Heterozygous loss of Engrailed-1 and α-synucleinopathy (En1/SYN): A dual-hit preclinical mouse model of Parkinson's disease, analyzed with artificial intelligence.

In this study, we develop and validate a new Parkinson's disease (PD) mouse model that can be used to better understand how the disease progresses and to test the effects of new, potentially disease-modifying, PD therapies. Our central hypothesis is that mitochondrial dysfunction intercalates with misfolded α-synuclein (α-syn) accumulation in a vicious cycle, leading to the loss of nigral neurons. Our hypothesis builds on the concept that PD involves multiple molecular insults, including mitochondrial dysfunction and aberrant α-syn handling. We predicted that mitochondrial deficits, due to heterozygous loss of Engrailed-1 (En1+/-), combined with bilateral injections of pathogenic α-syn fibrils (PFFs), will act to generate a highly relevant PD model - the En1/SYN model. Here, En1+/- mice received bilateral intrastriatal stereotaxic injections of either PBS or α-syn fibrils and were analyzed using automated behavioral tests and deep learning-assisted histological analysis at 2, 4, and 6 months post-injection. We observed significant and progressive Lewy body-like inclusion pathology in the amygdala, motor cortex, and cingulate cortex, as well as the loss of tyrosine hydroxylase-positive (TH+) cells in the substantia nigra. The En1/SYN model also exhibited significant motor impairments at 6 months post-injection, which were however not exacerbated as we had expected. Still, this model has a comprehensive number of PD-like phenotypes and is therefore superior when compared to the α-syn PFF or En1+/- models alone.

EGFR-dependent endocytosis of Wnt9a and Fzd9b promotes ß-catenin signaling during hematopoietic stem cell development in zebrafish.

Cell-to-cell communication through secreted Wnt ligands that bind to members of the Frizzled (Fzd) family of transmembrane receptors is critical for development and homeostasis. Wnt9a signals through Fzd9b, the co-receptor LRP5 or LRP6 (LRP5/6), and the epidermal growth factor receptor (EGFR) to promote early proliferation of zebrafish and human hematopoietic stem cells during development. Here, we developed fluorescently labeled, biologically active Wnt9a and Fzd9b fusion proteins to demonstrate that EGFR-dependent endocytosis of the ligand-receptor complex was required for signaling. In human cells, the Wnt9a-Fzd9b complex was rapidly endocytosed and trafficked through early and late endosomes, lysosomes, and the endoplasmic reticulum. Using small-molecule inhibitors and genetic and knockdown approaches, we found that Wnt9a-Fzd9b endocytosis required EGFR-mediated phosphorylation of the Fzd9b tail, caveolin, and the scaffolding protein EGFR protein substrate 15 (EPS15). LRP5/6 and the downstream signaling component AXIN were required for Wnt9a-Fzd9b signaling but not for endocytosis. Knockdown or loss of EPS15 impaired hematopoietic stem cell development in zebrafish. Other Wnt ligands do not require endocytosis for signaling activity, implying that specific modes of endocytosis and trafficking may represent a method by which Wnt-Fzd specificity is established.