Graduation Year

2015

Document Type

Dissertation

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Edwin J. Weeber, Ph.D.

Committee Member

Dave Morgan, Ph.D.

Committee Member

Maria Gieron, Ph.D.

Committee Member

Mike Schoenberg, Ph.D.

Committee Member

Matthew During, Ph.D., D.Sc.

Keywords

Angelman, Ataxia, Autism, Imprinting, Minocycline, Tetracycline

Abstract

Angelman syndrome (AS) is a rare genetic disorder affecting 1/10,000 to 1/20,000 births. This disorder arises through the genetic disruption of the maternal UBE3A allele, which when coupled with epigenetic silencing of the paternal allele UBE3A allele, gives rise to an absence of UBE3A protein in the central nervous system. Clinical manifestations of the syndrome vary in severity and include poor motor function, deficits in language and severe intellectual impairments. Previous research in the Angelman syndrome mouse model revealed abnormalities in dendritic spine density and morphology of hippocampal pyramidal cells. As seen in humans with AS, mice show abnormal behavioral characteristics that include deficits in motor coordination and ability as well as hippocampal dependent associative fear conditioning. Physiologically, these animals exhibit severe deficits in long-term potentiation (LTP) when compared to wildtype littermates.

In an attempt to reduce the time from laboratory study to human translation, we began to search a small molecule library for established compounds with the ability to improve the behavioral and physiological defects normally associated with the AS mouse. One compound, minocycline, was found to normalize the density of dendritic spines in the hippocampus as well as recover the associative memory of AS mice. Moreover, a significant increase in LTP after theta-burst stimulation was also observed in area CA1 hippocampal pyramidal neurons of AS mice treated with minocycline when compared to saline vehicle control mice. These results suggest treatment with minocycline improves synaptic function and learning and memory of AS mice and may provide similar improvements to patients with Angelman syndrome.

Twenty-five participants ages 4-12 were enrolled in a clinical trial examining the safety and tolerability of minocycline in children with Angelman syndrome. Patients were evaluated at 3 time points, baseline (T1), after 8 weeks of treatment (T2) and again 8 weeks after the drug was discontinued (T3). Each evaluation was identical and included laboratory testing, EEG recording and neuropsychological examination. Results of the study showed minocycline was safe and well tolerated with only minor adverse effects reported. While no change was observed in EEG recordings, significant increases in the mean clinical global impressions severity scale score were observed after treatment with minocycline. Moreover, participants showed significant improvement in the raw scores of the communication and self-care domains of the Bayley Scale of Infant and Toddler Development, 3rd Edition. These results show for the first time, a therapeutic with the ability to improve the characteristic behaviors of Angelman syndrome.

Unfortunately, currently available neuropsychological measures were found to be insensitive to the behavioral phenotype of AS. The primary outcome measure, the Bayley Scale of Infant and Toddler Development, 3rd Edition relies on verbal communication and for the examinee to perform specific tasks on command. These testing methods are not compatible with this patient population and resulted in raw scores outside of 2 standard deviations from the mean. The inability of the participants to perform on these exams led us to develop a novel outcome measure; one that relies on observation rather than verbal communication. 9 children with AS and 7 healthy children were enrolled in an observational study in which 30 minutes of free play activity was video recorded. Using behavioral coding software, 3 independent raters quantified stereotypical AS behaviors as well as communication methods. Speech attempts were categorized into five difference types of vocalizations and revealed children with AS used less advance forms of vocalization consisting mostly of phonation control. Phonetic inventories show mostly front or back vowel usage suggesting little tongue movement occurs during speech production. These results suggest deficits in verbal ability may be related to a childhood apraxia of speech.

Impairments in balance and motor coordination have been associated with AS. In an attempt to measure gross motor function, spatiotemporal gait parameters were collected using an electronic walkway and gait analysis software. Results show the gait of children with AS most resembles that of patients with ataxia but without cognitive impairment. In an attempt to develop a single quantitative measure able to describe the severity of gait-related disability, statistical methods were used to create a gait index for patients with AS. The results of this study provides AS researchers with the tools necessary to accurately measure changes in behavior and gait during the clinical evaluation of potential therapeutics

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