Tese de Doutoramento Medicina 2023 Faculdade de Ciências Médicas, Universidade NOVA de Lisboa

In the central nervous system, excitatory synaptic transmission mainly takes place at dendritic spines, structures rich in actin. Actin stabilizes and regulates dendritic spine structures. The stabilization of spines is essential for the maintenance of long-term memory and proper neural circuit formation. Therefore, actin plays a central role in synapse assembly and function. In late-onset Alzheimer’s disease (LOAD) the earliest memory alterations are due to synaptic dysfunction, yet the causal mechanisms are not known. Amyloid-β (Aβ) accumulation plays a central role in AD pathology. Aβ is produced by sequential cleavage of amyloid precursor protein (APP) by BACE1 and γ-secretase at the earlyendosomal membrane during intracellular trafficking of APP through the endocytic pathways. Altered trafficking of APP can disrupt Aβ production. For the past decade, several rare coding variants were identified in LOAD genetic risk factors that have created a broader picture of the cellular mechanisms involved in AD risk. One of which is CD2-associated protein (CD2AP), an adaptor molecule with a binding site for actin that regulates membrane trafficking and has been reported to be involved in the intracellular trafficking of APP. The role of the CD2AP mutation associated with a higher risk of AD however is not yet understood. Here, we present evidence that the LOAD risk gene CD2AP and its AD-associated genetic variant K633R play an important role in the postsynaptic development of neurons by regulating spinal actin. We found that CD2AP is enriched in dendritic spines and colocalizes with post-synaptic density protein. Its knockdown impairs spine development and synapse formation by actin-related mechanisms. Importantly, the C-terminal domain is required for CD2AP localization to spines. These findings identify CD2AP as a novel regulator of synaptic function and neuronal networks. We investigated the effect of CD2AP overexpression and KD/KO on intracellular Aβ and APP distribution and APP degradation in neuronal cells. We observed that CD2AP, both wild-type and mutant increased intracellular Aβ42 levels. CD2AP increases APP sorting to the lumen of the Rab5-positive endosomes. While in cells expressing the mutant form of the protein, APP does not sort into the lumen and remains at the endosomal membrane. Moreover, the CD2AP mutant reduced the degradation of APP which happens at a faster rate compared to the mutant-expressing cells. We showed that increased intracellular APP levels in cells expressing the mutant protein may be the reason for the increased accumulation of Aβ42 at synaptic sites, observed in primary neurons. These results may explain the impaired cellular function of the CD2AP mutation the AD risk factor, in APP processing and degradation, leading to Aβ42 accumulation and creation of the senile plaques, an important hallmark in AD. In summary, we discovered that the CD2AP(K633R) LOAD rare coding mutation contributes to AD pathology through two distinct yet related mechanisms.CD2AP LOAD mutation can contribute to AD pathology by impairing APP degradation and increasing the accumulation of Aβ42 in the cells and at the synapses. We also found that CD2AP LOAD mutation can impact neurons independent of Aβ, and through actin by regulating synaptic architecture. Thus, CD2AP mutation exerts its dual function in neurons and on synapses by first, regulating spinal actin, and second, by altering vesicular trafficking and APP degradation, yet both mechanisms are related since elevated Aβ levels produced due to impaired APP degradation, accumulate at synaptic sites, the spines, which are found in higher density in mutant cells, a synergy that can potentially cause an overexcitability of neurons, an early pathological event in AD