Abstract
Background: Cholinergic basal forebrain neurons, important in memory formation, require nerve growth factor (NGF) for their continued maintenance and are vulnerable in Alzheimer’s disease (AD). Application of NGF, through its interaction with tyrosine kinase receptor A (TrkA), has proven beneficial in both AD patients and animal models of AD. Levels of the NGF precursor protein proNGF are increased early in AD brain. Since proNGF has been shown capable of inducing apoptosis, via the p75NTR neurotrophin receptor and its co-receptor sortilin, it has been suggested that this may constitute an important factor in AD pathology.
Objectives: Our aims were twofold; to produce small molecule agonists at TrkA and to use proNGF to generate a cell model of sporadic AD in which to test these.
Methods: Small molecule TrkA binders were identified using in silico screening; and structural activity relationships determined
using data collected from radioligand binding and agonist/antagonist assays in HEK cells over-expressing TrkA. Nuclear magnetic
resonance (NMR) was used to confirm small molecule binding at TrkA. Non-cleavable proNGF (proNGF-nc) was added to PC12 cells, which were characterized for downstream responses including stimulation of intracellular signaling pathways (ERK and AKT), cell viability, neuritogenesis and caspase activation.
Results:We have identified a compound which displaces NGF with an IC50 of 3μM and can activate ERK in HEK-TrkA cells. It also binds to the isolated NGF-binding domain of TrkA (TrkAd5). PC12 cells express TrkA, p75NTR and sortilin and differentiate in response to NGF, as do cholinergic neurons. Contrary to expected outcomes, we demonstrate here that although less potent than NGF, proNGF-nc acted as a low affinity, but full agonist at TrkA. Moreover, at high concentrations proNGF-nc had equivalent efficacy to NGF.We show that blocking sortilin did not affect assay outcome. Similar results were obtained with differentiated PC12 cells. Conclusions: We have produced a potential lead compound towards generation of a small molecule therapeutic for AD. Our
results also suggest that an increase in proNGF alone may not be sufficient to induce the AD pathological process, and that other concurrent changes may be necessary
Objectives: Our aims were twofold; to produce small molecule agonists at TrkA and to use proNGF to generate a cell model of sporadic AD in which to test these.
Methods: Small molecule TrkA binders were identified using in silico screening; and structural activity relationships determined
using data collected from radioligand binding and agonist/antagonist assays in HEK cells over-expressing TrkA. Nuclear magnetic
resonance (NMR) was used to confirm small molecule binding at TrkA. Non-cleavable proNGF (proNGF-nc) was added to PC12 cells, which were characterized for downstream responses including stimulation of intracellular signaling pathways (ERK and AKT), cell viability, neuritogenesis and caspase activation.
Results:We have identified a compound which displaces NGF with an IC50 of 3μM and can activate ERK in HEK-TrkA cells. It also binds to the isolated NGF-binding domain of TrkA (TrkAd5). PC12 cells express TrkA, p75NTR and sortilin and differentiate in response to NGF, as do cholinergic neurons. Contrary to expected outcomes, we demonstrate here that although less potent than NGF, proNGF-nc acted as a low affinity, but full agonist at TrkA. Moreover, at high concentrations proNGF-nc had equivalent efficacy to NGF.We show that blocking sortilin did not affect assay outcome. Similar results were obtained with differentiated PC12 cells. Conclusions: We have produced a potential lead compound towards generation of a small molecule therapeutic for AD. Our
results also suggest that an increase in proNGF alone may not be sufficient to induce the AD pathological process, and that other concurrent changes may be necessary
| Original language | English |
|---|---|
| Title of host publication | Alzheimer's & Dementia |
| Publisher | Amsterdam:Elsevier |
| Pages | S802 |
| Number of pages | 1 |
| Volume | 7 |
| DOIs | |
| Publication status | Published - 1 Jul 2011 |
Research Groups and Themes
- Cerebrovascular and Dementia Research Group