Implication of thermal signaling in neuronal differentiation revealed by manipulation and measurement of intracellular temperature.

Neuronal differentiation-the development of neurons from neural stem cells-involves neurite outgrowth and is a key process during the development and regeneration of neural functions. In addition to various chemical signaling mechanisms, it has been suggested that thermal stimuli induce neuronal differentiation. However, the function of physiological subcellular thermogenesis during neuronal differentiation remains unknown. Here we create methods to manipulate and observe local intracellular temperature, and investigate the effects of noninvasive temperature changes on neuronal differentiation using neuron-like PC12 cells. Using quantitative heating with an infrared laser, we find an increase in local temperature (especially in the nucleus) facilitates neurite outgrowth. Intracellular thermometry reveals that neuronal differentiation is accompanied by intracellular thermogenesis associated with transcription and translation. Suppression of intracellular temperature increase during neuronal differentiation inhibits neurite outgrowth. Furthermore, spontaneous intracellular temperature elevation is involved in neurite outgrowth of primary mouse cortical neurons. These results offer a model for understanding neuronal differentiation induced by intracellular thermal signaling.

Epstein-Barr virus-derived vector suitable for long-term expression in neurons.

Exogenous gene expression is a fundamental and indispensable technique for testing gene function in neurons. Several ways to express exogenous genes in neurons are available, but each method has pros and cons. The lentivirus vector is useful for high efficiency gene transfer to neurons and stabilizes gene expression via genome integration, but this integration may destroy the host genome. The Epstein-Barr virus (EBV)-derived vector (EB vector) is an accessible and useful vector in human cell lines because the vector is not integrated into the host genome but stays in the nucleus as an episome. However, there has been no report on this process in rodent neurons. We examined the usefulness of the EB vector for testing gene function in neurons. We found that EB vector-derived exogenous proteins such as green fluorescent protein (GFP) and GFP-tagged actin were easily detectable even after three weeks of transfection. Second, a tetracycline-induced gene expression system in the EB vector was active after three weeks of transfection, indicating that plasmids were retained in neurons for up to three weeks. Third, we determined that only Family of repeat element of the plasmid vector is essential for its long-term presence in neurons. These results show that the modified EB vector is a useful tool for examining gene function in neurons.

New Alzheimer's disease model mouse specialized for analyzing the function and toxicity of intraneuronal Amyloid ß oligomers.

Oligomers of intracellular amyloid ß protein (Aß) are strongly cytotoxic and play crucial roles in synaptic transmission and cognitive function in Alzheimer's disease (AD). However, there is currently no AD model mouse in which to specifically analyze the function of Aß oligomers only. We have now developed a novel AD model mouse, an Aß-GFP transgenic mouse (Aß-GFP Tg), that expresses the GFP-fused human Aß1-42 protein, which forms only Aß oligomers within neurons throughout their life. The fusion proteins are expressed mainly in the hippocampal CA1-CA2 region and cerebral cortex, and are not secreted extracellularly. The Aß-GFP Tg mice exhibit increased tau phosphorylation, altered spine morphology, decreased expressions of the GluN2B receptor and neuroligin in synaptic regions, attenuated hippocampal long-term potentiation, and impaired object recognition memory compared with non-Tg littermates. Interestingly, these dysfunctions have already appeared in 2-3-months-old animals. The Aß-GFP fusion protein is bioactive and highly toxic, and induces the similar synaptic dysfunctions as the naturally generated Aß oligomer derived from postmortem AD patient brains and synthetic Aß oligomers. Thus, Aß-GFP Tg mouse is a new tool specialized to analyze the function of Aß oligomers in vivo and to find subtle changes in synapses in early symptoms of AD.

Dysferlinopathy Fibroblasts Are Defective in Plasma Membrane Repair.

BACKGROUND: Dysferlin is a sarcolemmal protein that is defective in Miyoshi myopathy and limb-girdle muscular dystrophy type 2B, and is involved in sarcolemmal repair. Primary cultured myoblasts and myotubes established from patient muscle biopsies have been widely utilized to explore the molecular mechanism of dysferlinopathy. OBJECTIVES: The purpose of this study was to explore the possible utility of dermal fibroblasts from dysferlin-deficient patients and SJL mice as a tool for studying dysferlinopathy. METHODS: Dysferlin protein expression in fibroblasts from dysferlin-deficient patients and SJL mice was analyzed by immunoblotting and immunocytochemistry. The membrane wound-repair assay was performed on the fibroblasts using a confocal microscope equipped with a UV-laser. The membrane blebbing assay using hypotonic shock, in which normal membrane blebbing is detected only in the presence of dysferlin, was also performed using human and mouse fibroblasts. RESULTS: Mis-sense mutated dysferlin was expressed at a very low level in fibroblasts from a dysferlinopathy patient, and lower expression level of truncated dysferlin was observed in SJL mouse fibroblast. Fibroblasts from patients with dysferlinopathy and SJL mice showed attenuated membrane repair and did not form membrane blebs in response to hypoosmotic shock. Proteosomal inhibitior increased mis-sense mutated or truncated dysferlin levels, and restored membrane blebbing, however, proteosomal inhibition failed to improve levels of dysferlin with non-sense or frame-shift mutation. CONCLUSION: Fibroblasts from dysferlinopathy patients and SJL mice showed attenuated plasma membrane repair, and could be a tool for studying dysferlinopathy.

Multiple functions of precursor BDNF to CNS neurons: negative regulation of neurite growth, spine formation and cell survival.

BACKGROUND: Proneurotrophins and mature neurotrophins elicit opposite effects via the p75 neurotrophin receptor (p75(NTR)) and Trk tyrosine kinase receptors, respectively; however the molecular roles of proneurotrophins in the CNS are not fully understood. RESULTS: Based on two rare single nucleotide polymorphisms (SNPs) of the human brain-derived neurotrophic factor (BDNF) gene, we generated R125M-, R127L- and R125M/R127L-BDNF, which have amino acid substitution(s) near the cleavage site between the pro- and mature-domain of BDNF. Western blot analyses demonstrated that these BDNF variants are poorly cleaved and result in the predominant secretion of proBDNF. Using these cleavage-resistant proBDNF (CR-proBDNF) variants, the molecular and cellular roles of proBDNF on the CNS neurons were examined. First, CR-proBDNF showed normal intracellular distribution and secretion in cultured hippocampal neurons, suggesting that inhibition of proBDNF cleavage does not affect intracellular transportation and secretion of BDNF. Second, we purified recombinant CR-proBDNF and tested its biological effects using cultured CNS neurons. Treatment with CR-proBDNF elicited apoptosis of cultured cerebellar granule neurons (CGNs), while treatment with mature BDNF (matBDNF) promoted cell survival. Third, we examined the effects of CR-proBDNF on neuronal morphology using more than 2-week cultures of basal forebrain cholinergic neurons (BFCNs) and hippocampal neurons. Interestingly, in marked contrast to the action of matBDNF, which increased the number of cholinergic fibers and hippocampal dendritic spines, CR-proBDNF dramatically reduced the number of cholinergic fibers and hippocampal dendritic spines, without affecting the survival of these neurons. CONCLUSION: These results suggest that proBDNF has distinct functions in different populations of CNS neurons and might be responsible for specific physiological cellular processes in the brain.

Optical microscopic observation of fluorescence enhanced by grating-coupled surface plasmon resonance.

On the substrate carrying a sub-wavelength grating covered with a thin metal layer, a fluorescent dye-labeled cell was observed by fluorescence microscope. The fluorescence intensity was more than 20 times greater than that on an optically flat glass substrate. Such a great fluorescence enhancement from labeled cells bound to the grating substrate was due to the excitation by grating coupled surface plasmon resonance. The application of a grating substrate to two-dimensional detection and fluorescence microscopy appears to offer a promising method of taking highly sensitive fluorescence images.

Brain-derived neurotrophic factor regulates cholesterol metabolism for synapse development.

Brain-derived neurotrophic factor (BDNF) exerts multiple biological functions in the CNS. Although BDNF can control transcription and protein synthesis, it still remains open to question whether BDNF regulates lipid biosynthesis. Here we show that BDNF elicits cholesterol biosynthesis in cultured cortical and hippocampal neurons. Importantly, BDNF elicited cholesterol synthesis in neurons, but not in glial cells. Quantitative reverse transcriptase-PCR revealed that BDNF stimulated the transcription of enzymes in the cholesterol biosynthetic pathway. BDNF-induced cholesterol increases were blocked by specific inhibitors of cholesterol synthesis, mevastatin and zaragozic acid, suggesting that BDNF stimulates de novo synthesis of cholesterol rather than the incorporation of extracellular cholesterol. Because cholesterol is a major component of lipid rafts, we investigated whether BDNF would increase the cholesterol content in lipid rafts or nonraft membrane domains. Interestingly, the BDNF-mediated increase in cholesterol occurred in rafts, but not in nonrafts, suggesting that BDNF promotes the development of neuronal lipid rafts. Consistent with this notion, BDNF raised the level of the lipid raft marker protein caveolin-2 in rafts. Remarkably, BDNF increased the levels of presynaptic proteins in lipid rafts, but not in nonrafts. An electrophysiological study revealed that BDNF-dependent cholesterol biosynthesis plays an important role for the development of a readily releasable pool of synaptic vesicles. Together, these results suggest a novel role for BDNF in cholesterol metabolism and synapse development.

Development of new screening system for Alzheimer disease, in vitro Abeta sink assay, to identify the dissociation of soluble Abeta from fibrils.

Abeta is one of the primary therapeutic targets for Alzheimer disease (AD). Abeta vaccination induces the disappearance of Abeta deposits. Since few reports have focused on the reverse phase of Abeta aggregation, we established a new screening system, the in vitro Abeta sink assay, to clarify the process of dissociation of soluble forms from fibrils. Abeta42 was more resistant to dissociation from fibrils to monomers and/or low molecular weight (LMW) soluble oligomers than Abeta40. We applied this system to find a potential therapy for AD. Ultrasound irradiation significantly enhanced the dissociation of soluble Abeta from fibrils, while ultrasound experiments also confirmed the difference between Abeta40 and Abeta42. We found that some compounds enhanced the dissociation of Abeta from fibrils. Here, we proposed that Abeta42 was more resistant to dissociation from fibrils to monomers and/or LMW soluble oligomers than Abeta40, and this system might be useful to identify dissociation of soluble Abeta from fibrils.

Diminished neuronal activity increases neuron-neuron connectivity underlying silent synapse formation and the rapid conversion of silent to functional synapses.

Neuronal activity regulates the synaptic strength of neuronal networks. However, it is still unclear how diminished activity changes connection patterns in neuronal circuits. To address this issue, we analyzed neuronal connectivity and relevant mechanisms using hippocampal cultures in which developmental synaptogenesis had occurred. We show that diminution of network activity in mature neuronal circuit promotes reorganization of neuronal circuits via NR2B subunit-containing NMDA-type glutamate receptors (NR2B-NMDARs), which mediate silent synapse formation. Simultaneous double whole-cell recordings revealed that diminishing neuronal circuit activity for 48 h increased the number of synaptically connected neuron pairs with both silent and functional synapses. This increase was accompanied by the specific expression of NR2B-NMDARs at synaptic sites. Analysis of miniature EPSCs (mEPSCs) showed that the frequency of NMDAR-mediated, but not AMPAR-mediated, mEPSCs increased, indicating that diminished neuronal activity promotes silent synapse formation via the surface delivering NR2B-NMDARs in mature neurons. After activation of neuronal circuit by releasing from TTX blockade (referred as circuit reactivation), the frequency of AMPAR-mediated mEPSCs increased instead, and this increase was prevented by ifenprodil. The circuit reactivation also caused an increased colocalization of glutamate receptor 1-specfic and synaptic NR2B-specific puncta. These results indicate that the circuit reactivation converts rapidly silent synapses formed during activity suppression to functional synapses. These data may provide a new example of homeostatic circuit plasticity that entails the modulation of neuron-neuron connectivity by synaptic activity.

Micropatterned composite membranes of polymerized and fluid lipid bilayers.

Micropatterned composite membranes of polymerized and fluid lipid bilayers were constructed on solid substrates. Lithographic photopolymerization of a diacetylene-containing phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC), and subsequent removal of nonreacted monomers by a detergent solution (0.1 M sodium dodecyl sulfate (SDS)) yielded a patterned polymeric bilayer matrix on the substrate. Fluid lipid bilayers of phosphatidylcholine from egg yolk (egg-PC) were incorporated into the lipid-free wells surrounded by the polymeric bilayers through the process of fusion and reorganization of suspended small unilamellar vesicles. Spatial distribution of the fluid bilayers in the patterned bilayer depended on the degree of photopolymerization that in turn could be modulated by varying the applied UV irradiation dose. The polymeric bilayer domains blocked lateral diffusion of the fluid lipid bilayers and confined them in the defined areas (corrals), if the polymerization was conducted with a sufficiently large UV dose. On the other hand, lipid molecules of the fluid bilayers penetrated into the polymeric bilayer domains, if the UV dose was relatively small. A direct correlation was observed between the applied UV dose and the lateral diffusion coefficient of fluorescent marker molecules in the fluid bilayers embedded within the polymeric bilayer domains. Artificial control of lateral diffusion by polymeric bilayers may lead to the creation of complex and versatile biomimetic model membrane arrays.

Re-expression of NR2B-containing NMDA receptors in vitro by suppression of neuronal activity.

N-methyl-D-aspartate receptors (NMDARs) are known to play critical roles in the development of the nervous system, and their expression is regulated in an activity-dependent fashion during development. However, the regulation of NMDAR expression after circuit formation is less well understood. To examine this, we performed patch-clamp recordings from chick cerebral neurons in an activity-controlled culture. Analysis of NMDAR channels from neurons before synapse formation showed that there are two components in channel open kinetics. The major slow component is clearly blocked by ifenprodil, a specific inhibitor of NR2B-containing NMDARs. In contrast, slow component of NMDAR channel opening from neurons after synapse formation became minor and ifenprodil had little effect on the NMDAR channel openings. Furthermore, this change is reversibly regulated by neuronal activity, in that suppression induces the re-expression of NR2B-containing NMDARs, even after circuit formation.

Development of two transmitter release components during the critical period for imprinting in the chick IMHV.

Transmitter release at an excitatory synapse has two components, fast synchronous and slow asynchronous transmitter release. Using the whole cell recording technique, we investigated the developmental properties of neurotransmitter release, which is composed of the two components in the intermediate and medial part of the hyperstriatum ventral (IMHV) of chicks during the critical period for imprinting. Analysis of the paired-pulse responses revealed that the depression of the excitatory postsynaptic currents (EPSCs), driven mainly by fast synchronous release, was frequently observed in P0-1 chicks but not in those at P5-8. The spontaneous excitatory postsynaptic currents (sEPSCs) after the paired-pulse stimulation, which were thought to be driven by asynchronous transmitter releases, were observed more frequently in P0-1 chicks than P5-8 chicks. Furthermore, examination of Ca2+ dependency in the evoked EPSCs showed that the amplitudes in P5-8 chicks were more sensitive to reduction of the extracellular Ca2+ concentration than younger chicks. Considering that the Ca2+ dependency of EPSCs is defined by both Ca2+ sensitivity and the proportion of each type of release machineries at the release site, these results indicate that the ratio of fast synchronous to slow asynchronous transmitter release machinery changed during the critical period. These changes may play critical roles in the capacity of the avian brain to consolidate novel experience in the immediate period after hatching.

A synaptic potentiation by a protein factor distinct from those induced by neurotrophins.

We report a protein factor(s) contained in the conditioned medium (CM) of the Mg(2+)-free treatment induced the synaptic potentiation. This type of potentiation shared a different pathway from those induced by neurotrophins. Neurotrophins were confirmed to induce a synaptic potentiation in the dissociated chick neurons. Furthermore, K252a, an inhibitor of tyrosine kinase, abolished this potentiation. Nevertheless, the potentiation induced by the CM was not blocked by K252a. In addition, the CM prepared from the chick neurons induced a similar potentiation in rat and mouse neurons. These results suggest that the protein factor is a novel protein molecule for inducing the potentiation and it plays a critical role in the common mechanism for the potentiation between avian and mammal.

Basic fibroblast growth factor evokes a rapid glutamate release through activation of the MAPK pathway in cultured cortical neurons.

We examined the possibility that basic fibroblast growth factor (bFGF) is involved in synaptic transmissions. We found that bFGF rapidly induced the release of glutamate and an increase in the intracellular Ca2+ concentration through voltage-dependent Ca2+ channels in cultured cerebral cortical neurons. bFGF also evoked a significant influx of Na+. Tetanustoxin inhibited the bFGF-induced glutamate release, revealing that bFGF triggered exocytosis. The mitogen-activated protein kinase (MAPK) pathway was required for these acute effects of bFGF. We also found that pretreatment with bFGF significantly enhanced high K+-elicited glutamate release also in a MAPK activation-dependent manner. Therefore, we propose that bFGF exerts promoting effects on excitatory neuronal transmission via activation of the MAPK pathway.