The aspartyl protease BACE  -Amyloid cleaving enzyme

BACE is expressed mostly in the brain 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 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 TM Propeptide sequence DTGDSG DDISLLK furin aa 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 Jun 7;54(5): Ischemic patients have increased levels of BACE in the brain…

Tesco et al., Neuron Jun 7;54(5): …and decreased levels of GGA3

Tesco et al., Neuron Jun 7;54(5): AD patients have increased levels of BACE and decreased levels of GGA3 in the brain

Tesco et al., Neuron Jun 7;54(5): GGA3 siRNA causes increase of BACE expression and accumulation of C99

What happens during apoptosis?

Tesco et al., Neuron Jun 7;54(5): APP contains caspase cleavage sites in its sequence Although apoptosis increases C99 and A  levels, this effects do not depend on caspase-mediated cleavage of APP (Tesco et al., 2003). HOWEVER

Tesco et al., Neuron Jun 7;54(5): Apoptosis increases levels of C99…..

Tesco et al., Neuron Jun 7;54(5): …and BACE

Tesco et al., Neuron Jun 7;54(5): 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 Jun 7;54(5): Review. Model of BACE stabilization during apoptosis

 -secretase complex and the phenomenon of INTRAMEMBRANE PROTEOLYSIS

The  -secretase complex -PS1 and PS2: 7- to 8- transmebrane domain proteins, are the catalytic unit of the  -secretase complex (they will cleave the substrates). -Nicastrin: a type 1a transmembrane protein, approximately 130kDa molecular weight, binds to active PS1, and can sort to the plasma membrane with PS1. Nicastrin has a large extracellular domain crucial for the identification of the substrate. -APH1: (antherior pharinx defective phenotype). Its knockout leads to developmental deficit in C.elegans. It is required for the correct localization of the mature form of nicastrin at the cell surface. Pen2: (presenilin enhancer 2). Stabilizes mature presenilin and nicastrin. The gene knockout for one of any protein of the complex will result in lack of  -secretase activity.

The members of the  -secretase complex Bart Der Strooper

Evidences that the only presenilin is not enough to generate  -secretase activity 1-Overexpression of PS1 in cells does not increase  -secretase dependent cleavage of APP. 2-In yeast, a system known to have NO ENDOGENOUS  -secretase related activity, processing of APP can be observed ONLY when all the four protein of the complex are exogenously expressed.

Model for intramembrane proteolysis Christian Haass

Presenilin: an active heterodimer in the  -secretase complex The Regulated Intramembrane Proteolysis Modified from Michael Wolfe Loop domain Endoproteolysis Loop domain H2O, substrates

 -secretase cleaves different substrates Bart De Strooper

 -secretase dependent intramembrane proteolysis on multiple substrates Xia and Wolfe

Notch is a protein regulating transcription of proteins crucially involved during early and late stages of development Selkoe and Kopan

At the plasma membrane  -secretase After endocytosis  -secretase Like APP, Notch is processed via  - and  -secretase, while trafficking within the cell Selkoe and Kopan

Roles of PS1 in the cell: a switch between physiological and pathological functions in AD Catalytic subunit of the  -secretase: in AD gain of toxic function APP: generation of beta amyloid peptides in AD. NOTCH: production of NICD and control on protein transcription, might be disrupted in AD. Cadherins: control over signaling for cell growth, might be disrupted in AD. Erb4: EGF signaling, might be disrupted in AD. Other mechanisms of toxicity by loss of physiological function?

Consequences of Macroautophagy failure during aging

Autophagic Vacuoles (AVs) accumulate in AD A defective fusion of AVs to the membrane of the lysosomes caused by a loss of function of PS1?

PS1 deletion selectively inhibits macrophagic turnover of proteins (A-D) PS1 KO causes Inhibition of proteolysis that cannot be further reduced by treatment with 3MA PS1 is involved in the autophagic- dependent PROTEOLYSIS of substrates. Short lived proteins Long lived proteins 3MA blocks the formation of AVs, thus selectively inhibits macroautophagy NH 4 Cl neutralizes lysosomal pH, thus blocks lysosomal proteolysis

(F,G) PS1 KO increases the number of AVs, without affecting the pathways that lead to formation of the autophagosomes (H) PS1 deletion is insensitive to macroautophagy stimuli Serum withdrawal induces autophagy

Non-mature autophagosomes or autolysosomes accumulate in PS1KO blastocysts } Engulfed material in autolysosomes from PS1KO

Defective autophagy in PS1KO is due to improper fusion of the AVs to the lysosomes

Lysosomes of PS1KO cells have lower pH

Impairment of PS1 activity in PS1KO cells results in defective acidification of the lysosomes (increased pH), impairing the autophagosomes to fuse with the lysosomes and to allow degradation of the phagosomes’ content.

What’s the implication in AD? Evidences for improper lysosomal proteolysis in PS1 FAD A possible loss of physiological function of PS1 in AD

Defective Autophagy in PS1-FAD human fibroblasts

Autophagy could be protective from AD, PS1 loss of physiological function may be involved in AD pathology

Abeta-42 induces formation of large autophagic vacuoles wt A  42

Abeta oligomers

APP TMD NH2 COOH  -secretase BACE  -secretase AA AICDN-terminal domain  Amyloid peptide=A  -exists in 3 forms, A  40; A  42 (the most aggressive one, as it tends to aggregate more than the A  0), and A  43. The size of the different A  peptides depends on where  -secretase cuts within the sequence of A  -A  peptides  aggregate forming the core of the plaque in AD

Where are Abeta peptides formed? At an intracellular level, either in late endosomes, ER or in lipid rafts

How can A  peptides reach the extracellular space to form the plaque? 2 hypothesis: 1- Secretion. A  peptides are not retained at the phospholipidic membrane, can be secreted in an hydrophilic environment such as the cytosol or the extracellular medium. In vitro, A  40 is very well harvetsed in the medium of cultured cells, very low levels of A  42 are detectable. 2-Formation of toxic aggregates in the cell. A  peptides, formed within the cell, tend to form aggregates (A  oligomers) which may have toxic properties and may lead cell death. Intracellualr material including the A  aggregates will be released from the cell, and can now be available in the extracellular space to form the core of the plaque.

Role of A  in modulating synaptic transmission Neuronal electrical activity modulates APP processing and A  release APP modulates neuronal signaling A  as a negative regulator of excitatory transmission

1-Excitatory glutamatergic transmission in the hippocampus increases A  release: study in hippocampal slices using either GABA-A inhibitors or stimulators. 2- Excitatory glutamatergic transmission enhances either BACE intrinsic activity or the amount of APP that is available for BACE cleavage. Neuronal electrical activity modulates APP processing and A  release

1-APP overexpression inhibits glutamatergic signaling; gamma-secretase processing of APP is required for the depressive effects played by APP and the resulting A  peptide release depresses, in a reversible way, glutamatergic activity. A  as a negative regulator of excitatory transmission APP modulates neuronal signaling Thus, early A  production could be an EARLY EVENT that initiates cognitive decline, characteristic of AD. This would lead to neuronal death, and eventually to formation of  -amyloid plaques.

WHO IS THE ENEMY TO FIGHT IN AD? Plaques or early A  formation and deposition? This is a crucial point in the treatment of AD.

Evidences that A  oligomers-induced toxicity is an early event preceding plaque formation -In humans, levels of cortical soluble A  correlate with cognitive decline -In AD cerebral cortex, monomeric an stable A  oligomers (up to ~12kDa in SDS-PAGE). -Similar oligomers have been detected in the hippocampus and cortex of humans in absence of amyloid plaques, and in many cases in absence of NFT (is A  pathology preceding tau pathology?). -Transgenic animal models overexpressing double mutant forms of APP (Swedish and a modified London mutation, Val717Phe) develop cognitive and behevioral deficit months before the deposition of  -amyloid plaques. -In neuronal cultures, cell derived soluble A  oligomers impair synaptic plasticity.

Nat Med Aug;14(8): Soluble monomeric and oligomeric A  is found specifically in AD

Nat Med Aug;14(8): Membrane associated monomeric and oligomeric A  is found mainly in AD

Nat Med Aug;14(8): Insoluble monomeric and oligomeric A  is not specific of AD

Nat Med Aug;14(8): Soluble A  extracted from AD brain alters synaptic activity specifically in AD patients

Soluble A  is toxic to the neuron causing excitotoxicity

Insoluble A  is the component of the core of the plaques. Does insoluble A  affect synaptic physiology and activity?

Nat Med Aug;14(8): Only plaque cores exposed to FA reduce synaptic activity

Insoluble A  in the core is not toxic per se. Soluble A  monomers and oligomers are toxic causing early defects in glutamatergic signaling and synaptic activity.

Extracellular Abeta42 oligomers are toxic to synaptic activity Abeta42 is produced intracellularly Is intracellular Abeta42 toxic?

Neuronal Abeta42 immunoreactivity increases with aging in AD mice 2 months 10 months

Abeta42 accumulates within soma and dendrites in human AD brain