No description available.

Recent epidemiological data have shown that metabolic disease is known to increase the propensity for developing cognitive decline and dementia, particularly Alzheimer’s disease (AD). While this interaction is not completely understood, clinical studies suggest that both hyper- and hypoinsulinemia are associated with an increased risk for developing AD. Indeed, insulin signaling is altered in post-mortem brain tissue from AD patients and insulin and treatments known to enhance insulin signaling, can improve cognitive function. Furthermore, clinical evidence has shown that AD patients and mouse models of AD often display alterations in peripheral metabolism. Since insulin is primarily derived from the periphery it is likely that peripheral alterations can lead to alterations in central nervous system (CNS) insulin signaling and that these changes contribute to cognitive decline. Recent results from our laboratory have shown that in both the APP/PS1 and 3xTg-AD mouse models of AD, peripheral metabolic alterations exist at an early age. Specifically, 3xTg-AD mice demonstrate impaired glucose tolerance at 1 month of age associated with a decrease in insulin and insulin secretion in response to a glucose challenge. This led to the hypothesis that insulin signaling in the CNS would be decreased as a result of decreased peripheral insulin and insulin transport into the CNS. Indeed, insulin signaling through the PI3K/AKT signaling pathway, but not the MAPK/ERK pathway, was decreased in the hippocampus of old, but not young, 3xTg-AD mice. PI3K/AKT signaling can affect several downstream molecules including glycogen synthase kinase 3 (GSK3), glucose transporters (GLUTs), and ATP dependent potassium (KATP) channels. We first examined GSK3 and pTau and found that both GSK3β and pTau were increased in aged 3xTg-AD mice. Next we looked at the translocation of GLUT3 and GLUT4 since both are found in the hippocampus and have recently been shown to be insulin sensitive. Our results showed that GLUT3 translocation, but not GLUT4, was decreased in the hippocampus of aged 3xTg-AD mice. Finally, since KATP channels are found in intracellular organelles as well as in the plasma membrane we examined crude plasma membrane and total fractions of KATP channel subunits Kir6.1 and Kir6.2 and found that the plasma membrane fraction of Kir6.2 was significantly increased. To assess how these changes corresponded with the time course of pathology and cognitive deficits we additionally looked at these changes in 6-8 month and 14-16 month animals. Interestingly, though peripheral insulin was decreased early on, changes in CNS PI3K/AKT insulin signaling did not occur until 18-20 months of age. Changes in GSK3β (but not pTau) and GLUT3 were consistent with this time point suggesting that they were potentially due to the decrease in PI3K/AKT signaling. Since these changes were not consistent with a decrease in peripheral insulin levels it suggests that another factor must be at play. One such factor is inflammation. The AD brain is characterized by inflammation and inflammatory compounds are known to block insulin signaling. KATP channels are not only insulin sensitive but have been shown to play a role in cognition, AD and epilepsy. Thus, to follow up the studies on KATP channels we used immunohistochemistry (IHC), to examine regional and cell specific changes. To our surprise we found that Kir6.2, a subunit typically found primarily in neurons, was present in reactive astrocytes. This finding was further examined in human AD tissue and a similar change was seen. Astrocytes become reactive during damage or under inflammatory conditions, such as AD, diabetes, traumatic brain injury (TBI), epilepsy and in normal aging. When they become reactive both gene expression and functions can change. Since reactive astrocytes and inflammation are a common finding among many neuropathological changes we looked at another neuropathological condition with several similarities to AD, epilepsy. These studies revealed that epileptic mice displayed a similar change in Kir6.2 in reactive astrocytes. Since both conditions are characterized by inflammation we next hypothesized that chronic peripheral inflammation induced by LPS would be enough to drive this change. These studies revealed that while 1 day of LPS treatment was not enough to induce a change in astrogliosis and Kir6.2 expression, three days caused a significant increase in Kir6.2 in reactive astrocytes. This suggests that an increase in Kir6.2 in reactive astrocytes could contribute to the difference in function in these cells and subsequently contribute to altered function in neuropathological disease. Taken together, these studies demonstrate an intricate balance between metabolism and inflammation in the CNS and further suggest that metabolic alterations could be a common link in neuropathological diseases that share similar phenotypic changes, as occurs in AD and epilepsy (i.e. cognitive decline, enhanced seizure susceptibility). Developing a better understanding of metabolism, inflammation, and cortical function/dysfunction could potentially lead to the identification of better treatment options for several neuropathological conditions including AD.

Title: Hypersensitive and circadian effects of acebutolol administration in Scn1b-/- mice. Rationale: Dravet syndrome (DS) is a severe form of pediatric epilepsy with characterizations of pharmacoresistant seizures and developmental delay. A rarer variant of the DS model is caused by homozygous loss-of-function mutations in SCN1B, which is essential in regulating sodium channel gating, expression, localization, and the firing of action potentials. Mutations in SCN1B result in severe seizures as well as a higher risk of Sudden Unexpected Death in EPilepsy (SUDEP). Factors underlying SUDEP are poorly understood, although cardiac arrhythmias have been implicated. Acebutolol (ACE) is a common beta-blocker used in the treatment of arrhythmias and hypertension. We hypothesized that treating mice with ACE will decrease cardiac arrhythmias and the incidence of SUDEP, prolonging lifespan of Scn1b null mice. Methods: Wild-type (WT) and null (KO) mice were given daily injections of 10 mg/kg ACE or saline starting at postnatal day 15 (after typical seizure onset) either during the day (09:00) or at night (21:00). In the day group, ECG was recorded daily from P13 until animal death. Starting at P15 mice were recorded both pre- and post- injection to analyze the long-term and acute effects of treatment. Results: A modest, but significant, increase in survival curves in our KO animals was observed compared to saline treated mice for those given injections during the day (a 2 day increase in median survival). In addition, in this group, the onset of animal death was delayed. To investigate the timing of drug delivery, a subset of mice was given injections at night. In this group there was actually a decrease in lifespan, with an earlier onset of death compared to saline treated mice. On a daily basis from P13, the heart rate (HR) of KO mice was significantly lower than WT but remained steady until the day prior to animal death. HR the day prior to death consistently dropped ~50% (average 414 bpm to 193 bpm) in our saline group; this was prevented in KO animals treated with ACE (421 bpm). Analysis of acute recordings following ACE administration showed that KO mice had a significantly larger reduction in heart rate compared to WT (38% vs. 11%). Further analysis of heart rate variability in these recordings demonstrated that RMSSD (a measure of vagal control of the heart) was reduced in KO mice, with differences in both baseline and following ACE administration. Conclusions: Leading up to death, we believe it is possible ACE assisted in decreased cardiovascular deficits that could lead to SUDEP and contributed to the modestly increased lifespan. In addition, our results demonstrate the importance of timing in delivery of drugs targeted at SUDEP. Finally, these results suggest that there is a possible hypersensitivity to beta-adrenergic blockade in Scn1b-/- mice. Funding: This work was supported by a grant from the Research Development Committee at East Tennessee State University and NIH grant R21NS116647 to C.R.F.

No description available.

No description available.

This study investigated the β-subunit of the GABAa receptor and determine if there are changes in the primary sequence of the extracellular N-Terminal domain, which likely regulates GABA binding.

Dravet syndrome (DS) is a severe, pediatric-onset epilepsy disorder linked to loss-of-function mutations in the sodium channel gene SCN1B. DS patients have a high risk of Sudden Unexpected Death in Epilepsy (SUDEP). Cardiac arrhythmias have been implicated as a potential cause underlying SUDEP. An exact pathway for how mutations in SCN1B leads to arrhythmia in DS is unclear. One cellular component linked to regulation of cardiac homeostasis are mitochondria, known as “the powerhouse of the cell” due to their ability to produce cellular energy (ATP) via the electron transport chain (ETC). The ETC is a major producer of reactive oxygen species (ROS). Typically, ROS are buffered by cellular antioxidants, to prevent oxidative stress, an imbalance of ROS that can lead to cell damage. Our previous work indicates that cardiac arrhythmias may result from mitochondrial instability and imbalances between ROS production and buffering. We analyzed whether Scn1b-/-mice are susceptible to arrhythmias due to altered mitochondrial ATP generation, ROS production, and compromised cellular antioxidant defenses. We isolated cardiac mitochondria from postnatal day (P) 15-20 KO and Scn1b+/+ (WT) mice. To assess mitochondrial ATP and ROS production, high-resolution respirometry (O2k, Oroboros) was used to measure mitochondrial O2 and H2O2 flux. We used a substrate-uncoupler inhibitor (SUIT) protocol to elucidate flux under different ETC pathways, including Complex I- and II-linked respiration. As a next step, we evaluated expression of superoxide dismutase (Sod) proteins associated with mitochondrial antioxidant defenses, including Cu/Zn-Sod (Sod1) and Mn-Sod (Sod2) in hearts from KO and WT mice pre- (P10) and post- (P17) seizure development. After addition of substrates supporting Complex-II linked respiration (succinate, ADP) there were no differences in O2 flux between mitochondria isolated from KO and WT hearts. Upon further addition of pyruvate to mitochondria to stimulate Complex I, O2 flux was significantly reduced (p < 0.0001) in mitochondria from KO mice, when compared to WT. Moreover, upon titration of rotenone (a Complex I inhibitor) its negative effect on O2 flux was not as substantial in KO mitochondria as in WT, suggesting that mitochondria from KO have deficits in Complex-I linked respiration. Furthermore, we detected significant differences in ROS production by mitochondria isolated from KO animals. Under conditions of reverse electron flow (succinate as substrate), a state where ROS production is highest, H2O2 flux was elevated significantly (p = 0.048) in mitochondria isolated from KO mice, compared to those isolated from WT. During our analysis of Sod expression, we found that Sod1 (p = 0.01) and Sod2 (p = 0.01) expression is significantly decreased at P17 in KO hearts compared to WT. Overall, our results suggest imbalances between mitochondrial activity and antioxidant defenses, which may underlie increased arrhythmia susceptibility in KO mice.

Part I. Development of an isoxylitone analog as an antiepileptic drug candidate. Delphinium denudatum is a medicinal plant traditionally used to treat a variety of conditions in Central Asia. Its interesting anticonvulsant effects were determined to be a property of the compound isoxylitone. Prior work from our group in collaboration with the Poulter group from Western University investigated this compound and generated a large number of isoxylitone analogs in order to optimize its antiepileptic activity. This led to the discovery of the prodrug 13 and the active form 15 shown below, which emerged as the most potent. In this work, the library of analogs was further expanded with 22 new compounds with several which matched the activity of 13 and 15, such as compounds 22 and 37, which led to valuable new insights on the activity of these analogs, and suggested other possible future improvements. In addition, efforts were continued regarding developing compound 15 as a clinical trial candidate. Optimization of the synthesis was performed to drastically reduce costs and waste of chemicals, as well as accelerating the duration of the synthesis. The purification of the final product was also greatly facilitated by the direct synthesis of 15, compared to the prior process of first preparing 13 and hydrolyzing the ester. Efforts were exerted to gather additional knowledge on the characteristics of the compound, with structural and conformational analysis via X-ray crystallography and NOE NMR as well as accelerated stability studies to test the viability of 15 in long-term storage under various conditions. All the information gathered throughout this work supported 15 and its sodium salt as excellent clinical trial candidates as treatments for epilepsy. Part II. Synthesis of SOX9 inhibitors as promoters of recovery from spinal cord injury. According to the World Health Organization, 250 to 500 thousand people develop a spinal cord injury each year with a large portion resulting in tetraplegia. A common misconception is that this is permanent because the damaged nerves cannot be repaired. In fact, nerves can and do regrow after being damaged, but cannot do so after spinal cord injuries due to formation of scar tissues which physically and chemically prevents the healing. The Brown group at Western University identified the SOX9 transcription factor as an important promoter of the formation of this scar and showed that SOX9 inhibitors could improve recovery and mobility in mice affected by spinal cord injuries. In collaboration with their group, previous work in our lab performed and SAR study on the lead compounds ZO2(1) and STL26 (2), shown below. The different sections of the molecule have been designated units A to D, to simplify discussion. Initial work by our group established an efficient method to prepare a library of analogs of the lead compounds. A number of compounds were prepared, which primarily investigated small amines as unit A and phenols with small aliphatic substituents as unit D. The initial SAR data confirmed the validity of STL26 as lead compound, as most alterations to the structure were detrimental to the SOX9 inhibitory activity. The objective of this work was to build on these preliminary SAR results, and expand the library of analogs. Larger substituents were introduced in unit A and D and showed that any group larger or smaller than diethylamide in unit A was detrimental to the activity, but that there seemed to be ample space to increase the size of the unit D isopropyl group. Analogs investigating unit B showed that adding substituents at most of the positions was detrimental, as well as changing the relative positions of unit A and B to be ortho or para to each other. However, the C4 on ring B seemed to be very tolerant to various electron donating or withdrawing functional groups. During this SAR study, a recurring theme was the awful solubility of the compounds in water, which heavily complicated their administration to mice during the bioassays. While none of the analogs tested proved superior to 2, the knowledge accrued during this work painted a clear path forward on which areas of the structure could be safely altered to improve solubility without negative impacts on SOX9 inhibition. Some additional efforts were put into obtaining an accurate three-dimensional structure of an active STL26 (2) analog, and information on the primary conformation in solution. Achieving these goals required the use of NOE NMR experiments and X-ray crystallography. One conformation was discovered to be strongly favoured as a result of an intramolecular hydrogen bond even in protic solvents. Subsequently, a small number of additional analogs were prepared containing modifications that would strongly favor or hinder the preferred conformation, in order to better understand its role in the inhibitory activity. The presence of this hydrogen bond appeared to be key to the activity of the compounds.

Thesis (Ed.D.)--Boston University / PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you. / The performance of a group of twenty brain-injured boys (mean IQ 117.4; mean chronological age 120.6 months) was compared with that of a control group of non-brain-injured boys (mean IQ 118.2; mean chronological age 128.6 months) on a battery of tests designed to measure perceptual processes underlying conceptual thinking. The experimental group was selected from the out-patient files of the Seizure Unit at Ohildrens Medical Center in Boston. The control group was drawn from a large elementary school in the greater Boston area. All children in both groups were enrolled in regular classrooms and groups were matched on the variable• ot WISC Full Scale IQ, chronological age, sex and socio-economic level. The battery of tests consisting of the WISC, the Grahm-Kendall Memory for Design, the Wisconsin Card sorting Teat, the Porteua Mazes and the Gilmore Sentence Completion Test was administered to each child in his own home. [TRUNCATED] / 2031-01-01

No description available.