Deficits in hippocampal neurogenesis in obesity-dependent and -independent type-2 diabetes mellitus mouse models

Abstract

Hippocampal neurogenesis plays an important role in learning and memory function throughout life. Declines in this process have been observed in both aging and Alzheimer's disease (AD). Type 2 Diabetes mellitus (T2DM) is a disorder characterized by insulin resistance and impaired glucose metabolism. T2DM often results in cognitive decline in adults, and significantly increases the risk of AD development. The pathways underlying T2DM-induced cognitive deficits are not known. Some studies suggest that alterations in hippocampal neurogenesis may contribute to cognitive deterioration, however, the fate of neurogenesis in these studies is highly controversial. To address this problem, we utilized two models of T2DM: (1) obesity-independent MKR transgenic mice expressing a mutated form of the human insulin-like growth factor 1 receptor (IGF-1R) in skeletal muscle, and (2) Obesity-dependent db/db mice harboring a mutation in the leptin receptor. Our results show that both models of T2DM display compromised hippocampal neurogenesis. We show that the number of new neurons in the hippocampus of these mice is reduced. Clone formation capacity of neural progenitor cells isolated from the db/db mice is deficient. Expression of insulin receptor and epidermal growth factor receptor was reduced in hippocampal neurospheres isolated from db/db mice. Results from this study warrant further investigation into the mechanisms underlying decreased neurogenesis in T2DM and its link to the cognitive decline observed in this disorder.

Figure 1

Reduced number of new neurons in the SGZ of db/db mice. (A–C) Quantification of the number of neural progenitor cells (Nestin + DCX−, A), neuroblasts (Nestin + DCX + , B) and immature neurons (Nestin− DCX + , C) in the subgranular zone of the dentate gyrus of db/db and wild type mice using unbiased stereology. The number of neural progenitor cells was similar between genotypes. There was a trending decrease in the number of proliferating neuroblasts (P = 0.0668, N = 4). A significant decrease was observed in the number of immature neurons (*P < 0.05) in the db/db mice (Unpaired t-test, 12 weeks of age, N = 4). (D) Representative images of wild type and db/db brain sections immunolabeled with antibodies raised against doublecortin (DCX) or nestin and counterstained with DAPI. Scale bar = 75 μm. (E–G) Clonogenic assay of primary neural progenitor cells isolated from the dentate gyrus of db/db and wild type mice. Analysis of the number (E) and diameter (F) of neurospheres formed, as well as the total number of cells (G) show a significant decrease in all parameters in the db/db compared to wild type. (H) Representative images of neurospheres cultured from wild type and db/db mice (N = 3, unpaired t-test, *P < 0.05). Inserts show high power images of a single neurosphere. (I) Real time PCR analysis of mRNA expression of epidermal growth factor receptor (EGFR) in primary neurospheres shows a significant decrease in the db/db genotype (N = 3, unpaired t-test, *P < 0.05). (J,K) Western blot analysis (J) and semi-quantification using image J (K) of the expression of insulin receptor 1α (IR1α) in protein lysate of neurospheres isolated from db/db and wild type mice. Expression of IR1α is severely compromised in the db/db genotype (N = 3, unpaired t-test, *P < 0.05).

Figure 2

Reduced number of new neurons in the subgranular zone is recapitulated in MKR mice. (A–C) Stereological analysis of neural progenitor cells (Nestin + DCX−, A), neuroblasts (Nestin + DCX + , B) and immature neurons (Nestin−DCX + , C) in the subgranular zone of the dentate gyrus of MKR and wild type FVB mice. The number of neural progenitor cells was similar between genotypes. A trending decrease was observed in proliferating neuroblasts (Nestin⁺ DCX⁺; P = 0.056) and a significant decrease of immature neuroblasts (Nestin⁻ DCX⁺) in MKR mice compared to FVB control (N = 6, Unpaired t-test, *P < 0.05). (D) Representative confocal images of brain sections of MKR and wild type mice stained with antibodies raised against Nestin and DCX. Scale bar = 75 μm.

Figure 3

Alterations in neural stem cell number in db/db mice. (A–C) Quantitative analysis of the number of neural stem cells (Nestin + GFAP + , A), neural progenitor cells (Nestin + GFAP−, B) and mature astrocytes (Nestin− GFAP + , C) in the subgranular zone of the dentate gyrus in db/db and wild type mice shows a trending decrease in the number of neural stem cells in db/db mice (P = 0.0524). The number of neural progenitors or mature astrocytes was similar in both genotypes (N = 3, 12 weeks of age, Unpaired t-test). (D) Representative confocal images of neural stem cells in the dentate gyrus in brain sections of db/db and wild type mice. Scale bar = 75 μ.

Figure 4

MKR mice display trending loss of mature astrocytes in the subgranular zone of the dentate gyrus. (A–C) The number of neural stem cell (Nestin + GFAP + , A) and neural progenitor cells (Nestin + GFAP−, B) was similar in db/db and wild type mice at 12 weeks of age, as determined by unbiased stereology. However, MKR mice display a trending decrease in the number of mature astrocytes (Nestin− GFAP + , P = 0.052), (N = 6, Unpaired t-test). (D) Representative confocal images of Nestin + and GFAP + cells in the subgranular zone of the dentate gyrus of MKR and wild type mice. Scale bar = 75 μm.