Evgeny Pryazhnikov

Endoplasmic Reticulum Stress Inhibition Protects against Excitotoxic Neuronal Injury in the Rat Brain

Elevated brain glutamate with activation of neuronal glutamate receptors accompanies neurological disorders, such as epilepsy and brain trauma. However, the mechanisms by which excitotoxicity triggers neuronal injury are not fully understood. We have studied the glutamate receptor agonist kainic acid (KA) inducing seizures and excitotoxic cell death. KA caused the disintegration of the endoplasmic reticulum (ER) membrane in hippocampal neurons and ER stress with the activation of the ER proteins Bip, Chop, and caspase-12. Salubrinal, inhibiting eIF2α (eukaryotic translation initiation factor 2 subunit α) dephosphorylation, significantly reduced KA-induced ER stress and neuronal death in vivo and in vitro. KA-induced rise in intracellular calcium was not affected by Salubrinal. The results show that ER responses are essential parts of excitotoxicity mediated by glutamate receptor activation and that Salubrinal decreases neuronal death in vivo. Inhibition of ER stress by small molecular compounds may be beneficial for treatment of various neuronal injuries and brain disorders.

Exocytotic release of ATP from cultured astrocytes

Astrocytes appear to communicate with each other as well as with neurons via ATP. However, the mechanisms of ATP release are controversial. To explore whether stimuli that increase [Ca2+]i also trigger vesicular ATP release from astrocytes, we labeled ATP-containing vesicles with the fluorescent dye quinacrine, which exhibited a significant co-localization with atrial natriuretic peptide. The confocal microscopy study revealed that quinacrine-loaded vesicles displayed mainly non-directional spontaneous mobility with relatively short track lengths and small maximal displacements, whereas 4% of vesicles exhibited directional mobility. After ionomycin stimulation only non-directional vesicle mobility could be observed, indicating that an increase in [Ca2+]i attenuated vesicle mobility. Total internal reflection fluorescence (TIRF) imaging in combination with epifluorescence showed that a high percentage of fluorescently labeled vesicles underwent fusion with the plasma membrane after stimulation with glutamate or ionomycin and that this event was Ca2+-dependent. This was confirmed by patch-clamp studies on HEK-293T cells transfected with P2X3 receptor, used as sniffers for ATP release from astrocytes. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small transient inward currents in sniffers, reminiscent of postsynaptic quantal events observed at synapses. Their incidence was highly dependent on extracellular Ca2+. Collectively, these findings indicate that glutamate-stimulated ATP release from astrocytes was most likely exocytotic and that after stimulation the fraction of quinacrine-loaded vesicles, spontaneously exhibiting directional mobility, disappeared.

Sub‐micromolar increase in [Ca2+]i triggers delayed exocytosis of ATP in cultured astrocytes

Astrocytes release a variety of transmitter molecules, which mediate communication between glial cells in the brain and modulate synaptic transmission. ATP is a major glia‐derived transmitter, but the mechanisms and kinetics of ATP release from astrocytes remain largely unknown. Here, we combined epifluorescence and total internal reflection fluorescence microscopy to monitor individual quinacrine‐loaded ATP‐containing vesicles undergoing exocytosis in cultured astrocytes. In resting cells, vesicles exhibited three‐dimensional motility, spontaneous docking and release at low rate. Extracellular ATP application induced a Ca2+‐dependent increase in the rate of exocytosis, which persisted for several minutes. Using UV flash photolysis of caged Ca2+, the threshold [Ca2+]i for ATP exocytosis was found to be ∼350 nM. Subthreshold [Ca2+]i transients predominantly induced vesicle docking at plasma membrane without subsequent release. ATP exocytosis triggered either by purinergic stimulation or by Ca2+ uncaging occurred after a substantial delay ranging from tens to hundreds of seconds, with only ∼4% of release occurring during the first 30 s. The time course of the cargo release from vesicles had two peaks centered on ≤10 s and 60 s. These results demonstrate that: (1) [Ca2+]i elevations in cultured astrocytes trigger docking and release of ATP‐containing vesicles; (2) vesicle docking and release have different Ca2+ thresholds; (3) ATP exocytosis is delayed by several minutes and highly asynchronous; (4) two populations of ATP‐containing vesicles with distinct (fast and slow) time course of cargo release exist in cultured astrocytes.

Transmembrane Prostatic Acid Phosphatase (TMPAP) Interacts with Snapin and Deficient Mice Develop Prostate Adenocarcinoma

The molecular mechanisms underlying prostate carcinogenesis are poorly understood. Prostatic acid phosphatase (PAP), a prostatic epithelial secretion marker, has been linked to prostate cancer since the 1930s. However, the contribution of PAP to the disease remains controversial. We have previously cloned and described two isoforms of this protein, a secretory (sPAP) and a transmembrane type-I (TMPAP). The goal in this work was to understand the physiological function of TMPAP in the prostate. We conducted histological, ultra-structural and genome-wide analyses of the prostate of our PAP-deficient mouse model (PAP−/−) with C57BL/6J background. The PAP−/− mouse prostate showed the development of slow-growing non-metastatic prostate adenocarcinoma. In order to find out the mechanism behind, we identified PAP-interacting proteins byyeast two-hybrid assays and a clear result was obtained for the interaction of PAP with snapin, a SNARE-associated protein which binds Snap25 facilitating the vesicular membrane fusion process. We confirmed this interaction by co-localization studies in TMPAP-transfected LNCaP cells (TMPAP/LNCaP cells) and in vivo FRET analyses in transient transfected LNCaP cells. The differential gene expression analyses revealed the dysregulation of the same genes known to be related to synaptic vesicular traffic. Both TMPAP and snapin were detected in isolated exosomes. Our results suggest that TMPAP is involved in endo-/exocytosis and disturbed vesicular traffic is a hallmark of prostate adenocarcinoma.

Reversible disruption of neuronal mitochondria by ischemic and traumatic injury revealed by quantitative two-photon imaging in the neocortex of anesthetized mice.

Mitochondria play a variety of functional roles in cortical neurons, from metabolic support and neuroprotection to the release of cytokines that trigger apoptosis. In dendrites, mitochondrial structure is closely linked to their function, and fragmentation (fission) of the normally elongated mitochondria indicates loss of their function under pathological conditions, such as stroke and brain trauma. Using in vivo two-photon microscopy in mouse brain, we quantified mitochondrial fragmentation in a full spectrum of cortical injuries, ranging from severe to mild. Severe global ischemic injury was induced by bilateral common carotid artery occlusion, whereas severe focal stroke injury was induced by Rose Bengal photosensitization. The moderate and mild traumatic injury was inflicted by focal laser lesion and by mild photo-damage, respectively. Dendritic and mitochondrial structural changes were tracked longitudinally using transgenic mice expressing fluorescent proteins localized either in cytosol or in mitochondrial matrix. In response to severe injury, mitochondrial fragmentation developed in parallel with dendritic damage signified by dendritic beading. Reconstruction from serial section electron microscopy confirmed mitochondrial fragmentation. Unlike dendritic beading, fragmentation spread beyond the injury core in focal stroke and focal laser lesion models. In moderate and mild injury, mitochondrial fragmentation was reversible with full recovery of structural integrity after 1–2 weeks. The transient fragmentation observed in the mild photo-damage model was associated with changes in dendritic spine density without any signs of dendritic damage. Our findings indicate that alterations in neuronal mitochondria structure are very sensitive to the tissue damage and can be reversible in ischemic and traumatic injuries. SIGNIFICANCE STATEMENT During ischemic stroke or brain trauma, mitochondria can either protect neurons by supplying ATP and adsorbing excessive Ca2+, or kill neurons by releasing proapoptotic factors. Mitochondrial function is tightly linked to their morphology: healthy mitochondria are thin and long; dysfunctional mitochondria are thick (swollen) and short (fragmented). To date, fragmentation of mitochondria was studied either in dissociated cultured neurons or in brain slices, but not in the intact living brain. Using real-time in vivo two-photon microscopy, we quantified mitochondrial fragmentation during acute pathological conditions that mimic severe, moderate, and mild brain injury. We demonstrated that alterations in neuronal mitochondria structural integrity can be reversible in traumatic and ischemic injuries, highlighting mitochondria as a potential target for therapeutic interventions.

Feasibility study of the permeability and uptake of mesoporous silica nanoparticles across the blood-brain barrier

Drug delivery into the brain is impeded by the blood-brain-barrier (BBB) that filters out the vast majority of drugs after systemic administration. In this work, we assessed the transport, uptake and cytotoxicity of promising drug nanocarriers, mesoporous silica nanoparticles (MSNs), in in vitro models of the BBB. RBE4 rat brain endothelial cells and Madin-Darby canine kidney epithelial cells, strain II, were used as BBB models. We studied spherical and rod-shaped MSNs with the following modifications: bare MSNs and MSNs coated with a poly(ethylene glycol)-poly(ethylene imine) (PEG-PEI) block copolymer. In transport studies, MSNs showed low permeability, whereas the results of the cellular uptake studies suggest robust uptake of PEG-PEI-coated MSNs. None of the MSNs showed significant toxic effects in the cell viability studies. While the shape effect was detectable but small, especially in the real-time surface plasmon resonance measurements, coating with PEG-PEI copolymers clearly facilitated the uptake of MSNs. Finally, we evaluated the in vivo detectability of one of the best candidates, i.e. the copolymer-coated rod-shaped MSNs, by two-photon in vivo imaging in the brain vasculature. The particles were clearly detectable after intravenous injection and caused no damage to the BBB. Thus, when properly designed, the uptake of MSNs could potentially be utilized for the delivery of drugs into the brain via transcellular transport.

Flat-floored Air-lifted Platform: A New Method for Combining Behavior with Microscopy or Electrophysiology on Awake Freely Moving Rodents

It is widely acknowledged that the use of general anesthetics can undermine the relevance of electrophysiological or microscopical data obtained from a living animal’s brain. Moreover, the lengthy recovery from anesthesia limits the frequency of repeated recording/imaging episodes in longitudinal studies. Hence, new methods that would allow stable recordings from non-anesthetized behaving mice are expected to advance the fields of cellular and cognitive neurosciences. Existing solutions range from mere physical restraint to more sophisticated approaches, such as linear and spherical treadmills used in combination with computer-generated virtual reality. Here, a novel method is described where a head-fixed mouse can move around an air-lifted mobile homecage and explore its environment under stress-free conditions. This method allows researchers to perform behavioral tests (e.g., learning, habituation or novel object recognition) simultaneously with two-photon microscopic imaging and/or patch-clamp recordings, all combined in a single experiment. This video-article describes the use of the awake animal head fixation device (mobile homecage), demonstrates the procedures of animal habituation, and exemplifies a number of possible applications of the method.

Low micromolar Ba2+ potentiates glutamate transporter current in hippocampal astrocytes

Glutamate uptake, mediated by electrogenic glutamate transporters largely localized in astrocytes, is responsible for the clearance of glutamate released during excitatory synaptic transmission. Glutamate uptake also determines the availability of glutamate for extrasynaptic glutamate receptors. The efficiency of glutamate uptake is commonly estimated from the amplitude of transporter current recorded in astrocytes. We recorded currents in voltage-clamped hippocampal CA1 stratum radiatum astrocytes in rat hippocampal slices induced by electrical stimulation of the Schaffer collaterals. A Ba2+-sensitive K+ current mediated by inward rectifying potassium channels (Kir) accompanied the transporter current. Surprisingly, Ba2+ not only suppressed the K+ current and changed holding current (presumably, mediated by Kir) but also increased the transporter current at lower concentrations. However, Ba2+ did not significantly increase the uptake of aspartate in cultured astrocytes, suggesting that increase in the amplitude of the transporter current does not always reflect changes in glutamate uptake.

Unusually Strong Temperature Dependence of P2X3 Receptor Traffic to the Plasma Membrane

ATP-gated P2X3 receptors are expressed by nociceptive neurons and participate in transduction of pain. Responsiveness of P2X3 receptors is strongly reduced at low temperatures, suggesting a role for these receptors in analgesic effects of cooling. Since sustained responsiveness depends on receptor trafficking to the plasma membrane, we employed total internal reflection fluorescence (TIRF) microscopy to highlight perimembrane pool of DsRed-tagged P2X3 receptors and studied the effects of temperature on perimembrane turnover of P2X3-DsRed. Patch-clamp recordings confirmed membrane expression of functional, rapidly desensitizing P2X3-DsRed receptors. By combining TIRF microscopy with the technique of fluorescence recovery after photobleaching (FRAP), we measured the rate of perimembrane turnover of P2X3-DsRed receptors expressed in hippocampal neurons. At room temperature, the P2X3-DsRed perimembrane turnover as measured by TIRF–FRAP had a time constant of ∼2 min. At 29°C, receptor turnover was strongly accelerated (0.6 min), yielding an extremely high temperature dependence coefficient Q10 ∼4.5. In comparison, AMPA receptor turnover measured with TIRF–FRAP was only moderately sensitive to temperature (Q10 ∼1.5). The traffic inhibitor Brefeldin A selectively decelerated P2X3-DsRed receptor turnover at 29°C, but had no effect at 21°C (Q10 ∼1.0). This indicates that receptor traffic to plasma membrane is the key temperature-sensitive component of P2X3 turnover. The selective inhibitor of the RhoA kinase Y27632 significantly decreased the temperature dependence of P2X3-DsRed receptor turnover (Q10 ∼2.0). In summary, the RhoA kinase-dependent membrane trafficking of P2X3 receptors to plasma membrane has an exceptionally high sensitivity to temperature. These findings suggest an important role of P2X3 receptor turnover in hypothermia-associated analgesia.

KV4.2 channels tagged in the S1-S2 loop for alpha-bungarotoxin binding provide a new tool for studies of channel expression and localization

We report the first successful insertion of an engineered, high-affinity alpha-bungarotoxin (Bgtx) binding site into a voltage-gated ion channel, KV4.2, using a short, intra-protein embedded sequence (GGWRYYESSLEPYPDGG), derived from a previously described mimotope peptide, HAP. A major benefit to this approach is the ability to live-image the distribution and fate of functional channels on the plasma membrane surface. The Bgtx binding sequence was introduced into the putative extracellular loop between the S1 and S2 transmembrane domains of KV4.2. Following co-expression with KChIP3 in tsA201 cells, S1-S2 HAP-tagged channels express at levels comparable to wild-type KV4.2, and their activation and inactivation kinetics are minimally altered under most conditions. Binding assays, as well as live staining of surface-expressed KV4.2 channels with fluorescent-Bgtx, readily demonstrate specific binding of Bgtx to HAP-tagged KV4.2 expressed on the surface of tsA201 cells. Similar live-imaging results were obtained with HAP-tagged KV4.2 transfected into hippocampal neurons in primary culture suggesting applicability for future in vivo studies. Furthermore, the activation kinetics of S1-S2-tagged KV4.2 channels are minimally affected by the binding of Bgtx, suggesting a limited role if any for the S1-S2 loop in voltage sensing or gating associated conformational changes. Successful functional insertion of the HAP sequence into the S1-S2 linker of KV4.2 suggests that other related channels may similarly be amenable to this tagging strategy.