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The tauopathy of Alzheimer’s disease (AD) is first observed in the brainstem and entorhinal cortex, spreading trans-synaptically along specific pathways to other brain regions with recognizable patterns. Tau propagation occurs retrogradely and anterogradely (trans-synaptically) along a given pathway and through exosomes and microglial cells. Some aspects of in vivo tau spreading have been replicated in transgenic mice models expressing a mutated human MAPT (tau) gene and in wild-type mice. In this study, we aimed to characterize the propagation of different forms of tau species in non-transgenic 3–4 months old wild-type rats after a single unilateral injection of human tau oligomers and tau fibrils into the medial entorhinal cortex (mEC). We determined whether different variants of the inoculated human tau protein, tau fibrils, and tau oligomers, would induce similar neurofibrillary changes and propagate in an AD-related pattern, and how tau- related pathological changes would correlate with presumed cognitive impairment. We injected human tau fibrils and tau oligomers stereotaxically into the mEC and examined the distribution of tau- related changes at 3 days and 4, 8, and 11 months post-injection using antibodies AT8 and MC1, which reveal early phosphorylation and aberrant conformation of tau, respectively, HT7, anti- synaptophysin, and the Gallyas silver staining method. Human tau oligomers and tau fibrils exhibited some similarities and some differences in their ability to seed and propagate tau-related changes. Both human tau fibrils and tau oligomers rapidly propagated from the mEC anterogradely into the hippocampus and various parts of the neocortex. However, using a human tau-specific HT7 antibody, 3 days post-injection we found inoculated human tau oligomers in the red nucleus, primary motor, and primary somatosensory cortex, a finding not seen in animals inoculated with human tau fibrils. In animals inoculated with human tau fibrils, 3 days post-injection the HT7 antibody showed fibrils in the pontine reticular nucleus, a finding explained only by uptake of human tau fibrils by incoming presynaptic fibers to the mEC and retrograde transport of inoculated human tau fibrils to the brainstem. Rats inoculated with human tau fibrils showed as early as 4 months after inoculation a spread of phosphorylated tau protein at the AT8 epitopes throughout the brain, dramatically faster propagation of neurofibrillary changes than with human tau oligomers. The overall severity of tau protein changes 4, 8, and 11 months after inoculation of human tau oligomers and tau fibrils correlated well with spatial working memory and cognition impairments, as measured by the T-maze spontaneous alternation, novel object recognition, and object location tests. We concluded that this non-trangenic rat model of tauopathy, especially when using human tau fibrils, demonstrates rapidly developing pathologic alterations in neurons, synapses, and identifiable pathways together with cognitive and behavioral changes, through the anterograde and retrograde spreading of neurofibrillary degeneration. Therefore, it represents a promising model for future experimental studies of primary and secondary tauopathies, especially AD.

The role of metals in the pathogenesis of Alzheimer’s disease (AD) is still debated. Although previous research has linked changes in essential metal homeostasis and exposure to environmental heavy metals to the pathogenesis of AD, more research is needed to determine the relationship between metals and AD. In this review, we included human studies that (1) compared the metal concentrations between AD patients and healthy controls, (2) correlated concentrations of AD cerebrospinal fluid (CSF) biomarkers with metal concentrations, and (3) used Mendelian randomization (MR) to assess the potential metal contributions to AD risk. Although many studies have examined various metals in dementia patients, understanding the dynamics of metals in these patients remains difficult due to considerable inconsistencies among the results of individual studies. The most consistent findings were for Zn and Cu, with most studies observing a decrease in Zn levels and an increase in Cu levels in AD patients. However, several studies found no such relation. Because few studies have compared metal levels with biomarker levels in the CSF of AD patients, more research of this type is required. Given that MR is revolutionizing epidemiologic research, additional MR studies that include participants from diverse ethnic backgrounds to assess the causal relationship between metals and AD risk are critical.

Background: The primary histological characteristic of Alzheimer's disease is the presence of neurofibrillary tangles, which are large aggregates of tau protein. Aging is the primary risk factor for the development of Alzheimer's disease, however, the underlying causes of tau protein aggregation and toxicity are unclear. Aims: Here we investigated tau aggregation and toxicity under the conditions of compromised protein homeostasis. Methods: We used heterologous expression of human tau protein in the unicellular eukaryote yeast Saccharomyces cerevisiae with evolutionarily conserved protein quality control pathways and examined tau-dependent toxicity and aggregation using growth assays, fluorescence microscopy, and a split luciferase-based reporter NanoBiT. Results: Tau protein expressed in yeast under mild proteotoxic stress, or in mutants with impaired pathways for proteotoxic stress response, did not lead to synthetic toxicity or the formation of obvious aggregates. Chronologically old cells also did not develop observable tau aggregates. Our examination of tau oligomerization in living cells using NanoBiT reporter suggests that tau does not form significant levels of oligomers under basal conditions or under mild proteotoxic stress. Conclusion: Together our data suggest that human tau protein does not represent a major burden to the protein quality control system in yeast cells.

Age-dependent protein aggregation is a conserved phenomenon that is associated with many neurodegenerative diseases, including Alzheimer's disease (AD). AD is characterized by aggregation of Tau, a microtubule-binding protein that is normally soluble and mainly localized to neuronal axons, but can form oligomers and higher-order amyloid-like aggregates that accumulate in soma and dendrites and eventually lead to neuronal death. Although the main risk factor for the onset of AD is aging, the exact causes of Tau protein aggregation are largely unclear. To investigate factors that influence Tau protein aggregation, we expressed human Tau protein fused with fluorescent proteins in yeast Saccharomyces cerevisiae. We examined intracellular localization of Tau in young and aged cells, and in cells under different stress conditions, such as glucose starvation, elevated temperature, hyperosmotic and proteotoxic stress caused by toxic amino acid analogue. Furthermore, to study factors affecting Tau oligomerization, we used luminescent reporter NanoBiT in which protein-protein interaction results in the complementation of the luciferase NanoLuc. Results show basal levels of Tau-NanoBiT reporter signal in logarithmically growing wild-type cells, suggesting that Tau oligomerization doesn't occur under normal growth conditions.

Age-dependent protein aggregation is a conserved phenomenon that is associated with many neurodegenerative diseases, including Alzheimer's disease (AD). AD is characterized by aggregation of Tau, a microtubule-binding protein that is normally soluble and mainly localized to neuronal axons, but which can form oligomers and higher order amyloid-like aggregates that accumulate in soma and dendrites and eventually lead to neuronal death. Although the main risk factor for the onset of AD is aging, the exact causes of Tau protein aggregation are still largely unclear. To investigate factors that influence Tau protein aggregation, we expressed human Tau protein fused with fluorescent proteins in yeast Saccharomyces cerevisiae. We examined the intracellular localization of Tau in young and aged cells, and in cells under different stress conditions, such as glucose starvation, hyperosmotic stress, elevated temperature and proteotoxic stress caused by a toxic amino acid analogue. Furthermore, to study the factors affecting Tau oligomerization, which is considered to be an early step in Tau pathology, we used luminescent reporter NanoBiT in which protein-protein interaction results in the complementation of the luciferase NanoLuc. Our results show basal levels of Tau-NanoBiT reporter signal in logarithmically growing wild-type cells, suggesting that Tau oligomerization does not occur under normal growth conditions.

Age-dependent protein aggregation is a conserved phenomenon that is associated with many neurodegenerative diseases, including Alzheimer's disease (AD). AD is characterized by aggregation of Tau, a microtubule-binding protein that is normally soluble and mainly localized to neuronal axons, but which can form oligomers and higher-order amyloid-like aggregates that accumulate in soma and dendrites and eventually lead to neuronal death. Although the main risk factor for the onset of AD is aging, the exact causes of tau protein aggregation are still largely unclear. To investigate factors that influence tau protein aggregation, we expressed human tau protein fused with fluorescent proteins in yeast Saccharomyces cerevisiae. We examined its intracellular localization in young, logarithmically growing cells, in chronologically aged cells, and under different stress conditions, such as glucose starvation, hyperosmotic stress, elevated temperature, and proteotoxic stress caused by a toxic amino acid analog. Furthermore, to study the factors affecting Tau oligomerization, which is considered to be an early step in Tau pathology, we used luminescent reporter NanoBiT in which protein-protein interaction results in the complementation of the luciferase NanoLuc. Our results show basal levels of Tau-NanoBiT reporter signal in logarithmically growing wild-type cells, suggesting that Tau oligomerization does not occur under normal growth conditions.

Nakupljanje agregata tau proteina u neurofibrilarne snopiće najkarakterističnije je histopatološko obilježje Alzheimerove bolesti. U zdravim neuronima tau ima karakteristike topljivog proteina te je uglavnom vezan za mikrotubule aksona, dok u neuronima i glija stanicama zahvaćenim patološkim neurofibrilarnim promjenama tvori agregate i akumulira se u somi i dendritima te jezgrinim porama. Količina agregata i širenje neurofibrilarnih promjena u mozgu u izravnoj su korelaciji s napredovanjem bolesti i stupnjem kognitivnog oštećenja. Glavni rizični čimbenik za nastanak Alzheimerove bolesti je starenje, no točni uzroci agregacije tau proteina nisu razjašnjeni. Budući da su putevi agregacije proteina evolucijski konzervirani, radi genetičkog pristupa otkrivanju čimbenika agregacije i toksičnosti tau proteina , u ovom smo istraživanju kao stanični model odabrali kvasac Saccharomyces cerevisiae. Kako bismo bolje razumjeli mehanizme putem kojih dolazi do ranih patoloških promjena, uspostavili smo i upotrijebili molekularni alat za proučavanje oligomerizacije tau proteina u živim stanicama. Taj se alat temelji na luminiscenciji, gdje interakcija dviju molekula tau proteina rezultira komplementacijom luciferaze NanoLuc i luminiscencijom. Istražili smo dovode li različiti čimbenici, napose proteotoksični stres, do agregacije taua te ispitali toksičnost tau proteina u stanicama kvasca u uvjetima stresa. Pokazali smo da proteotoksični stres ne doprinosi oligomerizaciji i toksičnosti tau proteina eksprimiranog u stanicama kvasca. Uspostavljeni reporter Tau-NanoBiT omogućit će genetički probir čimbenika agregacije i toksičnosti tau proteina u daljnjim istraživanjima.

Age-dependent protein aggregation is a conserved phenomenon that is associated with many neurodegenerative diseases, including Alzheimer's disease (AD). AD is characterized by aggregation of Tau, a microtubule-binding protein that is normally soluble and mainly localized to neuronal axons, but which can form oligomers and higher- order amyloid-like aggregates that accumulate in soma and dendrites and eventually lead to neuronal death. Although the main risk factor for the onset of AD is aging, the exact causes of Tau protein aggregation are still largely unclear. To investigate factors that influence Tau protein aggregation, we expressed human Tau protein fused with fluorescent proteins in yeast Saccharomyces cerevisiae. We examined its intracellular localization in young, logarithmically growing cells, in chronologically aged cells, and under different stress conditions, such as glucose starvation, hyperosmotic stress, elevated temperature, and proteotoxic stress caused by a toxic amino acid analog. Furthermore, to study the factors affecting Tau oligomerization, which is considered to be an early step in Tau pathology, we used luminescent reporter NanoBiT in which protein-protein interaction results in the complementation of the luciferase NanoLuc. Our results show basal levels of Tau-NanoBiT reporter signal in logarithmically growing wild-type cells, suggesting that Tau oligomerization does not occur under normal growth conditions.

The homeostasis of metals is altered in the brain of Alzheimer’s disease (AD) patients. It was proposed that changes in metal homeostasis are related to AD pathology. Additionally, metals that are not present in the brain under normal circumstances (such as mercury, lead and cadmium) were associated with AD pathogenesis. The aim of this study was to test if macro and microelements (Li, B, Na, Mg, Al, S, K, Ca, Cr, Fe, Co, Mn, Ni, Cu, Zn, As, Se, Sr, Mo, Cd, Ba, Tl, Pb, and Hg) measured in cerebrospinal fluid (CSF) and plasma of patients with dementia are associated with CSF biomarkers of AD. CSF AD biomarkers reflect pathological changes in AD brain, while the involvement of different metals in AD pathogenesis is still the matter of debate. Macro and microelements were determined by inductively coupled plasma mass spectroscopy (ICP-MS), while CSF biomarkers were measured by enzyme-linked immunosorbent assays (ELISA). Macro and microelements were measured in CSF of 194 subjects and in plasma of 144 subjects (study included overall 125 AD patients, 50 patients with mild cognitive disorder and 19 healthy controls). We used three different statistical methods to test the association of macro and microelements with CSF biomarkers of AD. All three methods (simple correlation and two machine learning algorithms ; redescription mining and principal component analysis [PCA]) demonstrated the association of macro and microelements with CSF biomarkers of AD. Possible explanations for association of macro and microelements with CSF biomarkers of AD await elucidation of their environmental sources or detection of their release from brain tissue due to cell death. Also, these results should be further validated on larger cohorts.

Alzheimer’s disease (AD) is a common neurodegenerative disorder associated with accumulation of Tau protein aggregates in neurons of the affected brain regions and the consequent neuronal death. Despite a vast number of studies, the causes of tau protein aggregation and toxicity are still largely unclear. Considering that the main risk factor for the onset of AD is aging, and that the ability of a cell to maintain protein homeostasis decreases with aging, impaired protein quality control pathways are considered a possible factor in the development of AD. In order to take advantage of the power of yeast genetics in identifying modifiers of Tau protein aggregation, we expressed human Tau protein in a cell model, yeast Saccharomyces cerevisiae. To study the factors affecting Tau oligomerization, which is considered an early step in Tau pathology, we used luminescent reporter NanoBiT in which protein-protein interaction results in the complementation of the luciferase NanoLuc. We expressed Tau-NanoBiT fusion constructs in a wild-type strain, mutants with impaired protein quality control pathways and mutants reported to have increased Tau phosphorylation and increased levels of sarkosyl-insoluble Tau, and measured reporter activation. Further, we examined Tau intracellular localization by fusing it with a fluorescent protein and tested how this is affected by chronological aging.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of tau protein aggregates in cells of the affected brain regions and the consequent neuronal death. In healthy neurons, tau is a soluble, intrinsically disordered protein that is mostly bound to the microtubules in the axon, however, in the affected neurons, tau accumulates in the soma and dendrites within amyloid-like aggregates, presumably via an intermediary step involving toxic oligomeric structures. The main risk factor for the onset of AD is aging, however, despite a vast number of studies, the causes of tau protein aggregation and toxicity are still largely unclear. Since the ability of a cell to maintain protein homeostasis decreases with aging, impaired protein quality control pathways are considered a possible factor in the development of AD. Yeast is a single cell eukaryotic organism that is amenable to genetic analysis, and many of its cellular pathways, including protein quality control are evolutionarily conserved. Moreover, quiescent yeast can be used to study processes characteristic for non-dividing cells, such as chronological aging. To investigate factors that influence tau protein aggregation and toxicity, in particular cellular aging and proteotoxic stress, we expressed human tau protein in yeast Saccharomyces cerevisiae. We will present a characterization of human tau protein expressed in quiescent yeast. Acknowledgments: Supported by the Research Cooperability Program of the Croatian Science Foundation funded by the European Union from the European Social Fund under the Operational Program Efficient Human Resources 2014-2020 (grant PZS-2019-02-3610) ; Croatian Science Foundation grants IP-2019-04-3584 and DOK- 01-2018/ European Social Fund ; Centre of Excellence for Basic, Clinical and Translational Neuroscience (project “Experimental and clinical research of hypoxic-ischemic damage in perinatal and adult brain” ; GA KK01.1.1.01.0007 funded by the European Union through the European Regional Development Fund).

One of the main neuropathological hallmarks of Alzheimer's disease are neurofibrillary tangles (NFTs), large aggregates of microtubule binding protein tau that form within the affected neurons. The formation of NFTs is preceded by early-stage tau oligomers, however the molecular pathways that initiate tau aggregation are unclear. To better understand early steps in tau pathology, we constructed a tool for studying tau oligomerization in living cells, based on the luminiscent reporter NanoBiT, in which protein-protein interaction results in the complementation of the luciferase NanoLuc, and consequently in generation of luminescence. Since molecular pathways of protein aggregation are largely evolutionary conserved, we selected a simple cell model, yeast Saccharomyces cerevisiae. We separately fused two luciferase subunits to the human tau protein C-terminus. Expression of tau constructs was verified using western blot. Cells expressing tau separately fused to two luciferase subunits exhibited an increased luminescence, as compared to controls, indicating the formation of tau oligomers. In order to test whether tau-NanoBiT reporter is able to detect sarkosyl-insoluble tau aggregates, we measured tau- NanoBiT luminescence in pho85delta, rim1delta, and sod2delta mutants that were previously reported to have increased levels of sarkosyl-insoluble tau. Our preliminary data showed that the tau-NanoBiT luminescence was elevated in the sod2delta mutant, while the signal in pho85delta and rim1delta mutants was similar to the wild-type levels. In conclusion, we constructed a luminescent reporter for human tau protein oligomerization in living yeast cells. Our future experiments will address whether tau-NanoBiT reporter correlates with the levels of sarkosyl- insoluble tau aggregates.

One of the main neuropathological hallmarks of Alzheimer’s disease are neurofibrillary tangles (NTFs), large aggregates of microtubule-binding protein tau that form within the affected neurons. The formation of NTFs is preceded by early-stage tau oligomers, however the molecular pathways that initiate tau aggregation are unclear. To better understand early steps in tau pathology, we constructed a tool for studying tau oligomerization in living cells, based on the luminescent reporter NanoBiT, in which protein- protein interaction results in the complementation of the luciferase NanoLuc, and consequently in generation of luminescence that can be measured in living cells. Since molecular pathways of protein aggregation are largely evolutionarily conserved, we selected a simple cell model, yeast Saccharomyces cerevisiae. We separately fused two luciferase subunits to the human tau protein C- terminus. In order to test whether the observed luminescence is a specific result of tau-tau interaction, we constructed negative controls, including cells that carry an empty vector, and cells that express tau fused to only one of the luciferase subunits. Expression of tau constructs was verified using western blot. By measuring luciferase activity in living cells, we showed that cells expressing tau separately fused to two luciferase subunits exhibited an increased luminescence, as compared to controls, indicating the formation of tau oligomers. In order to test whether tau-NanoBiT reporter is able to detect sarkosyl- insoluble tau aggregates, we measured tau-NanoBiT luminescence in pho85Δ, rim1Δ and sod2Δ mutants that were previously reported to have increased levels of sarkosyl-insoluble tau. Our preliminary data showed that the tau- NanoBiT luminescence was elevated in the sod2Δ mutant, while the signal in pho85Δ, and rim1Δ mutants was similar to the wild-type levels. In conclusion, we constructed a luminescent reporter for human tau protein oligomerization in living yeast cells. Our future experiments will address whether tau- NanoBiT reporter correlates with the levels of sarkosyl-insoluble tau aggregates. Research was co-financed by the Scientific Centre of Excellence for Basic, Clinical and Translational Neuroscience (project “Experimental and clinical research of hypoxic-ischemic damage in perinatal and adult brain” ; GA KK01.1.1.01.0007 funded by the European Union through the European Regional Development Fund) and Croatian Science Foundation (grant IP-2014-09-9730 and DOK-01- 2018/ European Social Fund).

Tauopathies, including Alzheimer’s disease, are neurodegenerative disorders characterized by tau protein aggregates in brain cells. Degeneration is characterized by a pathological accumulation of hyperphosphorylated tau, called neurofibrillary tangles, which show a high positive correlation with the degree of dementia. However, tau aggregation pathways leading to tau filament formation and toxicity are unclear. To better understand molecular pathways leading to tau pathology, we constructed a simple tool for studying tau oligomerization in living cells, based on the luminescent reporter NanoBiT. In short, proteins of interest are fused to luciferase subunits 11S and 114. Protein- protein interaction results in enzyme complementation, generating luminescence that can be measured in living cells. Since molecular pathways that prevent accumulation of protein aggregates in the cell are largely evolutionarily conserved, we selected a simple model, yeast Saccharomyces cerevisiae, to look for the factors that influence tau oligomerization. We constructed C-terminal fusions of human 2N4R-tau protein with the luciferase subunits 11S and 114. The DNA sequence encoding human tau protein was codon optimized for yeast and placed under the control of a weak promoter and short synthetic terminators. To be able to distinguish between the expression of tau- 11S and tau-114 within the same cell, we tagged the constructs with two different epitope tags. We will validate the reporter by comparing the luminescent signal of cells expressing constructs tau-11S and tau-114 with the cells additionally overexpressing untagged tau, and with the cells carrying constructs 11S and tau- 114. This work is supported by Croatian Science Foundation (grant IP-2014-09-9730 and DOK-01- 2018/European Social Fund).

Mnoga istraživanja pokazala su povezanost promijenjene regulacije homeostaze bakra s nastankom i progresijom neurodegenerativnih bolesti. Toksična svojstva bakra uglavnom se pripisuju povećanoj proizvodnji reaktivnih kisikovih vrsta i posljedičnom oksidacijskom stresu. Cilj istraživanja bio je analizirati učinak biljnog flavonoida kvercetina na neurone P19 u kojima je stanje oksidacijskog stresa izazvano suviškom iona bakra. U najvećoj primijenjenoj koncentraciji (150 μM) kvercetin je povećao preživljenje neurona P19 tretiranih s 0,5 mM CuSO4 te smanjio bakrom izazvan porast nakupljanja reaktivnih kisikovih vrsta. Analiza metodom Western blotting pokazala je da suvišak bakra dovodi do porasta ekspresije proapoptotskog proteina PUMA, te da je u prisutnosti kvercetina taj porast smanjen. Ovakvi rezultatu upućuje na antiapoptotski mehanizam djelovanja kvercetina. U neuronima P19 kvercetin je također inducirao ekspresiju nukleozid difosfat kinaze NME1. Promjene u ekspresiji proteina p53 i Bax nisu nađene. Dobiveni rezultati pokazuju neuroprotektivni učinak kvercetina u uvjetima oksidacijskog stresa izazvanog ionima bakra i upućuju na antioksidacijski i antiapoptotski mehanizam njegova djelovanja. A growing number of studies suggest that the deregulation of copper homeostasis is an important factor in the onset and progression of neurodegenerative diseases. Toxic effects of copper are mainly attributed to the increased production of reactive oxygen species (ROS) and subsequent oxidative stress. The aim of this study was to analyse the effect of the plant-derived flavonoid quercetin on P19 neurons, in which the oxidative stress was induced by exposure to the excess copper ions. In the highest applied concentration (150 μM) of quercetin the viability of P19 neurons treated with 0.5 mM CuSO4 was increased and the copper-induced accumulation of reactive oxygen species was prevented. Western blotting analysis showed that copper increases expression of proapoptotic protein PUMA, whereas in the presence of quercetin the obtained increase was diminished. These results indicate anti-apoptotic mechanism of quercetin action. In P19 neurons quercetin also induced the expression of nucleoside diphosphate kinase NME1. The expression of p53 and Bax remained unchanged at the protein level. The obtained results demonstrate neuroprotective effect of quercetin against oxidative stress induced by exposure to copper ions, and suggest antioxidative and antiapoptotic mechanism of its action.

Copper is an essential transition metal with multiple cellular functions mainly related to diverse types of oxidation and reduction reactions. Although mechanisms of copper storage and availability are tightly regulated, some factors, such as occupational hazard, environmental contamination and inborn errors of Cu metabolism, may overwhelm intracellular storage capacity, leading to copper accumulation and undesirable cellular effects. Deregulation of copper homeostasis is recognized as an important factor in the etiology of a number of devastating neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis. The aim of the present study was to investigate potential neuroprotective effects of flavonoid quercetin, one of the most potent scavengers of reactive oxygen species (ROS), against copper-induced toxicity and to elucidate mechanisms of its action at the molecular and cellular level in the culture of differentiated P19 neurons. P19 neurons were obtained by the differentiation procedure from the P19 mouse embryonal carcinoma cells in the presence of retinoic acid, and were exposed to quercetin and copper for 24 h. Survival of P19 neurons in the presence of copper, quercetin and inhibitors of signalling pathways was determined by the colorimetric 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. ROS accumulation was measured by the assay that utilizes the cell permeable substrate, 2’, 7’- dichlorofluorescin diacetate. Changes in the nuclear condensation that are characteristic for the programmed type of cell death were assessed by Hoechst staining. Measurement of the activities of key apoptotic markers caspase-3 and -7 was performed with Apo-ONE® Homogeneous Caspase-3/7 Assay (Promega). Copper- and quercetin-induced changes in the protein expression were determined by the Western blot method. In a dose-dependent manner, a concomitant treatment with quercetin improved viability of moderately-injured P19 neurons exposed to 0.5 mM CuSO4 for 24 h. Quercetin decreased ROS formation and prevented caspase-3/7 activation and chromatin condensation. Neuroprotective effects of quercetin were pharmacologically prevented by the inhibition of ERK1/2 and phosphoinositide 3-kinase/Akt signalling by exposure to UO126 and wortmannin, respectively. Preliminary results indicate that treatment with copper and quercetin also affected expression of transcription factor p53 at the protein level. In neurons that were more severely damaged by exposure to 1 mM CuSO4, quercetin exerted strong pro-oxidative action and further exacerbated cytotoxic effects. Quercetin exerted beneficial and desirable pro-survival effects in moderately injured neurons by affecting intracellular signalling pathways and attenuating ROS production, but was also able to enhance pro-oxidative copper-effects with detrimental biological consequences at higher copper concentrations. As herbal formulations are nowadays widely used in the prevention and therapy of oxidative-stress driven neuronal injuries, observed findings indicate that caution is required about taking dietary antioaxidative supplements and also for the future development of novel therapeutic drugs that are based on the structural backbone of the natural polyphenolic compounds.

Glikanske strukture terapeutskih proteina utječu na njihova farmakodinamička i farmakokinetička svojstva. Optimalna glikozilacija povećala bi učinkovitost i sigurnost rekombinantnih farmaceutskih proteina. S obzirom na jeftiniji uzgoj i procesiranje, jednostavan N-glikozilacijski put i odsutnost O-glikozilacijskog puta karakterističnog za sisavce, biljke su se posljednjih godina nametnule u modeliranju ekspresijskog sustava za proizvodnju potpuno humaniziranih rekombinantnih terapeutskih proteina. Ciljevi pri konstruiranju takvog optimalnog sustava su inaktivacija gena za enzime karakteristične za biljnu glikozilaciju te uvođenje gena za enzime koji su karakteristični za čovjeka. Glycan structures of therapeutic proteins affect their pharmacodynamics and pharmacokinetic characteristics. Optimal glycosylation would enhance efficiency and safety of recombinant pharmaceutical proteins. Due to inexpensive and simple cultivation, simple Nglycosylation pathway and absence of typical mammalian O-glycosylation pathway, in recent years plants have emerged as interesting new hosts for the production of fully humanized recombinant pharmaceutical proteins. Aims in engineering optimal expression system are inactivation of genes for plant-specific enzymes and introduction of enzymes that are humanspecific.

Glikanske strukture terapeutskih proteina utječu na njihova farmakodinamička i farmakokinetička svojstva. Optimalna glikozilacija povećala bi učinkovitost i sigurnost rekombinantnih farmaceutskih proteina. S obzirom na jeftiniji uzgoj i procesiranje, jednostavan N-glikozilacijski put i odsutnost O-glikozilacijskog puta karakterističnog za sisavce, biljke su se posljednjih godina nametnule u modeliranju ekspresijskog sustava za proizvodnju potpuno humaniziranih rekombinantnih terapeutskih proteina. Ciljevi pri konstruiranju takvog optimalnog sustava su inaktivacija gena za enzime karakteristične za biljnu glikozilaciju te uvođenje gena za enzime koji su karakteristični za čovjeka.