Neuropathology and Therapeutic Intervention in Spinal and Bulbar Muscular Atrophy

Spinal and bulbar muscular atrophy (SBMA) is a hereditary motor neuron disease caused by the expansion of a polyglutamine tract in the androgen receptor (AR). The histopathological finding in SBMA is loss of lower motor neurons in the anterior horn of the spinal cord as well as in the brainstem motor nuclei. Animal studies have revealed that the pathogenesis of SBMA depends on the level of serum testosterone, and that androgen deprivation mitigates neurodegeneration through inhibition of nuclear accumulation of the pathogenic AR. Heat shock proteins, ubiquitin-proteasome system and transcriptional regulation are also potential targets of therapy development for SBMA

Current Status of Treatment of Spinal and Bulbar Muscular Atrophy

Spinal and bulbar muscular atrophy (SBMA) is the first member identified among polyglutamine diseases characterized by slowly progressive muscle weakness and atrophy of the bulbar, facial, and limb muscles pathologically associated with motor neuron loss in the spinal cord and brainstem. Androgen receptor (AR), a disease-causing protein of SBMA, is a well-characterized ligand-activated transcription factor, and androgen binding induces nuclear translocation, conformational change and recruitment of coregulators for transactivation of AR target genes. Some therapeutic strategies for SBMA are based on these native functions of AR. Since ligand-induced nuclear translocation of mutant AR has been shown to be a critical step in motor neuron degeneration in SBMA, androgen deprivation therapies using leuprorelin and dutasteride have been developed and translated into clinical trials. Although the results of these trials are inconclusive, renewed clinical trials with more sophisticated design might prove the effectiveness of hormonal intervention in the near future. Furthermore, based on the normal function of AR, therapies targeted for conformational changes of AR including amino-terminal (N) and carboxy-terminal (C) (N/C) interaction and transcriptional coregulators might be promising. Other treatments targeted for mitochondrial function, ubiquitin-proteasome system (UPS), and autophagy could be applicable for all types of polyglutamine diseases

Current Status of Treatment of Spinal and Bulbar Muscular Atrophy

Spinal and bulbar muscular atrophy (SBMA) is the first member identified among polyglutamine diseases characterized by slowly progressive muscle weakness and atrophy of the bulbar, facial, and limb muscles pathologically associated with motor neuron loss in the spinal cord and brainstem. Androgen receptor (AR), a disease-causing protein of SBMA, is a well-characterized ligandactivated transcription factor, and androgen binding induces nuclear translocation, conformational change and recruitment of coregulators for transactivation of AR target genes. Some therapeutic strategies for SBMA are based on these native functions of AR. Since ligand-induced nuclear translocation of mutant AR has been shown to be a critical step in motor neuron degeneration in SBMA, androgen deprivation therapies using leuprorelin and dutasteride have been developed and translated into clinical trials. Although the results of these trials are inconclusive, renewed clinical trials with more sophisticated design might prove the effectiveness of hormonal intervention in the near future. Furthermore, based on the normal function of AR, therapies targeted for conformational changes of AR including amino-terminal (N) and carboxy-terminal (C) (N/C) interaction and transcriptional coregulators might be promising. Other treatments targeted for mitochondrial function, ubiquitin-proteasome system (UPS), and autophagy could be applicable for all types of polyglutamine diseases

ADAMTS13 gene deletion enhances plasma high-mobility group box1 elevation and neuroinflammation in brain ischemia-reperfusion injury

Highly adhesive glycoprotein von Willebrand factor (VWF) multimer induces platelet aggregation and leukocyte tethering or extravasation on the injured vascular wall, contributing to microvascular plugging and inflammation in brain ischemia-reperfusion. A disintegrin and metalloproteinase with thrombospondin type-1 motifs 13 (ADAMTS13) cleaves the VWF multimer strand and reduces its prothrombotic and proinflammatory functions. Although ADAMTS13 deficiency is known to amplify post-ischemic cerebral hypoperfusion, there is no report available on the effect of ADAMTS13 on inflammation after brain ischemia. We investigated if ADAMTS13 deficiency intensifies the increase of extracellular HMGB1, a hallmark of post-stroke inflammation, and exacerbates brain injury after ischemia-reperfusion. ADAMTS13 gene knockout (KO) and wild-type (WT) mice were subjected to 30-min middle cerebral artery occlusion (MCAO) and 23.5-h reperfusion under continuous monitoring of regional cerebral blood flow (rCBF). The infarct volume, plasma high-mobility group box1 (HMGB1) level, and immunoreactivity of the ischemic cerebral cortical tissue (double immunofluorescent labeling) against HMGB1/NeuN (neuron-specific nuclear protein) or HMGB1/MPO (myeloperoxidase) were estimated 24h after MCAO. ADAMTS13KO mice had larger brain infarcts compared with WT 24h after MCAO (p<0.05). The rCBF during reperfusion decreased more in ADAMTS13KO mice. The plasma HMGB1 increased more in ADAMTS13KO mice than in WT after ischemia-reperfusion (p<0.05). Brain ischemia induced more prominent activation of inflammatory cells co-expressing HMGB1 and MPO and more marked neuronal death in the cortical ischemic penumbra of ADAMTS13KO mice. ADAMTS13 deficiency may enhance systemic and brain inflammation associated with HMGB1 neurotoxicity, and aggravate brain damage in mice after brief focal ischemia. We hypothesize that ADAMTS13 protects brain from ischemia-reperfusion injury by regulating VWF-dependent inflammation as well as microvascular pluggin

Plasminogen Tochigi mice exhibit phenotypes similar to wild-type mice under experimental thrombotic conditions.

Plasminogen (Plg) is a precursor of plasmin that degrades fibrin. A race-specific A620T mutation in Plg, also known as Plg-Tochigi, originally identified in a patient with recurrent venous thromboembolism, causes dysplasminogenemia with reduced plasmin activity. The Plg-A620T mutation is present in 3-4% of individuals in East Asian populations, and as many as 50,000 Japanese are estimated to be homozygous for the mutant 620T allele. In the present study, to understand the changes of thrombotic phenotypes in individuals with the mutant 620T allele, we generated knock-in mice carrying the homozygous Plg-A622T mutation (PlgT/T), an equivalent to the A620T mutation in human Plg. PlgT/T mice grew normally but showed severely reduced plasmin activity activated by urokinase, equivalent to ~8% of that in wild-type mice. In vitro fibrin clot lysis in plasma was significantly slower in PlgT/T mice than in wild-type mice. However, all experimental models of electrolytic deep vein thrombosis, tissue factor-induced pulmonary embolism, transient focal brain ischaemic stroke, or skin-wound healing showed largely similar phenotypes between PlgT/T mice and wild-type mice. Protein S-K196E mutation (Pros1E/E) is a race-specific genetic risk factor for venous thromboembolism. Coexistence in mice of PlgT/T and Pros1E/E did not affect pulmonary embolism symptoms, compared with those in Pros1E/E mice. Hence, the present study showed that the Plg-A622T mutation, which confers ~8% plasmin activity, does not increase the risk of thrombotic diseases in mice under experimental thrombotic conditions and does not modify the thrombotic phenotype observed in Pros1E/E mice. PlgT/T mice can be used to investigate the potential pathophysiological impact of the Plg-A620T mutation

No effects of <i>Plg</i>^T/T mutation in the skin-wound healing model.

<p>The time course of the wound area was measured every other day for two weeks. Data are the means ± SDs of wild-type (WT, n = 10) and <i>Plg</i>^T/T (n = 9) mice. No significant differences (<i>p</i> > 0.05) were observed between groups.</p

Plasma Plg antigen levels and plasmin activities of wild-type and <i>Plg</i>^T/T mice.

<p>(<b><i>A</i></b>) Plg antigen levels. Data are the means ± SDs of wild-type (WT, n = 10) and <i>Plg</i>^T/T (n = 10) mice. The levels measured in WT mice were defined as 1 U/ml. (<b><i>B</i></b>) Plasmin activities. Plasma from the indicated mice was preincubated with human uPA and reacted with a synthetic substrate, S-2403, for plasmin. Data are the means ± SDs of 10 mice for each genotype. The mean activity measured in WT mice was defined as 100%. (<b><i>C</i></b>) Western blot analysis of uPA-treated mouse plasma. Two mouse plasma samples of each genotype were incubated with human uPA and separated by SDS-PAGE under reducing conditions. Plg (<i>black triangle</i>) and heavy chains of plasmin (<i>white triangles</i>) were detected with anti-mouse Plg antibodies.</p

Generation of Plg-A622T mice.

<p>(<b><i>A</i></b>) Structure of the targeted locus in the mouse <i>Plg</i> gene. Exons are represented by <i>filled boxes</i>. A <i>lox</i>P-flanked (<i>filled triangles</i>) neomycin-resistance cassette (<i>NEO</i>) and a diphtheria toxin A fragment expression cassette (<i>DT-A</i>) are indicated by <i>open boxes</i> with <i>arrows</i> that represent the transcriptional orientation. The A622T mutant allele was produced by homologous recombination and <i>NEO</i> deletion mediated by Cre recombinase. The c.1864G>A (p.A622T) mutation and three translationally silent mutations (c.1857T>G, c.1858C>T, c.1860G>A) creating a new <i>Hpa</i>I site (<i>GTTAAC</i>) were introduced into exon 15. Homologous fragments are indicated by <i>dotted lines</i>, while the <i>Mfe</i>I-<i>Hpa</i>I fragments detected by Southern blot analysis of the wild-type (WT) and Plg-A622T alleles are indicated by <i>double-headed arrows</i>. (<b><i>B</i></b>) Southern blot analysis. Genomic DNA from targeted ES cells was digested with <i>Mfe</i>I/<i>Hpa</i>I and detected with the specific probe (WT allele: 9.5 kb; Plg-A622T allele: 6.6 kb). (<b><i>C</i></b>) Quantitative RT-PCR analysis. Total RNA was extracted from mouse liver and subjected to real-time RT-PCR with dual-labeled probes for mouse <i>Plg</i> and <i>Rn18s</i>. Expression levels of <i>Plg</i> mRNA were normalized to <i>Rn18s</i> mRNA. Data are the means ± SDs of WT (n = 8) and <i>Plg</i>^T/T (n = 7) mice. The levels measured in WT mice were defined as 100%.</p