Kazunori Sano

Ultrasensitive human prion detection in cerebrospinal fluid by real-time quaking-induced conversion

The development of technologies for the in vitro amplification of abnormal conformations of prion protein (PrPSc) has generated the potential for sensitive detection of prions. Here we developed a new PrPSc amplification assay, called real-time quaking-induced conversion (RT-QUIC), which allows the detection of ≥1 fg of PrPSc in diluted Creutzfeldt-Jakob disease (CJD) brain homogenate. Moreover, we assessed the technique first in a series of Japanese subjects and then in a blind study of 30 cerebrospinal fluid specimens from Australia, which achieved greater than 80% sensitivity and 100% specificity. These findings indicate the promising enhanced diagnostic capacity of RT-QUIC in the antemortem evaluation of suspected CJD.

Early detection of abnormal prion protein in genetic human prion diseases now possible using real-time QUIC assay.

Introduction The definitive diagnosis of genetic prion diseases (gPrD) requires pathological confirmation. To date, diagnosis has relied upon the finding of the biomarkers 14-3-3 protein and total tau (t-tau) protein in the cerebrospinal fluid (CSF), but many researchers have reported that these markers are not sufficiently elevated in gPrD, especially in Gerstmann-Sträussler-Scheinker syndrome (GSS). We recently developed a new in vitro amplification technology, designated “real-time quaking-induced conversion (RT-QUIC)”, to detect the abnormal form of prion protein in CSF from sporadic Creutzfeldt-Jakob disease (sCJD) patients. In the present study, we aimed to investigate the presence of biomarkers and evaluate RT-QUIC assay in patients with gPrD, as the utility of RT-QUIC as a diagnostic tool in gPrD has yet to be determined. Method/Principal Findings 56 CSF samples were obtained from gPrD patients, including 20 cases of GSS with P102L mutation, 12 cases of fatal familial insomnia (FFI; D178N), and 24 cases of genetic CJD (gCJD), comprising 22 cases with E200K mutation and 2 with V203I mutation. We subjected all CSF samples to RT-QUIC assay, analyzed 14-3-3 protein by Western blotting, and measured t-tau protein using an ELISA kit. The detection sensitivities of RT-QUIC were as follows: GSS (78%), FFI (100%), gCJD E200K (87%), and gCJD V203I (100%). On the other hand the detection sensitivities of biomarkers were considerably lower: GSS (11%), FFI (0%), gCJD E200K (73%), and gCJD V203I (67%). Thus, RT-QUIC had a much higher detection sensitivity compared with testing for biomarkers, especially in patients with GSS and FFI. Conclusion/Significance RT-QUIC assay is more sensitive than testing for biomarkers in gPrD patients. RT-QUIC method would thus be useful as a diagnostic tool when the patient or the patients family does not agree to genetic testing, or to confirm the diagnosis in the presence of a positive result for genetic testing.

Cannabidiol potentiates pharmacological effects of Δ9-tetrahydrocannabinol via CB1 receptor-dependent mechanism

Cannabidiol, a non-psychoactive component of cannabis, has been reported to have interactions with Delta(9)-tetrahydrocannabinol (Delta(9)-THC). However, such interactions have not sufficiently been clear and may have important implications for understanding the pharmacological effects of marijuana. In the present study, we investigated whether cannabidiol modulates the pharmacological effects of Delta(9)-THC on locomotor activity, catalepsy-like immobilisation, rectal temperature and spatial memory in the eight-arm radial maze task in mice. In addition, we measured expression level of cannabinoid CB(1) receptor at striatum, cortex, hippocampus and hypothalamus. Delta(9)-THC (1, 3, 6 and 10 mg/kg) induced hypoactivity, catalepsy-like immobilisation and hypothermia in a dose-dependent manner. In addition, Delta(9)-THC (1, 3 and 6 mg/kg) dose-dependently impaired spatial memory in eight-arm radial maze. On the other hand, cannabidiol (1, 3, 10, 25 and 50 mg/kg) did not affect locomotor activity, catalepsy-like immobilisation, rectal temperature and spatial memory on its own. However, higher dose of cannabidiol (10 or 50 mg/kg) exacerbated pharmacological effects of lower dose of Delta(9)-THC, such as hypoactivity, hypothermia and impairment of spatial memory. Moreover, cannabidiol (50 mg/kg) with Delta(9)-THC (1 mg/kg) enhanced the expression level of CB(1) receptor expression in hippocampus and hypothalamus. Cannabidiol potentiated pharmacological effects of Delta(9)-THC via CB(1) receptor-dependent mechanism. These findings may contribute in setting the basis for interaction of cannabinoids and to find a cannabinoid mechanism in central nervous system.

Real-time quaking-induced conversion

We recently developed a new in vitro amplification technology, designated “real-time quaking-induced conversion (RT-QUIC)”, for detection of the abnormal form of prion protein (PrPSc) in easily accessible specimens such as cerebrospinal fluid (CSF). After assessment of more than 200 CSF specimens from Japanese and Australian patients, we found no instance of a false positive, and more than 80% accuracy for the correct diagnosis of sporadic Creutzfeldt–Jakob disease (sCJD). Furthermore, the RT-QUIC can be applied to other prion diseases, including scrapie, chronic wasting disease (CWD), and bovine spongiform encephalopathy (BSE), and is able to quantify prion seeding activity when combined with an end-point dilution of samples. These results indicate that the RT-QUIC, with its high sensitivity and specificity, will be of great use as an early, rapid and specific assay for prion diseases.

Antipsychotics improve Δ9-tetrahydrocannabinol-induced impairment of the prepulse inhibition of the startle reflex in mice

Recently, cannabinoid receptor agonists have been reported to impair prepulse inhibition (PPI) of the startle reflex. In the current study, we examined the effect of Delta9-tetrahydrocannabinol (THC), the principal psychoactive component of cannabis, on the PPI, and found that THC (10 mg/kg, i.p.) impaired the PPI concomitant with a decrease in the startle response. Antipsychotics such as haloperidol (0.3 mg/kg, i.p.) and risperidone (0.1 mg/kg, i.p.), which are potent dopamine D2 receptor antagonists, and SR141716 (10 mg/kg, i.p.), a CB1 cannabinoid receptor antagonist, reversed these THC-induced PPI deficits. Moreover, THC (10 mg/kg) increased dopamine (DA) release in the nucleus accumbens but not medial prefrontal cortex over a 50-100-min period (time of PPI test) after treatment, and SR141716 (10 mg/kg) reversed this increase in DA release induced by THC. These results suggest that dopaminergic hyperfunction in the nucleus accumbens may be involved in THC-induced PPI deficits.

Decreased acetylcholine release is correlated to memory impairment in the Tg2576 transgenic mouse model of Alzheimer's disease

Acetylcholine (ACh) release is one of the key factors in memory mechanisms. To clarify whether beta-amyloid (Abeta) induces a disturbance of the cholinergic system leading to memory impairment, we examined memory impairment and measured hippocampal ACh release in Tg2576 (Tg) mice that over-express the Swedish mutant amyloid precursor protein (APPsw). Furthermore, we examined Abeta burden with aging. Tg mice aged 9-11 months, but not aged 4-6 months, showed memory impairment in the 8-arm radial maze behavior test. Spontaneous ACh release was not altered in Tg mice compared with age-matched control mice at 4-6 or 9-11 months of age. On the other hand, high-K(+)-evoked ACh release was decreased in Tg mice aged 9-11 months, but not in Tg mice aged 4-6 months. Hippocampal Abeta increased in an age-dependent manner, but evident amyloid plaques were not found in the hippocampus of Tg mice aged 11 months. These results suggest that memory impairment in Tg mice could be attributed to cholinergic synapse dysfunction that could not be caused predominantly by amyloid plaques. Measuring ACh release in this model might be a useful index for the screening of new drugs to treat the early-phase of Alzheimers disease.

Disruption of the Prepulse Inhibition of the Startle Reflex in Vasopressin V1b Receptor Knockout Mice: Reversal by Antipsychotic Drugs

In the present study, we investigated whether mice lacking the arginine vasopressin (AVP) V1b receptor (V1bR) exhibit deficits of prepulse inhibition (PPI) of the startle reflex, reminiscent of the sensorimotor gating deficits observed in a large majority of schizophrenic patients. V1bR knockout (KO) mice displayed significantly reduced levels of PPI of the startle reflex. In addition to PPI deficits, V1bR KO mice showed increased acoustic startle response. However, acoustic startle response was not significantly correlated to the PPI of the startle reflex in V1bR KO mice. V1bR KO mice also showed a decrease in basal levels of extracellular dopamine (DA) in the medial prefrontal cortex, which is thought to be an important brain region for PPI. Moreover, PPI deficits observed in the V1bR KO mice are significantly reversed by atypical antipsychotics such as risperidone and clozapine but not by a typical neuroleptic haloperidol, like in schizophrenic patients. By contrast, we did not observe any significant differences between V1bR KO mice and wild-type mice in the open-field, light/dark, elevated plus maze, and forced swimming tests. The results of the present study indicate that V1bR may be involved in the regulation of PPI of the startle reflex. The V1bR has been considered an important molecular target for the development of antipsychotic drugs.

FK506 reduces abnormal prion protein through the activation of autolysosomal degradation and prolongs survival in prion-infected mice

Prion diseases are fatal neurodegenerative disorders and no effective treatment has been established to date. In this study, we evaluated the effect of FK506 (tacrolimus), a macrolide that is known to be a mild immunosuppressant, on prion infection, using cell culture and animal models. We found that FK506 markedly reduced the abnormal form of prion protein (PRNPSc) in the cell cultures (N2a58 and MG20) infected with Fukuoka-1 prion. The levels of autophagy-related molecules such as LC3-II, ATG12–ATG5 and ATG7 were significantly increased in the FK506-treated cells, and resulted in the increased formation of autolysosomes. Upregulation of the autophagy-related molecules was also seen in the brains of FK506-treated mice and the accumulation of PRNPSc was delayed. The survival periods in mice inoculated with Fukuoka-1 were significantly increased when FK506 was administered from day 20 post-inoculation. These findings provide evidence that FK506 could constitute a novel antiprion drug, capable of enhancing the degradation of PRNPSc in addition to attenuation of microgliosis and neuroprotection.

Δ9-Tetrahydrocannabinol-induced catalepsy-like immobilization is mediated by decreased 5-HT neurotransmission in the nucleus accumbens due to the action of glutamate-containing neurons

Delta(9)-tetrahydrocannabinol (THC) has been reported to induce catalepsy-like immobilization, but the mechanism underlying this effect remains unclear. In the present study, in order to fully understand the neural circuits involved, we determined the brain sites involved in the immobilization effect in rats. THC dose-dependently induced catalepsy-like immobilization. THC-induced catalepsy-like immobilization is mechanistically different from that induced by haloperidol (HPD), because unlike HPD-induced catalepsy, animals with THC-induced catalepsy became normal again following sound and air-puff stimuli. THC-induced catalepsy was reversed by SR141716, a selective cannabinoid CB(1) receptor antagonist. Moreover, THC-induced catalepsy was abolished by lesions in the nucleus accumbens (NAc) and central amygdala (ACE) regions. On the other hand, HPD-induced catalepsy was suppressed by lesions in the caudate putamen (CP), substantia nigra (SN), globus pallidus (GP), ACE and lateral hypothalamus (LH) regions. Bilateral microinjection of THC into the NAc region induced catalepsy-like immobilization. This THC-induced catalepsy was inhibited by serotonergic drugs such as 5-hydroxy-L-tryptophan (5-HTP), a 5-HT precursor, and 5-methoxy-N,N-dimethyltryptamine (5-MeODMT), a 5-HT receptor agonist, as well as by anti-glutamatergic drugs such as MK-801 and amantadine, an N-methyl-d-aspartate (NMDA) receptor antagonist. THC significantly decreased 5-HT and glutamate release in the NAc, as shown by in vivo microdialysis. SR141716 reversed and MK-801 inhibited this decrease in 5-HT and glutamate release. These findings suggest that the THC-induced catalepsy is mechanistically different from HPD-induced catalepsy and that the catalepsy-like immobilization induced by THC is mediated by decreased 5-HT neurotransmission in the nucleus accumbens due to the action of glutamate-containing neurons.

Increased expression of p62/SQSTM1 in prion diseases and its association with pathogenic prion protein

Prion diseases are neurodegenerative disorders characterized by the aggregation of abnormally folded prion protein (PrPSc). In this study, we focused on the mechanism of clearance of PrPSc, which remains unclear. p62 is a cytosolic protein known to mediate both the formation and degradation of aggregates of abnormal proteins. The levels of p62 protein increased in prion-infected brains and persistently infected cell cultures. Upon proteasome inhibition, p62 co-localized with PrPSc, forming a large aggregate in the perinuclear region, hereafter referred to as PrPSc-aggresome. These aggregates were surrounded with autophagosome marker LC3 and lysosomes in prion-infected cells. Moreover, transient expression of the phosphomimic form of p62, which has enhanced ubiquitin-binding activity, reduced the amount of PrPSc in prion-infected cells, indicating that the activation of p62 could accelerate the clearance of PrPSc. Our findings would thus suggest that p62 could be a target for the therapeutic control of prion diseases.