Yale Scientists Study Death of Neurons Using "Knockout" Mice, Develop New Strategy for Treating Stroke, Alzheimer's Disease

Using “knockout” mice – a strain of mice in which a single gene is disabled in order to observe its role during development – researchers at the Yale School of Medicine and Vertex Pharmaceuticals have discovered that an enzyme called Caspase-9 plays a key role in the genetically programmed death of neurons. Blocking the enzyme could become a viable strategy for treating Alzheimer’s disease, Parkinson’s disease, strokes and other age-related neurological diseases, the researchers suggest.

Published in the Aug. 7 issue of the journal Cell, the research was conducted by Drs. Richard A. Flavell, a Howard Hughes Medical Institute investigator at Yale; Pasko Rakic, Tarik Haydar and Chai-Yi Kuan of the Yale School of Medicine; Choji Taya and Hajime Karasuyama of Tokyo Metropolitan Institute of Medical Sciences; and Keisuke Kuida, Yong Gu and Michael Su, all of Vertex Pharmaceuticals.

Caspases are a family of enzymes that are thought to play a role in a number of diseases, based on their involvement in biochemical pathways of inflammation. Caspases also are important in apoptosis, which is programmed cell death, or cell suicide, that is essential in many normal biological processes, including tissue remodeling, immune system regulation and embryonic development.

Abnormal activation of apoptosis, however, is implicated in many human diseases, and links between specific caspases and specific diseases are beginning to be established. The Cell paper is the first to describe the in vivo function of Caspase-9, and suggests apparent tissue-selective activity among the 11 reported human caspases.

“When mitochondria, the energy factories of cells, are damaged, Caspase-9 is activated, leading to cell death. In cells lacking Caspase-9, this damage did not give rise to cell death,” said Flavell, a Yale professor of immunobiology and biology.

Kuida, a staff investigator at Vertex, said: “Our results also indicate that Caspase-9 is activated early and is essential for apoptosis in neuronal cells, and that deletion of Caspase-9 does not interfere with embryonic development of non-neuronal tissues. This suggests that a therapeutic approach designed to interrupt the apoptotic pathway triggered by Caspase-9 could block unwanted cell death linked to several neurological diseases.”

The researchers inserted a defective Caspase-9 gene into mice and then bred them to produce offspring with two defective copies of the gene. Biochemical experiments demonstrated that neuronal apoptosis was specifically blocked in the transgenic mice deficient in Caspase-9.

Experiments also suggested that Caspase-9 is active upstream of Caspase-3 in the neuronal apoptosis pathway. The Caspase-9 knockout mice showed alterations in central nervous system development that were more profound than those observed in Caspase-3 knockout mice. Both enzymes appear to be selectively active in neuronal tissues during development.

“The balance between cell production and cell death is important for normal brain development,” said Rakic, professor of neurobiology and neurology at the Yale School of Medicine. “Too much or too little cell death can cause severe malformations leading to disorders such as mental retardation and childhood epilepsy. This study shows that Caspase-9 is essential for cell death and, therefore, gives new insight into how the brain develops in normal and pathological conditions.”

The researchers noted that removing the Caspase-9 enzyme prevented brain cells from dying during the early stages of mouse development. By clarifying the relationship between caspases and establishing their activity in body tissues, the research will help scientists better identify which caspases are appropriate targets for drug discovery and development.

Vertex and Yale/HHMI researchers were the first to describe the biochemical and physiological effects of the ICE (Caspase-1) enzyme through a gene knockout mouse experiment, and Vertex researchers were the first to elucidate the three-dimensional atomic structure of ICE. Dr. Kuida, together with Flavell and Rakic of Yale, reported creating the first Caspase-3 gene knockout mouse, while Vertex researchers solved the atomic structure of the enzyme. Later this year, Vertex plans to begin Phase I clinical trials of an ICE inhibitor targeting inflammatory diseases.