The Alzheimer's β-secretase BACE1 localizes to normal presynaptic terminals and to dystrophic presynaptic terminals surrounding amyloid plaques

Abstract

β-Site amyloid precursor protein (APP) cleaving enzyme-1 (BACE1) is the β-secretase that initiates Aβ production in Alzheimer's disease (AD). BACE1 levels are increased in AD, which could contribute to pathogenesis, yet the mechanism of BACE1 elevation is unclear. Furthermore, the normal function of BACE1 is poorly understood. We localized BACE1 in the brain at both the light and electron microscopic levels to gain insight into normal and pathophysiologic roles of BACE1 in health and AD, respectively. Our findings provide the first ultrastructural evidence that BACE1 localizes to vesicles (likely endosomes) in normal hippocampal mossy fiber terminals of both non-transgenic and APP transgenic (5XFAD) mouse brains. In some instances, BACE1-positive vesicles were located near active zones, implying a function for BACE1 at the synapse. In addition, BACE1 accumulated in swollen dystrophic autophagosome-poor presynaptic terminals surrounding amyloid plaques in 5XFAD cortex and hippocampus. Importantly, accumulations of BACE1 and APP co-localized in presynaptic dystrophies, implying increased BACE1 processing of APP in peri-plaque regions. In primary cortical neuron cultures, treatment with the lysosomal protease inhibitor leupeptin caused BACE1 levels to increase; however, exposure of neurons to the autophagy inducer trehalose did not reduce BACE1 levels. This suggests that BACE1 is degraded by lysosomes but not by autophagy. Our results imply that BACE1 elevation in AD could be linked to decreased lysosomal degradation of BACE1 within dystrophic presynaptic terminals. Elevated BACE1 and APP levels in plaque-associated presynaptic dystrophies could increase local peri-plaque Aβ generation and accelerate amyloid plaque growth in AD.

Fig. 1

BACE1 is localized within presynaptic terminals at the light microscopic level. Representative images of coronal brain sections from 2- to 3-month-old wild-type mice co-stained with BACE1 (red) and synaptophysin or MAP2 (green) antibodies and imaged by laser scanning confocal microscopy. a–c At low magnification, BACE1 immunoreactivity is observed in the hilar region of the dentate gyrus (H), and in the infrapyramidal bundle (IPB) and stratum lucidum (SL) of the hippocampal mossy fiber pathway, where extensive co-labeling with synaptophysin also occurred, denoting presynaptic localization of BACE1 within these brain regions. d–f Higher magnification of BACE1 and synaptophysin immunoreactivity within the stratum lucidum of the wild-type mouse shown in a–c. BACE1 and synaptophysin signals significantly co-localize within mossy fiber terminals in f. Note the punctate BACE1 signals within neuronal soma (examples indicated by white arrowheads), which do not overlap with synaptophysin immunoreactivity and likely represent TGN and endosomes. g–l BACE1 immunoreactivity does not overlap with that of the somatodendritic marker MAP2 (green) within the stratum lucidum in CA3 (g–i) or the stratum radiatum in CA1 (j–l). Scale bar a–c, 200 μm; d–l, 25 μm

Fig. 2

BACE1^−/− mossy fiber terminals have normal ultrastructure. Serial ultrathin sections of the stratum lucidum from 5-month-old BACE1^+/+ (a–c) and 11-month-old BACE1^−/− (d–f) mice were imaged by electron microscopy. Shown are representative single presynaptic mossy fiber terminals (MFTs) contacting postsynaptic thorny excrescences (TE) with clearly recognizable synapses (bands of electron-dense material). Note the compact pool of synaptic vesicles distributed throughout the terminals. A high abundance of small, clear presynaptic vesicles can be resolved, as well as a lower abundance of large clear vesicles (red arrowheads) and dense-core vesicles (blue arrows). Mitochondria (m) of various shapes and sizes are located near the plasma membranes of the MFTs. No morphological differences in MFTs are evident between BACE1^+/+ and BACE1^−/− mice. Scale bars 200 nm

Fig. 3

BACE1 is localized within mossy fiber terminals at the electron microscopic level. Coronal brain sections from 4- to 14-month-old BACE1^+/+ (a–m) and BACE1^−/− (n–p) mice were processed for BACE1 pre-embedding silver-intensified ultrasmall immunogold and then serial ultrathin sections of the stratum lucidum were imaged by electron microscopy. a–p Representative serial images of single mossy fiber terminals. BACE1 immunoreactivity is clearly enriched within presynaptic terminals, although there is heterogeneity in the abundance of gold particles within a given terminal as well as distance from synapses (bands of electron-dense material). In some cases, BACE1 immunogold particles are located in close proximity to an active zone (f, g red arrowheads). Postsynaptic regions (dendrites and thorny excrescences, shaded yellow) contain little to no BACE1 immunoreactivity. n–p BACE1 immunogold particles are absent from presynaptic terminals in BACE1^−/− mice, although rare background particles (in this case, postsynaptic) are present. Scale bars 200 nm

Fig. 4

BACE1 accumulates presynaptically around amyloid plaques in 5XFAD transgenic mouse and human AD brains. Representative images of sections from 5XFAD transgenic mouse (~6 months old) or human AD brains co-stained with BACE1 (red) and synaptophysin or MAP2 (green) antibodies and imaged by laser scanning confocal microscopy. a–c At low magnification, in addition to normal BACE1 localization in hippocampal mossy fibers, BACE1 immunoreactivity (red) displays a plaque-like staining pattern that overlaps with synaptophysin (green) immunoreactivity in the 5XFAD brain (white arrowheads indicate representative plaque). d–f Higher magnification images showing extensive co-localization of BACE1 and synaptophysin around amyloid plaques (white asterisks) in 5XFAD brain, demonstrating accumulation of BACE1 in abnormal presynaptic structures, likely dystrophic terminals (white arrowheads indicate representative dystrophies with BACE1-synaptophysin co-localization). g–i No overlap between BACE1 and the somatodendritic marker, MAP2 (green), is apparent in dystrophic structures surrounding plaque cores stained with thioflavin S (blue). Superior temporal gyrus or entorhinal cortex brain sections of Braak stage V–VI human AD brains were co-stained with antibodies against BACE1 (red) and synaptophysin (j–l) or MAP2 (m–o) (green). BACE1 immunoreactivity overlaps with that of synaptophysin (j–l) but not MAP2 (m–o) within some of the abnormal structures near plaques in human AD (e.g., white arrowheads). Although immunostaining was somewhat weaker in AD compared to 5XFAD brain sections, both exhibited qualitatively similar patterns of presynaptic BACE1 accumulation around plaques. Scale bars a–c, 200 μm; d–f, j–o, 20 μm; g–i, 25 μm

Fig. 5

BACE1 co-localizes with APP and neurofilament, but not tubulin, around plaques in the 5XFAD brain. Coronal brain sections from ~6-month-old 5XFAD mice were co-stained with antibodies against BACE1 and either APP, β-tubulin, α-tubulin, or medium neurofilament (NFT160) and imaged by laser scanning confocal microscopy. a–c BACE1 immunoreactivity (red) exhibits variable but significant overlap with that of APP (green) within dystrophies around thioflavin S-positive plaques (blue), suggesting that APP could be processed by BACE1 in these abnormal structures. In contrast, BACE1 and β-tubulin (d–f) and α-tubulin (g–i) immunoreactivities display largely non-overlapping patterns around plaques. e, h Note that irregular spherical accumulations of tubulin are observed nearby plaques (arrowheads). j Sequential z axis images of BACE1 (white) and medium neurofilament (red) immunoreactivities were taken and subsequently stacked in Nikon Elements with alpha-blending. BACE1 co-localizes with neurofilament in a dystrophic neurite (dotted white line). Importantly, the process of the neurite, immunolabeled for neurofilament, ends in a dystrophy, likely an axon terminal. Scale bars a–i, 20 μm; Dimensions for box in j: width, 181.41 μm; height, 181.41 μm; depth, 22.80 μm

Fig. 6

Swollen dystrophic neurites form near amyloid plaques in the 5XFAD brain. a Low magnification electron microscope image of a plaque (P) surrounded by dystrophic neurites (dotted black lines) in the stratum lucidum of the CA3 hippocampal subregion of a 14-month-old 5XFAD mouse. Note that many of the dystrophic neurites are filled with vesicles that contain electron-dense material. Neighboring dendrites (d), preterminal mossy fiber bundles (PTB), and presynaptic terminals (white asterisks) appear normal. b, c High magnification electron microscope serial images of a single normal appearing mossy fiber terminal from the 5XFAD mouse. The vesicle-rich presynaptic terminal makes synaptic contact with a thorny excrescence (TE) and a dendrite (d) and contains an abundance of small clear vesicles, as well as some large clear vesicles (red arrowheads) and dense-core vesicles (blue arrows). Mitochondria (m) align near the plasma membrane of the terminal. Scale bars a, 1 μm; b–c, 200 nm

Fig. 7

BACE1 is localized within an electron-translucent subtype of plaque-associated dystrophic neurite. Coronal brain sections from 4- to 14-month-old 5XFAD mice were processed for BACE1 pre-embedding silver-intensified ultrasmall immunogold and then serial ultrathin sections of the stratum lucidum and cortex were imaged by electron microscopy. a Low magnification image of an amyloid plaque (P) surrounded by dystrophic neurites in the hippocampal CA3 subregion of the 5XFAD brain. b Higher magnification of boxed region in a showing BACE1-positive and BACE1-negative dystrophic neurites. Two types of dystrophic neurites are apparent, which we refer to as Type I (red dotted line) and Type II (black dotted lines). The different subtypes are distinguishable by the amount of electron density (Type I, less; Type II, more) and the size of vesicular structures (Type I, smaller vesicles; Type II, larger vesicles). Note that BACE1 immunogold labeling is more prominent in Type I dystrophic neurites (red dotted lines). c–e Representative images of serial ultrathin sections of BACE1 immunogold-labeled cortical neuritic dystrophies from the 5XFAD brain, with Type I dystrophic neurites predominantly enriched with BACE1 immunogold particles (red dotted lines) adjacent to Type II dystrophic neurites (black dotted lines). f–h Representative serial images of a BACE1-immunopositive Type I dystrophic neurite (red dotted lines) adjacent to an aberrant dendrite (d) and Type II dystrophic neurite (black dotted lines) within CA3. Note the Type II dystrophic neurite contains very few BACE1 immunogold particles. Close examination of the aberrant dendrite (d) reveals abnormal microtubule organization exhibiting a swirling pattern. Scale bars a, 2 μm; b, 1 μm; c–e, 200 nm; f–h, 1 μm

Fig. 8

Type I dystrophic neurites are immunoreactive for the presynaptic marker synaptophysin, but not β-tubulin. Coronal brain sections from ~6-month-old 5XFAD mice were processed for pre-embedding silver-intensified ultrasmall immunogold for synaptophysin (a–d) or β-tubulin (e–n) and then serial ultrathin sections of the stratum lucidum were imaged by electron microscopy. a–c Representative serial images of a single normal mossy fiber terminal exhibiting synaptophysin immunogold particle labeling. Note that synaptophysin immunoreactivity is absent in the adjacent postsynaptic dendrite (yellow shading). d Representative image of Type I dystrophic neurites (black dotted lines) that are labeled with synaptophysin immunogold particles, indicating that they are presynaptic structures. e–j Images of serial ultrathin sections labeled with β-tubulin immunogold particles. Note that dendrites (yellow shading) are positive for β-tubulin, while the dystrophic neurite (black dotted lines) lacks β-tubulin immunoreactivity. k–n Other serial images of a representative β-tubulin immunogold labeled dendrite (yellow shading). β-Tubulin immunoreactivity is absent from surrounding mossy fiber terminals. Scale bars 500 nm

Fig. 9

BACE1 co-localizes with the endosomal marker transferrin receptor, but not with the lysosomal marker LAMP1, in dystrophic neurites. Representative images of brain sections from ~6-month-old 5XFAD mice co-stained with BACE1 (red) and transferrin receptor (TfR) or LAMP1 (green) antibodies and imaged by laser scanning confocal microscopy. a–c BACE1 exhibits significant co-localization with TfR in endosomes (e.g., white arrowheads) within dystrophic neurites surrounding an amyloid plaque (white asterisk). d–f In contrast, BACE1 immunoreactivity occurs in a largely non-overlapping pattern with that of the late endosome/early lysosome marker, LAMP1, in dystrophic neurites that surround an amyloid plaque (white asterisk). Note that dystrophic neurites immunopositive for LAMP1 typically display minimal BACE1 immunoreactivity, and vice versa. The reason for this is unknown, but could occur if BACE1 undergoes degradation in lysosomes, thus destroying the BACE1 epitope. Blue in c, f indicates DAPI stain for nuclei and plaque cores. Scale bar 20 μm

Fig. 10

BACE1 is degraded by lysosomes, but not by autophagy. a–f The autophagy marker LC3B-II is elevated in the 5XFAD brain. a Homogenates of individual hippocampi from ~6-month-old 5XFAD (+) and non-transgenic (−) mice were analyzed by immunoblot for BACE1 and LC3B. Note the increased intensity of the LC3B-II band in 5XFAD compared to non-transgenic hippocampi. β-Tubulin was used as a loading control. b Densitometric analysis of LC3B-II signal intensity in a, normalized to that of LC3B-I, shows ~700 % increase in the LC3B-II:LC3B-I ratio in 5XFAD hippocampi compared to non-transgenic controls. c Densitometric analysis of BACE1 signal intensity in a, normalized to that of β-tubulin, shows ~50 % increase in BACE1 levels in 5XFAD hippocampi compared to non-transgenic controls (**p < 0.01, t test, n = 5 for b, c). d–f Representative images of brain sections from ~6-month-old 5XFAD mice co-stained with BACE1 (red) and LC3B (green) antibodies and imaged by laser scanning confocal microscopy. BACE1 and LC3B immunoreactivities largely occur in non-overlapping patterns in dystrophic neurites surrounding amyloid plaques (white asterisks). Note that dystrophic neurites immunopositive for LC3B typically display minimal BACE1 immunoreactivity, and vice versa. g Primary cortical neuron cultures from wild-type E15.5–16.5 mouse embryos were treated with either vehicle, 20 μM leupeptin (a lysosomal protease inhibitor), 150 mM trehalose (an inducer of autophagy), or leupeptin plus trehalose for 24 or 48 h, and then analyzed for BACE1, LC3B-I and LC3B-II levels by immunoblot. Ponceau staining was used as a loading control. h Densitometric analysis of the ratio of LC3B-II to LC3B-I levels in g shows a dramatic increase in LC3B-II:LC3B-I with trehalose and leupeptin plus trehalose treatment, demonstrating that trehalose is a strong inducer of autophagy in neurons. In contrast, leupeptin alone causes only a relatively modest elevation of neuronal LC3B-II:LC3B-I ratio. i Densitometric analysis of BACE1 levels in g, normalized to ponceau, shows a marked increase of BACE1 levels with leupeptin treatment in neurons, while trehalose does not decrease BACE1 levels. These results suggest that BACE1 is not cleared by autophagy but instead is likely degraded in the lysosomal pathway in neurons, at least in culture (*p < 0.05, **p < 0.01, ***p < 0.001 ANOVA, n = 3 for h, i). Scale bar d–f, 20 μm