Activity-dependent synaptic GRIP1 accumulation drives synaptic scaling up in response to action potential blockade

Synaptic scaling is a form of homeostatic plasticity that stabilizes neuronal firing in response to changes in synapse number and strength. Scaling up in response to action-potential blockade is accomplished through increased synaptic accumulation of GluA2-containing AMPA receptors (AMPAR), but the receptor trafficking steps that drive this process remain largely obscure. Here, we show that the AMPAR-binding protein glutamate receptor-interacting protein-1 (GRIP1) is essential for regulated synaptic AMPAR accumulation during scaling up. Synaptic abundance of GRIP1 was enhanced by activity deprivation, directly increasing synaptic GRIP1 abundance through overexpression increased the amplitude of AMPA miniature excitatory postsynaptic currents (mEPSCs), and shRNA-mediated GRIP1 knockdown prevented scaling up of AMPA mEPSCs. Furthermore, knockdown and replace experiments targeting either GRIP1 or GluA2 revealed that scaling up requires the interaction between GRIP1 and GluA2. Finally, GRIP1 synaptic accumulation during scaling up did not require GluA2 binding. Taken together, our data support a model in which activity-dependent trafficking of GRIP1 to synaptic sites drives the forward trafficking and enhanced synaptic accumulation of GluA2-containing AMPAR during synaptic scaling up.Proper development of neuronal circuits, as well as efficient information storage during learning and memory, are thought to depend upon the presence of homeostatic mechanisms that stabilize neuronal excitability (1–3). One such mechanism is synaptic scaling, which compensates for perturbations in average firing by scaling up or down the postsynaptic strength of all of a neuron’s excitatory synapses (4). Synaptic scaling is a cell-autonomous process in which neurons detect changes in their own firing through a set of calcium-dependent sensors that then regulate receptor trafficking to increase or decrease the accumulation of AMPA receptors (AMPAR) at synaptic sites and thus increase or decrease synaptic strength (4–6). Despite great recent interest, the AMPA receptor-trafficking events that underlie synaptic scaling remain largely obscure. Defects in synaptic scaling have been postulated to contribute to disorders as diverse as Alzheimer’s disease (7) and epilepsy (8), so illuminating the underlying AMPAR trafficking steps could shed light into the genesis of a wide range of neurological disorders.Most neocortical AMPAR are heteromeric receptors composed of both GluA1 and GluA2 subunits, which have unique phosphorylation sites and interact with distinct trafficking proteins (9). During synaptic scaling up in response to action potential blockade, synaptic strength is increased through enhanced synaptic accumulation of GluA1 and GluA2-containing AMPAR (5, 10–13) and requires the C-terminal domain of the GluA2 subunit (12), but which subunit-specific interactions underlie synaptic scaling remain controversial (12, 14). Several trafficking proteins are known to interact with the GluA2, but not the GluA1, C-tail, including glutamate receptor interacting protein-1 (GRIP1) (15) and protein interacting with C-kinase-1 (PICK1) (16). Many studies have examined the role of GRIP1 and PICK1 in AMPAR trafficking and surface accumulation (15, 17–22), but little is known about their potential roles in regulating AMPAR synaptic accumulation during synaptic scaling. It was recently shown that deletion of PICK1, which competes with GRIP1/2 for binding to GluA2, enhances AMPAR accumulation and occludes synaptic scaling up (23), suggesting GRIP/PICK1-GluA2 interactions as possible critical players in synaptic scaling.GRIP1 was one of the first AMPAR-binding proteins identified (15), and yet its exact function in synaptic transmission and plasticity remains controversial. GRIP1 is an abundant multi-PDZ domain-containing protein that interacts with GluA2 through its fourth and fifth PDZ domains (15) and has known interactions with several other signaling and trafficking proteins, including itself (24), ABP (25), EphB receptors (26); the rasGEF GRASP-1 (27), the scaffold protein liprin-α (28), and the microtubule motor protein KIF5, or kinesin 1 (29). The role of GRIP1 in AMPAR trafficking is complicated and may involve AMPAR trafficking to and stabilization at synapses (17), as well as microtubule-based transport into dendrites (29) and the regulation of AMPAR movement between intracellular recycling compartments and the cell surface (22, 30). How GRIP1 influences basal AMPAR trafficking is not entirely clear. Overexpression of GRIP1 or gain-of-function GRIP1 mutants have been consistently observed to enhance surface AMPAR levels (21, 31), but knockout or dominant-negative GRIP1 constructs have had inconsistent effects, with slower synaptic AMPAR accumulation observed in one study (17) but no effects on basal AMPAR recycling and transmission in others (22, 32). Interestingly, GRIP1 and -2 are critical for the expression of cerebellar long-term depression (LTD), where they play redundant roles in regulated AMPAR endocytosis (33, 34). Currently no direct role for GRIP1 in activity-dependent synaptic strengthening or homeostatic plasticity has been established.Here, we show that the GRIP1–GluA2 interaction plays an essential role in the activity-dependent synaptic AMPAR accumulation and enhanced excitatory synaptic strength that underlies synaptic scaling up. Activity blockade with TTX increased the accumulation of GRIP1 at synaptic sites, whereas directly enhancing synaptic GRIP1 accumulation through overexpression (OE) was sufficient to mimic synaptic scaling. GRIP1 was necessary for synaptic scaling, because scaling up was prevented by shRNA-mediated knock down (KD) of endogenous GRIP1 and rescued by replacement with an RNAi-insensitive (RNAiI) GRIP1 but not a GRIP1 mutant that lacks the GluA2 interaction domain. We showed previously that GluA2 KD blocks synaptic scaling (12). Here, we show that synaptic scaling after GluA2 KD can be rescued by wild-type (RNAiI) GluA2 or point mutants that do not interfere with GRIP1 binding but not by GluA2 point mutants (Y876E and S880E) that reduce GluA2- GRIP1 binding, strongly suggesting that GRIP1 mediates synaptic scaling through interactions with GluA2. Finally, TTX still induced GRIP1 synaptic accumulation even when AMPAR accumulation was prevented by expression of GluA2 Y876E; thus, during synaptic scaling GluA2 synaptic accumulation depends on GRIP1 binding, but GRIP1 translocation and synaptic accumulation occur independently of GluA2 binding. Together our data show that activity-dependent regulation of synaptic GRIP1 abundance is critical for the forward trafficking and accumulation of AMPA receptors at synapses during synaptic scaling.     

Automated diagnosis of diabetic retinopathy and glaucoma using fundus and OCT images

ABSTRACT: We describe a system for the automated diagnosis of diabetic retinopathy and glaucoma using fundus and optical coherence tomography (OCT) images. Automatic screening will help the doctors to quickly identify the condition of the patient in a more accurate way. The macular abnormalities caused due to diabetic retinopathy can be detected by applying morphological operations, filters and thresholds on the fundus images of the patient. Early detection of glaucoma is done by estimating the Retinal Nerve Fiber Layer (RNFL) thickness from the OCT images of the patient. The RNFL thickness estimation involves the use of active contours based deformable snake algorithm for segmentation of the anterior and posterior boundaries of the retinal nerve fiber layer. The algorithm was tested on a set of 89 fundus images of which 85 were found to have at least mild retinopathy and OCT images of 31 patients out of which 13 were found to be glaucomatous. The accuracy for optical disk detection is found to be 97.75%. The proposed system therefore is accurate, reliable and robust and can be realized.     

Systemic lupus erythematosus and small vessel coronary infarction: who’s to blame?

Systemic lupus erythematosus (SLE) with or without accompanying antiphospholipid antibody syndrome (APLS) is known to cause myocardial ischemia via multiple mechanisms, including accelerated coronary atherosclerosis, impaired coronary vasomotor function, spontaneous intracoronary thrombus formation, or endothelial dysfunction in the context of cardiac syndrome X (CSX). We present the case of a young woman with SLE and APLS who presented with myocardial ischemia and peculiar echocardiographic evidence of multiple small septal perforator infarcts despite a normal coronary angiogram, a rare combination.