Probes for CRE

The potassium channel Kv1.6 has recently been implicated as a major modulatory channel subunit expressed in primary nociceptors. Furthermore, its expression at juxtaparanodes (JXP) of myelinated primary afferents is induced following traumatic nerve injury as part of an endogenous mechanism to reduce hyperexcitability and pain-related hypersensitivity. In this study we compared two mouse models of constitutive Kv1.6 knock-out achieved by different methods: traditional gene trap via homologous recombination, and CRISPR-mediated excision. Both Kv1.6 knock-out mouse lines exhibited an unexpected reduction in sensitivity to noxious heat stimuli, to differing extents: the Kv1.6 mice produced via gene trap had a far more significant hyposensitivity. These mice (Kcna6lacZ ) expressed the bacterial reporter enzyme LacZ in place of Kv1.6 as a result of the gene trap mechanism and we found that their central primary afferent presynaptic terminals developed a striking neurodegenerative phenotype involving accumulation of lipid species, development of 'meganeurites' and impaired transmission to dorsal horn wide dynamic range (WDR) neurons. The anatomical defects were absent in CRISPR-mediated Kv1.6 knock-out mice (Kcna6 -/-) but were present in a third mouse model expressing exogenous LacZ in nociceptors under the control of a Nav1.8-promoted Cre recombinase. LacZ reporter enzymes are thus intrinsically neurotoxic to sensory neurons and may induce pathological defects in transgenic mice, which has confounding implications for the interpretation of gene knock-outs using lacZ Nonetheless, in Kcna6 -/- mice not affected by LacZ, we demonstrated a significant role for Kv1.6 regulating acute noxious thermal sensitivity, and both mechanical and thermal pain-related hypersensitivity after nerve injury.SIGNIFICANCE STATEMENTIn recent decades the expansion of technologies to experimentally manipulate the rodent genome has contributed significantly to the field of neuroscience. While introduction of enzymatic or fluorescent reporter proteins to label neuronal populations is now commonplace, often potential toxicity effects are not fully considered. We show a role of Kv1.6 in acute and neuropathic pain states through analysis of two mouse models lacking Kv1.6 potassium channels, one with additional expression of LacZ and one without. We show that LacZ reporter enzymes induce unintended defects in sensory neurons, with an impact on behavioural data outcomes. To summarise we highlight the importance of: Kv1.6 in recovery of normal sensory function following nerve injury, and careful interpretation of data from LacZ reporter models.

Purpose: As skeletal maturity is approached, long bone elongation draws to a close and the cartilaginous growth plate (GP) ossifies and fuses as bone bridges form. This is likely a pivotal moment for the appendicular skeleton, but our mechanistic appreciation of how this process is orchestrated is limited. We have been studying how chondrocytes integrate biological cues, such as growth factor signalling, and mechanical forces, and have investigated the mechanosensitivity of epiphyseal fusion and roles for putative mechanotransduction machinery, including the primary cilium, in these contexts. Here we asked whether primary cilia have a mechanotransduction role in the juvenile GP and adolescent epiphyseal fusion. Methods: We used an inducible aggrecan (ACAN) Cre mouse model, enabling temporal deletion of the core ciliary protein IFT88 in cartilage to investigate GP narrowing dynamics and closure from 4 - 10 weeks of age. Both control (Ift88fl/fl) and cKO (Ift88fl/fl;ACANCreERT2) were injected with tamoxifen (I.P.). Cre activity was validated using a ROSA26TdTomato reporter line. Animals were exposed to (i) sciatic and femoral double neurectomy (DN) to off-load the right hind limb (immobilised DN) whilst the left bears full weight (contralateral DN) at 8 weeks of age, or (ii) voluntary wheel exercise between 8 and 10 weeks of age. Joints were scanned by μCT before histomorphometric analyses of tibial GP using Safranin-O/fast green, TUNEL, Collagen type X (ColX) immunohistochemistry, Von Kossa and TRAP. Cryosections of mouse GPs were analysed by confocal microscopy to investigate primary cilia prevalence and RNA scope was used to identify molecular mechanisms in situ. Medians +/- 95% confidence intervals quoted throughout below, Two-way ANOVA statistical comparisons. Results: We have, for the first time, investigated the role of cilia beyond 4 weeks of age. μCT analysis showed GP length in wild-type mice reduces from ∼260 μm to 130 μm between 4 and 10 weeks of age. Deletion of IFT88 in juvenile mice at 4 or 6 weeks of age resulted in longer GPs in cKO mice at every timepoint compared with control mice (Fig. 1A, 1st and 2nd panel and 1B). Thus, two weeks after tamoxifen, cKO GP lengths were not statistically significantly different to controls at time of treatment, indicating inhibition of GP closure. Deleting IFT88 at 8 weeks of age also resulted in longer GPs (p< 0.0001, n=12 controls, n=23 cKO). Interestingly, some cKO mice exhibited extremely elongated GPs at the edges of the tibia, which appeared as large holes by μCT (Fig. 1A), whilst the centre of the GP appeared less affected. Histology confirmed longer GPs were predominantly characterised by increases in hypertrophic chondrocyte populations. The large, often bi-lateral “holes”, observed by μCT were largely filled with disorganised hypertrophic chondrocytes, as indicated by IHC labelling for ColX. Interestingly limb immobilisation, (DN), at 8 weeks of age, rescued the GP phenotype observed in IFT88 cKO mice (Fig. 1A, 2nd and 3rd panels, and 1C), whilst the contralateral, unoperated (increased load-bearing) limb exhibited bi-lateral failure of ossification, similar to that observed in IFT88 cKO mice. Compared with naïve controls, wheel-exercised mice also displayed elongated GP (p< 0.0001, n=12 controls, n=10 wheel exercised) (Fig. 1A, 4th panel, and 1C) at 10 weeks of age. These expanded GP were, again, most pronounced at the edges of the tibia, whilst the centre of the GP appeared less affected and again was largely filled with disorganised, differentiated, ColX positive hypertrophic chondrocytes. In both wheel exercised and IFT88 cKO mice, regions of failed ossification, but not middle regions, were associated with loss of osteoclast activity. Confocal imaging and analysis revealed a statistically significant (p< 0.001) decrease in cilia positive cells in wheel exercised mice (32.9%, n=5) compared with control (40.7%, n=4) and IFT88cKO mice (p< 0.0001, (23.4%, n=4) compared with controls (40.7% n=4) at 10 weeks of age. Ongoing experiments are investigating 3D spatial analysis of fusion mechanisms, and the status of ciliary Hh signalling (Gli1, by RNAscope) within GP from control, DN, exercised, and cKO mice to dissect the apparently negative, regulatory role IFT88 is plays in the mechanical regulation of epiphyseal fusion. Conclusions: We conclude that IFT88 unequivocally plays a role in GP closure, its removal resulting in failed ossification of the GP, without disruption to chondrocytic lineage differentiation. This phenomenon, observed in cKO animals, is mechanosensitive with limb immobilisation rescuing the phenotype, suggesting, paradoxically, that IFT88 is dampening a mechanically-induced signal in the GP. Wheel exercise also resulted in impaired ossification thus these data collectively unveil both the acute response of the adolescent mouse GP to exercise and, through Ift88 deletion (cKO), a novel mechanoregulatory mechanism orchestrated by ciliary IFT. The effects of altered mechanics and mechanotransduction are most pronounced in the hypertrophic zone where cells are apparently trapped short of transdifferentiation. Osteoclast recruitment and/or activity is impaired, and bone formation inhibited. These Results may have implications for our understanding of hypertrophic chondrocyte biology in articular cartilage in OA. Moreover, it has been proposed that changes to mechanical inputs during adolescence and associated cam morphology contribute to hip OA development. In adolescent patient cohorts, high levels of exercise lead to cartilaginous hypertrophy, epiphyseal extension, cam development, and reduced rates of GP closure. Femoral and tibial epiphyseal extension has also been observed in adolescent athletes that sustain repetitive trauma through high intensity exercise. This research is crucial to a holistic understanding of skeletal mechano-biological health, and the effects of exercise, on the maturing appendicular skeleton

Purpose: The integration of external cues, such as mechanics, with intrinsic cell signalling programmes, such as hedgehog (Hh) signalling, is crucial for the development, maturation and homeostasis of articular cartilage. Activation of Hh signalling in adulthood and pathophysiological mechanics, have both been associated with the development of murine and human OA. But, how chondrocytes might transduce and integrate these cues remains unknown. A microtubule-based organelle, the primary cilium, most noted for its crucial role in Hh signalling, is assembled by chondrocytes and possesses a devoted trafficking machinery, IntraFlagellar Transport or IFT. In vitro studies indicate chondrocyte helps tune the anabolic matrix response to compression and the response to Hh ligand. In vivo, the primary cilium has been proposed to be a platform for the integration of mechanics and Hh signalling in musculoskeletal tissues. While constitutive and peri-natal disruption of ciliary proteins, Hh signalling and altered mechanics, all drastically alter joint development in vivo, the influence of IFT in adult cartilage homeostasis remains unknown. Methods: IFT88 was targeted using a cartilage-specific, inducible mouse line (ACANCreERT2;Ift88fl/fl : cKO hereafter). Cre activity was validated by qPCR, RNA scope and a ROSA26tdtomato reporter line. Ift88fl/fl mice, also receiving I.P injections of tamoxifen, were used as controls. Tibial articular cartilage was assessed 2, 14 or 26 weeks-post tamoxifen, at 8, 10, 22 and 34 weeks of age respectively, using histomorphometric analyses, including measurements of articular cartilage thickness, relative calcification, subchondral bone thickness, and OARSI score by means of immunohistochemistry (IHC). The surgical DMM model, which destabilises the joint, was performed at 10 weeks of age. To explore the role of physiological mechanics, mice were allowed two weeks of voluntary wheel exercise immediately following tamoxifen administration at 8 weeks of age. qPCR was performed on micro dissected articular cartilage at 10 weeks of age in control and cKO. RNAscope was performed on cryosections of articular cartilage from 10 week old mice control and cKO. Means ± S.D are quoted throughout, Mann-Whitney U-test or Fisher’s test were used for statistical comparisons. Results: In our previous OARSI abstract of 2020 we described the phenotype arising in IFT88 cKO mice. Here we outline this in further detail and with an exploration of underlying mechanisms. Tamoxifen treatment of cKO mice resulted in a 50% reduction of Ift88 mRNA in articular cartilage (p=0.02, n=6 control, 14 cKO). Ift88 (cKO) mice had thinner medial articular cartilage (MAC), compared with controls, at all 5 time-points (Fig 1.A) In control mice, MAC thickness increased from 102.57μm (95% CI [94.30, 119.80]) at 8 weeks of age to 108.68 +/- (95% CI [101.32, 116.42]) at 10 weeks of age. Tamoxifen treatment, at 8 weeks of age, inhibited this increase in cKO mice (MC thickness at 10 weeks was 96.20 μm (95% CI [90.04, 102.36]), p=0.02, compared with 10 week ctrl, n=7). By 22 weeks of age mean MAC thickness in cKO was 90.16μm (95% CI [87.11, 93.22]) compared with 111.60μm (95% CI [104.34, 118.79]) in control animals (p=0.0002, n= 7 and 10 respectively). By 34 weeks MAC had continued to thin to 84.55μm (95% CI [75.43, 93.67]) in cKO, but this was now associated with surface damage and osteophyte formation. In the most extreme case, MAC was completely lost (Fig.1B). In contrast, lateral plateau thickness and OARSI score were unaffected. Calcified cartilage (below the tidemark) progressively increases on both plateaus, between 6 and 22 weeks of age and at all time-points thinning was attributable to the relative loss of calcified cartilage implying a failure of calcification. IHC analyses revealed no striking differences in collagen X expression, NITEGE neoepitope. There were no measurable increases in subchondral bone thickness or changes in osteoclastic activity in cKO mice. 12 weeks post DMM, OARSI scores were statistically significantly higher in cKO (29.83 +/- 7.69) than control (22.08 +/- 9.30, p< 0.05, n= 15 both groups). Two weeks of voluntary wheel exercise rescued cartilage atrophy in cKO mice (p< 0.0001), whilst no change was observed in controls. RNA isolated from microdissected articular cartilage of 10 week old control and cKO mice, two weeks post tamoxifen, revealed a statistically significant correlation between Ift88 and Tcf7l2 expression after Bonferroni correction (p=0.026). Ctgf, Gli2 and Enpp1 were also positively correlated with Ift88 expression before correction (p=0.002, p=0.0037, and p=0.009 respectively). RNA scope analysis of AC found a statistically significant (p< 0.0001, n=4 in both groups) decrease in Ift88 positive cells in cKO (27.78%) compared with controls (45.18%), whilst also showing an increase in Gli1 positive cells in cKO (50.42%) compared with controls (23.63%) (p< 0.0001, n=4 in both groups). Conclusions: Progressive thickening and calcification in the mouse medial compartment illustrates the continued mechanoadaptation of adolescent and adult articular cartilage. Depletion of the ciliary gene Ift88 inhibits medial articular cartilage thickening, leading to atrophy, which then predisposes the joint to spontaneous OA. The lateral compartment is relatively unaffected. We propose this may be due, in part, to disruption of mechanotransduction and downstream anabolic remodelling in medial cartilage. Deletion of Ift88 impairs the progressive calcification of articular cartilage, in both compartments, which may be due to disruption of Hh signalling, which is also mechanosensitive. Ift88 expression was correlated with Tcf7l2, previously shown to interact and influence Hh signalling pathways in cartilage. On-going experiments are aiming to dissect the relative roles of IFT, mechanics and Hh in the context of adult cartilage. We conclude that Ift88 is crucial to post-natal articular cartilage homeostasis and chondroprotective against OA.