Dissertations, Theses, and Capstone Projects

Date of Degree

9-2024

Document Type

Dissertation

Degree Name

Ph.D.

Program

Biochemistry

Advisor

Chang-Hui Shen

Committee Members

Greg Phillips

Qia-Sheng Hu

Vasanthan Nadarajah

Fei Liu

Subject Categories

Biochemistry | Biochemistry, Biophysics, and Structural Biology

Abstract

The neurodegenerative disorder is an umbrella term for a range of conditions that affects the neurons in the human brain causing neuronal cell death. The common pathway is the excitotoxicity, a phenomenon that describes the toxic actions of excitatory neurotransmitters, primarily glutamate, where the exacerbated or prolonged activation of glutamate receptors starts a cascade of neurotoxicity leading to the cell death. One of the most common neurodegenerative disorders is Alzheimer’s disease (AD), which represents 60 – 70% of cases and it ranked fourth cause of mortality in the USA. In Alzheimer’s disease, glutamate released from astrocytes and activates extrasynaptic N-methyl-D-aspartate (NMDA) receptors and triggers pro-apoptotic signaling that overcomes synaptic NMDA receptors mediated survival signaling that is already undermined by other mechanisms such as the endocytosis of NMDA receptors leading to further synaptic damage by the accumulation of glutamate causing neurotoxicity and apoptosis by activation of caspase-3. Glutamate- mediated neurotoxicity is finding increasing acceptance. Central to this assumption is that glutamate receptors of the NMDA type, are overactivated in a tonic rather than a phasic manner. Such continuous, mild, chronic activation ultimately leads to neuronal damage and impairment of synaptic plasticity(learning) and cell death. Thus, NMDA receptor antagonist could be beneficial in many neurodegenerative disorders.

It was accepted by FDA, Food Drug administration, that memantine, an uncompetitive NMDA antagonist as a Cognition-enhancing medication. The molecular mechanism of memantine (MEM) is the blockade of glutamate, the principal excitatory neurotransmitter, at NMDA receptors.

It was proposed that the combination of MEM and amide pyroglutamate, an excitatory antagonist acting at non-NMDA antagonist receptors as a single chemical agent with both pharmacophores will likely lead to neuroprotectivity and neuronal plasticity.

Our model proposes that the amide bond between the MEM drug and the pyroglutamic acid may reduce the neurotoxicity that induces impairment of synaptic plasticity and neuronal damage.

In the current study, we propose that part of the mechanism of the newly synthesized pyroglutamate amide, NSP, may promote molecular modifications that induce neuroprotection initiating protection and preventing neurotoxicity. Amides play a major role as neuroprotective group in medicinally important compounds not only because they are particularly stable, but also because they are polar (they contain regions of high positive and negative electrical charge density), which allows amide-containing drugs to interact with biological receptors and enzymes.

In the previous studies, it was shown that MEM reduces the actions of chemicals in the brain that may contribute to the symptoms of Alzheimer’s disease. In this study, we synthesized a compound using MEM as a starting material, which combined with the pyroglutamate amide, and the final product NSP was purified and identified using H-NMR and C-NMR. Our Findings indicates that NSP has a lower half-life maximal inhibitory concentration (IC50) compared to MEM, which clearly indicated that NSP has more potent effect in inhibiting N2a and PC12 cells than the potent effect of mem due to the amide function in NSP. We also demonstrate that NSP has an inhibitory effect at a great extent on glutamate induced N2a and PC12 cells death. The inhibition of the activity of glutamate was shown through the decrease in the of reactive oxygen species (ROS) and reduction in the production of malondialdehyde (MDA). In addition to reducing glutamate toxicity, NSP also shows an antioxidant effect by improving the superoxide dismutase (SOD) activity, and glutathione (GSH) content in N2a and PC12 cells.

We also demonstrate that NSP pretreatment improved the genotoxic effect of glutamate in both cells’ lines, N2a and PC12 cells. This antigenotoxic effect was investigated using Single Cell Gel Electrophoresis Effect (SCGE) or Comet assay. Importantly, NSP has shown an antiapoptotic effect by reducing caspase 3activity through the reduction of excitotoxicity. The reduction in caspase 3 activity was also enhanced by the antioxidative effect of NSP against H2O2. These results suggest that NSP show a decrease in the cleavage of caspase-3, leading to the decrease of glutamate mediated neurotoxicity and apoptosis.

Download

Included in

Biochemistry Commons

Share

COinS