A target discovery pipeline identified ILT3 as a target for immunotherapy of multiple myeloma.

Multiple myeloma (MM) is an incurable malignancy of plasma cells. To identify targets for MM immunotherapy, we develop an integrated pipeline based on mass spectrometry analysis of seven MM cell lines and RNA sequencing (RNA-seq) from 900+ patients. Starting from 4,000+ candidates, we identify the most highly expressed cell surface proteins. We annotate candidate protein expression in many healthy tissues and validate the expression of promising targets in 30+ patient samples with relapsed/refractory MM, as well as in primary healthy hematopoietic stem cells and T cells by flow cytometry. Six candidates (ILT3, SEMA4A, CCR1, LRRC8D, FCRL3, IL12RB1) and B cell maturation antigen (BCMA) present the most favorable profile in malignant and healthy cells. We develop a bispecific T cell engager targeting ILT3 that shows potent killing effects in vitro and decreased tumor burden and prolonged mice survival in vivo, suggesting therapeutic relevance. Our study uncovers MM-associated antigens that hold great promise for immune-based therapies of MM.

G-protein-coupled estrogen receptor 1 is anatomically positioned to modulate synaptic plasticity in the mouse hippocampus.

Both estrous cycle and sex affect the numbers and types of neuronal and glial profiles containing the classical estrogen receptors α and ß, and synaptic levels in the rodent dorsal hippocampus. Here, we examined whether the membrane estrogen receptor, G-protein-coupled estrogen receptor 1 (GPER1), is anatomically positioned in the dorsal hippocampus of mice to regulate synaptic plasticity. By light microscopy, GPER1-immunoreactivity (IR) was most noticeable in the pyramidal cell layer and interspersed interneurons, especially those in the hilus of the dentate gyrus. Diffuse GPER1-IR was found in all lamina but was most dense in stratum lucidum of CA3. Ultrastructural analysis revealed discrete extranuclear GPER1-IR affiliated with the plasma membrane and endoplasmic reticulum of neuronal perikarya and dendritic shafts, synaptic specializations in dendritic spines, and clusters of vesicles in axon terminals. Moreover, GPER1-IR was found in unmyelinated axons and glial profiles. Overall, the types and amounts of GPER1-labeled profiles were similar between males and females; however, in females elevated estrogen levels generally increased axonal labeling. Some estradiol-induced changes observed in previous studies were replicated by the GPER agonist G1: G1 increased PSD95-IR in strata oriens, lucidum, and radiatum of CA3 in ovariectomized mice 6 h after administration. In contrast, estradiol but not G1 increased Akt phosphorylation levels. Instead, GPER1 actions in the synapse may be due to interactions with synaptic scaffolding proteins, such as SAP97. These results suggest that although estrogen's actions via GPER1 may converge on the same synaptic elements, different pathways are used to achieve these actions.

Post-synaptic density-95 (PSD-95) binding capacity of G-protein-coupled receptor 30 (GPR30), an estrogen receptor that can be identified in hippocampal dendritic spines.

The estrogen 17ß-estradiol (E2) modulates dendritic spine plasticity in the cornu ammonis 1 (CA1) region of the hippocampus, and GPR30 (G-protein coupled estrogen receptor 1 (GPER1)) is an estrogen-sensitive G-protein-coupled receptor (GPCR) that is expressed in the mammalian brain and in specific subregions that are responsive to E2, including the hippocampus. The subcellular localization of hippocampal GPR30, however, remains unclear. Here, we demonstrate that GPR30 immunoreactivity is detected in dendritic spines of rat CA1 hippocampal neurons in vivo and that GPR30 protein can be found in rat brain synaptosomes. GPR30 immunoreactivity is identified at the post-synaptic density (PSD) and in the adjacent peri-synaptic zone, and GPR30 can associate with the spine scaffolding protein PSD-95 both in vitro and in vivo. This PSD-95 binding capacity of GPR30 is specific and determined by the receptor C-terminal tail that is both necessary and sufficient for PSD-95 interaction. The interaction with PSD-95 functions to increase GPR30 protein levels residing at the plasma membrane surface. GPR30 associates with the N-terminal tandem pair of PDZ domains in PSD-95, suggesting that PSD-95 may be involved in clustering GPR30 with other receptors in the hippocampus. We demonstrate that GPR30 has the potential to associate with additional post-synaptic GPCRs, including the membrane progestin receptor, the corticotropin releasing hormone receptor, and the 5HT1a serotonin receptor. These data demonstrate that GPR30 is well positioned in the dendritic spine compartment to integrate E2 sensitivity directly onto multiple inputs on synaptic activity and might begin to provide a molecular explanation as to how E2 modulates dendritic spine plasticity.

Estrogen effects on the brain: actions beyond the hypothalamus via novel mechanisms.

From its origins in how the brain controls the endocrine system via the hypothalamus and pituitary gland, neuroendocrinology has evolved into a science that now includes hormone action on many aspects of brain function. These actions involve the whole central nervous system and not just the hypothalamus. Advances in our understanding of cellular and molecular actions of steroid hormones have gone beyond the important cell nuclear actions of steroid hormone receptors to include signaling pathways that intersect with other mediators such as neurotransmitters and neuromodulators. This has, in turn, broadened the search for and identification of steroid receptors to include nonnuclear sites in synapses, dendrites, mitochondria, and glial cells, as well as cell nuclei. The study of estrogen receptors and estrogen actions on processes related to cognition, mood, autonomic regulation, pain, and neuroprotection, among other functions, has led the way in this new view of hormone actions on the brain. In this review, we summarize past and current work in our laboratory on this topic. This exciting and growing field involving many laboratories continues to reshape our ideas and approaches to neuroendocrinology both at the bench and the bedside.

Ovarian hormones influence corticotropin releasing factor receptor colocalization with delta opioid receptors in CA1 pyramidal cell dendrites.

Stress interacts with addictive processes to increase drug use, drug seeking, and relapse. The hippocampal formation (HF) is an important site at which stress circuits and endogenous opioid systems intersect and likely plays a critical role in the interaction between stress and drug addiction. Our prior studies demonstrate that the stress-related neuropeptide corticotropin-releasing factor (CRF) and the delta-opioid receptor (DOR) colocalize in interneuron populations in the hilus of the dentate gyrus and stratum oriens of CA1 and CA3. While independent ultrastructural studies of DORs and CRF receptors suggest that each receptor is found in CA1 pyramidal cell dendrites and dendritic spines, whether DORs and CRF receptors colocalize in CA1 neuronal profiles has not been investigated. Here, hippocampal sections of adult male and proestrus female Sprague-Dawley rats were processed for dual label pre-embedding immunoelectron microscopy using well-characterized antisera directed against the DOR for immunoperoxidase and against the CRF receptor for immunogold. DOR-immunoreactivity (-ir) was found presynaptically in axons and axon terminals as well as postsynaptically in somata, dendrites and dendritic spines in stratum radiatum of CA1. In contrast, CRF receptor-ir was predominantly found postsynaptically in CA1 somata, dendrites, and dendritic spines. CRF receptor-ir frequently was observed in DOR-labeled dendritic profiles and primarily was found in the cytoplasm rather than at or near the plasma membrane. Quantitative analysis of CRF receptor-ir colocalization with DOR-ir in pyramidal cell dendrites revealed that proestrus females and males show comparable levels of CRF receptor-ir per dendrite and similar cytoplasmic density of CRF receptor-ir. In contrast, proestrus females display an increased number of dual-labeled dendritic profiles and an increased membrane density of CRF receptor-ir in comparison to males. We further examined the functional consequences of CRF receptor-ir colocalization with DOR-ir in the same neuron using the hormone responsive neuronal cell line NG108-15, which endogenously expresses DORs, and assayed intracellular cAMP production in response to CRF receptor and DOR agonists. Results demonstrated that short-term application of DOR agonist SNC80 inhibited CRF-induced cAMP accumulation in NG108-15 cells transfected with the CRF receptor. These studies provide new insights on opioid-stress system interaction in the hippocampus of both males and females and establish potential mechanisms through which DOR activation may influence CRF receptor activity.

Effects of estrogen and aging on the synaptic distribution of phosphorylated Akt-immunoreactivity in the CA1 region of the female rat hippocampus.

The estrogen 17ß-estradiol (E) increases the axospinous synaptic density and plasticity in the hippocampal CA1 region of young female rats but fails to do so in aged female rats. This E stimulus on synaptic plasticity is associated with the phosphorylation-dependent activation of Akt kinase. Our previous findings demonstrated that increased estrogen levels subsequently increase phosphorylated Akt (pAkt)-immunoreactivity (-IR) within the dendritic shafts and spines of pyramidal neurons in young female rats. Therefore, because Akt can promote cell survival and growth, we tested the hypothesis that the less plastic synapses of aged female rats would contain less E-stimulated pAkt-IR. Here, young (3-4 months) and aged (22-23 months) female rats were ovariectomized 7 days prior to a 48-h administration of either vehicle or E. The pAkt-IR synaptic distribution was then analyzed using post-embedding electron microscopy. In both young and aged rats, pAkt-IR was found in dendritic spines and terminals, and pAkt-IR was particularly abundant at the post-synaptic density. Quantitative analyses revealed that the percentage of pAkt-labeled synapses was significantly greater in young rats compared to aged rats. Nonetheless, E treatment significantly increased pAkt-IR in pre- and post-synaptic profiles of both young and aged rats, although the stimulus in young rats was notably more widespread. These data support the evidence that hormone-activated signaling associated with cell growth and survival is diminished in the aged brain. However, the observation that E can still increase pAkt-IR in aged synapses presents this signaling component as a candidate target for hormone replacement therapies.

LIM kinase mediates estrogen action on the actin depolymerization factor Cofilin.

The ovarian hormone estrogen increases the axospinous synapse density in the hippocampal CA1 region of young female rats but fails to do so in aged rats. This estrogen-mediated alteration of spine synapse structures suggests the coincident requirement for the structural reorganization of the underlying actin cytoskeleton network. Actin reorganization is known to require the deactivation of Cofilin, an actin depolymerization factor. Cofilin is deactivated by LIM kinase (LIMK), and LIMK activity is modulated by the phosphorylation of specific residues. We have previously demonstrated that estrogen is able to increase phosphorylated LIMK (pLIMK) immunoreactivity (IR) in the hippocampus in vivo and that this estrogen-stimulated pLIMK-IR is decreased in the aged brain. Because Cofilin phosphorylation allows for actin filament elongation and spine synapse growth, we sought to determine if estrogen acts through Cofilin and if such estrogen action requires the observed LIMK activity. Using both hippocampal neurons and the NG108-15 neuroblastoma cell line, we demonstrate here that estrogen stimulates the phosphorylation of Cofilin in vitro. Furthermore, this estrogen action on Cofilin requires LIMK. Lastly, while initiating the phosphorylation of LIMK and Cofilin, estrogen can also stimulate the formation of filopodial extensions, an early step in the formation of nascent spines, demonstrating that estrogen can alter the actin-dependent neuronal morphology. This linkage of estrogen communication to Cofilin via LIMK provides the functionality to the age-sensitive pLIMK-IR that we have observed in vivo.

Effects of acute diuresis stress on egr-1 (zif268) mRNA levels in brain regions associated with motivated behavior.

Stressors evoke a well-studied physiological stress-response, namely, an immediate systemic release of catecholamines from the adrenals followed shortly afterwards by the release of adrenal steroids. The intensity of that response can often be inferred by the amount of adrenal steroids released into the circulatory system. It is still unclear however how the intensity and duration of the stressor affect a number of brain regions, including those in the motivational system. The present study sought to determine whether a brief stressor, such as an isotonic saline injection, activated the brain's motivational system in mesolimbic regions compared with a more intense stressor exemplified by pharmacological challenges caused by the administration of a diuretic. Adult male Sprague-Dawley rats were either injected (s.c.) with isotonic saline or 5mg of the diuretic, furosemide. Controls did not receive any injections. Animals were sacrificed at 30, 60, 120, and 240 min after injection and trunk blood and brains were collected. Serum corticosterone and aldosterone levels were assessed through radioimmunoassay and mesolimbic brain activity was determined through in situ hybridization of mRNA expression of the immediate-early gene egr-1 in the caudate-putamen and nucleus accumbens. While both adrenal steroids demonstrated an initial peak in both stress groups, levels were higher and longer lasting in rats treated with furosemide. Interestingly, egr-1 mRNA levels were significantly higher only in the furosemide-treated group compared with controls. These findings suggest that a selective activation of motivational circuits occurs under thirst and salt-appetite-induced conditions such as those caused by diuresis.

Thinking outside the pyramidal cell: unexplored contributions of interneurons and neuropeptide Y to estrogen-induced synapse formation in the hippocampus.

Since the first finding that 17beta-estradiol (E) can regulate CA1 pyramidal cell synapse formation, subsequent studies have explored many potential E-dependent mechanisms occurring within CA1 pyramidal cells. Fewer studies have focused on E-dependent processes outside of the pyramidal cell that may influence events activity of the pyramidal cells. This review considers hippocampal interneurons, which can potently regulate the excitability of simultaneously firing pyramidal cells. In particular, we discuss neuropeptide Y (NPY) expression by these interneurons because our published findings show that NPY expression is increased by E in a subset of interneurons which coincidentally exhibit E-regulated increase in GABA synthesis and are uniquely situated anatomically such that they may regulate synaptic activity. Here we review the role of different phenotypes of CA1 interneurons, and we propose a model in which E-stimulated NPY gene expression and the release of NPY by interneurons inhibits glutamate release presynaptically and alters glutamate-dependent synaptic events in the rat hippocampus during adulthood.

Gene therapy to bet on: protecting neurons from stress hormones.

Glucocorticoids are adrenal steroid 'stress' hormones that are important in normal brain function. However, during conditions of excitotoxic injury to neurons, glucocorticoids can exacerbate cell death. A recent study has developed a 'trifecta' of ingenious viral vector-based approaches to modify the neuronal stress response and to diminish this exacerbation both in vitro and in vivo. Stress hormone gene therapy in the brain is now off and running.

Estrogen and ovariectomy regulate mRNA and protein of glutamic acid decarboxylases and cation-chloride cotransporters in the adult rat hippocampus.

17beta-Estradiol spatiotemporally regulates the gamma-aminobutyric acid (GABAergic) tone in the adult hippocampus. However, the complex estrogenic effect on the GABAergic system is still unclear. In adult central nervous system (CNS) neurons, GABA can induce both inhibitory and excitatory actions, which are predominantly controlled by the cation-chloride cotransporters NKCC1 and KCC2. We therefore studied the estrogenic regulation of two glutamate decarboxylase (GAD) isoforms, GAD65 and GAD67, as well as NKCC1 and KCC2 in the adult female rat hippocampus by immunohistochemistry and in situ hybridization. First, we focused on the duration after ovariectomy (OVX) and its effects on GAD65 protein levels. The basal number of GAD65-immunoreactive cells decreased after long-term (10 days) OVX compared to short-term (3 days) OVX. We found that, only after long-term OVX but not after short-term OVX, estradiol increased the number of GAD65-immunoreactive cells in the CA1 pyramidal cell layer. Furthermore, estradiol did not alter the GAD65-immunoreactive cell population in any other CA1 subregion. Second, we therefore focused on long-term OVX and the estrogenic regulation of GAD and cation-chloride cotransporter mRNA levels. In the pyramidal cell layer, estradiol affected GAD65, GAD67 and NKCC1 mRNA levels, but not KCC2 mRNA levels. Both GAD65 and NKCC1 mRNA levels increased within 24 h after estradiol treatment, followed by a subsequent increase in GAD67 mRNA levels. These findings suggest that basal levels of estrogen might contribute to a balance between the excitatory and inhibitory synaptic transmission onto CA1 pyramidal cells by regulating perisomatic GAD and NKCC1 expression in the adult hippocampus.

Estrogen stimulates postsynaptic density-95 rapid protein synthesis via the Akt/protein kinase B pathway.

Estrogens induce synaptogenesis in the CA1 region of the dorsal hippocampus during the estrous cycle of the female rat. Functional consequences of such estrogen-mediated synaptogenesis include cyclic changes in neurotransmission and memory. At the molecular level, estrogen stimulates the rapid activation of specific signal transduction pathways, and of particular interest is the activation of Akt (protein kinase B), a key signal transduction intermediate that initiates protein translation by alleviating the downstream translational repression of eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). Using a well established in vitro model system of differentiated NG108-15 neurons to investigate such rapid signaling effects of estrogen, we show that estrogen stimulates the phosphorylation of Akt, an indication of kinase activation, as well as the phosphorylation of 4E-BP1. In turn, the activation of these signaling intermediates suggests a non-genomic mechanism by which estrogen might likewise lead to protein translation of dendrite-localized mRNA transcripts in the hippocampus in vivo. We therefore considered the translation of the dendritic spine scaffolding protein postsynaptic density-95 (PSD-95). Although estrogen does not stimulate a rapid increase in PSD-95 mRNA levels in NG108-15 neurons, we show here that estrogen does however stimulate a rapid increase in PSD-95 new protein synthesis in vitro and that this new protein synthesis is Akt dependent. These results demonstrate an essential role for Akt in estrogen-stimulated dendritic spine protein expression, describe for the first time a signal transduction pathway in PSD-95 expression, and delineate a novel, molecular mechanism by which ovarian hormones might translationally regulate synaptogenesis via activating protein synthesis for dendritic function.

Estrogen levels regulate the subcellular distribution of phosphorylated Akt in hippocampal CA1 dendrites.

In addition to genomic pathways, estrogens may regulate gene expression by activating specific signal transduction pathways, such as that involving phosphatidylinositol 3-kinase (PI3-K) and the subsequent phosphorylation of Akt (protein kinase B). The Akt pathway regulates various cellular events, including the initiation of protein synthesis. Our previous studies showed that synaptogenesis in hippocampal CA1 pyramidal cell dendritic spines is highest when brain estrogen levels are highest. To address the role of Akt in this process, the subcellular distribution of phosphorylated Akt immunoreactivity (pAkt-I) in the hippocampus of female rats across the estrous cycle and male rats was analyzed by light microscopy (LM) and electron microscopy (EM). By LM, the density of pAkt-I in stratum radiatum of CA1 was significantly higher in proestrus rats (or in estrogen-supplemented ovariectomized females) compared with diestrus, estrus, or male rats. By EM, pAkt-I was found throughout the shafts and in select spines of stratum radiatum dendrites. Quantitative ultrastructural analysis identifying pAkt-I with immunogold particles revealed that proestrus rats compared with diestrus, estrus, and male rats contained significantly higher pAkt-I associated with (1) dendritic spines (both cytoplasm and plasmalemma), (2) spine apparati located within 0.1 microm of dendritic spine bases, (3) endoplasmic reticula and polyribosomes in the cytoplasm of dendritic shafts, and (4) the plasmalemma of dendritic shafts. These findings suggest that estrogens may regulate spine formation in CA1 pyramidal neurons via Akt-mediated signaling events.

Decreases in neurokinin-3 tachykinin receptor-immunoreactive and -mRNA levels are associated with salt appetite in the deoxycorticosterone-treated rat.

Tachykinins are a family of neuropeptides that inhibit salt appetite. Although decreased tachykinin-mRNA levels are observed in natriorexic sodium-deplete rats, no decrease is seen in natriorexic sodium-replete rats that are administered the aldosterone-mimetic deoxycorticosterone acetate (DOCA). Since reduced synthesis of tachykinins could not account for increased appetite, we hypothesized that increased salt appetite was due to a downregulation of tachykinin receptors. Thus, we injected rats with DOCA once daily for 11 days and analyzed tachykinin receptor subtype, neurokinin 3 (NK3r)-immunoreactivity by Western blot analysis since intracerebroventricular injection of senktide (NK3r agonist) attenuates salt intake in DOCA-treated animals. We examined NK3r-immunoreactivity in several brain regions thought to be associated with the control of water and electrolyte balance including the bed nucleus of the stria terminalis, central nucleus of the amygdala, diagonal band of Broca, hippocampus, nucleus tractus solitarius, parabrachial nucleus, paraventricular nucleus of the hypothalamus, and supraoptic nucleus. Consistent with our hypothesis, we found decreased NK3r-immunoreactivity in all brain regions analyzed except for increases in the amygdala and no changes in the paraventricular nucleus of the hypothalamus. To examine whether DOCA's effects on NK3r synthesis are direct, we used differentiated N1E-115 neuroblastoma cells that express NK3r and treated them with a range of concentrations of DOCA and found a dose-dependent decrease in NK3r-mRNA abundance via Northern blotting. The present results suggest that the tachykinin receptors are downregulated after subchronic DOCA treatment and this finding is consistent with the hypothesis that suppressed inhibition of salt appetite as mediated through the tachykininergic system.