Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid-β peptide (Aβ) in the brain and intraneuronal hyperphosphorylated tau. Microglia in the brain adopt M1 (pro-inflammatory) or M2 (anti-inflammatory) phenotypes similar to peripheral monocytes. M1 microglia negatively affect neurogenesis and have reduced phagocytic capabilities whereas M2 microglia can enhance neurogenesis and support phagocytosis. Cluster of Differentiation-200 (CD200) is an anti-inflammatory glycoprotein physiologically expressed on neurons and lymphocytes, and its receptors (CD200R1 and CD200R3) are expressed on glia. Both AD patients and mouse models of AD show an age-related or Aβ-induced reduction in neural CD200 that may contribute to M1-skewing of microglia in AD. We hypothesize that CD200 skews microglia to an M2 phenotype, and that genetic over-expression of CD200 in transgenic mice expressing the Swedish familial AD mutation of human β-amyloid precursor protein (APP mice) can restore neurogenesis and enhance Aβ clearance in the hippocampus. In this study, we constructed a tetracycline-controlled transactivator-inducible adeno-associated virus serotype 2/1 expressing full-length CD200 (AAV2/1-CD200) or green fluorescent protein (AAV2/1- GFP). These were bilaterally injected into the hippocampi at 6 months of age, and mice were sacrificed at 12 months of age. AAV2/1-GFP-injected APP mice showed a reduction in number of proliferating neural stem cells (NSCs) by 65.0% and differentiating NSCs by 70.5% in the dentate gyrus compared to wild-type controls. AAV2/1-CD200 restored these neurogenic deficits to those of wild-type mouse levels. AAV2/1-CD200 reduced diffuse Aβ plaques in the hippocampal region by 65.5% compared to AAV2/1-GFP-injected APP mice, but did not alter thioflavin-S-positive compact plaques as measured by protein and immunohistochemical assays. In vitro studies demonstrated that CD200-stimulated microglia co-cultured in transwells increased differentiation and complexity of neural stem cells. CD200 also directly enhanced Aβ phagocytosis by microglia. CD200 enhanced expression of the adaptor protein TYRO protein tyrosine kinase binding protein (TYROBP), suggesting this may be the mechanism by which CD200 enhances phagocytosis of Aβ. Overall, the data presented here indicate that CD200 is a plausible therapeutic agent in patients with AD to enhance neural differentiation and microglial-mediated clearance of Aβ.

Most cancers rely disproportionately on glycolysis for energy even in the presence of adequate oxygen supply, a condition known as "aerobic glycolysis", or the Warburg effect. Pharmacological reversal of the Warburg effect has been shown to cause selective apoptosis of tumor cells, presumably by stimulating mitochondrial respiratory chain activity and production of reactive oxygen species that, in turn, induce a caspase-mediated series of reactions leading to cell death. We reasoned that a similar effect on tumor cells might result from up-regulation of the E1α subunit gene (pda1) of the pyruvate dehydrogenase complex (PDC) that catalyzes the rate-limiting step in aerobic glucose oxidation and thus plays a major role in the control of oxidative phosphorylation. To test this postulate, we employed a self-complementary adeno-associated virus (scAAV)-based delivery and expression system for targeting pda1 to the mitochondria of primary cultures of human hepatoblastoma (HB) and hepatocellular carcinoma (HCC) cells. Serotypes 1-10 scAAV vectors that included enhanced green fluorescent (egfp) reporter gene driven by either cytomegalovirus (CMV) or chicken beta-actin (CBA) promoters were analyzed for transduction ability of HB (Huh-6) and HCC (Huh-7 and HepG2) cell lines and primary cultures of normal human hepatocytes. Serotype 3 scAAV-egfp (scAAV3-egfp) vector was the most efficient and transduced up to 90% of cells. We limited the transgene expression primarily to liver cancer cells by generating scAAV3 vectors that contained the human alpha-fetoprotein promoter (AFP)-driven reporter gene (scAAV3.AFP-egfp) and the potentially therapeutic gene scAAV3.AFP-pda1. Infection of Huh-6 cells by the scAAV3.AFP-pda1 vector increased protein expression of E1α, PDC catalytic activity, and late-stage apoptotic cell death. Apoptosis was also associated with increased protein expression of Bcl-X/S, an early marker of apoptosis, and release of cytochrome c into the cytosol of infected HB cells. These data indicate that molecular targeting of mitochondrial oxidative metabolism in liver cancer cells by AAV3-mediated delivery of pda1 holds promise as a novel and effective therapeutic approach for human hepatic tumors.

Parkinson’s disease is an incurable neurodegenerative disease characterized by motor deficits, owing to dopaminergic denervation in the nigrostriatal pathway. The abnormal formation of hallmark Lewy bodies underlies the disease process. The pre-synaptic protein alpha- synuclein (⍺-syn) has prion-like properties arising from its propensity to propagate, seed misfolding, and self-aggregate. Pathogenesis is postulated to arise in olfactory and enteric regions, exploiting connected neuronal pathways to ultimately propagate to the substantia nigra pars compacta. There is little known about the earliest stages of ⍺-syn aggregation and its prion-like propagation mechanisms. Bimolecular fluorescence complementation of ⍺-syn aggregates has allowed us to directly visualize aggregation in transgenic mice and mice transduced with an adeno-associated virus vector. Although our transgenic mice expressed BiSyn in a mosaic fashion that limited utility, we were successful in transducing neurons in the mouse striatum. This work has validated the AAV2/9-CMV-BiSyn approach as groundwork for future systematic studies.

Les Tauopathies sont un ensemble de maladies neurodégénératives caractérisées par l’agrégation de la protéine Tau dans les neurones, astrocytes et autres types cellulaires. Toutefois, les mécanismes sous-tendant la présence de Tau dans les astrocytes et les conséquences de Tau sur les astrocytes restent peu connus. L’objectif de ce projet a été d’étudier l’interaction entre les neurones porteurs de Tau soluble et/ou agrégée et les astrocytes avoisinants. Nous avons généré trois modèles de Tauopathie par transfert de gènes et avons caractérisé la pathologie Tau neuronale et astrocytaire par histologie et biologie moléculaire. Dans l’hippocampe, la surexpression de l’isoforme mutante hTAUP301L ou d’une forme pro-agrégeante hTAUProAggr, mais pas celle de la protéine sauvage hTAUWT, a entraîné la formation progressive d’agrégats aussi bien dans les neurones que dans les astrocytes. Nous avons montré que les inclusions astrocytaires de Tau étaient secondaires à une pathologie neuronale par différents schémas expérimentaux. En utilisant des vecteurs spécifiques de types cellulaires, nous avons également démontré le transfert bi-directionnel d’espèces de Tau entre neurones et astrocytes. Curieusement, nous avons observé une perte astrocytaire uniquement dans le subiculum du groupe hTAUWT qui ne présente pas d’inclusions astrocytaires de Tau. Nos données ont montré que, dans nos modèles, la tauopathie astrocytaire est secondaire à la présence de dégénérescences neurofibrillaires et ne résulte pas d’une agrégation liée à une surexpression réactionnelle de Tau endogène dans l’astrocyte. De plus, des amorces de Tau neuronales peuvent promouvoir l’agrégation de Tau astrocytaire et la Tau astrocytaire peut être transférée aux neurones. Par ailleurs, si les agrégats de Tau paraissent plutôt bénins pour les astrocytes, les conséquences fonctionnelles d’une telle astrogliopathie restent encore à évaluer. / Tauopathies are neurodegenerative diseases characterized by the aggregation of Tau protein in neurons, astrocytes and other cell types. However, the mechanisms leading to the presence of Tau aggregates in astrocytes and the consequences of Tau on astrocytes are poorly understood. The aim of this project was to study the relationship between neurons bearing soluble and/or aggregated Tau species and their neighboring astrocytes.We thus generated three gene transfer-based Tauopathy models and used immunohistological and molecular biology methods to characterize Tau pathology in neurons and astrocytes.In the hippocampus, overexpression of mutant hTAUP301L or that of a pro-aggregating variant hTAUProAggr, but not that of wild-type Tau hTAUWT, led to a gradual increase in the formation of aggregates not only in neurons but also in astrocytes. Using different experimental paradigms, we showed that astrocytic Tau was secondary to neuronal pathology. Using cell type-specific AAV- Tau vectors, we further demonstrated the bi-directional transfer of Tau species between neurons and astrocytes. Interestingly, we observed astrocyte loss in the subiculum only in the hTauWT group in the absence of any astrocytic Tau inclusions.Our data show that astroglial tauopathy is secondary to the presence of neurofibrillary tangles in our models and does not result from aggregation of overexpressed endogenous Tau in astrocytes. In addition, neuronal Tau seeds can promote the aggregation of astrocytic Tau and astrocytic Tau can be transferred to neurons. Furthermore, if Tau aggregates appear fairly innocuous for astrocytes, the functional consequences of such astroglial tauopathy still remain to be further assessed.

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Thesis advisor: Vassilios Bezzerides / The aim of this study was to utilize a massively parallel reporter assay (MPRA) to identify organ-specific microRNA (miRNA) target sequences to refine the timing and expression of transgene expression for gene therapy. We previously had developed a cardiac gene therapy for Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) using a systemically delivered adeno-associated virus (AAV9) vector. We hypothesized that incorporation of organ specific miRNA target sites into our vector construct could improve our therapy’s tissue specificity due to the ability of miRNAs to silence transgene expression. Initially, we attempted to incorporate mir-124 target sequences into our vector to detarget the brain. Although these initial attempts were unsuccessful, the study allowed us to develop a protocol to test the effectiveness of miRNA target sequences. Thereafter, we developed a method to screen thousands of putative miRNA target sequences simultaneously. In this study, target sequences of miRNAs specific to the heart, brain and liver were incorporated into a plasmid library. This plasmid library was subsequently made into AAV and injected into mice from a CPVT transgenic line. Total DNA and RNA was later extracted from the target organs, converted into genomic DNA (gDNA) and complementary DNA (cDNA) libraries respectively, and sent for amplicon sequencing. We analyzed the results using Comparative Microbiome Analysis 2.0 software (CoMA) and a custom python script to count the occurrence of each specified barcode per sample. In doing so, we showed that the miRNA suppression mechanism is not only effective but also organ specific. Furthermore, we developed a second script to create a combinatorial library from a set list of miRNA target sequences enabling us to efficiently test thousands of target sequence combinations at once. In doing so, we will be able to identify effective miRNA target sequence combinations to further improve gene therapy specificity. / Thesis (BS) — Boston College, 2021. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

The hypocretin/orexin (HCRT) system is associated with compulsive stimulant drug use, involving both HCRT-receptor 1 (-R1) and HCRT-receptor 2 (-R2). Few studies, however, have examined the role of HCRT-R2 or combined HCRT-R1/2 on compulsive methamphetamine (METH) taking behavior. In this study, we examined the effects of HCRT-R1, -R2, and -R1/2 antagonists on compulsive METH self-administration, as modeled by escalated intake in adult male Wistar rats allowed extended access to METH. Three cohorts of rats were allowed either short (1h; ShA; n=7-10/cohort) or long (6h; LgA; n=7-9/cohort) access to METH intravenous self-administration for 14 sessions (fixed ratio 1 schedule). Each cohort was then systemically administered a single- or dual-HCRT-R antagonist 30 min prior to METH self-administration testing: cohort 1, selective HCRT-R1 antagonist (RTIOX-276; RTI-R1; 0, 10, and 20 mg/kg); cohort 2, selective HCRT-R2 antagonist (JNJ-10397049; JNJ-R2; 0, 10, and 20 mg/kg); and cohort 3, dual HCRT-R1/2 antagonist (Suvorexant; SUV-R1/2; 0, 30, and 60 mg/kg). RTI-R1 elicited a dose-dependent reduction in METH intake in LgA, but not ShA, in the first hour. Administration of JNJ-R2 had no effect on METH intake in the first hour in neither ShA nor LgA rats, but reduced METH intake during the full 6 h session at the lowest dose. SUV-R1/2 administration had no effect on METH intake in ShA rats, but showed significant attenuation of METH-taking at the highest dose in both the first hour and full 6h session for LgA rats. Locomotor activity was significantly reduced following RTI-R1 and SUV-R1/2 in ShA rats only. To further explore the role that HCRT plays in METH dependence after a period of abstinence, we used a shRNA-encoding adeno-associated viral vector (AAV) to silence Hcrt in a separate cohort of previously-escalated METH-dependent rats. Following an initial escalation phase, and prior to a 3-week period of drug abstinence, rats were injected with either a control scramble-RNA AAV (AAV-Scram; n= 4) or a Hcrt-knockdown AAV (AAV-HCRT-KD; n= 5). AAV-Scram rats showed a significant decrease in METH self-administration post-abstinence, and a subsequent increase in METH-taking following a re-escalation period. In contrast, AAV-HCRT-KD rats showed a significant attenuation of METH self-administration following the re-escalation period. Combined, these results suggest HCRT neurotransmission at both HCRT-R1 and -R2 may contribute to compulsive METH-taking behavior.

Gene therapy is advancing rapidly, with Recombinant Adeno-associated virus (rAAV) being investigated for potential use in treating cancer and neurological disorders. Plasmid DNA transfection and viral infection are standard methods for producing large-scale rAAV vectors. However, improving yield production requires careful monitoring and control of process state variables, which can be expensive and time-consuming. This thesis proposes a model predictive control (MPC) model that can efficiently monitor, predict, and optimize the final product by controlling state variables like DOT and temperature. The model relies on an unstructured mechanistic kinetic model designed explicitly based on rAAV upstream production. Monitoring viral vector production based on substrate or biomass concentration enhances bioprocess production efficiency. However, other state variables like dissolved oxygen (DO), pH, and temperature should also be considered. The objective of this thesis is to enhance cell growth in bioreactors by regulating dissolved oxygen and temperature levels using a Model Predictive Control (MPC) system. This model can be employed in different processes to enhance cell growth and examine the impact of control measures. The goal is to achieve a high cell density, increase productivity, and lower costs in a shorter duration. Simulink, a software tool developed by MATLAB, seamlessly integrates Ordinary Differential Equations (ODEs) to optimize bioprocesses in bioreactors. The Model Predictive Control (MPC) controller expertly regulates Dissolved Oxygen Tension (DOT) and temperature, thereby increasing cell growth concentrations. This sophisticated controller efficiently manages multiple variables simultaneously and exceeds the Proportional Integral Derivative (PID) controller. The model is straightforward to comprehend and promptly responds to anomaly data. To evaluate the suggested resolution, we conducted tests on both PID and MPC controllers by introducing measurement noise to the DOT. Our analysis indicated that MPC demonstrated superior performance based on the ISE (Integral of Squared Error), IAE (Integral of Absolute Error), and ITAE (Integral of Time-weighted Absolute Error), all of which were substantially higher for the PID controller. Regardless of changing conditions, MPC adeptly tracks the setpoint and optimizes the variable to enhance production efficiency.

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No description available.