CCR3 plays a role in murine age-related cognitive changes and T-cell infiltration into the brain.

Targeting immune-mediated, age-related, biology has the potential to be a transformative therapeutic strategy. However, the redundant nature of the multiple cytokines that change with aging requires identification of a master downstream regulator to successfully exert therapeutic efficacy. Here, we discovered CCR3 as a prime candidate, and inhibition of CCR3 has pro-cognitive benefits in mice, but these benefits are not driven by an obvious direct action on central nervous system (CNS)-resident cells. Instead, CCR3-expressing T cells in the periphery that are modulated in aging inhibit infiltration of these T cells across the blood-brain barrier and reduce neuroinflammation. The axis of CCR3-expressing T cells influencing crosstalk from periphery to brain provides a therapeutically tractable link. These findings indicate the broad therapeutic potential of CCR3 inhibition in a spectrum of neuroinflammatory diseases of aging.

Safety and Tolerability of GRF6019 Infusions in Severe Alzheimer's Disease: A Phase II Double-Blind Placebo-Controlled Trial.

BACKGROUND: The plasma fraction GRF6019 shows multiple benefits on brain aging in mice, including enhanced cognition, neurogenesis, and synaptic density, as well as reduced neuroinflammation. OBJECTIVE: To evaluate the safety, tolerability, and preliminary efficacy of GRF6019 in patients with severe Alzheimer's disease (AD). METHODS: A phase II, double-blind, placebo-controlled study in patients with severe AD (Mini-Mental State Examination score 0-10). Patients were randomized 2 : 1 to GRF6019 (N = 18) or placebo (N = 8) and received daily 250 mL intravenous infusions over 5 days. The primary endpoints were the rates of adverse events (AEs) and the tolerability of GRF6019 as assessed by the number of patients completing the study. Change from baseline in cognitive and functional assessments was also evaluated. RESULTS: All patients completed 100%of study visits and infusions. The rate of AEs was similar in the GRF6019 (8/18 patients [44.4%]) and placebo (3/8 patients [37.5%]) groups, and there were no deaths or serious AEs. The most common AEs considered related to treatment were mild, transient changes in blood pressure in the GRF6019 group (hypotension: 2 patients [11.1%]; hypertension: 1 patient [5.6%]); there were no related AEs in the placebo group. The trial was not powered to detect statistically significant differences between treatment groups. At the end of the study, patients in both treatment groups remained stable or improved on all cognitive and functional endpoints. CONCLUSION: GRF6019 demonstrated excellent safety, feasibility, and tolerability. Future trials designed to characterize the potential functional benefits of GRF6019 and related plasma fractions in severe AD are warranted.

Safety and tolerability of GRF6019 in mild-to-moderate Alzheimer's disease dementia.

INTRODUCTION: This phase 2 trial evaluated the safety, tolerability, and feasibility of repeated infusions of the plasma fraction GRF6019 in mild-to-moderate Alzheimer's disease. METHODS: In this randomized, double-blind, dose-comparison trial, 47 patients were randomized 1:1 to receive daily infusions of 100 mL (n = 24) or 250 mL (n = 23) of GRF6019 for 5 consecutive days over two dosing periods separated by a treatment-free interval of 3 months. RESULTS: The mean (standard deviation [SD]) age of the enrolled patients was 74.3 (6.9), and 62% were women. Most adverse events (55%) were mild, with no clinically significant differences in safety or tolerability between the two dose levels. The mean (SD) baseline Mini-Mental State Examination score was 20.6 (3.7) in the 100 mL group and 19.6 (3.7) in the 250 mL group; at 24 weeks, the within-patient mean change from baseline was -1.0 points (95% confidence interval [CI], -3.1 to 1.1) in the 100 mL group and +1.5 points (95% CI, -0.4 to 3.3) in the 250 mL group. The within-patient mean change from baseline on the Alzheimer's Disease Assessment Scale-Cognitive subscale was -0.4 points (95% CI, -2.9 to 2.2) in the 100 mL group, while in the 250 mL group it was -0.9 points (95% CI, -3.0 to 1.2). The within-patient mean change from baseline on the Alzheimer's Disease Cooperative Study-Activities of Daily Living was -0.7 points in the 100 mL group (95% CI, -4.3 to 3.0) and -1.3 points (95% CI, -3.4 to 0.7) in the 250 mL group. The mean change from baseline on the Category Fluency Test, Clinical Dementia Rating Scale-Sum of Boxes, Alzheimer's Disease Cooperative Study-Clinical Global Impression of Change, and Neuropsychiatric Inventory Questionnaire was similar for both treatment groups and did not show any worsening. DISCUSSION: GRF6019 was safe and well tolerated, and patients experienced no cognitive decline and minimal functional decline. These results support further development of GRF6019.

Safety, Tolerability, and Feasibility of Young Plasma Infusion in the Plasma for Alzheimer Symptom Amelioration Study: A Randomized Clinical Trial.

Importance: Young mouse plasma restores memory in aged mice, but, to our knowledge, the effects are unknown in patients with Alzheimer disease (AD). Objective: To assess the safety, tolerability, and feasibility of infusions of young fresh frozen plasma (yFFP) from donors age 18 to 30 years in patients with AD. Design, Setting, and Participants: The Plasma for Alzheimer Symptom Amelioration (PLASMA) study randomized 9 patients under a double-blind crossover protocol to receive 4 once-weekly infusions of either 1 unit (approximately 250 mL) of yFFP from male donors or 250 mL of saline, followed by a 6-week washout and crossover to 4 once-weekly infusions of an alternate treatment. Patients and informants were masked to treatment and subjective measurements. After an open-label amendment, 9 patients received 4 weekly yFFP infusions only and their subjective measurements were unmasked. Patients were enrolled solely at Stanford University, a tertiary academic medical center, from September 2014 to December 2016, when enrollment reached its target. Eighteen consecutive patients with probable mild to moderate AD dementia, a Mini-Mental State Examination (score of 12 to 24 inclusive), and an age of 50 to 90 years were enrolled. Thirty-one patients were screened and 13 were excluded: 11 failed the inclusion criteria and 2 declined to participate. Interventions: One unit of yFFP from male donors/placebo infused once weekly for 4 weeks. Main Outcome and Measures: The primary outcomes were the safety, tolerability, and feasibility of 4 weekly yFFP infusions. Safety end point analyses included all patients who received the study drug/placebo. Results: There was no difference in the age (mean [SD], 74.17 [7.96] years), sex (12 women [67%]), or baseline Mini-Mental State Examination score (mean [SD], 19.39 [3.24]) between the crossover (n = 9) and open-label groups (n = 9). There were no related serious adverse events. One patient discontinued participation because of urticaria and another because of an unrelated stroke. There was no statistically significant difference between the plasma (17 [94.4%]) and placebo (9 [100.0%]) cohorts for other adverse events, which were mild to moderate in severity. The most common adverse events in the plasma group included hypertension (3 [16.7%]), dizziness (2 [11.1%]), sinus bradycardia (3 [16.7%]), headache (3 [16.7%]), and sinus tachycardia (3 [16.7%]). The mean visit adherence (n = 18) was 86% (interquartile range, 87%-100%) and adherence, accounting for a reduction in the total visit requirement due to early patient discontinuation, was 96% (interquartile range, 89%-100%). Conclusions and Relevance: The yFFP treatment was safe, well tolerated, and feasible. The study's limitations were the small sample size, short duration, and change in study design. The results warrant further exploration in larger, double-blinded placebo-controlled clinical trials. Trial Registration: ClinicalTrials.gov Identifier: NCT02256306.

A pharmacological screening approach for discovery of neuroprotective compounds in ischemic stroke.

With the availability and ease of small molecule production and design continuing to improve, robust, high-throughput methods for screening are increasingly necessary to find pharmacologically relevant compounds amongst the masses of potential candidates. Here, we demonstrate that a primary oxygen glucose deprivation assay in primary cortical neurons followed by secondary assays (i.e. post-treatment protocol in organotypic hippocampal slice cultures and cortical neurons) can be used as a robust screen to identify neuroprotective compounds with potential therapeutic efficacy. In our screen about 50% of the compounds in a library of pharmacologically active compounds displayed some degree of neuroprotective activity if tested in a pre-treatment toxicity assay but just a few of these compounds, including Carbenoxolone, remained active when tested in a post-treatment protocol. When further examined, Carbenoxolone also led to a significant reduction in infarction size and neuronal damage in the ischemic penumbra when administered six hours post middle cerebral artery occlusion in rats. Pharmacological testing of Carbenoxolone-related compounds, acting by inhibition of 11-ß-hydroxysteroid dehydrogenase-1 (11ß-HSD1), gave rise to similarly potent in vivo neuroprotection. This indicates that the increase of intracellular glucocorticoid levels mediated by 11ß-HSD1 may be involved in the mechanism that exacerbates ischemic neuronal cell death, and inhibiting this enzyme could have potential therapeutic value for neuroprotective therapies in ischemic stroke and other neurodegenerative disorders associated with neuronal injury.

The sigma-1 receptor enhances brain plasticity and functional recovery after experimental stroke.

Stroke leads to brain damage with subsequent slow and incomplete recovery of lost brain functions. Enriched housing of stroke-injured rats provides multi-modal sensorimotor stimulation, which improves recovery, although the specific mechanisms involved have not been identified. In rats housed in an enriched environment for two weeks after permanent middle cerebral artery occlusion, we found increased sigma-1 receptor expression in peri-infarct areas. Treatment of rats subjected to permanent or transient middle cerebral artery occlusion with 1-(3,4-dimethoxyphenethyl)-4-(3-phenylpropyl)piperazine dihydrochloride, an agonist of the sigma-1 receptor, starting two days after injury, enhanced the recovery of lost sensorimotor function without decreasing infarct size. The sigma-1 receptor was found in the galactocerebroside enriched membrane microdomains of reactive astrocytes and in neurons. Sigma-1 receptor activation increased the levels of the synaptic protein neurabin and neurexin in membrane rafts in the peri-infarct area, while sigma-1 receptor silencing prevented sigma-1 receptor-mediated neurite outgrowth in primary cortical neuronal cultures. In astrocytic cultures, oxygen and glucose deprivation induced sigma-1 receptor expression and actin dependent membrane raft formation, the latter blocked by sigma-1 receptor small interfering RNA silencing and pharmacological inhibition. We conclude that sigma-1 receptor activation stimulates recovery after stroke by enhancing cellular transport of biomolecules required for brain repair, thereby stimulating brain plasticity. Pharmacological targeting of the sigma-1 receptor provides new opportunities for stroke treatment beyond the therapeutic window of neuroprotection.

KIBRA: A New Gateway to Learning and Memory?

The genetic locus encoding KIBRA, a member of the WWC family of proteins, has recently been shown to be associated with human memory performance through genome-wide single nucleotide polymorphism screening. Gene expression analysis and a variety of functional studies have further indicated that such a role is biologically plausible for KIBRA. Here, we review the existing literature, illustrate connections between the different lines of evidence, and derive models based on KIBRA's function(s) in the brain that can be further tested experimentally.

Expression and function of striatal enriched protein tyrosine phosphatase is profoundly altered in cerebral ischemia.

Striatal enriched protein tyrosine phosphatase (STEP) acts in the central nervous system to dephosphorylate a number of important proteins involved in synaptic function including ERK and NMDA receptor subunits. These proteins are also linked to stroke, in which cerebral ischemia triggers a complex cascade of events. Here we demonstrate that STEP is regulated at both the transcriptional and the post-transcriptional levels in rat models of cerebral ischemia and that its regulation may play a role in the outcome of ischemic insults. After transient middle cerebral artery occlusion, there are profound decreases in the levels of STEP mRNA, whilst in global ischemia STEP mRNA is selectively down-regulated in areas susceptible to ischemic damage. In a neuroprotective preconditioning paradigm, and in regions of the brain that are relatively resistant to ischemic damage, STEP mRNA levels are increased. Furthermore, there is a significant processing of STEP after ischemia to generate a novel species, STEP(33), resulting in a redistribution of STEP from membrane-bound to soluble compartments. Concomitant with the cleavage of mature forms of STEP, there are changes in the phosphorylation state of ERK. We show that the cleavage of STEP leads to a catalytically active form, but this cleaved form no longer binds to and dephosphorylates its substrate pERK. Therefore, in response to ischemic insults, there are profound reductions in both the amount and the activity of STEP, its localization, as well as the activity of one of its key substrates, pERK. These changes in STEP may reflect a critical role in the outcomes of ischemic brain injury.

Npas4, a novel helix-loop-helix PAS domain protein, is regulated in response to cerebral ischemia.

Basic helix-loop-helix PAS domain proteins form a growing family of transcription factors. These proteins are involved in the process of adaptation to cellular stresses and environmental factors such as a change in oxygen concentration. We describe the identification and characterization of a recently cloned PAS domain protein termed Npas4 in ischemic rat brain. Using gene expression profiling following middle cerebral artery occlusion, we showed that the Npas4 mRNA is differentially expressed in ischemic tissue. The full-length gene was cloned from rat brain and its spatial and temporal expression characterized with in situ hybridization and Northern blotting. The Npas4 mRNA is specifically expressed in the brain and is highly up-regulated in ischemic tissues following both focal and global cerebral ischemic insults. Immunohistochemistry revealed a strong expression in the limbic system and thalamus, as well as in layers 3 and 5 in the cortex of the unchallenged brain. When overexpressed in HEK 293 cells, Npas4 appears as a protein of approximately 100 kDa. In brain samples, however, in addition to the 100 kDa band a specific 200 kDa immunoreactive band was also detected. Ischemic challenge lead to a decrease in the 200 kDa form and a simultaneous increase in the 100 kDa immunoreactivity. This could indicate a novel regulatory mechanism for activation and/or deactivation of this protein in response to ischemic brain injury.

Regulation of NMDA receptor trafficking and function by striatal-enriched tyrosine phosphatase (STEP).

Regulation of N-methyl-D-aspartate (NMDA) receptors is critical for the normal functioning of the central nervous system. There must be precise mechanisms to allow for changes in receptor function required for learning and normal synaptic transmission, but within tight constraints to prevent pathology. Tyrosine phosphorylation is a major means by which NMDA receptors are regulated through the equilibrium between activity of Src family kinases and tyrosine phosphatases. Identification of NMDA receptor phosphatases has been difficult, the best candidate being striatal-enriched tyrosine phosphatase (STEP). Here we demonstrate that STEP is a critical regulator of NMDA receptors and reveal that the action of this tyrosine phosphatase controls the constitutive trafficking of NMDA receptors and leads to changes in NMDA receptor activity at the neuronal surface. We show that STEP binds directly to NMDA receptors in the absence of other synaptic proteins. The activity of STEP selectively affects the expression of NMDA receptors at the neuronal plasma membrane. The result of STEP's action upon the NMDA receptor affects the functional properties of the receptor and its downstream signaling. These effects are evident when STEP levels are chronically reduced, indicating that there is no redundancy amongst phosphatases to compensate for altered STEP function in the CNS. STEP may have evolved specifically to fill a pivotal role as the NMDA receptor phosphatase, having a distinct and restricted localization and compartmentalization, and unique activity towards the NMDA receptor and its signaling pathway.

Mechanisms of neural plasticity following brain injury.

Brain insults cause rapid cell death, and a disruption of functional circuits, in the affected regions. As the injured tissue recovers from events associated with cell death, regenerative processes are activated that over months lead to a certain degree of functional recovery. Factors produced by new neurons and glia, axonal sprouting of surviving neurons, and new synapse formation help to re-establish some of the lost functions. The timing and location of such events is crucial in the success of the regenerative process. Comprehensive gene expression profiling and proteomic analyses have enabled a deeper molecular and cellular mechanistic understanding of post-injury brain regeneration. These new mechanistic insights are aiding the design of novel therapeutic modalities that enhance regeneration.

Targeting of the receptor protein tyrosine phosphatase beta with a monoclonal antibody delays tumor growth in a glioblastoma model.

The receptor protein tyrosine phosphatase beta (RPTPbeta) is a functional biomarker for several solid tumor types. RPTPbeta expression is largely restricted to the central nervous system and overexpressed primarily in astrocytic tumors. RPTPbeta is known to facilitate tumor cell adhesion and migration through interactions with extracellular matrix components and the growth factor pleiotrophin. Here, we show that RPTPbeta is expressed in a variety of solid tumor types with low expression in normal tissue. To assess RPTPbeta as a potential target for treatment of glioblastoma and other cancers, antibodies directed to RPTPbeta have been developed and profiled in vitro and in vivo. The recombinant extracellular domain of human short RPTPbeta was used to immunize mice and generate monoclonal antibodies that selectively recognize RPTPbeta and bind to the antigen with low nanomolar affinities. Moreover, these antibodies recognized the target on living tumor cells as measured by flow cytometry. These antibodies killed glioma cells in vitro when coupled to the cytotoxin saporin either directly or via a secondary antibody. Finally, in vivo studies showed that an anti-RPTPbeta immunotoxin (7E4B11-SAP) could significantly delay human U87 glioma tumors in a mouse xenograft model. Unconjugated 7E4B11 provides a modest but statistically significant tumor growth delay when delivered systemically in mice bearing U87 glioma tumors.

Comprehensive regional and temporal gene expression profiling of the rat brain during the first 24 h after experimental stroke identifies dynamic ischemia-induced gene expression patterns, and reveals a biphasic activation of genes in surviving tissue.

In order to identify biological processes relevant for cell death and survival in the brain following stroke, the postischemic brain transcriptome was studied by a large-scale cDNA array analysis of three peri-infarct brain regions at eight time points during the first 24 h of reperfusion following middle cerebral artery occlusion in the rat. K-means cluster analysis revealed two distinct biphasic gene expression patterns that contained 44 genes (including 18 immediate early genes), involved in cell signaling and plasticity (i.e. MAP2K7, Sprouty2, Irs-2, Homer1, GPRC5B, Grasp). The first gene induction phase occurred at 0-3 h of reperfusion, and the second at 9-15 h, and was validated by in situ hybridization. Four gene clusters displayed a progressive increase in expression over time and included 50 genes linked to cell motility, lipid synthesis and trafficking (i.e. ApoD, NPC1, G3P-dehydrogenase1, and Choline kinase) or cell death-regulating genes such as mitochondrial CLIC. We conclude that a biphasic transcriptional up-regulation of the brain-derived neurotrophic factor (BDNF)-G-protein coupled receptor (GPCR)-mitogen-activated protein (MAP) kinase signaling pathways occurs in surviving tissue, concomitant with a progressive and persistent activation of cell proliferation signifying tissue regeneration, which provide the means for cell survival and postischemic brain plasticity.

Discoidin domain receptor-1a (DDR1a) promotes glioma cell invasion and adhesion in association with matrix metalloproteinase-2.

Invasion of glioma cells involves the attachment of invading tumor cells to extracellular matrix (ECM), disruption of ECM components, and subsequent cell penetration into adjacent brain structures. Discoidin domain receptor 1 (DDR1) tyrosine kinases constitute a novel family of receptors characterized by a unique structure in the ectodomain (discoidin-I domain). These cell surface receptors bind to several collagens and facilitate cell adhesion. Little is known about DDR1 expression and function in glioblastoma multiforme. In this study we demonstrate that DDR1 is overexpressed in glioma tissues using cDNA arrays, immunohistochemistry and Western blot analysis. Functional comparison of two splice variants of DDR1 (DDR1a and DDR1b) reveal novel differences in cell based glioma models. Overexpression of either DDR1a or DDR1b caused increased cell attachment. However, glioma cells overexpressing DDR1a display enhanced invasion and migration. We also detect increased levels of matrix metalloproteinase-2 in DDR1a overexpressing cells as measured by zymography. Inhibition of MMP activity using MMP inhibitor suppressed DDR1a stimulated cell-invasion. Similarly, an antibody against DDR1 reduced DDR1a mediated invasion as well as the enhanced adhesion of DDR1a and DDR1b overexpressing cells. These results suggest that DDR1a plays a critical role in inducing tumor cell adhesion and invasion, and this invasive phenotype is caused by activation of matrix metalloproteinase-2.

Death-associated protein kinase is activated by dephosphorylation in response to cerebral ischemia.

Death-associated protein kinase (DAPK) is a calcium calmodulin-regulated serine/threonine protein kinase involved in ischemic neuronal death. In situ hybridization experiments show that DAPK mRNA expression is up-regulated in brain following a global ischemic insult and down-regulated in ischemic tissues after focal ischemia. DAPK is inactive in normal brain tissues, where it is found in its phosphorylated state and becomes rapidly and persistently dephosphorylated and activated in response to ischemia in vivo. A similar dephosphorylation pattern is detected in primary cortical neurons subjected to oxygen glucose deprivation or N-methyl-D-aspartate (NMDA)-induced toxicity. Both a calcineurin inhibitor, FK506, and a selective NMDA receptor antagonist, MK-801, inhibit the dephosphorylation of DAPK after in vitro ischemia. This indicates that DAPK could be activated by NMDA receptor-mediated calcium flux, activation of calcineurin, and subsequent DAPK dephosphorylation. Moreover, concomitantly to dephosphorylation, DAPK is proteolytically processed by cathepsin after ischemia. Furthermore, a selective DAPK inhibitor is neuroprotective in both in vitro and in vivo ischemic models. These results indicate that DAPK plays a key role in mediating ischemic neuronal injury.

FAS associated phosphatase (FAP-1) blocks apoptosis of astrocytomas through dephosphorylation of FAS.

Astrocytomas are the most common primary tumor of the adult human central nervous system. Despite efforts to develop more effective clinical treatment strategies, median survival time for patients with the most severe form of astrocytoma, glioblastoma multiforme (GBM), remains about one year. Astrocytomas are resistant to cytotoxic therapy in general and radiation therapy in particular, greatly limiting treatment options. One reason for this seems to be defects in the pathways controlling apoptosis. We have characterized the role of the tyrosine phosphatase FAP-1 (FAS-associated phosphatase 1) in astrocytomas. Our studies demonstrate that FAP-1 is overexpressed in astrocytomas and this contributes to the resistance of the tumor cells to FAS-mediated apoptosis. We demonstrate that knockdown of FAP-1 by RNA interference leads to increased apoptosis and increased sensitivity of astrocytoma cells to FAS-induced cell death. FAP-1 binds to FAS in a ligand-dependent manner and forms a signaling complex that modulates the ability of astrocytoma cells to undergo FAS ligand (FASL)-mediated cell death. In astrocytoma cells, FASL treatment induces tyrosine phosphorylation of FAS. FAP-1 dephosphorylates phospho-tyrosine 275 in the carboxyl terminus of FAS. This is the first direct evidence that FAS activity can be regulated by reversible phosphorylation and suggests a mechanism for astrocytoma resistance to apoptosis.

Functional comparison of long and short splice forms of RPTPbeta: implications for glioblastoma treatment.

The receptor protein tyrosine phosphatase beta (RPTPbeta/PTPzeta) is overexpressed in glioblastoma tumors and plays a functional role in tumor cell migration and adhesion. Glioblastomas express at least three splice variants of RPTPbeta, including long and short receptor forms and a secreted chondroitin sulfate proteoglycan called phosphacan. Here we explore the differences in the expression pattern and function of long RPTPbeta and short RPTPbeta. The short form of RPTPbeta lacks exon 12, which encodes 860 amino acids located in the extracellular domain. Until now, functional differences between long and short RPTPbeta have been difficult to elucidate. In this study, antibodies specific to the splice junction, unique to short RPTPbeta, allowed for the discrimination of the two receptors. A study of normal brain tissue and graded astrocytomas indicates that long and short RPTPbeta forms have an overlapping expression pattern. In order to study functional differences between long and short RPTPbeta, we created stable U87 glioblastoma cells that expressed these receptors. U87 stable cell lines overexpressing long or short RPTPbeta migrate faster and adhere more robustly than parental U87 cells. The two forms differ in that long-RPTPbeta-overexpressing cells migrate and adhere better than short-RPTPbeta-overexpressing cells. A study of the extracellular domain of short RPTPbeta indicates that it retains much of the functional capacity of phosphacan. Indeed, the action of recombinant, short-RPTPbeta extracellular domain protein is similar to that of phosphacan as a repulsive substrate for glioblastoma cells. Comparison of the signaling capacity of long RPTPbeta to that of short RPTPbeta reveals very similar abilities to activate transcription pathways. Moreover, transient transfection with either long or short RPTPbeta activates NF-kappaB reporter gene transcription. Because of their tumor-restricted and largely overlapping expression patterns in glioblastoma, both RPTPbeta splice forms are potential therapeutic targets. The involvement of long and short RPTPbeta in glioma tumor cell biology also contributes to the value of RPTPbeta as a cancer target.

GPR56 is a GPCR that is overexpressed in gliomas and functions in tumor cell adhesion.

GPR56 (also known as TM7XN1) is a newly discovered orphan G-protein-coupled receptor (GPCR) of the secretin family that has a role in the development of neural progenitor cells and has been linked to developmental malformations of the human brain. GPR56 diverges from other secretin-like family members in that it has an extremely large N-terminal extracellular region (381 amino acids) and contains a novel feature among this new subclass, consisting of four cysteine residues that define a GPCR proteolytic site (GPS motif) located just before the first transmembrane spanning domain. The rest of the amino-terminal domain contains a large number of possible N- and O-linked glycosylation sites similar to mucin-like proteins. These features suggest a role in cell-cell, or cell-matrix interactions. Here, we demonstrate upregulation of GPR56 in glioblastoma multiforme tumors using functional genomics. Immunohistochemistry studies confirmed the expression of GPR56 protein in a majority of glioblastoma/astrocytoma tumor samples with undetectable levels of expression in normal adult brain tissue. Immunofluorescence analysis of human glioma cells using anti-GPR56 antibodies demonstrate that GPR56 is expressed on the leading edge of membrane filopodia and colocalizes with alpha-actinin. Purified recombinant GPR56 extracellular domain protein inhibits glioma cell adhesion and causes abnormal cytoskeletal morphology and cell rounding. These results indicate that the extracellular domain may compete for unidentified ligand(s), and block the normal function of GPR56 in cell attachment. In reporter assays, overexpression of GPR56 activates the NF-kappaB, PAI-1 and TCF transcriptional response elements. These pathways have been implicated in cytoskeletal signaling, adhesion and tumor biology. The above results indicate that GPR56 serves as an adhesion GPCR and is involved in adhesion signaling.