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, 49 (1), 13-22

Neurofibromatosis-1 Heterozygosity Impairs CNS Neuronal Morphology in a cAMP/PKA/ROCK-dependent Manner

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Neurofibromatosis-1 Heterozygosity Impairs CNS Neuronal Morphology in a cAMP/PKA/ROCK-dependent Manner

Jacquelyn A Brown et al. Mol Cell Neurosci.

Abstract

Children with the neurofibromatosis-1 (NF1) cancer predisposition syndrome exhibit numerous clinical problems that reflect defective central nervous system (CNS) neuronal function, including learning disabilities, attention deficit disorder, and seizures. These clinical features result from reduced NF1 protein (neurofibromin) expression in NF1+/- (NF1 heterozygosity) brain neurons. Previous studies have shown that mouse CNS neurons are sensitive to the effects of reduced Nf1 expression and exhibit shorter neurite lengths, smaller growth cone areas, and attenuated survival, reflecting attenuated neurofibromin cAMP regulation. In striking contrast, Nf1+/- peripheral nervous system (PNS) neurons are nearly indistinguishable from their wild-type counterparts, and complete neurofibromin loss leads to increased neurite lengths and survival in a RAS/Akt-dependent fashion. To gain insights into the differential responses of CNS and PNS neurons to reduced neurofibromin function, we designed a series of experiments to define the molecular mechanism(s) underlying the unique CNS neuronal sensitivity to Nf1 heterozygosity. First, Nf1 heterozygosity decreases cAMP levels in CNS, but not in PNS, neurons. Second, CNS neurons exhibit Nf1 gene-dependent increases in RAS pathway signaling, but no further decreases in cAMP levels were observed in Nf1-/- CNS neurons relative to their Nf1+/- counterparts. Third, neurofibromin regulates CNS neurite length and growth cone areas in a cAMP/PKA/Rho/ROCK-dependent manner in vitro and in vivo. Collectively, these findings establish cAMP/PKA/Rho/ROCK signaling as the responsible axis underlying abnormal Nf1+/- CNS neuronal morphology with important implications for future preclinical and clinical studies aimed at improving cognitive and behavioral deficits in mice and children with reduced brain neuronal NF1 gene expression.

Figures

Figure 1
Figure 1. Nf1 heterozygosity decreases cAMP levels only in CNS neurons
(A) Schematic representation of the proposed neurofibromin-regulated signaling pathways. (B) Both Nf1+/− hippocampal (CNS) neurons and DRG (PNS) neurons exhibit a greater than 2-fold increase in ERK1/2 activation (phospho-ERK normalized to total ERK) relative to their respective WT controls (P=.0001, N=6). (C) Nf1 heterozygosity reduces cAMP levels only in Nf1+/− hippocampal neurons (CNS) relative to WT controls (P=.003, N=8). No differences in cAMP levels are found between Nf1+/− and WT DRG (PNS) neurons. (D) A representative phospho-PKA (p-PKA) substrate Western blot of CNS (hippocampal) and PNS (DRG) neurons (left panel). While all four p-PKA protein bands exhibit reduced phosphorylation in Nf1+/− CNS neurons relative to their WT counterparts, Nf1+/− PNS neurons exhibited increased levels of p-PKA substrate expression (protein bands 1, 2, and 4). p-PKA substrate protein band 3 is reduced in Nf1+/− PNS neurons compared to WT controls (best visualized with the original uninverted image; left panel, bottom). Changes in Nf1+/− neurons are graphically illustrated relative to their respective WT controls (right panel). Asterisks denote statistically significant differences.
Figure 2
Figure 2. cAMP reduction in PNS neurons has no effect on neurite length or growth cone spreading
(A) Nf1+/− and WT DRG neurite lengths (following Tuj-1 immunostaining) are unchanged following DDA treatment while WT hippocampal neurite lengths were reduced (P=.83, N=33; P=.001, N=30). (B) No changes in growth cone areas were observed in WT and Nf1+/− DRG neurons following DDA treatment, unlike the smaller growth cones areas observed in WT hippocampal neurons (P=.86, N=33; P=.001, N=30). Scale bar, 100 μm.
Figure 3
Figure 3. CNS neurons exhibit Nf1 gene dose-dependent changes in ERK1/2 activity, but not cAMP levels
(A) Embryonic brains (E11) from WT, Nf1+/−, and Nf1−/− littermates were analyzed for ERK1/2 activation. Increasing ERK activity (p-ERK normalized to total ERK) was observed with decreasing Nf1 expression, such that Nf1+/− and Nf1−/− neurons exhibit 2.1-fold and 2.7-fold increased ERK1/2 activation, respectively. (B) cAMP levels are significantly reduced in Nf1+/− forebrain preparations (E11) compared to those from WT (P=.001, N=10), however no additional reduction was observed in Nf1−/− mouse brains. Statistical significance between groups was assessed by ANOVA followed by Bonferroni post-hoc comparisons.
Figure 4
Figure 4. cAMP regulation of PKA is responsible for the growth cone and neurite length defects seen in Nf1+/− CNS neurons
(A) Nf1+/− RGC growth cones (labeled with Tuj-1) are significantly smaller than their WT counterparts (P=.0001, N=33). Scale bar =50 μm. Whereas treatment with the Me-cAMP analog (Epac activator) fails to restore Nf1+/− CNS neuron growth cone areas to WT levels, Br-cAMP (activates Epac and PKA) and Phe-cAMP (activates PKA) both rescue the Nf1+/− neuronal growth cone defect. (B) Nf1+/− hippocampal neurite lengths, as identified by Tuj-1 immunostaining, are shorter than their WT counterparts (P=.0004, N=33). Similarly, only Br-cAMP and Phe-cAMP restore the Nf1+/− neurite length defects to WT levels. (C) Neurons in the retinal ganglion layer (bracketed region) of 3-month-old Nf1+/− retinas demonstrate reduced p-PKA substrate immunostaining relative to their WT counterparts, which is ameliorated following treatment with Rolipram for two weeks. In contrast, activated KRas expression (KRasBLBP mouse) has no effect on p-PKA substrate immunostaining in the retinal ganglion layer (mean fluorescent intensities: WT 23.6±1.9; Nf1+/− 11.7±0.5; Nf1+/− +Rol 25.6±2.1; KRasBLBP 22.1±0.5). (D) Transmitted light images of the same p-PKA immunostained retinas in panel C are shown.
Figure 5
Figure 5. Nf1+/− CNS neurons exhibit reduced Rho activation
(A) Microtubule architecture as revealed by tubulin (Tuj1) staining is largely unaffected in Nf1+/− hippocampal neurons, whereas the actin cytoskeleton (phalloidin) is disrupted in Nf1+/− CNS neurons. Scale bar = 20 μm. (B) Decreased ROCK activation (phospho-ROCK normalized to total ROCK levels) is observed in Nf1+/− hippocampal (CNS) neurons (2.8-fold decrease, P=.0001, N=8). No change in ROCK activity was observed in Nf1+/− DRG neurons relative to their WT counterparts. (C) Decreased MLC activation (phospho-MLC normalized to total MLC levels) is observed in Nf1+/− hippocampal (CNS) neurons (1.6-fold decrease, P=.0001, N=8). No change in MLC activity was observed in Nf1+/− DRG neurons relative to their WT counterparts.
Figure 6
Figure 6. Rho activation restores Nf1+/− CNS neuronal growth cone areas to wild-type levels
(A) Incubation of WT and Nf1+/− hippocampal tissue (E13.5) in media containing CN01 (1U/ml for 10min) increased ROCK phosphorylation by 3-fold in WT and 1.7-fold in Nf1+/− neurons (N=6). (B) Rho activation using CN01 restores Nf1+/− CNS (hippocampal) neuronal growth cone areas to WT levels (P=.83, N=30). CN01-mediated Rho activation in WT neurons results in a slight decrease in growth cone area (P=.003, N=30). Scale bar = 20 μm
Figure 7
Figure 7. cAMP/PKA activation restores ROCK and MLC phosphorylation in Nf1+/− CNS neurons to wild-type levels
(A) Growth cones were double labeled for Tuj-1 (green) and phospho-MLC (p-MLC; red), and the p-MLC fluorescence intensity was normalized to total protein using the cell dye, Cy3. Treatment with Phe-cAMP significantly increased p-MLC levels in Nf1+/− growth cones (P=0.02, N=30), whereas Me-cAMP had no significant effect. Scale bar = 50 μm. Treatment of Nf1+/− mice for 2 weeks with Rolipram (5 μg/g/day) restores (B) p-ROCK (mean fluorescent intensities: WT 32.1±3.1; Nf1+/− 10.1±0.4; Nf1+/− + Rol 35.0±1.4) and (C) p-MLC (mean fluorescent intensities: WT 36.9±1.2; Nf1+/− 21.7±2.7; Nf1+/− + Rol 36.5±4.0) levels in the retinal ganglion layer (bracketed region) of retinal neuronal preparations.

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