CX3CR1 deficiency exacerbates neuronal loss and impairs early regenerative responses in the target-ablated olfactory epithelium
Introduction
Olfactory sensory neurons (OSNs) in the nasal neuroepithelium are bipolar sensory neurons with ciliated dendritic nerve endings that are in direct contact with the external environment (Farbman and Buchholz, 1992). This cytoarchitectural feature allows for the immediate sampling of olfactory cues from the environment but also creates a vulnerability against pathogens and olfactory toxicants that may enter the nasal cavity. Indeed, OSN lifespan appears to depend on environmental conditions and the death of these cells is a relatively common event in the neuroepithelium (Moulton, 1974, Hinds et al., 1984, Mackay-Sim and Kittel, 1991). To counter continuous OSN degeneration, and thus maintain a receptive field for the sense of smell, the olfactory neuroepithelium has sustained the ability to effectively replace its neurons throughout adult life from a basal stem/progenitor cell compartment (Harding et al., 1977, Graziadei et al., 1979, Leung et al., 2007). Adult-born OSNs successfully regrow an axon into their central nervous system (CNS) target, the olfactory bulb, and restore appropriate connectivity with second-order neurons. Because of its accessibility, ease of manipulation and unsurpassed regenerative potential, the olfactory pathway provides an attractive model to study various aspects of neuronal injury and regeneration.
Tissue macrophages play an important role during injury and subsequent regenerative events in the olfactory neuroepithelium. Macrophage numbers rapidly increase following OSN degeneration and the influx of these phagocytes coincides with the time course of neuronal death (Graziadei et al., 1979, Suzuki et al., 1995, Cowan et al., 2001). In addition to removing degenerating OSNs from the neuroepithelium, macrophage activation and infiltration is also thought to be important for the induction and regulation of regenerative responses from the basal stem/progenitor cell compartment. Appropriately activated macrophages are a rich source of factors that influence neuronal survival and stem/progenitor cell proliferation (e.g. Nan et al., 2001, Borders et al., 2007a). In line with this, macrophage depletion exacerbates neuronal death and impairs regenerative tissue responses (Borders et al., 2007b). However, the molecular mechanisms involved in the cross-talk between neurons and macrophages as well as the regulation of macrophage activation profiles remain largely unclear.
We recently reported that CX3CL1/fractalkine is expressed on OSNs while its receptor, CX3CR1, is expressed on intraepithelial macrophages and putative dendritic cells (Ruitenberg et al., 2008, Vukovic et al., 2010); this allows for direct communication between epithelial neurons and these phagocytic immune cells. An important feature of CX3CL1 is that it is produced in a membrane-bound form (Bazan et al., 1997), from which the chemokine domain can be released through shedding by TACE/ADAM17 and ADAM10 (Garton et al., 2001, Tsou et al., 2001, Hundhausen et al., 2003). ADAM10 is thought to contribute to constitutive cleavage whereas TACE seems to mediate inducible shedding following injury or stress (Hundhausen et al., 2003). Together, these findings suggest that the shedding of CX3CL1 and release of the chemokine module may function as an early neuronal distress signal. Here, we used comparative histological and molecular analysis between wild-type and CX3CR1-deficient mice to test this hypothesis and further elucidate the physiological importance of CX3CL1-signaling in the olfactory neuroepithelium.
Section snippets
OSN death in response to bulbectomy
Immunofluorescent staining for OMP was performed to visualize mature OSN numbers in tissue sections from Cx3cr1+/+ and Cx3cr1gfp/gfp mice at 48 h after unilateral olfactory bulbectomy (OB-X; Figs. 1A–D). As expected, numerous OMP+ cells were found in the intermediate (neuronal) layer of the OE on the uninjured side, i.e. contralateral to olfactory bulbectomy, for both Cx3cr1+/+ and Cx3cr1gfp/gfp mice (Figs. 1A and C). Ipsilateral to the ablated olfactory bulb, a dramatic decline in OMP+ neurons
Discussion
The present study shows that the chemokine receptor CX3CR1 plays an important role in the macrophage response to injury within the primary olfactory pathway by maintaining an environment that reduces secondary cell death and allows for neuronal replacement from endogenous stem/progenitor cells. After bulbectomy, the vast majority of the mature (OMP+) olfactory sensory neurons in the olfactory epithelium dies via retrograde apoptosis (Cowan et al., 2001), resulting in macrophage recruitment and
Experimental animals
A total of 74 C57BL/6J mice (♂/♀; 2–4 months of age) were used in this study. Wild-type (Cx3cr1+/+), heterozygous (Cx3cr1gfp/+) and homozygous knock-in (Cx3cr1gfp/gfp) mice were generated from heterozygous Cx3cr1gfp breeding pairs (Jung et al., 2000). In Cx3cr1gfp mice, the coding sequence for enhanced green fluorescent protein (eGFP) was inserted into the Cx3cr1 coding region via targeted deletion, i.e. rendering the CX3CL1/fractalkine receptor non-functional and placing eGFP under Cx3cr1
Acknowledgments
The authors are grateful to Dr. Peter Mark and Mr. Greg Cozens (UWA) for assistance with quantitative PCR experiments. Work in the laboratory of M.J.R. was financially supported by the Australian Research Council (Discovery Grant DP0774113 to M.J.R.), The School of Biomedical Sciences at The University of Queensland and an Enabling Grant from the office of the DVC(R) of The University of Queensland (to M.J.R.). M.J.R. was additional supported by an ARC Postdoctoral Fellowship (2007–2009;
References (43)
- et al.
Tumor necrosis factor-alpha-converting enzyme (ADAM17) mediates the cleavage and shedding of fractalkine (CX3CL1)
J. Biol. Chem.
(2001) - et al.
Plasticity of connections of the olfactory sensory neuron: regeneration into the forebrain following bulbectomy in the neonatal mouse
Neuroscience
(1979) - et al.
Cues for apoptotic cell engulfment: eat-me, don't eat-me and come-get-me signals
Trends Cell Biol.
(2003) - et al.
Denervation in the primary olfactory pathway of mice. IV. Biochemical and morphological evidence for neuronal replacement following nerve section
Brain Res.
(1977) The disintegrin-like metalloproteinase ADAM10 is involved in constitutive cleavage of CX3CL1 (fractalkine) and regulates CX3CL1-mediated cell–cell adhesion
Blood
(2003)- et al.
CX3CR1 deficiency alters microglial activation and reduces beta-amyloid deposition in two Alzheimer's disease mouse models
Am. J. Pathol.
(2010) - et al.
Fractalkine-upregulated milk-fat globule EGF factor-8 protein in cultured rat microglia
J. Neuroimmunol.
(2005) - et al.
Production and neuroprotective functions of fractalkine in the central nervous system
Brain Res.
(2003) Interleukin-1 and neuronal injury: mechanisms, modification, and therapeutic potential
Brain Behav. Immun.
(2003)CX3CL1/fractalkine regulates branching and migration of monocyte-derived cells in the mouse olfactory epithelium
J. Neuroimmunol.
(2008)
Chemokines and chemokine receptors in neurological disease: raise, retain, or reduce?
Neurotherapeutics
Tumor necrosis factor-alpha-induced apoptosis in olfactory epithelium in vitro: possible roles of caspase 1 (ICE), caspase 2 (ICH-1), and caspase 3 (CPP32)
Exp. Neurol.
Phagocytic cells in the rat olfactory epithelium after bulbectomy
Exp. Neurol.
CX3CL1/fractalkine is released from apoptotic lymphocytes to stimulate macrophage chemotaxis
Blood
Tumor necrosis factor-alpha-converting enzyme mediates the inducible cleavage of fractalkine
J. Biol. Chem.
Lack of fibulin-3 alters regenerative tissue responses in the primary olfactory pathway
Matrix Biol.
Interleukin-1beta mediates proliferation and differentiation of multipotent neural precursor cells through the activation of SAPK/JNK pathway
Mol. Cell. Neurosci.
Fractalkine and CX(3)CR1 regulate hippocampal neurogenesis in adult and aged rats
Neurobiol. Aging
A new class of membrane-bound chemokine with a CX3C motif
Nature
Microglia-mediated neurotoxicity: uncovering the molecular mechanisms
Nat. Rev. Neurosci.
Macrophage depletion in the murine olfactory epithelium leads to increased neuronal death and decreased neurogenesis
J. Comp. Neurol.
Cited by (33)
Synthetic fragment (60–76) of RAGE improves brain mitochondria function in olfactory bulbectomized mice
2020, Neurochemistry InternationalCitation Excerpt :The expression reached a maximum level on the 20th day after bulbectomy and decreased in the neocortex on the 33rd day. We confirmed the previously published data about proinflammatory action of olfactory bulbectomy (Blomster et al., 2011). Simultaneously we found no evidence of decreased expression of inflammatory cytokines IL-1α, IL-1β and TNFα in the brain regions of OBX animals treated with either P1 or P2 (Fig. 3).
Fractalkine (CX3CL1) signaling and neuroinflammation in Parkinson's disease: Potential clinical and therapeutic implications
2020, Pharmacological ResearchCitation Excerpt :These findings further confirm that the effects of fractalkine signaling are greatly dependent on the model used, at least in neurodegeneration. Interestingly, a study has shown that the lack of CX3CR1 can promote neuronal death, decrease the proliferation of stem cell progenitors and enhance microglial activation in the olfactory epithelium upon bulbectomy [125]. Since hyposmia is an early prodromal manifestation of PD and that olfactory mucosa stem cells have increasingly gained attention as potential therapeutic PD targets [126], the role of fractalkine signaling in the olfactory bulb should be further investigated.
Genes dysregulated in the blood of people with Williams syndrome are enriched in protein-coding genes positively selected in humans
2020, European Journal of Medical GeneticsCitation Excerpt :The gene has been related to the pathophysiology of diabetes (Citro et al., 2015), hypertension (Martynowicz et al., 2014), and recurrent urinary tract infections (Zaffanello et al., 2010), which are common symptoms in WS. The gene is also widely expressed in neurons (Goczalik et al., 2008), being involved in neurogenesis and neuron differentiation (Edman et al., 2008; Blomster et al., 2011). NOTO.
NLRP3 inflammasome activation promotes the development of allergic rhinitis via epithelium pyroptosis
2020, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Subsequently, inflammatory microenvironment results in the injury and impaired regeneration of nasal epithelium, which further interrupts the function of olfactory region [5]. Except for eosinophils, innate immune response has been reported to play an important role on the survival and regeneration of olfactory neuroepithelium [6]. However, the molecular mechanism of innate immune molecules in the development and progression of AR has not been thoroughly studied.
Hesperidin reverses cognitive and depressive disturbances induced by olfactory bulbectomy in mice by modulating hippocampal neurotrophins and cytokine levels and acetylcholinesterase activity
2016, European Journal of PharmacologyCitation Excerpt :These data were in accordance with the data from Rinwa et al. (2013) and Poretti et al. (2015), showing that depressive disorder is associated with an increase of these pro-inflammatory cytokines. Moreover, this increase could be correlated with a decrease of neuroplasticity, as shown previously by Blomster et al. (2011) and Johnson and Sharma (2003), wherein an increase of pro-inflammatory cytokines was linked with a decrease of NGF and BDNF expression in mice. Together with the literature results, the current study supports and extends the thought that an inflammatory response, reflected by cytokine dysregulation in the brain regions implicated with memory and depressive alterations, could be an important neurobiological mechanism of OB-induced cognitive impairment and emotional alterations in mice.
Panax quinquefolium involves nitric oxide pathway in olfactory bulbectomy rat model
2014, Physiology and BehaviorCitation Excerpt :Apart from increased oxidative and nitrosative stress, we also found a significant enhancement in the markers of inflammation (TNF-α) and apoptosis (caspase-3) in both the cerebral cortex and hippocampal regions of OBX rats, suggesting neuro-inflammation induced apoptosis. Earlier, reports suggested that olfactory bulbectomy is linked with the generation of pro-inflammatory cytokines like TNF-α and IL-6 [48]. Studies from Hall and Macrides [49] found neuronal cell death (apoptosis) in different brain regions following olfactory bulbectomy.