Trafficking and processing of APP-secretase

Intracellular trafficking of APP: relation to its physiological

function?

APP

NH2

5A3/1G7TMD

APP localizes to the plasma membrane, Golgi and endosomes

Pastorino, unpublished data

APP

NH2

TMD C-termAPP Ab

Staining for C-term domain of APP is detected also in the nucleus

Pastorino, unpublished data

COOH

APP

NH2

5A3/1G7TMD

APP internalizes from the plasma membrane into intracellular compartments, endosomes and Golgi

Koo and Squazzo, 1994

Protein trafficking and endocytosis

APP co-localizes with the endosomes

Pastorino et al., 2006

Because, the intracellular localization of APP INFLUENCES the production of Amyloid peptide

Why we want to study the trafficking of APP?

Processing of APP

-secretase

Protectivenon-amyloidogenic

pathway

Pathogenicamyloidogenic

pathway

APPTMD

sAPPs C83sAPPs

C99

-secretase

NH2 COOH

AAICDp3AICD

-secretase

-secretase-secretase

-secretase -secretase

Intracellular compartments and processing of APP

-secretase activity: in the plasma membrane (where metalloproteases, known to have like TACE and ADAM10/ADAM17, reside).

-secretase activity: mostly in the endosomes, possible also in the ER and Golgi

-secretase activity: mostly in the ER, also in lysosomes and possible at the plasma membrane (still under debate).

Trafficking of APP

-secretase activity

-secretase activity

-secretase activity

APP

C83

APP

C99

APPs

APP

AICD C99

A

APP retained @ plasma membrane =

Internalization of full length APP =

GOOD!!

BAD!!!

Protective non-amyloidogenic processing

pathogenic amyloidogenic processing

Products dowstream of non-amyloidogenic processing:

APPs: soluble stub of APP deriving from the -secretase cleavage : possible neurotrophic function

p3: c-terminal truncated portion of the sequence of b-amyloid, deriving from the subsequent action of - and -secretase. DOES NOT aggregate. Unknown function.

AICD: APP Intra Cellular Domain, deriving from the cleavage of -secretase. Known regulation of transcriptional activity.

Products downstream of the amyloidogenic processing:

APPs: soluble stub of APP deriving from the -secretase cleavage : unknown function

C99: c-terminal stub containing the entire intact sequence of the -amyloid peptide, deriving from the action of -secretase. It is the substrate from where -amyloid peptides derive.

-Amyloid peptides: generated by the subsequent action of - and -secretases. At very low concentration could be neurotrophic, however, when forming aggregates they are VERY TOXIC and lead to the formation of the core of the -amyloid plaque in AD

AICD: APP Intra Cellular Domain, deriving from the cleavage of -secretase. Known regulation of transcriptional activity.

Loss of non-amyloidogenic activity as a possible way to develop AD?

Alzheimer’s pathology and depression

Selective Serotonin reuptake inhibitors (SSRI) reduce ISF Abeta…

…and activate protective pathways

Chronic SSRI treatment reduces the load of Abeta plaques in AD mice

Chronic SSRI treatment reduces the load of Abeta peptides in AD mice…

…and increases alpha-secretase activity

Use of antidepressant associates to reduced PIB uptake in humans

Activation of serotoninergic receptors leads to increased non-amyloidogenic pathway

Activation of non-amyloidogenic pathway as protective from AD?

Sirtuin: deacetylation and control on protein transcription

Sirtuins are involved in different diseases

Sirtuins may protect from AD

Sirtuins levels are reduced in aging

Is sirtuins activity lost in AD?

Could their activity be protective?

SIRT1 Tg AD mice show reduced plaque and Abeta load

Sirt1 expression regulates non-amyloidogenic processing of APP in AD mice

Sirt1 expression regulates levels of the -secretase ADAM10 in AD mice both as protein….

…and as mRNA

SIRT1 regulates ADAM10 expression by deacetylating Retinoic Acid Receptor beta (RAR

Levels of the transcription factor HES1 are regulated by SIRT1

Model: SIRT1 controls expression of ADAM10

The aspartyl protease BACE -Amyloid cleaving enzyme

BACE is expressed mostly in the brain

Vassar et al., 1999

Vassar et al., 1999

In the cell, BACE localizes to Golgi apparatus and Endosomes

1-In vitro, BACE is mostly active at an acidic pH range between 4.5-5.5.

2-BACE is supposed to be mostly active in the endosomes, due to BACE co-localization and to the acidic pH of these organelles.

Although in vivo, interaction between BACE and APP was observed at the plasma membrane and in the endosomes, in cell culture, BACE was active also in the ER and in the Golgi apparatus.

BACE activity

It was previously reported that BACE interacts with GGAs in yeast two hybrid system

GGA1, 2 and 3 are monomeric adaptors that are recruited to the trans-Golgi network.

GGAs’ function relates to regulation of the trafficking and degradation of proteins

GGA consists of 4 distinct domains:

VHS domain that binds DxxxLL residue in proteins

GAT domain which binds Arf:GTP

Hinge region to recruit clathrin

A gamma adapton ear homology domain

Tesco et al., Neuron. 2007 Jun 7;54(5):721-37.

BACE co-localizes and is degraded within lysosomes

Koh et al., 2005

Tesco et al., Neuron. 2007 Jun 7;54(5):721-37.

APP contains caspase cleavage sites in its sequence

However, although apoptosis increases C99 and A levels, this effects do not depend on caspase-mediated cleavage of APP (Tesco et al., 2003).

Does apoptosis lead to increased amyloidogenic processing of APP up-regulating BACE?

In this paper:

Apoptosis increases levels of BACE and C99 in different cell lines

These effects are reverted by the apoptosis inhibitor zVAD.

Apoptosis regulates levels of BACE affecting its degradation not its synthesis, suggesting a post-translational mechanism.

During apoptosis GGA3 is cleaved generating a Dominant Negative fragment that further affects BACE trafficking and degradation.

Tesco et al., Neuron. 2007 Jun 7;54(5):721-37.

Apoptosis increases levels of C99…..

Tesco et al., Neuron. 2007 Jun 7;54(5):721-37.

…and BACE

Apoptosis regulates levels of BACE affecting its degradation not its synthesis

The BACE trafficking molecule GGA3 is cleaved during apoptosis

A dominant negative truncated form of GGA3 is generated under apoptotic stimuli, increasing

levels of A and BACE

What happens reducing the levels of GGA3?

Tesco et al., Neuron. 2007 Jun 7;54(5):721-37.

GGA3 siRNA causes increase of BACE expression and accumulation of C99

Tesco et al., Neuron. 2007 Jun 7;54(5):721-37.

Ischemic patients have increased levels of BACE in the brain…

Tesco et al., Neuron. 2007 Jun 7;54(5):721-37.

And decreased levels of GGA3

Tesco et al., Neuron. 2007 Jun 7;54(5):721-37.

AD patients have increased levels of BACE and decreased levels of GGA3 in the brain…

Apoptosis stabilizes BACE causing elevated -secretase activity.

This mechanism is regulated via the caspase-dependent cleavage of GGA3, that reduces the amount of BACE targeted to the lysosomes for degradation.

AD, but not NAD patients show increased levels of BACE paralleled by decreased levels of GGA3.

Vassar, Neuron. 2007 Jun 7;54(5):671-3. Review.

Model of BACE stabilization during apoptosis

BACE activity preceding AD?

Brain Trauma?

Levels of BACE increase following Traumatic Brain Injury

BACE KO protects from Traumatic Brain injury

Hippocampal lesions are reduced in BACE KO

Can BACE trafficking contribute to amylodogenic pathology?

Could BACE trafficking be targeted for therapeutic purposes?

Nature Reviews Molecular Cell Biology 10, 513-525

RAB5 involved in early endosomes formation

ARF6 regulates endocytosis

A dominant positive RAB5 mutant that leads to formation of large endosomes

Question: is BACE localized to and active in RAB5 vesicles?

BACE colocalizes to and is active in RAB5 vesicles

BACE internalizes also independently on clathrin mediated endocytosis

Localization of BACE to RAB5 positive vesicles is independent on clathrin

BACE co-localizes with ARF6 cargo molecules

BACE colocalizes with ARF6-Q67L, a mutant that blocks the sorting of the vesicles to the early endosomes

Reduced levels of BACE at the early endosomes result in decreased Abeta: regulation of the ARF6 sorting pathway as a way to control BACE activity and reduce Abeta load

ARF6 regulates the sorting of intracellular BACE

Model: control of clathrin independent endocytosis to reduce BACE levels in the endosomes, thus to reduce BACE activity