KCC2 downregulation after sciatic nerve injury enhances motor function recovery

Injury to mature neurons induces downregulated KCC2 expression and activity, resulting in elevated intracellular [Cl−] and depolarized GABAergic signaling. This phenotype mirrors immature neurons wherein GABA-evoked depolarizations facilitate neuronal circuit maturation. Thus, injury-induced KCC2 downregulation is broadly speculated to similarly facilitate neuronal circuit repair. We test this hypothesis in spinal cord motoneurons injured by sciatic nerve crush, using transgenic (CaMKII-KCC2) mice wherein conditional CaMKIIα promoter-KCC2 expression coupling selectively prevents injury-induced KCC2 downregulation. We demonstrate, via an accelerating rotarod assay, impaired motor function recovery in CaMKII-KCC2 mice relative to wild-type mice. Across both cohorts, we observe similar motoneuron survival and re-innervation rates, but differing post-injury reorganization patterns of synaptic input to motoneuron somas—for wild-type, both VGLUT1-positive (excitatory) and GAD67-positive (inhibitory) terminal counts decrease; for CaMKII-KCC2, only VGLUT1-positive terminal counts decrease. Finally, we recapitulate the impaired motor function recovery of CaMKII-KCC2 mice in wild-type mice by administering local spinal cord injections of bicuculline (GABAA receptor blockade) or bumetanide (lowers intracellular [Cl−] by NKCC1 blockade) during the early post-injury period. Thus, our results provide direct evidence that injury-induced KCC2 downregulation enhances motor function recovery and suggest an underlying mechanism of depolarizing GABAergic signaling driving adaptive reconfiguration of presynaptic GABAergic input.


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Item 2 Statistics Summary: Figure

A.
In CaMKII-KCC2 mice, the tetO-tTA conditional expression system additionally couples KCC2 expression to the CaMKIIα promoter. Here, the tetracycline transactivator (tTA) gene is knocked-in downstream of the CaMKIIα promoter and the tetracycline response element (tetO) promoter is knocked-in upstream of the KCC2 gene. Thus, CaMKIIα driven tTA expression results in KCC2 expression. This gene expression setup can be reversibly disabled by doxycycline (DOX) as DOX-tTA binding prevents tTA-TRE binding.

B.
CaMKIIα expression in healthy mature motoneurons is normally very low but dramatically increases after injury. Thus, CaMKIIα driven KCC2 expression in motoneurons remains inactive after switching CaMKII-KCC2 mice from on-doxycycline (DOX ON) to off-doxycycline (DOX OFF), but becomes active after subsequent motoneuron injury. In this way, CaMKIIα driven KCC2 expression selectively compensates for injury induced KCC2 downregulation in motoneurons.

Supplementary Figure 2
A B KCC2 mRNA expression in the spinal cord ventral horn measured by RT-qPCR before and at various time-points after SNC.
KCC2 mRNA expression (normalized to GAPDH) in the L4-L5 ventral horns is plotted as a ratio between the injured-side ventral horn (inj) and uninjured-side ventral ( s2B p-value n/a n/a n/a 0.3125 n/a n/a >0.9999 significance n/a n/a n/a ns n/a n/a ns Sample size n, mice n/a n/a n/a 6 n/a n/a 4 Because characterization of KCC2 expression by immunohistochemistry is highly labour intensive, RT-qPCR was used as a preliminary screening approach to suggest a time-point where SNC-induced KCC2 expression downregulation would be most pronounced. Based on these results, the 3-days post-SNC time-point was selected as a time-point for the KCC2 immunohistochemistry experiments (Fig. 2 main text). RT-qPCR was also used to preliminarily check that SNC-induced KCC2 expression downregulation was selectively prevented in CaMKII-KCC2 mice. Note that the RT-qPCR data reflects KCC2 mRNA expression from all cell-types (not motoneurons exclusively) in the spinal cord ventral horn.  Quantification of VGLUT1-positive and GAD67-positive terminals VGLUT1-positive and GAD67-positive terminals synapsing onto motoneuron somas could be resolved as distinct punctae along the soma perimeter. To quantify these, the soma perimeter as demarcated by ChAT immunofluorescence was traced (top left panel) to generate a line plot profile of VGLUT1 or GAD67 immunofluorescence intensity (top middle and right panels).

Supplementary
From this fluorescence intensity plot (bottom panel), peaks larger than 2x the baseline and wider than 2 µm were counted as individual VGLUT1/GAD67-positive terminals. To account for variations in motoneuron size, the VGLUT1/GAD67-positive terminal counts were divided by the motoneuron soma perimeter length and expressed as terminal density per 100 μm of soma perimeter.

Quantification of GlyT2-positive terminals
GlyT2-positive terminals synapsing onto motoneuron somas were unable to be resolved as distinct punctae and were instead quantified as the (%) area coverage of soma perimeter ROI.

A.
Representative motoneurons from the injured-side (inj) and uninjured-side (non) ventral horn (L4-L5, Rexed lamina IX), for wild-type (WT) and CaMKII-KCC2 (KCC2) mice at 42-days post-SNC. Immunofluorescence for GlyT2 (green), ChAT (red) and DAPI (blue). Scale bar, 20 µm. GlyT2 terminals cannot be individually resolved and appear as a green border along the red soma perimeter.  Impaired motor function recovery in CaMKII-KCC2 mice is attributed to the prevention of injury induced KCC2 downregulation during the early post-injury period ( Fig. 2 main text; Supp. Fig. 1). However, subtle KCC2 overexpression in the late post-injury period is a potential alternative contributor to impaired motor function recovery. One approach for ruling this out is to delay the on-doxycycline (DOX ON) to off-doxycycline (DOX OFF) switch to the late postinjury period. If subtle KCC2 overexpression in the late post-injury meaningfully contributes to impaired motor function recovery, CaMKII-KCC2 mice should still have impaired motor function recovery compared to wild-type mice under this delayed doxycycline schedule.

A.
In a subset of wild-type and CaMKII-KCC2 mice, the on-doxycycline (DOX ON) to offdoxycycline (DOX OFF) switch was delayed from 2-3 weeks before SNC to 14-days post-SNC.

B.
Motor performance scores (rpm at falling), measured before (pre), and at 1 and 42-days after SNC, for wild-type (WT) and CaMKII-KCC2 (KCC2) mice, with both cohorts on-doxycycline until 14-days post-SNC.  The causal relationship between injury induced KCC2 downregulation and motor function recovery is correlated with the long-term reorganization of GAD67 (but not VGLUT1) synaptic inputs to motoneurons ( Fig. 7 main text). At the same time, injury induced KCC2 downregulation is limited to the early post-injury period. Thus, the most prominent features of these long-term synaptic reorganization trends should start to appear during the early postinjury period. To test this, VGLUT1 and GAD67 terminals were quantified at 1, 3 and 7-days post-SNC in wild-type and CaMKII-KCC2 mice. Full length western blot corresponding to Figure 1D in the main text. Figure 1D in the main text displays cropped KCC2 and β-actin protein bands which correspond to lanes 4-7 of the full length western blot shown here. In this western blot, proteins samples were extracted from the injured-side (inj) and uninjured-side (non) L4-L5 spinal cord ventral horn of an individual wild-type mouse (WT-inj, WT-non) and an individual CaMKII-KCC2 mouse (KCC2-inj, KCC2-non) at 3-days post-SNC. Protein samples were also extracted from the brains of these mice as an additional positive control. Precision Plus Protein WesternC Standards (BioRad, #161-0376) were used for the protein size ladder. The identity of the lanes are as follows: [1, 2, 3, 4, 5, 6, 7, 8] = [ladder, KCC2 (brain), WT (brain), WT-non, WT-inj, KCC2-non, KCC2-inj, ladder].

A-F.
As shown in the left panel, the western blot membrane was cut into two sections between the 75 kDa and 50 kDa size markers. The 75-250 kDa and 25-50 kDa membrane sections were separately incubated with the primary antibodies for KCC2 and β-actin respectively.