Rapid Behavioral Pharmacology Reveals Disrupted Signaling Pathways and Candidate Therapeutics from Zebrafish Mutants of Alzheimer's Disease Risk Genes

Core Concepts

Combining rapid loss-of-function mutagenesis of Alzheimer's risk genes and behavioral pharmacology in zebrafish can predict disrupted biological processes and identify candidate therapeutics.

Abstract

The authors demonstrate a strategy to rapidly link Alzheimer's disease (AD) risk genes to druggable biological processes using zebrafish. They first identified that around 75% of human AD risk genes have clear orthologues in zebrafish, and most are expressed early in larval brain development.
The authors then developed a high-throughput video-tracking system and analysis pipeline (FramebyFrame) to measure sleep/wake behaviors of zebrafish larvae with F0 knockouts of AD risk genes at high temporal resolution. This revealed distinct behavioral phenotypes for different gene knockouts:

psen2 knockouts were less active and slept more during the day
sorl1 knockouts were less active during the day but more active and slept less at night
Knockouts of late-onset AD risk genes (apoea/apoeb, cd2ap, clu, sorl1) had common effects of increased nighttime activity and reduced sleep
The authors suggest these behavioral changes reflect disruption of specific biological processes that contribute to AD pathogenesis. As proof-of-concept, they showed that sorl1 mutants have disrupted serotonin signaling, and identified betamethasone as a drug that can normalize the sleep phenotype of presenilin-2 knockouts.
This behavioral pharmacology approach provides a general framework to rapidly link disease-associated genes to druggable pathways, which could accelerate the discovery of new therapeutic targets.

Stats

Across F0 knockout samples for psen1, 99.0 ± 2.7% of reads were mutated and 78.6 ± 29.7% of all reads had a frameshift mutation.
Across F0 knockout samples for psen2, 99.9 ± 0.1% of reads were mutated and 82.0 ± 33.6% of all reads had a frameshift mutation.
Across F0 knockout samples for sorl1, 97.7 ± 4.2% of reads were mutated and 80.1 ± 20.8% of all reads had a frameshift mutation.

Quotes

"Predictive behavioural pharmacology offers a general framework to rapidly link disease-associated genes to druggable pathways."
"psen2 F0 knockout larvae were substantially less active and sleeping more than controls during the day."
"sorl1 F0 knockout larvae were less active during the day but more active and slept less at night."

Key Insights Distilled From

Behavioural pharmacology predicts disrupted signalling pathways and candidate therapeutics from zebrafish mutants of Alzheimer’s disease risk genes

by Krol... at www.biorxiv.org 11-29-2023

https://www.biorxiv.org/content/10.1101/2023.11.28.568940v1

Deeper Inquiries

How could this behavioral pharmacology approach be extended to other complex diseases beyond Alzheimer's?

This behavioral pharmacology approach can be extended to other complex diseases by identifying disease-associated genes through genomic studies and then using rapid loss-of-function mutagenesis in zebrafish to predict disrupted processes and potential therapeutics. By selecting genes associated with other diseases and generating F0 knockouts in zebrafish, researchers can analyze the behavioral phenotypes to uncover disrupted pathways and potential drug targets. This approach can be applied to a wide range of diseases by adapting the gene selection criteria and utilizing the behavioral analysis tools developed for zebrafish larvae. Additionally, the online tool ZOLTAR, which compares behavioral fingerprints to a library of compounds, can be expanded to include compounds relevant to other diseases, enabling the prediction of candidate therapeutics for various conditions.

What are the potential limitations or caveats of using zebrafish larvae as a model system to study Alzheimer's disease pathogenesis?

While zebrafish larvae offer numerous advantages as a model system for studying Alzheimer's disease pathogenesis, there are also some limitations and caveats to consider. One limitation is the evolutionary distance between zebrafish and humans, which may result in differences in disease mechanisms and responses to treatments. Additionally, zebrafish larvae have a simpler brain structure compared to humans, which may not fully capture the complexity of Alzheimer's disease pathogenesis. Another caveat is the potential for off-target effects when using CRISPR-Cas9 to generate F0 knockouts, which could lead to unintended mutations and phenotypic variations. Furthermore, the behavioral assays conducted on zebrafish larvae may not fully represent the cognitive and memory deficits characteristic of Alzheimer's disease in humans. It is essential to consider these limitations when interpreting results from zebrafish studies and to complement findings with data from other model systems and clinical studies.

What other types of high-throughput phenotyping assays could be combined with this strategy to provide a more comprehensive understanding of the biological consequences of Alzheimer's risk gene disruption?

In addition to the behavioral pharmacology approach using zebrafish larvae, other high-throughput phenotyping assays can be combined to gain a more comprehensive understanding of the biological consequences of Alzheimer's risk gene disruption. One potential assay is transcriptomics, which can provide insights into gene expression changes associated with the knockout of Alzheimer's risk genes. By analyzing the transcriptome of zebrafish larvae with disrupted genes, researchers can identify dysregulated pathways and molecular mechanisms underlying the observed behavioral phenotypes. Another valuable assay is proteomics, which can reveal alterations in protein expression and post-translational modifications that contribute to the disease phenotype. By integrating transcriptomic and proteomic data with behavioral analysis, researchers can uncover the molecular pathways affected by Alzheimer's risk gene disruption and identify potential therapeutic targets. Additionally, advanced imaging techniques, such as confocal microscopy and functional imaging, can provide detailed insights into the structural and functional changes in the brain of zebrafish larvae with Alzheimer's risk gene mutations. By combining multiple high-throughput assays, researchers can obtain a more holistic view of the biological consequences of Alzheimer's risk gene disruption and accelerate the discovery of novel therapeutic strategies.