CPT-cAMP Restores Lysosomal Acidification and Reduces Seizures in ATP6V1B2 Mutant Mice
Background
The cyclic adenosine monophosphate (cAMP) analogue CPT-cAMP, which improved lysosomal acidification defects in ATP6V1B2-mutant cells, also improved seizure and cognitive behavioral measures in mutant mice. These are preclinical findings examining therapeutic potential in cells and animals; the study did not establish treatment efficacy in humans.
ATP6V1B2 encodes a protein component of vacuolar H+-ATPase (V-ATPase), a proton pump that keeps the lysosomal interior acidic. Pathogenic variants in this gene can be associated with neurological symptoms, including intellectual disability and seizures. The researchers analyzed clinical data from five families in China alongside previously published reports and investigated whether a drug could alleviate lysosomal acidification defects in gene-edited cells and mutant mice.
Key findings
For the cell experiments, the team used HEK293T cells edited with CRISPR/Cas9 to introduce the ATP6V1B2 R506X variant. Mean lysosomal pH was 4.26 in wild-type cells, 4.59 in heterozygous mutant cells, and 4.90 in homozygous mutant cells. A higher pH indicates lower acidity inside the lysosomes.
CPT-cAMP treatment lowered lysosomal pH in mutant cells in a concentration-dependent manner. A significant effect was observed at concentrations of 100 nM and above in heterozygous mutant cells; at 1 μM, pH fell by as much as 0.7 units. Autophagy-related proteins and electron microscopy findings also supported improved intracellular degradation and clearance functions.
Homozygous mutant mice received CPT-cAMP at 20 mg/kg by intraperitoneal injection once a week from 4 to 12 weeks of age. During continuous 72-hour electroencephalographic monitoring at 12 weeks of age, spontaneous seizures occurred in 4 of 6 mice that did not receive CPT-cAMP, whereas none were detected in the 6 treated mice. Seizure severity also decreased in experiments using a seizure-inducing substance, and improved performance was observed in object recognition, passive avoidance, and spatial learning tests. These results are limited to the experimental conditions and observation period and do not mean that seizures were cured.
Implications and outlook
The study suggests that targeting lysosomal acidification defects could be a candidate therapeutic strategy for ATP6V1B2-related neurological disorders. However, the detailed pathways linking the pH changes observed in cells to behavioral improvements in mice require further investigation. The explanation that CPT-cAMP promotes V-ATPase assembly also cannot be regarded as a mechanism directly established by this study.
Lysosomal pH was not measured directly in the mouse brain, and reproducibility in neurons derived from human induced pluripotent stem cells has not been confirmed. Long-term toxicity, the risk of excessive acidification, and whether the effects extend to other ATP6V1B2 variants remain to be investigated.
Source: Article published in Cell Death Discovery.
Pathogenic variants in ATP6V1B2, which encodes a critical subunit of vacuolar-type H+-ATPases (V-ATPases), disrupt lysosomal acidification via haploinsufficiency and clinically manifest as intellectual disability and seizure disorders. Despite significant morbidity, mechanism-based therapies remain an unmet need. Through integrated clinical analysis of a Chinese cohort and systematic literature review, we delineated genotype-phenotype correlations in ATP6V1B2-related syndromes. Isogenic HEK293T models (ATP6V1B2
These findings provide a basis for assessing candidate compounds for ATP6V1B2-related neurological disorders using lysosomal pH together with seizure and cognitive behavioral measures. In subsequent development, CPT-cAMP could be tested in neurons generated from patient-derived induced pluripotent stem cells to determine whether improvements in acidification and degradation functions can be reproduced. Long-term dosing toxicity, an appropriate dose, and direct pH changes within the brain would then need to be assessed. These results alone cannot determine a dose or establish safety for patients, and effects across different variants also require separate evaluation.