Efficient entry of cell-penetrating peptide nona-arginine into adherent cells involves a transient increase in intracellular calcium.

Understanding the mechanism of entry of cationic peptides such as nona-arginine (R9) into cells remains an important challenge to their use as efficient drug-delivery vehicles. At nanomolar to low micromolar R9 concentrations and at physiological temperature, peptide entry involves endocytosis. In contrast, at a concentration ≥10 µM, R9 induces a very effective non-endocytic entry pathway specific for cationic peptides. We found that a similar entry pathway is induced at 1-2 µM concentrations of R9 if peptide application is accompanied by a rapid temperature drop to 15°C. Both at physiological and at sub-physiological temperatures, this entry mechanism was inhibited by depletion of the intracellular ATP pool. Intriguingly, we found that R9 at 10-20 µM and 37°C induces repetitive spikes in intracellular Ca(2+) concentration. This Ca(2+) signalling correlated with the efficiency of the peptide entry. Pre-loading cells with the Ca(2+) chelator BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) inhibited both Ca(2+) spikes and peptide entry, suggesting that an increase in intracellular Ca(2+) precedes and is required for peptide entry. One of the hallmarks of Ca(2+) signalling is a transient cell-surface exposure of phosphatidylserine (PS), a lipid normally residing only in the inner leaflet of the plasma membrane. Blocking the accessible PS with the PS-binding domain of lactadherin strongly inhibited non-endocytic R9 entry, suggesting the importance of PS externalization in this process. To conclude, we uncovered a novel mechanistic link between calcium signalling and entry of cationic peptides. This finding will enhance our understanding of the properties of plasma membrane and guide development of future drug-delivery vehicles.

Control of extracellular cleavage of ProBDNF by high frequency neuronal activity.

Pro- and mature neurotrophins often elicit opposing biological effects. For example, mature brain-derived neurotrophic factor (mBDNF) is critical for long-term potentiation induced by high-frequency stimulation, whereas proBDNF facilitate long-term depression induced by low-frequency stimulation. Because mBDNF is derived from proBDNF by endoproteolytic cleavage, mechanisms regulating the cleavage of proBDNF may control the direction of BDNF regulation. Using methods that selectively detect proBDNF or mBDNF, we show that low-frequency stimulation induced predominant proBDNF secretion in cultured hippocampal neurons. In contrast, high-frequency stimulation preferentially increased extracellular mBDNF. Inhibition of extracellular, but not intracellular cleavage of proBDNF greatly reduced high-frequency stimulation-induced extracellular mBDNF. Moreover, high-frequency, but not low-frequency stimulation selectively induced the secretion of tissue plasminogen activator, a key protease involved in extracellular proBDNF to mBDNF conversion. Thus, high-frequency neuronal activity controls the ratio of extracellular proBDNF/mBDNF by regulating the secretion of extracellular proteases. Our study demonstrates activity-dependent control of extracellular proteolytic cleavage of a secretory protein, and reveals an important mechanism that controls diametrically opposed functions of BDNF isoforms.

Sorting and activity-dependent secretion of BDNF require interaction of a specific motif with the sorting receptor carboxypeptidase e.

Activity-dependent secretion of BDNF is important in mediating synaptic plasticity, but how it is achieved is unclear. Here we uncover a sorting motif receptor-mediated mechanism for regulated secretion of BDNF. X-ray crystal structure analysis revealed a putative sorting motif, I(16)E(18)I(105)D(106), in BDNF, which when mutated at the acidic residues resulted in missorting of proBDNF to the constitutive pathway in AtT-20 cells. A V20E mutation to complete a similar motif in NGF redirected a significant proportion of it from the constitutive to the regulated pathway. Modeling and binding studies showed interaction of the acidic residues in the BDNF motif with two basic residues in the sorting receptor, carboxypeptidase E (CPE). (35)S labeling experiments demonstrated that activity-dependent secretion of BDNF from cortical neurons was obliterated in CPE knockout mice. Thus, we have identified a mechanism whereby a specific motif I(16)E(18)I(105)D(106) interacts with CPE to sort proBDNF into regulated pathway vesicles for activity-dependent secretion.

Regulation of TrkB receptor tyrosine kinase and its internalization by neuronal activity and Ca2+ influx.

Internalization of the neurotrophin-Trk receptor complex is critical for many aspects of neurotrophin functions. The mechanisms governing the internalization process are unknown. Here, we report that neuronal activity facilitates the internalization of the receptor for brain-derived neurotrophic factor, TrkB, by potentiating its tyrosine kinase activity. Using three independent approaches, we show that electric stimulation of hippocampal neurons markedly enhances TrkB internalization. Electric stimulation also potentiates TrkB tyrosine kinase activity. The activity-dependent enhancement of TrkB internalization and its tyrosine kinase requires Ca2+ influx through N-methyl-d-aspartate receptors and Ca2+ channels. Inhibition of internalization had no effect on TrkB kinase, but inhibition of TrkB kinase prevents the modulation of TrkB internalization, suggesting a critical role of the tyrosine kinase in the activity-dependent receptor endocytosis. These results demonstrate an activity- and Ca2+-dependent modulation of TrkB tyrosine kinase and its internalization, and they provide new insights into the cell biology of tyrosine kinase receptors.

Fusion Stage of HIV-1 Entry Depends on Virus-Induced Cell Surface Exposure of Phosphatidylserine.

HIV-1 entry into host cells starts with interactions between the viral envelope glycoprotein (Env) and cellular CD4 receptors and coreceptors. Previous work has suggested that efficient HIV entry also depends on intracellular signaling, but this remains controversial. Here we report that formation of the pre-fusion Env-CD4-coreceptor complexes triggers non-apoptotic cell surface exposure of the membrane lipid phosphatidylserine (PS). HIV-1-induced PS redistribution depends on Ca2+ signaling triggered by Env-coreceptor interactions and involves the lipid scramblase TMEM16F. Externalized PS strongly promotes Env-mediated membrane fusion and HIV-1 infection. Blocking externalized PS or suppressing TMEM16F inhibited Env-mediated fusion. Exogenously added PS promoted fusion, with fusion dependence on PS being especially strong for cells with low surface density of coreceptors. These findings suggest that cell-surface PS acts as an important cofactor that promotes the fusogenic restructuring of pre-fusion complexes and likely focuses the infection on cells conducive to PS signaling.

Hyper-recombinogenic RecA protein from Pseudomonas aeruginosa with enhanced activity of its primary DNA binding site.

According to one prominent model, each protomer in the activated nucleoprotein filament of homologous recombinase RecA possesses two DNA-binding sites. The primary site binds (1) single-stranded DNA (ssDNA) to form presynaptic complex and (2) the newly formed double-stranded (ds) DNA whereas the secondary site binds (1) dsDNA of a partner to initiate strand exchange and (2) the displaced ssDNA following the strand exchange. RecA protein from Pseudomonas aeruginosa (RecAPa) promotes in Escherichia coli hyper-recombination in an SOS-independent manner. Earlier we revealed that RecAPa rapidly displaces E.coli SSB protein (SSB-Ec) from ssDNA to form presynaptic complex. Here we show that this property (1) is based on increased affinity of ssDNA for the RecAPa primary DNA binding site while the affinity for the secondary site remains similar to that for E.coli RecA, (2) is not specific for SSB-Ec but is also observed for SSB protein from P.aeruginosa that, in turn, predicts a possibility of enhanced recombination repair in this pathogenic bacterium.

Multiple functions of precursor BDNF to CNS neurons: negative regulation of neurite growth, spine formation and cell survival.

BACKGROUND: Proneurotrophins and mature neurotrophins elicit opposite effects via the p75 neurotrophin receptor (p75(NTR)) and Trk tyrosine kinase receptors, respectively; however the molecular roles of proneurotrophins in the CNS are not fully understood. RESULTS: Based on two rare single nucleotide polymorphisms (SNPs) of the human brain-derived neurotrophic factor (BDNF) gene, we generated R125M-, R127L- and R125M/R127L-BDNF, which have amino acid substitution(s) near the cleavage site between the pro- and mature-domain of BDNF. Western blot analyses demonstrated that these BDNF variants are poorly cleaved and result in the predominant secretion of proBDNF. Using these cleavage-resistant proBDNF (CR-proBDNF) variants, the molecular and cellular roles of proBDNF on the CNS neurons were examined. First, CR-proBDNF showed normal intracellular distribution and secretion in cultured hippocampal neurons, suggesting that inhibition of proBDNF cleavage does not affect intracellular transportation and secretion of BDNF. Second, we purified recombinant CR-proBDNF and tested its biological effects using cultured CNS neurons. Treatment with CR-proBDNF elicited apoptosis of cultured cerebellar granule neurons (CGNs), while treatment with mature BDNF (matBDNF) promoted cell survival. Third, we examined the effects of CR-proBDNF on neuronal morphology using more than 2-week cultures of basal forebrain cholinergic neurons (BFCNs) and hippocampal neurons. Interestingly, in marked contrast to the action of matBDNF, which increased the number of cholinergic fibers and hippocampal dendritic spines, CR-proBDNF dramatically reduced the number of cholinergic fibers and hippocampal dendritic spines, without affecting the survival of these neurons. CONCLUSION: These results suggest that proBDNF has distinct functions in different populations of CNS neurons and might be responsible for specific physiological cellular processes in the brain.

The BDNF val66met polymorphism affects activity-dependent secretion of BDNF and human memory and hippocampal function.

Brain-derived neurotrophic factor (BDNF) modulates hippocampal plasticity and hippocampal-dependent memory in cell models and in animals. We examined the effects of a valine (val) to methionine (met) substitution in the 5' pro-region of the human BDNF protein. In human subjects, the met allele was associated with poorer episodic memory, abnormal hippocampal activation assayed with fMRI, and lower hippocampal n-acetyl aspartate (NAA), assayed with MRI spectroscopy. Neurons transfected with met-BDNF-GFP showed lower depolarization-induced secretion, while constitutive secretion was unchanged. Furthermore, met-BDNF-GFP failed to localize to secretory granules or synapses. These results demonstrate a role for BDNF and its val/met polymorphism in human memory and hippocampal function and suggest val/met exerts these effects by impacting intracellular trafficking and activity-dependent secretion of BDNF.

Cleavage of proBDNF by tPA/plasmin is essential for long-term hippocampal plasticity.

Long-term memory is thought to be mediated by protein synthesis-dependent, late-phase long-term potentiation (L-LTP). Two secretory proteins, tissue plasminogen activator (tPA) and brain-derived neurotrophic factor (BDNF), have been implicated in this process, but their relationship is unclear. Here we report that tPA, by activating the extracellular protease plasmin, converts the precursor proBDNF to the mature BDNF (mBDNF), and that such conversion is critical for L-LTP expression in mouse hippocampus. Moreover, application of mBDNF is sufficient to rescue L-LTP when protein synthesis is inhibited, which suggests that mBDNF is a key protein synthesis product for L-LTP expression.