Reelin

Reelin is a protein found mainly in the brain, but also in the spinal cord, blood and other body organs and tissues. Reelin is crucial for regulating the processes of neuronal migration and positioning in the developing brain. Besides this important role in the early period, reelin continues to work in the adult brain. It modulates the synaptic plasticity by enhancing LTP induction and maintenance. It also stimulates dendrite development and regulates the continuing migration of neuroblasts generated in adult neurogenesis sites like subventricular and subgranular zones.

Reelin is implicated in pathogenesis of several brain diseases: significantly lowered expression of the protein have been found in schizophrenia and psychotic bipolar disorder. Total lack of reelin causes a form of lissencephaly; reelin also may 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 found to have a deficiency of this brain protein and were homozygous for the RELN gene, which encodes reelin synthesis. The primary phenotype associated with loss of reelin function is inverted cortex, a neuroanatomical defect in which the six cortical layers are inverted. Heterozygous mice for the reelin gene have very little obvious neuroanatomical defect but those that they have resemble the changes of the human schizophrenic brain.



History
Mutant mice provide insight into the underlying molecular mechanisms of the development of the CNS. These spontaneous mutations were first identified by scientists interested in motor behavior, and it proved relatively easy to screen litter mates 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 first described in the 1951 edition of Journal of Genetics by Douglas Scott Falconer. Histopathological studies in the 1960's revealed that the reeler cerebellum is dramatically decreased in size and the normal laminar organization found in several brain regions is disrupted. 1970's brought the discovery of cellular layers inversion in the mice neocortex, which attracted more attention to the reeler mutation.

In 1995, the RELN gene and protein were discovered at chromosome 7q22 by Gabriella D'Arcangelo and colleagues. Almost immediately, Japanese scientists at Kochi Medical School had successfully created the first monoclonal antibody for reelin, called CR-50. They noted that CR-50 reacted specifically with Cajal-Retzius neurons, whose functional role was unknown till then.

The downstream pathway of Reelin was clarified using other mutant mice, including yotari and scrambler. These mice have phenotypes similar to that of reeler but have no mutation in reelin. It was then demonstrated that the mouse disabled homologue 1 (Dab1) gene, which encodes a homolog of Drosophila disabled, is the gene responsible for the phenotypes of these mutant mice, and Dab1 protein was absent (yotari) or only barely (scrambler) detectable in these mutants. Targeted disruption of Dab1 also caused a phenotype similar to that of reeler.

The Reelin receptors, apolipoprotein E receptor 2 and very-low-density lipoprotein receptor, were discovered serendipitously by Trommsdorff et al, who found that the double knockout mice for apolipoprotein E receptor 2 and very-low-density lipoprotein receptor, which they generated for another experiment, showed defects in cortical layering similar to that in reeler.

In the July of 2006, a group of Japanese scientists published the first report of X-ray crystallography and electron tomography investigation of reelin structure.

Secretion and localization of reelin
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 ECM proteins.

In the cortex and hippocampus, reelin is secreted by Cajal-Retzius cells, Cajal cells, and Retzius cells during brain development. In the cerebellum, Reelin is expressed first in the external granule cell layer (EGL) before the granule cell migration to the internal granule cell layer (IGL). In the adult brain, Reelin is expressed by GABA-ergic interneurons of the cortex and glutamatergic cerebellar neurons. Among GABAergic interneurons, Reelin seems to be detected predominantly in those expressing calretinin and calbindin, like bitufted, horisontal, and Martinotti cells, but not parvalbumin-expressing cells, like chandelier or basket neurons. 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. Its expression goes up when the liver is damaged, and returns to normal following its repair.



Structure
Reelin is a secreted extracellular matrix glycoprotein composed of 3461 amino acids with a relative molecular mass of 388 kDa.

Reelin molecule starts with a signaling peptide 27 amino acids in length, followed by a region bearing similarity to F-spondin, marked as "SP" on the scheme, and by a region unique to reelin, marked as "H". Next come the 8 repeats of 300-350 amino acids. These are called reelin repeats and have an EGF motif at their center, dividing each repeat into two subrepeats, A and B. Despite this interruption, the two subdomains make direct contact, resulting in a compact overall structure.

The last comes a highly basic and short C-terminal region (CTR, marked "+") with a length of 32 amino acids. This region is extremely conservative, being 100% identical in all investigated mammals. It was thought that CTR is necessary for reelin secretion, because 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, one recent study has shown that the CTR is not essential for secretion, which is most probably hindered then reelin is cut along one of the repeats.

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.

Function and mechanism of action
In the process of neural development, Reelin acts on migrating neuronal precursors and controls correct cell positioning in the cortex and other brain structures. The proposed role is one of a dissociation signal for neuronal groups, allowing them to separate and go from tangential chain-migration to radial individual migration. Dissociation detaches migrating neurons from the glial cells that are acting as their guides, converting them into individual cells that can strike out alone to find their final position.

In the adult brain, Reelin plays an important role by modulating cortical pyramidal neuron dendritic spine expression density, the branching of dendrites, and the expression of long-term potentiation.

Mechanism of action
Reelin acts on two receptors: which are members of the Low density lipoprotein receptor gene family.
 * VLDLR (very-low-density lipoprotein receptor) and the
 * ApoER2 (apolipoprotein E receptor 2),

The intracellular adaptor DAB1 binds to the VLDLR and ApoER2 through an NPxY motif and is involved in transmission of Reelin signals through these lipoprotein receptors.

The proposal that the protocadherin CNR1 behaves as a Reelin receptor has been disproved.

It has been shown that alpha-3-beta-1 integrin binds to the N-terminal region of reelin, a site distinct from the region of reelin shown to associate with other reelin receptors such as VLDLR/ApoER2.

Reelin molecules have been shown to form a large protein complex, a disulfide-linked homodimer. If the homodimer fails to form, efficient tyrosine phosphorylation of DAB1 also fails.

Reelin-dependent strengthening of long-term potentiation is caused by ApoER2 interaction with NMDA receptor. This interaction happens when ApoER2 has a region coded by exon 19. ApoER2 gene is alternatively spliced, with the exon 19-containing variant more actively produced during periods of activity.

Lissencephaly
Disruptions of the RELN gene are condsidered to be the cause of the rare form of lissencephaly with cerebellar hypoplasia called Norman-Roberts syndrome. The mutations disrupt splicing of RELN cDNA, resulting in low or undetectable amounts of reelin protein. The phenotype in these patients was characterized by hypotonia, ataxia, and developmental delay, with lack of unsupported sitting and profound mental retardation with little or no language development. Seizures and congenital lymphedema were also present.

Schizophrenia
Reduced expression of reelin and its mRNA levels in the brains of schizophrenia sufferers had been reported in 1998 and 2000 and independently confirmed in the postmortem studies of hippocampus samples and in the cortex studies. The reduction may reach up to 50% in some brain regions and is coupled with reduced expression of GAD-67 enzyme, which catalyses the transition of glutamate to GABA. Blood levels of reelin and its isoforms are also altered in schizophrenia, along with other mood disorders, according to one study. Reduced reelin mRNA prefrontal expression in schizophrenia was found to be the most statistically relevant disturbance found in the multicenter study conducted in 14 separate laboratories in 2001 by Stanley Foundation Neuropathology Consortium.

Epigenetic hypermethylation of DNA in schizophrenia patients is proposed as a cause of the reduction, in accordance with the knowledge that  administration of methionine to schizophrenic patients results in a profound exacerbation of schizophrenia symptoms in sixty to seventy percent of patients, a fact discovered in the 1960's.    A postmortem study comparing DNMT1 and Reelin mRNA expression in cortical layers I and V of schizophrenic patients and normal controls demonstrated that in the layer V both DNMT1 and Reelin levels were normal, while in the layer I DNMT1 was threefold higher, probably leading to the twofold decrease in the Reelin expression. Methylation inhibitors and histone deacetylase inhibitors, such as valproic acid, increase reelin mRNA levels, while L-methionine treatment downregulates the phenotypic expression of reelin.

Heterozygous reeler mouse, which is haploinsufficient for the reeler gene, shares several neurochemical and behavioral abnormalities with schizophrenia and bipolar disorder, but considered as not suitable for use as a genetic mouse model of schizophrenia.

Bipolar disorder
Decrease in RELN expression is typical of bipolar disorder with psychosis, but is not characteristic of patients with major depression without psychosis.

Autism
A number of studies have shown an association between the reelin gene and autism. A couple of studies were unable to duplicate linkage findings, however.

Temporal Lobe Epilepsy
Decreased reelin expression in the hippocampal tissue samples from patients with temporal lobe epilepsy was found to be directly correlated to the extent of granule cell dispersion, a major feature of the disease. According to one study, prolonged seizures in a rat model of mesial temporal lobe epilepsy have led to the loss of reelin-expressing interneurons and subsequent ectopic chain migration and aberrant integration of newborn dentate granule cells. Without reelin, the chain-migrating neuroblasts failed to detach properly.

Alzheimer's disease
According to one study, reelin expression and glycosylation patterns are altered in Alzheimer's disease. In the cortex of the patients, reelin levels were 40% higher compared with controls, but the cerebellar levels of the protein remain normal in the same patients. This finding correlates with an earlier study showing the presence of Reelin associated with amyloid plaques in a transgenic AD mouse model.

Recommended reading

 * 1) Forster E, Jossin Y, Zhao S, Chai X, Frotscher M, Goffinet AM. (2006) Recent progress in understanding the role of Reelin in radial neuronal migration, with specific emphasis on the dentate gyrus. Eur J Neurosci. 23(4):901-9. Review. PMID 16519655 (free full text)

Articles, publications, webpages

 * The real role of reelin - publication in The Journal of Cell Biology, 2002
 * Pleiotropic Action of Reelin in Psychosis - Web-lecture by Erminio Costa, MD., linking the reelin disfunction to schizophrenia and bipolar disorder.
 * Gabriella D'Arcangelo - the scientist who discovered the reelin gene and protein.
 * Neuronal Migration in Cortical Development - article from the Medscape website. By Shigeaki Kanatani, MIS; Hidenori Tabata, PhD; Kazunori Nakajima MD, PhD. (2005) J Child Neurol. 20(4):274-279


 * A short biography of the scientist who discovered the reeler mouse mutation - Mackay TF (2004) Douglas Scott Falconer (1913-2004). Heredity. 93(2):119-21. PMID 15241449

Figures and images

 * Schematic representation of signaling through the LDLR family members apoER2 and VLDL receptor - figure from an article.
 * Proposed mechanism by which mouse RELN promoter hypermethylation and recruitment of chromatin remodeling complexes (MeCP2, HDACs, and corepressors) regulate reelin gene expression - a figure from scientific publication by Dong et al.
 * Figure from an article. Corticogenesis in wild-type, reeler mutant and β1 deficient mice. - a pictorial rendition of the difference that the lack of reelin brings to the cortical structure.
 * Reelin gene expression in mice - images from BGEM (Brain Gene Expression Map) site.
 * Effects of human and naturally occurring mouse RELN mutations on the predicted protein - figure from an article by Susan E. Hong et al.
 * MRI analysis of chromosome 7q22-linked lissencephaly with cerebellar hypoplasia - Brain images from the same article.