Trafficking and processing of APPand-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Pastorino, unpublished data

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.

Alpha-secretase: ADAM10, ADAM17, TACE

ADAM10 and ADAM17 expression: colocalization with APP and BACE

Higher expression in the brain

ADAM10’s expression profile is similar to the one of BACE and APP

ADAM10 and ADAM17 have a protective role: Implication in AD and cell growth

ADAM10 is essential for non-amyloidogenic processing of APP: Evidences in vitro

Characterization of ADAM10 transgenic mice

18 weeks old

ADAM10 regulates non-amyloidogenic APP processing in vivo

10 months old

ADAM10 protects from plaque deposition in APPTg V717I (Indiana) mice…

17-19 months old

…in an age-dependent fashion

ADAM10XAPPtg

ADAM10 DN XAPPtg

Overexpression of ADAM10 in double transgenic mice ADAM10 X APPV717I rescues behavioral impairment

Could ADAM10 levels decrease during aging causing AD?

Sirtuins levels are reduced in aging

DO sirtuins regulate ADAM10 expression and/or activity?

Sirtuin: deacetylation and control on protein transcription

SIRT1 Tg AD mice show reduced plaque and Abeta load

Sirt1 expression in AD miceregulates non-amyloidogenic processing of APP…

..and also levels of the -secretase ADAM10 in AD mice both as protein….

…and as mRNA

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

4 months treatment

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

…and increases alpha-secretase activity

Use of antidepressant associates with reduced PIB uptake in humans

Activation of serotoninergic receptors leads to increased non-amyloidogenic pathway

Activation of non-amyloidogenic pathway as protective from AD!

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

BACE KO mice lack amyloidgenic processing of APP

Abeta levels are reduced in BACE KO mice

Levels of BACE protein are increased in AD

BACE enzymatic activity is increased in AD brain

BACE Domains and trafficking

TMPropeptidesequence DTG DSG

DDISLLKfurin

1 501aa460-476

Regulation of BACE Trafficking

Abeta?

The LL motif, but not the S (that can be phoshorylated) regulates the amount of BACE retained at the plasma membrane…..

Pastorino et al., MCN 2002

BACE LL motif determines lysosomal colocalization for degradation

Koh et al., 2005

GGA proteins: a crucial role in the regulation of BACE trafficking and degradation through BACE LL domain

Do GGA3 and BACE levels change during neurodegenerative pathologies?

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…

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

GGA3 siRNA causes increase of BACE expression and accumulation of C99

What happens during apoptosis?

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).

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

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

During apoptosis GGA3 levels are destabilized

Apoptotic mechanisms associated with neurodegeneration stabilize BACE via the inhibition of GGA3, therefore inhibiting

GGA3-mediated BACE degradation

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

Model of BACE stabilization during apoptosis