Paul J. Mathews - US grants

Action Potentials; Auditory; Auditory Localization; Auditory Pathways; Auditory pathway structure; Brain Stem; Brainstem; Cell Body; Cells; Code; Coding System; Cognitive Discrimination; Complex; Data; Development; Discrimination; Discrimination (Psychology); Exhibits; Fellowship; Figs; Figs - dietary; Frequencies (time pattern); Frequency; Individual; Ion Channels, Potassium; K channel; Kinetic; Kinetics; Lead; Localized; Medial; Names; Nerve Cells; Nerve Unit; Nervous; Neural Cell; Neurocyte; Neurons; Olives; Olives - dietary; Pattern; Pb element; Physiologic; Physiologic pulse; Physiological; Play; Potassium Channel; Process; Property; Property, LOINC Axis 2; Pulse; Pulse taking; Relative; Relative (related person); Role; Shapes; Sound Localization; Spatial Distribution; Stimulus; Synapses; Synaptic; Time; Training; V (voltage); Voltage-Gated K+ Channels; Voltage-Gated Potassium Channel; Work; auditory pathway; cell body (neuron); experiment; experimental research; experimental study; heavy metal Pb; heavy metal lead; in vivo; neural; neural cell body; neuronal; neuronal cell body; postsynaptic; relating to nervous system; research study; response; social role; soma; voltage; voltage clamp

DESCRIPTION (provided by applicant): Experimental lesions and blunt force traumas to the cerebellum result in behavioral abnormalities that indicate this brain region plays an important role in controlling smooth coordinated movement and motor memory (Fine, Ionita, & Lohr, 2002). Specifically, researchers believe the cerebellum evaluates the disparities between intention and action, and then adjusts the motor output to correct for these disparities in order to generate a desired, smooth-motor behavior. Experiments suggest these corrections arise from dynamic changes in the strength of synaptic connections in both the cerebellar cortex and deep nuclei. In addition, these changes are likely driven by the association or coincident detection of signals from two specific pathways, one by way of the mossy fibers (MF, carrying sensory information) and the other by way of the climbing fibers (CF, indicating a disparity or error in the motor command). Originating in the inferior olive the climbing fiber delivers a unique and powerful input that generates a complex spike in the sole output of the cerebellar cortex, the Purkinje cells (PCs). This input, when paired with parallel fiber (PF; mossy fiber relay) activation decreases the somatosensory receptive fields of PCs (Jvrntell & Ekerot, 2002). This change in receptive field is due to the depression of a subset of PF-PC synapses, a mechanism believed to remove sensory signals producing undesired motor behaviors. Similar experiments also demonstrate CFs drive associative changes in the receptive fields of molecular layer inhibitory interneurons (MLI) that synapse onto PCs. However, the nature of the CF-MLI connection remains unclear, nor are the mechanisms driving the associative plasticity that result in receptive field changes known. This deficiency in the current state of cerebellar knowledge is the result of an inability to reliably stimulate CFs without activating neighboring axons from other neuron types. To overcome this technical challenge, a novel optogenetic approach has been developed to allow robust stimulation of isolated CFs. The first aim of this proposal will further confirm preliminary results demonstrating the reliability and specificity of photostimulating CFs expressing Channelrhodopsin 2 by systematically exploring the optical stimulation and viral injection parameters necessary for robust CF stimulation. Using this technique, I propose to describe both the nature of CF-MLI transmission as well as the mechanisms and rules governing the CF- driven associative plasticity between parallel fibers and MLIs. This will be accomplished through whole-cell patch clamp recordings from MLIs in acute slices during selective CF photostimulation. These experiments will be the first of their kind to illustrate the effectiveness of optogenetic techniques in exploring the cerebellar cortex. In the end results from these experiments will allow for better predictions of how the cerebellar cortex evaluates and corrects for disparities between intention and action.

Ataxia-Telangiectasia (A-T) is rare (~ 1 in every 100,000) but a catastrophic and deadly disease that is often caused by a nonsense mutation (that is, a premature termination codon [PTC]) in the Atm (Ataxia- Telangiectasia mutated) gene. Children born with A-T suffer a progressive loss of motor function and coordination, become wheelchair-bound, and typically die by age 25. No effective treatments are available, in part because no satisfactory animal model exists. Atm-/- mice are not a good model because they do not develop ataxia. However, recognizing that the Atm gene product participates in DNA repair pathways, our collaborator Dr. McKinnon reasoned that inflicting a second hit ? an additional knockout mutation in a second DNA repair gene ? might produce ataxia. To test this notion, he selected to knockout the Aptx (aprataxin) gene because: 1) APTX participates in DNA repair: 2) humans lacking APTX protein develop a disease similar to A-T called AOA (Ataxia with Oculomotor Apraxia); and 3) like Atm-/- mice, Aptx-/- mice do not develop ataxia. He crossed the single mutant mice to generate a double mutant Atm-/-; Aptx-/- mouse that, as anticipated, exhibits the progressive ataxia observed in A-T and AOA patients. Our LONG TERM GOAL is to utilize these mice to test the safety and effectiveness of potential treatments for A-T and deploy them for clinical use. However, due to the nature of the genetic mutation in these mice (total knockout of the Atm gene), this model is not suitable for testing our recently developed Small Molecule Read-Through (SMRT) compounds, which efficiently read- through the mutation in Atm genes causing the PTCs in A-T (Lee et al., 2013). We have synthesized, purified, and patented a number of derivatives from our original screen that read through all three types of PTCs (TAG, TAA, and TGA) with similar efficiency in Atm and other PTC-containing genes as well as cross the blood brain barrier. To test whether SMRT compounds effectively treat A-T caused by a PTC, we need to create and validate a mouse model of A-T that incorporates a patient-derived PTC and displays ataxia. As a first step, we have created a mutant mouse that expresses a PTC in the Atm gene at the location on exon 15 that it occurs in many patients with A-T. These mice are viable and fertile. Like the Atm knockout, this AtmN/N genotype (?N? for nonsense mutation) is not ataxic. To induce ataxia, we will cross AtmN/N mice with Aptx-/- mice. We predict that like the Atm-/-; Aptx-/- mice, the resultant AtmN/N; Aptx-/- mice will exhibit ataxia and will also contain a PTC amenable to treatment with our SMRT compounds. We propose here to breed, genotype, characterize, and phenotype AtmN/N; Aptx-/- compound mutant mice. FUTURE DIRECTIONS: Once the AtmN/N; Aptx-/- mice are fully phenotyped, we will then proceed to perform safety and efficacy studies for our SMRT compounds. This treatment should restore production of ATM protein to sufficient quantities to normalize (or at least alleviate) the symptoms of A-T, including ataxia, immunodeficiency, and high susceptibility to cancer. Our approach could in principle be applied to hundreds of inherited human diseases that are similarly caused by a PTC mutation.

PROJECT SUMMARY Ataxia-Telangiectasia (A-T) is a rare (~ 1 in every 100,000) but catastrophic and deadly disease that causes progressive loss of motor function and coordination and death by age 25. In about one-third of cases, the cause is a nonsense mutation in the ATM (Ataxia-Telangiectasia mutated) gene that encodes a premature termination codon (PTC). No effective treatments are available, in part because no satisfactory animal model exists. Atm-/- mice are not a good model because they do not develop ataxia. However, recognizing that the Atm gene product participates in DNA repair pathways, our collaborator Dr. McKinnon reasoned that inflicting a second hit ? an additional knockout mutation in a second DNA repair gene that is also linked to ataxia ? might produce ataxia. To test this notion, he knocked out the Aptx (aprataxin) gene because: 1) APTX participates in DNA repair; 2) humans lacking APTX protein develop a disease similar to A-T called AOA (Ataxia with Oculomotor Apraxia); and 3) like Atm-/- mice, Aptx-/- mice do not develop ataxia. He crossed the single mutant mice to generate double mutant Atm-/-; Aptx-/- mice that, as anticipated, exhibit the progressive ataxia observed in A-T and AOA patients. Our LONG-TERM GOAL is to: 1) produce a comprehensive understanding of the histopathologic sequence of A-T disease development; 2) explain mechanistically how loss of DNA repair proteins leads to ataxia; and 3) utilize this new mouse model to test new treatments for A-T. AIM 1 proposes hypothesis-generating studies that will dissect the underlying neuropathological and electrophysiological abnormalities that accompany development of ataxia. These experiments, carried out in collaboration with Dr. Jon Cooper (Co-I), will concentrate on the cerebellum and brain regions that project to and from that structure, since it controls motor coordination functions. We will also characterize a new AtmN/N; Aptx-/- genotype that contain a nonsense mutation in the same exon (exon 15) as patients with A-T and that, we predict, will phenocopy Atm-/-; Aptx-/- mice and develop progressive ataxia similar to clinical A-T. AIM 2 will test AtmN/N; Aptx-/- mice and Atm-/-; Aptx-/- controls with our recently developed Small Molecule Read-Through (SMRT) compounds, which efficiently read-through nonsense mutations in the ATM gene (Lee et al., 2013). We will compare our lead candidate (GJ103) with other compounds that can also readthrough nonsense mutations. Dependent variables will include mRNA and protein expression analyses for ATM and downstream molecules, and assessment of motor function using standard tests. We predict that SMRT compounds (but not other readthrough compounds that do not cross the blood-brain barrier) will both restore ATM production in the brain and ameliorate the ataxic phenotype. FUTURE STUDIES: Our results will: 1) provide essential hypothesis-generating preliminary data to fuel mechanistic follow-on studies directed at explaining development of A-T in molecular, cellular, and brain region-specific terms; and 2) provide justification and support for more definitive IND-enabling preclinical development of SMRT compounds to create the first effective treatment for A-T caused by nonsense mutations.