Development and Characterization of a Novel In Vivo Model of BPAN Using CRISPR/Cas9-based Knockout of wdr45 in Zebrafish
(A) Generate wdr45-/- zebrafish using CRISPR/Cas9 mediated introduction of early frameshifting mutations.
(B) Characterize wdr45-/- zebrafish on a morphological, biochemical and behavioral level relevant to better understand the molecular biology of WDR45 and Beta-propeller Protein-associated Neurodegeneration (BPAN) in
humans. We anticipate that these phenotypes will:
(1) provide insight into WDR45 biology in vivo
(2) establish a human-to-zebrafish paradigm for small molecule screens and translational research
(3) inform the in vivo modeling of other forms of neurodegeneration with brain iron accumulation
Background and Significance:
Neurodegeneration with brain iron accumulation (NBIA) is a heterogeneous group of single gene disorders that share the feature of high brain iron content. Beta-propeller protein- associated neurodegeneration (BPAN) is a rare but prototypical form of NBIA caused by X-linked mutations in the WDR45 gene (1-3). Clinically this syndrome is characterized by early-onset developmental delay and intellectual disability, childhood onset epilepsy followed by dystonia, parkinsonism and progressive cognitive decline in early adulthood (3). WDR45 (also known as WIPI4), a member of the WD40 repeat family of proteins, is a beta-propeller scaffold with a putative role in autophagy through its interaction with phospholipids and autophagy-related proteins (4-6).
While the precise role of WDR45 in autophagy, iron metabolism and axonal homeostasis remain to be elucidated(7),
dysregulation of any of these pathways offers the opportunity for phenotypic high throughput screens in vitro and in
vivo. Characterization of primary cell lines and Wdr45 knockout mice have established that loss of WDR45 leads to
impaired autophagy, as well as lysosomal and mitochondrial function (2, 8, 9). CNS-specific Wdr45 knockout mice
further exhibit poor motor coordination, reduced learning and memory, and extensive axon swelling (9, 10).
The discovery of treatments for BPAN has been limited by the slow disease progression in knockout mouse models
(9, 10), their unsuitability for large-scale in vivo screens, and their relatively high costs. Zebrafish (Danio rerio) is a
small vertebrate model that offers key benefits to circumvent these issues (11-14). wdr45 in zebrafish is an autosomal
gene with~90% homology to human WDR45 and is ubiquitously expressed with robust expression in the CNS, making it a
strong candidate for modeling in zebrafish (15).
The development and characterization of a wdr45 knockout zebrafish model using CRISPR/Cas9 technology has several advantages over morpholino oligonucleotide-based models (16, 17) and allows for the generation of a robust genetic model (18-20). By establishing and validating the first zebrafish model for BPAN using state-of-the-art techniques we will address a significant unmet need for a small vertebrate model of BPAN that will enable us to elucidate basic disease mechanisms and to build a future screening platform for novel therapeutics in vivo. This study is a translational study that aims at generating and characterizing the first zebrafish model of BPAN. This model will be subjected to several proof-of-concept experiments evaluating wdr45-related phenotypes in vivo. Through this zebrafish model, we will investigate the neurodevelopmental role of wdr45 and its impact on motor and social behavior.
WANT TO LEARN MORE ABOUT ZEBRAFISH IN THE STUDY OF HUMAN DISEASE?
THE RESEARCH TALENT:
Angelica D’Amore, Ph.D.
Following training in Biotechnology, Angelica D’Amore received her PhD in Molecular Medicine from the University of Siena, Italy, in 2020. She joined Dr. Darius Ebrahimi-Fakhari at Boston Children’s Hospital/Harvard Medical School in March 2019 to work on disease models for rare neurodegenerative diseases in children. Her current work focuses on the generation and characterization of CRISPR/Cas9-engineered zebrafish to understand neurodegenerative mechanisms in vivo and to build a platform for drug screens. This has led to a first zebrafish model of AP-4 related hereditary spastic paraplegia. With support and funding from BPAN Warriors, Dr. D’Amore will develop a zebrafish model of BPAN, the first of its kind.
Darius Ebrahimi-Fakhari, M.D., Ph.D.
Dr. Ebrahimi-Fakhari is a Pediatric Neurologist and Neuroscientist with expertise in childhood-onset neurogenetic, neurodegenerative, and movement disorders. Trained as a physician-scientist, the objective of his research is to understand the genetic and molecular mechanisms of protein trafficking and the autophagy-lysosomal pathway in neurons, and to use this knowledge to develop novel therapeutic approaches to treat and cure a variety of neurological diseases. At Boston Children’s Hospital, he is leading several clinical research projects on childhood-onset forms of hereditary spastic paraplegia and other movement disorders. In the laboratory, his team is pursuing several projects aimed at developing iPSC-derived neurons and zebrafish as a new platform for high-throughput small molecule and genetic screens. The goal is to employ these models of rare childhood-onset diseases to identify novel therapeutic targets.
“The measure of greatness in a scientific idea is the extent to which it stimulates thought and opens up new lines of research.”