title: Olfactory Stem Cells as Disease Models for Schizophrenia
Creators: name: Tee, Jing Yang
Publisher: name: Griffith University
Year: 2019
Subjects: subject: Olfactory stem cells
subject: Schizophrenia
subject: Mental disorder
subject: Disease models

description: Schizophrenia is a strongly heritable multifactorial mental disorder that affects 1% of any population, but disease etiology is not yet fully understood. The limitations of using animal models, post-mortem brain studies and difficulty to obtain human brain biopsies have impeded the understanding of this disease from the biological perspective. There is a consensus, based on epidemiology and post-mortem human brains that schizophrenia is a neurodevelopmental disorder, but cellular and molecular mechanisms for this hypothesis are lacking. We have developed a model to address this using human olfactory neurosphere-derived (hONS) ‘stem’ cells, which were obtained non-invasively from human participants, expressed neural stem cell markers and allows comparative analyses of age- and sex-matched cell lines from patients diagnosed with schizophrenia with cell lines from healthy participants. Patient cells from various sources have a common dysregulation to gene pathway networks involved in axonal guidance, cell adhesion, cytoskeletal remodeling, focal adhesion signaling and reelin signaling - all important pathways that regulate cell migration. A high throughput live cell tracking technique was developed to unbiasly quantify cell motility in vitro. At baseline on uncoated tissue culture plastic (TCP), patient cells moved longer distances than control cells. Patient cells were unable to respond to their microenvironment when increasing concentrations of well-defined ECM proteins were coated on TCP cell culture surfaces. This was despite patient cells expressing the necessary cell surface integrin receptors measured by flow cytometry, which meant they have the capacity to bind and recognize all tested ECM proteins. Patient cells have consistently lower levels of cytoskeletal proteins in filamentous actin (F-actin) and stable microtubules, which became more evident when cultured on ECM proteins. Despite being able to adjust their cell sizes and shapes, patient cells were consistently smaller than control cells. Focal adhesion kinase (FAK) levels were consistently lower in patient cells and they were unable to manufacture enough focal adhesions of the correct sizes upon contact with ECM proteins. These findings highlighted a global deficit to cell migration in schizophrenia that was not limited to just Fibronectin but also on all ECM proteins, which were caused by disease-dependent intracellular deficits in patient cell cytoskeleton and focal adhesions. Reelin is an important brain ECM protein that orchestrates neuronal migration during neurodevelopment and its expression is reduced in post-mortem brain tissues from patients with schizophrenia. In this study, reelin protein levels were also reduced in patient hONS cells compared to control cells. Despite having the capabilities to bind to reelin through expression of key reelin signaling pathway accessory protein Dab1 and reelin-binding receptors ApoER2 and VLDLR, patient cells were unable to respond to extracellular reelin. Patient cells were unable to adjust their movement track lengths when tracked on surfaces coated with full length reelin. Similar to our previous findings on other ECM proteins, patient cells had lower levels of F-actin and stable microtubules, and were consistently smaller in size compared to control cells. Our findings suggested that non-responsive cell migration deficits were caused by defective focal adhesion responses to extracellular reelin. Instead of producing more focal adhesions that were larger in response to reelin, patient cells reduced both the densities and sizes of their focal adhesions at the peripheral region of the cell where lamellipodia forms. This was the first study to demonstrate a biological link between reelin signaling and schizophrenia, with a focus on the effects of reelin on cell migration. Published computer programs were used in silico to investigate the mechanistic components of cell migration and to further investigate reasons why patient cells were unable to respond to their ECM microenvironment. X-Y displacement coordinates for each individually tracked cell were analyzed by the DiPer and migration phase/cell turning analysis programs to compute cell movement variables in directionality, diffusivity, persistence and idling and turning. At baseline on TCP, patient cells did not move at the correct trajectories, failed to make directional changes, paused less and turned less when tracked for 24 h. When tracked on ECM proteins, it was difficult to pinpoint one explanation to explain our non-responsive cell migration findings as various mechanistic variables played a collective part to potentially give rise to the observed non-responsive cell migration phenotype. One explanation was that patient cells spent more time in persistent movement and were unable to make necessary changes to their directions. Another explanation was the unchanged cell pausing tendencies on Type I Collagen as a plausible explanation for observed non-responsive changes to motility on that ECM protein. The same in silico computational analyses showed that patient cells did not respond to extracellular reelin because they could not reduce the rate at which they changed the straightness of their movement trajectories. Patient cells were also unable to make well-timed changes to the direction of their movement trajectories by being more persistent in one direction for longer. Decreased densities and sizes of focal adhesions in patient cells upon contact with extracellular reelin resulted in patient cells not decreasing their turn angles, but instead made larger uncoordinated turns. The way that control cells responded negatively to extracellular reelin supported the hypothesis in the field that reelin acts as a “stop signal” for actively moving neuronal cells, which was disrupted in patient cells. In conclusion, this thesis has uncovered a novel non-responsive cell migration phenotype in schizophrenia, which was broadly relevant on different ECM proteins, to suggest consistent defects in how patient cells respond to their surrounding microenvironment. Findings from this thesis also showed that observed cell migration deficits were caused by differences to intracellular components such as cell cytoskeleton and focal adhesions, coupled to previously published findings that FAK, integrin and actin signaling pathways were all dysregulated in schizophrenia. Using an analogy of patient cells as a moving vehicle, our findings showed a global defect to many parts of this faulty “vehicle”, such as defective “ignition” in FAK signaling, uncoordinated “wheels” in the lamellipodia and consistent faults to the “clutch” mechanism in the focal adhesions. All working in concert to cause patient cells to move in a less coordinated fashion and unable to make controlled mechanistic changes to their movement, causing them to ultimately lose their way.