Jae-Wan Huh

Okadaic acid induces JNK activation, bim overexpression and mitochondrial dysfunction in cultured rat cortical neurons.

Apoptosis via tau phosphorylation has been implicated in the selective neuronal losses seen in Alzheimers disease (AD). Previous studies in vivo and in cultured neurons have shown that okadaic acid (OA) evokes tau phosphorylation to initiate a neurodegeneration that resembles the pathogenesis of AD. In an effort to identify additional key molecules in this neurodegeneration, we treated cultured rat neurons with OA and examined the apoptosis-related effects, such as changes in mitochondrial activity and expression levels of JNK, Bim, Bad, Bax and caspase-3. Western blotting revealed that phosphorylation of JNK and c-jun occurred first, followed by increased expression of Bim and subsequent caspase-3 activation in OA-treated neurons. In contrast, Bad levels decreased as early as 4 h after OA treatment. Immunocytochemistry showed that the increased phospho-JNK immunoreactivity was localized in the cytosol of degenerating neurons, while increased phospho-c-jun was localized in the nucleus. The mitochondria showed decreased membrane potential and increased swelling after OA treatment. Collectively, these data suggest that JNK- and Bim-related mitochondrial dysfunction is involved in OA-induced neurodegeneration.

BACE inhibitor reduces APP-β-C-terminal fragment accumulation in axonal swellings of okadaic acid-induced neurodegeneration

Emerging evidence suggests that not only beta-amyloid but also other amyloid precursor protein (APP) fragments, such as the beta-C-terminal fragment (betaCTF), might be involved in Alzheimers disease (AD). Treatment of neurons with okadaic acid (OA), a protein phosphatase-2A inhibitor, has been used to induce tau phosphorylation and neuronal death to create a research model of AD. In this study, we analyzed axonopathy and APP regulation in cultured rat neurons treated with OA. After OA treatment, the neurons presented with axonal swellings filled with vesicles, microtubule fragments, and transport molecules such as kinesin and synapsin-I. Western blotting showed that intracellular APP levels were increased and immunocytochemistry using antibodies against the APP C-terminus showed that APP accumulated in the axonal swellings. This APP C-terminus immunoreactivity disappeared when neurons were cotreated with a beta-secretase inhibitor, but not with alpha- or gamma-secretase inhibitors, indicating that the accumulation was primarily composed of APP-betaCTF. These findings provide the first evidence that APP-betaCTF can accumulate in the axons of OA-treated neurons, and may suggest that APP-betaCTF is involved in the pathogenesis of AD.

Identification of ADP-ribosylation site in human glutamate dehydrogenase isozymes

When the influence of ADP‐ribosylation on the activities of the purified human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was measured in the presence of 100 μM NAD+ for 60 min, hGDH isozymes were inhibited by up to 75%. If incubations were performed for longer time periods up to 3 h, the inhibition of hGDH isozymes did not increased further. This phenomenon may be related to the reversibility of ADP‐ribosylation in mitochondria. ADP‐ribosylated hDGH isozymes were reactivated by Mg2+‐dependent mitochondrial ADP‐ribosylcysteine hydrolase. The stoichiometry between incorporated ADP‐ribose and GDH subunits shows a modification of one subunit per catalytically active homohexamer. Since ADP and GTP had no effects on the extent of modification, it would appear that the ADP‐ribosylation is unlikely to occur in allosteric sites. It has been proposed that Cys residue may be involved in the ADP‐ribosylation of GDH, although identification of the reactive Cys residue has not been reported. To identify the reactive Cys residue involved in the ADP‐ribosylation, we performed cassette mutagenesis at three different positions (Cys59, Cys119, and Cys274) using synthetic genes of hGDH isozymes. Among the Cys residues tested, only Cys119 mutants showed a significant reduction in the ADP‐ribosylation. These results suggest a possibility that the Cys119 residue has an important role in the regulation of hGDH isozymes by ADP‐ribosylation.

Sitagliptin increases tau phosphorylation in the hippocampus of rats with type 2 diabetes and in primary neuron cultures

Increasing evidence supports an association between Alzheimers disease (AD) and diabetes. In this context, anti-diabetic agents such as rosiglitazone and glucagon-like peptide (GLP)-1 have been reported to reduce pathologies associated with AD, including tau hyperphosphorylation, suggesting that such agents might be used to treat AD. One such anti-diabetic agent is sitagliptin, which acts through inhibition of dipeptidyl peptidase (DPP)-IV to increase GLP-1 levels. Given this action, sitagliptin would be predicted to reduce AD pathology. Accordingly, we investigated whether sitagliptin is effective in attenuating AD pathologies, focusing on tau phosphorylation in the OLETF type 2 diabetic rat model. Unexpectedly, we found that sitagliptin was not effective against pathological tau phosphorylation in the hippocampus of OLETF type 2 diabetes rats, and instead aggravated it. This paradoxically increased tau phosphorylation was attributed to activation of the tau kinase, GSK3β (glycogen synthase kinase 3β). Sitagliptin also increased ser-616 phosphorylation of the insulin receptor substrate (IRS)-1, suggesting increased insulin resistance in the brain. These phenomena were recapitulated in primary rat cortical neurons treated with sitagliptin, further confirming sitagliptins effects on AD-related pathologies in neurons. These results highlight the need for caution in considering the use of sitagliptin in AD therapy.

Important role of Ser443 in different thermal stability of human glutamate dehydrogenase isozymes1

Molecular biological studies confirmed that two glutamate dehydrogenase isozymes (hGDH1 and hGDH2) of distinct genetic origin are expressed in human tissues. hGDH1 is heat‐stable and expressed widely, whereas hGDH2 is heat‐labile and specific for neural and testicular tissues. A selective deficiency of hGDH2 has been reported in patients with spinocerebellar ataxia. We have identified an amino acid residue involved in the different thermal stability of human GDH isozymes. At 45°C (pH 7.0), heat inactivation proceeded faster for hGDH2 (half life=45 min) than for hGDH1 (half‐life=310 min) in the absence of allosteric regulators. Both hGDH1 and hGDH2, however, showed much slower heat inactivation processes in the presence of 1 mM ADP or 3 mM L‐Leu. Virtually most of the enzyme activity remained up to 100 min at 45°C after treatment with ADP and L‐Leu in combination. In contrast to ADP and L‐Leu, the thermal stabilities of the hGDH isozymes were not affected by addition of substrates or coenzymes. In human GDH isozymes, the 443 site is Arg in hGDH1 and Ser in hGDH2. Replacement of Ser by Arg at the 443 site by cassette mutagenesis abolished the heat lability of hGDH2 with a similar half‐life of hGDH1. The mutagenesis at several other sites (L415M, A456G, and H470R) having differences in amino acid sequence between the two GDH isozymes did not show any change in the thermal stability. These results suggest that the Ser443 residue plays an important role in the different thermal stability of human GDH isozymes.

Inhibitory effects of gallic acid and quercetin on UDP-glucose dehydrogenase activity.

We have examined polyphenols as potential inhibitors of UDP‐glucose dehydrogenase (UGDH) activity. Gallic acid and quercetin decreased specific activities of UGDH and inhibited the proliferation of MCF‐7 human breast cancer cells. Western blot analysis showed that gallic acid and quercetin did not affect UGDH protein expression, suggesting that UGDH activity is inhibited by polyphenols at the post‐translational level. Kinetics studies using human UGDH revealed that gallic acid was a non‐competitive inhibitor with respect to UDP‐glucose and NAD+. In contrast, quercetin showed a competitive inhibition and a mixed‐type inhibition with respect to UDP‐glucose and NAD+, respectively. These results indicate that gallic acid and quercetin are effective inhibitors of UGDH that exert strong antiproliferative activity in breast cancer cells.

Regulation of glutamate level in rat brain through activation of glutamate dehydrogenase by Corydalis ternata.

When treated with protopine and alkalized extracts of the tuber of Corydalis ternata for one year, significant decrease in glutamate level and increase in glutamate dehydrogenase (GDH) activity was observed in rat brains. The expression of GDH between the two groups remained unchanged as determined by Western and Northern blot analysis, suggesting a post-translational regulation of GDH activity in alkalized extracts treated rat brains. The stimulatory effects of alkalized extracts and protopine on the GDH activity was further examined in vitro with two types of human GDH isozymes, hGDH1 (house-keeping GDH) and hGDH2 (nerve-specific GDH). Alkalized extracts and protopine activated the human GDH isozymes up to 4.8-fold. hGDH2 (nervespecific GDH) was more sensitively affected by 1 mM ADP than hGDH1 (house-keeping GDH) on the activation by alkalized extracts. Studies with cassette mutagenesis at ADP-binding site showed that hGDH2 was more sensitively regulated by ADP than hGDH1 on the activation by Corydalis ternata. Our results suggest that prolonged exposure to Corydalis ternata may be one of the ways to regulate glutamate concentration in brain through the activation of GDH.

Importance of Gly-13 for the Coenzyme Binding of Human UDP-glucose Dehydrogenase

UDP-glucose dehydrogenase (UGDH) is the unique pathway enzyme furnishing in vertebrates UDP-glucuronate for numerous transferases. In this report, we have identified an NAD+-binding site within human UGDH by photoaffinity labeling with a specific probe, [32P]nicotinamide 2-azidoadenosine dinucleotide (2N3 NAD+), and cassette mutagenesis. For this work, we have chemically synthesized a 1509-base pair gene encoding human UGDH and expressed it in Escherichia coli as a soluble protein. Photolabel-containing peptides were generated by photolysis followed by tryptic digestion and isolated using the phosphopeptide isolation kit. Photolabeling of these peptides was effectively prevented by the presence of NAD+ during photolysis, demonstrating a selectivity of the photoprobe for the NAD+-binding site. Amino acid sequencing and compositional analysis identified the NAD+-binding site of UGDH as the region containing the sequence ICCIGAXYVGGPT, corresponding to Ile-7 through Thr-19 of the amino acid sequence of human UGDH. The unidentified residue, X, can be designated as a photolabeled Gly-13 because the sequences including the glycine residue in question have a complete identity with those of other UGDH species known. The importance of Gly-13 residue in the binding of NAD+ was further examined with a G13E mutant by cassette mutagenesis. The mutagenesis at Gly-13 had no effects on the expression or stability of the mutant. Enzyme activity of the G13E point mutant was not measurable under normal assay conditions, suggesting an important role for the Gly-13 residue. No incorporation of [32P]2N3NAD+ was observed for the G13E mutant. These results indicate that Gly-13 plays an important role for efficient binding of NAD+ to human UGDH.

Inactivation of GSK-3beta in okadaic acid-induced neurodegeneration: relevance to Alzheimer's disease.

Hyperphosphorylation of tau is a characteristic feature of the neurodegenerative pathology in Alzheimers disease (AD). Okadaic acid is used as a research model of AD to increase the tau phosphorylation and neuronal death. Using Western blotting, we found that the amounts of activated PKB[pS-473] and inactivated GSK-3beta[pS-9] were increased in proportion to the progress of okadaic acid induced tau phosphorylation. Immunocytochemistry showed that PKB[pS-473] and GSK-3beta[pS-9] immunoreactivity increased in dystrophic neurites and cell bodies in degenerating neurons after okadaic acid treatment. Double staining with phosphospecific tau antibodies showed that PKB[pS-473] and GSK-3beta[pS-9] were colocalized with phosphospecific tau in response to okadaic acid. Taken together, our data suggest that inhibition of protein phosphatase results in the hyperphosphorylation of tau without GSK-3beta overactivation.

Amino Acid Changes within Antenna Helix Are Responsible for Different Regulatory Preferences of Human Glutamate Dehydrogenase Isozymes

Human glutamate dehydrogenase (hGDH) exists in hGDH1 (housekeeping isozyme) and in hGDH2 (nerve-specific isozyme), which differ markedly in their allosteric regulation. Because they differ in only 16 of their 505 amino acids, the regulatory preferences must arise from amino acid residues that are not common between hGDH1 and hGDH2. To our knowledge none of the mutagenesis studies on the hGDH isozymes to date have identified the amino acid residues fully responsible for the different regulatory preferences between hGDH1 and hGDH2. In this study we constructed hGDH1(hGDH2390–448)hGDH1 (amino acid segment 390–448 of hGDH1 replaced by the corresponding hGDH2 segment) and hGDH2(hGDH1390–448)hGDH2 (amino acid segment 390–448 of hGDH2 replaced by the corresponding hGDH1 segment) by swapping the corresponding amino acid segments in hGDH1 and hGDH2. The chimeric enzymes by reciprocal swapping resulted in double mutations in amino acid sequences at 415 and 443 residues that are not common between hGDH1 and hGDH2 and are located in the C-terminal 48-residue “antenna” helix, which is thought to be part of the regulatory domain of mammalian GDHs. Functional analyses revealed that the doubly mutated chimeric enzymes almost completely acquired most of the different regulatory preferences between hGDH1 and hGDH2 for electrophoretic mobility, heat-stability, ADP activation, palmitoyl-CoA inhibition, and l-leucine activation, except for GTP inhibition. Our results indicate that substitutions of the residues in the antenna region may be important evolutionary changes that led to the adaptation of hGDH2 to the unique metabolic needs of the nerve tissue.