Analysis of tau haplotypes in Pick's disease.

Pick's disease (PiD) is characterized by the deposition of tau protein as three-repeat tau Pick bodies, whereas progressive supranuclear palsy (PSP) involves the deposition of four-repeat tau neurofibrillary tangles. PSP is associated with the tau H1 haplotype. The authors investigated a possible association between PiD and the tau H1 or H2 haplotype. There was no difference between the tau H2 haplotype or H2H2 genotype frequency in PiD cases and control subjects. No tau mutations were identified in pathologically typical cases of PiD, with antibody 12-E8-negative Pick bodies.

High stability of mRNAs postmortem and protocols for their assessment by RT-PCR.

Measurement of gene expression is a major area of brain research. We report on the remarkable postmortem stability of a selection of brain mRNAs in both fresh and frozen brain tissue. We describe techniques for extracting total RNA, synthesizing cDNAs from the mRNAs, amplifying specific cDNAs by the polymerase chain reaction technique, and quantitating the products. We chose five genes to study: the housekeeping gene cyclophilin; the complement components C3 and C4; the microtubule associated protein-2 (MAP-2); and the strongly inducible cyclooxygenase COX-2. We found little deterioration in total RNA or in any of the mRNAs in postmortem tissue up to 96 h. When tissue was frozen, stored at -70 degrees C for 15 years and then thawed, there was no evidence of deterioration from storage, but there was gradual deterioration post thawing. All the mRNAs were stable for 1-2 h at 4 degrees C following thawing. Cyclophilin, C3 and C4 mRNAs were still stable after 8 h, MAP-2 and COX-2 mRNAs showed significant deterioration between 2 and 4 h, and COX-2 mRNA showed drastic deterioration between 4 and 8 h. The data give no indication of rapid postmortem degeneration of RNA. Reliable mRNA values may be obtained from postmortem brain with long autolysis times provided the tissue has been kept in the cold, and from frozen tissues for 1-2 h after thawing.

Relationship between beta amyloid peptide generating molecules and neprilysin in Alzheimer disease and normal brain.

beta-Amyloid peptide (Abeta) is generated by two cleavages of amyloid precursor protein (APP). The initial cleavage by BACE is followed by gamma-secretase cleavage of the C-terminal APP fragment. Presenilin-1 (PS-1) is intimately related to gamma-secretase. Once formed, Abeta is mainly broken down by neprilysin. To estimate vulnerability to Abeta senile plaque formation, we measured the relative mRNA levels of APP695, APP751, APP770, BACE, presenilin-1 (PS-1) and neprilysin in nine brain areas and in heart, liver, spleen and kidney in a series of Alzheimer disease (AD) and control cases. Each of the mRNAs was expressed in every tissue examined. APP695 was the dominant APP isoform in brain. Compared with controls, APP695 and PS-1 mRNA levels were significantly elevated in high plaque areas of AD brain, while neprilysin mRNA levels were significantly reduced. BACE levels were not significantly different in AD compared with control brain. In peripheral organs, there were no significant differences in any of the mRNAs between AD and control cases. APP isoforms were differently expressed in the periphery than in brain, with APP 751>770>695. Neprilysin mRNA levels were much higher, while APP695 and PS-1 mRNA levels were much lower in the periphery than in brain. The data suggest that, in the periphery, the capacity to degrade Abeta is srong, accounting for the failure of Abeta deposits to form. In plaque prone areas of AD brain, the capacity to degrade Abeta is weak, while the capacity to generate Ab is upregulated. In plaque resistant areas of brain, a closer balance exists, but there is some tendency towards lower degrading and higher synthesizing capacity in AD brain compared with control brain. Overall, the data indicate that effectiveness of degradation by neprilysin may be a key factor in determining whether Abeta deposits develop.

3-hydroxy-3-methylglutaryl-coenzyme A reductase mRNA in Alzheimer and control brain.

Statins are widely used pharmaceutical agents which lower plasma cholesterol by inhibiting the rate controlling enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase. One epidemiological study suggests that statin therapy may provide protection against Alzheimer disease (AD). The aim of the present study was to determine the relative expression of HMG-CoA reductase mRNAs in various areas of brain as well as in peripheral organs and to compare values in AD and control cases. High levels of the mRNA were found in all areas of brain but no obvious differences were found between AD and controls. We conclude that brain has a robust capacity to synthesize cholesterol which appears to be unaffected by AD pathology.

Marked increase in cyclooxygenase-2 in ALS spinal cord: implications for therapy.

OBJECTIVE: To evaluate the hypothesis that cyclooxygenase-2 (COX-2) is linked to the pathology of ALS by determining whether COX-2 mRNA levels are upregulated in ALS spinal cord. METHODS: Spinal cord from 11 ALS cases and 27 controls consisting of 15 cases of Alzheimer disease (AD), six cases of Parkinson disease (PD), three cases of cerebrovascular disease, and three control cases were analyzed. Total RNA was extracted and reverse transcriptase-PCR analysis performed for the mRNA of COX-2, COX-1, the microglial marker CD11b, and the housekeeping gene cyclophilin. RESULTS: In ALS compared with non-ALS spinal cord, COX-2 mRNA was upregulated 7.09-fold (p < 0.0001), COX-1 1.14-fold (p = 0.05), and CD11b 1.85-fold (p = 0.0012). COX-2 mRNA levels in AD, PD, cerebrovascular disease, and control cases were each significantly lower than in ALS and were not significantly different from each other. Western blots of the protein products were in general accord with the mRNA data, with COX-2 protein levels being upregulated 3.79-fold compared with non-ALS cases (p = 0.015). CONCLUSIONS: The strong upregulation of COX-2 mRNA in ALS is in accord with studies in the superoxide dismutase transgenic mouse model in which COX-2 upregulation occurs. Taken in conjunction with evidence of a neuroprotective effect of COX-2 inhibitors in certain animal models and in organotypic cultures, the data are supportive of a possible future role for COX-2 inhibitors in the treatment of ALS.

Complement components, but not complement inhibitors, are upregulated in atherosclerotic plaques.

Complement activation occurs in atherosclerotic plaques. The capacity of arterial tissue to inhibit this activation through generation of the complement regulators C1 inhibitor, decay accelerating factor, membrane cofactor protein (CD46), C4 binding protein (C4BP), and protectin (CD59) was evaluated in pairs of aortic atherosclerotic plaques and nearby normal artery from 11 human postmortem specimens. All 22 samples produced mRNAs for each of these proteins. The ratios of plaque versus normal artery pairs was not significantly different from unity for any of these inhibitors. However, in plaques, the mRNAs for C1r and C1s, the substrates for the C1 inhibitor, were increased 2.35- and 4.96-fold, respectively, compared with normal artery; mRNA for C4, the target for C4BP, was elevated l.34-fold; and mRNAs for C7 and C8, the targets for CD59, were elevated 2.61- and 3.25-fold, respectively. By Western blotting and immunohistochemistry, fraction Bb of factor B, a marker of alternative pathway activation, was barely detectable in plaque and normal arterial tissue. These data indicate that it is primarily the classical, not the alternative pathway, that is activated in plaques and that key inhibitors are not upregulated to defend against this activation.

Generation of C-reactive protein and complement components in atherosclerotic plaques.

C-reactive protein (CRP) and complement are hypothesized to be major mediators of inflammation in atherosclerotic plaques. We used the reverse transcriptase-polymerase chain reaction technique to detect the mRNAs for CRP and the classical complement components C1 to C9 in both normal arterial and plaque tissue, establishing that they can be endogenously generated by arteries. When the CRP mRNA levels of plaque tissue, normal artery, and liver were compared in the same cases, plaque levels were 10.2-fold higher than normal artery and 7.2-fold higher than liver. By Western blotting, we showed that the protein levels of CRP and complement proteins were also up-regulated in plaque tissue and that there was full activation of the classical complement pathway. By in situ hybridization, we detected intense signals for CRP and C4 mRNAs in smooth muscle-like cells and macrophages in the thickened intima of plaques. By immunohistochemistry we showed co-localization of CRP and the membrane attack complex of complement. We also detected up-regulation in plaque tissue of the mRNAs for the macrophage markers CD11b and HLA-DR, as well as their protein products. We showed by immunohistochemistry macrophage infiltration of plaque tissue. Because CRP is a complement activator, and activated complement attacks cells in plaque tissue, these data provide evidence of a self-sustaining autotoxic mechanism operating within the plaques as a precursor to thrombotic events.

Reduced neprilysin in high plaque areas of Alzheimer brain: a possible relationship to deficient degradation of beta-amyloid peptide.

Neprilysin is an enzyme capable of degrading beta-amyloid protein. We measured neprilysin mRNA and protein levels in brain and peripheral organs of Alzheimer disease (AD) and control cases. Neprilysin mRNA levels were lowest in the hippocampus and temporal gyrus, which are vulnerable to senile plaque development. They were highest in the caudate and peripheral organs which are resistant to senile plaque development. Levels in AD were significantly lower than controls in the hippocampus and midtemporal gyrus but not in other brain areas or peripheral organs. We also measured levels of the mRNA for the neuronal marker microtubule-associated protein-2. They were remarkably constant in all brain areas and were not lowered in AD, indicating that the neprilysin mRNA reduction in the hippocampus and temporal gyrus was not correlated with simple neuronal loss. Relative levels of neprilysin protein generally paralleled those of the mRNA. These results suggest that deficient degradation of beta-amyloid protein caused by low levels of neprilysin may contribute to AD pathogenesis.

The pentraxins: possible role in Alzheimer's disease and other innate inflammatory diseases.

Two short pentraxins, C-reactive protein and amyloid P, are found in association with the senile plaques and neurofibrillary tangles of Alzheimer disease (AD). Formerly thought to be made primarily if not solely in liver, recent work has shown that they are made not only in the brain but in other tissues such as heart and arteries. Their synthesis is markedly upregulated in affected brain regions in AD. Since they are known to activate the complement cascade in an antibody-independent fashion and chronic activation can cause destruction of host tissue, these pentraxins may be important initiators of an autodestructive process. As such, they may be prime targets for therapeutic intervention.

Immunoidentification of gonadotropin releasing hormone receptor in human sperm, pituitary and cancer cells.

PROBLEM: To generate monoclonal antibodies specific to the receptor of gonadotropin releasing hormone (GnRH) for immunoidentification of the GnRH receptor (RGnRH) in human sperm, pituitary and cancer cells. METHODS: Four oligopeptides corresponding to RGnRH amino acid residues 1-29, 182-193, 195-206 and 293-306 in the extracellular domains were synthesized, conjugated to hemocyanin from Keyhole Limpet and used as immunogens to generate specific monoclonal antibodies. Enzyme-linked immunosorbent assay was used for initial screening of hybridomas. RESULTS: A total of 15 hybrid cell lines secreting RGnRH-specific monoclonal antibodies were initially established. By using some of these monoclonal antibodies as immunohistochemical probes, RGnRH was localized in the acrosomal region of human sperm, in the anterior pituitary tissue and in cancer cells of human ovarian and cervical origins. RGnRH was shown to have a molecular size of 45,000 +/- 5,000 kDa by Western blot assay. Expression of RGnRH mRNA in several human tissues and cancer cells was established by the reverse transcriptase-polymerase chain reaction method. CONCLUSION: RGnRH-specific monoclonal antibodies may be a valuable tool of identifying the presence of RGnRH in various normal and malignant human tissues.

Preconditioning reduces myocardial complement gene expression in vivo.

This investigation examined the effect of preconditioning in an in vivo model of ischemia-reperfusion injury. Anesthetized New Zealand White rabbits underwent 30 min of regional myocardial ischemia followed by 2 h of reperfusion. Hearts preconditioned with two cycles of 5 min ischemia-10 min reperfusion (IPC) or with the ATP-sensitive K (K(ATP)) channel opener, diazoxide (10 mg/kg), exhibited significantly (P < 0.05) smaller infarcts compared with control. These treatments also significantly (P < 0.001 to P < 0.05) reduced C1q, C1r, C3, C8, and C9 mRNA in the areas at risk (AAR). The K(ATP) channel blocker 5-hydroxydecanoate (5-HD; 10 mg/kg) attenuated infarct size reduction elicited by IPC and diazoxide treatment. 5-HD partially reversed the decrease in complement expression caused by IPC but not diazoxide. There were no significant differences in complement gene expression in the nonrisk regions and livers of all groups. Western blot analysis revealed that IPC also reduced membrane attack complex expression in the AAR. The data demonstrate that preconditioning significantly decreases reperfusion-induced myocardial complement expression in vivo.

Alzheimer disease and neuroinflammation.

It is now generally accepted that the lesions of Alzheimer disease (AD) are associated with a host of inflammatory molecules, including complement proteins, as well as with many activated microglia. Most inflammatory components are synthesized by brain cells. In order to estimate the intensity of the inflammatory reaction, we have measured the levels of the mRNAs for complement proteins, two complement regulators (CD59 and C1 inhibitors), an acute phase reactant (C-reactive protein, CRP) and two microglial markers, (HLA-DR and CD11b), in normal and AD brain. The mRNAs for inflammatory mediators are markedly upregulated in AD tissue while those of the complement inhibitors are almost unchanged. The upregulations for CRP and CD11b in AD hippocampus are comparable to those in osteoarthritic joints. This lends further support to the hypothesis that chronic inflammation may be causing neuronal death in AD.

Free radicals upregulate complement expression in rabbit isolated heart.

Both free radicals and complement activation can injure tissue. Our study determined whether free radicals alter complement production by the myocardium. Isolated hearts from New Zealand White rabbits were perfused on a Langendorff apparatus and exposed to xanthine (X; 100 microM) plus xanthine oxidase (XO; 8 mU/ml) (X/XO). The free radical-generating system significantly (P < 0.05) increased C1q and also increased C1r, C3, C8, and C9 transcription compared with controls. Immunohistological examination revealed augmented membrane attack complex deposition on X/XO-treated tissue. X/XO-treated hearts also exhibited significant (P < 0.05) increases in coronary perfusion pressure and left ventricular end-diastolic pressure and a decrease in left-ventricular developed pressure. N-(2-mercaptopropionyl)-glycine (3 mM), in conjunction with the superoxide dismutase mimetic SC-52608 (100 microM), significantly (P < 0.05) reduced the upregulation of C1q, C1r, C3, C8, and C9 mRNA expression elicited by X/XO. The antioxidants also ameliorated the deterioration in function caused by X/XO. Local complement activation may represent a mechanism by which free radicals mediate tissue injury.

Casein kinase 1 delta mRNA is upregulated in Alzheimer disease brain.

The casein kinase-1 (Ck1) family are serine/threonine specific protein kinases. They are highly associated with Alzheimer disease (AD) brain-derived tau filaments and granulovacuolar bodies. Recently we have demonstrated that one family member, Ckidelta, colocalizes with tau containing neurofibrillary tangles (NFTs) and other tau deposits in a number of neurodegenerative diseases. Here we show that the association in AD is accompanied by a sharp upregulation of Ckidelta mRNA in brain but not in peripheral organs. The degree of upregulation in AD brain is correlated with the degree of regional pathology. There was a 24.4-fold increase of Ckidelta mRNA in AD hippocampus compared with control, 8.04-fold in the amygdala, 7.45 in the entorhinal cortex and 7.30-fold in the midtemporal gyrus. These are areas with a high burden of NFTs, neuropil threads and dystrophic neurites. In areas almost devoid of this tau pathology, such as the caudate nucleus, occipital cortex and cerebellum, the increases in AD compared to control brain were only 2.21-, 1.89- and 1.87-fold, respectively. Western blot analysis showed that the upregulation of Ckidelta mRNA was paralleled by an upregulation of Ckidelta protein. These data establish that the association of Ckidelta with the tau pathology of AD is reflective of an increase in gene transcription. Since Alzheimer-like phosphoepitopes of tau can be generated by Ck1, the Ckidelta isoform may play an important role in this fundamental aspect of AD pathology.

Human neurons generate C-reactive protein and amyloid P: upregulation in Alzheimer's disease.

C-reactive protein (CRP) and amyloid P (AP) are pentraxins which are associated with many pathological lesions, including the amyloid deposits and neurofibrillary tangles (NFTs) of Alzheimer disease (AD). It has always been assumed that they are generated by liver and delivered to their sites of action by serum. Here we report by in situ hydridization, reverse transcriptase-polymerase chain reaction analysis, Western blotting and immunohistochemistry that the mRNAs and proteins of both CRP and AP are concentrated in pyramidal neurons and are upregulated in affected areas of AD brain. Controlling pentraxin production at the tissue level may be important in reducing inflammatory damage in AD.

A set of temperature sensitive-replication/-segregation and temperature resistant plasmid vectors with different copy numbers and in an isogenic background (chloramphenicol, kanamycin, lacZ, repA, par, polA).

A set of plasmid vectors conferring chloramphenicol resistance (Cm(R)), 3064bp in size, or kanamycin resistance (Km(R)), 2972bp in size, were developed, having multiple cloning sites in lacZ' genes for alpha-complementation. pTH18cs1, pTH19cs1, pTH18ks1 and pTH19ks1 are temperature-sensitive (ts) in DNA replication (ts-Rep); pTH18cs5, pTH19cs5, pTH18ks5 and pTH19ks5 are ts in plasmid segregation (ts-Seg); and pTH18cr, pTH19cr, pTH18kr and pTH19kr are temperature resistant (tr) in both. They are based on the pSC101 replicon consisting merely of the replication origin and repA gene, compatible with ColE1/pMB1/p15-derived plasmids, and thus do not require polA function of host cells. The copy numbers of the ts-Rep, tr and ts-Seg plasmids were 14, 5 and 1 per chromosome at 30 degrees C, respectively. These plasmids are fairly stable when inherited at 30 degrees C, but not above 37 degrees C or 41.5 degrees C, depending on the repA mutations and host strains. They are isogenic apart from the ts mutations in the repA gene, and thus provide with useful tools for having appropriate controls in various experiments including bacterial gene-targeting, transposon mutagenesis, toxic gene expression, differential substitution on host functions, gene dosage analysis and so on.

Acute cocaine exposure up-regulates complement expression in rabbit heart.

The exact mechanism of the cardiotoxic actions of cocaine remains unclear. The finding that the heart may be a source of injurious complement components led us to investigate whether cocaine promotes myocardial expression of complement. Rabbit isolated hearts were perfused for 70 min with either cocaine hydrochloride (1 or 10 microM), the synthetic isomer (+)-cocaine (10 microM), or procaine hydrochloride (10 or 30 microM). Compared with controls perfused with drug-free buffer, both cocaine and procaine significantly (P <. 05) increased myocardial C1q, C1r, C8, and C9 mRNA expression, whereas 10 microM (+)-cocaine had no effect on complement mRNA expression. Cocaine also significantly increased (P <.05) C3 mRNA transcription. In addition, in vivo administration of cocaine (1 mg/kg) for three consecutive days significantly increased myocardial complement mRNA expression. Cocaine and procaine also increased membrane attack complex (MAC) formation in the myocardium. The antioxidant 2-N-mercaptopropionyl glycine, attenuated the increases in complement mRNA expression induced by 1 microM cocaine and 10 microM procaine. In vivo heparin administration (300 U/kg i.v.), 2 h before removal of the heart and exposure to 1 microM cocaine, did not inhibit C1q, C1r, C3, and C8 mRNA transcription, but decreased MAC expression. It was determined previously that heparin reduces myocardial ischemia/reperfusion injury. Our results suggest that cocaine may cause myocardial injury by up-regulating local complement expression, possibly via the production of reactive oxygen species. Furthermore, the glycosaminoglycan heparin may modulate the cytotoxic effects of cocaine by impeding formation of the MAC.

Preconditioning reduces tissue complement gene expression in the rabbit isolated heart.

Both preconditioning and inhibition of complement activation have been shown to ameliorate myocardial ischemia-reperfusion injury. The recent demonstration that myocardial tissue expresses complement components led us to investigate whether preconditioning affects complement expression in the isolated heart. Hearts from New Zealand White rabbits were exposed to either two rounds of 5 min global ischemia followed by 10 min reperfusion (ischemic preconditioning) or 10 microM of the ATP-dependent K+ (KATP) channel opener pinacidil for 30 min (chemical preconditioning) before induction of 30 min global ischemia followed by 60 min of reperfusion. Both ischemic and chemical preconditioning significantly (P < 0.05) reduced myocardial C1q, C1r, C3, C8, and C9 mRNA levels. Western blot and immunohistochemistry demonstrated a similar reduction in C3 and membrane attack complex protein expression. The K(ATP) channel blocker glyburide (10 microM) reversed the depression of C1q, C1r, C3, C8, and C9 mRNA expression observed in the pinacidil-treated hearts. The results suggest that reduction of local tissue complement production may be one means by which preconditioning protects the ischemic myocardium.