James F. Hatcher

Altered lipid metabolism in brain injury and disorders.

Deregulated lipid metabolism may be of particular importance for CNS injuries and disorders, as this organ has the highest lipid concentration next to adipose tissue. Atherosclerosis (a risk factor for ischemic stroke) results from accumulation of LDL-derived lipids in the arterial wall. Pro-inflammatory cytokines (TNF-alpha and IL-1), secretory phospholipase A2 IIA and lipoprotein-PLA2 are implicated in vascular inflammation. These inflammatory responses promote atherosclerotic plaques, formation and release of the blood clot that can induce ischemic stroke. TNF-alpha and IL-1 alter lipid metabolism and stimulate production of eicosanoids, ceramide, and reactive oxygen species that potentiate CNS injuries and certain neurological disorders. Cholesterol is an important regulator of lipid organization and the precursor for neurosteroid biosynthesis. Low levels of neurosteroids were related to poor outcome in many brain pathologies. Apolipoprotein E is the principal cholesterol carrier protein in the brain, and the gene encoding the variant Apolipoprotein E4 is a significant risk factor for Alzheimers disease. Parkinsons disease is to some degree caused by lipid peroxidation due to phospholipases activation. Niemann-Pick diseases A and B are due to acidic sphingomyelinase deficiency, resulting in sphingomyelin accumulation, while Niemann-Pick disease C is due to mutations in either the NPC1 or NPC2 genes, resulting in defective cholesterol transport and cholesterol accumulation. Multiple sclerosis is an autoimmune inflammatory demyelinating condition of the CNS. Inhibiting phospholipase A2 attenuated the onset and progression of experimental autoimmune encephalomyelitis. The endocannabinoid system is hypoactive in Huntingtons disease. Ethyl-eicosapetaenoate showed promise in clinical trials. Amyotrophic lateral sclerosis causes loss of motorneurons. Cyclooxygenase-2 inhibition reduced spinal neurodegeneration in amyotrophic lateral sclerosis transgenic mice. Eicosapentaenoic acid supplementation provided improvement in schizophrenia patients, while the combination of (eicosapentaenoic acid + docosahexaenoic acid) provided benefit in bipolar disorders. The ketogenic diet where >90% of calories are derived from fat is an effective treatment for epilepsy. Understanding cytokine-induced changes in lipid metabolism will promote novel concepts and steer towards bench-to-bedside transition for therapies.

Cytidine 5'-diphosphocholine (CDP-choline) in stroke and other CNS disorders.

Brain phosphatidylcholine (PC) levels are regulated by a balance between synthesis and hydrolysis. Pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1α/β) activate phospholipase A2 (PLA2) and PC-phospholipase C (PC-PLC) to hydrolyze PC. PC hydrolysis by PLA2 releases free fatty acids including arachidonic acid, and lyso-PC, an inhibitor of CTP-phosphocholine cytidylyltransferase (CCT). Arachidonic acid metabolism by cyclooxygenases/lipoxygenases is a significant source of reactive oxygen species. CDP-choline might increase the PC levels by attenuating PLA2 stimulation and loss of CCT activity. TNF-α also stimulates proteolysis of CCT. TNF-α and IL-1β are induced in brain ischemia and may disrupt PC homeostasis by increasing its hydrolysis (increase PLA2 and PC-PLC activities) and inhibiting its synthesis (decrease CCT activity). The beneficial effects of CDP-choline may result by counteracting TNF-α and/or IL-1 mediated events, integrating cytokine biology and lipid metabolism. Re-evaluation of CDP-choline phase III stroke clinical trial data is encouraging and future trails are warranted. CDP-choline is non-xenobiotic, safe, well tolerated, and can be considered as one of the agents in multi-drug treatment of stroke.

Role of lipids in brain injury and diseases

Lipid metabolism is of particular interest due to its high concentration in CNS. The importance of lipids in cell signaling and tissue physiology is demonstrated by many CNS disorders and injuries that involve deregulated metabolism. The long suffering lipid field is gaining reputation and respect as evidenced through the Center of Biomedical Research Excellence in Lipidomics and Pathobiology (COBRE), Lipid MAPS (Metabolites And Pathways Strategy) Consortium sponsored by NIH, European initiatives for decoding the lipids through genomic approaches, and Genomics of Lipid-associated Disorder (GOLD) project initiated by Austrian government. This review attempts to provide an overview of the lipid imbalances associated with neurological disorders (Alzheimers, Parkinsons; Niemann-Pick; Multiple sclerosis, Huntington, amyotrophic lateral sclerosis, schizophrenia, bipolar disorders and epilepsy) and CNS injury (Stroke, traumatic brain injury; and spinal cord injury) and a few provocative thoughts. Lipidomic analyses along with RNA silencing will provide new insights into the role of lipid intermediates in cell signaling and hopefully open new avenues for prevention or treatment options.

Tissue Plasminogen Activator (tPA) and Matrix Metalloproteinases in the Pathogenesis of Stroke: Therapeutic Strategies

Today there exists only one FDA-approved treatment for ischemic stroke; i.e., the serine protease tissue-type plasminogen activator (tPA). In the aftermath of the failed stroke clinical trials with the nitrone spin trap/radical scavenger, NXY-059, a number of articles raised the question: are we doing the right thing? Is the animal research truly translational in identifying new agents for stroke treatment? This review summarizes the current state of affairs with plasminogen activators in thrombolytic therapy. In addition to therapeutic value, potential side effects of tPA also exist that aggravate stroke injury and offset the benefits provided by reperfusion of the occluded artery. Thus, combinational options (ultrasound alone or with microspheres/nanobubbles, mechanical dissociation of clot, activated protein C (APC), plasminogen activator inhibitor-1 (PAI-1), neuroserpin and CDP-choline) that could offset tPA toxic side effects and improve efficacy are also discussed here. Desmoteplase, a plasminogen activator derived from the saliva of Desmodus rotundus vampire bat, antagonizes vascular tPA-induced neurotoxicity by competitively binding to low-density lipoprotein related-receptors (LPR) at the blood-brain barrier (BBB) interface, minimizing the tPA uptake into brain parenchyma. tPA can also activate matrix metalloproteinases (MMPs), a family of endopeptidases comprised of 24 mammalian enzymes that primarily catalyze the turnover and degradation of the extracellular matrix (ECM). MMPs have been implicated in BBB breakdown and neuronal injury in the early times after stroke, but also contribute to vascular remodeling, angiogenesis, neurogenesis and axonal regeneration during the later repair phase after stroke. tPA, directly or by activation of MMP-9, could have beneficial effects on recovery after stroke by promoting neurovascular repair through vascular endothelial growth factor (VEGF). However, any treatment regimen directed at MMPs must consider their pleiotropic nature and the likelihood of either beneficial or detrimental effects that might depend on the timing of the treatment in relation to the stage of brain injury.

Lipids and lipidomics in brain injury and diseases

Lipidomics is systems-level analysis and characterization of lipids and their interacting moieties. The amount of information in the genomic and proteomic fields is greater than that in the lipidomics field, because of the complex nature of lipids and the limitations of tools for analysis. The main innovation during recent years that has spurred advances in lipid analysis has been the development of new mass spectroscopic techniques, particularly the “soft ionization” techniques electrospray ionization and matrix-assisted laser desorption/ionization. Lipid metabolism may be of particular importance for the central nervous system, as it has a high concentration of lipids. The crucial role of lipids in cell signaling and tissue physiology is demonstrated by the many neurological disorders, including bipolar disorders and schizophrenia, and neurodegenerative diseases such as Alzheimers, Parkinsons, and Niemann-Pick diseases, that involve deregulated lipid metabolism. Altered lipid metabolism is also believed to contribute to cerebral ischemic (stroke) injury. Lipidomics will provide a molecular signature to a certain pathway or a disease condition. Lipidomic analyses (characterizing complex mixtures of lipids and identifying previously unknown changes in lipid metabolism) together with RNA silencing, using small interfering RNA (siRNA), may provide powerful tools to elucidate the specific roles of lipid intermediates in cell signaling and open new opportunities for drug development.

Glial glutamate transporter GLT-1 down-regulation precedes delayed neuronal death in gerbil hippocampus following transient global cerebral ischemia

Glial (GLT-1 and GLAST) and neuronal (EAAC1) high-affinity transporters mediate the sodium dependent glutamate reuptake in mammalian brain. Their dysfunction leads to neuronal damage by allowing glutamate to remain in the synaptic cleft for a longer duration. The purpose of the present study is to understand their contribution to the ischemic delayed neuronal death seen in gerbil hippocampus following transient global cerebral ischemia. The protein levels of these three transporters were studied by immunoblotting as a function of reperfusion time (6 h to 7 days) following a 10 min occlusion of bilateral common carotid arteries in gerbils. In the vulnerable hippocampus, there was a significant decrease in the protein levels of GLT-1 (by 36-46%, P < 0.05; between 1 and 3 days of reperfusion) and EAAC1 (by 42-68%, P < 0.05; between 1 and 7 days of reperfusion). Histopathological evaluation showed no neuronal loss up to 2 days of reperfusion but an extensive neuronal loss (by approximately 84%, P < 0.01) at 7 days of reperfusion in the hippocampal CA1 region. The time frame of GLT-1 dysfunction (1-3 days of reperfusion) precedes the initiation of delayed neuronal death (2-3 days of reperfusion). This suggests GLT-1 dysfunction as a contributing factor for the hippocampal neuronal death following transient global cerebral ischemia. Furthermore, decreased EAAC1 levels may contribute to GABAergic dysfunction and excitatory/inhibitory imbalance following transient global ischemia.

Citicoline mechanisms and clinical efficacy in cerebral ischemia

Citicoline, an intermediate in the biosynthesis of phosphatidylcholine (PtdCho), has shown beneficial effects in various CNS injury models and neurodegenerative diseases. PtdCho hydrolysis by phospholipase A2 (PLA2) after cerebral ischemia and reperfusion yields arachidonic acid (ArAc) and lyso‐PtdCho. ArAc oxidative metabolism results in formation of reactive oxygen species and lipid peroxides. Lyso‐PtdCho could inhibit activity of cytidine triphosphate‐phosphocholine cytidylyltransferase (the rate‐limiting enzyme in PtdCho biosynthesis), resulting in impaired PtdCho synthesis. Citicoline significantly increased glutathione levels and attenuated release of ArAc and the loss of PtdCho, cardiolipin, and sphingomyelin following transient cerebral ischemia. These effects could be explained by an effect of citicoline on PLA2. Based on these observations, a mechanism has been hypothesized. This Mini‐Review summarizes recent experimental data on the effects of citicoline in cerebral ischemia and evaluates several factors that might have hindered efficacy of citicoline in stroke clinical trials in the United States. Clinical stroke trials of citicoline in Europe and Japan have demonstrated beneficial effects. U.S. trials shown only marginal effects, which might be due to the 24 hr time window, the dose and route of administration, and the stringency of the primary outcome parameters. Recent evaluation of U.S. clinical data suggests that reduction of infarct growth may be a more sensitive measure of the citicoline effect than improvement on the NIH Stroke Scale (NIHSS) by ≥7 points. The citicoline neuroprotective mechanism has not been clearly identified, and its potential in stroke treatment might still be fully recognized in the United States. The clinical efficacy of citicoline should be examined further in light of the recent phase III stroke clinical trials and experimental data for cerebral ischemia.

Citicoline decreases phospholipase A2 stimulation and hydroxyl radical generation in transient cerebral ischemia.

Neuroprotection by citicoline (CDP‐choline) in transient cerebral ischemia has been demonstrated previously. Citicoline has undergone several Phase III clinical trials for stroke, and is being evaluated for treatment of Alzheimers and Parkinsons diseases. Phospholipid degradation and generation of reactive oxygen species (ROS) are major factors causing neuronal injury in CNS trauma and neurodegenerative diseases. Oxidative metabolism of arachidonic acid (released by the action of phospholipases) contributes to ROS generation. We examined the effect of citicoline on phospholipase A2 (PLA2) activity in relation to the attenuation of hydroxyl radical (OH·) generation after transient forebrain ischemia of gerbil. PLA2 activity (requires mM Ca2+) increased significantly (P < 0.05) in both membrane (50.2 ± 2.2 pmol/min/mg protein compared to sham 35.9 ± 3.2) and mitochondrial fractions (77.0 ± 1.2 pmol/min/mg protein compared to sham 33.9 ± 1.2) after cerebral ischemia and 2 hr reperfusion in gerbil, which was significantly attenuated (P < 0.01) by citicoline (membrane, 39.9. ± 2.2 and mitochondria, 41.9 ± 3.2 pmol/min/mg protein). In vitro, citicoline and its components cytidine and choline had no effect on PLA2 activity, and thus citicoline as such is not a PLA2 inhibitor. Ischemia/reperfusion resulted in significant OH· generation (P < 0.01) and citicoline significantly (P < 0.01) attenuated their formation (expressed as 2,3‐dihydroxybenzoic acid/salicylate ratio; ischemia/24 hr reperfusion, 6.30 ± 0.23; sham, 2.56 ± 0.27; ischemia/24 hr reperfusion + citicoline, 4.85 ± 0.35). These results suggest that citicoline affects PLA2 stimulation and decreases OH· generation after transient cerebral ischemia.

CDP-choline: neuroprotection in transient forebrain ischemia of gerbils.

CDP‐choline is a rate‐limiting intermediate in the biosynthesis of phosphatidylcholine (PtdCho), an important component of the neural cell membrane. The ability of CDP‐choline to alter phospholipid metabolism is an important function in the treatment of ischemic injury. Exogenous treatment with CDP‐choline stimulates PtdCho synthesis and prevents release of free fatty acids (FFA), especially arachidonic acid (AA), after ischemia/reperfusion. Phase III clinical trials of CDP‐choline in the treatment of stroke are currently underway. Here we report the neuroprotection by CDP‐choline in transient forebrain ischemia of gerbils. CDP‐choline significantly attenuated the blood‐brain barrier (BBB) dysfunction after ischemia with 6‐hr reperfusion, and considerably reduced the increase of AA in FFA and leukotriene C4 (LTC4) synthesis at 1 day. Edema was significantly elevated after 1 and 2 days, but attained maximum at 3‐day reperfusion. CDP‐choline substantially attenuated edema at 3 days. Ischemia resulted in 80 ± 8% CA1 hippocampal neuronal death after 6‐day reperfusion, and CDP‐choline provided 65 ± 6% neuroprotection. CDP‐choline may act by increasing PtdCho synthesis via two pathways: (1) conversion of 1,2‐diacylglycerol to PtdCho, and (2) biosynthesis of S‐adenosyl‐L‐methionine, thus stabilizing the membrane and reducing AA release and metabolism to leukotriene C4. This would result in decreased toxicity due to AA, leukotrienes, oxygen radicals, lipid peroxidation, and altered glutamate uptake, thus limiting BBB dysfunction, edema and providing neuroprotection. J. Neurosci. Res. 58:697–705, 1999.

Effects of MDL 72527, a specific inhibitor of polyamine oxidase, on brain edema, ischemic injury volume, and tissue polyamine levels in rats after temporary middle cerebral artery occlusion

Abstract The possible effects of the polyamine interconversion pathway on tissue polyamine levels, brain edema formation, and ischemic injury volume were studied by using a selective irreversible inhibitor, MDL 72527, of the interconversion pathway enzyme, polyamine oxidase. In an intraluminal suture occlusion model of middle coerebral artery in spontaneously hypertensive rats, 100 mg/kg MDL 72527 changed the brain edema formation from 85.7 ± 0.3 to 84.5 ± 0.9% in cortex (P < 0.05) and from 79.9 ± 1.7 to 78.4 ± 2.0% in subcortex (difference not significant). Ischemic injury volume was reduced by 22% in the cortex (P < 0.05) and 17% in the subcortex (P < 0.05) after inhibition of polyamine oxidase by MDL 72527. There was an increase in tissue putrescine levels together with a decrease in spermine and spermidine levels at the ischemic site compared with the nonischemic site compared with the nonischemic site after ischemia‐reperfusion injury. The increase in putrescine levels at the ischemic cortical and subcortical region was reduced by a mean of 45% with MDL 72527 treatment. These results suggest that the polyamine interconversion pathway has an important role in the postischemic increase ini putrescine levels and that blocking of this pathway can be neuroprotective against neuronal cell damage after temporary focal cerebral ischemia.