Metabolic Assessment of Human Induced Pluripotent Stem Cells-Derived Astrocytes and Fetal Primary Astrocytes: Lactate and Glucose Turnover.

Astrocytes represent one of the main cell types in the brain and play a crucial role in brain functions, including supplying the energy demand for neurons. Moreover, they are important regulators of metabolite levels. Glucose uptake and lactate production are some of the main observable metabolic actions of astrocytes. To gain insight into these processes, it is essential to establish scalable and functional sources for in vitro studies of astrocytes. In this study, we compared the metabolic turnover of glucose and lactate in astrocytes derived from human induced pluripotent stem cell (hiPSC)-derived Astrocytes (hiAstrocytes) as a scalable astrocyte source to human fetal astrocytes (HFAs). Using a user-friendly, commercial flow-based biosensor, we could verify that hiAstrocytes are as glycogenic as their fetal counterparts, but their normalized metabolic turnover is lower. Specifically, under identical culture conditions in a defined media, HFAs have 2.3 times higher levels of lactate production compared to hiAstrocytes. In terms of glucose, HFAs have 2.1 times higher consumption levels than hiAstrocytes at 24 h. Still, as we describe their glycogenic phenotype, our study demonstrates the use of hiAstrocytes and flow-based biosensors for metabolic studies of astrocyte function.

Cocaine Self-administration and Extinction Inversely Alter Neuron to Glia Exosomal Dynamics in the Nucleus Accumbens.

Alteration of glutamatergic synaptic plasticity in the Nucleus Accumbens (NAc) has been implicated in cocaine-seeking behaviors. Astroglial mechanisms for maintaining extracellular glutamate homeostasis through cysteine/glutamate exchanger (xCT) and glutamate transporter GLT1 are dysregulated following cocaine exposure and contribute to altered glutamatergic synaptic plasticity. However, how these astroglial proteins become dysregulated in cocaine addiction remains unknown. We recently showed that neuron to astroglial exosome signaling is essential to maintain GLT1 protein expression by transferring neuronal miR-124-3p into astrocytes to suppress GLT1-inhibiting microRNAs (miRs) in astrocytes. In the current study, by selectively labeling neuronal exosomes using CD63-GFPf/+ exosome reporter mice, we examined how the self-administration and extinction stages of the mouse cocaine self-administration model alter neuronal exosome signaling to astrocytes and microglia in the NAc. We found that cocaine (but not food) self-administration strongly reduces the internalization of neuronal exosomes, particularly in astrocytes in the NAc (but not in motor cortex), which can be effectively reversed by extinction training. In parallel, cocaine self-administration alone specifically and differentially affects activation of glial cells by decreasing GFAP expression in astrocytes but increasing Iba1 expression in microglia. However, extinction training fully reverses the increased Iba1 expression in microglia but only partially reverses the reduction of GFAP in astrocytes. Taken together, our study reveals altered in vivo dynamics of NAc neuronal exosomes in the cocaine addiction model, providing new insights about how altered neuron to glial exosome signaling may contribute to astroglial dysfunction in cocaine addiction.

Exercise-Induced Changes to the Macrophage Response in the Dorsal Root Ganglia Prevent Neuropathic Pain after Spinal Cord Injury.

Spinal cord injury (SCI) induces neuropathic pain that is refractory to treatment. Central and peripheral immune responses to SCI play critical roles in pain development. Although immune responses in the dorsal horn have been implicated in SCI-pain, immune mechanisms in the periphery, especially in the dorsal root ganglia (DRG), where nociceptor cell bodies reside, have not been well studied. Exercise is an immunomodulator, and we showed previously that early exercise after SCI reduces pain development. However, the mechanisms of exercise-mediated pain reduction are not understood. Therefore, we examined the 1) underlying immune differences in the spinal cord and DRG between rats with and without pain and 2) immunomodulatory effects of exercise in pain reduction. Rats were subjected to a unilateral contusion at C5 and tested for pain development using von Frey and mechanical conflict-avoidance paradigms. A subgroup of rats was exercised on forced running wheels starting at 5 days post-injury for 4 weeks. We observed greater microglial activation in the C7-C8 dorsal horn of rats with SCI-induced pain compared to rats with normal sensation, and early exercise reduced this activation independently of pain behavior. Further, abnormal pain sensation strongly correlated with an increased number of DRG macrophages. Importantly, exercise-treated rats that maintain normal sensation also have a lower number of macrophages in the DRG. Our data suggest that macrophage presence in the DRG may be an important effector of pain development, and early wheel walking exercise may mediate pain prevention by modulating the injury-induced macrophage response in the DRG. Further supportive evidence demonstrated that rats that developed pain despite exercise intervention still displayed a significantly elevated number of macrophages in the DRG. Collectively, these data suggest that macrophage presence in the DRG may be an amenable cellular target for future therapies.