Motor deficits in TDP-43Q331K mice correlate with increases in astrocytes and microglia during disease progression
To monitor the decline in motor performance during disease onset and progression in TDP-43Q331K mice, we performed hind-limb grip strength tests in animals. At 3 months, there was no difference in hind-limb grip strength between NTg, TDP-43WT and TDP-43Q331K mice (Fig. 1a). However, by 10 months, we observed a significant reduction in hind-limb grip strength in TDP-43Q331K mice when compared with NTg and TDP-43WT mice (~ 30% reduction, n = 15, ****P < 0.0001, ++++P < 0.0001; Fig. 1a). Furthermore, at 16 months, there was a progressive decline in hind-limb grip strength in TDP-43Q331K mice (~ 45% reduction, n = 15, ****P < 0.0001, ++++P < 0.0001; Fig. 1a) when compared with NTg and TDP-43WT mice. Importantly, we found that the decline in hind-limb grip strength in TDP-43Q331K mice closely correlated with the increase in immunoreactive area of astrocytes using immunofluorescence staining in the lumbar spinal cords at 10 months (~ 200% increase, n = 4, **P < 0.01; Fig. 1b, c) and 16 months (~ 380% increase, n = 4, ****P < 0.0001; Fig. 1b, c). Similarly, we also found that the decline in hind-limb grip strength in TDP-43Q331K mice closely correlated with the increase in immunoreactive area of microglia and number of activated microglia in the lumbar spinal cord at 10 months (140~230% increase, n = 4, **P < 0.01, ***P < 0.001, ****P < 0.0001; Fig. 1d–f) and 16 months (130~280% increase, n = 4, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; Fig. 1d–f). Taken together, these data reveal an age-related decline in hind-limb grip strength of TDP-43Q331K mice associated with increased glial activation in the lumbar spinal cord.
Components of the classical/lectin pathways of complement are upregulated along with decreased expression levels of complement regulator CD55 in TDP-43Q331K mice
The complement system is part of the innate immune system that can contribute to neuroinflammation in many neurodegenerative diseases, including ALS [8]. Previous studies, including our own, have identified major complement components are upregulated in the lumbar spinal cord of hSOD1G93A mice [15]. However, there is no comprehensive overview of complement system in different animal models of ALS other than hSOD1 transgenic mice [8]. Therefore, we measured the mRNA levels of key components of the classical/lectin pathway (C1qB and C4), alternative pathway (fB), the central component to all pathways (C3) and the complement regulators (CD55 and CD59a) in the lumbar spinal cord of NTg, TDP-43WT and TDP-43Q331K mice using quantitative real-time PCR during disease progression of ALS (3, 10 and 16 months).
Quantitative real-time PCR analyses showed significant increases of the C1qB transcript in TDP-43Q331K mice by 1.3-fold at 10 months of age when compared to NTg and TDP-43WT mice; at 16 months of age, the increase was 1.5-fold and 1.4-fold when compared to NTg and TDP-43WT mice, respectively (blue bar compared to orange and green bars respectively in Fig. 2a; n = 5, ***P < 0.001, ****P < 0.0001). C4 transcript was also increased by 2.4-fold and 3.3-fold at 10 months of age and 2.0-fold and 2.7-fold at 16 months of age when compared to NTg and TDP-43WT mice, respectively (blue bar compared to orange and green bars respectively in Fig. 2b; n = 5, ***P < 0.001, ****P < 0.0001). By contrast, fB did not show any significant changes at 10 and 16 months of age in TDP-43Q331K mice when compared to NTg and TDP-43WT mice (blue bar compared to orange and green bars respectively in Fig. 2c; n = 5, P > 0.05). The central component of complement, C3, was also increased in the lumbar spinal cord of TDP-43Q331K mice, with a 1.6-fold and 1.4-fold increase at 10 months of age when compared to NTg and TDP-43WT mice, respectively; at 16 months of age, the increase was 1.7-fold when compared to both genotypes (blue bar compared to orange and green bars respectively in Fig. 2d; n = 5, ***P < 0.001, ****P < 0.0001).
The negative regulators of the complement system, CD55 and CD59a, were also investigated due to their importance in maintaining homeostasis and keeping the complement system in its proper physiological state in response to altered physiological conditions (i.e. infection and/or neurodegeneration). CD55 mRNA expression in TDP-43Q331K mice was decreased at 10 months of age by 0.3-fold and 0.4-fold, and by 0.4-fold and 0.5-fold at 16 months of age, when compared with NTg and TDP-43WT mice (blue bar compared to orange and green bars respectively in Fig. 2e; n = 5, *P < 0.05, **P < 0.01, ****P < 0.0001). By contrast, CD59a mRNA expression did not significantly alter at 10 and 16 months of age in TDP-43Q331K mice when compared to NTg and TDP-43WT mice (blue bar compared to orange and green bars respectively in Fig. 2f; n = 5, P > 0.05). These results suggest widespread complement perturbation occurs in the lumbar spinal cord of TDP-43Q331K mice, which may contribute to glial activation and neuroinflammation, and ultimately disease progression in this model.
Upregulation of C1q at 16 months of age was also confirmed using immunofluorescence, where there was marked increase in TDP-43Q331K mice compared with NTg and TDP-43WT mice (blue bar compared to orange and green bars in Fig. 3a; n = 4, *P < 0.05). We also observed that the marked increase of C1q in TDP-43Q331K mice was localised to motor neurons and microglia (white arrows in Fig. 3d, g), compared with NTg and TDP-43WT mice where little to no C1q was observed (Fig. 3b, c, e, f). We did not observe C1q on astrocytes in either NTg, TDP-43WT or TDP-43Q331K mice (Fig. 3h–j).
The terminal complement pathway C5a receptor, C5aR1, is upregulated and expressed on motor neurons and microglia in the lumbar spinal cord of TDP-43Q331K mice
Previous studies have shown increases in C5aR1 expression in the central nervous system of multiple rodent models of ALS, with many studies suggesting a pathogenic role for C5aR1 in the disease progression of ALS in hSOD1G93A mice [9, 10, 12, 20]. C5a, the ligand for C5aR1, is an activation fragment of the terminal complement cascade that is rapidly generated following complement cascade initiation [21]. We therefore examined the protein levels of C5a in the spinal cord of TDP-43Q331K mice using enzyme-linked immunosorbent assay as a biomarker for terminal complement activation. Interestingly, the results showed no change in C5a protein levels in TDP-43Q331K mice at 16 months of age when compared to NTg and TDP-43WT mice (blue bar compared to orange and green bars in Fig. 4a; n = 5, P > 0.05). In contrast to protein levels of C5a, C5aR1 mRNA expression was significantly increased by 1.4-fold and 1.2-fold at 10 months of age and by 1.8-fold and 1.6-fold at 16 months of age, when compared to NTg and TDP-43WT mice (blue bar compared to orange and green bars in Fig. 4b; n = 5, *P < 0.05, ***P < 0.001, ****P < 0.0001).
We next aimed to determine the cellular localisation of C5aR1 that could explain the increased expression seen in TDP-43Q331K mice. To achieve this, we performed immunolabelling for C5aR1 on the lumbar spinal cord from NTg, TDP-43WT and TDP-43Q331K mice. These sections were immunostained for C5aR1 with specific cellular markers to identify motor neurons (anti-ChAT), astrocytes (anti-GFAP) and microglia (anti-Iba-1). C5aR1 localised to ChAT-positive motor neurons and Iba-1-positive microglia in NTg, TDP-43WT and TDP-43Q331K mice at 16 months of age (white arrows in Fig. 4c–e for motor neurons and Fig. 4f–h for microglia), whereas it was not observed in GFAP-positive astrocytes (Fig. 4i–k). Together, the results above indicate that C5a-C5aR1 signalling could play a role in facilitating microglia activation and phagocytosis ultimately leading to motor neuron death in these animals.
Dysregulation of complement in the tibialis anterior muscle of TDP-43Q331K mice
In addition to showing altered levels of complement components in the spinal cord of TDP-43Q331K mice, we also investigated the level of major complement components in the TA muscle of TDP-43Q331K mice, as it has been shown previously in hSOD1G93A mice that complement is upregulated in this muscle. To investigate this, we measured the mRNA levels of key components of the complement pathways, which include the classical/lectin (C1qB and C4), alternative (fB) and terminal pathways (C5a and C5aR1), as well as the major complement regulators (CD55 and CD59a) using quantitative real-time PCR and enzyme-linked immunosorbent assay for C5a in TA muscle of TDP-43Q331K mice during disease progression of ALS (3, 10 and 16 months).
C1qB and C4 transcripts were significantly increased by 1.6-fold and 1.9-fold when compared to NTg mice and by 1.5-fold when compared to TDP-43WT mice at 10 months of age, respectively (blue bar compared to orange and green bars in Fig. 5a, b; n = 5, *P < 0.05, **P < 0.01, ***P < 0.001). C1qB and C4 transcripts were also increased by 1.7-fold and 1.5-fold when compared to NTg mice and by 1.7-fold and 1.9-fold when compared to TDP-43WT mice at 16 months of age, respectively (blue bar compared to orange and green bars in Fig. 5a, b; n = 5, *P < 0.05, **P < 0.01, ***P < 0.001). In addition to C1qB and C4, fB also displayed a marked increase in mRNA levels by 1.4-fold and 1.3-fold at 10 months of age when compared to NTg and TDP-43WT mice, whereas 1.5-fold increase at 16 months of age when compared to TDP-43WT mice (blue bar compared to orange and green bars in Fig. 5c; n = 5, *P < 0.05, **P < 0.01). By contrast, C3 was surprisingly decreased by 0.3-fold in TA muscle of TDP-43Q331K mice when compared to NTg and TDP-43WT mice at 10 months of age (blue bar compared to orange and green bars in Fig. 5d; n = 5, *P < 0.05, **P < 0.01). The regulator, CD55, was increased by 1.3-fold at 10 months of age in TDP-43Q331K mice when compared to NTg mice (blue bar compared to orange bar in Fig. 5e; n = 5, *P < 0.05), while there was no significant change in CD59a in TDP-43Q331K mice when compared to NTg and TDP-43WT controls (Fig. 5f; n = 5, P > 0.05). Lastly, the terminal complement pathway component, C5a, and its receptor C5aR1 were investigated. The results showed significant increases in C5a protein at 16 months of age by 1.8-fold and 2.0-fold when compared to NTg and TDP-43WT mice respectively (blue bar compared to orange and green bars in Fig. 5g; n = 5, ***P < 0.001). C5aR1 mRNA expression was also significantly increased by 1.7-fold and 1.6-fold at 10 months of age and by 1.7-fold and 2.0-fold at 16 months of age when compared to NTg and TDP-43WT controls, respectively (blue bar compared to orange and green bars in Fig. 5h; n = 5, *P < 0.05, **P < 0.01, ***P < 0.001). Taken together, these results suggest that dysregulation of the complement system also occurs in the TA muscle of TDP-43Q331K mice, which could contribute to the disease pathology in these animals.
Motor dysfunction in TDP-43Q331K mice strongly correlates with lumbar spinal cord levels of C1qb, C3 and C5aR1
We next examined if there was any correlation between major complement transcript levels to the hind-limb grip strength of TDP-43Q331K mice. To investigate this aspect, we performed a Pearson correlation to measure the strength of the linear relationship between C1qB, C3 and C5aR1 mRNA levels in the lumbar spinal cord and TA muscle of NTg, TDP-43WT and TDP-43Q331K mice with their hind-limb grip strength. We found a strong negative correlation between lumbar spinal cord mRNA expression of C1qB (r = − 0.6824), C3 (r = − 0.8282) and C5aR1 (r = − 0.7428) to hind-limb grip strength of these animals, with C3 presenting the strongest correlation (n = 30, ****P < 0.0001; Fig. 6a–c). Furthermore, a moderate to strong negative correlation between TA mRNA levels of C1qB (r = − 0.6624) and C5aR1 (r = − 0.4116) to hind-limb grip strength was observed, whereas a moderate positive correlation was identified between C3 mRNA levels (r = 0.4149) to hind-limb grip strength (n = 25, *P < 0.05, ***P < 0.001; Fig. 6d–f). These results indicate that changes in complement transcript levels in the spinal cord and TA muscle directly correlates with the decrease in hind-limb grip strength (i.e. increased in ALS symptoms) in this ALS model.