Potential therapeutic target for Parkinson's disease.

PD is a highly prevalent neurodegenerative condition, with no precise mechanisms for midbrain dopaminergic neurons (mDANs) loss in the substantia nigra. Nurr1, a nuclear receptor, is a potential therapeutic target for PD; however, its role in PD pathogenesis is unclear.

The present study's authors previously reported a structure-activity link between Nurr1 agonists (amodiaquine, glafenine, and chloroquine) and 4-amino-7-chloroquinoline (4A7C) in PD patients.

About the study

In the present study, researchers investigated whether genetic [such as alpha-synuclein (αSyn)] and environmental [such as mitochondrial complex I inhibitor, 1-methyl-4-phyenylpyridinium (MPP) positivity] risk factors for Parkinson's disease affect Nurr1 expression and function.

The team performed a systematic search and generated more than 570 4A7C-chloroquine derivatives, which were characterized based on the hypothesis that 4A7C is a SAR that promotes Nurr1 activation and binding.

The potential SAR (4A7C)-based characterization resulted in the development of 4A7C-301, an optimal agonist. It was then investigated for its effects on several in vitro and in vivo PD models with chloroquine.

BV2 mouse microglia (ATCC), SK-N-BE(2)C human neuroblastoma, MN9D, and HeLa cells were used for the cell culture experiments, and transactivity, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cytotoxicity or lactate dehydrogenase (LDH), and autophagolysosome (APL) formation assays were performed.

For Nurr1 stability determination, MN9D cells were incubated with MPP+ or transduced with lentivirus expressing green fluorescent protein (GFP) only or αSyn.

Protein samples were prepared from cell harvests or brain tissues, and autophagic flux was determined by Western blot analysis, following which the immunoreactive bands were quantified. Ribonucleic acid (RNA) was extracted from the samples, and a quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed.

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Subsequently, site-directed mutagenesis, binding and competition assays, and time-resolved fluorescence resonance energy transfer (TR-FRET) assays were performed, followed by Nurr1 overexpression, knockdown, and lentivirus production and transduction.

Furthermore, cellular oxidative stress, cell viability, and mitochondrial activity assays were performed. Primary ventral mesencephalic (VM) dopaminergic neurons were obtained from embryonic C57BL/6 mice and treated with 4A7C-301 or chloroquine before being extracted for real-time dopaminergic gene expression PCR analysis.

Primary rat VM neuron-glia co-cultures were generated, and immunocytochemistry (ICC) was performed.

Mice were randomly allocated to five groups, i.e., vehicle (VEH), MPTP neurotoxin, MPTP + L-DOPA, MPTP + chloroquine, and MPTP + 4A7C-301. To test dyskinetic involuntary behavior, an L-DOPA-injected group was included.

Adeno-associated viruses (AAV)-αSyn-induced PD mice were subjected to various tests, assessing motor (rotarod, pole, and cylinder tests), as well as non-motor behaviors (olfactory discrimination tests).

The murine tissues were subjected to immunohistochemistry (IHC), immunofluorescence (IF), stereological analyses, enzyme-linked immunosorbent assays (ELISA), and blood-brain barrier (BBB) permeability assays.

Results

The researchers discovered 4A7C-301, a brain penetrant with strong neuroprotective properties in vitro. It preserved mDANs in an MPTP-induced murine model of Parkinson's disease and alleviated motor and non-motor olfactory impairments without causing dyskinesia.

Further, in AAV2-mediated αSyn-overexpressing male mice models, 4A7C-301 dramatically alleviated neuropathological abnormalities. Alpha-synuclein overexpression and MPP+ treatment robustly downregulated Nurr1 expression, while the treatments showed no impacts on beta-actin or other mDAN transcription factors' expression.

Pathogenic αSyn overexpression led to a larger decrease in Nurr1 levels compared to the wild-type. The findings indicated that compromised Nurr1 expression and function might underlie mDAN degeneration in sporadic and familial Parkinson's disease, contributing to disease pathogenesis. Nurr1 ligands could regulate Nurr1 levels at the post-transcriptional level.

Chloroquine or 4A7C-301 therapy effectively restored Nurr1 protein levels, and cellular autophagy activities decreased by MPP+ and αSyn without affecting messenger ribonucleic acid (mRNA) levels.

4A7C-301 and chloroquine (less potently) corrected the observed olfactory loss in both MPTP-induced and αSyn overexpression paradigms. The findings indicated that αSyn and Nurr1 reciprocally regulated each other in vitro as well as in vivo, with 4A7C-301 prominently influenced by its expression.

The murine model explained the age-associated mDANs loss and the genetic- and environmental factors-induced loss, concordant with prior animal and human studies.

4A7C-301 effectively enhanced Nurr1's transcriptional activator and repressor functions. 4A7C-301 showed 20.0-fold greater binding energy than chloroquine or amodiaquine, strongly potentiated the transcriptional activities of the Nurr1 protein, and protected midbrain dopaminergic neurons with neurotoxin (such as lipopolysaccharide and MPP) exposure more potently than chloroquine (>20.0-fold).

4A7C-301 considerably protected midbrain dopaminergic neurons by reducing cytotoxicity and oxidative stress, preserving mitochondrial function, and inducing mDAN-targeted genetic expression.

Conclusion

To summarize, 4A7C-301 showed neuroprotective effects via Nurr1 activation, restored Nurr1 levels reduced following exposure to genetic and environmental risk factors of PD, and restored autophagy against MPP+ in the in vitro settings.

The compound also suppressed neuroinflammation in VM neuron-glia co-cultures, improved motor and non-motor behaviors in MPTP toxin-induced mice, and rescued PD pathology in αSyn-induced murine animals.

Based on the study findings, 4A7C-301 could widen the therapeutic landscape of PD due to its disease-modifying properties. However, further research is required to elucidate the mechanisms through which 4A7C-301 regulates Nurr1's transcriptional function.