p75 neurotrophin receptor interacts with and promotes BACE1 localization in endosomes aggravating amyloidogenesis.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive deposition of amyloid beta (Aß) and dysregulation of neurotrophic signaling, causing synaptic dysfunction, loss of memory, and cell death. The expression of p75 neurotrophin receptor is elevated in the brain of AD patients, suggesting its involvement in this disease. However, the exact mechanism of its action is not yet clear. Here, we show that p75 interacts with beta-site amyloid precursor protein cleaving enzyme-1 (BACE1), and this interaction is enhanced in the presence of Aß. Our results suggest that the colocalization of BACE1 and amyloid precursor protein (APP) is increased in the presence of both Aß and p75 in cortical neurons. In addition, the localization of APP and BACE1 in early endosomes is increased in the presence of Aß and p75. An increased phosphorylation of APP-Thr668 and BACE1-Ser498 by c-Jun N-terminal kinase (JNK) in the presence of Aß and p75 could be responsible for this localization. In conclusion, our study proposes a potential involvement in amyloidogenesis for p75, which may represent a future therapeutic target for AD. Cover Image for this Issue: doi. 10.1111/jnc.14163.

Edaravone alleviates Alzheimer's disease-type pathologies and cognitive deficits.

Alzheimer's disease (AD) is one of most devastating diseases affecting elderly people. Amyloid-ß (Aß) accumulation and the downstream pathological events such as oxidative stress play critical roles in pathogenesis of AD. Lessons from failures of current clinical trials suggest that targeting multiple key pathways of the AD pathogenesis is necessary to halt the disease progression. Here we show that Edaravone, a free radical scavenger that is marketed for acute ischemic stroke, has a potent capacity of inhibiting Aß aggregation and attenuating Aß-induced oxidation in vitro. When given before or after the onset of Aß deposition via i.p. injection, Edaravone substantially reduces Aß deposition, alleviates oxidative stress, attenuates the downstream pathologies including Tau hyperphosphorylation, glial activation, neuroinflammation, neuronal loss, synaptic dysfunction, and rescues the behavioral deficits of APPswe/PS1 mice. Oral administration of Edaravone also ameliorates the AD-like pathologies and memory deficits of the mice. These findings suggest that Edaravone holds a promise as a therapeutic agent for AD by targeting multiple key pathways of the disease pathogenesis.

Development of mature BDNF-specific sandwich ELISA.

Mature brain-derived neurotrophic factor (mBDNF) plays a vital role in the nervous system, whereas proBDNF elicits neurodegeneration and neuronal apoptosis. Although current enzyme-linked immunosorbent assay (ELISA) has been widely used to measure BDNF levels, it cannot differentiate mBDNF from proBDNF. As the function of proBDNF differs from mBDNF, it is necessary to establish an ELISA assay specific for the detection of mBDNF. Therefore, we aimed to establish a new mBDNF-specific sandwich ELISA. In this study, we have screened and found a combination of antibodies for a sandwich ELISA. A monoclonal antibody and sheep anti-BDNF were chosen as capture and detection antibody for sandwich ELISA respectively. The new ELISA showed no cross-reactivity to human recombinant NT-3, NT-4, nerve growth factor and negligible cross-reactivity (0.99-4.99%) for proBDNF compared to commercial ELISA kits (33.18-91.09%). The application of the new mBDNF ELISA was shown through the measurement of mBDNF levels in different brain regions of rats and in the brain of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1)(-/-) and WT mice and compared to western blot. Overall, this new ELISA will be useful for the measurement of mBDNF levels with high specificity. As the function of proBDNF differs from mBDNF (mature BDNF), it is necessary to establish an ELISA specific for the detection of mBDNF. Here, we present a novel sandwich ELISA which detects mBDNF with high specificity. This new ELISA will be useful for the measurement of mBDNF levels with high specificity in various human and animal tissues. proBDNF, precursor of BDNF; BDNF, brain-derived neurotrophic factor; NT-3, neurotrophin-3; NT-4, neurotrophin-4; NGF, nerve growth factor.

Amyloid beta1₋42 (Aß42) up-regulates the expression of sortilin via the p75(NTR)/RhoA signaling pathway.

Sortilin, a Golgi sorting protein and a member of the VPS10P family, is the co-receptor for proneurotrophins, regulates protein trafficking, targets proteins to lysosomes, and regulates low density lipoprotein metabolism. The aim of this study was to investigate the expression and regulation of sortilin in Alzheimer's disease (AD). A significantly increased level of sortilin was found in human AD brain and in the brains of 6-month-old swedish-amyloid precursor protein/PS1dE9 transgenic mice. Aß42 enhanced the protein and mRNA expression levels of sortilin in a dose- and time-dependent manner in SH-SY5Y cells, but had no effect on sorLA. In addition, proBDNF also significantly increased the protein and mRNA expression of sortilin in these cells. The recombinant extracellular domain of p75(NTR) (P75ECD-FC), or the antibody against the extracellular domain of p75(NTR), blocked the up-regulation of sortilin induced by Amyloid-ß protein (Aß), suggesting that Aß42 increased the expression level of sortilin and mRNA in SH-SY5Y via the p75(NTR) receptor. Inhibition of ROCK, but not Jun N-terminal kinase, suppressed constitutive and Aß42-induced expression of sortilin. In conclusion, this study shows that sortilin expression is increased in the AD brain in human and mice and that Aß42 oligomer increases sortilin gene and protein expression through p75(NTR) and RhoA signaling pathways, suggesting a potential physiological interaction of Aß42 and sortilin in Alzheimer's disease.

p75NTR regulates Abeta deposition by increasing Abeta production but inhibiting Abeta aggregation with its extracellular domain.

Accumulation of toxic amyloid-ß (Aß) in the cerebral cortex and hippocampus is a major pathological feature of Alzheimer's disease (AD). The neurotrophin receptor p75NTR has been proposed to mediate Aß-induced neurotoxicity; however, its role in the development of AD remains to be clarified. The p75NTR/ExonIII-/- mice and APPSwe/PS1dE9 mice were crossed to generate transgenic AD mice with deletion of p75NTR gene. In APPSwe/PS1dE9 transgenic mice, p75NTR expression was localized in the basal forebrain neurons and degenerative neurites in neocortex, increased with aging, and further activated by Aß accumulation. Deletion of the p75NTR gene in APPSwe/PS1dE9 mice reduced soluble Aß levels in the brain and serum, but increased the accumulation of insoluble Aß and Aß plaque formation. There was no change in the levels of amyloid precursor protein (APP) and its proteolytic derivatives, or α-, ß-, and γ-secretase activities, or in levels of BACE1, neprilysin (NEP), and insulin-degrading enzyme (IDE) proteins. Aß production by cortical neurons of APPSwe/PS1dE9 mice was reduced by deletion of p75NTR gene in vitro. Recombinant extracellular domain of p75NTR attenuated the oligomerization and fibrillation of synthetic Aß(42) peptide in vitro, and reduced local Aß plaques after hippocampus injection in vivo. In addition, deletion of p75NTR attenuated microgliosis but increased the microhemorrhage profiles in the brain. The deletion of p75NTR did not significantly change the cognitive function of the mice up to the age of 9 months. Our data suggest that p75NTR plays a critical role in regulating Aß levels by both increasing Aß production and attenuating its aggregation, and they caution that a therapeutic intervention simply reducing p75NTR may exacerbate AD pathology.

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.

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 co-immunoprecipitation. 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 proBDNF·HAP1 complex in co-transfected HEK293 cells, helping to prevent proBDNF degradation. Furthermore, the complex facilitates furin cleavage to release mature BDNF.

Huntingtin associated protein 1 regulates trafficking of the amyloid precursor protein and modulates amyloid beta levels in neurons.

Amyloid precursor protein (APP) is involved in the pathogenesis of Alzheimer's disease. It is axonally transported, endocytosed and sorted to different cellular compartments where amyloid beta (Aß) is produced. However, the mechanism of APP trafficking remains unclear. We present evidence that huntingtin associated protein 1 (HAP1) may reduce Aß production by regulating APP trafficking to the non-amyloidogenic pathway. HAP1 and APP are highly colocalized in a number of brain regions, with similar distribution patterns in both mouse and human brains. They are associated with each other, the interacting site is the 371-599 of HAP1. APP is more retained in cis-Golgi, trans-Golgi complex, early endosome and ER-Golgi intermediate compartment in HAP1-/- neurons. HAP1 deletion significantly alters APP endocytosis and reduces the re-insertion of APP into the cytoplasmic membrane. Amyloid precursor protein-YFP(APP-YFP) vesicles in HAP1-/- neurons reveal a decreased trafficking rate and an increased number of motionless vesicles. Knock-down of HAP1 protein in cultured cortical neurons of Alzheimer's disease mouse model increases Aß levels. Our data suggest that HAP1 regulates APP subcellular trafficking to the non-amyloidogenic pathway and may negatively regulate Aß production in neurons.

Endogenous proBDNF is a negative regulator of migration of cerebellar granule cells in neonatal mice.

The majority of newborn neurons migrate from their birthplace to final destination in the developing brain. Migration of cerebellar granule cells (CGCs) requires multiple factors. Mature brain-derived neurotrophic factor (BDNF) positively regulates the proliferation, migration, survival and differentiation of CGCs in rodents. However, the role of the BDNF precursor, proBDNF, in neuronal development remains unknown. In this study, we investigated the effect of proBDNF in vivo and in vitro on migration of CGCs. We demonstrate that proBDNF and its receptors p75 neurotrophin receptor (p75NTR) and sortilin are highly expressed in the cerebella as determined by immunohistochemistry and Western blot. ProBDNF is released from cultured cerebellar neurons, and this release is increased by high potassium stimulation. ProBDNF inhibits migration of CGCs in vitro, and the neutralizing antibodies to proBDNF enhance such migration as assayed by transwell culture. In addition, proBDNF incorporated into an agarose plug reduces granule cell migration from such plugs, whereas the neutralizing antibodies attract these cells towards the plug. The application of proBDNF into the lateral ventricle significantly inhibits migration of CGCs out of the proliferative zone into the internal granular cell layer, whereas the neutralizing antibodies enhance this migration. Furthermore, the effects of proBDNF on cell migration are lost in p75NTR(-/-) mice. Our data suggest that proBDNF negatively regulates migration of CGCs and this effect is mediated by p75NTR. We conclude that proBDNF has an opposing role in migration of CGCs to that of mature BDNF.

Intramuscular delivery of a single chain antibody gene prevents brain Aß deposition and cognitive impairment in a mouse model of Alzheimer's disease.

Anti-beta-amyloid (Aß) immunotherapy is effective in removing brain Aß, but has shown to be associated with detrimental effects. We have demonstrated that Adeno-associated virus (AAV)-mediated delivery of an anti-Aß single chain antibody (scFv) gene was effective in clearing brain Aß without eliciting any inflammatory side effects in old APP(Swe)/PS1dE9 transgenic mice. In the present study, we tested the efficacy and safety of intramuscular delivery of the scFv gene in preventing brain Aß deposition. The scFv gene was intramuscularly delivered to APP(Swe)/PS1dE9 transgenic mice at 3 months of age, prior to Aß deposition in the brain. Six months later, we found that the transgenes were expressed in a stable form at the delivered sites, with a small amount of ectopic expression in the liver and olfactory bulb. Brain Aß plaque formation, Aß accumulation, AD-type pathologies and cognitive impairment were significantly attenuated in scFv-treated APP(Swe)/PS1dE9 transgenic mice relative to EGFP-treated mice. Intramuscular delivery of scFv gene was well tolerated by the animals, did not cause inflammation or microhemorrhage at the gene expression site and in the brain, and did not induce neutralizing antibodies in the animals. These findings suggest that peripheral application of scFv is effective and safe in preventing the development of Alzheimer's disease (AD), and would be a promising non-inflammatory immunological modality for prevention and treatment of AD.

Preconditioning selective ventral root injury promotes plasticity of ascending sensory neurons in the injured spinal cord of adult rats--possible roles of brain-derived neurotrophic factor, TrkB and p75 neurotrophin receptor.

Preconditioning sciatic nerve injury enhances axonal regeneration of ascending sensory neurons after spinal cord injury. A key question is whether direct injury of sensory nerves is necessary for the enhanced regeneration. The lumbar 5 ventral root transection (L5 VRT) model, a model of selective motor nerve injury, provides a useful tool to address this question. Here we examined the effects of a preconditioning L5 VRT on the regeneration after a subsequent dorsal column transection (DCT) in adult Sprague-Dawley rats. We found that L5 VRT 1 week before DCT increased the number of Fast Blue (FB)-labeled neurons in the L5 dorsal root ganglia (DRG) and promoted sprouting/regenerating axons to grow into the glial scar. L5 VRT also induced a dramatic upregulation of expression of brain-derived neurotrophic factor (BDNF) in the preconditioned DRG and in the injured spinal cord. Moreover, almost all of the FB-labeled sprouting/regenerating neurons expressed BDNF, and approximately 55% of these neurons were surrounded by p75 neurotrophin receptor-positive glial cells. This combined injury led to an increase in the number of BDNF- and TrkB-immunoreactive nerve fibers in the dorsal column caudal to the lesion site. Taken together, these findings demonstrate that L5 VRT promotes sprouting/regeneration of ascending sensory neurons, indicating that sensory axotomy may not be essential for the plasticity of injured dorsal column axons. Thus, the sensory neurons could be preprimed in the regenerative milieu of Wallerian degeneration and neuroinflammation, which might alter the expression of neurotrophic factors and their receptors, facilitating sprouting/regeneration of ascending sensory neurons.

Grape seed polyphenols and curcumin reduce genomic instability events in a transgenic mouse model for Alzheimer's disease.

The study set out to determine (a) whether DNA damage is elevated in mice that carry mutations in the amyloid precursor protein (APP695swe) and presenilin 1 (PSEN1-dE9) that predispose to Alzheimer's disease (AD) relative to non-transgenic control mice, and (b) whether increasing the intake of dietary polyphenols from curcumin or grape seed extract could reduce genomic instability events in a transgenic mouse model for AD. DNA damage was measured using the micronucleus (MN) assay in both buccal mucosa and erythrocytes and an absolute telomere length assay for both buccal mucosa and olfactory bulb tissue. MN frequency tended to be higher in AD mice in both buccal mucosa (1.7-fold) and polychromatic erythrocytes (1.3-fold) relative to controls. Telomere length was significantly reduced by 91% (p=0.04) and non-significantly reduced by 50% in buccal mucosa and olfactory bulbs respectively in AD mice relative to controls. A significant 10-fold decrease in buccal MN frequency (p=0.01) was found for AD mice fed diets containing curcumin (CUR) or micro-encapsulated grape seed extract (MGSE) and a 7-fold decrease (p=0.02) for AD mice fed unencapsulated grape seed extract (GSE) compared to the AD group on control diet. Similarly, in polychromatic erythrocytes a significant reduction in MN frequency was found for the MGSE cohort (65.3%) (p<0.05), whereas the AD CUR and AD GSE groups were non-significantly reduced by 39.2 and 34.8% respectively compared to the AD Control. A non-significant 2-fold increase in buccal cell telomere length was evident for the CUR, GSE and MGSE groups compared to the AD control group. Olfactory bulb telomere length was found to be non-significantly 2-fold longer in mice fed on the CUR diet compared to controls. These results suggest potential protective effects of polyphenols against genomic instability events in different somatic tissues of a transgenic mouse model for AD.

proBDNF inhibits the proliferation and migration of OLN­93 oligodendrocytes.

In contrast with mature brain­derived neurotrophic factor (mBDNF), proBDNF induces cell apoptosis. However, the function of proBDNF in oligodendrocytes remains unclear. In the present study, the OLN­93 oligodendroglia cell line was utilized as an in vitro model to analyse the functions of proBDNF in oligodendroglia. p75NTR, sortilin and proBDNF were expressed in cultured OLN­93 cells. It was indicated that proBDNF inhibited OLN­93 cell proliferation in a dose­dependent manner as determined using the MTT assay and BrdU staining. Furthermore, proBDNF suppressed the migration of OLN­93 cells as demonstrated using the scratch assay. proBDNF also decreased cell viability and promoted apoptosis as indicated by activated cysteine­aspartic acid protease­3 (caspase­3) immunocytochemistry. Notably, anti­proBDNF treatment neutralized the effect of proBDNF and resulted in increased cell proliferation and migration and decreased apoptosis. However, these effects were not observed in the presence of recombinant p75NTR extracellular domain­human FC fusion protein and p75NTR antibody, indicating that proBDNF imparts its inhibitory effects on oligodendrocytes through the p75NTR signal pathway.

Upregulation of brain-derived neurotrophic factor in the sensory pathway by selective motor nerve injury in adult rats.

Selective motor nerve injury by lumbar 5 ventral root transection (L5 VRT) induces neuropathic pain, but the underlying mechanisms remain unknown. Previously, increased expression and secretion of brain-derived neurotrophic factor (BDNF) had been implicated in injury-induced neuropathic pain in the sensory system. In this study, as a step to examine potential roles of BDNF in L5 VRT-induced neuropathic pain, we investigated BDNF gene and protein expression in adult rats with L5 VRT. L5 VRT induced a dramatic upregulation of BDNF mRNA in intact sensory neurons in the ipsilateral L5 dorsal root ganglia (DRG), in non-neuronal cells in the ipsilateral sciatic nerve, and in motoneurons in the ipsilateral spinal cord. L5 VRT also induced de novo synthesis of BDNF mRNA in spinal dorsal horn neurons and in glial cells in the white matter of the ipsilateral spinal cord. Consistent with the mRNA expression pattern, BDNF protein was also mainly upregulated in all populations of sensory neurons in the ipsilateral L5 DRG and in spinal neurons and glia. Quantitative analysis by ELISA showed that the BDNF content in the DRG and sciatic nerve peaked on day 1 and remained elevated 14 days after L5 VRT. These results suggest that increased BDNF expression in intact primary sensory neurons and spinal cord may be an important factor in the induction of neuropathic pain without axotomy of sensory neurons.

Suppression of p75NTR does not promote regeneration of injured spinal cord in mice.

The neurotrophin receptor p75NTR is the coreceptor for Nogo receptor, mediating growth cone collapse in vitro by MAG, myelin oligodendrocyte glycoprotein (Omgp), and Nogo. Whether p75NTR plays any role in the failure of nerve regeneration in vivo is not known. Immunohistochemical data showed that p75NTR was expressed in only a very small subset of ascending sensory axons but not in any corticospinal axons in the dorsal column of either normal or injured spinal cord. Using p75NTR-deficient mice, we showed that the depletion of the functional p75NTR did not promote the regeneration of the descending corticospinal tract and ascending sensory neurons in the spinal cord 2 weeks after spinal cord injury. Local administration of p75NTR-Fc fusion molecule, the dominant-negative receptor to block the function of neurite outgrowth inhibitors, did not improve regeneration of ascending sensory neurons in the injured spinal cord. Our results suggest that p75NTR may not be a critical molecule mediating the function of myelin-associated inhibitory factors in vivo.

Treatment of spinal cord injury with co-grafts of genetically modified Schwann cells and fetal spinal cord cell suspension in the rat.

Fetal spinal cord cells, Schwann cells and neurotrophins all have the capacity to promote repair of injured spinal cord in animal models. To explore the possibility of using these approaches to treat clinical patients, we have examined whether a combination of these protocols produces functional and anatomical improvement. The spinal cords of adult rats (n=16) were injured with a modified New York University (NYU) device (10 gram.5cm). One week after injury, the injured cords were injected with Dulbecco-modified Eagles Medium (DMEM, control group), or fetal spinal cord cell suspension (FSCS) plus nerve growth factor (NGF) gene-modified Schwann cells (SC) and brain-derived neurotrophic factor (BDNF) gene-modified SC (treatment group). The rats were subjected to BBB (Basso, Beattie, Bresnahan, Exp. Neurol. 139:244, 1996) behavioral tests. Anterograde tracing of corticospinal tract was performed before sacrifice 3 months after the treatment. The results showed that the combination treatment elicited a robust growth of corticospinal axons within and beyond the injury site. A dramatic functional recovery in the treatment group was observed compared with the control group. We conclude that the combination of FSCS with genetically modified Schwann cells over-expressing NGF and BDNF was an effective protocol for the treatment of severe spinal cord injury.

Upregulation of blood proBDNF and its receptors in major depression.

BACKGROUND: In recent decades, the role of brain-derived neurotrophic factor (BDNF) in depression has received intensive attention. However, the relationship between proBDNF and depression has not been clearly elucidated. METHODS: Forty drug-free women patients diagnosed with major depression and 50 healthy female controls were enrolled in our study. Peripheral blood was sampled from all the subjects. With the blood samples, we assessed the relationship between BDNF and major depression from following aspects: the levels of BDNF, proBDNF and their receptors in the sera and lymphocytes. The mRNA levels of these factors in lymphocytes were also examined. Furthermore, the correlations between each factor and the severity of major depression were tested. RESULTS: It was found that: (a) the protein and serum levels of proBDNF, sortilin and p75NTR were higher in major depressive patients than in healthy controls while mature BDNF and TrkB levels were lower; (b) the BDNF, TrkB, sortilin and p75NTR mRNA levels changed in line with their protein levels; (c) The levels of mature BDNF and TrkB had negative correlations with the major depression severity, and the levels of proBDNF, p75NTR and sortilin were positively correlated with the scores of HRSD-21; (d) the ratio of proBDNF and mBDNF was imbalanced in major depressive patients. CONCLUSION: The balance between the proBDNF/p75NTR/sortilin and mBDNF/TrkB signaling pathways appears dysregulated in major depression and both pathways should be considered as biomarkers for the major depression LIMITATIONS: More cases on both genders should be enrolled in our study. And further works on the mechanisms of how BDNF and its receptors are regulated in depression should also be carried out.

ProBDNF and its receptors are upregulated in glioma and inhibit the growth of glioma cells in vitro.

BACKGROUND: High-grade glioma is incurable, with a short survival time and poor prognosis. The increased expression of p75 neurotrophin receptor (NTR) is a characteristic of high-grade glioma, but the potential significance of increased p75NTR in this tumor is not fully understood. Since p75NTR is the receptor for the precursor of brain-derived neurotrophic factor (proBDNF), it is suggested that proBDNF may have an impact on glioma. METHODS: In this study we investigated the expression of proBDNF and its receptors p75NTR and sortilin in 52 cases of human glioma and 13 cases of controls by immunochemistry, quantitative real-time PCR, and Western blot methods. Using C6 glioma cells as a model, we investigated the roles of proBDNF on C6 glioma cell differentiation, growth, apoptosis, and migration in vitro. RESULTS: We found that the expression levels of proBDNF, p75NTR, and sortilin were significantly increased in high-grade glioma and were positively correlated with the malignancy of the tumor. We also observed that tumors expressed proBDNF, p75NTR, and sortilin in the same cells with different subcellular distributions, suggesting an autocrine or paracrine loop. The ratio of proBDNF to mature BDNF was decreased in high-grade glioma tissues and was negatively correlated with tumor grade. Using C6 glioma cells as a model, we found that proBDNF increased apoptosis and differentiation and decreased cell growth and migration in vitro via p75NTR. CONCLUSIONS: Our data indicate that proBDNF and its receptors are upregulated in high-grade glioma and might play an inhibitory effect on glioma.

The intracellular domain of sortilin interacts with amyloid precursor protein and regulates its lysosomal and lipid raft trafficking.

The processing of Amyloid precursor protein (APP) is multifaceted, comprising of protein transport, internalization and sequential proteolysis. However, the exact mechanism of APP intracellular trafficking and distribution remains unclear. To determine the interaction between sortilin and APP and the effect of sortilin on APP trafficking and processing, we studied the binding site and its function by mapping experiments, colocalization, coimmunoprecipitation and sucrose gradient fractionation. We identified for the first time that sortilin interacts with APP at both N- and C-terminal regions. The sortilin-FLVHRY (residues 787-792) and APP-NPTYKFFE (residues 759-766) motifs are crucial for the C-terminal interaction. We also found that lack of the FLVHRY motif reduces APP lysosomal targeting and increases APP distribution in lipid rafts in co-transfected HEK293 cells. These results are consistent with our in vivo data where sortilin knockout mice showed a decrease of APP lysosomal distribution and an increase of APP in lipid rafts. We further confirmed that overexpression of sortilin-FLVHRY mutants failed to rescue the lysosomal degradation of APP. Thus, our data suggests that sortilin is implicated in APP lysosomal and lipid raft targeting via its carboxyl-terminal F/YXXXXF/Y motif. Our study provides new molecular insights into APP trafficking and processing.

ProBDNF collapses neurite outgrowth of primary neurons by activating RhoA.

BACKGROUND: Neurons extend their dendrites and axons to build functional neural circuits, which are regulated by both positive and negative signals during development. Brain-derived neurotrophic factor (BDNF) is a positive regulator for neurite outgrowth and neuronal survival but the functions of its precursor (proBDNF) are less characterized. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that proBDNF collapses neurite outgrowth in murine dorsal root ganglion (DRG) neurons and cortical neurons by activating RhoA via the p75 neurotrophin receptor (p75NTR). We demonstrated that the receptor proteins for proBDNF, p75NTR and sortilin, were highly expressed in cultured DRG or cortical neurons. ProBDNF caused a dramatic neurite collapse in a dose-dependent manner and this effect was about 500 fold more potent than myelin-associated glycoprotein. Neutralization of endogenous proBDNF by using antibodies enhanced neurite outgrowth in vitro and in vivo, but this effect was lost in p75NTR(-/-) mice. The neurite outgrowth of cortical neurons from p75NTR deficient (p75NTR(-/-)) mice was insensitive to proBDNF. There was a time-dependent reduction of length and number of filopodia in response to proBDNF which was accompanied with a polarized RhoA activation in growth cones. Moreover, proBDNF treatment of cortical neurons resulted in a time-dependent activation of RhoA but not Cdc42 and the effect was absent in p75NTR(-/-) neurons. Rho kinase (ROCK) and the collapsin response mediator protein-2 (CRMP-2) were also involved in the proBDNF action. CONCLUSIONS: proBDNF has an opposing role in neurite outgrowth to that of mature BDNF. Our observations suggest that proBDNF collapses neurites outgrowth and filopodial growth cones by activating RhoA through the p75NTR signaling pathway.

Effects of proNGF on neuronal viability, neurite growth and amyloid-beta metabolism.

Alzheimer's disease (AD) is characterized pathologically by the deposition of amyloid-beta peptides (Abeta), neurofibrillary tangles, distinctive neuronal loss and neurite dystrophy. Nerve growth factor (NGF) has been suggested to be involved in the pathogenesis of AD, however, the role of its precursor (proNGF) in AD remains unknown. In this study, we investigated the effect of proNGF on neuron death, neurite growth and Abeta production, in vitro and in vivo. We found that proNGF promotes the death of different cell lines and primary neurons in culture, likely dependent on the expression of p75(NTR). We for the first time found that proNGF has an opposite role in neurite growth to that of mature NGF, retarding neurite growth in both cell lines and primary neurons. proNGF is localized to the Abeta plaques in AD mice brain, however, it had no significant effect on Abeta production in vitro and in vivo. Our findings suggest that proNGF is an important factor involving AD pathogenesis.