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Unpacking Reelin's Role in Neuronal Migration

22 February 2011. When neurons migrate by glia-independent means in developing mouse cortex, reelin signals help them reach their proper destination, according to a study published February 10 in Neuron. The study finds that the protein Disabled-1 (Dab1), a key part of the reelin pathway, is required for stages of migration that take place without the guidance of radial glial cells (RGCs). In contrast, glia-guided movement occurred normally without Dab1. Disrupting the glia-independent stages resulted in cell-layering defects just as severe as when reelin itself was lost, which suggests that these processes are critical for proper brain development. These findings offer clues to what goes awry in neurodevelopment disorders like schizophrenia.

Once a highly promising lead in schizophrenia pathology, the reelin gene (RELN) continues to produce findings of small effects on risk, leading some to suggest that it modifies features of the disorder (Wedenoja et al., 2010). In mice, reelin mutations disrupt neuron layering in the brain, leaving newborn neurons unable to migrate past their earlier-born predecessors as they should. Figuring out how this comes about has been a complicated endeavor, as reelin participates in an elaborate signaling pathway in multiple cell types. For example, reelin, located in the extracellular matrix, binds to receptors in RGCs and contributes to their shape. Because RGCs provide a kind of highway for migrating neurons, reelin-induced changes to RGC shape provide a widely accepted account for reelin-induced brain abnormalities.

But reelin also binds to receptors located on the migrating neurons themselves, triggering phosphorylation of Dab1, which then interacts with other molecules. Indeed, other studies have demonstrated a role for Dab1 within neurons during migration (Olson et al., 2006). To explore the contribution of this neuron-specific pathway to neuronal migration, lead researcher Ulrich Müller and colleagues at The Scripps Research Institute in San Diego, California, inactivated Dab1 in migrating neurons at key time points in development.

Trains, planes, and somal translocation
The timing of Dab1 inactivation matters because neurons born at different times use different modes of transportation to reach their final destinations. An early-born neuron headed for the deeper layers of the cortex not far from where neurons are born uses "somal translocation" to reach its destination: it puts out a leading process, fixes it in place, and then its cell body follows. A later-born neuron has farther to go, traveling past the early-born ones to form the more superficial layers of the cortex. It first uses multipolar migration, followed by locomotion along RGCs, and then somal translocation to reach its final position.

To inactivate Dab1 in newborn neurons, first authors Santos Franco and Isabel Martinez-Garay used a conditional knockout approach. They generated mice with Cre recombinase (CRE) splicing sites flanking one copy of the Dab1 gene. Next, they electroporated a neuron-specific vector containing CRE into mouse embryos in utero, resulting in the loss of Dab1 in newborn neurons, but not in glia.

When Dab1 was inactivated early on, the early-born neurons failed to reach their final destination. While 92 percent of control neurons made it into the cortical plate, only 4 percent of Dab1-inactivated neurons did. Instead, most of them were stuck below the subplate, closer to their birthplace. Although these mutant neurons seemed to extend processes successfully into the cortical plate, their cell bodies did not follow.

Inactivating Dab1 later, as neurons destined for superficial layers of the cortex were born, also perturbed migration. Though the multipolar and RGC migration portions of their journey appeared normal, these later-born neurons did not make it into the superficial layers of the cortical plate, as control neurons with normal Dab1 did. Time-lapse microscopy showed that Dab1-inactivated neurons traveled along RGCs normally, but in the final somal translocation phase of their journey, their cell bodies never followed their leading processes. This left later-born neurons in the wrong place in the brain.

Dab1's molecular machinery
Reelin affects both neurons and glia, but the researchers asked whether perturbing somal translocation by neurons alone could account for the reeler phenotype, in which reelin-deficient mice show abnormal brain development (see Katsuyama and Terashima, 2009). To explore this issue, the researchers generated mice that had a constitutive loss of Dab1 in migrating neurons but not in RGCs. Strikingly, the abnormal cell positioning in the brains of these mice was indistinguishable from that observed in reeler-like mice that had Dab1 inactivated in both glia and newborn neurons. This argues that somal translocation is not just fine-tuning a neuron's position, but rather is as critical to cell layering as glia-dependent migration.

Further experiments outlined the pathway of reelin signaling involved in somal translocation, ultimately connecting to cadherins. Reelin activation of Dab1 phosphorylation recruits PI3K and Crk/CrkL molecules, which in turn activate Limk1, a regulator of the leading process in migrating cells, and Akt1 and Rap1, which regulate cell adhesion. When the researchers inactivated each of these downstream players in early-born neurons that were migrating via somal translocation, they found migration defects only for Rap1 suppression. Similar to Dab1-inactivated neurons, the Rap1-deficient ones had leading processes that extended normally into the cortical plate, but their cell bodies did not follow. Rap1, in turn, acted through cadherins to maintain the leading process and/or help fix it in place upon reaching its destination. Interestingly, cadherin overexpression rescued Rap1-deficient migration anomalies, but could not correct the Dab1-deficient ones. This suggests that Dab1 acts through other molecules and may integrate several players involved in different aspects of somal translocation.

These findings not only illuminate how reelin acts, but they offer new insights into the multistep process of neuronal migration. Ultimately, disentangling the mechanisms behind the different parts of a neuron's journey in a developing brain will yield a deeper understanding of how brain development normally proceeds, and how it can be derailed, as suspected in schizophrenia.—Michele Solis.

Reference:
Franco SJ, Martinez-Garay I, Gil-Sanz C, Harkins-Perry SR, Müller U. Reelin Regulates Cadherin Function via Dab1/Rap1 to Control Neuronal Migration and Lamination in the Neocortex. Neuron. 2011 Feb 10; 69:482-97. Abstract

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Related News: Bad Timing: Prenatal Exposure to Maternal STDs Raises Risk of Schizophrenia

Comment by:  Paul Patterson
Submitted 22 May 2006
Posted 22 May 2006

Over the past six years, Alan Brown and colleagues have published an impressive series of epidemiological findings on schizophrenia in the offspring of a large cohort of carefully studied pregnant women (reviewed by Brown, 2006). Their work has confirmed and greatly extended prior findings linking maternal infection in the second trimester with increased risk for schizophrenia in the offspring. Moreover, Brown et al. found an association between anti-influenza antibodies in maternal serum and increased risk for schizophrenia, as well as a similar association with elevated levels of a cytokine in maternal serum. In a new paper (Babulas et al., 2006), this group reports a fivefold increase in risk for schizophrenia spectrum disorders in the offspring of women who experienced a genital/reproductive infection during the periconception period. The infections considered were endometritis, cervicitis, pelvic inflammatory disease, vaginitis, syphilis, condylomata, “venereal disease,” and gonorrhea. Strengths of the study include physician documentation of the infections and face-to-face assessments of schizophrenia. Although sample size was modest, these results extend a prior finding that elevated maternal anti-herpes simplex type 2 antibodies are associated with increased risk of psychotic disorders, including schizophrenia (Buka et al., 2001).

The mechanism of how maternal infection increases risk for schizophrenia could involve pathogens invading the fetus. Although this is certainly possible in the case of some of the infections studied by Babulas et al., in the case of a respiratory virus such as influenza, this explanation appears unlikely. A more parsimonious mechanism would involve activation of the maternal immune system, and action of soluble mediators such as cytokines at the level of the placenta or the fetus. Support for this hypothesis comes from animal studies. An antiviral immune response can be evoked in the absence of the pathogen by injection of synthetic double-stranded RNA (polyI:C). When this is done in pregnant rats or mice, the adult offspring display a number of behavioral abnormalities reminiscent of those observed in schizophrenia. These include deficits in prepulse inhibition, latent inhibition, and social interaction, as well as enhanced amphetamine-induced locomotion and anxiety under mildly stressful conditions (Shi et al., 2003; Zuckerman et al., 2003; Ozawa et al., 2005). Moreover, some of these deficits are ameliorated by treatment with antipsychotic drugs and exacerbated by psychotomimetics (Shi et al., 2003; Ozawa et al., 2005), and the offspring also exhibit dopaminergic hyperfunction (Zuckerman et al., 2003; Ozawa et al., 2005). Some of these abnormalities are also seen in the offspring of influenza-infected mothers or mothers injected with the bacterial cell wall component, LPS (Borrell et al., 2002; Fatemi et al., 2002; Shi et al., 2003).

The most recent advance in this growing cottage industry is the finding that there are critical periods of maternal immune activation that determine the type of adult behavioral dysfunction and neuropathology found in the offspring (Meyer et al., 2006). Injection of polyI:C during stages of mouse gestation corresponding to first-to-second versus second-to-third trimesters of human pregnancy yields different deficits in exploratory and perseverative behavior, postnatal reelin expression, and hippocampal apoptosis. Moreover, these two different stages of injection evoke diverse cytokine responses in the fetal brain. It would further be interesting to know which of these abnormalities is specific to the period corresponding to the human second trimester, as this is the key time of vulnerability for risk of schizophrenia associated with maternal infection.

Other fascinating questions for this increasingly popular model are, what mediates the effects of maternal immune activation (e.g., cytokines, antibodies, corticosteroids), and do they act directly on the fetus or via the placenta? Can imaging be used with the rodents to explore dopamine receptor occupancy? Which of the observed pathologies are most relevant for each of the behavioral abnormalities?

References:
Babulas V, Factor-Litvak P, Goetz R, Schaefer CA, Brown AS. Prenatal exposure to maternal genital and reproductive infections and adult schizophrenia. Am J Psychiatry. 2006 May;163(5):927-9. Abstract Borrell J, Vela JM, Arevalo-Martin A, Molina-Holgado E, Guaza C. Prenatal immune challenge disrupts sensorimotor gating in adult rats. Implications for the etiopathogenesis of schizophrenia. Neuropsychopharmacology. 2002 Feb;26(2):204-15. Abstract Brown AS. Prenatal infection as a risk factor for schizophrenia. Schizophr Bull. 2006 Apr;32(2):200-2. Epub 2006 Feb 9. Abstract Buka SL, Tsuang MT, Torrey EF, Klebanoff MA, Bernstein D, Yolken RH. Maternal infections and subsequent psychosis among offspring. Arch Gen Psychiatry. 2001 Nov;58(11):1032-7. Abstract Fatemi SH, Earle J, Kanodia R, Kist D, Emamian ES, Patterson PH, Shi L, Sidwell R. Prenatal viral infection leads to pyramidal cell atrophy and macrocephaly in adulthood: implications for genesis of autism and schizophrenia. Cell Mol Neurobiol. 2002 Feb;22(1):25-33. Abstract Meyer U, Feldon J, Schedlowski M, Yee BK. Towards an immuno-precipitated neurodevelopmental animal model of schizophrenia. Neurosci Biobehav Rev. 2005;29(6):913-47. Abstract Meyer U, Nyffeler M, Engler A, Urwyler A, Schedlowski M, Knuesel I, Yee BK, Feldon J. The time of prenatal immune challenge determines the specificity of inflammation-mediated brain and behavioral pathology. J Neurosci. 2006 May 3;26(18):4752-62. Abstract Ozawa K, Hashimoto K, Kishimoto T, Shimizu E, Ishikura H, Iyo M. Immune activation during pregnancy in mice leads to dopaminergic hyperfunction and cognitive impairment in the offspring: a neurodevelopmental animal model of schizophrenia. Biol Psychiatry. 2006 Mar 15;59(6):546-54. Epub 2005 Oct 26. Abstract Shi L, Fatemi SH, Sidwell RW, Patterson PH. Maternal influenza infection causes marked behavioral and pharmacological changes in the offspring. J Neurosci. 2003 Jan 1;23(1):297-302. Abstract Zuckerman L, Rehavi M, Nachman R, Weiner I. Immune activation during pregnancy in rats leads to a postpubertal emergence of disrupted latent inhibition, dopaminergic hyperfunction, and altered limbic morphology in the offspring: a novel neurodevelopmental model of schizophrenia. Neuropsychopharmacology. 2003 Oct;28(10):1778-89. Abstract

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Related News: Bad Timing: Prenatal Exposure to Maternal STDs Raises Risk of Schizophrenia

Comment by:  Jürgen Zielasek
Submitted 3 June 2006
Posted 3 June 2006

Meyer and coworkers provide interesting new data on the role of the immune system in mediating the damage caused by viral infections during pregnancy on the developing nervous system of the fetus. Not just the timing of the infection appears to be critical, but the developing fetal immune system appears to play a role, too.

Polyinosinic-polycytidylic acid (polyI:C), which was employed by Meyer et al., is frequently used to mimic viral infections. It is a synthetic double-stranded RNA and has adjuvant-effects (Salem et al., 2005). PolyI:C binds to target cells via the "Toll-like receptor 3" (TLR3). TLR3 serves as a receptor in trophoblast cells and uterine epithelial cells mediating local immune activation at the maternal-fetal interface after viral infections (Abrahams et al., 2005; Schaefer et al., 2005). Glial cells like microglia and astrocytes also express functional TLR3 (Farina et al., 2005; Park et al., 2006; Town et al., 2006). Thus, TLR3 plays an important role in immune responses, and its natural function appears to be immune activation in addition to cross-priming the immune system to virus-infected cells (Schulz et al., 2005). Given the expression of TLR3 at the maternal-fetal interface and on glial cells, the polyI:C-TLR3-model appears to be useful to study the basic mechanisms of viral infections and their consequences for brain development in animal models.

However, several limitations are evident: PolyI:C is not a virus, and different immunological pathways may be activated by intact viruses after binding to their appropriate receptors. Findings from the immune system of rodents cannot be directly transferred to humans, and it may be difficult to dissect—on a molecular level—the protective aspects of an immune response against a viral infection from its putative detrimental effects on human neurodevelopment. Still, such mechanisms may now be studied in the rodent models used by Meyer and coworkers and other groups, and this will help to pave the way for future studies in humans. This will hopefully lead to a better understanding of the role of the immune system and viral infections in the pathogenesis of schizophrenia.

References:

Abrahams VM, Visintin I, Aldo PB, Guller S, Romero R, Mor G. A role for TLRs in the regulation of immune cell migration by first trimester trophoblast cells. J Immunol. 2005 Dec 15;175(12):8096-104. Abstract

Farina C, Krumbholz M, Giese T, Hartmann G, Aloisi F, Meinl E. Preferential expression and function of Toll-like receptor 3 in human astrocytes. J Neuroimmunol. 2005 Feb;159(1-2):12-9. Epub 2004 Nov 11. Abstract

Park C, Lee S, Cho IH, Lee HK, Kim D, Choi SY, Oh SB, Park K, Kim JS, Lee SJ. TLR3-mediated signal induces proinflammatory cytokine and chemokine gene expression in astrocytes: differential signaling mechanisms of TLR3-induced IP-10 and IL-8 gene expression. Glia. 2006 Feb;53(3):248-56. Abstract

Salem ML, Kadima AN, Cole DJ, Gillanders WE. Defining the antigen-specific T-cell response to vaccination and poly(I:C)/TLR3 signaling: evidence of enhanced primary and memory CD8 T-cell responses and antitumor immunity. J Immunother. 2005 May-Jun;28(3):220-8. Abstract

Schaefer TM, Fahey JV, Wright JA, Wira CR. Innate immunity in the human female reproductive tract: antiviral response of uterine epithelial cells to the TLR3 agonist poly(I:C). J Immunol. 2005 Jan 15;174(2):992-1002. Abstract

Schulz O, Diebold SS, Chen M, Naslund TI, Nolte MA, Alexopoulou L, Azuma YT, Flavell RA, Liljestrom P, Reis e Sousa C. Toll-like receptor 3 promotes cross-priming to virus-infected cells. Nature. 2005 Feb 24;433(7028):887-92. Epub 2005 Feb 13. Abstract

Town T, Jeng D, Alexopoulou L, Tan J, Flavell RA. Microglia recognize double-stranded RNA via TLR3. J Immunol. 2006 Mar 15;176(6):3804-12. Abstract

View all comments by Jürgen Zielasek