Effect Of New Compound FLZ On Neurodegenerative Diseases (AD And PD) And Its Mechanism

Neurodegenerative disease is characterized as an age-related neuronal degeneration which induces individual function disorder, including Alzheimer's disease (AD) and Parkinson's disease (PD). While the specific clinical presentation varies with such diseases, the common feature of the neurodegenerative disease is the progressive accumulation of misfolded proteins with the formation of toxic oligomers and neural cell death. To date both of AD and PD are incurable. Although major advances in the understanding of the mechanism of the disease have been made effective treatment for them remains one of the most challenges in medicine today.Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by accumulation ofβ-amyloid (Aβ) and neurofibrillary tangles, progressive neuron loss, and cognitive deficits. The possible etiology of AD remains unclear. AD is associated with the interactions of APP processing abnormality, tau hyperphosphorylation, neural apoptosis, oxidative stress, inflammation, cholinergic dysfunction, loss of neurotrophins and neuroplasticity failure.PD is a progressive neurodegenerative disease which is characterized by movement disorder accompanied by tremor, rigidity and so on. Loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNC) and appearance of Lewy Bodies (LBs) are two main pathologic hallmarks of PD. Several mechanisms have been implicated as crucial to PD pathogenesis:oxidative stress, mitochondrial dysfunction, protein aggregation and misfolding, inflammation, excitotoxicity, apoptosis and loss of trophic support. These pathogenic mechanisms appear to act synergistically through complex interactions to promote neurodegeneration.FLZ is a synthetic cyclic derivative of squamosamide from annona glabra. Our previous studies have demonstrated that compound FLZ has strong neuroprotective activity and improves the behavior deficits in experimental dementia and PD mouse models. In vitro assay, FLZ markedly reduced damage and apoptosis of primary cultured neurons and SH-SY5Y cells exposed to several kinds of toxins. FLZ also has anti-inflammation activity in vitro and in vivo. In this thesis, the neuroprotective activity and its possible mechanism of FLZ were further studied on APP695 (Wt and Part 1 The inhibitory effect of FLZ on Aβoverproduction and tau hyperphosphorylation in APP695 (Wt and Swe) transfected SH-SY5Y cells and its possible mechanismThe aim of this part was to study whether compound FLZ can attenuate Aβproduction and tau hyperphosphorylation The cells were incubated with nontoxic concentrations of FLZ (0.1,1,10μM) for 24h. The results showed that (1) FLZ reduced:the level of Aβ, tau hyperphosphorylation at the sites of Ser202, Ser396 and Thr231,β-secretase (BACE1) expression, total APP and APP phosphorylation at the site of Thr668 in APP695 (Wt and Swe) transfected SH-SY5Y cells. Meanwhile, FLZ increased Akt activity by increasing the levels of phosphorylation at Ser473 and decreased GSK3βand Cdk5 activites by increasing the level of phosphorylation GSK3βat Ser9 and decreasing the p25/p35 expression in APP695 (Wt and Swe) transfected SH-SY5Y cells. The results suggest that FLZ not only reduces Aβlevel via inhibition of P-secretase and APP phosphorylation, but also attenuates tau hyperphosphoylation through mediating AKT/GSK3βpathway and Cdk5 activity in APP695 transfected SH-SY5Y cells.Part 2 Improvement of FLZ treatment on the Alzheimer-like behavior deficit and neuropathology in APP/PS-1 (PAP) mouse model and its possible mechanismThe present study was to further assess the effect of FLZ in cognitive deficit and neuropathology in the amyloid precursor protein (APP)/presenilin-1 (PS-1) double transgenic mice. The mice were orally treated with FLZ 150mg/kg or Donepezil 5 mg/kg once daily for 20 weeks. The cognitive deficit in PAP mice was tested with Passage Water Maze (PWM). The neuropathology in the hippocampus and/or frontal cortex of PAP mice were detected by immunohistochemical and western blotting assay. As a result, PAP mice showed longer escape tendency and more error number compared with WT mice in the PWM test. Chronic treatment of FLZ 150mg/kg daily for 20 weeks significantly improved the cognitive deficit in PAP mice without affecting the spontaneously motor activity of the mice. While chronic treatment with Donepezil 5 mg/kg had no significant improvement effect on behavior deficit of PAP mice. The results of pathology showed that (1) Aβaccumulation and tau hyperphosphorylation were found in hippocampus and cortex of PAP mice at 10 months of age. Meanwhile, APP, pAPP (Thr668), BACE1, PS1, IDE which are related to A(3 overproduction or clearance showed alternations in the hippocampus of PAP mice. The activity of GSK3β, the most important kinase phosphorylates APP and tau, increased. Chronic treatment of FLZ 150mg/kg decreased both the deposits and the soluble Aβlevel in the hippocampus and cortex of PAP mice and the level of tau hyperphosphorylated at Ser396 were also reduced. Furthermore, the treatment of FLZ 150mg/kg decreased the level of the CTF fragment of PS1, and inhibited the activity of GSK3βby increasing the expression of pGSK3β(Ser9). FLZ also showed a tendency to reduce the level of BACE1 and elevate the expression of IDE. (2) Chronic treatment of FLZ 150mg/kg significantly increased Bcl-2 expression and decreased the levels of Bax and active Caspase-3 fragments simultaneously. The results suggested that FLZ has anti-apoptotic activity in PAP mice. (3) The levels of neurotrophins such as NGF, BDNF and NT-3 decreased in hippocampus of PAP mice. Chronic treated with FLZ 150mg/kg increased the levels of BDNF and NT-3 significantly. Since both BDNF and NT3 are bind to TrkB receptor, the FLZ treatment was showed to promote the TrkB receptor autophosphorylation process, and activate the downstream ERK pathway, thereby exerts its neurotrophic action on neurons. (4) The immunohistochemical staining showed that chronic treatment with FLZ 150mg/kg increased the population of ChAT-positive neurons in the frontal cortex of PAP mice, whereas FLZ had no effect on the population of AChE-positive neurons. The results suggested that FLZ might enhance the synthesis of acetylcholine in brain. All the results suggested that FLZ has effects on several links of AD pathogenesis, and has the potential to be developed as a drug for treatment of AD.Part 3 Improvement of FLZ treatment on the motor deficit and neuropathology in A53T transgenic mouse model and its possible mechanismIn this study, A53T mutant human a-synuclein (SYN) transgenic mice (M83) at 9 months of age, which developed the motor deficit and a-synuclein neuropathology, were treated with FLZ 150 mg/kg daily for 14 weeks. The motor ability was tested with rod climbing and footprint tasks. The results showed that chronic treatment of M83 mice with FLZ 150mg/kg improved the motor deficit of the mice significantly. Immunohistochemical and western blot analysis showed that human SYN and total SYN in the midbrain of FLZ-treated M83 group decreased as compared with the M83 control group. Compared with WT mice the levels of BDNF and GDNF in the SNC of M83 mice markedly decreased. Chronic treatment with FLZ 150mg/kg increased both of the two neurotrophins levels in SNC of M83 mice, and also enhanced the TrkB receptor autophosphorylation, and activated of the downstream pathway, thereby facilitated the action of BDNF and GDNF on neuroplasticity. The results showed that the improvement of FLZ on motor deficit of M83 mice might partly attribute to its inhibition of SYN accumulation and increase of neurotrophins BDNF and GDNF.ConclusionsThe effects of FLZ may be summarized as follows:1. FLZ significantly improves cognitive deficit and neuropathology of double transgenic APP/PS-1 mice as indicated in:(1), FLZ not only reduced Aβlevel via inhibition ofβ-secretase and APP phosphorylation, but also attenuated tau hyperphosphoylation through mediating Akt/GSK3βpathway and CDK5 activity in APP695 transfected SH-SY5Y cells;(2), FLZ improved the cognitive deficit of PAP mice.(3), FLZ also decreased Aβaccumulation and reduced tau hyperphosphorylation in the brains of PAP mice and it might via the inhibition of PS1 activity and GSK3βactivity;(4), FLZ had neuroprotective effect by modulating apoptosis-related protein in the hippocampus of PAP mice.2. FLZ also significantly improved impairment of behavior and inhibited accumulation of a-synuclein in midbrain of A53T transgenic mice.3. FLZ markedly increases neurotrophins BNDF, NT3 and GDNF levels, enhanced TrkB receptor autophosphorylation, actived downstream ERK pathway, and facilite the action of neurotrophins on neural pasticity in transgenic AD and PD mice.All these results suggest that FLZ is a potential candidate drug for AD and PD treatment.