Intraneuronal Abeta42 accumulation in human brain

Abstract

Alzheimer's disease (AD) is characterized by the deposition of senile plaques (SPs) and neurofibrillary tangles (NFTs) in vulnerable brain regions. SPs are composed of aggregated beta-amyloid (Abeta) 40/42(43) peptides. Evidence implicates a central role for Abeta in the pathophysiology of AD. Mutations in betaAPP and presenilin 1 (PS1) lead to elevated secretion of Abeta, especially the more amyloidogenic Abeta42. Immunohistochemical studies have also emphasized the importance of Abeta42 in initiating plaque pathology. Cell biological studies have demonstrated that Abeta is generated intracellularly. Recently, endogenous Abeta42 staining was demonstrated within cultured neurons by confocal immunofluorescence microscopy and within neurons of PS1 mutant transgenic mice. A central question about the role of Abeta in disease concerns whether extracellular Abeta deposition or intracellular Abeta accumulation initiates the disease process. Here we report that human neurons in AD-vulnerable brain regions specifically accumulate gamma-cleaved Abeta42 and suggest that this intraneuronal Abeta42 immunoreactivity appears to precede both NFT and Abeta plaque deposition. This study suggests that intracellular Abeta42 accumulation is an early event in neuronal dysfunction and that preventing intraneuronal Abeta42 aggregation may be an important therapeutic direction for the treatment of AD.

Figure 1.

Intraneuronal Aβ42 accumulation occurs in AD-vulnerable neurons before the formation of senile plaques. A, Left: Neuronal Aβ42 staining (RU antibody) in the CA1 region of hippocampus derived from a 64-year-old patient with mild (CDR 0.5) cognitive dysfunction. Right: Aβ40 staining from the same CA1 region shows only slight immunoreactivity compared with the more pronounced intracytoplasmic staining seen with Aβ42. Antibody concentrations and time of development were equivalent. Bar = 60 μm. B, Left: Aβ42 immunoreactivity (RU antibody) in basal forebrain magnocellular neurons. Right: This staining is abolished by Aβ1–42 peptide competition; a blue filter was used to highlight negatively staining neurons. Bar = 60 μm. C, Left: Aβ42 staining (QCB) in the CA4 region of hippocampus from a neurologically normal 3-year-old patient (control); only faint neuronal staining can be seen (left), Bar = 60 μm. Center: Pronounced CA4 Aβ42 immunoreactivity (QCB) in a 3-year-old with Down’s syndrome. The arrow indicates a neuron with intracellular staining. Bar = 40 μm. Right: Aβ42 staining (QCB) in a 79-year-old without dementia indicates marked Aβ42 intracellular staining in layer II neurons (arrows) of the entorhinal cortex. Bar = 100 μm. D, Left: In this 83-year-old cognitively impaired subject (CDR1), the absence of intranuclear Aβ42 staining is evident in neurons stained for Aβ42 (RU). Early Aβ42 aggregates appear to be present within a neuron marked by an arrow; the inset provides another example of such seemingly intracellular Aβ42 accumulation in (RU) in a 94-year-old CDR 2 case. Bar = 40 μm. Center: “Neuronal” shaped SP (arrow) adjacent to a more conventional spherical SP in a 72-year-old subject with advanced AD (RU Aβ42). Bar = 60 μm. Right: The CA1 region of a 79-year-old cognitively impaired subject (CDR1) demonstrates both intraneuronal Aβ42 immunoreactivity (QCB) and apparent extraneuronal diffuse plaque-like staining (arrow) adjacent to a few neurons. Bar = 40 μm.

Figure 2.

A: Intraneuronal Aβ42 immunoreactivity (QCB) in layer II (islands of Calleja) of the entorhinal cortex (arrow) in a 90-year-old CDR1 patient, compared with the absence of staining (arrow) in an 83-year-old CDR2 patient; Aβ42 immunoreactive plaques can be seen above. In the CDR 2 patient, note the emergence of Aβ40 SPs. Bar = 100 μm. B: Abundant Aβ42 immunoreactivity (RU) compared with only occasional AT8 staining for hyperphosphorylated tau in the CA1 region of a 94-year-old patient (CDR 2). Bar = 60 μm. C: Adjacent sections of CA4 (below) and dentate gyrus (above) immunostained with antibodies to Aβ40, Aβ42 (QCB), and apoE in an 83-year-old cognitively impaired patient (CDR 2). Noticeable intraneuronal apoE staining is evident (inset, enlarged ×5). Bar = 100 μm.

Figure 3.

Metabolic labeling and immunoprecipitation of intraneuronal Aβ40 and Aβ42. A: Primary mouse neuronal cultures. Top: IP of conditioned medium indicates significantly lower secretion of Aβ42 compared with Aβ40. Bottom: Comparable amounts of Aβ40 and Aβ42 species in neuronal cell lysate. B: Aβ40 and Aβ42 species in sucrose density gradients from neuroblastoma cells harboring the Δ10eFAD PS1 mutation. Aβ1–40, Aβx-40, and Aβ1–42 species predominate in the Golgi-enriched fraction, whereas Aβx-42 predominates in the ER-enriched fraction.