A novel super-resolution microscopy platform for cutaneous alpha-synuclein detection in Parkinson's disease.

Alpha-synuclein (aSyn) aggregates in the central nervous system are the main pathological hallmark of Parkinson's disease (PD). ASyn aggregates have also been detected in many peripheral tissues, including the skin, thus providing a novel and accessible target tissue for the detection of PD pathology. Still, a well-established validated quantitative biomarker for early diagnosis of PD that also allows for tracking of disease progression remains lacking. The main goal of this research was to characterize aSyn aggregates in skin biopsies as a comparative and quantitative measure for PD pathology. Using direct stochastic optical reconstruction microscopy (dSTORM) and computational tools, we imaged total and phosphorylated-aSyn at the single molecule level in sweat glands and nerve bundles of skin biopsies from healthy controls (HCs) and PD patients. We developed a user-friendly analysis platform that offers a comprehensive toolkit for researchers that combines analysis algorithms and applies a series of cluster analysis algorithms (i.e., DBSCAN and FOCAL) onto dSTORM images. Using this platform, we found a significant decrease in the ratio of the numbers of neuronal marker molecules to phosphorylated-aSyn molecules, suggesting the existence of damaged nerve cells in fibers highly enriched with phosphorylated-aSyn molecules. Furthermore, our analysis found a higher number of aSyn aggregates in PD subjects than in HC subjects, with differences in aggregate size, density, and number of molecules per aggregate. On average, aSyn aggregate radii ranged between 40 and 200 nm and presented an average density of 0.001-0.1 molecules/nm2. Our dSTORM analysis thus highlights the potential of our platform for identifying quantitative characteristics of aSyn distribution in skin biopsies not previously described for PD patients while offering valuable insight into PD pathology by elucidating patient aSyn aggregation status.

Biochemical and neurophysiological effects of deficiency of the mitochondrial import protein TIMM50.

TIMM50, an essential TIM23 complex subunit, is suggested to facilitate the import of ∼60% of the mitochondrial proteome. In this study, we characterized a TIMM50 disease causing mutation in human fibroblasts and noted significant decreases in TIM23 core protein levels (TIMM50, TIMM17A/B, and TIMM23). Strikingly, TIMM50 deficiency had no impact on the steady state levels of most of its putative substrates, suggesting that even low levels of a functional TIM23 complex are sufficient to maintain the majority of TIM23 complex-dependent mitochondrial proteome. As TIMM50 mutations have been linked to severe neurological phenotypes, we aimed to characterize TIMM50 defects in manipulated mammalian neurons. TIMM50 knockdown in mouse neurons had a minor effect on the steady state level of most of the mitochondrial proteome, supporting the results observed in patient fibroblasts. Amongst the few affected TIM23 substrates, a decrease in the steady state level of components of the intricate oxidative phosphorylation and mitochondrial ribosome complexes was evident. This led to declined respiration rates in fibroblasts and neurons, reduced cellular ATP levels and defective mitochondrial trafficking in neuronal processes, possibly contributing to the developmental defects observed in patients with TIMM50 disease. Finally, increased electrical activity was observed in TIMM50 deficient mice neuronal cells, which correlated with reduced levels of KCNJ10 and KCNA2 plasma membrane potassium channels, likely underlying the patients' epileptic phenotype.

Multi-Sensor Origami Platform: A Customizable System for Obtaining Spatiotemporally Precise Functional Readouts in 3D Models.

Bioprinting technology offers unprecedented opportunities to construct in vitro tissue models that recapitulate the 3D morphology and functionality of native tissue. Yet, it remains difficult to obtain adequate functional readouts from such models. In particular, it is challenging to position sensors in desired locations within pre-fabricated 3D bioprinted structures. At the same time, bioprinting tissue directly onto a sensing device is not feasible due to interference with the printer head. As such, a multi-sensing platform inspired by origami that overcomes these challenges by "folding" around a separately fabricated 3D tissue structure is proposed, allowing for the insertion of electrodes into precise locations, which are custom-defined using computer-aided-design software. The multi-sensing origami platform (MSOP) can be connected to a commercial multi-electrode array (MEA) system for data-acquisition and processing. To demonstrate the platform, how integrated 3D MEA electrodes can record neuronal electrical activity in a 3D model of a neurovascular unit is shown. The MSOP also enables a microvascular endothelial network to be cultured separately and integrated with the 3D tissue structure. Accordingly, how impedance-based sensors in the platform can measure endothelial barrier function is shown. It is further demonstrated the device's versatility by using it to measure neuronal activity in brain organoids.

9-cis beta-carotene-enriched diet significantly improved cognition and decreased Alzheimer's disease neuropathology and neuroinflammation in Alzheimer's disease-like mouse models.

A significant progressive decline in beta-carotene (ßC) levels in the brain is associated with cognitive impairment and a higher prevalence of Alzheimer's disease (AD). In this study, we investigated whether the administration of 9-cis beta-carotene (9CBC)-rich powder of the alga Dunaliella bardawil, the best-known source of ßC in nature, inhibits the development of AD-like neuropathology and cognitive deficits. We demonstrated that in 3 AD mouse models, Tg2576, 5xFAD, and apoE4, 9CBC treatment improved long- and short-term memory, decreased neuroinflammation, and reduced the prevalence of ß-amyloid plaques and tau hyperphosphorylation. These findings suggest that 9CBC has the potential to be an effective preventive and symptomatic AD therapy.

The guanine nucleotide exchange factor RapGEF2 is required for ERK-dependent immediate-early gene (Egr1) activation during fear memory formation.

The MAP kinase ERK is important for neuronal plasticity underlying associative learning, yet specific molecular pathways for neuronal ERK activation are undetermined. RapGEF2 is a neuron-specific cAMP sensor that mediates ERK activation. We investigated whether it is required for cAMP-dependent ERK activation leading to other downstream neuronal signaling events occurring during associative learning, and if RapGEF2-dependent signaling impairments affect learned behavior. Camk2α-cre+/-::RapGEF2fl/fl mice with depletion of RapGEF2 in hippocampus and amygdala exhibit impairments in context- and cue-dependent fear conditioning linked to corresponding impairment in Egr1 induction in these two brain regions. Camk2α-cre+/-::RapGEF2fl/fl mice show decreased RapGEF2 expression in CA1 and dentate gyrus associated with abolition of pERK and Egr1, but not of c-Fos induction, following fear conditioning, impaired freezing to context after fear conditioning, and impaired cAMP-dependent long-term potentiation at perforant pathway and Schaffer collateral synapses in hippocampal slices ex vivo. RapGEF2 expression is largely eliminated in basolateral amygdala, also involved in fear memory, in Camk2α-cre+/-::RapGEF2fl/fl mice. Neither Egr1 nor c-fos induction in BLA after fear conditioning, nor cue-dependent fear learning, are affected by ablation of RapGEF2 in BLA. However, Egr1 induction (but not that of c-fos) in BLA is reduced after restraint stress-augmented fear conditioning, as is freezing to cue after restraint stress-augmented fear conditioning, in Camk2α-cre+/-::RapGEF2fl/fl mice. Cyclic AMP-dependent GEFs have been genetically associated as risk factors for schizophrenia, a disorder associated with cognitive deficits. Here we show a functional link between one of them, RapGEF2, and cognitive processes involved in associative learning in amygdala and hippocampus.

Super-Resolution-Chip: an in-vitro platform that enables super-resolution microscopy of co-cultures and 3D systems.

The development of organs-on-a-chip platforms has revolutionized in-vitro cellular culture by allowing cells to be grown in an environment that better mimics human physiology. However, there is still a challenge in integrating those platforms with advanced imaging technology. This is extremely important when we want to study molecular changes and subcellular processes on the level of a single molecule using super-resolution microscopy (SRM), which has a resolution beyond the diffraction limit of light. Currently, existing platforms that include SRM have certain limitations, either as they only support 2D monocultures, without flow or as they demand a lot of production and handling. In this study, we developed a Super-Res-Chip platform, consisting of a 3D-printed chip and a porous membrane, that could be used to co-culture cells in close proximity either in 2D or in 3D while allowing SRM on both sides of the membrane. To demonstrate the functionality of the device, we co-cultured in endothelial and epithelial cells and used direct stochastic optical reconstruction microscopy (dSTORM) to investigate how glioblastoma cells affect the expression of the gap-junction protein Connexin43 in endothelial cells grown in 2D and in 3D. Cluster analysis of Connexin43 distribution revealed no difference in the number of clusters, their size, or radii, but did identify differences in their density. Furthermore, the spatial resolution was high also when the cells were imaged through the membrane (20-30 nm for x-y) and 10-20 nm when imaged directly both for 2D and 3D conditions. Overall, this chip allows to characterize of complex cellular processes on a molecular scale in an easy manner and improved the capacity for imaging in a single molecule resolution complex cellular organization.

Brain-Targeted Liposomes Loaded with Monoclonal Antibodies Reduce Alpha-Synuclein Aggregation and Improve Behavioral Symptoms in Parkinson's Disease.

Monoclonal antibodies (mAbs) hold promise in treating Parkinson's disease (PD), although poor delivery to the brain hinders their therapeutic application. In the current study, it is demonstrated that brain-targeted liposomes (BTL) enhance the delivery of mAbs across the blood-brain-barrier (BBB) and into neurons, thereby allowing the intracellular and extracellular treatment of the PD brain. BTL are decorated with transferrin to improve brain targeting through overexpressed transferrin-receptors on the BBB during PD. BTL are loaded with SynO4, a mAb that inhibits alpha-synuclein (AS) aggregation, a pathological hallmark of PD. It is shown that 100-nm BTL cross human BBB models intact and are taken up by primary neurons. Within neurons, SynO4 is released from the nanoparticles and bound to its target, thereby reducing AS aggregation, and enhancing neuronal viability. In vivo, intravenous BTL administration results in a sevenfold increase in mAbs in brain cells, decreasing AS aggregation and neuroinflammation. Treatment with BTL also improve behavioral motor function and learning ability in mice, with a favorable safety profile. Accordingly, targeted nanotechnologies offer a valuable platform for drug delivery to treat brain neurodegeneration.

cAMP-Dependent Synaptic Plasticity at the Hippocampal Mossy Fiber Terminal.

Cyclic adenosine monophosphate (cAMP) is a crucial second messenger involved in both pre- and postsynaptic plasticity in many neuronal types across species. In the hippocampal mossy fiber (MF) synapse, cAMP mediates presynaptic long-term potentiation and depression. The main cAMP-dependent signaling pathway linked to MF synaptic plasticity acts via the activation of the protein kinase A (PKA) molecular cascade. Accordingly, various downstream putative synaptic PKA target proteins have been linked to cAMP-dependent MF synaptic plasticity, such as synapsin, rabphilin, synaptotagmin-12, RIM1a, tomosyn, and P/Q-type calcium channels. Regulating the expression of some of these proteins alters synaptic release probability and calcium channel clustering, resulting in short- and long-term changes to synaptic efficacy. However, despite decades of research, the exact molecular mechanisms by which cAMP and PKA exert their influences in MF terminals remain largely unknown. Here, we review current knowledge of different cAMP catalysts and potential downstream PKA-dependent molecular cascades, in addition to non-canonical cAMP-dependent but PKA-independent cascades, which might serve as alternative, compensatory or competing pathways to the canonical PKA cascade. Since several other central synapses share a similar form of presynaptic plasticity with the MF, a better description of the molecular mechanisms governing MF plasticity could be key to understanding the relationship between the transcriptional and computational levels across brain regions.

Clathrin-mediated endocytosis cooperates with bulk endocytosis to generate vesicles.

Clathrin-mediated endocytosis, the most prominent endocytic mode, is thought to be generated primarily from relatively flat patches of the plasma membrane. By employing conventional and platinum replica electron microscopy and super-resolution STED microscopy in neuroendocrine chromaffin cells, we found that large Ω-shaped or dome-shaped plasma membrane invaginations, previously thought of as the precursor of bulk endocytosis, are primary sites for clathrin-coated pit generation after depolarization. Clathrin-coated pits are more densely packed at invaginations rather than flat membranes, suggesting that invaginations are preferred sites for clathrin-coated pit formation, likely because their positive curvature facilitates coated-pit formation. Thus, clathrin-mediated endocytosis closely collaborates with bulk endocytosis to enhance endocytic capacity in active secretory cells. This direct collaboration between two classically independent endocytic pathways is of broad importance given the central role of both clathrin-mediated endocytosis and bulk endocytosis in neurons, endocrine cells, immune cells, and many other cell types throughout the body.

Hyperbaric Oxygen Treatment: Effects on Mitochondrial Function and Oxidative Stress.

Hyperbaric oxygen treatment (HBOT)-the administration of 100% oxygen at atmospheric pressure (ATA) greater than 1 ATA-increases the proportion of dissolved oxygen in the blood five- to twenty-fold. This increase in accessible oxygen places the mitochondrion-the organelle that consumes most of the oxygen that we breathe-at the epicenter of HBOT's effects. As the mitochondrion is also a major site for the production of reactive oxygen species (ROS), it is possible that HBOT will increase also oxidative stress. Depending on the conditions of the HBO treatment (duration, pressure, umber of treatments), short-term treatments have been shown to have deleterious effects on both mitochondrial activity and production of ROS. Long-term treatment, on the other hand, improves mitochondrial activity and leads to a decrease in ROS levels, partially due to the effects of HBOT, which increases antioxidant defense mechanisms. Many diseases and conditions are characterized by mitochondrial dysfunction and imbalance between ROS and antioxidant scavengers, suggesting potential therapeutic intervention for HBOT. In the present review, we will present current views on the effects of HBOT on mitochondrial function and oxidative stress, the interplay between them and the implications for several diseases.

Hyperbaric Oxygen Treatment-From Mechanisms to Cognitive Improvement.

Hyperbaric oxygen treatment (HBOT)-the medical use of oxygen at environmental pressure greater than one atmosphere absolute-is a very effective therapy for several approved clinical situations, such as carbon monoxide intoxication, incurable diabetes or radiation-injury wounds, and smoke inhalation. In recent years, it has also been used to improve cognition, neuro-wellness, and quality of life following brain trauma and stroke. This opens new avenues for the elderly, including the treatment of neurological and neurodegenerative diseases and improvement of cognition and brain metabolism in cases of mild cognitive impairment. Alongside its integration into clinics, basic research studies have elucidated HBOT's mechanisms of action and its effects on cellular processes, transcription factors, mitochondrial function, oxidative stress, and inflammation. Therefore, HBOT is becoming a major player in 21st century research and clinical treatments. The following review will discuss the basic mechanisms of HBOT, and its effects on cellular processes, cognition, and brain disorders.

Effect of SARS-CoV-2 proteins on vascular permeability.

Severe acute respiratory syndrome (SARS)-CoV-2 infection leads to severe disease associated with cytokine storm, vascular dysfunction, coagulation, and progressive lung damage. It affects several vital organs, seemingly through a pathological effect on endothelial cells. The SARS-CoV-2 genome encodes 29 proteins, whose contribution to the disease manifestations, and especially endothelial complications, is unknown. We cloned and expressed 26 of these proteins in human cells and characterized the endothelial response to overexpression of each, individually. Whereas most proteins induced significant changes in endothelial permeability, nsp2, nsp5_c145a (catalytic dead mutant of nsp5), and nsp7 also reduced CD31, and increased von Willebrand factor expression and IL-6, suggesting endothelial dysfunction. Using propagation-based analysis of a protein-protein interaction (PPI) network, we predicted the endothelial proteins affected by the viral proteins that potentially mediate these effects. We further applied our PPI model to identify the role of each SARS-CoV-2 protein in other tissues affected by coronavirus disease (COVID-19). While validating the PPI network model, we found that the tight junction (TJ) proteins cadherin-5, ZO-1, and ß-catenin are affected by nsp2, nsp5_c145a, and nsp7 consistent with the model prediction. Overall, this work identifies the SARS-CoV-2 proteins that might be most detrimental in terms of endothelial dysfunction, thereby shedding light on vascular aspects of COVID-19.

Hyperbaric oxygen therapy alleviates vascular dysfunction and amyloid burden in an Alzheimer's disease mouse model and in elderly patients.

Vascular dysfunction is entwined with aging and in the pathogenesis of Alzheimer's disease (AD) and contributes to reduced cerebral blood flow (CBF) and consequently, hypoxia. Hyperbaric oxygen therapy (HBOT) is in clinical use for a wide range of medical conditions. In the current study, we exposed 5XFAD mice, a well-studied AD model that presents impaired cognitive abilities, to HBOT and then investigated the therapeutical effects using two-photon live animal imaging, behavioral tasks, and biochemical and histological analysis. HBOT increased arteriolar luminal diameter and elevated CBF, thus contributing to reduced hypoxia. Furthermore, HBOT reduced amyloid burden by reducing the volume of pre-existing plaques and attenuating the formation of new ones. This was associated with changes in amyloid precursor protein processing, elevated degradation and clearance of Aß protein and improved behavior of 5XFAD mice. Hence, our findings are consistent with the effects of HBOT being mediated partially through a persistent structural change in blood vessels that reduces brain hypoxia. Motivated by these findings, we exposed elderly patients with significant memory loss at baseline to HBOT and observed an increase in CBF and improvement in cognitive performances. This study demonstrates HBOT efficacy in hypoxia-related neurological conditions, particularly in AD and aging.

miR-128 as a Regulator of Synaptic Properties in 5xFAD Mice Hippocampal Neurons.

Alzheimer's disease (AD) is characterized by progressive synaptic dysfunction, deterioration of neuronal transmission, and consequently neuronal death. Although there is no treatment for AD, exposure to enriched environment (EE) in mice, as well as physical and mental activity in human subjects have been shown to have a protective effect by slowing the disease's progression and reducing AD-like cognitive impairment. However, the molecular mechanism of this mitigating effect is still not understood. One of the mechanisms that has recently been shown to be involved in neuronal degeneration is microRNAs (miRNAs) regulation, which act as a post-transcriptional regulators of gene expression. miR-128 has been shown to be significantly altered in individuals with AD and in mice following exposure to EE. Here, we focused on elucidating the possible role of miR-128 in AD pathology and found that miR-128 regulates the expression of two proteins essential for synaptic transmission, SNAP-25, and synaptotagmin1 (Syt1). Clinically relevant, in 5xFAD mouse model for AD, this miRNA's expression was found as downregulated, resembling the alteration found in the hippocampi of individuals with AD. Interestingly, exposing WT mice to EE also resulted in downregulation of miR-128 expression levels, although EE and AD conditions demonstrate opposing effects on neuronal functioning and synaptic plasticity. We also found that miR-128 expression downregulation in primary hippocampal cultures from 5xFAD mice results in increased neuronal network activity and neuronal excitability. Altogether, our findings place miR-128 as a synaptic player that may contribute to synaptic functioning and plasticity through regulation of synaptic protein expression and function.

PI(4,5)P2-dependent regulation of exocytosis by amisyn, the vertebrate-specific competitor of synaptobrevin 2.

The functions of nervous and neuroendocrine systems rely on fast and tightly regulated release of neurotransmitters stored in secretory vesicles through SNARE-mediated exocytosis. Few proteins, including tomosyn (STXBP5) and amisyn (STXBP6), were proposed to negatively regulate exocytosis. Little is known about amisyn, a 24-kDa brain-enriched protein with a SNARE motif. We report here that full-length amisyn forms a stable SNARE complex with syntaxin-1 and SNAP-25 through its C-terminal SNARE motif and competes with synaptobrevin-2/VAMP2 for the SNARE-complex assembly. Furthermore, amisyn contains an N-terminal pleckstrin homology domain that mediates its transient association with the plasma membrane of neurosecretory cells by binding to phospholipid PI(4,5)P2 However, unlike synaptrobrevin-2, the SNARE motif of amisyn is not sufficient to account for the role of amisyn in exocytosis: Both the pleckstrin homology domain and the SNARE motif are needed for its inhibitory function. Mechanistically, amisyn interferes with the priming of secretory vesicles and the sizes of releasable vesicle pools, but not vesicle fusion properties. Our biochemical and functional analyses of this vertebrate-specific protein unveil key aspects of negative regulation of exocytosis.

Depopulation of dense α-synuclein aggregates is associated with rescue of dopamine neuron dysfunction and death in a new Parkinson's disease model.

Parkinson's disease (PD) is characterized by the presence of α-synuclein aggregates known as Lewy bodies and Lewy neurites, whose formation is linked to disease development. The causal relation between α-synuclein aggregates and PD is not well understood. We generated a new transgenic mouse line (MI2) expressing human, aggregation-prone truncated 1-120 α-synuclein under the control of the tyrosine hydroxylase promoter. MI2 mice exhibit progressive aggregation of α-synuclein in dopaminergic neurons of the substantia nigra pars compacta and their striatal terminals. This is associated with a progressive reduction of striatal dopamine release, reduced striatal innervation and significant nigral dopaminergic nerve cell death starting from 6 and 12 months of age, respectively. In the MI2 mice, alterations in gait impairment can be detected by the DigiGait test from 9 months of age, while gross motor deficit was detected by rotarod test at 20 months of age when 50% of dopaminergic neurons in the substantia nigra pars compacta are lost. These changes were associated with an increase in the number and density of 20-500 nm α-synuclein species as shown by dSTORM. Treatment with the oligomer modulator anle138b, from 9 to 12 months of age, restored striatal dopamine release, prevented dopaminergic cell death and gait impairment. These effects were associated with a reduction of the inner density of large α-synuclein aggregates and an increase in dispersed small α-synuclein species as revealed by dSTORM. The MI2 mouse model recapitulates the progressive dopaminergic deficit observed in PD, showing that early synaptic dysfunction is associated to fine behavioral motor alterations, precedes dopaminergic axonal loss and neuronal death that become associated with a more consistent motor deficit upon reaching a certain threshold. Our data also provide new mechanistic insight for the effect of anle138b's function in vivo supporting that targeting α-synuclein aggregation is a promising therapeutic approach for PD.

Golden Exosomes Selectively Target Brain Pathologies in Neurodegenerative and Neurodevelopmental Disorders.

Exosomes, nanovesicles that are secreted by different cell types, enable intercellular communication at local or distant sites. Alhough they have been found to cross the blood brain barrier, their migration and homing abilities within the brain remain unstudied. We have recently developed a method for longitudinal and quantitative in vivo neuroimaging of exosomes based on the superior visualization abilities of classical X-ray computed tomography (CT), combined with gold nanoparticles as labeling agents. Here, we used this technique to track the migration and homing patterns of intranasally administrated exosomes derived from bone marrow mesenchymal stem cells (MSC-exo) in different brain pathologies, including stroke, autism, Parkinson's disease, and Alzheimer's disease. We found that MSC-exo specifically targeted and accumulated in pathologically relevant murine models brains regions up to 96 h post administration, while in healthy controls they showed a diffuse migration pattern and clearance by 24 h. The neuro-inflammatory signal in pathological brains was highly correlated with MSC-exo accumulation, suggesting that the homing mechanism is inflammatory-driven. In addition, MSC-exo were selectively uptaken by neuronal cells, but not glial cells, in the pathological regions. Taken together, these findings can significantly promote the application of exosomes for therapy and targeted drug delivery in various brain pathologies.

Hyperbaric oxygen therapy ameliorates pathophysiology of 3xTg-AD mouse model by attenuating neuroinflammation.

There is a real need for new interventions for Alzheimer's disease (AD). Hyperbaric oxygen therapy (HBOT), the medical administration of 100% oxygen at conditions greater than 1 atmosphere absolute, has been used successfully to treat several neurological conditions, but its effects on AD pathology have never been thoroughly examined. Therefore, we exposed old triple-transgenic (3xTg) and non-transgenic mice to HBOT followed by behavioral, histological, and biochemical analyses. HBOT attenuated neuroinflammatory processes by reducing astrogliosis, microgliosis, and the secretion of proinflammatory cytokines (IL-1ß and TNFα) and increasing expression of scavenger receptor A, arginase1, and antiinflammatory cytokines (IL-4 and IL-10). Moreover, HBOT reduced hypoxia, amyloid burden, and tau phosphorylation in 3xTg mice and ameliorated their behavioral deficits. Therefore, we suggest that HBOT has multifaceted effects that reduce AD pathologies, even in old mice. Given that HBOT is used in the clinic to treat various indications, including neurological conditions, these results suggest HBOT as a novel therapeutic intervention for AD.

Phosphatidylinositol (4, 5)-bisphosphate targets double C2 domain protein B to the plasma membrane.

Double C2 domain protein B (DOC2B) is a high-affinity Ca2+ sensor that translocates from the cytosol to the plasma membrane (PM) and promotes vesicle priming and fusion. However, the molecular mechanism underlying its translocation and targeting to the PM in living cells is not completely understood. DOC2B interacts in vitro with the PM components phosphatidylserine, phosphatidylinositol (4, 5)-bisphosphate [PI(4, 5)P2 ] and target SNAREs (t-SNAREs). Here, we show that PI(4, 5)P2 hydrolysis at the PM of living cells abolishes DOC2B translocation, whereas manipulations of t-SNAREs and other phosphoinositides have no effect. Moreover, we were able to redirect DOC2B to intracellular membranes by synthesizing PI(4, 5)P2 in those membranes. Molecular dynamics simulations and mutagenesis in the calcium and PI(4, 5)P2 -binding sites strengthened our findings, demonstrating that both calcium and PI(4, 5)P2 are required for the DOC2B-PM association and revealing multiple PI(4, 5)P2 -C2B interactions. In addition, we show that DOC2B translocation to the PM is ATP-independent and occurs in a diffusion-like manner. Our data suggest that the Ca2+ -triggered translocation of DOC2B is diffusion-driven and aimed at PI(4, 5)P2 -containing membranes.