Lien Beckers

Large-scale genetic studies have identified numerous genetic risk factors that suggest a central role for innate immune cells in susceptibility to Alzheimer’s disease (AD). CD33, an immunomodulatory transmembrane sialic acid binding protein expressed on myeloid cells, was identified as one such genetic risk factor associated with Alzheimer’s disease. Several studies explored the molecular outcomes of genetic variation at the CD33 locus. It has been determined that the risk variant associated with AD increases the expression of the large isoform of CD33 (CD33M) in innate immune cells and alters its biological functions. CD33 is thought to signal via the interaction of its ITIM motif and the protein tyrosine phosphatase, SHP-1. Here, we utilize different molecular and computational approaches to investigate how AD-associated genetic variation in CD33 affects its interaction with SHP-1 in human microglia and microglia-like cells. Our findings demonstrate a genotype-dependent interaction between CD33 and SHP-1, which may functionally contribute to the AD risk associated with this CD33 variant. We also found that CD33-PTPN6 (SHP-1) gene–gene interactions impact AD-related traits, while CD33-PTPN11 (SHP-2) interactions do not.

Large-scale genetic studies have identified numerous genetic risk factors that suggest a central role for innate immune cells in susceptibility to Alzheimer’s disease (AD). CD33, an immunomodulatory transmembrane sialic-acid binding protein expressed on myeloid cells, was identified as one such genetic risk factor associated with Alzheimer’s disease. Several studies explored the molecular outcomes of genetic variation at the CD33 locus. It has been determined that the risk variant associated with AD increases the expression of the large isoform of CD33 (CD33M) in innate immune cells and alters its biological functions. CD33 is thought to signal via the interaction of its ITIM domain and the protein tyrosine phosphatase, SHP-1. Here, we utilize different molecular and computational approaches to investigate how AD-associated genetic variation in CD33 affects its interaction with SHP-1 in human microglia and microglia-like cells. Our findings demonstrate a genotype-dependent interaction between CD33 and SHP-1, which may functionally contribute to the AD risk associated with this CD33 variant. We also found that gene-gene interactions impact AD-related traits.

Microglia play a central role in most neurological disorders, but the impact of microgliosis on brain environment and clinical functions is not fully understood. Mice lacking multifunctional protein-2 (MFP2), a pivotal enzyme in peroxisomal β-oxidation, develop a fatal disorder characterized by motor problems similar to the milder form of MFP2 deficiency in humans. The hallmark of disease in mice is the chronic proliferation of microglia in the brain, but molecular pathomechanisms that drive rapid clinical deterioration in human and mice remain unknown. In the present study, we identified the effects of specific deletion of MFP2 from microglia in the brain on immune responses, neuronal functioning, and behavior. We created a novel Cx3cr1-Mfp2−/− mouse model and studied the impact of MFP2 deficiency on microglial behavior at different ages using immunohistochemistry and real-time PCR. Pro- and anti-inflammatory responses of Mfp2−/− microglia were assessed in vitro and in vivo after stimulation with IL-1β/INFγ and IL-4 (in vitro) and LPS and IL-4 (in vivo). Facial nerve axotomy was unilaterally performed in Cx3cr1-Mfp2−/− and control mice, and microglial functioning in response to neuronal injury was subsequently analyzed by histology and real-time PCR. Finally, neuronal function, motor function, behavior, and cognition were assessed using brainstem auditory evoked potentials, grip strength and inverted grid test, open field exploration, and passive avoidance learning, respectively. We found that Mfp2−/− microglia in a genetically intact brain environment adopt an inflammatory activated and proliferative state. In addition, we found that acute inflammatory and neuronal injury provoked normal responses of Mfp2−/− microglia in Cx3cr1-Mfp2−/− mice during the post-injury period. Despite chronic pro-inflammatory microglial reactivity, Cx3cr1-Mfp2−/− mice exhibited normal neuronal transmission, clinical performance, and cognition. Our data demonstrate that MFP2 deficiency in microglia causes intrinsic dysregulation of their inflammatory profile, which is not harmful to neuronal function, motor function, and cognition in mice during their first year of life.

It is becoming evident that microglia, the resident immune cells of the central nervous system (CNS), are active contributors in neurological disorders. Nevertheless, the impact of microgliosis on neuropathology, behavior and clinical decline in neuropathological conditions remains elusive. A mouse model lacking multifunctional protein-2 (MFP2), a pivotal enzyme in peroxisomal β-oxidation, develops a fatal disorder characterized by motor problems similar to the milder form of human disease. The molecular mechanisms underlying neurological decline in men and mice remain unknown. The hallmark of disease in the mouse model is chronic proliferation of microglia in the brain without provoking neuronal loss or demyelination. In order to define the contribution of Mfp2^−/− neural cells to development of microgliosis and clinical neuropathology, the constitutive Mfp2^−/− mouse model was compared to a neural selective Nestin-Mfp2^−/− mouse model. We demonstrate in this study that, in contrast to early-onset and severe microgliosis in constitutive Mfp2^−/− mice, Mfp2^+/+ microglia in Nestin-Mfp2^−/− mice only become mildly inflammatory at end stage of disease. Mfp2^−/− microglia are primed and acquire a chronic and strong inflammatory state in Mfp2^−/− mice whereas Mfp2^+/+ microglia in Nestin-Mfp2^−/− mice are not primed and adopt a minimal activation state. The inflammatory microglial phenotype in Mfp2^−/− mice is correlated with more severe neuronal dysfunction, faster clinical deterioration and reduced life span compared to Nestin-Mfp2^−/− mice. Taken together, our study shows that deletion of MFP2 impairs behavior and locomotion. Clinical decline and neural pathology is aggravated by an early-onset and excessive microglial response in Mfp2^−/− mice and strongly indicates a cell-autonomous role of MFP2 in microglia.

Purpose Activation of the innate immune system plays a significant role in pathologies of the central nervous system (CNS). In order to follow disease progression and evaluate effectiveness of potential treatments involved in neuroinflammation, it is important to track neuroinflammatory markers in vivo longitudinally. The translocator protein (TSPO) is used as a target to image neuroinflammation as its expression is upregulated in reactive glial cells during CNS pathologies. However, it remains unclear in which microglial phenotypes TSPO levels are upregulated, as microglia can display a plethora of activation states that can be protective or detrimental to the CNS. Procedures We assessed the levels of TSPO transcripts in cultured microglia that were polarized into pro- and anti-inflammatory states in vitro and in the brain of mice in which an anti-inflammatory environment was induced in vivo. In addition, we used a mouse model of peroxisomal multifunctional protein-2 (MFP2) deficiency that exhibits widespread neuroinflammation despite no neuronal loss and monitored TSPO expression by immunohistochemistry and by imaging using the TSPO radiotracer [¹⁸F]DPA-714. Results TSPO expression was selectively increased in so-called classically activated or M1 microglia but not in alternatively activated or M2 microglia in vitro. In agreement, TSPO transcript levels were not induced in an anti-inflammatory brain environment. We found that both transcript and protein levels of TSPO are significantly increased in the brain of Mfp2 ^−/− compared to those of the control mice and TSPO immunoreactivity colocalized predominantly with microglia in Mfp2 ^−/− brain. In vitro and ex vivo autoradiography in Mfp2 ^−/− mice using the TSPO radiotracer [¹⁸F]DPA-714 confirmed increased expression of TSPO. These data demonstrate that TSPO imaging reveals microgliosis in non-neurodegenerative brain pathologies. Conclusions We show that induced TSPO expression marks a pro-inflammatory brain environment that is not necessarily accompanied by neuronal loss.

The functional diversity and molecular adaptations of reactive microglia in the chronically inflamed central nervous system (CNS) are poorly understood. We previously showed that mice lacking multifunctional protein 2 (MFP2), a pivotal enzyme in peroxisomal β‐oxidation, persistently accumulate reactive myeloid cells in the gray matter of the CNS. Here, we show that the increased numbers of myeloid cells solely derive from the proliferation of resident microglia and not from infiltrating monocytes. We defined the signature of Mfp2^−/− microglia by gene expression profiling after acute isolation, which was validated by quantitative polymerase reaction (qPCR), immunohistochemical, and flow cytometric analysis. The features of Mfp2^−/− microglia were compared with those from SOD1^G93A mice, an amyotrophic lateral sclerosis model. In contrast to the neurodegenerative milieu of SOD1^G93A spinal cord, neurons were intact in Mfp2^−/− brain and Mfp2^−/− microglia lacked signs of phagocytic and neurotoxic activity. The chronically reactive state of Mfp2^−/− microglia was accompanied by the downregulation of markers that specify the unique microglial signature in homeostatic conditions. In contrast, mammalian target of rapamycin (mTOR) and downstream glycolytic and protein translation pathways were induced, indicative of metabolic adaptations. Mfp2^−/− microglia were immunologically activated but not polarized to a pro‐ or anti‐inflammatory phenotype. A peripheral lipopolysaccharide challenge provoked an exaggerated inflammatory response in Mfp2^−/− brain, consistent with a primed state. Taken together, we demonstrate that chronic activation of resident microglia does not necessarily lead to phagocytosis nor overt neurotoxicity. GLIA 2015;63:1606–1620