Leann H. Brennaman

NCAM in Neuropsychiatric and Neurodegenerative Disorders

The neural cell adhesion molecule (NCAM) has roles in normal brain development, including axonal/dendritic growth and branching and synaptic plasticity. A growing body of evidence has implicated NCAM as a susceptible risk for neuropsychiatric disorders such as schizophrenia, bipolar disorder, depression, and anxiety disorder, as well as the most prevalent neurodegenerative disease, Alzheimer’s disease. While individuals with these diseases vary in symptoms, age of onset, treatment, and neuronal systems affected, they share cognitive dysfunction as a core feature. This review will highlight the evidence for NCAM in the cause or progression of each of these disorders.

L1 and CHL1 Cooperate in Thalamocortical Axon Targeting

Neural cell adhesion molecule close homolog of L1 (CHL1) is a regulator of topographic targeting of thalamic axons to the somatosensory cortex (S1) but little is known about its cooperation with other L1 class molecules. To investigate this, CHL1(-/-)/L1(-/y) double mutant mice were generated and analyzed for thalamocortical axon topography. Double mutants exhibited a striking posterior shift of axons from motor thalamic nuclei to the visual cortex (V1), which was not observed in single mutants. In wild-type (WT) embryos, L1 and CHL1 were coexpressed in the dorsal thalamus (DT) and on fibers along the thalamocortical projection in the ventral telencephalon and cortex. L1 and CHL1 colocalized on growth cones and neurites of cortical and thalamic neurons in culture. Growth cone collapse assays with WT and mutant neurons demonstrated a requirement for L1 and CHL1 in repellent responses to EphrinA5, a guidance factor for thalamic axons. L1 coimmunoprecipitated with the principal EphrinA5 receptors expressed in the DT (EphA3, EphA4, and EphA7), whereas CHL1 associated selectively with EphA7. These results implicate a novel mechanism in which L1 and CHL1 interact with individual EphA receptors and cooperate to guide subpopulations of thalamic axons to distinct neocortical areas essential for thalamocortical connectivity.

NrCAM deletion causes topographic mistargeting of thalamocortical axons to the visual cortex and disrupts visual acuity.

NrCAM is a neural cell adhesion molecule of the L1 family that has been linked to autism spectrum disorders, a disease spectrum in which abnormal thalamocortical connectivity may contribute to visual processing defects. Here we show that NrCAM interaction with neuropilin-2 (Npn-2) is critical for semaphorin 3F (Sema3F)-induced guidance of thalamocortical axon subpopulations at the ventral telencephalon (VTe), an intermediate target for thalamic axon sorting. Genetic deletion of NrCAM or Npn-2 caused contingents of embryonic thalamic axons to misproject caudally in the VTe. The resultant thalamocortical map of NrCAM-null mutants showed striking mistargeting of motor and somatosensory thalamic axon contingents to the primary visual cortex, but retinogeniculate targeting and segregation were normal. NrCAM formed a molecular complex with Npn-2 in brain and neural cells, and was required for Sema3F-induced growth cone collapse in thalamic neuron cultures, consistent with a vital function for NrCAM in Sema3F-induced axon repulsion. NrCAM-null mice displayed reduced responses to visual evoked potentials recorded from layer IV in the binocular zone of primary visual cortex (V1), particularly when evoked from the ipsilateral eye, indicating abnormal visual acuity and ocularity. These results demonstrate that NrCAM is required for normal maturation of cortical visual acuity, and suggest that the aberrant projection of thalamic motor and somatosensory axons to the visual cortex in NrCAM-null mutant mice impairs cortical functions.

Developmental regulation of GABAergic interneuron branching and synaptic development in the prefrontal cortex by soluble neural cell adhesion molecule

Neural cell adhesion molecule, NCAM, is an important regulator of neuronal process outgrowth and synaptic plasticity. Transgenic mice that overexpress the soluble NCAM extracellular domain (NCAM-EC) have reduced GABAergic inhibitory and excitatory synapses, and altered behavioral phenotypes. Here, we examined the role of dysregulated NCAM shedding, modeled by overexpression of NCAM-EC, on development of GABAergic basket interneurons in the prefrontal cortex. NCAM-EC overexpression disrupted arborization of basket cells during the major period of axon/dendrite growth, resulting in decreased numbers of GAD65- and synaptophysin-positive perisomatic synapses. NCAM-EC transgenic protein interfered with interneuron branching during early postnatal stages when endogenous polysialylated (PSA) NCAM was converted to non-PSA isoforms. In cortical neuron cultures, soluble NCAM-EC acted as a dominant inhibitor of NCAM-dependent neurite branching and outgrowth. These findings suggested that excess soluble NCAM-EC reduces perisomatic innervation of cortical neurons by perturbing axonal/dendritic branching during cortical development.

Developmental Regulation of Neural Cell Adhesion Molecule in Human Prefrontal Cortex

Neural cell adhesion molecule (NCAM) is a membrane-bound cell recognition molecule that exerts important functions in normal neurodevelopment including cell migration, neurite outgrowth, axon fasciculation, and synaptic plasticity. Alternative splicing of NCAM mRNA generates three main protein isoforms: NCAM-180, -140, and -120. Ectodomain shedding of NCAM isoforms can produce an extracellular 105-115 kilodalton soluble neural cell adhesion molecule fragment (NCAM-EC) and a smaller intracellular cytoplasmic fragment (NCAM-IC). NCAM also undergoes a unique post-translational modification in brain by the addition of polysialic acid (PSA)-NCAM. Interestingly, both PSA-NCAM and NCAM-EC have been implicated in the pathophysiology of schizophrenia. The developmental expression patterns of the main NCAM isoforms and PSA-NCAM have been described in rodent brain, but no studies have examined NCAM expression across human cortical development. Western blotting was used to quantify NCAM in human postmortem prefrontal cortex in 42 individuals ranging in age from mid-gestation to early adulthood. Each NCAM isoform (NCAM-180, -140, and -120), post-translational modification (PSA-NCAM) and cleavage fragment (NCAM-EC and NCAM-IC) demonstrated developmental regulation in frontal cortex. NCAM-180, -140, and -120, as well as PSA-NCAM, and NCAM-IC all showed strong developmental regulation during fetal and early postnatal ages, consistent with their identified roles in axon growth and plasticity. NCAM-EC demonstrated a more gradual increase from the early postnatal period to reach a plateau by early adolescence, potentially implicating involvement in later developmental processes. In summary, this study implicates the major NCAM isoforms, PSA-NCAM and proteolytically cleaved NCAM in pre- and postnatal development of the human prefrontal cortex. These data provide new insights on human cortical development and also provide a basis for how altered NCAM signaling during specific developmental intervals could affect synaptic connectivity and circuit formation, and thereby contribute to neurodevelopmental disorders.

Transgenic mice overexpressing the extracellular domain of NCAM are impaired in working memory and cortical plasticity.

The neural cell adhesion molecule, NCAM, is a pivotal regulator of neural development, with key roles in axonal and dendritic growth and synaptic plasticity. Alterations in NCAM expression or proteolytic cleavage have been linked to human neuropsychiatric disorders such as schizophrenia, bipolar disorder and Alzheimers disease, and may contribute to cognitive dysfunction. We have generated mice overexpressing the NCAM extracellular (EC) proteolytic cleavage fragment which has been reported to be increased in schizophrenic versus normal brains. These mice show impaired GABAergic innervation and reduced number of apical dendritic spines on pyramidal neurons in the prefrontal cortex (PFC). Here, these NCAM-EC transgenic mice were subjected to behavioral tasks and electrophysiological measurements to determine the impact of structural abnormalities in the PFC on synaptic and cognitive functions. NCAM-EC mice exhibited impaired working memory in a delayed non-match-to-sample task, which requires PFC function, but showed no differences in anxiety, olfactory abilities, or sociability. Transgenic mice displayed impaired long- and short-term potentiation in the PFC but normal synaptic plasticity in the hippocampus, suggesting that the abnormal synaptic innervation in NCAM-EC mice impairs PFC plasticity and alters working memory. These findings may have implications for cognitive dysfunctions observed in neuropsychiatric disorders.

EphrinA/EphA-induced ectodomain shedding of neural cell adhesion molecule regulates growth cone repulsion through ADAM10 metalloprotease

EphrinA/EphA‐dependent axon repulsion is crucial for synaptic targeting in developing neurons but downstream molecular mechanisms remain obscure. Here, it is shown that ephrinA5/EphA3 triggers proteolysis of the neural cell adhesion molecule (NCAM) by the metalloprotease a disintegrin and metalloprotease (ADAM)10 to promote growth cone collapse in neurons from mouse neocortex. EphrinA5 induced ADAM10 activity to promote ectodomain shedding of polysialic acid‐NCAM in cortical neuron cultures, releasing a ~ 250 kDa soluble fragment consisting of most of its extracellular region. NCAM shedding was dependent on ADAM10 and EphA3 kinase activity as shown in HEK293T cells transfected with dominant negative ADAM10 and kinase‐inactive EphA3 (K653R) mutants. Purified ADAM10 cleaved NCAM at a sequence within the E‐F loop of the second fibronectin type III domain (Leu671‐Lys672/Ser673‐Leu674) identified by mass spectrometry. Mutations of NCAM within the ADAM10 cleavage sequence prevented EphA3‐induced shedding of NCAM in HEK293T cells. EphrinA5‐induced growth cone collapse was dependent on ADAM10 activity, was inhibited in cortical cultures from NCAM null mice, and was rescued by WT but not ADAM10 cleavage site mutants of NCAM. Regulated proteolysis of NCAM through the ephrin5/EphA3/ADAM10 mechanism likely impacts synapse development, and may lead to excess NCAM shedding when disrupted, as implicated in neurodevelopmental disorders such as schizophrenia.

Neural cell adhesion molecule NrCAM regulates semaphorin 3F-induced dendritic spine remodeling

Neuron-glial related cell adhesion molecule (NrCAM) is a regulator of axon growth and repellent guidance, and has been implicated in autism spectrum disorders. Here a novel postsynaptic role for NrCAM in Semaphorin3F (Sema3F)-induced dendritic spine remodeling was identified in pyramidal neurons of the primary visual cortex (V1). NrCAM localized to dendritic spines of star pyramidal cells in postnatal V1, where it was coexpressed with Sema3F. NrCAM deletion in mice resulted in elevated spine densities on apical dendrites of star pyramidal cells at both postnatal and adult stages, and electron microscopy revealed increased numbers of asymmetric synapses in layer 4 of V1. Whole-cell recordings in cortical slices from NrCAM-null mice revealed increased frequency of mEPSCs in star pyramidal neurons. Recombinant Sema3F-Fc protein induced spine retraction on apical dendrites of wild-type, but not NrCAM-null cortical neurons in culture, while re-expression of NrCAM rescued the spine retraction response. NrCAM formed a complex in brain with Sema3F receptor subunits Neuropilin-2 (Npn-2) and PlexinA3 (PlexA3) through an Npn-2-binding sequence (TARNER) in the extracellular Ig1 domain. A trans heterozygous genetic interaction test demonstrated that Sema3F and NrCAM pathways interacted in vivo to regulate spine density in star pyramidal neurons. These findings reveal NrCAM as a novel postnatal regulator of dendritic spine density in cortical pyramidal neurons, and an integral component of the Sema3F receptor complex. The results implicate NrCAM as a contributor to excitatory/inhibitory balance in neocortical circuits.

Neural cell adhesion molecule, NCAM, regulates thalamocortical axon pathfinding and the organization of the cortical somatosensory representation in mouse.

To study the potential role of neural cell adhesion molecule (NCAM) in the development of thalamocortical (TC) axon topography, wild type, and NCAM null mutant mice were analyzed for NCAM expression, projection, and targeting of TC afferents within the somatosensory area of the neocortex. Here we report that NCAM and its α-2,8-linked polysialic acid (PSA) are expressed in developing TC axons during projection to the neocortex. Pathfinding of TC axons in wild type and null mutant mice was mapped using anterograde DiI labeling. At embryonic day E16.5, null mutant mice displayed misguided TC axons in the dorsal telencephalon, but not in the ventral telencephalon, an intermediate target that initially sorts TC axons toward correct neocortical areas. During the early postnatal period, rostrolateral TC axons within the internal capsule along the ventral telencephalon adopted distorted trajectories in the ventral telencephalon and failed to reach the neocortex in NCAM null mutant animals. NCAM null mutants showed abnormal segregation of layer IV barrels in a restricted portion of the somatosensory cortex. As shown by Nissl and cytochrome oxidase staining, barrels of the anterolateral barrel subfield (ALBSF) and the most distal barrels of the posteromedial barrel subfield (PMBSF) did not segregate properly in null mutant mice. These results indicate a novel role for NCAM in axonal pathfinding and topographic sorting of TC axons, which may be important for the function of specific territories of sensory representation in the somatosensory cortex.

Neuron glia-related cell adhesion molecule (NrCAM) promotes topographic retinocollicular mapping.

NrCAM (Neuron-glial related cell adhesion molecule), a member of the L1 family of cell adhesion molecules, reversibly binds ankyrin and regulates axon growth, but it has not been studied for a role in retinotopic mapping. During development of retino-collicular topography, NrCAM was expressed uniformly in retinal ganglion cells (RGCs) along both mediolateral and anteroposterior retinal axes, and was localized on RGC axons within the optic tract and superior colliculus (SC). Anterograde tracing of RGC axons in NrCAM null mutant mice at P10, when the map resembles its mature form, revealed laterally displaced ectopic termination zones (eTZs) of axons from the temporal retina, indicating defective mediolateral topography, which is governed by ephrinB/EphBs. Axon tracing at P2 revealed that interstitial branch orientation of ventral-temporal RGC axons in NrCAM null mice was compromised in the medial direction, likely accounting for displacement of eTZs. A similar retinocollicular targeting defect in EphB mutant mice suggested that NrCAM and EphB interact to regulate mediolateral retino-collicular targeting. We found that EphB2 tyrosine kinase but not an EphB2 kinase dead mutant, phosphorylated NrCAM at a conserved tyrosine residue in the FIGQY ankyrin binding motif, perturbing ankyrin recruitment in NrCAM transfected HEK293 cells. Furthermore, the phosphorylation of NrCAM at FIGQY in SC was decreased in EphB1/3 and EphB1/2/3 null mice compared to WT, while phospho-FIGQY of NrCAM in SC was increased in EphB2 constitutively active (F620D/F620D) mice. These results demonstrate that NrCAM contributes to mediolateral retinocollicular axon targeting by regulating RGC branch orientation through a likely mechanism in which ephrinB/EphB phosphorylates NrCAM to modulate linkage to the actin cytoskeleton.