This report describes an adult onset neurodegeneration phenotype in a mouse model of Gigyf2 gene disruption. Gigyf2 null mice had normal intrauterine growth, and there were no evident anatomic or histological abnormalities in full-term fetuses. However, newborn mice lacking GIGYF2 failed to feed, as evidenced by reduced gastric milk spots together with post-natal weight loss, and exhibited 85% mortality during the first day after birth. The homologous GIGYF1 protein, which is encoded by a distinct gene, was shown to be present but not up-regulated in the mice with Gigyf2 gene disruption, and the lethal phenotype confirmed a lack of compensation for GIGYF2 function by GIGYF1.
null animals had normal self-righting behavior, an absence of gross abnormalities in motility, normal induced suckling movements and no apparent defects in oropharyngeal or nasal structures that might explain the failure to feed. Previous studies from other groups have demonstrated a phenotype in mice with olfactory dysfunction similar to the Gigyf2
null mice, including absent gastric milk spot, perinatal weight loss and high early mortality (22
). Olfactory sensory map development is regulated by IGF-I (24
), and defects in olfaction frequently develop early in the course of both Parkinson's disease and Alzheimer's disease (25
). Investigation of olfactory system development and function in Gigyf2
null mice would be of interest in future studies using techniques such as electrophysiological olfactory neuronal mapping (22
Heterozygous mice with gene trap disruption of a single Gigyf2 allele have an ~50% decrease in the abundance of GIGYF2 mRNA and protein. The Gigyf2+/− mice survive embryonic life at the expected Mendelian ratio and are indistinguishable from Gigyf2+/+ littermate controls in growth and development. Neurological deficits were not evident in growing or young adult mice. However, by 15 months of age, ~50% of the Gigyf2+/− mice developed motor dysfunction, which was quantifiable as a 50% decrease in balance time on a horizontal rotating rod compared with wild-type controls.
Histological examination of 15-month-old Gigyf2+/− animals demonstrated central nervous system abnormalities that included coarse neurites in the cerebellar granule cell layer, α-synuclein positive neuritic plaques in the brain and spinal cord, scattered inclusion body-like structures and spinal cord motor neurons with eosinophilic-stained cell bodies and swollen axons. Neurons appeared grossly normal in the substantia nigra, and nigral abnormalities were not evident by α-synuclein or tyrosine hydroxylase immunostaining. Quantitative histomorphometry confirmed a significant increase in α-synuclein positive neurites in the cerebrum and spinal cord, and the number of total brain α-synuclein plaques in individual mice correlates with the extent of decrease in motor function. These findings are consistent with a neurodegenerative process in the Gigyf2+/− mice that either has its onset in adulthood, or progresses to detectable levels with aging. Although we confirmed a statistically significant decrease in the ratio of neurons to glial cells in the lumbar spinal cord, this was associated with a non-significant decrease in neuron number and increase in glial cell number per cross-sectional area. It thus is possible that decreased GIGYF2 may affect glial as well as neuronal cell populations. Consistent with the observed effects of Gigyf2 gene disruption on cells in the cerebrum, midbrain, cerebellum and spinal cord, GIGYF2 mRNA is expressed at multiple sites throughout the central nervous system.
Although the Gigyf2+/−
mice manifest motor dysfunction, they do not have other features typical for human Parkinson's disease, such as an evident tremor or neuronal loss in the substantia nigra. Other mouse models with disruption of genes with established links to Parkinson's disease also do not fully recapitulate the functional and pathological features of the human disorder. For example, disruption of the Parkin
genes in mice results in neurodegeneration and motor function deficits, but these animals do not have cellular deficits in the nigrostriatal dopaminergic pathway (27
). In contrast, transgenic mice over-expressing human SNCA
have dopaminergic neuronal loss, especially when the transgene is specifically driven by a dopaminergic neuron-targeted tyrosine hydroxylase promoter (30
). The observation of inclusion body-like structures suggestive of Lewy bodies in the brain and spinal cord is a distinct feature of the Gigyf2+/−
mice, which has not been described in other mouse models with disruption of Parkinson's disease-associated genes. When an adequate antibody probe becomes available, it will be important to determine if GIGYF2 is present in the α-synuclein positive inclusion bodies or neuritic plaques in the Gigyf2+/−
mice. The anti-GIGYF2 antibodies that we have generated are effective for immunoblotting, but do not have adequate specificity for the assessment of GIGYF2 by immunohistochemistry.
We previously demonstrated in a yeast two-hybrid assay that the protein encoded by the GIGYF2
gene is a binding partner for the Grb10 adapter protein (1
). Grb10 is known to interact with intracellular domains of activated insulin and IGF-I receptors and negatively regulate insulin and IGF signaling (3
). As a protein that may be interactive with receptors via the adapter function of Grb10, GIGYF2 is of interest as a potential mediator or regulator of hormone signaling. We therefore compared IGF-I signaling in cultured MEFs from Gigyf2−/−
and control +/+ mice. This demonstrated a modest but significant decrease in IGF-I-stimulated receptor phosphorylation in Gigyf2−/−
cells, with no change in receptor abundance in comparison with control cells. In contrast, there was a significant increase in IGF-I-stimulated ERK1/2 phosphorylation and a suggestive, non-significant increase in Akt phosphorylation in the Gigyf2
null cells. We also observed increased numbers of randomly cycling Gigyf2
null MEFs in S-phase following treatment with the DNA cross-linking agent etoposide. In future studies, it will be of interest to determine whether this results from dysregulation of IGF-I effects on cell cycle control, which might increase susceptibility to apoptosis.
In previous work, Grb10 knockdown in cultured fibroblasts resulted in a similar decrease in IGF-I receptor tyrosine phosphorylation and an increase in downstream signaling from the IGF-I receptor (4
), as well as similar effects on insulin receptor tyrosine phosphorylation and its downstream signaling (3
). Recruitment of endogenous Grb10 to activated IGF-I and insulin receptors is thought to block receptor dephosphorylation by phosphoprotein phosphatases and simultaneously inhibit downstream signals that are generated by the tyrosine kinase activity of the receptors (4
). In future studies, it will be important to determine whether the observed effects of GIGYF2 on IGF-I receptor phosphorylation and signaling are mediated via a GIGYF2-receptor protein complex with Grb10 functioning as a linker or by GIGYF2 modulating the capacity of Grb10 to bind the receptors.
The identification of GIGYF2
as a gene linked to neurodegeneration as well as IGF-I and insulin signaling is of interest because of emerging evidence for a role of both of these hormones in human neurodegenerative disorders, including Parkinson's disease (6
). IGF-I stimulates brain growth in vivo
), promotes neuronal proliferation and differentiation and inhibits neuronal apoptosis (8
). IGF-I receptors are abundantly expressed in the midbrain, and IGF-I has specifically been shown to decrease dopamine-induced cell death in rat cerebellar cell cultures and a human neuroblastoma cell line (9
). Insulin receptors are abundant at various sites in the central nervous system, including the substantia nigra and basal ganglia (34
). A role for diminished insulin signaling in Alzheimer's disease has been proposed (32
), and a selective decrease in insulin receptor mRNA and protein in the substantia nigra in Parkinson's disease has been described (10
). Diabetes has been associated with increased risk of Alzheimer's disease (35
), and an approximately two-fold increased risk of Parkinson's disease in patients with type 2 diabetes mellitus (11
). Further studies will be needed to determine whether the GIGYF2
gene is functionally linked to Parkinson's disease. More data on of the molecular function of the GIGYF2 protein may provide insight into mechanisms through which mutations or altered expression of GIGYF2 result in neurodegeneration.