ADAMTS2 promotes radial migration by activating TGF-ß signaling in the developing neocortex.

The mammalian neocortex is formed by sequential radial migration of newborn excitatory neurons. Migrating neurons undergo a multipolar-to-bipolar transition at the subplate (SP) layer, where extracellular matrix (ECM) components are abundantly expressed. Here, we investigate the role of the ECM at the SP layer. We show that TGF-ß signaling-related ECM proteins, and their downstream effector, p-smad2/3, are selectively expressed in the SP layer. We also find that migrating neurons express a disintegrin and metalloproteinase with thrombospondin motif 2 (ADAMTS2), an ECM metalloproteinase, just below the SP layer. Knockdown and knockout of Adamts2 suppresses the multipolar-to-bipolar transition of migrating neurons and disturbs radial migration. Time-lapse luminescence imaging of TGF-ß signaling indicates that ADAMTS2 activates this signaling pathway in migrating neurons during the multipolar-to-bipolar transition at the SP layer. Overexpression of TGF-ß2 in migrating neurons partially rescues migration defects in ADAMTS2 knockout mice. Our data suggest that ADAMTS2 secreted by the migrating multipolar neurons activates TGF-ß signaling by ECM remodeling of the SP layer, which might drive the multipolar to bipolar transition.

Reelin regulates the migration of late-born hippocampal CA1 neurons via cofilin phosphorylation.

Reelin, a large secreted glycoprotein, plays an important role in neuronal migration during brain development. The C-terminal region (CTR) of Reelin is involved in the efficient activation of downstream signaling and its loss leads to abnormal hippocampal layer formation. However, the molecular mechanism by which Reelin CTR regulates hippocampal development remains unknown. Here, we showed that the migration of late-born, but not early-born, neurons is impaired in the knock-in mice in which Reelin CTR is deleted (ΔC-KI mice). The phosphorylation of cofilin, an actin-depolymerizing protein, was remarkably decreased in the hippocampus of the ΔC-KI mice. Exogenous expression of pseudo-phosphorylated cofilin rescued the ectopic positioning of neurons in the hippocampus of ΔC-KI mice. These results suggest that Reelin CTR is required for the migration of late-born neurons in the hippocampus and that this event involves appropriate phosphorylation of cofilin.

The Plasma Membrane Polarity Is Higher in the Neuronal Growth Cone than in the Cell Body of Hippocampal and Cerebellar Granule Neurons.

The polarity of the biological membrane, or lipid order, regulates many cellular events. It is generally believed that the plasma membrane polarity is regulated according to cell type and function, sometimes even within a cell. Neurons have a variety of functionally specialized subregions, each of which bears distinct proteins and lipids, and the membrane polarity of the subregions may differ accordingly. However, no direct experimental evidence of it has been presented to date. In the present study, we used a cell-impermeable solvatochromic membrane probe NR12A to investigate the local polarity of the plasma membrane of neurons. Both in hippocampal and cerebellar granule neurons, growth cones have higher membrane polarity than the cell body. In addition, the overall variation in the polarity value of each pixel was greater in the growth cone than in cell bodies, suggesting that the lateral diffusion and/or dynamics of the growth cone membrane are greater than other parts of the neuron. These tendencies were much less notably observed in the lamellipodia of a non-neuronal cell. Our results suggest that the membrane polarity of neuronal growth cones is unique and this characteristic may be important for its structure and function.

Reelin-Nrp1 Interaction Regulates Neocortical Dendrite Development in a Context-Specific Manner.

Reelin plays versatile roles in neocortical development. The C-terminal region (CTR) of Reelin is required for the correct formation of the superficial structure of the neocortex; however, the mechanisms by which this position-specific effect occurs remain largely unknown. In this study, we demonstrate that Reelin with an intact CTR binds to neuropilin-1 (Nrp1), a transmembrane protein. Both male and female mice were used. Nrp1 is localized with very-low-density lipoprotein receptor (VLDLR), a canonical Reelin receptor, in the superficial layers of the developing neocortex. It forms a complex with VLDLR, and this interaction is modulated by the alternative splicing of VLDLR. Reelin with an intact CTR binds more strongly to the VLDLR/Nrp1 complex than to VLDLR alone. Knockdown of Nrp1 in neurons leads to the accumulation of Dab1 protein. Since the degradation of Dab1 is induced by Reelin signaling, it is suggested that Nrp1 augments Reelin signaling. The interaction between Reelin and Nrp1 is required for normal dendritic development in superficial-layer neurons. All of these characteristics of Reelin are abrogated by proteolytic processing of the six C-terminal amino acid residues of Reelin (0.17% of the whole protein). Therefore, Nrp1 is a coreceptor molecule for Reelin and, together with the proteolytic processing of Reelin, can account for context-specific Reelin function in brain development.SIGNIFICANCE STATEMENT Reelin often exhibits a context-dependent function during brain development; however, its underlying mechanism is not well understood. We found that neuropilin-1 (Nrp1) specifically binds to the CTR of Reelin and acts as a coreceptor for very-low-density lipoprotein receptor (VLDLR). The Nrp1/VLDLR complex is localized in the superficial layers of the neocortex, and its interaction with Reelin is essential for proper dendritic development in superficial-layer neurons. This study provides the first mechanistic evidence of the context-specific function of Reelin (>3400 residues) regulated by the C-terminal residues and Nrp1, a component of the canonical Reelin receptor complex.

The Secreted Glycoprotein Reelin Suppresses the Proliferation and Regulates the Distribution of Oligodendrocyte Progenitor Cells in the Embryonic Neocortex.

Oligodendrocyte (OL) progenitor cells (OPCs) are generated, proliferate, migrate, and differentiate in the developing brain. Although the development of OPCs is prerequisite for normal brain function, the molecular mechanisms regulating their development in the neocortex are not fully understood. Several molecules regulate the tangential distribution of OPCs in the developing neocortex, but the cue molecule(s) that regulate their radial distribution remains unknown. Here, we demonstrate that the secreted glycoprotein Reelin suppresses the proliferation of OPCs and acts as a repellent for their migration in vitro These functions rely on the binding of Reelin to its receptors and on the signal transduction involving the intracellular protein Dab1. In the late embryonic neocortex of mice with attenuated Reelin signaling [i.e., Reelin heterozygote-deficient, Dab1 heterozygote-deficient mutant, or very low-density lipoprotein receptor (VLDLR)-deficient mice], the number of OPCs increased and their distribution shifted toward the superficial layers. In contrast, the number of OPCs decreased and they tended to distribute in the deep layers in the neocortex of mice with abrogated inactivation of Reelin by proteolytic cleavage, namely a disintegrin and metalloproteinase with thrombospondin type 1 motifs 3 (ADAMTS-3)-deficient mice and cleavage-resistant Reelin knock-in mice. Both male and female animals were used. These data indicate that Reelin-Dab1 signaling regulates the proliferation and radial distribution of OPCs in the late embryonic neocortex and that the regulation of Reelin function by its specific proteolysis is required for the normal development of OPCs.SIGNIFICANCE STATEMENT Here, we report that Reelin-Dab1 signaling regulates the proliferation and radial distribution of OPCs in the late embryonic mouse neocortex. Oligodendrocyte (OL) progenitor cells (OPCs) express Reelin signaling molecules and respond to Reelin stimulation. Reelin-Dab1 signaling suppresses the proliferation of OPCs both in vitro and in vivo Reelin repels OPCs in vitro, and the radial distribution of OPCs is altered in mice with either attenuated or augmented Reelin-Dab1 signaling. This is the first report identifying the secreted molecule that plays a role in the radial distribution of OPCs in the late embryonic neocortex. Our results also show that the regulation of Reelin function by its specific proteolysis is important for the normal development of OPCs.

Microinjection of Reelin into the mPFC prevents MK-801-induced recognition memory impairment in mice.

Reelin, a large extracellular matrix protein, helps to regulate neuronal plasticity and cognitive function. Several studies have shown that Reelin dysfunction, resulting from factors such as mutations in gene RELN or low Reelin expression, is associated with schizophrenia (SCZ). We previously reported that microinjection of Reelin into cerebral ventricle prevents phencyclidine-induced cognitive and sensory-motor gating deficits. However, it remains unclear whether and how Reelin ameliorates behavioral abnormalities in the animal model of SCZ. In the present study, we evaluated the effect of recombinant Reelin microinjection into the medial prefrontal cortex (mPFC) on abnormal behaviors induced by MK-801, an N-methyl-D-aspartate receptor antagonist. Microinjection of Reelin into the mPFC prevented impairment of recognition memory of MK-801-treated mice in the novel object recognition test (NORT). On the other hand, the same treatment had no effect on deficits in sensory-motor gating and short-term memory in the pre-pulse inhibition and Y-maze tests, respectively. To establish the neural substrates that respond to Reelin, the number of c-Fos-positive cells in the mPFC was determined. A significant increase in c-Fos-positive cells in the mPFC of MK-801-treated mice was observed when compared with saline-treated mice, and this change was suppressed by microinjection of Reelin into the mPFC. A K2360/2467A Reelin that cannot bind to its receptor failed to ameliorate MK-801-induced cognitive deficits in NORT. These results suggest that Reelin prevents MK-801-induced recognition memory impairment by acting on its receptors to suppress neural activity in the mPFC of mice.

A disintegrin and metalloproteinase with thrombospondin motifs 2 cleaves and inactivates Reelin in the postnatal cerebral cortex and hippocampus, but not in the cerebellum.

Reelin plays important roles in regulating neuronal development, modulating synaptic function, and counteracting amyloid ß toxicity. A specific proteolytic cleavage (N-t cleavage) of Reelin abolishes its biological activity. We recently identified ADAMTS-3 (a disintegrin and metalloproteinase with thrombospondin motifs 3) as the major N-t cleavage enzyme in the embryonic and early postnatal brain. The contribution of other proteases, particularly in the postnatal brain, has not been demonstrated in vivo. ADAMTS-2, -3 and -14 share similar domain structures and substrate specificity, raising the possibility that ADAMTS-2 and -14 may cleave Reelin. We found that recombinant ADAMTS-2 protein expressed in cultured cell lines cleaves Reelin at the N-t site as efficiently as ADAMTS-3 while recombinant ADAMTS-14 hardly cleaves Reelin. The disintegrin domain is necessary for the Reelin-cleaving activity of ADAMTS-2 and -3. ADAMTS-2 is expressed in the adult brain at approximately the same level as ADAMTS-3. We generated ADAMTS-2 knockout (KO) mice and found that ADAMTS-2 significantly contributes to the N-t cleavage and inactivation of Reelin in the postnatal cerebral cortex and hippocampus, but much less in the cerebellum. Therefore, it was suggested that ADAMTS-2 can be a therapeutic target for adult brain disorders such as schizophrenia and Alzheimer's disease.

Detachment of Chain-Forming Neuroblasts by Fyn-Mediated Control of cell-cell Adhesion in the Postnatal Brain.

In the rodent olfactory system, neuroblasts produced in the ventricular-subventricular zone of the postnatal brain migrate tangentially in chain-like cell aggregates toward the olfactory bulb (OB) through the rostral migratory stream (RMS). After reaching the OB, the chains are dissociated and the neuroblasts migrate individually and radially toward their final destination. The cellular and molecular mechanisms controlling cell-cell adhesion during this detachment remain unclear. Here we report that Fyn, a nonreceptor tyrosine kinase, regulates the detachment of neuroblasts from chains in the male and female mouse OB. By performing chemical screening and in vivo loss-of-function and gain-of-function experiments, we found that Fyn promotes somal disengagement from the chains and is involved in neuronal migration from the RMS into the granule cell layer of the OB. Fyn knockdown or Dab1 (disabled-1) deficiency caused p120-catenin to accumulate and adherens junction-like structures to be sustained at the contact sites between neuroblasts. Moreover, a Fyn and N-cadherin double-knockdown experiment indicated that Fyn regulates the N-cadherin-mediated cell adhesion between neuroblasts. These results suggest that the Fyn-mediated control of cell-cell adhesion is critical for the detachment of chain-forming neuroblasts in the postnatal OB.SIGNIFICANCE STATEMENT In the postnatal brain, newly born neurons (neuroblasts) migrate in chain-like cell aggregates toward their destination, where they are dissociated into individual cells and mature. The cellular and molecular mechanisms controlling the detachment of neuroblasts from chains are not understood. Here we show that Fyn, a nonreceptor tyrosine kinase, promotes the somal detachment of neuroblasts from chains, and that this regulation is critical for the efficient migration of neuroblasts to their destination. We further show that Fyn and Dab1 (disabled-1) decrease the cell-cell adhesion between chain-forming neuroblasts, which involves adherens junction-like structures. Our results suggest that Fyn-mediated regulation of the cell-cell adhesion of neuroblasts is critical for their detachment from chains in the postnatal brain.

Differential binding of anti-Reelin monoclonal antibodies reveals the characteristics of Reelin protein under various conditions.

Reelin is a large secreted protein that is essential for the development and function of the central nervous system. Dimerization and/or oligomerization is required for its biological activity, but the underlying mechanism is not fully understood. There are several widely used anti-Reelin antibodies and we noticed that their reactivity to monomeric or dimeric Reelin protein is different. We also found that their reactivity to Reelin in the solution or in fixed brain tissues also differs. Our results provide the information regarding how the N-terminal region of Reelin folds and contributes to the formation of higher order structure. We also provide a caveat that appropriate use of anti-Reelin antibody is necessary for quantitative analyses.

Reducing ADAMTS-3 Inhibits Amyloid ß Deposition in App Knock-in Mouse.

Reelin is a secreted protein that antagonizes the deposition and toxicity of amyloid ß peptide (Aß). Therefore, augmentation of Reelin activity may ameliorate Alzheimer's disease (AD). We have recently reported that a disintegrin and metalloproteinase with thrombospondin motifs 3 (ADAMTS-3) cleaves and inactivates Reelin in the mouse brain. In the present study, we investigated the effect of reducing ADAMTS-3 on deposition of Aß by crossbreeding drug-inducible ADAMTS-3 conditional knock-out (cKO) mice with "next-generation" AD model mice. We found that reducing ADAMTS-3 inhibited deposition of Aß significantly in AppNL-F mice, which produce human wild-type Aß. On the other hand, reducing ADAMTS-3 had no effect in AppNL-G-F mice, which produce the Arctic mutant Aß (E22G) that forms protofibrils more efficiently than does wild-type Aß. Thus, the findings suggest that the administration of an inhibitor against ADAMTS-3 will prevent the progression of AD pathology caused by deposition of wild-type Aß.

Secreted Metalloproteinase ADAMTS-3 Inactivates Reelin.

The secreted glycoprotein Reelin regulates embryonic brain development and adult brain functions. It has been suggested that reduced Reelin activity contributes to the pathogenesis of several neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease; however, noninvasive methods that can upregulate Reelin activity in vivo have yet to be developed. We previously found that the proteolytic cleavage of Reelin within Reelin repeat 3 (N-t site) abolishes Reelin activity in vitro, but it remains controversial as to whether this effect occurs in vivo Here we partially purified the enzyme that mediates the N-t cleavage of Reelin from the culture supernatant of cerebral cortical neurons. This enzyme was identified as a disintegrin and metalloproteinase with thrombospondin motifs-3 (ADAMTS-3). Recombinant ADAMTS-3 cleaved Reelin at the N-t site. ADAMTS-3 was expressed in excitatory neurons in the cerebral cortex and hippocampus. N-t cleavage of Reelin was markedly decreased in the embryonic cerebral cortex of ADAMTS-3 knock-out (KO) mice. Importantly, the amount of Dab1 and the phosphorylation level of Tau, which inversely correlate with Reelin activity, were significantly decreased in the cerebral cortex of ADAMTS-3 KO mice. Conditional KO mice, in which ADAMTS-3 was deficient only in the excitatory neurons of the forebrain, showed increased dendritic branching and elongation in the postnatal cerebral cortex. Our study shows that ADAMTS-3 is the major enzyme that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. Therefore, inhibition of ADAMTS-3 may be an effective treatment for neuropsychiatric and neurodegenerative disorders.SIGNIFICANCE STATEMENT ADAMTS-3 was identified as the protease that cleaves and inactivates Reelin in the cerebral cortex and hippocampus. ADAMTS-3 was expressed in the excitatory neurons of the embryonic and postnatal cerebral cortex and hippocampus. Cleavage by ADAMTS-3 is the major contributor of Reelin inactivation in vivo Tau phosphorylation was decreased and dendritic branching and elongation was increased in ADAMTS-3-deficient mice. Therefore, inhibition of ADAMTS-3 upregulates Reelin activity and may be a potential therapeutic strategy for the prevention or treatment of neuropsychiatric and neurodegenerative disorders, such as schizophrenia and Alzheimer's disease.

Reelin deficiency leads to aberrant lipid composition in mouse brain.

Reelin is a secreted protein essential for the development and function of the mammalian brain. The receptors for Reelin, apolipoprotein E receptor 2 and very low-density lipoprotein receptor, belong to the low-density lipoprotein receptor family, but it is not known whether Reelin is involved in the brain lipid metabolism. In the present study, we performed lipidomic analysis of the cerebral cortex of wild-type and Reelin-deficient (reeler) mice, and found that reeler mice exhibited several compositional changes in phospholipids. First, the ratio of phospholipids containing one saturated fatty acid (FA) and one docosahexaenoic acid (DHA) or arachidonic acid (ARA) decreased. Secondly, the ratio of phospholipids containing one monounsaturated FA (MUFA) and one DHA or ARA increased. Thirdly, the ratio of phospholipids containing 5,8,11-eicosatrienoic acid, or Mead acid (MA), increased. Finally, the expression of stearoyl-CoA desaturase-1 (SCD-1) increased. As the increase of MA is seen as an index of polyunsaturated FA (PUFA) deficiency, and the expression of SCD-1 is suppressed by PUFA, these results strongly suggest that the loss of Reelin leads to PUFA deficiency. Hence, MUFA and MA are synthesized in response to this deficiency, in part by inducing SCD-1 expression. This is the first report of changes of FA composition in the reeler mouse brain and provides a basis for further investigating the new role of Reelin in the development and function of the brain.

Importance of Reelin C-terminal region in the development and maintenance of the postnatal cerebral cortex and its regulation by specific proteolysis.

During brain development, Reelin exerts a variety of effects in a context-dependent manner, whereas its underlying molecular mechanisms remain poorly understood. We previously showed that the C-terminal region (CTR) of Reelin is required for efficient induction of phosphorylation of Dab1, an essential adaptor protein for canonical Reelin signaling. However, the physiological significance of the Reelin CTR in vivo remains unexplored. To dissect out Reelin functions, we made a knock-in (KI) mouse in which the Reelin CTR is deleted. The amount of Dab1, an indication of canonical Reelin signaling strength, is increased in the KI mouse, indicating that the CTR is necessary for efficient induction of Dab1 phosphorylation in vivo. Formation of layer structures during embryonic development is normal in the KI mouse. Intriguingly, the marginal zone (MZ) of the cerebral cortex becomes narrower at postnatal stages because upper-layer neurons invade the MZ and their apical dendrites are misoriented and poorly branched. Furthermore, Reelin undergoes proteolytic cleavage by proprotein convertases at a site located 6 residues from the C terminus, and it was suggested that this cleavage abrogates the Reelin binding to the neuronal cell membrane. Results from ectopic expression of mutant Reelin proteins in utero suggest that the dendrite development and maintenance of the MZ require Reelin protein with an intact CTR. These results provide a novel model regarding Reelin functions involving its CTR, which is not required for neuronal migration during embryonic stages but is required for the development and maintenance of the MZ in the postnatal cerebral cortex.

Cleavage within Reelin repeat 3 regulates the duration and range of the signaling activity of Reelin protein.

Reelin is a secreted glycoprotein that plays essential roles in the brain. Reelin is specifically cleaved at two distinct sites, called N-t and C-t, with the former being the major one. N-t cleavage can occur both in the extracellular space and in the endosomes, although the physiological importance of endosomal N-t cleavage has not been investigated. In this study, we first determined the exact N-t cleavage site catalyzed by a protease secreted by cerebral cortical neurons. Cleavage occurred between Pro-1244 and Ala-1245 within Reelin repeat 3. A Reelin mutant in which Pro-1244 was replaced with aspartate (Reelin-PD) was resistant to a protease secreted by cultured cerebral cortical neurons, and its biological activity stayed active longer than that of wild-type Reelin. Interestingly, Reelin-PD remained in the intracellular compartments longer than wild-type Reelin and persistently activated downstream signaling. Therefore, N-t cleavage of Reelin is required for halting the signaling machinery in the extracellular space as well as within endosomes of target neurons. We established a monoclonal antibody specific to uncleaved Reelin protein and found that it is localized in the vicinity of Reelin-producing cells, whereas the N-terminal fragment diffuses, or is transported, to distant regions. These data demonstrate that N-t cleavage of Reelin plays critical roles in regulating the duration and range of Reelin functions both in the extracellular milieu and in the intracellular compartments.

Exogenous Reelin modifies the migratory behavior of neurons depending on cortical location.

Malformations of cortical development can arise when projection neurons generated in the germinal zones fail to migrate properly into the cortical plate. This process is critically dependent on the Reelin glycoprotein, which when absent leads to an inversion of cortical layers and blurring of borders. Reelin has other functions including supporting neuron migration and maintaining their trajectories; however, the precise role on glial fiber-dependent or -independent migration of neurons remains controversial. In this study, we wish to test the hypothesis that migrating cortical neurons at different levels of the cortical wall have differential responses to Reelin. We exposed neurons migrating across the cortical wall to exogenous Reelin and monitored their migratory behavior using time-lapse imaging. Our results show that, in the germinal zones, exogenous Reelin retarded neuron migration and altered their trajectories. This behavior is in contrast to the response of neurons located in the intermediate zone (IZ), possibly because Reelin receptors are not expressed in this zone. In the reeler cortex, Reelin receptors are expressed in the IZ and exposure to exogenous Reelin was able to rescue the migratory defect. These studies demonstrate that migrating neurons have nonequivalent responses to Reelin depending on their location within the cortical wall.

Type 1 inositol trisphosphate receptor regulates cerebellar circuits by maintaining the spine morphology of purkinje cells in adult mice.

The structural maintenance of neural circuits is critical for higher brain functions in adulthood. Although several molecules have been identified as regulators for spine maintenance in hippocampal and cortical neurons, it is poorly understood how Purkinje cell (PC) spines are maintained in the mature cerebellum. Here we show that the calcium channel type 1 inositol trisphosphate receptor (IP3R1) in PCs plays a crucial role in controlling the maintenance of parallel fiber (PF)-PC synaptic circuits in the mature cerebellum in vivo. Significantly, adult mice lacking IP3R1 specifically in PCs (L7-Cre;Itpr1(flox/flox)) showed dramatic increase in spine density and spine length of PCs, despite having normal spines during development. In addition, the abnormally rearranged PF-PC synaptic circuits in mature cerebellum caused unexpectedly severe ataxia in adult L7-Cre;Itpr1(flox/flox) mice. Our findings reveal a specific role for IP3R1 in PCs not only as an intracellular mediator of cerebellar synaptic plasticity induction, but also as a critical regulator of PF-PC synaptic circuit maintenance in the mature cerebellum in vivo; this mechanism may underlie motor coordination and learning in adults.

Dab1-mediated colocalization of multi-adaptor protein CIN85 with Reelin receptors, ApoER2 and VLDLR, in neurons.

Reelin-Dab1 signaling is indispensable for proper positioning of neurons in mammalian brain. Reelin is a glycoprotein secreted from Cajal-Reztuis cells in marginal zone of cerebral cortex, and its receptors are Apolipoprotein E receptor 2 (ApoER2) or very low density lipoprotein receptor (VLDLR) expressed on migrating neurons. When Reelin binds to ApoER2 or VLDLR, an adaptor protein Dab1 bound to the receptors undergoes Tyr phosphorylation that is essential for Reelin signaling. We reported previously that Cdk5-p35 phosphorylates Dab1 at Ser400 and Ser491 and the phosphorylation regulates its binding to CIN85, which is an SH3-containing multiadaptor protein involved in endocytic downregulation of receptor-tyrosine kinases. However, the interaction of CIN85 with Dab1 has not been addressed in neurons. We examined here a possibility that CIN85 has a role in Reelin signaling. We found nonpho-sphorylated Dab1-mediated colocalization of CIN85 with ApoER2. The colocalization of CIN85 with ApoER2 was increased in neurons stimulated with Reelin repeats 3-6, an active Reelin fragment. The stimulation recruited CIN85 to domains in plasma membrane where it colocalized with ApoER2 and Dab1 and then to EEA1-labeled early endosomes in the cytoplasm. In addition, Tyr phosphorylation of Dab1 strengthened the binding to CIN85. These results suggest that CIN85 participates in Reelin signaling through the binding to Dab1.

Postnatal injection of Reelin protein into the cerebellum ameliorates the motor functions in reeler mouse.

Reelin is a large secreted protein important for brain development and functions. In both humans and mice, the lack of Reelin gene causes cerebellar hypoplasia and ataxia. Treatment against Reelin deficiency is currently unavailable. Here, we show that the injection of recombinant Reelin protein into the cerebellum of Reelin-deficient reeler mice at postnatal day 3 ameliorates the forelimb coordination and mice are noted to stand up along cage wall more frequently. A mutant Reelin protein resistant to proteases has no better effect than the wild-type Reelin. Such ameliorations were not observed when a mutant Reelin protein that does not bind to Reelin receptors was injected and the injection of Reelin protein did not ameliorate the behavior of Dab1-mutant yotari mice, indicating that its effect is dependent on the canonical Reelin receptor-Dab1 pathway. Additionally, a Purkinje cell layer in reeler mice was locally induced by Reelin protein injection. Our results indicate that the reeler mouse cerebellum retains the ability to react to Reelin protein in the postnatal stage and that Reelin protein has the potential to benefit Reelin-deficient patients.

Functional importance of covalent homodimer of reelin protein linked via its central region.

Reelin is a 3461-residue secreted glycoprotein that plays a critical role in brain development through its action on target neurons. Although it is known that functional reelin protein exists as multimer formed by interchain disulfide bond(s) as well as through non-covalent interactions, the chemical nature of the multimer assembly has been elusive. In the present study, we identified, among 122 cysteines present in full-length reelin, the single critical cysteine residue (Cys(2101)) responsible for the covalent multimerization. C2101A mutant reelin failed to assemble into disulfide-bonded multimers, whereas it still exhibited non-covalently associated high molecular weight oligomeric states in solution. Detailed analysis of tryptic fragments produced from the purified reelin proteins revealed that the minimum unit of the multimer is a homodimeric reelin linked via Cys(2101) present in the central region and that this cysteine does not connect to the N-terminal region of reelin, which had been postulated as the primary oligomerization domain. A surface plasmon resonance binding assay confirmed that C2101A mutant reelin retained binding capability toward two neuronal receptors apolipoprotein E receptor 2 and very low density lipoprotein receptor. However, it failed to show signaling activity in the assay using the cultured neurons. These results indicate that an intact higher order architecture of reelin multimer maintained by both Cys(2101)-mediated homodimerization and other non-covalent association present elsewhere in the reelin primary structure are essential for exerting its full biological activity.

Differential localization of sphingomyelin synthase isoforms in neurons regulates sphingomyelin cluster formation.

Sphingomyelin (SM) plays important roles in regulating structure and function of plasma membrane, but how intracellular localization of SM is regulated in neuronal cells is not understood. Here we show that two isoforms of SM synthase (SMS) are differentially expressed in neuronal subtypes and that only SMS2 proteins localize in neurites of hippocampal neurons. Moreover, SMS proteins induce Lysenin-binding SM clusters exclusively in their vicinity although neurons hardly contain such cluster under control condition. These findings indicate three important notions about SM metabolism in neurons. First, the activity of SMS is the rate-limiting step of SM cluster formation. Second, the SM content or clustering can be modulated by SMS activity. Third, SMS1 and SMS2 play distinct roles in regulating local SM clustering. Particularly, SMS2, rather than SMS1, is likely to be the major enzyme that is important for SM synthesis in the long neurites and its tip, the growth cone.