The Autism Spectrum Disorders (ASDs) are neurodevelopmental conditions that are diagnosed based on a child’s impaired social and communication development, restricted interests, and repetitive and behaviors. ASDs impact individuals for a lifetime and result in needs across a variety of domains. Previous research suggests that genetic factors contribute significantly to the development of ASD, with a clinical diagnostic yield as high as 17-21%. While research has been done to characterize the genetic abnormalities associated with autism in a pediatric population, no study has yet been conducted on genetic abnormalities in adults with ASDs because the majority of ASD adults have either never had genetic testing or the testing was done long ago and did not benefit from the increased yield of modern genetic testing. This study describes the prevalence of clinical genetic test abnormalities in adults with ASDs as well as the physical and demographic features of individuals with these genetic abnormalities compared to those without abnormalities. We performed both retrospective and prospective medical chart analysis on adults seen in the UW Adult Genetics Clinic and abstracted data from the charts such as age, sex, ASD diagnostic subtype, genetic test results, brain physiology test results, neuropsychological test results, physical exam features, medical history, medications, family history, developmental history, and social demographics. We then used statistical analyses to compare the clinical and demographic features between individuals with and without genetic abnormalities. This study can help to provide information to guide clinicians in making decisions regarding genetic testing for adults with ASDs. Identification of genetic test abnormalities can also inform medical treatment and health screening for individuals with ASDs, and also influence family planning for these individuals and their siblings.

Alzheimer's disease (AD) is the leading cause of dementia, and its precise cause is still unknown. Mutations in the two Presenilin (PS) genes, Presenilin 1 and Presenilin 2 (PS2), cause AD. Understanding how AD mutations impact PS function may help us understand AD pathogenesis. One function of PS involves calcium (Ca2+) transfer between the endoplasmic reticulum (ER) and mitochondria. Presenilin 2 (PS2) and mutant PS2 (mtPS2) in particular have been shown to increase this transfer, possibly causing mitochondrial Ca2+ overload. This could lead to metabolic dysfunction, which has been suggested as a key pathogenic mechanism in AD. Establishing a link between PS mutations and mitochondrial dysfunction would support this hypothesis. One symptom of mitochondrial dysfunction is changes in mitochondrial fragmentation and localization. "Fragmented," rather than interconnected, "branchlike" mitochondrial networks are associated with increased ER-mitochondria Ca2+-transfer and apoptosis. Furthermore, while healthy mitochondria move towards energy-demanding neuronal processes, mitochondria with reduced ATP production stay near the nucleus. To determine the effects of PS2 and mtPS2 on mitochondrial fragmentation and localization, wild-type (wt) and mtPS2 (N141I) transgenic mouse cortical neurons were infected with a lentiviral vector expressing mitochondrial targeted red fluorescent protein (DsRed). Fixed cultures were imaged using confocal microscopy and analyzed using Huygens software. Currently, we have plated, infected, and analyzed sample preparations for mitochondrial length, sphericity, and other characteristics associated with fragmentation, as well as mitochondrial volume as a function of distance from the soma. Using this paradigm, we will also challenge cultured wt and mtPS2 neurons with camptothecin and staurosporine, inducers of mitochondrial fission and fusion, respectively. If mtPS2 induces mitochondrial dysfunction, mtPS2-expressing neurons should exhibit more mitochondrial fragmentation and mitochondria localized nearer to the soma. This would provide evidence for mitochondrial dysfunction in AD, supporting further research into mitochondrial changes in AD and a better understanding of AD’s etiology.

Spinocerebellar ataxia type 7 (SCA7) belongs to a family of neurodegenerative diseases characterized by degeneration of Purkinje cell neurons (PC) within the cerebellum, which leads to motor dysfunction. The disease results from a CAG tract expansion mutation of the ataxin-7 gene. This type of expansion is common within the SCA family along with other neurological disorders like Huntington’s disease. Recent research has provided evidence for a non-cell autonomous degeneration of PCs, which means that other cell types cause the PCs to degenerate. One population of cells that may be involved is the inferior olive (IO). The IO, which is known to degenerate in humans, is made up of neurons and is located within the brainstem. The axons of the IO, known as climbing fibers, extend to the cerebellum and synapse with PCs. These climbing fibers transport neurotrophic factors to the PCs. We hypothesize that loss of neurotrophic factors due to the degeneration of climbing fibers may contribute to the disease state of SCA7.
To support this belief, we seek to characterize the climbing fibers by immunohistochemistry. I have stained cerebellar sections of non-transgenic and SCA7 positive mice for VGLUT2, a marker of climbing fiber terminals. In order to understand whether a change in climbing fibers occurs before or after presentation of the disease I have also stained sections from multiple time points. To analyze the differences between the groups I have focused on measuring the amount of VGLUT2 positive puncta in regions proximal and distal to the Purkinje cell soma. Preliminary results indicate that a change in distribution of puncta occurs in mice in the late stages of disease.
 

Ischemic preconditioning (IPC) is a robust neuroprotective phenomenon in which a brief period of ischemia leads to tolerance to subsequent ischemic attacks. Characterizing the mechanisms of IPC is a major focus in stroke basic research. Prior IPC investigations focused primarily on gray matter – infarct volume reduction in rodents following a surgical stroke model, middle cerebral artery occlusion (MCAO). IPC reduces infarct volume by greater than 50%. This effect has been shown to be dependent on immune cell receptor Toll-like receptor 4 (TLR4). However, human brains have a far greater percentage of WM than rodents and WM injury is a major component in human stroke. Preliminary studies from our laboratory have shown that IPC in vivo can protect optic nerve (a pure WM structure) from subsequent prolonged ischemic injury in vitro. This effect is also TLR4-dependent. We hypothesize here that specific IPC-induced changes in optic nerve gene and protein expression are responsible for WM protection. Based on prior work from our laboratory, we hypothesize that two sets of genes in particular: (i) AMPA class glutamate receptors and (ii) Interferon stimulated genes play a role in IPC-induced WM protection. To test this hypothesis, we are using our standard model of WM IPC (15 minute common carotid artery ligation) to generate preconditioned (ipsilateral) and control (contralateral) optic nerves. Three days later, the optic nerves are dissected out and homogenized. We then isolate either total RNA or total protein. We then use targeted PCR arrays and immune- (Western) blotting techniques to quantify expression of the AMPA glutamate receptors and interferon-stimulated genes. We anticipate that IPC will induce selective down regulation of the former and broad-based, TLR4-dependent up-regulation of the latter. Based on our results we hope to identify novel molecular pathways for therapeutic intervention in WM injury following either stroke or ischemic optic neuropathy.

Why do smokers with epilepsy suffer from cooking and not cigatte-induced burns? 21% of our patients smoke, yet cigarette-induced burns are uncommon. We studied whether nicotine might change seizure thresholds in patients undergoing video EEG telemetry (VEEG). We tested NP effects on duration of stay, rate of seizure aquisition and number of seizures collected in patients undergoing VEEG. 762 consecutive patients charts were screened for proven epilepsy (EPI) and NP use during their VEEG hospitalization (n=33). Randomized controls, EPI patients without NP (n=33), were also selected. Outcome variables included duration of stay number of seizures collected on day 2, and total number of seizures. T-test comparisons were done. Patients with psychogenic non-epileptic events (PNES) with NP (n=52) were compared to a randomized NP-free PNES group (n=52). The duration of VEEG in EPI patients with NP was 5.42 days (s.d=1.66) vs. 4.33 (s.d=1.74) days in patients without NP (p= 0.01). EPI patients with NP had a total of 3.88 (s.d.=8.82) seizures, while those without had 7.58 (s.d.=1.44, p=0.21). On day 2, 0.94 (s.d.=1.44) events were captured in patients with NP, and 2.18 (s.d.=5.38) events in patients without (p=0.21). PNES patients with NP spent 3.73 days for VEEG, those without 3.98 (p=0.46). No relationships were seen in PNES patients for day 2 or for total events captured. No differences in provider-specific stay durations were seen. In epilepsy subgroups, partial-onset EPI patients without NP stayed 4.41 days, those with NP 5.56 days (p < 0.05). Generalized EPI patients showed no differences. In conclusion, NP use during VEEG may prolong length of hospital stay in EPI patients, but not for those with PNES, this effect may be more notable in patients with partial onset epilepsy.

Toll-like receptor 4 (TLR4) is an important modulator in the pathophysiology of acute ischemic stroke (AIS).  Specific Single Nucleotide Polymorphisms (SNPs) in TLR4 alter the immune cell responsiveness to bacterial lipopolysaccharide and various endogenous activators, such as heat shock protein (HSP), tumor necrosis factor (TNF) and nitric oxide (NO).  This project utilized data generated from patients presenting with AIS to University of Washington Medical Center (UWMC) and Harborview Medical Center (HMC) between September 2005 and May 2009.  DNA was first extracted from patient serum samples collected at regular intervals following AIS.  The DNA was then analyzed using Direct-PCR for the presence of various TLR4 SNPs, such as 1063 A/G (Asp299Gly) and 1363 C/T (Thr399Ile).  In an analysis adjusted for both initial stroke severity and age, the presence of either TLR4 SNP was associated with an increased rate of infection and a decreased likelihood of favorable outcome at 3 months following stroke.  In addition to pointing to a functionally and clinically significant genetic variation in TLR4 SNPs for AIS patients, these results suggest a direct connection between TLR4 function and stroke pathophysiology.  

Spinocerebellar ataxia 7 (SCA7) is a heritable disease caused by a mutation in the gene for the protein ataxin-7. SCA7 results in progressive neurodegeneration in the cerebellum, brain stem, and retina, which causes patients to lose motor control and vision. The disease disrupts the intricate circuitry of the cerebellum, impacting cells such as Purkinje cell (PC) neurons and the Bergmann glia (BG) that support them. BG and PC function is interdependent such that dysfunction in one cell type causes damage in the other. I set out to characterize how BG function is altered in SCA7 by examining brain tissue from 40 week old mice with or without the ataxin-7 mutation. The tissue was analyzed for various markers of BG function and morphology. I addressed whether mutant ataxin-7 expression in BG leads to changes in their morphology and identity as radial glia using markers for radial glia such as Sox9. I also used the marker for GLAST, a glutamate transporter, to study changes in their ability to support proper PC neurotransmission through glutamate uptake from the synapses. BG distribution in the cerebellum was studied using the markers Calbindin and S100β to mark PC and BG respectively. The APC/β-catenin intracellular signaling pathway in BG was studied using markers for APC and β-catenin, as this pathway may impact BG’s ability to maintain its structure and thus, its function. The results of this study may suggest which BG functions are affected by SCA7. These functional changes may then provide insight into possible mechanisms for cerebellar neurodegeneration in SCA7.