Rapid and Direct Transport of Cell Surface APP to the Lysosome defines a novel selective pathway

Lorenzen, Angela; Samosh, Jonathan; Vandewark, Kenneth; Anborgh, Pieter H.; Seah, Claudia; Magalhães, Ana C.; Cregan, Sean P.; Ferguson, Stephen S. G.; Pasternak, Stephen

doi:10.1186/1756-6606-3-11

Cited by 61 publications

(92 citation statements)

References 58 publications

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“…Our shortened constructs also lack the N-terminal ectodomain, which has undefined cleavages and sorting signals 38 . Despite these N-terminal signals and cleavages, we find that our shortened construct has similar localization and trafficking as a full-length APPpaGFP 30,33 .…”

Section: Discussionmentioning

confidence: 92%

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Tam¹,

Pasternak

2015

JoVE

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Beta-amyloid (Aβ) is the major constituent of senile plaques found in the brains of Alzheimer's disease patients. Aβ is derived from the sequential cleavage of Amyloid Precursor Protein (APP) by β and γ-secretases. Despite the importance of Aβ to AD pathology, the subcellular localization of these cleavages is not well established. Work in our laboratory and others implicate the endosomal/lysosomal system in APP processing after internalization from the cell surface. However, the intracellular trafficking of APP is relatively understudied.While cell-surface proteins are amendable to many labeling techniques, there are no simple methods for following the trafficking of membrane proteins from the Golgi. To this end, we created APP constructs that were tagged with photo-activatable GFP (paGFP) at the C-terminus. After synthesis, paGFP has low basal fluorescence, but it can be stimulated with 413 nm light to produce a strong, stable green fluorescence. By using the Golgi marker Galactosyl transferase coupled to Cyan Fluorescent Protein (GalT-CFP) as a target, we are able to accurately photoactivate APP in the trans-Golgi network. Photo-activated APP-paGFP can then be followed as it traffics to downstream compartments identified with fluorescently tagged compartment marker proteins for the early endosome (Rab5), the late endosome (Rab9) and the lysosome (LAMP1). Furthermore, using inhibitors to APP processing including chloroquine or the γ-secretase inhibitor L685, 458, we are able to perform pulse-chase experiments to examine the processing of APP in single cells.We find that a large fraction of APP moves rapidly to the lysosome without appearing at the cell surface, and is then cleared from the lysosome by secretase-like cleavages. This technique demonstrates the utility of paGFP for following the trafficking and processing of intracellular proteins from the Golgi to downstream compartments. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 92%

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Tam¹,

Pasternak

2015

JoVE

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“…The exact mechanism behind the increase remains unknown, but it might involve altered activity of Ab-generating enzymes, impaired vesicular trafficking, lysosomal dysfunction, or combinations of these. Further links between NPC and AD are provided by accumulating evidence of lysosomal dysfunction in AD (Lee et al 2010;Liu et al 2010;Lorenzen et al 2010) and AD patients have increased levels of NPC1 in degenerated brain regions (Kagedal et al 2010).…”

Section: Introductionmentioning

confidence: 99%

Miglustat Treatment May Reduce Cerebrospinal Fluid Levels of the Axonal Degeneration Marker Tau in Niemann–Pick Type C

et al. 2011

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Introduction: Niemann-Pick disease type C (NPC) is a lysosomal storage disorder that leads to progressive neurodegeneration. The glucosylceramide synthase blocker miglustat is being used to treat NPC, but monitoring of disease progression and treatment response is difficult. NPC patients have elevated cerebrospinal fluid (CSF) levels of total-tau (T-tau) indicating axonal degeneration, and increased CSF amyloid b (Ab) indicating abnormal brain amyloid metabolism, but it is unknown if start of miglustat treatment affects these biomarker levels.Methods: Biomarkers were measured in serial CSF samples from NPC patients who started miglustat between samplings (N ¼ 5), were untreated at both samplings (N ¼ 5) or received treatment during the whole study (N ¼ 6) (median time between samplings 309 days [range 175-644]). CSF was analyzed for Ab 38 , Ab 40 , Ab 42 , a-cleaved soluble APP, b-cleaved soluble APP, T-tau and phospho-tau.Results: T-tau levels decreased in patients who started miglustat treatment (median 955 [range 338-1,271] ng/L at baseline vs. 382 ng/L at follow-up, p ¼ 0.043). Untreated patients and continuously treated patients had stable levels (p > 0.05). No changes were seen in the other biomarkers.Conclusion: Reduced CSF T-tau suggests that miglustat treatment might affect axonal degeneration in NPC. However, the results must be interpreted with caution and verified in future studies, since this pilot study was small, treatment was not randomized, and patients starting treatment had higher baseline CSF T-tau than untreated patients.

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“…APP is internalized from plasma membrane by endocytosis and moves through the endosomal pathway to lysosomes where it is cleaved by secretases. However, APP can also enter a novel, rapid transport pathway to move directly from the cell surface to lysosomes (Lorenzen et al, 2010). It is not known whether this trafficking is altered by oxidativenitrosative stress or what effect this could have on the function of its binding partner CHT, but this raises important questions about the regulation of cholinergic presynaptic function by APP disposition and trafficking in neuropathology.…”

mentioning

confidence: 98%

“…To further assess the amount of CHT in late endosomes and lysosomes under SIN-1 and vehicle treatments, we used a quantitative approach to determine the relative amount of CHT colocalization with Rab9-YFP or endogenous Lamp-1 by a method described by Lorenzen et al (2010). An example of this analysis is shown in Figure 6 for CHT and Rab9-YFP.…”

Section: Cht Internalizes Into Rab5a-positive Organelles In Control Amentioning

confidence: 99%

“…Colocalization analysis was performed on confocal images using Imaris 7.0 with the Imaris Colocalization module (bit-plane) to examine the colocalization of the brightest 2% of pixels in each channel, as described previously (Lorenzen et al, 2010). Graphing and statistical analysis of the data were performed with GraphPad Prism and Instat using one-way ANOVA with Tukey's post hoc test.…”

Section: Materials Sin-1 Was From Biomol Researchmentioning

confidence: 99%

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Peroxynitrite Donor SIN-1 Alters High-Affinity Choline Transporter Activity by Modifying Its Intracellular Trafficking

Cuddy¹,

Gordon²,

Black³

et al. 2012

J. Neurosci.

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Sodium-coupled, high-affinity choline transporters (CHTs) are inhibited by 3-morpholinosydnonimine (SIN-1) [peroxynitrite (ONOO Ϫ ) donor]; ONOOϪ can be produced from nitric oxide and reactive oxygen species during neurodegeneration. SIN-1 rapidly increases CHT internalization from the cell surface, and this correlates with decreased choline uptake. This study addresses mechanisms by which SIN-1 inhibits CHT function in human neuronal SH-SY5Y cells. Thus, mutant L531A-CHT, which does not constitutively internalize into cells by a clathrin-mediated process, is resistant to SIN-1 effects. This suggests that CHT inhibition is not due to oxidative-nitrosative inactivation of the protein and that decreased levels of cell surface CHT in SIN-1-treated cells is related to alterations in its trafficking and subcellular disposition. Dominant-negative proteins AP180C and dynamin-K44A, which interfere with clathrin-mediated and dynamindependent endocytosis, respectively, attenuate CHT inhibition by SIN-1. CHT in both vehicle-and SIN-1-treated cells colocalizes with Rab7, Rab9, and Lamp-1 in late endosomes and lysosomes to a similar extent. Lysosome inhibitors increase choline uptake, suggesting that CHT proteins are normally degraded by lysosomes, and this is not altered by oxidative stress. Unexpectedly, inhibitors of proteasomes, but not lysosomes, attenuate SIN-1-mediated inhibition of choline uptake, indicating that proteasomal degradation plays a role in regulating CHT disposition in SIN-1-treated cells. SIN-1 treatment also enhances CHT ubiquitination. Thus, CHT inhibition in SIN-1-treated cells is mediated by proteasomal degradation, which differs from inhibitory mechanisms for some neurotransmitter transporters under similar conditions. Increased oxidative-nitrosative stress in the microenvironment of cholinergic nerve terminals would diminish cholinergic transmission by reducing choline availability for ACh synthesis.

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Rapid and Direct Transport of Cell Surface APP to the Lysosome defines a novel selective pathway

Cited by 61 publications

References 58 publications

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Imaging the Intracellular Trafficking of APP with Photoactivatable GFP

Miglustat Treatment May Reduce Cerebrospinal Fluid Levels of the Axonal Degeneration Marker Tau in Niemann–Pick Type C

Peroxynitrite Donor SIN-1 Alters High-Affinity Choline Transporter Activity by Modifying Its Intracellular Trafficking

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