Browsing by Author "Neagu, George"
Now showing 1 - 2 of 2
- Results Per Page
- Sort Options
Item Discovery-driven Label-free Quantitative Proteomics Study to Understand Estradiol-mediated Neuronal Processes in the Hippocampus and its Implication in Alzheimer’s Disease in Ovariectomized Rats(2024-03-21) Neagu, George; Zaman, Khadiza; Nguyen, Vien; Kapic, Ammar; Prokai-Tatrai, Katalin; Prokai, LaszloPurpose: Alzheimer’s disease (AD) is a debilitating neurodegenerative disorder that affects millions of people globally. Studies report an increased susceptibility to the development of AD in post-menopausal women. There is renewed interest in utilizing estrogen therapies due to its neuroprotective effects on the brain; however, the mechanisms of these neuroprotective effects are poorly understood. The hippocampus is involved in memory formation and is a critical region where early damage in AD is often seen. This discovery-driven proteomics study elucidates several candidate proteins and biological pathways mediated via E2 and implicated in neurological signaling in the hippocampus. Methods: Ovariectomized female Sprague-Dawley rats were treated with daily subcutaneous injections of either vehicle or 50μg/kg E2 for five days before the rats were sacrificed with the hippocampus collected for proteomics. Protein extracts were taken from centrifugated hippocampal tissue and prepared via a series of steps including urea incubation, disulfide bond reduction, carbamidomethylation of thiol groups, and digestion via trypsin with subsequent quenching. The digested proteins were dried, reconstituted in solvent, and processed via nano-LC-MS/MS. The MS/MS spectra were searched against a Rattus norvegicus proteome database for peptide fragment and protein identification via ProteomeDiscover (Thermo Fisher Scientific) using Mascot as a search engine and validated using Scaffold (Proteome Software). Bioinformatic analysis using Ingenuity Pathway Analysis (Quiagen) allowed the construction of associative and predicted protein networks. Results: The processed MS/MS data proteins revealed several candidates for future targeted validation. Among those proteins, calcium/calmodulin-protein kinase II implicated in memory and learning processes, such as long-term signal potentiation in the hippocampus, was more abundant in the treatment group (p = 0.00052) with fold changes in the protein cluster abundances ranging from 1.3 to 1.5 versus control. Other proteins, such as microtubule-associated protein tau, implicated in AD, also had a marked fold change of –2.5 abundance in treatment versus control (p = 0.00014). There substantial overall difference in protein abundances for neurological disease pathways, including AD, identified in Ingenuity Pathway Analysis (p = 8.9e-7) as well as in pathways involving nervous system development and function (p= 4.57e-7) between E2-treated and vehicle-treated rats. Conclusion: This dataset analysis aims to evaluate the effects of E2 on the proteome of the hippocampus in ovariectomized rats. The evident increased fold changes in calcium-dependent and calcium-associated proteins in the context of neuronal processes suggest increased downstream modulation of synaptic signaling, which could be further examined by microdialysis assay of neurotransmitters. Future studies utilizing microdialysis may examine E2’s estrogen receptor-mediated effects on cholinergic neuronal signaling in the hippocampus, which could further understand AD in the context of cholinergic neuron loss.Item Neuroprotective Effects of DHED Eyedrops Protects Visual Function Despite Elevated IOP in an Ocular Hypertension Animal Model(2024-03-21) Kapic, Ammar; Neagu, George; Nguyen, Vien; Zaman, Khadiza; Prokai, Laszlo; Prokai-Tatrai, KatalinPurpose: Glaucoma remains the second leading cause of irreversible blindness and is often associated with chronically elevated intraocular pressure (IOP) leading to ocular hypertension (OHT). All of the currently accepted therapies attempt to reduce the elevated IOP. However, despite intervention, studies show progressive neuronal damage continues in the retina and may extend to the rest of the visual system, leading to additional neuropathologies. Interest in utilizing 17β-estradiol (E2) for its neuroprotective effects has become increasingly recognized; however, due to its side effects, such as cancer risk and feminization in males, its application as a therapy is limited. Our lab has developed the estrogen prodrug, 10β, 17β-dihydroxyestra-1,4-dien-3-one (DHED), which remains inactive until its CNS-specific metabolism via short-chain reductase into the active compound E2. This study aims to elucidate the pleiotropic effects of E2 derived from DHED as a potential therapy for preserving the visual system under OHT. We hypothesize that topical application of DHED will prevent the neurodegenerative effects of chronic OHT on the retina and maintain visual function. Methods: OHT was induced in 8 to 10-month-old male Brown Norway rats via hypertonic saline injection into an episcleral vein. IOP was measured via a tonometer (Tonolab) to confirm sustained elevated IOP post-surgery and throughout the treatment period. DHED was topically delivered through eyedrops (20% 2-hydroxylpropyl-beta-cyclodextrin, 0.1% DHED, and saline) once per day. Visual acuity (VA) and contrast sensitivity (CS) were measured using the OptoMotry system with the OptoMotry 1.7 software (Cerebral Mechanics Inc). VA and CS were assessed using the "Rat" preset, and gratings were adjusted using a simple staircase progression. A fixed frequency of 0.272 c/D was chosen for the CS based on prior studies. Observers for the OptoMotry tests were blinded. The eyes and optic nerves were collected and fixed for RGC and axon counts, respectively. Seminal vesicles were collected and weighed to assess peripheral estrogenic effects. Results: The IOP was elevated by 53 % ± 15% and was sustained in both vehicle and DHED-treated groups with no differences between treatment groups. The vehicle-treated group gradually lost visual function, retaining only 60% ± 5% and 30% ± 4% of their VA and CS, respectively, by the end of the treatment period. However, the DHED-treated group maintained significantly better visual performance, retaining 91% ± 3% and 75% ± 7% of their VA and CS compared to the baseline. No differences in the mass of the seminal vesicles between treatment groups. Comparison of RGC and axon counts in the optic nerve are ongoing. Conclusion: This study demonstrates the neuroprotective effects of DHED-derived E2 on the visual system without peripheral side effects. Despite sustained OHT, the VA and CS of the topically administered DHED reduced the impact of injury compared to the vehicle control group. Future studies will investigate DHED administration's impact on the retina and visual cortex proteome.