Periodic Reporting for period 1 - FIXNALCN (Exploring neuronal functions of NALCN in health and disease)
Reporting period: 2023-07-01 to 2025-06-30
Every living cell relies on a tiny electrical gradient across its membrane to function. This gradient, known as the resting membrane potential, regulates essential processes, such as communication between neurons, muscle contraction, cell growth, and even breathing. One of the key players in maintaining the membrane potential is NALCN, a sodium leak channel that helps maintain the delicate balance of ions across the cell membrane. When NALCN does not work properly, children develop rare but severe neurological disorders, including developmental delay, low muscle tone, and breathing irregularities. Interestingly, both gain-of-function (GOF, overactive channel) and loss-of-function (LOF, underactive channel) mutations in NALCN lead to very similar symptoms, despite their opposite effects on the channel itself. This makes it extremely difficult to understand the disease mechanism and to design effective treatments, which are currently not available for patients.
The overall aim of this project was to understand how patient variants in the sodium leak channel NALCN alter neuronal activity and to explore ways to correct these defects. First, we generated human stem cell models carrying both GOF and LOF NALCN variants. We then studied how these changes affected the electrical properties and communication of neurons. In the near future, we will we test newly developed compounds designed to restore normal NALCN function, laying groundwork for future therapeutic strategies.
The overall aim of this project was to understand how patient variants in the sodium leak channel NALCN alter neuronal activity and to explore ways to correct these defects. First, we generated human stem cell models carrying both GOF and LOF NALCN variants. We then studied how these changes affected the electrical properties and communication of neurons. In the near future, we will we test newly developed compounds designed to restore normal NALCN function, laying groundwork for future therapeutic strategies.
The project aimed to understand how GOF and LOF NALCN mutations impact human cellular physiology and to explore potential pharmacological interventions. The first objective was to generate humand induced pluripotent stem cell (hiPSC) models of NALCN GOF and LOF patient mutations. This was achieved, as hiPSC lines were successfully generated from both CRISPR-edited and patient-derived cells, validated, and differentiated into neurons. The second objective focused on characterizing the functional impact of NALCN mutations on neuronal activity. Electrophysiological recordings revealed variant-specific effects on resting membrane potential, action potential threshold and amplitude, and overall neuronal firing patterns. The third objective aimed to identify and test modulators capable of rescuing NALCN dysfunction. Novel modulators were engineered by the laboratory and validated in heterologous systems, with neuronal testing planned. Overall, key milestones in cell model generation and preliminary functional analysis were achieved, and the remaining computational simulations and neuronal testing of modulators were expected to complete the objectives within or just beyond the project timeline.
This project established the first-ever human disease models for NALCN GOF and LOF variants using patient-derived and CRISPR-engineered hiPSCs. These models overcome limitations of animal systems and allow the study of NALCN-related disorders in a physiologically relevant human context. Differentiated neurons revealed variant-specific effects on excitability, clarifying how opposite molecular mutations can lead to overlapping patient symptoms.
The laboratory also developed the first de novo NALCN binders, including engineered linker constructs and miniproteins, which selectively modulate channel activity in heterologous systems. These tools provide a platform for pharmacological rescue.
Overall, these advances combine human stem cell disease modeling, functional characterization, and pharmacological intervention, enabling both mechanistic insight and translational opportunities for therapeutic development in NALCN-related diseases.
The laboratory also developed the first de novo NALCN binders, including engineered linker constructs and miniproteins, which selectively modulate channel activity in heterologous systems. These tools provide a platform for pharmacological rescue.
Overall, these advances combine human stem cell disease modeling, functional characterization, and pharmacological intervention, enabling both mechanistic insight and translational opportunities for therapeutic development in NALCN-related diseases.