Celastrol alleviates comorbid obesity and depression by directly binding amygdala HnRNPA1 in a mouse model

Background and Purpose: Obesity and depression are highly comorbid and
far from effective treating. Celastrol was reported useful for obesity,
but its role in the obesity-depression comorbidity remains unknown. This
study aims to investigate the efficacy and associated mechanism of
celastrol in this comorbidity.

family, especially HnRNPA1, caught our attention (Figure 2A). As reported, HnRNPA1 mediates the proteasomedependent degradation of IKBα, the maximal activation of NFκB, and transcription of TNF-α. 5 Recently, GWAS found associations between HnRNPA1 and BMI-adjusted waist circumference and waist-hip ratio, indicating role of HnRNPA1 in energy metabolism. Third, the direct binding relationship between celastrol and HnRNPA1 was certified by pull-down assay, cellular thermal shift assay, and competitive binding test ( Figure 2B-D), while binding affinity was measured by surface plasmon resonance test ( Figure 2E).
Then, we validated celastrol-mediated modulation of HnRNPA1-IKBα-NFκB-TNF-α pathway in BV2 and BLA ( Figure 2F, G, I, and K). By RT-PCR and Western blot, celastrol-mediated downregulation of HnRNPA1 in BV2 and amygdala were proven on the protein, but not mRNA level ( Figure 2H and J). As shown by immunofluorescence staining and Western blot, the addition of proteasome inhibitor-MG132 was sufficient to offset celastrolmediated degradation of HnRNPA1, indicating the dependence of proteasome ( Figure 2H).
Furthermore, experiments were designed to validate the role HnRNPA1 assumed in the curation. First, the protein expression level of HnRNPA1 was downregulated by adeno-associated virus (AAV) encoding shRNA specific for HnRNPA1 in BLA of COM mice (A1-sh-AAV) ( Figure 3A). As shown, effective inhibition of microglia and TNF-α ( Figure 3B), alleviation of obesity and depressive behaviors ( Figure 3C), and reconstruction of neuropeptide Y (NPY) and TPH2 projections (Figure 3D) were achieved in A1-sh-AAV mice as compared to Com-non ones. These results indicate that downregulation of HnRNPA1 is sufficient to alleviate obesity-depression comorbidity.
Second, HnRNPA1 was upregulated by HnRNPA1encoding AAV in BLA ( Figure 3E). No significant difference of HnRNPA1 was detected between the Com-non and Cel-A1 AAV groups, indicating the overexpression of  Schematic diagram of forward experiments to validate curative effects by the downregulation of HnRNPA1. AAV was injected into BLA. In detail, AAV encoding nonsense shRNA-GFP was injected into chow (Chow-non) and comorbidity (Com-non) mice; AAV encoding hnRNPA1 induced by AAV just offset the downregulation by celastrol. Activation of the microglia was found in Cel-A1 AAV mice as compared with Cel-non ones and in Com-A1 AAV mice as compared with Com-non ( Figure 3F). Increase of weight, intolerance to glucose, and increase of immobility time in tail suspension test were observed in Cel-A1 AAV mice as compared with Cel-non ones (Figure 3G). In accordance with the morphological and behavioral changes, increases of TNF-α expression and NPY positive neurons, as well as decrease in TPH2 positive neurons were found in Com-A1-AAV mice as compared with Chow-non ones, and in Cel-A1-AAV mice as compared with Cel-non ones ( Figure 3H). These results indicate that HnRNPA1 is important in celastrol-mediated curation of obesity-depression comorbidity.
Metabolic cages were used to quantify energy intake and expenditure. In consistent with previous studies, celastrol may modulate energy intake rather than energy expenditure ( Figures 1B and 3G). Besides, indicated by comparison between A1-sh-AAV and Com-non, the downregulation of HnRNPA1 seems more likely to inhibit energy intake (Figure 3C). However, the upregulation of HnRNPA1 seems more likely to inhibit energy expenditure, indicated by comparisons between Com-A1-AAV and Com-non, Cel-A1-AAV and Cel-non ( Figure 3G).
In general, this study demonstrates that by the direct binding with HnRNPA1, celastrol accelerates the degradation of HnRNPA1, which may explain its curation of comorbid obesity and depression in COM mice. These findings indicate that celastrol may carry therapeutic potential for this complicated comorbidity in clinical patients who are in despair. Besides, HnRNPA1 could be a potential and effective target to combat this comorbidity.