Phospholipase A 2 Modulates Different Subtypes of Excitatory Amino Acid Receptors: Autoradiographic Evidence

Exogenous phospholipases have been used extensively as tools to study the role of membrane lipids in receptor mechanisms. We used in vitro quantitative autoradiography to evaluate the effect of phospholipase A 2 (PLA 2 ) on N -methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in rat brain. PLA 2 pretreatment induced a significant increase in Α-[ 3 H]amino-3-hydroxy-5-methylisoxazole-4-propionate ([ 3 H]AMPA) binding in the stratum radiatum of the CA1 region of the hippocampus and in the stratum moleculare of the cerebellum. No modification of [ 3 H]AMPA binding was found in the stratum pyramidale of the hippocampus at different ligand concentrations. [ 3 H]-Glutamate binding to the metabotropic glutamate receptor and the non-NMDA-, non-kainate-, non-quisqualate-sensitive [ 3 H]glutamate binding site were also increased by PLA 2 pretreatment. [ 3 H]Kainate binding and NMDA-sensitive [ 3 H]glutamate binding were minimally affected by the enzyme pretreatment. The PLA 2 effect was reversed by EGTA, the PLA 2 inhibitor p -bromophenacyl bromide, and prolonged pretreatment with heat. Bovine serum albumin (1%) prevented the increase in metabotropic binding by PLA 2 . Arachidonic acid failed to mimic the PLA 2 effect on metabotropic binding. These results indicate that PLA 2 can selectively modulate certain subtypes of excitatory amino acid receptors. This effect is due to the enzymatic activity but is probably not correlated with the formation of arachidonic acid metabolites. Independent of their possible physiological implications, our results provide the first autoradiographic evidence that an enzymatic treatment can selectively affect the binding properties of excitatory amino acid receptors in different regions of the CNS.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66358/1/j.1471-4159.1993.tb05843.x.pd

Quisqualate- and NMDA-sensitive [ 3 H]glutamate binding in primate brain

Excitatory amino acids (EAA) such as glutamate and aspartate are probably the neurotransmitters of a majority of mammalian neurons. Only a few previous studies have been concerned with the distribution of the subtypes of EAA receptor binding in the primate brain. We examined NMDA- and quisqualate-sensitive [ 3 H]glutamate binding using quantitative autoradiography in monkey brain (Macaca fascicularis) . The two types of binding were differentially distributed. NMDA-sensitive binding was most dense in dentate gyrus of hippocampus, stratum pyramidale of hippocampus, and outer layers of cerebral cortex. Quisqualate-sensitive binding was most dense in dentate gyrus of hippocampus, inner and outer layers of cerebral cortex, and molecular layer of cerebellum. In caudate nucleus and putamen, quisqualate- and NMDA-sensitive binding sites were nearly equal in density. However, in globus pallidus, substantia nigra, and subthalamic nucleus, quisqualate-sensitive binding was several-fold greater than NMDA-sensitive binding. In thalamus, [ 3 H]glutamate binding was generally low for both subtypes of binding except for the anterior ventral, lateral dorsal, and pulvinar nuclei. In the brainstem, low levels of binding were found, and strikingly the red nucleus and pons, which are thought to receive glutamatergic projections, had approximately 1/20 the binding observed in cerebral cortex. These results demonstrate that NMDA- and quisqualate-sensitive [ 3 H]glutamate binding are observed in all regions of primate brain, but that in some regions one subtype predominates over the other. In addition, certain areas thought to receive glutamatergic projections have low levels of both types of binding.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50225/1/490270412_ftp.pd

Inhibitory and excitatory amino acid neurotransmitter binding sites in cynomolgus monkey (Macaca fascicularis) cervical spinal cord

Autoradiography of inhibitory and excitatory amino acid neurotransmitter binding sites in the cervical spinal cord of M. fascicularis spinal cord revealed inhomogenous distribution of all binding sites in spinal gray matter. Quisqualate-sensitive [3H]glutamate binding, [3H]MK-801 binding, benzodiazepine binding, kainate binding, and GABAB binding had highest levels in the superficial layers of the dorsal horn (laminae 1 and 2) and substantially lower levels in other laminae. [3H]Strychnine binding was more uniformly distributed throughout all laminae with highest levels in the superficial layers of the dorsal horn. These results are similar to those found in other mammals.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30961/1/0000633.pd

Contamination of commercially available quisqualic acid by glutamate-like and aspartate-like substances

Six different batches of the glutamic acid analogue quisqualic acid were analyzed with high-pressure liquid chromatography (HPLC). All batches examined showed contaminant peaks. Different batches had different contaminant peaks and differing amounts of each contaminant. Every batch of quisqualic acid tested demonstrated a contaminant peak which co-eluted with exogenously added glutamic acid. Certain batches possessed a contaminant which co-eluted with aspartic acid. The levels of glutamate-like contamination ranged from 0.08 to 0.60%, and the levels of aspartate-like contamination ranged from undetectable amounts to 0.80%. The amount of combined glutamate- and aspartate-like contamination of each batch of quisqualate correlated very highly with the ability of that batch to interact with non-quisqualate receptors in an autoradiographic binding assay. These non-quisqualate receptors are likely (NMDA) receptors. Thus, when high concentrations of quisqualate are used experimentally, contamination is likely to produce spurious effects at non-quisqualate glutamate receptors. Quisqualate itself may be a more specific agonist than assumed previously.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28030/1/0000469.pd

Excitatory amino acid binding sites in the basal ganglia of the rat: A quantitative autoradiographic study

Quantitative receptor autoradiography was used to determine the distribution of excitatory amino acid binding sites in the basal ganglia of rat brain. [alpha]-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid, , kainate, quisqualate-sensitive metabotropic and , non-kainate, non-quisqualate glutamate binding sites had their highest density in striatum, nucleus accumbens, and olfactory tubercle. Kainate binding was higher in the lateral striatum but there was no medial-lateral striatal gradient for other binding sites. and [alpha]-amino-3-hydroxy-5-methylisoxazole-4-propionic acid binding sites were most dense in the nucleus accumbens and olfactory tubercle. There was no dorsal-ventral gradient within the striatal complex for the other binding sites. Other regions of the basal ganglia had lower densities of ligand binding. To compare binding site density within non-striatal regions, binding for each ligand was normalized to the striatal binding density. When compared to the striatal complex, [alpha]-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and metabotropic binding sites had higher relative density in the globus pallidus, ventral pallidum, and subthalamic nucleus than other binding sites. Metabotropic binding also had a high relative density in the substantia nigra. , non-kainate, non-quisqualate glutamate binding sites had a high relative density in globus pallidus, ventral pallidum, and substantia nigra. binding sites had a low relative density in pallidum, subthalamic nucleus, substantia nigra and ventral tegmental area.Our data indicate heterogeneous distribution of excitatory amino acid binding sites within rat basal ganglia and suggest that the character of excitatory amino acid-mediated neurotransmission within the basal ganglia is also heterogeneous.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30265/1/0000666.pd

Regional and cellular gene expression changes in human Huntington's disease brain

Huntington's disease (HD) pathology is well understood at a histological level but a comprehensive molecular analysis of the effect of the disease in the human brain has not previously been available. To elucidate the molecular phenotype of HD on a genome-wide scale, we compared mRNA profiles from 44 human HD brains with those from 36 unaffected controls using microarray analysis. Four brain regions were analyzed: caudate nucleus, cerebellum, prefrontal association cortex [Brodmann's area 9 (BA9)] and motor cortex [Brodmann's area 4 (BA4)]. The greatest number and magnitude of differentially expressed mRNAs were detected in the caudate nucleus, followed by motor cortex, then cerebellum. Thus, the molecular phenotype of HD generally parallels established neuropathology. Surprisingly, no mRNA changes were detected in prefrontal association cortex, thereby revealing subtleties of pathology not previously disclosed by histological methods. To establish that the observed changes were not simply the result of cell loss, we examined mRNA levels in laser-capture microdissected neurons from Grade 1 HD caudate compared to control. These analyses confirmed changes in expression seen in tissue homogenates; we thus conclude that mRNA changes are not attributable to cell loss alone. These data from bona fide HD brains comprise an important reference for hypotheses related to HD and other neurodegenerative disease

Levodopa-Induced Dyskinesia Is Associated with Increased Thyrotropin Releasing Hormone in the Dorsal Striatum of Hemi-Parkinsonian Rats

Background Dyskinesias associated with involuntary movements and painful muscle contractions are a common and severe complication of standard levodopa (L-DOPA, L-3,4-dihydroxyphenylalanine) therapy for Parkinson's disease. Pathologic neuroplasticity leading to hyper-responsive dopamine receptor signaling in the sensorimotor striatum is thought to underlie this currently untreatable condition. Methodology/Principal Findings Quantitative real-time polymerase chain reaction (PCR) was employed to evaluate the molecular changes associated with L-DOPA-induced dyskinesias in Parkinson's disease. With this technique, we determined that thyrotropin releasing hormone (TRH) was greatly increased in the dopamine-depleted striatum of hemi-parkinsonian rats that developed abnormal movements in response to L-DOPA therapy, relative to the levels measured in the contralateral non-dopamine-depleted striatum, and in the striatum of non-dyskinetic control rats. ProTRH immunostaining suggested that TRH peptide levels were almost absent in the dopamine-depleted striatum of control rats that did not develop dyskinesias, but in the dyskinetic rats, proTRH immunostaining was dramatically up-regulated in the striatum, particularly in the sensorimotor striatum. This up-regulation of TRH peptide affected striatal medium spiny neurons of both the direct and indirect pathways, as well as neurons in striosomes. Conclusions/Significance TRH is not known to be a key striatal neuromodulator, but intrastriatal injection of TRH in experimental animals can induce abnormal movements, apparently through increasing dopamine release. Our finding of a dramatic and selective up-regulation of TRH expression in the sensorimotor striatum of dyskinetic rat models suggests a TRH-mediated regulatory mechanism that may underlie the pathologic neuroplasticity driving dopamine hyper-responsivity in Parkinson's disease.Morris K. Udall Center for Excellence in Parkinson’s Research at MGH/MITNational Institutes of Health (U.S.) (NIH NS38372)American Parkinson Disease Association, Inc.University of Alabama at BirminghamMassachusetts General HospitalNational Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (NIDDK/NIH grant R01 DK58148)National Institute of Neurological Disorders and Stroke (U.S.) (R01 NINDS/NIH grant NS045231)Stanley H. and Sheila G. Sydney FundMichael J. Fox Foundation for Parkinson's Researc

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Excitatory amino acid binding sites in the periaqueductal gray of the rat

We used receptor autoradiography to determine the distribution of excitatory amino acid (EAA) binding site subtypes in the periaqueductal gray (PAG) of the rat. (NMDA), kainate, quisqualate-ionotropic, and quisqualate-metabotropic binding sites were all present in the PAG. Distribution was inhomogeneous with greatest density of all binding site subtypes in the dorsolateral subdivision and lowest density in the ventrolateral subdivision. Relative to regions of brain with high densities of EAA binding site subtypes, quisqualate-metabotropic binding sites had the highest relative density and NMDA binding sites the least. The presence of all subtypes of EAA binding sites in the PAG suggests that EAA action within the PAG is likely to be complex.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28357/1/0000120.pd