Research reportRegion-specific astrogliosis in brains of mice heterozygous for mutations in the neurofibromatosis type 1 (Nf1) tumor suppressor
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).
References (57)
- et al.
Astrocytosis in the molecular layer of the dentate gyrus: a study in Alzheimer's disease and schizophrenia
Psychiatric. Res.
(1990) - et al.
The protein product of the neurofibromatosis type 1 gene is expressed at highest abundance in neurons, Schwann cells, and oligodendrocytes
Neuron
(1992) - et al.
Molecular profile of reactive astrocytes implication for their role in neurologic disease
Neuroscience
(1993) - et al.
Intellectual impairment in NF1
J. Neurol. Sci.
(1996) - et al.
Neurofibromatosis type 1: the cognitive phenotype
J. Pediatrics
(1994) - et al.
Expression of neurofibromin, the neurofibromatosis 1 gene product: studies in human neuroblastoma cells and rat brain
Neurosci. Lett.
(1992) - et al.
Fibrillary astrocytes proliferate in response to brain injury
Dev. Bio.
(1979) - et al.
A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples
Anal. Biochemistry
(1978) - et al.
Reactive astrocytes and molecular cues to biological function
Trends Neurosci.
(1997) - et al.
Mutations in GDI1 are responsible for X-linked non-specific mental retardation
Nature Genet.
(1998)
Neurofibromatosis types 1 and 2: cranial MR findings
Radiology
Spinal cord astrocytes in vitro: phenotypic diversity and sodium channel immunoreactivity
Glia
Targeted disruption of the neurofibromatosis type-1 gene leads to developmental abnormalities in heart and various neural crest-derived tissues
Genes and Development
Neurofibromin, a predominantly neuronal GTPase activating protein in the adult, is ubiquitously expressed during development
Dev. Dynamics
Neurofibromatosis type 1: magnetic resonance imaging findings
Child Neurol.
Neurofibromatosis type 1: pathologic substrate of high signal intensity foci in the brain
Neuroradiology
The significance of MRI abnormalities in children with neurofibromatosis
Neurology
Evaluation of learning difficulties and incoordination in neurofibromatosis type 1
Neurofibromatosis
Increased expression of the neurofibromatosis 1 (NF1) gene product, neurofibromin, in astrocytes in response to cerebral ischemia
J. Neurosci. Res.
Catalytic properties, tissue distribution and intracellular distribution of neurofibromin
Oncogene
Requirement of Drosophila NF1 for activation of adenlyl cyclase by PACAP38-like neuropeptides
Science
The diagnostic evaluation and multidisciplinary management of neurofibromatosis 1 and neurofibromatosis 2
J. Am. Med. Assoc.
Expression of the neurofibromatosis 1 (NF1) gene in reactive astrocytes in vitro
NeuroReport
A genetic study of von Recklinghausen neurofibromatosis in South East Wales: I. Prevalence, fitness, mutation, rate and effect of parental transmission on severity
J. Med. Genetics
Cited by (42)
Insight into the transcription factors regulating Ischemic stroke and glioma in response to shared stimuli
2023, Seminars in Cancer BiologyQuantitative MRI demonstrates abnormalities of the third ventricle subventricular zone in neurofibromatosis type-1 and sporadic paediatric optic pathway glioma
2020, NeuroImage: ClinicalCitation Excerpt :Post-mortem analyses of NF1 brains have reported increased fluid within the myelin, associated with hyper- or dysplastic glial cell proliferation (DiPaolo et al., 1995), as well as increased expression of glial fibrillary acid protein, indicative of reactive astrogliosis in the NF1 brain (Nordlund et al., 1995). This is supported by animal studies reporting global astrogliosis in NF1-knockout mice (Gutmann et al., 1999; Rizvi et al., 1999). In addition, a previous study identified reduced CBF in paediatric NF1 subjects compared to age-matched controls (Yeom et al., 2013b), most significantly in the posterior circulation and border zones of the middle and posterior cerebral arteries.
The effect of apamin, a small conductance calcium activated potassium (SK) channel blocker, on a mouse model of neurofibromatosis 1
2013, Behavioural Brain ResearchCitation Excerpt :Homozygous mice (Nf1−/−) lacking both copies of the Nf1 gene die in the embryonic stage due to cardiovascular problems or neural tube defects [11]. On the other hand mice that are heterozygous for the mutation have many common attributes with the human disease such as astrocyte proliferation, tumor predisposition and learning disabilities [12,13]. The learning deficits in Nf1+/− mice parallel those seen in humans and tend to be primarily visuospatial, as exhibited through deficits on the Morris water maze task [14].
Neurofibromatosis type 1 (NF1): Diagnosis and management
2013, Handbook of Clinical NeurologyCitation Excerpt :It is possible that NF1-related epilepsy is linked with an underlying cortical dysgenesis and a pathology study of patients with NF1 and cognitive impairment revealed abnormal cerebral architecture, subcortical heterotopias, and glial nodules (Rosman and Pearce, 1967). Furthermore, the NF1 gene product neurofibromin has a role in central nervous system formation and loss of Nf1 function in neurons of transgenic mice results in astrogliosis and hippocampal dysplasia (Gutmann et al., 1999a; Rizvi et al., 1999). Neuroimaging of patients with epilepsy has detected mesial temporal sclerosis, and cortical dysplasia (Vivarelli et al., 2003).
The Genetic and Molecular Pathogenesis of NF1 and NF2
2006, Seminars in Pediatric NeurologyMouse models of neurofibromatosis type I: Bridging the GAP
2003, Trends in Molecular Medicine
- 1
Current address: Kellogg Eye Center, University of Michigan, 1000 Wall Street, Ann Arbor, MI 48105.