ABSTRACT
BACKGROUND: Evidence from mice suggests that brain infiltrating immune cells contribute to neurodegeneration, and we previously identified a deleterious lymphocyte infiltration in Parkinson's disease mice. However, this remains controversial for monocytes, due to artifact-prone techniques used to distinguish them from microglia. Our aim was to reassess this open question, by taking advantage of the recent recognition that chemokine receptors CCR2 and CX3CR1 can differentiate between inflammatory monocytes and microglia, enabling to test whether CCR2+ monocytes infiltrate the brain during dopaminergic (DA) neurodegeneration and whether they contribute to neuronal death. This revealed unexpected insights into possible regulation of monocyte-attracting CCL2 induction. METHODS: We used acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mice and assessed monocyte infiltration by combining laser microdissection-guided chemokine RNA profiling of the substantia nigra (SN) with immunohistochemistry and CCR2-GFP reporter mice. To determine contribution to neuronal loss, we used CCR2-deletion and CCL2-overexpression, to reduce and increase CCR2+ monocyte infiltration, and CX3CR1-deletion to assess a potential implication in CCL2 regulation. RESULTS: Nigral chemokine profiling revealed early CCL2/7/12-CCR2 axis induction, suggesting monocyte infiltration in MPTP mice. CCL2 protein showed early peak induction in nigral astrocytes, while CCR2-GFP mice revealed early but limited nigral monocyte infiltration. However, blocking infiltration by CCR2 deletion did not influence DA neuronal loss. In contrast, transgenic astrocytic CCL2 over-induction increased CCR2+ monocyte infiltration and DA neuronal loss in MPTP mice. Surprisingly, CCL2 over-induction was also detected in MPTP intoxicated CX3CR1-deleted mice, which are known to present increased DA neuronal loss. Importantly, CX3CR1/CCL2 double-deletion suggested that increased neurotoxicity was driven by astrocytic CCL2 over-induction. CONCLUSIONS: We show that CCR2+ monocytes infiltrate the affected CNS, but at the level observed in acute MPTP mice, this does not contribute to DA neuronal loss. In contrast, the underlying astrocytic CCL2 induction seemed to be tightly controled, as already moderate CCL2 over-induction led to increased neurotoxicity in MPTP mice, likely due to the increased CCR2+ monocyte infiltration. Importantly, we found evidence suggesting that during DA neurodegeneration, this control was mediated by microglial CX3CR1 signaling, which protects against such neurotoxic CCL2 over-induction by astrocytes, thus hinting at an endogenous mechanism to limit neurotoxic effects of the CCL2-CCR2 axis.
Subject(s)
Astrocytes/metabolism , Cell Movement/drug effects , Chemokine CCL2/metabolism , MPTP Poisoning/pathology , Microglia/metabolism , Receptors, Interleukin-8A/deficiency , Animals , Astrocytes/drug effects , CD11b Antigen/metabolism , Calcium-Binding Proteins/metabolism , Cell Movement/genetics , Disease Models, Animal , Gene Expression Regulation/drug effects , Lipopolysaccharides/pharmacology , MPTP Poisoning/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microfilament Proteins/metabolism , Microglia/drug effects , Monocytes/drug effects , Monocytes/metabolism , Receptors, CCR2/genetics , Receptors, CCR2/metabolism , Receptors, Interleukin-8A/genetics , Substantia Nigra/drug effects , Substantia Nigra/pathology , Time Factors , Tyrosine 3-Monooxygenase/metabolismABSTRACT
Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by a loss of dopaminergic neurons (DN) in the substantia nigra (SN). Several lines of evidence suggest that apoptotic cell death of DN is driven in part by non-cell autonomous mechanisms implicating microglial cells and inflammatory processes. Yet, how apoptotic DNs get removed by professional phagocytes and how this process modulates inflammatory processes are still unresolved issues. In this study, we investigated the role of MFGE8, a soluble factor involved in phagocytic recognition, in apoptotic DN clearance and neuroinflammation in PD. We report that glial expression of MFGE8 is enhanced in post-mortem PD brains compared to control individuals. Then, in vivo functional analysis of Mfge8 was assessed in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mouse model of PD using wild-type (WT) and Mfge8-deficient mice. Neuropathological analysis consisted in evaluating (i) the loss of nigral DN and striatal DN terminals, (ii) the extent of glial cell activation and (iii) the number of apoptotic profiles. In vivo microglial phagocytic activity was further assessed by measuring the engulfment of apoptotic DN preloaded with fluorescent latex beads. Here we show that Mfge8 deficiency neither impact the phagocytic clearance of apoptotic bodies nor change the overall neuropathological parameters (DN cell loss and glial cell activation). In summary, our data argue that MFGE8 is not likely involved in the phagocytic clearance of neuronal debris associated with nigrostriatal pathway injury.
Subject(s)
Antigens, Surface/metabolism , Apoptosis/physiology , Milk Proteins/metabolism , Neurons/pathology , Parkinson Disease/metabolism , Animals , Astrocytes/metabolism , Brain/metabolism , Brain/pathology , Humans , Immunohistochemistry , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Parkinson Disease/pathology , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
GH plays important pleiotropic roles in development, growth, metabolism, and aging of vertebrate species. Mouse mutants with altered GH signaling have been increasingly instrumental in studying somatotropic pathophysiology. However, the pulsatile characteristics of GH secretion are difficult to study in mice because catheterization is cumbersome and long-term serial sampling is limited by small body size and blood volume. We therefore developed an approach routinely applicable to mice, which detects endogenous, physiological GH pattern from randomly obtained spot samples. We determined individual hormone concentration in large groups of mice, ranked the data by magnitude, and statistically analyzed the resulting profiles. This revealed that the nadir-to-peak distribution of plasma GH concentration in mice was similar to other mammals, and that nycthemeral and sex differences existed as well. We found handling stress to be a potent immediate downregulator of circulating GH. We showed that samples need to be taken within seconds to reflect true endogenous levels, unaffected by stress. GH receptor/Janus kinase 2/signal transducer and activator of transcription 5 activation measured in the liver correlated strongly with plasma GH levels, but peak concentrations did not further increase the pathway activation. We applied this rank plot analysis to the GH-deficient and long-lived brain-specific IGF-1 receptor knockout (bIGF1RKO(+/-)) mouse mutant and found a high proportion of low GH concentrations, indicative of extended trough periods and rare peaks. Taken together, we showed that rank plot analysis is a useful method that allows straightforward studies of circadian endogenous GH levels in mice.
