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

Koshimizu H, Kiyosue K, Hara T, Hazama S, Suzuki S, Uegaki K, Nagappan G, Zaitsev E, Hirokawa T, Tatsu Y, Ogura A, Lu B, Kojima M - Mol Brain (2009)

Bottom Line: Second, we purified recombinant CR-proBDNF and tested its biological effects using cultured CNS 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute of Advanced Industrial Science and Technology (AIST), Ikeda, 563-8577 Japan. koshimih@mail.nih.gov

ABSTRACT

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.

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proBDNF reduces dendritic spine density in hippocampal neurons. Cultured neurons were maintained for 3–4 weeks and treated with the indicated reagents (50 ng/ml) for 2 (A, B, and D) or 3 days (C). In A and B, neurons were cultured in serum-containing medium. (A) CR-proBDNF reduced the density of DiI-labeled spines. Representative images of DiI-labeled neurons treated with the indicated reagents for 2 days (left). Summary of spine density (middle) and length (right). Data were collected from 52 (Mock), 59 (matBDNF), and 38 (CR-proBDNF) independent cells (non-parametric test, *P < 0.05, compared to Mock). Note that matBDNF increased spine density, while proBDNF reduced spine density (arrows and arrowheads). (B) proBDNF promoted the shrinkage of phalloidin-labeled spines and p75NTR was involved in the proBDNF action. Application of a functionally blocking antibody against p75NTR, AB1554 was performed and fixed neurons were stained with an anti-MAP2 antibody and FITC-labeled phalloidin. Representative images of the double-stained neurons (left) and a quantitative analysis of phalloidin-labeled spiny structures (right) are shown, n = 30–40 independent cells from three independent coverslips. t-test, *P < 0.05, **P < 0.01, compared to Mock (100% as control). (C) CR-proBDNF decreases spine density in low-density hippocampal neurons cultured in serum-free medium. The cultures were incubated with CR-proBDNF in serum-free medium for 2 days and double-stained with an anti-MAP2 antibody and FITC-labeled phalloidin. n = 24–32 cells from three independent coverslips. t-test, *P < 0.05, **P < 0.01, compared to Mock. (D) CR-proBDNF decreased spine density in hippocampal slices. Representative images of DiI-labeled neurons at low and high magnification are shown (left). A decrease in spine density was observed in CR-proBDNF-treated slices (arrow heads). Quantification of spine density (right). Data were collected from 7 (Mock) and 6 (CR-proBDNF) independent slices. Note that CR-proBDNF-treated slices showed a decrease in spine density when compared with matBDNF. t-test, *P < 0.05. Scale bar, 10 μm (A, B, and D).
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Figure 5: proBDNF reduces dendritic spine density in hippocampal neurons. Cultured neurons were maintained for 3–4 weeks and treated with the indicated reagents (50 ng/ml) for 2 (A, B, and D) or 3 days (C). In A and B, neurons were cultured in serum-containing medium. (A) CR-proBDNF reduced the density of DiI-labeled spines. Representative images of DiI-labeled neurons treated with the indicated reagents for 2 days (left). Summary of spine density (middle) and length (right). Data were collected from 52 (Mock), 59 (matBDNF), and 38 (CR-proBDNF) independent cells (non-parametric test, *P < 0.05, compared to Mock). Note that matBDNF increased spine density, while proBDNF reduced spine density (arrows and arrowheads). (B) proBDNF promoted the shrinkage of phalloidin-labeled spines and p75NTR was involved in the proBDNF action. Application of a functionally blocking antibody against p75NTR, AB1554 was performed and fixed neurons were stained with an anti-MAP2 antibody and FITC-labeled phalloidin. Representative images of the double-stained neurons (left) and a quantitative analysis of phalloidin-labeled spiny structures (right) are shown, n = 30–40 independent cells from three independent coverslips. t-test, *P < 0.05, **P < 0.01, compared to Mock (100% as control). (C) CR-proBDNF decreases spine density in low-density hippocampal neurons cultured in serum-free medium. The cultures were incubated with CR-proBDNF in serum-free medium for 2 days and double-stained with an anti-MAP2 antibody and FITC-labeled phalloidin. n = 24–32 cells from three independent coverslips. t-test, *P < 0.05, **P < 0.01, compared to Mock. (D) CR-proBDNF decreased spine density in hippocampal slices. Representative images of DiI-labeled neurons at low and high magnification are shown (left). A decrease in spine density was observed in CR-proBDNF-treated slices (arrow heads). Quantification of spine density (right). Data were collected from 7 (Mock) and 6 (CR-proBDNF) independent slices. Note that CR-proBDNF-treated slices showed a decrease in spine density when compared with matBDNF. t-test, *P < 0.05. Scale bar, 10 μm (A, B, and D).

Mentions: As proBDNF facilitates LTD [15], proBDNF may have distinct biological roles in synaptic plasticity [10]. To test this possibility, we cultured hippocampal neurons (6–8 × 104 cells/cm2) for 3–4 weeks and then applied 50 ng/ml of matBDNF or proBDNF for 2 days. We first performed DiI labeling of dendritic spine structures [31]. Consistently with a previous report [32], matBDNF dramatically increased spine density in hippocampal neurons (Fig. 5A, matBDNF, arrows). However, CR-proBDNF appeared to promote the density of spine protrusions (Fig. 5A, CR-proBDNF, arrowheads), which suggests that proBDNF negatively regulates dendritic spine density in CNS neurons. To evaluate this effect of CR-proBDNF in a quantitative manner, we assessed the density of dendritic spines with a typical mushroom head and length (> 1 μm) on the proximal region of dendrites, as described [31,33]. Quantitative analysis demonstrated that matBDNF increased dendritic spine density by 34.9 ± 8.9%, whereas CR-proBDNF decreased spine density by 41.6 ± 9.0% (Fig. 5A, left graph, nonparametric test; *P < 0.05 when compared with Mock). Finally, cultured hippocampal neurons treated with heat-denatured CR-proBDNF (50 ng/ml, 2 days) had a dendritic spine density that was 77.2 ± 0.1% higher than that observed with CR-proBDNF (t-test, **P < 0.001, compared to CR-proBDNF-treated cells, n = 41 (CR-proBDNF), 77 (heat-denatured CR-proBDNF) independent cells), suggesting that the reduction of dendritic spine density is a biological activity of proBDNF protein.


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

Koshimizu H, Kiyosue K, Hara T, Hazama S, Suzuki S, Uegaki K, Nagappan G, Zaitsev E, Hirokawa T, Tatsu Y, Ogura A, Lu B, Kojima M - Mol Brain (2009)

proBDNF reduces dendritic spine density in hippocampal neurons. Cultured neurons were maintained for 3–4 weeks and treated with the indicated reagents (50 ng/ml) for 2 (A, B, and D) or 3 days (C). In A and B, neurons were cultured in serum-containing medium. (A) CR-proBDNF reduced the density of DiI-labeled spines. Representative images of DiI-labeled neurons treated with the indicated reagents for 2 days (left). Summary of spine density (middle) and length (right). Data were collected from 52 (Mock), 59 (matBDNF), and 38 (CR-proBDNF) independent cells (non-parametric test, *P < 0.05, compared to Mock). Note that matBDNF increased spine density, while proBDNF reduced spine density (arrows and arrowheads). (B) proBDNF promoted the shrinkage of phalloidin-labeled spines and p75NTR was involved in the proBDNF action. Application of a functionally blocking antibody against p75NTR, AB1554 was performed and fixed neurons were stained with an anti-MAP2 antibody and FITC-labeled phalloidin. Representative images of the double-stained neurons (left) and a quantitative analysis of phalloidin-labeled spiny structures (right) are shown, n = 30–40 independent cells from three independent coverslips. t-test, *P < 0.05, **P < 0.01, compared to Mock (100% as control). (C) CR-proBDNF decreases spine density in low-density hippocampal neurons cultured in serum-free medium. The cultures were incubated with CR-proBDNF in serum-free medium for 2 days and double-stained with an anti-MAP2 antibody and FITC-labeled phalloidin. n = 24–32 cells from three independent coverslips. t-test, *P < 0.05, **P < 0.01, compared to Mock. (D) CR-proBDNF decreased spine density in hippocampal slices. Representative images of DiI-labeled neurons at low and high magnification are shown (left). A decrease in spine density was observed in CR-proBDNF-treated slices (arrow heads). Quantification of spine density (right). Data were collected from 7 (Mock) and 6 (CR-proBDNF) independent slices. Note that CR-proBDNF-treated slices showed a decrease in spine density when compared with matBDNF. t-test, *P < 0.05. Scale bar, 10 μm (A, B, and D).
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Figure 5: proBDNF reduces dendritic spine density in hippocampal neurons. Cultured neurons were maintained for 3–4 weeks and treated with the indicated reagents (50 ng/ml) for 2 (A, B, and D) or 3 days (C). In A and B, neurons were cultured in serum-containing medium. (A) CR-proBDNF reduced the density of DiI-labeled spines. Representative images of DiI-labeled neurons treated with the indicated reagents for 2 days (left). Summary of spine density (middle) and length (right). Data were collected from 52 (Mock), 59 (matBDNF), and 38 (CR-proBDNF) independent cells (non-parametric test, *P < 0.05, compared to Mock). Note that matBDNF increased spine density, while proBDNF reduced spine density (arrows and arrowheads). (B) proBDNF promoted the shrinkage of phalloidin-labeled spines and p75NTR was involved in the proBDNF action. Application of a functionally blocking antibody against p75NTR, AB1554 was performed and fixed neurons were stained with an anti-MAP2 antibody and FITC-labeled phalloidin. Representative images of the double-stained neurons (left) and a quantitative analysis of phalloidin-labeled spiny structures (right) are shown, n = 30–40 independent cells from three independent coverslips. t-test, *P < 0.05, **P < 0.01, compared to Mock (100% as control). (C) CR-proBDNF decreases spine density in low-density hippocampal neurons cultured in serum-free medium. The cultures were incubated with CR-proBDNF in serum-free medium for 2 days and double-stained with an anti-MAP2 antibody and FITC-labeled phalloidin. n = 24–32 cells from three independent coverslips. t-test, *P < 0.05, **P < 0.01, compared to Mock. (D) CR-proBDNF decreased spine density in hippocampal slices. Representative images of DiI-labeled neurons at low and high magnification are shown (left). A decrease in spine density was observed in CR-proBDNF-treated slices (arrow heads). Quantification of spine density (right). Data were collected from 7 (Mock) and 6 (CR-proBDNF) independent slices. Note that CR-proBDNF-treated slices showed a decrease in spine density when compared with matBDNF. t-test, *P < 0.05. Scale bar, 10 μm (A, B, and D).
Mentions: As proBDNF facilitates LTD [15], proBDNF may have distinct biological roles in synaptic plasticity [10]. To test this possibility, we cultured hippocampal neurons (6–8 × 104 cells/cm2) for 3–4 weeks and then applied 50 ng/ml of matBDNF or proBDNF for 2 days. We first performed DiI labeling of dendritic spine structures [31]. Consistently with a previous report [32], matBDNF dramatically increased spine density in hippocampal neurons (Fig. 5A, matBDNF, arrows). However, CR-proBDNF appeared to promote the density of spine protrusions (Fig. 5A, CR-proBDNF, arrowheads), which suggests that proBDNF negatively regulates dendritic spine density in CNS neurons. To evaluate this effect of CR-proBDNF in a quantitative manner, we assessed the density of dendritic spines with a typical mushroom head and length (> 1 μm) on the proximal region of dendrites, as described [31,33]. Quantitative analysis demonstrated that matBDNF increased dendritic spine density by 34.9 ± 8.9%, whereas CR-proBDNF decreased spine density by 41.6 ± 9.0% (Fig. 5A, left graph, nonparametric test; *P < 0.05 when compared with Mock). Finally, cultured hippocampal neurons treated with heat-denatured CR-proBDNF (50 ng/ml, 2 days) had a dendritic spine density that was 77.2 ± 0.1% higher than that observed with CR-proBDNF (t-test, **P < 0.001, compared to CR-proBDNF-treated cells, n = 41 (CR-proBDNF), 77 (heat-denatured CR-proBDNF) independent cells), suggesting that the reduction of dendritic spine density is a biological activity of proBDNF protein.

Bottom Line: Second, we purified recombinant CR-proBDNF and tested its biological effects using cultured CNS 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: National Institute of Advanced Industrial Science and Technology (AIST), Ikeda, 563-8577 Japan. koshimih@mail.nih.gov

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus