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(American Journal of Pathology. 2000;156:15-20.)
© 2000 American Society for Investigative Pathology

Short Communications

Intraneuronal Aß42 Accumulation in Human Brain

Gunnar K. Gouras^*, Julia Tsai, Jan Naslund^*, Bruno Vincent^*, Mark Edgar^§, Frederic Checler^¶, Jeffrey P. Greenfield^*, Vahram Haroutunian^||, Joseph D. Buxbaum^||, Huaxi Xu^*, Paul Greengard^* and Norman R. Relkin

From the Laboratory of Molecular and Cellular Neuroscience^*
and Fisher Center for Research on Alzheimer’s Disease,
The Rockefeller University; the Departments of Neurology and Neuroscience
and Pathology,||
Weill Medical College of Cornell University; Mount Sinai School of Medicine,||
New York, New York; and Institute de Pharmacologie Moleculaire et Cellulaire,^¶
Valbonne, France

	Abstract

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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 ß-amyloid (Aß) 40/42(43) peptides. Evidence implicates a central role for Aß in the pathophysiology of AD. Mutations in ßAPP and presenilin 1 (PS1) lead to elevated secretion of Aß, especially the more amyloidogenic Aß42. Immunohistochemical studies have also emphasized the importance of Aß42 in initiating plaque pathology. Cell biological studies have demonstrated that Aß is generated intracellularly. Recently, endogenous Aß42 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 Aß in disease concerns whether extracellular Aß deposition or intracellular Aß accumulation initiates the disease process. Here we report that human neurons in AD-vulnerable brain regions specifically accumulate -cleaved Aß42 and suggest that this intraneuronal Aß42 immunoreactivity appears to precede both NFT and Aß plaque deposition. This study suggests that intracellular Aß42 accumulation is an early event in neuronal dysfunction and that preventing intraneuronal Aß42 aggregation may be an important therapeutic direction for the treatment of AD.

	Introduction

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Over the past few years cell biological studies support the view that Aß is generated intracellularly^1,4-10 from the endoplasmic reticulum (ER)^1,7,8 to the trans-Golgi network (TGN),⁴ and the endosomal-lysosomal system.¹⁰ Recently, endogenous Aß42 staining was demonstrated within cultured primary neurons by confocal immunofluorescence microscopy⁹ and within neurons of human PS1 mutant transgenic mice by immunocytochemical light microscopy.¹¹ A central question on the role of Aß in AD is whether extracellular Aß deposition or intracellular Aß accumulation is initiating the disease process. Several groups had postulated the presence of intraneuronal Aß immunostaining. However, the Aß immunoreactivity observed in these studies was compromised by that of full-length ßAPP, because these Aß antibodies also recognize full-length ßAPP.^12-14 In addition, NFTs had previously been reported to be immunoreactive to Aß.^15-16 This association of Aß with NFTs was subsequently believed to be the result of artifactual "shared" epitopes.¹⁷

We now report that human neurons in AD-vulnerable brain regions specifically accumulate -cleaved Aß42 but not the more abundantly secreted Aß40. We also demonstrate intraneuronal Aß42 staining in neurons in both the absence and presence of NFTs. Our observations in adjacent sections of intraneuronal Aß42 staining and hyperphosphorylated tau staining suggest that neuronal Aß42 staining is more abundant and therefore may precede NFTs, which would exclude the possibility of cross-reactivity of shared epitopes. Furthermore, we observe the earliest Aß42 immunoreactive SPs developing along the projections and at terminals of early Aß42 accumulating neurons, suggesting a mechanism for the previously hypothesized regional specificity of AD disease progression within the brain.¹⁸

	Materials and Methods

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Antibodies

Polyclonal rabbit Aß40 (RU226) and Aß42 (RU228) C-terminal specific antibodies were generated at Rockefeller University (RU). Polyclonal rabbit Aß40 and Aß42 C-terminal antibodies were also obtained commercially (QCB). The results obtained with these two sets of antibodies were similar and were confirmed using well-characterized polyclonal rabbit Aß40 (FCA3340) and Aß42 (FCA3542) antibodies¹⁹ (kindly provided by F. Checler). Antibody 4G8 recognizes amino acids 17–24 of Aß (Senetek). Hyperphosphorylated tau was recognized by antibody AT8 (Polymedco). ApoE was visualized with a mouse monoclonal anti-ApoE antibody (Boehringer-Mannheim).

Immunocytochemistry

Postmortem brain tissue was examined from representative neurologically normal controls (ages 3 months and 3, 30, 44, 58, and 79 years); elderly nursing home residents without dementia (Clinical Dementia Rating (CDR) 0; ages 64, 69, 71, 72, 82, and 91 years) or with mild cognitive dysfunction (CDR 0.5; ages 67, 81, 87, 93, and 94 years) or mild (CDR 1; ages 79, 83, 84, 87, and 90 years), moderate (CDR 2; ages 83, 85, 90, 93, 94, and 94 years), or severe dementia (ages 64, 72, 79 years); and subjects with DS of varying ages (3 months and 3, 12, 13, and 24 years). The normal control and DS tissue were from New York Hospital, and the CDR tissue was from Mt. Sinai Medical Center. Postmortem intervals ranged from 6 to 18 hours. Ten percent formalin-fixed, paraffin-embedded brain sections (8 µm) were deparaffinized, washed in phosphate-buffered saline (PBS), incubated for 30 minutes at room temperature in 90% formic acid, washed again in PBS, incubated in 0.4% Triton X-100 (Tx) for 30 minutes, quenched for endogenous peroxidase with 3% hydrogen peroxide for 5 minutes, and preincubated in 3% serum from the species of the secondary antibody in 0.1% Tx/PBS for 1 hour to prevent nonspecific staining. Thereafter, slides were incubated with the appropriate antibody in 3% serum from the species of the secondary antibody/0.1%Tx/PBS overnight: anti-ApoE antibody (1:500), AT8 antibody (1:500), anti-Aß40, or 42 C-terminal specific antibodies (typically 1:500 for RU and 1:100 for QCB antibodies). Slides were washed with PBS and incubated with secondary antibody (anti-primary antibody species antibody) (Vectastain ABC kit; Vector) in 1.5% serum from the species of the secondary antibody/0.1%Tx/PBS at room temperature for 1 hour. Slides were incubated with avidin-biotin and developed with diaminobenzidine (DAB) (ABC kit) for 2 minutes. Except for some representative sections counterstained with hematoxylin and eosin (H&E), most sections were not counterstained, so as not to obscure the immunohistochemical staining.

Primary Neuronal Cultures

Primary neuronal cultures were derived from the cerebral cortices of embryonic day 15 (E15) CD1 mice (Charles River) as previously described.²⁰ Brains were removed, cortices were isolated, and the meninges were removed. Cortices were triturated in glass pipettes until cells were dissociated. Cells were counted in a hemocytometer and plated in serum-free Neurobasal media with N2 supplement (Gibco) and 0.5 mmol/L L-glutamine on poly-D-lysine-treated (0.1 mg/ml; Sigma) 100-mm dishes.

Metabolic Labeling and Immunoprecipitation

Cortical cultures plated 3–4 days previously or murine N2a neuroblastoma cells doubly transfected with human ßAPP₆₉₅ and the 10e FAD mutant human PS1²¹ were washed with PBS and incubated at 37°C for 20–30 minutes in methionine-free/glutamine-free Dulbecco’s minimum essential medium (Gibco). Cells were labeled with 750 µCi/ml [³⁵S]methionine (NEN/Dupont) (1 Ci = 37 GBq) in methionine-free medium supplemented with N2 and L-glutamine for 4 hours. Cells were scraped into ice-cold PBS with a rubber policeman. The supernatant was aspirated after brief centrifugation, and lysis buffer (100 µl) (0.5% deoxycholate, 0.5% NP-40, Trasylol (5 µg/ml), leupeptin (5 µg/ml), and phenylmethylsulfonyl fluoride (0.25 mmol/L)) was added. The lysate was subjected to agitation, repeat centrifugation, and collection of supernatant. Samples were treated with 0.5% sodium dodecyl sulfate, and the solutions were heated for 2 minutes at 75°C. Samples were adjusted to 190 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 8.3), 6 mmol/L EDTA, and 2.5% Triton X-100. Samples were incubated overnight with either antibody 4G8 or Aß40/42 antibodies, followed by secondary rabbit anti-mouse antibody (Cappell) for 1 hour and protein A-Sepharose (Pharmacia) beads for 2 hours (all at 4°C). Proteins were analyzed with 10–20% tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography on Kodak X-OMAT AR5 film.

Sucrose gradients used to prepare ER- and Golgi-enriched fractions were prepared as previously described.⁹ Metabolically labeled cells were homogenized in 0.25 mol/L sucrose, 10 mmol/L Tris-HCl (pH 7.4), 1 mmol/L MgAc₂, and a protease inhibitor cocktail (Boehringer-Mannheim). The homogenate was loaded on a step gradient of 2 mol/L, 1.3 mol/L, 1.16 mol/L, and 0.8 mol/L sucrose. Gradients were centrifuged for 2.5 hours at 100,000 x g. Fractions were collected from the top of each gradient, immunoprecipitated with Aß40/42 antibodies, and visualized as described above.

	Results

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Brain tissue from a 64-year-old representative subject with mild cognitive impairment (Clinical Dementia Rating Scale 0.5 (CDR 0.5); n = 5), stained with antibodies specific to the C-terminus of Aß42, revealed significant amounts of region-specific intraneuronal immunoreactivity (Figure 1a , left), compared with relatively little Aß40 immunoreactivity (Figure 1a , right). This intraneuronal Aß42 staining was especially evident within pyramidal neurons of areas such as the hippocampus/entorhinal cortex, which are prone to developing early AD neuropathology. Aß42 staining was less evident in sections from brain regions less affected by AD, such as primary sensory and motor cortices. The non-ß-pleated nature of this intracellular Aß42 is supported by a lack of Bielschowsky silver staining, the absence of Congo red birefringence under polarized light, the lack of thioflavin S staining, and the presence of Aß42 immunostaining without formic acid pretreatment. The Aß42 immunoreactivity was seen equivalently by three different Aß42 antibodies, was abolished by synthetic Aß1–42 peptide competition (Figure 1b) , and was not detected with the use of preimmune serum or in the absence of the primary antibody (data not shown). These Aß42 antibodies have negligible cross-reactivity to full-length ßAPP. Intraneuronal Aß42 immunoreactivity in a representative normal 3-month-old brain (Figure 1c , left) was of markedly less intensity than that in the brain of a 3-year-old with DS (Figure 1c , center) or the brain of a nondemented 76-year-old (Figure 1c , right). Thus neurons from neurologically normal controls (n = 6, ages 3 months to 79 years) showed intraneuronal Aß42 staining that appeared to increase in relation to the subject’s age at death. Analogous to the variability of SP deposition that exists among anatomical regions in an individual and between the same anatomical regions of different subjects, there was variability in the degree of intraneuronal Aß42 immunoreactivity between anatomical regions (ie, CA1 compared with CA4) within and between individuals.

View larger version (109K): [in this window] [in a new window]   Figure 1. Intraneuronal Aß42 accumulation occurs in AD-vulnerable neurons before the formation of senile plaques. A, Left: Neuronal Aß42 staining (RU antibody) in the CA1 region of hippocampus derived from a 64-year-old patient with mild (CDR 0.5) cognitive dysfunction. Right: Aß40 staining from the same CA1 region shows only slight immunoreactivity compared with the more pronounced intracytoplasmic staining seen with Aß42. Antibody concentrations and time of development were equivalent. Bar = 60 µm. B, Left: Aß42 immunoreactivity (RU antibody) in basal forebrain magnocellular neurons. Right: This staining is abolished by Aß1–42 peptide competition; a blue filter was used to highlight negatively staining neurons. Bar = 60 µm. C, Left: Aß42 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 Aß42 immunoreactivity (QCB) in a 3-year-old with Down’s syndrome. The arrow indicates a neuron with intracellular staining. Bar = 40 µm. Right: Aß42 staining (QCB) in a 79-year-old without dementia indicates marked Aß42 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 Aß42 staining is evident in neurons stained for Aß42 (RU). Early Aß42 aggregates appear to be present within a neuron marked by an arrow; the inset provides another example of such seemingly intracellular Aß42 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 Aß42). Bar = 60 µm. Right: The CA1 region of a 79-year-old cognitively impaired subject (CDR1) demonstrates both intraneuronal Aß42 immunoreactivity (QCB) and apparent extraneuronal diffuse plaque-like staining (arrow) adjacent to a few neurons. Bar = 40 µm.

It is of particular interest that with increasing cognitive dysfunction and Aß plaque deposition (CDR 2 subjects, n = 6, and severe AD, n = 3), we observed that intraneuronal Aß42 immunoreactivity tended to become less apparent. For example, in layer 2 neurons (islands of Calleja) of the entorhinal cortex from a CDR 1 patient, marked intraneuronal Aß42 immunoreactivity was observed (Figure 2a) , whereas in the patient with more advanced CDR 2 this staining was lost, presumably resulting from death or severe dysfunction of these neurons. In contrast, the emergence of Aß40 immunoreactive plaques can be seen in the patient with more advanced CDR 2 compared to the CDR1 patient, which is known to occur with disease progression.

View larger version (76K): [in this window] [in a new window]   Figure 2. A: Intraneuronal Aß42 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; Aß42 immunoreactive plaques can be seen above. In the CDR 2 patient, note the emergence of Aß40 SPs. Bar = 100 µm. B: Abundant Aß42 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 Aß40, Aß42 (QCB), and apoE in an 83-year-old cognitively impaired patient (CDR 2). Noticeable intraneuronal apoE staining is evident (inset, enlarged x5). Bar = 100 µm.

To corroborate our light microscopic observations of intraneuronal Aß42 immunoreactivity, we used metabolic labeling-immunoprecipitation to demonstrate endogenous Aß42 in primary rodent neuronal cultures. Pulse-labeling of these neuronal cultures, followed by immunoprecipitation of conditioned media by the use of Aß40 and Aß42 C-terminus-specific antibodies, revealed the expected predominance of secreted Aß40 over secreted Aß42 species (Figure 3a , top). In agreement with observations made using Aß40/Aß42 enzyme-linked immu-nosorbent assay in NT2 cells,⁶ we observed relatively greater ratios of intracellular Aß1–42/Aß1–40 and of Aßx-42/Aßx-40 in neuronal lysates than in conditioned media. In fact, almost equal amounts of Aßx-40 and Aßx-42 species were detected with the use of a standard detergent lysis buffer (Figure 3a , bottom). To more readily detect intracellular Aß42, we used a murine neuroblastoma N2a cell line harboring the human e10 FAD PS1 mutation, which is known to produce elevated levels of Aß42.²¹ Aß42 was readily detected in the ER- and Golgi-enriched fractions, with most of the secreted Aß1–42 in the Golgi-enriched fraction and most of the Aßx-42 in the ER-enriched fraction (Figure 3b) . Aß40 species were detected mainly in the Golgi-enriched fraction (Figure 3b) .⁹

View larger version (20K): [in this window] [in a new window]   Figure 3. Metabolic labeling and immunoprecipitation of intraneuronal Aß40 and Aß42. A: Primary mouse neuronal cultures. Top: IP of conditioned medium indicates significantly lower secretion of Aß42 compared with Aß40. Bottom: Comparable amounts of Aß40 and Aß42 species in neuronal cell lysate. B: Aß40 and Aß42 species in sucrose density gradients from neuroblastoma cells harboring the 10eFAD PS1 mutation. Aß1–40, Aßx-40, and Aß1–42 species predominate in the Golgi-enriched fraction, whereas Aßx-42 predominates in the ER-enriched fraction.

	Discussion

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The subcellular compartment(s) within which Aß42 peptides accumulate remains to be identified. One interesting study reported disruption of the Golgi apparatus as an early event in AD neuropathology and postulated that this may even proceed NFT development.²⁴ Given the growing body of evidence that both Aß40 and Aß42 formation occurs in the Golgi,^4,9 it is conceivable that Aß42 may begin accumulating abnormally within this organelle. However, more recent evidence indicates that Aß42 cleavage can also occur earlier in the secretory pathway in the ER, with retention of the peptide within this compartment.^7-9

Accumulating Aß42 may cause disruption of the cytoskeleton and initiate the formation of aggregated intracellular tau. Our proposal that intracellular accumulation of Aß42 disrupts the normal functioning of neurons is supported by increasing reports of cellular dysfunction within AD-susceptible neurons, such as the presence of markers of apoptosis¹⁴ and oxidative injury,²⁵ even before senile plaque and NFT formation. This proposal is further supported by the recent report of intraneuronal Aß42 accumulation and neural degeneration in FAD PS1 mutant transgenic mice in the absence of Aß plaque deposition.¹¹ Neuronal dysfunction arising from aggregating intraneuronal Aß42 may also explain recent studies reporting plaque-independent functional and structural disruption of neural circuits in ßAPP transgenic mice.^26,27

The role of apoE in AD remains incompletely understood. The decrease in plaque load of ßAPP transgenic mice crossed to apoE knockouts suggests an important relationship between apoE and aggregated Aß.²⁸ With Aß accumulation and neuronal dysfunction, neuronal or astrocyte-generated apoE may potentially bind to Aß intraneuronally and/or extracellularly with subsequent neuronal internalization, explaining the observation of apparent increased apoE immunoreactivity in Aß42 immunoreactive neurons.

Our observations of early intraneuronal accumulation ofAß42 within those brain areas that are affected earliest by AD suggest a mechanism that may explain AD disease progression within the brain. Intraneuronal Aß42 may act as a nidus for Aß deposition, intraneuronally and extracellularly, at the soma and along processes and terminals of affected neurons. The resultant accumulation of Aß in the parenchyma may hasten the pathological process, providing a potential mechanism for the "spread" of Aß-related pathology.

	Footnotes

 
Address reprint requests to Dr. Gunnar K. Gouras, Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, 1230 York Avenue, New York, NY 10021. E-mail: gkgouras{at}mail.med.cornell.edu

Supported by U.S.Public Health Service grants AG09464 (to P. G.), AG05138 (to V. H. and J. D. B.), and NS02037 (to G. K. G.); the American Health Assistance Foundation (to H. X.); the Alzheimer’s Association (to G. K. G.); and the Ellison Medical Foundation (to H. X. and P. G.).

Accepted for publication September 27, 1999.

	References

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