Brief and diverse excitotoxic insults increase the neuronal nuclear membrane permeability in the neonatal brain, resulting in neuronal dysfunction and cell death.

Abstract

Neuronal cytotoxic edema is implicated in neuronal injury and death, yet mitigating brain edema with osmotic and surgical interventions yields poor clinical outcomes. Importantly, neuronal swelling and its downstream consequences during early brain development remain poorly investigated, and new treatment approaches are needed. We explored Ca²⁺-dependent downstream effects after neuronal cytotoxic edema caused by diverse injuries in mice of both sexes using multiphoton Ca²⁺ imaging in vivo (P12-17) and in acute brain slices (P8-12). After different excitotoxic insults, cytosolic GCaMP6s translocated into the nucleus after a few minutes in a subpopulation of neurons, persisting for hours. We used an automated morphology-detection algorithm to detect neuronal soma and quantified the nuclear translocation of GCaMP6s as the nuclear to cytosolic intensity (N/C ratio). Elevated neuronal N/C ratios occurred concurrently with persistent elevation in Ca²⁺ loads and could also occur independently from neuronal swelling. Electron microscopy revealed that the nuclear translocation was associated with increased nuclear pore size. The nuclear accumulation of GCaMP6s in neurons led to neocortical circuit dysfunction, mitochondrial pathology, and increased cell death. Inhibiting calpains, a family of Ca²⁺-activated proteases, prevented elevated N/C ratios and neuronal swelling. In summary, in the developing brain, we identified a calpain-dependent alteration of nuclear transport in a subpopulation of neurons after disease-relevant insults leading to long-term circuit dysfunction and cell death. The nuclear translocation of GCaMP6 and other cytosolic proteins after acute excitotoxicity can be an early biomarker of brain injury in the developing brain.

Significance Statement Our study demonstrates that different insults that model diverse neurological conditions enlarge the neuronal nuclear pores in the developing brain. This enlargement of nuclear pores caused a rapid nuclear translocation of cytosolically localized proteins like GCaMP6s. The increased nuclear translocation in neurons was associated with attenuated neocortical circuit activity and neuronal death. Excitotoxicity-induced nuclear translocation of GCaMP6s and neuronal swelling was prevented by calpain inhibition. These findings have clinical implications for treating brain injury and neuronal swelling during early brain development, which currently lack direct pharmacological treatments. Since GCaMP6 variants are widely used in neuroscience, their abnormal nuclear translocation can be used to study early neuronal injury.