Homeostatic p62 levels and inclusion body formation in CHCHD2 knockout mice.

Inactivation of constitutive autophagy results in the formation of cytoplasmic inclusions in neurones, but the relationship between impaired autophagy and Lewy bodies (LBs) remains unknown. α-Synuclein and p62, components of LBs, are the defining characteristic of Parkinson's disease (PD). Until now, we have analyzed mice models and demonstrated p62 aggregates derived from an autophagic defect might serve as 'seeds' and can potentially be a cause of LB formation. P62 may be the key molecule for aggregate formation. To understand the mechanisms of LBs, we analyzed p62 homeostasis and inclusion formation using PD model mice. In PARK22-linked PD, intrinsically disordered mutant CHCHD2 initiates Lewy pathology. To determine the function of CHCHD2 for inclusions formation, we generated Chchd2-knockout (KO) mice and characterized the age-related pathological and motor phenotypes. Chchd2 KO mice exhibited p62 inclusion formation and dopaminergic neuronal loss in an age-dependent manner. These changes were associated with a reduction in mitochondria complex activity and abrogation of inner mitochondria structure. In particular, the OPA1 proteins, which regulate fusion of mitochondrial inner membranes, were immature in the mitochondria of CHCHD2-deficient mice. CHCHD2 regulates mitochondrial morphology and p62 homeostasis by controlling the level of OPA1. Our findings highlight the unexpected role of the homeostatic level of p62, which is regulated by a non-autophagic system, in controlling intracellular inclusion body formation, and indicate that the pathologic processes associated with the mitochondrial proteolytic system are crucial for loss of DA neurones.

Development of a novel tau propagation mouse model endogenously expressing 3 and 4 repeat tau isoforms.

The phenomenon of 'prion-like propagation' in which aggregates of abnormal amyloid-fibrilized protein propagate between neurons and spread pathology, is attracting attention as a new mechanism in neurodegenerative diseases. There is a strong correlation between the accumulation or spread of abnormal tau aggregates and the clinical symptoms of tauopathies. Microtubule-associated protein tau (MAPT) contains a microtubule-binding domain that consists of three or four repeats (3R/4R) due to alternative mRNA splicing of transcripts for the MAPT gene. Although a number of models for tau propagation have been reported, most use 4R human tau transgenic mice or adult wild-type mice expressing only endogenous 4R tau and these models have not been able to reproduce the pathology of Alzheimer's disease in which 3R and 4R tau accumulate simultaneously, or that of Pick's disease in which only 3R tau is aggregated. These deficiencies may reflect differences between human and rodent tau isoforms in the brain. To overcome this problem, we used genome editing techniques to generate mice that express an equal ratio of endogenous 3R and 4R tau, even after they become adults. We injected these mice with sarkosyl-insoluble fractions derived from the brains of human tauopathy patients such as those afflicted with Alzheimer's disease (3R and 4R tauopathy), corticobasal degeneration (4R tauopathy) or Pick's disease (3R tauopathy). At 8-9 months following intracerebral injection of mice, histopathological and biochemical analyses revealed that the abnormal accumulation of tau was seed-dependent, with 3R and 4R tau in Alzheimer's disease-injected brains, 4R tau only in corticobasal degeneration-injected brains and 3R tau only in Pick disease-injected brains, all of which contained isoforms related to those found in the injected seeds. The injected abnormal tau was seeded, and accumulated at the site of injection and at neural connections, predominantly within the same site. The abnormal tau newly accumulated was found to be endogenous in these mice and to have crossed the species barrier. Of particular importance, Pick's body-like inclusions were observed in Pick's disease-injected mice, and accumulations characteristic of Pick's disease were reproduced, suggesting that we have developed the first model that recapitulates the pathology of Pick's disease. These models are not only useful for elucidating the mechanism of propagation of tau pathology involving both 3R and 4R isoforms, but can also reproduce the pathology of tauopathies, which should lead to the discovery of new therapeutic agents.

GyrA and/or ParC alterations of Haemophilus influenzae strains isolated from the urethra of men with acute urethritis.

Of 73 clinical strains of Haemophilus influenzae isolated from the urethra of men with urogenital infections, we enrolled 6 strains (8.2%) with levofloxacin (LVFX) minimum inhibitory concentrations (MICs) of ≥0.03 µg/ml in this study. All the strains were isolated from non-gonococcal urethritis (NGU). We amplified the quinolone resistance-determining region of the gyrA gene and the analogous region of the parC gene from bacterial DNAs by PCR and sequenced the PCR products. Two strains with a LVFX MIC of 0.03 µg/ml had an amino acid change of Asp88 to Gly in GyrA. One with a LVFX MIC of 0.06 µg/ml had a change of Asp88 to Tyr in GyrA. Two with respective LVFX MICs of 0.12 and 0.25 µg/ml had a change of Ser84 to Leu in GyrA. One with a LVFX MIC of 1 µg/ml had changes of Ser84 to Leu in GyrA and of Ser84 to Ile in ParC. Multilocus sequence typing showed two strains with a change of Asp88 to Gly in GyrA had the same sequence type, but the others had sequence types different from each other. Single amino acid changes in GyrA alone or single changes in both GyrA and ParC could contribute to decreased susceptibility to fluoroquinolones in H. influenzae isolates from NGU. Most of the isolates with GyrA and/or ParC alterations would be multiclonal. The prevalence of such isolates would be relatively low, and they would still be susceptible to fluoroquinolones commonly prescribed for treatment of NGU.

Macrolide and fluoroquinolone resistance is uncommon in clinical strains of Chlamydia trachomatis.

We analyzed the 23S rRNA, gyrA and parC genes of Chlamydia trachomatis DNAs from men with urethritis and determined microbiological outcomes of an extended-release azithromycin (azithromycin-SR) regimen (2 g once daily for 1 day) and a sitafloxacin regimen (100 mg twice daily for 7 days) for chlamydial urethritis to clarify the macrolide and fluoroquinolone resistance status of clinical strains of C. trachomatis. We amplified the portions of 2 alleles of the 23S rRNA gene and the gyrA and parC genes from C. trachomatis DNAs in 284 first-voided urine specimens from men with chlamydial urethritis by PCR and sequenced their PCR products. We enrolled 369 men with chlamydial urethritis, comprising 314 and 55 treated with the azithromycin-SR regimen and the sitafloxacin regimen, respectively. Alleles 1 and/or 2 of the 23S rRNA gene were analyzed in 162 specimens. No mutations were found in the sequenced regions, including the central portion of domain V. The gyrA and parC genes were analyzed in 118 and 113 specimens, respectively. No amino acid changes were found within the quinolone resistance-determining region of the gyrA gene and in the sequenced region of the parC gene. The microbiological outcomes of the azithromycin-SR and sitafloxacin regimens were assessed in 176 and 30 men, respectively. The eradication rates were 96.0% (95% CI 93.1%-98.9%) for the azithromycin-SR regimen and 100% for the sitafloxacin regimen. Clinical strains of C. trachomatis with macrolide and/or fluoroquinolone resistance would be uncommon, and azithromycin or fluoroquinolone regimens could be recommended as treatments for chlamydial infections.

Haemophilus influenzae Isolated From Men With Acute Urethritis: Its Pathogenic Roles, Responses to Antimicrobial Chemotherapies, and Antimicrobial Susceptibilities.

BACKGROUND: There have been few comprehensive studies on Haemophilus influenza-positive urethritis. METHODS: In this retrospective study, we enrolled 68 men with H. influenzae-positive urethritis, including coinfections with Neisseria gonorrhoeae, Chlamydia trachomatis, and/or genital mycoplasmas: 2, 3, 20, and 43 treated with ceftriaxone, levofloxacin, sitafloxacin, and extended-release azithromycin (azithromycin-SR), respectively. We assessed microbiological outcomes in 54 men and clinical outcomes in 46 with H. influenzae-positive monomicrobial nongonococcal urethritis. We determined minimum inhibitory concentrations (MICs) of 6 antimicrobial agents for 59 pretreatment isolates. RESULTS: H. influenzae was eradicated from the men treated with ceftriaxone, levofloxacin, or sitafloxacin. The eradication rate with azithromycin-SR was 85.3%. The disappearance or alleviation of urethritis symptoms and the decreases in leukocyte counts in first-voided urine were significantly associated with the eradication of H. influenzae after treatment. For the isolates, ceftriaxone, levofloxacin, sitafloxacin, azithromycin, tetracycline, and doxycycline MICs were ≤0.008-0.25, 0.008-0.5, 0.001-0.008, 0.12-1, 0.25-16, and 0.25-2 µg/mL, respectively. The azithromycin MICs for 3 of 4 strains persisting after azithromycin-SR administration were 1 µg/mL. H. influenzae with an azithromycin MIC of 1 µg/mL increased chronologically. CONCLUSIONS: H. influenzae showed good responses to the chemotherapies for urethritis. The significant associations of the clinical outcomes of the chemotherapies with their microbiological outcomes suggested that H. influenzae could play pathogenic roles in urethritis. All isolates, except for one with decreased susceptibility to tetracyclines, were susceptible to the examined agents. However, the increase in H. influenzae with an azithromycin MIC of 1 µg/mL might threaten efficacies of azithromycin regimens on H. influenzae-positive urethritis.

Emergence of Mycoplasma genitalium with clinically significant fluoroquinolone resistance conferred by amino acid changes both in GyrA and ParC in Japan.

We observed fluoroquinolone treatment failures in 2 men with Mycoplasma genitalium-positive non-gonococcal urethritis in Japan. A fluoroquinolone regimen of sitafloxacin 100 mg twice daily for 7 days failed to eradicate M. genitalium. In both cases, M. genitalium had fluoroquinolone resistance-associated amino acid changes both in GyrA and ParC and a macrolide resistance-associated mutation in the 23S rRNA gene. The emergence of such multi-drug resistant strains can threaten antimicrobial chemotherapy for M. genitalium infections in Japan, because we will lose the first- (azithromycin) and second-line (sitafloxacin) antimicrobial agents to treat M. genitalium infections. We prescribed an extended minocycline regimen of minocycline 100 mg twice daily for 14 days for our patients, and the regimen was successful in eradicating the M. genitalium. The extended minocycline regimen might be an option that we can try when treating multi-drug resistant M. genitalium infections in clinical practice.

Clinical courses of herpes simplex virus-induced urethritis in men.

We retrieved clinical data of 13 men having herpes simplex virus (HSV)-induced non-gonococcal urethritis (NGU) without visible herpetic lesions. They visited a clinic in Sendai, Japan, between April 2013 and December 2015. All the men complained of dysuria. Meatitis was observed in 9 of the 13 men. Mononuclear cells were observed in the urethral smears from 9 men. The 13 men were treated with azithromycin or sitafloxacin regimen. First-voided urine (FVU) specimens became negative for HSV in 8 of the 10 men who returned to the clinic after antibacterial treatment, and urethritis symptoms were alleviated. However, herpetic lesions were observed at the follow-up visits in 3 men, and 2 of them were still positive for HSV in their FVU. HSV could be a cause of acute urethritis without causing visible herpetic lesions. The shedding of HSV from the urethra would spontaneously cease with alleviation of urethritis symptoms in most cases of HSV-induced NGU without antiviral therapy. However, new herpetic lesions could be developed in some cases. Early antiviral therapy is beneficial for patients with HSV infections. The development of meatitis and the mononuclear cell response in the urethral smear could be helpful to diagnose HSV-induced NGU. Therefore, we should presumptively initiate anti-HSV therapy for patients with signs and symptoms suggestive of HSV-induced NGU at their first presentation.

Long-term oral intake of aluminium or zinc does not accelerate Alzheimer pathology in AßPP and AßPP/tau transgenic mice.

Whether or not the oral intake of metals such as aluminium (Al) and zinc (Zn) is a risk for Alzheimer's disease (AD) has been a matter of controversy. Lack of AD pathology in patients with Al encephalopathy indicates Al does not cause AD. On the other hand, some epidemiological studies have suggested high Al increases the occurrence of AD. Our purpose is to test if high Al in drinking water is a risk factor for AD. We administered Al and Zn in drinking water to Tg2576, a transgenic mouse model for amyloid ß-protein (Aß) deposition with the Aß precursor protein (AßPP) mutations (K670N/M671L), and Tg2576/tau(P301L), a model for Aß and tau deposition. Deionized water was given to the control Tg2576 and Tg2576/tau. After administration for 4-10 months of approximately 100 mg/kg body weight Al or Zn per day, we were not able to find by quantitative immunohistochemical analyses differences in the deposition of Aß and tau between the treated and untreated groups. Nor did the Al or Zn treatment affect the amount of soluble Aß and Aß*56, an Aß oligomer, measured by ELISA or immunoblot. The oral intake of excess Al or Zn does not accelerate AD pathology in the transgenic mouse models for Aß and tau accumulation. Such results do not seem to support the notion that excessive oral intake of Al or Zn is a risk factor for AD.

Progressive nonfluent aphasia: a rare clinical subtype of FTLD-TDP in Japan.

Progressive nonfluent aphasia (PNFA) is a clinical subtype of frontotemporal lobar degeneration (FTLD). FTLD with tau accumulation (FTLD-tau) and FTLD with TDP-43 accumulation (FTLD-TDP) both cause PNFA. We reviewed clinical records of 29 FTLD-TDP cases in the brain archive of our institute and found only one case of PNFA. The patient was an 81-year-old male at death. There was no family history of dementia or aphasia. He presented with slow, labored and nonfluent speech at age 75. Behavioral abnormality and movement disorders were absent. MRI at age 76 demonstrated atrophy of the perisylvian regions, including the inferior frontal gyrus, insular gyrus and superior temporal gyrus. The atrophy was more severe in the left hemisphere than the right. On post mortem examinations, neuronal loss was evident in these regions as well as in the substantia nigra. There were abundant TDP-43-immunoreactive neuronal cytoplasmic inclusions and round or irregular-shaped structures in the affected cerebral cortices. A few dystrophic neurites and neuronal intranuclear inclusions were also seen. FTLD-TDP showing PNFA seems to be rare but does exist in Japan, similar to that in other countries.

Spatial frequency differentially affects habituation in migraineurs: a steady-state visual-evoked potential study.

A lack of habituation in visual-evoked potentials (VEPs) is the main abnormality observed in migraineurs. However, no study of steady-state VEPs has yet evaluated pattern-reversal stimuli with respect to habituation behavior or spatial frequency. The aim of this study was to clarify habituation behavior in migraineurs between attacks and to establish characteristics of VEPs in these patients. Steady-state VEPs were sequentially recorded as checkerboard patterns in four consecutive blocks from 12 patients with migraine without aura (MO), 12 patients with migraine with aura (MA), and 12 healthy controls (HC) at four spatial frequencies of 0.5, 1.0, 2.0, and 4.0 cycles per degree (cpd) with a stimulus rate of 7.5 Hz (15 reversal/s). VEP amplitudes were consistently higher in migraineurs. However, habituation was not demonstrated in HCs, and migraineurs did not reveal a clear lack of habituation. MAs exhibited high-amplitude VEPs, depending on spatial frequency. In the MA patients, amplitude differences reached statistical significance at 2.0 cpd. The sequential amplitude changes at 0.5 cpd were significantly different in MAs compared with HCs. Migraine patients exhibited high-amplitude VEPs, which were dependent on spatial frequency, and may be related to altered excitability in pre-cortical and cortical visual processing.

[Infected solitary renal cyst of the graft in a renal transplant recipient : a case report].

A 59-year-old woman with end-stage renal disease of diabetic nephropathy who had been on maintenance hemodialisis for 4 years, underwent a living-unrelated renal transplantation 6 years ago. She was admitted to our hospital, because of a low grade fever and edema. Ultrasonography revealed the cyst with heterogeneity structure in the upper pole of the transplanted kidney. Magnetic resonance imaging showed a high-intensity cystic mass measuring 68×53 mm. As fever and laboratory data did not improve sufficiently by the treatment with antibiotics, echo-guided puncture and drainage were performed for the abnormal structure in the upper pole of the transplanted kidney. In the culture of the purulent aspirate drained from renal cyst, Escherichia coli was isolated. To our knowledge, this is the first report of infected renal cyst of the graft in a renal transplant recipient in the world.

Usefulness of robot-assisted radical prostatectomy in a patient with oligometastatic castration-resistant prostate cancer.

INTRODUCTION: The patients with prostate cancer and low-volume osseous metastases who underwent local definitive therapies had lower risks of cancer-specific mortality. The usefulness of local definitive therapy for metastatic prostate cancer remains unclear. CASE PRESENTATION: A 76-year-old man visited a private hospital with a chief complaint of left lower limb pain. His serum prostate-specific antigen level was 365.156 ng/mL. Histological evaluation led to the initial diagnosis of adenocarcinoma of Gleason score 4 + 4 and clinical stage T3a N1 M1b. Although androgen deprivation therapy was performed, he developed metastatic castration-resistant prostate cancer 6 months after the initial treatment. Therefore, he received enzalutamide and attained a serum prostate-specific antigen level of 0.002 ng/mL 7 months after the second treatment. We performed robot-assisted radical prostatectomy 1 year after diagnosis. Histopathological examination revealed that prostate cancer cells disappeared into the prostate. CONCLUSION: Robot-assisted radical prostatectomy in selected patients with metastatic castration-resistant prostate cancer may improve oncological outcomes.

Author Correction: Electroretinographic recordings with skin electrodes to assess effects of vitrectomy with gas tamponade on eyes with rhegmatogenous retinal detachment.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correction: Electroretinograms recorded with skin electrodes in silicone oil-filled eyes.

[This corrects the article DOI: 10.1371/journal.pone.0216823.].

Electroretinographic recordings with skin electrodes to assess effects of vitrectomy with gas tamponade on eyes with rhegmatogenous retinal detachment.

The purpose of this study was to evaluate the retinal function by electroretinograms (ERGs) recorded with the RETeval system using skin electrodes after pars plana vitrectomy (PPV) with gas tamponade in eyes with a rhegmatogeneous retinal detachment (RRD). Flicker ERGs were recorded from 17 eyes with an RRD before (baseline), within 2 weeks after the PPV when the size of the tamponade gas was approximately one-half of the vitreous cavity (P1), and when the gas had been completely absorbed (P2). The amplitudes of the flicker ERGs at each phase were compared to that at the baseline. The median (25th, 75th percentile) of the amplitude was 10.0 µV (5.5, 13.0 µV) at the baseline, 11.7 µV at P1 (7.8, 14.8 µV; P = 0.003), and 17.1 µV at P2 (11.7 23.3 µV; P < 0.001). The ratio of the amplitudes in the affected eye to that in the fellow eye at the baseline and at each phase was calculated, and the ratio of the amplitudes at P1 and P2 were significantly and positively correlated (P = 0.723, P = 0.001; Spearman's rank correlation coefficient). We conclude that recordings the flicker ERGs with skin electrodes can be used to assess the physiology of eyes even with the vitreous cavity half-filled with the gas used to tamponade the retina.

Electroretinograms recorded with skin electrodes in silicone oil-filled eyes.

PURPOSE: To assess the physiology of the retina by electroretinography (ERG) with skin electrodes in eyes that had undergone vitrectomy with silicone oil (SO) tamponade. DESIGN: Retrospective case series. METHOD: ERGs were recorded from eleven eyes with complex vitreoretinal disorders and from the normal fellow eyes. The affected eyes underwent pars plana vitrectomy (PPV) with SO tamponade. ERGs were recorded before and after the SO was removed. The amplitudes and implicit times of the a- and b-waves of the affected eyes were compared to those of the normal fellow eyes. In addition, the ratios of the amplitudes of the b-waves of the affected eyes to those of the fellow eyes were compared before and after the SO was removed. RESULTS: ERGs were recordable from 7 eyes (63.6%) before the SO was removed and 11 eyes (100%) after the SO was removed. The a- and b-wave amplitudes were significantly smaller in the affected eyes than those of the fellow eyes at the baseline. The b-wave amplitude before the removal of the SO was significantly and positively correlated with that after the SO removal. The ratios of the b-waves of the affected/normal fellow eye significantly increased after the SO was removed. CONCLUSION: The results indicate that ERGs picked up by skin electrode can be used to assess the physiology of the retina in eyes with a SO tamponade. The amplitude of the b-waves of the ERGs in silicone-filled eyes can be used to predict the amplitude after the silicone is removed.

Effect of L-DOPA/Benserazide on Propagation of Pathological α-Synuclein.

Parkinson's disease (PD) and related disorders are characterized by filamentous or fibrous structures consisting of abnormal α-synuclein in the brains of patients, and the distributions and spread of these pathologies are closely correlated with disease progression. L-DOPA (a dopamine precursor) is the most effective therapy for PD, but it remains unclear whether the drug has any effect on the formation and propagation of pathogenic abnormal α-synuclein in vivo. Here, we tested whether or not L-DOPA influences the prion-like spread of α-synuclein pathologies in a wild-type (WT) mouse model of α-synuclein propagation. To quantitative the pathological α-synuclein in mice, we prepared brain sections stained with an anti-phosphoSer129 (PS129) antibody after pretreatments with autoclaving and formic acid, and carefully analyzed positive aggregates on multiple sections covering the areas of interest using a microscope. Notably, a significant reduction in the accumulation of phosphorylated α-synuclein was detected in substantia nigra of L-DOPA/benserazide (a dopamine decarboxylase inhibitor)-treated mice, compared with control mice. These results suggest that L-DOPA may slow the progression of PD in vivo by suppressing the aggregation of α-synuclein in dopaminergic neurons and the cell-to-cell propagation of abnormal α-synuclein. This is the first report describing the suppressing effect of L-DOPA/benserazide on the propagation of pathological α-synuclein. The experimental protocols and detection methods in this study are expected to be useful for evaluation of drug candidates or new therapies targeting the propagation of α-synuclein.

Progranulin haploinsufficiency reduces amyloid beta deposition in Alzheimer's disease model mice.

Granulin (Grn) mutations were identified in familial frontotemporal lobar degeneration (FTLD) patients with TAR DNA-binding protein of 43 kd (TDP-43) pathology. Grn transcript haploinsufficiency is proposed as a disease mechanism that leads to the loss of functional progranulin (PGRN) protein. Thus, these mutations are strongly involved in FTLD pathogenesis. Moreover, recent findings indicate that Grn mutations are associated with other neurodegenerative disorders with tau pathology, including Alzheimer's disease. To investigate the influence of PGRN on amyloid beta (Aß) accumulation, amyloid precursor protein (APP) transgenic mice were interbred with Grn-deficient mice, producing APP transgenic mice harboring the Grn hemizygote (APP/Grn+/-). Brains were collected from 16-18-month-old APP and APP/Grn+/- mice and sequential extraction of proteins, immunoblotting and immunohistochemical analysis were performed. Immunohistochemical analysis showed that the number and area of Aß plaque was significantly decreased in APP/Grn+/- mice as compared to APP mice. Immunoblotting analysis revealed that Aß was reduced in the sarkosyl-insoluble fraction of 16-18-month-old APP/Grn+/- mice as compared with that of APP transgenic mice. Our data suggest that PGRN haploinsufficiency may decrease accumulation of Aß.

Loss of autophagy in dopaminergic neurons causes Lewy pathology and motor dysfunction in aged mice.

Inactivation of constitutive autophagy results in the formation of cytoplasmic inclusions in neurons, but the relationship between impaired autophagy and Lewy bodies (LBs) as well as the in vivo process of formation remains unknown. Synuclein, a component of LBs, is the defining characteristic of Parkinson's disease (PD). Here, we characterize dopamine (DA) neuron-specific autophagy-deficient mice and provide in vivo evidence for LB formation. Synuclein deposition is preceded by p62 and resulted in the formation of inclusions containing synuclein and p62. The number and size of these inclusions were gradually increased in neurites rather than soma with aging. These inclusions may facilitate peripheral failures. As a result, DA neuron loss and motor dysfunction including the hindlimb defect were observed in 120-week-old mice. P62 aggregates derived from an autophagic defect might serve as "seeds" and can potentially be cause of LB formation.