Kainate receptor auxiliary subunit NETO2 is required for normal fear expression and extinction

NETO1 and NETO2 are auxiliary subunits of kainate receptors (KARs). They interact with native KAR subunits to modulate multiple aspects of receptor function. Variation in KAR genes has been associated with psychiatric disorders in humans, and in mice, knockouts of the Grik1 gene have increased, while Grik2 and Grik4 knockouts have reduced anxiety-like behavior. To determine whether the NETO proteins regulate anxiety and fear through modulation of KARs, we undertook a comprehensive behavioral analysis of adult Neto1(-/-) and Neto2(-/-) mice. We observed no differences in anxiety-like behavior. However, in cued fear conditioning, Neto2(-/-), but not Neto1(-/-) mice, showed higher fear expression and delayed extinction compared to wild type mice. We established, by in situ hybridization, that Neto2 was expressed in both excitatory and inhibitory neurons throughout the fear circuit including the medial prefrontal cortex, amygdala, and hippocampus. Finally, we demonstrated that the relative amount of synaptosomal KAR GLUK2/3 subunit was 20.8% lower in the ventral hippocampus and 36.5% lower in the medial prefrontal cortex in Neto2(-/-) compared to the Neto2(+/+) mice. The GLUK5 subunit abundance was reduced 23.8% in the ventral hippocampus and 16.9% in the amygdala. We conclude that Neto2 regulates fear expression and extinction in mice, and that its absence increases conditionability, a phenotype related to post-traumatic stress disorder and propose that this phenotype is mediated by reduced KAR subunit abundance at synapses of fear-associated brain regions.Peer reviewe

Kainate Receptors Coexist in a Functional Complex with KCC2 and Regulate Chloride Homeostasis in Hippocampal Neurons

Peer reviewe

Recent Fertility and Mortality Trends among Aboriginal and Nonaboriginal Populations of Central Siberia

We examine mortality and fertility patterns of aboriginal (primarily Evenki and Keto) and Russian (i.e., nonaboriginal) populations from the Baykit District of Central Siberia for the period 1982—1994. Mortality rates in the aboriginal population of Baykit are substantially greater than those observed in the Russians and are comparable to levels recently reported for other indigenous Siberian groups. Infant mortality rates average 48 per 1000 live births among Baykit aboriginals, three times greater than the Russians of the district (15 per 1000 births) and more than double the rates for Inuit and Indian populations of Canada. Similarly, crude death rates of the Baykit aboriginals are twice as high as those observed in either the Baykit Russians or the Canadian aboriginal populations (13 vs. 6-7 deaths per 1000 individuals). Birth rates of the indigenous population of Baykit are higher than those of the Russians (33 vs. 15 births per 1000 individuals) but are comparable to those of Canadian aboriginal groups. Violence and accidents are the leading causes of adult male mortality in both ethnic groups, whereas circulatory diseases have emerged as the prime cause of death in women. The greater male mortality resulting from violence and accidents is a widely observed cross-cultural phenomenon. The emergence of circulatory diseases as a major mortality risk for women, however, appears to be linked to specific lifestyle changes associated with Soviet reorganization of indigenous Siberian societies. Marked declines in mortality and increases in fertility were observed in the Baykit aboriginal population during the mid to late 1980s with the government’s implementation of anti-alcohol policies. The decline in mortality, however, was largely erased during the early 1990s, as the region became increasingly isolated and marginalized following the collapse of the Soviet Union. Demographic trends in the Baykit District suggest that because the indigenous groups have become more isolated, many are returning to a more traditional subsistence lifestyle

Neto2 interacts with the scaffolding protein GRIP and regulates synaptic abundance of kainate receptors.

Kainate receptors (KARs) are a class of ionotropic glutamate receptors that are expressed throughout the central nervous system. The function and subcellular localization of KARs are tightly regulated by accessory proteins. We have previously identified the single-pass transmembrane proteins, Neto1 and Neto2, to be associated with native KARs. In the hippocampus, Neto1, but not Neto2, controls the abundance and modulates the kinetics of postsynaptic KARs. Here we evaluated whether Neto2 regulates synaptic KAR levels in the cerebellum where Neto1 expression is limited to the deep cerebellar nuclei. In the cerebellum, where Neto2 is present abundantly, we found a ~40% decrease in GluK2-KARs at the postsynaptic density (PSD) of Neto2-null mice. No change, however, was observed in total level of GluK2-KARs, thereby suggesting a critical role of Neto2 on the synaptic localization of cerebellar KARs. The presence of a putative class II PDZ binding motif on Neto2 led us to also investigate whether it interacts with PDZ domain-containing proteins previously implicated in regulating synaptic abundance of KARs. We identified a PDZ-dependent interaction between Neto2 and the scaffolding protein GRIP. Furthermore, coexpression of Neto2 significantly increased the amount of GRIP associated with GluK2, suggesting that Neto2 may promote and/or stabilize GluK2:GRIP interactions. Our results demonstrate that Neto2, like Neto1, is an important auxiliary protein for modulating the synaptic levels of KARs. Moreover, we propose that the interactions of Neto1/2 with various scaffolding proteins is a critical mechanism by which KARs are stabilized at diverse synapses

Neto2 increases the interaction between GRIP and GluK2.

<p>Immunoblot of immunoprecipitates from lysates of transfected COS-7 cells. The cDNAs used for transfection are shown above each lane. Neto2Δ7, Neto2 lacking the last seven C-terminal residues. Blot, antibody used for immunoblot analysis; IP, antibody used for immunoprecipitation.</p

Neto1 is an auxiliary subunit of native synaptic kainate receptors

Ionotropic glutamate receptors of AMPA, NMDA and kainate receptor (KAR) subtypes mediate fast excitatory synaptic transmission in the vertebrate CNS. Auxiliary proteins have been identified for AMPA and NMDA receptor complexes, but little is known about KAR complex proteins. We previously identified the CUB-domain protein, Neto1, as an NMDA receptor-associated polypeptide. Here, we show that Neto1 is also an auxiliary subunit for endogenous synaptic KARs. We found that Neto1 and KARs co-immunoprecipitated from brain lysates, from post-synaptic densities (PSDs) and, in a manner dependent on Neto1 CUB domains, when co-expressed in heterologous cells. In Neto1-null mice, there was an ~50% reduction in the abundance of GluK2-KARs in hippocampal PSDs. Neto1 strongly localized to CA3 stratum lucidum and loss of Neto1 resulted in a selective deficit in KAR-mediated neurotransmission at mossy fiber-CA3 pyramidal cell synapses (MF-CA3): KAR-mediated EPSCs in Neto1-null mice were reduced in amplitude and decayed more rapidly than did those in wild-type mice. In contrast, the loss of Neto2, which also localizes to stratum lucidum and interacts with KARs, had no effect on KAR synaptic abundance or MF-CA3 transmission. Indeed MF-CA3 KAR deficits in Neto1/2 double null mutant mice were indistinguishable from Neto1 single null mice. Thus, our findings establish Neto1 as an auxiliary protein required for synaptic function of KARs. The ability of Neto1 to regulate both NMDARs and KARs reveals a unique dual role in controlling synaptic transmission by serving as an auxiliary protein for these two classes of ionotropic glutamate receptors in a synapse specific fashion

KARs are reduced in the cerebellar PSD of Neto2-null mice.

<p>(A) Immunoblots (representative of three experiments) of proteins from cerebellar homogenates and cerebellar PSD fractions of wild-type (Wt) and Neto2-null (<i>Neto2^−/−</i>) mice. Antibodies used for detection are indicated on the left. (B) Histogram showing normalized levels of different proteins in Neto2-null cerebellar homogenates relative to that of wild-type (white bars), and in Neto2-null cerebellar PSD fractions relative to that of wild-type (black bars); **, p<0.01, paired t-test, n = 3.</p

Neto2 is associated with KARs in the cerebellum.

<p>(A) Confocal micrographs of immunostained cerebellar slices. Antibodies used for immunostaining are indicated in the top left corner of each image. In the cerebellum, the NeuN antibody stains the neuronal nuclei of granule cells but does not recognize Purkinje cells. MCL, molecular cell layer; GCL, granule cell layer; Wt, wild-type sections; <i>Neto2^−/−</i>, Neto2-null sections. Scale bar, 100 μm. (B) High-magnification confocal microscopy of the cerebellar granule cell layer immunostained with Neto2, GluK2, NeuN, or synaptophysin antibodies. Scale bar, 20 μm; scale bar (small panels on the right), 5 μm (C) Immunoblot of immunoprecipitates from the cerebellum. Blot: antibody used for immunoblot analysis; IP: immunoprecipitate. The input represents 2% of the material used in the immunoprecipitation experiment.</p

Neto2 associates with GRIP <i>in vivo</i>.

<p>(A, B) Immunoblots of immunoprecipitates from cerebellar (A) or whole brain (B) membrane fractions. Samples were subjected to immunoprecipitation with an anti-GRIP antibody, or with normal rabbit IgGs (IgG), as the negative control. Blot, antibody used for immunoblot analysis; IP, immunoprecipitate.</p