Impact of copper and zinc oral chronic exposure on Carniolan honey bee survival and feeding preference.

Honey bees are important plant pollinators and honey producers. Contamination of the environment with metals can lead to a decline in honey bee populations. Copper (Cu) and zinc (Zn) salts are commonly used as fungicides and foliar fertilizers. In this study, we investigated the effects of 10-day chronic oral exposure to different concentrations of Cu (CuSO4) and Zn (ZnCl2) on survival and feeding rates of Carniolan honey bees in laboratory conditions. We found that mortality in honey bee workers increased in a concentration-dependent manner and that Cu (lethal concentration [LC50] = 66 mg/l) was more toxic than Zn (LC50 = 144 mg/l). There was no difference in the feeding rate of Cu-treated bees for the different concentrations tested, but the feeding rate decreased with the increase in Zn concentration. To determine feeding preference or avoidance for Cu and Zn, we conducted 2-choice 24-h feeding experiments. We demonstrated that honey bees preferred Zn-containing solutions compared to the control diet. A two-choice experiment with Cu showed a tendency for honey bees to be deterred by Cu at high concentrations; however, it was not statistically significant. In summary, our results suggest that honey bee workers may suffer adverse effects when exposed to ecologically relevant concentrations of Cu and Zn.

First Detection of Honeybee Pathogenic Viruses in Butterflies.

Several pathogens are important causes of the observed pollinator decline, some of which could be transmitted between different pollinator species. To determine whether honeybee viruses can be transmitted to butterflies, a total of 120 butterflies were sampled at four locations in Slovenia. At each location, butterflies from three families (Pieridae, Nymphalidae, Hesperiidae/Lycenidae) and Carniolan honeybees (Apis mellifera carnica) were collected. The RNA of six honeybee viruses, i.e., acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus A (DWV-A), Sacbrood bee virus (SBV), and Lake Sinai virus 3 (LSV3), was detected by a specific quantitative method (RT-PCR). The presence of ABPV, BQCV, LSV3, and SBV was detected in both butterflies and honeybees. All butterfly and bee samples were negative for CBPV, while DWV-A was detected only in honeybees. The viral load in the positive butterfly samples was much lower than in the positive bee samples, which could indicate that butterflies are passive carriers of bee viruses. The percentage of positive butterfly samples was higher when the butterflies were collected at sampling sites with a higher density of apiaries. Therefore, we believe that infected bees are a necessary condition for the presence of viruses in cohabiting butterflies. This is the first study on the presence of pathogenic bee viruses in butterflies.

Effects of Bioinsecticidal Aegerolysin-Based Cytolytic Complexes on Non-Target Organisms.

Aegerolysin proteins ostreolysin A6 (OlyA6), pleurotolysin A2 (PlyA2) and erylysin A (EryA) produced by the mushroom genus Pleurotus bind strongly to an invertebrate-specific membrane sphingolipid, and together with a protein partner pleurotolysin B (PlyB), form transmembrane pore complexes. This pore formation is the basis for the selective insecticidal activity of aegerolysin/PlyB complexes against two economically important coleopteran pests: the Colorado potato beetle and the western corn rootworm. In this study, we evaluated the toxicities of these aegerolysin/PlyB complexes using feeding tests with two ecologically important non-target arthropod species: the woodlouse and the honey bee. The mammalian toxicity of the EryA/PlyB complex was also evaluated after intravenous administration to mice. None of the aegerolysin/PlyB complexes were toxic against woodlice, but OlyA6/PlyB and PlyA2/PlyB were toxic to honeybees, with 48 h mean lethal concentrations (LC50) of 0.22 and 0.39 mg/mL, respectively, in their food. EryA/PlyB was also tested intravenously in mice up to 3 mg/kg body mass, without showing toxicity. With no toxicity seen for EryA/PlyB for environmentally beneficial arthropods and mammals at the tested concentrations, these EryA/PlyB complexes are of particular interest for development of new bioinsecticides for control of selected coleopteran pests.

Neuroinflammation-Induced Upregulation of Glial Cathepsin X Expression and Activity in vivo.

Neuroinflammation is an important factor in the pathogenesis of neurodegenerative diseases. Microglia-derived lysosomal cathepsins have been increasingly recognized as important inflammatory mediators that trigger signaling pathways that aggravate neuroinflammation. In vitro, a contribution to neuroinflammation processes has been shown for cathepsin X: however, the expression patterns and functional role of cathepsin X in neuroinflammatory brain pathology remain elusive. In this study we analyzed the expression, activity, regional distribution and cellular localization of cathepsin X in the rat brain with neuroinflammation-induced neurodegeneration. The unilateral injection of lipopolysaccharide (LPS) induced a strong upregulation of cathepsin X expression and its activity in the ipsilateral striatum. In addition to the striatum, cathepsin X overexpression was detected in other brain areas such as the cerebral cortex, corpus callosum, subventricular zone and external globus pallidus, whereas the upregulation was mainly restricted to activated microglia and reactive astrocytes. Continuous administration of the cathepsin X inhibitor AMS36 indicated protective effects against LPS-induced striatal degeneration, as seen by the attenuated LPS-mediated dilation of the lateral ventricles and partial decreased extent of striatal lesion. Taken together, our results indicate that cathepsin X plays a role as a pathogenic factor in neuroinflammation-induced neurodegeneration and represents a potential therapeutic target for neurodegenerative diseases associated with neuroinflammation.

Comparison of sublethal effects of natural acaricides carvacrol and thymol on honeybees.

The parasitic mite Varroa destructor is a threat to beekeeping colonies. Among naturally derived acaricides, the monoterpenoid essential oil compound thymol is used in beekeeping for varroa mite control, but adverse impacts on honeybees has been already documented. Carvacrol, another monoterpenoid, also has a high acaricidal potential and could thus be promising for regular use in beekeeping, but information is scarce regarding the effects of prolonged systemic administration of carvacrol on honeybees. In this study, we evaluate and compared the sublethal effects of long term consumption of carvacrol and thymol on Carnolian honeybee workers (Apis mellifera carnica). Survival and feeding rate were determined preliminary to assess sublethal concentrations. The sublethal effects were analysed by the activity of the acetylcholinesterase (AChE), enzyme involved in the control of neurotransmission, and the activity of detoxifying enzyme glutathione S-transferase (GST) in heads and thoraces. We found that, thymol and carvacrol, caused mortality only at the highest concentrations tested, 1% and 5% respectively. As demonstrated by others, both substances could be effective against varroa at concentrations ten times lower than those causing significant honeybee mortality. However, we demonstrated sublethal effects at the 0.05% carvacrol and thymol exposure concentrations evidenced as increased activity of AChE and GST in the honeybee heads. In conclusion, prolonged treatment with thymol and carvacrol affects bee nervous system and induce detoxification processes possibly resulting in a limited use for acaricidal purposes. We postulate that under the same chronic exposure conditions carvacrol and thymol will have similar sublethal effects on honeybees.

Upregulation of Cysteine Protease Cathepsin X in the 6-Hydroxydopamine Model of Parkinson's Disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by loss of midbrain dopaminergic neurons in the substantia nigra pars compacta (SNc). In vitro, a contribution to neuroinflammation and neurotoxicity has been shown for the lysosomal protease cathepsin X; however, its expression and its role in PD remain unknown. Therefore, the current study was designed to address the regional, cellular, and subcellular localization and activity of cathepsin X in hemi-parkinsonian rats with 6-hydroxydopamine (6-OHDA)-induced excitotoxicity in the unilateral medial forebrain bundle (MFB) lesion. We report for the first time that cathepsin X expression and activity are rapidly increased in the ipsilateral SNc after injection of 6-OHDA into the MFB reaching a maximum after 12 h but seem to stay strongly upregulated after 4 weeks after injection. At early time points of 6-OHDA injection into the MFB, the increased cathepsin X is localized in the lysosomes in the neuronal, predominantly tyrosine hydroxylase-positive dopaminergic cells. After 12 h of 6-OHDA induced lesion, only a few activated microglial cells are positive for cathepsin X whereas, in 4 weeks post-lesion accompanied with complete loss of dopaminergic neurons, there is persistent cathepsin X upregulation restricted to activated glia cells. Taken together, our results demonstrate that cathepsin X upregulation in the lesioned dopaminergic system may play a role as a pathogenic factor in PD. Moreover, inhibition of cathepsin X expression or activity may be useful in protecting the nigrostriatal dopaminergic projection in the PD.

Different response of acetylcholinesterases in salt- and detergent-soluble fractions of honeybee haemolymph, head and thorax after exposure to diazinon.

Organophosphate pesticide diazinon is a specific inhibitor of acetylcholinesterase (AChE), which is a common neurotoxicity biomarker in environmental studies. In honeybees, AChE exists in two forms having different physiological roles, one existing as a soluble form and the other as membrane-bound. In most studies AChE activity has been analysed without paying considerable attention to different forms of AChE. In this study, we exposed honeybees Apis mellifera carnica for 10days to diazinon via oral exposure and analysed the total AChE activities in salt soluble (SS) and detergent soluble (DS) fractions. We assumed that SS fraction would preferentially contain the soluble AChE, but the DS fraction would contain only membrane AChE. On the contrary, our results showed that SS and DS fractions both contain soluble and membrane AChE and the latter has considerably higher activity. Despite this we obtained a differential response of AChE activity in SS and DS fractions when exposed to diazinon. The head/thorax AChE activity in DS fraction decreased, while the head/thorax AChE activity in SS fraction increased at sublethal concentrations. The AChE activity in honeybee hemolymph shown here for the first time is a salt soluble enzyme. Its activity remained unaltered after diazinon treatment. In conclusion, we provide evidence that varying results regarding AChE activity alterations upon stressor exposure are obtained when extracted through different procedures. In further environmental studies with honeybees this differential response of AChE activity should be given considerable attention because this affects the outcome of ecotoxicity study.

Immune related gene expression in worker honey bee (Apis mellifera carnica) pupae exposed to neonicotinoid thiamethoxam and Varroa mites (Varroa destructor).

Varroa destructor is one of the most common parasites of honey bee colonies and is considered as a possible co-factor for honey bee decline. At the same time, the use of pesticides in intensive agriculture is still the most effective method of pest control. There is limited information about the effects of pesticide exposure on parasitized honey bees. Larval ingestion of certain pesticides could have effects on honey bee immune defense mechanisms, development and metabolic pathways. Europe and America face the disturbing phenomenon of the disappearance of honey bee colonies, termed Colony Collapse Disorder (CCD). One reason discussed is the possible suppression of honey bee immune system as a consequence of prolonged exposure to chemicals. In this study, the effects of the neonicotinoid thiamethoxam on honey bee, Apis mellifera carnica, pupae infested with Varroa destructor mites were analyzed at the molecular level. Varroa-infested and non-infested honey bee colonies received protein cakes with or without thiamethoxam. Nurse bees used these cakes as a feed for developing larvae. Samples of white-eyed and brown-eyed pupae were collected. Expression of 17 immune-related genes was analyzed by real-time PCR. Relative gene expression in samples exposed only to Varroa or to thiamethoxam or simultaneously to both Varroa and thiamethoxam was compared. The impact from the consumption of thiamethoxam during the larval stage on honey bee immune related gene expression in Varroa-infested white-eyed pupae was reflected as down-regulation of spaetzle, AMPs abaecin and defensin-1 and up-regulation of lysozyme-2. In brown-eyed pupae up-regulation of PPOact, spaetzle, hopscotch and basket genes was detected. Moreover, we observed a major difference in immune response to Varroa infestation between white-eyed pupae and brown-eyed pupae. The majority of tested immune-related genes were upregulated only in brown-eyed pupae, while in white-eyed pupae they were downregulated.

Feeding Preference and Sub-chronic Effects of ZnO Nanomaterials in Honey Bees (Apis mellifera carnica).

The extensive production of zinc oxide (ZnO) nanomaterials (NMs) may result in high environmental zinc burdens. Honeybees need to have special concern due to their crucial role in pollination. Our previous study indicated that low concentrations of ZnO NMs, corresponding to 0.8 mg Zn/mL, have a neurotoxic potential for honeybees after a 10-day oral exposure. Present study was designed to investigate the effect of a short, dietary exposure of honeybees to ZnO NMs at concentrations 0.8-8 mg Zn/mL on consumption rate, food preference, and two enzymatic biomarkers-a stress-related glutathione S-transferase (GST) and the neurotoxicity biomarker acetylcholinesterase (AChE). Consumption rate showed a tendency toward a decrease feeding with the increasing concentrations of ZnO NMs. None of Zn NMs concentrations caused alterations in mortality rate and in the activities of brain GST and AChE. To investigate if there is an avoidance response against Zn presence in food, 24-h two-choice tests were performed with control sucrose diet versus sucrose suspensions with different concentrations of ZnO NMs added. We demonstrated that honeybees prefer ZnO NMs ZnO NMs containing suspensions, even at highest Zn concentrations tested, compared with the control diet. This indicates that they might be able to perceive the presence of ZnO NMs in sucrose solution. Because honeybees feed frequently the preference towards ZnO NMs might have a high impact on their survival when exposed to these NMs.

Synaptotagmin 7 and SYNCRIP proteins are ubiquitously expressed in the rat brain and co-localize in Purkinje neurons.

Synaptotagmin 7 (SYT7) is ubiquitously expressed calcium sensor, involved in neuronal membrane trafficking. Immunoprecipitation experiments demonstrated that SYT7 interacts with Synaptotagmin-binding, cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a component of mRNA granules, which are transported to dendrites and are prerequisite for synaptic plasticity. Given the potential significance of SYT7 regulation in processes of neurodegeneration, which are characterized by high level of synaptic vulnerability, we aimed to analyse and compare the distribution of SYT7 and SYNCRIP proteins in the adult rat striatum, hippocampus, cerebral and cerebellar cortex. We investigated the degree of SYT7-SYNCRIP co-localization in order to examine possible functional interaction of these two proteins. We found that SYT7 is abundantly distributed in neuropil of all examined anatomical areas of the brain, most prominently in axons. On the contrary, SYNCRIP had cytoplasmic somatodendritic pattern of expression, which was most prominent in the hippocampus and cerebellum. In the striatum, hippocampus and cerebral cortex SYT7 and SYNCRIP immunofluorescent signals were mutually excluded, thus diminishing the probability for their physiological interaction. In somata of Purkinje neurons in the cerebellar cortex, both SYT7 and SYNCRIP were expressed and partially co-localized suggesting possible functional connection between SYT7 and SYNCRIP proteins in Purkinje neurons.

Prochloraz and coumaphos induce different gene expression patterns in three developmental stages of the Carniolan honey bee (Apis mellifera carnica Pollmann).

The Carniolan honey bee, Apis mellifera carnica, is a Slovenian autochthonous subspecies of honey bee. In recent years, the country has recorded an annual loss of bee colonies through mortality of up to 35%. One possible reason for such high mortality could be the exposure of honey bees to xenobiotic residues that have been found in honey bee and beehive products. Acaricides are applied by beekeepers to control varroosis, while the most abundant common agricultural chemicals found in honey bee and beehive products are fungicides, which may enter the system when applied to nearby flowering crops and fruit plants. Acaricides and fungicides are not intrinsically highly toxic to bees but their action in combination might lead to higher honey bee sensitivity or mortality. In the present study we investigated the molecular immune response of honey bee workers at different developmental stages (prepupa, white-eyed pupa, adult) exposed to the acaricide coumaphos and the fungicide prochloraz individually and in combination. Expression of 17 immune-related genes was examined by quantitative RT-PCR. In treated prepupae downregulation of most immune-related genes was observed in all treatments, while in adults upregulation of most of the genes was recorded. Our study shows for the first time that negative impacts of prochloraz and a combination of coumaphos and prochloraz differ among the different developmental stages of honey bees. The main effect of the xenobiotic combination was found to be upregulation of the antimicrobial peptide genes abaecin and defensin-1 in adult honey bees. Changes in immune-related gene expression could result in depressed immunity of honey bees and their increased susceptibility to various pathogens.

Upregulation and axonal transport of synaptotagmin-IV in the direct-pathway medium spiny neurons in hemi-parkinsonian rats induced by dopamine D1 receptor stimulation.

Synaptotagmin-IV (Syt-IV) may function as a regulator of Ca(2+) -dependent synaptic transmission. In the hemi-parkinsonian rats with unilateral lesions of dopaminergic nigrostriatal neurons Syt-IV and substance-P (SP) mRNAs could be upregulated within the dopaminergically hypersensitive striatum of the lesioned brain hemisphere via the stimulation of striatal dopamine D1 (D1-R), but not D2 receptors. The hypersensitive D1-R-mediated transmission may be the culprit for the undesired expression of levodopa-induced dyskinesia, implying the involvement of Syt-IV and SP in the process. First, striatal cellular phenotypes expressing Syt-IV were determined. It was found to be expressed in all striatal neurons and a small population of astrocytes. Then it was examined, if the D1-R-mediated upregulation of Syt-IV mRNA may result in the upregulation of the translated protein. It was found that, after acute stimulation with a selective D1 agonist, (±)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrobromide (SKF-82958), Syt-IV was elevated within the SP-expressing striatal neurons of the lesioned side. This was followed by the upregulation of Syt-IV, but not of its mRNA, within the ipsilateral target nuclei of the direct-pathway medium spiny neurons, indicating axonal transport of de novo synthesized protein to their SP-positive synaptic terminals. However, despite the striatal upregulation of SP and Syt-IV following a similar time-course, their subcellular co-localization within the axonal terminals was not found. It was therefore suggested that Syt-IV may regulate the hypersensitive striatal synaptic transmission, although via a SP-independent mechanism.

STAM2, a member of the endosome-associated complex ESCRT-0 is highly expressed in neurons.

STAM2 (signal transducing adaptor molecule 2), a subunit of the ESCRT-0 complex, is an endosomal protein acting as a regulator of receptor signaling and trafficking. To analyze STAM2 in the nervous system, its gene expression and protein localization in the mouse brain were identified using three methods: mRNA in situ hybridization, immunohistochemistry, and via lacZ reporter in frame with Stam2 gene using the gene trap mouse line Stam2(Gt1Gaj). STAM2 intracellular localization was analyzed by subcellular fractionation and co-immunofluorescence using confocal microscopy. Stam2 was strongly expressed in the cerebral and cerebellar cortex, hippocampal formation, olfactory bulb, and medial habenula. The majority of STAM2-positive cells co-stained with the neuronal markers. In neurons STAM2 was found in the early endosomes and also in the nucleus. The other members of the ESCRT-0 complex co-localized with STAM2 in the cytoplasm, but they were not present in the nucleus. The newly identified neuron-specific nuclear localization of STAM2, together with its high expression in the brain indicated that STAM2 might have a specific function in the mouse nervous system.

Neurotoxic potential of ingested ZnO nanomaterials on bees.

The honey bee is among most important pollinators threatened by environmental pollution, pest control and potentially, by products of nanotechnologies. The aim of the current study was an analysis of the neurotoxic potential of ingested zinc oxide nanomaterials (ZnO NMs) or zinc ions (Zn(2+)) on honey bees. We analysed a variety of biomarkers, including metabolic impairment, feeding rate, and survival, as well as the activities of a stress-related enzyme glutathione S-transferase, and the neurotoxicity biomarker acetylcholinesterase. Acetylcholinesterase activity was found to be elevated in bees exposed to either of the tested substances. In addition, we observed increased feeding rate in the group treated with Zn(2+) but not with ZnO NMs or control group. The observed effects we relate primarily to Zn(2+) ions. Here we provide evidence that zinc ions either originating from Zn salt or Zn-based NPs have a neurotoxic potential and thus might contribute to colony survival.

Up-regulation of Synaptotagmin IV within amyloid plaque-associated dystrophic neurons in Tg2576 mouse model of Alzheimer's disease.

AIM: To investigate the involvement of the vesicular membrane trafficking regulator Synaptotagmin IV (Syt IV) in Alzheimer's disease pathogenesis and to define the cell types containing increased levels of Syt IV in the ß-amyloid plaque vicinity. METHODS: Syt IV protein levels in wild type (WT) and Tg2576 mice cortex were determined by Western blot analysis and immunohistochemistry. Co-localization studies using double immunofluorescence staining for Syt IV and markers for astrocytes (glial fibrillary acidic protein), microglia (major histocompatibility complex class II), neurons (neuronal specific nuclear protein), and neurites (neurofilaments) were performed in WT and Tg2576 mouse cerebral cortex. RESULTS: Western blot analysis showed higher Syt IV levels in Tg2576 mice cortex than in WT cortex. Syt IV was found only in neurons. In plaque vicinity, Syt IV was up-regulated in dystrophic neurons. The Syt IV signal was not up-regulated in the neurons of Tg2576 mice cortex without plaques (resembling the pre-symptomatic conditions). CONCLUSIONS: Syt IV up-regulation within dystrophic neurons probably reflects disrupted vesicular transport or/and impaired protein degradation occurring in Alzheimer's disease and is probably a consequence but not the cause of neuronal degeneration. Hence, Syt IV up-regulation and/or its accumulation in dystrophic neurons may have adverse effects on the survival of the affected neuron.

Neuroprotective role of γ-enolase in microglia in a mouse model of Alzheimer's disease is regulated by cathepsin X.

γ-Enolase is a neurotrophic-like factor promoting growth, differentiation, survival and regeneration of neurons. Its neurotrophic activity is regulated by cysteine protease cathepsin X which cleaves the C-terminal end of the molecule. We have investigated the expression and colocalization of γ-enolase and cathepsin X in brains of Tg2576 mice overexpressing amyloid precursor protein. In situ hybridization of γ-enolase and cathepsin X revealed that mRNAs for both enzymes were expressed abundantly around amyloid plaques. Immunostaining demonstrated that the C-terminally cleaved form of γ-enolase was present in the immediate plaque vicinity, whereas the intact form, exhibiting neurotrophic activity, was observed in microglia cells in close proximity to senile plaque. The upregulation of γ-enolase in microglial cells in response to amyloid-ß peptide (Aß) was confirmed in mouse microglial cell line EOC 13.31 and primary microglia and medium enriched with γ-enolase proved to be neuroprotective against Aß toxicity; however, the effect was reversed by cathepsin X proteolytic activity. These results demonstrate an upregulation of γ-enolase in microglia cells surrounding amyloid plaques in Tg2576 transgenic mice and demonstrate its neuroprotective role in amyloid-ß-related neurodegeneration.

Differential patterns of synaptotagmin7 mRNA expression in rats with kainate- and pilocarpine-induced seizures.

Previous studies in rat models of neurodegenerative disorders have shown disregulation of striatal synaptotagmin7 mRNA. Here we explored the expression of synaptotagmin7 mRNA in the brains of rats with seizures triggered by the glutamatergic agonist kainate (10 mg/kg) or by the muscarinic agonist pilocarpine (30 mg/kg) in LiCl (3 mEq/kg) pre-treated (24 h) rats, in a time-course experiment (30 min-1 day). After kainate-induced seizures, synaptotagmin7 mRNA levels were transiently and uniformly increased throughout the dorsal and ventral striatum (accumbens) at 8 and 12 h, but not at 24 h, followed at 24 h by somewhat variable upregulation within different parts of the cerebral cortex, amigdala and thalamic nuclei, the hippocampus and the lateral septum. By contrast, after LiCl/pilocarpine-induced seizures, there was a more prolonged increase of striatal Synaptotagmin7 mRNA levels (at 8, 12 and 24 h), but only in the ventromedial striatum, while in some other of the aforementioned brain regions there was a decline to below the basal levels. After systemic post-treatment with muscarinic antagonist scopolamine in a dose of 2 mg/kg the seizures were either extinguished or attenuated. In scopolamine post-treated animals with extinguished seizures the striatal synaptotagmin7 mRNA levels (at 12 h after the onset of seizures) were not different from the levels in control animals without seizures, while in rats with attenuated seizures, the upregulation closely resembled kainate seizures-like pattern of striatal upregulation. In the dose of 1 mg/kg, scopolamine did not significantly affect the progression of pilocarpine-induced seizures or pilocarpine seizures-like pattern of striatal upregulation of synaptotagmin7 mRNA. In control experiments, equivalent doses of scopolamine per se did not affect the expression of synaptotagmin7 mRNA. We conclude that here described differential time course and pattern of synaptotagmin7 mRNA expression imply regional differences of pathophysiological brain activation and plasticity in these two models of seizures.

Exploiting microRNAs for cell engineering and therapy.

MicroRNAs (miRNAs) form a large class of non-coding RNAs that function in repression of gene expression in eukaryotes. By recognizing short stretches of nucleotides within the untranslated regions of mRNAs, miRNAs recruit partner proteins to individual transcripts, leading to mRNA cleavage or hindering of translation. Bioinformatic predictions and a wealth of data from wet laboratory studies indicate that miRNAs control expression of a large proportion of protein-coding genes, implying involvement of miRNAs in regulation of most biologic processes. In this review we discuss the biology of miRNAs and present examples of how manipulation of miRNA expression or activity can be exploited to attain the desired phenotypic traits in cell engineering as well as achieve therapeutic outcomes in treatment of a diverse set of diseases.

Synaptotagmins in neurodegeneration.

Synaptotagmins (Syts) are transmembrane proteins involved in the regulation of membrane trafficking. Here, we summarize literature data that provide growing evidence that several Syts are involved in the pathophysiological mechanisms of temporal lobe epilepsy and Parkinson's disease, as well as few reports related to brain ischemia and Alzheimer's disease (AD). We also report new data from our laboratories, showing changes of the expression of several Syts in Tg2576 mouse model of AD that may be related to neuroinflammation surrounding the beta-amyloid plaques. Furthermore, we demonstrate N-methyl-D-aspartate receptor-mediated upregulation of Syt 4 mRNA in a model of excitotoxic striatal lesion induced by unilateral striatal injection of quinolinic acid, associating the upregulation of Syt 4 with mechanisms of excitotoxicity. We propose that pharmacological manipulation of Syt expression in animal models of neurodegeneration should be further explored, as it may help to clarify the role of individual Syt isoforms in the regulation of membrane trafficking in neurodegeneration.

Intermittent L-DOPA treatment differentially alters synaptotagmin 4 and 7 gene expression in the striatum of hemiparkinsonian rats.

Long-term use of L-DOPA in Parkinson's disease (PD) is frequently associated with side effects that are reflected in changed neurotransmitter/neuropeptide secretion in basal ganglia. These side effects could be connected with synaptotagmins (syts) because syts are involved in regulation of membrane trafficking. We have previously reported that acute L-DOPA treatment upregulated the expression of Syt 4 and Syt 7 mRNAs in hypersensitive striatum of 6-OHDA rat model for PD. Here we investigate whether intermittent L-DOPA treatment that produces behavior sensitization affects the Syt 1, Syt 2, Syt 4, Syt 7 and Syt 10 mRNAs in striatum of 6-OHDA rats killed 4 and 12 h after the last L-DOPA injection. We verified behavioral sensitization by increased intensity of contralateral turning. 6-OHDA lesion caused Syt 2 mRNA downregulation and Syt 10 mRNA upregulation in striatum, but failed to alter Syt 4, Syt 7 and Syt 1 mRNAs. Acute l-DOPA induced an increase of Syt 4 and Syt 7 mRNAs in the denervated striatum leaving the levels of Syt 1, Syt 2 and Syt 10 mRNAs unaffected. Intermittent L-DOPA treatment did not alter Syt 1, Syt 2 and Syt 10 mRNA striatal levels, suggesting that 6-OHDA-induced Syt 2 and Syt 10 mRNA changes reflect a persistent striatal abnormality caused by dopamine depletion. On contrary, intermittent L-DOPA treatment downregulated Syt 4 mRNA and prolonged the elevation of Syt 7 mRNA in the denervated striatum. We conclude that Syt 4 and Syt 7 might be specifically involved in striatal plasticity caused by repeated L-DOPA administration that accompanies sensitization.