Investigating the Molecular Function of APP Interacting Proteins in Synaptic Neuromodulation

Alzheimer’s disease (AD) and autism spectrum disorder (ASD) are neurological disorders characterised by impairments in synaptic function and activity. Contactin-4 (CNTN4) is a synaptic adhesion molecule implicated in dendritic spine formation, neurite outgrowth, and synaptic transmission, often in association with its binding partner, amyloid precursor protein (APP). First identified as an ASD risk gene, evidence also suggests a potential role for CNTN4 in AD, but the exact molecular and cellular mechanisms through which CNTN4 influences synaptic function are not well understood. This thesis aimed to characterise CNTN4’s role in excitatory synapses and its significance to neurological disorders. We first investigated the effects of CNTN4 and APP on neurite outgrowth using neuronal-like cell models, assessing downstream effects on genes associated with synaptic plasticity. Secondly, we examined the interactions of CNTN4 with α-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid glutamate receptors (AMPARs), which mediate fast excitatory synaptic transmission and are often dysregulated in neurological disorders. Finally, we examined whether epigenetic methylation changes contribute to synaptic impairments by comparing Cntn4 knockout (KO) mice with a second mouse model of intellectual disability. In the prefrontal cortex of individuals with late-stage AD, CNTN4 mRNA levels were significantly downregulated compared to age-matched controls, and neuritogenesis was promoted in human neuroblastoma cell lines by CNTN4 and APP overexpression in a non-additive manner. Pull-down data indicated that CNTN4 interacts with the GluA1 and GluA2 subunits of AMPARs primarily via its extracellular fibronectin type III (FNIII) domain. Notably, this interaction occurs independently of APP. Acute knockdown of Cntn4 resulted in a reduction of both surface and total GluA1/2 levels, which could not be rescued by CNTN4 lacking its signal peptide. CNTN4 ASD-associated mutations, N178D and Y630C, failed to restore GluA2 surface and total expression, highlighting mechanisms underpinning CNTN4 dysfunction and synaptic phenotypes in ASD. Interestingly, GluA1 deficits, but not GluA2, were partially rescued by lysosomal inhibition, with GluA2 appearing retained in intracellular organelles, suggesting that CNTN4 specifically facilitates forward trafficking of GluA2. DNA methylation analyses in Cntn4 KO mice revealed widespread changes in synaptic genes, including AMPAR subunits and genes encoding synaptic scaffolding proteins such as Shank2 and Nrxn1/2. These results identify CNTN4 as a novel regulator of AMPAR trafficking, acting through distinct but potentially complementary mechanisms to APP. Collectively, this thesis demonstrates that CNTN4 is a regulator of excitatory synapses, whose dysfunction can contribute to ASD-related synaptic deficits and late-stage AD pathology.<p></p>

Author dc:creator

• Madie Eve (21053195)

• Autism
• Alzheimer's disease
• Cell adhesion molecule
• Synaptic plasticity
• AMPA receptor
• Amyloid precursor protein
• Contactin-4

Statement dc:rights

• All rights reserved
• Open Access after 2027-11-22

Identifier

10779/exe.32366454.v1

OAI identifier oai:identifier

oai:figshare.com:article/32366454