Multitargeted Molecular Docking and Dynamic Simulation Studies of Bioactive Compounds from Rosmarinus officinalis against Alzheimer's Disease.

Alzheimer's disease (AD) has been associated with the hallmark features of cholinergic dysfunction, amyloid beta (Aß) aggregation and impaired synaptic transmission, which makes the associated proteins, such as ß-site amyloid precursor protein cleaving enzyme 1 (BACE I), acetylcholine esterase (AChE) and synapsin I, II and III, major targets for therapeutic intervention. The present study investigated the therapeutic potential of three major phytochemicals of Rosmarinus officinalis, ursolic acid (UA), rosmarinic acid (RA) and carnosic acid (CA), based on their binding affinity with AD-associated proteins. Detailed docking studies were conducted using AutoDock vina followed by molecular dynamic (MD) simulations using Amber 20. The docking analysis of the selected molecules showed the binding energies of their interaction with the target proteins, while MD simulations comprising root mean square deviation (RMSD), root mean square fluctuation (RMSF) and molecular mechanics/generalized born surface area (MM/GBSA) binding free energy calculations were carried out to check the stability of bound complexes. The drug likeness and the pharmacokinetic properties of the selected molecules were also checked through the Lipinski filter and ADMETSAR analysis. All these bioactive compounds demonstrated strong binding affinity with AChE, BACE1 and synapsin I, II and III. The results showed UA and RA to be potential inhibitors of AChE and BACE1, exhibiting binding energies comparable to those of donepezil, used as a positive control. The drug likeness and pharmacokinetic properties of these compounds also demonstrated drug-like characteristics, indicating the need for further in vitro and in vivo investigations to ascertain their therapeutic potential for AD.

Subpial delivery of adeno-associated virus 9-synapsin-caveolin-1 (AAV9-SynCav1) preserves motor neuron and neuromuscular junction morphology, motor function, delays disease onset, and extends survival in hSOD1G93A mice.

Elevating neuroprotective proteins using adeno-associated virus (AAV)-mediated gene delivery shows great promise in combating devastating neurodegenerative diseases. Amyotrophic lateral sclerosis (ALS) is one such disease resulting from loss of upper and lower motor neurons (MNs) with 90-95% of cases sporadic (SALS) in nature. Due to the unknown etiology of SALS, interventions that afford neuronal protection and preservation are urgently needed. Caveolin-1 (Cav-1), a membrane/lipid rafts (MLRs) scaffolding and neuroprotective protein, and MLR-associated signaling components are decreased in degenerating neurons in postmortem human brains. We previously showed that, when crossing our SynCav1 transgenic mouse (TG) with the mutant human superoxide dismutase 1 (hSOD1G93A) mouse model of ALS, the double transgenic mouse (SynCav1 TG/hSOD1G93A) exhibited better motor function and longer survival. The objective of the current study was to test whether neuron-targeted Cav-1 upregulation in the spinal cord using AAV9-SynCav1 could improve motor function and extend longevity in mutant humanized mouse and rat (hSOD1G93A) models of familial (F)ALS. Methods: Motor function was assessed by voluntary running wheel (RW) in mice and forelimb grip strength (GS) and motor evoked potentials (MEP) in rats. Immunofluorescence (IF) microscopy for choline acetyltransferase (ChAT) was used to assess MN morphology. Neuromuscular junctions (NMJs) were measured by bungarotoxin-a (Btx-a) and synaptophysin IF. Body weight (BW) was measured weekly, and the survival curve was determined by Kaplan-Meier analysis. Results: Following subpial gene delivery to the lumbar spinal cord, male and female hSOD1G93A mice treated with SynCav1 exhibited delayed disease onset, greater running-wheel performance, preserved spinal alpha-motor neuron morphology and NMJ integrity, and 10% increased longevity, independent of affecting expression of the mutant hSOD1G93A protein. Cervical subpial SynCav1 delivery to hSOD1G93A rats preserved forelimb GS and MEPs in the brachial and gastrocnemius muscles. Conclusion: In summary, subpial delivery of SynCav1 protects and preserves spinal motor neurons, and extends longevity in a familial mouse model of ALS without reducing the toxic monogenic component. Furthermore, subpial SynCav1 delivery preserved neuromuscular function in a rat model of FALS. The latter findings strongly indicate the therapeutic applicability of SynCav1 to treat ALS attributed to monogenic (FALS) and potentially in sporadic cases (i.e., SALS).

Resveratrol ameliorates learning and memory impairments induced by bilateral hippocampal injection of streptozotocin in mice.

Resveratrol (RES) is a polyphenol with diverse beneficial pharmacological activities, and our previous results have demonstrated its neuroprotective potential. The purpose of this study was to investigate the therapeutic effect of RES in Alzheimer's disease (AD)-like behavioral dysfunction induced by streptozotocin (STZ) and explore it's potential mechanism of action. STZ was microinjected bilaterally into the dorsal hippocampus of C57BL/6J mice at a dose of 3 mg/kg, and RES was administered intragastrically at a dose of 25 mg/kg for 5 weeks. Neurobehavioral performance was observed, and serum concentrations of insulin and Nesfatin-1 were measured. Moreover, the protein expression of amyloid beta 1-42 (Aß1-42), Tau, phosphorylated Tau (p-Tau) (Ser396), synaptic ras GTPase activation protein (SynGAP), postsynaptic density protein 95 (PSD95), synapsin-1, synaptogomin-1, and key molecules of the Wnt/ß-catenin signaling pathway in the hippocampus and prefrontal cortex (PFC) were assessed. Finally, pathological damage to hippocampal tissue was examined by Nissl and immunofluorescence staining. The results showed that compared with the controls, bilateral hippocampal microinjections of STZ induced task-specific learning and memory impairments, as indicated by the disadvantaged performances in the novel object recognition test (NOR) and Morris water maze (MWM), but not the contextual fear conditioning test (CFC). Treatment with RES could improve these behavioral disadvantages. The serum concentrations of insulin and Nesfatin-1 in the model group were remarkably higher than those of the control group. In addition, protein expression of Aß1-42, Tau, and p-Tau (Ser396) was increased but expression of SynGAP, PSD95, brain-derived neurotrophic factor (BDNF), and p-GSK-3ß/GSK-3ß were decreased in the hippocampus. Although the protein expression of BDNF and SynGAP was also markedly decreased in the PFC of the model mice, there was no significant difference among groups in the protein expression of PSD95, BDNF, synapsin-1, synaptogomin-1, and p-GSK-3ß/GSK-3ß. RES (25 mg/kg) reversed the enhanced insulin level, the abnormal protein expression of Aß1-42, Tau, and p-Tau (Ser396) in the hippocampus and PFC, and the hippocampal protein expression of SynGAP, PSD95 and BDNF. In addition, RES reversed the STZ-induced decrease in the number of Nissl bodies and the increase in fluorescence intensity of IBA1 in the hippocampal CA1 region. These findings indicate that RES could ameliorate STZ-induced AD-like neuropathological injuries, the mechanism of which could be partly related to its regulation of BDNF expression and synaptic plasticity-associated proteins in the hippocampus.

Systemic AAV9 gene therapy using the synapsin I promoter rescues a mouse model of neuronopathic Gaucher disease but with limited cross-correction potential to astrocytes.

Gaucher disease is caused by mutations in the GBA gene, which encodes for the lysosomal enzyme ß-glucocerebrosidase (GCase), resulting in the accumulation of storage material in visceral organs and in some cases the brain of affected patients. While there is a commercially available treatment for the systemic manifestations, neuropathology still remains untreatable. We previously demonstrated that gene therapy represents a feasible therapeutic tool for the treatment of the neuronopathic forms of Gaucher disease (nGD). In order to further enhance the therapeutic affects to the central nervous system, we systemically delivered an adeno-associated virus (AAV) serotype 9 carrying the human GBA gene under control of a neuron-specific promoter to an nGD mouse model. Gene therapy increased the life span of treated animals, rescued the lethal neurodegeneration, normalized the locomotor behavioural defects and ameliorated the visceral pathology. Together, these results provided further indication of gene therapy as a possible effective treatment option for the neuropathic forms of Gaucher disease.

The hybrid between the ABC domains of synapsin and the B subunit of Escherichia coli heat-labile toxin ameliorates experimental autoimmune encephalomyelitis.

The B subunit of Escherichia coli heat-labile enterotoxin (LTB) acts as efficient mucosal carrier for conjugated antigens. We expressed two heterologous proteins using E. coli as a host: a hybrid consisting of LTB and the A, B and C domain of synapsin (LTBABC) and the separated ABC peptide of this synaptic protein. Refolded LTBABC and LTB bound to the GM1 receptor and internalized into CHO-K1(GM1+) cells. LTBABC showed enhanced solubility and cell binding ability respect to the former hybrid LTBSC. Several oral doses of LTBABC were administered to rats with experimental autoimmune encephalomyelitis (EAE) from induction to the acute stage of the disease. This treatment decreased disease severity, delayed type hypersensitivity reaction and lymph node cell proliferation stimulated by myelin basic protein. Amelioration of EAE was also associated with modulation of the Th1/Th2 cytokine ratio, increased TGF-ß secretion in mesenteric lymph nodes as well as expansion of CD4(+)CD25(+)Foxp3(+) regulatory T cell population. These results indicate that the fusion protein LTBABC is suitable for further exploration of its therapeutic effect on EAE development.

Expression of a bioactive fusion protein of Escherichia coli heat-labile toxin B subunit to a synapsin peptide.

The B subunit of Escherichia coli heat-labile toxin (LTB) may function as an efficient carrier molecule for the delivery of genetically coupled antigens across the mucosal barrier. We constructed vectors for the expression of LTB and LTBSC proteins. LTBSC is a fusion protein that comprises the amino acid sequence from the C-domain of rat synapsin fused to the C-terminal end of LTB. Both constructions have a coding sequence for a 6His-tag fused in-frame. LTBSC was expressed in E. coli as inclusion bodies. The inclusion bodies were isolated and purified by Ni2+-chelating affinity chromatography under denaturing condition. Purified LTBSC was diluted in several refolding buffers to gain a soluble and biologically active protein. Refolded LTBSC assembled as an active oligomer which binds to the GM1 receptor in an enzyme-linked immunosorbent assay (ELISA). Soluble LTB in the E. coli lysate was also purified by Ni2+-chelating affinity chromatography and the assembled pentamer was able to bind with high affinity to GM1 in vitro. LTBSC and LTB were fed to rats and the ability to induce antigen-specific tolerance was tested. LTBSC inhibited the specific delayed-type hypersensitivity (DTH) response and induced decreased antigen-specific in vivo and in vitro cell proliferation more efficiently than LTB. Thus, the novel hybrid molecule LTBSC when orally delivered was able to elicit a systemic immune response. These results suggest that LTBSC could be suitable for exploring further therapeutic treatment of autoimmune inflammatory diseases involving antigens from central nervous system.