The Alzheimer’s β-secretase BACE1 localizes to normal presynaptic terminals and to dystrophic presynaptic terminals surrounding amyloid plaques
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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.
KeywordsAlzheimer’s disease BACE1 β-Secretase Aβ Amyloid plaque Dystrophic neurite Lysosome Autophagy
Alzheimer’s disease (AD) is characterized by the presence of neurofibrillary tangles and cerebral amyloid plaques composed of the 40–42 amino acid β-amyloid peptide (Aβ; reviewed in Sisodia and Hyslop ). Much evidence implicates Aβ in the pathogenesis of AD (reviewed in ). Aβ is generated from the sequential proteolysis of amyloid precursor protein (APP) by the enzymes β-secretase and γ-secretase (reviewed in [21, 122]). β-Secretase initiates the cleavage of APP and has been identified as the transmembrane aspartic protease beta-site APP cleaving enzyme 1 (BACE1) [40, 64, 96, 106, 118]. As the initiator enzyme of the amyloidogenic pathway, BACE1 is a prime therapeutic target for reducing cerebral Aβ levels, and several BACE1 inhibitor drug candidates are currently in clinical trials for AD.
Elucidating the physiological functions of BACE1 is essential for predicting potential mechanism-based toxicities associated with BACE1 inhibition as a therapeutic approach for AD. BACE1-null mice display complex neurological phenotypes, including growth retardation , memory deficits [57, 77, 78], hypomyelination [37, 113], seizures [33, 39, 50], axon guidance defects [13, 32, 85], and schizophrenia-like behaviors . These BACE1−/− phenotypes likely reflect the functions of a diverse array of BACE1 substrates that include Golgi-localized membrane-bound α2,6-sialyltransferase , P-selectin glycoprotein ligand-1 , APP and the APP homolog proteins APLP1 and APLP2 [25, 61, 79], low density lipoprotein receptor-related protein , the voltage-gated sodium channel β2 subunit (Navβ2) [46, 47, 114], neuregulin-1 (NRG1) [37, 113], neuregulin-3 (NRG3) , and Close Homolog of L1 (CHL1) [32, 55, 123], among others [55, 123]. Additional as yet unknown BACE1 substrates are likely to exist and their identification will provide further insight into the biological functions of BACE1.
Understanding the subcellular localization of BACE1 may provide important clues as to the identities of key BACE1 substrates and the physiological functions of substrate processing by BACE1, especially in neurons of the brain where therapeutic inhibition of BACE1 will be targeted. Previously, using immunohistochemistry with a mono-specific anti-BACE1 antibody, we reported that BACE1 accumulates in swollen presynaptic neuronal structures that surround amyloid plaques in AD and APP transgenic brains . In that and another study, we also observed that BACE1 levels in the normal mouse brain were highest in axon terminal fields, especially within presynaptic terminals of the hippocampal mossy fiber pathway in CA3 [32, 121]. These results suggest that BACE1 has normal and aberrant presynaptic roles in non-demented and Alzheimer disease brains, respectively.
To further characterize the localization of BACE1 at both the light and electron microscopic levels and thereby infer potential normal and abnormal activities of BACE1 in the brain, we performed immunofluorescence confocal microscopy and immunogold electron microscopy (EM) of mossy fiber terminal regions in the hippocampal CA3 subregion in non-transgenic and APP transgenic (5XFAD) mouse brains. The high level of BACE1 in the mossy fiber rendered it an excellent model to investigate the subcellular localization of BACE1 in vivo. Our findings provide the first unequivocal demonstration that BACE1 localizes to vesicles in large presynaptic mossy fiber terminals within CA3 in both normal and AD model mouse brains. In some cases, BACE1-immunopositive vesicles were located near active zones, implying an important but as yet undetermined function of BACE1 at the synapse. Moreover, BACE1 was also enriched within a distinct subtype of dystrophic presynaptic neurite that surrounds the amyloid plaque in the APP transgenic brain. These BACE1-positive presynaptic dystrophies tended to contain fewer large electron-dense multilamellar autophagosomes. This was true for neuritic dystrophies observed within the hippocampus as well as in the cerebral cortex. Importantly, we observed that accumulations of BACE1 and APP co-localized in presynaptic dystrophies, implying increased BACE1 processing of APP in these abnormal peri-plaque regions. In addition, BACE1 partially co-localized with transferrin receptor, suggesting that BACE1 accumulates in endosomes of dystrophic terminals. In primary cortical neuron cultures, treatment with the lysosomal protease inhibitor leupeptin resulted in increased BACE1 levels; however, exposure of neurons to the autophagy inducer trehalose did not reduce BACE1 levels, suggesting that BACE1 is degraded in the lysosomal, but not the autophagic, pathway. Though the precise mechanism of BACE1 accumulation within aberrant presynaptic dystrophies in the APP transgenic brain is currently unknown, our data as well as other published reports imply a link to decreased lysosomal degradation of BACE1 within dystrophic axon terminals. An increase in BACE1 level in plaque-associated dystrophic presynaptic terminals, in conjunction with APP accumulation in these neuritic dystrophies [19, 20], may elevate local peri-plaque Aβ generation and exacerbate the progression of amyloid pathology in AD.
Materials and methods
5XFAD mice have been described previously  and were maintained on a B6/SJL F1 hybrid background. BACE1−/− mice on a C57/B6 background were obtained from Jackson Laboratories (strain #006554). Non-transgenic age-matched B6/SJL or C57/B6 mice were used as controls. Procedures were performed with Northwestern University IACUC approval.
Tissue collection for immunoblots and confocal microscopy
Mice were perfused with cold phosphate buffered saline (PBS) containing protease and phosphatase inhibitors [20 μg/ml phenylmethylsulfonyl fluoride (PMSF, Sigma), 500 ng/ml leupeptin (MP Biomedicals), 20 μM sodium orthovanadate (MP Biomedicals), 10 μM dithiothreitol (DTT, Sigma)]. A hemibrain from each mouse was dissected into hippocampus and cortex and snap-frozen separately for biochemical analyses; the other hemibrain was drop-fixed in 4 % paraformaldehyde overnight at 4 °C and cryopreserved in 30 % (w/v) sucrose/PBS at 4 °C for histology. Fixed cryopreserved human Braak stage V–VI brain tissues (entorhinal cortex and superior temporal gyrus) from three AD cases were obtained from the Cognitive Neurology and AD Center at Northwestern University.
5XFAD and non-transgenic (5 each) hippocampi were individually homogenized in 1 % Triton X-100/PBS with 1× protease inhibitor (Calbiochem) and 1× Halt Phosphatase Inhibitor cocktail (Thermo Scientific). Protein concentration was quantified by BCA (Thermo Scientific). Hippocampal homogenates (20 μg) and primary neuron lysates (10 μg) were separated by 12 % Tris–Glycine or 4–12 % Bis–Tris SDS-PAGE and transferred onto 0.45 μm PVDF membranes (Millipore) that were subsequently Ponceau stained, scanned, and probed with the following antibodies recognizing BACE1 (1:1000; BACE–Cat1 ), LC3B (1:1,000 or 1:4,000; Cell Signaling #3868), and β-III-tubulin (TuJ1) (1:10,000; gift of Dr. Lester Binder). Membranes were then washed with TBST and incubated with the appropriate HRP-conjugated secondary antibodies (1:10,000; Vector Laboratories), washed again and visualized using Luminata Crescendo (Millipore). Signals were quantified using a Kodak Image Station 4000R. Signal intensities were normalized to tubulin or ponceau staining as indicated.
Mouse immunofluorescence confocal microscopy
Free-floating hemibrain coronal sections (30 μm) from 5XFAD and non-transgenic mice (2–3 each) were cut on a freezing microtome, washed in TBS, and blocked in 5 % goat or donkey serum. Sections were incubated at 4 °C overnight on a shaker with the following primary antibodies: mouse monoclonal anti-BACE1 (1:250; BACE–Cat1 ) or rabbit monoclonal anti-BACE1 (1:250; Epitomics #EPR3956), goat polyclonal anti-synaptophysin (1:250 mouse brain, 1:50 human brain; R&D Systems #AF5555), goat polyclonal anti-APP (1:500; Karen, gift of Dr. Virginia Lee), rat monoclonal anti-transferrin receptor (1:500; Abcam #ab60344), rat monoclonal anti-LAMP1 (1:500; Abcam #ab25245), mouse monoclonal anti-β-III-tubulin (TuJ1) (1:100; gift of Dr. Lester Binder), chicken polyclonal anti-microtubule-associated protein 2 (MAP2) (1:250; Abcam #ab5392), mouse monoclonal anti-neurofilament NFT160 (1:250; Sigma #N5264), rabbit monoclonal anti-LC3B (1:500; Cell Signaling #3868). Sections treated with Epitomics anti-BACE1 primary antibody were incubated for 2 h at 37 °C on a shaker. Following primary antibodies, sections were washed in TBS and incubated with Alexa Fluor secondary antibodies at 1:1,000 (donkey anti-mouse or rabbit-488 or 594; goat anti-mouse or rabbit-488 or 594) and DAPI (Invitrogen), washed, mounted on charged slides and cover-slipped using ProLong Gold (Invitrogen). Images were captured on Nikon (Tokyo, Japan) A1R or Zeiss LSM 510 laser scanning confocal microscopes.
Human immunofluorescence confocal microscopy
Human AD sections were processed using the same method as mouse, with the addition of antigen retrieval and autofluorescence reduction steps. Free-floating sections (40 μm) from 3 Braak stage V–VI AD brains were washed in TBS and incubated for 1 h in 16 mM glycine on a shaker. After TBS washes, sections underwent antigen retrieval using 0.1 M sodium citrate, pH 9.0, for 1.5 h at 80–90 °C, TBS washed, and incubated in KMnO4 until brown. Sections were washed with DI water, treated with 0.5 % oxalic acid and 0.5 % K2S2O5 to remove brown color, washed again, and incubated on a shaker for 30 min in 0.25 % NaBH4. Following a 0.25 % Triton/TBS wash, sections were blocked and incubated with primary antibodies as in the mouse immunofluorescence procedure.
Pre-embedding ultrasmall silver-intensified immunogold electron microscopy
Two to three adult (4–14 months) 5XFAD, non-transgenic (C57Bl6/SJL), and BACE1−/− mice were anesthetized with isoflurane (Isothesia, Butler), transcardially perfused with 0.9 % saline followed by 50 ml ice-cold acidic fixative (2 % paraformaldehyde, 1 % glutaraldehyde in 0.1 M sodium acetate, pH 6.0), then slowly perfused with ice-cold basic fixative (2 % paraformaldehyde, 1 % glutaraldehyde in 0.1 M sodium borate buffer, pH 9.0) for 1 h. Brains were removed, placed in ice-cold basic fixative on a shaker at 4 °C overnight. The following day brains were bisected, washed 3 × 20 min in TBS, and cut into 70 μm coronal sections on a vibratome. Sections were washed in TBS 5 × 5 min, treated with 1 % NaBH4 in TBS for 30 min, washed in TBS 5 × 1 min and incubated in blocking solution (10 % NGS in TBS) for 30 min followed by incubation in primary antibody overnight at 4 °C. Primary antibody [rabbit monoclonal anti-BACE1 (1:500; Cell Signaling #5606 or Epitomics #EPR3956); mouse monoclonal anti-β-III-tubulin (TuJ1) (1:100; gift from Dr. Lester Binder); rabbit polyclonal anti-synaptophysin (1:500; Millipore #AB9272)] was diluted in 2 % NGS + 0.1 or 0.5 % Triton X-100 in TBS. Sections were washed 1 × 5 min in incubation buffer and 10 × 5 min in TBS, then incubated in secondary blocking buffer [2 % NGS + 1 % BSA + 0.3 % cold water fish skin gelatin (CWFSG) in TBS] for 1 h followed by incubation in Ultra Small Immunogold (Aurion) anti-rabbit secondary antibody at 1:100 in 2 % NGS + 1 % BSA-C + 0.3 % CWFSG at 4 °C for ~40 h. Sections were washed in incubation buffer 1 × 5 min, TBS 6 × 10 min, PBS 2 × 5 min then fixed in 2 % glutaraldehyde in PBS for 1 h, followed by washes 2 × 5 min in PBS, 4 × 10 min in TBS, and 3 × 10 min in enhancement conditioning solution (ECS). Sections were then incubated in R-Gent SE-EM Plus enhancement mixture for 90 min and washed in ECS 4 × 10 min, TBS 2 × 10 min, and PBS 2 × 10 min, osmicated with 0.4 % OsO4 in PBS for 15 min and rinsed in PBS 3 × 10 min and dH2O 2 × 5 min. Sections were stained in 1 % aqueous uranyl acetate for 10 min, rinsed 3 × 10 min in dH2O, dehydrated in graded ethanol and propylene oxide, infiltrated with 1:1 araldite:propylene oxide overnight at room temperature, followed by flat embedding between aclar sheets and curing for 48 h at 60 °C. Regions of interest were subdissected and re-embedded in Araldite and cured overnight at 60 °C. 500-nm-thick histological sections were cut by ultramicrotome and stained with toluidine blue to confirm the presence of the stratum lucidum. Serial ultrathin sections (63 nm) were cut with a diamond knife, placed onto formvar-coated slotted grids, stained with uranyl acetate–lead citrate (for 15 and 10 min, respectively), washed in ultrapure dH2O, and allowed to dry at room temperature. Images were taken with a JEOL 1200EX electron microscope (JEOL Ltd., IL, USA) at a magnification of 7500–20,000×. Electron micrographs from 8 to 30 serial sections containing mossy fiber terminals or dystrophic neurites were obtained between 1 and 8 microns from the tissue surface (i.e., the surface of the 70-μm section that was immunogold-labeled) . EM reagents were from Electron Microscopy Sciences (Hatfield, PA) unless otherwise noted.
Conventional electron microscopy
One adult 5XFAD, C57/Bl6, and BACE1−/− mouse was anesthetized with isofluorane (Isothesia, Butler), perfused transcardially with 0.12 M PBS (pH 7.4) for 1 min, then a dilute aldehyde mixture (1 % paraformaldehyde, 1.25 % glutaraldehyde, 0.02 mM CaCl2 in 0.1 M sodium cacodylate buffer) for 30 min, and a concentrated aldehyde mixture (2 % paraformaldehyde, 2.5 % glutaraldehyde, 0.04 mM CaCl2 in 0.1 M sodium cacodylate buffer) for 10 min. Brains were removed and placed in ice-cold concentrated fixative on a shaker at 4 °C overnight. The following day brains were bisected, rinsed 3 × 20 min in 0.12 M TBS and cut into 70 μm coronal sections as above. Sections were washed in 0.12 M phosphate buffer (PB) 3 × 10 min at 4 °C, treated with 2 % OsO4 in 0.12 M PB for 1 h at 4 °C, and washed 3 × 10 min in 0.12 M PB. The tissue was then dehydrated in graded ethanols and propylene oxide, infiltrated with 1:1 araldite:propylene oxide, flat embedded between aclar sheets and cured for 48 h at 60 °C. Regions of interest were subdissected and re-embedded as above. Serial ultrathin sections (65 nm) were cut, placed onto formvar-coated slotted grids, then stained with uranyl acetate–lead citrate (15 and 10 min, respectively) and rinsed in ultrapure dH2O. Images (7500–20,000×) were taken with a JEOL 1200EX electron microscope (JEOL Ltd., IL, USA) from 10–30 serial sections.
Primary neuron culture
Cortical neurons were isolated from day 15.5–16.5 C57/B6 mouse embryos via dissociation at 37 °C in 0.25 % trypsin. Neurons were plated at a density of 750,000 cells per well in poly-l-lysine coated 12-well plates containing neurobasal media supplemented with 2 % B-27, 500 μM glutamine, 10 % horse serum and 2.5 μM glutamate. After 2–3 h, the media was replaced with neurobasal media with 2 % B-27, 500 μM glutamine, and 2.5 μM glutamate. After 3 DIV, media was replaced with neurobasal media with 2 % B-27 and 500 μM glutamine. All cell culture reagents were from Invitrogen. After 6 DIV, neurons were exposed to 20 μM leupeptin (MP Biomedicals), 150 mM trehalose (Sigma), or both for 24 or 48 h. In a separate experiment, neurons were treated with 100 nM bafilomycin (Sigma) for 4 h. Neurons were lysed in RIPA buffer (150 mM NaCl, 1 % IGEPAL CA-630, 0.5 % sodium deoxycholate, 0.1 % SDS, 50 mM Tris pH 8, 1 mM PMSF) with 1× protease inhibitors (Calbiochem) and 1× Halt Phosphatase Inhibitor Cocktail (Thermo Scientific). Lysates were centrifuged at 10,000 rpm, 4 °C, 10 min and the supernatant protein was quantified by BCA (Thermo Scientific).
Densitometric analyses of immunoblots were performed using Kodak 1D 3.6 image analysis software. Statistical differences for immunoblot experiments were determined using two-tailed students t test or ANOVA (GraphPad Software, Inc., San Diego, CA). Graphed data are presented as the mean ± SEM, and p < 0.05 was considered significant.
BACE1 is localized in presynaptic terminals of normal brain at the ultrastructural level
Our initial attempts at defining BACE1 localization by immuno-EM proved futile, as our BACE–Cat1 antibody was not amenable to the fixation protocol used to preserve tissue for electron microscopic analysis. However, with the recent development of superior commercial anti-BACE1 antibodies, we were able to identify—with ultrastructural resolution—the precise subcellular location of BACE1 within the murine brain. To accomplish this, we incubated coronal sections of mouse hippocampus with a rabbit monoclonal anti-BACE1 antibody and goat anti-rabbit IgG conjugated to ultrasmall gold particles followed by silver enhancement, as previously described [60, 111]. As expected [57, 121], immunogold particles for BACE1 were concentrated within the hilar region of the dentate gyrus, the infrapyramidal bundle, and stratum lucidum of CA3, an exact match to the previously observed BACE1 immunofluorescence labeling pattern (Suppl. Fig. 1a, c). As a negative control to demonstrate antibody specificity, hippocampal sections from BACE1−/− mice treated with the anti-BACE1 antibody lacked BACE1 immunoreactivity using either the immunofluorescence or immunogold staining protocol (Suppl. Fig. 1b, d).
BACE1 is localized within endocytic vesicles of dystrophic presynaptic terminals in the APP transgenic brain at the ultrastructural level
Previous studies in both rodents and in humans have shown that many of the structures encircling plaques are dystrophic presynaptic terminals [9, 65, 66, 89, 121], though distended postsynaptic elements may also appear nearby plaques . We have previously shown that many BACE1-positive dystrophies are engorged presynaptic structures in the AD and 5XFAD brain . Using immunofluorescence confocal microscopy, we likewise demonstrated that BACE1 immunoreactivity overlapped that of synaptophysin within many of the neuritic dystrophies adjacent to amyloid plaques (Fig. 4d–f). Conversely, BACE1-positive structures did not co-localize with the somatodendritic marker MAP2 (Fig. 4g–i) . To determine the extent to which this pattern of BACE1 localization in 5XFAD brain recapitulated that in human AD brain, we performed BACE1 and synaptophysin (Fig. 4j–l) or MAP2 (Fig. 4m–o) co-staining of AD hippocampal sections followed by immunofluorescence confocal microscopy. Similar to the 5XFAD brain, BACE1 and synaptophysin displayed significant co-localization surrounding amyloid plaques, while MAP2 did not overlap with BACE1 in the AD brain. These results support our previous work  and confirm that 5XFAD mice are faithful models of amyloid-associated BACE1 elevation in AD.
To further characterize the pattern of BACE1 accumulation surrounding amyloid plaques, we co-stained 5XFAD brain sections with anti-BACE1 and antibodies that recognize either neuron-specific class III β-tubulin (Fig. 5d–f) or α-tubulin (Fig. 5g–i), which comprise microtubules. Interestingly, BACE1 showed minimal co-localization with β-tubulin or α-tubulin, suggesting that regions of BACE1 accumulation had reduced levels of microtubules. Moreover, a significant proportion of α- and β-tubulin staining around plaques was concentrated in aberrant spherical, oblate, or ring-like structures (Fig. 5e, h, arrowheads); such structures are unlikely to contain normal bundles of functional microtubules. Given the evidence of abnormal cytoskeletal elements in peri-plaque regions , we additionally co-labeled 5XFAD brain sections with antibodies against neurofilament (NFT), a major axonal cytoskeleton component, and BACE1. A 3-dimensional reconstruction of an immunofluorescence confocal microscopy z-series showed extensive labeling of axons coursing through brain tissue, with some axons ending as engorged bulbous structures that also contained BACE1 immunoreactivity (Fig. 5j). Thus far, our data indicated that (1) BACE1 localizes to normal mossy fiber terminals of hippocampal region CA3; (2) BACE1 accumulates in dystrophic presynaptic axon terminals surrounding amyloid plaques in the AD and 5XFAD brain; (3) little if any BACE1 localizes to postsynaptic structures, and (4) aberrant microtubule structures are present in regions that lack BACE1.
Upon further inspection of BACE1 immunogold-labeled 5XFAD sections, ultrastructurally there appeared to be two subtypes of dystrophic neurites that we will refer to as Type I and Type II (Fig. 7). Type I dystrophies contained numerous small round, oval, or irregularly shaped membraneous structures densely packed into an enlarged membrane-bound neurite. Type II dystrophies were distended membrane-bound neurites filled with large vacuolar structures of varying morphologies, many of which resembled autophagosomes with electron-dense centers that were encircled by at least a single or multiple concentric membranes. The amount of electron-dense material varied from one vacuolar structure to another, and in general Type II neurites appeared more electron dense than Type I neurites. The two neuritic subtypes surrounded amyloid plaques in both hippocampus and cortex of the 5XFAD brain. Mitochondria were also present in both subtypes. Although 5XFAD dystrophic neurites generally could be classified as either Type I or Type II, we observed that some dystrophies contained variable mixtures of vesicular structures found in both subtypes, suggesting that a continuum of neuritic morphologies might exist between Type I and Type II neurites. Interestingly, the smaller, less electron-dense vesicles of the Type I neurites were often immunoreactive for BACE1, whereas the larger, more electron-dense vesicles of the Type II neurites exhibited considerably less BACE1 immunogold labeling (Fig. 7). Overall, these findings support our immunofluorescence confocal microscopy results showing variable, non-uniform BACE1 immunosignal intensity in different dystrophic neurites surrounding plaques in the AD and 5XFAD brain (Fig. 4).
BACE1 is degraded by lysosomes, not by autophagy
A major goal of our study was to gain insight into the mechanism of BACE1 elevation in AD. Given our results that both autophagy and BACE1 were increased in the 5XFAD brain, we tested the hypothesis that BACE1 is a substrate of autophagy; if so, BACE1 elevation could arise from failure of autophagy to clear BACE1. To determine whether BACE1 is degraded by autophagy, we treated primary murine cortical neurons with two inducers of autophagy, rapamycin  and trehalose , and the lysosomal protease inhibitor leupeptin, followed by immunoblot analysis for BACE1 and LC3B. Leupeptin was used to test the alternative hypothesis that BACE1 is degraded by lysosomes. While rapamycin failed to induce autophagy in primary neurons (data not shown), as reported previously , trehalose treatment for either 24 or 48 h dramatically increased the LC3B-II:LC3B-I ratio (~7-fold by 48 h) compared to vehicle (Fig. 10g–h), an increase similar to that observed in the 5XFAD brain (Fig. 10a, b). Although leupeptin treatment also increased LC3B-II:LC3B-I ratio, supporting previous work that inhibition of lysosomal function results in decreased clearance of autophagosomes [48, 53, 101], it did so to a much lesser extent than trehalose (Fig. 10g–h). Combined leupeptin plus trehalose treatment did not markedly increase LC3B-II:LC3B-I ratio beyond that of trehalose alone. Importantly, immunoblot analysis revealed that, compared to vehicle, trehalose treatment did not reduce BACE1 levels in primary neurons (Fig. 10g, i), suggesting that BACE1 is not degraded in the autophagic pathway. Interestingly, BACE1 levels in primary neurons treated with leupeptin were increased to over 200 % of vehicle by 48 h (Fig. 10g, i), a BACE1 elevation similar in magnitude to that observed in the 5XFAD (Fig. 10a, c) and the AD brain. A similar increase in BACE1 level was obtained by treating neurons with bafilomycin, an inhibitor of endosome/lysosome acidification (data not shown). These results are consistent with previous reports showing that BACE1 is trafficked to the lysosome for degradation [51, 103]. Combined leupeptin plus trehalose treatment produced a small but significant increase of BACE1 level by 48 h compared to trehalose alone (Fig. 10g, i). Taken together, our findings support the conclusion that BACE1 is not cleared by autophagy, but instead is degraded in lysosomes, at least in primary cortical neurons in culture. Further, these cell culture data suggest that decreased lysosomal function could lead to both elevations of BACE1 and autophagy in AD.
BACE1 in the normal presynaptic terminal
BACE1 is required for Aβ generation. Thus, it is a promising AD therapeutic target (reviewed in [44, 107]). However, the numerous BACE1 substrates [30, 55, 123] and complex phenotypes of BACE1 null mice suggest that the inhibition of BACE1 for AD may not be free of mechanism-based toxicity. Thus, knowledge of BACE1 physiological functions is necessary to predict and potentially avert side effects of BACE1 inhibitor drugs.
One poorly understood question is the role of BACE1 in the brain, the target organ of BACE1 inhibitors. Although BACE1−/− mice indicate that BACE1 is involved in memory [57, 77, 78], myelination [37, 113], seizure [33, 39, 50], axon guidance [13, 32, 85], emotions , schizophrenia  and vision , the mechanisms of these BACE1 null neurological phenotypes are not fully understood. The subcellular localization of BACE1 in the brain may provide important clues as to the roles of BACE1 in the CNS and the molecular and cellular bases of BACE1 functions.
Thus, we have investigated BACE1 cerebral localization at both light and electron microscopic levels. To our knowledge, this is the first study to determine the subcellular localization of endogenous BACE1 in neurons of the brain. Our previous work suggested that BACE1 is concentrated in presynaptic terminals, especially in mossy fibers of the stratum lucidum in hippocampal CA3 . Using mono-specific anti-BACE1 antibodies, we performed immunofluorescence confocal microscopy and determined that BACE1 is highly localized within synaptophysin-positive puncta in large mossy fiber terminals (giant boutons) of CA3. Little if any BACE1 is localized to MAP2-positive somatodendritic postsynaptic sites in the hippocampus, although BACE1-positive puncta are found in neuronal soma (TGN, endosomes).
Immuno-EM revealed that BACE1 is localized to vesicles within mossy fiber terminals. BACE1-positive vesicles were located near synaptic active zones, although this was not always the case. The specific localization of BACE1 to membranous vesicular structures within presynaptic terminals suggests an important but as yet undetermined function of BACE1 substrate processing at the synapse. As with our immunofluorescence confocal microscopy, only background BACE1 immunogold was observed postsynaptically. Taken as a whole, our results demonstrate that the presynaptic terminal is the principal site of BACE1 localization in the brain.
The function of BACE1 in the presynaptic terminal is currently unknown. In tissue culture cells, overexpressed BACE1 mainly resides in acidic compartments (TGN, endosomes) where BACE1 substrate cleavage occurs. Endosomes within presynaptic terminals have been reported [42, 87, 110] and although they have not been extensively studied, evidence suggests that presynaptic endosomes are involved in neurotransmitter vesicle recycling [10, 28, 35, 82, 86]. Further investigation of presynaptic endosomes may reveal the role of BACE1 within this compartment, which may reflect the need for proteolytic processing of one or more BACE1 substrates at or near the synapse. Three BACE1 substrates that may be cleaved in the presynaptic terminal are Navβ2, CHL1, and neuregulin, which play a role in axon depolarization , neurite outgrowth/axon guidance  and myelination [37, 113], respectively. Presumably, BACE1-cleaved Navβ2 and neuregulin fragments would be trafficked from the presynaptic terminal to the axon via retrograde transport to exert their effects. However, BACE1 presynaptic localization argues that a different substrate(s) is processed by BACE1 in the terminal where it may perform a function required specifically at that location.
One such BACE1 substrate is APP, which has been hypothesized to play a role in neuroprotection , cell adhesion [99, 117], neurite outgrowth (reviewed in ), synapse formation or maintenance , as well as regulating synaptic transmission . APP is transported to the neuronal terminal [52, 97] where it is likely processed by BACE1 and γ-secretase to generate secreted APP ectodomain (APPsβ), APP C-terminal fragment (β-CTF), and Aβ [40, 64, 96, 106, 118]. It is possible that APPsβ released at the terminal may function to protect or maintain synaptic contacts. An intriguing alternative hypothesis is that Aβ itself may be involved in synaptic function or neurotransmission. Neuronal stimulation causes secretion of Aβ at the terminal , a process that requires endocytosis . Other studies suggest that Aβ may regulate glutamatergic synaptic transmission  and facilitate LTP [83, 84, 115, 116] at low endogenous Aβ concentrations normally released at the terminal. Thus, BACE1 in the presynaptic terminal may serve to process APP into Aβ for the latter’s presumptive role in synaptic function.
Another interesting BACE1 substrate that may function at the presynaptic terminal is CHL1 [32, 55, 123]. Importantly, BACE1−/− axon guidance defects in the hippocampus and olfactory bulb phenocopy axon targeting errors observed in CHL1−/− mice [31, 32, 68]. Moreover, CHL1 is processed by BACE1, and CHL1 and BACE1 co-localize in primary neuron growth cones and in presynaptic terminals in hippocampus and olfactory bulb , suggesting that BACE1 cleavage of CHL1 is necessary for proper axon guidance. The action of other BACE1 substrates at the terminal could also explain BACE1 presynaptic localization. Numerous BACE1 substrates have been identified [30, 55, 123] and others will likely be discovered in the future. Additional studies will be necessary to validate putative BACE1 substrates in vivo and thereby clarify the role of BACE1 in the presynaptic terminal.
BACE1 in the dystrophic presynaptic terminal surrounding the amyloid plaque
Several studies report that BACE1 levels are elevated in AD brains [27, 34, 62, 121]. These results raise two intriguing questions: (1) Does BACE1 elevation exacerbate AD pathogenesis? (2) What mechanism is responsible for BACE1 elevation? These questions have potential implications for AD mechanisms and novel therapeutics. To gain insight into the second question, we performed immunofluorescence confocal and immunogold electron microscopy to determine where cerebral BACE1 accumulates in the 5XFAD transgenic mouse model of amyloid pathology. BACE1 immunofluorescence confocal microscopy and immuno-EM of 5XFAD brain sections showed endogenous BACE1 localization within normal CA3 mossy fiber presynaptic terminals with occasional BACE1 labeling near active zones. In addition, BACE1 accumulates within presynaptic dystrophies that surround amyloid plaques within the hippocampus and cortex. Importantly, APP exhibited a high degree of co-localization with BACE1 in 5XFAD presynaptic dystrophic neurites, suggesting the intriguing possibility that BACE1 processing of APP might occur in these dystrophies to exacerbate Aβ generation and plaque formation. APP has been reported to localize within dystrophic neurites of AD brain [7, 8, 19, 22, 89]. In addition, we have shown here and in a previous study  that BACE1 co-localizes with synaptophysin, but not MAP2, in dystrophic neurites surrounding plaques in human AD brain in a pattern that is similar to that seen in 5XFAD brain. We conclude that 5XFAD mice recapitulate the pattern of BACE1 accumulation in plaque-associated dystrophies observed in human AD. Thus, investigation of BACE1 elevation in 5XFAD mice should provide valuable insight into the formation and progression of amyloid plaques in AD.
We observed different subtypes of dystrophic neurites in the 5XFAD brain, which we termed Type I and Type II, that were distinguishable based on the size, degree of electron density, and multi-lamellar nature of membrane-bound (autophagic intermediate-like) structures within a given neurite. Generally, Type I dystrophic neurites were less electron dense and contained smaller membraneous structures, while Type II dystrophies were more electron dense and contained larger, multi-lamellar structures. BACE1 immuno-EM signal was highest in Type I dystrophies that contain smaller, less electron-dense vesicles. Type II dystrophic neurites, which exhibit large multi-lamellar autophagosomes, had much lower BACE1 immunogold labeling. Although the identity of BACE1-positive vesicles in Type I dystrophies is unknown, we suspect they are endosomes based on the high degree of co-localization of BACE1 and transferrin receptor by immunofluorescence confocal microscopy. Poor co-localization of BACE1 with LAMP1 and LC3B allows us to exclude lysosomes and autophagosomes as primary organelles of BACE1 accumulation, respectively. These data concur with our immuno-EM results showing lack of BACE1 localization to multi-lamellar autophagosomes. We speculate that Type I and Type II dystrophies represent a continuum of the disease process, whereby Type I represents an earlier stage of disease. This notion is supported by EM studies that characterize dystrophic neurites at different stages of degeneration in AD mouse models [1, 23] and human AD , the latter in which two types of dystrophies were described having either large globoid APP and chromogranin-immunopositive or tau-immunopositive morphologies. The former may represent an early stage of dystrophy, while the latter are neurofibrillary tangle (NFT)-containing structures at a late stage of degeneration. 5XFAD mice do not have NFTs and therefore lack this late-stage dystrophic neurite. However, 5XFAD dystrophies may be related to early-stage globoid APP and chromogranin-positive AD dystrophic neurites, thus fitting with the general consensus that APP transgenic mice model an earlier phase of AD.
Roles of autophagy, lysosomes, and microtubule transport in BACE1 accumulation in AD
Dystrophic neurites are positive for markers of autophagy [14, 72] and at the ultrastructural level contain multi-lamellar autophagosomes [8, 23, 45, 71, 72, 89, 100, 119] observed in 5XFAD Type II dystrophies. Additionally, 5XFAD hippocampi had an elevated LC3B-II:LC3B-I ratio, indicating increased autophagy consistent with accumulation of autophagosomes by EM and concurring with elevated LC3B-II:LC3B-I ratio in another APP transgenic . The increased markers of autophagy correlate with elevated BACE1 levels in 5XFAD hippocampus, although both BACE1 immuno-EM and BACE1-LC3B co-localization by confocal microscopy suggest that BACE1 does not accumulate in autophagosomes.
BACE1 accumulation in endosomes of 5XFAD presynaptic dystrophic neurites suggests three potential mechanisms: (1) BACE1 is degraded by autophagy, but an earlier step of the autophagic pathway is impaired (e.g., fusion of endosomes with autophagosomes); (2) BACE1 is degraded by lysosomes, but an earlier step of the lysosomal pathway is impaired (e.g., lysosomal acidification or maturation); (3) BACE1 clearance requires microtubule transport, but microtubules are dysfunctional (e.g., failure of BACE1 transport back to the soma). To gain initial insights into these mechanisms, we investigated the roles of autophagy and the lysosomal pathway in BACE1 clearance in cultured primary neurons. Previous studies reported that axonal dystrophy could result from inhibition of lysosomal proteolysis [4, 5, 58, 59]. Treatment of primary neurons with the lysosomal protease inhibitor leupeptin resulted in elevated BACE1 levels and a small increase in the LC3B-II:LC3B-I ratio, the latter of which indicates reduced autophagosome clearance. These results support earlier work suggesting that BACE1 is degraded in lysosomes [51, 103]. In contrast, inducing autophagy in primary neurons with trehalose resulted in a large increase in LC3B-II:LC3B-I ratio, but had no effect on BACE1 level. If autophagy cleared BACE1 in neurons, then BACE1 should significantly decrease following trehalose treatment. Hypothetically, BACE1 could be an autophagy substrate under other conditions of autophagy induction; however, we find this possibility unlikely. Hence, BACE1 does not appear to be a target of autophagy, at least in primary neurons. Rather, our data indicate that BACE1 accumulation may result from impaired lysosomal degradation of BACE1.
Taken together, our in vitro and in vivo results indicate that BACE1 accumulates in endosomes within presynaptic dystrophic neurites surrounding plaques, suggesting that BACE1 degradation could be reduced due to decreased BACE1 flux through the endosomal–lysosomal pathway. Although the 5XFAD brain exhibits robust accumulation of autophagosomes and increased LC3B-II, autophagy does not appear to be directly involved in BACE1 clearance. The evidence for this is the following: (1) BACE1 co-localizes with synaptophysin and transferrin receptor but not MAP2, LAMP1 or LC3B, indicating that BACE1 accumulates in presynaptic endosomes but not lysosomes or autophagosomes; (2) BACE1 immunogold labels small electron translucent vesicles, not large electron-dense multi-lamellar autophagosomes; and (3) lysosomal protease inhibition elevates neuronal BACE1, but autophagy induction does not reduce BACE1.
The reason for reduced endosomal–lysosomal BACE1 flux is unclear, although aberrant accumulation of α- and β-tubulin in dystrophic neurites implies that dysfunctional microtubules could play a role. Lysosome maturation requires vesicles laden with lysosomal proteases that are derived from the Golgi apparatus in the soma. If microtubule-based axon transport is impaired by amyloid, deficient protease levels in presynaptic lysosomes may ensue resulting in reduced protein degradation. Although speculative, this model would provide a plausible mechanism for increased BACE1 levels in dystrophies near plaques. Similarly, organelle accumulation in dystrophic neurites has been postulated to arise from impaired axon transport . In addition, AD brains show reduced levels of dynein and kinesin, molecular motors critical for axoplasmic transport . Loss of retrograde transport in dystrophies could also contribute to accumulation of autophagosomes and BACE1-positive vesicles in axon terminals, causing neurites to swell as vesicles and mitochondria amass within. The molecular nature of the plaque-related toxic agent and mechanism responsible for neuritic dystrophy and BACE1 accumulation are unclear, but likely involve Aβ, given the intimate association of dystrophies with plaques. Alternatively, dystrophic neurite formation may involve reticulon/Nogo proteins (reviewed in ), since transgenic mice that overexpress reticulon-3 form plaque-independent neuritic dystrophies [38, 94, 95]. Future work should elucidate mechanisms of dystrophic neurite pathogenesis.
In summary, our study provides the first conclusive evidence, as shown by electron microscopy, that BACE1 resides predominantly within normal and dystrophic presynaptic terminals in wild-type and AD mouse model brain, consistent with our immunofluorescence microscopy of human AD. Presynaptic BACE1 localization suggests that BACE1 substrate processing has important consequences for axon terminal function. BACE1 and APP accumulate in presynaptic dystrophies surrounding amyloid plaques, implying a feed-forward mechanism of Aβ generation that may exacerbate AD pathogenesis. Deficient lysosomal degradation, but not impaired autophagy, of BACE1 could cause BACE1 accumulation in presynaptic endosomes. Although the molecular mechanism of BACE1 elevation around plaques is enigmatic, evidence of abnormal tubulin accumulation implies dysfunctional microtubule-based transport. Other mechanisms are possible, such as impaired lysosomal acidification or enzymatic activity . Future studies to elucidate mechanisms of BACE1 elevation in dystrophies may provide insights into potential therapeutic avenues to reduce BACE1 levels and ameliorate AD.
We wish to thank Drs. Lester Binder for the TuJ1 antibody and Virginia Lee for the anti-APP Karen antibody used in our immunofluorescence and immunoelectron microscopic analyses. Confocal imaging work was performed at the Northwestern University Cell Imaging Facility-Nikon Imaging Center (Chicago, IL) generously supported by NCI CCSG P30 CA060553 awarded to the Robert H. Lurie Comprehensive Cancer Center. Electron microscopy studies were performed at Rush University Medical Center, Chicago, IL. We thank Dr. Sangram Sisodia and members of the Vassar Laboratory for thoughtful comments on the manuscript. This study was supported by the Charles and M.R. Shapiro Foundation (DAN) and NIH Grants R00 AG031574 (DAN), R01 AG017139 (DAN), and R01 AG030142 (RV) and R01 AG022560 (RV).
Conflict of interest
The authors declare that they have no conflict of interest.
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