D-serine is an endogenous neurotransmitter that binds to the NMDA receptor, thereby increasing the affinity for glutamate, and the potential for excitotoxicity. The primary source of D-serine in vivo is enzymatic racemization by serine racemase (SR). Regulation of D-serine in vivo is poorly understood, but is thought to involve a combination of controlled production, synaptic reuptake by transporters, and intracellular degradation by D-amino acid oxidase (DAO). However, SR itself possesses a well-characterized eliminase activity which effectively degrades D-serine as well. D-serine is increased two-fold in spinal cords of G93A SOD1 mice – the standard model of amyotrophic lateral sclerosis (ALS). ALS mice with SR disruption show earlier symptom onset, but survive longer (progression phase is slowed), in an SR-dependent manner. Paradoxically, administration of D-serine to ALS mice dramatically lowers cord levels of D-serine, leading to changes in onset and survival very similar to SR deletion. D-serine treatment also increases cord levels of the transporter Asc-1. Although the mechanism by which SOD1 mutations increases D-serine is not known, these results strongly suggest that SR and D-serine are fundamentally involved in both the presymptomatic and progression phases of disease, and offer a direct link between mutant SOD1 and a glial-derived toxic mediator.

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 Gαi-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.

Theories about the initiation and progression of Alzheimer disease (AD) often consider potential roles played by elevations of β-amyloid precursor protein (βAPP). Since it is the source of amyloid β-peptide (Aβ), βAPP may simply contribute more pathogenic stimulus when elevated; some analyses have, however, reported a decline of βAPP in AD. We found a progressive increase in neuronal βAPP expression with increasing age in the brains of non-demented individuals, whereas in AD patient samples βAPP antigenicity decreased in neuronal somata in a manner that correlated with accumulation of mature Aβ plaques. By contrast, apolipoprotein E (ApoE) expression correlated with accumulation of plaques and even greater amounts of ApoE were detected in plaques. Induction of βAPP by glutamate in neuronal cell cultures was found to depend upon ApoE levels or activity. Thus, elevations in expression of ApoE and βAPP by cellular stresses are likely normally linked in vivo and uncoupling of this link, or other pathologic events in AD initiation, may leave neurons with diminished βAPP expression, which might in turn reduce their resistance to stressors.

This article discusses the potential role of the cytokine cycle and its corollary as drivers of the relentless progression of Alzheimer’s neuropathologies, whether they are the result of gene mutations, gene polymorphisms, and/or environmental and comorbid conditions. Based on the discovery of cytokine overexpression as an accompaniment to the dementia-related glial activation, the cytokine hypothesis was proposed. This states that in response to the negative impact on neurons of known and unknown risk factors—which include genetic inheritance, comorbid and environmental factors—microglia and astrocytes become activated and produce excess amounts of the immune-modulating cytokine interleukin-1 (IL-1) and the neuritogenic cytokine S100B, respectively. Finding that these glial events occur in fetuses and neonates with Down syndrome provided the first evidence that productive immune responses by activated glia precede rather than follow overt AD-related pathology. This finding can be added to the demonstration of IL-1 induction of amyloid β (Aβ) precursor protein and astrocyte activation with excess production of neuritogenic factor S100B. This combination suggests that IL-1 and S100B overexpression would favor the Aβ production and dystrophic neurite growth necessary for laying down neuritic Aβ plaques. This, together with demonstration of IL-1 induction of excessive production of the precursors of other features common in AD prompted a corollary to the cytokine hypothesis. The corollary states that regardless of the primary cause of the neuronal insult, the result will be chronic glial activation, which in turn will result in further neuronal injury, still more glial activation with excess cytokine expression and so on. This article discusses known causes, genetic and environmental risk factors, and comorbid conditions, and the potential contribution of glial activation with excessive cytokine expression to each.

Background

We have previously outlined functional interactions, including feedback cycles, between several of the gene products implicated in the pathogenesis of Alzheimer's disease. A number of Alzheimer-related stressors induce neuronal expression of apolipoprotein E (ApoE), β-amyloid precursor protein (βAPP), and fragments of the latter such as amyloid β-peptide (Aβ) and secreted APP (sAPP). These stressors include interleukin-1 (IL-1)-mediated neuroinflammation and glutamate-mediated excitotoxicity. Such circumstances are especially powerful when they transpire in the context of an APOE ε4 allele.

Methods

Semi-quantitative immunofluorescence imaging was used to analyze rat brains implanted with IL-1β slow-release pellets, sham pellets, or no pellets. Primary neuronal or NT2 cell cultures were treated with IL-1β, glutamate, Aβ, or sAPP; relative levels of ApoE mRNA and protein were measured by RT-PCR, qRT-PCR, and western immunoblot analysis. Cultures were also treated with inhibitors of multi-lineage kinases--in particular MAPK-p38 (SB203580), ERK (U0126), or JNK (SP600125)--prior to exposure of cultures to IL-1β, Aβ, sAPP, or glutamate.

Results

Immunofluorescence of tissue sections from pellet-implanted rats showed that IL-1β induces expression of βAPP, IL-1α, and ApoE; the latter was confirmed by western blot analysis. These protein changes were mirrored by increases in their mRNAs, as well as in those encoding IL-1β, IL-1β-converting enzyme (ICE), and tumor necrosis factor (TNF). IL-1β also increased ApoE expression in neuronal cultures. It stimulated release of sAPP and glutamate in these cultures too, and both of these agents--as well as Aβ--stimulated ApoE expression themselves, suggesting that they may contribute to the effect of IL-1β on ApoE levels. Inhibitors of MAPK-p38, ERK, and JNK inhibited ApoE induction by all these agents except glutamate, which was sensitive only to inhibitors of ERK and JNK.

Conclusion

Conditions of glial activation and hyperexcitation can elevate proinflammatory cytokines, ApoE, glutamate, βAPP, and its secreted fragments. Because each of these factors promotes glial activation and neuronal hyperexcitation, these relationships have the potential to sustain self-propagating neurodegenerative cycles that could culminate in a progressive neurodegenerative disorder such as Alzheimer's disease.

Background

D-serine is an important coagonist at the NR1 subunit of the NMDA receptor class of glutamate receptors. It is chiefly synthesized in the CNS by serine racemase (SR). Regulation of SR activity is still poorly understood. As step toward developing reagents and methods for investigating SR in vitro, we analyzed structure-function relationships of a recombinant enzyme of human sequence.

Results

Michaelis-Menten kinetic analysis indicated a KM value of 14 mM and Vmax value of 3.66 μmol·mg-1·hr-1 when L-serine was used as a substrate for purified SR. Gel-filtration chromatography and protein cross-linking experiments revealed that dimer is the major oligomeric form of recombinant SR in aqueous solution, though the proportions of monomer, tetramer, and larger aggregates differed somewhat with the specific buffer used. These buffers also altered activity in a manner correlating with the relative abundance of dimer. Activity assays showed that the dimeric gel-filtration fraction held the highest activity. Chemical reduction with DTT increased the activity of SR by elevating Vmax; cystamine, a reagent that blocks sulfhydryl groups, abolished SR activity. Gel-filtration chromatography and western blot analysis indicated that DTT enhanced the recovery of noncovalent SR dimer.

Conclusions

These data suggest that SR is most active as a noncovalent dimer containing one or more free sulfhydryls in the enzyme's active center or a modulatory site. Buffer composition and reduction/oxidation status during preparation can dramatically impact interpretations of SR activity. These findings also highlight the possibility that SR is sensitive to oxidative stress in vivo.

Background

Recent data indicate that inflammatory mechanisms contribute to diabetic retinopathy (DR). We have determined that serine racemase (SR) expression is increased by inflammatory stimuli including liposaccharide (LPS), amyloid β-peptide (A-beta), and secreted amyloid precursor protein (sAPP); expression is decreased by the anti-inflammatory drug, dexamethasone. We tested possibility that SR and its product, D-serine, were altered in a rat model of DR.

Methods

Intraperitoneal injection of streptozotocin (STZ; 70 mg/kg body weight) to Sprague-Dawley rats produced type-I diabetic mellitus (fasting blood sugar higher than 300 mg/dL). At 3 and 5 months after STZ or saline injection, retinas from some rats were subjected to cryosectioning for immunofluorescent analysis of SR and TUNEL assay of apoptosis. Retinal homogenates were used to detect SR levels and Jun N-terminal kinase (JNK) activation by immunoblotting. Aqueous humor and retina were also collected to assay for neurotransmitters, including glutamate and D-serine, by reverse-phase HPLC.

Results

Compared to saline-injected rats, STZ-injected (diabetic) rats showed elevation of SR protein levels in retinal homogenates, attributed to the inner nuclear layer (INL) by immunofluorescence. Aqueous humor fluid from STZ-injected rats contained significantly higher levels of glutamate and D-serine compared to controls; by contrast, D-serine levels in retinas did not differ. Levels of activated JNK were elevated in diabetic retinas compared to controls.

Conclusions

Increased expression of SR in retina and higher levels of glutamate and D-serine in aqueous humor of STZ-treated rats may result from activation of the JNK pathway in diabetic sequelae. Our data suggest that the inflammatory conditions that prevail during DR result in elevation of D-serine, a neurotransmitter contributing to glutamate toxicity, potentially exacerbating the death of retinal ganglion cells in this condition.

Abnormal N-methyl-d-aspartate receptor (NMDAR) function has been implicated in the pathophysiology of schizophrenia. d-serine is an important NMDAR modulator, and to elucidate the role of the d-serine synthesis enzyme serine racemase (Srr) in schizophrenia, we identified and characterized mice with an ENU-induced mutation that results in a complete loss of Srr activity and dramatically reduced d-serine levels. Mutant mice displayed behaviors relevant to schizophrenia, including impairments in prepulse inhibition, sociability and spatial discrimination. Behavioral deficits were exacerbated by an NMDAR antagonist and ameliorated by d-serine or the atypical antipsychotic clozapine. Expression profiling revealed that the Srr mutation influenced several genes that have been linked to schizophrenia and cognitive ability. Transcript levels altered by the Srr mutation were also normalized by d-serine or clozapine treatment. Furthermore, analysis of SRR genetic variants in humans identified a robust association with schizophrenia. This study demonstrates that aberrant Srr function and diminished d-serine may contribute to schizophrenia pathogenesis.

The unique physiology and function of neurons create differences in their cellular physiology, including their regulation of gene expression. We began several years ago exploring the relationships between the NFκB transcription factor, neuronal survival, and glutamate receptor activation in telencephalic neurons. These studies led us to conclude that this population of cells is nearly incapable of activating the NFκB that is nonetheless expressed at reasonable levels. A subset of the κB cis elements are instead bound by members of the Sp1 family in neurons. Also surprising was our discovery that Sp1 itself, typically described as ubiquitous, is severely restricted in expression within forebrain neurons; Sp4 seems to be substituted during neuronal differentiation. These findings and their implications for neuronal differentiation – as well as potential dedifferentiation during degenerative processes – are discussed here.

Accumulation of amyloid-β peptide (Aβ) appears to contribute to the pathogenesis of Alzheimer's disease (AD). Therapeutic hope for the prevention or removal of Aβ deposits has been placed in strategies involving immunization against the Aβ peptide. Initial Aβ immunization studies in animal models of AD showed great promise. However, when this strategy was attempted in human subjects with AD, an unacceptable degree of meningoencephalitis occurred. It is generally believed that this adverse outcome resulted from a T-cell response to Aβ. Specifically, CD4+ Th1 and Th17 cells may contribute to severe CNS inflammation and limit the utility of Aβ immunization in the treatment of AD. Interleukin (IL)-12 and IL-23 play critical roles in the development of Th1 and Th17 cells, respectively. In the present study, Aβ1−42 synergistically elevated the expression of IL-12 and IL-23 triggered by inflammatory activation of microglia, and the peroxisome proliferator-activated receptor (PPAR)-γ agonist 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) effectively blocked the elevation of these proinflammatory cytokines. Furthermore, 15d-PGJ2 suppressed the Aβ-related synergistic induction of CD14, MyD88, and Toll-like receptor 2, molecules that play critical roles in neuroinflammatory conditions. Collectively, these studies suggest that PPAR-γ agonists may be effective in modulating the development of AD.

In addition to their conventional G-C/T target sequences, Sp1 family transcription factors (Sp-factors) can interact with a subset of the target sequences for NFκB. Due to the low level of bona fide NFκB activity in most resting cells, this interaction between Sp-factors and κB-sites could play important roles in cell function. Here we used mutagenesis of a canonical κB element from the immunoglobulin and HIV promoters to identify the GC-rich sequences at each end required for Sp-factor targeting. Through screening of multiple κB-elements, a sequence element located in the second intron of superoxide dismutase-2 (SOD2) was identified as a good candidate for both NFκB and Sp-factor binding. In neurons, the prominent proteins interacting with this site were Sp3 and Sp4, whereas Sp1, Sp3, and NFκB were associated with this site in astroglia. The neuronal Sp-factors repressed transcriptional activity through this κB-site. In contrast, astroglial Sp-factors activated promoter activity through the same element. NFκB contributed to control of the SOD2 κB element only in astrocytes. These findings imply that cell-type specificity of transcription in the CNS—particularly with regard to κB elements—may include two unique aspects of neurons: 1) a recalcitrant NFκB and 2) the substitution of Sp4 for Sp1.

Nuclear factor κB (NFκB) is a dynamically modulated transcription factor with an extensive literature pertaining to widespread actions across species, cell types, and developmental stages. Analysis of NFκB in a complex environment such as neural tissue suffers from a difficulty in simultaneously establishing both activity and location. But much of the available data indicate a profound recalcitrance of NFκB activation in neurons, as compared to most other cell types. Few studies to date have sought to distinguish between the various combinatorial dimers of NFκB family members. Recent research has illustrated the importance of these problems, as well as opportunities to move past them to the nuances manifest through variable activation pathways, subunit complexity, and target sequence preferences.

When activated by proinflammatory stimuli, microglia release substantial levels of glutamate, and mounting evidence suggests this contributes to neuronal damage during neuroinflammation. Prior studies indicated a role for the Xc exchange system, an amino acid transporter that antiports glutamate for cystine. Because cystine is used for synthesis of glutathione (GSH), we hypothesized that glutamate release is an indirect consequence of GSH depletion by the respiratory burst, which produces superoxide from NADPH oxidase. Microglial glutamate release triggered by lipopolysaccharide was blocked by diphenylene iodonium chloride and apocynin, inhibitors of NADPH oxidase. This glutamate release was also blocked by vitamin E and elicited by lipid peroxidation products 4-hydroxynonenal and acrolein, suggesting that lipid peroxidation makes crucial demands on GSH. Although NADPH oxidase inhibitors also suppressed nitrite accumulation, vitamin E did not; moreover, glutamate release was largely unaffected by NO donors, inhibitors of NO synthase, or changes in gene expression. These findings indicate that a considerable degree of the neurodegenerative consequences of neuroinflammation may result from conversion of oxidative stress to excitotoxic stress. This phenomenon entails a biochemical chain of events initiated by a programmed oxidative stress and resultant mass-action amino acid transport. Indeed, some of the neuroprotective effects of antioxidants may be due to interference with these events rather than direct protection against neuronal oxidation.

Sp-family transcription factors (Sp1, Sp3 and Sp4) contain a zinc-finger domain that binds to DNA sequences rich in G-C/T. As assayed by RT-PCR analysis of mRNA, western blot analysis, immunofluorescence, and antibody-dependent “supershift” of DNA-binding assays, the prominent Sp-family factors in cerebral neurons were identified as Sp3 and Sp4. By contrast, glial cells were found to express Sp1 and Sp3. We previously showed that the pattern of G-C/T binding activity of Sp-family factors is rapidly and specifically altered by the calcium influx accompanying activation of glutamate receptors. Here, we demonstrate that Sp-factor activity is also lost after a cerebral ischemia/reperfusion injury in vivo. Consistent with its calcium-dependent nature, we found that glutamate’s effect on Sp-family factors could be blocked by inhibitors of calpains, neutral cysteine proteases activated by calcium. Purified calpain I cleaved Sp3 and Sp4 into products that retained G-C/T-binding activity, consistent with species observed in glutamate-treated neurons. These data provide details of an impact of glutamate-receptor activation on molecular events connected to gene expression.

Background

Clinical and neuropathological overlap between Alzheimer's (AD) and Parkinson's disease (PD) is now well recognized. Such cases of concurrent AD and Lewy body disease (AD/LBD) show neuropathological changes that include Lewy bodies (α-synuclein aggregates), neuritic amyloid plaques, and neurofibrillary tangles (hyperphosphorylated tau aggregates). The co-occurrence of these clinical and neuropathological changes suggests shared pathogenic mechanisms in these diseases, previously assumed to be distinct. Glial activation, with overexpression of interleukin-1 (IL-1) and other proinflammatory cytokines, has been increasingly implicated in the pathogenesis of both AD and PD.

Methods

Rat primary cultures of microglia and cortical neurons were cultured either separately or as mixed cultures. Microglia or cocultures were treated with a secreted fragment (sAPPα) of the β-amyloid precursor protein (βAPP). Neurons were treated with IL-1β or conditioned medium from sAPPα-activated microglia, with or without IL-1 receptor antagonist. Slow-release pellets containing either IL-1β or bovine serum albumin (control) were implanted in cortex of rats, and mRNA for various neuropathological markers was analyzed by RT-PCR. Many of the same markers were assessed in tissue sections from human cases of AD/LBD.

Results

Activation of microglia with sAPPα resulted in a dose-dependent increase in secreted IL-1β. Cortical neurons treated with IL-1β showed a dose-dependent increase in sAPPα release, an effect that was enhanced in the presence of microglia. IL-1β also elevated the levels of α-synuclein, activated MAPK-p38, and phosphorylated tau; a concomitant decrease in levels of synaptophysin occurred. Delivery of IL-1β by slow-release pellets elevated mRNAs encoding α-synuclein, βAPP, tau, and MAPK-p38 compared to controls. Finally, human cases of AD/LBD showed colocalization of IL-1-expressing microglia with neurons that simultaneously overexpressed βAPP and contained both Lewy bodies and neurofibrillary tangles.

Conclusion

Our findings suggest that IL-1 drives production of substrates necessary for formation of the major neuropathological changes characteristic of AD/LBD.

The role of inflammation in Alzheimer's disease (AD) has been controversial since its first consideration. As with most instances of neuroinflammation, the possibility must be considered that activation of glia and cytokine networks in AD arises merely as a reaction to neurodegeneration. Active, healthy neurons produce signals that suppress inflammatory events, and dying neurons activate phagocytic responses in microglia at the very least. But simultaneous with the arrival of a more complex view of microglia, evidence that inflammation plays a causal or exacerbating role in AD etiology has been boosted by genetic, physiological, and epidemiological studies. In the end, it may be that the semantics of "inflammation" and glial "activation" must be regarded as too simplistic for the advancement of our understanding in this regard. It is clear that elaboration of the entire repertoire of activated microglia – a phenomenon that may be termed "malactivation" – must be prevented for healthy brain structure and function. Nevertheless, recent studies have suggested that phagocytosis of Aβ by microglia plays an important role in clearance of amyloid plaques, a process boosted by immunization paradigms. To the extent that this clearance might produce clinical improvements (still an open question), this relationship thus obligates a more nuanced consideration of the factors that indicate and control the various activities of microglia and other components of neuroinflammation.

Background

Roles for excitotoxicity and inflammation in Alzheimer's disease have been hypothesized. Proinflammatory stimuli, including amyloid β-peptide (Aβ), elicit a release of glutamate from microglia. We tested the possibility that a coagonist at the NMDA class of glutamate receptors, D-serine, could respond similarly.

Methods

Cultured microglial cells were exposed to Aβ. The culture medium was assayed for levels of D-serine by HPLC and for effects on calcium and survival on primary cultures of rat hippocampal neurons. Microglial cell lysates were examined for the levels of mRNA and protein for serine racemase, the enzyme that forms D-serine from L-serine. The racemase mRNA was also assayed in Alzheimer hippocampus and age-matched controls. A microglial cell line was transfected with a luciferase reporter construct driven by the putative regulatory region of human serine racemase.

Results

Conditioned medium from Aβ-treated microglia contained elevated levels of D-serine. Bioassays of hippocampal neurons with the microglia-conditioned medium indicated that Aβ elevated a NMDA receptor agonist that was sensitive to an antagonist of the D-serine/glycine site (5,7-dicholorokynurenic acid; DCKA) and to enzymatic degradation of D-amino acids by D-amino acid oxidase (DAAOx). In the microglia, Aβ elevated steady-state levels of dimeric serine racemase, the apparent active form of the enzyme. Promoter-reporter and mRNA analyses suggest that serine racemase is transcriptionally induced by Aβ. Finally, the levels of serine racemase mRNA were elevated in Alzheimer's disease hippocampus, relative to age-matched controls.

Conclusions

These data suggest that Aβ could contribute to neurodegeneration through stimulating microglia to release cooperative excitatory amino acids, including D-serine.