Professor 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)
Dr. Witt was the Chair of the NIH Blueprint for Neuroscience Research Study Section. RFA-DA-24-039: Tools and Technologies to Explore Nervous System Biomolecular Condensates. ZRG1 NV-L (96)
Congratulations to Nirjhar Aloy for being awarded the 2nd year of the FWCC predoctoral fellowship.
Dr. Witt served on the NIH Study Section Member Conflict: Glia Function, Neurodegeneration, and Neuroregulation. ZRG1 BN-T (02).
Dr. Witt served as co-Chair of the NIH Fellowship Study Section: Biophysical, Physiological, Pharmacological, and Bioengineering Neuroscience. (F03B ZRG1 F03B-L (20) L)
Dr. Witt is part of a LSUHS team that showed that the menin tumor suppressor is lost in high grade cholangiocarcinomas. Cholangiocarcinoma is a rare and deadly cancer of the bile duct.
Loss of tumor suppressor menin expression in high grade cholangiocarcinomas.
Lairmore TC, Abdulsattar J, De Benedetti A, Shi R, Huang S, Khalil MI, Witt SN.BMC Res Notes. 2023 Feb 13;16(1):15. https://doi.org/10.1186/s13104-023-06282-6
Dr. Witt served on the NIH Study Section. Member Conflict: Topics in Neurobiology and Neuropharmacology. (ZRG1 BN-R (02))
Dr. Witt served on the NIH Fellowship Study Section: Biophysical, Physiological, Pharmacological, and Bioengineering Neuroscience (F03B ZRG1 F03B-L (20) L)
Santhan published a paper entitled “α-synuclein inhibits Snx3-retromer retrograde trafficking of the conserved membrane-bound proprotein convertase Kex2 in the secretory pathway of Saccharomyces cerevisiae” in Human Molecular Genetics. This study shows beautifully how alpha-synuclein can disrupt vesicular trafficking in cells.
Santhanasabapathy Rajasekaran et al.
Human Molecular Genetics, Volume 31, Issue 5, 1 March 2022, Pages 705717, https://doi.org/10.1093/hmg/ddab284
Dr. Witt served the NIH Fellowship Study Section: Biophysical, Physiological, Pharmacological, and Bioengineering Neuroscience. (F03B ZRG1 F03B-L (20) L)
Congratulations to Nirjhar Aloy for being awarded a FWCC predoctoral fellowship of $30,000.
Congratulations to the Witt lab for being awarded a FWCC Idea Award of $50,000 for their investigations of the mechanism by which alpha-synuclein modulates the cell surface expression of adhesion proteins.
Dr. Witt served on the NIH Fellowship Study Section: Biophysical, Physiological, Pharmacological, and Bioengineering Neuroscience. (F03B ZRG1 F03B-L (20) L)
Dr. Witt was awarded a R56 grant from the National Institute of Neurological Disorders and Stroke (NINDS) for his project entitled, “Examining the role of phosphatidylethanolamine and autophagic disruption in Lewy Body Dementias and Parkinson's disease.” Award amount: $642,408
Dr. Witt invited to comment on a paper published in PLOS BIOLOGY.
Trojan horses and tunneling nanotubes enable α-synuclein pathology to spread in Parkinson disease
Santhanasabapathy Rajasekaran, Stephan N. Witt
PLOS Biology: published July 20, 2021 | https://doi.org/10.1371/journal.pbio.3001331
Congratulations to Nirjhar for being awarded a competitive NSF-funded Fellowship to cover the tuition for a set of online summer modules over six days at the Summer Institute of Statistical Genetics at the University of Washington.
Dr. Witt served on the NIH Cellular and Molecular Biology of Neurodegeneration (CMND) Study Section.
Congratulations Sahar! Sahar Shekoohi, a member of the Witt lab, graduated with a PhD and is currently a postdoctoral fellow in the Department of Anesthesiology at LSUHS.
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
Dr. Witt served on the NIH Fellowship Study Section: Biophysical, Physiological, Pharmacological and Bioengineering Neuroscience. (2020/01 ZRG1 F03B-R (20) L)
Congratulations to Nirjhar for being awarded a competitive NSF-funded Fellowship to cover the tuition for a set of online summer modules over nine days at the Summer Institute of Statistical Genetics at the University of Washington.
α-Synuclein and Parkinson’s disease; α-synuclein and melanoma; protein aggregation; molecular bases of disease; vesicle trafficking
The Witt group uses several organisms to study the mechanism of toxicity of the human Parkinson’s disease-associated protein α-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 α-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. We also study the function of α-syn in melanoma. Examples of our ongoing projects include:
(1) Parkinson’s disease, phosphatidylethanolamine (PE), vesicular trafficking, and autophagy.
PE is the second most abundant phospholipid in mammalian cells. PE comprises about 15–25% of the total lipid in mammalian cells; it is enriched in the inner leaflet of membranes, and it is especially abundant in the inner mitochondrial membrane. PE has remarkable activities: it is a lipid chaperone that assists in the folding of certain membrane proteins, it is required for the activity of several of the respiratory complexes, it is an essential molecule for the synthesis of glycosylphosphatidylinositol-anchored proteins (GPI-AP), and it plays a key role in the initiation of autophagy. There are two main sources of PE in cells (Fig. 1A): (1) PE is synthesized via the Kennedy pathway, where the last step occurs in the ER, and (2) PE is also synthesized in mitochondria by the enzyme phosphatidylserine (PS) decarboxylase. Normal levels of PE can decline with age in the brain.
Yeast and worm models of PD
In one of our studies, we used yeast and worms to test the hypothesis that low levels of PE alter the homeostasis of α-syn. We chose to decrease the level of PE in cells by knocking out the gene coding for PS decarboxylase in yeast that express human α-syn; this mutant is called psd1Δ. Low levels of PE in psd1Δ cells cause endoplasmic reticulum (ER) stress, α-syn accumulation in foci (Fig. 1B), and slow growth. Supplemental ethanolamine (ETA), which can be converted to PE via the Kennedy pathway (Fig. 1A), partially eliminated the ER stress, decreased α-syn foci formation, and restored growth close to that of wild-type cells. Using a C. elegans PD model in which only DA neurons express human a α-syn, we proved genetically that ETA exerts its protective effect by boosting PE through the Kennedy pathway, and that, strikingly, ETA protects worm DA neurons from age-dependent neurodegeneration. Overall, a low level of PE causes ER stress, disrupts vesicle trafficking, and causes α-syn to accumulate. Supplemental ETA rescues α-syn toxicity because it converts to PE via the Kennedy pathway, and increasing PE improves the processing of GPI-APs, decreases ER stress, and increases autophagic flux.
Figure 1. α-syn forms foci and is toxic to psd1Δ cells. (A) PE synthesis in yeast and humans occurs via the Kennedy pathway in the ER, and by the decarboxylation of phosphatidylserine in mitochondria by Psd1. (B) Yeast cells transformed with indicated plasmids were induced for 5 h in mixed medium (1% Sucrose/1% Galactose) at 30 °C and then imaged by fluorescence microscopy. ETA (5 mM) was used for the rescue. (Scale bar, 5 μm) (Wang, 2014 PNAS)
iPSC model of PD
The findings from our yeast/worm studies (above) are the basis of an NIH grant that was awarded to Drs. Witt and Mazzulli (Northwestern University) entitled “Examining the role of phosphatidylethanolamine and autophagic disruption in Lewy body dementias and Parkinson’s disease” (1R56 NS114272-01A1). This is the major area of interest in our lab.
With funding from the R56 grant, we devised a cocktail composed of ETA, choline, uridine 5’-monophosphate, and DHA to increase the levels of PE in cells. The hypothesis was that stimulating PE synthesis will increase autophagic activity and rid cells of toxic forms of α-syn. We tested our ETA cocktail on H4 (± α-syn) neuroglioma cells, SH-SY5Y (± α-syn) cells, and midbrain DA neurons that express wt α-syn or A53T α-syn and conducted a lipidomics analysis after several days of treatment. We found:
a) Low levels of specific PE species: In day 60 A53T iPSn neurons and in SH-SY5Y cells, we found a decrease in PE species that directly correlate with autophagic dysfunction. These PE species are 16:0e / 22:6 and 18:1e / 22:6.
b) ETA cocktail increases PE species: In all cell lines tested, cells treated with the cocktail showed significant increases in PE species with C22:6 at the sn-2 position.
c) The ETA cocktail increased the sn-2 22:6 PE species, rescued autophagic flux, and accelerated the rate of α-syn clearance. (manuscript in preparation)
(2) α-Syn and iron homeostasis
Many studies over the last 10 years have found a link between α-syn and iron homeostasis. Red blood cells contain relatively high levels of α-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 α-syn and cellular iron homeostasis.
We discovered that in yeast α-syn inhibits the Snx3-retromer-mediated recycling of Ftr1-Fet3. Ftr1-Fet3 is a non-covalent complex that imports iron into cells. The human orthologs of Fet3 are hephestin and ceruloplasmin, and humans also express Snx3 and retromer orthologs. We proposed that α-syn inhibits Snx3-retromer-mediated recycling of Ftr1-Fet3 by inhibiting the binding of Snx3 to the phospholipid PI3P (Fig. 2).
Figure 2. Model for α-syn disrupting Snx3–retromer-mediated recycling of membrane proteins. Left panel: Snx3–retromer complex binds to the C-terminus of Ftr1, which results in the formation of tubular endosomes from which vesicles containing the Fet3/Ftr1 cargo bud off and transit to the trans-Golgi. From the trans-Golgi, Fet3/Ftr1 laden vesicles transit back to the plasma membrane. Right panel: α-syn inhibits the binding of Snx3 and Vps17 to vesicles containing PI3P. Lack of Snx3 and Vps17 binding to the surface of early endosomes results in the inability of retromer to retrieve Fet3/Ftr1 from the degradative pathway. (Rajasekaran, 2022 Hum Mol Genet; Patel, 2018 Hum Mol Genet)
(3) a-Syn and melanoma
Curiously, individuals with melanoma 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.
To probe the function of α-syn in melanoma, we knocked out SNCA, the gene that codes for α-syn, in the human SK-Mel-28 melanoma cell line using CRISPR/Cas9. The SNCA-knockout (KO) clones in culture exhibited a decrease in the transferrin receptor 1 (TfR1), an increase in ferritin, an increase of reactive oxygen species, and they proliferated slower than control cells. These SNCA-KO clones grafted into SCID mice grew significantly slower than the SK-Mel-28 control cells that expressed α-syn. Collectively, depletion of α-syn in SK-Mel-28 cells dysregulates cellular iron metabolism, especially in xenografts, yielding melanoma cells that are deficient in TfR1 and FPN1, that accumulate ferric iron and ferritin, and that undergo apoptosis relative to control cells expressing α-syn. Our model to explain these results is that α-syn promotes the anterograde and retrograde trafficking of cell-surface proteins to and from the plasma membrane, and that in the absence of α-syn nascent cell-surface proteins transit to the lysosome where they are degraded (Fig. 3).
Figure 3. Dysregulated intracellular trafficking in SNCA-KO melanoma cells dysregulates iron metabolism. (a) Iron handling in the control SK-Mel-28 cells. (b) Iron-mishandling in the SNCA-KO clones. The “mishandling” comes about because the SNCA-KO cells have a dysregulated level of the transferrin receptor (TfR1), which is involved in iron import, and a dysregulated level of ferroportin (FPN1), which is involved in iron export, as well las other perturbations. Ferric iron accumulates in such cells. (Shekoohi, 2021 Sci Rep)
(4) a-Syn and lipid elongases
We published a paper in 2011 entitled “Defects in Very Long Chain Fatty Acid Synthesis Enhance Alpha-Synuclein Toxicity in a Yeast Model of Parkinson’s Disease” (Lee YJ, et al., PLOS ONE 2011). In this paper, we reported the identification of three S. cerevisiae lipid elongase null mutants (elo1D, elo2D, and elo3D) that enhance the toxicity of α-syn. These elongases function in the endoplasmic reticulum to catalyze the elongation of medium chain fatty acids to very long chain fatty acids, which is a component of sphingolipids. Without α-syn expression, the various elo mutants showed no growth defects, no reactive oxygen species (ROS) accumulation, and a modest decrease in survival of aged cells compared to wild-type cells. With (WT, A53T or E46K) α-syn expression, the various elo mutants exhibited severe growth defects, ROS accumulation, and aberrant protein trafficking. Our work demonstrated beautifully that loss of elongase function shifts α-syn from being a non-toxic protein to a toxic protein that kills cells.
Strikingly, recent papers (Chang et al. Nat Genet 2017; Li et al Front Aging Neurosci 2018) showed an association between single nucleotide polymorphisms in ELOVL7 (a human elongase gene) and early-onset PD. This means that alterations in the expression of ELOVL7 are a risk factor for early-onset PD. We were the first group to demonstrate the importance of lipid elongases to maintaining α-syn homeostasis.
Arnold, M.R., Cohn, G.M., Oxe, K.C., Elliott, S.N., Moore, C., Laraia, P.V., Shekoohi, S., Brownell, D., Meshul, C.K., Witt, S.N., Larsen, D.H., Unni, V.K. (2024) Alpha-synuclein regulates nucleolar DNA double-strand break repair in melanoma. bioRxiv 2024 Jan 13:2024.01.13.575526. doi: 10.1101/2024.01.13.575526. Preprint. Submitted to Science Advances
Rajasekaran, S, Cheng, S., Gajendran, N., Shekoohi, S., Chesnokova, L., Yu, X., and Witt, S.N. (2023) Transcriptomic analysis of melanoma cells reveals an association of a-synuclein with regulation of the inflammatory response. bioRxiv 2023 Dec 23:2023.12.23.573196. doi: 10.1101/2023.12.23.573196. Preprint. Submitted to Scientific Reports
Rajasekaran, S., Peterson, P.P., Liu, Z., Robinson, L.C., and Witt, S.N. (2022) α-Synuclein inhibits Snx3-retromer retrograde trafficking of the conserved membrane-bound proprotein convertase Kex2 in the secretory pathway of Saccharomyces cerevisiae. Human Molecular Genetics Mar 3;31(5):705-717.
Graduate student
Email: nirjhar.aloy@lsuhs.edu
Nirjhar earned his MBBS (equivalent to MD in US) from Rajshahi Medical College, Bangladesh, which is one of the leading medical schools in Bangladesh. In his medical career, he practiced as a family physician and as an instructor of Anatomy and Microbiology. Later, he joined a residency program in Cardiovascular and thoracic surgery, where he was doing well. But his strong passion for basic science encouraged him to pursue a career of a physician-scientist and he joined the PhD program in Biochemistry and Molecular Biology at LSUHS.
Nirjhar’s dissertation research is focused on understanding the role of the Parkinson’s disease-associated protein alpha-synuclein in trafficking of multivesicular bodies (MVBs). The central hypothesis of his project is that alpha-synuclein promotes melanoma-metastasis by modulating the trafficking of MVBs and controlling the release of extracellular vesicles. This project has the potential to pave the way for new research in areas of cancer biology.
Post-doctoral Fellow
Email: liudmila.chesnokova@lsuhs.edu
Senior Scientist
Email: sergey.slepenkov@lsuhs.edu
We are currently accepting applications for Post-doctoral Fellows.
We are currently accepting applications for Graduate students interested in conducting research in the Witt lab.
We are currently accepting applications from undergraduates.
The Witt laboratory has a number of research projects available for any Medical Students, Residents, and Fellows interested. Contact the lab for more information.
LSU Health Shreveport
Department of Biochemistry and Molecular Biology
1501 Kings Hwy
Shreveport, LA 71103
Email:
stephan.witt@lsuhs.edu
Office:
(318) 675-7826
Fax:
(318) 675-5180
We look forward to speaking with you!
Department of Biochemistry & Molecular Biology
Phone: (318) 675-5160
Fax: (318) 675-5180
Mailing Address:
Department of Biochemistry & Molecular Biology
LSU Health Shreveport
1501 Kings Highway
Shreveport, LA 71103
David Gross, PhD
Professor of Biochemistry and Molecular Biology, Graduate Program Director
david.gross@lsuhs.edu
