Role of Mint family of Proteins in the nanoscale localization and real-time retention of surface Amyloid Precursor

Abstract

Amyloid beta, a key determinant in the pathology of Alzheimer's disease (AD), is formed by the sequential proteolysis of Amyloid Precursor Protein (APP) by β-and γ-secretase. Evidence over the last few decades indicates that synaptic dysfunction and loss is an early event in AD that correlates with amyloid production in the whole brain and at the synaptic level. Though not well understood, previous reports have shown that APP retention at the synaptic membrane undergoes a non-amyloidogenic pathway. On the contrary, the internalization of APP at the endocytic zones leads to enhanced amyloid-beta production. Therefore, it is vital to understand the molecular parameters that control the localization and trafficking of APP at these functional zones of synaptic compartments and how this could be regulated. The organization and trafficking of molecules to functional zones of synapses result from the coordination of several regulatory steps and is tightly controlled. APP shows the heterogeneous distribution in the neuronal processes, indicating differential regulation of APP at various sub-compartments. Recent studies have demonstrated differential nanoscale localization of APP and secretases in functional zones of the synapse. Furthermore, the instantaneous distribution of APP on the synaptic membrane can be controlled by passive lateral diffusion of APP between the synaptic membrane and endocytic zone and the active internalization of APP at the endocytic zone. The dynamics and biophysical properties of APP can be influenced by the proteins which interact with APP. It is known that the scaffolding proteins play a significant role in the trafficking of APP. Munc Interacting family of proteins (Mint) are scaffolding proteins known to regulate the trafficking and processing of APP. However, the mechanism by which it modulates these processes remains unclear. The Mint family of proteins has three isoforms- Mint1/X11α, Mint2/X11β, Mint3/X11γ, all of which are evolutionary conserved. These Mint isoforms interact with the internalization motif of APP through their phosphotyrosine binding (PTB) domain. To characterize the association with APP, it is essential to understand the differential expression, localization, and exchange kinetics of Mint isoforms. We observed that expression of mRNA transcripts of Mint isoforms is developmentally regulated in the hippocampus and cortex. Additionally, we studied the differential localization of Mint1 and Mint2 in the functional zones of the synapse with super-resolution microscopy. We observed that Mint1 and Mint2 are differentially distributed in the subsynaptic compartment indicating the differential role of Mint isoforms in APP regulation. They are highly enriched in inhibitory synapses compared to the excitatory synapse. Mint1 is enriched more in the endocytic zone and active zone, while Mint2 is highly enriched in the active zone than the endocytic zone. Further, we investigated if there is a difference in trafficking between Mint isoforms with fluorescence recovery after photobleaching and single particle tracking in heterologous cell lines. The results indicated strong confinement for Mint3 and Mint2 in comparison to Mint1. Elevation of Mint1 and Mint2 in heterologous cell lines significantly impacted the nanoscale clustering of endogenous APP. Mint1 and Mint2 increased the packing density of APP molecules per cluster, indicating that APP tends to aggregate more in the presence of Mint1 or Mint2. Additionally, we evaluated how co-expression of Mint2 with APP wild type and a detrimental variant of APP (APP Swedish) can affect the lateral mobility of APP on the plasma membrane. In cells with elevated levels of Mint2, confinement of both APP wild type and APP Swedish was higher and consistent with a reduction in lateral mobility. When the interaction of Mint2 with APP was perturbed by the deletion of the PTB domain from Mint2, it resulted in a disparate effect on the lateral mobility of APP wild type and APP Swedish. However, in cells expressing a variant of APP where the internalization motif (YENPTY) of APP was deleted, Mint2 did not affect the lateral mobility of APP. These observations support that, Mint2 is a molecular determinant in controlling lateral mobility and works independently of the mutations near the transmembrane and juxta membrane region resulting in an elevation of Amyloid-beta levels. These results implicate a broader role for C-terminal interactions of APP for regulating the molecular locus of canonical processing. Additionally, these observations highlight the role of such potential mechanisms that contribute to the sporadic onset of AD.