Distribution of the Vitamin D receptor and 1α-hydroxylase in human brain

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Abstract

Despite a growing body of evidence that Vitamin D is involved in mammalian brain functioning, there has been a lack of direct evidence about its role in the human brain. This paper reports, for the first time, the distribution of the 1,25-dihydroxyvitamin D3 receptor (VDR), and 1α-hydroxylase (1α-OHase), the enzyme responsible for the formation of the active vitamin in the human brain. The receptor and the enzyme were found in both neurons and glial cells in a regional and layer-specific pattern. The VDR was restricted to the nucleus whilst 1α-OHase was distributed throughout the cytoplasm. The distribution of the VDR in human brain was strikingly similar to that reported in rodents. Many regions contained equivalent amounts of both the VDR and 1α-OHase, however the macrocellular cells within the nucleus basalis of Meynert (NBM) and the Purkinje cells in the cerebellum expressed 1α-OHase in the absence of VDR. The strongest immunohistochemical staining for both the receptor and enzyme was in the hypothalamus and in the large (presumably dopaminergic) neurons within the substantia nigra. The observed distribution of the VDR is consistent with the proposal that Vitamin D operates in a similar fashion to the known neurosteroids. The widespread distribution of 1α-OHase and the VDR suggests that Vitamin D may have autocrine/paracrine properties in the human brain.

Introduction

The active form of Vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is a pluripotent seco-steroid with physiological functions that extend beyond its classical role in calcium homeostasis and bone metabolism (Jones et al., 1998).With respect to this, several recent studies have postulated functions for 1,25(OH)2D3 in both in the developing (Eyles et al., 2003) and adult brain (McGrath et al., 2001, Garcion et al., 2002). In particular our group has postulated that low levels of Vitamin D during early life may be relevant to several brain diseases such as schizophrenia and multiple sclerosis (McGrath, 2001). Although expression of the receptor for 1,25(OH)2D3 (Vitamin D receptor, VDR) has been described in the brains of several species, evidence for the presence of the receptor in human brain has been scant.

Previous studies have confirmed that the VDR is expressed in both developing (Veenstra et al., 1998a, Burkert et al., 2003) and adult rat brain (Stumpf and O’Brien, 1987, Clemens et al., 1988, Prufer et al., 1999) as well as in adult hamster brain (Musiol et al., 1992). The only previous investigation reporting VDR expression in human brain was by Sutherland et al. (1992) via in situ hybridization. Using radiolabeled cDNA probes, these researchers showed that VDR mRNA is expressed in the brains of patients with Alzheimer's or Huntington's disease. The VDR has also been shown to be expressed in a human neuroblastoma cell line (Moore et al., 1996). Evidence for VDR protein in human brain is still lacking.

Given the lack of data concerning the distribution of VDR in non-diseased human brain, we performed an immunohistochemical study in order to map the distribution of the VDR in multiple brain regions. The presence of a functional receptor however would mean little if the ligand could not actually gain access to the brain. The early autoradiographic studies would appear to indicate that this ligand partitions widely throughout the brain but this evidence is equivocal (Carswell, 1997). An alternative mechanism is that 1,25(OH)2D3 may be synthesized locally within the brain via expression of the enzyme 1α-hydroxylase (1α-OHase) which catalyzes conversion of 25OHD3 to 1,25(OH)2D3. The enzyme is classically expressed in the kidney (Zehnder and Hewison, 1999) but immunohistochemical techniques have been used to describe the distribution of this enzyme in non-renal human tissues as well (Zehnder et al., 2001). In the latter study only two regions of the brain were examined but nevertheless strong expression of 1α-OHase was observed in the cerebellar Purkinje cells as well as within neuronal cells of the cerebral cortex, thus indicating that local production of the active vitamin within human brain was possible. 1α-OHase has also been detected in foetal human brain (Fu et al., 1997). In addition, 1α-OHase has been shown previously to be both present within glial cells in culture and capable of the final hydroxylation of 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 (Neveu et al., 1994b). We have therefore elected to examine multiple regions of the human brain for this enzyme and compare these with the distribution for the VDR.

The aims of this study are threefold; firstly to confirm the presence of the VDR and 1α-OHase in human brain; secondly to map their distribution in selected brain regions and thirdly to determine if cells expressing either the receptor and/or the enzyme are neuronal or glial in origin.

Section snippets

Immunohistochemistry

Human brain tissue was obtained from the New South Wales tissue resource centre, Sydney, Australia. Formalin fixed brain tissue was selected from five males (34–58 years) all from individuals free of any psychopathology or history of alcohol or drug abuse. Tissue had been stored between 15 and 36 months in 10% formalin (sodium phosphate buffered, pH 7.0). Post-mortem intervals ranged from 12 to 25 h. Tissue segments were transferred to 0.32 M sucrose in 0.1 M phosphate buffer for 7 days before

General observations

The pattern and intensity of immunohistochemical responses to both antigens were similar in all five post-mortem samples. VDR immunoreactivity was present in both neurons and glia. Its sub-cellular distribution was strictly nuclear (Fig. 1A and B). Staining intensity for VDR varied widely throughout the brain. In general white matter regions were poorly immunoreactive. 1α-OHase was abundant in all brain regions examined. Like VDR its immunoreactivity was present in both neurons and glia.

Discussion

The Vitamin D receptor and the enzyme (1α-OHase) required for the production of the active form of Vitamin D, 1,25(OH)2D3, are found in the adult human brain. Both the receptor and the enzyme were identified in neuronal and glial cells. Most, but not all regions that were positive for 1α-OHase, were also positive for the VDR. A discrete distribution within layers and sub-regions frequently existed for both the receptor and the enzyme. Previous investigators have described a detailed pattern of

Acknowledgements

This work was supported in part by the Sylvia and Charles Viertel Charitable Foundation, the Stanley Foundation and the National Health and Medical Research Council of Australia. Dr. Hewison is supported by the Biotechnology and Biological Sciences Research Council (BBSRC), UK.

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