Early molecular events of autosomal-dominant Alzheimer's disease in marmosets with PSEN1 mutations

Abstract

Introduction: Fundamental questions remain about the key mechanisms that initiate Alzheimer's disease (AD) and the factors that promote its progression. Here we report the successful generation of the first genetically engineered marmosets that carry knock-in (KI) point mutations in the presenilin 1 (PSEN1) gene that can be studied from birth throughout lifespan.

Methods: CRISPR/Cas9 was used to generate marmosets with C410Y or A426P point mutations in PSEN1. Founders and their germline offspring are comprehensively studied longitudinally using non-invasive measures including behavior, biomarkers, neuroimaging, and multiomics signatures.

Results: Prior to adulthood, increases in plasma amyloid beta were observed in PSEN1 mutation carriers relative to non-carriers. Analysis of brain revealed alterations in several enzyme-substrate interactions within the gamma secretase complex prior to adulthood.

Discussion: Marmosets carrying KI point mutations in PSEN1 provide the opportunity to study the earliest primate-specific mechanisms that contribute to the molecular and cellular root causes of AD onset and progression.

Highlights: We report the successful generation of genetically engineered marmosets harboring knock-in point mutations in the PSEN1 gene. PSEN1 marmosets and their germline offspring recapitulate the early emergence of AD-related biomarkers. Studies as early in life as possible in PSEN1 marmosets will enable the identification of primate-specific mechanisms that drive disease progression.

Keywords: Alzheimer's disease; PSEN1; biomarkers; genetic engineering; marmosets.

FIGURE 1

Marmoset PSEN1 genetic engineering. (A, B) Partial marmoset PSEN1 normal control (NC) and knock‐in (KI) genomic DNA sequences showing relevant amino acids, the protospacer adjacent motif (PAM), and CRISPR gRNA binding sites. The DNA substitutions introduced by CRISPR gene editing and resulting amino acid changes are shown in red in the KI sequence. (C) DNA sequencing chromatograms from normal control and homozygous KI marmosets demonstrating the G to A substitution introduced to change cysteine at 410 to tyrosine. (D) DNA sequencing chromatograms from normal control and subcloned KI amplicon demonstrating the G to C substitution introduced to change alanine 426 to proline.

FIGURE 2

Growth and developmental trajectories of PSEN1 F1 germline (n = 2 males; n = 3 females) and age‐ and sex‐matched non‐mutation carriers (NC). Evaluations are conducted once per week beginning from postnatal week (PNW) 1 up to PNW12. (A) body weight; (B) body length (crown to rump); (C) tail length; (D) biparietal distance. Postnatal week at which specific developmental milestones are achieved are presented in (E) males and (F) females relative to age‐ and sex‐matched NC. Demographics for each subject are provided in Table 2, representing litters 2 and 3.

FIGURE 3

Elevations in plasma Aβ levels in PSEN1 mutation carriers. Top panel: longitudinal analysis of plasma from PSEN1 mutation founder marmosets from infancy through adulthood compared to age‐ and sex‐matched contemporaneous non‐carrier (NC) controls, (A) plasma Aβ42 (pg/mL); (B) plasma Aβ40 (pg/mL); (C) calculated Aβ42:40 ratio. (D–F) Longitudinal analysis of plasma from infancy to present age for germline offspring (F1) of PSEN1 founder mutation carrier marmosets compared to age‐ and sex‐matched contemporaneous NC controls, (D) plasma Aβ42 (pg/mL); (E) plasma Aβ40 (pg/mL); (F) calculated Aβ42:40 ratio. (G–I) Cross‐sectional evaluation of normative values of plasma Aβ levels across a population of aging NC marmosets (black symbols) in comparison to young PSEN1 mutation carrier founder marmosets (blue symbols; subjects from A to C); (G) plasma Aβ42 (pg/mL); (H) plasma Aβ40 (pg/mL); (I) calculated Aβ42:40 ratio. (J–L) Cross‐sectional evaluation of normative values of plasma biomarkers (J) GFAP (pg/mL); (K) NFL (pg/mL); and (L) Tau (total, fg/mL). Demographics for each subject are provided in Tables 1 and 2 for Subject ID no. in (A)–(F).

FIGURE 4

Western blot analysis of enzyme–substrate interactions of gamma–secretase complex from cortex of PSEN1 marmoset founders and age‐matched non‐carrier (NC) control tissues. (A) PSEN1; (B) nicastrin (NCT); (C) PEN‐2; (D) APH‐1; (E) membrane immunoblots of PSEN1, NCT, PEN‐2, APH‐1, PSEN2, and GAPDH; (F) NOTCH1 transmembrane domain (NTM); (G) NOTCH intracellular domain (NICD); (H) amyloid beta (Aβ) 6E10; (I) Membrane immunoblots of NTM, NICD, and GAPDH; (J) membrane immunoblots of APP antibody under long exposure (top) and short exposure (bottom), labeled for long‐form APP and CTP fragment (Aβ).

FIGURE 5

(A) Triple immunostaining of a 17‐month‐old founder marmoset homozygous for PSEN1 C410Y mutation, showing intra‐ and extracellular accumulation of amyloid beta 42 (Aβ42) (red), along with neuroinflammation (green: anti‐Iba1). Cell nuclei were stained with an anti‐NeuN antibody (blue). S1: primary somatosensory cortex. M1: primary motor cortex. VP: ventral pallidum. Am: amygdala. Ent: entorhinal cortex. Panel A1: sensorimotor cortex. S1‐1: intracellular Aβ. S1‐2: extracellular Aβ plaques (arrow). VP: Ventral Pallidum. Panel VP‐1: phagocytic microglia (green) within perivascular Aβ accumulation. Am: gliosis in the amygdala (green). (B) Intra‐ and extracellular Aβ accumulation shown with amyloid precursor protein/amyloid beta (NAB228) antibody in the same animal and coronal plane as in (A). M1 and S1 show two different fields within sensorimotor cortex, and GP shows a field within the globus pallidum region. M1: primary motor cortex, with insets M1‐1 and M1‐2 showing intracellular Aβ deposits. S1: primary somatosensory cortex, with inset S1‐1 showing intracellular Aβ deposits. GP shows extracellular Aβ plaque formation in globus pallidum. Scale bars show magnification for each panel.