Job ID: 118721

Project for potential PhD students working on a human-specific gene

Position: Ph.D. Student

Deadline: 15 May 2024

Employment Start Date: 1 October 2024

Contract Length: 3 years

City: Paris

Country: France

Institution: Institut Pasteur

Department: Neuroscience

Description:

We are inviting applications from prospective PhD students to apply together for competitive funding. This funding comes from Paris-based Doctoral Schools after competitive evaluation. Prospective students should have first experience in human iPSC cultures.

Send applications to umaskos@pasteur.fr and matthias.groszer@inserm.fr

 

Dissecting the impact of the human-specific CHRFAM7A gene on alpha7 nicotinic receptor signaling

We are proposing a comprehensive analysis of the role of CHRFAM7A, a human-specific gene in novel, in vitro, biomedical disease models, based on the use of human induced pluripotent stem cells (hiPSC). CHRFAM7A is present in humans for the last 200,000 years, but not in non-human primates, after a fusion between the CHRNA7 nicotinic acetylcholine receptor (nAChR) α7 gene, and ULK-4, encoding a serine/threonine kinase (1). The resulting protein is thought to integrate into the canonical nAChR pentamer, but due to its lack of ACh binding sites, reduces receptor signaling.  The importance of this human-specific, additional level of AchR signaling modulation/tuning has become clear in human genetic studies linking variation in CHRFAM7A to schizophrenia (SZ) (2, 3). These have identified copy number variation (CNV) and a two base-pair coding variation (2) in SZ. Upregulation of CHRFAM7A was observed in SZ brain samples (4). Further studies found genetic association with the presence of a two base-pair coding deletion (2bp variant)  leading to a further truncation of the protein. A further key finding: Expression patterns of the CHRFAM7A and CHRNA7 gene transcripts were quantified (5) in the prefrontal cortex (PFC) of 380 SZ subjects and 325 comparison subjects collected from fetal state to old age. The main findings were that the ratio of CHRFAM7A over CHRNA7 transcript was significantly increased in SZ. Increased CHRFAM7A/CHRNA7 ratios are also found in immature (incl prenatal) PFC samples, suggesting an abnormal persistence of neurodevelopmental expression pattern in the (adult) cortex. Together these data argue that the CHRFAM7A is an important modulator of SZ susceptibility and may significantly contribute to the heterogenous drug responses targeting the CHRNA7 nAChR in SZ. The study thus has fundamental as well as clinical implications for personalizing treatments. Our main aims are: 1. model CNV and the delta2bp variant of CHRFAM7A with isogenic hiPSC-derived cortical organoids and microglia; 2. study the consequences in biomedical disease models.

Experimental plans

The genetic manipulation will be supervised by Matthias Groszer: We are using human induced pluripotent stem cell (hiPSC) lines from the Sanger Centre collection. They are fully sequenced at the genomic and transcriptomic level and corresponding proteomics data are published. All genetic engineering and potential genetic drifts during long-term cultures can therefore be controlled for with respect to the starting material.

– A CHRFAM7A knock-out (KO) will be obtained using a CRISPR/Cas9 strategy in two iPSC lines.

– Generation of isogenic CHRFAM7Adelta2bp knock-in (KI) hiPSC.

– Generation of isogenic CHRFAM7Adelta2bp over-expression hiPSCs: We already generated lentiviral expression vectors (LVs) and validated them in NPCs. This need is based on human genetic findings that implicate the delta2bp variant in SZ(3).

The analysis will be supervised by Uwe Maskos. Our approach will employ recent methodological advances, including the transplantation of hiPSC-derived microglia into brain organoids (6)(7). We will use two-photon imaging of organoids to follow the morphological and functional maturation of the human neurons. Single-cell sequencing (10XGenomics) will identify corresponding changes in the transcriptome. CHRFAM7A is expressed in neurons and microglia, the latter are important for synaptic pruning in neurodevelopment. Thus hiPSC-derived microglia with different CHRFAM7A genotypes will be transplanted into the developing organoid. The consequences on neuronal and microglial function will be evaluated using KI of the transgenic calcium indicator GCaMP7b (8), where we have already generated the line.

 

  1. M. L. Sinkus, S. Graw, R. Freedman, R. G. Ross, H. A. Lester, S. Leonard, The human CHRNA7 and CHRFAM7A genes: A review of the genetics, regulation, and function. Neuropharmacology. 96, 274–288 (2015).
  2. M. L. Sinkus, M. J. Lee, J. Gault, J. Logel, M. Short, R. Freedman, S. L. Christian, J. Lyon, S. Leonard, A 2-base pair deletion polymorphism in the partial duplication of the alpha7 nicotinic acetylcholine gene (CHRFAM7A) on chromosome 15q14 is associated with schizophrenia. Brain Res. 1291, 1–11 (2009).
  3. K. Szigeti, B. Kellermayer, J. M. Lentini, B. Trummer, D. Lal, R. S. Doody, L. Yan, S. Liu, C. Ma, Ordered subset analysis of copy number variation association with age at onset of Alzheimer’s disease. J. Alzheimers. Dis. 41, 1063–71 (2014).
  4. A. R. Lew, T. R. Kellermayer, B. P. Sule, K. Szigeti, Copy Number Variations in Adult-onset Neuropsychiatric Diseases. Curr. Genomics. 19, 420–430 (2018).
  5. Y. Kunii, W. Zhang, Q. Xu, T. M. Hyde, W. McFadden, J. H. Shin, A. Deep-Soboslay, T. Ye, C. Li, J. E. Kleinman, K. H. Wang, B. K. Lipska, CHRNA7 and CHRFAM7A mRNAs: Co-localized and their expression levels altered in the postmortem dorsolateral prefrontal cortex in major psychiatric disorders. Am. J. Psychiatry. 172, 1122–1130 (2015).
  6. G. Popova, S. S. Soliman, C. N. Kim, M. G. Keefe, K. M. Hennick, S. Jain, T. Li, D. Tejera, D. Shin, B. B. Chhun, C. S. McGinnis, M. Speir, Z. J. Gartner, S. B. Mehta, M. Haeussler, K. B. Hengen, R. R. Ransohoff, X. Piao, T. J. Nowakowski, Human microglia states are conserved across experimental models and regulate neural stem cell responses in chimeric organoids. Cell Stem Cell. 28, 2153-2166.e6 (2021).
  7. M. L. Bennett, H. Song, G. Ming, Previews Microglia modulate neurodevelopment in human neuroimmune organoids. Cell Stem Cell. 28, 2035–2036 (2021).
  8. H. Dana, Y. Sun, B. Mohar, B. K. Hulse, A. M. Kerlin, J. P. Hasseman, G. Tsegaye, A. Tsang, A. Wong, R. Patel, J. J. Macklin, Y. Chen, A. Konnerth, V. Jayaraman, L. L. Looger, E. R. Schreiter, K. Svoboda, D. S. Kim, High-performance calcium sensors for imaging activity in neuronal populations and microcompartments. Nat. Methods. 16, 649–657 (2019).