Andrew Flores Abstracts
Andrew Flores
Ph.D. Candidate
Physiological Sciences GIDP
Society for Neuroscience Annual Meeting
Chicago, Illinois
October 17-21, 2016
Analysis of cellular morphology, growth dynamics and autophagic mechanisms in primary skin fibroblasts from individuals diagnosed with sporadic Parkinson’s disease
AUTHOR BLOCK: *A. J. FLORES1,2, M. J. CORENBLUM2, C. CURIEL3, S. J. SHERMAN2, L. MADHAVAN2,4;
1Grad. Interdisciplinary Program in Physiological Sci., 2Dept. of Neurol., 3Arizona Cancer Ctr., 4Evelyn F. McKnight Brain Inst., Univ. of Arizona, Tucson, AZ
Human primary skin fibroblasts are easily accessible peripheral cells that have Parkinson’s disease (PD)-relevant biochemical and gene expression profiles, and constitute a system which reflects the chronological and genetic aging of patients to form a patient-specific model of the disease. Recent data suggest that cytoskeletal and metabolic alterations play a role in the PD degenerative process. Because cytoskeletal and metabolic dynamics are also important determinants of cellular morphology, we systematically analyzed the morphological features and growth dynamics of primary fibroblasts generated from skin biopsies of individuals diagnosed with late-onset PD, as well as age-matched control subjects. Under phase contrast conditions, control and PD fibroblasts apparently differed with respect to spatial growth patterns, and cell size and shape. Therefore, to clearly visualize morphology, dermal fibroblasts were stained with a fluorescent phalloidin F-actin probe, and analyzed for five different shape and size parameters using CellProfiler software. In particular, comparisons of cell area, perimeter and minimum and maximum diameters revealed that fibroblasts from PD patients were significantly smaller than control fibroblasts. Additionally, PD fibroblasts displayed a significantly higher form factor, indicating that they were more circular than control fibroblasts. In terms of growth dynamics, comparisons between control and PD cell lines showed no significant differences in cell viability, time to reach 90% confluence, nor in total cell count at 90% confluence. However, a trend towards higher cell count and greater time to reach 90% confluence, was noted in the PD fibroblasts. Additionally, immunocytochemical analysis revealed that fibroblasts from Parkinson’s subjects had significantly higher expression of the PD-relevant protein alpha-synuclein (α-synuclein) than control fibroblasts, as reflected by higher intensity of α-synunclein staining per cell. Given that α-synuclein is degraded via chaperone-mediated autophagy (CMA, a selective form of autophagy), and also that aberrant α-synuclein may act as a CMA inhibitor, as a next step, we have begun to analyze autophagy-related mechanisms, in the patient fibroblasts. These studies provide a foundation for investigating PD-relevant structural and autophagic alterations in a patient-specific manner, and will complement future assessments in induced pluripotent stem cell (iPSC)-derived midbrain dopaminergic neurons generated from skin fibroblasts of PD subjects.
Presentation Preference (Complete): Poster Only
Theme and Topic (Complete): C.03.h. Cellular mechanisms ; C.03.b. Degeneration models
Keyword (Complete): PARKINSON'S DISEASE ; ALPHA-SYNUCLEIN ; AUTOPHAGY
Grant/Other Support: Arizona Center for the Biology of Complex Diseases Grant
Grant/Other Support: : University of Arizona Intramural Funds
Parkinson’s disease (PD) is a common neurodegenerative disorder that affects about 1 in every 100 people over age 60, and over 1 million people in the United States alone. PD is associated with the progressive loss of neurons that produce dopamine, a brain chemical necessary for movement control. Symptoms of PD are debilitating and typically include tremor, slowness of movement and an inability to initiate movement, rigidity of muscles, and the loss of balance reflexes. Although drugs are available that can help alleviate PD symptoms, there are currently no therapies that can prevent or slow the progression of the disease. The exact cause of PD is currently unknown, however evidence points to a complex interaction between genetics, aging, and environmental factors.
A major problem in understanding the underlying cause of PD is related to a lack of experimental models that can accurately replicate the disease pathology—especially patient-to- patient variability in disease pathology. The work described in this abstract uses primary skin fibroblast cells (also known as fibroblasts) as model to study Parkinson’s disease in an authentic and patient-specific manner. Primary fibroblasts are obtained directly from patients in a clinic and therefore reflect the unique aging and environmental history of each individual patient.
First, we visualized morphological differences in the primary fibroblasts by staining them with a fluorescent dye that binds to their actin cytoskeleton—an internal network of protein filaments that provides structural support to cells (Figure 1). This allowed us to accurately measure the size and shape of the fibroblasts using a computer program. Our analysis revealed that fibroblasts obtained from Parkinson’s patients were smaller in area and more rounded than fibroblasts from control individuals. This could mean that the actin cytoskeleton is destabilized in PD patient fibroblasts.
Next, we performed an analysis of alpha-synuclein. Alpha-synuclein (α-synuclein) is a protein whose normal function in the brain is currently unknown. It is of great interest in Parkinson's research because it misfolds and forms toxic aggregations (much like the infamous amyloid-beta aggregations seen in Alzheimer’s disease) that are the pathological hallmark of Parkinson’s disease. We found that expression of α-synuclein was highly expressed in fibroblasts from PD patients, and furthermore, that it exists in its pathological, aggregated form in patient fibroblasts.
As a next step, we have begun to analyze the process of autophagy in the fibroblasts. Autophagy or ‘self-eating’ is the process by which a cell digests damaged or unnecessary components of itself. Autophagy is a survival pathway turned on by stress or starvation and is thought to be dysregulated in a wide range of diseases, including neurodegenerative diseases like PD. Our preliminary findings indicate that autophagy may indeed be impaired in fibroblasts obtained from PD patients.
These data will be presented at the 2015 Society for Neuroscience annual meeting in Chicago, and provide a foundation for investigating cytoskeletal and autophagy-related alterations associated with PD, as well as alterations in the handling of α-synuclein, in a patient-specific manner. Future directions will involve the use of induced-pluripotent stem cell technology to derive dopamine neurons from patient fibroblasts so that we can study diseasemechanisms in a PD-relevant neural cell type.
Figure 1: A fibroblast with fluorescently stained actin cytoskeleton.
