Parkin accumulation in aggresomes due to proteasome impairment

Parkinson's disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra and by the presence of ubiquitinated cytoplasmic inclusions known as Lewy bodies. Alpha-synuclein and Parkin are two of the proteins associated with inherited forms of PD and are found in Lewy bodies. Whereas numerous reports indicate the tendency of alpha-synuclein to aggregate both in vitro and in vivo, no information is available about similar physical properties for Parkin. Here we show that overexpression of Parkin in the presence of proteasome inhibitors leads to the formation of aggresome-like perinuclear inclusions. These eosinophilic inclusions share many characteristics with Lewy bodies, including a core and halo organization, immunoreactivity to ubiquitin, alpha-synuclein, synphilin-1, Parkin, molecular chaperones, and proteasome subunit as well as staining of some with thioflavin S. We propose that the process of Lewy body formation may be akin to that of aggresome-like structures. The tendency of wild-type Parkin to aggregate and form inclusions may have implications for the pathogenesis of sporadic PD.     

Amyloid Cardiomyopathy in Hereditary Transthyretin V30M Amyloidosis - Impact of Sex and Amyloid Fibril Composition

Purpose

Transthyretin V30M (ATTR V30M) amyloidosis is a phenotypically diverse disease with symptoms ranging from predominant neuropathy to exclusive cardiac manifestations. The aims of this study were to determine the dispersion of the two types of fibrils found in Swedish ATTR V30M patients -Type A consisting of a mixture of truncated and full length ATTR fibrils and type B fibrils consisting of full length fibrils, and to estimate the severity of cardiac dysfunction in relation to fibril composition and sex.

Material and Methods

Echocardiographic data were analysed in 107 Swedish ATTR V30M patients with their fibril composition determined as either type A or type B. Measurements of left ventricular (LV) dimensions and evaluation of systolic and diastolic function including speckle tracking derived strain were performed. Patients were grouped according to fibril type and sex. Multivariate linear regression was utilised to determine factors of significant impact on LV thickness.

Results

There was no significant difference in proportions of the two types of fibrils between men and women. In patients with type A fibrils, women had significantly lower median septal (p = 0.007) and posterior wall thicknesses (p = 0.010), lower median LV mass indexed to height (p = 0.008), and higher septal strain (p = 0.037), as compared to males. These differences were not apparent in patients with type B fibrils. Multiple linear regression analysis revealed that fibril type, sex and age all had significant impact on LV septal thickness.

Conclusion

This study demonstrates a clear difference between sexes in the severity of amyloid heart disease in ATTR V30M amyloidosis patients. Even though type A fibrils were associated with more advanced amyloid heart disease compared to type B, women with type A fibrils generally developed less cardiac infiltration than men. The differences may explain the better outcome for liver transplanted late-onset female patients compared to males.     

Allele Specific Expression of the Transthyretin Gene in Swedish Patients with Hereditary Transthyretin Amyloidosis (ATTR V30M) Is Similar between the Two Alleles

Background

Hereditary transthyretin (TTR) amyloidosis (ATTR) is an autosomal dominant disease characterized by extracellular deposits of amyloid fibrils composed of misfolded TTR. The differences in penetrance and age at onset are vast, both between and within populations, with a generally late onset for Swedish carriers. In a recent study the entire TTR gene including the 3′ UTR in Swedish, French and Japanese ATTR patients was sequenced. The study disclosed a SNP in the V30M TTR 3′ UTR of the Swedish ATTR population that was not present in either the French or the Japanese populations (rs62093482-C>T). This SNP could create a new binding site for miRNA, which would increase degradation of the mutated TTR’s mRNA thus decrease variant TTR formation and thereby delay the onset of the disease. The aim of the present study was to disclose differences in allele specific TTR expression among Swedish V30M patients, and to see if selected miRNA had any effect upon the expression.

Methodology/Principal Findings

Allele-specific expression was measured on nine liver biopsies from Swedish ATTR patients using SNaPshot Multiplex assay. Luciferase activity was measured on cell lines transfected with constructs containing the TTR 3′ UTR. Allele-specific expression measured on liver biopsies from Swedish ATTR patients showed no difference in expression between the two alleles. Neither was there any difference in expression between cell lines co-transfected with two constructs with or without the TTR 3′ UTR SNP regardless of added miRNA.

Conclusions/Significance

The SNP found in the 3′ UTR of the TTR gene has no effect on degrading the variant allele’s expression and thus has no impact on the diminished penetrance of the trait in the Swedish population. However, the 3′ UTR SNP is unique for patients descending from the Swedish founder, and this SNP could be utilized to identify ATTR patients of Swedish descent.     

Promising potential of new generation translocator protein tracers providing enhanced contrast of arthritis imaging by positron emission tomography in a rat model of arthritis

Introduction

The role of popliteal cysts and subgastrocnemius bursitis in knee joint homeostasis is uncertain. The aim of this study is to describe cross-sectional associations between popliteal cysts, subgastrocnemius bursitis, knee symptoms and structural abnormalities in older adults.

Methods

A cross-sectional sample of 900 randomly-selected subjects (mean age 63 years, 48% female) were studied. Knee pain, stiffness and dysfunction were assessed by self-administered Western Ontario McMaster Osteoarthritis Index (WOMAC) questionnaire. Radiographic knee osteophyte and joint space narrowing (JSN) were recorded. Magnetic resonance imaging (MRI) was utilized to assess popliteal cysts, subgastrocnemius bursitis, cartilage defects and bone marrow lesions (BMLs).

Results

Popliteal cysts were present in 11.7% and subgastrocnemius bursitis in 12.7% of subjects. Subgastrocnemius bursitis was more common in those with popliteal cyst (36.2% versus 9.7%, P <0.01). In multivariable analyses, popliteal cysts were significantly associated with increased osteophytes in both medial and lateral tibiofemoral compartments while subgastrocnemius bursitis was associated with increased osteophytes and JSN in the medial tibiofemoral compartment. Both were significantly associated with cartilage defects in all compartments, and with BMLs in the medial tibiofemoral compartment. Furthermore, both popliteal cysts and subgastrocnemius bursitis were significantly associated with increased weight-bearing knee pain but these associations became non-significant after adjustment for cartilage defects and BMLs.

Conclusions

Popliteal cysts and subgastrocnemius bursitis are associated with increased symptoms as well as radiographic and MRI-detected joint structural abnormalities. Longitudinal data will help resolve if they are a consequence or a cause of knee joint abnormalities.     

Effect of hydroxyurea on the promoter occupancy profiles of tumor suppressor p53 and p73

Background  

The p53 tumor suppressor and its related protein, p73, share a homologous DNA binding domain, and mouse genetics studies have suggested that they have overlapping as well as distinct biological functions. Both p53 and p73 are activated by genotoxic stress to regulate an array of cellular responses. Previous studies have suggested that p53 and p73 independently activate the cellular apoptotic program in response to cytotoxic drugs. The goal of this study was to compare the promoter-binding activity of p53 and p73 at steady state and after genotoxic stress induced by hydroxyurea.     

Degradation of 4-chloro-2-methylphenoxyacetic acid in top- and subsoil is quantitatively linked to the class III tfdA gene

The tfdA gene is known to be involved in the first step of the degradation of the phenoxy acid herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) in several soil bacteria, but bacteria containing other tfdA-like genes have been isolated as well. A quantitative real-time PCR method was used to monitor the increase in the concentration of tfdA genes during degradation of MCPA in sandy topsoil and subsoil over a period of 115 days. Quantitative PCR revealed growth in the tfdA-containing bacterial community, from 500 genes g(-1) soil to approximately 3 x 10(4) genes g(-1) soil and to 7 x 10(5) genes g(-1) soil for topsoil initially added to 2.3 mg MCPA kg(-1) (dry weight) soil and 20 mg MCPA kg(-1) (dry weight) soil, respectively. We analyzed the diversity of the tfdA gene during the degradation experiment. Analyses of melting curves of real-time PCR amplification products showed that a shift in the dominant tfdA population structure occurred during the degradation period. Further denaturing gradient gel electrophoresis and sequence analysis revealed that the tfdA genes responsible for the degradation of MCPA belonged to the class III tfdA genes, while the tfdA genes present in the soil before the occurrence of degradation belonged to the class I tfdA genes. The implications of these results is that the initial assessment of functional genes in soils does not necessarily reflect the organisms or genes that would carry out the degradation of the compounds in question.     

Mitochondrial rejuvenation after induced pluripotency

Background

As stem cells of the early embryo mature and differentiate into all tissues, the mitochondrial complement undergoes dramatic functional improvement. Mitochondrial activity is low to minimize generation of DNA-damaging reactive oxygen species during pre-implantation development and increases following implantation and differentiation to meet higher metabolic demands. It has recently been reported that when the stem cell type known as induced pluripotent stem cells (IPSCs) are re-differentiated for several weeks in vitro, the mitochondrial complement progressively re-acquires properties approximating input fibroblasts, suggesting that despite the observation that IPSC conversion “resets” some parameters of cellular aging such as telomere length, it may have little impact on other age-affected cellular systems such as mitochondria in IPSC-derived cells.

Methodology/Principal Findings

We have examined the properties of mitochondria in two fibroblast lines, corresponding IPSCs, and fibroblasts re-derived from IPSCs using biochemical methods and electron microscopy, and found a dramatic improvement in the quality and function of the mitochondrial complement of the re-derived fibroblasts compared to input fibroblasts. This observation likely stems from two aspects of our experimental design: 1) that the input cell lines used were of advanced cellular age and contained an inefficient mitochondrial complement, and 2) the re-derived fibroblasts were produced using an extensive differentiation regimen that may more closely mimic the degree of growth and maturation found in a developing mammal.

Conclusions/Significance

These results — coupled with earlier data from our laboratory — suggest that IPSC conversion not only resets the “biological clock”, but can also rejuvenate the energetic capacity of derived cells.     

Diallyl sulfide enhances azoxymethane-induced preneoplasia in Fischer 344 rat colon

Azoxymethane (AOM) is an indirect-acting colon carcinogen that produces a high incidence of precancerous lesions, referred to as aberrant crypt foci (ACF), in rats. This study was undertaken to determine whether high dose gavage administration of the cytochrome P-450 2E1 (CYP2E1) inhibitor and chemopreventive agent, diallyl sulfide, would reduce the incidence and severity of ACF formation in the distal colons of AOM-treated Fischer 344 rats. Seven-week-old male rats received 150 or 50 mg/kg diallyl sulfide by gavage 24 and 2 h prior to two weekly i.p. injections of AOM (20 mg/kg). Ten weeks after the last injection of AOM the rats were sacrificed and the colons removed and stained with 0.2% methylene blue. ACF were visualized using stereomicroscopy. Rats pretreated with diallyl sulfide exhibited a significant increase in the number of ACF/cm in the distal colon compared with rats receiving AOM alone. This increase in ACF number was seen in ACF of all sizes. To examine the effects of diallyl sulfide on the initiation stage of AOM-induced carcinogenesis, mutations in the K-ras proto-oncogene were also investigated. ACF and normal appearing colonic mucosa (0.2-0.5 mm3) were microdissected for subsequent PCR-RFLP analysis of a codon 12 (GGT-GGA) activating mutation in the K-ras gene. Greater than 90% of ACF from AOM-treated animals, regardless of diallyl sulfide treatment, exhibited activating K-ras mutations. K-ras mutations were also detected in normal appearing mucosa of AOM-treated animals, although at a lesser frequency (15-35%). These studies demonstrate that diallyl sulfide given in large gavage doses enhances AOM-induced preneoplasia in rats and suggests that diallyl sulfide may alter the disposition of AOM intermediates and/or enhance colonic promotional activity in the rat.     

The G protein-coupled receptor GPR4 suppresses ERK activation in a ligand-independent manner

The lysophospholipids, lysophosphatidic acid, sphingosine-1-phosphate, and sphingosylphosphorylcholine (SPC), are bioactive lipid molecules that regulate diverse biological processes. Although the specific G protein-coupled receptors for lysophosphatidic acid and sphingosine-1-phosphate have been well-characterized, much less is known of the SPC receptors. It has been reported that ovarian cancer G protein-coupled receptor 1 (OGR1) is a high affinity receptor for SPC, and its closely related homologue GPR4 is a high affinity receptor for SPC with low affinity for lysophosphatidylcholine (LPC). However, in a functional assay to examine the specificity of ligand binding, we found that neither SPC nor LPC, or other related lysophospholipids, induced internalization of GPR4 from the plasma membrane. In agreement, these lysolipids also did not induce translocation of beta-arrestin2-GFP from the cytosol to the plasma membrane in GPR4 expressing cells. However, when these cells were cotransfected with G protein-coupled receptor kinase 2, in the absence of added ligands, beta-arrestin2-GFP accumulated in cytoplasmic vesicles, reminiscent of vesicular labeling usually observed after agonist stimulation of GPCRs. In addition, neither SPC nor LPC stimulated the binding of GTPgammaS to membranes prepared from GPR4 expressing cells and did not activate ERK1/2. Surprisingly, enforced expression of GPR4 inhibited activation of ERK1/2 induced by several stimuli, including SPC, sphingosine-1-phosphate, and even EGF. Collectively, our results suggest that SPC and LPC are not the ligands for GPR4 and that this receptor may constitutively inhibit ERK1/2 activation.