No description available.

<p> Alzheimer&rsquo;s disease (AD) is the leading cause of dementia, accounting for 50 to 80 percent of dementia cases, and the prevalence of the disease is projected to increase significantly with time. AD is characterized by severe cognitive decline with age, ultimately requiring continued caregiving and eventually death. The pathology of AD is characterized by the presence of extracellular amyloid plaques, intracellular neurofibrillary tangles (NFT) composed of hyperphosphorylated tau protein, neuron loss, and evidence of inflammation indicated by the presence of reactive microglia and astrocytes. Frontotemporal Lobe Dementia (FTLD) is a rare form of dementia that is related to AD, most notably in the pathology of hyperphosphorylated tau and macroscopic brain shrinkage. It has been defined as one of a host of tauopathies, and has a more rapid onset than AD. Symptoms that resemble personality changes, moreso than memory loss, are characteristic of these other tauopathies (FTLD is a representative of a whole class of neurological disorders). Like AD, there are no known treatments or cures for FTLD. AD and FTLD are two manifestations of a class of diseases known as tauopathies, due to the presence of toxic forms of tau. </p><p> Tau is a protein normally found in neurons. It functions as a stabilizer for microtubules, and has a role in the trafficking of materials from the cell body to the presynaptic terminal. In AD and FTLD, tau can become hyperphosphorylated, which causes it to form twisted fibrils called NFTs. An emerging area of research is to identify antibodies that target tau as a way to clear tau pathology and hopefully reduce synaptic and neuron loss (Boutajangout et al., 2011b). While these diseases have no known cure or treatment at present, immunotherapy is emerging as a promising approach for treatment. The studies presented here investigated a variety of antibodies directed against tau, and incorporated different timeframes and administration routes to identify the best candidate for future clinical investigation of tau immunotherapy. </p><p> The mouse model rTg4510, known for expressing cognitive-related tauopathy, was primarily used to evaluate tau antibody effectiveness prior to clinical consideration. Our investigations began by utilizing a more familiar mouse which was also reported to express tau pathology. </p><p> Our studies first examined intracranial injection of a variety of antibodies using a mouse model previously reported to demonstrate tau pathology, to identify short-term clearance of tau pathology and NFTs. Next, we examined a more robust tau-producing mouse line, to further identify a most effective antibody, as well as to examine the time course of effect, after administration. A longer-term administration, and different route of administration was tested using mini-osmotic pump implantation into the mice, which provided for 28-day continuous infusion. This approach was followed with administration of antibodies, systemically. Behavioral analysis, in addition to pathological testing, was incorporated into the longer-term administration studies.</p>

<p> Previous studies have shown that FMRFamide alters the contractions of the smooth muscle tissues in <i>Lumbricus terrestris</i> including muscles along the body wall. Recently, numerous new annelid FMRFamide-related peptides (FaRPs) have been identified across the annelid in both polychates and clitellates, including tetrapeptides and N-terminally extended peptides. The primary objectives of this study are to analyze and compare the effects of FMRFamide (phenylalanine-methionine-arginine-phenylalanine-NH2), YMRFamide (tyrosine-methionine-arginine-phenylalanine-NH₂), FVRFamide (phenylalanine-valine-arginine-NH₂), YVRFamide (tyrosine-valine-arginine-phenylalanine-NH₂), AGAYVRFamide (alanine-glycine-alanine-tyrosine-valine-arginine-phenylalanine-NH₂), PAKHYVRFamide (proline-alanine-lysine-histidine-tyrosine-valine-arginine-phenylalanine-NH₂), and APKQYVRFamide (alanine-proline-lysine-glutamine-tyrosine-valine-arginine-phenylalanine-NH₂) on the longitudinal muscles of the body wall, specifically contraction rate and contraction strength. A body wall strip without the ventral nerve cord was placed in a tissue bath and exposed to increasing concentrations of these neuropeptides. Mechanical contractions were recorded on a computer with a Grass force transducer attatched to an Iworx A/D converter. </p><p> FMRFamide decreased contraction rate at a threshold of 10^-7 to 10^-6 M and increased the contraction amplitude at 10^-7 to 10^-6 M. YMRFamide had no effect on contraction rate, while being excitatory on contraction strength with a threshold of 10^-7 M. FVRFamide had no effect on contraction rate, while being excitatory on contraction strength at 10^-9 M. YVRFamide inhibited contraction rate with a threshold of 10^-9 M and increased contraction strength at 10^-9 M. APKQYVRFamide showed a biphasic response with regards to rate, decreasing it at 10^-9 M and increasing at 10^-8 M; it increased amplitude with a threshold at 10^-9 M. PAKHYVRFamide decreased rate at a threshold of 10^-9 M; it showed a biphasic trend in regards to contraction amplitude, increasing strength at 10^-9 M and decreasing it at 10^-6 M. AGAYVRFamide demonstrated a biphasic effect on contraction rate decreasing it at 10^-9 M and increasing it at 10^-8 M; it also showed a biphasic effect on amplitude, decreasing at 10^-9 M and increasing at 10^-7 M. The data demonstrate that both tetrapeptides and N-terminally extended peptides were biologically active in a concentration dependent manner with similar trends. This seems to suggest that there is some conservation in either neuropeptide presence or FaRP receptor across the annelid phylum. Comparing the four tetrapeptide sequences the data suggested that the second position was important for determining potency of the peptide where valine containing peptides had a lower threshold for increasing contraction rate than did the methionine containing peptides. The N-terminally extended YVRFamide data suggest that the type of extension is more crucial than the extension itself. On contraction rate AGAYVRFamide and APKQYVRFamide are both biphasic while PAKHYVRFamide is only inhibitory and is the only extended YVRFamide to have a positively charged amino acid in the X position (as in ABCX-YVRFamide), suggesting that the positive charge can negate the effect of the N-terminal extension.</p>

<p> Stroke is the fifth leading cause of death in the United States and a major cause of long-term disability in industrialized countries. The core region of an ischemic stroke dies within minutes due to activation of necrotic pathways. Outside of this core region is the penumbral zone, where some perfusion is maintained via collateral arteries. Delayed cell death occurs in this area due to the triggering of apoptotic mechanisms, which expands the ischemic injury over time. The cellular and molecular events that produce the expansion of the ischemic core continue to be poorly understood. The increases in the amyloid precursor protein and pathogenic secretases lead to the increase in amyloid-&beta; (A&beta;) production. The relatively small amount of research in this area has hampered development of stroke therapy designed to prevent neuronal and glial cell degeneration in the penumbra. Currently, there is a significant lack of therapeutic options for acute ischemic stroke, and no drug has been approved for treating patients at delayed time points (&ge; 4.5 hr post-stroke). </p><p> Afobazole, an anxiolytic currently used clinically in Russia, has been shown to reduce neuronal and glial cell injury <i>in vitro</i> following ischemia, both of which have been shown to play important roles following an ischemic stroke. Treatment with afobazole decreased microglial activation in response to ATP and A&beta;, as indicated by reduced membrane ruffling and cell migration. Prolonged exposure of microglia to ischemia or A&beta; conditions resulted in glial cell death that was associated with increased expression of the pro-apoptotic protein, Bax, the death protease, caspase-3 and a reduced expression in Bcl-2. Co-application of afobazole decreased the number of cells expressing both Bax and caspase-3, while increasing the cells expressing Bcl-2 resulting in a concomitant enhancement in cell survival. While afobazole inhibited activation of microglia cells by A&beta;_25-35, it preserved normal functional responses in these cells following exposure to the amyloid peptide. Intracellular calcium increases induced by ATP were depressed in microglia after 24 hr exposure to A&beta;_25-35. However, co-incubation with afobazole returned these responses to near control levels. Therefore, stimulation of &sigma;-1 and &sigma;-2 receptors by afobazole prevents A&beta;_25-35 activation of microglia and inhibits A&beta;_25-35-associated cytotoxicity.</p><p> Examining the molecular mechanisms involved in the increased neuronal survival demonstrates that ischemia or A&beta; results in an increased expression of the pro-apoptotic protein Bax and the death protease caspase-3, while at the same time decreasing expression of the anti-apoptotic protein, Bcl-2. However, unlike observations made with microglia, afobazole was unable to modulate this ischemia-induced expression, but was able to modulate A&beta;-induced expression of apoptotic proteins while still rescuing neurons from death. Additional experiments were carried out to understand this disparity between the failures of afobazole to prevent the up-regulation of pro-apoptotic genes while retaining the ability to mitigate neuronal death. Although the neurons were still alive they were in a senescent state and were unresponsive to depolarization by high K⁺. However, these findings are still positive due to the ability of afobazole to delay neuron death, thus minimalizing the toxic environment of the penumbra. </p><p> These comorbidities of ischemia and A&beta; toxicity may lead to potentiated responses and increase the risk for various vascular dementias. It was of particular interest to study how the convergence of ischemia, acidosis and A&beta; influence cellular activity and survival within core and penumbral regions. Application of A&beta; increased the [Ca²⁺]_i overload produced by concurrent ischemia + acidosis application in isolated cortical neurons. We found that the acid-sensing ion channels 1a (ASIC1a) are involved in the potentiation of [Ca²⁺]_i overload induced by A&beta;. Furthermore, afobazole (100 &micro;M) abolished A&beta; potentiation of ischemia + acidosis evoked [Ca²⁺]_i overload, which may represent a therapeutic strategy for mitigating injury produced by A&beta; and stroke.</p>

<p> Abuse of psychostimulants including cocaine and new synthetic formulations remains an international public health problem and economic burden. Addiction develops consequential to positive and negative drives that underlie &ldquo;getting&rdquo; and &ldquo;staying&rdquo; high. Dopamine (DA), arising from ventral tegmental area (VTA), projects to ventral striatal targets to encode reward signals and reward prediction. Mesolimbic DA is implicated in both the immediate rewarding effects of psychostimulants, and its hypoactivity underlies negative affect as drug levels decline. Accordingly, modulating inputs to midbrain DA possesses capacity to mediate positive/rewarding and negative/aversive effects of drugs. Hypocretin/orexin (hcrt/ox) is a family of excitatory hypothalamic peptides that projects widely throughout the central nervous system including to VTA DA cells, and hcrt/ox mediates brain reward function and motivation for self-administered drugs. Notably, the first-in-class hcrt/ox receptor antagonist (suvorexant) was approved for management of insomnia in the summer of 2014. Also within the past decade, the caudal division of VTA (termed &ldquo;tail of VTA&rdquo; and &ldquo;rostromedial tegmental nucleus [RMTg]&rdquo;) was detailed for its ability to negatively regulate VTA DA. Functionally, stimulation of the GABA-producing RMTg population encodes aversion and responds to aversive cues. Curiously, anatomy work depicts the hypothalamus as a principal input to the RMTg although the cellular phenotypes and functions of hypothalamic projections to RMTg have not been fully resolved. </p><p> Work in this thesis was designed to map hcrt/ox projections to VTA and RMTg in effort to understand functionally-relevant topographical arrangement. In preliminary assessments, we test for the first time the ability of suvorexant to modulate reward and reinforcement associated with psychostimulant use in rats. Additionally, we profile how self-administered cocaine and &ldquo;bath salt&rdquo; synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV) influence affective states in rats by measuring ultrasonic vocalizations (USVs) and comparing patterns of responding. Subsequently, we test the ability of suvorexant to influence MDPV-taking and affective changes that promote self-administration. Finally, we utilize direct-site pharmacology to assess the degree to which hcrt/ox transmission within VTA and RMTg contributes to motivated responding for and affective processing of self-administered cocaine across two doses. Specifically, we hypothesized that intra-VTA suvorexant would suppress drug-taking by reducing the rewarding value of self-administered cocaine, whereas intra-RMTg hcrt/ox peptide injection would suppress drug-taking by increasing aversive value of self-administered cocaine. </p><p> We observed that systemic suvorexant effectively reduces motivated cocaine-taking, and that this reduction relates in part to reductions in subjective reward of self-administered cocaine as interpreted by reductions in positively-valenced 50-kHz USVs. Retrograde tracing supports that hcrt/ox projects to both VTA and RMTg without discernible topographical arrangement. Target-site pharmacology finds that intra-VTA suvorexant has no appreciable effects on motivated cocaine-taking but tends to elevate 50-kHz USVs during the pre-drug &ldquo;anticipation&rdquo; time epoch in low-dose cocaine self-administering rats (0.375 mg/kg/inf). While intra-RMTg hcrt/ox pre-treatment sparsely affected USVs, 0.3 nmol/hemisphere hcrt/ox significantly enhanced cocaine-taking in low-dose cocaine self-administering rats, and, in high-dose (0.750 mg/kg/inf) cocaine self-administering rats, intra-RMTg hcrt/ox significantly suppressed responding when pre-treated with 1.0 and 3.0 nmol/hemisphere. </p><p> Collectively, studies within this thesis promote the use of hcrt/ox receptor antagonists as adjunct pharmacotherapy in managing psychostimulant use disorders, although the circuitries through which aberrant motivated behaviors are modulated are not entirely clear. Future work will need to be performed to understand how hcrt/ox transmits to neurochemically-defined cell populations residing within VTA and RMTg&mdash;these pathways are recruited for processing stimuli as &ldquo;rewarding&rdquo; and &ldquo;aversive&rdquo; which are critical contributors in the development of substance use disorders and other psychiatric disorders characterized by dysregulated reward processing.</p><p>

<p> Improvements in anticancer pharmacotherapy over the past 40 years have led to a steady increase in the number of cancer survivors worldwide. The clinical effectiveness of anticancer agents like the microtubule-stabilizing agent, paclitaxel, ultimately led to their adoption into standard of care regimens for most cancers. What makes these drugs so effective is how they bind to their respective targets to disrupt fundamental cellular processes. For example, by binding to &beta;-tubulin, paclitaxel induces polymerization and stabilization of cellular microtubules, leading to impairments in cellular functions like mitosis and intracellular transport. While an ingenious approach to kill cancer cells, microtubules are ubiquitous in all cell types. Consequently, paclitaxel has a burdensome side effect profile due to its effects on noncancerous cells. The most prevalent nonhematologic side effect is chemotherapy-induced peripheral neuropathy, which arises due to paclitaxel-induced damage to peripheral afferent sensory neurons. Cancer patients with peripheral neuropathy often develop debilitating neuropathic pain and numbness that diminish everyday quality of life. Symptoms intensify as treatment progresses and can persist for months and years beyond treatment. There is no effective treatment or prevention for chemotherapy-induced peripheral neuropathy. Instead, patients must dose de-escalate or discontinue life-saving chemotherapy, which subsequently worsens cancer prognosis. Thus, there is a compelling need to identify novel therapeutic options to prevent or treat chemotherapy-induced peripheral neuropathy so to improve both anticancer treatment and patient quality of life. The goal of this dissertation is to evaluate the neuroprotective efficacy of a first-in-class stimulator of nicotinamide phosphoribosyltransferase, P7C3-A20, against paclitaxel-induced peripheral neurotoxicity. Nicotinamide phosphoribosyltransferase is the rate-limiting enzyme in the salvage pathway for nicotinamide adenine dinucleotide, an indispensable redox biomolecule that drives energy production. We first developed an aggressive model of paclitaxel-induced peripheral neuropathy in adult male rats. Treatment with a near maximally-tolerated dose of paclitaxel produced significant, but recoverable weight loss and leukopenia. Paclitaxel-treated rats exhibited differentially altered nociceptive thresholds to noxious stimuli, including the development of persistent allodynia to mechanical and cold stimulation as well as transient hyposensitivity to heat stimulation. Toxicity associated with paclitaxel treatment required that 25% of the rats be removed from the study. Histological analysis determined that paclitaxel triggered degeneration of intraepidermal nerve fibers and up-regulated expression of activating transcription factor 3 in nuclei of neuron cell bodies residing in the lumbar dorsal root ganglia. Remarkably, daily treatment with P7C3-A20 prevented mechanical allodynia and heat hypoalgesia, and reduced the cold allodynia associated with paclitaxel treatment. P7C3-A20 also prevented intraepidermal nerve fiber degeneration and partially decreased activating transcription factor 3 expression in lumbar dorsal root ganglia neurons. A randomized, double-blind trial determined that P7C3-A20, and another analogue, P7C3-S321, dose-dependently decreased paclitaxel-induced neuropathic pain and intraepidermal nerve fiber loss. Furthermore, P7C3-A20 improved indices of general health and prevented premature death that normally arose because of overt toxicity caused by paclitaxel. P7C3-A20 displayed superior neuroprotective efficacy, while an inhibitor of poly(adenosine diphosphate-ribose) polymerase was completely ineffective. P7C3-A20 stimulated nicotinamide adenine dinucleotide production in vitro following induction of damage with either hydrogen peroxide or paclitaxel, but not under normal conditions. Additionally, P7C3-A20 in vivo stimulated nicotinamide adenine dinucleotide recovery in the hindpaw and sciatic nerve of rats treated with paclitaxel. FK866 blocked P7C3-A20-mediated nicotinamide adenine dinucleotide production in vitro and the neuroprotective effects on peripheral nociceptive neurons in vivo. Although treatment with either nicotinamide or a subthreshold dose of P7C3-A20 alone was ineffective, the combination produced neuroprotection against paclitaxel that was equivalent to that of a maximal dose of P7C3-A20. We also investigated the effects of P7C3-A20 on cancer cell proliferation and on the anticancer efficacy of paclitaxel. Only MDA-MB-231 breast cancer cells demonstrated a slight increase in proliferation by P7C3-A20, but enhanced growth of implanted MDA-MB-231 tumor xenografts was not observed. Furthermore, P7C3-A20 did not diminish the antiproliferative effects of paclitaxel, despite preventing the development of mechanical allodynia in the tumored mice. In conclusion, these studies discovered robust neuroprotective efficacy of P7C3-A20 against paclitaxel -induced peripheral neurotoxicity, likely through the enhancement of nicotinamide phosphoribosyltransferase-mediated recovery of nicotinamide adenine dinucleotide. Based on these results, clinical investigation of P7C3-A20 as a potential treatment option for chemotherapy-induced peripheral neuropathy may be warranted. </p><p>

High doses of 3,4-methylenedioxymethamphetamine (MDMA; "Ecstasy") are known to reduce levels of various serotonergic markers outside of the raphe nuclei. To test the hypothesis that these deficits reflect a degeneration of distal axons/terminals, we investigated the effects of an MDMA binge (10mg/kg x 4) on the relative protein and genetic expression of several serotonergic markers in rats, as well as the effects of this compound on the quantity of serotonergic terminals in these animals. In experiment I, we examined whether MDMA alters serotonin transporter (SERT) levels as determined by lysate binding and immunoblotting analyses. Both methods of analysis revealed MDMA-induced reductions in regional SERT content. Experiment II investigated MDMA-induced changes in terminal-specific levels of SERT and the vesicular monoamine transporter 2 (VMAT-2) in the hippocampus, a region with sparse dopaminergic innervation, after lesioning noradrenergic input with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). Animals were administered 100 mg/kg DSP-4 or saline 1 week prior to MDMA (or saline). As determined by immunoblotting of synaptosomal tissue, the DSP-4/MDMA group showed little change in hippocampal VMAT-2 protein expression compared to DSP-4/Saline controls, despite large reductions in SERT levels in all regions examined in the MDMA-treated animals. Experiment III examined whether MDMA alters genetic expression of SERT and VMAT-2. When compared to saline-treated controls, animals given MDMA showed a striking decrease in SERT gene expression (and a lesser effect on VMAT-2) in dorsal/median raphe as assessed by quantitative RT-PCR. Experiment IV(a) investigated the effects of MDMA on gene and protein expression of tryptophan hydroxylase (TPH) in the hippocampus. Levels of TPH protein were unchanged between treatment groups, while transcript levels were decreased 15-fold in the dorsal/median raphe. In experiment IV(b), flow cytometry was used to measure whether MDMA alters the quantity of serotonergic terminals in the hippocampus. MDMA-treated animals showed an increase in the number of serotonergic synaptosomes identified by co-labeling for synaptosome-associated protein of 25 kDa (SNAP-25) and TPH. These results demonstrate that MDMA causes substantial regulatory changes in the expression of serotonergic markers with no evidence for synaptic loss, questioning the need to invoke distal axotomy as an explanation of MDMA-related serotonergic deficits. Keywords: MDMA, neurotoxicity, neurodegeneration, serotonin transporter, vesicular monoamine transporter 2, tryptophan hydroxylase, serotonin, immunoblotting, gene expression, flow cytometry.

Most recreational users of 3,4-methylenedioxymethamphetamine (MDMA; "ecstasy") also take cannabis, in part because cannabis can reduce the dysphoric symptoms of the ecstasy come-down, such as agitation and insomnia. Although previous animal studies have explored the acute effects of co-administering MDMA and Δ9-tetrahydrocannabinol (THC), the major psychoactive ingredient in cannabis, research on chronic exposure to this drug combination is lacking. The four experiments included in the current dissertation were designed to provide a wide breadth of information on the physiological, behavioral, and neurochemical effects of intermittent MDMA administration combined with daily THC exposure using a dosing regimen designed to reflect a clinically-relevant pattern of human ecstasy and cannabis co-usage. Because ecstasy and cannabis abuse usually starts during human adolescence, drug treatment was administered from postnatal day (PD) 35 to 60 in order to target the period of rat development lasting from approximately mid-adolescence to early adulthood. In addition, the dosing regimen in rats was also chosen to best correlate to patterns of human ecstasy and marijuana use. Drug-treated rats received two subcutaneous (s.c.) injections of 10 mg/kg of (±) MDMA-HCL every fifth day and/or a single daily intraperitoneal (i.p.) injection of 5 mg/kg of THC every day. The twice every fifth day MDMA dosing regimen was designed to simulate the intermittent weekend usage of ecstasy at "rave" parties and the "boosting" behavior (taking additional doses of MDMA in the same session to maintain desired effects) that has been noted in human users. THC was administered daily to simulate heavy cannabis usage in humans, which has been defined to mean using cannabis more than seven times per week. While THC helped to alleviate MDMA-induced anxiety-like, impulsivity-like, and exploratory behavior, co-administration of MDMA and THC additively produced depressive-like behavior and deficits in spatial memory. Furthermore, our experiments provide physiological and neurochemical evidence that helps to explain the behavioral outcomes, specifically as THC failed to protect against MDMA-induced neurotoxicity in the hippocampus, the brain region that is responsible for processing of spatial memory information, in both the SERT binding assay and in SERT autoradiography. Finally, our data suggested that male rats are more susceptible to MDMA-induced damages than females—which has significant implications for assessing the risks of recreational ecstasy and cannabis co-usage in humans.

Previous work has shown that the synthesis and release of dopamine may, under certain conditions, be influenced by an increase in the availability of its amino acid precursor, tyrosine. To examine whether exogenously supplied tyrosine could potentiate the methylphenidate-induced release of dopamine, seven Sprague-Dawley rats were implanted with microdialysis probes aimed at the right nucleus accumbens. Samples were collected from awake, freely moving animals beginning 20-24 hr after surgery. A repeated measures design was used involving the continuous collection of 20-min samples for a 4-hr period, once a day for 3 consecutive days. On a given day, the animal was infused with either 30 uM methylphenidate, 100 uM tyrosine or 30 uM methylphenidate plus 100 uM tyrosine. Periods of infusion with the active compound(s) were preceded and followed by baseline conditions and treatments were counterbalanced to control for possible order effects. Methylphenidate plus tyrosine significantly increased extracellular levels of dopamine in comparison to drug alone. Moreover, this effect was long-lasting, persisting into the post-treatment sampling period and peaking 40 min after the peak induced by methylphenidate alone. Tyrosine alone induced a small but steady rise in extracellular dopamine that did not reach statistical significance until the time of the first post-treatment sample. These findings have implications for the treatment of attention deficit hyperactivity disorder.