May 8th, BIOSE108

Therapeutic approaches in AD:

-Beta and gamma secretase inhibitors

-Abeta immunotherapy

-Tau

-Ongoing treatments in AD

- and -secretase : possible therapeutic targets in AD?

Could BACE be considered as a therapeutic target for AD?In favor:

1- BACE is the primary -secretase aspartyl protease that cleaves APP generating -amyloid species (secondary role of BACE2)

2-BACE KO mice DO NOT have amyloidogenic processing of APP

Against:

1-BACE+/- heterozygous mice do not show altered production of -amyloid and APP C-terminal fragments

2-BACE cleaves a number of other substrates, including *Amyloid Precursor Like Proteins APLP1 and APLP2*Low-density lipoprotein receptor (LDLR)-related protein LRPThus, targeting BACE for the treatment of AD would result in loss of BACE activity towards the other substrates, with possible consequent implications on their physiological function.

Hu et al.,

BACE KO mice show hypomyelination of hippocampal neurons…

Hu et al.,

…and of the optic and sciatic nerves

Optic

Sciatic

Hu et al.,

BACE KO- mediated hypomyelination is due to reduced activation of neuregulin

BACE inhibitor

ADAM inhibitor

BACE inhibition reduces or delays myelination in vitro

BACE1KO primary hippocampal neurons have impaired axonal outgrowth

BACE KO mice have axon guidance defects in the hippocampus

L1 (CHL1) t2 a substrate for BACE?

BACE KO mice have the same phenotype as the neuronal cell adhesion molecule close homolog of L1 (CHL1) t2 KO mice

CHL1 undergoes BACE1-dependent cleavage in hippocampus

CHL1 and BACE1 co-localize in growth cones of primary hippocampal neurons.

Thus reducing BACE levels (or activity) may lead to neurodegeneration as side effect

-secretase cleaves different substrates

Some of these substrates have crucial activity in regulating cell fate decision. In this respect, targeting -secretase for the treatment of AD is not an easy task, as blocking -secretase activity would have consequences on the physiological functions of the other protein substrates.

What about targeting -secretase for the treatment of AD?

Bart De Strooper

Model for -secretase complex and its interaction to the substrates

The bars represent the transmembrane domains of the the proteins constituting the -secretase complex

Aldehyde-type calpain and proteasome inhibitors.Weak potency and lack of selectivity:First studiedDifferential production of Abeta 40 to Abeta 42

Aldehydes are readily hydrated to a form that resembles the transition state of aspartyl protease catalysis.

Transition state analogs: whether aldehyde or keto forms, they can be active toward serine and cisteine proteases. Conversion of one of these difluoroalcohol peptidomimetics into an affinity labeling reagent led to identification of PS1 NTF and CTF as the direct targets of this type of inhibitor.

Hydroxyethylamines, more potent.Can isolate gamma-secretase in a detergent-solubilized state and demonstrate that immunoprecipitation of presenilin brought down gamma-secretase activity.

Gamma secretase inhibitors, peptide based inhibitors

DAPT“showed good inhibitory potency orally active in vivo, capable of lowering brain Abeta levels in an APP transgenic mice. The target is the CTF.”

Well known DAPT derivatives are the more potent Compound E and LY-411,575, the last one studied in vivo in humans.

Compound E: “effective in reducing brain Abeta levels in APP transgenic mice upon oral dosing. However, this compound also illustrated the toxicity issues that might be expected by GSI over Notch (gastrointestinal bleeding and immunosuppression caused by peripheral inhibition of Notch). Despite this ominous result, non-selective GSIs of this type continued to be pursued on evidence from animal studies that careful dosing couldidentify a therapeutic window (e.g. Hyde et al. 2006)”.

LY-411,575

Structure of the inhibitors LY-411,575 and LY-D

LY-411-575 reduces Abeta levels in the plasma and in the brain of TgCRND8 mice

Chronic administration of LY-411,575 causes atrophy of the thymus in TgCNDR8 mice (15 days)

One lobule One lobuleTwo lobules

Decreased Lymphocytes B generation in chronically treated TgCNRD8 mice with LY-411,575: Immunosuppression

Chronic administration of LY-411,575 causes deterioration of the intestinal epithelium

Higher doses of Semagacestat

Inactivation of Notch1 causes the formation of skin tumors

Notch1KO

Notch sparing GSMAbeta42 lowering GSMPS1-selective

g-secretase inhibitor

Other more selective gamma-secretase inhibitors

PS2 is part of the gamma-secretase too, and can actively cleave both APP and Notch

What could happen if GSIs were designed to target only PS1 and not PS2?

PS2-sparing GSI (MRK-560) reduces A40 and A42 levels in plasma and brain

plasma

brain

Lack of PS2 expression in PS2 KO mice, but not lack of endogenous PS2 expression, exacerbates guts histopathology

PS2 is present, although at moderate levels, in brain, spleen and thymus

Under conditions of PS1-specific inhibition, PS2 can compensate for PS1, although not up-regulated.

PS1 is not an easy therapeutic target for the treatment of AD

Could Nicastrin be a good target?

BACE-secretase

Mechanism by which Nicastrin participates in the “Regulated Intracellular Proteolysis” RIP

Steps:1-The substrates gets in close proximity with the -secretase complex (i.e. after internalization from the plasma membrane).2-Nicastrin specifically recognizes the substrate, and binds to it.3-Presenilin (PS1) cuts the substrate within the exposed sequence (in the case of APP will be the A sequence).

Shah et al.,

Chemical blocking of the N-terminal portion of the substrates will regulate nicastrin capability to recognize and to bind to it.

Implications for the treatment for AD

Will bind to Nicastrin

Will NOT bind to Nicastrin

Shah et al.,

How to target A oligomers?

Using molecules that interfere with the structure of the oligomer and break it up to single A monomers.

Advantages of this therapeutic approach would be:1-decreased accumulation of A oligomers, thus reduced formation of-amyloid plaques

2-the single A monomers have higher chances to be removed by the action of clearing enzymes like neprilysin or Insulin Degrading Enzyme IDE

3-both intracellular and extracellular formed A oligomers would be targeted and disrupted.

New approaches for future therapeutic intervention in AD

1-molecules that disrupt the structure of the A oligomers

2-use of selected - or -secretase inhibitors.

3-used of vaccines, to remove A deposits and plaques

Events leading to AD and roles of A immune-therapy

Active and Passive immunization in AD immuno-therapy

APC: antigen Presenting Cells

Abeta immuno-therapy reduces the plaque load in AD animal model

Nicoll et al.,

Before immunization, AD patients show plaques with dystrophic neurites (a) and tau staining (b)

Nicoll et al.,

Areas devoid of A plaques (c) do show NFTs but not dystrophic neurites (d)

Macrophages infiltrates the cerebral white matter in patients treated with A42 vaccine

Meningoencephalites in AD patients treated with AN1792

Vacuolation and refraction in myelinated fibers in the white matter

Infiltration of cerebral white matter by macrophages

Nicoll et al.,

Ongoing clinical trials for passive Abeta immuno-therapy

Ongoing clinical trials for active Abeta immuno-therapy

The protein tau and tau pathology in AD

Alzheimer’s disease: characterized by extracellular depositions, the -amyloid plaque, and intracellular depositions, the Neurofibrillary Tangles (NFT) comprised of Paired Helical Filaments (PHF), aggregates of hyperphosphorylated protein tau.

Deposition of fibrillar proteinacious material in Alzheimer’s disease

Bossy-Wetzel E, et al., Nat Med. 2004 Jul;10 Suppl:S2-9. Review.

The human tau gene and the 6 tau isoforms

Functional domains in tau

www.emdbiosciences.com

Tau binds to microtubules regulating their connections with other cytoskeletal components such as neurofilaments

This function is regulated by phospho/dephospho state of tau

Hyperphosphorylatd tau, antibodies and function

Normal and abnormal phosphorylation of tau

Tau hyperphosphorylation and NFT in AD

Tau hyperphosphorylation is not specific of AD, but occurs in other NADD

Tau hyperphosphorylation and NFT in FTDP-17 and Down’s syndrome

A pathology Tau pathology?

APPtgXtauP301Ltg tauP301Ltg

APP pathology increases tau pathology in APPtgXtauP301LtgAPP pathology increases tau hyperphosphorylation in an age-dependent fashion

A42 intracerebral injection in tau Tg mice increases NFTs numberA42 intracerebral injection in tau Tg mice causes tauopathy

phospho tau

Non injected Injected

amygdala

Br J Clin Pharmacol. 2011 Oct 28. doi: 10.1111/1365-2125.2011.04134.x

Therapeutic targets for A pathology

Br J Clin Pharmacol. 2011 Oct 28. doi: 10.1111/1365-2125.2011.04134.x

Other therapeutic approaches used in the treatment of AD

1-Use of cholinesterase inhibitors

2-NSAID (non steroidal antiinflammatory drugs)

3-Anti oxidant vitamins

All these approaches are used in the clinical treatment of AD.In vivo and in vitro, they reduce the amount of A release and slow down the progression of the disease. However, most of the times these are SYMPTOMATIC approaches, as they works in pathways related to AD, but not directly on those pathways that regulate formation and aggregation of A into oligomers and/or plaques.