Membrane contact sites between virtually any known organelle have been documented and, in the last decades, their study received momentum due to their importance for fundamental activities of the cell and for the subtle comprehension of many human diseases. The lack of tools to finely image inter-organelle proximity hindered our understanding on how these subcellular communication hubs mediate and regulate cell homeostasis. We develop an improved and expanded palette of split-GFP-based contact site sensors (SPLICS) for the detection of single and multiple organelle contact sites within a scalable distance range. We demonstrate their flexibility under physiological conditions and in living organisms.

Significance: The expression of genes involved in hippocampal synaptic plasticity, learning, and memory requires that Ca²⁺signals generated in spines, dendrites, or the soma by neuronal stimulation reach the nucleus. Here, we report that stimulation of hippocampal neurons induces Ca²⁺release mediated by RyR2 channels, which contributes to nuclear Ca²⁺signal generation. Suppression of RyR-mediated Ca²⁺release inhibited the activity-induced phosphorylation of the nuclear transcriptional regulator CREB and the expression of the Npas4 transcription factor and RyR2, which play crucial roles in hippocampal memory processes. We propose that RyR-mediated Ca²⁺release induced by neuronal stimulation, by promoting the sequential generation of nuclear Ca²⁺signals, CREB phosphorylation, and Npas4/RyR2 up-regulation, plays a significant role in hippocampal synaptic plasticity and spatial memory processes.

Nuclear calcium is an important signaling end-point in synaptic excitation-transcription coupling that is critical for long-term neuroadaptations. Here we show that nuclear calcium acting via a target gene VEGFD is required for hippocampus-dependent fear memory consolidation and extinction in mice. Nuclear calcium-VEGFD signaling upholds structural integrity and complexity of the dendritic arbor of CA1 neurons that renders those cells permissive for the efficient generation of synaptic input-evoked nuclear calcium transients driving the expression of plasticity-related genes. Thus, the gating of memory functions rests on the reciprocally reinforcing maintenance of an intact dendrite geometry and a functional synapse-to-nucleus communication axis. In psychiatric and neurodegenerative disorders, therapeutic application of VEGFD may help stabilize dendritic structures and network connectivity, which may prevent cognitive decline and could boost the efficacy of extinction-based exposure therapies. SIGNIFICANCE STATEMENT This study uncovers a reciprocal relationship between dendrite geometry, the ability to generate nuclear calcium transients in response to synaptic inputs, and the subsequent induction of expression of plasticity-related and dendritic structure-preserving genes. Insufficient nuclear calcium signaling in CA1 hippocampal neurons and, consequently, reduced expression of the nuclear calcium target gene, VEGFD, a dendrite maintenance factor, leads to reduced-complexity basal dendrites of CA1 neurons, which severely compromises the animals9 consolidation of both memory and extinction memory. The structure-protective function of VEGFD may prove beneficial in psychiatric disorders as well as neurodegenerative and ageing-related conditions that are associated with loss of neuronal structures, dysfunctional excitation-transcription coupling, and cognitive decline.

Ferroptosis, a newly described form of regulated cell death, is characterized by the iron-dependent accumulation of lipid peroxides, glutathione depletion, mitochondrial alterations, and enhanced lipoxygenase activity. Inhibition of glutathione peroxidase 4 (GPX4), a key intracellular antioxidant regulator, promotes ferroptosis in different cell types. Scant information is available on GPX4-induced ferroptosis in hippocampal neurons. Moreover, the role of calcium (Ca²⁺) signaling in ferroptosis remains elusive. Here, we report that RSL3, a selective inhibitor of GPX4, caused dendritic damage, lipid peroxidation, and induced cell death in rat primary hippocampal neurons. Previous incubation with the ferroptosis inhibitors deferoxamine or ferrostatin-1 reduced these effects. Likewise, preincubation with micromolar concentrations of ryanodine, which prevent Ca²⁺ release mediated by Ryanodine Receptor (RyR) channels, partially protected against RSL3-induced cell death. Incubation with RSL3 for 24 h suppressed the cytoplasmic Ca²⁺ concentration increase induced by the RyR agonist caffeine or by the SERCA inhibitor thapsigargin and reduced hippocampal RyR2 protein content. The present results add to the current understanding of ferroptosis-induced neuronal cell death in the hippocampus and provide new information both on the role of RyR-mediated Ca²⁺ signals on this process and on the effects of GPX4 inhibition on endoplasmic reticulum calcium content.

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder, characterized by cognitive decline and neuronal degeneration. The formation of amyloid β plaques and neurofibrillary tangles are key morphological features of AD pathology. However, the specific molecules responsible for the cell destruction triggered by amyloid β and tau proteinopathies in AD has not yet been identified. Here we use the 5xFAD mouse model of AD to investigate the role of a recently discovered death signaling complex which consists of the extrasynaptic N-methyl-D-aspartate receptor (NMDAR) and the transient receptor potential cation channel subfamily M member 4 (TRPM4). The NMDAR/TRPM4 death complex is responsible for toxic signaling of glutamate, which has been implicated in AD pathogenesis. We detected an increase in NMDAR/TRPM4 death complex formation in the brains of 5xFAD mice. This increase was blocked by the oral application of FP802, a small molecule TwinF interface inhibitor that can disrupt and thereby detoxify the NMDAR/TRPM4 death complex. FP802 treatment prevented the cognitive decline of 5xFAD mice assessed using a series of memory tasks. It also preserved the structural complexity of dendrites, prevented the loss of synapses, reduced amyloid β plaque formation, and protected against pathological alterations of mitochondria. These results identify the NMDAR/TRPM4 death complex as a major promoter of AD disease progression, amplifying potentially self-perpetuating pathological processes initiated by amyloid β. TwinF interface inhibitors offer a novel therapeutic avenue, serving as an alternative or complementary treatment to antibody-mediated clearing of amyloid β from AD brains.