The Higher Sensitivity of GABAergic Compared to Glutamatergic Neurons to Growth-Promoting C3bot Treatment Is Mediated by Vimentin

The current study investigates the neurotrophic effects of Clostridium botulinum C3 transferase (C3bot) on highly purified, glia-free, GABAergic, and glutamatergic neurons. Incubation with nanomolar concentrations of C3bot promotes dendrite formation as well as dendritic and axonal outgrowth in rat GABAergic neurons. A comparison of C3bot effects on sorted mouse GABAergic and glutamatergic neurons obtained from newly established NexCre;Ai9xVGAT Venus mice revealed a higher sensitivity of GABAergic cells to axonotrophic and dendritic effects of C3bot in terms of process length and branch formation. Protein biochemical analysis of known C3bot binding partners revealed comparable amounts of β1 integrin in both cell types but a higher expression of vimentin in GABAergic neurons. Accordingly, binding of C3bot to GABAergic neurons was stronger than binding to glutamatergic neurons. A combinatory treatment of glutamatergic neurons with C3bot and vimentin raised the amount of bound C3bot to levels comparable to the ones in GABAergic neurons, thereby confirming the specificity of effects. Overall, different surface vimentin levels between GABAergic and glutamatergic neurons exist that mediate neurotrophic C3bot effects

VGLUT1 binding to endophilin or intersectin1 and dynamin phosphorylation in a diurnal context

Glutamate is concentrated into synaptic vesicles (SV) by the vesicular glutamate transporters (VGLUT) 1 and 2. VGLUTs also harbor a Na+/Pi-transport activity when residing at the plasma membrane. Here we aimed to identify whether the diurnal switches of VGLUT1 parallels interactions with or modification of endocytic proteins.VGLUT1 and dynamin bind to SH3 domains of either endophilin (Enph) or intersectin 1 (ITSN1) harboring one or five SH3 domains A–E, respectively. We followed diurnal variations by pull down experiments using SH3 fusion protein and brains from mice entrained in a strict 24-h light–dark cycle (12-h light Zeitgeber (ZT) 0, 6; 12-h dark ZT 12 and 18). In pull downs with EnphSH3 interaction with VGLUT1 is high during the resting light and reduced during the active dark period while dynamin binding does not vary. This diurnal light/dark pattern depends on a functional period 2 gene and changes when animals are kept in complete darkness. Pull downs using ITSN1SH3 A reveal diurnally varying binding of VGLUT1 with slightly reduced VGLUT1/dynamin ratios at the beginning of the light (ZT 0) or the dark (ZT 12) period. Phosphorylation increases binding of VGLUT1 but not of dynamin to EnphSH3. In contrast binding of dynamin to ITSN1SH3 A decreases under phosphorylating conditions with no changes in VGLUT1 binding. Phosphorylation of dynamin at Ser 774 is high at ZT 6 and ZT 18 when more VGLUT1 is at the plasma membrane but low at ZT 0 and ZT 12 the diurnal peaks of VGLUT1 endocytosis. In conclusion the diurnally varying endocytosis of VGLUT1 involves differential interactions with the SH3 domains of Enph and ITSN1 and correlates with the de-phosphorylation of dynamin1

Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion

Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by Streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by Streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis

Synaptophysin is involved in resetting of the mammalian circadian clock

Background: Mammals can adapt to changing light/dark conditions by advancing or delaying their circadian clock phase. Light pulses evoke changes in gene expression and neuronal activity in the suprachiasmatic nuclei (SCN), the central pacemaker of the circadian system. Alterations in neuronal activity are partially mediated by changes in synaptic vesicle (SV) fusion at the presynaptic membrane, which modulates release of neurotransmitters. Methods: Male synaptophysin (Syp) knock-out and littermate control wild type mice were tested in an Aschoff type I resetting paradigm. Additionally, gene expression of 'cFos, Per1' and 'Per2' was assessed in the SCN. Finally, complexes between the synaptic vesicle proteins Syp and synaptobrevin (Syb) were studied in order to correlate behavior with protein complexes at synaptic vesicles. Results: Here we show that mice lacking Syp, a modulator of neurotransmitter release, are defective in delaying clock phase. In contrast, clock phase advances as well as clock period are normal in 'Syp-/-' knock-out mice. This correlates with the formation of Syp/Syb complexes. Conclusions: Our findings suggest that Syp is involved specifically in the response to a nocturnal light pulse occurring in the early night. It appears that the SV component Syp is critically involved in the delay portion of the resetting mechanism of the circadian clock

Further Characterization of Dopamine Release by Permeabilized PC 12 Cells

Rat pheochromocytoma cells (PC 12) permeabilized with staphylococcal α-toxin release [3H]dopamine after addition of micromolar Ca2+. This does not require additional Mg2+-ATP (in contrast to bovine adrenal medullary chromaffin cells). We also observed Ca2+-dependent [3H]-dopamine release from digitonin-permeabilized PC 12 cells. Permeabilization with α-toxin or digitonin and stimulation of the cells were done consecutively to wash out endogenous Mg2+-ATP. During permeabilization, ATP was removed effectively from the cytoplasm by both agents but the cells released [3H]dopamine in response to micromolar Ca2+ alone. Replacement by chloride of glutamate, which could sustain mitochondrial ATP production in permeabilized cells, does not significantly alter catecholamine release induced by Ca2+. However, Mg2+ without ATP augments the Ca2+-induced release. The release was unaltered by thiol-, hydroxyl-, or calmodulin-interfering substances. Thus Mg2+-ATP, calmodulin, or proteins containing -SH or -OH groups are not necessary for exocytosis in permeabilized PC 12 cells

Autoantibodies to synapsin I sequestrate synapsin I and alter synaptic function

Synapsin I is a phosphoprotein that coats the cytoplasmic side of synaptic vesicles and regulates their trafficking within nerve terminals. Autoantibodies against Syn I have been described in sera and cerebrospinal fluids of patients with numerous neurological diseases, including limbic encephalitis and clinically isolated syndrome; however, the effects and fate of autoantibodies in neurons are still unexplored. We found that in vitro exposure of primary hippocampal neurons to patient\u2019s autoantibodies to SynI decreased the density of excitatory and inhibitory synapses and impaired both glutamatergic and GABAergic synaptic transmission. These effects were reproduced with a purified SynI antibody and completely absent in SynI knockout neurons. Autoantibodies to SynI are internalized by Fc\u3b3II/III-mediated endocytosis, interact with endogenous SynI, and promote its sequestration and intracellular aggregation. Neurons exposed to human autoantibodies to SynI display a reduced density of SVs, mimicking the SynI loss-of-function phenotype. Our data indicate that autoantibodies to intracellular antigens such as SynI can reach and inactivate their targets and suggest that an antibody-mediated synaptic dysfunction may contribute to the evolution and progression of autoimmune-mediated neurological diseases positive for SynI autoantibodies

Intrathecal immunoglobulin A and G antibodies to synapsin in a patient with limbic encephalitis

To report on the identification of intrathecally synthesized immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies to synapsin, a synaptic vesicle-associated protein, in a patient with limbic encephalitis

A novel approach in the treatment of neuroendocrine gastrointestinal tumors: Additive antiproliferative effects of interferon-γ and meta-iodobenzylguanidine

BACKGROUND: Therapeutic options to effectively inhibit growth and spread of neuroendocrine gastrointestinal tumors are still limited. As both meta-iodobenzylguanidine (MIBG) and interferon-γ (IFNγ) cause antineoplastic effects in neuroendocrine gastrointestinal tumor cells, we investigated the antiproliferative effects of the combination of IFNγ and non-radiolabeled MIBG in neuroendocrine gut STC-1 and pancreatic carcinoid BON tumor cells. METHODS AND RESULTS: IFNγ receptors were expressed in both models. IFNγ dose- and time-dependently inhibited the growth of both STC-1 and of BON tumor cells with IC(50)-values of 95 ± 15 U/ml and 135 ± 10 U/ml, respectively. Above 10 U/ml IFNγ induced apoptosis-specific caspase-3 activity in a time-dependent manner in either cell line and caused a dose-dependent arrest in the S-phase of the cell cycle. Furthermore, IFNγ induced cytotoxic effects in NE tumor cells. The NE tumor-targeted drug MIBG is selectively taken up via norepinephrine transporters, thereby specifically inhibiting growth in NE tumor cells. Intriguingly, IFNγ treatment induced an upregulation of norepinephrine transporter expression in neuroendocrine tumors cells, as determined by semi-quantitative RT-PCR. Co-application of sub-IC(50 )concentrations of IFNγ and MIBG led to additive growth inhibitory effects, which were mainly due to increased cytotoxicity and S-phase arrest of the cell cycle. CONCLUSION: Our data show that IFNγ exerts antiproliferative effects on neuroendocrine gastrointestinal tumor cells by inducing cell cycle arrest, apoptosis and cytotoxicity. The combination of IFNγ with the NE tumor-targeted agent MIBG leads to effective growth control at reduced doses of either drug. Thus, the administration of IFNγ alone and more so, in combination with MIBG, is a promising novel approach in the treatment of neuroendocrine gastrointestinal tumors

Differential Sorting of the Vesicular Glutamate Transporter 1 into a Defined Vesicular Pool Is Regulated by Light Signaling Involving the Clock Gene Period2

Synaptic strength depends on the amount of neurotransmitter stored in synaptic vesicles. The vesicular transmitter content has recently been shown to be directly dependent on the expression levels of vesicular neurotransmitter transporters indicating that the transport capacity of synaptic vesicles is a critical determinant for synaptic efficacy. Using synaptic vesicles prepared from whole brain at different times of the day we now show that the amount of vesicular glutamate transporter (VGLUT) 1 undergoes strong diurnal cycling. VGLUT1 protein levels are high before the start of the light period, decline at noon, increase again before start of the dark period, and decline again at midnight. Mice kept in complete darkness showed within a 24-h period only a single peak of VGLUT1 expression in the middle of the rest phase. In contrast, mice lacking the period gene Period 2, a core component of the circadian clock, did not show any light-cycle-dependent changes of VGLUT1 levels. No other of several synaptic vesicle proteins examined underwent circadian cycling. Circadian cycling of VGLUT1 was not seen when analyzing homogenate or synaptosomes, the starting fraction for vesicle preparation. Circadian cycling of VGLUT1 was also not reflected at the mRNA level. We conclude that nerve terminals are endowed with mechanisms that regulate quantal size by changing the copy number of transporters in synaptic vesicles. A reduced amount of VGLUT1 per vesicle is probably achieved by means of selective sorting controlled by clock genes