Iryna Leshchyns'KA

Long-term potentiation (LTP), the persistent strengthening of synapses following high levels of stimulation, is a form of synaptic plasticity that has been studied extensively as a possible mechanism for learning and memory formation. The strengthening of the synapse that occurs during LTP requires cascades of complex molecular processes and the coordinated remodeling of pre-synaptic and post-synaptic neurons. Despite over four decades of research, our understanding of the transcriptional mechanisms and molecular processes underlying LTP remains incomplete. Identification of all the proteins and non-coding RNA transcripts expressed during LTP may provide greater insight into the molecular mechanisms involved in learning and memory formation.

Alzheimer’s disease (AD) is characterized by synapse loss due to mechanisms that remain poorly understood. We show that the neural cell adhesion molecule 2 (NCAM2) is enriched in synapses in the human hippocampus. This enrichment is abolished in the hippocampus of AD patients and in brains of mice overexpressing the human amyloid-β (Aβ) precursor protein carrying the pathogenic Swedish mutation. Aβ binds to NCAM2 at the cell surface of cultured hippocampal neurons and induces removal of NCAM2 from synapses. In AD hippocampus, cleavage of the membrane proximal external region of NCAM2 is increased and soluble extracellular fragments of NCAM2 (NCAM2-ED) accumulate. Knockdown of NCAM2 expression or incubation with NCAM2-ED induces disassembly of GluR1-containing glutamatergic synapses in cultured hippocampal neurons. Aβ-dependent disassembly of GluR1-containing synapses is inhibited in neurons overexpressing a cleavage-resistant mutant of NCAM2. Our data indicate that Aβ-dependent disruption of NCAM2 functions in AD hippocampus contributes to synapse loss.

In humans, deletions/mutations in the CHL1/CALL gene are associated with mental retardation and schizophrenia. Juvenile CHL1‐deficient (CHL1^−/−) mice have been shown to display abnormally high numbers of parvalbumin‐expressing (PV⁺) hippocampal interneurons and, as adults, display behavioral traits observed in neuropsychiatric disorders. Here, we addressed the question whether inhibitory interneurons and synaptic plasticity in the CHL1^−/− mouse are affected during brain maturation and in adulthood. We found that hippocampal, but not neocortical, PV⁺ interneurons were reduced with age in CHL1^−/− mice, from a surplus of +27% at 1 month to a deficit of ‐20% in adulthood compared with wild‐type littermates. This loss occurred during brain maturation, correlating with microgliosis and enhanced interleukin‐6 expression. In parallel with the loss of PV⁺ interneurons, the inhibitory input to adult CA1 pyramidal cells was reduced and a deficit in short‐ and long‐term potentiation developed at CA3–CA1 excitatory synapses between 2 and 9 months of age in CHL1^−/− mice. This deficit could be abrogated by a GABA_A receptor agonist. We propose that region‐specific aberrant GABAergic synaptic connectivity resulting from the mutation and a subsequently enhanced synaptic elimination during brain maturation lead to microgliosis, increase in pro‐inflammatory cytokine levels, loss of interneurons, and impaired synaptic plasticity. image Close homolog of L1‐deficient (CHL1^−/−) mice have abnormally high numbers of parvalbumin (PV)‐expressing hippocampal interneurons in juvenile animals, but in adult animals a loss of these cells is observed. This loss correlates with an increased density of microglia (M), enhanced interleukin‐6 (IL6) production and a deficit in short‐ and long‐term potentiation at CA3–CA1 excitatory synapses. Furthermore, adult CHL1^−/− mice display behavioral traits similar to those observed in neuropsychiatric disorders of humans.

Changes in expression of the neural cell adhesion molecule 2 (NCAM2) have been proposed to contribute to neurodevelopmental disorders in humans. The role of NCAM2 in neuronal differentiation remains, however, poorly understood. Using genetically encoded Ca²⁺reporters, we show that clustering of NCAM2 at the cell surface of mouse cortical neurons induces submembrane [Ca²⁺] spikes, which depend on the L-type voltage-dependent Ca²⁺channels (VDCCs) and require activation of the protein tyrosine kinase c-Src. We also demonstrate that clustering of NCAM2 induces L-type VDCC- and c-Src-dependent activation of CaMKII. NCAM2-dependent submembrane [Ca²⁺] spikes colocalize with the bases of filopodia. NCAM2 activation increases the density of filopodia along neurites and neurite branching and outgrowth in an L-type VDCC-, c-Src-, and CaMKII-dependent manner. Our results therefore indicate that NCAM2 promotes the formation of filopodia and neurite branching by inducing Ca²⁺influx and CaMKII activation. Changes in NCAM2 expression in Down syndrome and autistic patients may therefore contribute to abnormal neurite branching observed in these disorders.

The neural cell adhesion molecule (NCAM) is important during neural development, because it contributes to neurite outgrowth in response to its ligands at the cell surface. In the adult brain NCAM is involved in regulating synaptic plasticity. The molecular mechanisms underlying delivery of NCAM to the neuronal cell surface remain poorly understood. We used a protein macroarray and identified the kinesin light chain 1 (KLC1), a component of the kinesin-1 motor protein, as a binding partner of the intracellular domains of the two transmembrane isoforms of NCAM, NCAM140 and NCAM180. KLC1 binds to amino acids CGKAGPGA within the intracellular domain of NCAM and co-localizes with kinesin-1 in the Golgi compartment. Delivery of NCAM180 to the cell surface is increased in CHO cells and neurons co-transfected with kinesin-1. We further demonstrate that the p21-activated kinase 1 (PAK1) competes with KLC1 for binding to the intracellular domain of NCAM and contributes to the regulation of the membrane insertion of NCAM. Our results indicate that NCAM is delivered to the cell surface via a kinesin-1 mediated transport mechanism in a PAK1-dependent manner.

Glycogen synthase kinase 3 (GSK3) is essential for normal development and function of the central nervous system. It is especially important for regulating neurotransmission, although the downstream substrates mediating this function are not yet clear. In the present paper, we report the lipid kinase phosphatidylinositol 4-kinase II α (PI4KIIα) is a novel substrate of GSK3 that regulates trafficking and cell-surface expression of neurotransmitter receptors in neurons. GSK3 phosphorylates two distinct sites in the N-terminus of PI4KIIα (Ser5 and Ser47), promoting binding to the adaptor protein 3 (AP-3) complex for trafficking to the lysosome to be degraded. Blocking phosphorylation reduces trafficking to the lysosome, stabilizing PI4KIIα and its cargo proteins for redistribution throughout the cell. Importantly, a reduction in PI4KIIα expression or phosphorylation increases α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor expression at the surface of hippocampal neurons. These studies implicate signalling between GSK3 and PI4KIIα as a novel regulator of vesicular trafficking and neurotransmission in the brain.

Cell adhesion molecules (CAMs) play indispensable roles in the developing and mature brain by regulating neuronal migration and differentiation, neurite outgrowth, axonal fasciculation, synapse formation and synaptic plasticity. CAM-mediated changes in neuronal behavior depend on a number of intracellular signaling cascades including changes in various second messengers, among which CAM-dependent changes in intracellular Ca2+ levels play a prominent role. Ca2+ is an essential secondary intracellular signaling molecule that regulates fundamental cellular functions in various cell types, including neurons. We present a systematic review of the studies reporting changes in intracellular Ca2+ levels in response to activation of the immunoglobulin superfamily CAMs, cadherins and integrins in neurons. We also analyze current experimental evidence on the Ca2+ sources and channels involved in intracellular Ca2+ increases mediated by CAMs of these families, and systematically review the role of the voltage-dependent Ca2+ channels (VDCCs) in neurite outgrowth induced by activation of these CAMs. Molecular mechanisms linking CAMs to VDCCs and intracellular Ca2+ stores in neurons are discussed.

Newly formed synapses undergo maturation during ontogenetic development via mechanisms that remain poorly understood. We show that maturation of the presynaptic endocytotic machinery in CNS neurons requires substitution of the adaptor protein 3 (AP-3) with AP-2 at the presynaptic plasma membrane. In mature synapses, AP-2 associates with the intracellular domain of the neural cell adhesion molecule (NCAM). NCAM promotes binding of AP-2 over binding of AP-3 to presynaptic membranes, thus favoring the substitution of AP-3 for AP-2 during formation of mature synapses. The presynaptic endocytotic machinery remains immature in adult NCAM-deficient (NCAM−/−) mice accumulating AP-3 instead of AP-2 and its partner protein AP180 in synaptic membranes and vesicles. NCAM deficiency or disruption of the NCAM/AP-2 complex in wild-type (NCAM+/+) neurons by overexpression of AP-2 binding-defective mutant NCAM interferes with efficient retrieval of the synaptic vesicle v-SNARE synaptobrevin 2. Abnormalities in synaptic vesicle endocytosis and recycling may thus contribute to neurological disorders associated with mutations in NCAM.

The Neural cell adhesion molecule (NCAM) plays an important role in regulation of nervous system development. To expand our understanding of the molecular mechanisms via which NCAM influences differentiation of neurons, we used a yeast two-hybrid screening to search for new binding partners of NCAM and identified p21-activated kinase 1 (Pak1). We show that NCAM interacts with Pak1 in growth cones of neurons. The autophosphorylation and activity of Pak1 were enhanced when isolated growth cones were incubated with NCAM function triggering antibodies, which mimic the interaction between NCAM and its extracellular ligands. The association of Pak1 with cell membranes, the efficiency of Pak1 binding to its activators, and Pak1 activity were inhibited in brains of NCAM-deficient mice. NCAM-dependent Pak1 activation was abolished after lipid raft disruption, suggesting that NCAM promotes Pak1 activation in the lipid raft environment. Phosphorylation of the downstream Pak1 effectors LIMK1 and cofilin was reduced in growth cones from NCAM-deficient neurons, which was accompanied by decreased levels of filamentous actin and inhibited filopodium mobility in the growth cones. Dominant-negative Pak1 inhibited and constitutively active Pak1 enhanced the ability of neurons to increase neurite outgrowth in response to the extracellular ligands of NCAM. Our combined observations thus indicate that NCAM activates Pak1 to drive actin polymerization to promote neuronal differentiation.

Growth cones are motile structures at the tips of growing neurites, which play an essential role in regulation of growth and navigation of growing axons and dendrites of neurons in the developing nervous system. This protocol describes isolation of growth cones from the brain tissue from young mice. Growth cones isolated using this protocol have been extensively characterized using electron microscopy (Pfenninger et al., 1983) and may be used for any kind of subsequent biochemical and/or functional analyses, including Western blot analysis of protein expression (Westphal et al., 2010), analysis of the activity of growth cone-accumulated enzymes (Leshchyns’ka et al., 2003; Li et al., 2013), and analysis of the endocytosis and exocytosis rates (Chernyshova et al., 2011). Bio-protocol is an online peer-reviewed protocol journal. Its mission is to make life science research more efficient and reproducible by curating and hosting high quality, free access protocols.