LID Mice Research Articles

Shared GABA transmission pathology in dopamine agonist- and antagonist-induced dyskinesia

Dyskinesia is involuntary movement caused by long-term medication with dopamine-related agents: the dopamine agonist 3,4-dihydroxy-L-phenylalanine (L-DOPA) to treat Parkinson's disease (L-DOPA-induced dyskinesia [LID]) or dopamine antagonists to treat schizophrenia (tardive dyskinesia [TD]). However, it remains unknown why distinct types of medications for distinct neuropsychiatric disorders induce similar involuntary movements. Here, we search for a shared structural footprint using magnetic resonance imaging-based macroscopic screening and super-resolution microscopy-based microscopic identification. We identify the enlarged axon terminals of striatal medium spiny neurons in LID and TD model mice. Striatal overexpression of the vesicular gamma-aminobutyric acid transporter (VGAT) is necessary and sufficient for modeling these structural changes; VGAT levels gate the functional and behavioral alterations in dyskinesia models. Our findings indicate that lowered type 2 dopamine receptor signaling with repetitive dopamine fluctuations is a common cause of VGAT overexpression and late-onset dyskinesia formation and that reducing dopamine fluctuation rescues dyskinesia pathology via VGAT downregulation.

Amphiregulin blockade decreases the levodopa-induced dyskinesia in a 6-hydroxydopamine Parkinson's disease mouse model.

Levodopa (L-DOPA) is considered the most reliable drug for treating Parkinson's disease (PD) clinical symptoms. Regrettably, long-term L-DOPA therapy results in the emergence of drug-induced abnormal involuntary movements (AIMs) in most PD patients. The mechanisms underlying motor fluctuations and dyskinesia induced by L-DOPA (LID) are still perplexing. Here, we first performed the analysis on the microarray data set (GSE55096) from the gene expression omnibus (GEO) repository and identified the differentially expressed genes (DEGs) using linear models for microarray analysis (Limma) R packages from the Bioconductor project. 12 genes (Nr4a2, Areg, Tinf2, Ptgs2, Pdlim1, Tes, Irf6, Tgfb1, Serpinb2, Lipg, Creb3l1, Lypd1) were found to be upregulated. Six genes were validated on quantitative polymerase chain reaction and subsequently, Amphiregulin (Areg) was selected (based on log2 fold change) for further experiments to unravel its involvement in LID. Areg LV_shRNA was used to knock down Areg to explore its therapeutic role in the LID model. Western blotting and immunofluorescence results show that AREG is significantly expressed in the LID group relative to the control. Dyskinetic movements in LID mice were alleviated by Areg knockdown, and the protein expression of delta FOSB, the commonly attributable protein in LID, was decreased. Moreover, Areg knockdown reduced the protein expression of P-ERK. In order to ascertain whether the inhibition of the ERK pathway (a common pathway known to mediate levodopa-induced dyskinesia) could also impede Areg, the animals were injected with an ERK inhibitor (PD98059). Afterward, the AIMs, AREG, and ERK protein expression were measured relative to the control group. A group treated with ERK inhibitor had a significant decrease of AREG and phosphorylated ERK protein expression relative to the control group. Taken together, our results indicate unequivocal involvement of Areg in levodopa-induced dyskinesia, thus a target for therapy development.

Disparate Central and Peripheral Effects of Circulating IGF-1 Deficiency on Tissue Mitochondrial Function

Age-related decline in circulating levels of insulin-like growth factor (IGF)-1 is associated with reduced cognitive function, neuronal aging, and neurodegeneration. Decreased mitochondrial function along with increased reactive oxygen species (ROS) and accumulation of damaged macromolecules are hallmarks of cellular aging. Based on numerous studies indicating pleiotropic effects of IGF-1 during aging, we compared the central and peripheral effects of circulating IGF-1 deficiency on tissue mitochondrial function using an inducible liver IGF-1 knockout (LID). Circulating levels of IGF-1 (~ 75%) were depleted in adult male Igf1f/f mice via AAV-mediated knockdown of hepatic IGF-1 at 5 months of age. Cognitive function was evaluated at 18 months using the radial arm water maze and glucose and insulin tolerance assessed. Mitochondrial function was analyzed in hippocampus, muscle, and visceral fat tissues using high-resolution respirometry O2K as well as redox status and oxidative stress in the cortex. Peripherally, IGF-1 deficiency did not significantly impact muscle mass or mitochondrial function. Aged LID mice were insulin resistant and exhibited ~ 60% less adipose tissue but increased fat mitochondrial respiration (20%). The effects on fat metabolism were attributed to increases in growth hormone. Centrally, IGF-1 deficiency impaired hippocampal-dependent spatial acquisition as well as reversal learning in male mice. Hippocampal mitochondrial OXPHOS coupling efficiency and cortex ATP levels (~ 50%) were decreased and hippocampal oxidative stress (protein carbonylation and F2-isoprostanes) was increased. These data suggest that IGF-1 is critical for regulating mitochondrial function, redox status, and spatial learning in the central nervous system but has limited impact on peripheral (liver and muscle) metabolism with age. Therefore, IGF-1 deficiency with age may increase sensitivity to damage in the brain and propensity for cognitive deficits. Targeting mitochondrial function in the brain may be an avenue for therapy of age-related impairment of cognitive function. Regulation of mitochondrial function and redox status by IGF-1 is essential to maintain brain function and coordinate hippocampal-dependent spatial learning. While a decline in IGF-1 in the periphery may be beneficial to avert cancer progression, diminished central IGF-1 signaling may mediate, in part, age-related cognitive dysfunction and cognitive pathologies potentially by decreasing mitochondrial function.

Serum Insulin-Like Growth Factor I Deficiency Associates to Alzheimer's Disease Co-Morbidities.

Increasing evidence supports the notion that Alzheimer's disease (AD), a condition that presents heterogeneous pathological disturbances, is also associated to perturbed metabolic function affecting insulin and insulin-like growth factor I (IGF-I). While impaired insulin activity leading to insulin resistance has been associated to AD, whether altered IGF-I function affects the disease is not entirely clear. Despite the limitations of mouse models to mimic AD pathology, we took advantage that serum IGF-I deficient mice (LID mice) present many functional perturbations present in AD, most prominently cognitive loss, which is reversed by treatment with systemic IGF-I. We analyzed whether these mice display other pathological traits that are usual co-morbidities of AD. We found that LID mice not only display cognitive disturbances, but also show altered mood and sociability, increased susceptibility to epileptiform activity, and a disturbed sleep/wake cycle. Collectively, these data suggest that reduced IGF-I activity contributes to heterogeneous deficits commonly associated to AD. We suggest that impaired IGF-I activity needs to be taken into consideration when modeling this condition.

Chemogenetic stimulation of striatal projection neurons modulates responses to Parkinson's disease therapy.

Parkinson's disease (PD) patients experience loss of normal motor function (hypokinesia), but can develop uncontrollable movements known as dyskinesia upon treatment with L-DOPA. Poverty or excess of movement in PD has been attributed to overactivity of striatal projection neurons forming either the indirect (iSPNs) or the direct (dSPNs) pathway, respectively. Here, we investigated the two pathways' contribution to different motor features using SPN type-specific chemogenetic stimulation in rodent models of PD (PD mice) and L-DOPA-induced dyskinesia (LID mice). Using the activatory Gq-coupled human M3 muscarinic receptor (hM3Dq), we found that chemogenetic stimulation of dSPNs mimicked, while stimulation of iSPNs abolished the therapeutic action of L-DOPA in PD mice. In LID mice, hM3Dq stimulation of dSPNs exacerbated dyskinetic responses to L-DOPA, while stimulation of iSPNs inhibited these responses. In the absence of L-DOPA, only chemogenetic stimulation of dSPNs mediated through the Gs-coupled modified rat muscarinic M3 receptor (rM3Ds) induced appreciable dyskinesia in PD mice. Combining D2 receptor agonist treatment with rM3Ds-dSPN stimulation reproduced all symptoms of LID. These results demonstrate that dSPNs and iSPNs oppositely modulate both therapeutic and dyskinetic responses to dopamine replacement therapy in PD. We also show that chemogenetic stimulation of different signaling pathways in dSPNs leads to markedly different motor outcomes. Our findings have important implications for the design of effective antiparkinsonian and antidyskinetic drug therapies.

Abstract 2883: Genetic reduction of circulating IGF-1 inhibits growth of COX-2- and Kras-induced pancreatic tumors in orthotopic transplant models

Abstract Chronic pancreatitis dramatically increases the risk of pancreatic cancer, the 4th deadliest malignancy in the US. KRAS mutations, the predominant genetic lesion found in pancreatic cancer, are detectable in 30% of patients with chronic pancreatitis. We previously found that calorie restriction (CR) protected against the tumorigenic effects of chronic pancreatitis in the cyclooxygenase (COX)-2-driven (BK5.COX-2) transgenic mouse model of pancreatic cancer. The protection conferred by CR was associated with dramatically reduced circulating levels of insulin-like growth factor (IGF)-1, an established mitogen in a variety of cancer cell lines. We hypothesized that circulating IGF-1 levels have a crucial role in tumor progression in both pancreatitis- and KRAS-induced pancreatic cancer. To test this, mouse pancreatic cancer cells derived from either a BK5.COX-2 transgenic mouse (JC101) or a KrasG12D/INK4a+/− mouse (NB508) were orthotopically transplanted in liver-specific IGF-1-deficient (LID, n=16 per cell line) and wild-type (WT, n=11 per cell line) mice. LID mice were randomized to receive Alzet miniature osmotic pumps (implanted subcutaneously) continuously infusing either vehicle (n=8) or 1 µg/hr of recombinant human IGF-1 (rhIGF-1, Increlex®, Tercica, Inc., Brisbane, CA; n=8). WT mice were infused with vehicle. Mice were euthanized 28 days after tumor and pump implantation, and blood was collected and tumors were weighed and fixed in 10% normal buffered formalin. Immunohistochemical staining for Ki-67 was performed on paraffin-embedded tumors to assess proliferation. In LID mice serum IGF-1 levels were reduced approximately 70% relative to WT (p<0.05), while infusion of rhIGF-1 resulted in total serum IGF-1 levels (murine plus rhIGF-1) exceeding those in LID and WT mice. Insulin levels were higher in LID mice (p<0.01), relative to WT, but administration of rhIGF-1 reduced insulin to levels comparable to WT. Genetic reduction of circulating IGF-1 in LID mice reduced JC101 tumor burden by 60% (p<0.05) and NB508 tumor burden by 75% (p<0.05), relative to tumors in WT mice. Administration of rhIGF-1 to LID mice restored tumor burden to levels comparable to that of WT mice. Tumors from LID mice also displayed lower expression of the proliferation marker Ki-67 than tumors from WT mice or LID mice administered rhIGF-1. We conclude that reduction of circulating IGF-1 inhibits pancreatic cancer driven by alterations in two common but distinct protumorigenic pathways (chronic inflammation and KRAS alterations), and underscores the potential of IGF-1 pathway inhibition for prevention and treatment of pancreatic cancer. Work supported by NIH grants R25T CA57730 and CA135386. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2883.

Reduced Levels of IGF-I Mediate Differential Protection of Normal and Cancer Cells in Response to Fasting and Improve Chemotherapeutic Index

Inhibitors of the insulin-like growth factor-I (IGF-I) receptor have been widely studied for their ability to enhance the killing of a variety of malignant cells, but whether IGF-I signaling differentially protects the host and cancer cells against chemotherapy is unknown. Starvation can protect mice, but not cancer cells, against high-dose chemotherapy [differential stress resistance (DSR)]. Here, we offer evidence that IGF-I reduction mediates part of the starvation-dependent DSR. A 72-hour fast in mice reduced circulating IGF-I by 70% and increased the level of the IGF-I inhibitor IGFBP-1 by 11-fold. LID mice, with a 70% to 80% reduction in circulating IGF-I levels, were protected against three of four chemotherapy drugs tested. Restoration of IGF-I was sufficient to reverse the protective effect of fasting. Sixty percent of melanoma-bearing LID mice treated with doxorubicin achieved long-term survival whereas all control mice died of either metastases or chemotherapy toxicity. Reducing IGF-I/IGF-I signaling protected primary glia, but not glioma cells, against cyclophosphamide and protected mouse embryonic fibroblasts against doxorubicin. Further, S. cerevisiae lacking homologs of IGF-I signaling proteins were protected against chemotherapy-dependent DNA damage in a manner that could be reversed by expressing a constitutively active form of Ras. We conclude that normal cells and mice can be protected against chemotherapy-dependent damage by reducing circulating IGF-I levels and by a mechanism that involves downregulation of proto-oncogene signals.

OR13,76 Effects of metformin on hyperglycaemia-induced chemoresistance of DU145 and LNCaP prostate cancer cells

as amifostine, glutathione, mesna, and dexrazoxane have been investigated and shown to provide drug-dependent protection to specific tissues, but the use of these compounds has not been shown to increase disease-free or overall survival. We previously showed that fasting selectively protects mice, and possibly patients, but not tumors against high-dose chemotherapy (Differential Stress Resistance, DSR). Here we show that the beneficial effect of fasting extends beyond protecting normal cells to sensitizing malignant cells to chemotherapy. Further, we show that the differential protection of normal cells and sensitization of malignant cells by fasting is mediated by reduced IGF-I levels/signaling. A 72-hour fast provided remarkable protection against doxorubicin. We observed that fasting reduced circulating IGF-I by 70% and increased the level of the IGF-I inhibitor IGFBP-1 by 11-fold in mice. LID mice, with a 70–80% reduction in circulating IGF-I levels, were protected against 3 out of 4 chemotherapy drugs tested. Restoration of IGF-I during fasting was sufficient to reverse its protective effect. 60% of melanoma-bearing LID mice treated with doxorubicin reached long-term survival whereas all control mice died of either metastases or chemotoxicity. In both our fasted (72 hours) and LID mice, we observed a 40% decrease in IGFBP-3 levels. Therefore, to test if reduced IGFBP-3 levels mediated DSR, we challenge transgenic IGFBP-3 knockout mice (IGFBP3KO) with cyclophosphamide. We found no significant difference in survival between WT and IGFBP3KO mice, suggesting a larger role of IGF-I in mediating DSR independently of IGFBP-3. Reduction of IGF-I/IGF-I signaling protected primary glia, but not glioma cells against cyclophosphamide and protected mouse embryonic fibroblasts (MEFs) against doxorubicin-induced DNA damage. On the contrary, in malignant cells, the addition of IGF-I during fasting was sufficient to reverse the sensitization to chemotherapy in vitro. Similarly, S. cerevisiae lacking homologues of IGF-I signaling proteins displayed protection against chemotherapydependent DNA damage, which was reversed by expression of an oncogene homolog. Our studies suggest that fasting has the potential to protect patients and sensitize cancer cells, in part, by reducing IGF-I levels/signaling, thereby increasing the therapeutic index of toxic drugs commonly used in the treatment of a wide range of malignancies.

OR13,77 Insulin-like growth factors (IGFs) and IGF binding proteins in PSA-detected prostate cancer: a population-based case control study

as amifostine, glutathione, mesna, and dexrazoxane have been investigated and shown to provide drug-dependent protection to specific tissues, but the use of these compounds has not been shown to increase disease-free or overall survival. We previously showed that fasting selectively protects mice, and possibly patients, but not tumors against high-dose chemotherapy (Differential Stress Resistance, DSR). Here we show that the beneficial effect of fasting extends beyond protecting normal cells to sensitizing malignant cells to chemotherapy. Further, we show that the differential protection of normal cells and sensitization of malignant cells by fasting is mediated by reduced IGF-I levels/signaling. A 72-hour fast provided remarkable protection against doxorubicin. We observed that fasting reduced circulating IGF-I by 70% and increased the level of the IGF-I inhibitor IGFBP-1 by 11-fold in mice. LID mice, with a 70–80% reduction in circulating IGF-I levels, were protected against 3 out of 4 chemotherapy drugs tested. Restoration of IGF-I during fasting was sufficient to reverse its protective effect. 60% of melanoma-bearing LID mice treated with doxorubicin reached long-term survival whereas all control mice died of either metastases or chemotoxicity. In both our fasted (72 hours) and LID mice, we observed a 40% decrease in IGFBP-3 levels. Therefore, to test if reduced IGFBP-3 levels mediated DSR, we challenge transgenic IGFBP-3 knockout mice (IGFBP3KO) with cyclophosphamide. We found no significant difference in survival between WT and IGFBP3KO mice, suggesting a larger role of IGF-I in mediating DSR independently of IGFBP-3. Reduction of IGF-I/IGF-I signaling protected primary glia, but not glioma cells against cyclophosphamide and protected mouse embryonic fibroblasts (MEFs) against doxorubicin-induced DNA damage. On the contrary, in malignant cells, the addition of IGF-I during fasting was sufficient to reverse the sensitization to chemotherapy in vitro. Similarly, S. cerevisiae lacking homologues of IGF-I signaling proteins displayed protection against chemotherapydependent DNA damage, which was reversed by expression of an oncogene homolog. Our studies suggest that fasting has the potential to protect patients and sensitize cancer cells, in part, by reducing IGF-I levels/signaling, thereby increasing the therapeutic index of toxic drugs commonly used in the treatment of a wide range of malignancies.

Insulin-like growth factor-I regulates the liver microenvironment in obese mice and promotes liver metastasis.

Among the mechanisms implicated in the tumor-promoting effects of obesity, signaling by insulin-like growth factor-I (IGF-I) and insulin has received considerable attention. However, the emerging realization that obesity is associated with chronic inflammation has prompted other consideration of how the IGF-I axis may participate in cancer progression. In the present study, we used two mouse models of chronic (LID) and inducible (iLID) igf-1 gene deficiency in the liver to investigate the role of IGF-I in regulating the host microenvironment and colorectal carcinoma growth and metastasis in obese mice. Obese mice had a heightened inflammatory response in the liver, which was abolished in mice with chronic IGF-I deficiency (LID). In control animals changes to the hepatic microenvironment associated with obesity sustained the presence of tumor cells in the liver and increased the incidence of hepatic metastases after intrasplenic/portal inoculation of colon carcinoma cells. These changes did not occur in LID mice with chronic IGF-1 deficiency. In contrast, these changes occurred in iLID mice with acute IGF-1 deficiency, in the same manner as the control animals, revealing a fundamental difference in the nature of the requirement for IGF-1 on tumor growth and metastasis. In the setting of obesity, our findings imply that IGF-1 is critical to activate and sustain an inflammatory response in the liver that is needed for hepatic metastasis, not only through direct, paracrine effect on tumor cell growth, but also through indirect effects involving the tumor microenvironment.

Sugar and Bone: A Not-So Sweet Story

I predict that most readers of the article by Moyer-Mileur and colleagues in this month’s Journal of Bone and Mineral Research will be challenged not just by the results of their study, but also by the long term implications of their findings. In this relatively small, but well done CRU (Clinical Research Unit) investigation, several indices of the growth hormone- IGF-I axis in adolescent girls with at least 5 years of Type I diabetes mellitus (TIDM), were compared to appropriately age and tanner staged- matched controls (1). These parameters were then regressed against measures of skeletal size and content using areal and volumetric bone mass measurements. On first inspection, the results are not surprising, and are consistent with earlier studies from this group and others; i.e. adolescent girls with long standing TIDM have lower serum IGF-I, higher IGFBP-1, and greater growth hormone concentrations than their age-matched controls. Moreover, those girls with poor metabolic control had the lowest circulating IGF-I and highest IGFBP-1 concentrations. In respect to skeletal structure, increased urinary magnesium in the diabetic girls predicted a smaller, lighter skeleton, and overall disease duration accounted for the greatest variability in bone mineral content and cross sectional area. Higher blood glucoses and greater urinary magnesium were also associated with measures of increased bone resorption. Low serum IGF-I was a major predictor of tibial bone strength, as determined by pQCT.

The effects of exercise on spatial learning and anxiety-like behavior are mediated by an IGF-I-dependent mechanism related to hippocampal neurogenesis

Knowledge about the effects of physical exercise on brain is accumulating although the mechanisms through which exercise exerts these actions remain largely unknown. A possible involvement of adult hippocampal neurogenesis (AHN) in the effects of exercise is debated while the physiological and pathological significance of AHN is under intense scrutiny. Recently, both neurogenesis-dependent and independent mechanisms have been shown to mediate the effects of physical exercise on spatial learning and anxiety-like behaviors. Taking advantage that the stimulating effects of exercise on AHN depend among others, on serum insulin-like growth factor I (IGF-I), we now examined whether the behavioral effects of running exercise are related to variations in hippocampal neurogenesis, by either increasing or decreasing it according to serum IGF-I levels. Mutant mice with low levels of serum IGF-I (LID mice) had reduced AHN together with impaired spatial learning. These deficits were not improved by running. However, administration of exogenous IGF-I ameliorated the cognitive deficit and restored AHN in LID mice. We also examined the effect of exercise in LID mice in the novelty-suppressed feeding test, a measure of anxiety-like behavior in laboratory animals. Normal mice, but not LID mice, showed reduced anxiety after exercise in this test. However, after exercise, LID mice did show improvement in the forced swim test, a measure of behavioral despair. Thus, many, but not all of the beneficial effects of exercise on brain function depend on circulating levels of IGF-I and are associated to increased hippocampal neurogenesis, including improved cognition and reduced anxiety.

Does IGF-I stimulate pancreatic islet cell growth?

Both IGF-I and its receptor (IGF-IR) are specifically expressed in various cell types of the endocrine pancreas. IGF-I has long been considered a growth factor for islet cells as it induces DNA synthesis in a glucose-dependent manner, prevents Fas-mediated autoimmune beta-cell destruction and delays onset of diabetes in non-obese diabetic (NOD) mice. Islet-specific IGF-I overexpression promotes islet cell regeneration in diabetic mice. However, in the last few years, results from most gene-targeted mice have challenged this view. For instance, combined inactivation of insulin receptor and IGF-IR or IGF-I and IGF-II genes in early embryos results in no defect on islet cell development; islet beta-cell-specific inactivation of IGF-IR gene causes no change in beta-cell mass; liver- and pancreatic-specific IGF-I gene deficiency (LID and PID mice) suggests that IGF-I exerts an inhibitory effect on islet cell growth albeit indirectly through controlling growth hormone release or expression of Reg family genes. These results need to be evaluated with potential gene redundancy, model limitations, indirect effects and ligand-receptor cross-activations within the insulin/IGF family. Although IGF-I causes islet beta-cell proliferation and neogenesis directly, what occur in normal physiology, pathophysiology or during development of an organism might be different. Locally produced and systemic IGF-I does not seem to play a positive role in islet cell growth. Rather, it is probably a negative regulator through controlling growth hormone and insulin release, hyperglycemia, or Reg gene expression. These results complicate the perspective of an IGF-I therapy for beta-cell loss.

The ternary IGF complex influences postnatal bone acquisition and the skeletal response to intermittent parathyroid hormone

The role of circulating IGF-I in skeletal acquisition and the anabolic response to PTH is not well understood. We generated IGF-I-deficient mice by gene deletions of IGF ternary complex components including: (1) liver-specific deletion of the IGF-I gene (LID), (2) global deletion of the acid-labile (ALS) gene (ALSKO), and (3) both liver IGF-I and ALS inactivated genes (LA). Twelve-week-old male control (CTL), LID, ALSKO, and LA mice were treated with vehicle (VEH) or human PTH(1-34) for 4 weeks. VEH-treated IGF-I-deficient mice (i.e. LID, ALSKO and LA mice) exhibited reduced cortical cross-sectional area (P = 0.001) compared with CTL mice; in contrast, femoral trabecular bone volume fractions (BV/TV) of the IGF-I-deficient mice were consistently greater than CTL (P < 0.01). ALSKO mice exhibited markedly reduced osteoblast number and surface (P < 0.05), as well as mineral apposition rate compared with other IGF-I-deficient and CTL mice. Adherent bone marrow stromal cells, cultured in beta-glycerol phosphate and ascorbic acid, showed no strain differences in secreted IGF-I. In response to PTH, there were both compartment- and strain-specific effects. Cortical bone area was increased by PTH in CTL and ALSKO mice, but not in LID or LA mice. In the trabecular compartment, PTH increased femoral and vertebral BV/TV in LID, but not in ALSKO or LA mice. In conclusion, we demonstrated that the presentation of IGF-I as a circulating complex is essential for skeletal remodeling and the anabolic response to PTH. We postulate that the ternary complex itself, rather than IGF-I alone, influences bone acquisition in a compartment-specific manner (i.e. cortical vs trabecular bone).