Defective cAMP generation underlies the sensitivity of CNS neurons to neurofibromatosis-1 heterozygosity

Abstract

Individuals with the neurofibromatosis type 1 (NF1) inherited cancer syndrome exhibit neuronal dysfunction that predominantly affects the CNS. In this report, we demonstrate a unique vulnerability of CNS neurons, but not peripheral nervous system (PNS) neurons, to reduced Nf1 gene expression. Unlike dorsal root ganglion neurons, Nf1 heterozygous (Nf1+/-) hippocampal and retinal ganglion cell (RGC) neurons have decreased growth cone areas and neurite lengths, and increased apoptosis compared to their wild-type counterparts. These abnormal Nf1+/- CNS neuronal phenotypes do not reflect Ras pathway hyperactivation, but rather result from impaired neurofibromin-mediated cAMP generation. In this regard, elevating cAMP levels with forskolin or rolipram treatment, but not MEK (MAP kinase kinase) or PI3-K (phosphatidylinositol 3-kinase) inhibition, reverses these abnormalities to wild-type levels in vitro. In addition, Nf1+/- CNS, but not PNS, neurons exhibit increased apoptosis in response to excitotoxic or oxidative stress in vitro. Since children with NF1-associated optic gliomas often develop visual loss and Nf1 genetically engineered mice with optic glioma exhibit RGC neuronal apoptosis in vivo, we further demonstrate that RGC apoptosis resulting from optic glioma in Nf1 genetically engineered mice is attenuated by rolipram treatment in vivo. Similar to optic glioma-induced RGC apoptosis, the increased RGC neuronal death in Nf1+/- mice after optic nerve crush injury is also attenuated by rolipram treatment in vivo. Together, these findings establish a distinctive role for neurofibromin in CNS neurons with respect to vulnerability to injury, define a CNS-specific neurofibromin intracellular signaling pathway responsible for neuronal survival, and lay the foundation for future neuroprotective glioma treatment approaches.

Figure 1.

Nf1+/− hippocampal neurons have reduced growth cone areas, neurite lengths, and cell survival in vitro. A, Nf1+/− hippocampal neuron growth cones (labeled with Tuj-1) are smaller than their WT counterparts (p = 0.0001; N = 40). Scale bar, 50 μm. B, Neurite lengths, identified by Tuj-1 immunostaining, are significantly shorter in Nf1+/− hippocampal neurons (p = 0.02; N = 47). Scale bar, 50 μm. C, Nf1+/− hippocampal neurons exhibit more TUNEL+ labeling compared to their WT counterparts (p = 0.05). Error bars indicate SEM. Asterisks denote statistically significant differences (*p < 0.05, **p < 0.01).

Figure 2.

Nf1+/− DRG neurons have normal growth cone areas, neurite lengths, and cell survival in vitro. A, Nf1+/− DRG neuronal growth cones (labeled with Tuj-1) are marginally larger than their WT counterparts (p = 0.0001; N = 40). B, Neurite lengths, identified by Tuj-1, are equivalent in WT and Nf1+/− DRG neurons (N = 42). Scale bar, 20 μm. C, Nf1+/− and WT DRG neurons have equivalent numbers of TUNEL+ neuronal cells. Error bars indicate SEM. The asterisks denotes a statistically significant difference (p < 0.05).

Figure 3.

Nf1+/− RGCs exhibit reduced growth cone areas, neurite lengths, and cell survival in vitro. A, Nf1+/− RGC neuronal growth cones (labeled with Tuj-1) are smaller than their WT counterparts (p = 0.0001; N = 40). Scale bar, 20 μm. B, Nf1+/− RGC neurite lengths, after magnetic cell sorting and Tuj-1 immunostaining, are significantly shorter than their WT counterparts (p = 0.0001; N = 40). Scale bar, 20 μm. C, Nf1+/− retinal explants exhibit more TUNEL+ neuronal cells regardless of astrocyte genotype (p = 0.0001 for WT astrocytes; p = 0.0004 for Nf1−/− astrocytes). Error bars indicate SEM. Asterisks denote statistically significant differences (*p < 0.05, **p < 0.01).

Figure 4.

Nf1+/− forebrain neurons have increased cell death in response to excitotoxic and oxidative stress in vitro. A, Nf1+/− forebrain neurons exhibit increased LDH release (left; p = 0.001; N = 5) and apoptosis (percentage of TUNEL+ cells; right; p = 0.0001; N = 5) after exposure to 50 μm glutamate compared to their WT counterparts. B, Nf1+/− forebrain neurons exhibit increased LDH release (left; p = 0.05; N = 9) and apoptosis (percentage of TUNEL+ cells; right; p = 0.001; N = 9) after exposure to 100 μm H₂O₂ compared to their WT counterparts. C, Nf1+/− and WT DRG neurons exhibit similar levels of LDH release and percentage of TUNEL+ neuronal cells after exposure to 60 μm H₂O₂. Error bars indicate SEM. Asterisks denote statistically significant differences (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 5.

MAPK pathway activation is not responsible for the abnormal Nf1+/− RGC neuronal phenotypes. A, No differences in the number or intensity of pMAPK+, NeuN+ Nf1+/− RGC neurons were observed compared to their WT counterparts (arrows). B, Treatment with the MEK inhibitor U0126 (10 μm) does not restore Nf1+/− RGC neuronal growth cone areas to WT levels (N = 24). C, Treatment of whole retina explants with U0126 (10 μm) is sufficient to reduce pMAPK levels. Error bars indicate SEM. Asterisks denote statistically significant differences (**p < 0.01).

Figure 6.

Ras pathway hyperactivation is not responsible for the abnormal CNS Nf1+/− neuronal phenotypes. A, Treatment with the PI3-K inhibitor LY294002 (30 μm) did not restore Nf1+/− RGC neuronal growth cone areas to WT levels (p = 0.0001; N = 40). B, Expression of constitutively active KRas^G12D in neurons had no effect on RGC growth cone area (N = 40). Error bars indicate SEM. Asterisks denote a statistically significant difference (**p < 0.01).

Figure 7.

Nf1+/− neurite outgrowth and growth cone spreading defects are cAMP dependent. A, cAMP levels are significantly reduced in Nf1+/− whole retinal preparations compared to those from WT retinas (p = 0.02; N = 8). B, Forskolin (10 μm) treatment rescues Nf1+/− RGC neurite outgrowth lengths to WT levels (N = 40). Scale bar, 20 μm. C, Rolipram (200 μm) treatment of Nf1+/− retinal explant RGCs restores growth cone areas to WT levels (N = 40). D, Inhibition of adenylyl cyclase with 100 μm DDA reduces WT RGC explant growth cone areas to levels observed in Nf1+/− RGC neurons (N = 40). Error bars indicate SEM. Asterisks denote statistically significant differences (*p < 0.05, **p < 0.01).

Figure 8.

Rolipram treatment rescues Nf1+/− RGC death in vitro. A, Nf1+/− retinal explant neurons show reduced neurite numbers (normalized to the distance around the explant perimeter; p = 0.0035). Rolipram (200 μm) treatment restores Nf1+/− explant neurite numbers to WT levels. B, Forskolin (10 μm) or rolipram (200 μm) treatment reduces Nf1+/− retinal explant neuronal apoptosis (p = 0.0001). Error bars indicate SEM. Asterisks denote statistically significant differences (*p < 0.05, **p < 0.01).

Figure 9.

Nf1+/− RGC neuronal apoptosis is induced by optic nerve crush and optic glioma formation in vivo. A, Three days after optic nerve crush, Nf1+/− mice have greater numbers of TUNEL+ (green) and NeuN+ (red) neurons in the RGC layer (white arrows) than their WT counterparts (p = 0.01). B, The increased retinal ganglion layer neuronal apoptosis associated with optic glioma formation in Nf1+/−^GFAPCKO mice (white arrows) is reduced by treatment with rolipram in vivo (p = 0.0001). C, The increased retinal ganglion layer neuronal apoptosis associated with optic nerve crush injury 6 d after injury in Nf1+/− mice (white arrows) is reduced by treatment with rolipram in vivo (p = 0.006). Error bars indicate SEM. Asterisks denote statistically significant differences (**p < 0.01).