Marianna Jung - US grants

[unreadable] DESCRIPTION (provided by applicant): Alcoholism is a consequence of prolonged ethanol abuse and has been associated with the aging of cognition and psychomotor function. Because alcoholics go through repeated rounds of ethanol withdrawal (EW), we propose using repeated EW to investigate whether aging with exacerbates the effects of EW on cognition and psychomotor function through oxidative insults to mitochondria. We also propose to investigate whether a loss of estrogen contributes to this problem. This work may have particular significance for female alcoholics, who simultaneously undergo EW and estrogen deficiency in their advanced age. Our pilot study has demonstrated that estrogen protects against EW toxicity in both young and old female rats. In the proposed study, we will use three age groups of ovary-intact female rats: 5 months old, 12 months old, and 16 months old, when the ethanol diet begins. We propose to use a diet cycle of 25 days of ethanol followed by 5 days of withdrawal repeated for 5 cycles. These cycles will be timed such that rats will be at the estrus phase (high estrogen levels) when diet is removed and EW signs peak. In Aim 1, we will determine the effects of EW on cognition and psychomotor functions during aging. Rats at the estrus phase will be withdrawn from an intermittent ethanol diet and tested for psychomotor function using established behavioral methods. In Aim 2, we will characterize the mitochondrial oxidative mechanisms underlying the deleterious interaction between age and EW. We will measure oxidative markers (reactive oxygen species, lipid peroxidation) and consequent mitochondrial dysfunction (collapse of mitochondrial membrane potential and ATP loss). In Aim 3, we will characterize oxidatively modified mitochondrial enzyme cytochrome c oxidase (COX) which we hypothesize mediates the deleterious interaction between age and EW. We will quantify carbonylation of COX and lipid-peroxide-modified COX using liquid chromatography-mass spectrometry and tandem mass spectrometry. In Aim 4, we will determine whether estrogen deficiency during aging contributes to EW-induced oxidative damage to mitochondria and psychomotor deficit. The same psychomotor and oxidative markers will be measured in ovary-intact and ovariectomized rats with or without estrogen replacement. These studies may ultimately contribute to a better understanding of and strategy for female alcoholics undergoing EW during brain aging. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The cessation of ethanol consumption is difficult and often unsuccessful, even for highly motivated patients. Abrupt termination of ethanol-exposure renders the brain vulnerable to neurological sequelae, including sensorimotor impairment, tremor, and convulsions, which discourage the patient's efforts to stop consuming alcohol. The only available treatment for the ethanol withdrawal (EW) distress is benzodiazepines but the side effects (e.g. dependency and respiratory depression) limit clinical use of this treatment. In a rat model of ethanol intoxication and withdrawal, we recently demonstrated profound brain protection by daily cycles of 5-10 minutes, intermittent, normobaric, moderate (FIO2 9.5-10%) hypoxia (IH). In our pilot studies, when IH treatment was applied during the last 20 days of an ethanol diet, it attenuated the overt behavioral signs of EW (e.g. tremor and rigidity);EW-induced mitochondrial membrane swelling;and cytotoxicity. In particular, EW provoked a more than 6-fold increase in the proapoptotic kinase P38;IH conditioning almost completely suppressed this increase in ethanol withdrawn rats. Interestingly, all of these IH protections were more effective during EW than during ethanol-exposure, implicating that IH counteracts factors mediating EW. These remarkable results suggest that adaptation to intermittent normobaric hypoxia could provide a safe and effective treatment complementary to conventional benzodiazepines. Using a repeated withdrawal paradigm, we intend to define behavioral phenotypes of IH protection with various treatment windows of IH and cellular mechanisms involving P38 by which IH protects against repeated EW. We hypothesize that IH conditioning applied during the initial cycle of EW exerts persistent protection against behavioral and brain injury throughout repeated EW. Rats will receive two cycles of an ethanol diet (7.5%) for 5 weeks and abrupt withdrawal for 20 days. IH conditioning will be applied to the initial cycle of the EW or the ethanol-exposure phase. We will determine 1) whether IH treatment during the initial cycle of EW protects against overt behavioral signs of EW and cerebellum-/hippocampus-related behavioral deficit and whether the protection persists throughout the repeated EW;2) whether IH-induced suppression of P38 activity mediates protection against apoptotic (programmed cell death) brain injury and whether the protection persists throughout the repeated EW in a manner that correlates with behavioral protection. Some animals will receive the P38 inhibitor SB203580 to define the role of P38-inhibition in the IH protection. Defining the IH-induced behavioral and brain protection against EW insults and identifying an optimal treatment window may establish foundation data necessary for the development of an alternative management of EW. PUBLIC HEALTH RELEVANCE: Alcoholics failed to achieve complete abstinence due to distress associated with sudden termination of drinking. We observed that cycles of intermittent brief (5 to 8 min) and moderate hypoxia (9 to 10% oxygen) conditioning reduced signs of ethanol withdrawal (e.g. tremor and rigidity) and oxidative stress in rats. Information on intermittent hypoxia-evoked brain protection from ethanol withdrawal distress resulting will establish an empirical foundation to support eventual clinical application for a better management of alcoholism in which ethanol withdrawal plays a key role.

Abstract Benzodiazepines (BZDs) are a widely used class of sedative and anti-anxiety medications, and their usage increases with age (NIH news, 2014). While BZDs are powerfully effective for treating hyper-excitatory disorders, they can have serious side effects, such as motor deficits. The vast majority of elderly BZD users stays in BZD therapy for a prolonged period (NIH news, 2014), which exacerbates the motoric deficits. The adverse effects and long-term use of BZD are seen more in elderly women than other age/gender population groups. However, how chronic BZD (cBZD) usage impairs the motoric function of elderly women remains virtually unknown. The objective for this proposal is to establish a transgenic mouse model that is capable of identifying the mechanism by which cBZD induces motoric aging in female subjects. BZD binds to the GABA- BZD receptor complex, thereby eliciting CNS depressant effects. It is also well known that BZDs including diazepam, lorazepam, and alprazolam, bind to mitochondrial BZD receptors (mBZD-R), located in mitochondrial membranes. The excessive stimulation of mBZD-R damages mitochondrial membranes where mitochondrial respiration takes place, consequently overproducing reactive O2 species (ROS). The binding of BZD to mBZD-R was significantly increased in the cerebellum of the elderly with cognitive disorders (Yasuno et al., 2012), suggesting that mBZD-R plays a role in brain aging. In particular, mBZD-R activity involves pro- apoptotic protein p38 such that a p38 inhibitor attenuates the apoptosis induced by mBZD-R ligands (Sutter et al., 2004). Since ROS activates p38 by phosphorylation, the apoptotic effect of p38 would be more severe in elderly women who lack 17ß-estradiol (E2), which directly scavenges ROS. Purkinje cells are major cerebellar neurons that are particularly vulnerable to apoptotic p38 (see Approach, Guan et al., 2005). Purkinje neurons show an age-dependent accumulation of p38 in a manner that is mitigated by E2 (Jung et al., 2011). We have generated mice that lack p38 in Purkinje neurons, and observed that these mice are more resistant to cBZD- induced motoric deficit and mitochondrial respiratory suppression than wild-type mice. This protective effect of Purkinje p38 downregulation is more prominent in female mice than male mice, suggesting that a female hormone, especially E2, may play a role in that protection. These observations suggest that cBZD provokes an adverse interaction between the mBZD-R and p38 at an E2-deficient age, resulting in Purkinje apoptosis and motoric aging. We will pursue our objective by achieving Specific Aim 1: Determine the mechanism by which cBZD induces motoric aging. We will test a two-step hypothesis: 1A) cBZD's binding to mBZD-R age- dependently suppresses mitochondrial respiration and 1B) cBZD induces Purkinje neuronal apoptosis and motoric deficit through ROS-activated p38 at an E2-deficient age. The proposed research is absolutely critical because it is expected to establish a solid model and foundational mechanism that will be a first step toward lessening the motoric deterioration of elderly women receiving cBZD.