Elsevier

Brain Research

Volume 816, Issue 1, 16 January 1999, Pages 111-123
Brain Research

Research report
Region-specific astrogliosis in brains of mice heterozygous for mutations in the neurofibromatosis type 1 (Nf1) tumor suppressor

https://doi.org/10.1016/S0006-8993(98)01133-0Get rights and content

Abstract

Brains from human neurofibromatosis type 1 (NF1) patients show increased expression of glial fibrillary acidic protein (GFAP), consistent with activation of astrocytes (M.L. Nordlund, T.A. Rizvi, C.I. Brannan, N. Ratner, Neurofibromin expression and astrogliosis in neurofibromatosis (type 1) brains, J. Neuropathol. Exp. Neurology 54 (1995) 588–600). We analyzed brains from transgenic mice in which the Nf1 gene was targeted by homologous recombination. We show here that, in all heterozygous mice analyzed, there are increased numbers of astrocytes expressing high levels of GFAP in medial regions of the periaqueductal gray and in the nucleus accumbens. More subtle, but significant, changes in the number of GFAP positive astrocytes were observed in the hippocampus in 60% of mutant mice analyzed. Astrocytes with elevated GFAP were present at 1 month, 2 months, 6 months and 12 months after birth. Most brain regions, including the cerebellum, basal ganglia, cerebral cortex, hypothalamus, thalamus, cortical amygdaloid area, and white matter tracts did not show any gliotic changes. No evidence of degenerating neurons was found using de Olmos' cupric silver stain. We conclude that Nf1/nf1 mice provide a model to study astrogliosis associated with neurofibromatosis type 1.

Introduction

Type 1 neurofibromatosis is one of the most common inherited human diseases. NF1 is inherited as an autosomal dominant trait 6, 24, 25, 56, and affects about 1 in 3500 people worldwide. Common manifestations of NF1 include hyperpigmentation, bone abnormalities, and peripheral nerve sheath tumors (neurofibromas) (reviewed in Refs. 20, 49). NF1 patients are also at increased risk to develop malignant tumors including pheochromocytomas, childhood myelogenous leukemia, and malignant peripheral nerve sheath tumors. NF1 is characterized by extreme variability in disease severity, even among members of the same family (reviewed in Ref. [47]).

NF1 patients show changes associated with the central nervous system. For example, benign astrocytomas of the optic nerve are common in children with NF1 (reviewed in Ref. [36]). In addition, learning disabilities are a frequent and disabling problem in NF1 families. Between 30 and 45% of children who inherit one defective NF1 allele have learning disabilities (reviewed in Ref. [41]). School-aged children with NF1 can show organizational, visual spatial, memory, attentional and\or fine motor problems 17, 23, 24. Mean IQ scores of NF1 children are lower than the general population, but within one standard deviation of the mean [42].

Because of these CNS manifestations of NF1, it is important to define the cellular and molecular changes that exist in the brain as a consequence of NF1 mutation. Rosman and Pearce [50]described abnormal cortical lamination and heterotopic neurons in cortical gray and white matter as well as glial nodules that most closely resembled astrocytes in cerebral white matter in NF1 patients. A second abnormality is observed in a sub-population of children with NF1, who show foci of increased T2 signal on brain magnetic resonance imaging that are not enhanced by gadolinium, visible by CT, or associated with focal neurologic deficits. These have been called unidentified bright objects (UBOs) 12, 38, 46, and are found primarily in the cerebellum, subcortical white matter, brainstem, and basal ganglia 2, 10, 12, 13, 51. UBOs disappear with increasing age. DiPaulo et al. [11]studied brains from three children with UBO's at autopsy. They concluded that UBOs represent areas of demyelination or edema.

Another brain abnormality associated with NF1 is astrogliosis. Astrogliosis, or astrocyte activation, is a common response to brain injury, resulting in up-regulation of more than 100 proteins including cytokines, growth factors, adhesion molecules and transcription factors (reviewed in Refs. 13, 47). Astrogliosis is commonly marked by up-regulation of the intermediate filament protein GFAP, a 51 kd type III intermediate filament (reviewed in Refs. 15, 16, 35). We described astrogliosis in three of three NF1 adult brains examined, using GFAP as a marker for astrocyte activation [39].

The NF1 gene product, neurofibromin, is expressed at high levels in the brain as compared to other tissues 7, 19. Neurofibromin is present in subpopulations of adult brain neurons 26, 40. Astrocytes in neither rodent nor human brain express detectable neurofibromin 7, 39, 40. However, astrocytes up-regulate neurofibromin expression in vitro [22]and in vivo in response to cerebral ischemia [18].

The relevance of UBOs, astrogliosis, or abnormal cortical lamination to decreases in NF1 expression and learning problems in NF1 is not known; model systems in which to study these changes have not been described. Mice with targeted mutations at Nf1 were developed. Homozygous null embryos die at mid-gestation and are therefore unavailable for brain analysis 4, 27. Adult heterozygous Nf1 mice are predisposed to certain types of malignant tumors and show hyperplasia of some neuronal populations 4, 27. While Nf1/nf1 (heterozygous) mice do not mimic many features of human NF1, learning deficits have been defined in about 60% of the mutant mice suggesting the use of these mice to study brain abnormalities [53]. With additional training, Nf1 mice become comparable to wild-type controls, implying that Nf1 mutations affect rate of learning. It is not yet known whether Nf1/nf1 mice show the physical brain changes characteristic of human NF1, including UBO's, abnormal cortical lamination, nor whether the brains of mutant mice are gliotic. We therefore analyzed GFAP expression as a measure of astrocyte function in the mice. Our data demonstrate that astrogliosis occurs in Nf1/nf1 mice, providing a model system in which to study one of the features of human NF1.

Section snippets

Animals

Male C57Bl/6 mice were used in this study. Mice heterozygous for the Nf1 mutation [4]had been backcrossed onto C57Bl/6 for seven to nine generations at the time of these experiments. Mice were genotyped by PCR as described by Brannan et al. [4]. A total of 48 mice were fixed and analyzed for brain histology. Six wild type and six heterozygous mice were analyzed at each time point: 1 month, 2 months, 6 months and 1 year of age. An additional three wild type and three heterozygous mice were

Analysis of Nf1/nf1 brain sections for abnormalities in cortical lamination and for glial nodules

Rosman and Pearce [50]found that a subpopulation of NF1 patient brains showed abnormal cortical lamination, displaced neurons in the white matter, and, in one of twenty brains analyzed, glial cell nodules in white matter. Nf1/nf1 brain sections stained with cresyl violet were carefully analyzed to discern if these phenotypes were detectable. Three wild type and 3 mutants were analyzed at one month of age; 2 wild type and 2 mutants were analyzed at six months of age; 1 wild type and 1 mutant

Discussion

This report demonstrates that Nf1/nf1 mouse brains contain discrete regions of increased GFAP. In contrast, no changes in cortical lamination or brain organization were detected in Nf1/nf1 mutant mouse brains. The current study indicates that mutation at the Nf1 locus results in astrocyte activation; a previous study using human brains, from patients who died of malignant disease, left open the possibility that gliosis in NF1 was a non-specific change associated with tumor burden, or other

Acknowledgements

This work was supported by NIH NS28840 (to NR).

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    Current address: Kellogg Eye Center, University of Michigan, 1000 Wall Street, Ann Arbor, MI 48105.

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