Platform II: (#9 – 16) 9. Developmental Mechanisms Contribute to Altered Ganglion Cell Activity in Mice with Retinal Degeneration Stasheff SF (Iowa City, IA) Objective: We report changes in retinal ganglion cell (RGC) physiology during hereditary retinal degeneration that are modulated by developmental changes in neural circuits. Methods: We compared spontaneous and light-evoked activity in two closely related mouse models of retinitis pigmentosa. In the rd1 mouse, degeneration proceeds rapidly, overlapping a peak of maturation in retinal circuits; in the rd10 mouse, photoreceptor loss begins after this “critical period.” Extracellular action potentials were recorded simultaneously from 30-90 RGCs in the in vitro retina of wild type (wt), rd1 or rd10 mice, using a multielectrode array. Spontaneous activity and responses to light flashes were monitored at various developmental ages. Results: In rd1 mice, spontaneous firing increases by postnatal day 14-15 (P14-15) and reaches ⬃10 times normal by P28, when all responses to light have disappeared. In rd10 retinas, spontaneous hyperactivity also has emerged by P1415, yet light-evoked responses remain vigorous through P18, and do not disappear until after P48. Selective pharmacologic blockade reveals that a subset of RGC maintains spontaneous hyperactivity independent of synaptic transmission in both strains. Blockade of ON pathway signals in mature rd10 retinas reveals “waves” of correlated activity such as during normal development. This suggests that the physiologic infrastructure generating these waves persists longer in rd10 mice. Conclusions: Despite similar genetic defects in rd1 and rd10 mice, developmental mechanisms active during an early “critical period” play distinct roles in different aspects of neural circuit reorganization during retinal degeneration. Future treatments may preserve or restore normal visual function by exploiting these mechanisms. 10. Merkel Cells are Essential for Light Touch Responses Maricich SM (Cleveland, OH), Wellnitz SA, Nelson AM, Lumpkin EA, Zoghbi HY (Houston, TX) Objective: A key function of the peripheral nervous system is to detect different types of somatosensory stimuli including pain, temperature and touch. Different qualities of touch are encoded by discrete touch receptors, each with their own distinctive coding properties. One form of light touch that is particularly important for tactile discrimination of shapes and textures is mediated by nerve fibers associated with Merkel cells (MCs), an enigmatic skin cell population first described in 1875. Merkel receptors are found in fingertips and other touch-sensitive skin areas, and exhibit a characteristic response to light skin indentation. The role that MCs play in light touch responses has been the center of controversy for over 125 years. Our goal was to definitively determine whether MCs are needed for light touch responses. Methods: We used a genetic Cre-loxP system to selectively and completely ablate MCs from the body skin and footpads of mice. We then examined peripheral nerve responses to skin indentation using ex vivo skin/nerve preparations from control and conditional knockout animals. S100 Annals of Neurology Vol 66 (suppl 13) 2009 Results: We demonstrate that Atoh1, a bHLH transcription factor, is necessary for the production of MCs. Loss of the MC population does not affect the structure of the Merkel receptor or the presence of innervation. However, the characteristic Merkel receptor response to skin indentation is lost in MC-deficient mice. Conclusions: MCs are required for the proper encoding of Merkel receptor responses, suggesting that these cells form an indispensible part of the somatosensory system. 11. Congenital LCM Virus Infection can Induce Cerebellar Hypoplasia in Humans: an Animal Model Reveals its Mechanisms. Bonthius DJ, Rabe G, Klein de Licona HW, Ordoubadian A, Karacay B (Iowa City, IA) Objective: Congenital infection with lymphocytic choriomeningitis virus (LCMV) severely damages the fetal brain. We sought to determine whether congenital LCMV infection can induce cerebellar hypoplasia in humans and utilized a rat model to study its mechanisms. Methods: 21 human infants with serologically-proven congenital LCMV infection underwent MRI scanning of the brain and were clinically followed for up to 10 years. As an animal model of congenital infection, neonatal rats were inoculated with LCMV. The rats were sacrificed at a series of ages to evaluate viral load, cellular targets of infection, time course of lymphocytic infiltration, and patterns of cerebellar pathology. To determine the role of T-lymphocytes in the pathology, wild type rats were compared with congenitally athymic (nude) rats. Results: Five congenitally infected infants had cerebellar hypoplasia, either as an isolated finding or in combination with other pathology. All of these infants presented with jitteriness in the newborn period and had long-term ataxia and cognitive deficits. In the rats, LCMV specifically and heavily infected cerebellar glial cells, granule neurons, and Purkinje cells. Within the cerebellum, infected wild type rats had lobular hypoplasia, porencephalic cysts, neuronal migration disturbances, corrupted Bergman glia structure, and a robust infiltration of lymphocytes. In contrast, nude rats had only cerebellar hypoplasia and lacked the other forms of pathology. Conclusions: Congenital LCMV infection can induce cerebellar hypoplasia in humans, where it presents with neonatal jitteriness and induces long-term ataxia and cognitive deficits. The destructive lesions and neuronal migration disturbances are T-cell-mediated, while the hypoplasia is virusmediated. 12. Effect of Developmental Hypoxia on CNS Connectivity Bonkowsky JB, Kogelschatz C, Pavia K, Fujimoto E, Chien CB (Salt Lake City, UT) Objectives: Hypoxic injury to the developing CNS is associated with chronic neurodevelopmental sequelae including cerebral palsy, seizures, learning impairments, and behavioral problems. The pathophysiology of these multiple outcomes is poorly understood. Recent evidence suggests that hypoxic insults can cause changes in CNS connectivity. Our goal was to develop a vertebrate model for analysis of effects of hypoxia on the development of CNS connectivity. Methods: We developed transgenic zebrafish lines to express GFP or GFP-caax (targeted to axons) in defined sets of neurons. Hypoxia was induced using a sealed chamber at O2 levels of 2% for varying lengths of time, at different embryonic stages. Normoxic conditions were otherwise maintained. Effects on pathfinding, CNS patterning, neuron numbers and type, apoptosis, and proliferation were assayed using the transgenic zebrafish, and in situ and antibody markers. Results: We generated several transgenic zebrafish lines to visualize pathfinding of distinct neuron subsets in the CNS including telencephalic, retinal, and diencephalic neurons. Disruptions in axon tracts were found following hypoxia exposure, compared to normoxic embryos. Anterior commissure formation, and extension of longitudinal pathways, were particularly disrupted. Hypoxia during stages of pioneer axon extension, but not neuron patterning, proliferation, or synaptogenesis, had the most profound effects on pathfinding. Conclusions: We have developed a zebrafish model to study alterations in neuronal connectivity following developmental hypoxia insults. Discrete CNS pathfinding errors were caused by hypoxia, particularly at stages of early axon extension. These findings imply the disruption of specific genetic pathways. 13. Developmental Analysis of Neuronal Dendrite Structure in the MeCP2 A140V “knock-in” Mutant Mouse Model of Rett/XLMR Jentarra GM, Olfers SS, Rice SG, Narayanan V (Phoenix, AZ) Rett syndrome (RTT) is the most common cause of mental retardation in girls and is caused by mutation of the MeCP2 gene. We generated a mouse model of RTT that differs from other models in two key features. First, it reproduces a known human mutation (A140V). Second, the mice appear to have relatively normal life spans. Other mouse models either do not reproduce human mutations [the exon 4 and/or 3 knock-out (Bird/Jaenisch), MeCP2 over-expression via a transgene, and C-terminal truncating 308/y (Zoghbi)] or have very short life spans [the knock-in R168X (Coyle)]. Results: Male A140V mice survive past 8 months and do not display obvious neurological symptoms or seizures, allowing studies to be performed over longer developmental time periods. Male A140V mice exhibit deficits in motor coordination by Rotarod and open field testing and there are indications of deficits in spatial learning and memory by Morris water maze. Histological studies show an increase in packing density of neurons in the brain. GolgiCox studies in 4 month old mice show quantitative abnormalities of dendrite branching in the cortex by Scholl analysis. The apical and basal dendrites of layer II-III pyramidal neurons in mutant animals demonstrate a decreased branching complexity in comparison to WT controls. Quantitative Scholl analysis of dendrite complexity at additional ages will be presented. Conclusions: This new mouse model for Rett/XLMR displays abnormalities allowing us to investigate the function of MeCP2 in dendrite development as well as serving as a preclinical model for the testing of drug therapies. 14. Homozygous Loss-of-Function Mutations in the Dopamine Transporter (DAT), SLC6A3, are Associated With Infantile Parkinsonism-Dystonia (IPD) Kurian MA (Birmingham, UK), Zhen J (NY, USA), Cheng S-Y (NY, USA), Li Y (NY, USA), Mordekar SR (Sheffield, UK), Jardine P (Bristol, UK), Morgan NV (Birmingham, UK), Meyer E (Birmingham, UK), Tee L (Birmingham, UK), Pasha S (Birmingham, UK), Wassmer E (Birmingham UK), Assmann B (Dusseldorf, Germany), Heales SJR (London,UK), Gissen P (Birmingham, UK), Reith MEA (NY, USA) , Maher ER (Birmingham, UK) Objective: IPD is a severe parkinsonian syndrome of infancy with unique biochemical features. In order to define this condition, we undertook clinical and molecular genetic investigations. Methods: Children affected with IPD were clinically phenotyped. Autozygosity mapping studies were undertaken in consanguineous families to identify the disease-causing gene. To determine the effects of the identified mutations, functional analysis of mutant proteins was performed. Results: Clinical Presentation: Parkinsonian symptoms heralded disease onset in early infancy, followed rapidly by the development of pyramidal tract features and dystonia. There was no evidence of psychiatric or behavioural disorders. CSF neurotransmitter studies revealed markedly elevated concentrations of HVA (with normal 5-HIAA levels). Molecular Genetic Investigation: Genetic linkage studies mapped a novel locus to chromosome 5p15.3. Homozygous missense SLC6A3 mutations (p.Leu368Gln and p.Pro395Leu) were identified in two families. Functional studies demonstrated that both mutations severely reduced levels of mature (85 kDa) dopamine transporter while differentially impacting dopamine’s apparent binding affinity. SLC6A3 analysis in other IPD cases has identified further mutations (deletion and splice site mutations) Conclusions: Infantile parkinsonism is usually associated with reduced dopamine metabolites and neurotransmitter enzyme defects. However, we describe the first parkinsonian disorder linked to an inherited abnormality of dopamine transport and homeostasis. Although genetic variants in SLC6A3 have been linked to numerous neuropsychiatric disorders (particularly ADHD), our study suggests that loss-offunction SLC6A3 mutations are actually associated with an early onset movement disorder. SLC6A3 may thus be a candidate susceptibility gene for other movement disorders associated with parkinsonian or dystonic features. 15. Mitochondrial Dysfunction and Morphological Abnormalities in Cellular Models of Spinal Muscular Atrophy Acsadi G, Li X, Brenner CA, Parker GC, Silver RB (Detroit, MI) Objective: To investigate mitochondrial abnormalities associated with survival motor neuron (SMN) loss. A loss of SMN protein is responsible for lower motor neuron (LMN) death in spinal muscular atrophy (SMA). SMN protein is a component of RNA processing complexes and axonal growth cones. It is unclear which processes participate in SMA-related LMN degeneration. Previously, we identified energy failure and increased mitochondrial free radical production in SMN depleted cells. Methods: Two live cell types (neuronal NSC34 cell clones with decreased SMN expression by stable RNA interference, SMA patient and control fibroblasts) were observed with and without fluorescent markers at high resolution using a Zeiss AxioObserver epifluorescence microscope and AxioVision software. Results: Mitochondria in human fibroblasts appear as long Program and Abstracts, Child Neurology Society S101 rod-like organelles distributed along cytoskeletal tracks. In contrast, mitochondria from type 1 SMA patient fibroblasts appear as more numerous, short, ellipsoid bodies clustered substantially in the perinuclear region. NSC34 cells with normal SMN levels grow faster than SMN hypomorphic clones. Mitochondria appear as abnormal “caps” adjacent to nuclei of the SMN hypomorphic clones, and are more plentiful than in controls. Neurite outgrowth is more robust in control NSC34 cells where mitochondria show greater distribution than in the SMN hypomorphic clones, where mitochondrial clustering is associated with neuritic swelling. Conclusions: Abnormal distribution and proliferation of smaller mitochondria in both SMA patient fibroblasts and neuronal cells with low SMN levels was observed. These observations parallel and further characterize our previously reported observation of mitochondrial dysfunction in cellular models of SMA. 16. Phase II Trial of Pirfenidone for Progressive Plexiform Neurofibromas in Patients with Neurofibromatosis Type 1 Packer RJ (Washington, DC), Babovic-Vuksanovic D (Rochester, MN), Dombi E (Bethesda, MD), Goldman S (Chicago, IL), Goodspeed W (Bethesda, MD), Goodwin A (Bethesda, MD), Wolter P (Bethesda, MD), Martin S (Bethesda, MD), Balis F (Bethesda, MD), Fox E (Bethesda, MD), Kieran M (Boston, MA), Cohen B (Cleveland, OH), Blaney S (Houston, TX), Reddy A (Birmingham, AL), King A (St. Louis, MO), Solomon F (Bethesda, MD), Petronas N (Bethesda, MD), Steinberg S (Bethesda, MD), and Widemann B (Bethesda, MD) S102 Annals of Neurology Vol 66 (suppl 13) 2009 Objective: Plexiform neurofibromas (PNs) are a common, often debilitating feature of NF1. For those PNs which are symptomatic, progressive and nonresectable, there is no proven effective therapy. Pirfenidone is an orally bioavailable antifibrotic agent, with anti-growth factor properties, which inhibits fibroblastic proliferation and collagen formation and has completed phase I testing for optimal dosing in children with NF1. Methods: Patients between 3 and 21 years of age with symptomatic, radiographically progressive PNs were eligible for treatment with three times daily pirfenidone. Volumetric analysis of PN size was centrally reviewed and time-toprogression (TTP) was the primary endpoint, compared to data obtained on a concurrent, placebo controlled trial. Pirfenidone was to be considered active if it doubled TTP. Results: 36 patients (26 male/10 female), a median age of 8 years (range 3-18 years), with 49 measurable PNs were treated. Therapy was well tolerated with only 3 patients requiring a dose reduction. Twenty-nine patients progressed, 5 were removed at patient/parent/physician preference and 2 remain on study for greater than 2 years. The median TTP was 13.2 months, compared to the placebo arm of 10.6 months (p⫽0.92). Conclusions: Although Pirfenidone was well tolerated, it did not prolong TTP as compared to a placebo controlled arm. However, this study demonstrated the feasibility of performing studies in children with progressive PNs, the utility of central volumetric radiographic analysis and the ability to utilize a historical control arm, if identical eligibility criteria are utilized.