Aurea Pimenta

THE LIMBIC SYSTEM-ASSOCIATED MEMBRANE PROTEIN IS AN IG SUPERFAMILY MEMBER THAT MEDIATES SELECTIVE NEURONAL GROWTH AND AXON TARGETING

The formation of brain circuits requires molecular recognition between functionally related neurons. We report the cloning of a molecule that participates in these interactions. The limbic system-associated membrane protein (LAMP) is an immunoglobulin (Ig) superfamily member with 3 Ig domains and a glycosyl-phosphatidylinositol anchor. In the developing forebrain, lamp is expressed mostly by neurons comprising limbic-associated cortical and subcortical regions that function in cognition, emotion, memory, and learning. The unique distribution of LAMP reflects its functional specificity. LAMP-transfected cells selectively facilitate neurite outgrowth of primary limbic neurons. Most striking, administration of anti-LAMP in vivo results in abnormal growth of the mossy fiber projection from developing granule neurons in the dentate gyrus of the hippocampal formation, suggesting that LAMP is essential for proper targeting of this pathway. Rather than being a general guidance cue, LAMP likely serves as a recognition molecule for the formation of limbic connections.

The predominant form in which neurofilament subunits undergo axonal transport varies during axonal initiation, elongation, and maturation.

The forms in which neurofilament (NF) subunits undergo axonal transport is controversial. Recent studies from have provided real-time visualization of the slow axonal transport of NF subunits by transfecting neuronal cultures with constructs encoding green fluorescent protein (GFP)-conjugated NF-M subunits. In our studies in differentiated NB2a/d1 cells, the majority NF subunits underwent transport in the form of punctate NF precursors, while studies in cultured neurons have demonstrated transport of NF subunits in predominantly filamentous form. Although different constructs were used in these studies, transfection of the same cultured neurons with our construct yielded the filamentous pattern observed by others, while transfection of our cultures with their construct generated punctate structures, confirming that the observed differences did not reflect variances in assembly-competence among the constructs. Manipulation of intracellular kinase, phosphatase, and protease activities shifted the predominant form of GFP-conjugated subunits between punctate and filamentous, confirming, as shown previously for vimentin, that punctate structures represent precursors for intermediate filament formation. Since these prior studies were conducted at markedly differing neuronal differentiation states, we tested the alternate hypothesis that these differing results reflected developmental alterations in NF dynamics that accompany various stages of neuritogenesis. We conducted time-course analyses of transfected NB2a/d1 cells, including monitoring of transfected cells over several days, as well as transfecting cells at varying intervals prior to and following induction of differentiation and axonal neurite outgrowth. GFP-conjugated subunits were predominantly filamentous during the period of most robust axonal outgrowth and NF accumulation, and presented a mixed profile of punctate and filamentous forms prior to neuritogenesis and following the developmental slowing of neurite outgrowth. These analyses demonstrate that NF subunits are capable of undergoing axonal transport in multiple forms, and that the predominant form in which NF subunits undergo axonal transport varies in accord with the rate of axonal elongation and accumulation of NFs within developing axons.

Mitogen-activated protein kinase regulates neurofilament axonal transport

Mitogen-activated protein kinase (MAP) kinase plays a pivotal role in the development of the nervous system by mediating both neurogenesis and neuronal differentiation. Here we examined whether p42/44 MAP kinase plays a role in axonal transport and the organization of neurofilaments (NFs) in axonal neurites. Dominant-negative p42/44 MAP kinase, anti-MAP kinase antisense oligonucleotides and the MAP kinase inhibitor PD98059 all reduced NF phospho-epitopes and inhibited anterograde NF axonal transport of GFP-tagged NF subunits in differentiated NB2a/d1 neuroblastoma cells. Expression of constitutively active MAP kinase and intracellular delivery of active enzyme increased NF phospho-epitopes and increased NF axonal transport. Longer treatment with PD98059 shifted NF transport from anterograde to retrograde. PD98059 did not inhibit overall axonal transport nor compromise overall axonal architecture or composition. The p38 MAP kinase inhibitor SB202190 did not inhibit NF transport whereas the kinase inhibitor olomoucine inhibited both NF and mitochondrial transport. Axonal transport of NFs containing NF-H whose C-terminal region was mutated to mimic extensive phosphorylation was substantially less affected by PD98059 compared to a wild-type construct. These data suggest that p42/44 MAP kinase regulates NF anterograde transport by NF C-terminal phosphorylation. MAP kinase may therefore stabilize developing axons by promoting the accumulation of NFs within growing axonal neurites.

Dynamic expression suggests multiple roles of the eph family receptor brain-specific kinase (Bsk) during mouse neurogenesis

The eph family ligands and receptors have been implicated in mediating topographic neuron-target interactions. We recently isolated Bsk, a new member of the eph family receptors, and showed that it is expressed primarily in the brain. To investigate the role of Bsk in the development of the nervous system, we examined the temporal and spatial patterns of Bsk expression using in situ hybridization. We report here that Bsk expression exhibits dynamic changes during embryogenesis. In early embryos, Bsk is widely transcribed in the nervous system, including the forebrain, midbrain, hindbrain and spinal cord. Bsk expression in the midbrain, hindbrain and spinal cord, however, gradually decreases while in the forebrain increases over time. By embryonic day 18, the most intense Bsk expression was found in the limbic system. High levels of the expression in the limbic system persisted throughout post-natal development and remained stable in the adult up to 24 month. The topography of Bsk expression is in the form of gradients in several regions of the brain, including the lateral septum, spinal cord, as well as the hippocampus. Selective expression was also observed in Purkinje cells. Our findings on the topography of Bsk expression provide support to potential roles of Bsk in topographic projection. Our analyses further suggest that there may be other novel functions of Bsk in early neurogenesis in addition to potential roles in topographic mapping.

Neurofilament Transport Is Dependent on Actin and Myosin

Real-time analyses have revealed that some newly synthesized neurofilament (NF) subunits translocate into and along axonal neurites by moving along the inner plasma membrane surface, suggesting that they may translocate against the submembrane actin cortex. We therefore examined whether or not NF axonal transport was dependent on actin and myosin. Perturbation of filamentous actin in NB2a/d1 cells with cytochalasin B inhibited translocation of subunits into axonal neurites and inhibited bidirectional translocation of NF subunits within neurites. Intravitreal injection of cytochalasin B inhibited NF axonal transport in optic axons in a dose-response manner. NF subunits were coprecipitated from NB2a/d1 cells by an anti-myosin antibody, and myosin colocalized with NFs in immunofluorescent analyses. The myosin light chain kinase inhibitor ML-7 and the myosin ATPase inhibitor 2,3-butanedione-2-monoxime perturbed NF translocation within NB2a/d1 axonal neurites. These findings suggest that some NF subunits may undergo axonal transport via myosin-mediated interactions with the actin cortex.

Increased transcription and activity of glutathione synthase in response to deficiencies in folate, vitamin E, and apolipoprotein E

Oxidative stress is a major contributing factor in neurodegeneration and can arise from dietary, environmental, and genetic sources. Here we examine the separate and combined impact of deprivation of folate and vitamin E, coupled with dietary iron as a prooxidant, on normal mice and transgenic mice lacking apolipoprotein E (ApoE–/– mice). Both mouse strains exhibited increased levels of glutathione when deprived of folate and vitamin E, but a substantial further increase was observed in ApoE–/– mice. To determine the mechanism(s) underlying this increase, we quantified transcription and activity of glutathione synthase (GS). Both normal and ApoE–/– mice demonstrated increased GS activity when deprived of folate and vitamin E. However, transcription was increased only in ApoE–/– mice deprived of folate and vitamin E. These findings demonstrate that deficiency in one gene can result in compensatory up‐regulation in a second relevant gene and, furthermore, indicate that compensation for oxidative stress can occur in brain tissue at epigenetic and genetic levels depending on the nature and/or extent of oxidative stress.

Polymorphic GGC repeat differentially regulates human reelin gene expression levels

Summary.The human gene encoding Reelin (RELN), a pivotal protein in neurodevelopment, includes a polymorphic GGC repeat in its 5′ untranslated region (UTR). CHO cells transfected with constructs encompassing the RELN 5′UTR with 4-to-13 GGC repeats upstream of the luciferase reporter gene show declining luciferase activity with increasing GGC repeat number (P < 0.005), as predicted by computer-based simulations. Conversely, RELN 5′UTR sequences boost reporter gene expression above control levels in neuronal SN56 and N2A cell lines, but 12- and 13-repeat alleles still yield 50–60% less luciferase activity compared to the more common 8- and 10-repeat alleles (P < 0.0001). RELN “long” GGC alleles significantly blunt gene expression and may, through this effect, confer vulnerability to human disorders, such as schizophrenia and autism.

cDNA cloning and structural analysis of the human limbic-system-associated membrane protein (LAMP)

The limbic-system-associated membrane protein (LAMP) is a 64-68-kDa neuronal surface glycoprotein distributed in cortical and subcortical regions of the limbic system. The human LAMP gene was cloned by RT-PCR using human cerebral cortex mRNA and oligodeoxyribonucleotide (oligo) primers derived from the rat lamp cDNA sequence. The human and rat LAMP cDNAs showed 94% identity at the nucleotide (nt) level, and the encoded 338-amino-acid (aa) polypeptides shared 99% sequence identity. All the important features of LAMP were conserved: (i) the deduced aa sequence reflecting a glycosyl-phosphatidylinositol (GPI)-anchor, (ii) eight putative N-linked glycosylation sites, and (iii) conserved pairs of Cys forming three internal repeats characteristic of the immunoglobulin superfamily (IgSF). Northern blot analysis indicated the presence of two mRNA transcripts in the human brain of a size identical to those identified in adult rat brain. These data indicate that LAMP is a highly conserved new member of the IgSF which, together with the opioid-binding cell adhesion molecule (OBCAM) and neurotrimin, comprises a new subfamily that has been designated as IgLONs. With a unique distribution in limbic structures, LAMP may play an important role in limbic system development and function, as suggested by previous in vitro and in vivo functional studies.

Genetic deletion of Lsamp causes exaggerated behavioral activation in novel environments.

The limbic system-associated membrane protein (LAMP) is a GPI-anchored cell adhesion molecule expressed heavily in limbic and limbic-associated regions of the developing and adult brain. Experimental studies show that LAMP promotes the growth of limbic neurons and guides the projections of limbic fibers. In order to examine the functional consequences of disrupting limbic circuit assembly, we generated a mouse line in which the Lsamp gene encoding LAMP was deleted. Basic neuroanatomical organization and sensory and motor development are normal in Lsamp(-/-) mice. The most profound change in behavior in both male and female Lsamp(-/-) mice is a heightened reactivity to novelty exhibited in several behavioral tests. Lsamp(-/-) mice display hyperactivity in a novel arena and both sexes habituate to the same activity levels as their wild type littermates, but at different rates. In the elevated plus maze, Lsamp(-/-) mice exhibit increased total arm entries, with a bias towards the open arms; they spend more time in the open arms and have a substantial increase in the amount of risk assessment in unprotected areas of the maze. In the y-maze, Lsamp(-/-) mice exhibit characteristic hyperactivity and a decreased level of spontaneous alternation during the period when their novelty-induced hyperactivity is at its peak. We hypothesize that Lsamp(-/-) mice may not simply exhibit a decrease in anxiety, but may have a heightened, and possibly maladaptive, response to novel environmental stressors. Genetic deletion of Lsamp may thus cause circumscribed changes in the fine connectivity of specific circuits that underlie these behaviors.

Loss of limbic system-associated membrane protein leads to reduced hippocampal mineralocorticoid receptor expression, impaired synaptic plasticity and spatial memory deficit

BACKGROUND The limbic system-associated membrane protein (LAMP) promotes development of neurons of limbic origin. We have previously shown that genetic deletion of LAMP results in heightened reactivity to novelty and reduced anxiety-like behaviors in mice. Here, we demonstrate a critical role of LAMP in hippocampal-dependent synaptic physiology and behavior. METHODS We tested spatial memory performance, hippocampal synaptic plasticity, and stress-related modalities in Lsamp(-/-) mice and their littermate control mice. RESULTS Lsamp(-/-) mice exhibit a pronounced deficit in spatial memory acquisition and poorly sustained CA1 long-term potentiation. We found reduced expression of mineralocorticoid receptor (MR) transcripts in the hippocampus and reduction in the corticosterone-induced, MR-mediated nongenomic modulatory effects on CA1 synaptic transmission. Importantly, the impaired long-term potentiation in Lsamp(-/-) mice can be rescued by stress-like levels of corticosterone in a MR-dependent manner. CONCLUSIONS Our study reveals a novel functional relationship between a cell adhesion molecule enriched in developing limbic circuits, glucocorticoid receptors, and cognitive functioning.