Elevated TGFß signaling contributes to cerebral small vessel disease in mouse models of Gould syndrome.

Cerebral small vessel disease (CSVD) is a leading cause of stroke and vascular cognitive impairment and dementia. Studying monogenic CSVD can reveal pathways that are dysregulated in common sporadic forms of the disease and may represent therapeutic targets. Mutations in collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) cause highly penetrant CSVD as part of a multisystem disorder referred to as Gould syndrome. COL4A1 and COL4A2 form heterotrimers [a1α1α2(IV)] that are fundamental constituents of basement membranes. However, their functions are poorly understood and the mechanism(s) by which COL4A1 and COL4A2 mutations cause CSVD are unknown. We used histological, molecular, genetic, pharmacological, and in vivo imaging approaches to characterize central nervous system (CNS) vascular pathologies in Col4a1 mutant mouse models of monogenic CSVD to provide insight into underlying pathogenic mechanisms. We describe developmental CNS angiogenesis abnormalities characterized by impaired retinal vascular outgrowth and patterning, increased numbers of mural cells with abnormal morphologies, altered contractile protein expression in vascular smooth muscle cells (VSMCs) and age-related loss of arteriolar VSMCs in Col4a1 mutant mice. Importantly, we identified elevated TGFß signaling as a pathogenic consequence of Col4a1 mutations and show that genetically suppressing TGFß signaling ameliorated CNS vascular pathologies, including partial rescue of retinal vascular patterning defects, prevention of VSMC loss, and significant reduction of intracerebral hemorrhages in Col4a1 mutant mice aged up to 8 months. This study identifies a novel biological role for collagen α1α1α2(IV) as a regulator of TGFß signaling and demonstrates that elevated TGFß signaling contributes to CNS vascular pathologies caused by Col4a1 mutations. Our findings suggest that pharmacologically suppressing TGFß signaling could reduce the severity of CSVD, and potentially other manifestations associated with Gould syndrome and have important translational implications that could extend to idiopathic forms of CSVD.

[Tetrodotoxin Content of the Small Gastropod Nassarius sufflatus].

Severe tetrodotoxin (TTX) poisoning due to small gastropods has been documented in Japan. In this study, we investigated the TTX content of the muscles and viscera of Nassarius sufflatus collected off the coast of Futaoi Island, Shimonoseki, Yamaguchi Prefecture, Japan, to prevent the occurrence of TTX poisoning caused by this small gastropod. Live specimens were obtained, and their muscles and viscera were collected. Test solutions were prepared from tissues of specimens and analyzed for TTX by HPLC-fluorescence detection. TTX was detected in both tissues at concentrations ranging from <0.1 to 18.2 µg/g for muscle and <0.1 to 130.7 µg/g for viscera. These results suggested that N. sufflatus accumulates TTX not only in its viscera but also in its muscles, and that precautions should be taken to prevent food poisoning due to this gastropod.

COL4A1 Mutations Cause Neuromuscular Disease with Tissue-Specific Mechanistic Heterogeneity.

Collagen type IV alpha 1 and alpha 2 chains form heterotrimers ([α1(IV)]2α2(IV)) that represent a fundamental basement membrane constituent. Dominant COL4A1 and COL4A2 mutations cause a multisystem disorder that is marked by clinical heterogeneity and variable expressivity and that is generally characterized by the presence of cerebrovascular disease with ocular, renal, and muscular involvement. Despite the fact that muscle pathology is reported in up to one-third of individuals with COL4A1 and COL4A2 mutations and in animal models with mutations in COL4A1 and COL4A2 orthologs, the pathophysiological mechanisms underlying COL4A1-related myopathy are unknown. In general, mutations are thought to impair [α1(IV)]2α2(IV) secretion. Whether pathogenesis results from intracellular retention, extracellular deficiency, or the presence of mutant proteins in basement membranes represents an important gap in knowledge and a major obstacle for developing targeted interventions. We report that Col4a1 mutant mice develop progressive neuromuscular pathology that models human disease. We demonstrate that independent muscular, neural, and vascular insults contribute to neuromyopathy and that there is mechanistic heterogeneity among tissues. Importantly, we provide evidence of a COL4A1 functional subdomain with disproportionate significance for tissue-specific pathology and demonstrate that a potential therapeutic strategy aimed at promoting [α1(IV)]2α2(IV) secretion can ameliorate or exacerbate myopathy in a mutation-dependent manner. These data have important translational implications for prediction of clinical outcomes based on genotype, development of mechanism-based interventions, and genetic stratification for clinical trials. Collectively, our data underscore the importance of the [α1(IV)]2α2(IV) network as a multifunctional signaling platform and show that allelic and tissue-specific mechanistic heterogeneities contribute to the variable expressivity of COL4A1 and COL4A2 mutations.

Use of sodium 4-phenylbutyrate to define therapeutic parameters for reducing intracerebral hemorrhage and myopathy in Col4a1 mutant mice.

Collagen type IV alpha 1 (COL4A1) and alpha 2 (COL4A2) form heterotrimers that constitute a major component of nearly all basement membranes. COL4A1 and COL4A2 mutations cause a multisystem disorder that includes variable cerebrovascular and skeletal muscle manifestations. The pathogenicity of COL4A1 and COL4A2 mutations is generally attributed to impaired secretion into basement membranes. Sodium 4-phenylbutyrate (4PBA) is a US Food and Drug Administration-approved drug that promotes mutant heterotrimer secretion in vitro and in vivo Here, we use different 4PBA treatment paradigms to define therapeutic parameters for preventing cerebrovascular and muscular pathologies in Col4a1 mutant mice. We show the efficacy of long-term 4PBA treatment in reducing the severity of intracerebral hemorrhages (ICHs) in Col4a1 mutant mice aged up to 8 months. In addition, we demonstrate that maximal efficacy of 4PBA on ICH and myopathy was achieved when treatment was initiated prenatally, whereby even transient 4PBA administration had lasting benefits after being discontinued. Importantly, postnatal treatment with 4PBA also reduced ICH and skeletal myopathy severities in Col4a1 mutant mice, which has significant clinical implications for patients with COL4A1 and COL4A2 mutations.This article has an associated First Person interview with the first author of the paper.

Dimethyl fumarate mediates Nrf2-dependent mitochondrial biogenesis in mice and humans.

The induction of mitochondrial biogenesis could potentially alleviate mitochondrial and muscle disease. We show here that dimethyl fumarate (DMF) dose-dependently induces mitochondrial biogenesis and function dosed to cells in vitro, and also dosed in vivo to mice and humans. The induction of mitochondrial gene expression is more dependent on DMF's target Nrf2 than hydroxycarboxylic acid receptor 2 (HCAR2). Thus, DMF induces mitochondrial biogenesis primarily through its action on Nrf2, and is the first drug demonstrated to increase mitochondrial biogenesis with in vivo human dosing. This is the first demonstration that mitochondrial biogenesis is deficient in Multiple Sclerosis patients, which could have implications for MS pathophysiology and therapy. The observation that DMF stimulates mitochondrial biogenesis, gene expression and function suggests that it could be considered for mitochondrial disease therapy and/or therapy in muscle disease in which mitochondrial function is important.

Lymphoblast Oxidative Stress Genes as Potential Biomarkers of Disease Severity and Drug Effect in Friedreich's Ataxia.

There is no current approved therapy for the ultimately lethal neuro- and cardio-degenerative disease Friedreich's ataxia (FA). Finding minimally-invasive molecular biomarkers of disease progression and drug effect could support smaller, shorter clinical trials. Since we and others have noted a deficient oxidative stress response in FA, we investigated the expression of 84 genes involved in oxidative stress, signaling, and protection in control and FA lymphoblasts ranging from 460 to 1122 GAA repeats. Several antioxidant genes responded in a dose-dependent manner to frataxin expression at the mRNA and protein levels, which is inversely correlated with disease progression and severity. We tested the effect of experimental Friedreich's ataxia therapies dimethyl fumarate (DMF) and type 1 histone deacetylase inhibitor (HDACi) on biomarker mRNA expression. We observed that exposure of lymphoblasts to DMF and HDACi dose-dependently unsilenced frataxin expression and restored the potential biomarkers NCF2 and PDLIM1 expression to control levels. We suggest that in addition to frataxin expression, blood lymphoblast levels of NCF2 and PDLIM1 could be useful biomarkers for disease progression and drug effect in future clinical trials of Friedreich's ataxia.

Oxidative stress in inherited mitochondrial diseases.

Mitochondria are a source of reactive oxygen species (ROS). Mitochondrial diseases are the result of inherited defects in mitochondrially expressed genes. One potential pathomechanism for mitochondrial disease is oxidative stress. Oxidative stress can occur as the result of increased ROS production or decreased ROS protection. The role of oxidative stress in the five most common inherited mitochondrial diseases, Friedreich ataxia, LHON, MELAS, MERRF, and Leigh syndrome (LS), is discussed. Published reports of oxidative stress involvement in the pathomechanisms of these five mitochondrial diseases are reviewed. The strongest evidence for an oxidative stress pathomechanism among the five diseases was for Friedreich ataxia. In addition, a meta-analysis was carried out to provide an unbiased evaluation of the role of oxidative stress in the five diseases, by searching for "oxidative stress" citation count frequency for each disease. Of the five most common mitochondrial diseases, the strongest support for oxidative stress is for Friedreich ataxia (6.42%), followed by LHON (2.45%), MELAS (2.18%), MERRF (1.71%), and LS (1.03%). The increased frequency of oxidative stress citations was significant relative to the mean of the total pool of five diseases (p<0.01) and the mean of the four non-Friedreich diseases (p<0.0001). Thus there is support for oxidative stress in all five most common mitochondrial diseases, but the strongest, significant support is for Friedreich ataxia.

Frataxin deficiency increases cyclooxygenase 2 and prostaglandins in cell and animal models of Friedreich's ataxia.

An inherited deficiency of the mitochondrial protein frataxin causes Friedreich's ataxia (FRDA); the mechanism by which this deficiency triggers neuro- and cardio-degeneration is unclear. Microarrays of neural tissue of animal models of the disease showed decreases in antioxidant genes, and increases in inflammatory genes. Cyclooxygenase (COX)-derived oxylipins are important mediators of inflammation. We measured oxylipin levels using tandem mass spectrometry and ELISAs in multiple cell and animal models of FRDA. Mass spectrometry revealed increases in concentrations of prostaglandins, thromboxane B2, 15-HETE and 11-HETE in cerebellar samples of knockin knockout mice. One possible explanation for the elevated oxylipins is that frataxin deficiency results in increased COX activity. While constitutive COX1 was unchanged, inducible COX2 expression was elevated over 1.35-fold (P < 0.05) in two Friedreich's mouse models and Friedreich's lymphocytes. Consistent with higher COX2 expression, its activity was also increased by 58% over controls. COX2 expression is driven by multiple transcription factors, including activator protein 1 and cAMP response element-binding protein, both of which were elevated over 1.52-fold in cerebella. Taken together, the results support the hypothesis that reduced expression of frataxin leads to elevation of COX2-mediated oxylipin synthesis stimulated by increases in transcription factors that respond to increased reactive oxygen species. These findings support a neuroinflammatory mechanism in FRDA, which has both pathomechanistic and therapeutic implications.

Frataxin deficiency leads to defects in expression of antioxidants and Nrf2 expression in dorsal root ganglia of the Friedreich's ataxia YG8R mouse model.

AIMS: Oxidative stress is thought to be involved in Friedreich's ataxia (FRDA), yet it has not been demonstrated in the target neurons that are first to degenerate. Using the YG8R mouse model of FRDA, microarray and neuritic growth experiments were carried out in the dorsal root ganglion (DRG), the primary site of neurodegeneration in this disease. RESULTS: YG8R hemizygous mice exhibited defects in movement, and DRG neurites had growth defects. Microarray of DRG tissue identified decreased transcripts encoding the antioxidants, including peroxiredoxins, glutaredoxins, and glutathione S-transferase, and these were confirmed by immunoblots and quantitative real-time PCR. Because the decreased gene transcripts are the known targets of the antioxidant transcription factor nuclear factor-E2-related factor-2 (Nrf2), Nrf2 expression was measured; it was significantly decreased at the transcript and protein level in both the DRG and the cerebella of the YG8R hemizygous mouse; further, frataxin expression was significantly correlated with Nrf2 expression. Functionally, in YG8R hemizygous DRG, the total glutathione levels were reduced and explanted cells were more sensitive to the thioredoxin reductase (TxnRD) inhibitor auranofin, a thiol oxidant. In cell models of FRDA, including Schwann and the DRG, frataxin deficiency caused a decreased expression of the Nrf2 protein level in the nucleus, but not a defect in its translocation from the cytosol. Further, frataxin-deficient cells had decreased enzyme activity and expression of TxnRD, which is regulated by Nrf2, and were sensitive the TxnRD inhibitor auranofin. INNOVATION AND CONCLUSION: These results support a mechanistic hypothesis in which frataxin deficiency decreases Nrf2 expression in vivo, causing the sensitivity to oxidative stress in target tissues the DRG and the cerebella, which contributes to the process of neurodegeneration.

Psoriasis patients are enriched for genetic variants that protect against HIV-1 disease.

An important paradigm in evolutionary genetics is that of a delicate balance between genetic variants that favorably boost host control of infection but which may unfavorably increase susceptibility to autoimmune disease. Here, we investigated whether patients with psoriasis, a common immune-mediated disease of the skin, are enriched for genetic variants that limit the ability of HIV-1 virus to replicate after infection. We analyzed the HLA class I and class II alleles of 1,727 Caucasian psoriasis cases and 3,581 controls and found that psoriasis patients are significantly more likely than controls to have gene variants that are protective against HIV-1 disease. This includes several HLA class I alleles associated with HIV-1 control; amino acid residues at HLA-B positions 67, 70, and 97 that mediate HIV-1 peptide binding; and the deletion polymorphism rs67384697 associated with high surface expression of HLA-C. We also found that the compound genotype KIR3DS1 plus HLA-B Bw4-80I, which respectively encode a natural killer cell activating receptor and its putative ligand, significantly increased psoriasis susceptibility. This compound genotype has also been associated with delay of progression to AIDS. Together, our results suggest that genetic variants that contribute to anti-viral immunity may predispose to the development of psoriasis.

Rare and common variants in CARD14, encoding an epidermal regulator of NF-kappaB, in psoriasis.

Psoriasis is a common inflammatory disorder of the skin and other organs. We have determined that mutations in CARD14, encoding a nuclear factor of kappa light chain enhancer in B cells (NF-kB) activator within skin epidermis, account for PSORS2. Here, we describe fifteen additional rare missense variants in CARD14, their distribution in seven psoriasis cohorts (>6,000 cases and >4,000 controls), and their effects on NF-kB activation and the transcriptome of keratinocytes. There were more CARD14 rare variants in cases than in controls (burden test p value = 0.0015). Some variants were only seen in a single case, and these included putative pathogenic mutations (c.424G>A [p.Glu142Lys] and c.425A>G [p.Glu142Gly]) and the generalized-pustular-psoriasis mutation, c.413A>C (p.Glu138Ala); these three mutations lie within the coiled-coil domain of CARD14. The c.349G>A (p.Gly117Ser) familial-psoriasis mutation was present at a frequency of 0.0005 in cases of European ancestry. CARD14 variants led to a range of NF-kB activities; in particular, putative pathogenic variants led to levels >2.5× higher than did wild-type CARD14. Two variants (c.511C>A [p.His171Asn] and c.536G>A [p.Arg179His]) required stimulation with tumor necrosis factor alpha (TNF-α) to achieve significant increases in NF-kB levels. Transcriptome profiling of wild-type and variant CARD14 transfectants in keratinocytes differentiated probably pathogenic mutations from neutral variants such as polymorphisms. Over 20 CARD14 polymorphisms were also genotyped, and meta-analysis revealed an association between psoriasis and rs11652075 (c.2458C>T [p.Arg820Trp]; p value = 2.1 × 10(-6)). In the two largest psoriasis cohorts, evidence for association increased when rs11652075 was conditioned on HLA-Cw*0602 (PSORS1). These studies contribute to our understanding of the genetic basis of psoriasis and illustrate the challenges faced in identifying pathogenic variants in common disease.

Protective effect of human endogenous retrovirus K dUTPase variants on psoriasis susceptibility.

Previous genetic and functional studies have implicated the human endogenous retrovirus K (HERV-K) dUTPase located within the PSORS1 locus in the major histocompatibility complex region as a candidate psoriasis gene. Here, we describe a variant discovery and case-control association study of HERV-K dUTPase variants in 708 psoriasis cases and 349 healthy controls. Five common HERV-K dUTPase variants were found to be highly associated with psoriasis, with the strongest association occurring at the missense single-nucleotide polymorphism (SNP) rs3134774 (K158R, P=3.28 × 10(-15), odds ratio =2.36 (95% confidence interval: 1.91-2.92)). After adjusting the association of the HERV-K dUTPase variants for the potential confounding effects of HLA alleles associated with psoriasis, the HERV-K SNPs rs9264082 and rs3134774 remained significantly associated. Haplotype analysis revealed that HERV-K haplotypes containing the non-risk alleles for rs3134774 and rs9264082 significantly reduced the risk of psoriasis. Functional testing showed higher antibody responses against recombinant HERV-K dUTPase in psoriasis patients compared with controls (P<0.05), as well as higher T-cell responses against a single HERV-K dUTPase peptide (P<0.05). Our data support an independent role for the HERV-K dUTPase on psoriasis susceptibility, and suggest the need for additional studies to clarify the role of this dUTPase in the pathogenesis of psoriasis.

Association analysis identifies ZNF750 regulatory variants in psoriasis.

BACKGROUND: Mutations in the ZNF750 promoter and coding regions have been previously associated with Mendelian forms of psoriasis and psoriasiform dermatitis. ZNF750 encodes a putative zinc finger transcription factor that is highly expressed in keratinocytes and represents a candidate psoriasis gene. METHODS: We examined whether ZNF750 variants were associated with psoriasis in a large case-control population. We sequenced the promoter and exon regions of ZNF750 in 716 Caucasian psoriasis cases and 397 Caucasian controls. RESULTS: We identified a total of 47 variants, including 38 rare variants of which 35 were novel. Association testing identified two ZNF750 haplotypes associated with psoriasis (p < 0.05). We also identified an excess of rare promoter and 5'untranslated region (UTR) variants in psoriasis cases compared to controls (p = 0.041), whereas there was no significant difference in the number of rare coding and rare 3' UTR variants. Using a promoter functional assay in stimulated human primary keratinocytes, we showed that four ZNF750 promoter and 5' UTR variants displayed a 35-55% reduction of ZNF750 promoter activity, consistent with the promoter activity reduction seen in a Mendelian psoriasis family with a ZNF750 promoter variant. However, the rare promoter and 5' UTR variants identified in this study did not strictly segregate with the psoriasis phenotype within families. CONCLUSIONS: Two haplotypes of ZNF750 and rare 5' regulatory variants of ZNF750 were found to be associated with psoriasis. These rare 5' regulatory variants, though not causal, might serve as a genetic modifier of psoriasis.