PgmNr M5036: Investigating how cytoskeletal protein mutations cause Amyotrophic Lateral Sclerosis disease using neuronal cells differentiated from mouse embryonic stem cells.

Authors:
Kim Nguyen; Lisa Schneper; Kathryn Sheldon; Zhonghua Gao; James Broach


Institutes
Penn State Hershey Coll of Medicine, Hershey, PA.


Abstract:

Amyotrophic Lateral Sclerosis (ALS) affects 15,000 to 21,000 Americans at any one time (ALS association). Unfortunately, this destructive motor-neurodegenerative disease still remains untreatable largely due to a lack of knowledge in the disease mechanisms and a lack of model organism for drug screen. Currently, known causative genetic mutations of ALS only account for ~6% of ALS cases despite the total ALS heritability being 50%. Not only is there a tremendous knowledge gap in the genetic basis of ALS, but also the associated clinical phenotypes are complicated and disconnected to the known genetics. Therefore, the goal of our study is to identify and characterize the genetic factors underlying the disease and specifically correlating these to various groups of ALS clinical features. To understand genetic mechanism underlying ALS disease, we have recruited and exome-sequenced more than one hundred ALS patients. In addition to rare damaging variants in ALS causative genes such as SOD1 and TARDBP (TDP43) we have found de novo compound heterozygous mutations in cytoskeletal-related proteins such as DNAH2, DYNC2LI1, STARD9 and NRP2. The ex vivo functional characterization of mutations were established in mouse embryonic stem cells  (mESC) by CRISPR knockout technology with the presumption that compound heterozygous mutations abolish protein function. Two CRISPR targets per gene were generated and transfected into neuronal-fluorescent mESC. Single positive knockout cell were FACS-sorted and propagated for validations. Validated knockout clones then are used for differentiation into motor neurons, astrocytes, and oligodendrocytes. Phenotypes of neuronal cell viability and neurite outgrowth length and numbers are then accessed by live-cell staining, fixed-cell staining and qRT-PCR. The results of the ex vivo characterization will establish the roles of cytoskeletal-related proteins in ALS and will also allow us to generate mouse models that could be great contributions to the ALS field.