Microglial neurotransmitter receptors trigger superoxide production in microglia; consequences for microglial-neuronal interactions

Microglia express three isoforms of the NADPH oxidase, Nox1, Nox2 and Nox4, with the potential to produce superoxide (O(2) ˙(-) ). Microglia also express neurotransmitter receptors, which can modulate microglial responses. In this study, microglial activity of Nox1, Nox2 and Nox4 in primary rat cultured microglia or the rodent BV2 cell line were altered by microglial neurotransmitter receptor modulation. Glutamate, GABA or ATP triggered microglial O(2) ˙(-) production via Nox activation. Nox activation was elicited by agonists of metabotropic mGlu3 receptors and by group III receptors, by GABA(A) but not GABA(B) receptors, and by purinergic P2X(7) or P2Y(2/4) receptors but not P2Y(1) receptors, and inhibited by metabotropic glutamate receptor 5 antagonists. The neurotransmitters also modulated Nox mRNA expression and NADPH activity. The activation of Nox by BzATP or GABA promoted a neuroprotective phenotype whilst the activation of Nox by glutamate promoted a neurotoxic phenotype. Taken together, these data indicate that microglial neurotransmitter receptors can signal via Nox to promote neuroprotection or neurotoxicity. This has implications for the subsequent neurotoxic profile of microglia when neurotransmitter levels may become skewed in neurodegeneration.     

Microglial apoptosis induced by chromogranin A is mediated by mitochondrial depolarisation and the permeability transition but not by cytochrome c release

Chromogranin A is up-regulated in the senile plaques of Alzheimer's brain and is a novel activator of microglia, transforming them to a neurotoxic phenotype. Treatment of primary cultures of rat brain microglia or the murine N9 microglial cell line with chromogranin A resulted in nitric oxide production, which triggered microglial apoptosis. Exposure of microglia to chromogranin A resulted in a fall in mitochondrial membrane potential. Mitochondrial depolarisation and apoptosis were reduced significantly by cyclosporin A, but not by the calcineurin inhibitor FK506. Cytochrome c did not translocate from the mitochondria to the cytosol, but its expression became significantly enhanced within the mitochondria. Inhibition of caspase 1 attenuated chromogranin A-induced microglial apoptosis, but did not prevent mitochondrial depolarisation, indicating that apoptosis occurred downstream of mitochondrial depolarisation. Conversely, staurosporine-induced microglial apoptosis led to mitochondrial cytochrome c release, but not caspase 1 activation. Our findings provide insight into the pathways controlling activation-triggered microglial apoptosis and may point to routes for the modulation of microglial evoked neurotoxicity.     

Myelin-induced microglial neurotoxicity can be controlled by microglial metabotropic glutamate receptors

Microglia are present in an activated state in multiple sclerosis lesions. Incubation of primary cultured rat microglia with rat-brain derived myelin (0.1–1 μg/mL) for 24 h induced microglial activation; cells displayed enhanced ED1 staining, expression of inducible nitric oxide synthase, production and release of the cytokine tumour necrosis factor-α and glutamate release. Exposure of microglia to myelin induced the expression of neuronal caspases and ultimately neuronal death in cultured cerebellar granule cell neurons; neurotoxicity was directly because of microglial-derived soluble toxins. Co-incubation of microglia with agonists or antagonists of different metabotropic glutamate receptor (mGluR) subtypes ameliorated microglial neurotoxicity by inhibiting soluble neurotoxin production. Activation of microglial mGluR2 exacerbated myelin-evoked neurotoxicity whilst activation of mGluR3 was protective as was activation of group III mGluRs. These data show that myelin-induced microglial neurotoxicity can be prevented by regulation of mGluRs and suggest these receptors on microglia may be promising targets for therapeutic intervention in multiple sclerosis.     

Apoptotic Pathways Mobilized in Microglia and Neurones as a Consequence of Chromogranin A-Induced Microglial Activation

Senile plaques of Alzheimer's brain are characterized by activated microglia and immunoreactivity for the peptide chromogranin A. We have investigated the mechanisms by which chromogranin A activates microglia, producing modulators of neuronal survival. Primary cultures of rat brain-derived microglia display a reactive phenotype within 24 h of exposure to 10 nM chromogranin A, culminating in microglial death via apoptotic mechanisms mediated by interleukin-1beta converting enzyme. The signalling cascade initiated by chromogranin A triggers nitric oxide production followed by enhanced microglial glutamate release, inhibition of which prevents microglial death. The plasma membrane carrier inhibitor aminoadipate and the type II/III metabotropic glutamate receptor antagonist (RS)-alpha-methyl-4-sulphonophenylglycine are equally protective. A significant amount of the released glutamate occurs from bafilomycin-sensitive stores, suggesting a vesicular mode of release. Inhibition of this component of release affords significant microglial protection. Conditioned medium from activated microglia kills cerebellar granule cells by inducing caspase-3-dependent neuronal apoptosis. Brain-derived neurotrophic factor is partially neuroprotective, as are ionotropic glutamate receptor antagonists, and, when combined with boiling of conditioned medium, full protection is achieved; nitric oxide synthase inhibitors are ineffective.     

Scavenger receptor control of chromogranin A-induced microglial stress and neurotoxic cascades

Chromogranin A (CgA), a neuroactive glycoprotein, is associated with microglial activation cascades implicated in neurodegeneration. Here we show that CgA-dependent inducible nitric oxide synthase (iNOS) expression and stress responses in microglia involved signalling via scavenger receptors (SR), since SR class-A (SR-A) ligands blocked iNOS expression, mitochondrial depolarisation, apoptosis and glutamate release. Furthermore, block of SR-A ameliorated CgA-induced microglial neurotoxicity. In contrast, block of CD36, or the receptor for advanced glycation end products (RAGE) did not prevent CgA-induced microglial activation and neurotoxicity. Thus, manipulation of specific scavenger receptor-coupled signalling pathways may provide avenues for therapeutic intervention in neurodegenerative diseases implicating microglial activation with chromogranin peptides.     

Activation of group II mGlu receptors inhibits voltage-gated Ca2+ currents in myenteric neurons

The enteric nervous system (ENS) contains functional ionotropic and group I metabotropic glutamate (mGlu) receptors. In this study, we determined whether enteric neurons express group II mGlu receptors and the effects of mGlu receptor activation on voltage-gated Ca(2+) currents in these cells. (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC), a group II mGlu receptor agonist, reversibly suppressed the Ba(2+) current in myenteric neurons isolated from the guinea pig ileum. Significant inhibition was also produced by L-glutamate and the group II mGlu receptor agonists, (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG-IV) and (2S,1'S,2'S)-2-(2-carboxycyclopropyl)glycine (L-CCG-I), with a rank order potency of 2R,4R-APDC > DCG-IV > L-glutamate > L-CCG-I, and was reduced by the group II mGlu receptor antagonist LY-341495. Pretreatment of neurons with pertussis toxin (PTX) reduced the action of mGlu receptor agonists, suggesting participation of G(i)/G(o) proteins. Finally, omega-conotoxin GVIA blocked current suppression by DCG-IV, suggesting modulation of N-type calcium channels. mGlu2/3 receptor immunoreactivity was displayed by neurons in culture and in the submucosal and myenteric plexus of the ileum. A subset of these cells displayed a glutamatergic phenotype as shown by the expression of vesicular glutamate transporter 2. These results provide the first evidence for functional group II mGlu receptors in the ENS and show that these receptors are PTX sensitive and negatively coupled to N-type calcium channels. Inhibition of N-type calcium channels produced by activation of group II mGlu receptors may modulate enteric neurotransmission.     

Localization and function of group III metabotropic glutamate receptors in rat pancreatic islets

Pancreatic islets contain ionotropic glutamate receptors that can modulate hormone secretion. The purpose of this study was to determine whether islets express functional group III metabotropic glutamate (mGlu) receptors. RT-PCR analysis showed that rat islets express the mGlu8 receptor subtype. mGlu8 receptor immunoreactivity was primarily displayed by glucagon-secreting alpha-cells and intrapancreatic neurons. By demonstrating the immunoreactivities of both glutamate and the vesicular glutamate transporter 2 (VGLUT2) in these cells, we established that alpha-cells express a glutamatergic phenotype. VGLUT2 was concentrated in the secretory granules of islet cells, suggesting that glutamate might play a role in the regulation of glucagon processing. The expression of mGlu8 by glutamatergic cells also suggests that mGlu8 may function as an autoreceptor to regulate glutamate release. Pancreatic group III mGlu receptors are functional because mGlu8 receptor agonists inhibited glucagon release and forskolin-induced accumulation of cAMP in isolated islets, and (R,S)-cyclopropyl-4-phosphonophenylglycine, a group III mGlu receptor antagonist, reduced these effects. Because excess glucagon secretion causes postprandial hyperglycemia in patients with type 2 diabetes, group III mGlu receptor agonists could be of value in the treatment of these patients.     

Activation of microglia by secreted amyloid precursor protein evokes release of glutamate by cystine exchange and attenuates synaptic function

Microglial activation as part of a chronic inflammatory response is a prominent component of Alzheimer's disease. Secreted forms of the beta-amyloid precursor protein (sAPP) previously were found to activate microglia, elevating their neurotoxic potential. To explore neurotoxic mechanisms, we analyzed microglia-conditioned medium for agents that could activate glutamate receptors. Conditioned medium from primary rat microglia activated by sAPP caused a calcium elevation in hippocampal neurons, whereas medium from untreated microglia did not. This response was sensitive to the NMDA receptor antagonist, aminophosphonovaleric acid. Analysis of microglia-conditioned by HPLC revealed dramatically higher concentrations of glutamate in cultures exposed to sAPP. Indeed, the glutamate levels in sAPP-treated cultures were substantially higher than those in cultures treated with amyloid beta-peptide. This sAPP-evoked glutamate release was completely blocked by inhibition of the cystine-glutamate antiporter by alpha-aminoadipate or use of cystine-free medium. Furthermore, a sublethal concentration of sAPP compromised synaptic density in microglia-neuron cocultures, as evidenced by neuronal connectivity assay. Finally, the neurotoxicity evoked by sAPP in microglia-neuron cocultures was attenuated by inhibitors of either the neuronal nitric oxide synthase (N(G)-propyl-L-arginine) or inducible nitric oxide synthase (1400 W). Together, these data indicate a scenario by which microglia activated by sAPP release excitotoxic levels of glutamate, probably as a consequence of autoprotective antioxidant glutathione production within the microglia, ultimately causing synaptic degeneration and neuronal death.     

Tumor necrosis factor-alpha induces neurotoxicity via glutamate release from hemichannels of activated microglia in an autocrine manner

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-alpha (TNF-alpha) secreted from activated microglia may elicit neurodegeneration through caspase-dependent cascades and silencing cell survival signals. However, direct neurotoxicity of TNF-alpha is relatively weak, because TNF-alpha also increases production of neuroprotective factors. Accordingly, it is still controversial how TNF-alpha exerts neurotoxicity in neurodegenerative diseases. Here we have shown that TNF-alpha 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.     

Metabotropic glutamate receptors in neurodegeneration/neuroprotection: Still a hot topic?

Moving from early studies, we here review the most recent evidence linking metabotropic glutamate (mGlu) receptors to processes of neurodegeneration/neuroprotection. The use of knockout mice and subtype-selective drugs has increased our knowledge of the precise role played by individual mGlu receptor subtypes in these processes. Activation of mGlu1 and mGlu5 receptors may either amplify or reduce neuronal damage depending on the context and the nature of the toxic insults. In contrast, mGlu1 and mGlu5 receptors antagonists are consistently protective in in vitro and in vivo models of neuronal death. A series of studies suggest that mGlu1 receptor antagonists or negative allosteric modulators (NAMs) are promising candidates for the treatment of ischemic brain damage, whereas mGlu5 receptor NAMs, which have been clinically developed for the treatment of Parkinson's disease (PD) and l-DOPA-induced dyskinesias, protect nigro-striatal dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) toxicity in mice and monkeys. Activation of glial mGlu3 receptors promotes the formation of various neurotrophic factors, such as transforming growth factor-β (TGF-β), glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF). Hence, selective mGlu3 receptor agonists or positive allosteric modulators (PAMs) (not yet available) are potentially helpful in the treatment of chronic neurodegenerative disorders such as PD, Alzheimer's disease (AD), and amyotrophic lateral sclerosis. Selective mGlu2 receptor PAMs should be used with caution in AD patients because these drugs are shown to amplify β-amyloid neurotoxicity. Finally, mGlu4 receptor agonists/PAMs share with mGlu5 receptor NAMs the ability to improve motor symptoms associated with PD and attenuate nigro-striatal degeneration at the same time. No data are yet available on the role of mGlu7 and mGlu8 receptors in neurodegeneration/neuroprotection.     

Metabotropic Glutamate Receptors in Glial Cells

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS) and exerts its actions via a number of ionotropic glutamate receptors/channels and metabotropic glutamate (mGlu) receptors. In addition to being expressed in neurons, glutamate receptors are expressed in different types of glial cells including astrocytes, oligodendrocytes, and microglia. Astrocytes are now recognized as dynamic signaling elements actively integrating neuronal inputs. Synaptic activity can evoke calcium signals in astrocytes, resulting in the release of gliotransmitters, such as glutamate, ATP, and d-serine, which in turn modulate neuronal excitability and synaptic transmission. In addition, astrocytes, and microglia may play an important role in pathology such as brain trauma and neurodegeneration, limiting or amplifying the pathologic process leading to neuronal death. The present review will focus on recent advances on the role of mGlu receptors expressed in glial cells under physiologic and pathologic conditions. Special issue article in honor of Dr. Anna Maria Giuffrida-Stella.     

Stimulation of group II metabotropic glutamate receptors or inhibition of group I ones exerts anxiolytic-like effects in rats

Using the conflict drinking Vogel test in rats as a model we examined the anxiolytic-like activity of (S)-4-carboxyphenylglycine (S-4CPG), an antagonist of group I metabotropic glutamate receptors (mGlu receptors), of (RS)-a-methylserine-O-phosphate-monophenyl ester (MSOPPE), an antagonist of group II mGlu receptors, and of (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine (L-CCG-I), an agonist of group II mGlu receptors. The obtained results indicate that intrahippocampal administration of S-4CPG and L-CCG-I, but not MSOPPE to rats produces a dose-dependent anticonflict effect, which is unrelated to the reduced perception of the stimulus or to an increased thirst drive. The hippocampus may be one of the neuroanatomical sites of the anxiolytic-like effects of either agent.     

RanBPM is expressed in synaptic layers of the mammalian retina and binds to metabotropic glutamate receptors

In the central nervous system, synaptic signal transduction depends on the regulation of neurotransmitter receptors by interacting proteins. Here, we searched for proteins interacting with two metabotropic glutamate receptor type 8 isoforms (mGlu8a and mGlu8b) and identified RanBPM. RanBPM is expressed in several brain regions, including the retina. There, RanBPM is restricted to the inner plexiform layer where it co-localizes with the mGlu8b isoform and processes of cholinergic amacrine cells expressing mGlu2 receptors. RanBPM interacts with mGlu2 and other group II and group III receptors, except mGlu6. Our data suggest that RanBPM might be associated with mGlu receptors at synaptic sites.     

Microglial secreted cathepsin B induces neuronal apoptosis

Activated microglia release a number of substances that can influence neuronal signalling and survival. Here we report that microglia stimulated with the peptide chromogranin A (CGA), secreted the cysteine protease, cathepsin B. Conditioned medium from CGA exposed microglia was neurotoxic to the HT22 hippocampal cell line and to primary cultures of cerebellar granule neurones. In both neuronal cell types, the neurotoxicity could be significantly attenuated with z-FA-fmk or by depletion of microglial conditioned medium with cathepsin B antibody. Conditioned medium from activated microglia or cathepsin B alone induced neuronal apoptosis and caspase 3 activation. Our data indicate that CGA-activated microglia can trigger neuronal apoptosis and that this may be mediated through the secretion of cathepsin B. Since cathepsins may also play a role in the amyloidogenic processing of amyloid precursor protein, these results may have significance for tissue damage and neuronal loss in the neuropathology of Alzheimer's disease.     

Blockade of gap junction hemichannel suppresses disease progression in mouse models of amyotrophic lateral sclerosis and Alzheimer's disease

Background

Glutamate released by activated microglia induces excitotoxic neuronal death, which likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases, including amyotrophic lateral sclerosis and Alzheimer''s disease. Although both blockade of glutamate receptors and inhibition of microglial activation are the therapeutic candidates for these neurodegenerative diseases, glutamate receptor blockers also perturbed physiological and essential glutamate signals, and inhibitors of microglial activation suppressed both neurotoxic/neuroprotective roles of microglia and hardly affected disease progression. We previously demonstrated that activated microglia release a large amount of glutamate specifically through gap junction hemichannel. Hence, blockade of gap junction hemichannel may be potentially beneficial in treatment of neurodegenerative diseases.

Methods and Findings

In this study, we generated a novel blood-brain barrier permeable gap junction hemichannel blocker based on glycyrrhetinic acid. We found that pharmacologic blockade of gap junction hemichannel inhibited excessive glutamate release from activated microglia in vitro and in vivo without producing notable toxicity. Blocking gap junction hemichannel significantly suppressed neuronal loss of the spinal cord and extended survival in transgenic mice carrying human superoxide dismutase 1 with G93A or G37R mutation as an amyotrophic lateral sclerosis mouse model. Moreover, blockade of gap junction hemichannel also significantly improved memory impairments without altering amyloid β deposition in double transgenic mice expressing human amyloid precursor protein with K595N and M596L mutations and presenilin 1 with A264E mutation as an Alzheimer''s disease mouse model.

Conclusions

Our results suggest that gap junction hemichannel blockers may represent a new therapeutic strategy to target neurotoxic microglia specifically and prevent microglia-mediated neuronal death in various neurodegenerative diseases.     

Amyloid neurotoxicity is attenuated by metallothionein: dual mechanisms at work

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive loss of memory and cognition. One of the hallmarks of AD is the accumulation of beta-amyloid (Aβ). Although endoplasmic reticulum stress, mitochondrial dysfunction, and oxidative stress have been implicated in Aβ toxicity, the molecular mechanism(s) of Aβ-induced neurotoxicity are not fully understood. In this study, we present evidence that the glia-derived stress protein metallothionein (MT) attenuates Aβ-induced neurotoxicity by unique mechanisms. MT expression was increased in brain astrocytes of a NSE-APPsw transgenic mouse model of AD. Astrocyte-derived MT protected N2a neuroblastoma cells and primary cortical neurons against Aβ toxicity with concurrent reduction of reactive oxygen species levels. MT reversed Aβ-induced down-regulation of Bcl-2 and survival signaling in neuroblastoma cells. Moreover, MT inhibited Aβ-induced proinflammatory cytokine production from microglia. The neurotoxicity of Aβ-stimulated microglia was significantly attenuated by MT-I. The results indicate that MT released from reactive astrocytes may antagonize Aβ neurotoxicity by direct inhibition of Aβ neurotoxicity and indirect suppression of neurotoxic microglial activation. These findings broaden the understanding of neurotoxic mechanisms of Aβ and the crosstalk between Aβ and MT in AD.     

Neurotoxicity of microglial cathepsin D revealed by secretome analysis

Microglia-driven inflammatory responses have both neuroprotective and neurotoxic effects in the CNS. The excessive and chronic activation of microglia, however, may shift the balance towards neurotoxic effects. In this regard, proteins secreted from activated microglia likely play a key role in the neurotoxic effects. To characterize secreted proteins of activated microglia, conditioned media obtained from BV-2 mouse microglia cells were analyzed by two-dimensional gel electrophoresis or liquid chromatography coupled with tandem mass spectrometry. Among many proteins identified in the secretome of activated microglia, an aspartic endoprotease cathepsin D has been found to mediate microglial neurotoxicity based on the following results: (i) the expression of cathepsin D protein was markedly increased in lipopolysaccharide/interferon-γ-stimulated microglia compared with resting microglia as determined by western blot analysis of conditioned media; (ii) knockdown of cathepsin D expression in microglia using short hairpin RNA diminished the neurotoxicity in the coculture of microglia and neuroblastoma cells and (iii) recombinant procathepsin D protein exerted cytotoxic effects toward cultured neurons. In conclusion, cathepsin D appears to play a central role in the microglial neurotoxicity, and could be a potential biomarker or drug target for the diagnosis and treatment of neurodegenerative diseases that are associated with excessive microglial activation and subsequent neurotoxic inflammation.     

Metabotropic glutamate receptors inhibit microglial glutamate release

Pro-inflammatory stimuli evoke an export of glutamate from microglia that is sufficient to contribute to excitotoxicity in neighbouring neurons. Since microglia also express various glutamate receptors themselves, we were interested in the potential feedback of glutamate on this system. Several agonists of mGluRs (metabotropic glutamate receptors) were applied to primary rat microglia, and the export of glutamate into their culture medium was evoked by LPS (lipopolysaccharide). Agonists of group-II and -III mGluR ACPD [(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid] and L-AP4 [L-(+)-2-amino-4-phosphonobutyric acid] were both capable of completely blocking the glutamate export without interfering with the production of NO (nitric oxide); the group-I agonist tADA (trans-azetidine-2,4-dicarboxylic acid) was ineffective. Consistent with the possibility of feedback, inhibition of mGluR by MSPG [(R,S)-α-2-methyl-4sulfonophenylglycine] potentiated glutamate export. As the group-II and -III mGluR are coupled to Gα_i-containing G-proteins and the inhibition of adenylate cyclase, we explored the role of cAMP in this effect. Inhibition of cAMP-dependent protein kinase [also known as protein kinase A (PKA)] by H89 mimicked the effect of ACPD, and the mGluR agonist had its actions reversed by artificially sustaining cAMP through the PDE (phosphodiesterase) inhibitor IBMX (isobutylmethylxanthine) or the cAMP mimetic dbcAMP (dibutyryl cAMP). These data indicate that mGluR activation attenuates a potentially neurotoxic export of glutamate from activated microglia and implicate cAMP as a contributor to this aspect of microglial action.     

Neuroprotective and symptomatic effects of targeting group III mGlu receptors in neurodegenerative disease

Neurodegenerative disorders possess common pathological mechanisms, such as protein aggregation, inflammation, oxidative stress (OS) and excitotoxicity, raising the possibility of shared therapeutic targets. As a result of the selective cellular and regional expression of group III metabotropic glutamate (mGlu) receptors, drugs targeting such receptors have demonstrated both neuroprotective properties and symptomatic improvements in several models of neurodegeneration. In recent years, the discovery and development of subtype‐selective ligands for the group III mGlu receptors has gained pace, allowing further research into the functions of these receptors and revealing their roles in health and disease. Activation of this class of receptors results in neuroprotection, with a variety of underlying mechanisms implicated. Group III mGlu receptor stimulation prevents excitotoxicity by inhibiting glutamate release from neurons and microglia and increasing glutamate uptake by astrocytes. It also attenuates the neuroinflammatory response by reducing glial reactivity and encourages neurotrophic phenotypes. This article will review the current literature with regard to the neuroprotective and symptomatic effects of group III mGlu receptor activation and discuss their promise as therapeutic targets in neurodegenerative disease.