Aging brain lesions of a 60-year-old Bornean orangutan (Pongo pygmaeus)

Abstract

A female Bornean orangutan (Pongo pygmaeus) raised in a zoo died at 60 years of age. The animal showed no clinical signs besides decreased gait speed. Pathological examination revealed small hemorrhagic foci in the cerebral cortex and liver fibrosis with fatty degeneration of the hepatocytes. To evaluate aging lesions, the brain was examined histologically and immunohistochemically. Severe amyloid β (Aβ) deposition was observed in the cerebral cortex and vascular walls. Parenchymal Aβ deposits in the cerebral cortex appeared as diffuse plaques. Loss of smooth muscle was observed in the vascular walls affected by Aβ deposition. In contrast, only a few phosphorylated tau aggregates were observed. This report describes severe Aβ deposition in the brain of the oldest orangutan ever reported, which did not exhibit significant tau pathologies.

Alzheimer’s disease (AD) is the most prevalent age-related neurodegenerative disease in humans and causes dementia. The pathology of AD is characterized by parenchymal amyloid β (Aβ) deposits in the cerebral cortex, called senile plaques (SPs), and phosphorylated tau (p-tau) aggregates in neurons, called neurofibrillary tangles (NFTs) [7, 33]. In addition, Aβ deposits in the vascular walls, known as cerebral amyloid angiopathy (CAA), are observed in approximately 80% of AD patient brains [1, 9]. These pathological changes have been reported in non-human primates, including great apes such as orangutans, chimpanzees, and gorillas. Aβ deposits and p-tau aggregates have been observed in the brains of aged chimpanzees and gorillas; however, most p-tau aggregates were non-fibrillar pretangles, an early form of NFT [8, 10, 12, 17, 20, 21]. In the brains of 28–46-year-old orangutans, Aβ deposits were observed without p-tau aggregates [11, 25]. In this study, we examined Aβ deposits and p-tau aggregates in the brain of a 60-year-old orangutan and compared them with those in humans and other mammalian species.

A female orangutan (studbook ID: 0726) born and raised in a zoo died at 60 years and three months of age. The animal showed decreased gait speed but no signs suggestive of dementia. A necropsy at the zoo revealed no significant gross lesions in the brain (Fig. 1A–C). Among the visceral organs, the liver was diffusely firm and had an irregular surface. The brain and visceral tissues were collected, fixed in 10% neutral-buffered formalin, and sent to the Laboratory of Veterinary Pathology, The University of Tokyo, for histological examination. Fixed tissues were routinely embedded in paraffin, and 2 μm-thick sections were stained with hematoxylin and eosin. Histopathological examination revealed small hemorrhagic foci in the frontal, temporal, and occipital cortices and severe deposition of lipofuscin in neuronal cells. The liver exhibited diffuse fibrosis and fatty degeneration of hepatocytes.

Fig. 1.

Orangutan, gross findings, brain, formalin fixed. No significant atrophy on the dorsal view (A), ventral view (B), and coronal planes (C). Scale bars: 2 cm.

Brain tissue sections were subjected to several special staining, including Congo red, periodic acid-methenamine silver (PAM), periodic acid-Schiff (PAS), and immunohistochemistry. The details of the primary antibodies and antigen retrieval procedures used for immunohistochemistry are summarized in Supplementary Table 1. The severity of SPs and CAA in each brain region (frontal cortex, parietal cortex, temporal cortex, occipital cortex, entorhinal cortex, hippocampus, striatum, thalamus, midbrain, cerebral medulla, and meninges) was evaluated using Aβ40-, Aβ42-, and Aβ43-stained slides as follows: -, no SPs and CAA; +, SPs and CAA were detected in <25% of the cortical circumference (frontal cortex, parietal cortex, temporal cortex, occipital cortex, entorhinal cortex, and hippocampus) or area (striatum, thalamus, midbrain, cerebral medulla, and meninges); ++, SPs and CAA were detected in 25–50% of the cortical circumference or area; +++, SPs and CAA were detected in >50% of the cortical circumference or area. The severity of p-tau aggregates in each brain region was evaluated using AT8-stained slides as follows: -, no p-tau aggregates; +, <5 p-tau aggregates in an area (0.64 mm²); ++, ≥5 p-tau aggregates in an area (0.64 mm²).

Immunohistochemistry revealed the presence of SPs and CAA in all brain regions examined (Fig. 2A–C, Table 1). Severe Aβ deposition was observed in the frontal, parietal, temporal, occipital, and entorhinal cortices and hippocampus. Mild Aβ deposition was observed in the striatum, thalamus, midbrain, and cerebral medulla. The immunohistochemical reactions for Aβ40 and Aβ42 were moderate, whereas the reaction for Aβ43 was very weak.

Fig. 2.

Orangutan, cerebral cortex. Aβ40-, Aβ42-, and Aβ43-positive senile plaques and cerebral amyloid angiopathy. Immunohistochemistry for Aβ40 (A), Aβ42 (B), Aβ43 (C). Scale bars: 200 μm.

Table 1. Immunohistochemical scoring of Aβ deposition and p-tau aggregation in the brain.

		FC	PC	TC	OC	EC	HP	STR	TH	MB	CM	MG
Senile plaques	Aβ40	+++	+++	+++	+++	+++	++	+	++	+	+
	Aβ42	+++	+++	+++	+++	+++	+++	+	++	+	+
	Aβ43	+	+	+	+	+	+	+	+	+	-
Cerebral amyloid angiopathy	Aβ40	+++	++	+++	++	+++	+	+	+	+	+	+++
	Aβ42	+++	++	+++	++	+++	+	+	++	+	+	+++
	Aβ43	+	+	+	+	+	+	+	+	+	-	+
Phophorylated tau aggregates	AT8	+	-	+	-	-	-	-	-	-	-

Aβ deposition: -, no SPs, CAA; +, SPs and CAA were detected in less than 25% of the cortical circumference (frontal cortex, parietal cortex, temporal cortex, occipital cortex, entorhinal cortex, and hippocampus) or area (striatum, thalamus, midbrain, cerebral medulla, and meninges); ++, SPs and CAA were detected in 25% to 50% of the cortical circumference or area; +++, SPs and CAA were detected in more than 50% of the cortical circumference or area. P-tau aggregation: -, no p-tau aggregates; +, <5 p-tau aggregates in an area (0.64 mm²); ++, ≥5 p-tau aggregates in an area (0.64 mm²). Abbreviations: Aβ, amyloid β; p-tau; phosphorylated tau; FC, frontal cortex; PC, parietal cortex; TC, temporal cortex; OC. occipital cortex; EC, entorhinal cortex; HA, hippocampus; STR, striatum; TH, thalamus; MB, midbrain; CM, cerebral medulla; MG, meninges.

Diffuse amorphous and demarcated round SPs were visualized using PAM staining. Diffuse amorphous SPs were positive for Aβ40 and Aβ42 but negative for Aβ43 (Fig. 3A–D). Demarcated round SPs were positive for Aβ40, Aβ42, and Aβ43 (Fig. 3E–H). Neither type of SPs was detected on HE-, Congo red-, or PAS-stained sections.

Fig. 3.

Orangutan, cerebral cortex. Diffuse amorphous senile plaque and demarcated round senile plaque were positive for periodic acid-methenamine silver (A and E). Diffuse amorphous senile plaque is positive for Aβ40 (B) and Aβ42 (C), but negative for Aβ43 (D). Demarcated round senile plaque is positive for Aβ40 (F), Aβ42 (G), Aβ43 (H). (A and E) periodic acid-methenamine silver, (B–D and F–H) Immunohistochemistry for Aβ40 (B and F), Aβ42 (C and G), Aβ43 (D and H). Scale bars: 50 μm (A–D), 20 μm (E–H).

On HE-stained sections, eosinophilic deposits were found in the arterial walls of the meninges and the small arterial and capillary walls of the cortices, which were stained with PAS and Congo red, showing apple-green birefringence under polarized light (Fig. 4A–C). Immunohistochemistry revealed that these deposits were positive for Aβ40, Aβ42, and Aβ43. Aβ40 deposition was observed in a circumferential pattern in the most affected vascular walls (Fig. 4D). Aβ42 deposition was observed partially within the vascular wall and in a circumferential pattern (Fig. 4E). Aβ43 deposition was mainly observed in the capillary walls and rarely observed in the meningeal and cortical arterial walls (Fig. 4F). Smooth muscle actin (SMA) was lost in the Aβ-affected arterial walls of the meninges and cortex (Fig. 4G). Furthermore, argyrophilic Aβ deposition was observed in PAM-stained sections in areas adjacent to or surrounding the CAA (Fig. 4H and 4I).

Fig. 4.

Orangutan, cerebral cortex. Cerebral amyloid angiopathy. Eosinophilic deposits in the vascular wall (A) were positive for periodic acid-Schiff (B) and Congo red (C), showing apple-green birefringence under polarized light (inset). Circumferential Aβ40 deposition in the vascular wall (D). Partial Aβ42 deposition restricted to portions of the vascular wall (E). Aβ43 deposition in the capillary walls (F). Loss of smooth muscle actin (SMA) in the small arterial wall with Aβ deposition (G). Aβ deposition in areas adjacent to or surrounding CAA (arrowhead), showing argyrophilic (H and I). (A) Hematoxylin and eosin, (B) periodic acid-Schiff, (C) Congo red, (D–I) Immunohistochemistry for Aβ40 (D and H), Aβ42 (E), Aβ43 (F), SMA (G), (I) Periodic acid-methenamine silver. Scale bars: 50 μm.

In contrast to severe Aβ deposition, only a few AT8-positive p-tau aggregates were observed in the neurites of the frontal and temporal cortices (Table 1), but not in the neuronal soma and glial cells (Fig. 5A and 5B). AT8-positive neurites associated with SPs, such as in neuritic plaques in human AD, were not observed. Phosphorylated α-synuclein and phosphorylated TDP43 associated with Parkinson’s disease and amyotrophic lateral sclerosis, respectively, were not detected in any examined regions.

Fig. 5.

Orangutan. Cerebral cortex. Phosphorylated tau (p-tau) aggregation. Only rare and non-clustered p-tau aggregates were observed in neurites and axons (A and B). (A and B) Immunohistochemistry for p-tau (AT8). Scale bar: 100 μm (A), 20 μm (B).

Molecular subtypes of the C-terminal region of Aβ are classified according to the amino acid cleavage sites (Aβ40, Aβ42, and Aβ43) [23]. Among these subtypes, Aβ42 is the primary component of SPs in AD, whereas Aβ40 is predominant in CAA. This difference in deposition patterns is associated with the higher aggregation potential of Aβ42 than that of Aβ40 [13, 15, 22]. In several Caniformia species, such as dogs and sea lions, the degree of Aβ42 positivity is more severe than that of Aβ40 in SPs and CAA [26]. In contrast, the degree of Aβ40 positivity was comparable to or even more severe than that of Aβ42 in orangutans [11, 12]. In this orangutan, the severity of Aβ40 and Aβ42 positivity was comparable in both SPs and CAA. Therefore, Aβ40 in orangutans may have a different aggregation potential for forming SPs and CAA than in humans and Caniformia species. In addition, Aβ43 has recently attracted attention for its high aggregation potential and neurotoxicity [5, 6, 24] and acts as a core component in the formation of SPs in AD patients [16, 32]. Similarly, the role of Aβ43 in Aβ deposition in animals has received increasing attention. Aβ43-positive SPs and CAA equivalent to Aβ42 have been observed in dogs, cats, sea lions, gorillas, and rhesus monkeys, suggesting that Aβ43 deposition, like Aβ42 deposition, may play an important role in the formation of SPs and CAA in animals [14, 17, 26]. However, in this orangutan, Aβ43 deposition was limited to demarcated round SPs and CAA of capillary walls. This restricted distribution suggests that the contribution of Aβ43 to Aβ deposition may vary among species and may be associated with patterns of SPs and CAA deposition, particularly in the maturation and structural organization of these deposits.

SPs are known to spread progressively in the human brain with aging, and the Thal phase, based on SP distribution, is widely used for staging AD pathology [27, 29]. The distribution of SPs in the Thal phase is as follows: phase 1, neocortex; phase 2, entorhinal cortex, hippocampal area, amygdala, and cingulate gyrus; phase 3, striatum, thalamus, and cerebral medulla; phase 4, midbrain and medulla oblongata; and phase 5, pons and cerebellum. Phases 4 and 5 are considered symptomatic. In the present orangutan, the degrees of SPs were severe in the neocortex, entorhinal cortex, and hippocampal area, moderate in the thalamus, and mild in the striatum, midbrain, and cerebral medulla, corresponding to Thal phases 3–4. Moreover, mature plaques with a central core and neuritic plaques containing dystrophic neurites, both of which are associated with cognitive decline in humans [31, 35], were not observed in the present study. Only plaques without a central core, including diffuse amorphous and demarcated round SPs, on PAM-stained sections were detected. Demarcated round SPs in the orangutan were positive for Aβ43, which has also been documented in mature plaques in an aged Gorilla [17], suggesting maturation of SPs compared to diffuse plaques. In addition, coarse-grained plaques and primitive plaques containing vessel structures have been observed in humans and dogs, and these plaques have been suggested to indicate a relationship between SPs and CAA [2, 26]. In this orangutan, Aβ deposition was observed in areas adjacent to or surrounding CAA, and some SPs contained blood vessel-like structures. In addition, the distribution of CAA was similar to that of SPs. These findings suggest that the expansion of CAA throughout the brain may lead to the formation of additional SPs.

CAA is associated with increased vulnerability of the vascular wall in the brain [18]. The severity of CAA is graded according to the degree of destruction of the vascular smooth muscle [28, 30]: grade 1, focal Aβ deposition in the vascular wall without major destruction of the lamina media; grade 2, significant destruction of the lamina media; and grade 3, CAA associated with hemorrhage. In this orangutan, circumferential Aβ deposition in the vascular wall with loss of SMA in the tunica media was observed, corresponding to grade 2 lesions. Although no CAA-associated hemorrhage was observed, Aβ deposition in the vascular wall with loss of SMA can be a risk factor for brain hemorrhage in aged orangutans.

Despite severe Aβ deposition, p-tau aggregates were rare, and pretangles and NFTs were not formed. The degree of p-tau aggregates did not exceed that of Braak stage 1 describes in humans [3, 4], consistent with previous reports in orangutans [11, 25]. In humans, Aβ Thal phase 2–3 and p-tau can be observed in the hippocampus and entorhinal cortex of elderly individuals without dementia [31]. There is currently no established method for evaluating cognitive dysfunction in non-human primates. Behavioral changes, such as disorientation, reduced interactions with other individuals, altered sleep-wake cycle, reduced activity level, and gait abnormalities, have been reported in a super-aged rhesus macaque with SPs and p-tau aggregates in its brain [34]. In the present case of an orangutan, no behavioral changes other than a decrease in gait speed were observed. The association between decreased gait speed and Aβ and p-tau pathologies could not be determined, as it may be attributable to multiple etiologies, including age-related frailty. In chimpanzees and gorillas, which are closely related to orangutans, p-tau aggregates have been observed, but most are pretangles, and NFTs are rare [8, 10, 12, 17, 20, 21]. In contrast, in humans, NFTs are prominent in the later Thal phase [19]. Therefore, despite significant Aβ deposition in the brains of non-human great apes, they may have a lower potential to form NFTs than humans. Further analysis of p-tau aggregation in great apes are expected to elucidate the reasons for the rarity of p-tau aggregation. Phosphorylated α-synuclein and phosphorylated TDP43 were not detected in the present orangutan. Humans and orangutans share a high degree of amino acid sequence identity in α-synuclein and TDP43 (Supplementary Table 2), suggesting that the antibodies used in the present study cross-react with orangutan proteins.

In conclusion, this study described severe Aβ deposition in a super-aged orangutan without significant p-tau aggregation. In addition, the detailed distribution and morphology of SPs and CAA are described. Further studies are necessary to clarify the relationship between Aβ deposition and cognitive decline in orangutans.

Conflicts of Interest

The authors declare no conflict of interest.