Background
Fractalkine signaling in the CNS represents a unique microglial-neuron receptor-ligand pair, where fractalkine (CX
3CL1) is expressed by neurons and its cognate receptor CX
3CR1 is exclusively expressed by the CNS resident microglia [
1]. CX
3CL1 is a 373-amino acid protein, which contains an extracellular chemokine domain linked to a mucin-like stalk [
2,
3]. CX
3CL1 is functional in its membrane-bound form but can also be cleaved through metalloprotease (ADAM10/ADAM17) activity to produce a ~ 95-kDa soluble moiety [
4,
5]. It has been proposed that the heavily glycosylated mucin-like stalk of fractalkine provides rigidity to the chemokine domain for the adhesive potency of the chemokine domain during patrolling/crawling behavior [
6]. Several mouse models have been used to elucidate the role of fractalkine in mediating neurodegenerative and neuroinflammatory processes [
7‐
11].
CX
3CL1-CX
3CR1 signaling is regulated through direct neuron-microglia interaction, which acts to tether microglia until pathological activation, via an inflammatory influence, or through normal physiological activity, which disrupts this interaction through the cleavage of CX
3CL1 [
12,
13]. Disruption of CX
3CL1-CX
3CR1 signaling by chemical or genetic manipulation induces dramatic morphological activation and altered levels of scavenger/inflammatory receptors on the cell surface, alterations in pro-inflammatory chemokine production, and over-sensitization to pathological insults [
14‐
17].
Previous studies from our group have explored the role of CX
3CL1 signaling in the context of Alzheimer’s disease (AD) and related dementias. Notably, we found that disrupting the CX
3CL1-CX
3CR1 signaling axis reduces Aβ burden with concomitant increases in pro-inflammatory IL-1 and heightened microglial activation in both APPPS1/
Cx3cr1−/− and APPPS1/
Cx3cl1−/− transgenic mouse models of AD [
18]. Interestingly, this phenomenon was unaffected by the presence of soluble CX
3CL1 [
18]. In a separate study, converse to the protective anti-amyloid phenotype observed in APPPS1/
Cx3cr1−/− mice, deletion of
Cx3cr1 in hTau mice resulted in hyperphosphorylation and aggregation of tau, worsened cognitive function, and increased microglial inflammation [
17]. This effect was regulated via the same IL-1-p38 MAPK axis [
17,
19]. The dichotomy between the two studies likely stems from the type of pathological insults present, namely Aβ is extracellular whereas hyperphosphorylated tau exists primarily intraneuronally [
20]. The precise mechanism of how disrupting the CX
3CL1-CX
3CR1 signaling affects the microglia either to a beneficial (in the case of Aβ study) or to a detrimental degree (in the hTau study) is still unclear. However, it is possible that the IL-1β promotes phagocytic phenotype of microglia in clearing Aβ (in case of APPPS1/
Cx3cr1−/− and APPPS1/
Cx3cl1−/− mice), while causing collateral damage (for example, over-activation of p38 MAPK) in neurons and leading to tau hyperphosphorylation [
17‐
19]. Seemingly, contrary work demonstrated that
Cx3cl1 overexpression through viral transfection models reduces tau and α-synuclein pathology [
10,
21]. The present study seeks to determine if genetically expressing only the soluble chemokine domain of CX
3CL1 could prevent tau pathology in both chemical (LPS) and genetic (hTau) mouse models of tauopathy.
Discussion
CX
3CL1-CX
3CR1 represents a unique signaling axis between the microglia and neurons, which is profoundly involved in the suppression of innate inflammatory responses. Alterations in fractalkine signaling by chemical or genetic manipulations have dichotomous consequences within the context of canonical AD pathological outcomes. Notably, the absence of fractalkine signaling ameliorates Aβ plaque burden in APPPS1 transgenic mice [
30], but exacerbates intraneuronal tau pathology in hTau mouse model of pure tauopathy [
17], even though both events likely occur via dysregulation of IL-1β-p38 MAPK signaling pathway [
19]. Here, we demonstrate that the expression of the chemokine domain of CX
3CL1 does not suppress inflammation-induced tau pathology or mitigate microglial responses.
Earlier studies suggested that
Cx3cr1 deficiency increased tau phosphorylation in both LPS and hTau models of tau pathology [
17]. This suggests that the presence of CX
3CR1 may downregulate microglial pro-inflammatory signaling and mitigate inflammation-induced tau hyperphosphorylation. For reasons currently unknown, unlike
Cx3cr1−/− mice, the
Cx3cl1−/− mice demonstrate only a modest increase in AT8
+ tau following LPS administration. Tau phosphorylation in hTau/
Cx3cl1−/− mice seems to mimic hTau/
Cx3cr1−/− mice as previously reported [
17]. Furthermore, the expression of chemokine domain of fractalkine has virtually no beneficial effects on either LPS-mediated microglial morphological alterations or AT8/AT180 site tau phosphorylation. This observation suggests that in the absence of membrane-bound form, chemokine domain of the fractalkine may, in fact, disrupt normal microglia-neuron signaling, leading to downregulation and/or internalization of fractalkine receptor on the microglial cell surface. Our flow cytometry analysis reveals decreased microglial CX
3CR1 levels in the
Cx3cl1105Δ mice compared with Non-Tg or
Cx3cl1−/− and supports this hypothesis. Interestingly, a previous study observed prolonged downregulation of cell surface CX
3CR1 on aged microglia in response to LPS [
31]. This reduced CX
3CR1 on the CD11b
+ microglia corresponded with delayed recovery from sickness behavior, elevated IL-1β induction, and reduced TGFβ [
31]. Reduced
Cx3cr1 expression (both mRNA and protein) in monocytes was also reported following septic shock [
32]. This loss of monocyte-specific CX
3CR1, which causes sepsis-induced lethality in humans, compromised this cell’s ability to respond to a fractalkine challenge [
32]. While these and our current results suggested pro-inflammatory and pathological effects of reduced CX
3CR1 expression on the microglial cell surface, the exact intra-microglial alterations in
Cx3cl1105Δ mice will need to be further explored using isolated microglia and high-throughput single-cell RNA sequencing. Surprisingly, the
Cx3cl1105Δ mice also had an increased baseline expression of total tau. Finally, there may be the remote possibility of ligand-independent CX
3CR1 negatively influencing TLR4 signaling in immune cells (in
Cx3cl1−/− mice) and reducing pro-inflammatory cytokine secretion. Indeed, such un-liganded receptor function was recently reported for progesterone receptor B (without progesterone, acting alone) in the regulation of the function of estrogen receptor-α affecting the proliferation and survival of breast cancer cells following estradiol stimulation [
33]. Alternatively, the chemokine domain of CX
3CL1 has also been shown to induce intracellular signaling independent of CX
3CR1 via binding to αvβ3 integrins [
34].
Similar to our previously reported exacerbation of tau pathology in hTau/
Cx3cr1−/− mice [
17], neuronal fractalkine deletion seems to worsen tau pathology in hTau mice, although differences in tau phosphorylation were only detected at the AT8 (S202) site. Fractalkine deletion elevates the pro-inflammatory response from microglia in hTau/
Cx3cl1−/− and hTau/
Cx3cl1105Δ mice compared to hTau mice. Further, cognitive abnormalities are evident in aged hTau/
Cx3cl1−/− or hTau/
Cx3cl1105Δ mice regardless of increased production of the soluble chemokine domain of fractalkine in the latter group. Given that hTau mice display impaired performance in the Morris water maze at 12 months of age [
35], it is plausible that
Cx3cl1105Δ overexpression fails to prevent cognitive impairment in hTau mice.
Our results contrast with a previous report where AAV-transduced overexpression of soluble fractalkine rescued several pathological phenomena including the hyperphosphorylation of tau at multiple epitopes and microglial phenotypes in a mouse model of tauopathy, rTg4510 [
10]. The discrepancies between our study and Nash et al. could be due to a number of factors including the following: (1) Inducible AAV approach vs. our germline genetic system—their animal model had intact membrane-bound CX
3CL1, while the
Cx3cl1105Δ mice did not. (2) Differences in the structure of the soluble fractalkine moiety—in the AAV study, the mucin stalk of fractalkine was included, whereas, in our germline
Cx3cl1105Δ mice, only the soluble chemokine domain, without the mucin stalk, was present. A previous 3D structural analysis of different domains of CX
3CL1 has suggested that mucin stalk of CX
3CL1 is important for the presentation of the chemokine domain to the outer cell membrane and increases adhesive interaction between CX
3CL1 and CX
3CR1 [
6]. Therefore, lack of the mucin stalk in CX
3CL
105Δ may not be sufficient to restrict LPS-induced or hTau-mediated microglial activation [
36]. (3) Presence of endogenous CX
3CL1 in rTg4510 mice vs. the lack of it in
Cx3cl1105Δ mice—because of this, the levels of soluble CX
3CL1
105Δ levels in
Cx3cl1105Δ mice (which is comparable to that of Non-Tg (see Additional file
1: Figure S1B)) may be insufficient compared to significantly higher levels of soluble CX
3CL1 levels in the AAV study. (4) rTg4510 vs. hTau are two different types of tauopathy mouse models. In rTg4510 only, 4R-Tau with a P301L mutation is expressed and pathological tau is present at 13-fold higher than endogenous levels (AAV study), vs. only an approximate two- to threefold higher expression of all six isoforms, including both 3R and 4R tau, in hTau mice (current study). Based on the data from these two studies, we hypothesize that when there is a robust tau pathology (like in rTg4510 mice), the effect of soluble CX
3CL1 (containing the mucin stalk) may be beneficial and the benefits are discernable. We also speculate that this beneficial effect could be due, in part, to the contributions from the membrane-bound form of endogenous CX
3CL1, present in the rTg4510 mice, and the rigidity of the soluble form containing the mucin stalk facilitating the “anti”-inflammation. In contrast, hTau mice do not display as robust tau pathology as rTg4510 mice. Due to the complete lack of membrane-bound CX
3CL1 in our hTau/
Cx3cl1105Δ mice, CX
3CL
105Δ may not be as efficient and therefore leads to the downregulation of CX
3CR1 and exacerbation of neuroinflammation/tau pathology. Together, these interpretations suggest that both membrane-bound CX
3CL1 and the soluble form of fractalkine may make a concerted effort together to mediate both neuroinflammation and tau pathology.
Acknowledgements
We would like to acknowledge the Cleveland Clinic and University New Mexico Flow Cytometry, Imaging, and Behavioral Cores for their continued valuable assistance in acquiring and analyzing the data. We would like to thank Dr. Richard Ransohoff for his invaluable input in preparing this manuscript. We also like to acknowledge Dr. Surojit Paul and Mr. Sathyanarayanan Rajagopal for their assistance in performing the CX3CL1 ELISAs.