Alzheimer's Disease: simultaneous modulation of NMDAR, BACE1, APP and Fyn activity by palmitoylation-targeting CIRC825 - PROJECT SUMMARY/ABSTRACT
Alzheimer’s Disease (AD) has predominantly been characterized by amyloid plaques and neurofibrillary tau
tangles, and soluble oligomers of these peptides are thought to be toxic; hence, targeting these histopathological
hallmarks has led to a long series of clinical trials, but to date they have all failed. Intriguingly, aberrant
palmitoylation of multiple proteins has been reported in AD. Accordingly, we seek here to simultaneously
modulate the location and activity of multiple AD-associated proteins (neurotransmitter receptor NMDAR,
amyloid plaque-associated BACE1 and APP, and neurofibrillary tangle-associated Fyn kinase) by altering their
shared post-translational modification, palmitoylation. To this effort, we will transfer learnings from a drug
development program of a small molecule that removes palmitate from proteins, N-(tert-butyl)-hydroxylamine
(NtBuHA, called CIRC825) in the well-defined neurodegenerative disease that is associated with over-
palmitoylation, CLN1 Batten (CLN1). Of great importance to this AD-related grant, extrasynaptic (e)NMDARs
containing predominantly GluN2B subunits have been implicated to be overactivated in the pathogenesis of AD.
In a feasibility study demonstrating our approach will work in the context of human AD, our preliminary findings
have recently shown that CIRC825 treatment virtually eliminates the aberrant hyperexcitable phenotype in
human AD hiPSC cerebrocortical neurons compared to WT. Additionally as feasibility data, we investigated
CIRC825 in CHOAPP cells (which overexpress APP), and found it lowered palmitoylation of APP, and it decreased
Aβ42 generation (the pathogenic form) without decreasing soluble Aβ40. The aims of this Phase 1 work are
focused on expanding our understanding the pathomolecular importance of the palmitoylation of APP, BACE1,
Fyn, and NMDAR in both Cln1-/- mouse neurons and in human (h)iPSC-derived 2D cortical neuronal cultures
and 3D cerebral organoids that have familial AD mutations. Human iPSCs to be used include wPSEN1
M146V/WT and APPswe/WT hiPSC lines as well as the PSEN1 ΔE9/WT hiPSC line (with WT isogenic controls).
We will modulate palmitoylation in these models by treating them with CIRC825. Finally, in a Cln1-/- mouse
model, we will compare physiologic benefits of CIRC825 to those of memantine. Specifically, in aim 1 we will
evaluate CIRC825 in cultured CLN1-/- neurons and (h)iPSC-derived AD neurons for 1) effects on decreasing
palmitoylation of APP, BACE1, Fyn, and NMDAR; 2) suppression of extra-synaptic dendritic Ca2+ influxes by
calcium imaging, and 3) mitigation of glutamate-induced cell death in an excitotoxicity assay. In aim 2 we will
test concentration-dependent physiologic effects of CIRC825 on (h)iPSC-derived 3D cerebral organoids (with
familial AD mutations and WT controls), using multiple assays including patch-clamp electrophysiology, calcium
imaging, multi-electrode array experiments, and immunohistochemistry. In aim 3 we will investigate the dose-
dependent response of CIRC825 and memantine in CLN1-/- mice, assessing for motor deficits by a rotarod
analysis, cognitive deficits by a water-maze and Y-maze assessment, and control of seizures by EEG. On
completion of this work, we aim to demonstrate with greater confidence that palmitoylation is a druggable target
in AD, which, to our knowledge, is a novel pharmacologic approach in AD. Further, as CIRC825 is being
investigated in IND-enabling studies, we aim to develop a data package on CIRC825 that justifies more rigorous
investigations of its efficacy in AD models, with the ultimate goal of clinical translation of CIRC825 (or other
hydroxylamine derivatives) in AD. Results of this work will guide a Phase II program that is focused on a more
targeted dose range assesment in Cln1-/- sheep and investigations in a broader set of AD models, including in
hAPP-J20 and 5XfAD transgenic mice.
