Exogenous ketone bodies (KBs), acetoacetate (AA), and β-hydroxybutyrate (BHB) act as alternative energy substrates in neural cells under starvation. The present study examined the endogenous ketogenic capacity of astroglia under hypoxia with/without glucose and the possible roles of KBs in neuronal energy metabolism. Cultured neurons and astroglia were prepared from Sprague-Dawley rats. Palmitic acid (PAL) and l-carnitine (LC) were added to the assay medium. The 4- to 24-hr production of AA and BHB was measured using the cyclic thio-NADH method. 14C-labeled acid-soluble products (KBs) and 14CO2 produced from [1-14C]PAL were also measured. l-[U-14C]lactic acid ([14C]LAC), [1-14C]pyruvic acid ([14C]PYR), or β-[1-14C]hydroxybutyric acid ([14C]BHB) was used to compare the oxidative metabolism of the glycolysis end products with that of the KBs. Some cells were placed in a hypoxic chamber (1% O2). PAL and LC induced a higher production of KBs in astroglia than in neurons, while the CO2 production from PAL was less than 5% of the KB production in both astroglia and neurons. KB production in astroglia was augmented by the AMP-activated protein kinase activators, AICAR and metformin, as well as hypoxia with/without glucose. Neuronal KB production increased under hypoxia in the absence of PAL and LC. In neurons, [14C]LAC and [14C]PYR oxidation decreased after 24 hr of hypoxia, while [14C]BHB oxidation was preserved. Astroglia responds to ischemia in vitro by enhancing KB production, and astroglia-produced KBs derived from fatty acid might serve as a neuronal energy substrate for the tricarboxylic acid cycle instead of lactate, as pyruvate dehydrogenase is susceptible to ischemia.

BackgroundToll-like receptor 4 (TLR4) plays a pivotal role in the pathophysiology of stroke-induced inflammation. Both astroglia and microglia express TLR4, and endogenous ligands produced in the ischemic brain induce inflammatory responses. Reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines produced by TLR4 activation play harmful roles in neuronal damage after stroke. Although astroglia exhibit pro-inflammatory responses upon TLR4 stimulation by lipopolysaccharide (LPS), they may also play cytoprotective roles via the activation of the pentose phosphate pathway (PPP), reducing oxidative stress by glutathione peroxidase. We investigated the mechanisms by which astroglia reduce oxidative stress via the activation of PPP, using TLR4 stimulation and hypoxia in concert with microglia.MethodsIn vitro experiments were performed using cells prepared from Sprague–Dawley rats. Coexisting microglia in the astroglial culture were chemically eliminated using l-leucine methyl ester (LME). Cells were exposed to LPS (0.01 μg/mL) or hypoxia (1 % O2) for 12–15 h. PPP activity was measured using [1-14C]glucose and [6-14C]glucose. ROS and NO production were measured using 2′,7′-dichlorodihydrofluorescein diacetate and diaminofluorescein-FM diacetate, respectively. The involvement of nuclear factor-erythroid-2-related factor 2 (Nrf2), a cardinal transcriptional factor under stress conditions that regulates glucose 6-phosphate dehydrogenase, the rate-limiting enzyme of PPP, was evaluated using immunohistochemistry.ResultsCultured astroglia exposed to LPS elicited 20 % increases in PPP flux, and these actions of astroglia appeared to involve Nrf2. However, the chemical depletion of coexisting microglia eliminated both increases in PPP and astroglial nuclear translocation of Nrf2. LPS induced ROS and NO production in the astroglial culture containing microglia but not in the microglia-depleted astroglial culture. LPS enhanced astroglial ROS production after glutathione depletion. U0126, an upstream inhibitor of mitogen-activated protein kinase, eliminated LPS-induced NO production, whereas ROS production was unaffected. U0126 also eliminated LPS-induced PPP activation in astroglial–microglial culture, indicating that microglia-derived NO mediated astroglial PPP activation. Hypoxia induced astroglial PPP activation independent of the microglia–NO pathway. Elimination of ROS and NO production by sulforaphane, a natural Nrf2 activator, confirmed the astroglial protective mechanism.ConclusionsAstroglia in concert with microglia may play a cytoprotective role for countering oxidative stress in stroke.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0564-0) contains supplementary material, which is available to authorized users.

Tau aggregates represent a key pathologic feature of Alzheimer’s disease and other neurodegenerative diseases. Recently, PET probes have been developed for in vivo detection of tau accumulation; however, they are limited because of off-target binding and a reduced ability to detect tau in non-Alzheimer’s disease tauopathies. The novel tau PET tracer, [18F]PI-2620, has a high binding affinity and specificity for aggregated tau; therefore, it was hypothesized to have desirable properties for the visualization of tau accumulation in Alzheimer’s disease and non-Alzheimer’s disease tauopathies. To assess the ability of [18F]PI-2620 to detect regional tau burden in non-Alzheimer’s disease tauopathies compared with Alzheimer’s disease, patients with progressive supranuclear palsy (n = 3), corticobasal syndrome (n = 2), corticobasal degeneration (n = 1), or Alzheimer’s disease (n = 8), and healthy controls (n = 7) were recruited. All participants underwent MRI, amyloid β assessment, and [18F]PI-2620 PET (Image acquisition at 60–90 minutes post-injection). Cortical and subcortical tau accumulations were assessed by calculating standardized uptake value ratios using [18F]PI-2620 PET. For pathologic validation, tau pathology was assessed using tau immunohistochemistry and compared with [18F]PI-2620 retention in an autopsied case of corticobasal degeneration. In Alzheimer’s disease, focal retention of [18F]PI-2620 was evident in the temporal and parietal lobes, precuneus, and cingulate cortex. Standardized uptake value ratio analyses revealed that patients with non-Alzheimer’s disease tauopathies had elevated [18F]PI-2620 uptake only in the globus pallidus, as compared to patients with Alzheimer’s disease, but not healthy controls. A head-to-head comparison of [18F]PI-2620 and [18F]PM-PBB3, another tau PET probe for possibly visualizing the 4-repeat tau pathogenesis in non-Alzheimer’s disease, revealed different retention patterns in one subject with progressive supranuclear palsy. Imaging-pathology correlation analysis of the autopsied patient with corticobasal degeneration revealed no significant correlation between [18F]PI-2620 retention in vivo. High [18F]PI-2620 uptake at 60–90 minutes post-injection in the globus pallidus may be a sign of neurodegeneration in four-repeat tauopathy, but not necessarily practical for diagnosis of non- Alzheimer’s disease tauopathies. Collectively, this tracer is a promising tool to detect Alzheimer’s disease-tau aggregation. However, late acquisition PET images of [18F]PI-2620 may have limited utility for reliable detection of four-repeat tauopathy because of lack of correlation between postmortem tau pathology and different retention pattern than the non-Alzheimer’s disease-detectable tau radiotracer, [18F]PM-PBB3. A recent study reported that [18F]PI-2620 tracer kinetics curves in four-repeat tauopathies peak earlier (within 30 minutes) than Alzheimer’s disease; therefore, further studies are needed to determine appropriate PET acquisition times that depend on the respective interest regions and diseases.

Oxidative stress plays an important role in the onset and progression of Parkinson disease. Although released dopamine at the synaptic terminal is mostly reabsorbed by dopaminergic neurons, some dopamine is presumably taken up by astroglia. This study examined the dopamine-induced astroglial protective function through the activation of the pentose-phosphate pathway (PPP) to reduce reactive oxygen species (ROS). In vitro experiments were performed using striatal neurons and cortical or striatal astroglia prepared from Sprague-Dawley rats or C57BL/6 mice. The rates of glucose phosphorylation in astroglia were evaluated using the [14C]deoxyglucose method. PPP activity was measured using [1-14C]glucose and [6-14C]glucose after acute (60 min) or chronic (15 hr) exposure to dopamine. ROS production was measured using 2′,7′-dichlorodihydrofluorescein diacetate. The involvement of the Kelch-like ECH-associated protein 1 (Keap1) or nuclear factor-erythroid-2-related factor 2 (Nrf2) system was evaluated using Nrf2 gene knockout mice, immunohistochemistry, and quantitative reverse transcription polymerase chain reaction analysis for heme oxygenase-1. Acute exposure to dopamine elicited increases in astroglial glucose consumption with lactate release. PPP activity in astroglia was robustly enhanced independently of Na+-dependent monoamine transporters. In contrast, chronic exposure to dopamine induced moderate increases in PPP activity via the Keap1/Nrf2 system. ROS production from dopamine increased gradually over 12 hr. Dopamine induced neuronal cell damage that was prevented by coculturing with astroglia but not with Nrf2-deficient astroglia. Dopamine-enhanced astroglial PPP activity in both acute and chronic manners may possibly reduce neuronal oxidative stress.

Induced pluripotent stem cell (iPSC)-based disease modeling has a great potential for uncovering the mechanisms of pathogenesis, especially in the case of neurodegenerative diseases where disease-susceptible cells can usually not be obtained from patients. So far, the iPSC-based modeling of neurodegenerative diseases has mainly focused on neurons because the protocols for generating astrocytes from iPSCs have not been fully established. The growing evidence of astrocytes’ contribution to neurodegenerative diseases has underscored the lack of iPSC-derived astrocyte models. In the present study, we established a protocol to efficiently generate iPSC-derived astrocytes (iPasts), which were further characterized by RNA and protein expression profiles as well as functional assays. iPasts exhibited calcium dynamics and glutamate uptake activity comparable to human primary astrocytes. Moreover, when co-cultured with neurons, iPasts enhanced neuronal synaptic maturation. Our protocol can be used for modeling astrocyte-related disease phenotypes in vitro and further exploring the contribution of astrocytes to neurodegenerative diseases.