Repurposing Visible-Light-Excited Ene-Reductases for Diastereo- and Enantioselective Lactones Synthesis.

Repurposing enzymes to catalyze non-natural asymmetric transformations that are difficult to achieve using traditional chemical methods is of significant importance. Although radical C-O bond formation has emerged as a powerful approach for constructing oxygen-containing compounds, controlling the stereochemistry poses a great challenge. Here we present the development of a dual bio-/photo-catalytic system comprising an ene-reductase and an organic dye for achieving stereoselective lactonizations. By integrating directed evolution and photoinduced single electron oxidation, we repurposed engineered ene-reductases to steer non-natural radical C-O formations (one C-O bond for hydrolactonizations and lactonization-alkylations while two C-O bonds for lactonization-oxygenations). This dual catalysis gave a new approach to a diverse array of enantioenhanced 5- and 6-membered lactones with vicinal stereocenters, part of which bears a quaternary stereocenter (up to 99 % enantiomeric excess, up to 12.9 : 1 diastereomeric ratio). Detailed mechanistic studies, including computational simulations, uncovered the synergistic effect of the enzyme and the externally added organophotoredox catalyst Rh6G.

Researches of calcium-activated chloride channel ANO1 intervening amyotrophic lateral sclerosis progression by activating EGFR and CaMKII signaling.

BACKGROUND: ANO1 is closely correlated with the activation of EGFR and CaMKII, while EGFR and CaMKII show low activation in amyotrophic lateral sclerosis (ALS) models. Therefore, we designed experiments to verify that ANO1 may play a protective role on motor neurons in ALS by activating EGFR and CaMKII. METHODS: The expression changes of ANO1, EGFR, CaMKII, pEGFR, and pCaMKII, cell survival status, and apoptosis were studied by western blot, real-time quantitative PCR, immunofluorescence, immunohistochemistry, CCK-8, and flow cytometry. The role of ANO1 in the ALS model by activating EGFR and CaMKII was studied by applying corresponding activators, inhibitors, gene silencing, and overexpression. RESULTS: In hSOD1G93A transgenic animals and cell lines, low expression of ANO1 and low activation of EGFR and CaMKII were identified. ANO1 expression decreased gradually with the progression of ALS. Overexpression of ANO1 in the hSOD1G93A cell line and primary neurons of hSOD1G93A transgenic mice increased cell viability and decreased cell apoptosis. After the application of ANO1 inhibitor CaCC-inhA01 in hSOD1G93A cell line and primary neurons of hSOD1G93A transgenic mice, EGFR activator EGF and CaMKII activator Carbachol, increased cell viability and reduced cell apoptosis. After ANO1 was overexpressed in the hSOD1G93A cell line and primary neurons of hSOD1G93A transgenic mice, EGFR inhibitor AEE788 and CaMKII inhibitor KN93 decreased cell viability and increased cell apoptosis. CONCLUSIONS: Our results suggest that ANO1 plays an important role in the survival of ALS motor neurons. ANO1 can increase cell activity and reduce apoptosis by activating EGFR and CaMKII signals.

Metal-Free Stereoconvergent C-H Borylation of Enamides.

Enamides, functional derivatives of enamines, play a significant role as synthetic targets. However, the stereoselective synthesis of these molecules has posed a longstanding challenge in organic chemistry, particularly for acyclic enamides that are less thermodynamically stable. In this study, we present a general strategy for constructing ß-borylenamides by C-H borylation, which provides a versatile platform for generating the stereodefined enamides. Our approach involves the utilization of metalloid borenium cation, generated through the reaction of BBr3 and enamides in the presence of two different additives, avoiding any exogenous catalyst. Importantly, the stereoconvergent nature of this methodology allows for the use of starting materials with mixed E/Z configurations, thus highlighting the unique advantage of this chemistry. Mechanistic investigations have shed light on the pivotal roles played by the two additives, the reactive boron species, and the phenomenon of stereoconvergence.

PIII -Directed Late-Stage Ligation and Macrocyclization of Peptides with Olefins by Rhodium Catalysis.

Transition metal-catalyzed C-H activation is a step-economical strategy for peptide functionalization. Herein, we report the method of late-stage peptide ligation and macrocyclization through rhodium-catalyzed alkylation of tryptophan residues at the C7 position. This method utilizes a N-Pt Bu2 directing group and tolerates various peptide and alkene substrates. Utilizing internal olefins, this study represents the first example of site-selective peptide C-H alkylation through deconjugative isomerization. Furthermore, our method provides access to peptide macrocycles with unique Trp(C7)-alkyl crosslinks and potent cytotoxicity towards cancer cells.

Decreased Netrin-1 in Mild Cognitive Impairment and Alzheimer's Disease Patients.

BACKGROUND AND OBJECTIVE: Inflammatory mediators are closely associated with the pathogenesis of Alzheimer's disease (AD) and mild cognitive impairment (MCI). Netrin-1 is an axon guidance protein and despite its capacity to function as a neuroimmune guidance signal, its role in AD or MCI is poorly understood. In addition, the association among netrin-1, cognitive impairment and serum inflammatory cytokines such as interleukin-17 (IL-17) and tumor necrosis (TNF-α) remains unclear. The aim of this study was to determine serum levels of IL-17, TNF-α and netrin-1in a cohort of AD and MCI patients, and to study the relationship between these cytokines and cognitive status, as well as to assess the possible relationships between netrin-1 levels and inflammatory molecules. METHODS: Serum concentrations of netrin-1, TNF-α and IL-17 were determined in 20 AD patients, 22 MCI patients and 22 healthy controls using an enzyme-linked immunosorbent assay (ELISA). In addition, neuropsychological evaluations and psychometric assessments were performed in all subjects. RESULTS: Serum netrin-1 levels were decreased in AD and MCI patients and were positively correlated with Mini Mental State Examination (MMSE) scores. In contrast, serum TNF-α and IL-17 levels were elevated in AD and MCI cohorts and negatively correlated with MMSE scores. Serum netrin-1 levels were inversely related with TNF-α and IL-17 levels in AD, but not MCI, patients. CONCLUSION: Based on the findings reported here, netrin-1 may serve as a marker for the early recognition of dementia and predict cognitive impairment.

Wild-type p53-induced phosphatase 1 down-regulation promotes apoptosis by activating the DNA damage-response pathway in amyotrophic lateral sclerosis.

Accumulation of DNA damage has been detected in the spinal cord of patients as well as in the G93A mouse model of amyotrophic lateral sclerosis (ALS). Wild-type p53-induced phosphatase 1 (Wip1) is a p53-inducible serine/threonine phosphatase that terminates DNA-damage responses via dephosphorylation of DNA-damage response proteins, namely ataxia-telangiectasia mutated (ATM) kinase, checkpoint kinase 2, and p53, thus enhancing cell proliferation. However, the role of Wip1, DNA-damage responses, and their interaction in ALS development remains to be elucidated. Here, we showed that Wip1 expression levels were substantially decreased in ALS motor neurons compared with wild-type controls both in vivo and in vitro. The DNA-damage response was activated in superoxide dismutase 1 (SOD1) G93A-transfected cells. However, increased expression of Wip1 improved cell viability and inhibited the DNA-damage response in mutated SOD1G93A cells. Further studies demonstrated that decreased Wip1 expression reduced cell viability and further activated the DNA-damage response in chronic H2O2-treated NSC34 cells. In contrast, Wip1 promoted cell survival and suppressed DNA damage-induced apoptosis during persistent DNA damage conditions. Over-expression of Wip1 in the central nervous system (CNS) can delay the onset of disease symptoms, extended the survival, decreased MN loss improved motor function and inhibit the DNA-damage response in SOD1 G93A mice. Furthermore, homeodomain-interacting protein kinase 2 (HIPK2) promoted the degradation of Wip1 via the ubiquitin-proteasome system during chronic stress. These findings indicate that persistent accumulation of DNA damage and subsequent chronic activation of the downstream DNA damage-response ATM and p53 pro-apoptotic signaling pathways may trigger neuronal dysfunction and neuronal death in ALS. Wip1 may play a protective role by targeting the DNA-damage response in ALS motor neurons. Importantly, these findings provide a novel direction for therapeutic options for patients with ALS.

Lithium facilitates removal of misfolded proteins and attenuated faulty interaction between mutant SOD1 and p-CREB (Ser133) through enhanced autophagy in mutant hSOD1G93A transfected neuronal cell lines.

Abnormally protein aggregation and deposition are key pathological features of ALS, which may related with dysfunctional cellular autophagy. In the current study, we found that, compared with wtSOD1 cells, serum starvation treatment resulted in significant higher percentage of apoptosis in mutSOD1 cells; Lithium treatment exerted protection for those mutSOD1 cells, with decreased GFP-tagged mutant SOD1 protein aggregates deposition; Whereas, pre-treatment with Baf or 3-MA (autophagy inhibitors) blocked protection of lithium for mutant SOD1 cells, and induced increased GFP-tagged mutant SOD1 protein aggregation. Further, Western blots results showed that lithium treatment led to decrease of mutant hSOD1 protein levels in both Triton X-100 soluble and Triton X-100 insoluble fraction of mutSOD1 cells. Besides, improper binding of mutant SOD1 proteins' aggregates with p-CREB (Ser133) (transcription factor) in mutSOD1 cells were demonstrated; whereas lithium treatment attenuated this fault interaction. In conclusion, our results showed that, in mutSOD1 cells, mutSOD1 protein aggregates were related with abnormal autophagic regulation. Lithium treatment could induce autophagy and enhance clearance of protein aggregates, further exerting protection on mutSOD1 cells. More importantly, we uncovered another distinct pathological role of mutSOD1 protein aggregates, that is abnormal binding with p-CREB (Ser133), an important transcription factor, which may play crucial role in the PI3K-Akt-CREB-AEG-1 signaling pathway.

γ-Oryzanol mitigates oxidative stress and prevents mutant SOD1-Related neurotoxicity in Drosophila and cell models of amyotrophic lateral sclerosis.

Oxidative stress plays a critical role in mutant copper/zinc superoxide dismutase 1 (SOD1)-linked amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease characterized by selective loss of motor neurons. Thus, an anti-oxidative stress remedy might be a promising means for the treatment of ALS. The aim of the present study is to investigate the neuroprotective effects of γ-oryzanol (Orz) and elucidate its relevant molecular mechanisms in mutant hSOD1-linked Drosophila and cell models of ALS. Orz treatment provided neuroprotection in flies with expression of hSOD1-G85R in motor neurons, as demonstrated by the prolonged survival, improvement of motor deficits, reduced oxidative damage and regulated redox homeostasis when compared with those in controls. Moreover, Orz significantly decreased neuronal apoptosis and upregulated the nuclear factor erythroid 2-related factor 2 (Nrf2)/glutamate-cysteine ligase catalytic subunit (GCLC) antioxidant pathway via activating Akt in hSOD1-G93A-expressing NSC-34 cells. In addition, our results showed that both in vivo and in vitro, Akt served as an upstream regulator of signal transducers and activators of transcription (Stat) 3 stimulated by Orz, which further increased the level of another anti-oxidative stress factor heat-shock protein 70 (HSP70). Altogether, these findings provide evidence that Orz has potential neuroprotective effects that may be beneficial in the treatment of ALS disease with SOD1 mutations.

Decreased Netrin-1 and Correlated Th17/Tregs Balance Disorder in Aß1-42 Induced Alzheimer's Disease Model Rats.

There is increasing evidence indicating that inflammation represents a key pathological component of Alzheimer's disease (AD). A possible factor that may contribute to this process is netrin-1, a neuronal guidance molecule. This molecule has been shown to exert an unexpected immunomodulatory function. However, the potential changes and correlations of netrin-1 with T helper 17/regulatory T cells (Th17/Tregs) as related to inflammation in AD has yet to be examined. In this study, netrin-1 and Th17/Tregs balance were investigated, and the relationship among netrin-1, Th17/Tregs and cognitive function were analyzed in a rat model of AD. In this model, a bilateral intracerebroventricular administration of Amyloid ß1-42 (Aß1-42) was used to produce spatial learning and memory deficits, as well as increased neuronal apoptosis, which were detected 7 days after injection for AD7d group and 14 days for AD14d group. Netrin-1 concentrations were significantly down regulated in both serum and cerebrospinal fluid (CSF) of these AD rats, effects which were strongly correlated with cognitive deficits. Increased levels of interleukin (IL)-17 and deceased IL-10 were observed in both the circulation and CSF and were also correlated with the percent of time spent in the target quadrant of AD in these rats. These changes resulted in netrin-1 concentrations being negatively correlated with IL-17 but positively correlated with IL-10 concentrations in the serum and CSF. We also found that the Th17/Tregs balance was disrupted in these AD rats. Collectively, these findings reveal that the reduction in netrin-1 and the correlated disruption of Th17/Tregs balance in AD rats may diminish the immunosuppressive effect of netrin-1 on Th17/Tregs in AD pathogenesis.

Neuroprotection by urate on the mutant hSOD1-related cellular and Drosophila models of amyotrophic lateral sclerosis: Implication for GSH synthesis via activating Akt/GSK3ß/Nrf2/GCLC pathways.

Oxidative stress has been considered as a principal mechanism of motor neuron death in amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease which could be caused by dominant mutations in an antioxidant enzyme superoxide dismutase-1 (SOD1). The aim of the present study was to investigate the potential neuroprotective effects and mechanisms of urate, an important endogenous antioxidant and a biomarker of favorable ALS progression rates, in the mutant human SOD1-related cellular and Drosophila models of ALS. Our results showed that urate treatment provided neuroprotective effects as confirmed by enhanced survival, attenuated motor impairments, reduced oxidative damage and increased antioxidant defense in hSOD1-G85R-expressing Drosophila models of ALS. In vitro studies, we demonstrated that urate protected motor neurons (NSC-34 cells) against hSOD1-G93A-induced cell damage and apoptosis by decreasing reactive oxygen specials (ROS) production and oxidative damage. Moreover, urate markedly increased the expression and activation of nuclear factor erythroid 2-related factor 2 (Nrf2), stimulated Nrf2-targeted antioxidant gene glutathione cysteine ligase catalytic subunit (GCLC) expression and glutathione (GSH) synthesis by upregulating Akt/GSK3ß pathway. Furthermore, the inhibition of Akt pathway with LY294002 abolished urate-mediated elevation of GSH synthesis and neuroprotective effects both in vivo and in vitro. Overall, these results suggested that, in addition to its direct scavenging of ROS, urate markedly enhanced GSH expression by activating Akt/GSK3ß/Nrf2/GCLC pathway, and thus offering neuroprotective effects on motor neurons against oxidative stress.

Spy1, a unique cell cycle regulator, alters viability in ALS motor neurons and cell lines in response to mutant SOD1-induced DNA damage.

Increasing evidence indicates that DNA damage and p53 activation play major roles in the pathological process of motor neuron death in amyotrophic lateral sclerosis (ALS). Human SpeedyA1 (Spy1), a member of the Speedy/Ringo family, enhances cell proliferation and promotes tumorigenesis. Further studies have demonstrated that Spy1 promotes cell survival and inhibits DNA damage-induced apoptosis. We showed that the Spy1 expression levels were substantially decreased in ALS motor neurons compared with wild-type controls both in vivo and in vitro by qRT-PCR, western blotting, and Immunoassay tests. In addition, we established that over-expression of human SOD1 mutant G93A led to a decreased expression of Spy1. Furthermore, DNA damage response was activated in SOD1G93A-transfected cells (mSOD1 cells). Moreover, decreased Spy1 expression reduced cell viability and further activated the DNA damage response in mSOD1 cells. In contrast, increased Spy1 expression improved cell viability and inhibited the DNA damage response in mSOD1 cells. These results suggest that Spy1 plays a protective role in ALS motor neurons. Importantly, these findings provide a novel direction for therapeutic options for patients with ALS as well as for trial designs, such as investigating the role of oncogenic proteins in ALS.

α-Lipoic acid attenuates oxidative stress and neurotoxicity via the ERK/Akt-dependent pathway in the mutant hSOD1 related Drosophila model and the NSC34 cell line of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a degenerative disease with a progressive loss of motor neurons in the central nervous system (CNS). However, there are unsolved problems with the therapies for this disease. α-Lipoic acid (LA) is a natural, universal antioxidant capable of scavenging hydroxyl radicals as well as regenerating a series of antioxidant enzymes that has been widely used in clinical settings. This study aimed to evaluate the antioxidant and neuroprotective effects of LA in ALS cell and Drosophila models with mutant G85R and G93A hSOD1 genes. The biological effects of LA and the protein levels of several antioxidant factors were examined, as were those of phospho-Akt and phospho-ERK. Furthermore, specific inhibitors of the PI3K/Akt and MEK/ERK signaling pathways were used to analyze their effects on LA-induced antioxidant expression in vivo and in vitro. Evidences showed that the mutant hSOD1 resulted in the increased oxidative stress, abnormal antioxidant signaling and pathological behaviors in motor performance and survival compared with non-mutant hSOD1 models, treatment with LA improved motor activity and survival in transgenic flies, prevented NSC34 cells from mutant hSOD1 or H2O2 induced decreased antioxidant enzymes as well as increased ROS levels. In addition, LA regulated the expression levels of antioxidant proteins in a dose- and periodical time-dependent manner, which might be mediated by ERK/Akt pathway activation and independent from the mutant hSOD1 gene. Our observations suggest that LA exerts strong and positive antioxidant and neuroprotective effects through the activation of the ERK-Akt pathway in hSOD1 ALS models.

Astrocyte elevated gene-1 is a novel regulator of astrogliosis and excitatory amino acid transporter-2 via interplaying with nuclear factor-κB signaling in astrocytes from amyotrophic lateral sclerosis mouse model with hSOD1G93A mutation.

AEG-1 has received extensive attention on cancer research. However, little is known about its roles in astrogliosis of Amyotrophic lateral sclerosis (ALS). In this study, we detected AEG-1 expression in hSOD1G93A-positive (mut-SOD1) astrocytes and wild type (wt-SOD1) astrocytes, and intend to elucidate its potential functions in ALS related astrogliosis and the always accompanied dysregulated glutamate clearance. Results showed elevated protein and mRNA levels of AEG-1 in mut-SOD1 astrocytes; Also, NF-κB signaling pathway related proteins and inflammatory cytokines were upregulated in mut-SOD1 astrocytes; AEG-1 knockdown attenuated astrocytes proliferation and pro-inflammatory release; also we found that AEG-1 silence inhibited translocation of p65 from cytoplasma to nuclear, which was associated with inhibited NF-κB signaling. Besides, excitatory amino acid transporter-2 (EAAT2) expression levels were significantly decreased, accompanied by impaired glutamate clearance ability, in mut-SOD1 astrocytes; yin yang 1 (YY1), a transcriptional inhibitor for EAAT2, increased in nucleus of mut-SOD1 astrocytes. AEG-1 silence inhibited translocation of YY1 to nucleus, increased EAAT2 expression levels, and enhanced astrocytic ability of glutamate clearance, ultimately exerted the neuronal protection. Findings from this study implicate potential function of AEG-1 in mut-SOD1 related astrogliosis and the accompanied excitatory cytotoxic mechanism in ALS.

Downregulation of Homer1b/c in SOD1 G93A Models of ALS: A Novel Mechanism of Neuroprotective Effect of Lithium and Valproic Acid.

BACKGROUND: Mutations in the Cu/Zn superoxide dismutase (SOD1) gene have been linked to amyotrophic lateral sclerosis (ALS). However, the molecular mechanisms have not been elucidated yet. Homer family protein Homer1b/c is expressed widely in the central nervous system and plays important roles in neurological diseases. In this study, we explored whether Homer1b/c was involved in SOD1 mutation-linked ALS. RESULTS: In vitro studies showed that the SOD1 G93A mutation induced an increase of Homer1b/c expression at both the mRNA and protein levels in NSC34 cells. Knockdown of Homer1b/c expression using its short interfering RNA (siRNA) (si-Homer1) protected SOD1 G93A NSC34 cells from apoptosis. The expressions of Homer1b/c and apoptosis-related protein Bax were also suppressed, while Bcl-2 was increased by lithium and valproic acid (VPA) in SOD1 G93A NSC34 cells. In vivo, both the mRNA and protein levels of Homer1b/c were increased significantly in the lumbar spinal cord in SOD1 G93A transgenic mice compared with wild type (WT) mice. Moreover, lithium and VPA treatment suppressed the expression of Homer1b/c in SOD1 G93A mice. CONCLUSION: The suppression of SOD1 G93A mutation-induced Homer1b/c upregulation protected ALS against neuronal apoptosis, which is a novel mechanism of the neuroprotective effect of lithium and VPA. This study provides new insights into pathogenesis and treatment of ALS.