Pivotal role of the RanBP9-cofilin pathway in Aβ-induced apoptosis and neurodegeneration

Neurodegeneration associated with amyloid β (Aβ) peptide accumulation, synaptic loss, neuroinflammation, tauopathy, and memory impairments encompass the pathophysiological features of Alzheimer's disease (AD). We previously reported that the scaffolding protein RanBP9, which is overall increased in brains of AD patients, simultaneously promotes Aβ generation and focal adhesion disruption by accelerating the endocytosis of amyloid precursor protein (APP) and β1-integrin, respectively. Here, we show that RanBP9 protein levels are increased by fourfold in FAD mutant APP transgenic mice. Accordingly, RanBP9 transgenic mice demonstrate significantly increased synapse loss, neurodegeneration, gliosis, and spatial memory deficits. RanBP9 overexpression promotes apoptosis and potentiates Aβ-induced neurotoxicity independent of its capacity to promote Aβ generation. Conversely, RanBP9 reduction by siRNA or gene dosage mitigates Aβ-induced neurotoxicity. Importantly, RanBP9 activates/dephosphorylates cofilin, a key regulator of actin dynamics and mitochondria-mediated apoptosis, and siRNA knockdown of cofilin abolishes both Aβ and RanBP9-induced apoptosis. These findings implicate the RanBP9-cofilin pathway as critical therapeutic targets not only for stemming Aβ generation but also antagonizing Aβ-induced neurotoxicity.     

Characterization of a multidomain adaptor protein, p140Cap, as part of a pre-synaptic complex

p140Cap (Cas-associated protein) is an adaptor protein considered to play pivotal roles in cell adhesion, growth and Src tyrosine kinase-related signaling in non-neuronal cells. It is also reported to interact with a pre-synaptic membrane protein, synaptosome-associated protein of 25 kDa, and may participate in neuronal secretion. However, properties and precise functions of p140Cap in neuronal cells are almost unknown. Here we show, using biochemical analyses, that p140Cap is expressed in rat brain in a developmental stage-dependent manner, and is relatively abundant in the synaptic plasma membrane fraction in adults. Immunohistochemistry showed localization of p140Cap in the neuropil in rat brain and immunofluorescent analyses detected p140Cap at synapses of primary cultured rat hippocampal neurons. Electron microscopy further revealed localization at pre- and post-synapses. Screening of p140Cap-binding proteins identified a multidomain adaptor protein, vinexin, whose third Src-homology 3 domain interacts with the C-terminal Pro-rich motif of p140Cap. Immunocomplexes between the two proteins were confirmed in COS7 and rat brain. We also clarified that a pre-synaptic protein, synaptophysin, interacts with p140Cap. These results suggest that p140Cap is involved in neurotransmitter release, synapse formation/maintenance, and signaling.     

Gephyrin expression and clustering affects the size of glutamatergic synaptic contacts

We have recently shown that disrupting the expression and post-synaptic clustering of gephyrin in cultured hippocampal pyramidal cells, by either gephyrin RNAi (RNA interference) or over-expression of a dominant negative gephyrin-enhanced green fluorescent protein (EGFP) fusion protein, leads to decreased number of post-synaptic gephyrin and GABA_A receptor clusters and to reduced GABAergic innervation of these cells. On the other hand, increasing gephyrin expression led to a small increase in the number of gephyrin and GABA_A receptor clusters and to little or no effect on GABAergic innervation. We are now reporting that altering gephyrin expression and clustering affects the size but not the density of glutamatergic synaptic contacts. Knocking down gephyrin with gephyrin RNAi, or preventing gephyrin clustering by over-expression of the dominant negative gephyrin-enhanced green fluorescent protein fusion protein, leads to larger post-synaptic PSD-95 clusters and larger pre-synaptic glutamatergic terminals. On the other hand, over-expression of gephyrin leads to slightly smaller PSD-95 clusters and pre-synaptic glutamatergic terminals. The change in size of PSD-95 clusters were accompanied by a parallel change in the size of NR2-NMDA receptor clusters. It is concluded that the levels of expression and clustering of gephyrin, a protein that concentrates at the post-synaptic complex of the inhibitory synapses, not only has homotypic effects on GABAergic synaptic contacts, but also has heterotypic effects on glutamatergic synaptic contacts. We are proposing that gephyrin is a counterpart of the post-synaptic glutamatergic scaffold protein PSD-95 in regulating the number and/or size of the excitatory and inhibitory synaptic contacts.     

Immunological dynamics in response to two anthrax vaccines in mice

In order to understand the variation of humoral and cellular immune responses to A16R live spore and AVA vaccine and to identify efficient immunological parameters for the early evaluation of post immu- nization in mice, we dynamically monitored the antibody production and cellular responses after the vaccination of Balb/C mice with the anthrax vaccines. The results show that both anti-AVA and anti-Spore antibodies were detectable in the A16R live spore vaccinated group while high titers of anti-AVA antibodies but not anti-Spore antibodies existed in the AVA-immunized group. IgG1 and IgG2 were the major subtypes of IgG in both of the two groups. However, the IgG2a level was significantly higher in the A16R group than in the AVA group. At the cellular level, responses of antigen-specific TH2, TH1 and plasma cells were detected. The peripheral TH2 responses could be seen on day 5 after vac- cination, and remained at a high level throughout the experiment (from day 5 post primary immuniza- tion to day 60 post the tertiary immunization); the TH1 responses to A16R vaccine appeared on day 5, while the responses to AVA could only be detected by day 7 after the secondary immunization; a low level of TH1 responses could be observed at the end of the experiment. Antigen-specific plasma cells could be found in the peripheral blood of both the immunized groups, however, the responses in the A16R group appeared earlier, lasted longer, and shown an ascending tendency until the end of the ex- periment when the plasma cell responses in the AVA group were reduced to a very low level. The re- sults suggest that the multiple antigen containing A16R live spore vaccine induces better immune re- sponses than AVA. Combined with serum antibody titers, TH2, TH1 and plasma cell responses could be used as immunological parameters for the evaluation of vaccine efficacy. These findings may afford new insight into the early evaluation of vaccination as well as being a powerful strategy for vaccine development.     

Normal adult climbing fiber monoinnervation of cerebellar Purkinje cells in mice lacking MHC class I molecules

Some immune system proteins have recently been implicated in the development and plasticity of neuronal connections. Notably, proteins of the major histocompatibility complex 1 (MHC class 1) have been shown to be involved in synaptic plasticity in the hippocampus and the development of projection patterns in the visual system. We examined the possible role for the MHC class 1 proteins in one well-characterized example of synaptic exuberance and subsequent refinement, the climbing fiber (CF) to Purkinje cell (PC) synapse. Cerebella from adult mice deficient for two MHC genes, H2-D1 and H2-K1, and for beta2-microglobulin gene were examined for evidence of deficient elimination of supernumerary CF synapses on their PCs. Electrophysiological and morphological evidence showed that, despite the absence of these MHC class 1 molecules, adult PCs in these transgenic mice are monoinnervated as in wild-type animals. These findings indicate that, at the level of restriction of afferent number at this synapse, functional MHC class 1 proteins are not required.     

Ultrastructural analysis of chemical synapses and gap junctions between Drosophila brain neurons in culture

Dissociated cultures from many species have been important tools for exploring factors that regulate structure and function of central neuronal synapses. We have previously shown that cells harvested from brains of late stage Drosophila pupae can regenerate their processes in vitro. Electrophysiological recordings demonstrate the formation of functional synaptic connections as early as 3 days in vitro (DIV), but no information about synapse structure is available. Here, we report that antibodies against pre-synaptic proteins Synapsin and Bruchpilot result in punctate staining of regenerating neurites. Puncta density increases as neuritic plexuses develop over the first 4 DIV. Electron microscopy reveals that closely apposed neurites can form chemical synapses with both pre- and postsynaptic specializations characteristic of many inter-neuronal synapses in the adult brain. Chemical synapses in culture are restricted to neuritic processes and some neurite pairs form reciprocal synapses. GABAergic synapses have a significantly higher percentage of clear core versus granular vesicles than non-GABA synapses. Gap junction profiles, some adjacent to chemical synapses, suggest that neurons in culture can form purely electrical as well as mixed synapses, as they do in the brain. However, unlike adult brain, gap junctions in culture form between neuronal somata as well as neurites, suggesting soma ensheathing glia, largely absent in culture, regulate gap junction location in vivo. Thus pupal brain cultures, which support formation of interneuronal synapses with structural features similar to synapses in adult brain, are a useful model system for identifying intrinsic and extrinsic regulators of central synapse structure as well as function.     

Derailed regulates development of the Drosophila neuromuscular junction

Neural function is dependent upon the proper formation and development of synapses. We show here that Wnt5 regulates the growth of the Drosophila neuromuscular junction (NMJ) by signaling through the Derailed receptor. Mutations in both wnt5 and drl result in a significant reduction in the number of synaptic boutons. Cell-type specific rescue experiments show that wnt5 functions in the presynaptic motor neuron while drl likely functions in the postsynaptic muscle cell. Epistatic analyses indicate that drl acts downstream of wnt5 to promote synaptic growth. Structure-function analyses of the Drl protein indicate that normal synaptic growth requires the extracellular Wnt inhibitory factor domain and the intracellular domain, which includes an atypical kinase. Our findings reveal a novel signaling mechanism that regulates morphology of the Drosophila NMJ.     

Slow oscillations in neural networks with facilitating synapses

The synchronous oscillatory activity characterizing many neurons in a network is often considered to be a mechanism for representing, binding, conveying, and organizing information. A number of models have been proposed to explain high-frequency oscillations, but the mechanisms that underlie slow oscillations are still unclear. Here, we show by means of analytical solutions and simulations that facilitating excitatory (E _f) synapses onto interneurons in a neural network play a fundamental role, not only in shaping the frequency of slow oscillations, but also in determining the form of the up and down states observed in electrophysiological measurements. Short time constants and strong E _f synapse-connectivity were found to induce rapid alternations between up and down states, whereas long time constants and weak E _f synapse connectivity prolonged the time between up states and increased the up state duration. These results suggest a novel role for facilitating excitatory synapses onto interneurons in controlling the form and frequency of slow oscillations in neuronal circuits.     

Activity-dependent changes of the hippocampal CA3–CA1 synapse during the acquisition of associative learning in conscious mice

Contemporary neuroscientists are paying increasing attention to subcellular, molecular and electrophysiological mechanisms underlying learning and memory processes. Recent efforts have addressed the development of transgenic mice affected at different stages of the learning process, or emulating pathological conditions involving cognition and motor-learning capabilities. However, a parallel effort is needed to develop stimulating and recording techniques suitable for use in behaving mice, in order to grasp activity-dependent neural changes taking place during the very moment of the process. These in vivo models should integrate the fragmentary information collected by different molecular and in vitro approaches. In this regard, long-term potentiation (LTP) has been proposed as the neural mechanism underlying synaptic plasticity. Moreover, N -methyl- d -aspartate (NMDA) receptors are accepted as the molecular substrate of LTP. It now seems necessary to study the relationship of both LTP and NMDA receptors with the plastic changes taking place, in selected neural structures, during actual learning. Here, we review data on the involvement of the hippocampal CA3–CA1 synapse in the acquisition of classically conditioned eyelid conditioned responses (CRs) in behaving mice. Available data show that LTP, evoked by high-frequency stimulation of Schaffer collaterals, disturbs both the acquisition of CRs and the physiological changes that occur at the CA3–CA1 synapse during learning. Moreover, the administration of NMDA-receptor antagonists is able not only to prevent LTP induction in vivo , but also to hinder the formation of both CRs and functional changes in strength of the CA3–CA1 synapse. Thus, there is experimental evidence relating activity-dependent synaptic changes taking place during actual learning with LTP mechanisms and with the role of NMDA receptors in both processes.