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.

Focal swellings of dendrites (“dendritic blebbing”) together with structural damage of mitochondria and the endoplasmic reticulum (ER) are morphological hallmarks of glutamate neurotoxicity, also known as excitotoxicity. These pathological alterations are generally thought to be caused by the so-called “overactivation” of N-methyl-D-aspartate receptors (NMDARs). Here, we demonstrate that the activation of extrasynaptic NMDARs, specifically when forming a protein–protein complex with TRPM4, drives these pathological traits. In contrast, strong activation of synaptic NMDARs fails to induce cell damage despite evoking plateau-type calcium signals that are comparable to those generated by activation of the NMDAR/TRPM4 complex, indicating that high intracellular calcium levels per se are not toxic to neurons. Using confocal laser scanning microscopy and transmission electron microscopy, we show that disrupting the NMDAR/TRPM4 complex using the recently discovered small-molecule TwinF interface inhibitor FP802 inhibits the NMDA-induced neurotoxicity-associated dendritic blebbing and structural damage to mitochondria and the ER. It also prevents, at least in part, the disruption of ER–mitochondria contact sites. These findings establish the NMDAR/TRPM4 complex as the trigger for the structural damage of dendrites and intracellular organelles associated with excitotoxicity. They also suggest that activation of the NMDAR/TRPM4 complex, in addition to inducing high-amplitude, plateau-type calcium signals, generates a second signal required for glutamate neurotoxicity (“two-hit hypothesis”). As structural damage to organelles, particularly mitochondria, is a common feature of many human neurodegenerative diseases, including Alzheimer’s disease and amyotrophic lateral sclerosis (ALS), TwinF interface inhibitors have the potential to provide neuroprotection across a broad spectrum of these diseases.

The brain constantly adapts to environmental changes by modifying the expression of genes that enable synaptic plasticity, learning and memory. The expression of several of these genes requires nuclear calcium (Ca2+) signals, which in turn requires that Ca2+ signals generated by neuronal activity at the synapses or the soma propagate to the nucleus. Since cytoplasmic Ca2+ diffusion is highly restricted, Ca2+ signal propagation to the nucleus requires the participation of other cellular mechanisms. The inositol trisphosphate receptor (IP3R) and the ryanodine receptor (RyR) channels, both of which reside in the endoplasmic reticulum (ER) membrane, play key roles in cellular Ca2+ signal generation. Yet, their roles in the generation of nuclear and mitochondrial Ca2+ signals induced by neuronal activity require further investigation. Here, the impact of IP3R1 or RyR2 knockdown on gabazine-induced nuclear and mitochondrial Ca2+ signals in neurons was evaluated. To this aim, recombinant adeno-associated viruses (rAAVs) were used to introduce small hairpin RNAs (shRNAs) to knockdown type-1 (IP3R1) and type-2 (RyR2) channel expression in cultured rat hippocampal neurons. Additionally, synaptic contact numbers were assessed through immunocytochemistry. Knockdown of IP3R1 or RyR2 channels significantly reduced their protein contents and the generation of gabazine-induced nuclear and mitochondrial Ca2+ signals, without altering synaptic contact numbers. Our results highlight the contribution of IP3R1 and RyR2 channels to the generation of nuclear and mitochondrial Ca2+ signal induced by neuronal activity, reinforcing the role that these Ca2+ release channels play in hippocampal synaptic plasticity and memory formation.

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.

The expression of several hippocampal genes implicated in learning and memory processes requires that Ca2+ signals generated in dendritic spines, dendrites, or the soma in response to neuronal stimulation reach the nucleus. The diffusion of Ca2+ in the cytoplasm is highly restricted, so neurons must use other mechanisms to propagate Ca2+ signals to the nucleus. Here, we present evidence showing that Ca2+ release mediated by the ryanodine receptor (RyR) channel type-2 isoform (RyR2) contributes to the generation of nuclear Ca2+ signals induced by gabazine (GBZ) addition, glutamate uncaging in the dendrites, or high-frequency field stimulation of primary hippocampal neurons. Additionally, GBZ treatment significantly increased cyclic adenosine monophosphate response element binding protein (CREB) phosphorylation-a key event in synaptic plasticity and hippocampal memory-and enhanced the expression of Neuronal Per Arnt Sim domain protein 4 (Npas4) and RyR2, two central regulators of these processes. Suppression of RyR-mediated Ca2+ release with ryanodine significantly reduced the increase in CREB phosphorylation and the enhanced Npas4 and RyR2 expression induced by GBZ. We propose that RyR-mediated Ca2+ release induced by neuronal activity, through its contribution to the sequential generation of nuclear Ca2+ signals, CREB phosphorylation, Npas4, and RyR2 up regulation, plays a central role in hippocampal synaptic plasticity and memory processes.

Neuronal Ca2+ signals are fundamental for synaptic transmission and activity-dependent changes in gene expression. Voltage-gated Ca2+ channels and N-methyl-d-aspartate receptors play major roles in mediating external Ca2+ entry during action potential firing and glutamatergic activity. Additionally, the inositol-1,4,5-trisphosphate receptor (IP3R) and the ryanodine receptor (RyR) channels expressed in the endoplasmic reticulum (ER) also contribute to the generation of Ca2+ signals in response to neuronal activity. The ER forms a network that pervades the entire neuronal volume, allowing intracellular Ca2+ release in dendrites, soma and presynaptic boutons. Despite its unique morphological features, the contributions of ER structure and of ER-shaping proteins such as atlastin - an ER enriched GTPase that mediates homotypic ER tubule fusion - to the generation of Ca2+ signals in dendrites remains unreported. Here, we investigated the contribution of RyR-mediated Ca2+ release to IP3-generated Ca2+ signals in dendrites of cultured hippocampal neurons. We also employed GTPase activity-deficient atlastin-2 (ATL2) mutants to evaluate the potential role of atlastin on Ca2+ signaling and ER-resident Ca2+ channel distribution. We found that pharmacological suppression of RyR channel activity increased the rising time and reduced the magnitude and propagation of IP3-induced Ca2+ signals. Additionally, ATL2 mutants induced specific ER morphological alterations, delayed the onset and increased the rising time of IP3-evoked Ca2+ signals, and caused RyR2 and IP3R1 aggregation and RyR2 redistribution. These results indicate that both RyR and ATL2 activity regulate IP3-induced Ca2+ signal dynamics through RyR-mediated Ca2+-induced Ca2+ release, ER shaping and RyR2 distribution.

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.

Hepatitis C virus (HCV) RNA replication occurs in tight association with remodeled host cell membranes, presenting as cytoplasmic accumulations of single-, double-, and multimembrane vesicles in infected cells. Formation of these so-called replication organelles is mediated by a complex interplay of host cell factors and viral replicase proteins. Of these, nonstructural protein 4B (NS4B), an integral transmembrane protein, appears to play a key role, but little is known about the molecular mechanisms of how this protein contributes to organelle biogenesis. Using forward and reverse genetics, we identified glycine zipper motifs within transmembrane helices 2 and 3 of NS4B that are critically involved in viral RNA replication. Foerster resonance energy transfer analysis revealed the importance of the glycine zippers in NS4B homo- and heterotypic self-interactions. Additionally, ultrastructural analysis using electron microscopy unraveled a prominent role of glycine zipper residues for the subcellular distribution and the morphology of HCV-induced double-membrane vesicles. Notably, loss-of-function NS4B glycine zipper mutants prominently induced single-membrane vesicles with secondary invaginations that might represent an arrested intermediate state in double-membrane vesicle formation. These findings highlight a so-far-unknown role of glycine residues within the membrane integral core domain for NS4B self-interaction and functional as well as structural integrity of HCV replication organelles. IMPORTANCE Remodeling of the cellular endomembrane system leading to the establishment of replication organelles is a hallmark of positive-strand RNA viruses. In the case of HCV, expression of the nonstructural proteins induces the accumulation of double-membrane vesicles that likely arise from a concerted action of viral and coopted cellular factors. However, the underlying molecular mechanisms are incompletely understood. Here, we identify glycine zipper motifs within HCV NS4B transmembrane segments 2 and 3 that are crucial for the protein's self-interaction. Moreover, glycine residues within NS4B transmembrane helices critically contribute to the biogenesis of functional replication organelles and, thus, efficient viral RNA replication. These results reveal how glycine zipper motifs in NS4B contribute to structural and functional integrity of the HCV replication organelles and, thus, viral RNA replication.

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 the 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. Therefore, 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 to 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 animals' consolidation of both memory and extinction memory. The structure-protective function of VEGFD may prove beneficial in psychiatric disorders as well as neurodegenerative and aging-related conditions that are associated with loss of neuronal structures, dysfunctional excitation-transcription coupling, and cognitive decline.

CDK5 is a serine/threonine kinase that is involved in the normal function of the adult brain and plays a role in neurotransmission and synaptic plasticity. However, its over-regulation has been associated with Tau hyperphosphorylation and cognitive deficits. Our previous studies have demonstrated that CDK5 targeting using shRNA-miR provides neuroprotection and prevents cognitive deficits. Dendritic spine morphogenesis and forms of long-term synaptic plasticity—such as long-term potentiation (LTP)—have been proposed as essential processes of neuroplasticity. However, whether CDK5 participates in these processes remains controversial and depends on the experimental model. Using wild-type mice that received injections of CDK5 shRNA-miR in CA1 showed an increased LTP and recovered the PPF in deficient LTP of APPswe/PS1Δ9 transgenic mice. On mature hippocampal neurons CDK5, shRNA-miR for 12 days induced increased dendritic protrusion morphogenesis, which was dependent on Rac activity. In addition, silencing of CDK5 increased BDNF expression, temporarily increased phosphorylation of CaMKII, ERK, and CREB; and facilitated calcium signaling in neurites. Together, our data suggest that CDK5 downregulation induces synaptic plasticity in mature neurons involving Ca2+ signaling and BDNF/CREB activation.

In neurons, secretory organelles within the cell body are complemented by the dendritic endoplasmic reticulum (ER) and Golgi outposts (GOps), whose role in neurotransmitter receptor trafficking is poorly understood. γ-aminobutyric acid (GABA) type B metabotropic receptors (GABABRs) regulate the efficacy of synaptic transmission throughout the brain. Their plasma membrane availability is controlled by mechanisms involving an ER retention motif and assembly-dependent ER export. Thus, they constitute an ideal molecular model to study ER trafficking, but to what extent the dendritic ER participates in GABABR biosynthesis has not been thoroughly explored. Here we show that GABAB1 localizes preferentially to the ER in dendrites and moves long distances within this compartment. Not only diffusion, but also microtubule and dynein-dependent mechanisms control dendritic ER transport. GABABRs insert throughout the somatodendritic plasma membrane but dendritic post-ER carriers containing GABABRs do not fuse selectively with GOps. This study contributes to clarify the spatial selectivity of neurotransmitter receptors for dendritic organelles.

The synaptic efficacy of neurons depends on the number of neurotransmitter receptors in the plasma membrane. The availability of these receptors is controlled by their specific intracellular trafficking routes. γ-Aminobutyric acid type B receptors (GABABRs) are heteromeric proteins consisting of GABABR1 and GABABR2 subunits. These receptors are found at the plasma membrane of somatodendritic postsynaptic sites and in axons. It is unknown whether the assembly of the subunits occurs directly in the somatic endoplasmic reticulum (ER) followed by vesicular transport, or whether the assembly occurs after the separate transport of the subunits to the dendritic ER compartment. To address this question we have studied the assembly of the GABABRs in hippocampal neurons with dual-color, 3D super-resolution optical fluctuation imaging (SOFI). SOFI is a fluorescence imaging modality which yields superresolved spatial resolution, 3D-sectioning and high image contrast. We will use the SOFI images to quantify the distribution of the GABABR subunits in the plasma membrane and in the dendritic intracellular compartments. Finally, we want to apply quantitative co-localization analysis to determine the compartments in which the assembly of the GABABR subunits occurs.

Optical flow approaches calculate vector fields which determine the apparent velocities of objects in time-varying image sequences. They have been analyzed extensively in computer science using both natural and synthetic video sequences. In life sciences, there is an increasing need to extract kinetic information from temporal image sequences which reveals the interplay between form and function of microscopic biological structures. In this work, we test different variational optical flow techniques to quantify the displacements of biological objects in 2D fluorescent image sequences. The accuracy of the vector fields is tested for defined displacements of fluorescent point sources in synthetic image series which mimic protein traffic in neuronal dendrites, and for GABABR1 receptor subunits in dendrites of hippocampal neurons. Our results reveal that optical flow fields predict the movement of fluorescent point sources within an error of 3% for a maximum displacement of 160 nm. Displacement of agglomerated GABABR1 receptor subunits can be predicted correctly for maximum displacements of 640 nm. Based on these results, we introduce a criteria to derive the optimum parameter combinations for the calculation of the optical flow fields in experimental images. From these results, temporal sampling frequencies for image acquisition can be derived to guarantee correct motion estimation for biological objects.

The formation of the nervous systems requires processes that coordinate proliferation, differentiation and migration of neuronal cells, which extend axons, generate dendritic branching and establish synaptic connections during development. The structural organization and dynamic remodeling of the cytoskeleton and its association to the secretory pathway are critical determinants of cell morphogenesis and migration. Marlin-1 (Jakmip1) is a microtubule-associated protein predominantly expressed in neurons and lymphoid cells. Marlin-1 participates in polarized secretion in lymphocytes, but its functional association with the neuronal cytoskeleton and its contribution to brain development have not been explored. Combining in vitro and in vivo approaches we show that Marlin-1 contributes to the establishment of neuronal morphology. Marlin-1 associates to the cytoskeleton in neurites, is required for the maintenance of an intact Golgi apparatus and its depletion produces the down-regulation of kinesin-1, a plus-end directed molecular motor with a central function in morphogenesis and migration. RNA interference of Marlin-1 in vivo results in abnormal migration of newborn pyramidal neurons during the formation of the cortex. Our results support the involvement of Marlin-1 in the acquisition of the complex architecture and migration of pyramidal neurons, two fundamental processes for the laminar layering of the cortex.

Neurons are highly polarized, but the trafficking mechanisms that operate in these cells and the topological organization of their secretory organelles are still poorly understood. Particularly incipient is our knowledge of the role of the neuronal endoplasmic reticulum. Here we review the current understanding of the endoplasmic reticulum in neurons, its structure, composition, dendritic distribution and dynamics. We also focus on the trafficking of proteins through the dendritic endoplasmic reticulum, emphasizing the relevance of transport, retention, assembly of multi-subunit protein complexes and export. We additionally discuss the roles of the dendritic endoplasmic reticulum in synaptic plasticity.

Slow and persistent synaptic inhibition is mediated by metabotropic GABA(B) receptors (GABA(B)Rs). GABA(B)Rs are responsible for the modulation of neurotransmitter release from presynaptic terminals and for hyperpolarization at postsynaptic sites. Postsynaptic GABA(B)Rs are predominantly found on dendritic spines, adjacent to excitatory synapses, but the control of their plasma membrane availability is still controversial. Here, we explore the role of glutamate receptor activation in regulating the function and surface availability of GABA(B)Rs in central neurons. We demonstrate that prolonged activation of NMDA receptors (NMDA-Rs) leads to endocytosis, a diversion from a recycling route, and subsequent lysosomal degradation of GABA(B)Rs. These sorting events are paralleled by a reduction in GABA(B)R-dependent activation of inwardly rectifying K+ channel currents. Postendocytic sorting is critically dependent on phosphorylation of serine 783 (S783) within the GABA(B)R2 subunit, an established substrate of AMP-dependent protein kinase (AMPK). NMDA-R activation leads to a rapid increase in phosphorylation of S783, followed by a slower dephosphorylation, which results from the activity of AMPK and protein phosphatase 2A, respectively. Agonist activation of GABA(B)Rs counters the effects of NMDA. Thus, NMDA-R activation alters the phosphorylation state of S783 and acts as a molecular switch to decrease the abundance of GABA(B)Rs at the neuronal plasma membrane. Such a mechanism may be of significance during synaptic plasticity or pathological conditions, such as ischemia or epilepsy, which lead to prolonged activation of glutamate receptors.

Understanding the mechanisms that control synaptic efficacy through the availability of neurotransmitter receptors depends on uncovering their specific intracellular trafficking routes. gamma-Aminobutyric acid type B (GABA(B)) receptors (GABA(B)Rs) are obligatory heteromers present at dendritic excitatory and inhibitory postsynaptic sites. It is unknown whether synthesis and assembly of GABA(B)Rs occur in the somatic endoplasmic reticulum (ER) followed by vesicular transport to dendrites or whether somatic synthesis is followed by independent transport of the subunits for assembly and ER export throughout the somatodendritic compartment. To discriminate between these possibilities we studied the association of GABA(B)R subunits in dendrites of hippocampal neurons combining live fluorescence microscopy, biochemistry, quantitative colocalization, and bimolecular fluorescent complementation. We demonstrate that GABA(B)R subunits are segregated and differentially mobile in dendritic intracellular compartments and that a high proportion of non-associated intracellular subunits exist in the brain. Assembled heteromers are preferentially located at the plasma membrane, but blockade of ER exit results in their intracellular accumulation in the cell body and dendrites. We propose that GABA(B)R subunits assemble in the ER and are exported from the ER throughout the neuron prior to insertion at the plasma membrane. Our results are consistent with a bulk flow of segregated subunits through the ER and rule out a post-Golgi vesicular transport of preassembled GABA(B)Rs.

The cytoskeleton and cytoskeletal motors play a fundamental role in neurotransmitter receptor trafficking, but proteins that link GABAB receptors (GABABRs) to the cytoskeleton have not been described. We recently identified Marlin-1, a protein that interacts with GABABR1. Here, we explore the association of GABABRs and Marlin-1 to the cytoskeleton using a combination of biochemistry, microscopy and live cell imaging. Our results indicate that Marlin-1 is associated to microtubules and the molecular motor kinesin-I. We demonstrate that a fraction of Marlin-1 is mobile in dendrites of cultured hippocampal neurons and that mobility is microtubule-dependent. We also show that GABABRs interact robustly with kinesin-I and that intracellular membranes containing GABABRs are sensitive to treatments that disrupt a protein complex containing Marlin-1, kinesin-I and tubulin. Finally, we report that a kinesin-I mutant severely impairs receptor transport. We conclude that Marlin-1 and kinesin-1 link GABABRs to the tubulin cytoskeleton in neurons.