Stephan N. Witt, PhD
Professor and Chair of Biochemistry and Molecular Biology
Bachelor of Fine Arts, 1976-1979 Fine Arts, Tufts University, Medford, Massachusetts
Bachelor of Science, 1979-1981 Chemistry, Union College, Schenectady, New York
PhD, 1982-1988 Biophysical Chemistry, California Institute of Technology, Pasadena, California
(Mentor: Sunney I. Chan)
Postdoctoral Fellow, 1988-1993 Immunochemistry, Stanford University, Stanford, California
(Mentor: Harden M. McConnell)
2001 Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (July 16-August 4)
2009 Analytic and Quantitative Light Microscopy, Marine Biology Lab, Woods Hole, MA (May 5-15)
2014 Statistics in Medicine, Stanford University Open Online Course, (June 24-August 30), Certificate of Completion with Distinction. (9/3/14)
https://verify.class.stanford.edu/SOA/74cd08554f3845b28f43dd61a47f6990
News
Congratulations Sahar! Sahar Shekoohi, a member of the Witt lab, graduated with a PhD in May of 2021.
Sahar published a comprehensive characterization of SK-Mel-28, a melanoma cell line, with the gene SNCA knocked out. SNCA codes for the Parkinson’s disease-associated protein alpha-synuclein.
Knocking out alpha-synuclein in melanoma cells dysregulates cellular iron metabolism and suppresses tumor growth. Shekoohi, S., Rajasekaran, S., Patel, P., Yang, S., Liu, W., Huang, S., Yu, X and Witt, S.N. Scientific Reports 2021 Mar 4;11(1):5267. doi: 10.1038/s41598-021-84443-y
Sahar is currently a postdoctoral fellow in the Department of Anesthesiology at LSU Health Shreveport.
Congratulations Dhaval! Dhaval Patel graduated with a PhD in June of 2018, and he then stayed in the Witt lab for one more year as a postdoc.
Dhaval published two beautiful papers over the last two years:
- Sorting Out the Role of α-Synuclein in Retromer-Mediated Endosomal Protein Sorting. Patel D, Witt SN. J Exp Neurosci. 2018 Aug 23;12:1179069518796215.
- Alpha-synuclein inhibits Snx3-retromer-mediated retrograde recycling of iron transporters in S. cerevisiae and C. elegans models of Parkinson's disease. Patel D, Xu C, Nagarajan S, Liu Z, Hemphill WO, Shi R, Uversky VN, Caldwell GA, Caldwell KA, Witt SN. Hum Mol Genet. 2018 May 1;27(9):1514-1532.
In September of 2019, Dhaval started a postdoctoral fellowship in Dr. Heidi McBride’s lab at McGill University in Montreal.
Research
Major Research Interests:
α-Synuclein and Parkinson’s disease; α-synuclein and melanoma; vesicle trafficking; molecular bases of disease.
α-Synuclein and Parkinson disease
The WITT group uses several organisms (yeast, mice and human cells) to study the mechanism of toxicity of the human Parkinson’s disease-associated protein a-synuclein (α-syn). α-Syn is an intrinsically unfolded protein of unknown function that is the main protein component of Lewy bodies, which are proteinaceous cytoplasmic inclusions in dopamine-producing neurons in individuals who suffer from PD. High expression levels of a-syn or posttranslational modifications of the protein are thought to convert α-syn from a non-toxic protein into a toxic one. There is increasing evidence that the toxic conformation of α-syn is a prion: it acts as a template or seed that converts non-infectious α-syn monomers into infectious oligomers.
Examples of ongoing projects include:
(1) α-Syn intracellular trafficking and lipid dyshomeostasis: In eukaryotic cells, wild-type a-syn transits through the endoplasmic reticulum and Golgi apparatus on route to the inner leaflet of the plasma membrane. In neurons, a-syn is thought to promote the fusion of presynaptic vesicles with the presynaptic membrane. We have found that the trafficking of a-syn through the ER is exquisitely sensitive to the level of certain cellular phospholipids, and we are investigating how changes in phospholipid homeostasis alter, and in some cases blocks, a-syn transit through the ER. This work has implications to the mechanism of Lewy body formation.
The figure below (Wang, 2014 PNAS) shows how a-syn aggregates form in yeast cells that lack the mitochondrial enzyme phosphatidylserine decarboxylase (Psd1). The role of this ancient enzyme vis-à-vis a-syn solubility/toxicity is an area of our focus.
(2) Viruses and Lewy body formation: We have found that some viral proteins greatly accelerate the formation of a-syn fibrils in vitro. Usually, the half-time for fibril formation in vitro is 5 days, whereas in the presence of certain viral proteins the half-time is in the order of minutes! Using atomic force microscopy, we found that the fibrils formed in the absence of the viral protein are flat (A), whereas fibrils formed in the presence of the viral protein are helical (B). We are conducting a variety of kinetic and structural experiments to decipher how the viral peptides accelerate fibril formation.
(3) a-Syn, iron homeostasis, retromer: Many studies over the last 10 years have found a link between a-syn and iron homeostasis. Red blood cells contain relatively high levels of a-syn as well as neurons. Because many of the genes involved in iron homeostasis in human cells are also found in yeast, we are using yeast to investigate the connection between a disruption of cellular iron homeostasis and a-syn toxicity. The findings from yeast are being tested in worms and human cells.
The figure below (Patel, 2018 Hum Mol Genet) shows how a-syn inhibits the Snx3-retromer-mediated recycling of Ftr1-Fet3. In yeast, Ftr1-Fet3 is a complex that imports iron into cells. The human orthologs of Fet3 are hephestin and ceruloplasmin.
(4) a-Synuclein and melanoma: It is curious that individuals with melanoma (who live) have a 2-fold higher risk of being afflicted with PD than age-matched healthy controls. And it works the other way, that is, individuals with PD have a significantly higher risk of developing melanoma than age-matched individuals without PD. Melanocytes, like dopaminergic neurons, express α-syn. Melanocytes, like dopaminergic neurons, synthesize a pigment (melanin). Some of the most aggressive melanomas also express very high levels of α-syn, as if somehow α-syn promotes growth. Experiments are underway using α-syn knockout cells to decipher the function of this interesting protein in melanoma.
Publications
Selected Publications
- Patel, D, Xu, C., Nagarajan, S., Liu, Z., Hemphill, W.O., Shi, R., Uversky, V.N., Caldwell, G.A., Caldwell, K.A., & Witt, S.N. (2018) Alpha -synuclein inhibits Snx3-retromer-mediated retrograde recycling of iron transporters in S. cerevisiae and C. elegans models of Parkinson’s disease. Human Molecular Genetics 27(9), 1514–1532
- Wang, S., Zhang, S., Liou, L.-C., Ren, Q., Zhang, Z., Caldwell, G.A., Caldwell, K.A., & Witt, S.N. (2014) Phosphatidylethanolamine deficiency disrupts α-synuclein homeostasis in yeast and worm models of Parkinson disease. Proceedings of the National Academy of Sciences U S A 111(38): E3976–E3985
- Witt, S.N. (2013). Molecular chaperones, alpha-synuclein and neurodegeneration. Molecular Neurobiology 47, 552-560.
- Wang, S., Xu, B., Liou, L.-C., Ren, Q., Huang, S., Luo, Y., Zhang, Z. & Witt, S.N. (2012). α-Synuclein disrupts stress signaling by inhibiting polo-like kinase Cdc5/Plk2. Proceedings of the National Academy of Sciences U S A 109, 16119-16124.
- Liu, X., Lee, Y.L., Liou, L.-C., Ren, Q., Zhang, Z., Wang, S. & Witt, S.N. (2011). Alpha-synuclein protects cells from hydroxyurea-induced replication stress in a yeast model of Parkinson’s disease. Human Molecular Genetics 20, 3401-3414.
- Lee, Y.J., Wang, S., Slone, S.R., Yacoubian, T.A. & Witt, S.N. (2011). Defects in very long chain fatty acid synthesis enhance alpha-synuclein toxicity in a yeast model of Parkinson’s disease. PLoS ONE 6 (1):e15946
- Witt, S.N. (2010). Hsp70 molecular chaperones and Parkinson’s disease. Biopolymers 93, 218-28.
- Liang, J., Clark-Dixon, C., Wang, S., Flower, T.R., Williams-Hart, T., Zweig, R., Robinson, L.C., Tatchell, K. & Witt, S.N. (2008). Novel suppressors of alpha-synuclein toxicity identified using yeast. Human Molecular Genetics 17, 3784-3795.
- Flower, T.R., Clark-Dixon, C., Metoyer, C., Yang, H., Shi, R., Zhang, Z. & Witt, S.N. (2007). YGR198w (YPP1) targets A30P alpha-synuclein to the vacuole for degradation. Journal of Cell Biology 177, 1091-1104.
- Flower, T.R., Chesnokova, L.S., Froelich, C.A., Dixon, C. & Witt, S.N. (2005). Heat shock prevents alpha synuclein-triggered apoptosis in a yeast model of Parkinson’s disease. Journal of Molecular Biology 351, 1081-1100.
Team
Sahar Shekoohi
Graduate student
Email: ssheko@lsuhsc.edu
Sahar is elucidating the function of the Parkinson’s disease-associated protein alpha-synuclein (a-syn; SNCA) in melanoma. She has used clustered regularly interspaced short palindromic repeats (CRISPR-Cas9) to generate several homozygous SNCA knockout clones of the human melanoma cell line SK-Mel-28, and she thoroughly characterized the knockout clones by DNA sequencing, RT qPCR and western blotting. Sahar has found an intriguing connection between a-syn expression and iron accumulation, and she has also conducted RNAseq experiments on several of the SNCA knockout clones as well as the parental cells. Her experiments have uncovered unexpected connections between a-syn and cell signaling pathways.
Santhanasabapathy "Santhan" Rajasekaran
Post-doctoral Fellow
Email: sraja@lsuhsc.edu
Santhan has several projects. First, in his primary project, he uses yeast to study the mechanism by which a-syn disrupts Snx3-retromer-mediated recycling of two cell-surface proteins: Kex2 and Ste13. This is an extension of our published work on how a-syn affects the recycling of the iron import pair Fet3-Ftr1. This work is funded by GM131226 (The role of alpha-synuclein in Snx3-retromer mediated recycling of membrane proteins). Second, he is also elucidating the function of the Parkinson’s disease-associated protein alpha-synuclein (a-syn; SNCA) in melanoma. He has knocked SNCA back into the SNCA knockout clones to ensure that the knockout phenotype is reversed on re-expression of a-syn.
Nirjhar Aloy
Graduate student
Email: naloy@lsuhsc.edu
Nirjhar is a first-year graduate student who is studying cancer signaling via exosomes. He is using our collection of melanoma SNCA knockout cells. Given that alpha-synuclein (a-syn) has roles in endo- and exocytosis, Nirjhar hypothesizes that loss of SNCA will result in exosomes that are inefficient in priming the extracellular milieu towards invasion by melanoma cells. He is isolating exosomes from parental and knockout cells and characterizing the transcriptomes, lipidomes and proteomes of each set of vesicles.
Mekenzie M. Peshoff
Undergraduate student worker
Email: mpeshoff@my.centenary.edu
Mekenzie studies viral protein – a-syn interactions using purified recombinant human a-syn and purified viral protein. She follows the reaction between the viral protein and a-syn using a thioflavin T (ThT) binding assay. The ThT molecule is non-fluorescent and does not bind to the a-syn monomer (see figure). However, ThT avidly binds to amyloid structures, which are composed of b-sheets, and, upon binding, ThT strongly fluoresces. Mekenzie also monitors amyloid fibril formation using fluorescence spectroscopy. Mekenzie is trying to decipher how some small molecules/proteins trigger a-syn to form amyloid fibrils and how other small molecules inhibit fibril formation.
Positions
Post-doctoral Fellows
We are currently accepting applications for Post-doctoral Fellows.
Graduate Students
We are currently accepting applications for Graduate students interested in conducting research in the Witt lab.
Undergraduate Research Assistants
We are currently accepting applications from undergraduates.
Medical Students, Residents, and Fellows
The Witt laboratory has a number of research projects available for any Medical Students, Residents, and Fellows interested. Contact the lab for more information.
contact
Contact Us
LSU Health Shreveport
Department of Biochemistry and Molecular Biology
1501 Kings Hwy
Shreveport, LA 71103
Email:
stephan.witt@lsuhs.edu
Office:
(318) 675-5163
Fax:
(318) 675-5180