Emotional regulatory function of receptor interacting protein 140 revealed in the ventromedial hypothalamus

Abstract

Receptor-interacting protein (RIP140) is a transcription co-regulator highly expressed in macrophages to regulate inflammatory and metabolic processes. However, its implication in neurological, cognitive and emotional conditions, and the cellular systems relevant to its biological activity within the central nervous system are currently less clear. A transgenic mouse line with macrophage-specific knockdown of RIP140 was generated (MΦRIPKD mice) and brain-region specific RIP140 knockdown efficiency evaluated. Mice were subjected to a battery of tests, designed to evaluate multiple behavioral domains at naïve or following site-specific RIP140 re-expression. Gene expression analysis assessed TNF-α, IL-1β, TGF-1β, IL1-RA and neuropeptide Y (NPY) expression, and in vitro studies examined the effects of macrophage's RIP140 on astrocytes' NPY production. We found that RIP140 expression was dramatically reduced in macrophages within the ventromedial hypothalamus (VMH) and the cingulate cortex of MΦRIPKD mice. These animals exhibited increased anxiety- and depressive-like behaviors. VMH-targeted RIP140 re-expression in MΦRIPKD mice reversed its depressive- but not its anxiety-like phenotype. Analysis of specific neurochemical changes revealed reduced astrocytic-NPY expression within the hypothalamus of MΦRIPKD mice, and in vitro analysis confirmed that conditioned medium of RIP140-silnenced macrophage culture could no longer stimulate NPY production from astrocytes. The current study revealed an emotional regulatory function of macrophage-derived RIP140 in the VMH, and secondary dysregulation of NPY within hypothalamic astrocyte population, which might be associated with the observed behavioral phenotype of MΦRIPKD mice. This study highlights RIP140 as a novel target for the development of potential therapeutic and intervention strategies for emotional regulation disorders.

Keywords: Anxiety; Depression; Immune system; NPY; RIP140; Ventromedial hypothalamus.

Fig. 1

The Behavioral phenotype of MΦRIPKD mice. Map describing the generation (A) and representative genotyping data (B) of MΦRIPKD transgenic mice. Mean and SE of percentage of entries into (C) and time spent in (D) the open arms of the EPM, time spent out of ‘shelter’ in the emergence test (E), time spent in lit area (F) and number of entries into lit area (G) of the LDB, immobility time (H) and latency to immobility (I) in the FST, immobility time in the TST (J), distance traveled (K) and ambulatory count (L) in the open field, and Latency to fall in the Rotarod (M).

Fig. 2

RIP140 expression within brain samples of MΦRIPKD mice. Representative immunofluorescence data from hypothalamus (A, 20X and 60X magnification) and PFC (B, 4X, 10X and 60X magnification) of MΦRIPKD (empty arrow) and WT (full arrow) mice. Mean and SE of RIP140 fluorescence expression within macrophages of the VMH and Cg1 (C) and of RIP140 mRNA expression within the hypothalamus, PFC, hippocampus and amygdala of MΦRIPKD and WT mice (D) including representative mRNA data (E). Coronal diagrams adapted from the Franklin and Paxinos mouse brain atlas.

Fig. 3

The Behavioral phenotype of MΦRIPKD mice following intra-VMH administration of RIP140-OE-lentivirus. Schematic representation of the lentiviral construct, designed to over-express mouse RIP140 (RIP140-OE) together with a GFP protein (A), representative RT-PCR image of RIP140-OE in HEK293T cells infected with over-expressing lentivirus or with GFP-expressing negative control (GFP-control) (B), representative immunofluorescence of viruses expression in vivo (motor cortex, 20X and 60X magnification) (C), representative immunofluorescence image of Intra-VMH GFP visualization (10X magnification) and microinjection localization for mice included in the RIP140 re-expression experiment (D–E), mean and SE of immobility time in the FST (F) and the TST (G), percentage of entries into (H) and time spent in (I) the open arms of the EPM and time spent out of ‘shelter’ in the emergence test (J). CMV=cytomegalovirus. Coronal diagrams adapted from the Franklin and Paxinos mouse brain atlas

Fig. 4

Hypothalamic gene expression in MΦRIPKD mice. Mean and SE of TNF-α, IL-1β, TGF-1β, IL1-RA and NPY in hypothalamic samples from MΦRIPKD and WT mice (A), a correlation between hypothalamic RIP140 and NPY mRNA expression (B), NPY immunofluorescence in the hypothalamus of MΦRIPKD and WT mice (C), RIP140 silencing in RAW 264.7 cells (D) and NPY expression in astrocytes treated with RIP140-silenced RAW 264.7-conditioned medium (E–F).