Null mutation in P4h-tm leads to decreased fear and anxiety and increased social behavior in mice

doi:10.1016/j.neuropharm.2019.04.023

Neuropharmacology

Volume 153, 15 July 2019, Pages 63-72

https://doi.org/10.1016/j.neuropharm.2019.04.023 Get rights and content

Highlights

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P4H-TM is a transmembrane HIF prolyl 4-hydroxylase with an unknown function.
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P4H-TM expression is high in brain regions related to emotional and social behavior.
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P4h-tm deficient mice are social and show little anxiety or behavioral despair.
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P4H-TM is a promising new target for anxiolytic and antidepressant drugs.

Abstract

HIF prolyl 4-hydroxylases (HIF-P4Hs, also known as PHDs and EGLNs) are crucial enzymes that modulate the hypoxia inducible factor (HIF) response and help to maintain cellular oxygen homeostasis. This function is especially well-known for cytoplasmic or nuclear enzymes HIF-P4H-1–3 (PHDs 1–3, EGLNs 2, 1 and 3, respectively), but the physiological role is still obscure for a fourth suggested HIF-P4H, P4H-TM that is a transmembrane protein and resides in the endoplasmic reticulum. Recently however, both experimental and clinical evidence of the P4H-TM involvement in CNS physiology has emerged. In this study, we first investigated the expression pattern of P4H-TM in the mouse brain and found a remarkably selective abundance in brains areas that are involved in social behaviors and anxiety including amygdala, lateral septum and bed nucleus of stria terminalis. Next, we performed behavioral assays in P4h-tm^−/− mice to investigate a possible phenotype associated to these brain areas. In locomotor activity tests, we found that P4h-tm^−/− mice were significantly more active than their wild-type (WT) littermate mice, and habituation to test environment did not abolish this effect. Instead, spatial learning and memory seemed normal in P4h-tm^−/− mice as assessed by Morris swim task. In several tests assessing anxiety and fear responses, P4h-tm^−/− mice showed distinct courageousness, and they presented increased interaction towards fellow mice in social behavior tests. Most strikingly, P4h-tm^−/− mice practically lacked behavioral despair response, a surrogate marker of depression, in forced swim and tail suspension tests. Instead, mutant mice of all other Hif-p4h isoforms lacked such a behavioral phenotype. In summary, this study presents a remarkable anatomy-physiology association between the brain expression of P4H-TM and the behavioral phenotype in P4h-tm^−/− mice. Future studies will reveal whether P4H-TM may serve as a novel target for anti-depressant and anti-anxiety pharmacotherapy.

Introduction

Prolyl-hydroxylases are enzymes that modulate collagen synthesis and oxygen balance (Myllyharju and Koivunen, 2013). According to these roles, they are classified into two main categories: collagen prolyl 4-hydroxylases (collagen-P4Hs) and hypoxia-inducible factor (HIF) prolyl 4-hydroxylases (HIF-P4Hs). The common denominator for these two subtypes is that they both share structural similarity and utilize iron, ascorbate and 2-oxoglutarate as cofactors. Collagen-P4Hs reside inside the endoplasmic reticulum (ER) and catalyze the formation of hydroxyproline in collagen. HIF-P4Hs play a vital role in our bodies modulating the HIF response in an oxygen-dependent manner. In normoxia, HIFα is continuously and quickly degraded by HIF-P4Hs, but in hypoxia the catalytic activity of HIF-P4Hs is decreased leading to its stabilization and formation of a functional HIFαβ dimer. Once HIF is stabilized, it induces a robust modulation in a large number of genes, such as erythropoietin (EPO), vascular endothelial growth factor (VEGF), glycolytic enzymes and glucose transporters (GLUTs), leading to increased oxygen transport and decreased oxygen demand by cells. Four HIF-P4Hs have been found so far: HIF-P4H-1, HIF-P4H-2, HIF-P4H-3 and a transmembrane form known as P4H-TM [enzyme-family reviewed in (Myllyharju and Koivunen, 2013)]. P4H-TM is an oddball enzyme in the classification for several reasons. (1) HIF-P4Hs 1–3 are localized in the cytoplasm or nucleus, whereas P4H-TM resides in the ER and its catalytic side faces the lumen. (2) The amino acid sequence of P4H-TM resembles more that of collagen-P4Hs than HIF-P4Hs, but P4H-TM is unable to hydroxylate procollagen-polypeptides. (3) While P4H-TM can hydroxylate the prolines in HIFα targeted by HIF-P4Hs in vitro, it also hydroxylated to a small extent HIFα in which these prolines were mutated to alanines suggesting it may have additional substrates to HIFα (Koivunen et al., 2007). Finally, the physiological role of collagen-P4Hs and HIF-P4Hs 1–3 is established (Myllyharju and Koivunen, 2013), but the role and function of P4H-TM remain still largely unknown (Leinonen et al., 2016), although its inhibition associates with renal EPO production and induction of erythropoiesis similarly to inhibition of HIF-P4Hs 1–3 (Laitala et al., 2012).

Genetic studies on P4h-tm gene homolog in morpholino knockdown zebrafish provided the first evidence of the significance of P4H-TM in living vertebrates (Hyvarinen et al., 2010), later followed by mouse knockout studies (Leinonen et al., 2016). P4H-tm gene is not vital since P4h-tm^−/− mice are fertile and have a normal lifespan (Leinonen et al., 2016). However, both the morpholino knockdown zebrafish and P4h-tm^−/− mice display abnormalities that coexist between these species: structural alterations of the kidneys and proteinuria, and abnormalities in the eye structure and function (Hyvarinen et al., 2010; Leinonen et al., 2016). In both species, the expression level of P4H-TM mRNA is high in the eyes and brain, and moderate in kidneys. Specifically, the expression of P4H-TM is highest in the retinal pigment epithelium, and amygdala, hypothalamus and CA3 area of hippocampus in the mouse brain (Leinonen et al., 2016). Lack of P4H-tm stabilizes HIF-1α in mouse cortical neurons and leads to induction of HIF target genes in several tissues. In the mouse retina, it leads to an unusual phenotype where cone-mediated visual function shows an age-independent impairment that is associated with decelerated signal transmission in the cone-pathway with no clear-cut cone photoreceptor degeneration. Therefore, the previous study proposed that a universal defect might exist in neurotransmission and CNS physiology in P4h-tm^−/− mice.

In the only published clinical report on P4H-TM mutations, six patients presenting with a novel type of intellectual disability syndrome were clinically and genetically assessed (Kaasinen et al., 2014). In addition to the intellectual disability, the clinical features of these patients included hypotonia, strabismus, difficulty for visual focusing, planovalgus (flat feet), mild contractures in elbow joins, interphalangeal joint hypermobility and coarse facial features that developed during childhood. Therefore, the syndrome was coined HIDEA (Hypotonia-Intellectual Disability-Eye Abnormalities). Genetic mapping of HIDEA patients identified a novel ID predisposition locus with three candidate genes including P4H-TM. More recently, a whole exome sequence analysis found five HIDEA patients with gene variants only in the P4H-TM gene demonstrating that it indeed is the causative gene for HIDEA (Rahikkala et al., 2019).

We have earlier shown that P4h-tm^−/− mice recapitulate some aspects of the visual disturbances found in HIDEA patients (Leinonen et al., 2016). In the same study, we found a high expression of P4H-TM in limbic areas of the brain: amygdala, hypothalamus and CA3 area of hippocampus suggesting its direct involvement in fundamental behaviors such as fear, anxiety, motivation, learning and memory. In this study, we repeated the expression analysis of P4H-TM in the brain and performed a comprehensive behavioral characterization of P4H-tm^−/− mice. Our data suggests that P4H-TM is a potential novel target for anti-depressant and anti-anxiety pharmacotherapy.

Section snippets

Animals

We used P4h-tm^−/− mice generated by inserting a lacZneo cassette into exon 3 of the P4h-tm gene via homologous recombination leading to a truncated transcript (Leinonen et al., 2016). The mice had been back-crossed for 10 generations into C57BL/6JOlaHsd background. Most experiments were performed in 5-month-old male mice (cohort 1: WT, n = 10; −/−, n = 10 and cohort 2: WT, n = 6; −/−, n = 12). A few supplementary experiments (noted in figure legends and Table 1) were performed in other cohorts

High expression of P4H-TM is observed in brain areas involved in anxiety, social behavior and exploration

The use of LacZ motive for β-galactosidase in the inactivating gene construct for P4h-tm allowed us to observe the P4H-TM protein expression pattern using XGal staining. Fig. 1 summarizes the brain regions with the observed highest expression of P4H-TM. Notably, most nuclei of amygdala (Fig. 1A,E), lateral septum (Fig. 1A,D), and bed nucleus of stria terminalis (Fig. 1D) and cingulate cortex (Fig. 1 B,D), which are all linked to anxiety and social behavior (Lebow and Chen, 2016), showed a high

Discussion

We report here the first behavioral characterization of P4h-tm^−/− mice. We found that loss of P4H-TM function in mice causes a striking phenotype. P4h-tm^−/− mice appeared daring and distinctly explorative in all paradigms assessing fear, anxiety and social behavior. For instance, in a test where we triggered panic reaction by supplying 10% CO₂ into breathing air, WT mice showed substantially increased freezing behavior, an innate fear reaction in prey animals, compared to baseline situation

Disclosures and funding

J.M. owns equity in FibroGen Inc., which develops HIF-P4H inhibitors as potential therapeutics. This company supports research in the J.M. group. This work was in part funded by Biocenter Finland. H.L. was supported by Doctoral Program of Molecular Medicine at the University of Eastern Finland, and by Predoctoral Research Grants from Eye and Tissue Bank Foundation (Finland), Finnish Cultural Foundation and Kuopio University Foundation, and by Postdoctoral Research Grants from Fight for Sight

Acknowledgments

We thank the laboratory animal center staff in the University of Eastern Finland and University of Oulu for the excellent care of animals.

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Brown adipose tissue (BAT) plays a crucial role in regulating non-shivering thermogenesis under cold exposure. Proline hydroxylases (PHDs) were found to be involved in adipocyte differentiation and lipid deposition. However, the effects of PHDs on regulatory mechanisms of BAT thermogenesis are not fully understood.
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This gene is highly expressed in brain tissues (GTEx Consortium, 2020) and involved in many brain functions, including learning and memory (Mizuno et al., 2007), neuronal differentiation and migration (Kokubo et al., 2009), and synapse formation (Saneyoshi et al., 2008). P4HTM is highly expressed in brain regions related to emotional and social behavior, and P4htm deficient mice shows little anxiety or behavioral despair (Leinonen et al., 2019). Clinically, the diagnosis of MDD is primarily based on structured interview, whereas some of the symptoms in the diagnostic criteria are not specific to MDD, which results in the clinical heterogeneity of MDD and further hinders the genetic and pathological research (McIntosh et al., 2019).
Depression is a common and complex psychiatric illness with considerable heritability. Genome-wide association studies (GWAS) have been conducted among different definitions of depression based on different diagnostic criteria. However, the heritability explained by different depression GWAS and the identified loci varied widely. To understand the genetic architectures of different definitions of depression, we conducted a series of genetic analyses including linkage disequilibrium score regression (LDSC), Mendelian randomization, and polygenic overlap quantification and identification. Different definitions of depression and other common psychiatric traits were included in this analysis. We found that although genetic correlations between different definitions of depression were relatively high, they showed substantially different genetic correlation and causality with other psychiatric traits. Using bivariate causal mixture mode (MiXeR) and conjunctional false discovery rate (conjFDR) approach, we observed both shared and unique risk loci across different definitions of depression. Further functional mapping with expression quantitative trait loci (eQTL) information from multiple brain tissues and single cell types indicated distinct genes underlying different definitions of depression, and pathways associated with synapses were significantly enriched in the illness. Our study showed that the genetic architectures of different definitions of depression were distinct and genetic studies of depression should be conducted more cautious.
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Taken together, despite higher HIF1α immunoreactivity in the injured hemisphere of P4htm−/− mice relative to WT, no difference was observed between the genotypes in Bnip3 mRNA expression level, and the effect on the mRNA levels for EPO and VEGF was in fact opposite to HIF1α level (Fig. 4, A–F). Therefore, in line with previous studies (27–32), it is highly unlikely that the mechanistic effects of P4H-TM are solely mediated by effects on HIF. Notably however, the observed reduced mRNA expression of the neuroprotective growth factors EPO and VEGF-A in P4htm−/− mice under ischemic stroke may add to the increased MCAO-induced neuroinflammation in these mice relative to WT.
Hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) regulate the hypoxic induction of >300 genes required for survival and adaptation under oxygen deprivation. Inhibition of HIF-P4H-2 has been shown to be protective in focal cerebral ischemia rodent models, while that of HIF-P4H-1 has no effects and inactivation of HIF-P4H-3 has adverse effects. A transmembrane prolyl 4-hydroxylase (P4H-TM) is highly expressed in the brain and contributes to the regulation of HIF, but the outcome of its inhibition on stroke is yet unknown. To study this, we subjected WT and P4htm^−/− mice to permanent middle cerebral artery occlusion (pMCAO). Lack of P4H-TM had no effect on lesion size following pMCAO, but increased inflammatory microgliosis and neutrophil infiltration was observed in the P4htm^−/− cortex. Furthermore, both the permeability of blood brain barrier and ultrastructure of cerebral tight junctions were compromised in P4htm^−/− mice. At the molecular level, P4H-TM deficiency led to increased expression of proinflammatory genes and robust activation of protein kinases in the cortex, while expression of tight junction proteins and the neuroprotective growth factors erythropoietin and vascular endothelial growth factor was reduced. Our data provide the first evidence that P4H-TM inactivation has no protective effect on infarct size and increases inflammatory microgliosis and neutrophil infiltration in the cortex at early stage after pMCAO. When considering HIF-P4H inhibitors as potential therapeutics in stroke, the current data support that isoenzyme-selective inhibitors that do not target P4H-TM or HIF-P4H-3 would be preferred.
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Null mutation in P4h-tm leads to decreased fear and anxiety and increased social behavior in mice

Highlights

Abstract

Introduction

Section snippets

Animals

High expression of P4H-TM is observed in brain areas involved in anxiety, social behavior and exploration

Discussion

Disclosures and funding

Acknowledgments

Cell

Neuron

Pharmacol. Biochem. Behav.

Neurosci. Biobehav. Rev.

Trends Pharmacol. Sci.

Prog. Neuro-Psychopharmacol. Biol. Psychiatry

Curr. Biol.

J. Biol. Chem.

Eur. J. Med. Genet.

Trends Mol. Med.

J. Biol. Chem.

Blood

Behav. Brain Res.

Lancet Psychiatry

Eur. J. Pharmacol.

J. Anxiety Disord.

Neuron

Behav. Brain Res.