Inhibition of the deubiquitinase USP8 corrects a Drosophila PINK1 model of mitochondria dysfunction.

Aberrant mitochondrial dynamics disrupts mitochondrial function and contributes to disease conditions. A targeted RNA interference screen for deubiquitinating enzymes (DUBs) affecting protein levels of multifunctional mitochondrial fusion protein Mitofusin (MFN) identified USP8 prominently influencing MFN levels. Genetic and pharmacological inhibition of USP8 normalized the elevated MFN protein levels observed in PINK1 and Parkin-deficient models. This correlated with improved mitochondrial function, locomotor performance and life span, and prevented dopaminergic neurons loss in Drosophila PINK1 KO flies. We identified a novel target antagonizing pathologically elevated MFN levels, mitochondrial dysfunction, and dopaminergic neuron loss of a Drosophila model of mitochondrial dysfunction.

USP14 inhibition corrects an in vivo model of impaired mitophagy.

Mitochondrial autophagy or mitophagy is a key process that allows selective sequestration and degradation of dysfunctional mitochondria to prevent excessive reactive oxygen species, and activation of cell death. Recent studies revealed that ubiquitin-proteasome complex activity and mitochondrial membrane rupture are key steps preceding mitophagy, in combination with the ubiquitination of specific outer mitochondrial membrane (OMM) proteins. The deubiquitinating enzyme ubiquitin-specific peptidase 14 (USP14) has been shown to modulate both proteasome activity and autophagy. Here, we report that genetic and pharmacological inhibition of USP14 promotes mitophagy, which occurs in the absence of the well-characterised mediators of mitophagy, PINK1 and Parkin. Critical to USP14-induced mitophagy is the exposure of the LC3 receptor Prohibitin 2 by mitochondrial fragmentation and mitochondrial membrane rupture. Genetic or pharmacological inhibition of USP14 in vivo corrected mitochondrial dysfunction and locomotion behaviour of PINK1/Parkin mutant Drosophila model of Parkinson's disease, an age-related progressive neurodegenerative disorder that is correlated with diminished mitochondrial quality control. Our study identifies a novel therapeutic target that ameliorates mitochondrial dysfunction and in vivo PD-related symptoms.

Culture of Rat Mesencephalic Dopaminergic Neurons and Application to Neurotoxic and Neuroprotective Agents.

Dopaminergic neuronal cell degeneration is the principal characteristic feature of the neuropathology of Parkinson disease. Cultures of mesencephalic neurons are widely used as a source of dopaminergic neurons for the study of mechanisms implicated in dopaminergic cell death and for the evaluation of potential dopaminergic neuroprotective agents, including neurotrophic factors. This chapter presents a detailed protocol for the preparation of rat mesencephalic cell cultures and their application to evaluating the effect of the dopaminergic neurotoxin 1-methyl-4-phenylpyridinium and the neuroprotective action of brain-derived neurotrophic factor.

Analysis of the Catecholaminergic Phenotype in Human SH-SY5Y and BE(2)-M17 Neuroblastoma Cell Lines upon Differentiation.

Human cell lines are often used to investigate cellular pathways relevant for physiological or pathological processes or to evaluate cell toxicity or protection induced by different compounds, including potential drugs. In this study, we analyzed and compared the differentiating activities of three agents (retinoic acid, staurosporine and 12-O-tetradecanoylphorbol-13-acetate) on the human neuroblastoma SH-SY5Y and BE(2)-M17 cell lines; the first cell line is largely used in the field of neuroscience, while the second is still poorly characterized. After evaluating their effects in terms of cell proliferation and morphology, we investigated their catecholaminergic properties by assessing the expression profiles of the major genes involved in catecholamine synthesis and storage and the cellular concentrations of the neurotransmitters dopamine and noradrenaline. Our results demonstrate that the two cell lines possess similar abilities to differentiate and acquire a neuron-like morphology. The most evident effects in SH-SY5Y cells were observed in the presence of staurosporine, while in BE(2)-M17 cells, retinoic acid induced the strongest effects. Undifferentiated SH-SY5Y and BE(2)-M17 cells are characterized by the production of both NA and DA, but their levels are considerably higher in BE(2)-M17 cells. Moreover, the NAergic phenotype appears to be more pronounced in SH-SY5Y cells, while BE(2)-M17 cells have a more prominent DAergic phenotype. Finally, the catecholamine concentration strongly increases upon differentiation induced by staurosporine in both cell lines. In conclusion, in this work the catecholaminergic phenotype of the human BE(2)-M17 cell line upon differentiation was characterized for the first time. Our data suggest that SH-SY5Y and BE(2)-M17 represent two alternative cell models for the neuroscience field.

Use of serum amyloid A and milk amyloid A in the diagnosis of subclinical mastitis in dairy cows.

Mastitis is the most frequent and costly disease in dairy herds, as it negatively affects yield and milk quality. The presence of clinical mastitis is quite easy to asses, whereas the diagnosis of the subclinical form can be more difficult and requires laboratory assays. Somatic cell count (SCC) is widely used as a rapid and low-cost indicator of mastitis, even if is not useful in discriminating between the clinical and subclinical form. As amyloid A has been investigated as a marker of mastitis, the aim of this study was to assess the potential value of measuring amyloid A in serum and milk and the correlation with SCC in the diagnosis of subclinical mastitis. The reliability of two different ELISA kits for the measurement of amyloid A in milk was also tested. During a 1-month trial period, 21 cows were assigned to three experimental groups according to their health status: 6 cows with clinical mastitis (CM), 10 cows with subclinical mastitis (SM) and 5 healthy cows (HE). Amyloid A was measured both in serum (SAA) and in quarter milk samples (mAA) with a serum ELISA kit, and in quarter milk samples (MAA) with a milk ELISA kit. SCC, total microbial count (TMC) and bacterial examination of the milk were also carried out. After a log transformation, the data were submitted to ANOVA and linear regression. TMC was significantly higher in cows with clinical mastitis, while no differences were observed between the other two experimental groups. SCC and MAA levels were significantly different among the three groups. mAA concentrations were similar between cows with subclinical and clinical mastitis, and SAA was not affected by mastitis. A significant correlation between SCC and MAA or mAA was detected, while no correlation was recorded between SAA and mAA. A close relationship between MAA and mAA was noticeable even at low concentrations, suggesting MAA as a potential physiological marker of subclinical mastitis.

Effects of electromagnetic fields of low frequency and low intensity on rat metabolism.

A series of experiments on rats have been performed, to study the effects of long time (50 days) exposure to electromagnetic fields of extremely low frequency (ELF, i.e. less than 100 Hz) and amplitude (non thermal), testing whether the metabolic processes would be affected. The background lies on recent observations on the behaviour of isolated enzymes in vitro exposed to EFL fields. In these experiments, the cyclotron (or Larmor) frequency of the metallic ion has been used to "stimulate" the metalloproteins redox-active site, thus obtaining a clear variation of the enzyme functionality. In this paper we have extended for the first time the check to more complex animal metabolism. The novelty of this approach implies that a large amount of data had to be analyzed since it was not possible, in principle, to select only a few parameters among all the potential effects. Several biochemical parameters have been evaluated by comparing their values during the periods of exposure (field ON) and non exposure (field OFF). The evidence that long term exposure to electromagnetic fields with a well defined frequency may have relevant effects on parameters such as body weight, blood glucose and fatty acid metabolism has been obtained.