Monitoring alpha-synuclein oligomerization and aggregation using bimolecular fluorescence complementation assays: What you see is not always what you get.

Bimolecular fluorescence complementation (BiFC) was introduced a decade ago as a method to monitor alpha-synuclein (α-syn) oligomerization in intact cells. Since then, several α-syn BiFC cellular assays and animal models have been developed based on the assumption that an increase in the fluorescent signal correlates with increased α-syn oligomerization or aggregation. Despite the increasing use of these assays and models in mechanistic studies, target validation and drug screening, there have been no reports that (1) validate the extent to which the BiFC fluorescent signal correlates with α-syn oligomerization at the biochemical level; (2) provide a structural characterization of the oligomers and aggregates formed by the BiFC. To address this knowledge gap, we first analysed the expression level and oligomerization properties of the individual constituents of α-syn-Venus, one of the most commonly used BiFC systems, in HEK-293 & SH-SY5Y cells from three different laboratories using multiple biochemical approaches and techniques. Next, we investigated the biochemical and aggregation properties of α-syn upon co-expression of both BiFC fragments. Our results show that (1) the C-terminal-Venus fused to α-syn (α-syn-Vc) is present in much lower abundance than its counterpart with N-terminal-Venus fused to α-syn (Vn-α-syn); (2) Vn-α-syn exhibits a high propensity to form oligomers and higher-order aggregates; and (3) the expression of either or both fragments does not result in the formation of α-syn fibrils or cellular inclusions. Furthermore, our results suggest that only a small fraction of Vn-α-syn is involved in the formation of the fluorescent BiFC complex and that some of the fluorescent signal may arise from the association or entrapment of α-syn-Vc in Vn-α-syn aggregates. The fact that the N-terminal fragment exists predominantly in an aggregated state also indicates that one must exercise caution when using this system to investigate α-syn oligomerization in cells or in vivo. Altogether, our results suggest that cellular and animal models of oligomerization, aggregation and cell-to-cell transmission based on the α-syn BiFC systems should be thoroughly characterized at the biochemical level to ensure that they reproduce the process of interest and measure what they are intended to measure.

Between an ugly truth and a perfect lie: Wiping off fearful memories using beta-adrenergic receptors antagonists.

Psychiatric disorders such as anxiety, phobias, and post-traumatic stress disorder are considered of high global prevalence. Currently, a therapeutic approach to treat these disorders using beta-blockers, which antagonize the beta-adrenergic receptors (B1, B2, and B3) is being studied. This approach claims that beta-blockers, such as propranolol, inhibit fear memory reconsolidation. However, there are several studies refuting such claims by discrediting their experimental design and pointing out both the drugs pharmacokinetic properties and confounding factors. In this review, we explore the different effects of central beta-adrenergic agonists and antagonists on the fear memory consolidation providing mixed-evidence, limitations, and future directions.

Overexpression of mitochondrial fission or mitochondrial fusion genes enhances resilience and extends longevity.

The dynamicity of the mitochondrial network is crucial for meeting the ever-changing metabolic and energy needs of the cell. Mitochondrial fission promotes the degradation and distribution of mitochondria, while mitochondrial fusion maintains mitochondrial function through the complementation of mitochondrial components. Previously, we have reported that mitochondrial networks are tubular, interconnected, and well-organized in young, healthy C. elegans, but become fragmented and disorganized with advancing age and in models of age-associated neurodegenerative disease. In this work, we examine the effects of increasing mitochondrial fission or mitochondrial fusion capacity by ubiquitously overexpressing the mitochondrial fission gene drp-1 or the mitochondrial fusion genes fzo-1 and eat-3, individually or in combination. We then measured mitochondrial function, mitochondrial network morphology, physiologic rates, stress resistance, and lifespan. Surprisingly, we found that overexpression of either mitochondrial fission or fusion machinery both resulted in an increase in mitochondrial fragmentation. Similarly, both mitochondrial fission and mitochondrial fusion overexpression strains have extended lifespans and increased stress resistance, which in the case of the mitochondrial fusion overexpression strains appears to be at least partially due to the upregulation of multiple pathways of cellular resilience in these strains. Overall, our work demonstrates that increasing the expression of mitochondrial fission or fusion genes extends lifespan and improves biological resilience without promoting the maintenance of a youthful mitochondrial network morphology. This work highlights the importance of the mitochondria for both resilience and longevity.

Evolutionarily Conserved Role of Thioredoxin Systems in Determining Longevity.

Thioredoxin and thioredoxin reductase are evolutionarily conserved antioxidant enzymes that protect organisms from oxidative stress. These proteins also play roles in redox signaling and can act as a redox-independent cellular chaperone. In most organisms, there is a cytoplasmic and mitochondrial thioredoxin system. A number of studies have examined the role of thioredoxin and thioredoxin reductase in determining longevity. Disruption of either thioredoxin or thioredoxin reductase is sufficient to shorten lifespan in model organisms including yeast, worms, flies and mice, thereby indicating conservation across species. Similarly, increasing the expression of thioredoxin or thioredoxin reductase can extend longevity in multiple model organisms. In humans, there is an association between a specific genetic variant of thioredoxin reductase and lifespan. Overall, the cytoplasmic and mitochondrial thioredoxin systems are both important for longevity.

Endosomal trafficking protein TBC-2 modulates stress resistance and lifespan through DAF-16-dependent and independent mechanisms.

The FOXO transcription factor, DAF-16, plays an integral role in insulin/IGF-1 signaling (IIS) and stress response. In conditions of stress or decreased IIS, DAF-16 moves to the nucleus where it activates genes that promote survival. To gain insight into the role of endosomal trafficking in resistance to stress, we disrupted tbc-2, which encodes a GTPase activating protein that inhibits RAB-5 and RAB-7. We found that tbc-2 mutants have decreased nuclear localization of DAF-16 in response to heat stress, anoxia, and bacterial pathogen stress, but increased nuclear localization of DAF-16 in response to chronic oxidative stress and osmotic stress. tbc-2 mutants also exhibit decreased upregulation of DAF-16 target genes in response to stress. To determine whether the rate of nuclear localization of DAF-16 affected stress resistance in these animals, we examined survival after exposure to multiple exogenous stressors. Disruption of tbc-2 decreased resistance to heat stress, anoxia, and bacterial pathogen stress in both wild-type worms and stress-resistant daf-2 insulin/IGF-1 receptor mutants. Similarly, deletion of tbc-2 decreases lifespan in both wild-type worms and daf-2 mutants. When DAF-16 is absent, the loss of tbc-2 is still able to decrease lifespan but has little or no impact on resistance to most stresses. Combined, this suggests that disruption of tbc-2 affects lifespan through both DAF-16-dependent and DAF-16-independent pathways, while the effect of tbc-2 deletion on resistance to stress is primarily DAF-16-dependent. Overall, this work demonstrates the importance of endosomal trafficking for the proper nuclear localization of DAF-16 during stress and that perturbation of normal endosomal trafficking is sufficient to decrease both stress resistance and lifespan.

Endosomal trafficking protein TBC-2 is required for the longevity of long-lived mitochondrial mutants.

Mutations that result in a mild impairment of mitochondrial function can extend longevity. Previous studies have shown that the increase in lifespan is dependent on stress responsive transcription factors, including DAF-16/FOXO, which exhibits increased nuclear localization in long-lived mitochondrial mutants. We recently found that the localization of DAF-16 within the cell is dependent on the endosomal trafficking protein TBC-2. Based on the important role of DAF-16 in both longevity and resistance to stress, we examined the effect of disrupting tbc-2 on lifespan and stress resistance in the long-lived mitochondrial mutants nuo-6 and isp-1 in Caenorhabditis elegans. Loss of tbc-2 markedly reduced the long lifespans of both mitochondrial mutants. Disruption of tbc-2 also decreased resistance to chronic oxidative stress in nuo-6 and isp-1 mutants but had little or no detrimental effect on resistance to other stressors. In contrast, tbc-2 inhibition had no effect on oxidative stress resistance or lifespan in isp-1 worms when DAF-16 is absent, suggesting that the effect of tbc-2 on mitochondrial mutant lifespan may be mediated by mislocalization of DAF-16. However, this result is complicated by the fact that deletion of daf-16 markedly decreases both phenotypes in isp-1 worms, which could result in a floor effect. In exploring the contribution of DAF-16 further, we found that disruption of tbc-2 did not affect the nuclear localization of DAF-16 in isp-1 worms or prevent the upregulation of DAF-16 target genes in the long-lived mitochondrial mutants. This suggests the possibility that the effect of tbc-2 on lifespan and stress resistance in the long-lived mitochondrial mutants is at least partially independent of its effects on DAF-16 localization. Overall, this work demonstrates the importance of endosomal trafficking for the extended longevity and enhanced stress resistance resulting from mild impairment of mitochondrial function.

Biological resilience and aging: Activation of stress response pathways contributes to lifespan extension.

While aging was traditionally viewed as a stochastic process of damage accumulation, it is now clear that aging is strongly influenced by genetics. The identification and characterization of long-lived genetic mutants in model organisms has provided insights into the genetic pathways and molecular mechanisms involved in extending longevity. Long-lived genetic mutants exhibit activation of multiple stress response pathways leading to enhanced resistance to exogenous stressors. As a result, lifespan exhibits a significant, positive correlation with resistance to stress. Disruption of stress response pathways inhibits lifespan extension in multiple long-lived mutants representing different pathways of lifespan extension and can also reduce the lifespan of wild-type animals. Combined, this suggests that activation of stress response pathways is a key mechanism by which long-lived mutants achieve their extended longevity and that many of these pathways are also required for normal lifespan. These results highlight an important role for stress response pathways in determining the lifespan of an organism.

Effect of DMSO on lifespan and physiology in C. elegans : Implications for use of DMSO as a solvent for compound delivery.

Dimethyl sulfoxide (DMSO) is a solvent that has been used for basic and medical research based on its ability to dissolve both polar and non-polar compounds. In order to use DMSO to deliver compounds that may impact longevity or neurodegeneration, it is important to first determine the effects of DMSO on aging and physiology. We examined the effect of different concentrations of DMSO on lifespan and development time in C. elegans. We found that DMSO concentrations up to 2% DMSO did not affect longevity in wild-type worms, while concentrations of up to 0.5% DMSO were compatible with normal development times. 0.5% DMSO also had minimal effect on fertility and movement. In summary, our results show that concentrations of DMSO up to 0.5% can be safely used to deliver compounds to C. elegans with little or no modifying effects on lifespan or physiologic rates.

The consequences of viral infection on host DNA damage response: a focus on SARS-CoVs.

DNA damage and genome instability in host cells are introduced by many viruses during their life cycles. Severe acute respiratory syndrome coronaviruses (SARS-CoVs) manipulation of DNA damage response (DDR) is an important area of research that is still understudied. Elucidation of the direct and indirect interactions between SARS-CoVs and DDR not only provides important insights into how the viruses exploit DDR pathways in host cells but also contributes to our understanding of their pathogenicity. Here, we present the known interactions of both SARS-CoV and SARS-CoV-2 with DDR pathways of the host cells, to further understand the consequences of infection on genome integrity. Since this area of research is in its early stages, we try to connect the unlinked dots to speculate and propose different consequences on DDR mechanisms. This review provides new research scopes that can be further investigated in vitro and in vivo, opening new avenues for the development of anti-SARS-CoV-2 drugs.

Mitochondrial thioredoxin system is required for enhanced stress resistance and extended longevity in long-lived mitochondrial mutants.

Mild impairment of mitochondrial function has been shown to increase lifespan in genetic model organisms including worms, flies and mice. To better understand the mechanisms involved, we analyzed RNA sequencing data and found that genes involved in the mitochondrial thioredoxin system, trx-2 and trxr-2, are specifically upregulated in long-lived mitochondrial mutants but not other non-mitochondrial, long-lived mutants. Upregulation of trx-2 and trxr-2 is mediated by activation of the mitochondrial unfolded protein response (mitoUPR). While we decided to focus on the genes of the mitochondrial thioredoxin system for this paper, we identified multiple other antioxidant genes that are upregulated by the mitoUPR in the long-lived mitochondrial mutants including sod-3, prdx-3, gpx-6, gpx-7, gpx-8 and glrx-5. In exploring the role of the mitochondrial thioredoxin system in the long-lived mitochondrial mutants, nuo-6 and isp-1, we found that disruption of either trx-2 or trxr-2 significantly decreases their long lifespan, but has no effect on wild-type lifespan, indicating that the mitochondrial thioredoxin system is specifically required for their longevity. In contrast, disruption of the cytoplasmic thioredoxin gene trx-1 decreases lifespan in nuo-6, isp-1 and wild-type worms, indicating a non-specific detrimental effect on longevity. Disruption of trx-2 or trxr-2 also decreases the enhanced resistance to stress in nuo-6 and isp-1 worms, indicating a role for the mitochondrial thioredoxin system in protecting against exogenous stressors. Overall, this work demonstrates an important role for the mitochondrial thioredoxin system in both stress resistance and lifespan resulting from mild impairment of mitochondrial function.

A role for Rad5 in ribonucleoside monophosphate (rNMP) tolerance.

Ribonucleoside monophosphate (rNMP) incorporation in genomic DNA poses a significant threat to genomic integrity. In addition to repair, DNA damage tolerance mechanisms ensure replication progression upon encountering unrepaired lesions. One player in the tolerance mechanism is Rad5, which is an E3 ubiquitin ligase and helicase. Here, we report a new role for yeast Rad5 in tolerating rNMP incorporation, in the absence of the bona fide ribonucleotide excision repair pathway via RNase H2. This role of Rad5 is further highlighted after replication stress induced by hydroxyurea or by increasing rNMP genomic burden using a mutant DNA polymerase (Pol ε - Pol2-M644G). We further demonstrate the importance of the ATPase and ubiquitin ligase domains of Rad5 in rNMP tolerance. These findings suggest a similar role for the human Rad5 homologues helicase-like transcription factor (HLTF) and SNF2 Histone Linker PHD RING Helicase (SHPRH) in rNMP tolerance, which may impact the response of cancer cells to replication stress-inducing therapeutics.

Human-stimulated oocyte extract induces genetic and mitochondrial reprogramming of mesenchymal stromal cells.

SUMMARY: Reprogramming autologous adult cells to pluripotent cells allows for relatively safe cell replacement therapy. This can be achieved by nuclear transfer, cell fusion, or induced pluripotent stem cell technology However, the epigenetic memory of the cell is considered as a great challenge facing the complete reprograming of cells by these methods. Introducing oocyte-specific factors into differentiated cells may present a promising approach by mimicking cellular reprogramming during fertilization. METHODS: Human bone marrow mesenchymal stromal cells (hBM-MSCs) were cultured with different concentrations of human metaphase II (M II) oocyte extract (0.1, 1, 5, 10, 30 ng/µl). Reprogramming was assessed at various exposure times (1, 4, 7 days). Cells were tested for their proliferation rate, morphological changes, expression of pluripotency markers, expression of mesenchymal to epithelial transition markers, and mitochondrial rejuvenation. (mitochondrial localization, morphological changes, bioenergetics, transmembrane potential, and levels of reactive oxygen species, ROS). RESULTS: Treatment of human BM-MSCs with 10 ng/µl oocyte extract resulted in increased cell proliferation, which was associated with the upregulation of the pluripotency genes OCT-4, NANOG, and SOX-2 and a concomitant downregulation of mesenchymal-specific genes. MSCs exhibited small, immature round mitochondria with few swollen cristae localized proximal to the cell nucleus. This was accompanied by morphological cell changes, a metabolic shift towards oxidative phosphorylation, a high mitochondrial membrane potential, and increased ROS production. CONCLUSION: These data show that treatment with 10 ng/µl human MII-phase oocyte extract induced genetic and mitochondrial reprogramming of human BM-MSCs to a more embryonic phenotype.

Role of human oocyte-enriched factors in somatic cell reprograming.

Cellular reprograming paves the way for creating functional patient-specific tissues to eliminate immune rejection responses by applying the same genetic profile. However, the epigenetic memory of a cell remains a challenge facing the current reprograming methods and does not allow transcription factors to bind properly. Because somatic cells can be reprogramed by transferring their nuclear contents into oocytes, introducing specific oocyte factors into differentiated cells is considered a promising approach for mimicking the reprograming process that occurs during fertilization. Mammalian metaphase II oocyte possesses a superior capacity to epigenetically reprogram somatic cell nuclei towards an embryonic stem cell-like state than the current factor-based reprograming approaches. This may be due to the presence of specific factors that are lacking in the current factor-based reprograming approaches. In this review, we focus on studies identifying human oocyte-enriched factors aiming to understand the molecular mechanisms mediating cellular reprograming. We describe the role of oocyte-enriched factors in metabolic switch, chromatin remodelling, and global epigenetic transformation. This is critical for improving the quality of resulting reprogramed cells, which is crucial for therapeutic applications.

Tissue regeneration: Impact of sleep on stem cell regenerative capacity.

The circadian rhythm orchestrates many cellular functions, such as cell division, cell migration, metabolism and numerous intracellular biological processes. The physiological changes during sleep are believed to promote a suitable microenvironment for stem cells to proliferate, migrate and differentiate. These effects are mediated either directly by circadian clock genes or indirectly via hormones and cytokines. Hormones, such as melatonin and cortisol, are secreted in response to neural optic signals and act in harmony to regulate many biological functions during sleep. Herein, we correlate the effects of the main circadian genes on the expression of certain stem cell genes responsible for the regeneration of different tissues, including bone, cartilage, skin, and intestine. We also review the effects of different hormones and cytokines on stem cell activation or suppression and their relationship to the day/night cycle. The correlation of circadian rhythm with tissue regeneration could have implications in understanding the biology of sleep and tissue regeneration and in enhancing the efficacy and timing of surgical procedures.

Recent advances in stem cells therapy: A focus on cancer, Parkinson's and Alzheimer's.

Stem cells serve as potential therapeutics due to their high proliferative capacity, low immunogenic reactivity and their differentiating capabilities. Several pre-clinical and early-stage clinical studies are carried out to treat genetic diseases, cancers and neurodegenerative disorders with promising preliminary results. However, there are still many challenges that scientists are trying to overcome such as the unclear expression profile of stem cells in vivo, the homing of stem cells to the site of injury and their potential immune-reactivity. Prospective research lies in gene editing of autologous stem cells in vitro and safe injection of these modified cells back into patients. Here, we review the clinical trials executed using stem cell therapy in an attempt to cure challenging diseases like cancer, Parkinson's and Alzheimer's diseases.

Corrigendum to "Resolving Contributions of Oxygen-Consuming and ROS-Generating Enzymes at the Synapse".

[This corrects the article DOI: 10.1155/2016/1089364.].