Structural and functional coupling of Hsp90- and Sgt1-centred multi-protein complexes

Sgt1 is an adaptor protein implicated in a variety of processes, including formation of the kinetochore complex in yeast, and regulation of innate immunity systems in plants and animals. Sgt1 has been found to associate with SCF E3 ubiquitin ligases, the CBF3 kinetochore complex, plant R proteins and related animal Nod-like receptors, and with the Hsp90 molecular chaperone. We have determined the crystal structure of the core Hsp90–Sgt1 complex, revealing a distinct site of interaction on the Hsp90 N-terminal domain. Using the structure, we developed mutations in Sgt1 interfacial residues, which specifically abrogate interaction with Hsp90, and disrupt Sgt1-dependent functions in vivo, in plants and yeast. We show that Sgt1 bridges the Hsp90 molecular chaperone system to the substrate-specific arm of SCF ubiquitin ligase complexes, suggesting a role in SCF assembly and regulation, and providing multiple complementary routes for ubiquitination of Hsp90 client proteins.     

SGT1 encodes an essential component of the yeast kinetochore assembly pathway and a novel subunit of the SCF ubiquitin ligase complex.

We have identified SGT1 as a dosage suppressor of skp1-4, a mutation causing defects in yeast kinetochore function. Sgt1p physically associates with Skp1p in vivo and in vitro. SGT1 is an essential gene, and different sgt1 conditional mutants arrest with either a G1 or G2 DNA content. Genetic and phenotypic analyses of sgt1-3 (G2 allele) mutants support an essential role in kinetochore function. Sgt1p is required for assembling the yeast kinetochore complex, CBF3, via activation of Ctf13p. Sgt1p also associates with SCF (Skp1p/Cdc53p/F box protein) ubiquitin ligase. sgt1-5 (G1 allele) mutants are defective in Sic1p turnover in vivo and Cln1p ubiquitination in vitro. Human SGT1 rescues an sgt1 null mutation, suggesting that the function of SGT1 is conserved in evolution.     

Sgt1 associates with Hsp90: an initial step of assembly of the core kinetochore complex

The kinetochore, which consists of DNA sequence elements and structural proteins, is essential for high-fidelity chromosome transmission during cell division. In budding yeast, Sgt1, together with Skp1, is required for assembly of the core kinetochore complex (CBF3) via Ctf13 activation. Formation of the active Ctf13-Skp1 complex also requires Hsp90, a molecular chaperone. We have found that Sgt1 interacts with Hsp90 in yeast. We also have determined that Skp1 and Hsc82 (a yeast Hsp90 protein) bind to the N-terminal region of Sgt1 that contains tetratricopeptide repeat motifs. Results of sequence and phenotypic analyses of sgt1 mutants strongly suggest that the N-terminal region containing the Hsc82-binding and Skp1-binding domains of Sgt1 is important for the kinetochore function of Sgt1. We found that Hsp90's binding to Sgt1 stimulates the binding of Sgt1 to Skp1 and that Sgt1 and Hsp90 stimulate the binding of Skp1 to Ctf13, the F-box core kinetochore protein. Our results strongly suggest that Sgt1 and Hsp90 function in assembling CBF3 by activating Skp1 and Ctf13.     

Sgt1 is required for human kinetochore assembly

Budding yeast Sgt1 is required for kinetochore assembly, and its homologues have a role in cAMP signalling in fungi and pathogen resistance in plants. The function of mammalian Sgt1 is unknown. We report that RNA interference-mediated depletion of Sgt1 from HeLa cells causes dramatic alterations of the mitotic spindle and problems in chromosome alignment. Cells lacking Sgt1 undergo a mitotic delay due to activation of the spindle checkpoint. The checkpoint response, however, is significantly weakened in Sgt1-depleted cells, and this correlates with a dramatic reduction in kinetochore levels of Mad1, Mad2 and BubR1. These effects are explained by a problem in kinetochore assembly that prevents the localization of Hec1, CENP-E, CENP-F, CENP-I, but not CENP-C, to mitotic kinetochores. Our studies implicate Sgt1 as an essential protein and a critical assembly factor for the mammalian kinetochore, and lend credit to the hypothesis of a kinetochore assembly pathway that is conserved from yeast to man.     

Calcium-regulated interaction of Sgt1 with S100A6 (calcyclin) and other S100 proteins

S100A6 (calcyclin), a small calcium-binding protein from the S100 family, interacts with several target proteins in a calcium-regulated manner. One target is Calcyclin-Binding Protein/Siah-1-Interacting Protein (CacyBP/SIP), a component of a novel pathway of beta-catenin ubiquitination. A recently discovered yeast homolog of CacyBP/SIP, Sgt1, associates with Skp1 and regulates its function in the Skp1/Cullin1/F-box complex ubiquitin ligase and in kinetochore complexes. S100A6-binding domain of CacyBP/SIP is in its C-terminal region, where the homology between CacyBP/SIP and Sgt1 is the greatest. Therefore, we hypothesized that Sgt1, through its C-terminal region, interacts with S100A6. We tested this hypothesis by performing affinity chromatography and chemical cross-linking experiments. Our results showed that Sgt1 binds to S100A6 in a calcium-regulated manner and that the S100A6-binding domain in Sgt1 is comprised of 71 C-terminal residues. Moreover, S100A6 does not influence Skp1-Sgt1 binding, a result suggesting that separate Sgt1 domains are responsible for interactions with S100A6 and Skp1. Sgt1 binds not only to S100A6 but also to S100B and S100P, other members of the S100 family. The interaction between S100A6 and Sgt1 is likely to be physiologically relevant because both proteins were co-immunoprecipitated from HEp-2 cell line extract using monoclonal anti-S100A6 antibody. Phosphorylation of the S100A6-binding domain of Sgt1 by casein kinase II was inhibited by S100A6, a result suggesting that the role of S100A6 binding is to regulate the phosphorylation of Sgt1. These findings suggest that protein ubiquitination via Sgt1-dependent pathway can be regulated by S100 proteins.     

Effects of hCG on reduced numbers of hCG receptors in the prefrontal cortex and cerebellum of rat models of Alzheimer’s disease

ABSTRACT

Age-associated changes in the levels of luteinizing hormone and human chorionic gonadotropin (hCG) are potential risk factors for Alzheimer’s disease (AD); hCG concentration is related to the incidence of AD. The highest density of hCG receptors is in zones of the brain that are vulnerable to AD and streptozotocin (STZ) can decrease the density of this receptor. We investigated the effects of different doses of hCG on hCG receptor density in the prefrontal cortex and cerebellum in a rat model of STZ-induced AD. AD was induced by intracerebroventricular injection of 3 mg/kg STZ. The resulting AD rats were treated for 3 days with 50, 100 or 200 IU/200 μl hCG, or with saline as a control. Sections of prefrontal cortex and cerebellum were stained immunohistochemically and hCG receptor-immunoreactive (ir) neurons were counted. STZ injected into the lateral ventricles of rat brains reduced the density of hCG receptor-ir neurons in the prefrontal cortex and cerebellum. hCG administration resulted in a significant dose-dependent increase in the number of hCG receptor-ir neurons in the prefrontal cortex and cerebellum. The maximum increase in the number of receptors occurred following the 200 IU dose of hCG. Administration of hCG ameliorated the lowered density of hCG receptor-ir neurons in the cerebellum and prefrontal cortex in STZ-induced AD rats.     

Human Sgt1 binds HSP90 through the CHORD-Sgt1 domain and not the tetratricopeptide repeat domain

Sgt1 has been identified as a subunit of both core kinetochore and SCF (Skp1-Cul1-F-box) ubiquitin ligase complexes and is also implicated in plant disease resistance. Sgt1 has two putative HSP90 binding domains, a tetratricopeptide repeat and a p23-like CHORD and Sgt1 (CS) domain. Using NMR spectroscopy, we show that only the CS domain of human Sgt1 physically interacts with HSP90. The tetratricopeptide repeat domain does not bind to either HSP90 or HSP70. Determination of the three-dimensional structure showed that the Sgt1-CS domain shares the same beta-sandwich fold as p23 but lacks the last highly conserved beta-strand in p23. Analysis of the structures of Sgt1-CS and p23 revealed a similar charge distribution on one of two opposing surfaces that suggests that it is the binding region for HSP90 in Sgt1. Although ATP is absolutely required for p23 binding to HSP90, Sgt1 binds to HSP90 also in the absence of the non-hydrolyzable analog ATPgammaS. Our findings suggest the CS domain is a binding module for HSP90 distinct from p23-like domains, which implies that Sgt1 and related proteins function in recruiting heat shock protein activities to multiprotein assemblies.     

A frontal variant of Alzheimer's disease exhibits decreased calcium-independent phospholipase A2 activity in the prefrontal cortex

A frontal variant of Alzheimer's disease (AD) has recently been identified on neuropathological and neuropsychological grounds (Johnson, J.K., Head, E., Kim, R., Starr, A., Cotman, C.W., 1999. Clinical and pathological evidence for a frontal variant of Alzheimer Disease. Arch. Neurol. 56, 1233-1239). Frontal AD differs strikingly from typical AD by the occurrence of neurofibrillary tangle densities in the frontal cortex as high or higher than in the entorhinal cortex. Since cerebrocortical membranes are commonly abnormal in Alzheimer's disease (AD), we assayed frontal AD cases for enzymes regulating membrane phospholipid composition. We specifically measured activity of phospholipase A2s (PLA2s) in dorsolateral prefrontal and lateral temporal cortices of frontal AD cases (n=12), which have respectively high and low densities of neurofibrillary tangles. In neither cortical area was Ca(2+)-dependent PLA2 activity abnormal compared to controls (n=12). In contrast, a significant 42% decrease in Ca(2+)-independent PLA2 activity was found in the dorsolateral prefrontal, but not the lateral temporal, cortex of the frontal AD cases. Similarly, the dorsolateral prefrontal cortex, but not the lateral temporal cortex of the frontal AD cases suffered a 42% decrease in total free fatty acid content, though neither that decrease nor those in any one species of free fatty acid was significant. The observed biochemical changes probably occurred in neurons given (a) our finding that PLA2 activity of cultured human NT2 neurons is virtually all Ca(2+)-independent and (b) the finding of others that nearly all Ca(2+)-independent PLA2 in brain gray matter is neuronal. The decrease in Ca(2+)-independent PLA2 activity is not readily attributable to Group VI or VIII iPLA2s since neither NT2N neurons nor our brain homogenates were greatly inhibited by drugs potently suppressing those iPLA2s. Decreased Ca(2+)-independent PLA2 activity in frontal AD may reflect a compensatory response to pathologically accelerated phospholipid metabolism early in the disorder. That could cause an early elevation of prefrontal free fatty acids, which can stimulate polymerization of tau and thus promote the prefrontal neurofibrillary tangle formation characteristic of frontal AD.     

Glia Maturation Factor Expression in Entorhinal Cortex of Alzheimer’s Disease Brain

Alzheimer’s disease (AD) is characterized by the presence of neuropathological lesions containing amyloid plaques (APs) and hyperphosphorylated Tau containing neurofibrillary tangles (NFTs) and is associated with neuroinflammation and neurodegeneration. Entorhinal cortex (Brodmann’s area 28) is involved in memory associated functions and is one of the first brain areas targeted to form the neuropathological lesions and also severely affected cortical region in AD. Glia maturation factor (GMF), a central nervous system protein and a proinflammatory molecule is known to be up-regulated in the specific areas of AD brain. Our previous immunohistochemical studies using temporal cortex showed that GMF is expressed in the vicinity of APs and NFTs in AD brains. In the present study, we have analyzed the expression of GMF and its association with APs and NFTs in the entorhinal cortex of AD brains by using immunohistochemistry combined with thioflavin-S fluorescence labeling methods. Results showed that GMF immunoreactive glial cells, glial fibrillary acidic protein labeled reactive astrocytes and ionized calcium binding adaptor molecule-1 labeled activated microglia were increased in the entorhinal cortical layers especially at the sites of 6E10 labeled APs and Tau containing NFTs. In conclusion, increased expression of GMF by the glial cells in the entorhinal cortex region, and the co-localization of GMF with APs and NFTs suggest that GMF may play important proinflammatory roles in the pathogenesis of AD.     

MAP-2 immunolabeling can distinguish diffuse from dense-core amyloid plaques in brains with Alzheimer's disease

Alzheimer's disease (AD) neuropathology is characterized by the presence of diffuse and dense-core (neuritic) amyloid plaques in specific areas of the brain. The origin of these plaques and the relationship between them is poorly understood. Current methods to identify clearly these types of plaques in the AD brains are largely dependent upon morphological characteristics. Dense-core amyloid plaques in the entorhinal cortex and hippocampus of AD brains might arise from the lysis of neurons overburdened by excessive intracellular deposition of amyloid beta_1-42 (Aβ42) peptide. The local release of active lysosomal enzymes, which persist within these plaques, might degrade most of the released intracellular proteins, leaving behind only those that are resistant to proteolytic digestion, such as ubiquitin, tau, neurofilament proteins and amyloid. To test the possibility that proteins that are sensitive to proteolysis may be degraded selectively in plaques, we used immunohistochemistry to examine the distribution of microtubule-associated protein-2 (MAP-2), a protein localized primarily in neuronal dendrites and known to be sensitive to proteolysis. Uniform MAP-2 immunolabeling was detected throughout the somatodendritic compartment of neurons in age-matched control cortical brain tissues as well as throughout areas of Aβ42-positive diffuse plaques in AD brains. In contrast, analysis of serial sections revealed that MAP-2 was absent from Aβ42-positive dense-core plaques in AD brains. Our results indicate that this differential MAP-2 immunolabeling pattern among plaques may be employed as a reliable and sensitive method to distinguish dense-core plaques from diffuse plaques within AD brain tissue. Furthermore, this biochemical distinction indicates that dense-core and diffuse plaques are formed by different mechanisms.     

Comparative lipidomic analysis of mouse and human brain with Alzheimer disease

Lipids are key regulators of brain function and have been increasingly implicated in neurodegenerative disorders including Alzheimer disease (AD). Here, a systems-based approach was employed to determine the lipidome of brain tissues affected by AD. Specifically, we used liquid chromatography-mass spectrometry to profile extracts from the prefrontal cortex, entorhinal cortex, and cerebellum of late-onset AD (LOAD) patients, as well as the forebrain of three transgenic familial AD (FAD) mouse models. Although the cerebellum lacked major alterations in lipid composition, we found an elevation of a signaling pool of diacylglycerol as well as sphingolipids in the prefrontal cortex of AD patients. Furthermore, the diseased entorhinal cortex showed specific enrichment of lysobisphosphatidic acid, sphingomyelin, the ganglioside GM3, and cholesterol esters, all of which suggest common pathogenic mechanisms associated with endolysosomal storage disorders. Importantly, a significant increase in cholesterol esters and GM3 was recapitulated in the transgenic FAD models, suggesting that these mice are relevant tools to study aberrant lipid metabolism of endolysosomal dysfunction associated with AD. Finally, genetic ablation of phospholipase D(2), which rescues the synaptic and behavioral deficits of an FAD mouse model, fully normalizes GM3 levels. These data thus unmask a cross-talk between the metabolism of phosphatidic acid, the product of phospholipase D(2), and gangliosides, and point to a central role of ganglioside anomalies in AD pathogenesis. Overall, our study highlights the hypothesis generating potential of lipidomics and identifies novel region-specific lipid anomalies potentially linked to AD pathogenesis.     

Sgt1p and Skp1p modulate the assembly and turnover of CBF3 complexes required for proper kinetochore function

Kinetochores are composed of a large number of protein complexes that must be properly assembled on DNA to attach chromosomes to the mitotic spindle and to coordinate their segregation with the advance of the cell cycle. CBF3 is an inner kinetochore complex in the budding yeast Saccharomyces cerevisiae that nucleates the recruitment of all other kinetochore proteins to centromeric DNA. Skp1p and Sgt1p act through the core CBF3 subunit, Ctf13p, and are required for CBF3 to associate with centromeric DNA. To investigate the contribution of Skp1p and Sgt1p to CBF3 function, we have used a combination of in vitro binding assays and a unique protocol for synchronizing the assembly of kinetochores in cells. We have found that the interaction between Skp1p and Sgt1p is critical for the assembly of CBF3 complexes. CBF3 assembly is not restricted during the cell cycle and occurs in discrete steps; Skp1p and Sgt1p contribute to a final, rate-limiting step in assembly, the binding of the core CBF3 subunit Ctf13p to Ndc10p. The assembly of CBF3 is opposed by its turnover and disruption of this balance compromises kinetochore function without affecting kinetochore formation on centromeric DNA.     

Induction of excitatory and inhibitory presynaptic differentiation by GluD1

The δ subfamily of ionotropic glutamate receptor subunits consists of GluD1 and GluD2. GluD2, which is selectively expressed in cerebellar Purkinje neurons, has been shown to contribute to the formation of synapses between granule neurons and Purkinje neurons through interaction with Cbln1 (cerebellin precursor protein1) and presynaptic Neurexin. On the other hand, the synaptogenic activity of GluD1, which is expressed not in the cerebellum but in the hippocampus, remains to be characterized. Here, we report that GluD1 expressed in non-neuronal HEK cells, induced presynaptic differentiation of granule neurons through its N-terminal domain in co-cultures with cerebellar neurons, similarly to GluD2. We also show that GluD1 rescued the defect of synapse formation in GluD2-knockout Purkinje neurons, indicating the functional similarity of GluD1 and GluD2. In contrast, GluD1 expression alone did not induce presynaptic differentiation in co-cultures of HEK cells with hippocampal neurons. However, when Cbln1 was exogenously added to the culture medium, GluD1 induced presynaptic differentiation of not only glutamatergic presynaptic terminals but also GABAergic ones. Cbln1 is not expressed in hippocampal neurons but is expressed in entorhinal cortical neurons projecting to the hippocampus. In co-cultures of HEK cells expressing GluD1 and entorhinal cortical neurons, both glutamatergic and GABAergic presynaptic terminals were formed on the HEK cells without exogenous application of Cbln1. These results suggest that GluD1 might contribute to the formation of specific synapses in the hippocampus such as those formed by the projecting neurons of the entorhinal cortex.     

TPR-Mediated self-association of plant SGT1

The tetratricopeptide repeat (TPR) domain mediates inter-protein associations in a number of systems. The domain is also thought to mediate oligomerization of some proteins, but this has remained controversial, with conflicting data appearing in the literature. By way of investigating such TPR-mediated self-associations we used a variety of biophysical techniques to characterize purified recombinant Sgt1, a TPR-containing protein found in all eukaryotes that is involved in a broad range of biological processes, including kinetochore assembly in humans and yeast and disease resistance in plants. We show that recombinant Sgt1 from Arabidopsis, barley, and yeast self-associates in vitro while recombinant human Sgt1 does not. Further experiments on barley Sgt1 demonstrate unambiguously a TPR-mediated dimerization, which is concentration- and ionic-strength-dependent and results in a global increase in helical structure and stability of the protein. Dimerization is also redox sensitive, being completely abolished by the formation of an intramolecular disulfide bond where the contributing cysteines are conserved in plant Sgt1s. The dimer interface was mapped through cross-linking and mass spectrometry to the C-terminal region of the TPR domain. Our study, which provides the first biophysical characterization of plant Sgt1, highlights how TPR domains can mediate self-association in solution and that sequence variation in the regions involved in oligomerization affects the propensity of TPR-containing proteins to dimerize.     

Transglutaminase bonds in neurofibrillary tangles and paired helical filament tau early in Alzheimer's disease.

Transglutaminase-catalyzed epsilon(gamma-glutamyl)lysine cross-links exist in Alzheimer's disease (AD) paired helical filament (PHF) tau protein but not normal soluble tau. To test the hypothesis that these cross-links could play a role in the formation of neurofibrillary tangles (NFT), we used single- and double-label immunofluorescence confocal microscopy and immunoaffinity purification and immunoblotting to examine epsilon(gamma-glutamyl)lysine cross-links in AD and control brains. The number of neurons that are immunoreactive with an antibody directed at the epsilon-(gamma-glutamyl)lysine bond was significantly higher in AD cortex compared with age-matched controls and schizophrenics. PHF tau-directed antibodies AT8, MC-1 and PHF-1 co-localized with epsilon(gamma-glutamyl)lysine immunolabeling in AD NFT. Immunoaffinity purification and immunoblotting experiments demonstrated that PHF tau contains epsilon(gamma-glutamyl)lysine bonds in parietal and frontal cortex in AD. In control cases with NFT present in the entorhinal cortex and hippocampus, indicative of Braak and Braak stage II, epsilon(gamma-glutamyl)lysine bonds were present in PHF tau in parietal and frontal cortex, despite the lack of microscopically detectable NFT or senile plaques in these cortical regions. The presence of PHF tau with epsilon(gamma-glutamyl)lysine bonds in brain regions devoid of NFT in stage II (but regions, which would be expected to contain NFT in stage III) suggests that these bonds occur early in the formation of NFT.     

Sgt1p is a unique co-chaperone that acts as a client adaptor to link Hsp90 to Skp1p

Sgt1p is a conserved, essential protein required for kinetochore assembly in both yeast and animal cells. Sgt1p has homology to both TPR and p23 domains, sequences often found in proteins that interact with and regulate the molecular chaperone, Hsp90. The presence of these domains and the recent findings that Sgt1p interacts with Hsp90 has led to the speculation that Sgt1p and Hsp90 form a co-chaperone complex. To test this possibility, we have used purified recombinant proteins to characterize the in vitro interactions between yeast Sgt1p and Hsp82p (an Hsp90 homologue in yeast). We show that Sgt1p interacts directly with Hsp82p via its p23 homology region in a nucleotide-dependent manner. However, Sgt1p binding does not alter the enzymatic activity of Hsp82p, suggesting that it is distinct from other co-chaperones. We find that Sgt1p can form a ternary chaperone complex with Hsp82p and Sti1p, a well characterized Hsp90 co-chaperone. Sgt1p interacts with its binding partner Skp1p through its TPR domains and links Skp1p to the core Hsp82p-Sti1p co-chaperone complex. The multidomain nature of Sgt1p and its ability to bridge the interaction between Skp1p and Hsp82p argue that Sgt1p acts as a "client adaptor" recruiting specific clients to Hsp82p co-chaperone complexes.     

Localization of the CB1 cannabinoid receptor in the rat brain. An immunohistochemical study

The presence of central cannabinoid receptor (CB1), involving the N-terminal 14 amino acid peptide, was demonstrated in the rat brain by immunohistochemistry. Intensely stained neurons were observed in the principal neurons of the hippocampus, striatum, substantia nigra, cerebellar cortex, including the Purkinje cells. Moderate CB1-IR cell bodies and fibers were present in the olfactory bulb, cingulate, entorhinal and piriform cortical areas, amygdala and nucleus accumbens. The perivascular glial fibers have shown moderate to high density CB1-IR in olfactory and limbic structures. Low density was detected in the thalamus and hypothalamus and area postrema. The CB1 receptor was widely distributed in the forebrain and sparsely in the hindbrain. These new data support the view that the endogenous cannabinoids play an important role in different neuronal functions as neuromodulators or neurotransmitters.     

The interaction between Sgt1p and Skp1p is regulated by HSP90 chaperones and is required for proper CBF3 assembly

Sgt1p is a well-conserved protein proposed to be involved in a number of cellular processes. Genetic studies of budding yeast suggest a role for SGT1 in signal transduction, cell cycle advance, and chromosome segregation. Recent evidence has linked Sgt1p to HSP90 chaperones, although the precise relationship between these proteins is unclear. To further explore the role of Sgt1p in these processes, we have characterized the interactions among Sgt1p, the inner kinetochore complex CBF3, and HSP90 chaperones. We show that the amino terminus of Sgt1p interacts with CBF3 subunits Skp1p and Ctf13p. HSP90 interacts with Sgt1p and, in combination with the carboxy terminus of Sgt1p, regulates the interaction between Sgt1p and Skp1p in a nucleotide-dependent manner. While the Sgt1p-Skp1p interaction is required for CBF3 assembly, mutations that stabilize this interaction prevent the turnover of protein complexes important for CBF3 assembly. We propose that HSP90 and Sgt1p act together as a molecular switch, maintaining transient interactions required to balance protein complex assembly with turnover.     

Regional Heterogeneity of Cerebral Microvessels and Brain Susceptibility to Oxidative Stress

The hippocampus is one of the earliest and most affected regions in Alzheimer’s disease (AD), followed by the cortex while the cerebellum is largely spared. Importantly, endothelial dysfunction is a common feature of cerebral blood vessels in AD. In this study, we sought to determine if regional heterogeneity of cerebral microvessels might help explain the susceptibility of the hippocampus and cortex as compared to the cerebellum. We isolated microvessels from wild type mice from the cerebellum, cortex, and hippocampus to characterize their vascular phenotype. Superoxide anion was significantly higher in microvessels isolated from the cortex and hippocampus as compared to the cerebellum. Importantly, protein levels of NADPH oxidase (NOX)-2 and NOX-4 were significantly higher in the cortical and hippocampal microvessels as compared to microvessels from the cerebellum. In addition, expression of manganese superoxide dismutase protein was significantly lower in microvessels from the cortex and hippocampus as compared to cerebellum while other antioxidant enzymes were unchanged. There was no difference in eNOS protein expression between the microvessels of the three brain regions; however, bioavailability of tetrahydrobiopterin (BH₄), an essential cofactor for eNOS activity, was significantly reduced in microvessels from the hippocampus and cortex as compared to the cerebellum. Higher levels of superoxide and reduced tetrahydrobiopterin bioavailability may help explain the vulnerability of the hippocampus and cortical microvessels to oxidative stress and development of endothelial dysfunction.