Identification of a Nuclear Respiratory Factor 1 Recognition Motif in the Apolipoprotein E Variant APOE4 linked to Alzheimer's Disease.

Alzheimer's disease affects tens of millions of people worldwide and its prevalence continues to rise. It is caused by a combination of a subject's heredity, environment, lifestyle, and medical condition. The most significant genetic risk factor for late onset Alzheimer's disease is a variant of the apolipoprotein E gene, APOE4. Here we show that the single nucleotide polymorphism rs429358 that defines APOE4 is located in a short sequence motif repeated several times within exon 4 of apolipoprotein E, reminiscent of the structure of transcriptional enhancers. A JASPAR database search predicts that the T to C transition in rs429358 generates a binding motif for nuclear respiratory factor NRF1. This site appears to be part of a binding site cluster for this transcription factor on exon 4 of APOE. This de novo NRF1 binding site has therefore the potential to affect the expression of multiple genes in its genomic vicinity. Our in silico analysis, suggesting a novel function for APOE4 at the DNA level, offers a potential mechanism for the observed tissue specific neurodegeneration and the role of environmental factors in Alzheimer's disease etiology.

Phase II trial of the Sigma-1 receptor agonist cutamesine (SA4503) for recovery enhancement after acute ischemic stroke.

BACKGROUND AND PURPOSE: The σ-1 receptor (Sig-1R) agonist cutamesine (SA4503) enhanced functional recovery after experimental stroke with a treatment initiation window of 48 hours and chronic treatment for 28 days. We conducted a phase 2 clinical trial exploring the safety, tolerability, dose range, and functional effects of cutamesine in patients with ischemic stroke. METHODS: Subjects were randomized between 48 and 72 hours after stroke to receive cutamesine 1 mg/d, 3 mg/d, or placebo for 28 days. Effects on safety and function were assessed at baseline, at end of treatment (day 28), and at end of follow-up (day 56). RESULTS: In 60 patients, treatment with both cutamesine dosages was safe and well tolerated without significant differences in numbers of treatment emergent or serious adverse events. No significant effect was observed on the primary efficacy measure (change in National Institutes of Health Stroke Scale from baseline to day 56) or modified Rankin Scale and Barthel Index scores. Post hoc analysis of moderately and severely affected patients (baseline National Institutes of Health Stroke Scale, ≥7 and ≥10) showed greater National Institutes of Health Stroke Scale improvements in the 3 mg/d cutamesine group when compared with placebo (P=0.034 and P=0.038, respectively). A trend toward a higher proportion being able to complete a 10m timed walk was observed for cutamesine-treated subjects. CONCLUSIONS: Cutamesine was safe and well tolerated at both dosage levels. Although no significant effects on functional end points were seen in the population as a whole, greater improvement in National Institutes of Health Stroke Scale scores among patients with greater pretreatment deficits seen in post hoc analysis warrants further investigation. Additional studies should focus on the patient population with moderate-to-severe stroke. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov/show/NCT00639249. Unique identifier: NCT00639249. The EudraCT number is 2007-004840-60 (https://www.clinicaltrialsregister.eu/ctr-search/trial/2007-004840-60/GB).

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.

Sigma-1 receptor chaperones regulate the secretion of brain-derived neurotrophic factor.

The sigma-1 receptor (Sig-1R) is a novel endoplasmic reticulum (ER) molecular chaperone that regulates protein folding and degradation. The Sig-1R activation by agonists is known to improve memory, promote cell survival, and exert an antidepressant-like action in animals. Cutamesine (SA4503), a selective Sig-1R ligand, was shown to increase BDNF in the hippocampus of rats. How exactly the intracellular chaperone Sig-1R or associated ligand causes the increase of BDNF or any other neurotrophins is unknown. We examined here whether the action of Sig-1Rs may relate to the post-translational processing and release of BDNF in neuroblastoma cell lines. We used in vitro assays and confirmed that cutamesine possesses the bona fide Sig-1R agonist property by causing the dissociation of BiP from Sig-1Rs. The C-terminus of Sig-1Rs exerted robust chaperone activity by completely blocking the aggregation of BDNF and GDNF in vitro. Chronic treatment with cutamesine in rat B104 neuroblastoma caused a time- and dose-dependent potentiation of the secretion of BDNF without affecting the mRNA level of BDNF. Cutamesine decreased the intracellular level of pro-BDNF and mature BDNF whereas increased the extracellular level of mature BDNF. The pulse-chase experiment indicated that the knockdown of Sig-1Rs decreased the secreted mature BDNF in B104 cells without affecting the synthesis of BDNF. Our findings indicate that, in contrast to clinically used antidepressants that promote the transcriptional upregulation of BDNF, the Sig-1R agonist cutamesine potentiates the post-translational processing of neurotrophins. This unique pharmacological profile may provide a novel therapeutic opportunity for the treatment of neuropsychiatric disorders.

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.

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.

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.

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.

Linking solubility and permeability assays for maximum throughput and reproducibility.

Solubility and permeability are intimately linked in drug absorption processes. They have, however, been traditionally assayed separately. To support this linkage, a combined solubility/permeability assay was developed for determining absorption properties of chemical entities. First, solubility is determined at 4 pH values by comparing the concentration of a saturated compound solution to its dilute, known concentration. The filtered, saturated solution from the solubility assay is then used as input material for the membrane permeability determination. The permeability assay is a parallel artificial membrane technique whereby a membrane is created on a solid support parallel artificial membrane permeation assay (PAMPA). The 2 artificial membranes presented here model the gastrointestinal tract and the blood-brain barrier (BBB). Data are presented for control compounds, which are well documented in the literature and exemplify a range of solubility and membrane permeability. The advantages of the combination method are 1) reduction of sample usage and preparation time, 2) elimination of interference from compound precipitation in membrane permeability determination, 3) maximization of input concentration to permeability assay for improved reproducibility, and 4) optimization of sample tracking by streamlining data entry and calculations. BBB permeability ranking of compounds correlates well with literature CNS activity.

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.

A high-throughput turbidometric assay for screening inhibitors of protein disulfide isomerase activity.

Protein disulfide isomerase (PDI) plays a key role in protein folding by catalyzing rearrangements of disulfide bonds in substrate proteins following their synthesis in eukaryotic cells. Besides its major role in the processing and maturation of secretory proteins in the endoplasmic reticulum, this enzyme and its homologs have been implicated in multiple important cellular processes; however, they have not served as targets for the development of therapeutic agents. The authors developed a high-throughput screening assay for PDI and its homologous enzymes in 384-well microplates. The method is based on the enzyme-catalyzed reduction of insulin in the presence of dithiothreitol and measures the aggregation of reduced insulin chains at 650 nm. This kinetic assay was converted to an end-point assay by using hydrogen peroxide as a stop reagent. The feasibility of this high-throughput assay for screening chemical libraries was demonstrated in a pilot screen. The authors show that this homogenous turbidometric assay is robust and cost-effective and can be applied to identify PDI inhibitors from chemical libraries, opening this class of enzymes for therapeutic exploration.

Identification of cathepsin B as a mediator of neuronal death induced by Abeta-activated microglial cells using a functional genomics approach.

Alzheimer's disease is a progressive neurodegenerative disease characterized by senile plaques, neurofibrillary tangles, dystrophic neurites, and reactive glial cells. Activated microglia are found to be intimately associated with senile plaques and may play a central role in mediating chronic inflammatory conditions in Alzheimer's disease. Activation of cultured murine microglial BV2 cells by freshly sonicated Abeta42 results in the secretion of neurotoxic factors that kill primary cultured neurons. To understand molecular pathways underlying Abeta-induced microglial activation, we analyzed the expression levels of transcripts isolated from Abeta42-activated BV2 cells using high density filter arrays. The analysis of these arrays identified 554 genes that are transcriptionally up-regulated by Abeta42 in a statistically significant manner. Quantitative reverse transcription-PCR was used to confirm the regulation of a subset of genes, including cysteine proteases cathepsin B and cathepsin L, tissue inhibitor of matrix metalloproteinase 2, cytochrome c oxidase, and allograft inflammatory factor 1. Small interfering RNA-mediated silencing of the cathepsin B gene in Abeta-activated BV2 cells diminished the microglial activation-mediated neurotoxicity. Moreover, CA-074, a specific cathepsin B inhibitor, also abolished the neurotoxic effects caused by Abeta42-activated BV2 cells. Our results suggest an essential role for secreted cathepsin B in neuronal death mediated by Abeta-activated inflammatory response.