Characterization of Non-motor Phenotypes in Parkinson's Disease

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1. Specific Aims: Aim 1: Demonstrate striosome-dependent behavior in wildtype (WT) mice. The Aim 1 objective is to identify behaviors reliant on striosome activity, and will be accomplished using genetically targeted striosome lesions. Aim 2: Identify in vivo striosomal activity encoding behavior. The Aim 2 objective is to identify striosome activity patterns serving behavior, and will be accomplished using in vivo calcium imaging. Aim 3: Test whether striosome activity distinguishes Lrrk2 mutant mice. The Aim 3 objective is to test whether altered striosome dynamics accompany altered anxiety and choice behaviors in PD mouse models, and will be accomplished using Lrrk2 G2019S KI mice. 2. Scientific Premise and Significance Despite recognition as a movement disorder, PD includes important non-motor symptoms of elevated anxiety and impaired judgement. SPNs are divided into striosome and matrix compartments. Striosomes are a likely candidate to support affective and executive functions impaired in PD, since striosomal SPNs have stronger connections to limbic regions than those located in matrix, have newly indicated roles in stress- and cost-sensitive choice behaviors, and receive unique innervation from a nucleus controlling anxiety. The single most common genetic factor in PD is mutation in the gene encoding Leucine-rich repeat kinase 2 (LRRK2), which is highly expressed in striosomal SPNs. Lrrk2 G2019S KI mice show subtle abnormality in measures of cognition and anxiety that could model non-motor symptoms of PD. A hyper-kinetic phenotype may indicate a loss of executive control. Indeed, a recent genetic screen of mice with healthy (risk-sensitive) or else compulsive (risk-insensitive) behavior found that significant Lrrk2 upregulation distinguishes compulsive animals. LRRK2 G2019S mutation is reported to enhance LRRK2 kinase activity, but the influence of this common PD-linked mutation on striosome activity in vivo is untested. This research identifies neural dynamics underlying anxiety and choice behaviors with major significance for mental health. It is highly significant to PD as LRRK2 mutation is present from birth, and non-motor symptoms may precede PD onset. Therefore, any difference in neurophysiology corresponding to Lrrk2 mutation in mice could provide information on PD development, early diagnosis, and how physiology might be restored to prevent or treat disease. This research has potential to define a new pathophysiological phenotype for LRRK2 mutation carriers which may be targeted by new prophylactic therapies and disease treatments. 3. Innovation This is an innovative study of striosome neuron roles in anxiety and choice performance, first in wild-type mice and later in mice carrying Lrrk2 G2019S missense mutation. Conceptual innovation exists in the combined focus on striosome neurons and PD, and in the direct focus on neural activation serving non-motor PD. This is possible due to technically innovative miniature head-mounted microscopes and genetically encoded fluorescent calcium sensors to reveal dynamic neural activity in awake mice. 4. Research Plan In Aim 1, using Sepw1_NP67 (Sepw1) mice expressing Cre in striosomes, we bilaterally injected intra-striatal AAV expressing Cre-dependent diphtheria toxin A fragment (AAV-DTA) to create genetically targeted striosome lesions. We will compare lesioned and sham controls to test candidate behaviors for striosome-dependence. Open-field test assesses basic locomotion, and Elevated Zero Maze (EZM) and Light/Dark Box assess anxiety. Fixed-Interval 30second (FI30) operant food self-administration assesses motivated choice performance under standard and reward-devalued conditions. One cohort of animals has completed Aim 1 experiments, demonstrating the feasibility of the behavioral tests. Data from this cohort will be analyzed once histology confirms the lesion. In Aim 2, we introduced AAV with Cre-dependent fluorescent calcium sensor GCaMP6s to striosomes or matrix using Sepw1 or matrix-Cre mice (Plxnd1-OG1) mice, respectively, and optically recorded neural activity in behaving mice using custom MiniScopes above an implanted 1mm diameter GRIN lens. We related activity among striosome neurons to events in the same candidate behaviors studied in Aim 1, such as lever-press or area-transition, and will compare this to activity among matrix neurons. Imaging results from three Sepw1 mice suggest that striosomes weakly encode velocity in the Open-field, and that greater striosome activation accompanies the (presumably higher-anxiety) transitions in to the Light as opposed to Dark areas in the Light/Dark Box test. As mice navigate the Light/Dark box, neural activity can be aligned to the precise time of transition between areas. In this way we can identify individual striosome neurons which are reliably active (positive z-score) at these transition events. Cell maps indicating the position of identified neurons can be made for each mouse, and may be useful for identifying anatomical trends in engaged neurons. We find that many neurons engaged by one transition are not engaged by alternative transitions. This suggests that distinct striosome neurons may have distinct selectivity for area transitions. Finally, we find greater correlation between striosome neural activation and the behavior of exploring an area (pre-transition) as opposed to net behavior within an area. In behavior data, we see mice spend more time exploring the Light as opposed to the Dark area per transition, particularly in early trials. This measure of hesitation to transition may demonstrate anxiety. At the same time when hesitation is highest, our preliminary data suggest greater striosome activation during corresponding exploration of Light relative to exploration of Dark. In Aim 3, to test whether striosome activity distinguishes Lrrk2 mutant mice, we will repeat Aim 2 in vivo imaging of striosome dynamics in Sepw1-Cre mice with G2019S KI mutation and in their Lrrk2-WT littermates. We will compare behavior and in vivo striosome activity between mutant and WT animals to determine effects of PD-related Lrrk2 G2019S KI mutation. Our gene expression studies found significant upregulation in potassium channels and channel associated proteins in Lrrk2 KO mice, suggesting altered neural excitability. G2019S mutation is purported to enhance Lrrk2 activity 64,66. Aim 3 tests whether this influences striosome neuron activation.