In this study, we used adult-onset, tissue-specific knockouts of Igf1 to explore the endocrine and paracrine roles of IGF-1 in the genesis of depression. We confirmed that hippocampal IGF-1 protein levels are dependent upon serum IGF-1 levels in mice. Hippocampal IGF-1 levels have a significant effect on the forced swim measure of depressive behavior in mice, and differences in this test occurred in the absence of alterations in basal mobility. Furthermore, the open field test provided no evidence of differences in anxiety due to variation in IGF-1 levels, and this was also supported by data from the elevated plus maze. To our knowledge, this is the first demonstration that long-term IGF-1 deficiency initiated during adulthood is sufficient to induce depressive behavior.
Previous research examining the roles of IGF-1 in the genesis of depression has relied primarily on observational studies (Deuschle et al., 1997
), exogenous supplementation of the protein (C. H. Duman et al., 2009
; Hoshaw et al., 2005
), or short-term blockade of either IGF-1 (C. H. Duman et al., 2009
) or IGF-1 binding proteins (Malberg et al., 2007
). Such studies provide valuable, but inconclusive, evidence for the role of IGF-1 deficiency in depressive disorders. Studies involving supplementation of growth factors or neurotrophins (including brain-derived neurotrophic factor, BDNF) provide strong evidence that such proteins ameliorate some types of depression. However, these studies do not provide direct evidence that prolonged deficiencies of those proteins contribute to depression. Previous work with 3 month-old LID (liver IGF-1 deficient) mice has demonstrated similar results in the forced swim test (Trejo et al., 2008
), but it was unclear if the depressive phenotype was due to a primary IGF-1 deficiency during prepubertal development, secondary endocrine dysfunction, or if the phenotype was maintained into later adulthood. Our experimental design allows us to conclude that prolonged IGF-1 deficiency during adulthood contributes to depressive symptoms and that this effect is dependent on both serum and brain IGF-1 levels. Previous studies of short-term blockade of IGF-1 or its binding proteins provide compelling evidence that deficiencies of these proteins can affect mood within hours, but do not control for the possibility of homeostatic compensation over the course of weeks or months. Our study demonstrates that the brain does not compensate fully for adult-onset reduction in liver IGF-1 production by either increased IGF-1 production or transport of IGF-1 from the serum. Our methods do not exclude the possibility of other forms of compensation, but if present, they were insufficient to prevent the behavioral phenotype.
The possibility that other tissues may compensate for reductions in circulating IGF-1 is under considerable debate, which makes simultaneous control of endocrine and paracrine IGF-1 production an important experimental issue. Complete knockouts of Igf1
or its receptor in mice are generally lethal, and survivors are growth retarded and largely infertile (reviewed in (Liu et al., 2000
)). Early life knockout in the liver only does not appear to have significant effects on growth or fertility (Yakar et al., 1999
), and it was suggested that paracrine IGF-1, rather than circulating IGF-1 from the liver, exerted an important role in the growth compensation that occurs in this model. However, this conclusion was later challenged based on insufficient IGF-1 deficiency during an early critical growth period (Tang et al., 2005
) and the demonstration that mice which produce IGF-1 only
in their livers are fertile and have relatively mild growth phenotypes compared to their controls (Stratikopoulos et al., 2008
). Together, these reports suggest that IGF-1 production by the liver is not necessary, but is sufficient to maintain tissue functions that are dependent upon IGF-1.
In the absence of liver IGF-1, paracrine mechanisms may compensate for the reduction in circulating IGF-1. Indeed, some of the few phenotypes detected in LID mice disappear by two years of age, and/or are sexually dimorphic (Tang et al., 2005
). These results emphasize the need to consider the role of paracrine IGF-1 separately for different tissues and functions. However, analysis of Igf1
gene expression alone is insufficient to determine local IGF-1 deficiency, as the circulating protein may simply be transported into tissues at a greater rate. In this study, we clearly observed that a reduction in circulating IGF-1 decreases IGF-1 protein levels in the hippocampus, with little or no compensation even after 7 months. Additionally, Igf1
knockouts in CA1 neurons equivalently in all animals, independent of circulating IGF-1 knockdowns (two-way ANOVA test of interactions, p=0.826), demonstrating that there was little or no compensatory IGF-1 production by CA1 neurons. These results are in qualitative agreement with gene expression data from 6-week-old LID mice, which do not show detectable increases in Igf1
expression in fat, spleen, heart, muscle, or kidney (Yakar et al., 1999
). It is possible (albeit unlikely) that such compensation may appear later in life; nevertheless the appearance of other phenotypes of aging may confound interpretation in a more prolonged experiment. For our model, we note that lack of detectable paracrine compensation for endocrine IGF-1 deficiency (either through IGF-1 production or transport), and lack of detectable endocrine compensation for paracrine deficiency should not be construed to represent limited transport of IGF-1 across the blood-brain barrier. We doubt we could create a severe enough neuronal knockout of Igf1
to produce a detectable change in serum IGF-1 levels using our current methods, particularly if endocrine compensation occurs. Furthermore, the robust positive correlation between hippocampal and serum IGF-1 levels observed in this study largely precludes a hypothesis of limited IGF-1 transport. Though this correlation existed across experimental groups, our methods do not allow a direct measure of IGF-1 transport into or out of the brain, and further work is necessary to determine whether if IGF-1 transport is altered in IGF-1 deficient animals.
In the present study we were able to substantially reduce paracrine IGF-1 in the neurons of the CA1 region of the hippocampus. We chose this region because magnetic resonance imaging (Ballmaier et al., 2008
; Cho et al., 2010
; Cole et al., 2010
), physiological (Marchetti et al., 2010
), and ultrastructural (Hajszan et al., 2009
; Hajszan et al., 2005
) studies of depressed humans and animals have demonstrated changes in CA1 compared to controls. As tissue volume and neuronal/synaptic development (Kar et al., 1997
; Knusel & Hefti, 1991
; Lichtenwalner et al., 2001
; Seto et al., 2002
; Trejo et al., 2008
; Trejo et al., 2007
) are affected by IGF-1, we hypothesized that the effects of depression on the CA1 region may be related to local IGF-1 production. Furthermore, CA1 (and the hippocampus in general) has an important role in memory formation and maintenance. Since depression, reduced cognitive function, and IGF-1 decline in the geriatric population exhibit frequent coincidence, we reasoned that the effects of IGF-1 within various regions of the hippocampus should be examined experimentally. Our results are consistent with the hypothesis that the low IGF-1 levels present in older animals and humans have the potential to contribute to depressive disorders. The present study also complements studies of GH deficiency and GH replacement therapy, which associate GH deficiency with depression and reduced quality of life, and GH replacement with improved quality of life (Abe et al., 2009
; Deijen & Arwert, 2006
; Kelly et al., 2006
). It appears likely that these reported effects of GH may be largely due to IGF-1, but further study with other models is necessary to determine the contribution of IGF-1 independent effects of GH. For example, the IGF-2 pathway interacts with the GH pathway, and is known to have behavioral effects in the hippocampus (Chen et al., 2011
The degree of serum IGF-1 deficiency in the present study (roughly a 40% reduction from control levels) appears to be translationally relevant to the aging human phenotype (see, e.g., (Rudman et al., 1981
)). Taken together with studies of IGF-1 supplementation, our results suggest that depression in at least a subset of elderly humans may be due to the age-related decline in circulating IGF-1. Additionally, the effect of IGF-1 deficiency on the forced swim measure of depression was non-linear in this study, implying that there may be a threshold IGF-1 level below which humans are at greater risk for depression. Conversely, low IGF-1 was insufficient in this study to induce anxiety. It is possible that targeting different regions of the brain (or more extreme knockdowns of circulating IGF-1) could induce anxiety behaviors. However, we observed a highly significant correlation of depressive behavior with serum and hippocampal IGF-1 levels. Not even a modest correlation between anxiety measures and IGF-1 measures was observed. It is possible that IGF-1 deficiency may only compromise the anxiolytic effects of other interventions such as exercise (Trejo et al., 2008
), without directly modulating anxiety. Both explanations suggest that comorbidity of anxiety and IGF-1 related depression may occur due to unrelated factors.
The molecular mechanisms by which IGF-1 deficiency causes depression are unclear, but several possibilities have been identified. Previous studies indicate that blocking hippocampal IGF-1 receptors reduces or abolishes exercise-induced increases in hippocampal BDNF mRNA and protein (Ding et al., 2006
). As many studies have described a role for BDNF in the action of antidepressant medications, electroconvulsive shock therapy, and the effects of exercise on depression (reviewed in (R. S. Duman, 2004
)), reductions of IGF-1 in the hippocampus may lead to depression by interrupting already identified BDNF pathways. An additional, parallel action of IGF-1 deficiency may involve regulation of serotonin levels in the hippocampus. IGF-1 administration has been shown to increase extracellular serotonin levels in rat hippocampus in approximately the same time course that it ameliorates depression (measured by the forced swim test). In the same study, it was also demonstrated that depletion of serotonin blocks the antidepressant effects of IGF-1 administration (Hoshaw et al., 2008
). Yet, very late-onset depression in elderly humans (who have relatively low IGF-1 levels) can be resistant to antidepressants (Meyers & Jeste, 2010
). Furthermore, older rodents show a decreased behavioral and neurogenic response to SSRIs (selective serotonin reuptake inhibitors) (Herrera-Perez et al., 2010
), providing evidence that depression may be regulated through multiple pathways. Also, growth hormone is produced in the hippocampus (Donahue et al., 2006
), and its pathways may interact and/or have IGF-1 independent effects on mood. These issues emphasize the need for clinical studies of the interactions of serotonin, growth factors, and neurotrophins in geriatric depression. Our preliminary hippocampal gene expression data suggest no changes in Igf1
(the IGF-1 receptor), Igf2
(insulin-like growth factor II), Igf2r
(insulin-like growth factor II receptor), Gh
(growth hormone receptor), or Ghrh
(growth hormone releasing hormone; unpublished observations). It should be noted that in a similar model, the LID mouse, GH overexpression and other phenotypes disappear with age (Tang et al., 2005
), and a cross-sectional study through the lifespan of our model would be required to conclude these pathways were never affected by knockout of Igf1
. However, in our model Bdnf
gene expression appears to be upregulated in the hippocampi of hepatic, hippocampal, and double knockouts of Igf1
, and serotonergic pathways may be altered as well (unpublished observations). If these are compensatory changes, they were insufficient to prevent the behavioral effects of IGF-1 deficiency, and suggest that Bdnf
regulation in IGF-1 deficient animals is dominated by factors other than IGF-1.
Future studies will determine whether it is more important for neurons to produce their own IGF-1 (i.e., autocrine IGF-1), or if other local cell types (e.g., glia and vasculature) support IGF-1 production in the hippocampus. As the hippocampal circuitry is only a part of the putative depression pathway, the techniques used in this study have the potential to be applied to other brain regions as well. Additionally, IGF-1 production and IGF-1 cell signaling are interdependent issues in both experimental design and translational medicine. It is necessary to know which cells are most important for IGF-1 production in the aged brain in order to design viable treatments for paracrine IGF-1 deficiency. However, similar experiments targeting the IGF-1 receptor are necessary to understand the IGF-1-mediated depression pathway. It is likely that depressive effects caused by IGF-1 deficiency are not solely due to reduced IGF-1 receptor activation in neurons, but reduced activation of the receptor in other cell types. Animals from all such experiments should be tested for effects on learning and memory, and examined for physiological, genomic, and proteomic correlates to their behavioral profiles.
In conclusion, we have generated a novel model of tissue and age-specific IGF-1 deficiency, and demonstrated robust control over the production of both circulating and locally produced IGF-1. Using this system, we have demonstrated that IGF-1 deficiency alone is sufficient to induce depression-like behaviors in normally developed adult mice, and that no sufficient compensatory mechanisms appear within a timeframe of several months. Our results suggest that depression in a subset of the geriatric population may be due to the age-related decline in IGF-1 production.