Ahfeldt Lab

Neurodegenerative Disease Modeling

Research

Why are some cells are more vulnerable to neurodegenerative disease than others? This is a fundamental question for which research tools are limited. The answer to this question has broad implications for our understanding of pathological mechanisms and the development of pharmacological interventions. Human pluripotent stem cells (hPSCs), which can be differentiated into all cell types, provide an unparalleled tool for studying human neurodegenerative diseases in vitro.

For example, Parkinson’s Disease (PD) is characterized by the almost complete loss of midbrain dopaminergic neurons (DN) in the substantia nigra pars compacta (SNpc), while the closest relatives in the ventral tegmental area (VTA) are relatively spared. To study cell autonomous and non-cell autonomous contributions to DN vulnerability, we have developed knock-in human pluripotent stem cell (hPSC) reporter cell lines and differentiation protocols to derive and track not only midbrain DNs but also forebrain striatal medium spiny neurons (MSNs), midbrain astrocytes and microglia and in principle assemble a simplified nigrostriatal niche.

In another study we compare distinct hPSC derived midbrain and hypothalamic DN populations against each other to determine autonomous cellular functions that may explain vulnerability.

In previous and ongoing studies, we focused on understanding how genetic mutations cause or contribute to the development of Parkinson’s Disease (PD). In PD the interplay between risk genes and genetic or cellular background is poorly understood. hPSC isogenic models allow study of disease genes while controlling for genetic background and cell type. Using CRISPR gene editing techniques, we have introduced highly penetrant PD-causing mutations into hPSCs to study PD pathology in its most affected cell type, dopaminergic neurons (DNs). Transcriptomics and proteomics studies revealed how single gene edits can lead to dysregulation of hundreds of genes and we found PD relevant phenotypes including oxidative stress and specific cell death. We observed that PD relevant genes primarily affect various cellular pathways and functions but converge on common pathways such as oxidative stress and impaired lysosomal functions. This involves synergistic effects of PD risk or causative genes, feed-forward loops, and gene networks.

In the most recent studies, we began to explore how PD risk factors contribute to pathology. To achieve this, we are using various molecular tools taking advantage of CRISPR technologies including Cas9 knock-ins and misexpression approaches and RNA knockdown using CasRX, to simultaneously and bidirectionally modulate multiple PD relevant gene expression in a cell-type specific manner and to assess PD-phenotypes.

Our long-term goal is to utilize hPSC PD models to understand neurodegeneration and the interactions and pathological contributions of different cell types, familial and risk PD genes, and environmental toxins; and identify and test the impact of both candidate therapeutics and potential toxins through pharmacological perturbations.

Contact Us

Ahfeldt Laboratory

Tim Ahfeldt
Principal Investigator
Email

Tatyana Kareva
Associate Researcher/Lab Manager
Email

Publications

1.Fernando MB, Ahfeldt T, Brennand KJ. Modeling the complex genetic architectures of brain disease. Nat Genet. 2020 Apr;52(4):363-369. doi: 10.1038/s41588-020-0596-3. Epub 2020 Mar 23. Review. PMID:  32203467

2. Ahfeldt, T., Ordureau, A., Bell, C., Sarrafha, L., Sun, C., Piccinotti, S., Grass, T., Parfitt, G. M., Paulo, J. A., Yanagawa, F., Uozumi, T., Kiyota, Y., Harper, J. W. & Rubin, L. L. Pathogenic Pathways in Early-Onset Autosomal Recessive Parkinson’s Disease Discovered Using Isogenic Human Dopaminergic Neurons. Stem cell reports 14, 75-90, doi:10.1016/j.stemcr.2019.12.005 (2020).

3. Ordureau A, Paulo JA, Zhang W, Ahfeldt T, Zhang J, Cohn EF, Hou Z, Heo JM, Rubin LL, Sidhu SS, Gygi SP, Harper JW. Dynamics of PARKIN-Dependent Mitochondrial Ubiquitylation in Induced Neurons and Model Systems Revealed by Digital Snapshot Proteomics. Mol Cell. 2018 Apr 19;70(2):211-227.e8. doi: 10.1016/j.molcel.2018.03.012. Epub 2018 Apr 12. PubMed [citation] PMID: 29656925,
PMCID: PMC5910199

4. Ahfeldt T, Litterman NK, Rubin LL. Studying human disease using human neurons. Brain Res. 2017 Feb 1;1656:40-48. doi: 10.1016/j.brainres.2016.03.051. Epub 2016 Apr 6. Review. PubMed [citation] PMID: 27060768, PMCID: PMC5053850

5. Madison JM, Zhou F, Nigam A, Hussain A, Barker DD, Nehme R, van der Ven K, Hsu J, Wolf P, Fleishman M, O’Dushlaine C, Rose S, Chambert K, Lau FH, Ahfeldt T, Rueckert EH, Sheridan SD, Fass DM, Nemesh J, Mullen TE, Daheron L, McCarroll S, et al. Characterization of bipolar disorder patient-specific induced pluripotent stem cells from a family reveals neurodevelopmental and mRNA expression abnormalities. Mol Psychiatry. 2015 Jun;20(6):703-17. doi: 10.1038/mp.2015.7. Epub 2015 Mar 3. PubMed [citation] PMID: 25733313, PMCID: PMC4440839

6. Ding Q, Lee YK, Schaefer EA, Peters DT, Veres A, Kim K, Kuperwasser N, Motola DL, Meissner TB, Hendriks WT, Trevisan M, Gupta RM, Moisan A, Banks E, Friesen M, Schinzel RT, Xia F, Tang A, Xia Y, Figueroa E, Wann A, Ahfeldt T, et al. A TALEN genome-editing system for generating human stem cell-based disease models. Cell Stem Cell. 2013 Feb 7;12(2):238-51. doi: 10.1016/j.stem.2012.11.011. Epub 2012 Dec 13. PubMed [citation] PMID: 23246482, PMCID: PMC3570604

7. Mou H, Zhao R, Sherwood R, Ahfeldt T, Lapey A, Wain J, Sicilian L, Izvolsky K, Musunuru K, Cowan C, Rajagopal J. Generation of multipotent lung and airway progenitors from mouse ESCs and patient-specific cystic fibrosis iPSCs. Cell Stem Cell. 2012 Apr 6;10(4):385-97. doi: 10.1016/j.stem.2012.01.018. Erratum in: Cell Stem Cell. 2012 May 4;10(5):635. PubMed [citation] PMID: 22482504, PMCID: PMC3474327

8. Ahfeldt T, Schinzel RT, Lee YK, Hendrickson D, Kaplan A, Lum DH, Camahort R, Xia F, Shay J, Rhee EP, Clish CB, Deo RC, Shen T, Lau FH, Cowley A, Mowrer G, Al-Siddiqi H, Nahrendorf M, Musunuru K, Gerszten RE, Rinn JL, Cowan CA. Programming human pluripotent stem cells into white and brown adipocytes. Nat Cell Biol. 2012 Jan 15;14(2):209-19. doi: 10.1038/ncb2411. PubMed [citation] PMID: 22246346, PMCID: PMC3385947

9. Lau FH, Deo RC, Mowrer G, Caplin J, Ahfeldt T, Kaplan A, Ptaszek L, Walker JD, Rosengard BR, Cowan CA. Pattern specification and immune response transcriptional signatures of pericardial and subcutaneous adipose tissue. PLoS One. 2011;6(10):e26092. doi: 10.1371/journal.pone.0026092. Epub 2011 Oct 11. PubMed [citation] PMID: 22022522, PMCID: PMC3191160

10. Schinzel RT, Ahfeldt T, Lau FH, Lee YK, Cowley A, Shen T, Peters D, Lum DH, Cowan CA. Efficient culturing and genetic manipulation of human pluripotent stem cells. PLoS One. 2011;6(12):e27495. doi: 10.1371/journal.pone.0027495. Epub 2011 Dec 15. PubMed [citation] PMID: 22194785, PMCID: PMC3240614

11. Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal PK, Smith ZD, Meissner A, Daley GQ, Brack AS, Collins JJ, Cowan C, Schlaeger TM, Rossi DJ. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell. 2010 Nov 5;7(5):618-30. doi: 10.1016/j.stem.2010.08.012. Epub 2010 Sep 30. PubMed [citation] PMID: 20888316, PMCID: PMC3656821

12. Musunuru K, Strong A, Frank-Kamenetsky M, Lee NE, Ahfeldt T, Sachs KV, Li X, Li H, Kuperwasser N, Ruda VM, Pirruccello JP, Muchmore B, Prokunina-Olsson L, Hall JL, Schadt EE, Morales CR, Lund-Katz S, Phillips MC, Wong J, Cantley W, Racie T, Ejebe KG, et al. From noncoding variant to phenotype via SORT1 at the 1p13 cholesterol locus. Nature. 2010 Aug 5;466(7307):714-9. doi: 10.1038/nature09266. PubMed [citation] PMID: 20686566, PMCID: PMC3062476

13. Lau F, Ahfeldt T, Osafune K, Akustsu H, Cowan CA. Induced pluripotent stem (iPS) cells: an up-to-the-minute review. F1000 Biol Rep. 2009 Nov 16;1:84. doi: 10.3410/B1-84. PubMed [citation] PMID: 20948605, PMCID: PMC2948253

14. Eminli S, Foudi A, Stadtfeld M, Maherali N, Ahfeldt T, Mostoslavsky G, Hock H, Hochedlinger K. Differentiation stage determines potential of hematopoietic cells for reprogramming into induced pluripotent stem cells. Nat Genet. 2009 Sep;41(9):968-76. doi: 10.1038/ng.428. Epub 2009 Aug 9. PubMed [citation] PMID: 19668214, PMCID: PMC3987895

15. Maherali N, Ahfeldt T, Rigamonti A, Utikal J, Cowan C, Hochedlinger K. A high-efficiency system for the generation and study of human induced pluripotent stem cells. Cell Stem Cell. 2008 Sep 11;3(3):340-5. doi: 10.1016/j.stem.2008.08.003. PubMed [citation] PMID: 18786420, PMCID: PMC3987901

16. Park IH, Arora N, Huo H, Maherali N, Ahfeldt T, Shimamura A, Lensch MW, Cowan C, Hochedlinger K, Daley GQ. Disease-specific induced pluripotent stem cells. Cell. 2008 Sep 5;134(5):877-86. doi: 10.1016/j.cell.2008.07.041. Epub 2008 Aug 7. PubMed [citation] PMID: 18691744, PMCID: PMC2633781

17. Composition and methods for enhanced knock-in reporter gene expression Ahfeldt T, Rubin LL. USA 62/443,543,. My Bibliography [patent]

18. Differentiation into brown adipocytes Ahfeldt T, Cowan C, Lee Y, Schinzel R. USA WO2013082106 A1. My Bibliography [patent]

19. Compositions and methods of generating reprogrammed adipocyte cells and methods of use therefore Ahfeldt T, Lum DH, Cowan C. USA US20120219530 A1. My Bibliography [patent]

20. Compositions comprising hepatocyte-like cells and uses thereof Ahfeldt T, Cowan C, Lum DH, Pryor HI, Vacanti JP. USA WO2010014949 A3. My Bibliography [patent]

Meet the Team

noimageprofileTim Ahfeldt
Principal Investigator

noimageprofileTatyana Kareva
Associate Researcher / Lab Manager

Gustavo Parfitt
Postdoctoral Fellow

Elena Coccia
Postdoctoral Fellow

noimageprofileLily Sarrafha
PhD Student

Camille Goldman 
PhD Student

Ricardo Reyes
Masters Student