A Synthetic Agonist to Vasoactive Intestinal Peptide Receptor-2 Induces Regulatory T Cell Neuroprotective Activities in Models of Parkinson\u27s Disease

A paradigm shift has emerged in Parkinson\u27s disease (PD) highlighting the prominent role of CD4+ Tregs in pathogenesis and treatment. Bench to bedside research, conducted by others and our own laboratories, advanced a neuroprotective role for Tregs making pharmacologic transformation of immediate need. Herein, a vasoactive intestinal peptide receptor-2 (VIPR2) peptide agonist, LBT-3627, was developed as a neuroprotectant for PD-associated dopaminergic neurodegeneration. Employing both 6-hydroxydopamine (6-OHDA) and α-synuclein (α-Syn) overexpression models in rats, the sequential administration of LBT-3627 increased Treg activity without altering cell numbers both in naïve animals and during progressive nigrostriatal degeneration. LBT-3627 administration was linked to reductions of inflammatory microglia, increased survival of dopaminergic neurons, and improved striatal densities. While α-Syn overexpression resulted in reduced Treg activity, LBT-3627 rescued these functional deficits. This occurred in a dose-dependent manner closely mimicking neuroprotection. Taken together, these data provide the basis for the use of VIPR2 agonists as potent therapeutic immune modulating agents to restore Treg activity, attenuate neuroinflammation, and interdict dopaminergic neurodegeneration in PD. The data underscore an important role of immunity in PD pathogenesis

Salmon and human thrombin differentially regulate radicular pain, glial-induced inflammation and spinal neuronal excitability through protease-activated receptor-1.

Chronic neck pain is a major problem with common causes including disc herniation and spondylosis that compress the spinal nerve roots. Cervical nerve root compression in the rat produces sustained behavioral hypersensitivity, due in part to the early upregulation of pro-inflammatory cytokines, the sustained hyperexcitability of neurons in the spinal cord and degeneration in the injured nerve root. Through its activation of the protease-activated receptor-1 (PAR1), mammalian thrombin can enhance pain and inflammation; yet at lower concentrations it is also capable of transiently attenuating pain which suggests that PAR1 activation rate may affect pain maintenance. Interestingly, salmon-derived fibrin, which contains salmon thrombin, attenuates nerve root-induced pain and inflammation, but the mechanisms of action leading to its analgesia are unknown. This study evaluates the effects of salmon thrombin on nerve root-mediated pain, axonal degeneration in the root, spinal neuronal hyperexcitability and inflammation compared to its human counterpart in the context of their enzymatic capabilities towards coagulation substrates and PAR1. Salmon thrombin significantly reduces behavioral sensitivity, preserves neuronal myelination, reduces macrophage infiltration in the injured nerve root and significantly decreases spinal neuronal hyperexcitability after painful root compression in the rat; whereas human thrombin has no effect. Unlike salmon thrombin, human thrombin upregulates the transcription of IL-1β and TNF-α and the secretion of IL-6 by cortical cultures. Salmon and human thrombins cleave human fibrinogen-derived peptides and form clots with fibrinogen with similar enzymatic activities, but salmon thrombin retains a higher enzymatic activity towards coagulation substrates in the presence of antithrombin III and hirudin compared to human thrombin. Conversely, salmon thrombin activates a PAR1-derived peptide more weakly than human thrombin. These results are the first to demonstrate that salmon thrombin has unique analgesic, neuroprotective and anti-inflammatory capabilities compared to human thrombin and that PAR1 may contribute to these actions

Salmon and human thrombin have similar affinities for serum containing media and ATIII, but not hirudin.

<p>(<b>A</b>) Salmon thrombin (STh) and human thrombin (HTh) maintain similar enzymatic activity towards a fluorescent fibrinogen-like substrate over time in serum containing media kept at 37°C; their normalized cleavage rate is not different at any time point after thrombin addition. (<b>B</b>) The activity of salmon thrombin is inhibited significantly less (p = 0.005) than human thrombin by Antithrombin III (ATIII) overall, but not at any one individual concentration ranging from 0 to 45 nM. (<b>C</b>) Salmon thrombin activity towards fibriniogen is inhibited less than human thrombin overall (p<0.001) and at hirudin-to-thrombin ratios of 1 and 1.5 (*p<0.001). Data are shown as means with standard deviations (μ ± SD).</p

Salmon thrombin does not increase pro-inflammatory cytokines in cortical cultures at early time points.

<p>(<b>A</b>) IL-1β and TNFα mRNA are significantly upregulated in cortical cultures at 4 hours after their treatment with human thrombin (HTh) at 1 U/ml compared to untreated cultures (UT) (^#p<0.001). Salmon thrombin (STh), at the same concentration, is unchanged from UT and significantly less than human thrombin treatment (HTh) (*p<0.002). (<b>B</b>) IL-6 concentration is significantly greater in cultures treated with human thrombin overall at each individual concentration (^#p<0.01) for 6 hours compared to the untreated (UT) control. IL-6 secreted from cortical cultures is significantly less after treatment with salmon thrombin (STh) compared to human thrombin (HTh) at 6 hours after stimulation overall (p<0.001) and at each individual concentration of 0.2, 0.5 and 1 U/ml (*p<0.03). Data are shown as means with standard deviations (μ ± SD).</p

Myelin basic protein (MBP) organization in the injured nerve root.

<p>−striated MBP patterning;</p><p>+mostly striated MBP with some disruption in labeling;</p><p>++mostly disorganized MBP labeling;</p><p>+++highly disorganized MBP labeling with pockets of debris.</p

Salmon thrombin preserves nerve root health and prevents inflammation after painful compression in the rat.

<p>(<b>A</b>) Schematic depicting the spinal cord, nerve root and dorsal root ganglion (DRG); red box indicates location within the nerve root where the root was analyzed. (<b>B</b>) Uncompressed nerve roots from un-operated (normal) rats exhibit myelin basic protein (MBP; green) labeling in a striated pattern that is homogenous across the width of the root and no immunoreactivity for macrophages (Iba1; red). On day 7 after a painful root compression treated with neurobasal media (NB media), MBP is disrupted and Iba1 is more abundant. Human thrombin treated roots (HTh) have MBP and Iba1 labeling that is similar to those roots treated with NB media. Salmon thrombin treated roots (STh) exhibit the same striated MBP labeling with minimal Iba1 as normal roots and much less than roots treated with NB media or human thrombin. Scale bar is 100 µm. (<b>C</b>) Quantification of positive Iba1 labeling normalized to expression in normal un-operated tissue. Normal tissue exhibits very low levels of Iba1. Human thrombin significantly increases (^#p<0.001) Iba1 in the nerve root compared to normal. Roots treated with salmon thrombin are not different from normal levels or those treated with neurobasal media, but induces significantly less (*p = 0.035) Iba1 infiltration in the nerve root compared to human thrombin.</p

Salmon thrombin reduces spinal neuronal hyperexcitability after a painful root compression in the rat.

<p>(<b>A</b>) Schematic showing the location of recording in the deep laminae of the ipsilateral spinal dorsal horn. (<b>B</b>) Representative extracellular potentials evoked in response to five 1-second stimuli by a 26 g von Frey filament applied to the ipsilateral forepaw showing neurons in the human thrombin (HTh) group are more excitable than those in the salmon thrombin (STh) group. (<b>C</b>) Overall, salmon thrombin (STh) produces a significant decrease in the number of evoked spikes compared to both neurobasal media (NB media) (p = 0.002) and human thrombin (HTh) treatment (p<0.001). Salmon thrombin also significantly reduces responses compared to human thrombin in response to stimulation by the 4 and 26 g von Frey filaments (*p<0.029). Interestingly, human thrombin induces a significant increase in evoked firing compared to NB media treated rats overall (p = 0.015). Data are shown as means with standard error (μ ± SEM).</p

Salmon thrombin attenuates mechanical allodynia after painful nerve root compression in the rat.

<p>Mechanical allodynia was significantly elevated in rats after a painful nerve root compression treated with the vehicle, neurobasal media (NB media), compared to sham for all von Frey filament strengths (1.4 g, 4 g, 10 g) over all of the testing days (p<0.012). NB media was also significantly elevated on various individual testing days for various filament strengths (&p<0.036). Human thrombin (HTh) did not alter mechanical allodynia compared to NB media and remained significantly elevated over sham operated rats for all filaments over the entire testing period (p<0.009), on various days for the 1.4 and 4 g filaments (^#p<0.041) and on each testing day for the 10 g filament (^#p<0.027). Salmon thrombin (STh) significantly reduced mechanical allodynia compared to NB media (p<0.028) and was unchanged from sham for all filaments over all days. Notably, STh also significantly attenuated allodynia compared to HTh overall (p<0.047), on day 1 for the 1.4 g filament (*p = 0.003) and on each individual testing day for the 10 g filament (*p<0.027). Data are shown as means with standard deviations (μ ± SD).</p