Neurodegeneration Cell Model Research Articles

BAG2 prevents Tau hyperphosphorylation and increases p62/SQSTM1 in cell models of neurodegeneration.

Protein aggregates are pathological hallmarks of many neurodegenerative diseases, however the physiopathological role of these aggregates is not fully understood. Protein quality control has a pivotal role for protein homeostasis and depends on specific chaperones. The co-chaperone BAG2 can target phosphorylated Tau for degradation by an ubiquitin-independent pathway, although its possible role in autophagy was not yet elucidated. In view of this, the aim of the present study was to investigate the association among protein aggregation, autophagy and BAG2 levels in cultured cells from hippocampus and locus coeruleus as well as in SH-SY5Y cell line upon different protein aggregation scenarios induced by rotenone, which is a flavonoid used as pesticide and triggers neurodegeneration. The present study showed that rotenone exposure at 0.3nM for 48h impaired autophagy prior to Tau phosphorylation at Ser199/202 in hippocampus but not in locus coeruleus cells, suggesting that distinct neuron cells respond differently to rotenone toxicity. Rotenone induced Tau phosphorylation at Ser199/202, together with a decrease in the endogenous BAG2 protein levels in SH-SY5Y and hippocampus cell culture, which indicates that rotenone and Tau hyperphosphorylation can affect this co-chaperone. Finally, it has been shown that BAG2 overexpression, increased p62/SQSTM1 levels in cells from hippocampus and locus coeruleus, stimulated LC3II recycling as well as prevented the raise of phosphorylated Tau at Ser199/202 in hippocampus. Results demonstrate a possible role for BAG2 in degradation pathways of specific substrates and its importance for the study of cellular aspects of neurodegenerative diseases.

Human stem cell models of neurodegeneration: From basic science of amyotrophic lateral sclerosis to clinical translation.

Neurodegenerative diseases are characterized by progressive cell loss leading to disruption of the structure and function of the central nervous system. Amyotrophic lateral sclerosis (ALS) was among the first of these disorders modeled in patient-specific iPSCs, and recent findings have translated into some of the earliest iPSC-inspired clinical trials. Focusing on ALS as an example, we evaluate the status of modeling neurodegenerative diseases using iPSCs, including methods for deriving and using disease-relevant neuronal and glial lineages. We further highlight the remaining challenges in exploiting the full potential of iPSC technology for understanding and potentially treating neurodegenerative diseases such as ALS.

Human stem cell models of disease and the prognosis of academic medicine.

Rickie Patani is Professor of Human Stem Cells and Regenerative Neurology at University College London and The Francis Crick Institute, and is a consultant neurologist at The National Hospital for Neurology, Queen Square, London. He has over a decade of experience using human stem cell models of neurodegeneration, and his research contributions have been recognized by the International Paulo Gontijo Award in Medicine and the International 3Rs prize.

Inter-Species Differences in Regulation of the Progranulin-Sortilin Axis in TDP-43 Cell Models of Neurodegeneration.

Cytoplasmic aggregates and nuclear depletion of the ubiquitous RNA-binding protein TDP-43 have been described in the autoptic brain tissues of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTLD) patients and both TDP-43 loss-of-function and gain-of-function mechanisms seem to contribute to the neurodegenerative process. Among the wide array of RNA targets, TDP-43 regulates progranulin (GRN) mRNA stability and sortilin (SORT1) splicing. Progranulin is a secreted neurotrophic and neuro-immunomodulatory factor whose endocytosis and delivery to the lysosomes are regulated by the neuronal receptor sortilin. Moreover, GRN loss-of-function mutations are causative of a subset of FTLD cases showing TDP-43 pathological aggregates. Here we show that TDP-43 loss-of-function differently affects the progranulin–sortilin axis in murine and human neuronal cell models. We demonstrated that although TDP-43 binding to GRN mRNA occurs similarly in human and murine cells, upon TDP-43 depletion, a different control of sortilin splicing and protein content may determine changes in extracellular progranulin uptake that account for increased or unchanged secreted protein in murine and human cells, respectively. As targeting the progranulin–sortilin axis has been proposed as a therapeutic approach for GRN-FTLD patients, the inter-species differences in TDP-43-mediated regulation of this pathway must be considered when translating studies from animal models to patients.

Integrated Proteomics and Lipidomics Investigation of the Mechanism Underlying the Neuroprotective Effect of N-benzylhexadecanamide.

Macamides are very important secondary metabolites produced by Lepidium meyenii Walp, which possess multiple bioactivities, especially in the neuronal system. In a previous study, we observed that macamides exhibited excellent effects in the recovery of injured nerves after 1-methyl-4-phenylpyridinium (MPP+)-induced dopaminergic neuronal damage in zebrafish. However, the mechanism underlying this effect remains unclear. In the present study, we observed that N-benzylhexadecanamide (XA), which is a typical constituent of macamides, improved the survival rate of neurons in vitro. We determined the concentration of neurotransmitters in MN9D cells and used it in conjunction with an integrated proteomics and lipidomics approach to investigate the mechanism underlying the neuroprotective effects of XA in an MPP+-induced neurodegeneration cell model using QqQ MS, Q-TOF MS, and Orbitrap MS. The statistical analysis of the results led to the identification of differentially-expressed biomarkers, including 11 proteins and 22 lipids, which may be responsible for the neuron-related activities of XA. All these potential biomarkers were closely related to the pathogenesis of neurodegenerative diseases, and their levels approached those in the normal group after treatment with XA. Furthermore, seven lipids, including five phosphatidylcholines, one lysophosphatidylcholine, and one phosphatidylethanolamine, were verified by a relative quantitative approach. Moreover, four proteins (Scarb2, Csnk2a2, Vti1b, and Bnip2) were validated by ELISA. The neurotransmitters taurine and norepinephrine, and the cholinergic constituents, correlated closely with the neuroprotective effects of XA. Finally, the protein–lipid interaction network was analyzed. Based on our results, the regulation of sphingolipid metabolism and mitochondrial function were determined to be the main mechanisms underlying the neuroprotective effect of XA. The present study should help us to better understand the multiple effects of macamides and their use in neurodegenerative diseases.

Tau-Centric Multitarget Approach for Alzheimer's Disease: Development of First-in-Class Dual Glycogen Synthase Kinase 3β and Tau-Aggregation Inhibitors.

Several findings propose the altered tau protein network as an important target for Alzheimer's disease (AD). Particularly, two points of pharmacological intervention can be envisaged: inhibition of phosphorylating tau kinase GSK-3β and tau aggregation process. On the basis of this consideration and on our interest in multitarget paradigms in AD, we report on the discovery of 2,4-thiazolidinedione derivatives endowed with such a profile. 28 and 30 displayed micromolar IC50 values toward GSK-3β, together with the capacity of inhibiting AcPHF6 aggregation of 60% and 80% at 10 μM, respectively. In addition, they showed PAMPA-BBB permeability, together with a suitable cellular safety profile. 30 also displayed inhibition of both K18 and full-length tau aggregations. Finally, both compounds were able to improve cell viability in an okadaic acid-induced neurodegeneration cell model. To the best of our knowledge, 28 and 30 are the first balanced, nontoxic, dual-acting compounds hitting tau cascade at two different hubs.

The S100A4 Protein Signals through the ErbB4 Receptor to Promote Neuronal Survival.

Understanding the mechanisms of neurodegeneration is crucial for development of therapies to treat neurological disorders. S100 proteins are extensively expressed in the injured brain but S100's role and signalling in neural cells remain elusive. We recently demonstrated that the S100A4 protein protects neurons in brain injury and designed S100A4-derived peptides mimicking its beneficial effects. Here we show that neuroprotection by S100A4 involves the growth factor family receptor ErbB4 and its ligand Neuregulin 1 (NRG), key regulators of neuronal plasticity and implicated in multiple brain pathologies. The neuroprotective effect of S100A4 depends on ErbB4 expression and the ErbB4 signalling partners ErbB2/Akt, and is reduced by functional blockade of NRG/ErbB4 in cell models of neurodegeneration. We also detect binding of S100A4 with ErbB1 (EGFR) and ErbB3. S100A4-derived peptides interact with, and signal through ErbB, are neuroprotective in primary and immortalized dopaminergic neurons, and do not affect cell proliferation/motility - features which make them promising as potential neuroprotectants. Our data suggest that the S100-ErbB axis may be an important mechanism regulating neuronal survival and plasticity.

Chemical characterization and bioactivity of phytochemicals from Iberian endemic Santolina semidentata and strategies for ex situ propagation

Asteraceae family members are well-known for their medicinal potential, comprising several properties that make them unique among plants. Here we focus on Santolina semidentata, an endemic plant from the Iberian Peninsula, not yet described for its medicinal properties. Phytochemical characterization of S. semidentata was performed, concerning total phenol content, flavonoid content, antioxidant capacity, HPLC-DAD profile, acetylcholinesterase inhibitory capacity, cytotoxicity and neuroprotective effect in a human neurodegeneration cell model. Moreover, essential oil composition and antifungal activity were also analised. This oil might be useful for therapeutical purposes, particularly in the treatment of dermatophytosis. S. semidentata potential for neuroprotection was revealed by acetylcholinesterase inhibitory capacity and also by an effective protective effect in human neuronal cells. Furthermore, different seed conservation protocols, as well as successful in vitro propagation were established which may be useful when integrated in a broad strategy for the conservation of these endemic plants and their sustainable use for potential biotechnological applications. The results presented here greatly contribute to value this species regarding its potential as a source of phytochemicals with prospective neuroprotective health benefits, either as alternative neuroprotective drugs or as leads for synthetizing more effective molecules.

Human stem cell models of neurodegeneration: a novel approach to study mechanisms of disease development

The number of patients with neurodegenerative diseases is increasing significantly worldwide. Thus, intense research is being pursued to uncover mechanisms of disease development in an effort to identify molecular targets for therapeutic intervention. Analysis of postmortem tissue from patients has yielded important histological and biochemical markers of disease progression. However, this approach is inherently limited because it is not possible to study patient neurons prior to degeneration. As such, transgenic and knockout models of neurodegenerative diseases are commonly employed. While these animal models have yielded important insights into some molecular mechanisms of disease development, they do not provide the opportunity to study mechanisms of neurodegeneration in human neurons at risk and thus, it is often difficult or even impossible to replicate human pathogenesis with this approach. The generation of patient-specific induced pluripotent stem (iPS) cells offers a unique opportunity to overcome these obstacles. By expanding and differentiating iPS cells, it is possible to generate large numbers of functional neurons in vitro, which can then be used to study the disease of the donating patient. Here, we provide an overview of human stem cell models of neurodegeneration using iPS cells from patients with Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia, Huntington's disease, spinal muscular atrophy and other neurodegenerative diseases. In addition, we describe how further refinements of reprogramming technology resulted in the generation of patient-specific induced neurons, which have also been used to model neurodegenerative changes in vitro.

Neuroprotective effect of raspberry extract by inhibiting peroxynitrite-induced DNA damage and hydroxyl radical formation

Raspberry extract has recently been reported to show neuroprotective capacity in a neurodegeneration cell model. However, the mechanism underlying raspberry extract-mediated neuroprotection is unknown. Because peroxynitrite is involved in the pathogenesis of neurodegenerative diseases via its cytotoxic effects, this study was undertaken to investigate whether the neuroprotective effect of raspberry extract is associated with inhibition of peroxynitrite-induced DNA damage, a critical event leading to peroxynitrite-mediated cytotoxicity. Our results showed that peroxynitrite can cause DNA damage in φX-174 plasmid DNA and rat primary astrocytes. The presence of raspberry extract (50–100μg/ml) was found to significantly inhibit peroxynitrite-induced DNA cleavage. EPR spectroscopy demonstrated that the formation of DMPO-hydroxyl radical adduct (DMPO–OH) from peroxynitrite, and that raspberry extract potently diminished the adduct signal in a concentration‐dependent manner. Taken together, these results demonstrate for the first time that raspberry extract can protect against peroxynitrite-mediated DNA damage and hydroxyl radical formation.

The neuroprotective potential of phenolic-enriched fractions from four Juniperus species found in Portugal

The increase in population lifespan has enhanced the incidence of neurodegenerative diseases, for which there is, as yet, no cure.We aimed to chemically characterize phenolic-enriched fractions (PEFs) from four wild Juniperus sp. found in Portugal (Juniperus navicularis, Juniperus oxycedrus badia, Juniperus phoenicea and Juniperus turbinata) and address their potential as sources of natural products for treatment of neurodegenerative diseases.Leaves from the four Juniperus sp. evaluated contained a range of phenolic components which differed quantitatively between the species. The PEFs obtained were rich sources of phenolic compounds, exhibited acetylcholinesterase (AChE) inhibitory activity and also displayed effective intracellular radical scavenging properties in neurons submitted to oxidative injury but showed a different order of effectiveness compared to AChE inhibition. These properties made them good candidates for testing in a neurodegeneration cell model. Pre-incubation with J. oxycedrus badia PEF for 24h protected neurons from injury in the neurodegeneration cell model.

Neuroprotective effects of digested polyphenols from wild blackberry species

Blackberry ingestion has been demonstrated to attenuate brain degenerative processes with the benefits ascribed to the (poly)phenolic components. The aim of this work was to evaluate the neuroprotective potential of two wild blackberry species in a neurodegeneration cell model and compare them with a commercial variety. This work encompasses chemical characterization before and after an in vitro digestion and the assessment of neuroprotection by digested metabolites. Some studies targeting redox/cell death systems were also performed to assess possible neuroprotective molecular mechanisms. The three blackberry extracts presented some quantitative differences in polyphenol composition that could be responsible for the different responses in the neurodegeneration cell model. Commercial blackberry extracts were ineffective but both wild blackberries, Rubus brigantinus and Rubus vagabundus, presented neuroprotective effects. It was verified that a diminishment of intracellular ROS levels, modulation of glutathione levels and activation of caspases occurred during treatment. The last effect suggests a preconditioning effect since caspase activation was not accompanied by diminution in cell death and loss of functionality. This is the first time that metabolites obtained from an in vitro digested food matrix, and tested at levels approaching the concentrations found in human plasma, have been described as inducing an adaptative response.

Neuroprotective effect of blackberry (Rubus sp.) polyphenols is potentiated after simulated gastrointestinal digestion

Blackberry ingestion has been demonstrated to attenuate brain degenerative processes in rodents with the benefits ascribed to the (poly)phenolic components. The aim of this work was to assess the efficacy of blackberry polyphenolics in a neurodegeneration cell model before and after simulated gastrointestinal digestion.Digested blackberry metabolites protected neuroblastoma cells from H2O2-induced death at low, non-toxic levels that approach physiologically-relevant serum concentrations. However, the original extracts were not protective even at fivefold higher concentrations. This potentiation may reflect alterations in the polyphenolic composition caused by the digestion procedure, as detected by liquid-chromatography-mass spectrometric analysis. This protection was not caused by modulation of the intracellular antioxidant capacity or through alteration of glutathione levels, although the original extract influenced both of these parameters. This work reinforces the importance of evaluating digested metabolites in disease cell models and highlights the possible involvement of other mechanisms beyond antioxidant systems.

Mechanisms Controlling the Cellular Accumulation of Copper Bis(thiosemicarbazonato) Complexes

Copper (Cu) bis(thiosemicarbazonato) metal complexes [Cu(II)(btsc)s] have unique tumor-imaging and treatment properties and more recently have revealed potent neuroprotective actions in animal and cell models of neurodegeneration. However, despite the continued development of Cu(II)(btsc)s as potential therapeutics or diagnostic agents, little is known of the mechanisms involved in cell uptake, subcellular trafficking, and efflux of this family of compounds. Because of their high lipophilicity, it has been assumed that cellular accumulation is through passive diffusion, although this has not been analyzed in detail. The role of efflux pathways in cell homeostasis of the complexes is also largely unknown. In the present study, we investigated the cellular accumulation of the Cu(II)(btsc) complexes Cu(II)(gtsm) and Cu(II)(atsm) in human neuronal (M17) and glial (U87MG) cell lines under a range of conditions. Collectively, the data strongly suggested that Cu(II)(gtsm) and Cu(II)(atsm) may be taken into these cells by combined passive and facilitated (protein-carrier-mediated) mechanisms. This was supported by strong temperature-dependent changes to the uptake of the complexes and the influence of the cell surface protein on Cu accumulation. We found no evidence to support a role for copper-transporter 1 in accumulation of the compounds. Importantly, our findings also demonstrated that Cu from both Cu(II)(gtsm) and Cu(II)(atsm) was rapidly effluxed from the cells through active mechanisms. Whether this was in the form of released ionic Cu or as an intact metal complex is not known. However, this finding highlighted the difficulty of trying to determine the uptake mechanism of metal complexes when efflux is occurring concomitantly. These findings are the first detailed exploration of the cellular accumulation mechanisms of Cu(II)(btsc)s. The study delineates strategies to investigate the uptake and efflux mechanisms of metal complexes in cells, while highlighting specific difficulties and challenges that need to be considered before drawing definitive conclusions.

Antioxidant Properties and Neuroprotective Capacity of Strawberry Tree Fruit (Arbutus unedo)

Berries contain significant amounts of phytochemicals, including polyphenols, which are reported to reduce cancer risk, coronary heart disease and other degenerative diseases. These effects are mainly attributed to the antioxidant capacity of polyphenols found in berries. Strawberry tree (Arbutus unedo) berries are used in folk medicine but seldom eaten as fresh fruits. Their phenolic profile and antioxidant capacity reveal a high potential, but they are not well characterized as a “health promoting food”. The aim of this study was to assess the antioxidant properties of the edible strawberry tree fruit in vitro and in a neurodegeneration cell model. Raspberry (Rubus idaeus), a well documented health-promoting fruit, was used as a control for comparison purposes. A. unedo yielded a similar content in polyphenols and a slightly lower value of total antioxidant capacity in comparison to R. idaeus. Although the chemically-measured antioxidant activity was similar between both fruits, R. idaeus increased neuroblastoma survival in a neurodegeneration cell model by 36.6% whereas A. unedo extracts caused no effect on neuroblastoma viability. These results clearly demonstrate that a promising level of chemically-determined antioxidant activity of a plant extract is not necessarily correlated with biological significance, as assessed by the effect of A. unedo fruit in a neurodegeneration cell model.

Neuroprotective and MMP-9 inhibitory activity of hydroethanolic extract of Arbustus unedo leaves

Arbutus unedo L., the strawberry tree (Ericaceae family), is an endemic Mediterranean species. Its leaves have been employed for a long time in traditional and popular medicine as an astringent, diuretic, urinary anti-septic, and more recently, in the therapy of hypertension and diabetes [1]. The aim of this work is to evaluate the antioxidant properties of a hydroethanolic extract of A. unedo leaves in a neurodegeneration cell model and the inhibitory activity to human matrix metalloprotease (MMP-9), an enzyme involved in cancer invasion.