Ippei Sakakibara

Histopathological studies of senile plaques and cerebral amyloidosis in cynomolgus monkeys

Senile plaques (SPs) and cerebral amyloid angiopathy (CAA), pathological hallmarks of Alzheimers disease, have not been thoroughly investigated histopathologically in nonhuman primates. To determine the onset age and histopathological characteristics of SPs and CAA, we examined the brains of 64 cynomolgus monkeys (Macaca fascicularis) from 2 to 35 years old. Mature (classical and primitive) plaques appeared in 16 out of 25 monkeys that were >20 years old. Moreover, mature plaques were observed more frequently than diffuse plaques and were located in the temporal cortex of the superior or inferior gyri and amygdala. Diffuse plaques in contrast to mature plaques did not show definite tendencies in onset age and distribution. CAA appeared in more than 22‐year‐old monkeys in 10 out of 16 animals and was frequently observed in capillaries and often found adjoining mature plaques. During immunohistochemical examination, an antiserum for amyloid β protein (Aβ) 1–40 could detect all SPs, whereas a monoclonal antibody for Aβ 8–17 could not detect any diffuse plaques and only one third of the primitive plaques. As for CAA, the polyclonal antiserum was more sensitive than the monoclonal antibody. The present study describes the histopathological features of SPs and CAA in old cynomolgus monkeys.

Carboxyl end-specific monoclonal antibodies to amyloid β protein (Aβ) subtypes (Aβ40 and Aβ42(43)) differentiate Aβ in senile plaques and amyloid angiopathy in brains of aged cynomolgus monkeys

Abstract Senile plaques (SPs) and cerebral amyloid angiopathy (CAA) in the brains of five aged (20–26 years old) cynomolgus monkeys were investigated immunohistochemically using two monoclonal antibodies (anti-Aβ 40 (BA27) and anti-Aβ 42(43) (BC05)) that can differentiate the carboxyl termini of amyloid β protein (Aβ) subtypes. In four of five animals, all types of SPs (i.e. diffuse, primitive, and classical plaques; DPs, PPs, and CPs, respectively) were identified by BC05. However, BA27 did not label DPs and stained only about one third of PPs and CPs, mainly labeling granular structures and cored portions, respectively. In CAA, lesions of cortical capillaries reacted to BC05 in four of five cases, but rarely and weakly to BA27 in two of five cases. On the other hand, lesions of parenchymal and meningeal arterioles were stained by both BA27 and BC05. These staining profiles of SPs in cynomolgus monkeys correspond well to those in humans, although there are two remarkable features in cynomolgus monkeys. First, BA27 stained PPs associated with granular structures. Secondly, capillary Aβ reacted intensely to BC05 but only slightly to BA27. Despite these unique features, the results suggest that aged cynomolgus monkeys can be used to investigate the pathogenesis of Aβ deposition in SPs and CAA.

Age‐related changes of intracellular Aβ in cynomolgus monkey brains

To confirm the intracellular accumulation of amyloid β‐protein (Aβ), we carefully performed immunohistochemistry using brains of cynomolgus monkeys of various ages. Cortical neurones and their large neurites were immunostained with antibodies against Aβ in young monkey brains. In aged monkey brains, intracellular Aβ localized within cortical neurones; no clear association was found between the presence of intracellular Aβ and senile plaques (SPs). Interestingly, we did not observe Aβ‐immunoreactive cortical neurones in brains fixed with neutral buffered formalin. Western blot analyses of microsomal and nerve ending fractions derived from the brains of young to aged monkeys revealed that intracellular Aβ generation changed with age. In the microsomal fraction, the amount of Aβ42 significantly increased in brains from older monkeys (> 30 years of age), and the amount of Aβ43 significantly decreased with age in the microsomal fraction. The amount of Aβ40 remained the same regardless of age. Biochemical analyses also showed that intracellular levels of each of these Aβ molecules significantly increased with age in nerve ending fractions. As we previously observed that a similar accumulation of presenilin1, β‐amyloid precursor protein (APP) and APP C‐terminal fragment cleaved by β‐secretase in the nerve ending fractions obtained from brains with SPs, the accumulation of intracellular Aβ in this fraction may be closely related to formation of spontaneous SPs with age. Taken together, these results suggest that intensive investigation of age‐related changes in the nerve ending will contribute to a better understanding of the pathogenesis of age‐related neurodegenerative disorders such as sporadic Alzheimers disease.

Age-related changes in the localization of presenilin-1 in cynomolgus monkey brain.

Age-related changes in PS-1 localization were examined in the brains of 22 cynomolgus monkeys ranging in age from embryonic day 87 to 35 years. In embryonic monkey brains, anti-PS-1 antibody N12, which recognizes the PS-1 N-terminal fragment (Ntf) and holo protein, stained immature neuronal cells. In juvenile monkeys, N12 stained large pyramidal neurons, cerebral neocortical neurons, and cerebellar Purkinjes cells. Cytoplasmic staining of these cells was granular in appearance. In aged monkeys, N12 stained neurons in all layers of the neocortex. In contrast, regardless of the age of the animals examined, M5, an anti-PS-1 antibody that specifically recognizes only the PS-1 C-terminal fragment (Ctf), stained neurons in all layers of the neocortex and neurons in the cerebellum. M5 also stained neuropil and white matter, and in aged monkeys, M5 stained swollen neurites of mature senile plaques. Age-related changes in PS-1 expression were further examined using Western blot analysis of mitochondrial, myelin, microsomal, nuclear, synaptosomal, and cytosol fractions isolated from 10 monkey brains ranging in age from embryonic day 87 to 32 years. In all brains, Ntf and Ctf were expressed most abundantly in the microsome fraction. The amount of PS-1 in the nuclear fraction dramatically increased with age. We conclude that the transport of PS-1 diminished with age and that PS-1 fragments accumulated in endoplasmic reticulum (ER) associated with the nuclear membrane.

Immunohistochemical characteristics of the constituents of senile plaques and amyloid angiopathy in aged cynomolgus monkeys.

Abstract: In this study, we immunohistochemically examined the several constituents of senile plaques (SPs) and cerebral amyloid angiopathy (CAA) in aged cynomolgus monkeys. Apolipoprotein E (apoE) deposited in all mature plaques and CAA, and in half of the diffuse plaques. Alpha‐1‐antichymotripsin (αACT) deposited in half of the mature plaques and in one third of the CAA. Amyloid precursor protein (APP), ubiquitin (Ub), and microtubule‐associated protein‐2 (MAP‐2) accumulated in the swollen neurites of mature plaques. Glial fibrillary acidic protein (GFAP) was detected in the astrocytes and their processes surrounding the mature plaques. Tau was detected in neither the SPs nor CAA. Therefore, mature plaques involved extracellular Aβ, apoE, and αACT, and also astrocytes and swollen neurites. However, diffuse plaques involved only extracellular Aβ and apoE. Since these features, except for tau, were consistent with those in humans, this animal model will be useful for studying the pathogenesis of cerebral amyloid deposition.