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Tryptophan metabolism, through the kynurenine pathway (KP), produces neurotoxic intermediates that are implicated in the pathogenesis of Alzheimer disease (AD). In particular, oxidative stress via 3-hydroxykynurenine (3-HK) and its cleaved product 3-hydroxyanthranilic acid (3-HAA) significantly damages neuronal tissue and may potentially contribute to a cycle of neurodegeneration through consequent amyloid-β accumulation, glial activation, and upregulation of the KP. To determine the role of the KP in eliciting and continuing oxidative stress within AD brain, we used immunocytochemical methods to show elevated levels of 3-HK modifications and the upstream, rate-limiting enzyme indoleamine 2,3-dioxygenase (IDO-1) in AD brain when compared to controls. Importantly, the association of IDO-1 with senile plaques was confirmed, and for the first time, IDO-1 was shown to be specifically localized in conjunction with neurofibrillary tangles. As senile plaques and neurofibrillary tangles are the pathological hallmarks of AD, our study provides further evidence that the KP is involved with the destructive neurodegenerative pathway of AD.
The kynurenine pathway (KP) is the primary mode of L-tryptophan catabolism in mammalian tissues1, responsible for 95% of L-tryptophan degradation2 (Figure 1). Though chiefly carried out to yield the ubiquitous coenzymes nicotine adenine dinucleotide (NAD+) and nicotine adenine dinucleotide phosphate (NADP) for use in basic cellular processes3, the KP plays additional roles in cellular, particularly neuronal, physiology4-6. Notably, L-tryptophan is an essential amino acid, and thus must be retrieved in the diet7. Its balanced catabolism is therefore important for the survival of a given tissue: upregulation of the KP depletes the surrounding cells of L-tryptophan, while the opposite reduces cellular levels of NAD+ and NADP. Accordingly, the KP is utilized in a pathophysiological response to invading microorganisms; a depletion of L-tryptophan via the KP has antimicrobial, antiviral, and antiproliferative activities due to the basic cellular need for dietary tryptophan7-9. However, as some intermediates of the KP have neurotoxic effects, an upregulation of the pathway also threatens the surrounding tissue10, 11. Specifically, the L-tryptophan metabolites 3-hydroxykynurenine (3-HK) and 3-hydroxyanthranilic acid (3-HAA) are associated with the generation of the potent oxidative species superoxide (O2-), hydroxyl radical (H·) and hydrogen peroxide (H2O2), which frequently contribute to macromolecular damage within defective cells12-15. Moreover, quinolinic acid (QUIN), a downstream metabolite of 3-HAA, is also a potent neurotoxic element16, 17 that has been shown to exhibit excitotoxic effects via N-methyl D-aspartate (NMDA) receptor agonism, as well as oxidative stress via lipid peroxidation18-22. Consequently, any unbalanced upregulation of the KP is likely to elicit some degree of detriment to surrounding tissue, and this phenomenon is manifest in several inflammatory-associated diseases, such as multiple sclerosis23, AIDS-dementia complex24, and cerebral malaria25-27, as well as in Alzheimer disease (AD)28, 29.
AD is a complex neurodegenerative disease that is characterized by hippocampal neuronal loss and severe dementia in its later stages30. The pathological mechanisms that underlie the disease are a subject of moderate controversy, however it is known that oxidative stress and neuroinflammation play pivotal roles31. The mediators of neuroinflammation in AD are microglia and astrocytes32, 33, known sites of KP catabolism which contain all enzymes required for KP progression34-36. A recent study also implicated QUIN, the downstream component of the KP, with tau phosphorylation in AD through reduction of tau phosphatases PP2A, PP1, and PP537. Tau hyperphosphorylation, resulting in neurofibrillary tangle (NFT) formation, represents a hallmark feature of AD that induces severe neuronal detriment upon increasing deposition38. Consequently, AD pathophysiology is believed to involve an upregulation of the KP.
Specifically, serum studies measuring the ratio of 3-HK to L-tryptophan levels found an increased ratio (i.e., more 3-HK) in the serum of AD patients, compared to age-matched and younger controls28. Immunohistochemical studies of AD hippocampal sections similarly demonstrated elevated levels of the upstream, rate-limiting enzyme indoleamine 2,3-dioxygenase (IDO-1) and QUIN in microglia, astrocytes, and neurons, as compared to control subjects, with microglia and astrocytes showing the highest levels29. In this report, we analyzed the relative levels of 3-HK modified proteins in AD hippocampal tissue sections compared to age-matched controls, as well as the levels and localization of IDO-1 in order to discern the role of the KP in AD. Importantly, these results may open new insights into AD therapeutics, particularly via a modification of the catabolism of L-tryptophan.
Using Case Western Reserve University IRB approved protocols, brain samples were collected at the time of autopsy. Hippocampal sections from cases of pathologically confirmed AD (n= 12; age 69-96 yr, mean 84 ± 8.2 yr) and from young (n= 3; age 31-54 yr, mean 42 ± 11.5 yr) and age-matched (n= 7; age 66-86 yr, mean 71.5 ± 6.8 yr) controls were collected, fixed in methacarn (methanol: chloroform: acetic acid; 6:3:1), and embedded in paraffin. Sections (6 μm) were cut and processed for immunohistochemistry using the peroxidase-anti-peroxidase method39. Briefly, sections were deparaffinized in xylene, rehydrated in a series of graded ethanol, and endogenous peroxidase activity was removed by incubating 30 min in 3% H2O2. Following a 10-min incubation in 10% normal goat serum, the primary antibodies were applied for 16 h at 4°C. Following successive incubations in secondary antibody and peroxidase-anti-peroxidase complex, staining was developed using 3,3-diaminobenzidine as cosubstrate.
For some cases, the tissue was treated with 50% hydrofluoric acid before pre-embedding (Dako Cytomation, Carpinteria, CA, USA). Antibodies used include; monoclonal anti-IDO-1 (Chemicon, Temecula, CA, USA), anti 3-HK modified Nα-acetyl lysine, Nα-acetyl histidine, Nα-acetyl arginine, and Nα-acetyl cysteine14, anti-phosphorylated tau (Biosource, Carlsbad, CA, USA). As a negative control, the above method was repeated omitting the primary antibody.
Computer-assisted digital analysis was used to measure the level of immunoreaction in pyramidal neurons in three fields from the CA1/2 region of the hippocampus in the AD and control sections. Using a Zeiss axiocam and associated Axiovision software, pyramidal neurons were detected and the intensity of immunoreaction, with the background level of the surrounding neuropil subtracted, was determined for each case. An average neuronal staining level was determined for each case and the Student's t-test was used to statistically compare the different groups.
Western immunoblotting was carried out as previously described40. In brief, brain sections from AD (n = 7; age 65-89 yr) and control (n = 6; age 62-89 yr) cases were homogenized in 10 volume of lysis buffer [50 mM Tris-HCl (pH 7.6), 0.02% sodium azide, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 1% Nonidet P-40, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 1 mg/mL aprotinin, 2 mg/mL antipain, and 1 mM sodium orthovanadate], and centrifuged at 11745 g for 10 min at 4°C. Protein concentrations of each sample's supernatant were assessed by bicinchoninic acid assay (Pierce, Rockford, IL, USA). Separation of proteins was completed by SDS-polyacrylamide gel electrophoresis (20 μg/lane), and subsequently, proteins were electroblotted onto polyvinylidene difluoride membrane as previously described41. Transferred blots were incubated in sequence with blocking agent (10% nonfat milk in Tris-buffered saline solution with Tween 20), mouse monoclonal antibody to 3-HK modifications, and affinity purified goat anti-mouse immunoglobulin peroxidase conjugate was preabsorbed to eliminate human cross-reactivity. Development of blots was completed by using enhanced chemiluminescence reagents (Santa Cruz Biotechnology, Santa Cruz, CA, USA) according to the manufacturer's instructions. Blots were then stripped, as previously published42, and incubated with monoclonal antibody to the constitutively expressed actin protein. Densitometric analysis was performed on the scanned images using Quantity 1 software (Biorad) to determine precise relative protein levels of 3-HK modifications and actin. For each individual case, the 3-HK modification level was divided by the actin level to control for gel loading and allow for more accurate comparative analysis.
IDO-1 was detected in both control and AD tissue as previously described29. Intense labeling of neuronal (Figure 2A) and glial (Figure 2B) accumulations were detected in all cases of AD. Following treatment with hydrofluoric acid, intense labeling of neurofibrillary pathology was seen (Figure 2C). In confirmation that the IDO staining pattern observed indeed represented NFTs, adjacent tissue sections were stained with anti-tau antibody and a replicate staining pattern was observed (data not shown). Additionally, amyloid deposits (Figure 2D) including those with an intense core (Figure 2D, inset), were readily detected.
3-HK modifications are increased in hippocampal pyramidal neurons in cases of AD compared to controls (Figure 3). In AD, the cytoplasm in neurons extending throughout the entorhinal cortex showed much stronger immunoreactivity (Figure 3B) than age-matched controls (Figure 3A). Quantification of the neuronal levels of 3-HK modifications in the CA1 and CA2 regions of the hippocampus, revealed that the AD cases displayed significantly greater staining intensity than age-matched controls upon quantification (p<0.02) (Figure 3C), as well as controls under age 55 (data not shown). Further data analysis showed there is no significant correlation between the neuronal levels of 3-HK modifications with age in either the AD or control groups.
Western blot data demonstrated 3-HK modifications to be slightly elevated in AD hippocampal tissues, compared to controls (Figure 4A); however, statistical significance was not reached (Figure 4B). Of note, three cases with dual diagnosis of AD and diffuse Lewy body disease (DLBD) demonstrated relatively lower levels of 3-HK compared to AD cases, and, upon their removal from set of AD cases in the comparison between AD and control, the difference in 3-HK concentration between AD and controls reached statistical significance (p=0.0425) (Figure 4B).
The elevated expression of both 3-HK modifications and IDO-1 in AD tissue sections confirms the upregulation of the kynurenine pathway in AD. As IDO-1 facilitates the rate-limiting step in the catabolism of L-tryptophan, its increased presence strongly indicates the corresponding progression of the KP and its corresponding over-activation of the downstream intermediates 3-HK, ANA, KYNA, and QUIN (the latter three were not monitored in this study). In addition, the co-localization of IDO-1 with AD tauopathies in this study implicates the KP in NFT formation in the brain. Taken together, we believe that amyloid-β (Aβ), through its activation of microglia and astrocytes (discussed below), induces the upregulation of the toxic intermediates of the KP that produce further oxidative stress, excitotoxicity, and tau phosphorylation. Therefore, a potential therapeutic window is identified by the results of our research.
While exact role of Aβ in the pathogenesis of AD is a subject of controversy, accumulating evidence suggests it to be a secondary factor in disease, rather than a primary one31. That is, while Aβ was long regarded to be the primary, causal factor in the development and progression of AD-type dementia, it actually may accumulate as a compensatory response to age-induced increases in oxidative stress31, 43-45. The role of Aβ peptide is therefore initially one of neuronal protection. Briefly, in AD cases with the most extensive deposition of Aβ, as well as those containing NFTs, there are relatively low levels of the oxidative stress marker 8-hydroxyguanosine (8OHG), despite the apparent history of oxidative damage (i.e., advanced glycation endproducts or lipid peroxidation)46. Moreover, Aβ itself demonstrates strong antioxidant properties under appropriate cellular conditions: the peptide contains strong chelating properties for zinc, iron, and copper (highly active oxidative agents)47, 48. However, despite the initial benefits of Aβ secretion, the peptide's eventual extracellular aggregation into senile plaques (resulting from oxidative stress-inducing dityrosine cross-linkages49 and the corresponding insolubility of Aβ) stimulates inflammation that ultimately yields further oxidative stress. It is this latter phenomenon that we believe to be mediated through an upregulation of the KP.
Aβ plaques are known to activate microglial and astrocytic cells in the brain following accumulation and aggregation, and consequent neurotoxic effects. That is, reactive oxygen species production probably occurs as a result of astrocyte and microglia activation50, 51. For instance, the KP is primarily functional in microglia and astrocytes (as well as in infiltrating macrophages). Moreover, all of the enzymes that facilitate the catabolism of L-tryptophan via the KP are primarily contained in microglial cells. Therefore, it is logical that Aβ exerts its neurotoxic effects (at least partly) through the indirect activation of the KP, the result being the overactivation of KP intermediates with proven destructive capabilities (i.e., 3-HK, ANA, QUIN). Interestingly, in addition to its excitotoxic and ROS-generating effects52, 53, QUIN has recently been demonstrated to activate astrocytes and/or induce astrocytic apoptosis54, 55, as well as stimulate tau hyperphosphorylation in AD possibly through a reduction of tau phosphatases PP2A, PP1, and PP537. This finding provides another potential link between hallmark AD pathologies, specifically Aβ and hyperphosphorylated tau, and the KP.
Perhaps, then, a therapeutic approach aimed at IDO-1 inhibition could prevent the toxic intermediates of the KP from accumulating, thereby partially reducing overall oxidative stress, excitotoxicity, and tau phosphorylation in the brain. While several such inhibitors have been investigated2, none have been analyzed in their capacity to reduce AD stress. Consequently, it may be of therapeutic benefit to study the possibility of IDO-1 inhibition as an AD treatment. Though the inhibition of IDO-1, the upstream rate-limiting enzyme in the KP, may be the most efficacious target for outright prevention of downstream KP intermediates, other KP enzyme inhibitors may be similarly useful for disease modification. A recent study has in fact measured the effect of kynurenine aminotransferase II (KATII) inhibition, the enzyme responsible for the conversion of L-KYN into KYNA (Figure 1).56 These knockout mice, which ultimately express lower levels of KYNA, demonstrated improved cognitive performance, and hippocampal slices from these mice showed significant increases in long-term potentiation. These modifications represent important advances in AD treatment, though they neglect to attenuate the formation of the primary progenitor of oxidative stress in the kynurenine pathway, namely 3-HK.
The finding of significantly lower 3-HK modifications in concomitant AD and DLBD, compared to cases with strictly AD, is quite interesting and opens many additional questions. Notably, the Lewy body variant of AD tends to have significantly lower amounts of NFT pathology than pure AD57. Given our novel finding of the association of IDO with NFTs, perhaps sequestration of IDO-1, a primary KP enzyme (Figure 1), within NFTs interferes with its ability to participate in negative feedback of the expression of KP enzymes; this in turn may lead to an overactivation of the KP and consequent higher level of 3-HK modifications in pure AD. Future studies will focus on revealing the mechanisms behind the observed differences in 3-HK modifications in DLBD type AD and pure AD. As AD is an increasingly burdensome neurodegenerative disease growing in prevalence, any insight into possible therapeutics is of great value. Future research to further elucidate the role of oxidative stress via the KP in AD and other degenerative brain diseases is warranted.
This study was supported by the National Institutes of Health [R01 EY016219 (RHN), R01 EY09912 (RHN), P30 EY11373 to the Visual Sciences Research Center of CWRU, and R01 AG026151 (MAS)] and the Alzheimer's Association.