FoxO1 Alleviates the Mitochondrial ROS Levels Induced by α-Synuclein Preformed Fibrils in BV-2 Microglial Cells.

Parkinson's disease (PD) is a complex neurodegenerative disorder marked by the gradual deterioration of dopaminergic neurons, especially in the substantia nigra pars compacta (SNc). Dysregulation of the transcription factor FoxO1 is associated with various neurodegenerative conditions, including Alzheimer's disease and PD, though the specific mechanisms involved are not fully understood. This study explores the effects of α-Synuclein preformed fibrils (PFF) on BV-2 microglial cells, focusing on changes in molecular characteristics and their impact on neuronal degeneration. Our results demonstrate that PFF treatment significantly increases FoxO1 mRNA (p = 0.0443) and protein (p = 0.0216) levels, leading to its nuclear translocation (p = 0.0142) and enhanced expression of genes involved in the detoxification of reactive oxygen species (ROS), such as Catalase (Cat, p = 0.0249) and superoxide dismutase 2 (Sod2, p = 0.0313). Furthermore, we observed that PFF treatment elevates mitochondrial ROS levels. However, cells lacking FoxO1 or treated with FoxO1 inhibitors showed increased vulnerability to PFF-induced ROS, attributed to reduced expression of ROS detoxifying enzymes Cat and Sod2 (p < 0.0001). Besides enhancing ROS production, inhibiting FoxO1 also heightens neurotoxicity induced by PFF treatment in microglia-conditioned medium (p < 0.0001). Conversely, treatment with N-acetylcysteine or bacterial superoxide dismutase A mitigated the ROS increase induced by PFF (p < 0.0001). These findings suggest the essential role of FoxO1 in regulating ROS levels, which helps alleviate pathology in PFF-induced PD models. Our study provides insights into the genetic mechanisms of PD and suggests potential pathways for developing novel therapeutic strategies.

DJ-1 protects cell death from a mitochondrial oxidative stress due to GBA1 deficiency.

BACKGROUND: GBA1 mutations are the most common genetic risk factor for development of Parkinson's disease (PD). The loss of catalytic activity in GBA1, as well as the reduction of the GBA1 protein in certain cellular compartment, may increase disease progression. However, the mechanisms underlying cellular dysfunction caused by GBA1 deficiency are still mostly unknown. OBJECTIVE: In this study, we focus on the genetic interaction between GBA1 deficiency and PD-causing genes, such as DJ-1, in mitochondrial dysfunction. METHODS: GBA1 knockout (KO) SH-SY5Y cells were used to assess DJ-1 functions against oxidative stress in vitro. The levels of cellular reactive oxygen species were monitored with MitoSOX reagent. The expression of the PARK7 gene was analyzed using the quantitative real-time PCR (qRT-PCR). To understand the mechanism underlying DJ-1 upregulation in GBA1 KO cells, we assess ROS levels, antioxidant protein, and cell viability in GBA1 KO cells with treatment of ROS inhibitor N-acetyl-cysteine or miglustat, which is an inhibitor of glucosylceramide synthase. Dopaminergic degeneration was assessed from Gba1 L444P heterozygous mice mated with Park7 knockout mice. RESULTS: We find that DJ-1 is significantly upregulated in GBA1 KO cells. Elevated levels of DJ-1 are attributed to the transcriptional expression of PARK7 mRNA, but not the inhibition of DJ-1 protein degradation. Because DJ-1 expression is highly linked to oxidative stress, we observe cellular reactive oxygen species (ROS) in GBA1 KO cells. Moreover, several antioxidant gene expressions and protein levels are increased in GBA1 KO cells. To this end, GBA1 KO cells are more susceptible to H2O2-induced cell death. Importantly, there is a significant reduction in dopaminergic neurons in the midbrain from Gba1 L444P heterozygous mice mated with Park7 knockout mice, followed by mild motor dysfunction. CONCLUSION: Taken together, our results suggest that DJ-1 upregulation due to GBA1 deficiency has a protective role against oxidative stress. It may be supposed that mutations or malfunctions in the DJ-1 protein may have disadvantages in the survival of dopaminergic neurons in the brains of patients harboring GBA1 mutations.

Structural and Mechanistic Insights into Development of Chemical Tools to Control Individual and Inter-Related Pathological Features in Alzheimer's Disease.

To elucidate the involvement of individual and inter-related pathological factors [i.e., amyloid-ß (Aß), metals, and oxidative stress] in the pathogenesis of Alzheimer's disease (AD), chemical tools have been developed. Characteristics required for such tool construction, however, have not been clearly identified; thus, the optimization of available tools or new design has been limited. Here, key structural properties and mechanisms that can determine tools' regulatory reactivities with multiple pathogenic features found in AD are reported. A series of small molecules was built up through rational structural selection and variations onto the framework of a tool useful for in vitro and in vivo metal-Aß investigation. Variations include: (i) location and number of an Aß interacting moiety; (ii) metal binding site; and (iii) denticity and structural flexibility. Detailed biochemical, biophysical, and computational studies were able to provide a foundation of how to originate molecular formulas to devise chemical tools capable of controlling the reactivities of various pathological components through distinct mechanisms. Overall, this multidisciplinary investigation illustrates a structure-mechanism-based strategy of tool invention for such a complicated brain disease.

A Redox-Active, Compact Molecule for Cross-Linking Amyloidogenic Peptides into Nontoxic, Off-Pathway Aggregates: In Vitro and In Vivo Efficacy and Molecular Mechanisms.

Chemical reagents targeting and controlling amyloidogenic peptides have received much attention for helping identify their roles in the pathogenesis of protein-misfolding disorders. Herein, we report a novel strategy for redirecting amyloidogenic peptides into nontoxic, off-pathway aggregates, which utilizes redox properties of a small molecule (DMPD, N,N-dimethyl-p-phenylenediamine) to trigger covalent adduct formation with the peptide. In addition, for the first time, biochemical, biophysical, and molecular dynamics simulation studies have been performed to demonstrate a mechanistic understanding for such an interaction between a small molecule (DMPD) and amyloid-ß (Aß) and its subsequent anti-amyloidogenic activity, which, upon its transformation, generates ligand-peptide adducts via primary amine-dependent intramolecular cross-linking correlated with structural compaction. Furthermore, in vivo efficacy of DMPD toward amyloid pathology and cognitive impairment was evaluated employing 5xFAD mice of Alzheimer's disease (AD). Such a small molecule (DMPD) is indicated to noticeably reduce the overall cerebral amyloid load of soluble Aß forms and amyloid deposits as well as significantly improve cognitive defects in the AD mouse model. Overall, our in vitro and in vivo studies of DMPD toward Aß with the first molecular-level mechanistic investigations present the feasibility of developing new, innovative approaches that employ redox-active compounds without the structural complexity as next-generation chemical tools for amyloid management.

Synthetic Flavonoids, Aminoisoflavones: Interaction and Reactivity with Metal-Free and Metal-Associated Amyloid-ß Species.

Metal ion homeostasis in conjunction with amyloid-ß (Aß) aggregation in the brain has been implicated in Alzheimer's disease (AD) pathogenesis. To uncover the interplay between metal ions and Aß peptides, synthetic, multifunctional small molecules have been employed to modulate Aß aggregation in vitro. Naturally occurring flavonoids have emerged as a valuable class of compounds for this purpose due to their ability to modulate both metal-free and metal-induced Aß aggregation. Although, flavonoids have shown anti-amyloidogenic effects, the structural moieties of flavonoids responsible for such reactivity have not been fully identified. In order to understand the structure-interaction-reactivity relationship within the flavonoid family for metal-free and metal-associated Aß, we designed, synthesized, and characterized a set of isoflavone derivatives, aminoisoflavones (1-4), that displayed reactivity (i.e., modulation of Aß aggregation) in vitro. NMR studies revealed a potential binding site for aminoisoflavones between the N-terminal loop and central helix on prefibrillar Aß different from the non-specific binding observed for other flavonoids. The absence or presence of the catechol group differentiated the binding affinities and enthalpy/entropy balance between aminoisoflavones and Aß. Furthermore, having a catechol group influenced the binding mode with fibrillar Aß. Inclusion of additional substituents moderately tuned the impact of aminoisoflavones on Aß aggregation. Overall, through these studies, we obtained valuable insights on the requirements for parity among metal chelation, intermolecular interactions, and substituent variation for Aß interaction.

Tuning reactivity of diphenylpropynone derivatives with metal-associated amyloid-ß species via structural modifications.

A diphenylpropynone derivative, DPP2, has been recently demonstrated to target metal-associated amyloid-ß (metal-Aß) species implicated in Alzheimer's disease (AD). DPP2 was shown to interact with metal-Aß species and subsequently control Aß aggregation (reactivity) in vitro; however, its cytotoxicity has limited further biological applications. In order to improve reactivity toward Aß species and lower cytotoxicity, along with gaining an understanding of a structure-reactivity-cytotoxicity relationship, we designed, prepared, and characterized a series of small molecules (C1/C2, P1/P2, and PA1/PA2) as structurally modified DPP2 analogues. A similar metal binding site to that of DPP2 was contained in these compounds while their structures were varied to afford different interactions and reactivities with metal ions, Aß species, and metal-Aß species. Distinct reactivities of our chemical family toward in vitro Aß aggregation in the absence and presence of metal ions were observed. Among our chemical series, the compound (C2) with a relatively rigid backbone and a dimethylamino group was observed to noticeably regulate both metal-free and metal-mediated Aß aggregation to different extents. Using our compounds, cell viability was significantly improved, compared to that with DPP2. Lastly, modifications on the DPP framework maintained the structural properties for potential blood-brain barrier (BBB) permeability. Overall, our studies demonstrated that structural variations adjacent to the metal binding site of DPP2 could govern different metal binding properties, interactions with Aß and metal-Aß species, reactivity toward metal-free and metal-induced Aß aggregation, and cytotoxicity of the compounds, establishing a structure-reactivity-cytotoxicity relationship. This information could help gain insight into structural optimization for developing nontoxic chemical reagents toward targeting metal-Aß species and modulating their reactivity in biological systems.