Juha Kuja-Panula

AMIGO, a transmembrane protein implicated in axon tract development, defines a novel protein family with leucine-rich repeats

Ordered differential display identified a novel sequence induced in neurons by the neurite-promoting protein amphoterin. We named this gene amphoterin-induced gene and ORF (AMIGO), and also cloned two other novel genes homologous to AMIGO (AMIGO2 and AMIGO3). Together, these three AMIGOs form a novel family of genes coding for type I transmembrane proteins which contain a signal sequence for secretion and a transmembrane domain. The deduced extracellular parts of the AMIGOs contain six leucine-rich repeats (LRRs) flanked by cysteine-rich LRR NH2- and COOH-terminal domains and by one immunoglobulin domain close to the transmembrane region. A substrate-bound form of the recombinant AMIGO ectodomain promoted prominent neurite extension in hippocampal neurons, and in solution, the same AMIGO ectodomain inhibited fasciculation of neurites. A homophilic and heterophilic binding mechanism is shown between the members of the AMIGO family. Our results suggest that the members of the AMIGO protein family are novel cell adhesion molecules among which AMIGO is specifically expressed on fiber tracts of neuronal tissues and participates in their formation.

Increased expression of the dyslexia candidate gene DCDC2 affects length and signaling of primary cilia in neurons.

DCDC2 is one of the candidate susceptibility genes for dyslexia. It belongs to the superfamily of doublecortin domain containing proteins that bind to microtubules, and it has been shown to be involved in neuronal migration. We show that the Dcdc2 protein localizes to the primary cilium in primary rat hippocampal neurons and that it can be found within close proximity to the ciliary kinesin-2 subunit Kif3a. Overexpression of DCDC2 increases ciliary length and activates Shh signaling, whereas downregulation of Dcdc2 expression enhances Wnt signaling, consistent with a functional role in ciliary signaling. Moreover, DCDC2 overexpression in C. elegans causes an abnormal neuronal phenotype that can only be seen in ciliated neurons. Together our results suggest a potential role for DCDC2 in the structure and function of primary cilia.

Merlin Links to the cAMP Neuronal Signaling Pathway by Anchoring the RIβ Subunit of Protein Kinase A

The cAMP-protein kinase A (PKA) pathway, important in neuronal signaling, is regulated by molecules that bind and target PKA regulatory subunits. Of four regulatory subunits, RIβ is most abundantly expressed in brain. The RIβ knockout mouse has defects in hippocampal synaptic plasticity, suggesting a role for RIβ in learning and memory-related functions. Molecules that interact with or regulate RIβ are still unknown. We identified the neurofibromatosis 2 tumor suppressor protein merlin (schwannomin), a molecule related to the ezrin-radixin-moesin family of membrane-cytoskeleton linker proteins, as a binding partner for RIβ. Merlin and RIβ demonstrated a similar expression pattern in central nervous system neurons and an overlapping subcellular localization in cultured hippocampal neurons and transfected cells. The proteins were coprecipitated from brain lysates by cAMP-agarose and coimmunoprecipited from cellular lysates with specific antibodies. In vitro binding studies verified that the interaction is direct. The interaction appeared to be under conformational regulation and was mediated via the α-helical region of merlin. Sequence comparison between merlin and known PKA anchoring proteins identified a conserved α-helical PKA anchoring protein motif in merlin. These results identify merlin as the first neuronal binding partner for PKA-RIβ and suggest a novel function for merlin in connecting neuronal cytoskeleton to PKA signaling.

RAGE-mediated cell signaling.

RAGE (receptor for advanced glycation end products) is a multi-ligand receptor that belongs to the immunoglobulin superfamily of transmembrane proteins. RAGE binds AGEs (advanced glycation end products), HMGB1 (high-mobility group box-1; also designated as amphoterin), members of the S100 protein family, glycosaminoglycans and amyloid β peptides. Recent studies using tools of structural biology have started to unravel common molecular patterns in the diverse set of ligands recognized by RAGE. The distal Ig domain (V1 domain) of RAGE has a positively charged patch, the geometry of which fits to anionic surfaces displayed at least in a proportion of RAGE ligands. Association of RAGE to itself, to HSPGs (heparan sulfate proteoglycans), and to Toll-like receptors in the cell membrane plays a key role in cell signaling initiated by RAGE ligation. Ligation of RAGE activates cell signaling pathways that regulate migration of several cell types. Furthermore, RAGE ligation has profound effects on the transcriptional profile of cells. RAGE signaling has been mainly studied as a pathogenetic factor of several diseases, where acute or chronic inflammation plays a role. Recent studies have suggested a physiological role for RAGE in normal lung function and in neuronal signaling.

High Mobility Group Box-1 (HMGB1; Amphoterin) Is Required for Zebrafish Brain Development

Hmgb1 (high mobility group box-1; amphoterin) is highly expressed in brain during early development of vertebrate and nonvertebrate species. However, its role in brain development remains elusive. Here we have cloned the zebrafish Hmgb1 and specifically manipulated Hmgb1 expression using injection of morpholino antisense oligonucleotides or Hmgb1 cRNA. The HMGB1 knockdown morphants produced by injection of three different morpholino oligonucleotides display a characteristic phenotype with smaller size, smaller brain width, and shorter distance between the eyes. Closer examination of the phenotype reveals severe defects in the development of the forebrain that largely lacks catecholaminergic neural networks. The HMGB1 morphant is deficient in survival and proliferation of neural progenitors and displays fewer cell groups expressing the transcription factor Pax6a in the forebrain and aberrant Wnt8 signaling. The mechanism of HMGB1-dependent progenitor survival involves the neuronal transmembrane protein AMIGO (amphoterin-induced gene and orf), the expression of which is regulated by HMGB1 in vivo. Our data demonstrate that HMGB1 is a critical factor for brain development, enabling survival and proliferation of neural progenitors that will form the forebrain structures.

Crystal structure and role of glycans and dimerization in folding of neuronal leucine-rich repeat protein AMIGO-1.

AMIGO-1 is the parent member of a novel family of three cell surface leucine-rich repeat (LRR) proteins. Its expression is induced by the binding of HMGB1 (high-mobility group box 1 protein) to RAGE (receptor for advanced glycation end products) on neurons. Binding of HMGB1 to RAGE is known to have a direct effect on cellular growth regulation and mobility, and AMIGO-1 directly supports growth of neuronal processes and fasciculation of neurites. In addition, the second member of the AMIGO-family, AMIGO-2, has been implicated in adhesion of tumor cells in adenocarcinoma and survival of neurons. We have determined the crystal structure of AMIGO-1 at 2.0 Å resolution, which reveals a typical cell surface LRR domain arrangement with N- and C-terminal capping domains with disulfide bridges, followed by a C2-type Ig domain. AMIGO-1 is a dimer, with the LRR regions forming the dimer interface, and sequence conservation analysis and static light-scattering measurements suggest that all three AMIGO family proteins form similar dimers. Based on the AMIGO-1 structure, we have also modeled AMIGO-2 and present small-angle X-ray scattering data on AMIGO-2 and AMIGO-3. Our mutagenesis studies show that AMIGO-1 dimerization is necessary for proper cell surface expression and thus probably for proper or stable folding in the endoplastic reticulum and for the function of the protein. Based on the data presented earlier, we also suggest that dimerization through the LRR-LRR interface is likely to be involved in cell-cell adhesion by AMIGO-1, while extensive glycosylation may have a role.

AMIGO is an auxiliary subunit of the Kv2.1 potassium channel

Kv2.1 is a potassium channel α‐subunit abundantly expressed throughout the brain. It is a main component of delayed rectifier current (IK) in several neuronal types and a regulator of excitability during high‐frequency firing. Here we identify AMIGO (amphoterin‐induced gene and ORF), a neuronal adhesion protein with leucine‐rich repeat and immunoglobin domains, as an integral part of the Kv2.1 channel complex. AMIGO shows extensive spatial and temporal colocalization and association with Kv2.1 in the mouse brain. The colocalization of AMIGO and Kv2.1 is retained even during stimulus‐induced changes in Kv2.1 localization. AMIGO increases Kv2.1 conductance in a voltage‐dependent manner in HEK cells. Accordingly, inhibition of endogenous AMIGO suppresses neuronal IK at negative membrane voltages. In conclusion, our data indicate AMIGO as a function‐modulating auxiliary subunit for Kv2.1 and thus provide new insights into regulation of neuronal excitability.

Expression of GluK1c underlies the developmental switch in presynaptic kainate receptor function

Kainate-type glutamate receptors (KARs) regulate synaptic transmission and neuronal excitability via multiple mechanisms, depending on their subunit composition. Presynaptic KARs tonically depress glutamatergic transmission during restricted period of synapse development; however, the molecular basis behind this effect is unknown. Here, we show that the developmental and cell-type specific expression pattern of a KAR subunit splice variant, GluK1c, corresponds to the immature-type KAR activity in the hippocampus. GluK1c localizes to dendritic contact sites at distal axons, the distal targeting being promoted by heteromerization with the subunit GluK4. Presynaptic expression of GluK1c strongly suppresses glutamatergic transmission in cell-pairs in vitro and mimics the immature-type KAR activity at CA3-CA1 synapses in vivo, at a developmental stage when the endogenous expression is already downregulated. These data support a central role for GluK1c in mediating tonic inhibition of glutamate release and the consequent effects on excitability and activity-dependent fine-tuning of the developing hippocampal circuitry.

HB-GAM (pleiotrophin) reverses inhibition of neural regeneration by the CNS extracellular matrix

Chondroitin sulfate (CS) glycosaminoglycans inhibit regeneration in the adult central nervous system (CNS). We report here that HB-GAM (heparin-binding growth-associated molecule; also known as pleiotrophin), a CS-binding protein expressed at high levels in the developing CNS, reverses the role of the CS chains in neurite growth of CNS neurons in vitro from inhibition to activation. The CS-bound HB-GAM promotes neurite growth through binding to the cell surface proteoglycan glypican-2; furthermore, HB-GAM abrogates the CS ligand binding to the inhibitory receptor PTPσ (protein tyrosine phosphatase sigma). Our in vivo studies using two-photon imaging of CNS injuries support the in vitro studies and show that HB-GAM increases dendrite regeneration in the adult cerebral cortex and axonal regeneration in the adult spinal cord. Our findings may enable the development of novel therapies for CNS injuries.

AMIGO-Kv2.1 Potassium Channel Complex Is Associated With Schizophrenia-Related Phenotypes

The enormous variability in electrical properties of neurons is largely affected by a multitude of potassium channel subunits. Kv2.1 is a widely expressed voltage-dependent potassium channel and an important regulator of neuronal excitability. The Kv2.1 auxiliary subunit AMIGO constitutes an integral part of the Kv2.1 channel complex in brain and regulates the activity of the channel. AMIGO and Kv2.1 localize to the distinct somatodendritic clusters at the neuronal plasma membrane. Here we have created and characterized a mouse line lacking the AMIGO gene. Absence of AMIGO clearly reduced the amount of the Kv2.1 channel protein in mouse brain and altered the electrophysiological properties of neurons. These changes were accompanied by behavioral and pharmacological abnormalities reminiscent of those identified in schizophrenia. Concomitantly, we have detected an association of a rare, population-specific polymorphism of KV2.1 (KCNB1) with human schizophrenia in a genetic isolate enriched with schizophrenia. Our study demonstrates the involvement of AMIGO-Kv2.1 channel complex in schizophrenia-related behavioral domains in mice and identifies KV2.1 (KCNB1) as a strong susceptibility gene for schizophrenia spectrum disorders in humans.