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.

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.

Plasmids with a kanamycin-resistance gene for site-directed mutagenesis using the oligodeoxyribonucleotide-directed dual amber method.

Plasmid vectors carrying lacZ' and kanamycin-resistance (KmR) genes were constructed for site-directed mutagenesis (SDM) using the oligodeoxyribonucleotide (oligo)-directed dual amber (ODA) method [Hashimoto-Gotoh et al., Gene 152 (1995) 271-276]. The plasmids, designated pKF16k, pKF17k, pKF18k and pKF19k, correspond to the previously reported chloramphenicol resistant (CmR) ODA plasmids, pKF16c, pKF17c, pKF18c and pKF19c, respectively, but contain dual amber (am) codons in KmR instead of the CmR gene. The SDM procedure using the KmR ODA plasmids is essentially the same as that with CmR ODA plasmids, which utilizes two oligo primers for in vitro DNA synthesis, one (selection primer) for dual am reversions and the other (mutagenic primer) for the target site. The KmR ODA plasmids yield 5-10-times more DNA per culture volume as compared to the CmR ODA plasmids, and one can prepare selection agar medium simply by spreading Km solution on dried agar plate at a final concentration of 50-100 micrograms/ml; due to the broad range of selecting antibiotic resistance.

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.

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.

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.

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.

Tangled areas of Alzheimer brain have upregulated levels of exon 10 containing tau mRNA.

We measured the relative levels of exon 10 containing and exon 10 deleted tau mRNAs in multiple areas of Alzheimer disease (AD) and normal brain. Compared with normal brain, we found a 3.4-fold upregulation of exon 10 plus and a 1.9-fold downregulation of exon 10 minus mRNAs in areas of AD brain with a heavy burden of neurofibrillary tangles. These data suggest that tangle formation in AD is initially determined by transcriptional factors and is not exclusively caused by post-translational events.

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.

Complement regulators C1 inhibitor and CD59 do not significantly inhibit complement activation in Alzheimer disease.

Proteins characteristic of activated complement are associated with Alzheimer disease (AD) lesions. The classical complement pathway can be activated only when the influence of such endogenous regulators as C1-inhibitor (C1-inh) and CD59 are overcome. We used the techniques of reverse transcriptase-polymerase chain reaction and Western blotting to assess the mRNA and protein levels of C1-inh and CD59 in AD and control brains in comparison with levels of the complement components with which they interact. The inhibitors were only slightly upregulated and then only in heavily affected areas of AD brain such as the entorhinal cortex, hippocampus, midtemporal gyrus and midfrontal gyrus. The ratio of AD to control mRNAs in these four areas was 1.17 for C1-inh and 1.12 for CD59, compared to 3.06 for C1r, 2.67 for C1s, 2.35 for C5, 2.56 for C6, 2.42 for C7, 5. 08 for C8 and 16.3 for C9. Peripheral organ expression of C1-inh and CD59 mRNAs was no different in AD than controls but was slightly upregulated in infarcted heart tissue. Again, the increase was small compared with that of the competitive complement components. These data indicate that the forces which upregulate and activate complement in AD and myocardial infarction are not effectively suppressed by the endogenous regulators, C1-inh and CD59.

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.

Distribution of cyclooxygenase-1 and cyclooxygenase-2 mRNAs and proteins in human brain and peripheral organs.

We used the techniques of reverse transcriptase-polymerase chain reaction, Western blotting and immunohistochemistry to evaluate the expression of cyclooxygenase (COX)-1 and -2 in brain and peripheral organs of Alzheimer disease (AD) and control cases. We found both COX-1 and COX-2 to be constitutively expressed in all organs tested, i.e., brain, heart, liver, kidney, spleen and intestine. COX-2 was substantially upregulated in affected areas of AD brain and in infarcted areas of human heart. COX-1 was only mildly upregulated in AD brain. Immunohistochemically, COX-2 was strongly expressed in the perinuclear, dendritic and axonal areas of pyramidal neurons, with enhanced staining in AD. These data suggest a special role for COX-2 in neuronal function.

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.

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.

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.

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.

[Alexia-agraphia of kanji (Japanese morphogram) after left posterior-inferior temporal lesion]. / Alexie-agraphie pour l'écriture kanji après lésion temporale postéro-inférieure gauche.

Several cases of selective alexia with agraphia of kanji have been reported in Japan in this decade. It is well known that the lesion in the posterior inferior temporal lobe of the dominant hemisphere is responsible for this cognitive syndrome. Neuropsychological data in our patient suggest that the postero-inferior region of the temporal lobe of the dominant hemisphere may be the visuo-verbal association area for the analysis of the complex visuo-verbal information. The symptoms caused by the same lesion in western patients might be subangular alexia (alexia without agraphia). Alexia with agraphia of kanji and subangular alexia would appear to be distinct entities, but a dual processing hypothesis of visuo-verbal information and the concept of the visuo-verbal association area might well explain both syndromes.

Cloning of Xenopus presenilin-alpha and -beta cDNAs and their differential expression in oogenesis and embryogenesis.

Human presenilin (ps)-1 and -2 genes have recently been shown to be involved in genesis of early-onset familial Alzheimer's disease. By probing with human (H-) ps-1 cDNA, we isolated two types of cDNA clones, named X-ps-alpha and -beta, from a Xenopus brain cDNA library. The encoded proteins, X-PS-alpha and -beta, may correspond to H-PS-1 and -2 with 89.4 and 85.9% similarity, respectively. The strongest expression of these genes was observed in ovaries and in the early stages of oogenesis, although weak or moderate expression was detected ubiquitously for both X-ps-alpha and -beta genes in multiple tissues. Upon oocyte maturation, the X-ps-beta mRNA level was constant even after fertilization until the midblastula transition. Zygotic expression of these genes became evident only at the tailbud stage. We propose that presenilins may function in preventing cells from undergoing apoptotic degeneration particularly prior to embryonic development and in developmentally matured tissues.

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.