Metabolic Profiling of Suprachiasmatic Nucleus Reveals Multifaceted Effects in an Alzheimer's Disease Mouse Model

Abstract

Background: Circadian rhythm disturbance is commonly observed in Alzheimer's disease (AD). In mammals, these rhythms are orchestrated by the superchiasmatic nucleus (SCN). Our previous study in the Tg2576 AD mouse model suggests that inflammatory responses, most likely manifested by low GABA production, may be one of the underlying perpetrators for the changes in circadian rhythmicity and sleep disturbance in AD. However, the mechanistic connections between SCN dysfunction, GABA modulation, and inflammation in AD is not fully understood.

Objective: To reveal influences of amyloid pathology in Tg2576 mouse brain on metabolism in SCN and to identify key metabolic sensors that couple SCN dysfunction with GABA modulation and inflammation.

Methods: High resolution magic angle spinning (HR-MAS) NMR in conjunction with multivariate analysis was applied for metabolic profiling in SCN of control and Tg2576 female mice. Immunohistochemical analysis was used to detect neurons, astrocytes, expression of GABA transporter 1 (GAT1) and Bmal1.

Results: Metabolic profiling revealed significant metabolic deficits in SCN of Tg2576 mice. Reductions in glucose, glutamate, GABA, and glutamine provide hints toward an impaired GABAergic glucose oxidation and neurotransmitter cycling in SCN of AD mice. In addition, decreased redox co-factor NADPH and glutathione support a redox disbalance. Immunohistochemical examinations showed low expression of the core clock protein, Bmal1, especially in activated astrocytes. Moreover, decreased expression of GAT1 in astrocytes indicates low GABA recycling in this cell type.

Conclusion: Our results suggest that redox disbalance and compromised GABA signaling are important denominators and connectors between neuroinflammation and clock dysfunction in AD.

Keywords: 1H high-resolution magic angle spinning NMR; Alzheimer’s disease; GABA dysfunction; Tg2576 mouse model; metabolic deficit; suprachiasmatic nucleus.

Fig. 1

Representative high-resolution magic angle spin (HR-MAS) NMR spectra showing metabolic profile of SCN from (A) wild-type, and (B) Tg2576 mice. C, D) Multivariate analysis of the HR-MAS NMR spectra (n = 6 per group) using partial least square-discriminant analysis (PLS-DA) modelling (R2 = 0.907, Q2 = 0.989). C) Scores plots (PLS-DA1 versus PLS-DA2). The score plot explains 55.4%of total variance of WT SCN clustering in the negative PLSDA1 scores, and Tg2576 SCN in the positive PLSDA1 scores. D) Loading plots of PLS-DA1 for all buckets containing assigned peaks. Ala, alanine; Asp, aspartate; Chol, cholesterol; FA, fatty acids; GSH, glutathione; GABA, g-aminobutyric acid; Glu, glutamate; Gln, glutamine; GPC, glycerophosphocholine; Gly, glycine; Lac, lactate; m-Ins, myo-inositol; PC, phosphocholine; Tau, taurine; tCr, total creatine.

Fig. 2

Metabolic profile of intact SCN from wild-type (WT) and Tg2576 (TG) mice measured by HR-MAS NMR. Shown are concentrations of metabolites relative to total creatine (tCr): A) metabolites including amino acids and neurotransmitters; B) Carbohydrate and energy metabolites; C) Pyruvate and TCA cycle intermediates. For statistical analysis, one-way analysis of variance (ANOVA) with a Tukey post-hoc correction for multiple comparisons were performed using OriginPro v. 8 (Northampton, MA, USA). Values are average±SE of mean (n = 6). ^#p < 0.001, ^**p < 0.01, and ^*p < 0.05. Glu, glutamate; Gln, glutamine; Gly, glycine; Ala, alanine; Asp, aspartate; GSH, glutathione; GABA, γ-aminobutyric acid; NAA, N-acetyl aspartate; Tau, taurine; m-Ins, myo-inositol; Glc, glucose; Lac, lactate; Ace, acetate; ATP, adenosine triphosphate; ADP, adenosine diphosphate; NADH/NAD+, reduced/oxidized nicotinamide adenine dinucleotide; NADPH/NADP+, reduced/oxidized nicotinamide adenine dinucleotide phosphate; Pyr, pyruvate; Cit, citrate; Suc, succinate; Fum, fumarate; Mal, malate.

Fig. 3

Immunohistochemical analyses of Bmal1 and GFAP staining in SCN of Tg2576 (TG) and wild-type (WT) mice. A) Representative confocal images of Bmal1 and GFAP stained sections through SCN of 18-month-old WT and TG mice. Scale bar, 250μm (first and third column); 60μm (second and fourth column) and 20μm (in magnifications). B) Quantitative analysis of Bmal1 and GFAP staining in SCN of 18 months old WT and Tg2576 (TG) mice (n = 6 per group). ^**p < 0.01, ^*p < 0.05. GFAP, glial fibrillary acidic protein.

Fig. 4

Immunohistochemical analyses of GABA transporter 1 (GAT1) in SCN of Tg2576 (TG) and wild-type (WT) mice. A) Representative confocal images of GAT1 and overlayed images of GAT1 with GFAP stained sections through SCN of TG and WT mice. Scale bar, 250μm (first and third column); 60μm (second and fourth column) and 20μm (in magnifications). B) Quantitative analysis of GAT1 staining in SCN of 18 months old WT and Tg2576 (TG) mice (n = 6 per group). ^*p < 0.05.

Fig. 5

Proposed model of interconnection between redox dysregulation, compromised GABA signaling, neuroinflammation and SCN dysfunction in AD as evidenced by observed changes in metabolic profile and immunohistology in the present study. A) Observed increases and decreases in metabolites in SCN of AD mice is shown by arrows (i.e., ↑ and ↓, respectively). B) Summarized view of the interconnection between reduced Bmal1, redox disbalance, dysregulation of GABA signaling and inflammation that may be at the root of circadian rhythm disruption leading to sleep disturbance in AD. αKG, α-ketoglutarate; Asp, aspartate; GABA_A GABA_B, GABA receptors; GAT1 and GAT3/4, specific GABA transporter; GAD, glutamic acid decarboxylase; Glc, glucose; Gln, glutamine; Glu, glutamate; GS, glutamine synthetase; GSH, glutathione; Lac, lactate; NAA, N-acetyl aspartate; NMDAR, N-methyl-D-aspartate receptor (Glu receptor): OA, oxaloacetic acid; PPP, pentose phosphate pathway; SSA, succinate semialdehyde; TCA cycle, tricarboxylic acid cycle.