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Grb10-Interacting GYF Protein 2 (GIGYF2) was initially identified through its interaction with Grb10, an adapter protein that binds activated IGF-I and insulin receptors. The GIGYF2 gene maps to human chromosome 2q37 within a region linked to familial Parkinson's disease (PARK11 locus), and association of GIGYF2 mutations with Parkinson's disease has been described in some but not other recent publications. This study investigated the consequences of Gigyf2 gene disruption in mice. Gigyf2 null mice undergo apparently normal embryonic development, but fail to feed and die within the first 2 post-natal days. Heterozygous Gigyf2+/− mice survive to adulthood with no evident metabolic or growth defects. At 12–15 months of age, the Gigyf2+/− mice begin to exhibit motor dysfunction manifested as decreased balance time on a rotating horizontal rod. This is associated with histopathological evidence of neurodegeneration and rare intracytoplasmic Lewy body-like inclusions in spinal anterior horn motor neurons. There are α-synuclein positive neuritic plaques in the brainstem and cerebellum, but no abnormalities in the substantia nigra. Primary cultured embryo fibroblasts from Gigyf2 null mice exhibit decreased IGF-I-stimulated IGF-I receptor tyrosine phosphorylation and augmented ERK1/2 phosphorylation. These data provide further evidence for an important role of GIGYF2 in age-related neurodegeneration and IGF pathway signaling.
Grb10-Interacting GYF Protein 2 (GIGYF2) was initially identified in our laboratory through yeast two-hybrid screening for binding partners of the Grb10 adapter protein (1). Grb10 is recruited to activated IGF-I and insulin receptors, as well as other receptor tyrosine kinases (2). Studies on Grb10 null mice and cultured cells with siRNA knockdown of Grb10 strongly support a role for the endogenous Grb10 protein in negatively regulating IGF-I and insulin receptor signaling (3,4). GIGYF2 and the homologous GIGYF1 protein, which are encoded by distinct genes on human chromosomes 2 and 7, respectively, may be recruited to activated IGF-I and insulin receptors through binding to the N-terminus of Grb10, whereas the Grb10 C terminus interacts with the receptors. We have previously obtained evidence for IGF-I-stimulated GIGYF1 recruitment to Grb10, with resulting regulatory effects on IGF-I receptors (1). However, studies on GIGYF1 and GIGYF2 function have been limited by a propensity of these proteins to form toxic aggregates when over-expressed in cultured cells.
To gain insight into the potential functional role of GIGYF2, we examined human genome data for GIGYF2 disease associations and noted that the GIGYF2 gene is within the PARK11 Parkinson's disease linkage region on chromosome 2q37.1 (5). Since the IGFs and insulin have important effects in the central nervous system (6–8) and are potentially associated with Parkinson's disease (9–11), we examined the GIGYF2 gene for mutations in familial Parkinson's disease. Complete sequencing of the GIGYF2 coding region in 254 patients with familial Parkinson's disease and 227 controls from distinct Italian and French populations demonstrated seven different missense mutations in 12 unrelated Parkinson's disease patients (4.8%), one mutation specific to the control population and several polymorphisms (12). A subsequent study on two Asian populations described distinct non-synonymous GIGYF2 variants and confirmed a significant increase in these variants in Parkinson's disease patients compared with healthy controls (13). However, multiple other reports on additional populations with a mix of sporadic and familial Parkinson's disease have not found the association of GIGYF2 mutations with Parkinson's disease (14–19) or linkage at the PARK11 site via the GIGYF2 gene (20). Thus, there is uncertainty about the role of GIGYF2 as a contributory factor in Parkinson's disease (21).
We here report the generation of a mouse Gigyf2 gene disruption model as an independent approach to further investigating GIGYF2 function. The Gigyf2 null mice exhibit early post-natal lethality. Heterozygous Gigyf2+/− mice initially survive and grow normally, but develop adult-onset neurodegeneration. Primary cultured fibroblasts from embryonic Gigyf2 null mice manifest IGF-I receptor signaling abnormalities.
A gene trap strategy was used to generate a mouse line with inactivation of the Gigyf2 gene. For this purpose, mouse embryonic stem cells were obtained with a gene trap construct composed of a splice acceptor, beta-geo cassette and polyadenylation sequence inserted into the proximal coding region of the Gigyf2 gene (Bay Genomics, http://baygenomics.ucsf.edu). We mapped the insertion point of the construct within the 5 kb intron between Gigyf2 exons 4 and 5 using intronic and gene trap PCR primer pairs (Fig. 1A). This predicted a truncated transcript with deletion of 23 of the total 27 exons of Gigyf2. Genotyping of mouse colony progeny obtained from crosses of parental mice heterozygous for the gene trap insert demonstrated E17.5 embryos with each of the three expected genotypes (Fig. 1B). Northern blotting analysis of total RNA extracts from primary mouse embryonic fibroblast (MEF) cultures derived from E12.5 embryos using a probe corresponding to the first 4 exons of Gigyf2 is shown in Figure 1C. A single 7 kb GIGYF2 mRNA band was observed in the wild-type +/+ MEFs, a smaller 6 kb band corresponding to the reduced size expected for the truncated gene trap-containing transcript in the homozygous Gigyf2−/− MEFs, and both bands in MEFs from heterozygous +/− progeny. Similar data were obtained using total RNA extracts from whole E17.5 mouse embryos (data not shown). Northern blotting with a Gigyf2 cDNA probe corresponding to sequence downstream from the gene trap insert demonstrated a single band in the wild-type +/+ MEFs, a lower intensity single band of the same size in heterozygous +/− MEFs, and no hybridizing band in the −/− MEFs (Fig. 1D). This confirmed the absence of transcribed full-length mRNA sequence downstream from the gene trap. The failure to detect smaller bands on these blots further indicated that bypassing of the gene trap by alternative splicing or alternative start codons was not occurring. Northern blotting with a probe specific for the homologous Gigyf1 gene (1) demonstrated a single band of equal intensity in mice of all three genotypes (Fig. 1E), indicating a lack of upregulation of Gigyf1 mRNA in response to loss of Gigyf2 expression.
Immunoblotting of total cell lysates with an affinity-purified GIGYF2 anti-peptide antibody confirmed the presence of a GIGYF2 band at 180 kDa in wild-type MEFs (Fig. 1F). This band was confirmed to correspond to full-length GIGYF2 based on its recognition by both N-terminal and C-terminal anti-GIGYF2 peptide antibodies (data not shown). The GIGYF2 protein was undetectable in −/− MEFs, and a 180 kDa band of ~50% lower intensity compared with the control cells was present in the +/− heterozygotes. A similar partial decrease of GIGYF2 protein in heterozygotes and absence of expression in −/− animals was demonstrated by immunoblotting of extracts from whole E14.5 embryos (Fig. 1G). Immunoblotting of whole embryo extracts (data not shown) and MEF lysates (Fig. 1H) with GIGYF1 antibody identified a dominant band of the expected 150 kDa size for the full-length GIGYF1 protein, which was present at equal intensity in all three genotypes. Thus, consistent with the northern blotting data, there is a partial (~50%) decrease in abundance of the GIGYF2 protein in heterozygotes and a complete absence of detectable GIGYF2 protein in the −/− homozygotes. The homologous GIGYF1 protein is expressed and not changed in abundance in control versus GIGYF2-deficient mice.
Analysis of genotype frequency in late gestation embryos (E17.5–18.5) from heterozygous matings demonstrated the predicted Mendelian genotype distribution, consistent with the generation and survival of heterozygous and homozygous Gigyf2 gene-disrupted mice equivalent to the wild-type (Table 1). However, we observed that a substantial number of newborn animals from the heterozygous matings did not survive beyond the first post-natal day. When both surviving and non-surviving animals were collected during the first day after birth and analyzed, the cumulative genotype frequencies were close to the Mendelian ratio observed for the late gestation embryos (Table 1). Progeny deaths were limited almost entirely to the first post-natal day and specifically occurred in the Gigyf2 null (−/−) newborns. The modest (25%) decrease in the number of Gigyf2−/− compared with wild-type +/+ newborn mice observed at the first post-natal day (Table 1) likely reflected the failure to recover some non-surviving −/− animals as a consequence of post-mortem maternal cannibalism. At the time of weaning (day P21), genotyping revealed the predicted 2:1 ratio of Gigyf2+/− to +/+ animals, but a marked decrease in the Gigyf2 null (−/−) genotype, consistent with ~85% lethality of the Gigyf2 null mice during the first post-natal day (Table 1). The Gigyf2 heterozygotes were indistinguishable from the wild-type animals in growth curves and adult size and weight, and appeared to have normal fertility. There were not enough surviving Gigyf2−/− mice to define their growth characteristics.
Examination of fetal animals of all three genotypes in late gestation (E17.5) revealed no differences among the wild-type, heterozygous and Gigyf2 null mice in gross appearance, linear growth or body weight (Fig. 2A–C). In contrast, during the first post-natal day, crown-rump length and body weight were significantly lower in Gigyf2 null (−/−) animals (Fig. 2D–F). This correlated with a marked decrease in size of the gastric milk spot in the Gigyf2 null mice compared with wild-type and heterozygotes (Fig. 2D and G), indicating a failure of the Gigyf2 null newborns to feed. The rapid loss of weight and less marked decrease in crown-rump length likely were secondary to dehydration, and the magnitude of the feeding deficit appeared adequate to explain the observed mortality rate. The newborn Gigyf2 null mice had normal motility based on timed self-righting capacity and detailed anatomic and histological examination of P1 mice showed no abnormalities in nasal, oropharyngeal or gastrointestinal structures (data not shown). The Gigyf2 null mice also were not cyanotic and had no detectable pulmonary or cardiac abnormalities. The mean body weight and milk spot size of animals heterozygous for Gigyf2 gene disruption were slightly lower than wild-type controls (Fig. 2F–G). Although these differences were not statistically significant with the number of animals studied, they suggest a possible intermediate phenotype with partial loss of the GIGYF2 protein.
The gene trap construct contains a β-galactosidase sequence under control of the endogenous Gigyf2 promoter. By staining sections of Gigyf2+/− and −/− embryos with X-gal, it thus is possible to determine the tissue and cellular sites of Gigyf2 promoter activity as a surrogate for Gigyf2 gene expression at different stages of development. As shown in Figure 3A, Gigyf2 is broadly expressed in embryonic mouse tissues, including strong expression in the central nervous system. X-gal staining is more intense in −/− compared with +/− mice, consistent with the presence of two versus one copy of the reporter allele, respectively. Wild-type mice had absent X-gal staining (data not shown). Gigyf2 gene promoter activity was observed throughout the CNS in adult Gigyf2−/− mice (Fig. 3Ba). Animals of this genotype were used, since they have a strong Gigyf2 promoter-driven β-galactosidase signal, and they were confirmed to have no histological changes from wild-type or Gigyf2+/− mice by Luxol Fast Blue H&E staining. Gigyf2 expression was noted to be especially marked in cerebellar Purkinje cells, the pons and the choroid plexus (Fig. 3Bb–e). High intensity staining was evident in both neuronal and glial cells in multiple brain regions (Fig. 3C).
Given the potential association of the GIGYF2 gene with human Parkinson's disease (12), which most frequently has onset in adulthood and increases in prevalence with advancing age, we examined neurological function in post-natal Gigyf2+/− mice up to 18 months of age. An adequate number of surviving −/− mice were not available for investigation of neurological function or histopathology (see below). Abnormalities were not evident in the heterozygous Gigyf2+/− animals until ~12 months of age, at which point mild truncal and hindlimb motor dysfunction became apparent as an exaggerated splaying of the hindlimbs with delayed positional correction when animals were dropped 15 cm onto a flat surface. This abnormal motor response was present in ~50% of 12-month-old Gigyf2+/− mice and absent from wild-type (Gigyf2+/+) littermates, as assessed by an investigator unaware of the genotype of individual animals. As a more quantitative test, balance performance on a rotating horizontal rod then was determined. As shown for 15-month-old mice in Figure 4, animals with inactivation of one Gigyf2 gene allele exhibited an ~50% decrease in staying time on a rod rotating at 5 r.p.m. compared with wild-type controls (P < 0.05).
Histological sections were examined from the brain and spinal cord of 15-month-old Gigyf2+/− and control (+/+) mice. In Luxol Fast Blue H&E stained sections, there was evidence of motor neuron degeneration at multiple spinal cord levels in Gigyf2+/− mice. Specific pathological findings included eosinophilic degeneration with nuclear pyknosis, axonal swelling and rare inclusion body-like structures affecting predominantly large pyramidal neurons in the ventral horns of the spinal cord (Fig. 5A). Histomorphometric quantitation of neuronal and glial cell number in lumbar region spinal cord sections confirmed a significant decrease in the neuronal/glial cell ratio (Fig. 5B). This was associated with a non-significant decrease in neuron number and non-significant increase in glial cells, when assessed independently.
There were no overt neuronal or glial cellular abnormalities on Luxol Fast Blue H&E staining in the cerebrum, midbrain or cerebellum (data not shown), but immunostaining with α-synuclein antibody yielded additional evidence of neuronal abnormalities and neurodegeneration in these brain regions. This included α-synuclein positive, coarse neurites in the cerebellar granule cell layer of the Gigyf2+/− mice, but not in age-matched (Gigyf2+/+) controls (Fig. 5C). Increased numbers of α-synuclein-positive neurites were diffusely present throughout the brain and spinal cord of Gigyf2+/− animals in comparison with controls (Fig. 5D). Quantitative histomorphometry by an observer unaware of the genotypes of individual animals confirmed a statistically significant increase in α-synuclein-positive neuritic plaques in Gigyf2+/− cerebrum and spinal cord (Fig. 5E). The mean number of plaques also was higher in midbrain and cerebellum of Gigyf2+/− versus control animals, but this did not reach statistical significance. The total brain plaques per section and also the number of plaques per section in specific brain regions (midbrain, cerebellum or cerebrum) in the individual Gigyf2+/− animals correlated inversely with balance time on the horizontal rotating rod. Linear regression analysis of total brain plaques per section versus time on the rotating rod in seconds for the 10 heterozygous knockout mice demonstrated a slope of −0.0269 (P < 0.001).
Neuronal loss was not evident in the substantia nigra as assessed by Luxol Fast Blue H&E or anti-tyrosine hydroxylase immunofluorescent staining (data not shown). There also were no abnormalities evident in brain or spinal cord sections immunostained with antibodies to glial fibrillary acid protein (GFAP) or ubiquitin.
GIGYF2 was initially identified in a yeast two-hybrid screen as a binding partner for the Grb10 adapter protein (1). Since Grb10 is recruited to activated IGF-I and insulin receptors, and it negatively regulates IGF and insulin signaling (3,4), we considered the potential role of decreased GIGYF2 levels in modifying the actions of these hormones. As evident in Figure 2 A–C, both the Gigyf2+/− heterozygotes and the Gigyf−/− mice exhibited normal embryonic and fetal growth. When examined post-natally, the adult Gigyf2+/− heterozygotes and the small number of surviving Gigyf2−/− mice were indistinguishable from wild-type littermates in length, body weight and blood glucose levels (data not shown). Abnormalities in growth and glucose levels were not evident in the few Gigyf2−/− mice that survived to adulthood, but adequate numbers were not available to formally assess these animals.
As an initial approach to further examining the effects of GIGYF2 on cell survival, we determined the percentage of randomly cycling primary MEFs from Gigyf2+/+ and −/− mice in S-phase by flow cytometry in the absence or presence of the DNA cross-linking agent etoposide under serum-stimulated conditions. The percentage of cells in S-phase under control conditions was similar for Gigyf2+/+ and −/− MEFs (28.7 ± 4.1 versus 30.3 ± 3.5, respectively, n = 3). Etoposide resulted in a marked (80%) decrease in the number of Gigyf2+/+ cells in S-phase, consistent with the expected decrease in S-phase entry following DNA damage. There was a significantly higher percentage of Gigyf2−/− compared with +/+ cells in S-phase after etoposide (12.1% of −/− in S-phase versus 5.6% of +/+ in S-phase, P = 0.026). These findings suggest dysregulation of cell cycle control with Gigyf2 gene dysruption.
To more specifically examine the effects of decreased GIGYF2 protein on receptor signaling, IGF-I stimulation of IGF-I receptor tyrosine phosphorylation and the activation of the downstream signaling intermediates Akt and ERK1/2 were assessed in multiple isolates of primary MEFs from Gigyf2+/+ and −/− mice. IGF-I receptor abundance was similar in the wild-type and Gigyf2 null MEFs based on immunoblotting with receptor antibody, and IGF-I-stimulated receptor tyrosine phosphorylation as assessed by phospho-specific antibody in a time-dependent manner, with a maximum effect at 5 min (Fig. 6A). Quantitation at the 5 min time point in multiple MEF isolates confirmed a modest but statistically significant (~25%) lower level of IGF-I-stimulated receptor tyrosine phosphorylation in the Gigyf2 null cells compared with wild-type (Fig. 6B). Determination of Akt and ERK1/2 activation by immunoblotting of the same cell extracts with phospho-specific antibodies for each of these intracellular signaling proteins demonstrated a similar time course of activation by IGF-I with a maximum effect at 5 min (Fig. 6A). In contrast with the inhibition of IGF-I-stimulated receptor tyrosine phosphorylation in Gigyf2 null MEFs, disruption of the Gigyf2 gene resulted in a modest but significant increase in IGF-I stimulation of ERK1/2 (25%, P < 0.02) (Fig. 6B). There was a similar magnitude increase in IGF-I-stimulated Akt activation in the Gigyf2 null MEFs, with larger variation in the level of phospho-Akt such that the increase was not statistically significant (Fig. 6B). These experiments provide evidence of a role for the GIGYF2 protein in the regulation of IGF-I receptor signaling. The decrease in IGF-I-stimulated receptor tyrosine phosphorylation and oppositely directed augmentation of IGF-I effect on ERK1/2 and possibly Akt activation in Gigyf2 null MEFs is similar to the previously described effects of knockdown of the GIGYF2 binding partner, Grb10, in cultured fibroblasts (4).
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.
The Gigyf2 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 (22,23). 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 (22,26).
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 or DJ-1 genes in mice results in neurodegeneration and motor function deficits, but these animals do not have cellular deficits in the nigrostriatal dopaminergic pathway (27–29). 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 (3,4). 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 (6,7,31,32). IGF-I stimulates brain growth in vivo (33), 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 (35,36), 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.
Mice with disruption of the Gigyf2 gene on a mixed C57BL/6-Tyrc-Brd/129Ola background were generated using Gigyf2 gene trap 129Ola ES cells (XH076) from Bay Genomics (UC San Francisco). The gene trap integration site in the Gigyf2 gene was defined by sequencing of PCR products yielding a 290 bp fragment from intron 4 of the gene trap-containing Gigyf2 gene (P1-F: 5′-GGGTGCCAAACTCAGTCCATTC-3′ and GT2-R: 5′-CGTGTCCTACAACACACACTCCAACC-3′) and a 639 bp fragment from the wild-type allele (P2-F: 5′-AAGCAGGGCTGGAGGTAGTC-3′ and P2-R: 5′-TTCTCCCTCTGCCCTACATTC-3′). All breeding and animal procedures were carried out according to institutional regulations of the Rhode Island Hospital animal facility (IACUC protocol no. 0221-06).
Total RNA was isolated from primary cultured MEFs using TRI Reagent (Molecular Research Center, Cincinnati, OH, USA), and 30 µg of RNA was resolved on 1% agarose gels under denaturing conditions. After transfer to nitrocellulose, blots were hybridized with one of two probes labeled with [32P] deoxy-CTP by random priming (Multiple DNA Labeling Kit, Amersham, GE Healthcare-Life Science, Piscataway, NJ, USA). Probe A was generated by amplifying a 450 bp fragment from GIGYF2 cDNA using primers spanning exons 1 to 4 (NT-F: 5′-T T T G G G C C T G A A T G G C T C C G T G C-3′ and NT-R: 5′-C T T C T T G A A G C T G G A G A T C C T C G-3′), thus matching sequence 5′ from the gene trap insertion. Probe B, corresponding to a region downstream from the gene trap vector, was obtained by HindIII digestion of GIGYF2 cDNA (1738–2769 bp, exons 13–26). Blots were washed twice with 2× SSC-0.05% SDS (40 min each) and once with 0.1× SSC-0.1% SDS, and specific mRNA bands were identified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA, USA). The GIGYF1 probe for northern blotting was generated by restriction digestion of mouse GIGYF1 cDNA using BamHI–XhoI, generating a 450 bp fragment at the N terminus.
For total protein lysates, MEFs derived from Gigyf2+/+ and −/− mice or whole embryos from timed matings, were homogenized in a 1% NP-40 buffer containing 10% glycerol, 137 mm NaCl, 1 mm MgCl2, 1 mm CaCl2, 20 mm Tris–HCl at pH 7.6, 2 mm EDTA, 10 mm sodium pyrophosphate, 10 mm NaF, 2 mm Na2VO4, 2 mm PMSF and protease inhibitors (Sigma, St Louis, MO, USA). For IGF-I stimulation experiments, Gigyf2+/+ and −/− MEFs were cultured for 6 h in serum-free Dulbecco's modified Eagle's medium (4.5 g/l glucose) supplemented with 0.5% bovine serum albumin and 25 mm HEPES at pH 7.5. Cells were incubated with 10−7 M IGF-I (GroPep, Adelaide, Australia) for the indicated duration at 37°C prior to lysis in 1% NP-40 buffer. All lysates were cleared by centrifugation, protein concentrations were determined by Bradford assay, and 50–60 µg protein aliquots were subjected to SDS–PAGE and transferred to nitrocellulose membranes for immunoblotting. Membranes were blocked at room temperature for 1 h with 5% BSA in Tris-buffered saline Tween-20 followed by incubation overnight at 4°C with primary antibody. After washing, membranes were incubated with appropriate secondary antibodies conjugated to horseradish peroxidase (Jackson Immuno Research, West Grove, PA, USA). Immunoblots were developed by Western Lightning Chemiluminescence Reagent Plus (PerkinElmer, Boston, MA, USA), and digital image acquisition was performed on a FluorChem Imaging System (Alpha Innotech, San Leandro, CA, USA). Rabbit anti-mouse GIGYF2 and GIGYF1 polyclonal antibodies for immunoblotting were generated against 17 amino acid peptides corresponding to the C-terminal sequences of these proteins and affinity-purified with the corresponding peptides immobilized on Sulfolink resin (Pierce, Rockford, IL, USA). Addition antibodies included: anti-ERK1 (Transduction Laboratories), anti-Akt (Cell Signaling Technology, Beverly, MA, USA), anti-IGF-I receptor (kindly provided by Dr Kenneth Siddle, Cambridge, UK) and anti-β-actin (C4; Santa Cruz Biotechnology, Santa Cruz, CA, USA); phosphorylation was measured using anti-ACTIVE MAPK (Promega, Madison, WI, USA), anti-phospho-Akt (Thr308) (Cell Signaling Technology) and anti-phospho-IGF-I receptor (pY1158-specific) (BioSource, Camarillo, CA, USA).
Brain and spinal cord from 15-month-old Gigyf2+/+ and +/− mouse littermates were fixed overnight in 4% paraformaldehyde/PBS at 4°C, dehydrated with increasing concentrations of ethanol, embedded in paraffin and sectioned sagittally (7–10 µm). Luxol Fast Blue/Hematoxylin&Eosin staining was performed using standard methods. Adjacent sections were rehydrated, treated with Avidin–Biotin blocking solution (Vector Laboratories, Burlingame, CA, USA) and incubated overnight at 4°C in a humidified chamber with sheep anti-α-synuclein antibody (Chemicon, Temecula, CA, USA). Primary antibody detection was performed with biotinylated goat anti-sheep IgG and Vector Elite ABC Reagent (Vector Laboratories). Sections were counterstained with Hematoxylin.
Day 14.5 embryos from timed matings were obtained by Caesarean section, washed in PBS, fixed in fresh 2% paraformaldehyde/PBS for 1 h at 4°C, hemisected, fixed for an additional hour and then placed in 18% sucrose overnight at 4°C. Brains from adult Gigyf2−/− mice were also treated in a similar manner. After removal of sucrose by blotting, the specimens were equilibrated in OCT (Tissuetech, EMS, Hatfield, PA, USA), positioned in a plastic base mold filled with OCT and frozen. Sagittal sections (10 μm) were prepared, adhered to slides, washed in PBS and incubated for 24 h at 30°C in the dark in fresh X-Gal solution (1 mg/ml X-Gal, 35 mm potassium ferrocyanide, 35 mm potassium ferricyanide, 2 mm MgCl2, 0.02% NP-40 in PBS). After washing in double-distilled water, the sections were counterstained for 2 min with Vector Fast Red (Vector Laboratories), dehydrated and mounted under coverslips.
A horizontal rotating rod system was used to measure animal balance and coordination (37). Mice were tested at 12–15 months of age. Each animal was housed in a single cage and all experiments were performed during the light phase of a 12:12 h light:dark cycle. Animals were first conditioned on a stationary rod for 1 min and during this time any animal that fell was placed back on the rod. The next day, animals were conditioned at a constant speed of 5 r.p.m. for 90 s. If an animal fell during this time they were placed back on the rod until the end of the conditioning time (38). After conditioning, animals were subjected to one trial per day at 5 r.p.m. for 6 days, for a total of six trials. The length of time each mouse remained on the rod was recorded.
Gigyf2+/+ and −/− primary MEFs (passage 4) were plated onto 10 cm tissue culture plates at 1.5 × 106 cells/plate and cultured for 24 h in Dulbecco's modified Eagle's medium (4.5 g/l glucose) supplemented with 10% fetal bovine serum. Cells were incubated with 20 µm Etoposide (Sigma) dissolved in DMSO or DMSO alone for an additional 24 h prior to staining with propidium iodide and cell cycle analysis by flow cytometry.
Statistical analysis was conducted using Sigma Stat 3.0 and Sigma Plot software (Systat Software, San Jose, CA, USA). One-tailed or two-tailed t-tests and the Mann–Whitney rank sum test were performed as indicated to evaluate the significance of differences between means. Results with P < 0.05 were considered to be statistically significant.
Conflict of Interest statement. None declared.
Portions of this work were supported by the COBRE Genomics Center at Brown University (J.K.), the Hallett Center for Diabetes and Endocrinology at Rhode Island Hospital (R.J.S.) and NIH Grant DK43038 (R.J.S.).