Neuropathology and Biomarker Correlates of Late Onset Alzheimer’s Disease in Aged Old World Monkeys

Abstract

Non-invasive predictive biomarkers for preclinical Alzheimer’s Disease (AD) may inform future dementia risk and support early intervention for AD. In this review, we describe clinical, biomarker and neuropathologic characteristics in spontaneously aged Old World monkeys (OWMs) in the context of preclinical AD. Reliable age-related amyloid-β (Aβ) plaque deposition occurs in OWMs. Plaque composition is complex, signifying significant disruption of synaptic connectivity. Pretangle pTau pathology in brainstem nuclei and limbic system prevails, consistent with Braak Stage 1b in macaques. Soluble pTau distribution approximates Braak Stage III-IV stage in perfused frozen macaque tissue, and colocalizes with Aβ and acetylcholinesterase labeling in AD-vulnerable circuits. Tau and Aβ pathology in OWMs is accompanied by fluid biomarker changes consistent with Core 1 AD diagnosis in humans but cannot be visualized using amyloid or tau tracers. Despite age-related cognitive decline, aging OWMs do not experience significant hippocampal atrophy or neuropathologic co-morbidities. Minimal expression of senescence markers implicates differences in rates of biological brain aging between OWMs and humans. OWMs support mechanistic studies and biomarker discovery in the areas of Aβ plaque and pTau evolution and resolution following anti-amyloid or Tau-directed therapeutics, as well as effects of senotherapeutics, lifestyle intervention or co-morbidities on biological brain aging.

Central to the justification for the use of research animals is the capacity to discover biological phenomena that cannot be identified in patient populations. Interest in spontaneously aged Old World primates as models of Late Onset Alzheimer’s Disease (LOAD) has been enduring, ^{6,26,27,46,68,136} particularly as models of preclinical AD. ^6,136,155 In this stage of AD, ³⁸ cognitively normal adults experience heterogenous accumulation of AD biomarkers. ^72,75.Because early intervention has the greatest potential for delaying clinical trajectories of AD, significant effort is directed at developing non-invasive predictive biomarkers for preclinical AD ⁷⁵ that inform eventual AD risk. ¹¹⁵ In humans, identifying these is complicated by inconsistent access to tissues at stages that may provide insight into critical determinants of normal and pathologic aging. Consequently, developing predictive biomarkers has relied upon painstaking correlation between prospective fluid biomarkers, ^8,9,70 various imaging modalities, ^{4,64,65,95,96} neuropathology ¹⁰⁶ and future cognitive decline. ^76,93 Animal models of AD provide a means to accelerate this discovery process through access to tissues under controlled circumstances. This allows identification of AD relevant neuropathologic and biomarker phenomena at early AD disease stages in which patients are cognitively normal. In this review, we describe clinical, biomarker and neuropathologic characteristics in spontaneously aged Old World monkeys (OWMs) in the context of the AD framework in patients. ^73,75 We focus on the rhesus macaque (Macaca mulatta), cynomolgus macaque (Macaca fascicularis) and African green monkey (Chlorocebus sabaeus).

The neuropathologic basis of AD biomarker progression in AD patients

Because distinct neuropathologic co-morbidities accompany cognitive decline, ^21,45,77 definitive diagnosis of AD can only be confirmed at autopsy. Patient neuropathology is characterized according to criteria defined by the National Institute of Aging-Alzheimer’s Association (NIA-AA) Consensus Guidelines for the Neuropathologic Assessment of AD.¹⁰⁶ Diagnosis of AD is based upon semi-quantitative scoring of the primary hallmarks of AD (beta amyloid (Aβ) plaque load and accumulation of neurofibrillary tangles) through application of the ABC scoring system. ¹⁰⁶ Co-occurring neuropathologic phenomena are also recorded.

Spatial Aβ plaque load is characterized by progressive anatomic involvement of immunohistochemically detectable Aβ in cortical regions, limbic structures, midbrain and eventually cerebellum using Thal staging ¹³⁹ – this is converted to an A score. ¹⁰⁶ Density of neuritic plaques (defined as amyloid plaques with pTau positive dystrophic neurites with or without dense cores) is quantified semi-quantitatively to yield a CERAD (Consortium to Establish a Registry for Alzheimer’s Disease) or C score. ^63,103 Cerebral amyloid angiopathy (CAA) is scored but is not included in the final scoring rubric for AD risk. ¹⁰⁶

In patients, Aβ plaque deposition is reflected by a declining CSF and blood Aβ42/Aβ40 ^51,129 ratio and a positive amyloid PET signal constituting Core 1 biomarkers sufficient for diagnosis of AD (Figure 1). ^75,86,131 Patients with moderate to high Thal (A2–3) and CERAD (C2–3) scores can be distinguished from those with low (A0–1, C0–1) scores using 11C-PiB scans ⁸⁶ or 18F-flutemetamol. ²⁸

Figure 1:

Categorization of biomarker changes in relationship to cognitive decline in patients with AD neuropathologic change. Sequential accumulation of amyloid (A) and tau (T) is followed by neurodegeneration (N) and clinical symptoms (C). Presence of neuropathologic co-pathology may accelerate neurodegeneration and clinical decline, whereas cognitive reserve may delay onset of clinical decline despite presence of significant AD neuropathologic change. Time is shown on the x axis and magnitude of biomarker or clinical abnormality is shown on the y axis.

Categorization of fluid analyte (CSF or blood) and imaging biomarkers shown in the table beneath. Positive Core 1 fluid biomarkers are sufficient to diagnose AD. Neurodegeneration (N) and Inflammation (I) biomarkers frequently accompany AD neuropathologic change but are not specific for it. Vascular (V) and alpha-synuclein (S) biomarkers denote presence of co-pathologies that may influence cognitive state.

Adapted from: Jack CR Jr, et al., Revised criteria for diagnosis and staging of Alzheimer’s disease: Alzheimer’s Association Workgroup. Alzheimers Dement. 2024 Aug;20(8):5143–5169.

Abbreviations: Aβ, amyloid beta; AD, Alzheimer’s disease; αSyn-SAA, alpha-synuclein seed amplification assay; CSF, cerebrospinal fluid; CT, computed tomography; FDG, fluorodeoxyglucose; GFAP, glial fibrillary acidic protein; MRI, magnetic resonance imaging; MTBR, microtubule-binding region; NfL, neurofilament light chain; PET, positron emission tomography; SMBT-1: (S)-(2-methylpyrid-5-yl)-6-[(3–18F-fluoro-2-hydroxy)propoxy]quinoline PET tracer; SPECT, Single-photon emission computed tomography; SV2A, Synaptic Vesicle Glycoprotein 2A; TREM2, Triggering receptor expressed on myeloid cells 2.

Braak staging describes the expanding anatomical distribution of neurofibrillary tangles (NFT’s; currently detected using a pTau antibody) accompanying AD progression. ^17,18 Within neurons, abnormally phosphorylated pTau progresses through a series of histological appearances. Pretangles constitute the earliest finding and present as granular cytoplasmic non-argyrophilic pTau positive bodies occurring within morphologically normal neurons. ^104,105 These progress to mature neuropil threads (NTs) and NFTs, ¹⁶ seen as tightly packed coiled and aggregated argyrophilic fibers located in neurons with a dislocated or shrunken nucleus. ¹⁰⁴ Because histology of NFT’s is so characteristic in AD, tangles are diagnosed using pTau antibodies (usually AT8) in lieu of argyrophilia. ¹⁰⁴ However, pTau antibodies also detect pretangles, and in non-human primates, progression to tangle pathology requires demonstration of characteristic paired-helical filament ultrastructural appearance ^117,127 or positive Thioflavin-S staining (which will detect β-pleated sheet morphology of both amyloid plaques and mature tangles. ⁷ Eventually, affected neurons die and disintegrate, leaving residual NFTs suspended in surrounding neuropil (ghost tangles).

Patients with low Braak stages (I, II) exhibit dense NTs and mature NFTs in presubiculum and subiculum. With increasing Braak stages, these progress through CA1 hippocampal region, entorhinal cortex (III, IV), and progressive cortical regions (V, VI) in highest scoring individuals. ¹⁸ Braak stages are converted to B scores. ¹⁰⁶ Likelihood of AD (low, intermediate or high) is assigned on the basis of combined A, B and C scores. ¹⁰⁶ Braak NFT staging requires, by definition, the presence of NFTs. However, in humans, pretangles appear in brainstem nuclei in the first decade of life, and progress to include subcortical regions in young adults. ^19,20 This progression is captured using a pretangle scoring system that is not part of AD diagnosis.

Neuropathologic Braak staging can be detected in vivo using tau PET ligands. ⁹⁶ Altered fluid pTau biomarker profiles in patients typically occur prior to a positive tau PET signal. ⁷⁰ pTau profiles in cognitively normal and AD brains ^98,152 are quite similar, however, some pTau markers (e.g., pT217, pT231, pT181, and pT205) have discriminatory potential and constitute both Core 1 and Core 2 biomarkers (Figure 1).^{75 44,74,93} In cognitively intact patients, elevated fluid biomarkers pT217 and pT231 are more closely associated with Aβ-PET positivity (i.e. pTau signal originating from dystrophic neurites in neuritic plaques) than PET-detectable tangles. ^8,9,102 In later disease stages, these also increase due to tau tangle accumulation. ^44,107 The recently approved AD blood test (pTau217/β-Amyloid-42 Plasma Ratio) permits detection of early Aβ-plaque positivity ¹ and likelihood of progression of cognitive decline. ³⁰

Astro- and microgliosis are not scored but constitute important aspects of AD pathophysiology and biomarker development (Figure 1). ^{13,43,146,157} Astrocytosis is significantly associated with Aβ plaque load rather than pTau accumulation. This association is recapitulated in PET imaging ¹⁴⁶ and blood biomarker studies. ^119,137 Tau propagation is associated with a complex range of microglial activation states. ¹¹⁶ Microglia demonstrate a spectrum of active to senescent proteomic profiles depending on regional pathology and location. ^54,109. The microglial transmembrane protein Triggering receptor expressed on myeloid cells 2 (TREM2) has emerged as a potential fluid biomarker for early AD.¹³⁷ TREM2 radioligands are in preclinical development.²⁹

Additional neuropathologic changes that can impact cognition and complicate biomarker profiles are also recorded during neuropathologic assessment. ^45,66,91,106 These include gross and histologic indicators of cerebrovascular disease (atherosclerosis, infarction, macro- or microbleeds, arteriolosclerosis, vascular mineralization, red neurons and laminar cortical necrosis), nigral Parkinsonian pathology, presence of alpha-synuclein or TAR DNA-binding protein 43 (TDP-43) inclusions, hippocampal pathology such as hippocampal sclerosis and primary age-related tauopathy (PART) and aging-related tau astrogliopathy (ARTAG).

There are few comparative studies in which the NIA/AA Consensus Guidelines for the Neuropathologic Assessment of AD ¹⁰⁶ are used to assess AD pathology in NHP’s ^7,88,155 and only one in which these are directly compared in NHP and human cohorts. ¹⁵⁵

Detection of AD risk in living patients

Over the last two decades, the AD research framework ^73,134 has been an essential tool to describe increasingly earlier changes that reflect increased risk of progression to clinically evident AD (Figure 1). Beginning in middle age, ³⁸ cognitively normal adults may accumulate amyloid (A), tau (T), and neurodegeneration (N) biomarkers. ⁷⁵ An essential component of the most recent iteration of this framework ⁷⁵ is the diagnosis of AD on characteristic biomarkers rooted in AD neuropathology, rather than evidence of clinically evident dementia. This recognizes the contribution of other neuropathologic entities that may accelerate cognitive decline, as well as the capacity of cognitive reserve to delay decline. Clinical expression of dementia in the presence of AD neuropathologic change (ADNP) is in large part dependent on life-long exposures that that determine neuronal resilience and are estimated to contribute to approximately 40% of clinically emergent AD cases. ⁹² Accurate placement of NHPs within the AD research framework ⁷⁵ is essential to understanding their utility as models of early-stage AD.

Ageing and cognitive decline in OWMs

Declining age-related cognitive function is most consistently reported in rhesus macaques, ^56,108,122 but is also described in cynomolgus macaques ³³ and African Green monkeys. ^48,49 In rhesus, aged animals display impaired executive system function, ^87,154 working memory ¹³³ and cognitive flexibility. ^10,22

Beta-Amyloid (Aβ) accumulation and biomarker correlates in OWMs

Age-associated accumulation of Aβ plaques (Figure 2) is a consistent finding in rhesus macaques, ^46,69,155 cynomolgus macaques, ³² African green monkeys ^27,79 and baboons ¹¹¹. As in many mammalian species apart from rodents, ¹¹⁸ Aβ plaques also occur in aged mouse lemurs, ⁵³ common marmosets, ¹²⁰ squirrel monkeys, ¹⁴⁷ the bearded capuchin ³⁷ and great apes. ^40,52,121 Cerebral amyloid angiopathy (CAA) is also described in macaques and squirrel monkeys ^{68,110,143,147} with particular predisposition for this lesion in the latter. ^58,147 In rhesus macaques, CAA scores are lower compared to patients. ¹⁵⁵

Figure 2:

Amyloid-β pathology in AD patients and macaques

a. Amyloid-β plaque load (% area) in FFPE tissue of AD patients (blue) and aging macaques (red). Plaque load in patients increased with increasing ABC score, particularly in cortical regions. Plaque load increased with age in macaques and was negligible under 26 years.

b, c: Plaque morphology in FFPE tissue. A range of diffuse to dense core (arrows, b) plaques were evident in patient tissue. In macaques (c), most plaques were diffuse.

d-m: Amyloid-β plaque content in perfused frozen tissues from a 32.5 year-old rhesus. Constituents comprised phosphorylated Tau AT8 (d), pT217 (e), SMI-32 (f), GFAP (g), Iba1 (h), AchE (i), TH (j), SV2A (k), Nissl (l) and Prussian blue for iron (m). Not all plaques demonstrated all of these constituents.

Patient locations: MFG: middle frontal gyrus; S/MTG: superior and middle temporal gyrus; AC: anterior cingulate gyrus; ERC: entorhinal cortex; O: occipital cortex (BA17, 18). Macaque locations: 46d: dorsolateral prefrontal cortex area 46d; ST2: superior temporal gyrus area ST2; 24: anterior cingulate gyrus; Amyg: amygdala; ERC: entorhinal cortex; P: parietal cortex; O: occipital cortex (BA17, 18).

AchE: acetylcholinesterase, GFAP: glial fibrillary acidic protein, Iba1: ionized calcium-binding adapter molecule, SV2A: synaptic vesicle glycoprotein 2A, SMI-32: non-phosphorylated form of the neurofilament heavy protein, TH: tyrosine hydroxylase.

Bar = 50 μm (b, c); 20 μm (d-m).

Reprinted from: Zeiss CJ, Huttner A, Nairn AC, et al. The neuropathologic basis for translational biomarker development in the macaque model of late-onset Alzheimer’s disease. Journal of Alzheimer’s Disease. 2025;104(4):1243–1258. doi:10.1177/13872877251323787

While both mature and diffuse Aβ plaques are noted in cynomologus macaques, ¹¹⁰ plaques in rhesus macaques ^135,155 and African green monkeys ⁷⁹ most commonly assume diffuse rather than dense-core morphology (Figure 2). In rhesus macaques,¹⁵⁵ plaques contain dystrophic neurites immunoreactive for various antigens (AT8, pT217, pT181, AChE, and TH). Consequently, these meet the definition of neuritic plaques (defined as amyloid plaques with pTau positive dystrophic neurites with or without dense cores). ¹⁰⁶ Aβ plaques also exhibit depletion of some proteins (e.g. SV2A) and neurons, along with the accumulation of iron (Figure 2). Iron has long been considered contributory to Alzheimer’s disease pathogenesis. ¹⁵⁰ These findings suggest significant disruption of neural connectivity in regions with high Aβ load. Consequently, established AD patients are likely to retain an imprint of neuritic injury following therapeutic extraction of Aβ, potentially undermining significant cognitive recovery. ¹²⁵ Plaques are rare in rhesus under 25 years of age, ^135,155 but occur earlier in cynomolgus and African Green monkeys (< 20–21 years) ^48,82,110.

As in patients, Aβ plaque accumulation in OWMs is also reflected by declining Aβ42/Aβ40 ratios in CSF, ^{33,48,88,101,126,153} serum ³¹ and plasma (Table 1). ¹⁰¹ In contrast, attempts at detecting the imaging biomarker (Aβ PET) have been unsuccessful in macaques. ^{41,112,113,128} These studies used animals that ranged from maximum ages of 19– 24 years. As plaques are uncommon in rhesus under 25 years of age, ^135,155 Aβ loads may have been below the level of detection in these studies. In patients, correlation of Aβ immunohistochemical load (% area) at postmortem with in vivo retention of the amyloid ligand Pittsburgh Compound B (PiB) indicates that Aβ loads of 0.5–4% correlate with 11C-PiB distribution volume ratio (DVR) of 1.5–1.8. ⁶⁴ This level of PiB uptake is associated with future cognitive decline. ⁴² Using immunohistochemistry, ¹⁵⁵ comparison of Aβ load in macaques and patients demonstrated that amyloid load sufficient to be detectable by PET (0.5–3%) accumulates after 25 years in macaques. Composition of plaques in macaques has been proposed as a reason for relatively lower amyloid PET tracer uptake. ^69,128 In patients, both dense-core and diffuse plaques (which prevail in macaques) are detected by either 11C-PiB and 18F-flutemetamol, ^28,65 however dense-core plaques generate approximately three times the signal of diffuse plaques.

Table 1:

Comparison of biomarker changes in patients and OWMs within current criteria for diagnosis and staging of Alzheimer’s disease.⁷⁵

	Species	CSF/ blood	Imaging
Core biomarkers of AD neuropathologic change
Core 1 A (Aβ proteinopathy) T1: (p-Tau)	Patient	Declining CSF/blood Aβ42/40 ratio 51,129 Increased CSF/blood p-tau181; p-tau217; p-tau231 8,100,102	Amyloid PET distinguishes between absent/low and moderate/high neuropathologic amyloid load 86,131
	Rhesus macaque	Declining plasma Aβ42/40 ratio starting in middle age (16 years), 101 but no change in plasma p-tau181 with age 101 Increasing plasma p-tau217 with age (> 18 years) 36	PET studies fail to demonstrate amyloid 41,113
	Cynomolgus macaque	Declining serum Aβ42 with increasing age (over 20 years) 31,33,62,153 No change in CSF p-tau with age 31,153	No studies
	African green monkey	Declining CSF Aβ42/40 ratio with age (>21 years) 88 but no change in CSF p-tau with increasing age 88	No studies
Core 2 T2 (AD tau proteinopathy)	Patient	pT205, MTBR-243, non-phosphorylated tau fragments 9,59	Tau PET recapitulates Braak scoring 96
	Rhesus macaque	No studies	PET studies fail to demonstrate tau 41
	Cynomolgus macaque	No studies	No studies
	African green monkey	No studies	No studies
Biomarkers of non-specific processes involved in AD pathophysiology
N (neurodegeneration)	Patient	Increasing Nfl and total-tau 124 reflect neuronal injury	MRI: Cortical, temporal and hippocampal atrophy in AD 15,71 FDG-PET: Cerebral hypometabolism accompanies but is not specific for AD 24,75 SV2A-PET: Cortical and hippocampal synaptic loss accompanies AD 99
	Rhesus macaque	Increasing plasma Nfl by middle age (16 years), but no increase in plasma total tau with age 101	MRI: Minimal temporal or hippocampal atrophy with age 34,85 SV2A-PET: No synaptic loss 41
	Cynomolgus macaque	Increased CSF total tau (>20 years) 33; no change in CSF total tau with increasing age 31	MRI: Hippocampal atrophy in low cognitive performers (>20 years) 33
	African green monkey	No change in CSF total tau with increasing age 88	18F-FDG PET: Reduced activity associated with Aβ plaque density 88 MRI: Reduction in brain volume in some regions with age, correlated with Aβ plaque density 88
I (inflammation)	Patient	Increasing plasma GFAP correlates with amyloid neuropathology 80 Increasing CSF TREM2 correlates with tau neuropathology 137	Astrocytosis detected by (18)F-SMBT-1 146 TREM2 radioligands in preclinical development 29
	Rhesus macaque	No change in plasma GFAP with age 101	No studies
	Cynomolgus macaque	No studies	No studies
	African green monkey	No studies	No studies
Biomarkers of non-AD co-pathology
S (alpha-synuclein)	Patient	CSF αSyn-SAA detect co-occuring synucleopathies 25	αSyn-PET: used to detect a variety of synucleopathies 83 DAT-SPECT: reduced striatal DAT binding in Parkinsonian disorders 149
	Rhesus macaque	No studies	No studies; histologic evidence of age related nigral loss only 132
	Cynomolgus macaque	No studies	No studies
	African green monkey	No studies	No studies
V (vascular brain injury)	Patient	Broad, but non-specific array of fluid biomarkers accompany vascular injury 138	Imaging of small vessel disease, 151 Imaging of infarction and white matter hyperintensity 145
	Rhesus macaque	NA	No studies
	Cynomolgus macaque	NA	No studies
	African green monkey	NA	No studies

Biomarker changes in OWMs are reported in Table 1 are statistically significant (p<0.05).

Altered Core 1 fluid biomarkers reflect underlying AD neuropathologic change and are thus sufficient to diagnose AD ¹⁰ before amyloid and tau PET imaging abnormalities manifest. ¹² N and I biomarkers reflect non-specific processes involved in AD pathophysiology, whereas S and V biomarkers are used to detect non-AD co-pathology.

Abbreviations: Aβ, amyloid beta; AD, Alzheimer's disease; αSyn-SAA, alpha-synuclein seed amplification assay; CSF, cerebrospinal fluid; CT, computed tomography; DAT, dopamine transporter; FDG, fluorodeoxyglucose; GFAP, glial fibrillary acidic protein; MRI, magnetic resonance imaging; MTBR, microtubule-binding region; NA, not applicable: NfL, neurofilament light chain; PET, positron emission tomography; SMBT-1: (S)-(2-methylpyrid-5-yl)-6-[(3-18F-fluoro-2-hydroxy)propoxy]quinoline PET tracer; SPECT, Single-photon emission computed tomography; SV2A, Synaptic Vesicle Glycoprotein 2A; TREM2, Triggering receptor expressed on myeloid cells 2; WMH, white matter hyperintensity.

Tau pathology and biomarker correlates in OWMs

While Aβ plaque-associated pTau immunoreactivity is readily demonstrated in dystrophic neurites (Figure 3), neurofibrillary tangles (NFT’s) and neuropil threads (NT’s) central to Braak staging are typically absent in OWMs. ^{7,32,48,88,140,148} Consequently, NFT Braak score >0 cannot be assigned to OWMs. ^7,155 However, pretangle pathology in macaques can be scored according to Braak criteria defined in younger people. ^19,20 Granular cytoplasmic pretangle neuronal pTau ¹⁵⁵ immunoreactivity occurs in locus coeruleus and substantia nigra consistent with pretangle stage Braak a-c, and within isolated neurons within CA1 region, subiculum and entorhinal cortex consistent with Braak Stage 1a-b. ²⁰ In very old macaques (>30 years), biochemical and ultrastructural studies demonstrate increasing insoluble, fibrillated tau in the entorhinal cortex ¹¹⁷ indicating that pretangles can progress to NFTs in rhesus macaques.

Figure 3:

Immunostaining against pTau epitopes AT8, pT217 and pT181in FFPE macaque tissue.

a-d: Pretangle pTauT217 and AT8 immunoreactivity within individual neurons (arrow) in locus coeruleus (LC) and substantia nigra (SN) in FFPE (37.7 year old M. arctoides)

e-j: Immunostaining in FFPE tissue from a 37.7 year old M. arctoides against pTau epitopes AT8, pT217 and pT181. Within the subiculum (e, f, g), similar pretangle morphology in neurons was noted with all three antibodies. Within amyloid-β plaques, pTau positive dystrophic neurites were evident with all p-Tau antibodies. Double immunohistochemistry for amyloid-β and pT217 is illustrated in i.

Bar = 50 μm (b, d, e, g, i); 20 μm (a-d, h-j).

FFPE: formalin-fixed paraffin embedded

Reprinted from: Zeiss CJ, Huttner A, Nairn AC, et al. The neuropathologic basis for translational biomarker development in the macaque model of late-onset Alzheimer’s disease. Journal of Alzheimer’s Disease. 2025;104(4):1243–1258. doi:10.1177/13872877251323787

Human and NHP pathology described thus far is derived from formalin-fixed, paraffin embedded tissue. However, pTau detection in tissue may be reduced by the combined effects of post-mortem interval, immersion fixation and paraffin processing. ^98,117 In frozen sections of perfused macaques, diffuse age-related AT8 immunoreactivity is evident in neurons and processes of AD-vulnerable cortical and limbic regions approximating Braak III-IV staging in human FFPE material. ^117,155 In old macaques, high spatial correlation between AChE, Aβ plaques and pTau load occurs in the basal forebrain cholinergic system, amygdala, hippocampus, and medial temporal regions (Figure 4), consistent with the enduring roles of the Tau, ⁶⁷ amyloid ¹³⁰ and cholinergic ⁵⁵ hypotheses in AD pathogenesis. Age-related tau astrogliopathy (ARTAG) and primary-age-related tauopathy (PART) have not been demonstrated in macaques.

Figure 4:

Spatial relationship of acetylcholinesterase (AchE), amyloid-β and AT8 expression in perfused frozen tissue from a 32.4-year-old rhesus

a-g: AchE histochemistry, anterior to posterior coronal sections. AchE staining was evident in cortical regions, basal forebrain structures, amygdala and hippocampus that were also reactive for AT8. Some subcortical regions (basal ganglia, thalamus, midbrain) were only reactive for AchE.

h-n: AT8 immunohistochemistry, similar sections. Regions of AT8 and AchE co-expression included anterior cingulate cortex (areas 32, 24 and 25:a, h), orbitofrontal cortex (b, i), basal forebrain (c, j), amygdala and temporal cortex with emphasis on perirhinal and parahippocampal regions (c, d, j, k), entorhinal cortex and subiculum (e, f, l, m).

o-p: Higher magnification of AchE, amyloid-βand AT8 staining using area 32 as an example. AchE was evident with processes and within amyloid-βplaques (arrows, o). amyloid-β plaques (arrows, p) contained AT8 positive dystrophic neuritis (arrows, q). Diffuse AT8 immunoreactivity within cortical neuronal processes was evident (asterisk, q).

Bregma locations in mm are illustrated above.

Bar = 1500 μm (a, h); 1600 μm (b, i); 2700 μm (c-g, j-m); 100 μm (o-q), 20 μm (inset, q).

Reprinted from: Zeiss CJ, Huttner A, Nairn AC, et al. The neuropathologic basis for translational biomarker development in the macaque model of late-onset Alzheimer’s disease. Journal of Alzheimer’s Disease. 2025;104(4):1243–1258. doi:10.1177/13872877251323787

Some tau phosphorylation sites exhibit age-related cortical and fluid biomarker increases in rhesus macaques (pS202/T205, pT217, pT181, pS235, pS356, pS396, and pS214) ^11,35,36,89 and cynomolgus macaque cortex (pS202/T205, pT231). ⁷⁸ In rhesus macaques, elevated plasma pT217³⁶ and unchanged pT181¹⁰¹ with age appear to correlate with superior performance of the former in identification of AD pathology (Table 1). ¹⁰⁰ In light of the AD framework, ⁷⁵ increasing plasma pT217 levels ³⁶ inform on Core 1 biomarker events rather than Core 2 pathology (characterized by positive medial temporal Tau-PET ⁹⁶ and additional tau species ⁵⁹). Inability to detect AT8 positive non-argyrophilic pretangles is a consistent feature of current tau PET tracers. ^5,94,104 Therefore, predominant pretangle tau pathology in aged NHP brains is consistent with negative tau PET findings to date. ^41,60 Consequently, the established relationship between fluid pTau biomarkers and tau PET in patients ⁷⁰ cannot be currently assessed in the NHP model.

Neurodegeneration (N) in OWMs

The neurobiological basis for age-related cognitive decline has been most extensively explored in rhesus macaques ^56,123,133 and has been correlated with synaptic loss, neuronal loss and cortical thinning in prefrontal cortex, ^{39,122,123,133} cholinergic neuron loss ¹³³ and decreased cell density in cortically projecting brain stem nuclei. ¹²² Hippocampal spine density declines in aged animals, ¹⁴² however, in contrast to AD, neuronal numbers are preserved. ⁸¹ Hippocampal and temporo-cortical atrophy characteristic of late-stage AD in patients ^15,71 is limited on gross morphology ¹⁵⁵ and absent or inconsistent on MRI ^33,34,85,88 in aged OWMs, thus placing them much earlier in the AD trajectory. Synaptic loss detected by synaptic vesicle glycoprotein 2 A(SV2A) tracer [11C]UCB-J in AD patients ⁹⁹ is not evident in aged macaques. ⁴¹ Elevated neurofilament light (Nfl) levels (a marker of large-caliber axonal injury) in preclinical AD patients ²³ are recapitulated in older rhesus ¹⁰¹ but not evident in middle aged macaques ¹²⁶ or in Aβ oligomer-injected rhesus brain. ¹² Fluorodeoxyglucose hypometabolism detected using fluorodeoxyglucose positron emission tomography (FDG-PET) in AD patients ²⁴ is influenced by co-morbid neuropathologies, ⁹⁰ as well as non-neurologic multimorbidities independent of amyloid accumulation.¹⁴⁴ In contrast to Core biomarkers, neurodegeneration biomarkers, including total-tau ^75,124 are not considered specific for AD (Figure 1, Table 1), as they may occur in other conditions, including traumatic brain injury and ischemic injury.⁷⁵

Inflammation (I), vascular (V) and synucleinopathy (S) processes in OWMs

In patients, plasma glial fibrillary acidic protein (GFAP) is increased in preclinical AD. ^2,80 A comparative study identified significant correlation between GFAP and Aβ load in FFPE sections from patients, but not macaques ¹⁵⁵ despite accumulation of astrocytes within Aβ plaques in both species. ^47,57 A recent study in rhesus macaques failed to identify increased plasma GFAP with age (Table 1). ¹⁰¹ Increased glial cytoplasmic and nuclear expression of the senescence marker p16 is evident in brains of AD patients ⁶¹ and clearance of p16-expressing microglia improves cognitive function and brain immune aging in mice. ¹¹⁴ Compared to AD patients, both AT8 and p16 expression are scarce in aged macaque brain ¹⁵⁵ suggesting that macaques may experience relative protection from NFT accumulation and immune senescence compared to humans. On autopsy, patients with AD neuropathologic disease frequently have additional neuropathologic comorbidities, most commonly Lewy body disease and ischemic injury. ⁹⁷ These may be identified ante-mortem using fluid biomarkers for synucleopathies ²⁵ and more broadly with imaging modalities. ^{83,145,149,151} Aged macaques experience modest nigral neuronal loss, no significant synucleinopathy and limited vascular pathology. ^132,155 The relationship between hypertension, vascular disease, Type II diabetes and dementia risk is well-established ^3,91. Although arteriosclerosis may be noted histologically, spontaneous hypertension in aging macaques is more commonly noted in the context of metabolic syndrome ¹⁵⁶. Type II diabetes is a common comorbidity of OWMs and is accompanied by increased Aβ plaque load and pro-inflammatory biomarkers, ^62,84 as well as cerebral hypometabolism in African green monkeys.⁸⁴

Conclusions

Aging OWMs have been proposed as models of preclinical AD ^{6,134,136,155} i.e., the clinically asymptomatic state harboring the underlying AD pathophysiologic disease process. The combination of Aβ plaque accumulation, declining CSF or blood Aβ42/ Aβ40 ratios and increasing fluid pTau biomarkers place aging OWMs within the early preclinical spectrum of AD, specifically within Core 1 biomarker territory. ⁷⁵

Due to its complex neuritic plaque composition, the macaque may be a robust model to study retention or restoration of synaptic connectivity following removal of Aβ. Notably, detection of prefibrillar pTau in AD-vulnerable regions in perfused macaque brains recapitulates the pattern of NFT Braak III-IV staging. This finding implies that similar changes may occur in cognitively middle-aged humans and are likely to inform biomarker studies in early preclinical AD. pTau accumulation in regions that also express AChE and Aβ, allowing study of early amyloid-tau interactions in AD-vulnerable cholinergic circuits.

OWMs are largely spared from neuropathologic comorbidities, and experience relative protection from both fibrillar Tau accumulation and expression of senescence markers. These data imply that differences in genetics, lifespan and lifetime exposures may drive differential brain biologic aging ¹⁴¹ between OWMs and humans. Aged macaques and African Green monkeys present a singular opportunity to explore the effects of various co-morbidities (particularly diabetes) and interventions, ^14,50,135 discover early predictive fluid biomarkers and correlate these with in-vivo spatial expression in perfused brain.

Data availability:

Human and macaque digital slides are freely accessible (MacBrain: https://medicine.yale.edu/neuroscience/macbrain/) for further comparative studies.