Ricardo Mostany, Ph.D. - US grants

PROJECT SUMMARY/ABSTRACT The neural mechanisms that mediate the decline of brain performance with aging are poorly defined and affect many aspects of normal aging life: reductions in motor dexterity, sensory discrimination, executive function, and attention which impact the degree of independence, number of injuries, and fatal accidents. We will define mechanisms of age-related changes in synaptic plasticity and investigate their impact in memory and learning. Our hypothesis is that in the aged cerebral cortex, disruption of the excitation/inhibition balance at the level of the microcircuits of layer 5 (L5) pyramidal neurons leads to reduced formation of long-lasting stable synapses between excitatory neurons, resulting in impaired learning. We have recently described that dendritic spine density of aged mice is stable, but that their dynamics are elevated in somatosensory cortex. But, we do not if density and dynamics of dendritic spines are differentially affected by age in different brain areas. Also, the mechanisms underlying the alteration in synaptic dynamics in the aging brain are unexplored. One possibility is that the intracortical inhibition controlling synaptic plasticity in the adult brain is released with aging allowing the formation of excess synaptic contacts, many of them meaningless and subsequently be eliminated and making the handling and storing of information less effective. Thus, increasing levels of intracortical inhibition in the aged brain may prevent alterations in synaptic dynamics and preserve brain performance. We will test the following hypotheses: (a) elevated dendritic spine dynamics in the aged brain impedes the creation of memory-forming synaptic contacts and impairs the ability of cortical circuits to store/manage information; (b) age- related reduction in inhibitory transmission at the level of the local circuitry of L5 pyramidal neurons is responsible for the increased instability of dendritic spines; (c) restoring intracortical inhibition in the primary motor cortex of aged mice will stabilize dendritic spines of L5 pyramidal neurons and improve performance in a motor learning task. We will use transgenic mice for in vivo 2PE microscopy and optogenetics in the conditional expression of viral vectors, behavioral tasks, and electrophysiological recordings of synaptically connected neurons: Aim 1 will determine that the alteration of synaptic dynamics in the aged brain is a maladaptive mechanism impairing learning. Aim 2 will identify age-dependent changes in PV and SOM neurons of the L5 cortical microcircuit responsible for instability of dendritic spines in pyramidal neurons and impaired learning. Aim 3 will confirm that the age-related decrease of inhibition in L5 pyramidal neurons impairs synaptic plasticity and learning. By using state-of-the-art techniques and innovative experimental approaches will elucidate the effects of normal aging on the assembly and maintenance of cortical circuits to facilitate future development of therapeutic interventions designed to delay the onset of aging-related brain decline and prolong the quality of life and welfare of the elderly. Results from the proposed research may be applied and used for studies on other neurodegenerative disorders.

Neuroprotective Role of Parvalbumin Interneurons Following Ischemia in the Aged Brain PROJECT SUMMARY/ABSTRACT There is no cure for stroke. The lack of pharmacological treatments, restricted to acute clot-buster interventions aimed to restore blood flow to ischemic brain regions as soon as possible makes stroke the fifth leading cause of death and the leading cause of long-term adult disability in North America. Studies in animal models of stroke have thrown promising results, however interventions to prevent extended brain cell death have not translated yet into useful treatments for humans. Given that age is associated with worse functional outcome after stroke in both, humans and animals, a better understanding of the effects of normal aging in neuronal brain circuits will provide a foundation for the development of therapeutic interventions designed to protect the ischemic brain from further progression of the damage, regardless of the age on the patient. Our overall hypothesis is that inhibitory interneuron activity is a neuroprotective mechanism and an age-related reduction in the presence and/or activity of parvalbumin (PV) interneurons renders the aged brain more susceptible to ischemic damage. PV neurons are probably the most abundant inhibitory interneurons in the cortex and the inhibitory action that these cells exert on the principal cortical excitatory neurons could be utilized to prevent the massive release of glutamate by these neurons during stroke. Therefore, our goal is to elucidate the degree of vulnerability of PV interneurons with age and during brain ischemic conditions and whether these neurons could be used during the early stages of the ischemic cascade to attenuate the glutamate-induced excitoxicity characteristic of this brain injury. This will have the potential to open the door to new neuroprotective interventions aimed to minimize the severity of the stroke and improve functional outcome. We will test the following hypotheses: (a) PV interneuron density decreases with age and that these neurons are more susceptible to ischemia in the aged brain, which may account for poorer prognosis following stroke in the aged brain. In addition we will test the hypothesis that aging induces a selective alteration in the relative abundance of RNA species associated with inhibitory neurotransmission in PV neurons; (b) PV interneuron activity is reduced in the peri-infarct cortex and that the deficits in PV network activity after ischemia are exacerbated in the aged brain; (c) promoting PV activity early after MCAO will have a neuroprotective effect, preventing the loss of synaptic inputs of L5 pyramidal neurons, and will improve functional recovery in both young and aged mice. We will use transgenic mice for in vivo 2PE microscopy and optogenetics using conditional expression of viral vectors, behavioral tasks, and electrophysiological recordings. Specifically: Aim 1 will determine the extent to which the number of PV interneurons in the peri-infarct cortex is affected by stroke and by age as well as the age-related changes in gene expression in these neurons that increases their susceptibility to aging and ischemia. Aim 2 will characterize network inhibitory activity and PV interneuron excitability following stroke. Aim 3 will promote synaptic plasticity and functional recovery after stroke using optogenetic stimulation of PV interneurons in the peri-infarct cortex following stroke. By using state of the art techniques and innovative experimental models, we will elucidate the effects of normal aging and ischemia on the integrity of inhibitory cortical circuits for the future development of therapeutic interventions designed to protect and improve functional outcome after stroke, especially in the aged brain.

Summary Brain microvessels play an important role in the neurovascular coupling (NVC). Mitochondria are energy sensors of cells. My work for the first time demonstrated the link between mitochondrial depolarization and activation of nitric oxide synthases (NOS). Recently, we have made a novel discovery of a neuronal NOS (nNOS) isoform in endothelial cells that uniquely produce reactive oxygen species (ROS). The nNOS is co-expressed with NO- producing eNOS in endothelial cells and both isoforms are involved in the bidirectional regulation of mitochondria. Diabetes mellitus (DM) increases the risk of cerebrovascular dysfunction and dementia. Importantly, hypoglycemia is a dangerous side effect of DM treatments, particularly insulin-therapy. Patients with DM often experience mild hypoglycemia, but these episodes are unaccounted for in determining the cardiovascular morbidity and mortality. We achieved a technological breakthrough utilizing Seahorse XFe24 analyzer and determined the mitochondrial respiration and cellular bioenergetics of brain microvessels. We made a novel observation that five episodes of recurrent hypoglycemia (RH) impaired the microvascular mitochondrial function. Notably, single episode of acute mild or severe hypoglycemia as well as Impairments of NOS activity was found to mediate RH-induced alterations of cellular bioenergetics. Thus, we hypothesize that mild RH disrupts NVC by promoting microvascular mitochondrial dysfunction leading to impaired cognitive function. We further hypothesize that increased nNOS-induced oxidative stress coupled with reduced eNOS-derived NO contribute to the mitochondrial dysfunction following RH. We propose to use streptozotocin treated C57Bl/6 mice and db/db mice with leptin receptor mutation as models of diabetes with untreated mice as controls. In addition, we will employ eNOS knockout and inducible endothelial cell specific nNOS knockout mice to investigate the role of NOS isoforms in RH-induced microvascular dysfunction. Each animal will be subjected to five episodes (one per day) of mild (blood glucose 70-80 mg/dl) or severe (blood glucose 40-54 mg/dl) insulin-induced hypoglycemia or saline control. Aim 1 is to demonstrate that mild and severe RH (in vivo) can increase the production of NOS-derived ROS and display RH-induced functional mitochondrial respiration deficits in cerebral microvessels (ex vivo). Aim 2 is to establish the impact of RH on NVC in vivo. We will determine the RH-induced deficits in NVC by measuring the changes in arteriolar and capillary diameter in response to neuronal activation (whisker stimulation) in awake mice using two-photon laser scanning microscopy. Aim 3 is to determine the impact of RH on cognitive function using novel texture discrimination task and modified Y-maze test. The results of this proposal would identify the mechanistic link between mild RH and the cerebral microvascular mitochondria dysfunction and challenge the existing dogma to demonstrate that mild RH is equally detrimental as severe RH in contributing to the DM-induced cerebrovascular dysfunction, impaired NV, and cognitive dysfunction.

PROJECT SUMMARY/ABSTRACT Brain performance declines with Alzheimer?s disease (AD) progression. The massive loss of neurons observed at advances stages of the disease are confirmatory observations of the disruption of the brain circuits governing the brain tasks affected. This is, however, too late in the progression of the disease. Beta-amyloid (A?) progressively accumulates over many years, surpassing its physiological levels early in the disease. Unfortunately, little is known about the effects of A? before the first symptoms appear. By then, it has been reported, among other things, that there is an increase in the excitability of the neurons. We found that cortical pyramidal neurons of young APPNL-G-F mice, a relatively novel mouse model of AD that does not overexpress amyloid precursor protein, but accumulates A? aggressively after the second month of life, present with physiological features that indicate a reduction of their intrinsic excitability when compared with neurons from age-matched controls. The same indicators 3-4 months later, when the accumulation of A? is significant, show a swing in their excitability, and the neurons become more excitable than in control mice, results more in agreement with the data from the literature. We believe that sustained hypoexcitability results in impaired homeostatic mechanisms of intrinsic excitability in 6-month-old mice. Our hypothesis is that early accumulation of A? leads to hypoexcitability of cortical neurons resulting in a pathological hyperexcitability at later stages of the disease. This abnormal switch in excitability is a consequence of an impairment of the homeostatic mechanism caused by upregulation of CaMKIV activity. The questions that arise now are how early A? accumulation leads to hypoexcitability, what causes the rebound in excitability a few months later, and whether there is a manipulation that could correct the hypoexcitable state to prevent the hyperexcitable state. To answer these questions we will test the following hypotheses: (1) hypoexcitability in the young APPNL-G-F mice is caused by upregulation of voltage-gated potassium channels, downregulation of voltage-gated sodium channels changes, or both, (2) defective or saturated mechanisms of homeostatic plasticity lead to hypoexcitability at younger ages, (3) homeostatic plasticity dysregulation is a direct consequence of A? accumulation, (4) hypoexcitability occurring during young adulthood in the progression of pathology in the APPNL-G-F mouse model is a cause of hyperexcitability at later stages (middle age), (5) early hypoexcitability results in blunted homeostatic response at middle age, due to downregulation of CaMKIV, and (6) long-term block of K+ channels using FDA- approved drugs during early stages of the pathology will increase homeostatic downregulation of excitability. We will use APP knock-in (APPNL-G-F) transgenic mice, the most clinically relevant mouse model of AD, in vivo 2PE microscopy, optogenetics, chemogenetics, and electrophysiological recordings to test our hypotheses. Aim 1 will identify the mechanisms responsible for early, pre-plaque hypoexcitability of pyramidal neurons in APPNL-G-F mice and Aim 2 will determine if interventions aimed to correct early hypoexcitability of pyramidal neurons can prevent or decrease middle age hyperexcitability. By using state of the art techniques and innovative experimental and animal models, we will elucidate the effects of the progression of the AD pathology on neuronal homeostatic mechanisms. This study has the potential to generate novel knowledge on the deficits impacting brain function before the appearance of cognitive symptoms for the design and improvement of personalized or precision interventions aimed to prevent or delay cognitive disturbances in Alzheimer?s disease patients.

Summary Brain microvessels (BMVs) play an important role in the neurovascular coupling (NVC). Mitochondria are energy sensors of cells and impaired mitochondrial respiratory function initiate critical signaling detrimental to NVC leading to impaired cognitive function associated with Alzheimer's disease (AD). Our recent technological breakthrough utilizing Agilent Seahorse XFe extracellular flux analyzer developed a mitochondrial respiration assay in BMVs. Using this novel method, we observed age-dependent impairment of mitochondrial respiration and bioenergetics in BMVs from male and female C57Bl/6 mice. Notably, we found that BMVs from APP NL-G-F Knock-in model of AD display impaired mitochondrial respiration and accelerated senescence. Furthermore, we observed that young and aged female mice display sex-dependent differences in microvascular energetics related to the relative contribution of oxidative phosphorylation and glycolysis to overall energy production. Finally, we found that peroxynitrite scavenger (FeTMPyP) treatment enhanced non-mitochondrial respiration young female mice but enhanced proton leak in young male mice indicating that the differential peroxynitrite activity is sex-dependent. Therefore, we hypothesize that peroxynitrite differentially regulates microvascular mitochondrial function sex-dependently and is the molecular determinant of exaggerated age-related impairment of NVC and cognitive function in AD. We will employ male and female AD and C57Bl/6 mice of 8 months and 20 months age. Aim 1 will determine the sex dependent differential impact of peroxynitrite on the bioenergetics and enzyme activities (Krebs cycle, glycolysis, and antioxidants) in BMVs ex vivo. Aim 2 will determine the sex dependent differential impact of peroxynitrite on in vivo NVC responses to whisker deflections by two-photon excitation microscopy in awake mice. Aim 3 will determine the sex dependent differential impact of FeTMPyP on cognitive function by assessing whisker-dependent perceptual learning using the novel texture discrimination task. The results of this proposal would challenge the existing dogma and will demonstrate the sex-specific physiological role of peroxynitrite in regulating the microvascular bioenergetics and neurovascular unit. Furthermore, our results will firmly establish microvascular peroxynitrite as a potential therapeutic target in sex- dependent vulnerability and severity of AD and other neurodegenerative diseases.