Abnormal Chloride Homeostasis in the Substancia Nigra Pars Reticulata Contributes to Locomotor Deficiency in a Model of Acute Liver Injury

<div><p>Background</p><p>Altered chloride homeostasis has been thought to be a risk factor for several brain disorders, while less attention has been paid to its role in liver disease. We aimed to analyze the involvement and possible mechanisms of altered chloride homeostasis of GABAergic neurons within the substantia nigra pars reticulata (SNr) in the motor deficit observed in a model of encephalopathy caused by acute liver failure, by using glutamic acid decarboxylase 67 - green fluorescent protein knock-in transgenic mice.</p><p>Methods</p><p>Alterations in intracellular chloride concentration in GABAergic neurons within the SNr and changes in the expression of two dominant chloride homeostasis-regulating genes, KCC2 and NKCC1, were evaluated in mice with hypolocomotion due to hepatic encephalopathy (HE). The effects of pharmacological blockade and/or activation of KCC2 and NKCC1 functions with their specific inhibitors and/or activators on the motor activity were assessed.</p><p>Results</p><p>In our mouse model of acute liver injury, chloride imaging indicated an increase in local intracellular chloride concentration in SNr GABAergic neurons. In addition, the mRNA and protein levels of KCC2 were reduced, particularly on neuronal cell membranes; in contrast, NKCC1 expression remained unaffected. Furthermore, blockage of KCC2 reduced motor activity in the normal mice and led to a further deteriorated hypolocomotion in HE mice. Blockade of NKCC1 was not able to normalize motor activity in mice with liver failure.</p><p>Conclusion</p><p>Our data suggest that altered chloride homeostasis is likely involved in the pathophysiology of hypolocomotion following HE. Drugs aimed at restoring normal chloride homeostasis would be a potential treatment for hepatic failure.</p></div

Reduced level of KCC2 protein and unaltered level of NKCC1 protein in the SNr by TAA injection.

<p>Western blot experiments were performed for analysis of KCC2 (A and B) and NKCC1 (C and D) proteins in the SNr of TAA-injected and sham mice at different time points. All data have been normalized to actin levels within the same sample. Data are calculated as percentages of the average value of control mice (B and D). Note that a dramatic down-regulation of KCC2 protein was seen at 1 d and 2 d in the TAA group (B). No significant change of NKCC1 protein was seen at different time points in TAA group compared with those in sham group (D). * <i>P</i><0.05 vs sham controls.</p

Reduced targeting of KCC2 to membrane of GABAergic neuron within the SNr following TAA treatment.

<p>(A–D) Results of immunohistochemical staining with a KCC2 - specific antibody (red) in SNr GABAergic neurons (green) of control and TAA mice. Single optical sections showing normal KCC2 staining and the discontinuous clusters of stained spots (arrows) in GABAergic neuron membranes of control (A) and TAA (B) mice. Single optical sections showing the density of cytoplasmic KCC2 - immunolabeled clusters (arrows) in control (C) and TAA (D) mice. (E) Quantification of the density of membrane labeling (ratios of labeled pixel surface per somatic perimeter) in 24 GABAergic neurons from three control mice and 27 GABAergic neurons from three TAA mice. (F) Number of cytoplasmic KCC2-immunolabeled clusters per 100 µm² of somatic area. GABAergic neurons in both control and TAA mice. Bars in A, B, C and D = 10 µm. * in E and F <i>P</i><0.05 vs normal controls.</p

Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in different experimental groups.

<p>TAA treatment induced clear signs of hepatic failure with significant increases in plasma level of the LDH, accompanied by less dramatic but still significant increases in ALT and AST. The intranigral injection of DIOA (a KCC2 blocker, 20 µg/0.5 µl) did not affect any of the serum transaminases in normal mice. Similarly, the intranigral injection of bumetanide (BUM, a selective inhibitor of NKCC1, a 20 nmol/0.5 µl) could not alter the increases of plasma level of ALT and AST, while ameliorate LDH increase, in mice treated with TAA.</p>*<p><i>p</i><0.05 as compared to normal controls;</p>#<p><i>p</i><0.05 as compared to TAA group.</p

Enhancement of intracellular chloride concentration ([Cl⁻]_i) following TAA treatment.

<p>(A–H) Results of confocal microscopic images of a single neuron within the SNr loaded with the chloride indicator dye MQAE from normal controls (A, B, C and D) and TAA-treated mice (E, F, G and H). The white circles illustrate the area from which the mean fluorescence intensity was obtained. Background subtraction removed most of the pericellular fluorescence as well as the fluorescence originating from the nucleus. All cell sections with intact perimeters were evaluated. The fluorescence is inversely proportional to the Cl⁻ concentration. (I) The corresponding [Cl⁻]_i levels were shown as bar graphs. * <i>P</i><0.05 vs normal controls. Bars in A–H = 10 µm.</p

Hypolocomotion induced by TAA in GAD67-GFP knock-in mice, evaluated by open field test.

<p>Horizontal (A) and vertical (B) locomotor activity was measured. Data are expressed as the mean ± SEM of photocell counts during a 10 min period. * <i>P</i><0.05 vs normal controls.</p

Histology of liver stained with H&E of GAD67-GFP knock-in mice in different groups.

<p>Increased necrosis of hepatocytes observed in mice treated with TAA (B and b) compared to normal animals (A and a). Intranigral treatment with bumetanide (BUM) (a selective inhibitor of NKCC1) led to similar liver architecture as that of TAA-injected mice (C and c). a, b and c show a section from A, B and C, respectively, at high magnification. Bars in A, B and C = 30 µm; bars in a, b and c = 3 µm.</p

Loss of SLC9A3 decreases CFTR protein and causes obstructed azoospermia in mice

<div><p>Mutations in the cystic fibrosis transmembrane conductance regulator (<i>CFTR</i>) gene cause cystic fibrosis (CF) and are associated with congenital bilateral absence of the vas deferens (CBAVD), which is the major cause of infertility in male patients with CF. However, most Taiwanese patients with CBAVD do not carry major <i>CFTR</i> mutations. Some patients have a single copy deletion of the solute carrier family 9 isoform 3 (<i>SLC9A3</i>) gene. SLC9A3 is a Na⁺/H⁺ exchanger, and depleted <i>Slc9a3</i> in male mice causes infertility due to the abnormal dilated lumen of the rete testis and efferent ductules. Furthermore, SLC9A3 interacts with CFTR in the pancreatic duct and functions as a genetic modifier of CF. However, SLC9A3 function and its relation to CFTR expression in the male reproductive tract in vivo remain elusive. In the present study, we found that CFTR expression was dramatically decreased in the epididymis and vas deferens of <i>Slc9a3</i> knockout mice. Adult <i>Slc9a3</i>^<i>-/-</i> mice showed not only significantly decreased epididymis and vas deferens weight but also increased testis weight. Furthermore, <i>Slc9a3</i>^<i>-/-</i> mice developed obstructive azoospermia because of abnormal abundant secretions and calcification in the lumen of the reproductive tract. Ultrastructural analysis of the epithelium in <i>Slc9a3</i>^{<i>–/–</i>}epididymis and vas deferens displayed disorganized and reduced number of stereocilia and numerous secretory apparatuses. Our data revealed that interdependence between SLC9A3 and CFTR is critical for maintaining a precise microenvironment in the epithelial cytoarchitecture of the male reproductive tract. The <i>Slc9a3</i>-deficient mice with impaired male excurrent ducts in this study provide proof for our clinical findings that some Taiwanese of CBAVD carry <i>SLC9A3</i> deletion but without major <i>CFTR</i> mutations.</p></div

Schematic drawing placement of injectors for the mice receiving drugs (blue cylinders) in the left substantia nigra.

<p>Schematic drawing placement of injectors for the mice receiving drugs (blue cylinders) in the left substantia nigra.</p

Reduced expression of KCC2 mRNA within the SNr following TAA injection.

<p>Real-time quantitative RT-PCR experiments were performed using SYBR green for analysis of KCC2 (A and B) and NKCC1 (C and D) mRNAs expression in SNr of TAA-injected mice at different time points and sham controls. All data were normalized for levels of GAPDH expression within the same sample. Data are calculated as percentages of the average value of controls (B and D). Note that a dramatic down-regulation of KCC2 mRNA was seen at 1 d and maintained up to 3 d in TAA group (B). However, a significant increase in NKCC1 mRNA was observed at 1 d in both the sham and TAA groups, which disappeared at 2 d and 3 d after treatment (D). * <i>P</i><0.05 vs sham controls.</p