It has long been known that retinoids (vitamin A derivatives) are
required for normal development and growth; in excess however metabolites
such as all-trans retinoic acid are potent teratogens. Retinoids exert
their effects through transcription factors known as the retinoic acid
receptors (RARam -ß and -y) and retinoid X receptors (RXRa, -ß and -y).
Intracellularly, two types of binding proteins also appear to be involved
in retinoid homeostasis, cellular retinol binding protein I and II (CRBP
I and II) and cellular retinoic acid binding protein I and II (CRABP I
and II). RARs and RXRs belong to the nuclear receptor superfamily which
also includes receptors for the steroid and thyroid hormones, vitamin D3
and several other small lipophilic molecules. Moreover, the superfamily
includes a large group of receptors which lack identified ligands (termed
"orphan receptors"). Nurr1, NGFI-B and Nor1 are closely related members
of this latter group of proteins.
This study was undertaken in order to explore the roles of retinoids and
the Nurr1/NGFI-B/Norl subfamily of orphan receptors in the developing and
adult CNS. The expression patterns of CRBPs and CRABPs and RARs and RXRs
in the postnatal and adult CNS were investigated using
immunocytochemistry and in situ hybridization histochemistry (ISH). In
addition, an in vitro reporter assay was used to detect retinoids in
striatal tissue. It was found that many of the retinoid binding proteins
and receptors are present in the CNS. Striatum, a part of the basal
ganglia, contains all necessary components for retinoid signaling. Both
CRBP I and CRABP I as well as RARß RXRß and RXRy are highly expressed
within this region. In addition, it has also been possible to detect
retinoids in striatal tissue. The role of Nurr1, NGFI-B and Nor1 has been
explored. In particular, these studies have emphasized the role of NuTrl.
All three members have previously been shown to interact with DNA as
monomers, but Nurr1 and NGFI-B have also been shown to heterodimerize
with RXR, thereby defining a distinct pathway for retinoid signaling. The
most recently cloned member, Nor1, was shown to lack the ability to
heterodimerize with RXR. To begin to understand the function of these
orphan receptors, ISH was used to localize the mRNA expression patterns
in both developing and adult tissues. All three members are highly
expressed in the CNS; a striking observation was that Nurr1 mRNA was
present in the developing ventral midbrain as well as in the mature
dopamine (DA) neurons of the substantia nigra pars compacta (SNc) and the
ventral tegmental area (VTA).
To further investigate the function of Nurr1, a targeted mutation was
introduced into the Nurr1 locus. Mice lacking Nurr1 are born alive but
die within the first two days after birth. The mice were found to
completely lack DA neurons in the ventral midbrain as demonstrated by the
absence of several markers, such as the rate limiting enzyme in DA
synthesis, tyrosine hydroxylase, a postulated retinoic acid converting
enzyme, AHD2, and the DA transporter in SNC, the VTA as well as in the
retrorubral field. In addition, high-pressure liquid chromatography was
used to demonstrate the absence of DA itself in the major target area for
the DAergic midbrain neurons, striatum. The role of Nurrl during early
development of the ventral midbrain has been further studied and the
relation to other factors postulated to be important for DA cell
generation such as sonic hedgehog explored. Since survival could not be
prolonged by administration of either the DA precursor L-DOPA, the DA
receptor agonist apomorphine or DA itself despite apparently normal DA
receptor expression, it is hypothesized that the mice do not die from
lack of DA. Interestingly, brains of newborn and two months old
heterozygous, apparently healthy, mice contained less DA than wild type
littermates, indicating a function for Nurr1 not only in the generation
of the DA cells, but also for maturation and maintenance of a normal
DAergic phenotype. These studies demonstrate that retinoids most likely
are important signaling molecules in the postnatal and adult CNS and
establish Nurr1 as a key component for proper development of DA midbrain
neurons, known to degenerate in patients with Parkinson's disease