Genetic knockout of myosin light chain kinase (MLCK210) prevents cerebral microhemorrhages and attenuates neuroinflammation in a mouse model of vascular cognitive impairment and dementia

Abstract

The blood-brain barrier (BBB) is critical in maintenance of brain homeostasis, and loss of its functional integrity is a key feature across a broad range of neurological insults. This includes both acute injuries such as traumatic brain injury and stroke, as well as more chronic pathologies associated with aging, such as vascular cognitive impairment and dementia (VCID). A specific form of myosin light chain kinase (MLCK210) is a major regulator of barrier integrity in general, including the BBB. Studies have demonstrated the potential of MLCK210 as a therapeutic target for peripheral disorders involving tissue barrier dysfunction, but less is known about its potential as a target for chronic neurologic disorders. We report here that genetic knockout (KO) of MLCK210 protects against cerebral microhemorrhages and neuroinflammation induced by chronic dietary hyperhomocysteinemia. Overall, the results are consistent with an accumulating body of evidence supporting MLCK210 as a potential therapeutic target for tissue barrier dysfunction and specifically implicate it in BBB dysfunction and neuroinflammation in a model of VCID.

Keywords: Cerebrovascular; Knockout; Microhemorrhage; Myosin light chain kinase; Neuroinflammation; Vascular cognitive impairment and dementia.

Fig. 1

HHcy diet induces hyperhomocysteinemia in both strains but MLCK210 KO mice are protected from microhemorrhages. a Plasma homocysteine was measured in a subset of mice (n = 3) from each group. After 6 weeks of HHcy diet, plasma homocysteine was significantly elevated in both strains relative to control diet. There were no differences between WT and MLCK210 KO mice at baseline. b Average Prussian blue positive microhemorrhages per section were elevated in WT mice in response to HHcy diet, but unchanged in MLCK210 KO mice. ***p < .001, ****p < .0001 vs. within-strain control, two-way ANOVA with Sidak’s post hoc tests

Fig. 2

Differences in baseline gene expression levels between WT and MLCK210 KO mice on control diet. Cortical expression levels in MLCK210 KO mice relative to WT mice on control diet are shown. The basal mRNA levels of six genes (IL-10, IL-1β, MMP14, MMP3, MMP9, YM1) were significantly lower in MLCK210 KO mice compared to WT mice. *p < .05, **p < .01 vs. WT mice on control diet, multiple independent sample t tests, followed by two-stage step-up analyses (Benjamini et al. 2006) with Q = 10%

Fig. 3

MMP transcript levels are unchanged in WT and upregulated in MLCK210 KO mice on HHcy diet, with no changes in TIMP transcript levels. a Cortical MMP expression levels show that in WT mice, there were no HHcy-induced increases in any of the MMP transcripts quantified. In MLCK210 KO mice, all of the MMPs were significantly upregulated as a result of HHcy diet. b Transcript levels of tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 were unchanged by HHcy in either strain. *p < .05, **p < .01 vs. within-strain control, two-way ANOVA with Sidak’s post hoc tests

Fig. 4

MLCK210 KO mice are protected from HHcy-induced neuroinflammation. a Cortical levels of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 were upregulated in WT mice on HHcy diet, and IL-12A was downregulated. There was no significant change in these cytokines in the MLCK210 KO mice on HHcy diet. b Anti-inflammatory mediators IL-10, IL-1Ra, and YM1 were significantly upregulated in the MLCK210 KO mice with HHcy, but not in the WT mice. Arg1 was unchanged with HHcy in either strain. *p < .05, **p < .01 vs. within-strain control, two-way ANOVA with Sidak’s post hoc tests