Vivek joined the Department of Neurology and the Parkinson’s Center of Oregon as an Assistant Professor in September 2011. He has an affiliate appointment and laboratory space in the Jungers Center. Vivek received a BS and MS in Chemistry and Biological Sciences from Stanford in 1995. He then earned an MD and PhD from Columbia University in 2003, where his research in Steven Siegelbaum’s lab was on synaptic plasticity in the hippocampus. Specifically, Vivek used multiphoton imaging and dual whole-cell recording methods to examine the role of presynaptic ryanodine-sensitive calcium stores in long-term depression. After a medical internship at St Luke’s-Roosevelt Hospital in New York, he completed a neurology residency and movement disorders fellowship at Massachusetts General Hospital/Brigham & Women’s Hospital. Before joining OHSU, Vivek was an Instructor in Neurology at Harvard Medical School and a research fellow in the laboratory of Bradley Hyman at Mass General. In his recent work, Vivek has used in vivo multiphoton imaging to examine the trafficking and metabolism of alpha-synuclein in mouse models of Parkinson’s Disease, a focus that he plans to continue at OHSU. In 2010, Vivek received a K08 career development award from NINDS that will allow him the freedom to develop his laboratory. Vivek brings in vivo multiphoton imaging to the substantial imaging expertise already in the Jungers Center.
The Role of Protein Degradation and Aggregation in Neurologic Disease
The goal of our research is to understand how the protein alpha-synuclein is involved in Parkinson’s disease (PD) and related disorders like Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA). Although many neurodegenerative diseases are characterized by the abnormal aggregation and accumulation of specific proteins, the exact protein in each disease varies (e.g. alpha-synuclein in PD and DLB, beta-amyloid and tau in Alzheimer’s Disease, tau and TDP-43 in forms of Fronto-temporal Dementia and Amyotrophic Lateral Sclerosis, huntingtin in Huntington’s Disease, etc.). Of all these diseases, the best evidence that simply increasing the specific protein level can cause disease exists for PD and DLB. One strong piece of evidence for this is that duplication (or triplication) of the SCNA locus coding for alpha-synuclein, which increases alpha-synuclein levels by the seemingly modest amount of 50% (or 100%), causes patients to develop PD and/or DLB. The goal of our work is to use mouse models of PD and DLB, where alpha-synuclein is over-expressed, to understand in the living brain how increasing levels of this protein leads to its aggregation and subsequent neuronal dysfunction and cell death.
The ability to test hypotheses in the living brain could greatly benefit from the development of new experimental strategies to visualize alpha-synuclein in vivo, including its metabolism, aggregation and the consequences of these processes for neurons. For these reasons we have spent the past several years developing new approaches to try and tackle these problems. Our work allows us (for the first time) to visualize alpha-synuclein in the living brain in mouse models of PD and DLB using in vivo multiphoton fluorescence microscopy through “cranial windows.”
Our recent data demonstrate that we can visualize human wild-type alpha-synuclein fused to enhanced Green Fluorescent Protein (Syn-GFP) in the cortex of transgenic mice with cellular and synaptic resolution using this cranial window-based approach. Furthermore, we can follow individually labeled neurons and presynaptic terminals serially over a period of many months and have discovered relative stability in this expression. In addition, by pairing fluorescence recovery after photobleaching (FRAP) techniques with in vivo multiphoton imaging, we find evidence that at least two pools of Syn-GFP exist in terminals, with lower levels of mobility than measured previously. Interestingly, terminals with the highest amount of alpha-synuclein expression have the largest amount of the immobile protein, potentially represented the beginning of presynaptic microaggregate formation. We are building on these strategies to test the hypothesis that alpha-synuclein first begins to form pathological aggregates at presynaptic terminals, using a variety of genetic and pharmacological manipulations paired with in vivo imaging.
Figure 1. In vivo image of alpha-synuclein fused to enhanced Green Fluorescent Protein expressed in presynaptic terminals of mouse cortex. A: Photobleaching (FRAP) between time points 0 and 1 min of individual terminals shows transient loss of signal and recovery. B: FRAP of highly expressing terminals demonstrates less complete recovery and a larger immobile fraction of alpha-synuclein. C: Group data shows similar time course of recovery of mobile fraction but larger immobile pool in highly expressing terminals. (Adapted from Unni et al. 2
We have also paired in vivo multiphoton imaging with topical treatment of inhibitors of the two major protein degradation pathways in cells, the ubiquitin-proteasome system (UPS) and autophagy. Using this strategy we can recover tissue that was treated in vivo and measure alpha-synuclein protein or mRNA levels directly with standard immunoblot or quantitative PCR techniques. This approach allows us to answer an open question in the field of PD research about how alpha-synuclein is degraded by neurons in vivo. Our results demonstrate that under normal expression conditions, alpha-synuclein appears to be degraded solely by the UPS. Under conditions of over-expression, however, autophagy can be recruited to help degrade this protein. In addition, under conditions of over-expression, a bidirectional functional coupling exists in vivo between the cell’s two major degradation pathways, such that inhibiting the UPS can upregulate autophagy and vice versa. We are working to determine how different aggregate forms of alpha-synuclein are degraded by neurons in vivo, a critically important question for understanding the interplay between protein over-expression, aggregation and neurodegeneration.
Figure 2. Above: In vivo image of alpha-synuclein fused to enhanced Green Fluorescent Protein expressed in one cell body and presynaptic terminals of mouse cortex. Top: Baseline signal before application of ubiqutin-proteasome system inhibitor. Middle: Increased signal after 24 hours of exposure to proteasome inhibitor. Below: Immunoblot for alpha-synuclein from vehicle-treated and proteasome inhibitor-treated cortex shows increased levels after 24 hours of UPS inhibition.
These kinds of strategies, which test in vivo specific hypotheses about the causes and consequences of protein aggregation, hold the promise of providing new insights into the pathobiology of PD. We believe a detailed understanding of the mechanisms causing the aggregation of alpha-synuclein and their downstream impact on neurons in the living brain will suggest new therapeutic approaches to treat this devastating group of illnesses.
Schaser AJ, Osterberg VR, Dent SE, Stackhouse TL, Wakeham C, Boutros SW, Weston LJ, Owen N, Weissman TA, Luna E, Raber J, Luk KC, McCullough AK, Woltjer RL and Unni VK (2019) Alpha-synuclein is a DNA binding protein that modulates DNA repair with implications for Lewy body disorders. Scientific Reports 9(1):10919.
Brockway NL, Cook ZT, O'Gallagher MJ, Tobias ZJC, Gedi M, Carey KM, Unni VK, Pan YA, Metz MR, Weissman TA (2019) Multicolor lineage tracing using in vivo time-lapse imaging reveals coordinated death of clonally related cells in the developing vertebrate brain. Developmental Biology 453(2):130-140.
Volpicelli-Daley LA, Abdelmotilib H, Liu Z, Stoyka L, Daher JP, Milnerwood AJ, Unni VK, Hirst WD, Yue Z, Zhao HT, Fraser K, Kennedy RE, West AB (2016) G2019s-LRRK2 expression augments Î±-synuclein sequestration into inclusions in neurons. Journal of Neuroscience 36(28):7415-27.
Osterberg VR, Spinelli KJ, Weston LJ, Luk KC, Woltjer RL, Unni VK (2015) Progressive aggregation of alpha-synuclein and selective degeneration of Lewy inclusion-bearing neurons in a mouse model of parkinsonism. Cell Reports 10:1252-1260.
Spinelli KJ, Osterberg VR, Meshul CK, Soumyanath A, Unni VK (2015) Curcumin treatment improves motor behavior in alpha-synuclein transgenic mice. PLoS One 10:e0128510.
Spinelli KJ, Taylor JK, Osterberg VR, Churchill MJ, Pollock E, Moore C, Meshul CK, Unni VK (2014) Presynaptic alpha-synuclein aggregation in a mouse model of Parkinson's disease. J Neurosci, 34:2037-2050.
Ebrahimi-Fakhari D, McLean PJ, Unni VK (2012) Alpha-synuclein's degradation in vivo: Opening a new (cranial) window on the roles of degradation pathways in Parkinson disease. Autophagy. Feb 1;8(2). [Epub ahead of print]
Ebrahimi-Fakhari D, Cantuti-Castelvetri I, Fan Z, Rockenstein E, Masliah E, Hyman BT, McLean PJ and Unni VK. (2011) Distinct roles in vivo for the ubiquitin-proteasome system and the autophagy-lysosomal pathway in the degradation of α-synuclein. J Neurosci, 31:14508-14520.
Unni VK, Ebrahimi-Fakhari D, Vanderburg CR, McLean PJ, Hyman BT (2011). Studying
protein degradation pathways in vivo using a cranial window-based approach.
Methods. 2011 53:194-200.
Unni VK, Weissman TA, Rockenstein E, Masliah E, McLean PJ, and Hyman BT (2010)
In Vivo Imaging of α-Synuclein in Mouse Cortex Demonstrates Stable Expression and Differential Subcellular Compartment Mobility. PLoS ONE 5:e10589.
Valerie R. Osterberg
B.A., Concordia College Moorhead, MN
During my time at Concordia College, my academic interests covered many areas including art, philosophy, cultural studies, music, and science. These interests continue to be important to me and have invaluably enriched my professional scientific career. Initially, I thought that I wanted to go into medicine, but quickly got hooked on lab work. I have been working as a Research Assistant ever since. I have been in several laboratories over the years, including the University of Minnesota, University of Oregon, and the Western Ecology Division of the US-EPA in Corvallis, OR. My past work has ranged from studying the role of cytoskeletal motor-proteins during meiosis in C.elegans, T-RFLP DNA analysis of root competition in conifer species, to now looking at the protein dynamics involved in Parkinson’s Disease. Working as a Senior Research Assistant in Vivek’s lab combines two of my favorite things in science, innovative research techniques and in vivo fluorescence microscopy.
B.S., Miami University, 2007
M.S., University of Wisconsin-Madison, 2009
Ph.D., University of Wisconsin-Madison, 2015
I completed my undergraduate (Miami University) and graduate work (UW-Madison) in the field of Communication Sciences and Disorders. The focus of my research to date has been in the area of voice and swallowing. For the upper airway to function well for speech, voice, and swallowing, the elements of respiration, phonation, resonance, and articulation must work in harmony. As a researcher, my interest lies in gaining a better understanding of these individual elements and their coordinated sensorimotor interactions in both healthy and disordered populations. For example, in my dissertation work I examined the effect of age and exercise on neurotrophins within the cranial sensorimotor system using a rat model, with the goal of improving the effectiveness of exercise-based treatments for age-related voice and swallowing problems. As a post-doctoral fellow in the Unni Lab, I look forward to using my clinical background and knowledge of animal behavior and the cranial sensorimotor system to further characterize the synuclein-based transgenic animals models of Parkinson disease currently studied in the lab. My project will use in vivo cranial window imaging and fluorescently tagged preformed alpha-synuclein fibrils to study the progression of protein aggregation in a mouse model of Parkinson disease.