Collin Challis, Ph.D. - US grants

DESCRIPTION (provided by applicant): Major depression is the most prevalent mood disorder, yet there has not been major therapeutic progress in developing clinical treatments in the past 20 years. Most antidepressant strategies affect monoamine neurotransmitter systems with serotonin (5-HT) as the most common target. Recently, though, there has been a growing body of clinical and preclinical evidence that implicate the medial prefrontal cortex (mPFC) in depression and its treatment. Structural and functional imaging studies in patients have revealed consistent volume changes and hyperactivity of this region in mood disorders. Studies performed in animals have also shown that the neuroplastic changes in the mPFC may also be related to vulnerability and resiliency to stressors and depression-like behaviors. Additionally, deep brain stimulation (DBS) of the mPFC in both humans and animals produced an antidepressant-like effect. Interestingly, an intact 5-HT system was required for DBS to produce this effect. Previous work has shown that the mPFC sends projections to the dorsal raphe nucleus (DRN), which contains the largest population of 5-HT neurons in the brain. However, the DRN is heterogeneous and these projections actually appear to converge on GABAergic neurons, which suggests that these GABA neurons may act as filters of sensory control over 5-HT. Additionally, our lab and others have also shown that DRN GABA neurons are primarily activated in response to a variety of stressors, which would also suggest how dysregulation of this population might lead to mood disorders. The goal of this proposal is to show that neuroplastic adaptations mediated by DRN GABAergic neurons in the mPFC-DRN pathway affect depressive-like behaviors and antidepressant response. My first specific aim is to perform a neuroanatomical and functional dissection of the mPFC-DRN pathway and will allow me to better characterize the cellular architecture of this circuit. The second aim will use electrophysiological and morphological techniques to evaluate the adaptations induced by social stress in various cellular components of the mPFC-DRN pathway. In the third aim I will use optogenetic tools to dissect the behavioral impact of specific neural populations in the mPFC-DRN pathway in the social defeat model of depression. Together, these aims will provide a better understanding of the circuitry that underlies mood disorders and could lead to the development of more effective and efficient antidepressant strategies. PUBLIC HEALTH RELEVANCE: The proposed project will explore the functional cytoarchitecture of the pathway between the medial prefrontal cortex and the dorsal raphe nucleus and its role in depression-like behaviors. This work will investigate the physiological adaptations and behavioral contributions of specific neural populations that compose this circuit. Results from this work will contribute to the development of more effective and efficient antidepressant strategies.

PROJECT SUMMARY  The  aggregation  and  accumulation  of  alpha-­synuclein  (aSyn)  fibrils  results  in  histopathological  inclusions  called Lewy bodies and Lewy neurites and is observed in several neurodegenerative disorders. In Parkinson?s  disease (PD), this culminates in the degeneration of dopaminergic neurons in the substantia nigra and severe  motor  impairment.  Emerging  findings  suggest  that  PD  may  have  a  prodromal  phase  characterized  by  non-­ motor symptoms such as gastrointestinal disturbances. Thus, it is hypothesized that pathologic aSyn might first  accumulate  in  peripheral  tissue  before  propagating  to  the  brain  where  it  precipitates  PD  pathology.  This  is  supported  by  research  that  shows  that  aSyn  fibrils  are  capable  of  interneuronal  transport  and  can  seed  the  formation  of  additional  fibrils  from  endogenous  aSyn.  The  goal  of  this  proposal  is  to  provide  me  with  training  in  advanced  techniques  to  interrogate  peripheral  nervous  systems  in  mice.  I  will  use  these  techniques to test the hypothesis that pathologic aSyn propagates from enteric neurons in the gut to  the  brain  via  the  vagus  nerve  in  an  age-­  and  activity-­dependent  manner,  eventually  damaging  the  dopaminergic system that coordinates movement. To visualize aSyn fibrils, enteric populations, and nerve  tracts in high resolution, I will use the CLARITY tissue clearing method developed in our lab that renders whole  organs  and  organisms  optically  transparent  and  macromolecule  permeable.  I  will  also  use  novel  quantitative  measures  to  analyze  large  three-­dimensional  datasets.  I  will  use  a  novel  adeno  associated  virus  capsid  that  has increased affinity for peripheral neurons to deliver constructs to neurons and nerves in the enteric nervous  system.  I  will  use  whole  cell  patch  clamp  recordings  to  determine  the  effect  of  aSyn  fibrils  on  enteric  neuron  electrophysiology  and  neurotransmission.  Lastly,  I  will  use  optogenetics  to  explore  an  activity-­dependent  mechanism  of  aSyn  uptake  and  release.  Upon  completion,  the  experiments  detailed  in  this  proposal  will  contribute to our understanding of pathologic aSyn formation in the gut and their propagation to the brain, and  will be key in developing novel diagnostic and therapeutic strategies for PD. 

PROJECT SUMMARY/ABSTRACT Age is the biggest risk factor for neurodegenerative diseases such as Parkinson?s disease (PD) and Alzheimer?s disease. Homeostatic decline that occurs as part of aging might promote the accumulation of protein aggregates observed in these diseases, which can affect function and cause cell death. In a subset of diseases called synucleinopathies, the presynaptic protein alpha-synuclein (aSyn) oligomerizes into insoluble amyloid fibrils in neurons and can lead to distinct clinical phenotypes depending on the population affected. Accumulation in the substantia nigra pars compacta (SNc) occurs in PD, resulting in the death of midbrain dopamine neurons and deterioration of motor skills; however, motor dysfunction may actually represent a late stage of the disease. Clinicians observed a prodromal period of idiopathic PD marked by non-motor symptoms. Commonly, patients reported a loss of smell, and assessment of the nasal cavity found aSyn aggregates. This led to the hypothesis that aSyn pathology originates in the periphery before propagating to the brain. However, the nose to brain progression of aSyn pathology has not been directly demonstrated. Additionally, whether age-related factors increase susceptibility to aSyn fibril seeding and spread has not been determined. The goal of this proposal is to establish a training plan to learn and utilize techniques to interrogate neural pathways connecting the periphery to the brain. I will test the hypothesis that age-relevant factors underlie the susceptibility to peripheral synucleinopathy. I will also determine how aSyn pathology affects the olfactory system and progresses from the nose to the brain. Under the guidance of Dr. Viviana Gradinaru and Dr. David Chan, I will advance my technical and professional training, which will prepare me for independent research. In the mentored phase (K99), I will perform a focused analysis of PD-relevant proteins in olfactory pathway structures to see how age and seeding of pathologic aSyn in the nasal cavity affects expression. I will also perform in vivo imaging of mitochondrial dynamics in olfactory regions to visualize the impact of aging and aSyn pathology on cellular physiology. The techniques and results acquired in the mentored phase of the award will facilitate the transition to independent research (R00). In this phase, I will use tissue clearing methods we developed (Bone CLARITY, PACT) to visualize aSyn pathology and olfactory pathways in transparent mouse skulls. Using novel viral capsids that we engineered, I will manipulate aSyn homeostasis-relevant protein expression in the periphery to determine if they contribute to peripheral synucleinopathy pathogenesis. I will also image genetically encoded calcium and dopamine indicators in vivo to understand how peripherally seeded aSyn fibrils progress through the neurocircuitry responsible for olfaction and affect olfactory function. Completing this proposal will contribute to our understanding of synucleinopathy etiology in peripheral systems and its progression to the brain. The findings will be key in developing novel diagnostic and therapeutic strategies for synucleinopathies.