SARM1 is an injury-induced nicotinamide adenine dinucleotide nucleosidase (NADase) that was previously shown to promote axonal degeneration in response to traumatic, toxic, and excitotoxic stressors. This raises the question of whether a SARM1-dependent program of axonal degeneration is central to a common pathway contributing to disease burden in neurological disorders. The degree to and mechanism by which SARM1 inactivation decreases the pathophysiology of such disorders is of interest to establish the rationale to pursue SARM1 as a therapeutic target. In this study, we compare the course and pathology of experimental autoimmune encephalomyelitis (EAE) in Sarm1-knockout (KO) mice and wild-type littermates to test the contribution of SARM1-dependent axonal degeneration specifically in the context of chronic, immune-mediated central nervous system (CNS) inflammation. The question of whether SARM1 loss in Sarm1-KO mice would inhibit, promote, or have a negligible impact on EAE-induced axonal degeneration and more broadly CNS inflammation was explored using a variety of analyses: quantification of clinical score in a chronic EAE model, CNS immune infiltrate profile, axon initial segment morphology in layer V cortical neurons, axonal transport disruption and transection in the lumbar spinal cord. Additionally, we have proposed a method for detecting SARM1 activation in situusing a novel SARM1-mCitrine bimolecular fluorescence complementation (BiFC) technique. Successful implementation of such a molecular tool would allow for a detailed, mechanistic approach to enhance our understanding of upstream intracellular signals that trigger SARM1 activation.

The word "Prion" refers to self-perpetuating protein aggregates that cause neurodegenerative diseases in mammals. It is a protein isoform that has undergone a conformational change which converts the normal form of the protein into the infectious form with the same amino acid sequence. Yeast [PSI+] prion is the prion isoform of Sup35 protein, a translation termination factor eRF3. It has been suggested that prion [PSI+] is controlled by the ensemble of chaperones with Hsp104 playing the major role. The previous work performed in the Chernoffs lab showed that the defective GET pathway caused by get led to the defect in [PSI+] curing by excess Hsp104. The GET pathway is a system responsible for transporting newly synthesized TA-protein to the ER membrane, and the components which have been proven to be involved in this pathway include: Get1, Get2, Get3, Get4, Get5 and Sgt2. In this study we describe the mechanism underlying the effect of the defective GET pathway on [PSI+]. We demonstrate that Sgt2, one of the components of GET pathway, interacts with Sup35 in both [PSI+] and [psi-] strains through its prion domain. Overproduction of Sgt2 and Hsp70-Ssa is triggered by the defective GET pathway and leads to the protection of [PSI+] aggregates from curing by excess Hsp104. We show that the direct interaction between Sgt2 and Hsp70-Ssa is not required for this protective effect.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 126-144).

Disputats. Rheinische Friedrick-Wilhelms-Universität, 2002. / Haves kun i elektronisk udg.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 97-114).

Traumatic brain and spinal cord injury continue to be substantial clinical problems with few available treatment strategies. Individuals who are at a greater risk for sustaining a central nervous system (CNS) injury, such as professional athletes and military personnel, may benefit from a prophylactic supplement that would intervene in the neurodegenerative pathways immediately following injury. The high demand for selenium within the central nervous system, as well as the synthesis of selenoproteins by neurons and astrocytes suggests a critical role of selenium within the brain and spinal cord. Studies were designed to test the efficacy of enriched dietary selenium status in providing neuroprotective benefits in rodent models of spinal cord and traumatic brain injury. Levels of selenium storage within the CNS are increased relative to the amount of selenium present in the diet, indicating that selenium compounds effectively cross the blood brain barrier. In a model of moderate severity spinal cord contusion injury, dietary selenium supplementation reduced the number of days until recovery of independent bladder function following injury. These benefits did not translate to improvements in locomotor function during open field testing or reduction in overall lesion volume in the injured animal groups. Examination of gene expression changes 24 hours after spinal cord injury revealed that dietary selenium enrichment increased expression of genes involved in DNA repair, mitochondrial respiration, and transcriptional regulation. By expanding the scope of these studies to include models of traumatic brain injury, these data show the importance of selenium in the cortex as well. In particular, when compared to diets deficient in selenium, higher levels of dietary selenium improve spatial memory performance and mitochondrial respiration. The results of this dietary study show modest improvements following both traumatic brain and spinal cord injury and suggest that while selenium enrichment may not have a profound effect on the secondary injury cascade immediately following injury, the presence of adequate dietary selenium is critical for mitochondrial respiration. Together the results of these studies suggest that dietary supplementation may play a subtle role in injury mechanisms within the CNS and warrant further investigation.

No description available.

Inflammation of the central nervous system (CNS) is important in many human diseases, and is regulated by a multitude of factors, including the cytokine interferon-gamma (IFNgamma). The importance of IFNgamma is highlighted in experimental autoimmune encephalomyelitis (EAE), an animal model of CNS inflammation. Mice lacking IFNgamma show exaggerated disease, with a different pattern of chemokine expression than the wild-type. We administered IFNgamma to the CNS using intrathecal injection of a replication-defective adenoviral vector to ask about direct actions of IFNgamma on chemokine expression without the confounding factors present during CNS inflammation. AdIFNgamma induced expression of CXCL10 and CCL5, two chemokines strikingly absent in Ifng-/- EAE. Chemokine expression was not associated with inflammation, though when an infectious stimulus was administered, an influx of immune cells to the CNS was seen. Using AdIFNgamma to restore IFNgamma to Ifng-/- mice with EAE had a disease-limiting effect. We used vectors encoding CXCL10 or CCL5, to replace these chemokines which are absent during Ifng-/- EAE, attempting to modulate the disease into a form resembling that of the wild-type. AdCCL5 treatment showed a mild reduction in EAE severity in the Ifng-/-, though AdCXCL10 treatment had no effect. A principal inducer of IFNgamma is interleukin-18 (IL 18), and IFNgamma induces IL18-binding protein (IL18bp) which inhibits IL18, establishing a negative feedback loop. We found that ILl8bp expression is upregulated in wild-type mice with EAE, but not in the Ifng-/-, suggesting that the exaggerated disease of the Ifng -/- may be due in part to unrestrained actions of ILI8. Treatment with a vector encoding IL18bp (AdIL18bp) significantly inhibited EAE, without restricting immune cell entry to the CNS. Cytokine expression was shifted away from a pattern favouring Th17 development. AdIL18bp treatment inhibited EAE in Ifng-/- mice, indicating that IFNgamma was not required for this activity. We used a vector encoding M3, a chemokine-binding protein derived from MHV-68, to reduce EAE severity, showing the first use of a viral chemokine-binding protein in EAE.

Attention-deficit/hyperactivity disorder (ADHD) is one of the most common disorders of childhood. It is theorized to be caused by catecholamine dysfunction in the striatum (Str) and frontal cortex (FC). The spontaneously hypertensive rat (SHR) has been used as a model for ADHD because of its attention deficits, impulsiveness, and hyperactivity. Prior studies of dopamine (DA) in the Str and FC have revealed conflicting results in the SHR compared to control, indicative of a need for a better understanding of DA dynamics in this model. In addition to the DA hypothesis, studies have begun implicating glutamate in the etiology of ADHD. Previous evaluations of the SHR model of ADHD found that the SHR have increased α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor activity and elevated calcium levels in the FC, suggesting that altered glutamatergic neurotransmission exists in the SHR. The first set of studies presented here suggest that increased surface expression of DA transporters may exist in the SHR model of ADHD, lowering basal DA levels. Second, we discovered that the glutamate system in the FC of the SHR model of ADHD is hyperfunctional, thus raising the possibility that targeting glutamate dysfunction in the FC could lead to the development of novel therapeutics for the treatment of ADHD. The third and fourth set of studies explored glutamate signaling in the awake rodent to fully understand glutamate neurotransmission as well as the effects of methylphenidate (MPH) on glutamate signaling in the prelimbic cortex, a region heavily implicated in ADHD. The SHR displayed similar phasic glutamate signaling compared to control; however, in the SHR but not the WKY control, chronic treatment with MPH lowered phasic glutamate amplitude. Additionally, intermediate treatment with MPH increased tonic glutamate in the SHR only, whereas chronic MPH treatment increased tonic levels in both the SHR and WKY compared to saline. Taken together, this body of work characterizes DA and glutamate signaling in the anesthetized SHR model of ADHD. Additionally, glutamate dynamics and the effects of the stimulant medication MPH were explored in the awake animal, providing evidence that glutamate is a likely target for future neuropharmacology for the treatment of ADHD.

No description available.