Glutamate released by activated microglia induces excitoneurotoxicity and may contribute to neuronal damage in neurodegenerative diseases, including Alzheimer disease, Parkinson disease, amyotrophic lateral sclerosis, and multiple sclerosis. In addition, tumor necrosis factor-␣ (TNF-␣) secreted from activated microglia may elicit neurodegeneration through caspasedependent cascades and silencing cell survival signals. However, direct neurotoxicity of TNF-␣ is relatively weak, because TNF-␣ also increases production of neuroprotective factors. Accordingly, it is still controversial how TNF-␣ exerts neurotoxicity in neurodegenerative diseases. Here we have shown that TNF-␣ is the key cytokine that stimulates extensive microglial glutamate release in an autocrine manner by up-regulating glutaminase to cause excitoneurotoxicity. Further, we have demonstrated that the connexin 32 hemichannel of the gap junction is another main source of glutamate release from microglia besides glutamate transporters. Although pharmacological blockade of glutamate receptors is a promising therapeutic candidate for neurodegenerative diseases, the associated perturbation of physiological glutamate signals has severe adverse side effects. The unique mechanism of microglial glutamate release that we describe here is another potential therapeutic target. We rescued neuronal cell death in vitro by using a glutaminase inhibitor or hemichannel blockers to diminish microglial glutamate release without perturbing the physiological glutamate level. These drugs may give us a new therapeutic strategy against neurodegenerative diseases with minimum adverse side effects.

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Astroglia play active and diverse roles in modulating neuronal/synaptic functions in the CNS. How these astroglial functions are regulated, especially by neuronal signals, remains largely unknown. Exosomes, a major type of extracellular vesicles (EVs) that originate from endosomal intraluminal vesicles (ILVs), have emerged as a new intercellular communication process. By generating cell-type-specific ILVs/exosome reporter (CD63-GFPf/f) mice and immuno-EM/confocal image analysis, we found that neuronal CD63-GFP+ ILVs are primarily localized in soma and dendrites, but not in axonal terminals in vitro and in vivo. Secreted neuronal exosomes contain a subset of microRNAs (miRs) that is distinct from the miR profile of neurons. These miRs, especially the neuron-specific miR-124-3p, are potentially internalized into astrocytes. MiR-124-3p further up-regulates the predominant glutamate transporter GLT1 by suppressing GLT1-inhibiting miRs. Our findings suggest a previously undescribed neuronal exosomal miR-mediated genetic regulation of astrocyte functions, potentially opening a new frontier in understanding CNS intercellular communication.

The blood–brain barrier (BBB) is composed of capillary endothelial cells, pericytes, and perivascular astrocytes, which regulate central nervous system homeostasis. Sonic hedgehog (SHH) released from astrocytes plays an important role in the maintenance of BBB integrity. BBB disruption and microglial activation are common pathological features of various neurologic diseases such as multiple sclerosis, Parkinson’s disease, amyotrophic lateral sclerosis, and Alzheimer’s disease. Interleukin-1β (IL-1β), a major pro-inflammatory cytokine released from activated microglia, increases BBB permeability. Here we show that IL-1β abolishes the protective effect of astrocytes on BBB integrity by suppressing astrocytic SHH production. Astrocyte conditioned media, SHH, or SHH signal agonist strengthened BBB integrity by upregulating tight junction proteins, whereas SHH signal inhibitor abrogated these effects. Moreover, IL-1β increased astrocytic production of pro-inflammatory chemokines such as CCL2, CCL20, and CXCL2, which induce immune cell migration and exacerbate BBB disruption and neuroinflammation. Our findings suggest that astrocytic SHH is a potential therapeutic target that could be used to restore disrupted BBB in patients with neurologic diseases.

Interferon-gamma (IFN-gamma) is a proinflammatory cytokine that plays a pivotal role in pathology of diseases in the central nervous system (CNS), such as multiple sclerosis. However, the direct effect of IFN-gamma on neuronal cells has yet to be elucidated. We show here that IFN-gamma directly induces neuronal dysfunction, which appears as dendritic bead formation in mouse cortical neurons and enhances glutamate neurotoxicity mediated via alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptors but not N-methyl-D-aspartate receptors. In the CNS, IFN-gamma receptor forms a unique, neuron-specific, calcium-permeable receptor complex with AMPA receptor subunit GluR1. Through this receptor complex, IFN-gamma phosphorylates GluR1 at serine 845 position by JAK1.2/STAT1 pathway, increases Ca(2+) influx and following nitric oxide production, and subsequently decreases ATP production, leading to the dendritic bead formation. These findings provide novel mechanisms of neuronal excitotoxicity, which may occur in both inflammatory and neurodegenerative diseases in the CNS.

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A homozygous mutation in the gene for sigma 1 receptor (Sig1R) is a cause of inherited juvenile amyotrophic lateral sclerosis (ALS16). Sig1R localizes to the mitochondria‐associated membrane (MAM), which is an interface of mitochondria and endoplasmic reticulum. However, the role of the MAM in ALS is not fully elucidated. Here, we identified a homozygous p.L95fs mutation of Sig1R as a novel cause of ALS16. ALS‐linked Sig1R variants were unstable and incapable of binding to inositol 1,4,5‐triphosphate receptor type 3 (IP 3R3). The onset of mutant Cu/Zn superoxide dismutase (SOD1)‐mediated ALS disease in mice was accelerated when Sig1R was deficient. Moreover, either deficiency of Sig1R or accumulation of mutant SOD1 induced MAM disruption, resulting in mislocalization of IP 3R3 from the MAM, calpain activation, and mitochondrial dysfunction. Our findings indicate that a loss of Sig1R function is causative for ALS16, and collapse of the MAM is a common pathomechanism in both Sig1R‐ and SOD1‐linked ALS. Furthermore, our discovery of the selective enrichment of IP 3R3 in motor neurons suggests that integrity of the MAM is crucial for the selective vulnerability in ALS.

Neuroinflammation, which includes both neuroprotective and neurotoxic reactions by activated glial cells and infiltrated immune cells, is involved in the pathomechanism of amyotrophic lateral sclerosis (ALS). However, the cytokines that regulate the neuroprotective inflammatory response in ALS are not clear. Here, we identify transforming growth factor-β1 (TGF-β1), which is upregulated in astrocytes of murine and human ALS, as a negative regulator of neuroprotective inflammatory response. We demonstrate that astrocyte-specific overproduction of TGF-β1 in SOD1(G93A) mice accelerates disease progression in a non-cell-autonomous manner, with reduced IGF-I production in deactivated microglia and fewer T cells with an IFN-γ-dominant milieu. Moreover, expression levels of endogenous TGF-β1 in SOD1(G93A) mice negatively correlate with lifespan. Furthermore, pharmacological administration of a TGF-β signaling inhibitor after disease onset extends survival time of SOD1(G93A) mice. These findings indicate that astrocytic TGF-β1 determines disease progression and is critical to the pathomechanism of ALS.