Chung-Yin Lin

Anti-glutamatergic agents may have neuroprotective effects against excitotoxicity that is known to be involved in the pathogenesis of Parkinson's disease (PD). One of these agents is kynurenic acid (KYNA), a tryptophan metabolite, which is an endogenous N-methyl-D-aspartic acid (NMDA) receptor antagonist. However, its pharmacological properties of poor water solubility and limited blood-brain barrier (BBB) permeability rules out its systemic administration in disorders affecting the central nervous system. Our aim in the present study was to investigate the neuroprotective effects of KYNA-loaded micelles (KYNA-MICs) against PD in vitro and in vivo. Lipid-based micelles (MICs) in conjunction with KYNA drug delivery have the potential to enhance the penetration of therapeutic drugs into a diseased brain without BBB obstacles. KYNA-MICs were characterized by particle size (105.8 +/- 12.1 nm), loading efficiency (78.3 +/- 4.23%), and in vitro drug release (approximately 30% at 24 h). The in vitro experiments showed that KYNA-MICs effectively reduced 2-fold protein aggregation. The in vivo studies revealed that KYNA was successfully delivered by 5-fold increase in neurotoxin-induced PD brains. The results showed significant enhancement of KYNA delivery into brain. We also found that the KYNA-MICs exhibited several therapeutic effects. The KYNA-MICs reduced protein aggregation of an in vitro PD model, ameliorated motor functions, and prevented loss of the striatal neurons in a PD animal model. The beneficial effects of KYNA-MICs are probably explained by the anti-excitotoxic activity of the treatment's complex. As the KYNA-MICs did not induce any appreciable side-effects at the protective dose applied to a chronic PD mouse model, our results demonstrate that KYNA provides neuroprotection and attenuates PD pathology.

Neuroinflammation and oxidative stress have been emerging as important pathways contributing to Parkinson’s disease (PD) pathogenesis. In PD brains, the activated microglia release inflammatory factors such as interleukin (IL)-β, IL-6, tumor necrosis factor (TNF)-α, and nitric oxide (NO), which increase oxidative stress and mediate neurodegeneration. Using 1-methyl-4-phenylpyridinium (MPP⁺)-activated human microglial HMC3 cells and the sub-chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD, we found the potential of indole derivative NC009-1 against neuroinflammation, oxidative stress, and neurodegeneration for PD. In vitro, NC009-1 alleviated MPP⁺-induced cytotoxicity, reduced NO, IL-1β, IL-6, and TNF-α production, and suppressed NLR family pyrin domain containing 3 (NLRP3) inflammasome activation in MPP⁺-activated HMC3 cells. In vivo, NC009-1 ameliorated motor deficits and non-motor depression, increased dopamine and dopamine transporter levels in the striatum, and reduced oxidative stress as well as microglia and astrocyte reactivity in the ventral midbrain of MPTP-treated mice. These protective effects were achieved by down-regulating NLRP3, CASP1, iNOS, IL-1β, IL-6, and TNF-α, and up-regulating SOD2, NRF2, and NQO1. These results strengthen the involvement of neuroinflammation and oxidative stress in PD pathogenic mechanism, and indicate NC009-1 as a potential drug candidate for PD treatment.

Peptide conformational imprints (PCIs) offer a promising perspective to directly generate binding sites for preserving enzymes with high catalytic activity and stability. In this study, we synthesized a new chiral cross-linker cost-effectively for controlling the matrix morphology of PCIs on magnetic particles (PCIMPs) to stabilize their recognition capability. Meanwhile, based on the flank part of the sequences on papain (PAP), three epitope peptides were selected and synthesized. Molecularly imprinted polymers (MIPs) were then fabricated in the presence of the epitope peptide using our new cross-linker on magnetic particles (MPs) to generate PCIMPs. PCIMPs were formed with helical cavities that complement the PAP structure to adsorb specifically at the targeted position of PAP. PCIMPs^65–79 were found to have the best binding parameters to the PAP with K_d = 0.087 μM and B_max = 4.56 μM. Upon esterification of N-Boc-His-OH, proton nuclear magnetic resonance (¹H-NMR) was used to monitor the yield of the reaction and evaluate the activity of PAP/PCIMPs. The kinetic parameters of PAP/PCIMPs^65–79 were calculated as V_max = 3.0 μM s⁻¹, K_m = 5 × 10⁻² M, k_cat = 1.1 × 10^–1 s⁻¹, and k_cat/K_m = 2.2 M⁻¹ s⁻¹. In addition, PAP is bound tightly to PCIMPs to sustain its activity after four consecutive cycles.

In the present study, molecularly imprinted polymers (MIPs) were used as a tool to grasp a targeted α-helix or β-sheet of protein. During the fabrication of the hinge-mediated MIPs, elegant cavities took shape in a special solvent on quartz crystal microbalance (QCM) chips. The cavities, which were complementary to the protein secondary structure, acted as a peptide conformational imprint (PCI) for adenylate kinase 1 (AK1). We established a promising strategy to examine the binding affinities of human AK1 in conformational dynamics using the peptide-imprinting method. Moreover, when bound to AK1, PCIs are able to gain stability and tend to maintain higher catalytic activities than free AK1. Such designed fixations not only act on hinges as accelerators; some are also inhibitors. One example of PCI inhibition of AK1 catalytic activity takes place when PCI integrates with an AK1_9-23 β-sheet. In addition, conformation ties, a general MIP method derived from random-coil AK1_133-144 in buffer/acetonitrile, are also inhibitors. The inhibition may be due to the need for this peptide to execute conformational transition during catalysis.

Parkinson’s disease (PD) is characterized by α-synuclein (αSNCA) aggregation in dopaminergic neurons. Gradual accumulation of αSNCA aggregates in substantia nigra (SN) diminishes the normal functioning of soluble αSNCA, leading to a loss of dopamine (DA) neurons. In this study, we developed focused ultrasound-targeted microbubble destruction (UTMD)-mediated PD model that could generate the disease phenotype via αSNCA CNS gene delivery. The formation of neuronal aggregates was analyzed with immunostaining. To evaluate the DA cell loss, we used tyrosine hydroxylase immunostaining and HPLC analysis on DA and its two metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). This loss of DA was associated with a dose-dependent impairment in motor function, as assessed by the rotarod motor assessment. We demonstrate that UTMD-induced SNCA expression initiates αSNCA aggregation and results in a 50% loss of DA in SN. UTMD-related dose-dependent neuronal loss was identified, and it correlates with the degree of impairment of motor function. In comparison to chemical neurotoxin 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated and conventional intracerebral (IC)-injected animal models of PD, the UTMD-mediated αSNCA-based mouse model offers the advantage of mimicking the rapid development of the PD phenotype. The PD models that we created using UTMD also prove valuable in assessing specific aspects of PD pathogenesis and can serve as a useful PD model for the development of new therapeutic strategies.

The structural preferences of furanic compounds were studied using a combination of a molecularly imprinted film (MIF) on a piezoelectric-quartz chip. The furanic compounds and their derivatives were used as the templates. Owing to their similar heterocyclic structures, it is difficult to verify the structural differences between the templates. Therefore, a new cross-linker (Methacr-l-Cys-NHBn)₂, was employed to generate a platform on a quartz crystal microbalance (QCM) chip. The cross-linker self-assembled to link the surface of the chip to copolymerize with other functional monomers. A layered film with chiral hydrophobicity and rigidity was thus fabricated. Subsequently, Acr-l-Ser-NHBn was utilized as a chiral monomer to construct MIF on a QCM chip. Forcomparison, we synthesized a more hydrophobic monomer, Methacr-l-Ser-NHBn, to enhance the binding ability of the MIF. The QCM flow injection system was handled in an organic solvent system. The proportion of the monomers was adjusted to optimize the recognition ability of these films. As the binding ability of the MIF toward model templates and structurally-related furanic compounds was improved, a MIF derived from 2-furaldehyde (FUL) achieved a lower detection limit (10 ng/mL). The binding properties of MIFs prepared against furanic compounds exhibited strong similarities to the binding properties of other compounds with heterocyclic ring structures. For example, 2-furaldehyde is very similar to 2-formylthiazole, 2-acetylfuran is similar to 2-acetylthiazole, and 2-furfuryl alcohol is similar to imidazole-2-methanol. Such recognition ability can help distinguish between the structural counterparts of other small heterocyclic compounds.