HSPA12A controls cerebral lactate homeostasis to maintain hippocampal neurogenesis and mood stabilization

Abstract

Mood instability, a subjective emotional state defined as rapid mood oscillations of up and down, is a symptom that occurs in several psychiatric disorders, particularly major depressive disorder and bipolar disorder. Heat shock protein A12A (HSPA12A) shows decreased expression in the brains of schizophrenia patients. However, the causal effects of HSPA12A in any psychiatric disorders are completely unknown. To investigate whether HSPA12A affects mood stability, Hspa12a-knockout mice (Hspa12a^-/-) and wild-type (WT) littermates were subjected to tests of open field, forced swimming, elevated plus maze, and sucrose preference. Cerebral lactate levels were measured in cerebrospinal fluid (CSF). Adult hippocampal neurogenesis (AHN) was assessed by BrdU labeling. We found that acute mood stress increased hippocampal HSPA12A expression and CSF lactate levels in mice. However, Hspa12a^-/- mice exhibited behaviors of mood instability (anhedonia, lower locomotor activity, antidepression, and anxiety), which were accompanied by impaired AHN, decreased CSF lactate levels, and downregulated hippocampal glycolytic enzyme expression. By contrast, HSPA12A overexpression increased lactate production and glycolytic enzyme expression of primary hippocampal neurons. Intriguingly, lactate administration alleviated the mood instability and AHN impairment in Hspa12a^-/- mice. Further analyses revealed that HSPA12A was necessary for sustaining cerebral lactate homeostasis, which could be mediated by inhibiting GSK3β in hippocampal neurons, to maintain AHN and mood stabilization. Taken together, HSPA12A is defined as a novel regulator of mood stability and exerts therapeutic potential for mood disorder. Our findings establish a framework for determining mood disorder and AHN relevance of cerebral lactate homeostasis. HSPA12A is a novel mood stabilizer through inhibiting GSK3β in hippocampal neurons, thereby sustaining glycolysis-generated lactate to maintain cerebral lactate homeostasis, which ultimately leading to maintenance of hippocampal neurogenesis and mood stabilization.

HSPA12A is a novel mood stabilizer through inhibiting GSK3β in hippocampal neurons, thereby sustaining glycolysis-generated lactate to maintain cerebral lactate homeostasis, which ultimately leading to maintenance of hippocampal neurogenesis and mood stabilization.

Fig. 1. HSPA12A was upregulated in hippocampus following acute psychological stress.

A Experimental protocol. B HSPA12A expression. After ASS, the indicated tissues were collected for immunoblotting against HSPA12A. The blots against GAPDH served as loading controls. n = 7/group. C Brief illustration of glycolysis pathway. D Glycolysis-related gene expression. After ASS, the indicated gene expression was analyzed in hippocampus by immunoblotting analysis. n = 5/group. E Lactate in cerebrospinal fluid (CSF). After ASS, CSF was extracted from the mice for lactate content measurement. n = 5/group. Data are mean ± SD, **P < 0.01 and *P < 0.05 by Student’s two-tailed unpaired t test or Mann–Whitney U test.

Fig. 2. Hspa12a⁻/⁻ mice displayed impairment of neurogenesis, neuronal spinogenesis in hippocampus and displayed mood disorder.

A Neuronal dendrite length and dendrite spine density. Neuronal dendrite length (Scale bar = 50 μm) in dentate gurus and spine density (Scale bar = 20 μm) of neuronal dendrite in dentate gurus was analyzed using Golgi staining. n = 6/group. B Adult hippocampal neurogenesis (AHN). AHN was examined in granular layer and interspace of dentate gyrus (DG) by BrdU incorporation. DAPI was used to counter stain nuclei. The images showed the representative staining in DG, and the boxed areas were magnified in the down panels. n = 5/group. Scale bar = 100 μm. C BDNF expression. The expression of BNDF was examined in hippocampus by immunoblotting. n = 8/group. D Sucrose preference test. The total liquid consumption and sucrose preference ratio were measured in 24 h duration in mice. E Open field test. The time spent, distance travelled, moving speed and entries in each area of an open field were recorded within 20 min duration. F Forced swimming test. Immobility episodes and time were recorded within 4 min duration of test. G. Tail suspension test. Immobility episodes and time were recorded within 4 min duration of test. H Elevated plus maze test. The time spent in open arm was recorded within 5 min of test. I Self-grooming test. The events of self-grooming were recorded within 10 min of test. Data are mean ± SD, ^**P < 0.01 and ^*P < 0.05 by Student’s two-tailed unpaired t test or Mann–Whitney U test test, n = 14 for WT group and n = 13 for Hspa12a^−/− group.

Fig. 3. HSPA12A was required for maintaining cerebral lactate homeostasis and promoting lactate generation by hippocampal neurons.

A Experimental protocol of mice. B Brief illustration of glycolysis pathway. C Lactate in cerebrospinal fluid (CSF). CSF was extracted from the mice and lactate content was measured. n = 5/group. D Hippocampal expression of glycolysis-related genes. Hippocampi were collected for measuring the indicated gene expression by immunoblotting analysis. n = 6/group. E Experimental protocol of primary hippocampal neurons. F Extracellular lactate of neuron cultures. After HSPA12A overexpression for 24 h, culture medium of primary hippocampal neurons was collected for measuring lactate contents. n = 6/group. G Expression of glycolysis-related genes in hippocampal neurons. After HSPA12A overexpression for 24 h, primary hippocampal neurons were collected for measuring the indicated gene expression by immunoblotting analysis. n = 6/group. Data are mean ± SD, ^**P < 0.01 and ^*P < 0.05 by Student’s two-tailed unpaired t test or Mann–Whitney U test.

Fig. 4. Lactate administration alleviated mood disorder and rescued the impairment of hippocampal neurogenesis in Hspa12a⁻/⁻ mice.

A Experimental protocol. After lactate treatment for 21 days, the following tests were performed. B Lactate levels in cerebrospinal fluid (CSF). n = 5 for WT group, n = 6 for Hspa12a^−/− group and n = 8 for lactate-treated group. C–F Behavioral tests. n = 18 for WT group, n = 16 for Hspa12a^−/− group, and n = 7 for lactate-treated Hspa12a^−/− group. G Adult hippocampal neurogenesis (AHN). AHN was examined in granular layer and interspace of dentate gyrus (DG) by BrdU incorporation. DAPI was used to counter stain nuclei. The images showed the representative staining in dentate gyrus, and the boxed areas were magnified in the down panels. n = 5, 8 and 6 for WT, Hspa12a^−/−, and lactate-treated Hspa12a^−/− group, respectively. Scale bar=100 μm. H BDNF expression. The expression of BNDF was examined in hippocampus by immunoblotting. n = 5/group. Data are mean ± SD; ^**P < 0.01 and ^*P < 0.05 by one-way ANOVA followed by post-hoc test (C, F, G) and Kruskal–Wallis test (B, D–E, H). ns no significance.

Fig. 5. HSPA12A regulated GSK3β pathway, and GSK-3β inhibitor lithium rescued mood disorder and hippocampal neurogenic impairment in Hspa12a⁻/⁻ mice.

A Immunoblotting in hippocampus. The indicated gene expression was examined in hippocampus by immunoblotting analysis. n = 5/group. B Experimental protocol. Following lithium treatment for 10 days, the following experiment were performed. C Adult hippocampal neurogenesis (AHN). AHN was examined in granular layer and interspace of dentate gyrus (DG) by BrdU incorporation. DAPI was used to counter stain nuclei. The images showed the representative staining in dentate gyrus, and the boxed areas were magnified in the down panels. n = 5, 10 and 5 for WT, Hspa12a^−/−, and lithium-treated Hspa12a^−/− group, respectively. Scale bar= 100 μm. D–F Behavioral tests. n = 13 for WT group, n = 14 for Hspa12a^−/− group, and n = 6 for lithium-treated group. ns, no significance. G Mechanistic scheme. HSPA12A was required for sustaining cerebral lactate homeostasis to maintain hippocampal neurogenesis and mood stabilization through inhibiting GSK3β in hippocampal neurons. Hippocampal HSPA12A is identified as new regulator of mood behaviors that lends support for its therapeutic potential in mood stabilization. Data are mean ± SD, ^**P < 0.01 and ^*P < 0.05 by Mann–Whitney U test (A) and Kruskal–Wallis test (D–F).