S-nitrosylation of CRTC1 in Alzheimer’s Disease Impairs CREB-dependent Gene Expression Induced by Neuronal Activity

Academic Background

Alzheimer’s disease (AD) is a common neurodegenerative disorder characterized by the gradual loss of memory and cognitive function. The pathological mechanisms of AD are complex, involving various molecular and cellular processes, among which abnormal protein modifications are considered key factors in disease progression. S-nitrosylation, a posttranslational modification mediated by nitric oxide (NO), has been shown to play a significant role in multiple neurodegenerative diseases. However, the specific mechanism of S-nitrosylation in AD remains incompletely understood.

This study focuses on the S-nitrosylation of CREB-regulated transcription coactivator 1 (CRTC1) in AD. In the normal brain, CRTC1 plays a crucial role in regulating gene expression related to neuronal plasticity and memory consolidation. However, in the AD brain, excessive NO-related species disrupt the function of CRTC1 through S-nitrosylation, impairing its interaction with CREB and leading to disrupted gene expression patterns associated with synaptic plasticity and memory. By elucidating this mechanism, our study provides a potential therapeutic target for preserving synaptic function and memory in AD patients.

Source of the Paper

The research was conducted by Xu Zhang, Roman Vlkolinsky, Chongyang Wu, et al., from the Neurodegeneration New Medicines Center, Department of Molecular & Cellular Biology, Department of Translational Medicine at The Scripps Research Institute, and the Department of Neurosciences at the University of California San Diego School of Medicine. The paper was published in PNAS (Proceedings of the National Academy of Sciences) on February 27, 2025, titled “S-nitrosylation of CRTC1 in Alzheimer’s disease impairs CREB-dependent gene expression induced by neuronal activity.”

Research Workflow

1. S-nitrosylation of CRTC1 and Its Manifestation in AD Models

The study initially verified increased S-nitrosylation of CRTC1 (forming SNO-CRTC1) in cell-based, animal-based, and human-induced pluripotent stem cell (hiPSC)-derived AD models. It was found that the formation of SNO-CRTC1 disrupts the binding of CRTC1 with CREB and diminishes the activity-dependent gene expression mediated by the CRTC1/CREB pathway. Using CRISPR/Cas9 techniques, researchers mutated Cys216 to Ala in hiPSC-derived cerebrocortical neurons bearing one allele of the APPswe mutation (an AD-related mutation). Introduction of this non-nitrosylatable CRTC1 mutant significantly improved defects in AD-hiPSC neurons, including decreased neurite length and increased neuronal cell death.

2. Molecular Mechanism of CRTC1 S-nitrosylation

Researchers further identified Cys216 of CRTC1 as the primary target of S-nitrosylation by NO-related species. Site-directed mutagenesis experiments confirmed that Cys216 is the critical site for CRTC1 S-nitrosylation. Additionally, it was discovered that the NO donor SNOC (S-nitrosocysteine) could promote the translocation of CRTC1 from synapses and dendrites into the nucleus, a process dependent on calcium ions and calcineurin activation.

3. Impact of S-nitrosylation on CRTC1 Interaction with CREB

The study showed that the formation of SNO-CRTC1 disrupts the interaction between CRTC1 and CREB, leading to the downregulation of activity-dependent gene expression. Co-immunoprecipitation experiments revealed that while SNO-CRTC1 accumulates in the nucleus, its binding to CREB does not increase. Conversely, high potassium-induced neuronal depolarization significantly enhanced the binding of CRTC1 to CREB.

4. Protective Effects of Non-nitrosylatable CRTC1 Mutant in AD Models

In hiPSC-derived AD neurons, researchers introduced a non-nitrosylatable CRTC1 mutant (Cys216Ala) using CRISPR/Cas9 technology and found that this mutant significantly improved morphological defects and cell death in AD neurons. Moreover, the expression of non-nitrosylatable CRTC1 restored the expression of CREB-dependent genes in AD neurons, including BDNF (brain-derived neurotrophic factor), Arc (activity-regulated cytoskeleton-associated protein), Fos (immediate-early gene), and Egr1 (early growth response protein 1).

5. Therapeutic Effects of Non-nitrosylatable CRTC1 in AD Mouse Models

The study also validated the therapeutic effects of non-nitrosylatable CRTC1 in vivo by overexpressing it in the hippocampus of 5xFAD transgenic AD mice. Results showed that the expression of non-nitrosylatable CRTC1 significantly improved synaptic plasticity and long-term potentiation (LTP) in 5xFAD mice and increased the expression of the synaptic marker synaptophysin.

Key Results

1. Significant Increase in CRTC1 S-nitrosylation in AD Models: Using the biotin-switch assay, researchers detected significantly elevated levels of SNO-CRTC1 in AD mouse models and hiPSC-derived AD neurons.
2. Cys216 Is the Primary Site of CRTC1 S-nitrosylation: Site-directed mutagenesis experiments confirmed that Cys216 is the key site for CRTC1 S-nitrosylation, with approximately 80% reduction in SNO-CRTC1 formation after mutation.
3. SNO-CRTC1 Disrupts CRTC1 Interaction with CREB: Co-immunoprecipitation experiments showed that while SNO-CRTC1 accumulates in the nucleus, its binding to CREB does not increase, leading to the downregulation of CREB-dependent gene expression.
4. Non-nitrosylatable CRTC1 Mutant Improves Morphology and Function of AD Neurons: In hiPSC-derived AD neurons, the non-nitrosylatable CRTC1 mutant significantly improved neurite length and cell survival and restored the expression of CREB-dependent genes.
5. Protective Effects of Non-nitrosylatable CRTC1 in AD Mouse Models: In 5xFAD mice, the expression of non-nitrosylatable CRTC1 significantly improved synaptic plasticity and LTP and increased the expression of synaptic markers.

Conclusion and Significance

This study found that S-nitrosylation of CRTC1 plays a crucial role in the early stages of AD by disrupting the interaction between CRTC1 and CREB, leading to the dysregulation of gene expression related to neuronal plasticity and memory. By introducing a non-nitrosylatable CRTC1 mutant, researchers successfully reversed neuronal defects and synaptic dysfunction in AD models. This discovery provides a new therapeutic target for early intervention in AD, suggesting that inhibiting CRTC1 S-nitrosylation may be an effective treatment strategy.

Research Highlights

1. Revealing a Novel Mechanism of CRTC1 S-nitrosylation in AD: This study first elucidated how S-nitrosylation of CRTC1 disrupts its interaction with CREB, leading to AD-related gene expression disorders.
2. Therapeutic Potential of Non-nitrosylatable CRTC1 Mutant: The non-nitrosylatable CRTC1 mutant introduced via CRISPR/Cas9 technology demonstrated significant neuroprotective effects in both cellular and animal models, offering new directions for AD treatment.
3. Validation Across Multiple Models: The study comprehensively validated CRTC1 S-nitrosylation in cellular, hiPSC-derived neuron, and transgenic mouse models, enhancing the credibility of the research conclusions.

Other Valuable Information

The study also found that S-nitrosylation of CRTC1 might lead to synaptic loss and cognitive decline by downregulating BDNF expression. By restoring BDNF expression, the non-nitrosylatable CRTC1 mutant significantly improved synaptic function and cognitive performance in AD models. This finding further supports the potential application value of BDNF in AD treatment.