Toll-like receptor 4 stimulation with monophosphoryl lipid A ameliorates motor deficits and nigral neurodegeneration triggered by extraneuronal α-synucleinopathy

Abstract

Background: Alpha-synuclein (α-syn) aggregation represents the pathological hallmark of α-synucleinopathies like Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Toll-like receptors (TLRs) are a family of highly conserved molecules that recognize pathogen-associated molecular patterns and define the innate immunity response. It was previously shown that TLR4 plays a role in the clearance of α-syn, suggesting that TLR4 up-regulation in microglia may be a natural mechanism to improve the clearance of α-syn. However, administration of TLR4 ligands could also lead to dangerous adverse effects associated with the induction of toxic inflammatory responses. Monophosphoryl lipid A (MPLA) is a TLR4 selective agonist and a potent inducer of phagocytosis which does not trigger strong toxic inflammatory responses as compared to lipopolysaccharide (LPS). We hypothesize that MPLA treatment will lead to increased clearance of α-syn inclusions in the brain of transgenic mice overexpressing α-syn in oligodendrocytes under the proteolipid protein promoter (PLP-α-syn mouse model of MSA), without triggering toxic cytokine release, thus leading to a general amelioration of the pathology.

Methods: Six month old PLP-α-syn mice were randomly allocated to four groups and received weekly intraperitoneal injections of MPLA (50 or 100 μg), LPS or vehicle. After a 12-week treatment period, motor behavior was assessed with the pole test. Brains and plasma samples were collected for neuropathological and immunological analysis.

Results: Chronic systemic MPLA treatment of PLP-α-syn mice led to increased uptake of α-syn by microglial cells, a significant motor improvement, rescue of nigral dopaminergic and striatal neurons and region-specific reduction of the density of oligodendroglial α-syn cytoplasmic inclusions in the absence of a marked systemic inflammatory response.

Conclusion: Our findings demonstrate beneficial effects of chronic MPLA treatment in transgenic PLP-α-syn mice. MPLA appears to be an attractive therapeutic candidate for disease modification trials in MSA and related α-synucleinopathies.

Keywords: Inclusion pathology; Monophosphoryl lipid A; Neuroinflammation; Toll-like receptor; α-synuclein.

Fig. 1

MPLA treatment increases the phagocytic activity of microglia. a Murine microglia were exposed to various treatments and phagocytic activity was measured by the incorporation of fluorescent beads. At baseline (vehicle treatment) both MPLA and LPS significantly increased the phagocytosis. Pre-exposure to fibrils of α-syn (αSyn (PF) treatment) further elevated the phagocytic activity of microglia treated with MPLA or LPS, whereas monomeric soluble α-syn (αSyn (sol) treatment) had no such effect. Data are means ± SEM, n = 4 per group. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control. ^## p < 0.01, ^### p < 0.001. b Confocal image demonstrates the localization of human α-syn in the cytoplasm of Iba-1 positive microglial cell in a PLP-α-syn mouse treated with MPLA. Z-stack analysis showed more frequent detection of microglial cells incorporating α-syn after MPLA treatmentas compared to vehicle treated PLP-α-syn mice by Fischer’s exact test (***p < 0.001)

Fig. 2

LPS, but not MPLA, causes systemic toxic inflammatory response in PLP-α-syn mice. a Heatmap demonstrates the overall log2 foldchange in the cytokine/chemokine levels in the brain and plasma as measured with the ProcartPlex Multiplex immunoassay 2 weeks after the last application of systemic low-dose LPS, MPLA50 or MPLA100. The following cytokine/chemokines of the 36-plex panel -GM-CSF, CXCL1, INF-α, IL-12p70, IL-3- were excluded from the analysis because no detectable levels were measured with this kit. b The detailed analysis of the cytokines/chemokines in the brains showed significant decrease of CCL2 and CCL4 after treatment with LPS or MPLA 100 μg. Data are means ± SEM, n = 4 per group. **p < 0.01 compared to vehicle treated mice

Fig. 3

LPS, but not MPLA induces re-distribution of α-syn from soluble monomeric to insoluble and oligomeric species. a Brains of PLP-α-syn mice were lysed and three protein fractions were prepared – TX soluble fraction, SDS soluble fraction, and Urea fraction. Membranes were probed for four different α-syn antibodies (pS129, 4B12, Syn1, and C20). Relative density (RD) for monomeric α-syn (15 kDa) and oligomeric high molecular weight α-syn (HMW, over 30 kDa) was normalized to the intensity of the β-actin band (40 kDa). b No significant effects of the treatments on α-syn distribution were identified in the TX soluble fraction. c LPS treatment induced significant reduction of the SDS soluble monomeric α-syn and a tendency towards increase of the HMW SDS soluble α-syn species. MPLA treatment had no significant effect on the distribution of α-syn species in the SDS fraction. d In the urea fraction a significant increase of insoluble α-syn species was identified after LPS treatment, but not after MPLA systemic treatment of PLP-α-syn mice. Data are means ± SEM, n = 4 per group. * p < 0.05, ** p < 0.01 compared to vehicle treated mice

Fig. 4

Absolute levels of soluble α-syn in the brains of PLP- α-syn mice remain unchanged after systemic chronic TLR4 agonist treatment. a TX soluble and b SDS soluble levels of α-syn were unaffected by the LPS or MPLA treatments. c The increase in the concentration of α-syn in the SDS fraction positively correlated with the increase in the RD of SDS-soluble monomeric species. In contrast, the higher concentration of α-syn measured in the SDS fraction by ELISA correlated with the lower RD of insoluble oligomeric α-syn species in the urea fraction, suggesting that the tendency of increased levels of α-syn after MPLA 50 in the SDS fraction may reflect the reduction of toxic insoluble oligomeric species after this treatment in PLP-α-syn mice. Data are means ± SEM, n = 4 per group

Fig. 5

Systemic MPLA treatment of PLP-α-syn mice ameliorates the MSA-like phenotype. a Pole test showed significant improvement of the motor performance of PLP-α-syn after MPLA 100 μg treatment as compared to vehicle treated mice – both Tturn and Ttotal were significantly reduced. MPLA 50 μg and LPS treatment reduced T total but had no significant effect on Tturn. Data are means ± SEM, n_vehicle = 14, n_LPS = 7, n_MPLA50 = 17, n_MPLA100 = 16. b TH-immunohistochemistry was used to visualize dopaminergic neurons in SNc. c Stereology counts identified significant rescue of dopaminergic neurons in SNc after MPLA treatment (Data are means ± SEM, n_vehicle = 11, n_LPS = 3, n_MPLA50 = 9, n_MPLA100 = 7.* p < 0.05, ** p < 0.01 compared to vehicle treated mice). A historical healthy control group of age-, sex- and background-matched mice (n = 6) was added here to demonstrate the normal level of TH-positive neurons in SNc in non-tg mice (***p < 0.001 compared to vehicle treated PLP-α-syn mice, ##p < 0.01 compared to LPS treated PLP-α-syn mice). d The lower number of dopaminergic neurons in the transgenic SNc significantly correlated with the higher disability (increased Ttotal) in the pole test. e DARPP-32 immunoreactivity was used to define the number of GABAergic medium spiny neurons in the striatum (ce, capsula externa; Str, striatum. f Stereological analysis significant rescue of DARPP-32 neurons in the striatum after MPLA treatment (Data are means ± SEM, n_vehicle = 4, n_LPS = 3, n_MPLA50 = 4, n_MPLA100 = 4.* p < 0.05, compared to vehicle treated mice)

Fig. 6

Systemic MPLA treatment of PLP-α-syn mice reduces the density of GCIs. a Three different antibodies to α-syn (15G7, 5G4, and pS129) were used to identify GCI-like accumulation of α-syn (arrows) in the brains of PLP-α-syn. b Image analysis demonstrated significant reduction of the density of GCIs in the SNc of PLP-α-syn mice treated with MPLA 100 μg as compared to vehicle-treated animals (Data are means ± SEM, n_vehicle = 13, n_LPS = 3, n_MPLA50 = 13, n_MPLA100 = 12.* p < 0.05, ** p < 0.01). c The decrease in the nigral GCI density significantly correlated with the motor improvement of the PLP-α-syn mice as measured by Ttotal of the pole test