An immunocytochemical study of alpha 1-antichymotrypsin in the senescent cerebral amyloid

An immunocytochemical study of alpha 1-antichymotrypsin (alpha 1ACT) was performed in order to demonstrate its localization and the relationship between alpha 1ACT and senescent cerebral amyloid. We examined 5 brains with dementia of the Alzheimer type (SDAT), a peripheral nerves of familial amyloidotic polyneuropathy (variant transthyretin type, FAP) and dorsal root ganglions of a primary amyloidosis with peripheral neuropathy (AL type, PA). Avidin-biotin-peroxidase complex method and double immunoenzymatic staining method (peroxidase-antiperoxidase method combined with avidin-biotin-alkaline phosphatase complex method) were used. Anti-beta protein serum was used as the marker of cerebral amyloid. About 98% of senile plaques had alpha 1ACT like-immunoreactivity (alpha 1ACTI). All types of plaques showed the immunoreactivity: Core and peripheral of typical plaques, primitive plaques, core plaques and amorphous cerebral amyloid deposits. Although, a part of a senile plaque showed beta protein like-immunoreactivity alone and the other part had alpha 1ACT, many remainder part of a senile plaque had both immunoreactivity. Of the other pathological changes of SDAT, eosinophilic tangles and cerebrovascular amyloid were positive, in contrast, intracellular tangles, granulovacuolar degeneration and Hirano body were negative. The amyloid from FAP had weak alpha 1ACTI and diffusely stained. alpha 1ACTI was seen in the peripheral margin of the amyloid from PA. These results indicate that alpha 1ACT is closely associated with senile plaques formation.     

Association of α1-antichymotrypsin polymorphism with cerebral amyloid angiopathy

In search of genetic risk factors of sporadic cerebral amyloid angiopathy (CAA), we investigated the association of a polymorphism in the signal peptide sequence of α1-antichymotrypsin (ACT) with the severity of CAA in 155 autopsy cases of the elderly, including 48 patients with Alzheimer's disease. In the total cases, there was no significant association of the ACT genotypes (AA, AT, and TT) with the severity of CAA. Within the Alzheimer's disease group, however, a significant correlation was found between the ACT A allele frequency and the severity of CAA.     

Proteinase inhibitor alpha 1-antichymotrypsin has different expression in various forms of neuronal ceroid lipofuscinosis.

Defective proteolytic degradation is most widely maintained as the major pathogenetic factor in neuronal ceroid lipofuscinosis (NCL). The goal of the present study was to examine the expression in NCL brain tissue of one of the serine proteinase inhibitors, alpha 1-antichymotrypsin. Our study was based on previous findings of alpha 1-antichymotrypsin association with CNS amyloidoses related to amyloid beta protein deposits and our previous findings suggesting abnormal processing of amyloid beta-protein precursor (ABPP) in NCL brains. Immunocytochemical study was performed on formalin-fixed brain tissues collected from 15 NCL cases representing four different forms of the disorder and from 16 control cases comprising age-matched controls, older nondemented individuals, and Alzheimer disease cases. Our present study has shown that the expression of alpha 1-antichymotrypsin is generally higher in NCL cases than in control cases; however, it manifests in distinct variations of intensity and proportions of immunostained cells. The strongest immunoreactivity was found in the infantile form of NCL, which is characterized by a rapid clinical course and widespread tissue damage. We found no evidence of direct involvement of alpha 1-antichymotrypsin in either the ceroid lipopigment accumulation or the abnormal processing of ABPP in NCL. However, our findings may reflect the heterogeneity of the pathomechanism underlying this group of disorders and suggest that, similarly to blood circulation, alpha 1-antichymotrypsin can also represent an acute-phase protein in brain tissue.     

Serological alpha 1-antichymotrypsin in Down's syndrome and Alzheimer's disease.

alpha 1-Antichymotrypsin (ACT) is a serine protease inhibitor that is markedly elevated in the serum and cerebrospinal fluid of patients with Alzheimer's disease (AD). Patients with Down's syndrome are known to develop neuropathological changes of AD by age 40 years and many become demented. Therefore, in the present study, we obtained serum ACT levels from patients with Down's syndrome and AD, diagnosed by autopsy or clinically, and healthy control subjects. Newman-Keuls' multiple range test revealed a significantly greater (p less than 0.01) mean ACT level in the Autopsy AD (906.4 +/- 94.64 mg/L) and Clinical AD (745.00 +/- 59.95 mg/L) groups in contrast to the Old Control group (531.00 +/- 23.05 mg/L). The mean ACT level of the Down's Syndrome group (513.33 +/- 14.73 mg/L) was not significantly different from that of the Young Control subjects. Furthermore, we did not observe a positive correlation of ACT levels with age in the Down's Syndrome group, in spite of the age-dependent premature increase in neuropathological changes of AD that are known to occur in patients with Down's syndrome. A positive correlation between serum ACT levels and the density of plaques or tangles, neuropathological hallmarks of AD, in brains of patients with AD also did not exist. Thus, our results suggest that ACT levels may not parallel the development of the classical neuropathological hallmarks of AD.     

Alpha 1-antitrypsin and alpha 1-antichymotrypsin are in the lesions of Alzheimer's disease.

We performed immunocytochemistry to localize alpha 1-antichymotrypsin and alpha 1-antitrypsin in tissue sections of Alzheimer disease patients. Our results show that both serine protease inhibitors are localized in neurofibrillary tangles and senile plaques. Using various monoclonal and polyclonal antibodies, immunolabeling was evident in formalin, methacarn or acetone-fixed sections. Brief pretreatments of sections with either formic acid or guanidine-HCl were also necessary to reveal clear immunostaining of the lesions with two of the antibodies. We suggest that both alpha 1-antitrypsin and alpha 1-antichymotrypsin may be functionally involved in the pathogenesis of the lesions of Alzheimer's disease. Like alpha 1-antichymotrypsin, the major cell producing alpha 1-antitrypsin is likely to be astrocytes since the protein was localized there and astrocytes are involved in both lesions.     

Activation of the amyloid cascade by intracerebroventricular injection of the protease inhibitor phosphoramidon

We presently investigated the pathological effects of prolonged inhibition of brain beta-amyloid (Abeta) degradation in vivo. The results obtained revealed that intracerebroventricular injection of the protease inhibitor phosphoramidon into wild-type mice for up to a month elevated the soluble and deposited brain Abeta levels and concomitantly induced the neurodegeneration of distinct hippocampal neurons as well as neuroinflammation. These findings reproduce pathological effects associated with the initial stages of the amyloid cascade and provide a novel model system for studying their underlying mechanisms.     

Distribution of calcium-activated neutral protease inhibitor in the central nervous system of the rat.

The ubiquitous existence of calcium-activated neutral protease (CANP, calpain), an enzyme whose activity is regulated by calcium ions and a specific endogenous CANP inhibitor (calpastatin), is well known. Although there has been much investigation concerning the distribution and role of CANP, investigations of the distribution of the CANP inhibitor using immunohistochemical techniques are rare. We made antiserum against a 40K fragment of cDNA corresponding to two C-terminal repeats of rat liver CANP inhibitor expressed in Escherichia coli. Using this antiserum, we examined the distribution of CANP inhibitor in the rat central nervous system by the ABC technique and compared it with the distribution of CANP. Neurons and glias were stained, with the cytosol stained diffusely and the cell membranes stained clearly and strongly. Axons and myelin were stained faintly, but nuclei and vessels were not stained. The distribution of CANP inhibitor was thus found to be similar to that of CANP.     

Lack of association between alpha1-antichymotrypsin polymorphism and late-onset depressive disorder

Late-onset depressive disorder (LOD) has been thought to be associated with dementia. Recently, it was reported that the position-15 (alanine) polymorphism of the alpha1-antichymotrypsin gene (ACT*A) was a risk factor for Alzheimer's disease. We wondered whether the ACT*A allele frequency might be elevated in LOD. ACT genotyping was performed as described by Kamboh et al. (Kamboh, M.I., Sanghera D.K., Ferrell R.E., DeKosky, S.T., 1995. APO* E4-associated Alzheimer's disease risk is modified by alpha1-antichymotrypsin polymorphism. Nature Genetics 10, 486-488) in 153 patients with depressive disorders and 107 healthy controls. The patients were subdivided into those with early-onset and late-onset, using 50 years as the cut-off age. There was no statistically significant difference in the age of onset of depressive disorders according to the ACT genotype. There was also no significant association between early-/late-onset depressive disorders and the ACT genotype. In addition, there was no association between the apolipoprotein E epsilon 4 allele and the ACT genotype in LOD. Our results suggest that there is no association between the ACT*A allele and LOD.     

A deletion polymorphism of alpha(2)-macroglobulin gene and cerebral amyloid angiopathy.

BACKGROUND AND PURPOSE: alpha(2)-Macroglobulin may be implicated in amyloid beta protein deposition. A deletion in the exon 18 splice acceptor of the alpha(2)-macroglobulin gene (A2M) has been reported to be associated with risk for Alzheimer's disease (AD). In search of genetic risk factors for cerebral amyloid angiopathy (CAA), we investigated association of the A2M deletion polymorphism with CAA. METHODS: The association between the severity of CAA and A2M deletion polymorphism was investigated in 178 autopsy cases of the elderly including 68 patients with AD. RESULTS: There was no significant difference in the severity of CAA between individuals with the A2M deletion allele and those without in the AD, non-AD, or total cases. Status for the epsilon4 allele of the apolipoprotein E gene did not influence the results. CONCLUSIONS: Our results suggest that the A2M deletion polymorphism may not be a definitive risk factor of CAA in the elderly, although further study with larger samples is necessary to confirm this.     

Ubiquitin in cerebral amyloid angiopathy.

Immunohistological findings in cerebral blood vessels of 4 cases with cerebral amyloid angiopathy (CAA) were compared with those of 4 Alzheimer's (AD) cases. A panel of antibodies against 2 neurofilament subunits (BF10 and RT97), a microtubule-associated protein (TAU) and ubiquitin were used. CAA cases showed a strong immunoreactivity for ubiquitin in blood vessel wall. Senile plaques (SPs) in CAA cases showed strong ubiquitin positivity but the central amyloid core was negative. AD brains showed immunoreactivity with all antibodies in SPs and neurofibrillary tangles (NFTs); blood vessels were consistently negative for ubiquitin. Control brains showed few SPs and NFTs; these were positive for ubiquitin, but blood vessels were negative. These results indicate that vascular amyloid deposition in CAA and AD may have different pathophysiological mechanisms.     

Stroke related to cerebral amyloid angiopathy: the significance of systemic vascular disease

Summary A retrospective postmortem analysis of 25 cases of cerebral amyloid angiopathy (CAA) in the setting of Alzheimer's disease or senile dementia of the Alzheimer type (AD/SDAT) is reported. Seven patients experienced clinically significant cerebral infarcts or hemorrhages or both. There was no statistically significant difference in the incidence of infarcts or hemorrhages in hypertensive and nonhypertensive patients. Hypertension does not appear to be an additional risk factor in the causation of cerebral infarct or hemorrhage associated with CAA in the setting of AD/SDAT. Just over half of patients with CAA and significant ischemic and/or hemorrhagic brain lesions showed atherosclerosis of the circle of Willis, sometimes in the context of severe disseminated atheromatous disease.     

Upregulation of amyloid precursor protein isoforms containing Kunitz protease inhibitor in dementia with Lewy bodies

Amyloid precursor protein (APP) is involved in the accumulation of alpha-synuclein, the main component of Lewy bodies. It is currently unknown, however, whether any of the APP isoforms is instrumental in alpha-synuclein deposition in dementia with Lewy bodies (DLB). Using real-time RT-PCR, we have studied relative mRNA expression levels of APP isoforms in frozen postmortem frontal cortices of DLB patients, Alzheimer disease (AD) patients, and control subjects. Of the three main APP isoforms, the two with a Kunitz protease inhibitory (KPI) motif (APP770 and APP751) were found to be specifically overexpressed in the frontal cortices of DLB patients when compared with controls and AD patients. These findings suggest a specific role of APP isoforms containing Kunitz protease inhibitor in DLB pathogenesis.     

Plasma and cerebrospinal fluid alpha1-antichymotrypsin levels in Alzheimer's disease: correlation with cognitive impairment

alpha-1-Antichymotrypsin (ACT) is present in neuritic plaques in which it participates in the inflammatory cascade of Alzheimer's disease (AD). Reports of blood ACT levels in AD, and its usefulness as a disease biomarker, have been conflicting. In an effort to clarify this, we measured plasma ACT levels in 516 white subjects including 359 subjects with probable or possible AD, 44 subjects with other late-life dementias, and 113 nondemented people. Subjects with systemic inflammatory diseases or who were taking antiinflammatory medications were excluded. All patients underwent extensive medical and detailed neuropsychological examinations at the time their blood was drawn. We found that plasma ACT levels were elevated in AD patients compared with the control group (p = 0.01) and were associated with severity of AD dementia; there was a negative association with the Mattis Dementia Rating Scale (a global measure of cognition) and a positive association with the Clinical Dementia Rating Scale (a global functional assessment). These relationships remained significant after controlling for demographic and genetic variables. When AD subjects were stratified into subgroups by dementia severity, matched by age, education, and gender, increased serum ACT correlated with Clinical Dementia Rating Scale (p = 0.0041) or Mattis Dementia Rating Scale (p = 0.0031) scores. ACT measurements in cerebrospinal fluid from an additional 34 AD cases and 16 controls showed elevated levels (p = 0.02) in AD. There was a negative correlation (p = 0.037) between cerebrospinal fluid ACT levels and clinical severity as measured by the Mini-Mental State Examination. Our results demonstrate that peripheral ACT levels are elevated in AD, but not in dementias other than AD, and they increase with progression of AD dementia. Although not useful as a diagnostic biomarker, ACT may reflect disease severity and may be helpful as a within subject biomarker in interventions (particularly with antiinflammatory agents) directed at slowing or halting progression of disease.     

Imaging of amyloid burden and distribution in cerebral amyloid angiopathy

OBJECTIVE: Cerebrovascular deposition of beta-amyloid (cerebral amyloid angiopathy [CAA]) is a major cause of hemorrhagic stroke and a likely contributor to vascular cognitive impairment. We evaluated positron emission tomographic imaging with the beta-amyloid-binding compound Pittsburgh Compound B (PiB) as a potential noninvasive method for detection of CAA. We hypothesized that amyloid deposition would be observed with PiB in CAA, and based on the occipital predilection of CAA pathology and associated hemorrhages, that specific PiB retention would be disproportionately greater in occipital lobes. METHODS: We compared specific cortical PiB retention in 6 nondemented subjects diagnosed with probable CAA with 15 healthy control subjects and 9 patients with probable Alzheimer's disease (AD). RESULTS: All CAA and AD subjects were PiB-positive, both by distribution volume ratio measurements and by visual inspection of positron emission tomographic images. Global cortical PiB retention was significantly increased in CAA (distribution volume ratio 1.18 +/- 0.06) relative to healthy control subjects (1.04 +/- 0.10; p = 0.0009), but was lower in CAA than in AD subjects (1.41 +/- 0.17; p = 0.002). The occipital-to-global PiB ratio, however, was significantly greater in CAA than in AD subjects (0.99 +/- 0.07 vs 0.86 +/- 0.05; p = 0.003). INTERPRETATION: We conclude that PiB-positron emission tomography can detect cerebrovascular beta-amyloid and may serve as a method for identifying the extent of CAA in living subjects.     

The effect of 2-methoxyestradiol, a HIF-1 alpha inhibitor, in global cerebral ischemia in rats

Global cerebral ischemia is an important clinical problem with few effective treatments. The hippocampus, which is important for memory, is especially vulnerable during global ischemia. Brain-specific knockout of hypoxia inducible factor-1 alpha (HIF-1 alpha) has been shown to be protective in focal ischemia in vivo. 2-methoxyestradiol (2ME2) is a natural metabolite of estrogen that is known to inhibit HIF-1 alpha. We tested 2ME2 in a rat model of global cerebral ischemia. Global ischemia was induced with the two-vessel occlusion model (2VO) which entailed hemorrhagic hypotension to a mean arterial pressure of 38-42 mmHg with simultaneous bilateral common carotid artery occlusion for 8 minutes. Sprague-Dawley rats (male, 280-350 g) were randomly assigned to three groups: global ischemia (GI, n=17), global ischemia with 2ME2 treatment (GI + 2ME2, n=17) and sham surgery (sham, n=12). 2ME2 treatment (15 mg/kg in 1% DMSO) was rendered 10 minutes after reperfusion. Rats in the GI and sham groups received similar doses of the DMSO solvent. Rats were killed 24 hours, 72 hours and 7 days after reperfusion. Quantitative CA1 hippocampal cell counts demonstrated significantly lower cell survival in the GI + 2ME2 group compared to either the GI or sham groups, in spite of a statistically significant reduction in HIF-1 alpha by Western blotting analysis of the GI + 2ME2 group. We conclude that 2ME2 worsens outcomes after global ischemia in rats.