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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>Based on two rare single nucleotide polymorphisms (SNPs) of the <it>human brain-derived neurotrophic factor (BDNF) </it>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.</p> <p>Conclusion</p> <p>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.</p

Structure of the catalytic domain of the hyperthermophilic chitinase from Pyrococcus furiosus

The crystal structure of the catalytic domain of a chitinase from P. furiosus is reported

BDNF Binds Its Pro-Peptide with High Affinity and the Common Val66Met Polymorphism Attenuates the Interaction

Most growth factors are initially synthesized as precursors then cleaved into bioactive mature domains and pro-domains, but the biological roles of pro-domains are poorly understood. In the present study, we investigated the pro-domain (or pro-peptide) of brain-derived neurotrophic factor (BDNF), which promotes neuronal survival, differentiation and synaptic plasticity. The BDNF pro-peptide is a post-processing product of the precursor BDNF. Using surface plasmon resonance and biochemical experiments, we first demonstrated that the BDNF pro-peptide binds to mature BDNF with high affinity, but not other neurotrophins. This interaction was more enhanced at acidic pH than at neutral pH, suggesting that the binding is significant in intracellular compartments such as trafficking vesicles rather than the extracellular space. The common Val66Met BDNF polymorphism results in a valine instead of a methionine in the pro-domain, which affects human brain functions and the activity-dependent secretion of BDNF. We investigated the influence of this variation on the interaction between BDNF and the pro-peptide. Interestingly, the Val66Met polymorphism stabilized the heterodimeric complex of BDNF and its pro-peptide. Furthermore, compared with the Val-containing pro-peptide, the complex with the Met-type pro-peptide was more stable at both acidic and neutral pH, suggesting that the Val66Met BDNF polymorphism forms a more stable complex. A computational modeling provided an interpretation to the role of the Val66Met mutation in the interaction of BDNF and its pro-peptide. Lastly, we performed electrophysiological experiments, which indicated that the BDNF pro-peptide, when pre-incubated with BDNF, attenuated the ability of BDNF to inhibit hippocampal long-term depression (LTD), suggesting a possibility that the BDNF pro-peptide may interact directly with BDNF and thereby inhibit its availability. It was previously reported that the BDNF pro-domain exerts a chaperone-like function and assists the folding of the BDNF protein. However, our results suggest a new role for the BDNF pro-domain (or pro-peptide) following proteolytic cleave of precursor BDNF, and provide insight into the Val66Met polymorphism

Crystallization and X-ray diffraction analysis of a catalytic domain of hyperthermophilic chitinase from Pyrococcus furiosus

The expression, purification and preliminary X-ray diffraction analysis of a catalytic domain of a chitinase from P. furiosus is reported

Crystallization and preliminary X-ray diffraction analysis of thioredoxin peroxidase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1

The hyperthermostable thioredoxin peroxidase from the aerobic hyperthermophilic archaeon A. pernix K1 was crystallized. The crystal diffracted to 2.7 Å resolution

Crystallization and preliminary X-ray diffraction analysis of a chitin-binding domain of hyperthermophilic chitinase from Pyrococcus furiosus

The expression, purification and preliminary X-ray diffraction studies of a chitin-binding domain of the chitinase from P. furiosus are reported