Amantha Thathiah

The Disintegrin/Metalloproteinase ADAM10 Is Essential for the Establishment of the Brain Cortex

The metalloproteinase and major amyloid precursor protein (APP) α-secretase candidate ADAM10 is responsible for the shedding of proteins important for brain development, such as cadherins, ephrins, and Notch receptors. Adam10 −/− mice die at embryonic day 9.5, due to major defects in development of somites and vasculogenesis. To investigate the function of ADAM10 in brain, we generated Adam10 conditional knock-out (cKO) mice using a Nestin-Cre promotor, limiting ADAM10 inactivation to neural progenitor cells (NPCs) and NPC-derived neurons and glial cells. The cKO mice die perinatally with a disrupted neocortex and a severely reduced ganglionic eminence, due to precocious neuronal differentiation resulting in an early depletion of progenitor cells. Premature neuronal differentiation is associated with aberrant neuronal migration and a disorganized laminar architecture in the neocortex. Neurospheres derived from Adam10 cKO mice have a disrupted sphere organization and segregated more neurons at the expense of astrocytes. We found that Notch-1 processing was affected, leading to downregulation of several Notch-regulated genes in Adam10 cKO brains, in accordance with the central role of ADAM10 in this signaling pathway and explaining the neurogenic phenotype. Finally, we found that α-secretase-mediated processing of APP was largely reduced in these neurons, demonstrating that ADAM10 represents the most important APP α-secretase in brain. Our study reveals that ADAM10 plays a central role in the developing brain by controlling mainly Notch-dependent pathways but likely also by reducing surface shedding of other neuronal membrane proteins including APP.

Alteration of the microRNA network during the progression of Alzheimer's disease

An overview of miRNAs altered in Alzheimers disease (AD) was established by profiling the hippocampus of a cohort of 41 late‐onset AD (LOAD) patients and 23 controls, showing deregulation of 35 miRNAs. Profiling of miRNAs in the prefrontal cortex of a second independent cohort of 49 patients grouped by Braak stages revealed 41 deregulated miRNAs. We focused on miR‐132‐3p which is strongly altered in both brain areas. Downregulation of this miRNA occurs already at Braak stages III and IV, before loss of neuron‐specific miRNAs. Next‐generation sequencing confirmed a strong decrease of miR‐132‐3p and of three family‐related miRNAs encoded by the same miRNA cluster on chromosome 17. Deregulation of miR‐132‐3p in AD brain appears to occur mainly in neurons displaying Tau hyper‐phosphorylation. We provide evidence that miR‐132‐3p may contribute to disease progression through aberrant regulation of mRNA targets in the Tau network. The transcription factor (TF) FOXO1a appears to be a key target of miR‐132‐3p in this pathway.

The role of G protein-coupled receptors in the pathology of Alzheimer's disease

G protein-coupled receptors (GPCRs) are involved in numerous key neurotransmitter systems in the brain that are disrupted in Alzheimers disease (AD). GPCRs also directly influence the amyloid cascade through modulation of the α-, β- and γ-secretases, proteolysis of the amyloid precursor protein (APP), and regulation of amyloid-β degradation. Additionally, amyloid-β has been shown to perturb GPCR function. Emerging insights into the mechanistic link between GPCRs and AD highlight the potential of this class of receptors as a therapeutic target for AD.

The Orphan G Protein–Coupled Receptor 3 Modulates Amyloid-Beta Peptide Generation in Neurons

Deposition of the amyloid-β peptide is a pathological hallmark of Alzheimers disease. A high-throughput functional genomics screen identified G protein–coupled receptor 3 (GPR3), a constitutively active orphan G protein–coupled receptor, as a modulator of amyloid-β production. Overexpression of GPR3 stimulated amyloid-β production, whereas genetic ablation of GPR3 prevented accumulation of the amyloid-β peptide in vitro and in an Alzheimers disease mouse model. GPR3 expression led to increased formation and cell-surface localization of the mature γ-secretase complex in the absence of an effect on Notch processing. GPR3 is highly expressed in areas of the normal human brain implicated in Alzheimers disease and is elevated in the sporadic Alzheimers disease brain. Thus, GPR3 represents a potential therapeutic target for the treatment of Alzheimers disease.

ADAM10, the Rate-limiting Protease of Regulated Intramembrane Proteolysis of Notch and Other Proteins, Is Processed by ADAMS-9, ADAMS-15, and the γ-Secretase

ADAM10 is involved in the proteolytic processing and shedding of proteins such as the amyloid precursor protein (APP), cadherins, and the Notch receptors, thereby initiating the regulated intramembrane proteolysis (RIP) of these proteins. Here, we demonstrate that the sheddase ADAM10 is also subject to RIP. We identify ADAM9 and -15 as the proteases responsible for releasing the ADAM10 ectodomain, and Presenilin/γ-Secretase as the protease responsible for the release of the ADAM10 intracellular domain (ICD). This domain then translocates to the nucleus and localizes to nuclear speckles, thought to be involved in gene regulation. Thus, ADAM10 performs a dual role in cells, as a metalloprotease when it is membrane-bound, and as a potential signaling protein once cleaved by ADAM9/15 and the γ-Secretase.

β-arrestin 2 regulates Aβ generation and γ-secretase activity in Alzheimer's disease

β-arrestins are associated with numerous aspects of G protein–coupled receptor (GPCR) signaling and regulation and accordingly influence diverse physiological and pathophysiological processes. Here we report that β-arrestin 2 expression is elevated in two independent cohorts of individuals with Alzheimers disease. Overexpression of β-arrestin 2 leads to an increase in amyloid-β (Aβ) peptide generation, whereas genetic silencing of Arrb2 (encoding β-arrestin 2) reduces generation of Aβ in cell cultures and in Arrb2−/− mice. Moreover, in a transgenic mouse model of Alzheimers disease, genetic deletion of Arrb2 leads to a reduction in the production of Aβ40 and Aβ42. Two GPCRs implicated previously in Alzheimers disease (GPR3 and the β2-adrenergic receptor) mediate their effects on Aβ generation through interaction with β-arrestin 2. β-arrestin 2 physically associates with the Aph-1a subunit of the γ-secretase complex and redistributes the complex toward detergent-resistant membranes, increasing the catalytic activity of the complex. Collectively, these studies identify β-arrestin 2 as a new therapeutic target for reducing amyloid pathology and GPCR dysfunction in Alzheimers disease.

G Protein-Coupled Receptors, Cholinergic Dysfunction, and A beta Toxicity in Alzheimer's Disease

β-amyloid may induce neurodegeneration by affecting G protein–coupled receptor signaling. Alzheimer’s disease (AD) is the most common neurodegenerative disorder afflicting the elderly. Neuropathologically, the AD brain is characterized by amyloid plaques, which are mainly composed of the β-amyloid protein (Aβ) and neurofibrillary tangles (NFTs), which are comprised of hyperphosphorylated aggregates of the tau protein. Few treatments are available, but acetylcholinesterase inhibitors, which increase acetylcholine concentrations in the brain, provide some beneficial effects. Abnormal accumulation of Aβ in the brain is believed to drive disease progression. Although the mechanism of Aβ-mediated toxicity is not clearly understood, new studies provide insight into the effect that Aβ accumulation has on the clustering of a heterotrimeric GTP-binding protein (G protein)–coupled receptor (GPCR), the angiotensin type 2 (AT2) receptor, and the resulting effect on the Gαq/11 family of G proteins, which facilitate GPCR downstream signaling cascades. Assemblies of the AT2 receptor sequester Gαq/11, preventing it from initiating signaling cascades mediated by other GPCRs. The resulting reduction in Gαq/11 binding and signaling by the M1 muscarinic acetylcholine receptor (mAChR) has now been shown to accompany neurodegeneration in an AD mouse model. Thus, these studies provide a potential link between Aβ toxicity, AT2 receptor clustering, and disruption of the M1 mAChR and Gαq/11 signaling pathways, and they contribute to our understanding of the pathogenesis of AD. This Review with 1 figure and 100 citations highlights GPCR-mediated mechanisms of Aβ-induced toxicity in AD. The β-amyloid (Aβ) peptide is associated with the pathogenesis of Alzheimer’s disease (AD). Evidence gathered over the last two decades suggests that the gradual accumulation of soluble and insoluble Aβ peptide species triggers a cascade of events that leads to the clinical manifestation of AD. Aβ accumulation has also been associated with the cholinergic dysfunction observed in AD, which is characterized by diminished acetylcholine release and impaired coupling of the muscarinic acetylcholine receptors (mAChRs) to heterotrimeric GTP-binding proteins (G proteins). Although the mechanism of Aβ-mediated toxicity is not clearly understood, evidence shows that Aβ accumulation has an effect on the oligomerization of the angiotensin II (AngII) AT2 (angiotensin type 2) receptor and sequestration of the Gαq/11 family of G proteins. Sequestration of Gαq/11 results in dysfunctional coupling and signaling between M1 mAChR and Gαq/11 and accompanies neurodegeneration, tau phosphorylation, and neuronal loss in an AD transgenic mouse model. Collectively, these results provide a putative link among Aβ toxicity, AT2 receptor oligomerization, and disruption of the signaling pathway through M1 mAChR and Gαq/11 and potentially contribute to our understanding of the cholinergic deficit observed in AD.

Loss of GPR3 reduces the amyloid plaque burden and improves memory in Alzheimer’s disease mouse models

Loss of GPR3 reduced amyloid plaque burden and improved cognition in four mouse models of Alzheimer’s disease, suggesting that GPR3 may be a potential therapeutic target. GPR3, a therapeutic target for AD? Alzheimer’s disease (AD) is characterized by the degeneration of brain networks involved in cognitive function. AD mouse models are used to study disease pathogenesis, but no single model fully captures the pathological changes in AD patients. Thus, extensive validation of AD therapeutic targets in multiple animal models is required before advancing to clinical research. In new work, Huang et al. determined that the absence of the G protein–coupled receptor 3 (GPR3), a protein expressed in the brain, alleviated the cognitive deficits and reduced amyloid pathology in four different disease-relevant mouse models of AD. Furthermore, GPR3 was found to be elevated in postmortem brain tissue from a subset of AD patients. This study demonstrates that GPR3 is a potential AD therapeutic target and provides the validation needed for future development of GPR3 modulators. The orphan G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor (GPCR) GPR3 regulates activity of the γ-secretase complex in the absence of an effect on Notch proteolysis, providing a potential therapeutic target for Alzheimer’s disease (AD). However, given the vast resources required to develop and evaluate any new therapy for AD and the multiple failures involved in translational research, demonstration of the pathophysiological relevance of research findings in multiple disease-relevant models is necessary before initiating costly drug development programs. We evaluated the physiological consequences of loss of Gpr3 in four AD transgenic mouse models, including two that contain the humanized murine Aβ sequence and express similar amyloid precursor protein (APP) levels as wild-type mice, thereby reducing potential artificial phenotypes. Our findings reveal that genetic deletion of Gpr3 reduced amyloid pathology in all of the AD mouse models and alleviated cognitive deficits in APP/PS1 mice. Additional three-dimensional visualization and analysis of the amyloid plaque burden provided accurate information on the amyloid load, distribution, and volume in the structurally intact adult mouse brain. Analysis of 10 different regions in healthy human postmortem brain tissue indicated that GPR3 expression was stable during aging. However, two cohorts of human AD postmortem brain tissue samples showed a correlation between elevated GPR3 and AD progression. Collectively, these studies provide evidence that GPR3 mediates the amyloidogenic proteolysis of APP in four AD transgenic mouse models as well as the physiological processing of APP in wild-type mice, suggesting that GPR3 may be a potential therapeutic target for AD drug development.

Regulation of Amyloid Precursor Protein Processing by Serotonin Signaling

Proteolytic processing of the amyloid precursor protein (APP) by the β- and γ-secretases releases the amyloid-β peptide (Aβ), which deposits in senile plaques and contributes to the etiology of Alzheimers disease (AD). The α-secretase cleaves APP in the Aβ peptide sequence to generate soluble APPα (sAPPα). Upregulation of α-secretase activity through the 5-hydroxytryptamine 4 (5-HT4) receptor has been shown to reduce Aβ production, amyloid plaque load and to improve cognitive impairment in transgenic mouse models of AD. Consequently, activation of 5-HT4 receptors following agonist stimulation is considered to be a therapeutic strategy for AD treatment; however, the signaling cascade involved in 5-HT4 receptor-stimulated proteolysis of APP remains to be determined. Here we used chemical and siRNA inhibition to identify the proteins which mediate 5-HT4d receptor-stimulated α-secretase activity in the SH-SY5Y human neuronal cell line. We show that G protein and Src dependent activation of phospholipase C are required for α-secretase activity, while, unexpectedly, adenylyl cyclase and cAMP are not involved. Further elucidation of the signaling pathway indicates that inositol triphosphate phosphorylation and casein kinase 2 activation is also a prerequisite for α-secretase activity. Our findings provide a novel route to explore the treatment of AD through 5-HT4 receptor-induced α-secretase activation.

Regulation of neuronal communication by G protein‐coupled receptors

Neuronal communication plays an essential role in the propagation of information in the brain and requires a precisely orchestrated connectivity between neurons. Synaptic transmission is the mechanism through which neurons communicate with each other. It is a strictly regulated process which involves membrane depolarization, the cellular exocytosis machinery, neurotransmitter release from synaptic vesicles into the synaptic cleft, and the interaction between ion channels, G protein‐coupled receptors (GPCRs), and downstream effector molecules. The focus of this review is to explore the role of GPCRs and G protein‐signaling in neurotransmission, to highlight the function of GPCRs, which are localized in both presynaptic and postsynaptic membrane terminals, in regulation of intrasynaptic and intersynaptic communication, and to discuss the involvement of astrocytic GPCRs in the regulation of neuronal communication.