Nashaat Z. Gerges

Dual role of the exocyst in AMPA receptor targeting and insertion into the postsynaptic membrane

Intracellular membrane trafficking of glutamate receptors at excitatory synapses is critical for synaptic function. However, little is known about the specialized trafficking events occurring at the postsynaptic membrane. We have found that two components of the exocyst complex, Sec8 and Exo70, separately control synaptic targeting and insertion of AMPA‐type glutamate receptors. Sec8 controls the directional movement of receptors towards synapses through PDZ‐dependent interactions. In contrast, Exo70 mediates receptor insertion at the postsynaptic membrane, but it does not participate in receptor targeting. Thus, interference with Exo70 function accumulates AMPA receptors inside the spine, forming a complex physically associated, but not yet fused with the postsynaptic membrane. Electron microscopic analysis of these complexes indicates that Exo70 mediates AMPA receptor insertion directly within the postsynaptic density, rather than at extrasynaptic membranes. Therefore, we propose a molecular and anatomical model that dissects AMPA receptor sorting and synaptic delivery within the spine, and uncovers new functions of the exocyst at the postsynaptic membrane.

Local Control of AMPA Receptor Trafficking at the Postsynaptic Terminal by a Small GTPase of the Rab Family

The delivery of neurotransmitter receptors into the synaptic membrane is essential for synaptic function and plasticity. However, the molecular mechanisms of these specialized trafficking events and their integration with the intracellular membrane transport machinery are virtually unknown. Here, we have investigated the role of the Rab family of membrane sorting proteins in the late stages of receptor trafficking into the postsynaptic membrane. We have identified Rab8, a vesicular transport protein associated with trans-Golgi network membranes, as a critical component of the cellular machinery that delivers AMPA-type glutamatergic receptors (AMPARs) into synapses. Using electron microscopic techniques, we have found that Rab8 is localized in close proximity to the synaptic membrane, including the postsynaptic density. Electrophysiological studies indicated that Rab8 is necessary for the synaptic delivery of AMPARs during plasticity (long-term potentiation) and during constitutive receptor cycling. In addition, Rab8 is required for AMPAR delivery into the spine surface, but not for receptor transport from the dendritic shaft into the spine compartment or for delivery into the dendritic surface. Therefore, Rab8 specifically drives the local delivery of AMPARs into synapses. These results demonstrate a new role for the cellular secretory machinery in the control of synaptic function and plasticity directly at the postsynaptic membrane.

Adverse effect of the combination of hypothyroidism and chronic psychosocial stress on hippocampus-dependent memory in rats

Both hypothyroidism and stress interfere with cognitive function in patients. This study examined the effect of hypothyroidism and stress on hippocampus-dependent learning and memory in rats using the novel radial arm water maze (RAWM), which measures spatial working memory. Hypothyroidism was accomplished by thyroidectomy and 2 weeks later a form of intruder stress was used as the chronic psychosocial stressor. After 4-6 weeks of stress, rats were trained to learn (during the acquisition phase; four trials) and then remember (during two memory test trials occurring 15 and 120 min after the acquisition phase) the within-day location of a hidden escape platform, which was in different arm every day. The number of errors (entry into arms other than the platform arm) was noted. Within-day learning of the platform location was largely unaffected by the experimental manipulations, indicating that rats in all groups were equally capable of finding the platform to escape from the water with similar numbers of errors (P > 0.005). The number of days a rat took to reach a criterion (DTC; a maximum of one error in three consecutive days) indicated that chronic stress or hypothyroidism, alone, resulted in a mild impairment of spatial memory, and the combination of chronic stress and hypothyroidism resulted in a more severe and long-lasting memory impairment. The data indicated that the combination of stress and hypothyroidism produced more deleterious effects on hippocampal function than either chronic stress or hypothyroidism alone.

Levothyroxin restores hypothyroidism-induced impairment of hippocampus-dependent learning and memory: Behavioral, electrophysiological, and molecular studies.

Hypothyroidism induces cognitive impairment in experimental animals and patients. Clinical reports are conflicting about the ability of thyroid hormone replacement therapy to fully restore the hypothyroidism‐induced learning and memory impairment. In this study, we investigated the effects of L‐thyroxin (thyroxin) treatment on hippocampus‐dependent learning and memory in thyroidectomized adult rats. In the radial arm water maze (RAWM) task, thyroxin treated thyroidectomized animals made significantly fewer errors than the untreated hypothyroid animals in Trial 3 of the acquisition phase, short‐term memory and long‐term memory tests. In addition, the number of errors made by the thyroxin treated thyroidectomized animals was not different from that of the control group. Furthermore, the days‐to‐criterion (DTC) values for thyroxin treated thyroidectomized animals were not different from those of the control group but significantly lower than those of the untreated hypothyroid animals. In anesthetized rats, extracellular recording from hippocampal area CA1 of hypothyroid rats shows that thyroxin treatment restores impaired Late‐phase long‐term potentiation (L‐LTP). Immunoblot analysis of signaling molecules, including cyclic‐AMP response element binding protein (CREB), mitogen‐activated protein kinases (MAPKp44/42; ERK1/2), in area CA1 revealed that thyroxin treatment reversed hypothyroidism‐induced reduction of signaling molecules essential for learning and memory, and L‐LTP. This study shows that thyroxin treatment reverses hypothyroidism‐induced impairment of hippocampus‐dependent cognition, and L‐LTP, probably by restoring the levels of signaling molecule important for these processes.

Levothyroxin restores hypothyroidism-induced impairment of LTP of hippocampal CA1: electrophysiological and molecular studies.

Hypothyroidism impairs synaptic plasticity as well as learning and memory. Clinical reports are conflicting about the ability of thyroid hormone replacement therapy to fully restore the hypothyroidism-induced learning and memory impairment. Recently, we have shown that hypothyroidism impairs LTP and cognition in adult rats. We have studied the effect of thyroxin replacement therapy on hypothyroidism-induced LTP impairment using electrophysiological and molecular approaches. Recording from CA1 region of the hippocampus in anesthetized adult rat indicated that 6 weeks of thyroxin replacement therapy (20 microg/kg/day) fully restored LTP impaired by hypothyroidism. Western blotting showed reduction in phosphorylated (P)-CAMKII, total-CaMKII, neurogranin, and calmodulin basal levels in the CA1 region of the hippocampus of hypothyroid rats. The levels of these molecules were normalized by thyroxin replacement therapy. The hypothyroid-induced elevation of basal calcineurin levels and activity was also normalized by thyroxin treatment. However, thyroxin replacement therapy did not restore hypothyroidism-induced reduction in PKCgamma basal protein levels. Additionally, real-time PCR, showed a reduction in basal neurogranin mRNA level that was normalized by thyroxin replacement therapy. In the sham (control) rats, induction of LTP by high-frequency stimulation increases P-CaMKII, and total CaMKII levels as well as CaMKII phosphotransferase activity. However, in hypothyroid rats, the same stimulation protocol induced an increase only in total-CaMKII. Thyroxin treatment normalized the levels and activity of these molecules. The results demonstrated that thyroxin therapy normalized the electrophysiological and molecular effects of hypothyroidism on the CA1 region and emphasized the critical role P-CaMKII plays in hypothyroidism-induced LTP impairment.

Impaired long-term potentiation in obese zucker rats: possible involvement of presynaptic mechanism.

Electrophysiological investigation of basal synaptic transmission and synaptic plasticity in the CA1 region of the hippocampus was carried out in anesthetized obese Zucker rats (OZR). Comparison of the input/output curves of basal field excitatory postsynaptic potential indicates that these are similar in both the OZR and its lean counterpart suggesting that basal synaptic transmission is intact in the OZR. However, high frequency stimulation evokes long-term potentiation (LTP) in the lean rat but not in the OZR. Since post-tetanic potentiation and paired pulse facilitation, forms of short-term potentiation of presynaptic origin, are also severely impaired in the OZR, the results imply that impairment of CA1 hippocampal LTP in these obese rats may be due, in part, to impaired presynaptic function. The results emphasize the potential deleterious effect of obesity on learning and memory functions of the CNS.

GKAP orchestrates activity-dependent postsynaptic protein remodeling and homeostatic scaling

How does chronic activity modulation lead to global remodeling of proteins at synapses and synaptic scaling? Here we report that guanylate kinase–associated protein (GKAP; also known as SAPAP), a scaffolding molecule linking NMDA receptor–PSD-95 to Shank-Homer complexes, acts in these processes. Overexcitation removes GKAP from synapses via the ubiquitin-proteasome system, whereas inactivity induces synaptic accumulation of GKAP in rat hippocampal neurons. Bidirectional changes in synaptic GKAP amounts are controlled by specific CaMKII isoforms coupled to different Ca2+ channels. CaMKIIα activated by the NMDA receptor phosphorylates GKAP Ser54 to induce polyubiquitination of GKAP. In contrast, CaMKIIβ activation via L-type voltage-dependent calcium channels promotes GKAP recruitment by phosphorylating GKAP Ser340 and Ser384, which uncouples GKAP from myosin Va motor complex. Overexpressing GKAP turnover mutants not only hampers activity-dependent remodeling of PSD-95 and Shank but also blocks bidirectional synaptic scaling. Therefore, activity-dependent turnover of PSD proteins orchestrated by GKAP is critical for homeostatic plasticity.

Neurogranin enhances synaptic strength through its interaction with calmodulin

Learning‐correlated plasticity at CA1 hippocampal excitatory synapses is dependent on neuronal activity and NMDA receptor (NMDAR) activation. However, the molecular mechanisms that transduce plasticity stimuli to postsynaptic potentiation are poorly understood. Here, we report that neurogranin (Ng), a neuron‐specific and postsynaptic protein, enhances postsynaptic sensitivity and increases synaptic strength in an activity‐ and NMDAR‐dependent manner. In addition, Ng‐mediated potentiation of synaptic transmission mimics and occludes long‐term potentiation (LTP). Expression of Ng mutants that lack the ability to bind to, or dissociate from, calmodulin (CaM) fails to potentiate synaptic transmission, strongly suggesting that regulated Ng–CaM binding is necessary for Ng‐mediated potentiation. Moreover, knocking‐down Ng blocked LTP induction. Thus, Ng–CaM interaction can provide a mechanistic link between induction and expression of postsynaptic potentiation.

Independent Functions of hsp90 in Neurotransmitter Release and in the Continuous Synaptic Cycling of AMPA Receptors

The delivery of neurotransmitter receptors into synapses is essential for synaptic function and plasticity. In particular, AMPA-type glutamate receptors (AMPA receptors) reach excitatory synapses according to two distinct routes: a regulated pathway, which operates transiently during synaptic plasticity, and a constitutive pathway, which maintains synaptic function under conditions of basal transmission. However, the specific mechanisms that distinguish these two trafficking pathways are essentially unknown. Here, we evaluate the role of the molecular chaperone hsp90 (heat shock protein 90) in excitatory synaptic transmission in the hippocampus. On one hand, we found that hsp90 is necessary for the efficient neurotransmitter release at the presynaptic terminal. In addition, we identified hsp90 as a critical component of the cellular machinery that delivers AMPA receptors into the postsynaptic membrane. Using the hsp90-specific inhibitors radicicol and geldanamycin, we show that hsp90 is required for the constitutive trafficking of AMPA receptors into synapses during their continuous cycling between synaptic and nonsynaptic sites. In contrast, hsp90 function is not required for either the surface delivery of AMPA receptors into the nonsynaptic plasma membrane or for the acute, regulated delivery of AMPA receptors into synapses during plasticity induction (long-term potentiation). The synaptic cycling of AMPA receptors was also blocked by an hsp90-binding tetratricopeptide repeat (TPR) domain, suggesting that the role of hsp90 in AMPA receptor trafficking is mediated by a TPR domain-containing protein. These results demonstrate new roles for hsp90 in synaptic function by controlling neurotransmitter release and, independently, by mediating the continuous cycling of synaptic AMPA receptors.

Nicotine Reverses Adult-Onset Hypothyroidism-Induced Impairment of Learning and Memory: Behavioral and Electrophysiological Studies

Nicotine alleviates cognitive impairment associated with a variety of health conditions. We examined the effect of chronic nicotine treatment on adult‐onset hypothyroidism‐induced impairment of learning and memory in rats. Hypothyroidism was induced by surgical removal of thyroid glands (thyroidectomy). One month later, chronic nicotine treatment (1 mg/kg sc, twice/day) was instituted for 4–6 weeks. Test of hippocampus‐dependent spatial learning and memory in the radial arm water maze showed that hypothyroidism impaired learning as well as short‐term and long‐term memory retention. Chronic nicotine treatment reversed the hypothyroidism‐induced learning and memory impairment. In normal rats, chronic nicotine treatment had no effect on learning and memory. Extracellular recordings from the CA1 region of anesthetized hypothyroid rats showed severe reduction of both early‐phase and late‐phase long‐term potentiation (LTP) magnitude, which was reversed in nicotine‐treated hypothyroid rats. These results show that chronic nicotine treatment prevents hypothyroidism‐induced impairment of spatial cognition and LTP.