Ubiquitin Subproteome of Brain Mitochondria and Its Changes Induced by Experimental Parkinsonism and Action of Neuroprotectors.

The review summarizes the data of our research and published studies on the ubiquitination of brain mitochondrial proteins and its changes during the development of experimental parkinsonism and administration of the neuroprotector isatin (indole-2,3-dione) with special attention to the mitochondrial ubiquitin-conjugating system and location of ubiquitinated proteins in these organelles. Incubation of brain mitochondrial fraction with biotinylated ubiquitin in vitro resulted in the incorporation of biotinylated ubiquitin in both mitochondrial and mitochondria-associated proteins. According to the interactome analysis, the identified non-ubiquitinated proteins are able to form tight complexes with ubiquitinated proteins or their partners and components of mitochondrial membranes, in which interactions of ubiquitin chains with the ubiquitin-binding protein domains play an important role. The studies of endogenous ubiquitination in the total brain mitochondrial fraction of C57Bl mice performed in different laboratories have shown that mitochondrial proteins represent about 30% of all ubiquitinated proteins. However, comparison of brain subproteomes of mitochondrial ubiquitinated proteins reported in the literature revealed significant differences both in their composition and involvement of identified ubiquitinated proteins in biological processes listed in the Gene Ontology database. The development of experimental parkinsonism in C57Bl mice induced by a single-dose administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) resulted in a decrease in the total number of mitochondrial ubiquitinated proteins and increase in the number of oxidized mitochondrial proteins containing the ubiquitin signature (K-ε-GG). Comparison of ubiquitinated proteins associated with the mouse brain mitochondrial fraction and mouse brain mitochondrial proteins bound to the proteasome ubiquitin receptor (Rpn10 subunit) did not reveal any common proteins. This suggests that ubiquitination of brain mitochondrial proteins is not directly related to their degradation in the proteasomes. Proteomic profiling of brain isatin-binding proteins identified enzymes involved in the ubiquitin-conjugating system functioning. Mapping of the identified isatin-binding proteins to known metabolic pathways indicates their participation in the parkin (E3 ubiquitin ligase)-associated pathway (CH000000947). The functional links involving brain mitochondrial ubiquitinated proteins were found only in the group of animals with the MPTP-induced parkinsonism, but not in animals treated with MPTP/isatin or isatin only. This suggests that the neuroprotective effect of isatin may be associated with the impaired functional relationships of proteins targeted to subsequent degradation.

Effects of titanium dioxide nanoparticles exposure on parkinsonism in zebrafish larvae and PC12.

Nanomaterials hold significant potential for industrial and biomedical application these years. Therefore, the relationship between nanoparticles and neurodegenerative disease is of enormous interest. In this contribution, zebrafish embryos and PC12 cell lines were selected for studying neurotoxicity of titanium dioxide nanoparticles (TiO2 NPs). After exposure of different concentrations of TiO2 NPs to embryos from fertilization to 96 hpf, the hatching time of zebrafish was decreased, accompanied by an increase in malformation rate. However, no significant increases in mortality relative to control were observed. These results indicated that TiO2 NPs exposure hold a risk for premature of zebrafish embryos, but not fatal. The further investigation confirmed that TiO2 NPs could accumulate in the brain of zebrafish larvae, resulting in reactive oxygen species (ROS) generation and cell death of hypothalamus. Meanwhile, q-PCR analysis showed that TiO2 NPs exposure increased the pink1, parkin, α-syn and uchl1 gene expression, which are related with the formation of Lewy bodies. We also observed loss of dopaminergic neurons in zebrafish and in vitro. These remarkable hallmarks are all linked to these Parkinson's disease (PD) symptoms. Our results indicate that TiO2NPs exposure induces neurotoxicity in vivo and in vitro, which poses a significant risk factor for the development of PD.

Toxic equine parkinsonism: an immunohistochemical study of 10 horses with nigropallidal encephalomalacia.

Chronic ingestion of yellow star thistle (Centaurea solstitialis) or Russian knapweed (Acroptilon repens) causes nigropallidal encephalomalacia (NPE) in horses with an abrupt onset of neurologic signs characterized by dystonia of lips and tongue, inability to prehend food, depression, and locomotor deficits. The objectives of this study were to reexamine the pathologic alterations of NPE and to conduct an immunohistochemistry study using antibodies to tyrosine hydroxylase and α-synuclein, to determine whether NPE brains show histopathologic features resembling those in human Parkinson disease. Results confirm that the NPE lesions are located within the substantia nigra pars reticulata, sparing the cell bodies of the dopaminergic neurons in the substantia nigra pars compacta, and in the rostral portion of the globus pallidus, with partial disruption of dopaminergic (tyrosine hydroxylase-positive) fibers passing through the globus pallidus. No abnormal cytoplasmic inclusions like the Lewy bodies of human Parkinson disease were seen in these NPE brains. These findings indicate that equine NPE may serve as a large animal model of environmentally acquired toxic parkinsonism, with clinical phenotype directly attributable to lesions in globus pallidus and substantia nigra pars reticulata rather than to the destruction of dopaminergic neurons.

Retrospective comparison of rat recovery weights using inhalation and injectable anaesthetics, nutritional and fluid supplementation for right unilateral neurosurgical lesioning.

Current veterinary standards of biomedical research support include refinements in animal models that are targeted at enhancing humane care and decreasing inter-animal variability. This ultimately results in fewer numbers of animals being used and reduction in animal experimentation through mitigation of waste as well as faster research results. 6-hydroxydopamine-lesioning of the substantia nigra using a stereotactic frame device is a common procedure and is routinely performed under pentobarbital anaesthesia with monitoring by 8 h workforces. Our programme supports the humane care and use of several protocols involving the unilateral stereotactic-lesioning of rats for the purposes of creating research models of Parkinsonianism. Such procedures are commonly performed as unilateral in order to minimize aphagia and other untoward effects of the lesion. Generally, this procedure is considered minor because it involves a small incision, a cranial burrhole, and penetration of the dura. Inflammation and/or irritation of the ear canal can occur secondarily to the earbar placement procedure. Human patients undergoing similar procedures typically complain of headaches from loss of intracranial pressure; which is a transient outcome. Despite the perception of minor insult, we provided aggressive periprocedural care, and our veterinary staff documented weight loss that was often greater than 15% body weight during the first 3 days. The goal of this study was to evaluate refinements to improve this outcome. For humane concerns, and because of the need to begin experimental testing one week following surgery, a goal in the recent past has been to enhance outcome for researchers and animals by refining postoperative support modalities as well as by seeking the best anaesthetic regimen to shorten postoperative deviations from baseline. Analysis of three groups of rat patients lesioned by the same investigative group over the course of refinements made in our programme indicate that an early return of homeostasis was achieved by the use of inhalation anaesthetics as replacements for barbiturates in these models. Comparison with pentobarbital recipients also indicated that homeostasis is achieved earlier when barbiturates are utilized with fluid therapy and analgesics immediately after operative procedures rather than the next morning.