Proteomic analysis of synaptosomal protein expression reveals that cerebral ischemia alters lysosomal Psap processing.

Cerebral ischemia (CI) induces dramatic changes in synaptic structure and function that precedes delayed post-ischemic neuronal death. Here, a proteomic analysis was used to identify the effects of focal CI on synaptosomal protein levels. Contralateral and ipsilateral synaptosomes, prepared from adult mice subjected to 60 min middle cerebral artery occlusion, were isolated following 3, 6 and 20 h of reperfusion. Synaptosomal protein samples (n=3) were labeled using the cleavable ICAT system prior to analysis with nanoLC-MS/MS. Each sample was analyzed by LC-MS to identify differential expressions using InDEPT software and differentially expressed peptides were identified by targeted LC-MS/MS. A total of 62 differentially expressed proteins were identified and Gene Ontology classification (cellular component) indicated that the majority of the proteins were located in the mitochondria and other components consistent with synaptic localization. The observed alterations in synaptic protein levels poorly correlated with gene expression, indicating the involvement of post-transcriptional regulatory mechanisms in determining post-ischemic synaptic protein content. Additionally, immunohistochemistry analysis of prosaposin (Psap) and saposin C (SapC) indicates that CI disrupts Psap processing and glycosphingolipid metabolism. These results demonstrate that the synapse is adversely affected by CI and may play a role in mediating post-ischemic neuronal viability.

ABCG2 is upregulated in Alzheimer's brain with cerebral amyloid angiopathy and may act as a gatekeeper at the blood-brain barrier for Abeta(1-40) peptides.

Alzheimer's disease (AD) is characterized by accumulation and deposition of Abeta peptides in the brain. Abeta deposition in cerebrovessels occurs in many AD patients and results in cerebral amyloid angiopathy (AD/CAA). Since Abeta can be transported across blood-brain barrier (BBB), aberrant Abeta trafficking across BBB may contribute to Abeta accumulation in the brain and CAA development. Expression analyses of 273 BBB-related genes performed in this study showed that the drug transporter, ABCG2, was significantly upregulated in the brains of AD/CAA compared with age-matched controls. Increased ABCG2 expression was confirmed by Q-PCR, Western blot, and immunohistochemistry. Abcg2 was also increased in mouse AD models, Tg-SwDI and 3XTg. Abeta alone or in combination with hypoxia/ischemia failed to stimulate ABCG2 expression in BBB endothelial cells; however, conditioned media from Abeta-activated microglia strongly induced ABCG2 expression. ABCG2 protein in AD/CAA brains interacted and coimmunoprecipitated with Abeta. Overexpression of hABCG2 reduced drug uptake in cells; however, interaction of Abeta(1-40) with ABCG2 impaired ABCG2-mediated drug efflux. The role of Abcg2 in Abeta transport at the BBB was investigated in Abcg2-null and wild-type mice after intravenous injection of Cy5.5-labeled Abeta(1-40) or scrambled Abeta(40-1). Optical imaging analyses of live animals and their brains showed that Abcg2-null mice accumulated significantly more Abeta in their brains than wild-type mice. The finding was confirmed by immunohistochemistry. These results suggest that ABCG2 may act as a gatekeeper at the BBB to prevent blood Abeta from entering into brain. ABCG2 upregulation may serve as a biomarker of CAA vascular pathology in AD patients.

Cerebral ischemia causes dysregulation of synaptic adhesion in mouse synaptosomes.

Synaptic pathology is observed during hypoxic events in the central nervous system in the form of altered dendrite structure and conductance changes. These alterations are rapidly reversible, on the return of normoxia, but are thought to initiate subsequent neuronal cell death. To characterize the effects of hypoxia on regulators of synaptic stability, we examined the temporal expression of cell adhesion molecules (CAMs) in synaptosomes after transient middle cerebral artery occlusion (MCAO) in mice. We focused on events preceding the onset of ischemic neuronal cell death (<48 h). Synaptosome preparations were enriched in synaptically localized proteins and were free of endoplasmic reticulum and nuclear contamination. Electron microscopy showed that the synaptosome preparation was enriched in spheres (approximately 650 nm in diameter) containing secretory vesicles and postsynaptic densities. Forebrain mRNA levels of synaptically located CAMs was unaffected at 3 h after MCAO. This is contrasted by the observation of consistent downregulation of synaptic CAMs at 20 h after MCAO. Examination of synaptosomal CAM protein content indicated that certain adhesion molecules were decreased as early as 3 h after MCAO. For comparison, synaptosomal Agrn protein levels were unaffected by cerebral ischemia. Furthermore, a marked increase in the levels of p-Ctnnb1 in ischemic synaptosomes was observed. p-Ctnnb1 was detected in hippocampal fiber tracts and in cornu ammonis 1 neuronal nuclei. These results indicate that ischemia induces a dysregulation of a subset of synaptic proteins that are important regulators of synaptic plasticity before the onset of ischemic neuronal cell death.

Analysis of the pharmacological properties of JWH-122 isomers and THJ-2201, RCS-4 and AB-CHMINACA in HEK293T cells and hippocampal neurons.

Synthetic cannabinoids are marketed as legal alternatives to Δ9-THC, and are a growing worldwide concern as these drugs are associated with severe adverse effects. Unfortunately, insufficient information regarding the physiological and pharmacological effects of emerging synthetic cannabinoids (ESCs) makes their regulation by government authorities difficult. One strategy used to evade regulation is to distribute isomers of regulated synthetic cannabinoids. This study characterized the pharmacological properties of a panel of ESCs in comparison to Δ9-THC, as well as six JWH-122 isomers relative to its parent compound (JWH-122-4). Two cell-based assays were used to determine the potency and efficacy of ESCs and a panel of reference cannabinoids. HEK293T cells were transfected with human cannabinoid receptor 1 (CB1) and pGloSensor-22F, and the inhibition of forskolin-stimulated cyclic adenosine monophosphate (cAMP) levels was monitored in live cells. All ESCs examined were classified as agonists, with the following rank order of potency: Win 55,212-2 > CP 55,940 > JWH-122-4 > Δ9-THC ≈ RCS-4 ≈ THJ-2201 > JWH-122-5 > JWH-122-7 > JWH-122-2 ≈ AB-CHMINACA > JWH-122-8 > JWH-122-6 > JWH-122-3. Evaluation of ESC-stimulated Ca2+ transients in cultured rat primary hippocampal neurons confirmed the efficacy of four of the most potent ESCs (JWH-122-4, JWH-122-5, JWH-122-7 and AB-CHMINACA). This work helps regulatory agencies make informed decisions concerning these poorly characterized recreational drugs.

Cerebral ischemia induces neuronal expression of novel VL30 mouse retrotransposons bound to polyribosomes.

Mammalian genomes are burdened with a large heterogeneous group of endogenous replication defective retroviruses (retrotransposons). Previously, we identified a transcript resembling a virus-like 30S (VL30) retrotransposon increasing in mouse brain following transient cerebral ischemia. Paradoxically, this non-coding RNA was found bound to polyribosomes. Further analysis revealed that multiple retrotransposon species (BVL-1-like and mVL30-1-like) were bound to polyribosomes and induced by ischemia. These VL30 transcripts remained associated with polyribosomes in the presence of 0.5 M KCl, indicating that VL30 mRNA was tightly associated with ribosomal subunits. Furthermore, the profile of BVL-1 distribution on polyribosomal profiles was distinct from those of translated and translationally repressed mRNA. Consistent with expectations, 5.0 kb VL30 transcripts were detected in ischemic brain with a temporal pattern of expression that was distinct from c-fos. Expression of VL30 was localized in neurons using a combination of in situ hybridization and immunocytochemistry. 3'-RACE-PCR experiments yielded two unique sequences (VL30x-1 and VL30x-2) that were homologous to known VL30 genes. Phylogenetic analysis of VL30 promoter sequence (U3 region) resulted in the identification of two large VL30 subgroups. VL30x-1 and VL30x-2 were closely related and classified in a group that was distinct from the well-characterized VL30 genes BVL-1 and mVL30-1. The promoter regions of VL30x-1 and VL30x-2 did not possess the consensus sequences for either hypoxia or anoxia response elements, suggesting an alternative mechanism for induction. This is the first report that demonstrates ischemia-induced, neuronal expression of unique VL30 retrotransposons in mouse brain.

Pharmacological characterization of emerging synthetic cannabinoids in HEK293T cells and hippocampal neurons.

There has been a worldwide proliferation of synthetic cannabinoids that have become marketed as legal alternatives to cannabis (marijuana). Unfortunately, there is a dearth of information about the pharmacological effects of many of these emerging synthetic cannabinoids (ESCs), which presents a challenge for regulatory authorities that need to take such scientific evidence into consideration in order to regulate ECSs as controlled substances. We aimed to characterize the pharmacological properties of ten ESCs using two cell based assays that enabled the determination of potency and efficacy relative to a panel of well-characterized cannabinoids. Agonist-mediated inhibition of forskolin-stimulated cyclic adenosine monophosphate (cAMP) levels was monitored in live HEK293T cells transfected with human cannabinoid receptor 1 gene (CNR1) and pGloSensor-22F. Pharmacological analysis of this data indicated that all of the ESCs tested were full agonists, with the following rank order of potency: Win 55212-2≈5F-PB-22≈AB-PINACA≈EAM-2201≈MAM-2201>JWH-250≈ PB-22>AKB48 N-(5FP)>AKB-48≈STS-135>XLR-11. Assessment of agonist-stimulated depression of Ca(2+) transients was also used to confirm the efficacy of five ESCs (XLR-11, JWH-250, AB-PINACA, 5F-PB-22, and MAM-2201) in cultured primary hippocampal neurons. This work aims to help inform decisions made by regulatory agencies concerned with the profusion of these poorly characterized recreational drugs.

Translation-state analysis of gene expression in mouse brain after focal ischemia.

Confounding any genome-scale analysis of gene expression after cerebral ischemia is massive suppression of protein synthesis. This inefficient translation questions the utility of examining profiles of total transcripts. Our approach to such postischemic gene profiling in the mouse by microarray analysis was to concentrate on those mRNAs bound to polyribosomes. In our proof-of-principle study, polysomally bound and unbound mRNAs were subjected to microarray analysis: of the 1,161 transcripts that we found to increase after ischemia, only 36% were bound to polyribosomes. In addition to the expected increases in heat-shock proteins and metallothioneins, increases in several other bound transcripts involved in the promotion of cell survival or antiinflammatory behavior were noted, such as CD63 (Lamp3), Lcn2 (lipocalin-2), Msn (moesin), and UCP2 (uncoupling protein 2), all of which showed increases in cognate protein by Western blotting. The list of heretofore nonfunctionally annotated transcripts (RIKEN clones/ESTs) that increased appeared to be novel. How some transcripts are selected in ischemic brain for translation into protein, while others are rejected, is not clear. The length of the 5'-UTR in the ischemically induced transcripts that occur in the NCBI RefSeq database did not indicate any general tendency to be more than 200 nt, nor to be longer than the 5'-UTRs of the unbound transcripts. Thus, the presence of a complex 5'-UTR region with internal ribosome entry sites (IRES) or polypyrimidine tracts (TOP) does not appear to be the basis of selection for translation in ischemic brain.

Absence of the transcription factor E2F1 attenuates brain injury and improves behavior after focal ischemia in mice.

Because of observations that cultured neurons from mice deficient in the transcription factor E2F1 exhibit resistance after treatment with a wide variety of cell-death inducers, the authors investigated whether resistance extended to a cerebral ischemic insult. No differences in cerebral blood flow or physiologic parameters were observed in the mutant E2F1 littermates after the focal ligation. After 2 hours of left middle cerebral artery occlusion and 1 day of reperfusion, a 33% smaller infarct (P < 0.05) was observed by 2,3,5-triphenyltetrazolium staining in the brains of E2F1-null mice compared with their E2F1+/+ and +/- littermates. A milder ischemic insult produced by 20 minutes of middle cerebral artery occlusion and 7 days of reperfusion produced a greater difference in the E2F1-null animals with a 71% smaller infarct (P < 0.001) compared to littermate controls. A decrease in neuronal damage after mild ischemia in E2F1-null mice was observed by immunohistochemical monitoring of the loss in neuronal-specific microtubule-associated protein 2 cytoskeletal protein and the appearance of nuclear DNA fragmentation by terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate-biotin nick end labeling. This decreased brain damage was evidenced by improved behavior in motor function of E2F1 -/- mice compared with their E2F1 +/+ littermates by 7 days of reperfusion. In an effort to address the underlying molecular mechanism of the resistance of E2F1-null mice, the expression of several downstream proapoptotic target genes (p73, Apaf1, Arf) of the E2F1 transcription factor was measured by quantitative polymerase chain reaction. Although an attenuated increase in Hsp68 mRNA was found in E2F1 -/- mice, no changes in the proapoptotic transcripts were found after ischemia, and a mechanistic inference was not possible. The authors conclude that the transcription factor E2F1 does modulate neuronal viability in brain after cerebral ischemia and corroborates the findings with cultured neurons.