The methylation status of cytosines in a tau gene promoter region alters with age to downregulate transcriptional activity in human cerebral cortex.

Changes with age in the methylation status of cytosines in a promoter region of the tau gene were investigated in autopsy human cerebral cortex, using the bisulfite method, polymerase chain reaction (PCR) and direct sequencing of PCR products. While the total number of methylcytosines decreased with age, the changes in methylation status differed among transcription factor binding sites. Cytosines in the AP2-binding sites were never methylated in any of the cases studied at any age. Methylcytosines in the binding sites for Sp1, a transcriptional activator, significantly increased with age, whereas those in the binding sites for GCF, a repressor of GC-rich promoters, significantly decreased with age. These findings suggest that the methylation status of cytosines in the promoter region of the tau gene alters with age to decrease its transcriptional activity in the human cerebral cortex.

Local variation in expression of pro- and antithrombotic factors in vascular endothelium of human autopsy brain.

The expression of tissue factor (TF), tissue factor pathway inhibitor (TFPI), von Willebrand factor (vWF), endothelial nitric oxide (NO) synthase (eNOS), tissue plasminogen activator (tPA), its inhibitor (PAI-1), and myosin, an indicator of local shear stress, was examined in the endothelium of cerebral vessels according to vessel size and location in human autopsy brains, using immunohistochemistry. Expression of TF, vWF, eNOS, tPA/PAI-1, and myosin was much greater in intracerebral perforating arteries and the microvasculature than the pial and carotid arteries. Expression of all antigens studied was normally faint or negative in the pial and carotid arteries. However, TF, vWF, myosin, tPA, and PAI-1 were strongly expressed in the endothelium of the inner wall of the carotid bifurcation where flowing blood collides, but not in the outer wall. In the endothelium of arteries with fibrillary hyperplasia, vWF, myosin, eNOS, tPA, and PAI-1 were strongly expressed. Within the brain, microvascular expression of TFPI was very faint or negative, whereas that of vWF was intense throughout all brain regions. However, expression of TF and myosin was more intense in the basal gray matter and white matter than in the cortex. eNOS was expressed more strongly in the basal gray matter and cortex than the white matter, whereas tPA and PAI-1 expression was more intense in the white matter than the gray matter. In addition to intrinsic properties of individual vessels, these local variations in expression of pro- and antithrombotic factors in cerebral vessels may in part be due to differences in hemorheological and humoral environments to which they are exposed, and may result in local difference in vulnerability to ischemia. The present findings may in part account for the propensity of thrombus generation in the carotid inner wall, an usual source of artery-to-artery microemboli, frequent development of lacunar (small) infarcts in deep brain regions, and diffuse white matter lesions as seen in Binswanger's leukoencephalopathy.

Reduction with age in methylcytosine in the promoter region -224 approximately -101 of the amyloid precursor protein gene in autopsy human cortex.

Methylation status of cytosines and its changes with age in the promoter region (-226 approximately -101) of the amyloid precursor protein (APP) was analyzed using bisulfite genomic sequencing in the cerebral cortex of human autopsy brain. Cytosines at 13 locations were methylated in at least one of the cases studied. Methylcytosines at these locations was more frequent in cases 70 years old (8%) (p<0.05). Cytosines at -207, -204, -200, and -182 are frequently methylated, and the frequency of methylcytosine in these locations was significantly higher in cases 70 years old (5%) (p<0.01). These cytosines constituted one of the 9-bp-long GC-rich elements (GGGCGC G/A GG) or an 11-bp inverted repeat (GGCCGT CGGCC). The present findings indicate that some cytosines, particularly those at -207 approximately -182, in the promoter region of the APP gene are frequently methylated and suggest that their demethylation with age may have some significance in the development of Abeta deposition in the aged brain. The relative importance of these elements in the total promoter activity of the APP gene remains to be definitively established.

[The effect of lymphocytapheresis on multiple sclerosis].

We studied the effect of lymphocytapheresis (LCP) on the expanded disability status scale (EDSS) clinical score, lymphocyte subsets and Interleukin-2 (IL-2) production by peripheral blood mononuclear cell (PBM) in 5 patients with multiple sclerosis (MS). The EDSS clinical score significantly decreased after LCPs (p < 0.05). PBM IL-2 production and CD 4/8 ratio significantly decreased (p < 0.05, p < 0.05), and the number of neutrocytes and CD 11 b+CD 8+ (%) significantly increased immediately after LCP (p < 0.05, p < 0.05). Down-regulation of PBM IL-2 production and CD 4/8 ratio and up-regulation of CD 11 b+CD 8+ may account for therapeutic effect of LCP on MS. However, similar changes were observed in patients with CIDP and MG during immunoadsorbent therapy (IAT). It is possible that down-regulation of PBM IL-2 production and CD 4/8 ratio and up regulation of CD 11 b+CD 8+ and the number of neutrocytes may commonly result from apheresis therapy using extra-corporeal circulation.

Quantitation of nicotinic acetylcholine receptor subunits alpha 4 and beta 2 messenger RNA in postmortem human brain using a non-radioactive RT-PCR and CCD imaging system.

We present a simple and rapid procedure for quantifying mRNA in the brain after RT-PCR, in which the intensity of the ethidium bromide luminescence of PCR products is measured directly from electrophoretic gels by a highly sensitive CCD camera combined with an image analyzing computer system (Gel Doc 1000 system). The CCD camera allows the mRNA in the ethidium bromide-stained PCR-amplified bands to be quantified in a broad exponential range of PCR cycles. The proposed protocol enables standard curves to be constructed to examine the relationship between the number of reaction cycles and amplified log intensity and between the amount of sample RNA for RT-PCR and amplified intensity. The method was applied to nicotinic acetylcholine receptor (nAChR) subunits alpha 4 and beta 2 mRNA in postmortem human putamen in the present study, but is also applicable to mRNAs of other receptors and neurotransmitter precursor peptides.

Decrease with age in methylcytosines in the promoter region of receptor for advanced glycated end products (RAGE) gene in autopsy human cortex.

Changes with age in the methylation status of cytosines in the promoter region of the receptor for advanced glycated end products (RAGE) in autopsy human cortex were investigated, using the bisulfite method, polymerase chain reaction (PCR), and direct sequencing of PCR products. The total number of methylcytosines significantly decreased with age. While the number of methylated cytosines at CpG dinucleotides was stable throughout adult life, that at sites other than CpG dinucleotides significantly decreased with age in cases >/=70 years old. Of 13 transcription factor binding sites, cytosines in CpG doublets in NF-IL6 and SP-1 binding sites were methylated in all cases, suggesting that these sites are repressed throughout adulthood. In contrast, the number of methylcytosines in AP-2 or SP-1 binding sites located at CpC, CpA, or CTG was significantly lower or at least tended to be lower in cases >/=70 years than <70 years old. These reductions in the number of methylcytosines at transcription factor binding sites may increase expression of RAGE, which may in turn play a role in aging of the brain.