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RanBP9 at the intersection between cofilin and Aβ pathologies: rescue of neurodegenerative changes by RanBP9 reduction.

Woo JA, Boggess T, Uhlar C, Wang X, Khan H, Cappos G, Joly-Amado A, De Narvaez E, Majid S, Minamide LS, Bamburg JR, Morgan D, Weeber E, Kang DE - Cell Death Dis (2015)

Bottom Line: In this study, we found that endogenous RanBP9 positively regulates SSH1 levels and mediates Aβ-induced translocation of cofilin to mitochondria and induction of cofilin-actin pathology in cultured cells, primary neurons, and in vivo.Endogenous level of RanBP9 was also required for Aβ-induced collapse of growth cones in immature neurons (days in vitro 9 (DIV9)) and depletion of synaptic proteins in mature neurons (DIV21).Therefore, these results underscore the critical importance of endogenous RanBP9 not only in Aβ accumulation but also in mediating the neurotoxic actions of Aβ at the level of synaptic plasticity, mitochondria, and cofilin-actin pathology via control of the SSH1-cofilin pathway in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, USF Health Byrd Alzheimer's Institute, Tampa, FL, USA.

ABSTRACT
Molecular pathways underlying the neurotoxicity and production of amyloid β protein (Aβ) represent potentially promising therapeutic targets for Alzheimer's disease (AD). We recently found that overexpression of the scaffolding protein RanBP9 increases Aβ production in cell lines and in transgenic mice while promoting cofilin activation and mitochondrial dysfunction. Translocation of cofilin to mitochondria and induction of cofilin-actin pathology require the activation/dephosphorylation of cofilin by Slingshot homolog 1 (SSH1) and cysteine oxidation of cofilin. In this study, we found that endogenous RanBP9 positively regulates SSH1 levels and mediates Aβ-induced translocation of cofilin to mitochondria and induction of cofilin-actin pathology in cultured cells, primary neurons, and in vivo. Endogenous level of RanBP9 was also required for Aβ-induced collapse of growth cones in immature neurons (days in vitro 9 (DIV9)) and depletion of synaptic proteins in mature neurons (DIV21). In vivo, amyloid precursor protein (APP)/presenilin-1 (PS1) mice exhibited 3.5-fold increased RanBP9 levels, and RanBP9 reduction protected against cofilin-actin pathology, synaptic damage, gliosis, and Aβ accumulation associated with APP/PS1 mice. Brains slices derived from APP/PS1 mice showed significantly impaired long-term potentiation (LTP), and RanBP9 reduction significantly enhanced paired pulse facilitation and LTP, as well as partially rescued contextual memory deficits associated with APP/PS1 mice. Therefore, these results underscore the critical importance of endogenous RanBP9 not only in Aβ accumulation but also in mediating the neurotoxic actions of Aβ at the level of synaptic plasticity, mitochondria, and cofilin-actin pathology via control of the SSH1-cofilin pathway in vivo.

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RanBP9 mediates the depletion of postsynaptic proteins and F-actin induced by Aβ1-42O in mature primary hippocampal neurons. (a-d) DIV21 primary hippocampal neurons derived from P0 RanBP9+/− and WT littermate mice treated with or without Aβ1-42O (1 μM) for 2 h and subjected to immunocytochemistry for Drebrin, PSD95, and F-actin (Rhodamine-phalloidin). (a) Representative images showing Aβ1-42O-induced depletion of Drebrin, PSD95, and F-actin in WT neurons but not in RanBP9+/− neurons. (b) Quantitation of Drebrin intensity in secondary and tertiary spine-containing dendrites (ANOVA, post-hoc Tukey, ***P<0.0005, n=9 replicates from three pups per genotype). (c) Quantitation of PSD95 intensity in secondary and tertiary spine-containing dendrites (ANOVA, post-hoc Tukey, ***P<0.0005, n=10 replicates from three pups per genotype). (d) Quantitation of F-actin (Rhodamine-phalloidin) intensity in secondary and tertiary spine-containing dendrites (ANOVA, post-hoc Tukey, **P<0.005, *P<0.05, n=4 replicates from two pups per genotype). Error bars represent S.E.M. on graphs. (e and f) Scholl analysis of neurite arborization/elongation in WT and RanBP9+/− hippocampal neurons on DIV21. (e) Representative images of saturated F-actin stain. (f) Quantitation of neurite intersections on concentric circles from the soma in 10μm increments (10–220 μm) (two-way ANOVA, post-hoc Bonferroni, *P<0.05, #P<0.0005, n=3 replicates from two pups per genotype). Error bars represent S.E.M. in graphs
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fig3: RanBP9 mediates the depletion of postsynaptic proteins and F-actin induced by Aβ1-42O in mature primary hippocampal neurons. (a-d) DIV21 primary hippocampal neurons derived from P0 RanBP9+/− and WT littermate mice treated with or without Aβ1-42O (1 μM) for 2 h and subjected to immunocytochemistry for Drebrin, PSD95, and F-actin (Rhodamine-phalloidin). (a) Representative images showing Aβ1-42O-induced depletion of Drebrin, PSD95, and F-actin in WT neurons but not in RanBP9+/− neurons. (b) Quantitation of Drebrin intensity in secondary and tertiary spine-containing dendrites (ANOVA, post-hoc Tukey, ***P<0.0005, n=9 replicates from three pups per genotype). (c) Quantitation of PSD95 intensity in secondary and tertiary spine-containing dendrites (ANOVA, post-hoc Tukey, ***P<0.0005, n=10 replicates from three pups per genotype). (d) Quantitation of F-actin (Rhodamine-phalloidin) intensity in secondary and tertiary spine-containing dendrites (ANOVA, post-hoc Tukey, **P<0.005, *P<0.05, n=4 replicates from two pups per genotype). Error bars represent S.E.M. on graphs. (e and f) Scholl analysis of neurite arborization/elongation in WT and RanBP9+/− hippocampal neurons on DIV21. (e) Representative images of saturated F-actin stain. (f) Quantitation of neurite intersections on concentric circles from the soma in 10μm increments (10–220 μm) (two-way ANOVA, post-hoc Bonferroni, *P<0.05, #P<0.0005, n=3 replicates from two pups per genotype). Error bars represent S.E.M. in graphs

Mentions: Dendritic spines are structures critical for excitatory synaptic transmission and highly enriched in postsynaptic proteins such as Drebrin, PSD95, and F-actin.26 To assess synaptic perturbations induced by Aβ42O, we cultured P0 hippocampal neurons derived from RanBP9+/− and WT littermate mice. We cultured neurons to DIV21 to visualize mature dendritic spines and treated them with or without Aβ42O for 2 h. As expected, immunocytochemical analysis demonstrated that Aβ42O significantly depleted Drebrin, PSD95, and F-actin in dendritic spines and spine-containing neurites in WT neurons (Figures 3a and d). However, the same Aβ42O treatment had no significant effects in RanBP9+/− neurons (Figures 3a and d), indicating that endogenous level of RanBP9 is required for Aβ1-42O-induced depletion of postsynaptic proteins and F-actin in mature neurons. Aβ42 monomer treatment (1 μM, 2 h) did not appreciably alter Drebrin or F-actin levels (Supplementary Figure S1B). In DIV21 mature neurons, Scholl analysis demonstrated significantly increased complexity and length of neurites beyond 50 μm from the soma in RanBP9+/− versus WT neurons (Figures 3e and f), supportive of a role for endogenous RanBP9 in the inhibition of neurite outgrowth and maturation.


RanBP9 at the intersection between cofilin and Aβ pathologies: rescue of neurodegenerative changes by RanBP9 reduction.

Woo JA, Boggess T, Uhlar C, Wang X, Khan H, Cappos G, Joly-Amado A, De Narvaez E, Majid S, Minamide LS, Bamburg JR, Morgan D, Weeber E, Kang DE - Cell Death Dis (2015)

RanBP9 mediates the depletion of postsynaptic proteins and F-actin induced by Aβ1-42O in mature primary hippocampal neurons. (a-d) DIV21 primary hippocampal neurons derived from P0 RanBP9+/− and WT littermate mice treated with or without Aβ1-42O (1 μM) for 2 h and subjected to immunocytochemistry for Drebrin, PSD95, and F-actin (Rhodamine-phalloidin). (a) Representative images showing Aβ1-42O-induced depletion of Drebrin, PSD95, and F-actin in WT neurons but not in RanBP9+/− neurons. (b) Quantitation of Drebrin intensity in secondary and tertiary spine-containing dendrites (ANOVA, post-hoc Tukey, ***P<0.0005, n=9 replicates from three pups per genotype). (c) Quantitation of PSD95 intensity in secondary and tertiary spine-containing dendrites (ANOVA, post-hoc Tukey, ***P<0.0005, n=10 replicates from three pups per genotype). (d) Quantitation of F-actin (Rhodamine-phalloidin) intensity in secondary and tertiary spine-containing dendrites (ANOVA, post-hoc Tukey, **P<0.005, *P<0.05, n=4 replicates from two pups per genotype). Error bars represent S.E.M. on graphs. (e and f) Scholl analysis of neurite arborization/elongation in WT and RanBP9+/− hippocampal neurons on DIV21. (e) Representative images of saturated F-actin stain. (f) Quantitation of neurite intersections on concentric circles from the soma in 10μm increments (10–220 μm) (two-way ANOVA, post-hoc Bonferroni, *P<0.05, #P<0.0005, n=3 replicates from two pups per genotype). Error bars represent S.E.M. in graphs
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4385917&req=5

fig3: RanBP9 mediates the depletion of postsynaptic proteins and F-actin induced by Aβ1-42O in mature primary hippocampal neurons. (a-d) DIV21 primary hippocampal neurons derived from P0 RanBP9+/− and WT littermate mice treated with or without Aβ1-42O (1 μM) for 2 h and subjected to immunocytochemistry for Drebrin, PSD95, and F-actin (Rhodamine-phalloidin). (a) Representative images showing Aβ1-42O-induced depletion of Drebrin, PSD95, and F-actin in WT neurons but not in RanBP9+/− neurons. (b) Quantitation of Drebrin intensity in secondary and tertiary spine-containing dendrites (ANOVA, post-hoc Tukey, ***P<0.0005, n=9 replicates from three pups per genotype). (c) Quantitation of PSD95 intensity in secondary and tertiary spine-containing dendrites (ANOVA, post-hoc Tukey, ***P<0.0005, n=10 replicates from three pups per genotype). (d) Quantitation of F-actin (Rhodamine-phalloidin) intensity in secondary and tertiary spine-containing dendrites (ANOVA, post-hoc Tukey, **P<0.005, *P<0.05, n=4 replicates from two pups per genotype). Error bars represent S.E.M. on graphs. (e and f) Scholl analysis of neurite arborization/elongation in WT and RanBP9+/− hippocampal neurons on DIV21. (e) Representative images of saturated F-actin stain. (f) Quantitation of neurite intersections on concentric circles from the soma in 10μm increments (10–220 μm) (two-way ANOVA, post-hoc Bonferroni, *P<0.05, #P<0.0005, n=3 replicates from two pups per genotype). Error bars represent S.E.M. in graphs
Mentions: Dendritic spines are structures critical for excitatory synaptic transmission and highly enriched in postsynaptic proteins such as Drebrin, PSD95, and F-actin.26 To assess synaptic perturbations induced by Aβ42O, we cultured P0 hippocampal neurons derived from RanBP9+/− and WT littermate mice. We cultured neurons to DIV21 to visualize mature dendritic spines and treated them with or without Aβ42O for 2 h. As expected, immunocytochemical analysis demonstrated that Aβ42O significantly depleted Drebrin, PSD95, and F-actin in dendritic spines and spine-containing neurites in WT neurons (Figures 3a and d). However, the same Aβ42O treatment had no significant effects in RanBP9+/− neurons (Figures 3a and d), indicating that endogenous level of RanBP9 is required for Aβ1-42O-induced depletion of postsynaptic proteins and F-actin in mature neurons. Aβ42 monomer treatment (1 μM, 2 h) did not appreciably alter Drebrin or F-actin levels (Supplementary Figure S1B). In DIV21 mature neurons, Scholl analysis demonstrated significantly increased complexity and length of neurites beyond 50 μm from the soma in RanBP9+/− versus WT neurons (Figures 3e and f), supportive of a role for endogenous RanBP9 in the inhibition of neurite outgrowth and maturation.

Bottom Line: In this study, we found that endogenous RanBP9 positively regulates SSH1 levels and mediates Aβ-induced translocation of cofilin to mitochondria and induction of cofilin-actin pathology in cultured cells, primary neurons, and in vivo.Endogenous level of RanBP9 was also required for Aβ-induced collapse of growth cones in immature neurons (days in vitro 9 (DIV9)) and depletion of synaptic proteins in mature neurons (DIV21).Therefore, these results underscore the critical importance of endogenous RanBP9 not only in Aβ accumulation but also in mediating the neurotoxic actions of Aβ at the level of synaptic plasticity, mitochondria, and cofilin-actin pathology via control of the SSH1-cofilin pathway in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, USF Health Byrd Alzheimer's Institute, Tampa, FL, USA.

ABSTRACT
Molecular pathways underlying the neurotoxicity and production of amyloid β protein (Aβ) represent potentially promising therapeutic targets for Alzheimer's disease (AD). We recently found that overexpression of the scaffolding protein RanBP9 increases Aβ production in cell lines and in transgenic mice while promoting cofilin activation and mitochondrial dysfunction. Translocation of cofilin to mitochondria and induction of cofilin-actin pathology require the activation/dephosphorylation of cofilin by Slingshot homolog 1 (SSH1) and cysteine oxidation of cofilin. In this study, we found that endogenous RanBP9 positively regulates SSH1 levels and mediates Aβ-induced translocation of cofilin to mitochondria and induction of cofilin-actin pathology in cultured cells, primary neurons, and in vivo. Endogenous level of RanBP9 was also required for Aβ-induced collapse of growth cones in immature neurons (days in vitro 9 (DIV9)) and depletion of synaptic proteins in mature neurons (DIV21). In vivo, amyloid precursor protein (APP)/presenilin-1 (PS1) mice exhibited 3.5-fold increased RanBP9 levels, and RanBP9 reduction protected against cofilin-actin pathology, synaptic damage, gliosis, and Aβ accumulation associated with APP/PS1 mice. Brains slices derived from APP/PS1 mice showed significantly impaired long-term potentiation (LTP), and RanBP9 reduction significantly enhanced paired pulse facilitation and LTP, as well as partially rescued contextual memory deficits associated with APP/PS1 mice. Therefore, these results underscore the critical importance of endogenous RanBP9 not only in Aβ accumulation but also in mediating the neurotoxic actions of Aβ at the level of synaptic plasticity, mitochondria, and cofilin-actin pathology via control of the SSH1-cofilin pathway in vivo.

Show MeSH
Related in: MedlinePlus