Generation Of Astrocytes Research Articles

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.

A human fetal cerebellar map of the late second trimester reveals developmental molecular characteristics and abnormality in trisomy 21

Our understanding of human fetal cerebellum development during the late second trimester, a critical period for the generation of astrocytes, oligodendrocytes, and unipolar brush cells (UBCs), remains limited. Here, we performed single-cell RNA sequencing (scRNA-seq) in human fetal cerebellum samples from gestational weeks (GWs) 18-25. We find that proliferating UBC progenitors distribute in the subventricular zone of the rhombic lip (RLSVZ) near white matter (WM), forming a layer structure. We also delineate two trajectories from astrogenic radial glia (ARGs) to Bergmann glial progenitors (BGPs) and recognize oligodendrogenic radial glia (ORGs) as one source of primitive oligodendrocyte progenitor cells (PriOPCs). Additionally, our scRNA-seq analysis of the trisomy 21 fetal cerebellum at this stage reveals abnormal upregulated genes in pathways such as the cell adhesion pathway and focal adhesion pathway, which potentially promote neuronal differentiation. Overall, our research provides valuable insights into normal and abnormal development of the human fetal cerebellum.

Astrocytes in Amyloid Generation and Alcohol Metabolism: Implications of Alcohol Use in Neurological Disorder(s).

As per the National Survey on Drug Use and Health, 10.5% of Americans aged 12 years and older are suffering from alcohol use disorder, with a wide range of neurological disorders. Alcohol-mediated neurological disorders can be linked to Alzheimer's-like pathology, which has not been well studied. We hypothesize that alcohol exposure can induce astrocytic amyloidosis, which can be corroborated by the neurological disorders observed in alcohol use disorder. In this study, we demonstrated that the exposure of astrocytes to ethanol resulted in an increase in Alzheimer's disease markers-the amyloid precursor protein, Aβ1-42, and the β-site-cleaving enzyme; an oxidative stress marker-4HNE; proinflammatory cytokines-TNF-α, IL1β, and IL6; lncRNA BACE1-AS; and alcohol-metabolizing enzymes-alcohol dehydrogenase, aldehyde dehydrogenase-2, and cytochrome P450 2E1. A gene-silencing approach confirmed the regulatory role of lncRNA BACE1-AS in amyloid generation, alcohol metabolism, and neuroinflammation. This report is the first to suggest the involvement of lncRNA BACE1-AS in alcohol-induced astrocytic amyloid generation and alcohol metabolism. These findings will aid in developing therapies targeting astrocyte-mediated neurological disorders and cognitive deficits in alcohol users.

Restoring hippocampal glucose metabolism rescues cognition across Alzheimer's disease pathologies.

Impaired cerebral glucose metabolism is a pathologic feature of Alzheimer Disease (AD), and recent proteomic studies highlight a disruption of glial carbohydrate metabolism with disease progression. Here, we report that inhibition of indoleamine-2,3-dioxygenase 1 (IDO1), which metabolizes tryptophan to kynurenine (KYN) in the first step of the kynurenine pathway, rescues hippocampal memory function and plasticity in preclinical models of amyloid and tau pathology by restoring astrocytic metabolic support of neurons. Activation of IDO1 in astrocytes by amyloid-beta 42 and tau oligomers, two major pathological effectors in AD, increases KYN and suppresses glycolysis in an AhR-dependent manner. Conversely, pharmacological IDO1 inhibition restores glycolysis and lactate production. In amyloid-producing APP Swe -PS1 ΔE9 and 5XFAD mice and in tau-producing P301S mice, IDO1 inhibition restores spatial memory and improves hippocampal glucose metabolism by metabolomic and MALDI-MS analyses. IDO1 blockade also rescues hippocampal long-term potentiation (LTP) in a monocarboxylate transporter (MCT)-dependent manner, suggesting that IDO1 activity disrupts astrocytic metabolic support of neurons. Indeed, in vitro mass-labeling of human astrocytes demonstrates that IDO1 regulates astrocyte generation of lactate that is then taken up by human neurons. In co-cultures of astrocytes and neurons derived from AD subjects, deficient astrocyte lactate transfer to neurons was corrected by IDO1 inhibition, resulting in improved neuronal glucose metabolism. Thus, IDO1 activity disrupts astrocytic metabolic support of neurons across both amyloid and tau pathologies and in a model of AD iPSC-derived neurons. These findings also suggest that IDO1 inhibitors developed for adjunctive therapy in cancer could be repurposed for treatment of amyloid- and tau-mediated neurodegenerative diseases.

Mature astrocytes as source for astrocyte repopulation after deletion in the medial prefrontal cortex: Implications for depression.

The adult brain retains a high repopulation capacity of astrocytes after deletion, and both mature astrocytes in the neocortex and neural stem cells in neurogenic regions possess the potential to generate astrocytes. However, the origin and the repopulation dynamics of the repopulating astrocytes after deletion remain largely unclear. The number of astrocytes is reduced in the medial prefrontal cortex (mPFC) of patients with depression, and selective elimination of mPFC astrocytes is sufficient to induce depression-like behaviors in rodents. However, whether astrocyte repopulation capacity is impaired in depression is unknown. In this study, we used different transgenic mouse lines to genetically label different cell types and demonstrated that in the mPFC of normal adult mice of both sexes, mature astrocytes were a major source of the repopulating astrocytes after acute deletion induced by an astrocyte-specific toxin, L-alpha-aminoadipic acid (L-AAA), and astrocyte regeneration was accomplished within two weeks accompanied by reversal of depression-like behaviors. Furthermore, re-ablation of mPFC astrocytes post repopulation led to reappearance of depression-like behaviors. In adult male mice subjected to 14-day chronic restraint stress, a well-validated mouse model of depression, the number of mPFC astrocytes was reduced; however, the ability of mPFC astrocytes to repopulate after L-AAA-induced deletion was largely unaltered. Our study highlights a potentially beneficial role for repopulating astrocytes in depression and provides novel therapeutic insights into enhancing local mature astrocyte generation in depression.

Polar lipids modify Alzheimer's Disease pathology by reducing astrocyte pro-inflammatory signaling through platelet-activating factor receptor (PTAFR) modulation.

Pro-inflammatory processes triggered by the accumulation of extracellular amyloid beta (Aβ) peptides are a well-described pathology in Alzheimer's disease (AD). Activated astrocytes surrounding Aβ plaques contribute to inflammation by secreting proinflammatory factors. While astrocytes may phagocytize Aβ and contribute to Aβ clearance, reactive astrocytes may also increase Aβ production. Therefore, identifying factors that can attenuate astrocyte activation and neuroinflammation and how these factors influence pro-inflammatory pathways is important for developing therapeutic and preventive strategies in AD. Here, we identify the platelet-activating factor receptor (PTAFR) pathway as a key mediator of astrocyte activation. Intriguingly, several polar lipids (PLs) have exhibited anti-inflammatory protective properties outside the central nervous system through their inhibitory effect on the PTAFR pathway. Thus, we additionally investigated whether different PLs also exert inhibitory effects on the PAF pathway in astrocytes and whether their presence influences astrocytic pro-inflammatory signaling and known AD pathologies in vitro. PLs from salmon and yogurt were extracted using novel food-grade techniques and their fatty acid profile was determined using LC/MS. The effect of PLs on parameters such as astrocyte activation and generation of oxygen species (ROS) was assessed. Additionally, effects of the secretome of astrocytes treated with these polar lipids on aged neurons was measured. We show that PLs obtained from salmon and yogurt lower astrocyte activation, the generation of reactive oxygen species (ROS), and extracellular Aβ accumulation. Cell health of neurons exposed to the secretome of astrocytes treated with salmon-derived PLs and Aβ was less affected than those treated with astrocytes exposed to Aβ only. Our results highlight a novel underlying mechanism, why consuming PL-rich foods such as fish and dairy may reduce the risk of developing dementia and associated disorders.

KYNA Ameliorates Glutamate Toxicity of HAND by Enhancing Glutamate Uptake in A2 Astrocytes.

Reactive astrocytes are key players in HIV-associated neurocognitive disorders (HAND), and different types of reactive astrocytes play opposing roles in the neuropathologic progression of HAND. A recent study by our group found that gp120 mediates A1 astrocytes (neurotoxicity), which secrete proinflammatory factors and promote HAND disease progression. Here, by comparing the expression of A2 astrocyte (neuroprotective) markers in the brains of gp120 tgm mice and gp120+/α7nAChR-/- mice, we found that inhibition of alpha 7 nicotinic acetylcholine receptor (α7nAChR) promotes A2 astrocyte generation. Notably, kynurenine acid (KYNA) is an antagonist of α7nAChR, and is able to promote the formation of A2 astrocytes, the secretion of neurotrophic factors, and the enhancement of glutamate uptake through blocking the activation of α7nAChR/NF-κB signaling. In addition, learning, memory and mood disorders were significantly improved in gp120 tgm mice by intraperitoneal injection of kynurenine (KYN) and probenecid (PROB). Meanwhile, the number of A2 astrocytes in the mouse brain was significantly increased and glutamate toxicity was reduced. Taken together, KYNA was able to promote A2 astrocyte production and neurotrophic factor secretion, reduce glutamate toxicity, and ameliorate gp120-induced neuropathological deficits. These findings contribute to our understanding of the role that reactive astrocytes play in the development of HAND pathology and provide new evidence for the treatment of HAND via the tryptophan pathway.

Zika virus infection impairs synaptogenesis, induces neuroinflammation, and could be an environmental risk factor for autism spectrum disorder outcome

Zika virus (ZIKV) infection was first associated with Central Nervous System (CNS) infections in Brazil in 2015, correlated with an increased number of newborns with microcephaly, which ended up characterizing the Congenital Zika Syndrome (CZS). Here, we investigated the impact of ZIKV infection on the functionality of iPSC-derived astrocytes. Besides, we extrapolated our findings to a Brazilian cohort of 136 CZS children and validated our results using a mouse model. Interestingly, ZIKV infection in neuroprogenitor cells compromises cell migration and causes apoptosis but does not interfere in astrocyte generation. Moreover, infected astrocytes lost their ability to uptake glutamate while expressing more glutamate transporters and secreted higher levels of IL-6. Besides, infected astrocytes secreted factors that impaired neuronal synaptogenesis. Since these biological endophenotypes were already related to Autism Spectrum Disorder (ASD), we extrapolated these results to a cohort of children, now 6–7 years old, and found seven children with ASD diagnosis (5.14 %). Additionally, mice infected by ZIKV revealed autistic-like behaviors, with a significant increase of IL-6 mRNA levels in the brain. Considering these evidence, we inferred that ZIKV infection during pregnancy might lead to synaptogenesis impairment and neuroinflammation, which could increase the risk for ASD.

SRF-deficient astrocytes provide neuroprotection in mouse models of excitotoxicity and neurodegeneration.

Reactive astrogliosis is a common pathological hallmark of CNS injury, infection, and neurodegeneration, where reactive astrocytes can be protective or detrimental to normal brain functions. Currently, the mechanisms regulating neuroprotective astrocytes and the extent of neuroprotection are poorly understood. Here, we report that conditional deletion of serum response factor (SRF) in adult astrocytes causes reactive-like hypertrophic astrocytes throughout the mouse brain. These SrfGFAP-ERCKO astrocytes do not affect neuron survival, synapse numbers, synaptic plasticity or learning and memory. However, the brains of Srf knockout mice exhibited neuroprotection against kainic-acid induced excitotoxic cell death. Relevant to human neurodegenerative diseases, SrfGFAP-ERCKO astrocytes abrogate nigral dopaminergic neuron death and reduce β-amyloid plaques in mouse models of Parkinson's and Alzheimer's disease, respectively. Taken together, these findings establish SRF as a key molecular switch for the generation of reactive astrocytes with neuroprotective functions that attenuate neuronal injury in the setting of neurodegenerative diseases.

Activation of mitochondrial DNA-mediated cGAS-STING pathway contributes to chronic postsurgical pain by inducing type I interferons and A1 reactive astrocytes in the spinal cord

Chronic postsurgical pain (CPSP) is increasingly recognized as a public health issue. Recent studies indicated the innate immune pathway of cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) was involved in pain regulation. However, the detailed mechanisms remain unclear. Previous studies found A1 reactive astrocytes in the spinal cord contributed to CPSP. This study aimed to investigate the roles and mechanisms of the cGAS-STING pathway in regulating the generation of A1 reactive astrocytes during CPSP. First, CPSP model was established using skin/muscle incision and retraction (SMIR) in rats. We found that cGAS-STING pathway was activated accompanied with an increase in mitochondrial DNA in the cytosol in the spinal cord following SMIR. Second, a STING inhibitor C-176 was intrathecally administrated. We found that C-176 decreased the expression of type I interferons and A1 reactive astrocytes in the spinal cord, and alleviated mechanical allodynia in SMIR rats. Third, cyclosporin A as a mitochondrial permeability transition pore blocker was intrathecally administrated. We found that cyclosporin A decreased the leakage of mitochondrial DNA and inhibited the activation of cGAS-STING pathway. Compared with C-176, cyclosporin A exhibits similar analgesic effects. The expression of type I interferons and A1 reactive astrocytes in the spinal cord were also down-regulated after intervention with cyclosporin A. Moreover, simultaneous administration of cyclosporin A and C-176 did not show synergistic effects in SMIR rats. Therefore, our study demonstrated that the cGAS-STING pathway activated by the leakage of mitochondrial DNA contributed to chronic postsurgical pain by inducing type I interferons and A1 reactive astrocytes in the spinal cord.

Regulation of NAD+/NADH Redox Involves the Protective Effects of Ginsenoside Rb1 against Oxygen-Glucose Deprivation/Reoxygenation-Induced Astrocyte Lesions.

The aim of this study was to investigate NAD+/NADH redox regulation in astrocytes by Ginsenoside Rb1 subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) and to reveal the neuroprotective mechanism of ginseng. Neonatal mouse brain was used to culture primary astrocytes. The third generation of the primary astrocytes was used for the experiments. OGD/R was introduced by culturing the cells in a glucose-free media under nitrogen for 6 h followed by a regular culture for 24 h. Ginsenoside Rb1 attenuated OGD/R-induced astrocyte injury in a dose-dependent manner. It improved the mitochondrial function of OGD/R astrocytes indicated by improving mitochondrial distribution, increasing mitochondrial membrane potential, and enhancing mitochondrial DNA copies and ATP production. Ginsenoside Rb1 significantly lifted intracellular NAD+/NADH, NADPH/NADP+, and GSH/GSSG in OGD/R astrocytes. It inhibited the protein expression of both PARP1 and CD38, while attenuating the SIRT1 drop in OGD/R cells. In line with its effects on PARP1, Ginsenoside Rb1 significantly reduced the expression of poly-ADP-ribosylation (PARylation) proteins in OGD/R cells. Ginsenoside Rb1 also significantly increased the expression of NAMPT and NMNAT2, both of which are key players in NAD/NADH synthesis. The results suggest that the regulation of NAD+/NADH redox involves the protective effects of ginsenoside Rb1 against OGD/R-induced astrocyte injury.

Temporal-spatial Generation of Astrocytes in the Developing Diencephalon.

Astrocytes are the largest glial population in the mammalian brain. However, we have a minimal understanding of astrocyte development, especially fate specification in different regions of the brain. Through lineage tracing of the progenitors of the third ventricle (3V) wall via in-utero electroporation in the embryonic mouse brain, we show the fate specification and migration pattern of astrocytes derived from radial glia along the 3V wall. Unexpectedly, radial glia located in different regions along the 3V wall of the diencephalon produce distinct cell types: radial glia in the upper region produce astrocytes and those in the lower region produce neurons in the diencephalon. With genetic fate mapping analysis, we reveal that the first population of astrocytes appears along the zona incerta in the diencephalon. Astrogenesis occurs at an early time point in the dorsal region relative to that in the ventral region of the developing diencephalon. With transcriptomic analysis of the region-specific 3V wall and lateral ventricle (LV) wall, we identified cohorts of differentially-expressed genes in the dorsal 3V wall compared to the ventral 3V wall and LV wall that may regulate astrogenesis in the dorsal diencephalon. Together, these results demonstrate that the generation of astrocytes shows a spatiotemporal pattern in the developing mouse diencephalon.

Comparison of two protocols for the generation of iPSC-derived human astrocytes

BackgroundAstrocytes have recently gained attention as key contributors to the pathogenesis of neurodegenerative disorders including Parkinson’s disease. To investigate human astrocytes in vitro, numerous differentiation protocols have been developed. However, the properties of the resulting glia are inconsistent, which complicates the selection of an appropriate method for a given research question. Thus, we compared two approaches for the generation of iPSC-derived astrocytes. We phenotyped glia that were obtained employing a widely used long, serum-free (“LSF”) method against an in-house established short, serum-containing (“SSC”) protocol which allows for the generation of astrocytes and midbrain neurons from the same precursor cells.ResultsWe employed high-content confocal imaging and RNA sequencing to characterize the cultures. The astrocytes generated with the LSF or SSC protocols differed considerably in their properties: while the former cells were more labor-intense in their generation (5 vs 2 months), they were also more mature. This notion was strengthened by data resulting from cell type deconvolution analysis that was applied to bulk transcriptomes from the cultures to assess their similarity with human postmortem astrocytes.ConclusionsOverall, our analyses highlight the need to consider the advantages and disadvantages of a given differentiation protocol, when designing functional or drug discovery studies involving iPSC-derived astrocytes.

Exploring the effects of embryonic and neonatal exposure to lipopolysaccharides on oligodendrocyte differentiation in the rat hippocampus and the protective effect of alpha-glycosyl isoquercitrin

This study compared the effects of embryonic and neonatal lipopolysaccharides (LPS) exposure (E-LPS and N-LPS) on oligodendrocyte (OL) differentiation in the hippocampus of male rats and explored the protective effect of the antioxidant alpha-glycosyl isoquercitrin (AGIQ). Using SD rats, LPS exposure occurred either intraperitoneally in dams between gestational days 15 and 16 (50 µg/kg body weight/time) or in male pups on postnatal day (PND) 3 (1 mg/kg body weight). Under both regimens, AGIQ at 0.5% (w/w) was supplemented, to dams from the gestation period (before LPS exposure) until weaning on PND 21 and to male offspring from weaning until PND 77 (adulthood). Compared with a control treatment, E-LPS treatment resulted in fewer NG2+ OL progenitor cells (OPCs) and an upregulation of Tcf4 at PND 6; by PND 21, low NG2+ OPC number persisted, but OLIG2+ OL lineage cells increased, while CNPase+ mature OLs counts were unchanged. By contrast, N-LPS treatment resulted in fewer OLIG2+ cells and an upregulation of Bmp4 at PND 6; by PND 21, NG2+ OPCs decreased, while GFAP+ astrocytes increased at both PND 6 and 21. After N-LPS treatment, Kl and Yy1 were downregulated and there were fewer Klotho+ and CNPase+ cells at PND 21. Results suggest that E-LPS treatment facilitates OPC differentiation into pre- and immature OLs until weaning, while N-LPS treatment suppresses OPC differentiation into mature OLs but facilitates astrocyte generation; however, these changes spontaneously recovered by adulthood under both regimens. AGIQ treatment ameliorated the effects of LPS treatment of both regimens, suggesting that LPS-induced disruption of OPC/OL differentiation occurs via neuroinflammation.

Store-Operated Calcium Entry Inhibition and Plasma Membrane Calcium Pump Upregulation Contribute to the Maintenance of Resting Cytosolic Calcium Concentration in A1-like Astrocytes.

Highly neurotoxic A1-reactive astrocytes have been associated with several human neurodegenerative diseases. Complement protein C3 expression is strongly upregulated in A1 astrocytes, and this protein has been shown to be a specific biomarker of these astrocytes. Several cytokines released in neurodegenerative diseases have been shown to upregulate the production of amyloid β protein precursor (APP) and neurotoxic amyloid β (Aβ) peptides in reactive astrocytes. Also, aberrant Ca2+ signals have been proposed as a hallmark of astrocyte functional remodeling in Alzheimer's disease mouse models. In this work, we induced the generation of A1-like reactive astrocytes after the co-treatment of U251 human astroglioma cells with a cocktail of the cytokines TNF-α, IL1-α and C1q. These A1-like astrocytes show increased production of APP and Aβ peptides compared to untreated U251 cells. Additionally, A1-like astrocytes show a (75 ± 10)% decrease in the Ca2+ stored in the endoplasmic reticulum (ER), (85 ± 10)% attenuation of Ca2+ entry after complete Ca2+ depletion of the ER, and three-fold upregulation of plasma membrane calcium pump expression, with respect to non-treated Control astrocytes. These altered intracellular Ca2+ dynamics allow A1-like astrocytes to efficiently counterbalance the enhanced release of Ca2+ from the ER, preventing a rise in the resting cytosolic Ca2+ concentration.

Enhancement of the liver’s neuroprotective role ameliorates traumatic brain injury pathology

Traumatic brain injury (TBI) is a pervasive problem worldwide for which no effective treatment is currently available. Although most studies have focused on the pathology of the injured brain, we have noted that the liver plays an important role in TBI. Using two mouse models of TBI, we found that the enzymatic activity of hepatic soluble epoxide hydrolase (sEH) was rapidly decreased and then returned to normal levels following TBI, whereas such changes were not observed in the kidney, heart, spleen, or lung. Interestingly, genetic downregulation of hepatic Ephx2 (which encodes sEH) ameliorates TBI-induced neurological deficits and promotes neurological function recovery, whereas overexpression of hepatic sEH exacerbates TBI-associated neurological impairments. Furthermore, hepatic sEH ablation was found to promote the generation of A2 phenotype astrocytes and facilitate the production of various neuroprotective factors associated with astrocytes following TBI. We also observed an inverted V-shaped alteration in the plasma levels of four EET (epoxyeicosatrienoic acid) isoforms (5,6-, 8,9-,11,12-, and 14,15-EET) following TBI which were negatively correlated with hepatic sEH activity. However, hepatic sEH manipulation bidirectionally regulates the plasma levels of 14,15-EET, which rapidly crosses the blood-brain barrier. Additionally, we found that the application of 14,15-EET mimicked the neuroprotective effect of hepatic sEH ablation, while 14,15-epoxyeicosa-5(Z)-enoic acid blocked this effect, indicating that the increased plasma levels of 14,15-EET mediated the neuroprotective effect observed after hepatic sEH ablation. These results highlight the neuroprotective role of the liver in TBI and suggest that targeting hepatic EET signaling could represent a promising therapeutic strategy for treating TBI.

Robust induction of functional astrocytes using NGN2 expression in human pluripotent stem cells

Emerging evidence of species divergent features of astrocytes coupled with the relative inaccessibility of human brain tissue underscore the utility of human pluripotent stem cell (hPSC) technologies for the generation and study of human astrocytes. However, existing approaches for hPSC-astrocyte generation are typically lengthy or require intermediate purification steps. Here, we establish a rapid and highly scalable method for generating functional human induced astrocytes (hiAs). These hiAs express canonical astrocyte markers, respond to pro-inflammatory stimuli, exhibit ATP-induced calcium transients and support neuronal network development. Moreover, single-cell transcriptomic analyses reveal the generation of highly reproducible cell populations across individual donors, mostly resembling human fetal astrocytes. Finally, hiAs generated from a trisomy 21 disease model identify expected alterations in cell-cell adhesion and synaptic signaling, supporting their utility for disease modeling applications. Thus, hiAs provide a valuable and practical resource for the study of basic human astrocyte function and dysfunction in disease.

Serine metabolism during differentiation of human iPSC-derived astrocytes.

Astrocytes are essential players in development and functions, being particularly relevant as regulators of brain energy metabolism, ionic homeostasis, and synaptic transmission. They are also the major source of L-serine in the brain, which is synthesized from the glycolytic intermediate 3-phosphoglycerate through the phosphorylated pathway. L-serine is the precursor of the two main co-agonists of the N-methyl-D-aspartate receptor, glycine and D-serine. Strikingly, dysfunctions in both L- and D-serine metabolism are associated with neurological and psychiatric disorders. Here, we exploited a differentiation protocol, based on the generation of human mature astrocytes from neural stem cells, and investigated the modification of the proteomic and metabolomic profile during the differentiation process. We show that differentiated astrocytes are more similar to mature rather than to reactive ones, and that axogenesis and pyrimidine metabolism increase up to 30 days along with the folate cycle and sphingolipid metabolism. Consistent with the proliferation and cellular maturation processes that are taking place, also the intracellular level of L-serine, glycine, threonine, L- and D-aspartate (which level is unexpectedly higher than that of D-serine) show the same biosynthetic time course. A significant utilization of L-serine from the medium is apparent while glycine is first consumed and then released with a peak at 30 days, parallel to its intracellular level. These results underline how metabolism changes during astrocytes differentiation, highlight that D-serine synthesis is restricted in differentiated astrocytes, and provide a valuable model for developing potential novel therapeutic approaches to address brain diseases, especially the ones related to serine metabolism alterations.

Rescue of neurogenesis and age-associated cognitive decline in SAMP8 mouse: Role of transforming growth factor-alpha.

Neuropathological aging is associated with memory impairment and cognitive decline, affecting several brain areas including the neurogenic niche of the dentate gyrus of the hippocampus (DG). In the healthy brain, homeostatic mechanisms regulate neurogenesis within the DG to facilitate the continuous generation of neurons from neural stem cells (NSC). Nevertheless, aging reduces the number of activated neural stem cells and diminishes the number of newly generated neurons. Strategies that promote neurogenesis in the DG may improve cognitive performance in the elderly resulting in the development of treatments to prevent the progression of neurological disorders in the aged population. Our work is aimed at discovering targeting molecules to be used in the design of pharmacological agents that prevent the neurological effects of brain aging. We study the effect of age on hippocampal neurogenesis using the SAMP8 mouse as a model of neuropathological aging. We show that in 6-month-old SAMP8 mice, episodic and spatial memory are impaired; concomitantly, the generation of neuroblasts and neurons is reduced and the generation of astrocytes is increased in this model. The novelty of our work resides in the fact that treatment of SAMP8 mice with a transforming growth factor-alpha (TGFα) targeting molecule prevents the observed defects, positively regulating neurogenesis and improving cognitive performance. This compound facilitates the release of TGFα in vitro and in vivo and activates signaling pathways initiated by this growth factor. We conclude that compounds of this kind that stimulate neurogenesis may be useful to counteract the neurological effects of pathological aging.

Differentiation of astrocytes with characteristics of ventral midbrain from human embryonic stem cells.

Molecular and functional diversity among region-specific astrocytes is of great interest in basic neuroscience and the study of neurological diseases. In this study, we present the generation and characterization of astrocytes from human embryonic stem cells with the characteristics of the ventral midbrain (VM). Fine modulation of WNT and SHH signaling during neural differentiation induced neural precursor cells (NPCs) with high expression of EN1 and NKX6.1, but less expression of FOXA2. Overexpression of nuclear factor IB in NPCs induced astrocytes, thereby maintaining the expression of region-specific genes acquired in the NPC stage. When cocultured with dopaminergic (DA) precursors or DA neurons, astrocytes with VM characteristics (VM-iASTs) promoted the differentiation and survival of DA neurons better than those that were not regionally specified. Transcriptomic analysis showed that VM-iASTs were more closely related to human primary midbrain astrocytes than to cortical astrocytes, and revealed the upregulation of WNT1 and WNT5A, which supports their VM identity and explains their superior activity in DA neurons. Taken together, we hope that VM-iASTs can serve to improve ongoing DA precursor transplantation for Parkinson's disease, and that their transcriptomic data provide a valuable resource for investigating regional diversity in human astrocyte populations.