Reelin, encoded by the RELN gene, is a large secreted extracellular matrix glycoprotein that helps regulate processes of neuronal migration and positioning in the developing brain by controlling cell–cell interactions. Besides this important role in early development, reelin continues to work in the adult brain. It modulates synaptic plasticity by enhancing the induction and maintenance of long-term potentiation. It also stimulates dendrite and dendritic spine development in the hippocampus, and regulates the continuing migration of neuroblasts generated in adult neurogenesis sites of the subventricular and subgranular zones. It is found not only in the brain but also in the liver, thyroid gland, adrenal gland, fallopian tube, breast and in comparatively lower levels across a range of anatomical regions.
Reelin has been suggested to be implicated in pathogenesis of several brain diseases. The expression of the protein has been found to be significantly lower in schizophrenia and psychotic bipolar disorder, but the cause of this observation remains uncertain, as studies show that psychotropic medication itself affects reelin expression. Moreover, epigenetic hypotheses aimed at explaining the changed levels of reelin expression are controversial. Total lack of reelin causes a form of lissencephaly. Reelin may also play a role in Alzheimer's disease, temporal lobe epilepsy and autism.
Reelin's name comes from the abnormal reeling gait of reeler mice, which were later found to have a deficiency of this brain protein and were homozygous for mutation of the RELN gene. The primary phenotype associated with loss of reelin function is a failure of neuronal positioning throughout the developing central nervous system (CNS). The mice heterozygous for the reelin gene, while having little neuroanatomical defects, display the endophenotypic traits linked to psychotic disorders.
Discovery
Mutant mice have provided insight into the underlying molecular mechanisms of the development of the central nervous system. Useful spontaneous mutations were first identified by scientists who were interested in motor behavior, and it proved relatively easy to screen littermates for mice that showed difficulties moving around the cage. A number of such mice were found and given descriptive names such as reeler, weaver, lurcher, nervous, and staggerer.
The "reeler" mouse was described for the first time in 1951 by D. S. Falconer at Edinburgh University as a spontaneous variant arising in a colony of at least mildly inbred snowy-white bellied mice stock in 1948. Histopathological studies in the 1960s revealed that the cerebellum of reeler mice is dramatically reduced in size and the normal laminar organization found in several brain regions is disrupted. The 1970s brought about the discovery of cellular layer inversion in the mouse neocortex, which attracted more attention to the reeler mutation.
In 1994, a new allele of reeler was obtained by means of insertional mutagenesis. This provided the first molecular marker of the locus, permitting the RELN gene to be mapped to chromosome 7q22 and subsequently cloned and identified. Japanese scientists at Kochi Medical School successfully raised antibodies against normal brain extracts in reeler mice, later these antibodies were found to be specific monoclonal antibodies for reelin, and were termed CR-50 (Cajal–Retzius marker 50). They noted that CR-50 reacted specifically with Cajal–Retzius neurons, whose functional role was unknown until then.
The Reelin receptors, apolipoprotein E receptor 2 (ApoER2) and very-low-density lipoprotein receptor (VLDLR), were discovered by Trommsdorff, Herz and colleagues, who initially found that the cytosolic adaptor protein Dab1 interacts with the cytoplasmic domain of LDL receptor family members. They then went on to show that the double knockout mice for ApoER2 and VLDLR, which both interact with Dab1, had cortical layering defects similar to those in reeler.
The downstream pathway of reelin was further clarified with the help of other mutant mice, including yotari and scrambler mice. These mutants have phenotypes similar to that of reeler mice, but without mutation in reelin. It was then demonstrated that the mouse disabled homologue 1 (Dab1) gene is responsible for the phenotype of these mutant mice, as Dab1 protein was absent (yotari) or only barely detectable (scrambler) in these mutants. Targeted disruption of Dab1 also caused a phenotype similar to that of reeler. Pinpointing the DAB1 as a pivotal regulator of the reelin signaling cascade started the tedious process of deciphering its complex interactions.
There followed a series of speculative reports linking reelin's genetic variation and interactions to schizophrenia, Alzheimer's disease, autism and other highly complex dysfunctions. These and other discoveries, coupled with the perspective of unraveling the evolutionary changes that allowed for the creation of human brain, highly intensified the research. As of 2008, some 13 years after the gene coding the protein was discovered, hundreds of scientific articles address the multiple aspects of its structure and functioning.
Tissue distribution and secretion Studies show that reelin is absent from synaptic vesicles and is secreted via constitutive secretory pathway, being stored in Golgi secretory vesicles. Reelin's release rate is not regulated by depolarization, but strictly depends on its synthesis rate. This relationship is similar to that reported for the secretion of other extracellular matrix proteins.
During the brain development, reelin is secreted in the cortex and hippocampus by the so-called Cajal–Retzius cells, Cajal cells, and Retzius cells. Reelin-expressing cells in the prenatal and early postnatal brain are predominantly found in the marginal zone (MZ) of the cortex and in the temporary subpial granular layer (SGL), which is manifested to the highest extent in human, and in the hippocampal stratum lacunosum-moleculare and the upper marginal layer of the dentate gyrus.
In the developing cerebellum, reelin is expressed first in the external granule cell layer (EGL), before the granule cell migration to the internal granule cell layer (IGL) takes place.
Having peaked just after the birth, the synthesis of reelin subsequently goes down sharply, becoming more diffuse compared with the distinctly laminar expression in the developing brain. In the adult brain, reelin is expressed by GABA-ergic interneurons of the cortex and glutamatergic cerebellar neurons, the glutamatergic stellate cells and fan cells in the superficial entorhinal cortex that are supposed to carry a role in encoding new episodic memories, and by the few extant Cajal–Retzius cells. Among GABAergic interneurons, reelin seems to be detected predominantly in those expressing calretinin and calbindin, like bitufted, horizontal, and Martinotti cells, but not parvalbumin-expressing cells, like chandelier or basket neurons. In the white matter, a minute proportion of interstitial neurons has also been found to stain positive for reelin expression.
Outside the brain, reelin is found in adult mammalian blood, liver, pituitary pars intermedia, and adrenal chromaffin cells. In the liver, reelin is localized in hepatic stellate cells. The expression of reelin increases when the liver is damaged, and returns to normal following its repair. In the eyes, reelin is secreted by retinal ganglion cells and is also found in the endothelial layer of the cornea. Just as in the liver, its expression increases after an injury has taken place.
The protein is also produced by the odontoblasts, which are cells at the margins of the dental pulp. Reelin is found here both during odontogenesis and in the mature tooth. Some authors suggest that odontoblasts play an additional role as sensory cells able to transduce pain signals to the nerve endings. According to the hypothesis, reelin participates in the process by enhancing the contact between odontoblasts and the nerve terminals.
Structure
Reelin is composed of 3461 amino acids with a relative molecular mass of . It also has serine protease activity. Murine RELN gene consists of 65 exons spanning approximately 450 kb. One exon, coding for only two amino acids near the protein's C-terminus, undergoes alternative splicing, but the exact functional impact of this is unknown. Two transcription initiation sites and two polyadenylation sites are identified in the gene structure.
The reelin protein starts with a signaling peptide 27 amino acids in length, followed by a region bearing similarity to F-spondin (the reeler domain), marked as "SP" on the scheme, and by a region unique to reelin, marked as "H". Next comes 8 repeats of 300–350 amino acids. These are called reelin repeats and have an epidermal growth factor motif at their center, dividing each repeat into two subrepeats, A (the BNR/Asp-box repeat) and B (the EGF-like domain). Despite this interruption, the two subdomains make direct contact, resulting in a compact overall structure.
The final reelin domain contains a highly basic and short C-terminal region (CTR, marked "+") with a length of 32 amino acids. This region is highly conserved, being 100% identical in all investigated mammals. It was thought that CTR is necessary for reelin secretion, because the Orleans reeler mutation, which lacks a part of 8th repeat and the whole CTR, is unable to secrete the misshaped protein, leading to its concentration in cytoplasm. However, other studies have shown that the CTR is not essential for secretion itself, but mutants lacking the CTR were much less efficient in activating downstream signaling events.
Reelin is cleaved in vivo at two sites located after domains 2 and 6 – approximately between repeats 2 and 3 and between repeats 6 and 7, resulting in the production of three fragments. This splitting does not decrease the protein's activity, as constructs made of the predicted central fragments (repeats 3–6) bind to lipoprotein receptors, trigger Dab1 phosphorylation and mimic functions of reelin during cortical plate development. Moreover, the processing of reelin by embryonic neurons may be necessary for proper corticogenesis.
Function
The primary functions of Reelin are the regulation of corticogenesis and neuronal cell positioning in the prenatal period, but the protein also continues to play a role in adults. Reelin is found in numerous tissues and organs, and one could roughly subdivide its functional roles by the time of expression and by localisation of its action.
During development A number of non-nervous tissues and organs express reelin during development, with the expression sharply going down after organs have been formed. The role of the protein here is largely unexplored, because the knockout mice show no major pathology in these organs. Reelin's role in the growing central nervous system has been extensively characterized. It promotes the differentiation of progenitor cells into radial glia and affects the orientation of its fibers, which serve as the guides for the migrating neuroblasts. The position of reelin-secreting cell layer is important, because the fibers orient themselves in the direction of its higher concentration. For example, reelin regulates the development of layer-specific connections in hippocampus and entorhinal cortex.