科学研究

Seminars

The Conditional Knockout Approach: Cre/Lox Technology in Human Neurons

2019-11-29

SEMINAR TYPE

B-type


PREFERRED LOCATION

Third Floor Lecture Hall, Jianzan Building (Phase I)

Chinese Institute for Brain Research, Beijing


TIME

11:00-12:00  Monday,December 9th, 2019


SPEAKER

Christopher Patzke, Ph.D.


HOST

Dr. Minmin Luo


TOPIC

The Conditional Knockout Approach: Cre/Lox Technology in Human Neurons


ABSTRACT

The use of human pluripotent stem cells to model human diseases has become a new standard in biomedical sciences. To this end, patient-derived somatic cells are studied in vitro to mimic human pathological conditions. Particularly, the human synapse is involved in many congenital disorders, hence a systematic understanding of its function and structure is essential for the quest of therapies. In my presentation I describe as an experimental strategy the ‘Conditional Knockout Approach’ which allows to engineer disease-relevant mutations in human neurons. In combination with Cre/Lox technology, this method enables me to investigate the molecular causes of human diseases independent of the genetic background or of genetic alterations induced by clonal selection. I generated knockouts in human genes encoding for major neuronal and synaptic components including Munc18-1 (STXBP1), L1CAM, and Synapsin-1, which impact synaptic transmission, axon development, and synaptic plasticity, respectively. Strikingly, in the case of Synapsin-1 we uncovered a novel presynaptic mechanism of plasticity in human neurons: After stimulation by neuromodulators synapsins acutely and bi-directionally control in a cAMP-dependent manner the reserve pool size of synaptic vesicles in human neurons by acting downstream of neuromodulator G-protein coupled receptors for serotonin and noradrenaline.


SPEAKER BIOGRAPHY 

Christopher Patzke studied Biology at the Freie Universität in Berlin and the École Normale Supérieure in Paris (1998-2004). During his PhD training and initial postdoc time (2004-2011) in the laboratory of Dr. Fritz Rathjen at the Max-Delbrück-Center in Berlin he investigated the role of cell adhesion in neuronal cells. Since January 2012, he works as a postdoctoral research associate at Stanford in the laboratory of Dr. Thomas Südhof and focuses on developing new neurobiological disease and research models by utilizing human pluripotent stem cell derived neurons.

Initially in his work, Christopher helped developing and characterizing stem cell derived human neurons generated by overexpressing a single transcription factor (Neurogenin-2). Subsequently, he sought ways of applying these induced human neurons for disease modelling. In particular, he established a truly isogenic platform by combining cre/lox technology with induced neurons. This conditional knockout approach allows to study disease-relevant candidate mutations in a controlled genetic background, because mutant and wild type cell are derived from the same cell population of healthy stem cells.

He has successfully engineered heterozygous and homozygous mutant stem cell lines for Munc18-1 (STXBP1), a member of the SM-protein family, essential for synaptic vesicle fusion. Heterozygous mutations lead to a severe form of early infantile epileptic encephalopathy, namely Ohtahara Syndrome. Christopher’s work demonstrated that Munc18-1 deletions in a healthy genetic background leads to decreased synaptic transmission. Moreover, he generated conditionally hemizygous mutants for L1CAM and Synapsin-1, two X-chromosomal genes linked to severe intellectual disabilities and epilepsy. In the case of L1CAM, an immunoglobulin-domain containing cell adhesion molecule, he was able to reveal a new phenotype in loss-of-function mutant neurons: The decreased excitability of these neurons, due to an impaired axonal development, represents an underappreciated phenotype, which likely contributes to the disease pathology of L1-syndrome patients. In his main project, the Synapsin-1 conditional knockout, Christopher can compellingly show, that the presynaptic phosphoprotein Synapsin acts down-stream of neuromodulators such as Norepinephrine or Serotonin, as a bidirectional cAMP-dependent regulator of the synaptic vesicle pool size.

Combining technologies, such as genetic engineering, cell imaging, biochemistry, brain organoid cultures and physiology, he generates and studies Cre/Lox conditionally mutated human neurons, allowingin vitro analyses of cellular processes independent of the genetic background or genetic alterations induced by clonal selection. His main goal is to answer questions about the molecular underpinnings of human biology and pathology mechanisms in neurodevelopmental and neuropsychiatric disorders.