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 ...

Tufts University School of Medicine

Golgi-localized γ-ear-containing ARF binding protein 3 (GGA3) is a monomeric clathrin adaptor that has been shown to regulate the trafficking of the Beta-site APP-cleaving enzyme (BACE1), which is required for production of the Alzheimer's disease (AD)-associated amyloid βpeptide. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that depletion of GGA3 results in increased BACE1 levels and activity owing to impaired lysosomal trafficking and degradation. We further demonstrated the role of GGA3 in the regulation of BACE1 in vivo by showing that BACE1 levels are increased in the brain of GGA3 null mice. We report here that GGA3 deletion results in novelty-induced hyperactivity and decreased anxiety-like behaviors. Given the pivotal role of GABAergic transmission in the regulation of anxiety-like behaviors, we performed electrophysiological recordings in hippocampal slices and found increased phasic and decreased tonic inhibition in the dentate gyrus granule cells (DGGC). Moreover, we found that the number of inhibitory synapses is increased in the dentate gyrus of GGA3 null mice in further support of the electrophysiological data. Thus, the increased GABAergic transmission is a leading candidate mechanism underlying the reduced anxiety-like behaviors observed in GGA3 null mice. All together these findings suggest that GGA3 plays a key role in GABAergic transmission. Since BACE1 levels are elevated in the brain of GGA3 null mice, it is possible that at least some of these phenotypes are a consequence of increased processing of BACE1 substrates.

Genetic Deletion of the Clathrin Adaptor GGA3 Reduces Anxiety and Alters GABAergic Transmission.

β-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the rate-limiting enzyme in the production of amyloid beta (Aβ), the toxic peptide that accumulates in the brains of Alzheimer's disease (AD) patients. Our previous studies have shown that the clathrin adaptor Golgi-localized γ-ear-containing ARF binding protein 3 (GGA3) plays a key role in the trafficking of BACE1 to lysosomes, where it is normally degraded. GGA3 depletion results in BACE1 stabilization both in vitro and in vivo. Moreover, levels of GGA3 are reduced and inversely related to BACE1 levels in post-mortem brains of AD patients.

BACE1 elevation engendered by GGA3 deletion increases β-amyloid pathology in association with APP elevation and decreased CHL1 processing in 5XFAD mice.

The β-secretase β-site APP-cleaving enzyme 1 (BACE1) is deemed a major culprit in Alzheimer's disease, but accumulating evidence indicates that there is more to the enzyme than driving the amyloidogenic processing of the amyloid precursor protein. For example, BACE1 has emerged as an important regulator of neuronal activity through proteolytic and, most unexpectedly, also through nonproteolytic interactions with several ion channels. Here, we identify and characterize the voltage-gated K channel 3.4 (Kv3.4) as a new and functionally relevant interaction partner of BACE1. Kv3.4 gives rise to A-type current with fast activating and inactivating kinetics and serves to repolarize the presynaptic action potential. We found that BACE1 and Kv3.4 are highly enriched and remarkably colocalized in hippocampal mossy fibers (MFs). In BACE1 mice of either sex, Kv3.4 surface expression was significantly reduced in the hippocampus and, in synaptic fractions thereof, Kv3.4 was specifically diminished, whereas protein levels of other presynaptic K channels such as K1.1 and K2.3 remained unchanged. The apparent loss of presynaptic Kv3.4 affected the strength of excitatory transmission at the MF-CA3 synapse in hippocampal slices of BACE1 mice when probed with the Kv3 channel blocker BDS-I. The effect of BACE1 on Kv3.4 expression and function should be bidirectional, as predicted from a heterologous expression system, in which BACE1 cotransfection produced a concomitant upregulation of Kv3.4 surface level and current based on a physical interaction between the two proteins. Our data show that, by targeting Kv3.4 to presynaptic sites, BACE1 endows the terminal with a powerful means to regulate the strength of transmitter release. The β-secretase β-site APP-cleaving enzyme 1 (BACE1) is infamous for its crucial role in the pathogenesis of Alzheimer's disease, but its physiological functions in the intact nervous system are only gradually being unveiled. Here, we extend previous work implicating BACE1 in the expression and function of voltage-gated Na and K channels. Specifically, we characterize voltage-gated K channel 3.4 (Kv3.4), a presynaptic K channel required for action potential repolarization, as a novel interaction partner of BACE1 at the mossy fiber (MF)-CA3 synapse of the hippocampus. BACE1 promotes surface expression of Kv3.4 at MF terminals, most likely by physically associating with the channel protein in a nonenzymatic fashion. We advance the BACE1-Kv3.4 interaction as a mechanism to strengthen the temporal control over transmitter release from MF terminals.

β-Secretase BACE1 Promotes Surface Expression and Function of Kv3.4 at Hippocampal Mossy Fiber Synapses.

Axonal dystrophy, indicative of perturbed axonal transport, occurs early during Alzheimer's disease (AD) pathogenesis. Little is known about the mechanisms underlying this initial sign of the pathology. This study proves that Golgi-localized γ-ear-containing ARF binding protein 3 (GGA3) loss of function, due to  genetic deletion or a  rare variant that cosegregates with late-onset AD, disrupts the axonal trafficking of the β-site APP-cleaving enzyme 1 (BACE1) resulting in its accumulation in axonal swellings in cultured neurons and in vivo. We show that BACE pharmacological inhibition ameliorates BACE1 axonal trafficking and diminishes axonal dystrophies in  null neurons in vitro and in vivo. These data indicate that axonal accumulation of BACE1 engendered by GGA3 loss of function results in local toxicity leading to axonopathy.  deletion exacerbates axonal dystrophies in a mouse model of AD before β-amyloid (Aβ) deposition. Our study strongly supports a role for GGA3 in AD pathogenesis, where GGA3 loss of function triggers BACE1 axonal accumulation independently of extracellular Aβ, and initiates a cascade of events leading to the axonal damage distinctive of the early stage of AD.

 deletion and a  rare variant associated with late onset Alzheimer's disease trigger BACE1 accumulation in axonal swellings.

Neural cell adhesion molecules 1 (NCAM1) and 2 (NCAM2) belong to the cell adhesion molecules of the immunoglobulin superfamily and have been shown to regulate formation, maturation, and maintenance of synapses. NCAM1 and NCAM2 undergo proteolysis, but the identity of all the proteases involved and how proteolysis is used to regulate their functions are not known. We report here that NCAM1 and NCAM2 are BACE1 substrates in vivo. NCAM1 and NCAM2 overexpressed in HEK cells were both cleaved by metalloproteinases or BACE1, and NCAM2 was also processed by γ-secretase. We identified the BACE1 cleavage site of NCAM1 (at Glu 671) and NCAM2 (at Glu 663) using mass spectrometry and site-directed mutagenesis. Next, we assessed BACE1-mediated processing of NCAM1 and NCAM2 in the mouse brain during aging. NCAM1 and NCAM2 were cleaved in the olfactory bulb of BACE1+/+ but not BACE1-/- mice at postnatal day 10 (P10), 4 and 12 months of age. In the hippocampus, a BACE1-specific soluble fragment of NCAM1 (sNCAM1β) was only detected at P10. However, we observed an accumulation of full-length NCAM1 in hippocampal synaptosomes in 4-month-old BACE1-/- mice. We also found that polysialylated NCAM1 (PSA-NCAM1) levels were increased in BACE1-/- mice at P10 and demonstrated that BACE1 cleaves both NCAM1 and PSA-NCAM1 in vitro. In contrast, we did not find evidence for BACE1-dependent NCAM2 processing in the hippocampus at any age analyzed. In summary, our data demonstrate that BACE1 differentially processes NCAM1 and NCAM2 depending on the region of brain, subcellular localization, and age in vivo.

Spatiotemporal processing of neural cell adhesion molecules 1 and 2 by BACE1 in vivo.

Neurodegenerative diseases (NDs) are heterogeneous neurological disorders characterized by a progressive loss of selected neuronal populations. A significant risk factor for most NDs is aging. Considering the constant increase in life expectancy, NDs represent a global public health burden. Axonal transport (AT) is a central cellular process underlying the generation and maintenance of neuronal architecture and connectivity. Deficits in AT appear to be a common thread for most, if not all, NDs. Neuroinflammation has been notoriously difficult to define in relation to NDs. Inflammation is a complex multifactorial process in the CNS, which varies depending on the disease stage. Several lines of evidence suggest that AT defect, axonopathy and neuroinflammation are tightly interlaced. However, whether these impairments play a causative role in NDs or are merely a downstream effect of neuronal degeneration remains unsettled. We still lack reliable information on the temporal relationship between these pathogenic mechanisms, although several findings suggest that they may occur early during ND pathophysiology. This article will review the latest evidence emerging on whether the interplay between AT perturbations and some aspects of CNS inflammation can participate in ND etiology, analyze their potential as therapeutic targets, and the urge to identify early surrogate biomarkers.

Pathophysiology of neurodegenerative diseases: An interplay among axonal transport failure, oxidative stress, and inflammation?

Available models to study neuropathological diseases include cell cultures and animal models. Brain pathologies, however, are often poorly recapitulated in animal models. 2D cell culture systems are well established and have been used since the early 1900s to grow cells on flat dishes. However, conventional 2D neural culture systems, which lack key features of the brain's 3D microenvironment, often inaccurately represent the diversity and maturation of multiple cell types and their interaction under physiological and pathological conditions.To improve CNS modeling, we have designed a 3D bioengineered neural tissue model generated from human iPSC-derived neural precursor cells (NPCs). This NPC-derived biomaterial scaffold, composed of silk fibroin with an intercalated hydrogel, matches the mechanical properties of native brain tissue and supports the long-term differentiation of neural cells in a donut-shaped sponge within an optically clear central window. This chapter describes integrating iPSC-derived NPCs in these silk-collagen scaffolds and differentiating them into neural cells over time.

A 3D Bioengineered Neural Tissue Model Generated from Human iPSC-Derived Neural Precursor Cells.

Several iPSC-derived three-dimensional (3D) cultures have been generated to model Alzheimer's disease (AD). While some AD-related phenotypes have been identified across these cultures, none of them could recapitulate multiple AD-related hallmarks in one model. To date, the transcriptomic features of these 3D models have not been compared with those of human AD brains. However, these data are crucial to understanding the pertinency of these models for studying AD-related pathomechanisms over time. We developed a 3D bioengineered model of iPSC-derived neural tissue that combines a porous scaffold composed of silk fibroin protein with an intercalated collagen hydrogel to support the growth of neurons and glial cells into complex and functional networks for an extended time, a fundamental requisite for aging studies. Cultures were generated from iPSC lines obtained from two subjects carrying the familial AD (FAD) APP London mutation, two well-studied control lines, and an isogenic control. Cultures were analyzed at 2 and 4.5 months. At both time points, an elevated Aβ42/40 ratio was detected in conditioned media from FAD cultures. However, extracellular Aβ42 deposition and enhanced neuronal excitability were observed in FAD culture only at 4.5 months, suggesting that extracellular Aβ deposition may trigger enhanced network activity. Remarkably, neuronal hyperexcitability has been described in AD patients early in the disease. Transcriptomic analysis revealed the deregulation of multiple gene sets in FAD samples. Such alterations were strikingly similar to those observed in human AD brains. These data provide evidence that our patient-derived FAD model develops time-dependent AD-related phenotypes and establishes a temporal relation among them. Furthermore, FAD iPSC-derived cultures recapitulate transcriptomic features of AD patients. Thus, our bioengineered neural tissue represents a unique tool to model AD in vitro over time.

3D bioengineered neural tissue generated from patient-derived iPSCs mimics time-dependent phenotypes and transcriptional features of Alzheimer's disease.

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.

Selene Lomoio

Alzheimer's Disease Research Laboratory,
Department of Neuroscience,
Alzheimer's Disease Research Laboratory, Department of Neuroscience

Tufts University School of Medicine

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

Selene Lomoio

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Tufts University School of Medicine

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

Alzheimer's Disease Research Laboratory,
Department of Neuroscience,
Alzheimer's Disease Research Laboratory, Department of Neuroscience