Karen Rash

Amyloid precursor protein accumulates in regions of neurodegeneration following focal cerebral ischemia in the rat

The distribution of beta-amyloid precursor protein (APP) was examined immunocytochemically in rats subjected to focal cerebral ischemia by permanent occlusion of the middle cerebral artery. At 4 and 7 days post-occlusion, APP immunoreactivity was preferentially localized within axonal swellings, dystrophic neurites and neuronal perikarya all along the periphery of the infarct. Immunolabeling was observed with antibodies generated against N-terminal, midregion, and C-terminal domains of APP. No immunoreactivity was observed with antisera directed against beta-amyloid protein (beta A4) itself. This pathological accumulation of APP is consistent with alterations of APP recently described in other models of neurodegeneration and implies a role for this protein in the response to CNS injury.

Cytosolic phospholipase A2 is induced in reactive glia following different forms of neurodegeneration.

Many recent studies have emphasized the deleterious role of inflammation in CNS injury. Increases in free fatty acids, eicosanoids, and products of lipid peroxidation are known to occur in various conditions of acute and chronic CNS injury, including cerebral ischemia, traumatic brain injury, and Alzheimers disease. Although an inflammatory response can be induced by many different means, phospholipases, such as cytosolic phospholipase A2 (cPLA2), may play an important role in the production of inflammatory mediators and in the production of other potential second messengers. cPLA2 hydrolyzes membrane phospholipids and its activity liberates free fatty acids leading directly to the production of eicosanoids. We investigated the cellular localization of cytosolic phospholipase A2 in the CNS following: (1) focal and global cerebral ischemia, (2) facial nerve axotomy, (3) human cases of Alzheimers disease, (4) transgenic mice overexpressing mutant superoxide dismutase, a mouse model of amyotrophic lateral sclerosis, and (5) transgenic mice overexpressing mutant amyloid precursor protein, which exhibits age‐related amyloid deposition characteristic of Alzheimers disease. We show that in every condition evaluated, cytosolic phospholipase A2 is present in reactive glial cells within the precise region of neuron loss. In conditions where neurons did not degenerate or are protected from death, cytosolic phospholipase A2 is not observed. Both astrocytes and microglial cells are immunoreactive for cytosolic phospholipase A2 following injury, with astrocytes being the most consistent cell type expressing cytosolic phospholipase A2. The presence of cytosolic phospholipase A2 does not merely overlap with reactive astroglia, as reactive astrocytes were observed that did not exhibit cytosolic phospholipase A2 immunoreactivity. In most conditions evaluated, inflammatory processes have been postulated to play a pivotal role and may even participate in neuronal cell death. These results suggest that cytosolic phospholipase A2 may prove an attractive therapeutic target for neurodegeneration. GLIA 27:110–128, 1999.

Global cerebral ischemia activates nuclear factor-κB prior to evidence of DNA fragmentation

Abstract The oxidative stress responsive transcription factor nuclear factor-κB (NF-κB) consists of a p50 (50 kDa) and p65/RelA (65 kDa) component and can be activated in vitro by TNFα, IL1β, hydrogen peroxide and oxygen radicals. All of the above factors are also known to be elevated at certain times after transient global ischemia. The present study was performed to determine if NF-κB was activated in vivo by transient global forebrain ischemia. Adult male rats were subjected to 30 min of 4-vessel occlusion (4-VO) and sacrificed at selected post-ischemic time points. Levels of NF-κB p50 and p65 subunits were determined by immunocytochemistry, Western blot and electrophoretic mobility-shift analysis. The enhancer complex was also confirmed by immuno-gel-shift analysis. Specific labeling of DNA strand breaks and DNA fragmentation was examined in situ by means of the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. Western blot analysis of hippocampus showed induction of p50 and p65. A time course of NF-κB induction in hippocampus showed a p50-specific band at 6 h that increased in intensity over 12, 48 h and then decreased by 96 h post-ischemia. Immunocytochemistry revealed at 24 h post-ischemia that p65 and p50 immunoreactivity was present in neuronal nuclei of hippocampal CA1 neurons as well as all other hippocampal regions and several other forebrain regions which were not vulnerable to transient forebrain ischemia. At 72 h post-ischemia, nuclear NF-κB immunoreactivity had disappeared in all brain areas except in hippocampal CA1 neurons which were degenerating. No evidence for DNA fragmentation as revealed by TUNEL staining could be observed at 24 h. However, at 72 h, hippocampal CA1 neurons were heavily labeled. The results of this study demonstrate that global forebrain ischemia causes a transient activation of NF-κB in many forebrain regions. NF-κB remains persistently activated in the vulnerable hippocampal CA1 sector. Because of the persistent activation of NF-κB in these neurons, the possibility exists that NF-κB has a role in programmed cell death in hippocampal CA1 neurons.

Increased expression of peripheral benzodiazepine receptors in the facial nucleus following motor neuron axotomy.

Peripheral benzodiazepine receptors (PBRs) are expressed in a variety of tissues but are normally found at low levels in the brain. Following various types of nerve injury, a reactive gliosis results that exhibits a high expression of this receptor. To further characterize the expression of PBRs following neuronal injury, we evaluated PBR expression in the facial nucleus following facial nerve axotomy (FNA). Injury to a peripheral nerve results in a complex series of metabolic and morphological changes around the injured neuron. Transections of the facial nerve results in a rapid activation of both astrocytes and microglia around axotomized motor neurons. FNA resulted in an increase in the staining for both astrocytes (glial fibrillary acidic protein) and activated microglia (OX42). There was also a reduction in synaptic contacts with the motor nucleus as evidenced by reduced staining for the synaptic marker, synaptophysin. In sections labeled with [3H]-PK11195, the subsequent autoradiograms displayed marked increases in the labeling for PBRs. This increase was observed at 5, 7 and 10 days after nerve transection. The increase was primarily in the level of expression (Bmax), with no change in the affinity of the ligand (Kd). The increase in PBR expression after FNA supports the hypothesis that PBRs can be used as a sensitive marker for CNS injury.

Synthesis and Evaluation of 11C-LY2795050 as a κ-Opioid Receptor Antagonist Radiotracer for PET Imaging

Kappa-opioid receptors (KOR) are believed to be involved in the pathophysiology of depression, anxiety disorders, drug abuse, and alcoholism. To date, only 1 tracer, the KOR agonist 11C-GR103545, has been reported to be able to image KOR in primates. The goal of the present study was to synthesize the selective KOR antagonist 11C-LY2795050 and evaluate its potential as a PET tracer to image KOR in vivo. Methods: The in vitro binding affinity of LY2795050 was measured in radioligand competition binding assays. Ex vivo experiments were conducted using microdosing of the unlabeled ligand in Sprague–Dawley rats and in wild-type and KOR knockout mice, to assess the ligand’s potential as a tracer candidate. Imaging experiments with 11C-LY2795050 in monkeys were performed on the Focus-220 scanner with arterial blood input function measurement. Binding parameters were determined with kinetic modeling analysis. Results: LY2795050 displays full antagonist activity and high binding affinity and selectivity for KOR. Microdosing studies in rodents and ex vivo analysis of tissue concentrations with liquid chromatography–tandem mass spectrometry identified LY2795050 as an appropriate tracer candidate able to provide specific binding signals in vivo. 11C-LY2795050 was prepared in an average yield of 12% and greater than 99% radiochemical purity. In rhesus monkeys, 11C-LY2795050 displayed a moderate rate of peripheral metabolism, with approximately 40% of parent compound remaining at 30 min after injection. In the brain, 11C-LY2795050 displayed fast uptake kinetics (regional activity peak times of <20 min) and an uptake pattern consistent with the distribution of KOR in primates. Pretreatment with naloxone (1 mg/kg, intravenously) resulted in a uniform distribution of radioactivity. Further, specific binding of 11C-LY2795050 was reduced by the selective KOR antagonist LY2456302 in a dose-dependent manner. Conclusion: 11C-LY2795050 displayed favorable pharmacokinetic properties and binding profiles in vivo and therefore is a suitable ligand for imaging the KOR in primates. This newly developed KOR antagonist tracer has since been advanced to PET imaging of KOR in humans and constitutes the first successful KOR antagonist radiotracer.

In Vitro and in Vivo Evidence for a Lack of Interaction with Dopamine D2 Receptors by the Metabotropic Glutamate 2/3 Receptor Agonists 1S,2S,5R,6S-2-Aminobicyclo[3.1.0]hexane-2,6-bicaroxylate Monohydrate (LY354740) and (−)-2-Oxa-4-aminobicyclo[3.1.0] Hexane-4,6-dicarboxylic Acid (LY379268)

Some recently published in vitro studies with two metabotropic glutamate 2/3 receptor (mGluR2/3) agonists [(−)-2-oxa-4-aminobicyclo[3.1.0] hexane-4,6-dicarboxylic acid (LY379268) and 1S,2S,5R,6S-2-aminobicyclo[3.1.0]hexane-2,6-bicaroxylate monohydrate (LY354740)] suggest that these compounds may also directly interact with dopamine (DA) D2 receptors. The current in vitro and in vivo studies were undertaken to further explore this potential interaction with D2 receptors. LY379268 and LY354740 failed to inhibit D2 binding in both native striatal tissue homogenates and cloned receptors at concentrations up to 10 μM. LY379268 and LY354740 (up to 10 μM) also failed to stimulate [35S]GTPγS binding in D2L- and D2S-expressing clones in the presence of NaCl or N-methyl-d-glucamine. In an in vivo striatal D2 receptor occupancy assay, LY379268 (3–30 mg/kg) or LY354740 (1–10 mg/kg) failed to displace raclopride (3 μg/kg i.v.), whereas aripiprazole (10–60 mg/kg) showed up to 90% striatal D2 receptor occupancy. LY379268 (10 mg/kg) and raclopride (3 mg/kg) blocked d-amphetamine and phencyclidine (PCP)-induced hyperactivity in wild-type mice. However, the effects of LY379268 were lost in mGlu2/3 receptor knockout mice. In DA D2 receptor-deficient mice, LY379268 but not raclopride blocked both PCP and d-amphetamine-evoked hyperactivity. In the striatum and nucleus accumbens, LY379268 (3 and 10 mg/kg) was without effect on the DA synthesis rate in reserpinized rats and also failed to prevent S-(−)-3-(3-hydroxyphenyl)-N-propylpiperidine-induced reductions in DA synthesis rate. Taken together, the current data fail to show evidence of direct DA D2 receptor interactions of LY379268 and LY354740 in vitro or in vivo. Instead, these results provide further evidence for a novel antipsychotic mechanism of action for mGluR2/3 agonists.

Increase in Insulin-like Growth Factor II Receptor within Ischemic Neurons following Focal Cerebral Infarction

The mechanisms underlying the response of the brain to ischemia are not fully understood. Biochemical and morphological changes following neocortical infarction can be investigated in rats using a model of focal cerebral ischemia induced by unilateral occlusion of the middle cerebral artery (MCA). Evaluation of ischemic damage often employs conventional histologic stains. Immunocytochemistry can be used as a valuable tool in this model to define changes in specific proteins of interest. In this study, an antiserum raised against insulin-like growth factor II (IGF-II) receptor was used to evaluate changes of IGF-II receptor immunoreactivity in the cerebral cortex of rats 4 and 7 days following permanent MCA occlusion. IGF-II receptor immunoreactivity was found to be associated with neocortical pyramidal neurons within the core of the ischemic infarct itself. The staining intensity was markedly elevated above that observed in nonischemic neurons. Immunopositive neurons exhibited a punctate staining pattern. These neurons appeared to correspond to argentophilic neurons, as defined by modified Bielschowsky silver staining. Evaluation of other neuronal markers revealed the absense of immunoreactivity for neuron-specific enolase and for tyrosine hydroxylase within the ischemic area. These observations show an increase in a specific growth factor receptor within neurons in the ischemic core of a focal infarct several days following permanent focal infarction, a time when neurons are presumed to be dead. The significance and the potential role of IGF-II receptor in lesion-induced plasticity are discussed.

A click-chemistry based strategy for labeling an anti-phosphatidylserine (PS) antibody with copper-64 via a cross-bridged tetraazamacrocyclic chelator scaffold

The κ-opioid receptors (KORs) are implicated in several neuropsychiatric diseases and addictive disorders. PET with radioligands provides a means to image the KOR in vivo and investigate its function in health and disease. The purpose of this study was to develop the selective KOR antagonist 11C-LY2459989 as a PET radioligand and characterize its imaging performance in nonhuman primates. Methods: LY2459989 was synthesized and assayed for in vitro binding to opioid receptors. Ex vivo studies in rodents were conducted to assess its potential as a tracer candidate. 11C-LY2459989 was synthesized by reaction of its iodophenyl precursor with 11C-cyanide, followed by partial hydrolysis of the resulting 11C-cyanophenyl intermediate. Imaging experiments with 11C-LY2459989 were performed in rhesus monkeys with arterial input function measurement. Imaging data were analyzed with kinetic models to derive in vivo binding parameters. Results: LY2459989 is a full antagonist with high binding affinity and selectivity for KOR (0.18, 7.68, and 91.3 nM, respectively, for κ, μ, and δ receptors). Ex vivo studies in rats indicated LY2459989 as an appropriate tracer candidate with high specific binding signals and confirmed its KOR binding selectivity in vivo. 11C-LY2459989 was synthesized in high radiochemical purity and good specific activity. In rhesus monkeys, 11C-LY2459989 displayed a fast rate of peripheral metabolism. Similarly, 11C-LY2459989 displayed fast uptake kinetics in the brain and an uptake pattern consistent with the distribution of KOR in primates. Pretreatment with naloxone (1 mg/kg, intravenously) resulted in a uniform distribution of radioactivity in the brain. Further, specific binding of 11C-LY2459989 was dose-dependently reduced by the selective KOR antagonist LY2456302 and the unlabeled LY2459989. Regional binding potential values derived from the multilinear analysis-1 (MA1) method, as a measure of in vivo specific binding signal, were 2.18, 1.39, 1.08, 1.04, 1.03, 0.59, 0.51, and 0.50, respectively, for the globus pallidus, cingulate cortex, insula, caudate, putamen, frontal cortex, temporal cortex, and thalamus. Conclusion: The novel PET radioligand 11C-LY2459989 displayed favorable pharmacokinetic properties, a specific and KOR-selective binding profile, and high specific binding signals in vivo, thus making it a promising PET imaging agent for KOR.

Synthesis and evaluation of [C-11]LY2795050, an antagonist PET imaging tracer for the kappa opioid receptors

Introduction: The kappa opioid receptors (KOR) are believed to be involved in the pathophysiology of depression, anxiety disorders, drug abuse and alcoholism. To date only one tracer, the kappa opioid receptor agonist [C-11]GR103545, has been reported to be able to image KOR in nonhuman primates. The goal of the present study was to synthesize the selective KOR antagonist [C-11]LY2795050 and evaluate its potential as a PET tracer to image KOR in non-human primates.