SURP Final Paper [Final] DW

Regulation of synaptic BACE1 trafficking and Aβ generation through late endocytic transport David Wilkes, Xuan Ye, Qian Cai.

Transcript of SURP Final Paper [Final] DW

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Regulation of synaptic BACE1 trafficking and Aβ generation

through late endocytic transport

David Wilkes, Xuan Ye, Qian Cai.

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Cumulative evidence suggests that -amyloid (A) peptides play a key role in synaptic damage

and affect memory processes early in the course of Alzheimer's disease (AD). Amyloid-β (Aβ) peptide is

produced via β-secretase, mainly by β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1).

BACE1 aberrantly accumulates at the distal axons and synapses in AD neurons and relies on late

endocytic transport to be degraded within lysosomes in the soma. APP is primarily processed in the late

endocytic pathway, but the mechanism through which BACE1 is trafficked from the synapse to the

lysosome is still largely unknown. We hypothesized that snapin deficiency results in defective

retrograde transport of late endosomes and causes accumulation of BACE1 at the nerve terminal, thus

enhancing APP amyloidogenic processing. To test this hypothesis, synaptosomes were prepared from

conditional snapin knockout mice and protein levels were assayed by Western blotting. We showed that

deletion of snapin causes an abnormal accumulation of synaptic BACE1 and APP, increasing APP

processing and Aβ generation at the nerve terminal. Altogether, our study indicated that Snapin-

mediated retrograde transport is important for regulating synaptic BACE1 trafficking and its cleavage of



Alzheimer’s disease (AD) is a progressive neurodegenerative disease characterized by memory

loss and impaired cognitive function. The disease begins primarily in the highly plastic neurons of the

hippocampus, and then spreads to the cerebral cortex and other brain regions. The pathology of this

disease is characterized by synaptic dysfunction, axonal degeneration, neuronal loss, and the formation

of neurofibrillary tangles and senile plaques that are composed mostly of amyloid-beta (Aβ) peptide.

However, it has been found that the degree of cognitive impairment in AD is most correlated to the level

of the soluble form of oligomeric Aβ in neurons, and not to the amount or size of the extracellular

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plaques themselves (Hardy and Selkoe, 2002). Aβ is generated through the sequential cleaving of

amyloid precursor protein (APP) by a β-secretase and then by a γ-secretase. β-site amyloid precursor

protein cleaving enzyme 1 (BACE1) is the prominent β-secretase involved in amyloidogenesis in neurons

(Huse et al., 2000; Vassar et al., 2009).

The initial cleavage step involving BACE1 and APP is the rate-limiting step in Aβ production,

which occurs predominantly in late endosomes [also called “multi vesicular bodies” (MVBs)]. The low pH

and extensive surface area within late endosomes is optimal for BACE1 activity (Vassar et al., 2009; Wu

et al., 2011). BACE1 is a transmembrane aspartyl protease (Huse et al. 2000), which is endocytosed

from the plasma membrane into the endocytic pathway, where it is eventually trafficked by means of

retrograde transport to lysosomes for degradation (Sannerud et al., 2011). In AD brains, increased levels

of BACE1, especially when colocalized with APP in the endocytic pathway (Das et al., 2013), have been

directly correlated to a rise in Aβ production (Vassar et al., 2009; Ye and Cai, 2014), as well as to a

greater accumulation in acidic compartments—probably endosomes (Kandalepas et al., 2013).

Particularly of interest, BACE1 seems to concentrate aberrantly in dystrophic neurons around amyloid

plaques in APP transgenic mouse brains, which suggests an increase in synaptic APP processing. BACE1

levels increase with age and in the brains of patients with sporadic AD (Fukumoto et al., 2004). It is

important to note that a rise in BACE1 levels is due to its enhanced stability and/or reduced turnover,

and not by an increase in production of BACE1 (Lefort et al., 2012). Consequently, BACE1 levels

potentially could be primarily controlled by modulation of late endosomal retrograde transport.

Neuronal endocytosis moderates cell signaling by controlling how many receptors can actively

interact at the membrane surface (Yuzaki, 2010). Endocytosis begins with budding of the membrane,

which contains surface proteins (including BACE1), to form vesicles, which fuse with early endosomes.

Early endosomes mature into late endosomes by a regulated inward budding process to form a “multi

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vesicular body” (late endosome), increasing membrane surface area and allowing lysosome-delivered

hydrolase access (Nixon, 2005). The late endosome then fuses with a lysosome in order to degrade its

protein cargo. The process by which late endosomes and its BACE1 contents migrate to and then fuse

with lysosomes is still poorly understood (Ye and Cai, 2014). Upon fusion, acid hydrolases from the

lysosome become active and digest late endosomal cargo (Nixon, 2005). Mature lysosomes mainly

reside in the neuronal soma, far from the axon terminal (Cai et al., 2010). Therefore, late endosomes

containing BACE1 depend on retrograde transport along the axonal microtubules in order to deliver

their cargoes to the lysosomes for degradation in the soma (Cai et al., 2010).

Retrograde transport is driven by dynein, the major microtubule-based minus-end directed

motor protein in neurons. Deficits in dynein-related proteins such as dynein intermediate chain (DIC)

and Snapin have been shown to disrupt late-endocytic transport and lead to excess production of Aβ

(Cai et al., 2010; Ye and Cai, 2014). Snapin is an important adaptor protein for dynein motors, as it has

been found to coordinate late endocytic transport and maturation of lysosomes (Cai et al., 2010).

Snapin is also thought to interact with dynein intermediate chain (DIC), which allows recruitment of

dynein motors to late endocytic organelles for retrograde transport. Experiments involving snapin

knockout (KO) mice or DIC substitution resulted in decreased dynein-assisted transport of BACE1

containing vesicles, and enhanced APP processing (Ye and Cai, 2014). Distal accumulation of late

endosomes containing Aβ42 has also been found in AD mouse neurons, possibly due to impaired

retrograde transport of APP and BACE1, which may account for the excess production of Aβ observed in

AD neurons (Ye and Cai, 2014). In dendrites, APP and BACE1 converge mostly during the endocytic

pathway, triggering amyloidogenesis (Das et al., 2013).

One of the earliest symptoms in AD brains is the loss of synaptic functionality (Selkoe, 2002).

The loss of synapses has been discovered to be the best predictor of cognitive impairment in AD brains

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(Tampellini et al., 2011). BACE1 has been found to localize in vesicles at the presynaptic terminals near

active zones (Kandalepas et al., 2013). Excessive Aβ production and release at synaptic sites are tightly

correlated with increased amyloid deposition and synaptic dysfunction (Stokin and Goldstein, 2006;

Takahashi et al., 2002). Considering that abnormal accumulation of BACE1 and APP at presynaptic

terminals occurs before the onset of plaque (Zhang et al., 2009), intervention in the BACE1 trafficking

pathway could be a reliable means for halting amyloid pathology early in AD. This raises the central

question of whether enhancement of Snapin-mediated dynein motor-assisted BACE1 transport can

decrease its synaptic accumulation and Aβ generation at nerve terminals, reducing or delaying synaptic

pathology in AD mouse brains.

In this study, we sought to address whether disruption of retrograde transport accumulates

BACE1 at the synapse, thereby enhancing amyloidogenic processing and amplifying synaptic AD

pathology. Snapin KO mice will serve as a unique genetic tool that allows us to address whether snapin

deficiency heightens synaptic retention of BACE1, exacerbating synaptic A generation.

Materials and Methods


PCR genotyping assay was performed to identify homozygous snapin flox mice (snapinflox/flox; Thy-

1 Cre Tg) after the tissue-specific Cre was confirmed. DNA was extracted from mouse tail snips using

PCR lysate reagent (Viagen). The snapin loxP site forward primer 5 - GTGCAGCAGCTCGACTCTC -3 and ′ ′

loxP site reverse primer 5’- AGCCAACCTCAACTTCAAGG – 3’ were used to flox the snapin gene coding

region. The DNA segments were amplified by using the following PCR reaction system: 9.46µl H2O,

1.25µl 10X PCR Buffer, 0.25µl 10mM dNTPs, 0.38µl 50mM MgCl2, 0.3µl 10µM forward primer, 0.3µl

10µM reverse primer, 0.06µl (5U/µl) Taq Polymerase, and 0.5µl tail DNA. Thirty three cycles of PCR

were used with the following cycles: 94° C for 3 minutes (only first time), 94° C for 30 seconds, 55° C for

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30 seconds, 72° C for 45 seconds, repeat 33 times, 72° C for 5 minutes (only last time), then keep at 16°

C. By running PCR products on 2% agarose gel, homozygous snapin flox mice show a band at 275bp,

while wild-type mice can be identified by a band at 188bp.

Synaptosome Preparation

Synaptosome preparations from WT and snapin conditional KO mouse brains (snapinflox/flox; Thy-1

Cre Tg) were collected using Percoll gradient centrifugation as described in the protocol (Leenders et al.,

2004). Cortex tissue from WT and snapin mutant mice were homogenized in ice cold Isolation Buffer

(IB) [10mM Tris-HCl, 1mM EGTA, 1mM EDTA, 0.25 M sucrose and protease inhibitors (Roche), pH 7.4].

Homogenates were centrifuged at 1,330 g for 3 minutes, the supernatant was gathered, and the pellet

was resuspended with IB and centrifuged again at 1,330 g for 3 minutes. The first and second

supernatant were combined and then centrifuged at 21,000 g for 10 minutes and then resuspended in

12 ml of 15% Percoll. 2 ml of the 15% Percoll suspension was overlaid on Percoll gradient that has 3.5

ml of 15% Percoll gradient layered over 23% Percoll gradient. The gradient was then separated by

centrifugation for 5 minutes at 30,700 g. The synaptosomal fraction was collected from the 15%/23%

Percoll layers, and combined with 0.5 ml of 10 mg/ml bovine serum albumin (BSA) in 3 ml IB. The

mixture was then centrifuged at 16,700g for 10 minutes and resuspended in IB. Protein quantification

was performed by BCA assay (Pierce Chemical Co.). 15µg of protein from synaptosome and post nuclear

supernatant (PNS) homogenates were resolved by 4-12% SDS-PAGE for sequential Western blots on the

same membranes after stripping between each application of antibody.

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Figure 1Percoll gradient centrifugation (Sims, 1990)

Western blotting

Western blot was performed by first blocking for 30 minutes with 10% non-fat milk/TBST

solution, followed by incubation with primary and secondary antibodies for 1 hour each. SuperSignal

West Pico Chemiluminescence substrate (Thermo Scientific) was used to perform enhanced

chemiluminescence (ECL) using scientific imaging film (Lab Scientific, Inc.). NIH ImageJ software was

used for quantitative analysis. All immunoblots were from experiments done at least 4 times. Statistical

analyses were completed with unpaired Student's t test and are shown in figures as mean ± SEM. H. Cai

provided polyclonal anti-BACE1 antibody. Polyclonal anti-Snapin antibody was supplied by Z.H. Sheng.

Other sources of antibodies are as follows: polyclonal anti-APP c-terminal and polyclonal anti-APP-CTF

antibodies (Millipore/Chemicon), monoclonal anti-rab7 and polyclonal anti-SNAP25 antibodies (Sigma),

and monoclonal anti-p115 antibody (BD biosciences).


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Figure 2

Gel electrophoresis, performed after confirmation of the tissue specific Cre, of constitutional knockout PCR products obtained from mouse tails. Cre-Lox recombination was used on mice embryos to create a constitutional site-specific deletion at the snapin coding region. Homozygous snapin KO mutant mice (snapinflox/flox; Thy-1 Cre Tg) show a single band at 275bp (Lanes: 3 and 6), while WT mice (snapin+/+; Thy-1 Cre Tg) display a band at 188bp (Lanes: 8, 9 and 10). Heterozygous mutant mice (snapinflox/+; Thy-1 Cre Tg) show a band at both 188bp and 275bp (Lanes: 1, 2, 4, 5, 7 and 11). Lane L is a 100bp DNA ladder and lane Ctrl is a positive control for snapin KO.

Previously, site-specific Cre was tested for and confirmed in the brains of the mice used in the

following investigation. In order to confirm homozygous snapin-mutant mice, a gel electrophoresis assay

was performed for the purpose of visually distinguishing mouse mutations (Figure 2). After DNA

separation, a single band at 275bp for lanes 3 and 6 (Figure 2) reveals that the snapin gene encoding

region was completely floxed in both chromosomes of the mice meaning they are homozygous

(snapinflox/flox; Thy-1 Cre Tg). Lanes 1, 2, 4, 5, 7 and 11 each showed two bands at both 188bp and 275bp,

indicating that the mice are heterozygous (snapinflox/+; Thy-1 Cre Tg), and thus they are not viable for

experimental use. In lanes 8, 9, and 10, there is a single band at 188bp which specifies that these mice

contain an unaffected coding region (snapin+/+; Thy-1 Cre Tg) and are therefore wild-type mice that can

be utilized as experimental controls.

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Figure 3

Representative blots and quantitative analysis showing increased synaptic accumulation of APP, BACE1, and Rab7 in the brain of snapin KO mice. Following synaptosome fractionation, equal amounts (15µg) of synaptosome (SS) and post-nuclear supernatant (PNS) from WT and snapin KO mice were sequentially immunoblotted. The purity of the synaptosome was confirmed by the absence of p115, a Golgi marker protein. Snapin is shown to be absent in the synaptosome of snapin KO mice, confirming the precision of this mouse model. Relative protein levels in synaptosomes of snapin KO mice were compared with those of WT littermates. Data were analyzed from 4 pairs of mice for each genotype and expressed as mean ± SEM with Student’s t test: ∗∗p < 0.01; ∗p < 0.05.

To examine whether synaptic BACE1 accumulation occurs in snapin-deficient mice, sequential

immunoblots were performed on synaptosomes from the brains of snapin KO mice and WT littermates.

Sequential immunoblots for both the post-nuclear supernatant (PNS)—a homogenate of neural cells

with the nuclear compartment removed—and the purified synaptosome (SS) were performed on

separate membranes against the following antibodies: APP, CTFs [carboxyl-terminal fragments: C83,

C89, C99 (byproducts of APP processing)], BACE1, late-endosomal marker Rab7, presynaptic membrane

associated indicator SNAP25, and Golgi marker p115. The purity of the synaptosomes was determined

by the absence of p115. Relative protein levels in the synaptosome of WT and snapin KO mice were

quantitatively compared (Figure 3). The APP level detected in the synaptosome of snapin-deficient mice

was 4.08 ± 0.45 (p < 0.01) times more than the amount identified in WT littermates (Figure 3). Likewise,

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BACE1 levels in the synaptosome were markedly increased in the snapin KO mice, 5.1 ± .27 (p < 0.01)

times higher relative to the WT mice (Figure 3). Rab7 exhibited a significant rise in the synaptosome of

snapin-mutant mice, with a protein level 1.7 ± 0.12 (p < 0.05) times higher than in the WT control (Figure

3). CTFs, a by-product of amyloidogenesis, appeared to occur in the highest density at the synaptosomes

of snapin KO mice. Additionally, there was a higher concentration of BACE1 in snapin-deficient mice at

the synaptosome than in the PNS.


In previous studies, Snapin was shown to mediate late-endosomal trafficking through

interactions with dynein (Cai et al., 2010), and also to modulate BACE1 retrograde transport and

turnover (Ye and Cai, 2014). In this study, we hypothesized that disruption of late-endocytic transport

via snapin deletion causes accumulation of BACE1 at synaptic terminals, leading to enhanced APP

processing. This study expanded further on previous work, demonstrating that the amount of BACE1 in

snapin-mutant mouse brains was elevated in the synaptosome compared with in the PNS, lending

credence to this study’s hypothesis.

The snapin KO mice used in this study, identified by genotyping, were confirmed to function as

intended by the immunoblot in figure 3, showing a substantial decrease in Snapin expression level.

Small amounts of Snapin can still be seen in the immunoblot for the PNS knockout because the

homogenate included glial cells, which were not affected by Cre-Lox recombination. Further validation

for this study’s hypothesis is provided by the elevated amount of late-endosomal markers (rab7) in the

snapin-deficient synaptosome, indicating an accumulation of late endosomes in the KO condition. This

evidence suggests that without Snapin, late endosomes would be unable to associate with dynein and

be delivered to lysosomes for degradation. It is now clear that late-endocytic trafficking is vital to the

regulation of amyloidogenesis near the synapse, in agreement with previous studies that confirmed this

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pathway to be the primary location of APP processing and to be crucial in BACE1 turnover (Ye and Cai,

2014). The most direct piece of evidence to support this study’s hypothesis is the profound increase in

BACE1, as well as in APP, in the synaptosomes of snapin KO mice when compared to WT mice.

Previously, an increase of Aβ in snapin-deficient mice was demonstrated by using an ELISA assay (Ye and

Cai, 2014). Consistently, ELISA assay results have shown that Aβ levels in snapin KO mice are

significantly elevated in synaptic preparations compared to PNS from the total cortex (data not shown).

These findings implicate snapin deficiency in impaired late endocytic transport and BACE1 to

accumulation in late endosomes, enhancing BACE1 cleavage of APP and Aβ production at nerve


The significant increase in synaptic amyloid-related proteins due to an absence of Snapin

functionality is a solid confirmation that Snapin has a major role in regulating Aβ production through late

endocytic transport. These results, in conjunction with the previously described studies, support a

potential mechanism for synaptic AD pathology in which Aβ causes Snapin-dynein motor uncoupling.

Therefore, introducing excess Snapin into an AD brain could be a possible method for overcoming the

disruption of motor coupling induced by Aβ.

Conclusion and Future Plans

The results from this experiment demonstrate that dynein motor-driven transport of late

endosomes serves an important role in regulating BACE1 accretion and, consequently, synaptic APP

processing. Excessive amyloidogenesis, especially in axon terminals, is thought to cause axonal

degeneration and synaptic dysfunction (Stokin and Goldstein, 2006). Additionally, synaptic loss has

been found to be one of the earliest and best predictors of cognitive decline in AD (Tampellini et al.,

2011). Considering this, synaptic BACE1 trafficking is a potentially invaluable therapeutic target for

treating AD pathology early and effectively.

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Further experiments will need to be conducted to confirm the findings in this study, and to shed

more light on the regulatory mechanisms of synaptic BACE1 trafficking through dynein-associated

transport. AD neurons show reduced retrograde transport of BACE1, which is caused by Snapin-dynein

motor uncoupling (Ye and Cai, 2014). Dr. Qian Cai’s lab will next study the effects of defective late

endocytic BACE1 transport on synaptic retention of BACE1 and whether it will increase BACE1

processing of APP at the nerve terminal in AD mouse models and AD patient brains. This lab will also be

investigating the effects of enhancing Snapin-mediated synaptic BACE1 trafficking in AD transgenic

mouse models by overexpression of Snapin. This approach will potentially be used to reduce synaptic

Aβ generation and synapse loss. Neither AD mouse brains nor human AD synaptosomes have been

isolated and studied for synaptic BACE1 accumulation or defects in Snapin-dynein coupling. Looking

further ahead, human AD brains will most likely be studied in order to determine if there is potential for

Snapin-targeted or late-endocytic-related therapies in repressing or ameliorating the symptoms of AD

patients. The goal for this research is that human AD patients will respond positively to Snapin or

related treatments, as is the case so far in mouse models. However, current mouse models only

represent the hereditary form of AD, as opposed to the sporadic form of AD that is seen prevalently in

humans. Sporadic AD patients may have varied and distinct underlying causes for their disease not yet

considered, and so these patients may differ in the types of treatment that are effective for them.

Despite possible complications in targeting sporadic forms of AD, understanding abnormalities in

retrograde transport shows promise for the development of earlier diagnoses and more beneficial

interventions in the future.


I would like to thank Dr. Qian Cai for giving me the opportunity to be a member of her lab, and

for her taking the time out of her very busy schedule to be patient with me, and her commitment to

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helping me expand my thinking and succeed. I would also like to thank my lab mentor, Dr. Xuan Ye, as

well as all the lab members, for providing insight into laboratory techniques and experimental design. I

am also very grateful for the 2015 Neuroscience Summer Undergraduate Research Program, for the

program’s co-directors, Dr. Janet Alder, Dr. Michael Matise, and Dr. Mladen-Roko Rasin, and for the

NeuroSURP program administrative coordinator, Joan Mordes, all of whom have shown phenomenal

dedication to the students in the program. I appreciate the accommodations, resources, and

commitment to scientific research provided by The Robert Wood Johnson Medical School and by

Rutgers, The State University of New Jersey. Finally, I would like to thank all those who have helped

support me and have contributed positively to my life: family, friends, professors and mentors.


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