CEBPβ regulation of endogenous IGF-1 in adult sensory neurons can be mobilized to overcome diabetes-induced deficits in bioenergetics and axonal outgrowth

Abstract

Aberrant insulin-like growth factor 1 (IGF-1) signaling has been proposed as a contributing factor to the development of neurodegenerative disorders including diabetic neuropathy, and delivery of exogenous IGF-1 has been explored as a treatment for Alzheimer's disease and amyotrophic lateral sclerosis. However, the role of autocrine/paracrine IGF-1 in neuroprotection has not been well established. We therefore used in vitro cell culture systems and animal models of diabetic neuropathy to characterize endogenous IGF-1 in sensory neurons and determine the factors regulating IGF-1 expression and/or affecting neuronal health. Single-cell RNA sequencing (scRNA-Seq) and in situ hybridization analyses revealed high expression of endogenous IGF-1 in non-peptidergic neurons and satellite glial cells (SGCs) of dorsal root ganglia (DRG). Brain cortex and DRG had higher IGF-1 gene expression than sciatic nerve. Bidirectional transport of IGF-1 along sensory nerves was observed. Despite no difference in IGF-1 receptor levels, IGF-1 gene expression was significantly (P < 0.05) reduced in liver and DRG from streptozotocin (STZ)-induced type 1 diabetic rats, Zucker diabetic fatty (ZDF) rats, mice on a high-fat/ high-sugar diet and db/db type 2 diabetic mice. Hyperglycemia suppressed IGF-1 gene expression in cultured DRG neurons and this was reversed by exogenous IGF-1 or the aldose reductase inhibitor sorbinil. Transcription factors, such as NFAT1 and CEBPβ, were also less enriched at the IGF-1 promoter in DRG from diabetic rats vs control rats. CEBPβ overexpression promoted neurite outgrowth and mitochondrial respiration, both of which were blunted by knocking down or blocking IGF-1. Suppression of endogenous IGF-1 in diabetes may contribute to neuropathy and its upregulation at the transcriptional level by CEBPβ can be a promising therapeutic approach.

Keywords: Diabetic neuropathy; Dorsal root ganglia; IGF-1; Mitochondria; NFAT1; Neurite outgrowth; Neurotrophic factor.

Fig. 1

Cellular localization and axonal transport of endogenous IGF-1 in DRG. A, B DRG tissue sections or C cultured DRG neurons from control rats underwent RNA FISH assay for IGF-1 mRNA detection and localization. A punctate pattern of IGF-1 mRNA is evident in neurons and a limited number of associated glia. As a control, tissue sections and cells were exposed to RNase enzyme before hybridizing with IGF-1 probes. Images are magnified for clarification. As a control, tissue sections and cells were exposed to RNase enzyme before hybridizing with IGF-1 probes. IGF-1 mRNA punctate labeling is shown in A neurons and B glia in DRG tissue sections. In C, cultured DRG neurons and associated glia showed punctate staining for IGF-1. Boxes 1–3 are magnified parts of the figure shown for clarity. Arrows show glial and neuronal IGF-1 in box1, neuronal IGF-1 in box2 and glial IGF-1 in box3. In D, publicly available scRNA-Seq normalized data (RPM) from 240 to 480 cells was analyzed and relative transcript levels of Igf1 and Igf1r were plotted for each DRG subpopulation: NF cluster (myelinated neurons), PEP cluster (peptidergic nociceptors), NP cluster (non-peptidergic nociceptors), TH cluster (type-C low-threshold mechanoreceptors), SC cluster (Schwann cells) and SGC cluster (satellite glial cells). In E, a 12-h double ligature (1 cm width) experiment was carried out on rat sciatic nerve with four regions proximal 1 (P1), P2, distal 1 (D1) and D2 analyzed for IGF-1 levels. Data are mean ± SEM of N = 3–4. *p < 0.05 or **p < 0.01; analyzed by one-way ANOVA with Tukey’s post hoc test

Fig. 2

The level of endogenous IGF-1 was reduced in DRG tissue from type 1 and type 2 diabetic rodents, in part, via hyperglycemia-activated polyol pathway activity and restored by exogenous hIGF-1. In A–D DRG tissues from control (Ctrl), hIGF-1-treated (STZ-Db + hIGF-1), untreated STZ-diabetic (STZ-Db), Zucker diabetic fatty (ZDF) rats and db/db mice were homogenized and underwent A qRT-PCR or B–D ELISA for IGF-1 detection. In E, DRG neurons derived from Ctrl and STZ-Db rats were cultured and media was collected after 2 days to measure secreted IGF-1 protein. DRG neurons derived from control (F, G, H) or diabetic (I) rats were cultured in the presence of 25 mM glucose with/without insulin or hIGF-1 treatment or in the presence of 5 mM glucose. RNA was extracted and utilized for real-time PCR assay. In G, 25 mM mannitol was used to control for osmotic pressure compared with 25 mM D-glucose. In H, sorbinil (10 and 100 µM), an aldose reductase inhibitor (ARI), was applied. Data are mean ± SEM of N = 3–6; *p < 0.05 or **p < 0.01 or ****p < 0.0001; analyzed by Student’s t test or one-way ANOVA with Dunnett’s or Tukey’s post hoc test

Fig. 3

IGF-1 neutralizing antibody or IGF-1 targeting siRNAs reduced IGF-1Rβ and Akt phosphorylation and diminish neurite outgrowth. DRG neurons from control rats were cultured, treated with different doses of IGF-1 neutralizing antibody and underwent A, B Western blotting for Akt phosphorylation and IGF-1Rβ or C, D neurite outgrowth measurement. In A, B, total protein bands were used for normalization. In C, D, normal goat IgG was used as a control antibody. In E–G, DRG neurons from control rats were cultured, treated with different doses of two IGF-1 targeting siRNAs and underwent E real-time PCR assay for IGF-1 or F, G neurite outgrowth measurement. In E, four transcript variants (a pair of primers designed for the detection of two variants 1 and 2 or 3 and 4) of IGF-1 that produce protein were analyzed. In F, G, exogenous hIGF-1 was added alone or along with IGF-1 knock-down as control groups. Data are mean ± SEM of N = 4; *p < 0.05 or **p < 0.01 or ****p < 0.0001; analyzed by Student’s t test or one-way ANOVA with Dunnett’s post hoc test

Fig. 4

IGF-1-overexpressing plasmid enhanced glycolysis, mitochondrial respiration, and neurite outgrowth. DRG neurons from control rats were cultured, transfected with different doses of hIGF-1 (transcript variant 4)-overexpressing plasmid and 200 ng control GFP plasmid (ctrl). In A, B, mitochondrial OCR was measured in live neurons after 6 h. In C–F, glycolysis parameters and total neurite outgrowth were calculated. Total protein in mg was used to normalize OCR and ECAR data. Data are mean ± SEM of N = 4–6; *p < 0.05 or **p < 0.01 or ***p < 0.001; analyzed by Student’s t test or one-way ANOVA with Dunnett’s post hoc test

Fig.5

NFAT1 and CEBPβ transcription factors activated IGF-1 gene expression and were less enriched at the Igf1 promoter in DRG tissue derived from diabetic rats. About A 1.2 kb of IGF-1 gene promoter region was chosen to design ChIP assay. Five regions were used for amplification and each pair of primers was designed to include one transcription factor-binding site. In B, DRG tissues derived from adult control and STZ-diabetic rats underwent ChIP assay using NFAT1 and CEBPβ antibodies for pull down followed by IGF-1 promoter region (five regions in total stats for three are shown) amplification using ChIP-qRT-PCR analysis. In C, diagram of the mutated transcription factor (NFAT1 and CEBPβ) binding sites on promoter region of Igf1 gene is given. A total of 4 binding sites (3a for NFAT1-binding site 3, 3b for CEBPβ-binding site 3, 5 for NFAT1- and CEBPβ-binding site 5) from (A) were mutated. In D, luciferase activity of the mutated and wild type IGF-1 promoter was measured in HEK293 cells. In E, DRG neurons from STZ-diabetic (Db) rats were transfected with NFAT1, CEBPβ or both, and different transcript variants (Tv1,2,3,4) of IGF-1 were measured using qRT-PCR. Data are mean ± SEM of N = 4–5 animals or N = 4–6 culture groups; *p < 0.05 or **p < 0.01 or ****p < 0.0001; analyzed by Student’s t test or one-way ANOVA with Tukey’s post hoc test

Fig. 6

CEBPβ overexpression elevated mitochondrial ETS protein levels and mitochondrial respiration. DRG neurons from A–D STZ-diabetic rats were cultured, transfected with CEBPβ-overexpressing plasmid or control GFP plasmid. In A, B, mitochondrial OCR was measured in live neurons after 36 h. In C, D, mitochondrial ETS proteins were measured using Western blotting. Total protein band was used to normalize Western blot data. Data are mean ± SEM of N = 4–6; *p < 0.05 or **p < 0.01 or ***p < 0.001; analyzed by Student’s t test

Fig. 7

IGF-1 knock-down suppressed CEBPβ upregulation of mitochondrial function and neurite outgrowth. DRG neurons from STZ-diabetic (Db) rats were cultured, transfected with pCEBPβ–LNP, pGFP–LNP or siIGF1–LNP, and underwent A, B mitochondrial respiration assay or C, D neurite outgrowth measurement. In A, B, total protein in mg was used to normalize OCR data. In E, ELISA assay was used to measure IGF-1 levels in the same condition as in C. Data are mean ± SEM of N = 3–5; *p < 0.05 or **p < 0.01 or ***p < 0.001; analyzed by one-way ANOVA with Tukey’s post hoc test