Neto Auxiliary Subunits Regulate Interneuron Somatodendritic and Presynaptic Kainate Receptors to Control Network Inhibition.

Although Netos are considered auxiliary subunits critical for kainate receptor (KAR) function, direct evidence for their regulation of native KARs is limited. Because Neto KAR regulation is GluK subunit/Neto isoform specific, such regulation must be determined in cell-type-specific contexts. We demonstrate Neto1/2 expression in somatostatin (SOM)-, cholecystokinin/cannabinoid receptor 1 (CCK/CB1)-, and parvalbumin (PV)-containing interneurons. KAR-mediated excitation of these interneurons is contingent upon Neto1 because kainate yields comparable effects in Neto2 knockouts and wild-types but fails to excite interneurons or recruit inhibition in Neto1 knockouts. In contrast, presynaptic KARs in CCK/CB1 interneurons are dually regulated by both Neto1 and Neto2. Neto association promotes tonic presynaptic KAR activation, dampening CCK/CB1 interneuron output, and loss of this brake in Neto mutants profoundly increases CCK/CB1 interneuron-mediated inhibition. Our results confirm that Neto1 regulates endogenous somatodendritic KARs in diverse interneurons and demonstrate Neto regulation of presynaptic KARs in mature inhibitory presynaptic terminals.

Neto2-null mice have impaired GABAergic inhibition and are susceptible to seizures.

Neto2 is a transmembrane protein that interacts with the neuron-specific K(+)-Cl(-) cotransporter (KCC2) in the central nervous system (CNS). Efficient KCC2 transport is essential for setting the neuronal Cl(-) gradient, which is required for fast GABAergic inhibition. Neto2 is required to maintain the normal abundance of KCC2 in neurons, and increases KCC2 function by binding to the active oligomeric form of this cotransporter. In the present study, we characterized GABAergic inhibition and KCC2-mediated neuronal chloride homeostasis in pyramidal neurons from adult hippocampal slices. Using gramicidin perforated patch clamp recordings we found that the reversal potential for GABA (EGABA) was significantly depolarized. We also observed that surface levels of KCC2 and phosphorylation of KCC2 serine 940 (Ser940) were reduced in Neto2(-/-) neurons compared to wild-type controls. To examine GABAergic inhibition we recorded spontaneous inhibitory postsynaptic currents (sIPSCs) and found that Neto2(-/-) neurons had significant reductions in both their amplitude and frequency. Based on the critical role of Neto2 in regulating GABAergic inhibition we rationalized that Neto2-null mice would be prone to seizure activity. We found that Neto2-null mice demonstrated a decrease in the latency to pentylenetetrazole (PTZ)-induced seizures and an increase in seizure severity.

Disruption of Src Is Associated with Phenotypes Related to Williams-Beuren Syndrome and Altered Cellular Localization of TFII-I

Src is a nonreceptor protein tyrosine kinase that is expressed widely throughout the central nervous system and is involved in diverse biological functions. Mice homozygous for a spontaneous mutation in Src (Src (thl/thl) ) exhibited hypersociability and hyperactivity along with impairments in visuospatial, amygdala-dependent, and motor learning as well as an increased startle response to loud tones. The phenotype of Src (thl/thl) mice showed significant overlap with Williams-Beuren syndrome (WBS), a disorder caused by the deletion of several genes, including General Transcription Factor 2-I (GTF2I). Src phosphorylation regulates the movement of GTF2I protein (TFII-I) between the nucleus, where it is a transcriptional activator, and the cytoplasm, where it regulates trafficking of transient receptor potential cation channel, subfamily C, member 3 (TRPC3) subunits to the plasma membrane. Here, we demonstrate altered cellular localization of both TFII-I and TRPC3 in the Src mutants, suggesting that disruption of Src can phenocopy behavioral phenotypes observed in WBS through its regulation of TFII-I.

Transcription factor PRDM8 is required for rod bipolar and type 2 OFF-cone bipolar cell survival and amacrine subtype identity.

Retinal bipolar (BP) cells mediate the earliest steps in image processing in the visual system, but the genetic pathways that regulate their development and function are incompletely known. We identified PRDI-BF1 and RIZ homology domain containing 8 (PRDM8) as a highly conserved transcription factor that is abundantly expressed in mouse retina. During development and in maturity, PRDM8 is expressed strongly in BP cells and a fraction of amacrine and ganglion cells. To determine whether Prdm8 is essential to BP cell development or physiology, we targeted the gene in mice. Prdm8(EGFP/EGFP) mice showed nonprogressive b-wave deficits on electroretinograms, consistent with compromised BP cell function or circuitry resembling the incomplete form of human congenital stationary night blindness (CSNB). BP cell specification was normal in Prdm8(EGFP/EGFP) retina as determined by VSX2(+) cell numbers and retinal morphology at postnatal day 6. BP subtype differentiation was impaired, however, as indicated by absent or diminished expression of BP subtype-specific markers, including the putative PRDM8 regulatory target PKCα (Prkca) and its protein. By adulthood, rod bipolar (RB) and type 2 OFF-cone bipolar (CB) cells were nearly absent from Prdm8-null mice. Although no change was detected in total amacrine cell (AC) numbers, increased PRKCA(+) and cholinergic ACs and decreased GABAergic ACs were seen, suggesting an alteration in amacrine subtype identity. These findings establish that PRDM8 is required for RB and type 2 OFF-CB cell survival and amacrine subtype identity, and they present PRDM8 as a candidate gene for human CSNB.

STAT3 promotes survival of mutant photoreceptors in inherited photoreceptor degeneration models.

Inherited photoreceptor degenerations (IPDs), a group of incurable progressive blinding diseases, are caused by mutations in more than 200 genes, but little is known about the molecular pathogenesis of photoreceptor (PR) death. Increased retinal expression of STAT3 has been observed in response to many retinal insults, including IPDs, but the role of this increase in PR death is unknown. Here, we show that the expression of Stat3 is increased in PRs of the Tg(RHO P347S) and Prph2(rds) (/+) mouse models of IPD and is activated by tyrosine phosphorylation. PR-specific deletion of Stat3 substantially accelerated PR degeneration in both mutant strains. In contrast, increased PR-specific expression of ROSA26 (R26) alleles encoding either WT STAT3 (Stat3(wt)) or the gain-of-function variant STAT3(C) (Stat3(C)) improved PR survival in both models. Moreover, PR signaling in Tg(RHO P347S) mice carrying either a R26-Stat3(wt) or R26-Stat3(C) allele demonstrated increased a-wave amplitude of the scotopic electroretinogram. Phosphorylation of STAT3 at tyrosine 705 was required for the prosurvival effect because an R26-Stat3(Y705F) allele was not protective. The prosurvival role of enhanced Stat3 activity was validated using recombinant adenoassociated virus (rAAV) vector-mediated PR Stat3 expression in Tg(RHO P347S) mice. Our findings (i) establish that the increase in endogenous PR Stat3 expression is a protective response in IPDs, (ii) suggest that therapeutic augmentation of PR Stat3 expression has potential as a common neuroprotective therapy for these disorders, and (iii) indicate that prosurvival molecules whose expression is increased in mutant PRs may have promise as novel therapies for IPDs.

Kainate receptors coexist in a functional complex with KCC2 and regulate chloride homeostasis in hippocampal neurons.

KCC2 is the neuron-specific K+-Cl(-) cotransporter required for maintaining low intracellular Cl(-), which is essential for fast inhibitory synaptic transmission in the mature CNS. Despite the requirement of KCC2 for inhibitory synaptic transmission, understanding of the cellular mechanisms that regulate KCC2 expression and function is rudimentary. We examined KCC2 in its native protein complex in vivo to identify key KCC2-interacting partners that regulate KCC2 function. Using blue native-polyacrylamide gel electrophoresis (BN-PAGE), we determined that native KCC2 exists in a macromolecular complex with kainate-type glutamate receptors (KARs). We found that KAR subunits are required for KCC2 oligomerization and surface expression. In accordance with this finding, acute and chronic genetic deletion of KARs decreased KCC2 function and weakened synaptic inhibition in hippocampal neurons. Our results reveal KARs as regulators of KCC2, significantly advancing our growing understanding of the tight interplay between excitation and inhibition.

Neto auxiliary protein interactions regulate kainate and NMDA receptor subunit localization at mossy fiber-CA3 pyramidal cell synapses.

Neto1 and Neto2 auxiliary subunits coassemble with NMDA receptors (NMDARs) and kainate receptors (KARs) to modulate their function. In the hippocampus, Neto1 enhances the amplitude and prolongs the kinetics of KAR-mediated currents at mossy fiber (MF)-CA3 pyramidal cell synapses. However, whether Neto1 trafficks KARs to synapses or simply alters channel properties is unresolved. Therefore, postembedding electron microscopy was performed to investigate the localization of GluK2/3 subunits at MF-CA3 synapses in Neto-null mice. Postsynaptic GluK2/3 Immunogold labeling was substantially reduced in Neto-null mice compared with wild types. Moreover, spontaneous KAR-mediated synaptic currents and metabotropic KAR signaling were absent in CA3 pyramidal cells of Neto-null mice. A similar loss of ionotropic and metabotropic KAR function was observed in Neto1, but not Neto2, single knock-out mice, specifically implicating Neto1 in regulating CA3 pyramidal cell KAR localization and function. Additional controversy pertains to the role of Neto proteins in modulating synaptic NMDARs. While Immunogold labeling for GluN2A at MF-CA3 synapses was comparable between wild-type and Neto-null mice, labeling for postsynaptic GluN2B was robustly increased in Neto-null mice. Accordingly, NMDAR-mediated currents at MF-CA3 synapses exhibited increased sensitivity to a GluN2B-selective antagonist in Neto1 knockouts relative to wild types. Thus, despite preservation of the overall MF-CA3 synaptic NMDAR-mediated current, loss of Neto1 alters NMDAR subunit composition. These results confirm that Neto protein interactions regulate synaptic localization of KAR and NMDAR subunits at MF-CA3 synapses, with implications for both ionotropic and metabotropic glutamatergic recruitment of the CA3 network.

Endothelin-2 deficiency causes growth retardation, hypothermia, and emphysema in mice.

To explore the physiological functions of endothelin-2 (ET-2), we generated gene-targeted mouse models. Global Et2 knockout mice exhibited severe growth retardation and juvenile lethality. Despite normal milk intake, they suffered from internal starvation characterized by hypoglycemia, ketonemia, and increased levels of starvation-induced genes. Although ET-2 is abundantly expressed in the gastrointestinal tract, the intestine was morphologically and functionally normal. Moreover, intestinal epithelium-specific Et2 knockout mice showed no abnormalities in growth and survival. Global Et2 knockout mice were also profoundly hypothermic. Housing Et2 knockout mice in a warm environment significantly extended their median lifespan. However, neuron-specific Et2 knockout mice displayed a normal core body temperature. Low levels of Et2 mRNA were also detected in the lung, with transient increases soon after birth. The lungs of Et2 knockout mice showed emphysematous structural changes with an increase in total lung capacity, resulting in chronic hypoxemia, hypercapnia, and increased erythropoietin synthesis. Finally, systemically inducible ET-2 deficiency in neonatal and adult mice fully reproduced the phenotype previously observed in global Et2 knockout mice. Together, these findings reveal that ET-2 is critical for the growth and survival of postnatal mice and plays important roles in energy homeostasis, thermoregulation, and the maintenance of lung morphology and function.

Endothelin-2-mediated protection of mutant photoreceptors in inherited photoreceptor degeneration.

Expression of the Endothelin-2 (Edn2) mRNA is greatly increased in the photoreceptors (PRs) of mouse models of inherited PR degeneration (IPD). To examine the role of Edn2 in mutant PR survival, we generated Edn2(-/-) mice carrying homozygous Pde6b(rd1) alleles or the Tg(RHO P347S) transgene. In the Edn2(-/-) background, PR survival increased 110% in Pde6b(rd1/rd1) mice at post-natal (PN) day 15, and 60% in Tg(RHO P347S) mice at PN40. In contrast, PR survival was not increased in retinal explants of Pde6b(rd1/rd1) ; Edn2(-/-) mice. This finding, together with systemic abnormalities in Edn2(-/-) mice, suggested that the increased survival of mutant PRs in the Edn2(-/-) background resulted at least partly from the systemic EDN2 loss of function. To examine directly the role of EDN2 in mutant PRs, we used a scAAV5-Edn2 cDNA vector to restore Edn2 expression in Pde6b(rd1/rd1) ; Edn2(-/-) PRs and observed an 18% increase in PR survival at PN14. Importantly, PR survival was also increased after injection of scAAV5-Edn2 into Pde6b(rd1/rd1) retinas, by 31% at PN15. Together, these findings suggest that increased Edn2 expression is protective to mutant PRs. To begin to elucidate Edn2-mediated mechanisms that contribute to PR survival, we used microarray analysis and identified a cohort of 20 genes with >4-fold increased expression in Tg(RHO P347S) retinas, including Fgf2. Notably, increased expression of the FGF2 protein in Tg(RHO P347S) PRs was ablated in Tg(RHO P347S); Edn2(-/-) retinas. Our findings indicate that the increased expression of PR Edn2 increases PR survival, and suggest that the Edn2-dependent increase in PR expression of FGF2 may contribute to the augmented survival.

Neto2 is a KCC2 interacting protein required for neuronal Cl- regulation in hippocampal neurons.

KCC2 is a neuron-specific K(+)-Cl(-) cotransporter that is essential for Cl(-) homeostasis and fast inhibitory synaptic transmission in the mature CNS. Despite the critical role of KCC2 in neurons, the mechanisms regulating its function are not understood. Here, we show that KCC2 is critically regulated by the single-pass transmembrane protein neuropilin and tolloid like-2 (Neto2). Neto2 is required to maintain the normal abundance of KCC2 and specifically associates with the active oligomeric form of the transporter. Loss of the Neto2:KCC2 interaction reduced KCC2-mediated Cl(-) extrusion, resulting in decreased synaptic inhibition in hippocampal neurons.

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.

Bhlhb5 and Prdm8 form a repressor complex involved in neuronal circuit assembly.

Although transcription factors that repress gene expression play critical roles in nervous system development, their mechanism of action remains to be understood. Here, we report that the Olig-related transcription factor Bhlhb5 (also known as Bhlhe22) forms a repressor complex with the PR/SET domain protein, Prdm8. We find that Bhlhb5 binds to sequence-specific DNA elements and then recruits Prdm8, which mediates the repression of target genes. This interaction is critical for repressor function since mice lacking either Bhlhb5 or Prdm8 have strikingly similar cellular and behavioral phenotypes, including axonal mistargeting by neurons of the dorsal telencephalon and abnormal itch-like behavior. We provide evidence that Cadherin-11 functions as target of the Prdm8/Bhlhb5 repressor complex that must be repressed for proper neural circuit formation to occur. These findings suggest that Prdm8 is an obligate partner of Bhlhb5, forming a repressor complex that directs neural circuit assembly in part through the precise regulation of Cadherin-11.

A transgenic mouse line expressing cre recombinase in undifferentiated postmitotic mouse retinal bipolar cell precursors.

Approaches for manipulating cell type-specific gene expression during development depend on the identification of novel genetic tools. Here, we report the generation of a transgenic mouse line that utilizes Vsx2 upstream sequences to direct Cre recombinase to developing retinal bipolar cells. In contrast to the endogenous Vsx2 expression pattern, transgene expression was not detected in proliferating retinal progenitor cells and was restricted to post-mitotic bipolar cells. Cre immunolabeling was detected in rod bipolar cells and a subset of ON and OFF cone bipolar cells. Expression was first observed at postnatal day 3 and was detectable between 24 hours and 36 hours after the last S-phase of the cell cycle. The appearance of Cre-immunolabeled cells preceded the expression of bipolar cell type-specific markers such as PKCα and Cabp5 suggesting that transgene expression is initiated prior to terminal differentiation. In the presence of a constitutive conditional reporter transgene, reporter fluorescence was detected in Cre-expressing bipolar cells in the mature retina as expected, but was also observed in Cre-negative Type 2 bipolar cells and occasionally in Cre-negative photoreceptor cells. Together these findings reveal a new transgenic tool for directing gene expression to post-mitotic retinal precursors that are mostly committed to a bipolar cell fate.

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 (complement C1r/C1s, Uegf, Bmpl) 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 coimmunoprecipitated from brain lysates, from postsynaptic densities (PSDs) and, in a manner dependent on Neto1 CUB domains, when coexpressed 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 (MF-CA3) synapses: 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/Neto2-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.

Evidence of severe mitochondrial oxidative stress and a protective effect of low oxygen in mouse models of inherited photoreceptor degeneration.

The role of oxidative stress within photoreceptors (PRs) in inherited photoreceptor degeneration (IPD) is unclear. We investigated this question using four IPD mouse models (Pde6b(rd1/rd1), Pde6b(atrd1/atrd1), Rho(-/-) and Prph2(rds/rds)) and compared the abundance of reduced glutathione (GSH) and the activity of mitochondrial NADH:ubiquinone oxidoreductase (complex I), which is oxidative stress sensitive, as indirect measures of redox status, in the retinas of wild type and IPD mice. All four IPD mutants had significantly reduced retinal complex I activities (14-29% of wild type) and two showed reduced GSH, at a stage prior to the occurrence of significant cell death, whereas mitochondrial citrate synthase, which is oxidative stress insensitive, was unchanged. We orally administered the mitochondrially targeted anti oxidant MitoQ in order to reduce oxidative stress but without any improvement in retinal complex I activity, GSH or rates of PR degeneration. One possible source of oxidative stress in IPDs is oxygen toxicity in the outer retina due to reduced consumption by PR mitochondria. We therefore asked whether a reduction in the ambient O(2) concentration might improve PR survival in Pde6b(rd1/rd1) retinal explants either directly, by reducing reactive oxygen species formation, or indirectly by a neuroprotective mechanism. Pde6b(rd1/rd1) retinal explants cultured in 6% O(2) showed 31% less PR death than normoxic explants. We conclude that (i) mitochondrial oxidative stress is a significant early feature of IPDs; (ii) the ineffectiveness of MitoQ may indicate its inability to reduce some mediators of oxidative stress, such as hydrogen peroxide; and (iii) elucidation of the mechanisms by which hypoxia protects mutant PRs may identify novel neuroprotective pathways in the retina.

The genomic, biochemical, and cellular responses of the retina in inherited photoreceptor degenerations and prospects for the treatment of these disorders.

The association of more than 140 genes with human photoreceptor degenerations, together with studies of animal models of these monogenic diseases, has provided great insight into their pathogenesis. Here we review the responses of the retina to photoreceptor mutations, including mechanisms of photoreceptor death. We discuss the roles of oxidative metabolism, mitochondrial reactive oxygen species, metabolic stress, protein misfolding, and defects in ciliary proteins, as well as the responses of Müller glia, microglia, and the retinal vasculature. Finally, we report on potential pharmacologic and biologic therapies, the critical role of histopathology as a prerequisite to treatment, and the exciting promise of gene therapy in animal models and in phase 1 trials in humans.

Loss of inhibitory interneurons in the dorsal spinal cord and elevated itch in Bhlhb5 mutant mice.

Itch is the least well understood of all the somatic senses, and the neural circuits that underlie this sensation are poorly defined. Here we show that the atonal-related transcription factor Bhlhb5 is transiently expressed in the dorsal horn of the developing spinal cord and appears to play a role in the formation and regulation of pruritic (itch) circuits. Mice lacking Bhlhb5 develop self-inflicted skin lesions and show significantly enhanced scratching responses to pruritic agents. Through genetic fate-mapping and conditional ablation, we provide evidence that the pruritic phenotype in Bhlhb5 mutants is due to selective loss of a subset of inhibitory interneurons in the dorsal horn. Our findings suggest that Bhlhb5 is required for the survival of a specific population of inhibitory interneurons that regulate pruritus, and provide evidence that the loss of inhibitory synaptic input results in abnormal itch.

Maximizing functional photoreceptor differentiation from adult human retinal stem cells.

Retinal stem cells (RSCs) are present in the ciliary margin of the adult human eye and can give rise to all retinal cell types. Here we show that modulation of retinal transcription factor gene expression in human RSCs greatly enriches photoreceptor progeny, and that strong enrichment was obtained with the combined transduction of OTX2 and CRX together with the modulation of CHX10. When these genetically modified human RSC progeny are transplanted into mouse eyes, their retinal integration and differentiation is superior to unmodified RSC progeny. Moreover, electrophysiologic and behavioral tests show that these transplanted cells promote functional recovery in transducin mutant mice. This study suggests that gene modulation in human RSCs may provide a source of photoreceptor cells for the treatment of photoreceptor disease.