3D-printed scaffold, comprising a polypyrrole/polycaprolactone framework filled with biomimetic & neurotrophic extracellular matrix. 3D printing allowed inclusion of channels in the scaffold designed to mimic the size of human corticospinal tracts to direct electrostimulation to growing neurons. Scaffolds exhibited excellent biocompatibility with both neurons and human primary astrocytes and maintained electrical and biofunctionality when scaled to match the size of human corticospinal tracts. When neurons were cultured for 7 days on the scaffolds under continuous electrostimulation (200 mV/mm, 12 Hz), significantly longer neurites were observed on electrically stimulated electroconductive scaffolds. These results demonstrate that electrostimulation applied via an anatomically-mimetic, 3D-printed electroconductive scaffold drives neurite outgrowth and represents a promising approach for treatment of spinal cord injury.

Usutu virus and West Nile virus use a transcellular route of neuroinvasion across an in vitro model of the human blood–brain barrier

West Nile virus (WNV) leads to thousands of cases of severe neurological disease in humans each year. Usutu virus (USUV) is closely related to WNV, but rarely induces disease in humans. We hypothesised that USUV is less able to cross the blood-brain barrier (BBB) and, consequently, is less likely to infect the brain. Therefore, we developed an in vitro BBB model consisting of primary human brain microvascular endothelial cells, pericytes and astrocytes. Both USUV and WNV invaded across the in vitro BBB via a transcellular mechanism in the absence of barrier disruption. USUV replicated to lower titres than WNV but induced a comparable cytokine and chemokine response, with modulation of key factors associated with barrier function and immune-cell migration. In conclusion, USUV appears attenuated in its ability to replicate at this interface compared with WNV, but further work must be done to identify key determinants underlying the differing clinical presentations.

BATF2 is a regulator of interferon-γ signaling in astrocytes during neuroinflammation.

Astrocytic interferon (IFN)γ signaling is associated with a reduction in neuroinflammation. We have previously shown that the benefits of astrocytic IFNγ arise from a variety of mechanisms; however, downstream effectors responsible for regulating this protection are unknown. We address this by identifying a specific transcription factor that may play a key role in modulating the consequences of IFNγ signaling. RNA-sequencing of primary human astrocytes treated with IFNγ revealed basic leucine zipper ATF-like transcription factor ( BATF )2 as a highly expressed interferon-specific gene. Primarily studied in the periphery, BATF2 has been shown to exert both inflammatory and protective functions; however, its function in the central nervous system (CNS) is unknown. Here, we demonstrate that human spinal cord astrocytes upregulate BATF2 transcript and protein in an IFNγ-specific manner. Additionally, we found that BATF2 prevents overexpression of interferon regulatory factor (IRF)1 and IRF1 targets such as Caspase-1, which are known downstream pro-inflammatory mediators. We also show that Batf2 -/- mice exhibit exacerbated clinical disease severity in a murine model of CNS autoimmunity, characterized by an increase in both CNS immune cell infiltration and demyelination. Batf2 -/- mice also exhibit increased astrocyte-specific expression of IRF1 and Caspase-1, suggesting an amplified interferon response in vivo . Further, we demonstrate that BATF2 is expressed primarily in astrocytes in MS lesions and that this expression is co-localized with IRF1. Collectively, our results further support a protective role for IFNγ and implicate BATF2 as a key suppressor of overactive immune signaling in astrocytes during neuroinflammation.

SARS-CoV-2 infects neurons, astrocytes, choroid plexus epithelial cells and pericytes of the human central nervous system in vitro.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is associated with neurological sequelae including haemorrhage, thrombosis and ischaemic necrosis and encephalitis. However, the mechanism by which this occurs is unclear. Neurological disease associated with COVID-19 has been proposed to occur following direct infection of the central nervous system and/or indirectly by local or systemic immune activation. We evaluated the expression of angiotensin-converting enzyme-2 and transmembrane protease, serine 2 (TMPRSS2) in brain tissue from five healthy human donors and observed low-level expression of these proteins in cells morphologically consistent with astrocytes, neurons and choroidal ependymal cells within the frontal cortex and medulla oblongata. Primary human astrocytes, neurons, choroid plexus epithelial cells and pericytes supported productive SARS-CoV-2 infection with ancestral, Alpha, Delta and Omicron variants. Infected cells supported the full viral life cycle, releasing infectious virus particles. In contrast, primary brain microvascular endothelial cells and microglia were refractory to SARS-CoV-2 infection. These data support a model whereby SARS-CoV-2 can infect human brain cells, and the mechanism of viral entry warrants further investigation.

Multielectrode array characterization of human induced pluripotent stem cell derived neurons in co-culture with primary human astrocytes.

Human induced pluripotent stem cells (hiPSCs) derived into neurons offer a powerful in vitro model to study cellular processes. One method to characterize functional network properties of these cells is using multielectrode arrays (MEAs). MEAs can measure the electrophysiological activity of cellular cultures for extended periods of time without disruption. Here we used WTC11 hiPSCs with a doxycycline-inducible neurogenin 2 (NGN2) transgene differentiated into neurons co-cultured with primary human astrocytes. We achieved a synchrony index ∼0.9 in as little as six-weeks with a mean firing rate of ∼13 Hz. Previous reports show that derived 3D brain organoids can take several months to achieve similar strong network burst synchrony. We also used this co-culture to model aspects of blood-brain barrier breakdown by using human serum. Our fully human co-culture achieved strong network burst synchrony in a fraction of the time of previous reports, making it an excellent first pass, high-throughput method for studying network properties and neurodegenerative diseases.

Astrocytic stress response is induced by exposure to astrocyte-binding antibodies expressed by plasmablasts from pediatric patients with acute transverse myelitis

BackgroundPediatric acute transverse myelitis (ATM) accounts for 20–30% of children presenting with a first acquired demyelinating syndrome (ADS) and may be the first clinical presentation of a relapsing ADS such as multiple sclerosis (MS). B cells have been strongly implicated in the pathogenesis of adult MS. However, little is known about B cells in pediatric MS, and even less so in pediatric ATM. Our lab previously showed that plasmablasts (PB), the earliest B cell subtype producing antibody, are expanded in adult ATM, and that these PBs produce self-reactive antibodies that target neurons. The goal of this study was to examine PB frequency and phenotype, immunoglobulin selection, and B cell receptor reactivity in pediatric patients presenting with ATM to gain insight to B cell involvement in disease.MethodsWe compared the PB frequency and phenotype of 5 pediatric ATM patients and 10 pediatric healthy controls (HC) and compared them to previously reported adult ATM patients using cytometric data. We purified bulk IgG from the plasma samples and cloned 20 recombinant human antibodies (rhAbs) from individual PBs isolated from the blood. Plasma-derived IgG and rhAb autoreactivity was measured by mean fluorescence intensity (MFI) in neurons and astrocytes of murine brain or spinal cord and primary human astrocytes. We determined the potential impact of these rhAbs on astrocyte health by measuring stress and apoptotic response.ResultsWe found that pediatric ATM patients had a reduced frequency of peripheral blood PB. Serum IgG autoreactivity to neurons in EAE spinal cord was similar in the pediatric ATM patients and HC. However, serum IgG autoreactivity to astrocytes in EAE spinal cord was reduced in pediatric ATM patients compared to pediatric HC. Astrocyte-binding strength of rhAbs cloned from PBs was dependent on somatic hypermutation accumulation in the pediatric ATM cohort, but not HC. A similar observation in predilection for astrocyte binding over neuron binding of individual antibodies cloned from PBs was made in EAE brain tissue. Finally, exposure of human primary astrocytes to these astrocyte-binding antibodies increased astrocytic stress but did not lead to apoptosis.ConclusionsDiscordance in humoral immune responses to astrocytes may distinguish pediatric ATM from HC.

STEM-01. DEVELOPMENT OF A HUMAN-SPECIFIC 3DIN VITRO PAEDIATRIC CEREBELLAR TUMOUR MODEL USING DECELLULARISED BRAIN AUTOPSY TISSUE

Abstract BACKGROUND Medulloblastoma (MB) and Atypical Teratoid/ Rhabdoid Tumours (ATRT) represent paediatric cerebellar tumours with a dismal prognosis. For ATRT in particular, 5-year survival remains at less than 32%. We have developed a 3D in vitro model termed ‘tumoursphere matrix’ which recapitulates in vivo crosstalk between tumour and reactive brain. METHODS AND RESULTS We co-cultured D283 (Group 3-MYC amplified MB) and BT12 (ATRT-MYC) cells with primary human cerebellar astrocytes within a PEGDA hydrogel containing decellularised cerebellar extracellular matrix (ECM) derived from non-disease human autopsy brain. Cerebellar ECM was extensively characterised, showing a significant reduction in cellular material whilst retaining ECM ligands. Reduction of DNA content to 174 ng/mg was corroborated by an absence of nuclei in immunohistochemical staining. Colorimetric assays indicated collagen and glycosaminoglycan retention in decellularised ECM, and Immunofluorescence confirmed retention of fibronectin, laminin and hyaluronic acid. Detection of all ECM ligands was cross-validated by Orbitrap-Secondary Ion Mass Spectrometry. A solubilised ECM/PEGDA hydrogel was used to coat an AggreWell™ plate. Primary human cerebellar astrocytes, D283 and BT12 cells were cultured as spheroids within the ECM/PEGDA hydrogel for 7 days with no significant decrease in cellular viability. Cytokine and Human Matrix Metalloproteinase (MMP) Antibody Arrays showed cytokine and MMP expression in all three cell lines was retained in the presence of ECM material. Furthermore, D283 and BT12 cells were co-cultured with primary astrocytes for 7 days prior to separation using fluorescence activated cell sorting to generate individual cell populations. CONCLUSIONS We have developed a 3D in vitro model which can co-culture cells for over 7 days, whilst recapitulating the in vivo tumour environment. RNA sequencing is currently in progress to identify differentially expressed genes in cerebellar tumour cells upon astrocytic and brain ECM crosstalk. We hypothesise that biochemical signalling underlying this communication will reveal putative therapeutic vulnerabilities.

A convenient model of serum-induced reactivity of human astrocytes to investigate astrocyte-derived extracellular vesicles.

Extracellular vesicles (EVs) are secreted by all cells in the CNS, including neurons and astrocytes. EVs are lipid membrane enclosed particles loaded with various bioactive cargoes reflecting the dynamic activities of cells of origin. In contrast to neurons, the specific role of EVs released by astrocytes is less well understood, partly due to the difficulty in maintaining primary astrocyte cultures in a quiescent state. The aim of this study was to establish a human serum-free astrocyte culture system that maintains primary astrocytes in a quiescent state to study the morphology, function, and protein cargoes of astrocyte-derived EVs. Serum-free medium with G5 supplement and serum-supplemented medium with 2% FBS were compared for the culture of commercially available human primary fetal astrocytes. Serum-free astrocytes displayed morphologies similar to in vivo astrocytes, and surprisingly, higher levels of astrocyte markers compared to astrocytes chronically cultured in FBS. In contrast, astrocyte and inflammatory markers in serum-free astrocytes were upregulated 24 h after either acute 2% FBS or cytokine exposure, confirming their capacity to become reactive. Importantly, this suggests that distinct signaling pathways are involved in acute and chronic astrocyte reactivity. Despite having a similar morphology, chronically serum-cultured astrocyte-derived EVs (ADEVs) were smaller in size compared to serum-free ADEVs and could reactivate serum-free astrocytes. Proteomic analysis identified distinct protein datasets for both types of ADEVs with enrichment of complement and coagulation cascades for chronically serum-cultured astrocyte-derived EVs, offering insights into their roles in the CNS. Collectively, these results suggest that human primary astrocytes cultured in serum-free medium bear similarities with in vivo quiescent astrocytes and the addition of serum induces multiple morphological and transcriptional changes that are specific to human reactive astrocytes and their ADEVs. Thus, more emphasis should be made on using multiple structural, molecular, and functional parameters when evaluating ADEVs as biomarkers of astrocyte health.

Human Cytomegalovirus Dysregulates Cellular Dual-Specificity Tyrosine Phosphorylation-Regulated Kinases and Sonic Hedgehog Pathway Proteins in Neural Astrocyte and Placental Models.

Human cytomegalovirus (CMV) infection is the leading non-genetic cause of congenital malformation in developed countries, causing significant fetal injury, and in some cases fetal death. The pathogenetic mechanisms through which this host-specific virus infects then damages both the placenta and the fetal brain are currently ill-defined. We investigated the CMV modulation of key signaling pathway proteins for these organs including dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) and Sonic Hedgehog (SHH) pathway proteins using human first trimester placental trophoblast (TEV-1) cells, primary human astrocyte (NHA) brain cells, and CMV-infected human placental tissue. Immunofluorescence demonstrated the accumulation and re-localization of SHH proteins in CMV-infected TEV-1 cells with Gli2, Ulk3, and Shh re-localizing to the CMV cytoplasmic virion assembly complex (VAC). In CMV-infected NHA cells, DYRK1A re-localized to the VAC and DYRK1B re-localized to the CMV nuclear replication compartments, and the SHH proteins re-localized with a similar pattern as was observed in TEV-1 cells. Western blot analysis in CMV-infected TEV-1 cells showed the upregulated expression of Rb, Ulk3, and Shh, but not Gli2. In CMV-infected NHA cells, there was an upregulation of DYRK1A, DYRK1B, Gli2, Rb, Ulk3, and Shh. These in vitro monoculture findings are consistent with patterns of protein upregulation and re-localization observed in naturally infected placental tissue and CMV-infected ex vivo placental explant histocultures. This study reveals CMV-induced changes in proteins critical for fetal development, and identifies new potential targets for CMV therapeutic development.

Centenarian hippocampus displays high levels of astrocytic metallothioneins.

The hippocampus is a brain area linked to cognition. The mechanisms that maintain cognitive activity in humans are poorly understood. Centenarians display extreme longevity which is generally accompanied by better quality of life, lower cognitive impairment, and reduced incidence of pathologies including neurodegenerative diseases. We performed transcriptomic studies in hippocampus samples from individuals of different ages (centenarians [≥97 years], old, and young) and identified a differential gene expression pattern in centenarians compared to the other two groups. In particular, several isoforms of metallothioneins (MTs) were highly expressed in centenarians. Moreover, we identified that MTs were mainly expressed in astrocytes. Functional studies in human primary astrocytes revealed that MT1 and MT3 are necessary for their homeostasis maintenance. Overall, these results indicate that the expression of MTs specifically in astrocytes is a mechanism for protection during aging.

A novel PSMB8 isoform associated with multiple sclerosis lesions induces P-body formation.

Multiple sclerosis (MS) is an inflammatory and demyelinating disease of the central nervous system (CNS). Current therapies primarily target the inflammatory component of the disease and are highly effective in early stages of MS while limited therapies have an effect in the more chronic progressive stages of MS where resident glia have a larger role. MS lesions tend to be inflammatory even after the initial peripheral immune cell invasion has subsided and this inflammation is known to cause alternative splicing events. We used qPCR of normal-appearing white matter and white matter lesions from postmortem MS tissue, in vitro studies, and immunostaining in MS tissue to investigate the alternative splicing of one gene known to be important during recovery in an animal model of MS, PSMB8. We found a novel, intron-retained isoform which has not been annotated, upregulated specifically in MS patient white matter lesions. We found that this novel isoform activates the nonsense-mediated decay pathway in primary human astrocytes, the most populous glial cell in the CNS, and is then degraded. Overexpression of this isoform in astrocytes leads to an increased number of processing bodies in vitro, the primary site of mRNA decay. Finally, we demonstrated that MS white matter lesions have a higher burden of processing bodies compared to normal-appearing white matter, predominantly in GFAP-positive astrocytes. The increase in alternative splicing of the PSMB8 gene, the stress that this alternative splicing causes, and the observation that processing bodies are increased in white matter lesions suggests that the lesion microenvironment may lead to increased alternative splicing of many genes. This alternative splicing may blunt the protective or reparative responses of resident glia in and around white matter lesions in MS patients.