Ischemic Stroke, Intracerebral Hemorrhage (ICH), Subrachnoid Hemorrhage (SAH), Traumatic Brain Injury (TBI)
In a previous study, we demonstrated that neural damage following middle cerebral artery occlusion, a model of focal ischemia of vascular stroke, is substantially worsened in HO2-/- animals. These findings implicate HO as an endogenous neuroprotective system in the brain whose pharmacologic manipulation may have therapeutic relevance.
Stroke damage following blood vessel occlusion and reperfusion involves a variety of mechanisms. One component derives from the formation of oxygen-free radicals by hypoxic mitochondria. Hypoxia also triggers a massive release of glutamate, which causes neurotoxicity via stimulation of glutamate receptors, especially the NMDA subtype. We sought to ascertain which mechanisms of stroke damage might be reversed by HO activation and hence contribute to augmented stroke damage in HO2-/- animals. Neurotoxicity can be elicited by direct injections of NMDA into the brain, and we observed substantial neural damage following NMDA injections into the cerebral cortex. The extent of this damage is increased three- to four-fold in HO2-/- mice. Thus, it appears that physiologic HO2 activity is neuroprotective against glutamate excitotoxicity that involves NMDA receptor activation.
The onset and progression of Alzheimer disease remain to be elucidated, although it appears that free radical damage and neuroinflammation are likely to play a significant role. In familial cases of AD, it was found that specific mutation in the precursor of amyloid protein generates a toxic protein fragment, i.e., beta-amyloid, which triggers inflammation. Subsequently, toxins — free radicals — are generated and could lead to the death of neurons, causing decreases in a vital brain chemical, the neurotransmitter acetylcholine, affecting memory and concentration, and developing into debilitating clinical symptoms that are most common in AD.
Role of Neuroinflammation in AD
It has been suggested that loss of neurons could be due to vascular problems (silent strokes) and/or undetectable inflammation processes. Some theories in the development of Alzheimer disease have focused on the presence of beta-amyloid plaques and neurofibrillary tangles in the brain of AD patients. The only defense against these abnormal processes is to fight back, and, to do that, the brain activates immune cells, such as microglia. These reactive brain cells generate toxins, which under chronic conditions can become counterproductive.
Low-grade chronic inflammation (as opposed to acute), generally unnoticed, is believed to underlie the most serious neurodegenerative diseases. Consequently, anti-inflammatory drugs, such as the non-steroidal anti-inflammatory drugs (NSAIDs, e.g., aspirin, ibuprofen, and acetaminophen) and the newer COX-2 inhibitors, are being investigated. Another main objective of Dr. Doré’s research is to understand the actions of cyclooxygenase and its metabolites, the prostaglandins.
Role of Heme Oxygenase in AD
In addition, in order to discover factors that could modulate the neuroprotective activities found to be associated with HO2, Dr. Doré and collaborators have performed a yeast-2-hybrid technique, which allows the identification of protein-protein interactions. They found that an amyloid-like precursor protein and its homologue, amyloid precursor proteins (APP), can directly bind to heme oxygenase and slightly decrease its activity. With Dr. Snyder’s laboratory, they identified that mutants associated with the familial AD would have higher affinity, suggesting that a possible chronically lower HO activity can be one of the factors contributing to the etiology of Alzheimer disease. Dr. Doré’s goal now is to further characterize this observation using in vivo and in vitro models.
Brain Aging and Neuroinflammation
Recently, inflammation has been recognized as playing a central role in the cognitive decline too often associated with aging. Inflammation is now thought to play a major role in pathological conditions ranging from cardiovascular diseases to Alzheimer disease, implicating it in many more diseases than was previously thought.
Inflammation becomes particularly relevant in several neurological disorders, such as Alzheimer disease and vascular dementia. Although mental decline and memory loss have long been considered inevitable hallmarks of old age, new research suggests that such inflammation/age-associated decline is avoidable. Indeed, early intervention to limit damage associated with inflammation and free radicals may offer some protection against these dreaded brain diseases.
Heme Oxygenase in Neurodegenerative Diseases
Heme oxygenase is the only enzyme in the body that can degrade heme, and it generates different metabolites with specific actions: iron, biliverdin/bilirubin, and carbon monoxide. Heme, or iron protoporphyrin IX, is a prooxidant. Following subarachnoid hemorrhage, for example, it plays a significant role in detoxifying the brain. Within a cell, after different stress conditions, heme is released from heme-containing enzymes and can reach micromolar concentrations. Heme is not recycled and must be degraded. Using mice with deletion of the HO2 gene, we found an approximate twice greater infarct size after induced cerebral ischemia.
Biliverdin and Bilirubin
Biliverdin and bilirubin have been generally associated with neurotoxicity in newborn babies. Dr. Doré has proposed that, at low concentrations, these can be significant antioxidants. Using primary neuronal cultures, he showed that nanomolar concentrations of BR appear to be neuroprotective against oxidative stress. In order to test this hypothesis in vivo, he has shown that HO2 knockout mice have greater infarct after transient cerebral ischemia and have greater damage after stereotaxic injection of NMDA. He is now interested in testing new ways to deliver free bilirubin in the brain into order to stay within the physiological protective levels.
Another metabolite of HO reaction is carbon monoxide. The field of CO is still quite controversial. CO is a gas, which has been postulated to be an important neurotransmitter. Because it is a gas, it does not accumulate within vesicles and can readily diffuse to activate its targets. Due to its stability and its relatively long half-life, it can travel longer distances. Carbon monoxide can modulate soluble guanylate cyclase and influence the production of cGMP. For these reasons, it has been postulated that it plays a role in modulating long-term potentiation and influencing memory. Observation of significant differences in the expression levels of the HO2 in aged cognitively impaired rodents is interesting. Additionally, it has been demonstrated that CO can activate the MAP kinase pathway and limit apoptotic-like cell death. This could also partially explain the protective effect found in the HO2-/- mice using in vivo and in vitro models. The goal now is to test whether low levels of CO can be protective using in vivo models of neurotoxicity.
It is known that iron levels are tightly regulated in order to keep cellular homeostasis and that heme oxygenase appears to control iron efflux from the cell and determines the rate of programmed cell death. One of Dr. Doré’s future objectives is to investigate the cellular mechanism by which heme oxygenase can regulate the iron level within a cell.
Cyclooxygenase and Prostaglandins
Another main objective of Dr. Doré’s research is to understand the actions of cyclooxygenase and its metabolites. Inflammation and its consequences are suggested to play an important role in the loss of normal neuronal functions associated with aging and Alzheimer disease. Cyclooxygenase (COX) is the rate-limiting enzyme for the production of the prostaglandins (PGs) through metabolism of the arachidonic acid. COX-1 is constitutively expressed in most neuronal cells and has been suggested to respond to normal cellular functions. On the other hand, COX-2 expression and PG production increase markedly in neurons following a variety of brain insults, including hypoxia, inflammation, and excitotoxicity. Studies in rodent ischemic and excitotoxic models show that COX-2 enzymatic activity promotes neuronal injury and the administration of specific inhibitors reduces neuronal damage.
Epidemiologic studies have suggested a reduction in the incidence of AD in patients who were taking anti-inflammatory drugs, and numerous studies have reported the induction of COX-2 in AD brains. Clinical trials using selective COX-2 inhibitors have been designed, although the results reported so far have not satisfied the high expectations. Dr. Doré and his collaborators have recently tested transgenic mice overexpressing COX-2 selectively in neurons and observed an increased infarct size in these transgenic mice; however, they were not able to show significant reduction after administration of a specific COX-2 inhibitor. Their results in primary neuronal cultures indicate that biological actions of the COX inhibitors and the PGs often follow a very narrow bell-shaped curve.
All together, Dr. Doré’s research team believes that a better understanding of the PG receptors is of utmost importance and could explain several of the discrepancies and failures previously reported. The mechanism by which PGs promote neuronal injury in excitotoxic conditions has not yet been defined. Some PGs have been reported to promote injury, while others have been reported to be cytoprotective. PGs are diffusible signaling lipids whose effects are mediated through a diverse class of G-protein-coupled receptors that can have opposing effects on cAMP, IP turnover, and Ca2+ levels. Using in vivo studies, as well as in vitro culture techniques, Dr. Doré’s aim is to define the role of the respective PG receptors in regulating excitotoxic damage. The overall goal is to develop more selective action that would specifically relieve inflammation while minimizing the distressing side effects that are possible with chronic use of the present available drugs (either over-the-counter or prescribed).
Complementary and Alternative Medicine
Dr. Doré has shown interest in alternative and complementary medicine. People have used herbal medicines for centuries, and today, a vast majority of people have considered and used alternative medicine. Although many therapeutic functions have been attributed to these treatments, very few rigorous scientific studies have been completed.
Using primary neuronal cultures, Dr. Doré has observed that pre-treatment with some of these compounds (for example Ginkgo biloba) would provide neuroprotection, mainly by inducing heme oxygenase 1 in neurons. Use of protein inhibitor synthesis would block this protection. His future goal is to identify which proteins are expressed or post-transcriptionally modified in order to reveal possible mechanisms of protection associated with these “natural” compounds.