Indiana University-Purdue University Indianapolis (IUPUI) / Naltrexone (NTX) is clinically efficacious at attenuating alcohol intake in non-abstinent alcoholics and, to a lesser extent, craving, independent of intake. While generally regarded as a non-selective opioid antagonist, NTX has been shown to have concentration dependent selectivity with lower doses (< 1.0 mg/kg) selective for the mu receptor and doses exceeding 1.0 mg/kg capable of binding to delta and kappa receptors. Like the mu system, the delta receptor system has also been implicated in mediating the rewarding effects of EtOH. In contrast, the role of the kappa system is less clear though recent evidence suggests that kappa activation may mediate EtOH aversion. Thus, the present study sought to evaluate the effects of both mu-selective and non-selective doses of naltrexone, the selective delta antagonist naltrindole (NTI), and the selective kappa agonist U50,488H (U50) in a paradigm that procedurally separates the motivation to seek versus consume a reinforcer to assess whether these receptor-selective drugs differentially affects these behaviors in both selected (alcohol-preferring P rats) and non-selected (Long Evans) rats, and whether these effects are specific to EtOH. Rats were trained to complete a single response requirement that resulted in access to either 2% sucrose or 10% EtOH for a 20-min drinking session. In three separate experiments, rats were injected (using a balanced design) with either vehicle or 1 of 3 doses of drug: U50 (IP; 2.5, 5.0, or 10.0mg/kg), NTI (IP; 2.5, 5.0, or 10.0 mg/kg), low NTX (SC; 0.1, 0.3, or 1.0 mg/kg) or high NTX (SC; 1.0, 3.0, or 10.0 mg/kg) on both consummatory and appetitive treatment days. Following either a 20 (U50), 15 (NTI), or 30 minute (NTX) pretreatment, rats were placed into an operant chamber and intake (consummatory) or lever responses (appetitive) and response latencies were recorded. The results showed that overall: U50, NTI, and NTX attenuated intake and responding for sucrose and EtOH. Independent of reinforcer, LE rats were more sensitive to U50’s effects on intake while P rats were more sensitive to the effects on seeking. P rats reinforced with EtOH were more sensitive to NTI’s effects on intake and seeking than all other rat groups. P rats were more sensitive overall to lower doses of NTX than LE rats and lower doses of NTX were more selective in attenuating EtOH responding vs. sucrose. Higher doses of NTX suppressed intake and responding across both lines and reinforcers. These results demonstrate that craving and intake may be differentially regulated by the kappa, delta, and mu opioid receptor systems as a function of “family history” and suggest that different mechanisms of the same (opioid) system may differentially affect craving and intake.

Indiana University-Purdue University Indianapolis (IUPUI) / Down syndrome (DS) is the result of trisomy of human chromosome 21 (Hsa 21) and occurs in approximately 1/700 live births. Mouse models of DS have been crucial in understanding the gene-phenotype relationships that underlie many DS anomalies. The Ts65Dn mouse model, trisomic for half of the Hsa 21 orthologs replicates many DS phenotypes including craniofacial alterations such as a small, dysmorphic mandible, midface, and maxilla. Other mouse models, such as the Ts1Rhr which contains a triplication of 33 Hsa 21 orthologs, have been used to better understand the genes responsible for craniofacial alterations. Our laboratory has demonstrated that the postnatal mandibular phenotype found in Ts65Dn mice can be traced back to an original neural crest cell (NCc) deficit in the developing first pharyngeal arch (PA1) at embryonic day 9.5 (E9.5). Furthermore, evidence suggested that both a proliferation deficit in the PA1 and a migration deficit in the NCC from the neural tube (NT) could be the mechanism behind this deficit. However, the molecular mechanisms behind these deficits remain to be elucidated. Due to the involvement of the Hsa 21 genes DYRK1A and RCAN1 in regulation of signaling pathways including NFATc (NFAT2), a transcription factor known to influence cellular proliferation and, later, bone development, we hypothesized that dysregulation of these genes could underlie the cellular deficit in the PA1. Furthermore, we hypothesized that targeting Dyrk1a by decreasing activity or available protein could ameliorate the established deficits. Through the use of RNA isolation techniques and cell culture systems of cell from the PA1 and NT of E9.5 Ts65Dn, Ts1Rhr, and control embryos, we established that trisomic genes Dyrk1a and Rcan1 ara dysregulated in both structures and that these two genes may interact. Furthermore, we established that a proliferation deficit in the Ts65Dn PA1 and a migration deficit in the Ts65Dn PA1 and NT exists at E9.5 and can be rescued to euploid levels in vitro with the addition of the Dyrk1a inhibitor, EGCG, a green tea polyphenol. We also confirmed that harmine, a more highly studied and specific Dyrk1a inhibitor, is capable of similar effects on proliferation of PA1 cell from E9.5 Ts65Dn embryos. Furthermore, when Ts65Dn pregnant mothers were treated with EGCG in vivo, the cellular deficit found in the developing E9.5 embryonic PA1 was rescued to near euploid volume and NCC number. Treatment with EGCG did not adversely impact litter size or embryonic development. Interestingly, euploid embryonic volume increased with EGCG treatment. Expression analysis of the E9.5 PA1 of EGCG treated Ts65Dn and control embryos revealed dysregulation of several genes involved in craniofacial and developmental pathways including Dyrk1a, Rcan1, Ets2 and members of the sonic hedgehog pathways. Our novel results provide a foundation for better understanding the molecular mechanisms of craniofacial development and may provide evidence-based therapeutic options to improve the quality of life for individuals with DS.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Kryštof Dobečka Supervisor: PharmDr. Tomáš Smutný, Ph.D. Advisor: prof. PharmDr. Petr Pávek, Ph.D. Title of diploma thesis: Experimental approaches for studying hepatic enzyme induction mediated by pregnane X receptor The thesis focuses on hepatic pregnane X receptor (PXR)-mediated induction of biotransformation enzymes. Emphasis is placed on experimental models and methods which are used for the assessment of enzyme induction. In addition to summarizing its well- established role as a xenobiotic-sensing receptor, PXR is also presented as a transcription factor with an important role in endogenous pathways. Furthermore, cell and animal models are evaluated in terms of expression and function of PXR and its target xenobiotic-metabolising enzymes. Primary human hepatocytes in 2D cultures are considered to be the gold standard of in vitro hepatic models. However, 3D technologies are expected to be increasingly used in the future. The use of animal models is limited due to pronounced interspecies differences in PXR activation. Thus, humanized models have been established to overcome these limitations. Next, this thesis comments screening methods for an assessment of interaction between PXR and...

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Huntington diseases(HD) is a fatal autosomal neurodegenerative genetic disorder, caused by a CAG trinucleotide repeat expansion in the huntingtin (HTT) gene, resulting in a toxic gain-of-function in the mutant huntingtin protein(mHTT). To date, there is no approved treatment to either cure or halt the course of HD. It has been established that wild-type(wt) HTT protein is essential for development and has a critical role for maintaining neuronal health, thus, a preferable approach for treatment is an mHTT specific lowering maintaining the wild type HTT expression. The achievement of an allele specific therapies depends on targetable allele variation, hence in this project, was the allele frequency in the Swedish population investigated and compared with both the total population and the European population selective. The data demonstrated that there is significant differences between populations. Additionally, the gene expression in five human fibroblast from HD patients with CAG repeats varying from 40 up to180, was analyzed as well as the gene variation across tissue , where the human HD brain and two animal brains; a nonhuman primate and a transgenic minipig, was compared. The result demonstrated that there is similarity in the gene expression between the two models and the human brain, where the highest expression was seen in the prefrontal cortex. The results from the gene expression analyze in the cell lines of fibroblast demonstrated that there is difference in expression between CAG repeats. Furthermore could it be seen that there were only two cell lines, HD180 and HD70, that was heterozygous for dACTT, rs362307, and for the SNP, rs7223906, in exon 67. There are various therapeutic approaches in the pipeline for HD as shown in this thesis, and hopefully a treatment for the disease in the not too distant future. / Huntingtons sjukdom är en dödlig autosomal neurodegenerativ genetisk avvikelse, orsakad av en specifik DNA-sekvens, CAG, upprepning i arvsanlaget som kodar för proteinet huntingtin (HTT). Det muterade HTT skadar nervcellerna i hjärnan och leder till att cellerna bryts ner. Idag finns ännu inga godkända terapier för att bota eller stoppa förloppet av Huntingtons sjukdom. Det har konstaterats att det friska HTT protein är betydelsefullt för utvecklingen och att den har en kritisk roll för att upprätthålla hjärnans nervceller. Därför skulle det vara fördelaktigt att som behandling sänka nivåerna av det muterade HTT och samtidigt behålla nivåerna av det friska HTT i en så kallad allel-specifik strategi. Utförandet av en allel-specifik behandling är beroende allel variationen mellan den friska genen och den muterade. Därför undersöktes allel-frekvensen i den svenska populationen och jämfördes mellan den europiska populationens frekvens. Resultatet från denna undersökning påvisade att det finns tydliga skillnader mellan förekomst av allel-variationer mellan olika populationer. Utöver detta undersöktes även genuttrycket i fem mänskliga friboblaster från patienter med Huntingtons med varierande CAG längd, från 40 repetitioner upp till 180 repetitioner, samt genvariationen mellan vävnader i hjärnan. För den sistnämnda användes data från en mänsklig hjärnan med Huntingtons sjukdom och två djurhjärnor; en ifrån en icke-mänsklig primat och ifrån en transgen minigris. Resultatet påvisade likheter mellan genuttrycket mellan den mänskliga hjärnan och djurhjärnorna, och det högsta uttrycket återfanns i prefrontala cortex. Resultat från fibroblastproverna visade att det finns skillnader i genuttryck mellan patienter som innehar olika längd på CAG-sekvensen. D, dessutom var det endast två cellinjerna, HD180 och HD70, som var heterozygoter för dACTT, rs 362307, var det enda somoch variationen i exon 67, rs7223906. Det finns varierande en multitud av tillvägagångssätt som anges i denna uppsats för att behandla utvecklandet av Huntingtons sjukdom i utveckling, , som anges i denna uppsats, och förhoppningsvis är finns ett botemedel inte i en inte alltför avlägsen framtid.

A ballistic pendulum impulse generator was used to impact networks in primary culture growing on microelectrode arrays. This approach has the advantage of imparting pure tangential acceleration insults (50 to 300 g) with simultaneous morphological and electrophysiological multichannel monitoring for days before and after the impact. Action potential (AP) production, network activity patterns, and cell electrode coupling of individual units using AP waveshape templates were quantified. Network adhesion was maintained after tangential impacts up to 300g with minimal loss of pre-selected active units. Time lapse phase contrast microscopy revealed stable nuclei pre-impact, but post impact nuclear rotation in 95% of observations (n= 30). All recording experiments (n=31) showed a repeatable two-phase spike production response profile: recovery to near reference in 1-2 hrs, followed by a slow activity decay to a stable, level plateau approximately 30-40% below reference. Phase 1 consisted of a complex two-step recovery: rapid activity increase to an average 23.6% (range: 11-34%) below reference, forming a level plateau lasting from 5 to 20 min, followed by a climb to within 20% of reference where a second plateau was established for 1 to 2 hrs. Cross correlation profiles showed changes in firing hierarchy after impact, and in spontaneous network oscillatory activity. Native oscillations were found in the Delta band (2 to 3 Hz), and decreased by approximately 20% after impact. Under network disinhibition with bicuculline, oscillations were slower (0.8-1Hz) and decreased 40% after impact. These data link network performance deficits with microscopically observable subcellular changes.

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In Parkinson's disease, aggregates of α-synuclein within Lewy bodies and Lewy neurites represent neuropathological hallmarks. However, the cellular and molecular mechanisms triggering oligomeric and fibrillary α-synuclein aggregation are not fully understood. Recent evidence indicates that oxidative stress induced by metal ions and post-translational modifications such as phosphorylation, ubiquitination, nitration, glycation, and SUMOylation affect α-synuclein conformation along with its aggregation propensity and neurotoxic profiles. In addition, proteolytic cleavage of α-synuclein by specific proteases results in the formation of a broad spectrum of fragments with consecutively altered and not fully understood physiological and/or pathological properties. In the present review, we summarize the current knowledge on proteolytical α-synuclein cleavage by neurosin, calpain-1, cathepsin D, and matrix metalloproteinase-3 in health and disease. We also shed light on the contribution of the same enzymes to proteolytical processing of pathogenic proteins in Alzheimer's disease and report potential cross-disease mechanisms of pathogenic protein aggregation.

Parkinson’s disease (PD) is a progressive neurodegenerative disorder that is neuropathologically characterized by degeneration of dopaminergic neurons of the substantia nigra (SN) and formation of Lewy bodies and Lewy neurites composed of aggregated α-synuclein. Proteolysis of α-synuclein by matrix metalloproteinases was shown to facilitate its aggregation and to affect cell viability. One of the proteolysed fragments, Gln79-α-synuclein, possesses a glutamine residue at its N-terminus. We argue that glutaminyl cyclase (QC) may catalyze the pyroglutamate (pGlu)79-α-synuclein formation and, thereby, contribute to enhanced aggregation and compromised degradation of α-synuclein in human synucleinopathies. Here, the kinetic characteristics of Gln79-α-synuclein conversion into the pGlu-form by QC are shown using enzymatic assays and mass spectrometry. Thioflavin T assays and electron microscopy demonstrated a decreased potential of pGlu79-α-synuclein to form fibrils. However, size exclusion chromatography and cell viability assays revealed an increased propensity of pGlu79- α-synuclein to form oligomeric aggregates with high neurotoxicity. In brains of wild-type mice, QC and α-synuclein were co-expressed by dopaminergic SN neurons. Using a specific antibody against the pGlu-modified neo-epitope of α-synuclein, pGlu79-α-synuclein aggregates were detected in association with QC in brains of two transgenic mouse lines with human α-synuclein overexpression. In human brain samples of PD and dementia with Lewy body subjects, pGlu79-α-synuclein was shown to be present in SN neurons, in a number of Lewy bodies and in dystrophic neurites. Importantly, there was a spatial co-occurrence of pGlu79-α-synuclein with the enzyme QC in the human SN complex and a defined association of QC with neuropathological structures. We conclude that QC catalyzes the formation of oligomer-prone pGlu79-α-synuclein in human synucleinopathies, which may—in analogy to pGlu-Aβ peptides in Alzheimer’s disease—act as a seed for pathogenic protein aggregation.

Indiana University-Purdue University Indianapolis (IUPUI) / Tendinopathy is a common degenerative pathology that is characterized by activity related pain, focal tendon tenderness, intratendinous imaging changes, and typically results in changes in the histological, mechanical, and molecular properties of the tendon. Tendinopathy is difficult to study in humans, which has contributed to limited knowledge of the pathology, and thus a lack of appropriate treatment options. However, most believe that the pathology is degenerative as a result of a combination of both extrinsic and intrinsic factors. In order to gain understanding of this pathology, animal models are required. Because each tendon is naturally exposed to different conditions, a universal model is not feasible; therefore, an appropriate animal model must be established for each tendon susceptible to degenerative changes. While acceptable models have been developed for several tendons, a reliable model for the Achilles tendon remains elusive. The purpose of this dissertation was to develop an animal model of Achilles tendinopathy by investigating the individual and combined effects of an intrinsic and extrinsic factor on the rodent Achilles tendon. Rats selectively bred for high capacity running and Sprague Dawley rats underwent uphill treadmill running (an extrinsic factor) to mechanically overload the Achilles tendon or served as cage controls. Collagenase (intrinsic factor) was injected into one Achilles tendon in each animal to intrinsically break down the tendon. There were no interactions between uphill running and collagenase injection, indicating that the influence of the two factors was independent. Uphill treadmill running alone failed to produce any pathological changes in the histological or mechanical characteristics of the Achilles tendon, but did modify molecular activity. Intratendinous collagenase injection had negative effects on the histological, mechanical, and molecular properties of the tendon. The results of this dissertation demonstrated that the combined introduction of uphill treadmill running and collagenase injection did not lead to degenerative changes consistent with human Achilles tendinopathy. Intratendiouns collagenase injection negatively influenced the tendon; however, these changes were generally transient and not influenced by mechanical overload. Future studies should consider combinations of other intrinsic and extrinsic factors in an effort to develop an animal model that replicates human Achilles tendinopathy.