This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.1. Background and SignificanceThere are approximately 750,000 new strokes each year in the U.S and stroke patients are notorious in showing a variable degree of spontaneous recovery. Unfortunately, such recovery is often limited, making stroke the leading cause of adult disability in the U.S. There are limited therapeutic options and patients who survive stroke live an average of 7 years, leading to a prevalence of more than four million stroke survivors alive in the U.S, most patients are left with chronic neurological impairments. Hence, a better understanding of the basis for stroke recovery, and its inter-individual variability, could be of great importance to improving therapeutic options. There are currently no approved treatments that target patients with chronic deficits. Many aspects of neurological function can be impaired after a focal stroke. The most common domain of impairment is the motor system, with a large majority of patients having weakness on one or both sides early after stroke. This recovery is highly variable, however, leaving some degree of permanent motor deficit in many, and perhaps most stroke patients. As a result, stroke is the leading cause of adult disability in the U.S. and many other countries. A number of studies have described the time course by which motor function returns after a stroke. For example, it has been shown that the most dramatic improvements in neurological recovery occurred in the first 30 days post-stroke, but that many patients further improved until 90 days post-stroke. This recovery requires changes in cell function and structure, and is thus an energy-dependent process. The current collaborative study seeks to understand the extent to which key mitochondrial genetics factors influence these events.2. Mitochondria Processes: Energy production, Reactive Oxygen Species Production and ApoptosisGenetic variation is likely to be a key factor in influencing individual patterns of long term recovery after stroke. Specifically, variations in mitochondrial genetics are likely to be one of the more important factors in rehabilitation and outcome after stroke. Indeed, energy is provided by sub-cellular structures known as mitochondria. Each cell contains hundred of mitochondria and each of which produces a portion of the energy for our cells. Indeed, calories coming from our diet are burned with oxygen we breathe by mitochondria and in turn generate energy in the form of ATP (adenosine triphosphate) used to do work or maintain constant our body temperature. In each mitochondrion there are multiple copies of a small piece of DNA called mitochondrial genome (or mtDNA). Hence, mitochondria are under genetic control of 2 genomes: nuclear genome, genetic information coming from nucleus of the cell and hence from the mitochondrial genome. Furthermore, mitochondria also generate most of the reactive oxygen species (ROS) as a toxic by-product of energy production. These ROS can damage mitochondria and especially mitochondrial DNA within the mitochondrion. Energy production by mitochondria will decline because without good mtDNA, the damaged mitochondria can not be repaired.3. Genetic variations of mitochondrial genome are stratified into different mtDNA lineages The mtDNA is a small circular DNA molecule located inside mitochondria and encodes 37 essential proteins of the mitochondrial energy generating process. MtDNA is consistently inherited through the mother and has a very high mutation rate due to its direct exposure to the mitochondrial ROS generated by mitochondria. As a consequence of its high mutation rate, the mtDNA from human origins has accumulated sequential mutations as humans migrated out of Africa and into temperate Eurasia and cold arctic Siberian and North America. Consequently, all of the mtDNA types fit into a single dichotomous tree with an African root and with specific branches radiating into the different continents giving regionally-specific groups called haplogroups defined by all mitochondrial genetic changes (Wallace et al, 1999). The African mtDNAs are the most diverse and thus ancient, with an overall age of about 150,000 YBP. For instance, african mtDNAs fall into four major haplogroups called L0, L1, L2 and L3. During human migrations, one of these African lineages radiated in Europe to give all of the European mtDNAs encompassing 8 major Europeans haplogroups called H, I, K, J, T, U, V and W.4. Genetic influence of mtDNA haplogroups in stroke and neurodegenerative diseasesGenetic variation is likely one of the main parameter in determining potentiality of individuals for recovery after brain injury. Influence of the mitochondrial DNA (mtDNA) genetic background in a variety of clinical manifestations associated with increase risk of stroke, degenerative diseases and aging has been recently shown in different studies. Indeed, the main clinical characteristic of one of the well known mitochondrial syndrome called MELAS syndrome is recurrent cerebral insults resembling strokes causing neurological deficits. Numerous studies have showed that a genetic change in the mtDNA sequence at position A3243G was indeed the cause of this syndrome. Furthermore, specific mtDNA lineages also called haplogroups have been associated with disadvantageous effects. For example, mitochondrial haplogroup U was found as a risk factor for stroke in patients with migraine. This result suggests that this particular mtDNA lineage may harbor mutations that are involved in the pathogenesis of migraine-associated stroke. Indeed, the presence of an additional common mitochondrial variant in the mtDNA sequence at position A12308G has been suggested to increased risk of stroke in these patients. It is currently unknown the extent to which such mtDNA haplogroups influence the specific events underlying recovery from stroke. 5.
Aims of the studyThe proposed study aims to evaluate the influence of mitochondrial genetics on 90 day outcome in patients with stroke. Three major mitochondrial parameters might contribute to the modulation of the recovery process in patients after stroke : (1) production of most of the cellular energy in the form of ATP and heat, (2) generation of most of the cellular ROS as a by-product, oxidative stress is a leading cause of cellular damage after stroke and (3) incorporation of a self-destruct system called the mitochondrial permeability transition pore (mtPTP) an important factor contributing to ischemic brain damage, that monitors mitochondrial energy decline, ROS toxicity and ultimately destroys cells with defective mitochondria.These three aspects of mitochondrial physiology (energy generation, ROS production and programmed cell death) are modulated by mitochondrial genes encoded in either the mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). Since my colleagues including Dr Douglas Wallace and I have recently shown that mitochondrial genes are highly variable and that these variants predispose individuals to a variety of degenerative diseases, it follows that these same variants may be critical factors in individual response to stroke (Ruiz-Pesini et al , 2004). Indeed, each individual is born with an initial mitochondrial energy capacity depending on the mtDNA and nDNA base substitutions inherited. Furthermore, evidence have been accumulated that mtDNA mutations increased during life time in tissues like brain or skeletal muscle and erode bioenergetic capacities of the individual. Experimental models of mitochondrial neurodegeneration have shown that pharmacological agents that improve energy efficiency and reduce ROS production might prevent neuronal death after ischemic insults, suggesting that addressing the aims of the current study might open the door to new avenues of treatment for patients with ischemic stroke. We have been investigating the molecular basis of the striking regional-specific distribution of mtDNA lineages and have concluded that specific mtDNA variants permitted humans to move out of tropical Africa into the colder temperate Eurasia and arctic Siberia and North America. These 'cold' adaptation mutations change highly conserved amino acids and occur at key positions that would modulate the coupling efficiency of mitochondria, in other words a balance between energy production and heat generation. From these observations we have hypothesized mtDNA mutations that partially uncoupled mitochondria converted a higher percentage of the dietary calories into heat, instead of energy, and thus permitted humans to survive the colder northern latitudes. Such 'cold' adaptations could influence cellular events and energetic during critical times during the process of recovery from stroke. We now hypothesize that as a consequence of partial uncoupling of mitochondria due to the genetic background, will reduce mitochondrial ROS production and also reduce cellular oxidative stress after brain injury. This in turn will increase preservation of cells and thus increased protection of neuronal tissues from deleterious consequences of a stroke.
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