quantitative-analysis-mitochondria-associated-endoplasmic-reticulum

Quantitative Analysis of Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM) Stabilization in a Neural Model of Alzheimer's Disease (AD) 

A method to quantitate the stabilization of Mitochondria-Associated endoplasmic reticulum Membranes (MAMs) in a 3-dimensional (3D) neural model of ...

Massachusetts General Hospital, Harvard Medical School

Axonal generation of Alzheimer's disease (AD)-associated amyloid-β (Aβ) plays a key role in AD neuropathology, but the cellular mechanisms involved in its release have remained elusive. We previously reported that palmitoylated APP (palAPP) partitions to lipid rafts where it serves as a preferred substrate for β-secretase. Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are cholesterol-rich lipid rafts that are upregulated in AD. Here, we show that downregulating MAM assembly by either RNA silencing or pharmacological modulation of the MAM-resident sigma1 receptor (S1R) leads to attenuated β-secretase cleavage of palAPP. Upregulation of MAMs promotes trafficking of palAPP to the cell surface, β-secretase cleavage, and Aβ generation. We develop a microfluidic device and use it to show that MAM levels alter Aβ generation specifically in neuronal processes and axons, but not in cell bodies. These data suggest therapeutic strategies for reducing axonal release of Aβ and attenuating β-amyloid pathology in AD.

Axonal generation of amyloid-β from palmitoylated APP in mitochondria-associated endoplasmic reticulum membranes.

Genome-wide association studies have identified BIN1 (Bridging integrator 1) and RIN3 (Ras and Rab interactor 3) as genetic risk factors for late-onset Alzheimer's disease (LOAD). The neuronal isoform of BIN1 (BIN1V1), but not the non-neuronal isoform (BIN1V9), has been shown to regulate tau-pathology and Aβ generation via RAB5-mediated endocytosis in neurons. BIN1 directly interacts with RIN3 to initiate RAB5-mediated endocytosis, which is essential for β-secretase (BACE1)-mediated β-secretase cleavage of β-amyloid precursor protein (APP) to generate Amyloid-β (Aβ), the key component of senile plaques in AD. Understanding the regulatory roles of BIN1 (neuronal BIN1V1) and RIN3 in β-secretase mediated cleavage of APP and Aβ generation is key to developing novel therapeutics to delay or prevent AD progression. Neuronal and non-neuronal isoforms of BIN1 (BIN1V1 and BIN1V9, respectively) were introduced with RIN3 into an in vitro cell-based system to test RIN3-dependent effects of neuronal BIN1V1 and non-neuronal BIN1V9 on β-secretase-mediated cleavage of APP and Aβ generation. Confocal microscopy was performed to examine RIN3-dependent subcellular localization of BIN1V1 and BIN1V9. Western blot analysis was performed to assess the effects of RIN3 and BIN1V1/BIN1V9 on β-secretase mediated processing of APP. We enriched cells expressing BIN1V1 without or with RIN3 via FACS to measure Aβ generation using Aβ ELISA assay, and to evaluate APP internalization by chasing biotinylated or antibody-labeled cell surface APP. Neuronal BIN1V1 containing the CLAP domain and non-neuronal BIN1V9 lacking the CLAP domain are the major isoforms present in the brain. Employing confocal microscopy, we showed that RIN3 differentially regulates the recruitment of both BIN1V1 and BIN1V9 into RAB5-endosomes. We further showed that BIN1V1, but not BIN1V9, downregulates β-secretase (BACE1)-mediated processing of APP in a RIN3-dependent manner. Overexpression of BIN1V1 also attenuated Aβ generation in a RIN3-dependent manner. Using cell-based internalization assays, we show BIN1V1, but not BIN1V9, delays the endocytosis of APP, but not of BACE1, into early endosomes, thereby spatially and temporally separating these two proteins into different cellular compartments, resulting in reduced cleavage of APP by BACE1 and reduced Aβ generation-all in a RIN3-dependent manner. Finally, we show that RIN3 sequesters BIN1V1 in RAB5-positive early endosomes, likely via the CLAP-domain, resulting in attenuated β-secretase processing of APP and Aβ generation by delaying endocytosis of APP. Our findings provide new mechanistic data on how two AD-associated molecules, RIN3 and BIN1 (neuronal BIN1V1), interact to govern Aβ production, implicating these two proteins as potential therapeutic targets for the prevention and treatment of AD.

The neuronal-specific isoform of BIN1 regulates β-secretase cleavage of APP and Aβ generation in a RIN3-dependent manner.

Author Correction: The neuronal-specific isoform of BIN1 regulates β-secretase cleavage of APP and Aβ generation in a RIN3-dependent manner.

We previously demonstrated that regulating mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) affects axonal Aβ generation in a well-characterized three-dimensional (3D) neural Alzheimer's disease (AD) model. MAMs vary in thickness and length, impacting their functions. Here, we examined the effect of MAM thickness on Aβ in our 3D neural model of AD.

Stabilization of mitochondria-associated endoplasmic reticulum membranes regulates Aβ generation in a three-dimensional neural model of Alzheimer's disease.

Raja Bhattacharyya

Massachusetts General Hospital, Harvard Medical School

Quantitative Analysis of Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM) Stabilization in a Neural Model of Alzheimer's Disease (AD)