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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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Neurite growth of basal forebrain cholinergic neurons is inhibited by CR-proBDNF, but elicited by matBDNF. BFCNs were cultured in serum-containig (A-E) or serum-free (F) medium for 2 weeks and treated with 100 ng/ml CR-proBDNF or matBDNF in the same medium condition for 2 days. Histochemistry and quantitation of AChE-positve neurites were done as described in Methods. (A) BFCNs were double-stained using AChE histochemistry and Hoechst 33258 (arrows). (B) The survival rate of BFCNs (%) = 100 × [living cells]/([living cells] + [dead cells]). Data were normalized to mock cultures (100% as control). n = 159 (Mock), 151 (matBDNF), and 149 (CR-proBDNF) from three independent coverslips. Results were replicated in three independent experiments. (C) Low- and high-magnification images of AChE-stained BFCNs. (D) The number of neurites extending outwards from the cell body shown in C. n = 38 (Mock), 37 (matBDNF), and 40 (CR-proBDNF) cells from three independent coverslips. (E) Neurite complexity as revealed by Sholl analysis. n = 30 (Mock), 30 (matBDNF), and 30 (CR-proBDNF) cells from three independent coverslips. In multi-bar graphs, ANOVA followed by post-hoc analysis was used. **P < 0.01. Results were replicated in at least three independent experiments. (F) The opposing effects of matBDNF and CR-proBDNF on neurite fiber density were confirmed by a distinct quantitative method. The maximal threshold of AChE-positive cholinergic fiber intensity was defined as 70% above the background. The total intensity of the fibers was determined in an optical field and was divided by the number of AChE-positive neurons in the same field. Data were collected from four independent fields in a single chamber. t-test, **P < 0.01, compared to Mock (100% as control); n = 6 independent culture dishes. Scale bar, 5 μm (A and C). (G) Effect of CR-proBDNF on neurite density of BFCNs in serum-free condition and a dose-dependency test of CR-proBDNF. Cell survival (left) and the neurite number of BFCNs (right) were determined. Note that proBDNF negatively regulates the neurite density of BFCNs in serum-free conditions and at subnanomoler concentration. n = 3 independent coverslips. t-test, **P < 0.01, significantly different from Mock.
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Figure 4: Neurite growth of basal forebrain cholinergic neurons is inhibited by CR-proBDNF, but elicited by matBDNF. BFCNs were cultured in serum-containig (A-E) or serum-free (F) medium for 2 weeks and treated with 100 ng/ml CR-proBDNF or matBDNF in the same medium condition for 2 days. Histochemistry and quantitation of AChE-positve neurites were done as described in Methods. (A) BFCNs were double-stained using AChE histochemistry and Hoechst 33258 (arrows). (B) The survival rate of BFCNs (%) = 100 × [living cells]/([living cells] + [dead cells]). Data were normalized to mock cultures (100% as control). n = 159 (Mock), 151 (matBDNF), and 149 (CR-proBDNF) from three independent coverslips. Results were replicated in three independent experiments. (C) Low- and high-magnification images of AChE-stained BFCNs. (D) The number of neurites extending outwards from the cell body shown in C. n = 38 (Mock), 37 (matBDNF), and 40 (CR-proBDNF) cells from three independent coverslips. (E) Neurite complexity as revealed by Sholl analysis. n = 30 (Mock), 30 (matBDNF), and 30 (CR-proBDNF) cells from three independent coverslips. In multi-bar graphs, ANOVA followed by post-hoc analysis was used. **P < 0.01. Results were replicated in at least three independent experiments. (F) The opposing effects of matBDNF and CR-proBDNF on neurite fiber density were confirmed by a distinct quantitative method. The maximal threshold of AChE-positive cholinergic fiber intensity was defined as 70% above the background. The total intensity of the fibers was determined in an optical field and was divided by the number of AChE-positive neurons in the same field. Data were collected from four independent fields in a single chamber. t-test, **P < 0.01, compared to Mock (100% as control); n = 6 independent culture dishes. Scale bar, 5 μm (A and C). (G) Effect of CR-proBDNF on neurite density of BFCNs in serum-free condition and a dose-dependency test of CR-proBDNF. Cell survival (left) and the neurite number of BFCNs (right) were determined. Note that proBDNF negatively regulates the neurite density of BFCNs in serum-free conditions and at subnanomoler concentration. n = 3 independent coverslips. t-test, **P < 0.01, significantly different from Mock.

Mentions: Consistently with previous reports [28,29], 4-day treatment with matBDNF (100 ng/ml) markedly increased the number and complexity of acetylcholine esterase (AChE)-positive neurites of the 2-week cultured BFCNs (Fig. 4A and 4C, matBDNF). The cell viability of the AChE-positive BFCNs was assessed by DAPI staining (Fig. 4A). Unlike what was observed in CGNs, neither matBDNF nor CR-proBDNF (E. Coli-derived recombinant protein) has a significant effect on the number of the survived BFCNs and on the nuclear morphology (Fig. 4A, arrows, and 4B). However, CR-proBDNF caused the opposite effect on fiber density in BFCNs: treatment with 100 ng/ml CR-proBDNF for 4 days led to a marked reduction in the neurite number of AChE-positive neurites extending outwards from the cells (Fig. 4D, CR-proBDNF), without affecting the survival of these cells (Fig. 4C, CR-proBDNF). Quantitative analysis showed that CR-proBDNF decreased the number of primary fibers by more than 45% (Fig. 4D, CR-proBDNF). In contrast, matBDNF increased the number of the AChE-positive fibers by approximately 17% (Fig. 4D, mBDNF). We performed several methods to evaluate of neurite growth. Sholl analysis, which is used to assess the number and complexity of fibers extending out from the cell body [30], revealed that CR-proBDNF significantly reduced the number of cholinergic fibers in the first 10 μm from the cell body by 42% (Fig. 4E, CR-proBDNF). We also determined the neurite density of BFCNs in culture dishes (intensity of cholinergic fibers/number of cholinergic neurons). The opposing effects of matBDNF and CR-proBDNF on the fiber density were confirmed by this quantitative method (see Methods): Mock 100 ± 13.6% (as control); matBDNF, 149 ± 9.3%**; CR-proBDNF, 37.3 ± 9.4%** (t-test, **P < 0.01, compared to Mock; n = 6 independent culture dishes) (Fig. 4F). These results together suggest that CR-proBDNF inhibits neurite growth of BFCNs while matBDNF enhances this growth.


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)

Neurite growth of basal forebrain cholinergic neurons is inhibited by CR-proBDNF, but elicited by matBDNF. BFCNs were cultured in serum-containig (A-E) or serum-free (F) medium for 2 weeks and treated with 100 ng/ml CR-proBDNF or matBDNF in the same medium condition for 2 days. Histochemistry and quantitation of AChE-positve neurites were done as described in Methods. (A) BFCNs were double-stained using AChE histochemistry and Hoechst 33258 (arrows). (B) The survival rate of BFCNs (%) = 100 × [living cells]/([living cells] + [dead cells]). Data were normalized to mock cultures (100% as control). n = 159 (Mock), 151 (matBDNF), and 149 (CR-proBDNF) from three independent coverslips. Results were replicated in three independent experiments. (C) Low- and high-magnification images of AChE-stained BFCNs. (D) The number of neurites extending outwards from the cell body shown in C. n = 38 (Mock), 37 (matBDNF), and 40 (CR-proBDNF) cells from three independent coverslips. (E) Neurite complexity as revealed by Sholl analysis. n = 30 (Mock), 30 (matBDNF), and 30 (CR-proBDNF) cells from three independent coverslips. In multi-bar graphs, ANOVA followed by post-hoc analysis was used. **P < 0.01. Results were replicated in at least three independent experiments. (F) The opposing effects of matBDNF and CR-proBDNF on neurite fiber density were confirmed by a distinct quantitative method. The maximal threshold of AChE-positive cholinergic fiber intensity was defined as 70% above the background. The total intensity of the fibers was determined in an optical field and was divided by the number of AChE-positive neurons in the same field. Data were collected from four independent fields in a single chamber. t-test, **P < 0.01, compared to Mock (100% as control); n = 6 independent culture dishes. Scale bar, 5 μm (A and C). (G) Effect of CR-proBDNF on neurite density of BFCNs in serum-free condition and a dose-dependency test of CR-proBDNF. Cell survival (left) and the neurite number of BFCNs (right) were determined. Note that proBDNF negatively regulates the neurite density of BFCNs in serum-free conditions and at subnanomoler concentration. n = 3 independent coverslips. t-test, **P < 0.01, significantly different from Mock.
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Figure 4: Neurite growth of basal forebrain cholinergic neurons is inhibited by CR-proBDNF, but elicited by matBDNF. BFCNs were cultured in serum-containig (A-E) or serum-free (F) medium for 2 weeks and treated with 100 ng/ml CR-proBDNF or matBDNF in the same medium condition for 2 days. Histochemistry and quantitation of AChE-positve neurites were done as described in Methods. (A) BFCNs were double-stained using AChE histochemistry and Hoechst 33258 (arrows). (B) The survival rate of BFCNs (%) = 100 × [living cells]/([living cells] + [dead cells]). Data were normalized to mock cultures (100% as control). n = 159 (Mock), 151 (matBDNF), and 149 (CR-proBDNF) from three independent coverslips. Results were replicated in three independent experiments. (C) Low- and high-magnification images of AChE-stained BFCNs. (D) The number of neurites extending outwards from the cell body shown in C. n = 38 (Mock), 37 (matBDNF), and 40 (CR-proBDNF) cells from three independent coverslips. (E) Neurite complexity as revealed by Sholl analysis. n = 30 (Mock), 30 (matBDNF), and 30 (CR-proBDNF) cells from three independent coverslips. In multi-bar graphs, ANOVA followed by post-hoc analysis was used. **P < 0.01. Results were replicated in at least three independent experiments. (F) The opposing effects of matBDNF and CR-proBDNF on neurite fiber density were confirmed by a distinct quantitative method. The maximal threshold of AChE-positive cholinergic fiber intensity was defined as 70% above the background. The total intensity of the fibers was determined in an optical field and was divided by the number of AChE-positive neurons in the same field. Data were collected from four independent fields in a single chamber. t-test, **P < 0.01, compared to Mock (100% as control); n = 6 independent culture dishes. Scale bar, 5 μm (A and C). (G) Effect of CR-proBDNF on neurite density of BFCNs in serum-free condition and a dose-dependency test of CR-proBDNF. Cell survival (left) and the neurite number of BFCNs (right) were determined. Note that proBDNF negatively regulates the neurite density of BFCNs in serum-free conditions and at subnanomoler concentration. n = 3 independent coverslips. t-test, **P < 0.01, significantly different from Mock.
Mentions: Consistently with previous reports [28,29], 4-day treatment with matBDNF (100 ng/ml) markedly increased the number and complexity of acetylcholine esterase (AChE)-positive neurites of the 2-week cultured BFCNs (Fig. 4A and 4C, matBDNF). The cell viability of the AChE-positive BFCNs was assessed by DAPI staining (Fig. 4A). Unlike what was observed in CGNs, neither matBDNF nor CR-proBDNF (E. Coli-derived recombinant protein) has a significant effect on the number of the survived BFCNs and on the nuclear morphology (Fig. 4A, arrows, and 4B). However, CR-proBDNF caused the opposite effect on fiber density in BFCNs: treatment with 100 ng/ml CR-proBDNF for 4 days led to a marked reduction in the neurite number of AChE-positive neurites extending outwards from the cells (Fig. 4D, CR-proBDNF), without affecting the survival of these cells (Fig. 4C, CR-proBDNF). Quantitative analysis showed that CR-proBDNF decreased the number of primary fibers by more than 45% (Fig. 4D, CR-proBDNF). In contrast, matBDNF increased the number of the AChE-positive fibers by approximately 17% (Fig. 4D, mBDNF). We performed several methods to evaluate of neurite growth. Sholl analysis, which is used to assess the number and complexity of fibers extending out from the cell body [30], revealed that CR-proBDNF significantly reduced the number of cholinergic fibers in the first 10 μm from the cell body by 42% (Fig. 4E, CR-proBDNF). We also determined the neurite density of BFCNs in culture dishes (intensity of cholinergic fibers/number of cholinergic neurons). The opposing effects of matBDNF and CR-proBDNF on the fiber density were confirmed by this quantitative method (see Methods): Mock 100 ± 13.6% (as control); matBDNF, 149 ± 9.3%**; CR-proBDNF, 37.3 ± 9.4%** (t-test, **P < 0.01, compared to Mock; n = 6 independent culture dishes) (Fig. 4F). These results together suggest that CR-proBDNF inhibits neurite growth of BFCNs while matBDNF enhances this growth.

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