Parkinson's disease: Evaluation of a neuroprotective target and identification of candidate biomarker signatures using murine models

Parkinson's disease (PD) is one of the most common age-related neurologic diseases. While existing therapeutic approaches, focusing on dopamine replacement, can alleviate some of the cardinal symptoms, they are associated with severe adverse effects in the long-term. Therefore, identification of new therapeutic interventions to reverse, stop or slow down the progression of Parkinson’s disease is a major focus of PD research. Similarly, identifying reliable biomarkers that would enable early therapeutic intervention is another key area of current research. Here, we evaluated a recently proposed non-dopaminergic protein drug target for PD, Regulator of G-Protein Signaling 4 (RGS4), and performed preliminary studies aimed at the identification of novel biomarker signatures using two murine models of Parkinson’s disease. Recent research on new non-dopaminergic PD drug targets has indicated that inhibition of RGS4, a member of the RGS family of proteins that inactivate G-proteins, could be an effective adjuvant treatment option. However, the effectiveness of RGS4 inhibition for an array of PDlinked functional and structural neuroprotection endpoints had not yet been demonstrated. Here, we used the 6-Hydroxydopamine (6-OHDA) lesioning mouse model to address this question. We observed, using a battery of behavioral and pathological measures, that mice deficient for RGS4 are not protected from 6-OHDA induced injury, and showed enhanced susceptibility in some measures of motor function. Our results suggest that inhibition of RGS4 as a non-dopaminergic target for PD should be approached with caution. In the second part of this study, two alpha-synuclein based PD mouse models, human E46K mutated overexpressed alpha-synuclein and alpha-synuclein fibril spreading models, were used to investigate early pathological events in PD and identify novel candidate biomarker signatures for subsequent validation. Two different time points, before disease onset, and at peak disease manifestation, were analyzed in the two models. Using multiple histopathology and molecular biology techniques, we were able to identify complex changes in patterns of gene expression at early stages of the disease, well before neurodegeneration is detectable. These findings might open venues for new therapeutic strategies and provide insights on the molecular perturbations occurring during the earliest stages of the disease, paving the way for the development of a biomarker signatures for early diagnosis of Parkinson’s disease

Insights into the antiatherogenic molecular mechanisms of andrographolide against Porphyromonas gingivalis-induced atherosclerosis in rabbits

Atherosclerosis is the commonest and most important vascular disease. Andrographolide (AND) is the main bioactive component of the medicinal plant Andrographis paniculata and is used in traditional medicine. This study was aimed to evaluate the antiatherogenic effect of AND against atherosclerosis induced by Porphyromonas gingivalis in White New Zealand rabbits. Thirty rabbits were divided into five groups as follows: G1, normal group; G2-5, were orally challenged with P. gingivalis five times a week over 12 weeks; G2, atherogenic control group; G3, standard group treated with atorvastatin (AV) 5 mg/kg; and G4 and G5, treatment groups treated with AND 10 and 20 mg/kg, respectively over 12 weeks. Serums were subjected to antioxidant enzymatic and anti-inflammatory activities, and the aorta was subjected to histological analyses. Groups treated with AND showed a significant reversal of liver and renal biochemical changes, compared with the atherogenic control group. In the same groups, superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), total glutathione (GSH) levels in serum were significantly increased (P < 0.05), and lipid peroxidation (malondialdehyde (MDA)) levels were significantly decreased (P < 0.05), respectively. Furthermore, treated groups with AV and AND showed significant decrease in the level of VCAM-1 and ICAM-1 compared with the atherogenic control group. In aortic homogenate, the level of nitrotyrosine was significantly increased, while the level of MCP1 was significantly decreased in AV and AND groups compared with the atherogenic control group. In addition, staining the aorta with Sudan IV showed a reduction in intimal thickening plaque in AV and AND groups compared with the atherogenic control group. AND has showed an antiatherogenic property as well as the capability to reduce lipid, liver, and kidney biomarkers in atherogenic serum that prevents atherosclerosis complications caused by P. gingivalis

Absence of regulator of G-protein signaling 4 does not protect against dopamine neuron dysfunction and injury in the mouse 6-hydroxydopamine lesion model of Parkinson's disease.

Regulator of G-protein signaling 4 (RGS4), a member of the RGS family of proteins that inactivate G-proteins, has gained interest as a potential drug target for neurological disorders, such as epilepsy and Parkinson's disease (PD). In the case of PD, the main current options for alleviating motor symptoms are dopamine replacement therapies, which have limitations because of side effects and reduced effectiveness over the long term. Research on new nondopaminergic PD drug targets has indicated that inhibition of RGS4 could be an effective adjuvant treatment option. The effectiveness of RGS4 inhibition for an array of PD-linked functional and structural neuroprotection end points has not yet been demonstrated. Here, we use the 6-hydroxydopamine (6-OHDA) lesioning model of the nigrostriatal pathway in mice to address this question. We observe, using a battery of behavioral and pathological measures, that mice deficient for RGS4 are not protected from 6-OHDA-induced injury and show enhanced susceptibility in some measures of motor function. Our results suggest that inhibition of RGS4 as a nondopaminergic target for PD should be approached with caution

Characterization of Molecular Determinants of the Conformational Stability of Macrophage Migration Inhibitory Factor: Leucine 46 Hydrophobic Pocket

Macrophage Migration Inhibitory Factor (MIF) is a key mediator of inflammatory responses and innate immunity and has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. The oligomerization of MIF, more specifically trimer formation, is essential for its keto-enol tautomerase activity and probably mediates several of its interactions and biological activities, including its binding to its receptor CD74 and activation of certain signaling pathways. Therefore, understanding the molecular factors governing the oligomerization of MIF and the role of quaternary structure in modulating its structural stability and multifunctional properties is crucial for understanding the function of MIF in health and disease. Herein, we describe highly conserved intersubunit interactions involving the hydrophobic packing of the side chain of Leu46 onto the β-strand β3 of one monomer within a hydrophobic pocket from the adjacent monomer constituted by residues Arg11, Val14, Phe18, Leu19, Val39, His40, Val41, Val42, and Pro43. To elucidate the structural significance of these intersubunit interactions and their relative contribution to MIF’s trimerization, structural stability and catalytic activity, we generated three point mutations where Leu46 was replaced by glycine (L46G), alanine (L46A) and phenylalanine (L46F), and their structural properties, stability, oligomerization state, and catalytic activity were characterized using a battery of biophysical methods and X-ray crystallography. Our findings provide new insights into the role of the Leu46 hydrophobic pocket in stabilizing the conformational state of MIF in solution. Disrupting the Leu46 hydrophobic interaction perturbs the secondary and tertiary structure of the protein but has no effect on its oligomerization state

A New Synuclein-Transgenic Mouse Model for Early Parkinson's Reveals Molecular Features of Preclinical Disease.

Understanding Parkinson's disease (PD), in particular in its earliest phases, is important for diagnosis and treatment. However, human brain samples are collected post-mortem, reflecting mainly end-stage disease. Because brain samples of mouse models can be collected at any stage of the disease process, they are useful in investigating PD progression. Here, we compare ventral midbrain transcriptomics profiles from α-synuclein transgenic mice with a progressive, early PD-like striatal neurodegeneration across different ages using pathway, gene set, and network analysis methods. Our study uncovers statistically significant altered genes across ages and between genotypes with known, suspected, or unknown function in PD pathogenesis and key pathways associated with disease progression. Among those are genotype-dependent alterations associated with synaptic plasticity and neurotransmission, as well as mitochondria-related genes and dysregulation of lipid metabolism. Age-dependent changes were among others observed in neuronal and synaptic activity, calcium homeostasis, and membrane receptor signaling pathways, many of which linked to G-protein coupled receptors. Most importantly, most changes occurred before neurodegeneration was detected in this model, which points to a sequence of gene expression events that may be relevant for disease initiation and progression. It is tempting to speculate that molecular changes similar to those changes observed in our model happen in midbrain dopaminergic neurons before they start to degenerate. In other words, we believe we have uncovered molecular changes that accompany the progression from preclinical to early PD

Mutation of Leu46 does not alter the quaternary structure of MIF.

<p>Sedimentation rate distributions as determined by Analytical Ultracentrifugation/Sedimentation Velocity experiments indicating similar sedimentation rates for the wt and Leu46 huMIF mutants (15 µM in PBS 1X, pH 7.4 buffer).</p

X-ray crystallography demonstrates that the three-dimensional structure of Leu46 mutants is very similar to the wt protein.

<p>(A) Overlay of crystal structures of L46F (blue) L46A (green) and L46G (red). (B, C, D) Secondary structure disruptions induced by the Leu46 mutants are shown by superimposition of the wt human and L46F (B), L46A (C) and L46G (D) MIF monomers. Wt and Leu46 mutant monomers are represented in pink and cyan respectively. Black arrows highlight the structural changes induced in the Leu46 variants.</p