Reduction of matrix metalloproteinase 9 (MMP-9) protects motor neurons from TDP-43-triggered death in rNLS8 mice

Abstract

Therapeutic strategies are needed for the treatment of amyotrophic lateral sclerosis (ALS). One potential target is matrix metalloproteinase-9 (MMP-9), which is expressed only by fast motor neurons (MNs) that are selectively vulnerable to various ALS-relevant triggers. Previous studies have shown that reduction of MMP-9 function delayed motor dysfunction in a mouse model of familial ALS. However, given that the majority of ALS cases are sporadic, we propose preclinical testing in a mouse model which may be more clinically translatable: rNLS8 mice. In rNLS8 mice, neurodegeneration is triggered by the major pathological hallmark of ALS, TDP-43 mislocalization and aggregation. MMP-9 was targeted in 3 different ways in rNLS8 mice: by AAV9-mediated knockdown, using antisense oligonucleotide (ASO) technology, and by genetic modification. All 3 strategies preserved the motor unit during disease, as measured by MN counts, tibialis anterior (TA) muscle innervation, and physiological recordings from muscle. However, the strategies that reduced MMP-9 beyond the motor unit lead to premature deaths in a subset of rNLS8 mice. Therefore, selective targeting of MMP-9 in MNs could be beneficial in ALS, but side effects outside of the motor circuit may limit the most commonly used clinical targeting strategies.

Keywords: AAV; Amyotrophic lateral sclerosis (ALS); Antisense oligonucleotide; MMP-9; Motor neuron; TDP-43; rNLS8 mice.

Fig. 1.. Motor pool-specific MMP-9 knock-down protects the injected muscle from axonal dieback after 6 weeks of transgene expression.

(A) rNLS8 mice at P4 were injected unilaterally into the TA with AAV9.shMMP9 (titer: 9.47 × 10¹³ vg/ml, custom made by Virovek, Inc.) to achieve pool-specific knockdown of MMP-9. (B) Representative cryosection of lumbar SC, 2 months after AAV9.shMMP9 administration, shows that GFP transduced MNs (green, arrow) are not MMP-9 positive (red). (C) AAV9.shMMP9 i.m. administration leads to a 36% reduction in the total number of MMP9⁺ MNs (t₆=−4.5, p= 0.004), and a 60% reduction in the numbers of MMP-9^hi (t₆=9.6, p<0.001). Bars show mean ± S.D, n=3−5. (D-E) The amplitude of the maximum evoked CMAP was 23.9 ± 9% larger from the left TA (green, AAV9.shMMP-9-injected) compared to the right (black, control) TA. Individual points show the ratio of left to right CMAPs for each animal. Bars are mean ± S.D., n=3−5. (F) While control animals have symmetrical paw placement (ratio close to 1) as they run on a treadmill, gait analysis software shows an imbalance of motor function in left versus right hind limbs (ratio: 1.3) in rNLS8 mice injected unilaterally with AAV9.shMMP9 at 6 weeks off DOX (t₆ = −10.2, p<0.001). Bars show mean ± S.D, n=3−5. (G-H) The overlap of VAChT-positive motor terminals (red) with acetylcholine receptors stained using BTX (green) was used as an indicator of innervated motor endplates. The percent innervation of TAs following 6 weeks of transgene expression shows that the left TA is protected after injection with AAV9.shMMP9 (triangles) compared to its uninjected TA (squares). Note that the control rNLS8 mice had symmetric muscle denervation (circles). (I) Representative images of lumbar SC from an rNLS8 mouse that given a unilateral i.m. injection with AAV9.shMMP9 and 6 weeks off DOX immunostained for pTDP-43 (p409/410, white) shows a rare instance of a pTDP-43 inclusion in a neuron which is not a MN in the uninfected side of lumbar SC (top panels, indicated with a yellow arrow). Very few pTDP-43 inclusions were detected at the lumbar level of SC, regardless of the MMP-9 levels. Bars represent mean ± S.D, n=5 for AAV9.shMMP9 animals. Scale bar= 100 μm.

Fig 2.. A single i.c.v. injection of an ASO targeting Mmp9 results in widespread MMP-9 reduction in nTg controls.

(A) Representative pictures of nTg brains immunostained with MMP-9 (green) shows a marked decrease in the number of MMP-9-positive MNs in the facial nucleus at 2 weeks post injection. Similar staining was observed in 4 replicates. (B) There was also a decrease in the number of MMP-9-positive MNs (stained with VAChT, red) compared to the ASO-cntrltreated controls at the lumbar level of SC in the same mice. (C-D). In the TA motor pool, identified by an i.m. injection with CTB-594 (red), there was about a 35% increase in the number of MMP-9 negative MNs (light grey) and the majority of remaining MNs only expressed a low level of MMP-9 (~55%, dark grey). MMP-9 negative MNs are indicated with white arrows, and asterisks show MMP-9 positive MNs. Bars show mean ± S.D. n=3 animals with 2 TAs back-filled in each. Scale bars= 100 μm.

Fig. 3.. ASO-mediated MMP-9 knock-down attenuates neuromuscular defects in rNLS8 mice in mice at 4–6 weeks off DOX.

(A) Muscle recordings from the gastrocnemius muscle show more robust motor function in rNLS8 injected with the control ASO compared to those treated with ASOxMMP9 (**, t₁₁= −3.3, p= 0.007). (B) ASOxMMP9 treated rNLS8 mice have about 17% more intact NMJs in their TA muscles compared ASO-cntrl-treated rNLS8 mice (*, t7= −3.0, p= 0.02). (C). There are significantly more MNs in ASOxMMP9 mice compared to control (***,t₇ = −7.2, p<0.001). For all panels, individual data points are shown with bars indicating mean ± S.D to the right.

Fig 4.. MMP-9 reduction attenuates neuromuscular defects, but has unwanted side effects in rNLS8 animals.

(A) rNLS8;Mmp9^−/− mice have more premature deaths than littermates (Log-Rank test, p=0.03), with half dying of suspected seizures between 2 and 3 weeks off DOX. (B) rNLS8;Mmp9^+/+ mice can run faster than rNLS8;Mmp9^−/− mice at 5–6 weeks off DOX (*, One way ANOVA, F2,11=5.1, p=0.03). (C) However, reducing MMP-9 preserves evoked compound muscle action potentials in rNLS8 mice (**, F2,21=6.7,p = 0.006). (D-E) Finally, deleting Mmp9 attenuates MN death in rNLS8 mice(*, F2,11=4.1, p=0.04). Representative cryosections (D) from lumbar SC of rNLS8;Mmp9^+/+, rNLS8;Mmp9^+/−, and rNLS8;Mmp9^−/− littermates at 6 weeks off DOX immunostained with VAChT (red) to label MNs and MMP-9 (green) shows successful removal of MMP-9 in rNLS8;Mmp9^−/−. Scale bars= 100 μm. (E) Individual data points are shown with bars indicating mean ± S.D to the right.