Masayoshi Okada

Identification of a ubiquitin- and ATP-dependent protein degradation pathway in rat cerebral cortex

To investigate the existence of a ubiquitin-dependent protein degradation system in the brain, the proteolytic activity of the cerebral cortex was examined. The soluble extract of rat cerebral cortex degraded 125I-radiolabeled lysozyme in an ATP-dependent manner. The ATP-dependent proteolysis was suppressed with iodoacetamide, which inhibits ubiquitin conjugation, and was abolished by blocking of the amino residues of lysozyme. These results suggest the participation of ubiquitination in the proteolytic activity. An ATP-dependent 125I-ubiquitin-conjugating activity was detected in fraction II from the cerebral cortex. The presence of ATP-dependent proteolytic activity which acted preferentially on ubiquitinated lysozyme was demonstrated, using ubiquitin-125I-lysozyme conjugates as a substrate. The proteinase had a molecular mass of 1500 kDa and displayed nucleotide dependence and sensitivity to various proteinase inhibitors similar to those of the 26S proteinase complex found in reticulocytes. Dialysis of the soluble fraction caused a decrease in the proteolytic activity of ATP-dependent and preferential for ubiquitin-lysozyme conjugates and a reciprocal increase in the ATP-independent free 125I-lysozyme-degrading activity which was scarcely detected before dialysis. The former ATP-dependent proteolytic activity may play a physiological role in the brain.

Sprouting of CA3 pyramidal neurons to the dentate gyrus in rat hippocampal organotypic cultures

The understanding of the mechanisms of a functional synaptic plasticity in the hippocampus has been expanded greatly by the use of in vitro slice preparations. The question addressed in the present study was whether morphological plasticity observed in vivo can also be reproduced in hippocampal slices. In vivo, hippocampal commissural and association fibers are known to sprout and occupy synaptic sites vacated by deafferentation of the dentate gyrus (DG). In hippocampal slice preparations, the major input to the DG is eliminated, so that the DG is deafferented. Might intrinsic neurons sprout to the DG if the slice preparation is maintained for weeks? In this study hippocampal slices obtained from 6-day-old rats were cultured. Stimulation of the dentate stratum moleculare produced antidromic field potentials in the CA3 of the slices cultivated for more than 1 week. The antidromic response was not observed in CA1 pyramidal neurons. The CA3 to DG projection response was also observed in a CA3 mini-slice placed near a co-cultured whole hippocampal slice, when the DG in the latter was stimulated. Moreover, stimulation of the CA3 mini-slice induced synaptic responses in the DG of the whole-slice. The conclusion drawn is that deafferentation could induce axonal sprouting in a neuron-specific manner in hippocampal organotypic culture. This preparation would be potentially useful for the screening of chemical factors that influence sprouting of central neurons.

Correlation between anti-ubiquitin immunoreactivity and region-specific neuronal death in N-methyl-d-aspartate-treated rat hippocampal organotypic cultures

Neuronal degeneration appears to be associated with changes in anti-ubiquitin immunoreactivity (UIR). To elucidate the relationship between the two events, we examined the time course of changes in UIR in pyramidal neurons of hippocampal organotypic cultures following exposure to an excitotoxin, N-methyl-D-aspartate (NMDA). In nontreated cultures, weak UIR was confined to the nucleus. Exposure to 100 microM NMDA for 15 min induced degeneration of pyramidal neurons, within 24 h, in the CA1 and CA3c regions. In these neurons, the nuclear UIR was reduced, and instead, UIR developed in the cytoplasm. In response to the same procedure, CA3a,b pyramidal neurons showed slight shrinkage but otherwise virtually normal morphological features. Little perikaryal (cytoplasmic) UIR developed in CA3a,b neurons. Both degeneration and perikaryal UIR were observed in CA3a,b neurons, however, when the culture was exposed to 300 microM NMDA. Immunoblot analysis showed that changes in the amount of a ubiquitin protein conjugate (24 kDa), presumably ubiquitinated histone, are similar to those of nuclear UIR in the same time course. We propose that the changes in the expression of nuclear and perikaryal ubiquitinated proteins represent some process closely related to neuronal death.

Voltage-dependent block by internal spermine of the murine inwardly rectifying K + channel, Kir2.1, with asymmetrical K + concentrations

Effects of internal spermine on outward single‐channel currents through a strongly inwardly rectifying K+ channel (Kir2.1) were studied at asymmetrical K+ concentrations (30 mm external and 150 mm internal K+). The current–voltage (I–V) relation for the single channel was almost linear and reversed at −37 ± 3 mV (VR; n= 19). The channel conductance was 26.3 ± 1.3 pS (n= 24). The open‐time and closed‐time histograms were fitted with a single exponential function. Internal spermine at a concentration of 1–100 nm reduced the open time of the outward currents in a concentration‐dependent manner and produced a blocked state. The steady‐state open probability of the outward current decreased with larger depolarizations in both the absence and presence of internal spermine. The steady‐state open probability with asymmetrical K+ and symmetrical (150 mm external and internal K+) concentrations plotted against driving force (V−VR) coincided with smaller depolarizations in the absence of spermine and larger depolarizations and higher spermine concentrations in the presence of spermine. The blocking rate constants and unblock rates with 30 mm and 150 mm external K+ were similar at the same driving force. The dissociation constant–membrane potential relation for 30 mm external K+ was shifted in the negative direction from that for 150 mm external K+ by 36 mV. These results suggested that the blocking kinetics depends on driving force to produce driving force‐dependent inward rectification when the equilibrium potential for K+ is altered by changing external K+ and that the energy barriers and wells for blocking ions from passing or lodging are not stable but affected by external K+ ions.

Proliferating cell nuclear antigen in neurones: induction by u.v. irradiation.

Proliferating cell nuclear antigen (PCNA) plays a pleiotropic role in DNA replication, excision repair of injured DNA and apoptosis. We found that PCNA protein is expressed predominantly in CA3 pyramidal neurones in hippocampal slices cultured from 1-week-old rats. Immunoblotting analysis revealed that this PCNA expression is enhanced by u.v. irradiation with no effect on neuronal viability. These results suggest that expression of PCNA in non-proliferating central neurones is associated with repair of injured DNA.

Anti-ubiquitin immunoreactivity associates with pyramidal cell death induced by intraventricular infusion of leupeptin in rat hippocampus

Pathological studies on several neurodegenerative diseases including Alzheimers disease have revealed common deposition of ubiquitin in many inclusion bodies. This implies a possible association of ubiquitin with neurodegeneration. To address this possibility, we examined histochemically the effect of intraventricular infusion of leupeptin, a thiol proteinase inhibitor, which is known to elevate anti-ubiquitin immunoreactivity in rat Purkinje cells. In the leupeptin-infused rat, an intense anti-ubiquitin immunoreactivity in the cytoplasm of neurons occurred not only in cerebellar Purkinje cells but also elsewhere in a wide area of the rat brain. The increase in the immunoreactivity was followed by a gradual depletion of pyramidal neurons in the hippocampal CA1 and CA3 subfields. The immunoreactive neurons disappeared concurrently. The number of anti-ubiquitin immunoreactive neurons was negatively correlated with that of surviving neurons when the duration of leupeptin infusion was varied. These results suggest that increased anti-ubiquitin immunoreactivity associates with neuronal death in leupeptin-treated rat brain.

Increase in the titer of lentiviral vectors expressing potassium channels by current blockade during viral vector production

BackgroundHigh titers of lentiviral vectors are required for the efficient transduction of a gene of interest. During preparation of lentiviral the vectors, the protein of interest is inevitably expressed in the viral vector-producing cells. This expression may affect the production of the lentiviral vector.MethodsWe prepared lentiviral vectors expressing inwardly rectifying potassium channel (Lv-Kir2.1), its dominant-negative form (Lv-Kir-DN), and other K+ channels, using the ubiquitously active β-actin and neuron-specific synapsin I promoters.ResultsThe titer of Lv-Kir-DN was higher than that of Lv-Kir2.1, suggesting a negative effect of induced K+ currents on viral titer. We then blocked Kir2.1 currents with the selective blocker Ba2+ during Lv-Kir2.1 production, and obtained about a 5-fold increase in the titer. Higher extracellular K+ concentrations increased the titer of Lv-Kir2.1 about 9-fold. With a synapsin I promoter Ba2+ increased the titer because of the moderate expression of Kir2.1 channel. Channel blockade also increased the titers of the lentivirus expressing Kv1.4 and TREK channels, but not HERG. The increase in titer correlated with the K+ currents generated by the channels expressed.ConclusionIn the production of lentivirus expressing K+ channels, titers are increased by blocking K+ currents in the virus-producing cells. This identifies a crucial issue in the production of viruses expressing membrane channels, and should facilitate basic and gene therapeutic research on channelopathies.

The degradation of the inwardly rectifying potassium channel, Kir2.1, depends on the expression level: Examination with fluorescent proteins

The expression of ion channels is regulated by their synthesis as well as degradation, and some ion channels are degraded in an expression level-dependent way. Recently, new techniques of fluorescent proteins have been developed and seem to be useful to study protein degradation. To examine the regulation of the degradation of strongly inwardly rectifying potassium channel (Kir2.1) and the usefulness of the fluorescent proteins, we constructed Kir2.1 fusion proteins with SNAP tag and fluorescent timer (FT). The SNAP tag, which covalently binds to a specific membrane-permeable fluorescent dye, enables a pulse-chase experiment with fluorescence. When the SNAP-Kir2.1 proteins were expressed in 293T cells by low and high expression plasmids, the half-life of the fusion protein expressed by a high-expression plasmid was shorter (18.2±1.9 h) than that expressed by a low-expression plasmid (35.1+2.3h). The addition of Ba(2+), a selective blocker of Kir2.1, slowed the degradation, suggesting a current-dependency of degradation. Consistently, patch-clamp recording showed that cultivation in the presence of Ba(2+) increased the whole cell conductance of SNAP-Kir2.1. Since the fluorescence of FT changes gradually changes from green to red, the green/red ratio should allow us to monitor the changes in the degradation rate of FT-Kir2.1. Using this method, we confirmed the slower degradation by Ba(2+). The results suggest a homeostatic regulation of the degradation of Kir2.1 in the 293T cells, and the usefulness of fluorescence-based methods for examining the degradation of ion channels.

A pore forming peptide from spider Lachesana sp. venom induced neuronal depolarization and pain

BACKGROUND Arachnoid venoms contain numerous peptides with ion channel modifying and cytolytic activities. METHODS We developed a green fluorescent protein (GFP)-based assay that can monitor the changes in currents through overexpressed inwardly rectifying K(+) channels (Kir2.1), in which GFP expression was increased by blockade of Kir2.1 current. Using this assay, we screened venom of many spider species. A peptide causing GFP decreasing effect was purified and sequenced. Electrophysiological and pain-inducing effects of the peptide were analyzed with whole-cell patch-clamp recordings and hot-plate test, respectively. RESULTS Among venoms we screened, soluble venom from Lachesana sp. decreased the GFP expression. Purification and sequencing of the peptide showed that the peptide is identical to a pore-forming peptide purified from Lachesana tarabaevi venom. Whole cell patch-clamp recordings revealed that the peptide had no effect on Kir2.1 current. Instead, it induced a current that was attributable to the pore-formation of the peptide. The peptide was selectively incorporated into hyperpolarized, i.e., Kir2.1 expressing, cells and for this reason the peptide decreased GFP expression in our Kir2.1 assay. The pore-formation positively shifted the reversal potential and induced burst firings in the hippocampal neurons in a synaptic current-independent way. The application of the Lachesana sp. peptide induced pain-related behavior in mice. CONCLUSIONS The peptide, which was found in Lachesana sp. venom, formed pores and thereby depolarized neurons and induced pain. GENERAL SIGNIFICANCE Our data suggested an additional physiological role of the pore-forming peptides.

Antidepressive and anxiolytic effects of ostruthin, a TREK-1 channel activator

We screened a library of botanical compounds purified from plants of Vietnam for modulators of the activity of a two-pore domain K+ channel, TREK-1, and we identified a hydroxycoumarin-related compound, ostruthin, as an activator of this channel. Ostruthin increased whole-cell TREK-1 channel currents in 293T cells at a low concentration (EC50 = 5.3 μM), and also activity of the TREK-2 channel (EC50 = 3.7 mM). In contrast, ostruthin inhibited other K+ channels, e.g. human ether-à-go-go-related gene (HERG1), inward-rectifier (Kir2.1), voltage-gated (Kv1.4), and two-pore domain (TASK-1) at higher concentrations, without affecting voltage-gated potassium channel (KCNQ1 and 3). We tested the effect of this compound on mouse anxiety- and depression-like behaviors and found anxiolytic activity in the open-field, elevated plus maze, and light/dark box tests. Of note, ostruthin also showed antidepressive effects in the forced swim and tail suspension tests, although previous studies reported that inhibition of TREK-1 channels resulted in an antidepressive effect. The anxiolytic and antidepressive effect was diminished by co-administration of a TREK-1 blocker, amlodipine, indicating the involvement of TREK-1 channels. Administration of ostruthin suppressed the stress-induced increase in anti-c-Fos immunoreactivity in the lateral septum, without affecting immunoreactivity in other mood disorder-related nuclei, e.g. the amygdala, paraventricular nuclei, and dorsal raphe nucleus. Ostruthin may exert its anxiolytic and antidepressive effects through a different mechanism from current drugs.