Precursor of Brain-derived Neurotrophic Factor (proBDNF) Forms a Complex with Huntingtin-associated Protein-1 (HAP1) and Sortilin That Modulates proBDNF Trafficking, Degradation, and Processing

Abstract

proBDNF, a precursor of brain-derived neurotrophic factor (BDNF), is anterogradely transported and released from nerve terminals, but the mechanism underlying this process remains unclear. In this study, we report that proBDNF forms a complex with Huntingtin associated protein-1 (HAP1) and sortilin, which plays an important role in proBDNF intracellular trafficking and stabilization. The interaction of proBDNF with both HAP1A and sortilin in co-transfected HEK293 cells is confirmed by both fluorescence resonance energy transfer and coimmunoprecipitation. The frequent co-localization (>90%) of endogenous HAP1, sortilin, and proBDNF is also found in cultured cortical neurons. Mapping studies using GST pulldown and competition assays has defined the interacting region of HAP1 with proBDNF within amino acids 371-445 and the binding sequences of proBDNF to HAP1 between amino acids 65 and 90. Fluorescence recovery after photobleaching confirms the defective movement of proBDNF-containing vesicles in neurites of HAP1 ؊/؊ neurons, which can be partially restored by reintroducing HAP1 cDNA into the neurons. However, the effect is significantly increased by simultaneously reintroducing both HAP1 and sortilin. proBDNF and HAP1 are highly co-localized with organelle markers for the Golgi network, microtubules, molecular motor, or endosomes in normal neurons, but this co-localization is reduced in HAP1 ؊/؊ neurons.Co-immunoprecipitation and Western blot showed that sortilin stabilizes the proBDNF⅐HAP1 complex in co-transfected HEK293 cells, helping to prevent proBDNF degradation. Furthermore, the complex facilitates furin cleavage to release mature BDNF.

Disruption of the expression of the proprotein convertase PC7 reduces BDNF production and affects learning and memory in mice

“…One possible explanation for these findings is that PC7 affects proBDNF via two separate mechanisms. Processing of proSortilin would be reduced in PC7 KO mice, and this should increase degradation of proBDNF (22). By contrast, decreased processing of proBDNF may be expected to increase its levels.…”

“…This difference between proBDNF and mature BDNF levels suggested that the precursor may be selectively destabilized in PC7 KO tissues. Mature Sortilin has been reported to participate in the stabilization of proBDNF and to protect it from degradation (22). Sortilin is a type-1 membrane-bound receptor expressed in the central and peripheral nervous systems, which is synthesized as a precursor (proSortilin) that is converted to the mature receptor in vitro at the conserved RWRR 77 ↓SA site by Furin (23).…”

Pro-Brain-Derived Neurotrophic Factor Inhibits GABAergic Neurotransmission by Activating Endocytosis and Repression of GABAAReceptors

“…Thus, the expected outcomes are the demonstration that in vivo, a significant proportion of BDNF is secreted as a precursor, and that the extracellular proBDNF/mBDNF ratio modulates the strength of GABAergic synapses. Pathological alterations in BDNF processing have been reported in several cognitive dysfunctions in neurodevelopmental and neurodegenerative disorders (Barnes and Thomas, 2008;Unsain et al, 2008;Volosin et al, 2008;Carlino et al, 2011;Yang et al, 2011;Shulga et al, 2012). Because patients with cognitive disorders have abnormal GABA levels, it is plausible that alterations in BDNF processing produce a disturbance of GABAergic synaptic activity.…”

SPIG1 Negatively Regulates BDNF Maturation

“…Topographically appropriate connections may be established by selective branching along the axon shaft with a bias at the AP location of their future appropriate terminal zone (TZ) (Simon and O'Leary, 1992a;Yates et al, 2001) and preferential extension toward the TZ along the DV axis in the tectum (Nakamura and O'Leary, 1989;Hindges et al, 2002;McLaughlin et al, 2003a). Refined topography has then been shown to develop through the large-scale elimination of overshoots against ectopic branches and arbors (Nakamura and O'Leary, 1989;Simon andO'Leary, 1992a, 1992b;Yates et al, 2001). However, the mechanisms underlying the formation and refinement of axonal branches, which are requisite steps for the establishment of the final pattern of connections between RGCs and its targets, have not yet been examined in detail, although previous studies proposed the involvement of Ephs/ephrins and TrkB/BDNF in branch formation (Cohen-Cory and Fraser, 1995;Yates et al, 2001;Hindges et al, 2002;Sakurai et al, 2002;Marler et al, 2008).…”

“…These molecules regulate the expression of downstream genes, including Eph-ephrin systems, directly involved in the axon guidance of retinal ganglion cells (RGCs) to the optic tectum/superior colliculus (SC) along the anteroposterior (AP) and dorsoventral (DV) axes (McLaughlin et al, 2003b;. Topographically appropriate connections may be established by selective branching along the axon shaft with a bias at the AP location of their future appropriate terminal zone (TZ) (Simon and O'Leary, 1992a;Yates et al, 2001) and preferential extension toward the TZ along the DV axis in the tectum (Nakamura and O'Leary, 1989;Hindges et al, 2002;McLaughlin et al, 2003a). Refined topography has then been shown to develop through the large-scale elimination of overshoots against ectopic branches and arbors (Nakamura and O'Leary, 1989;Simon andO'Leary, 1992a, 1992b;Yates et al, 2001).…”