Long-range action of an HDAC inhibitor treats chronic pain in a spared nerve injury rat model.

Histone deacetylase inhibitors (HDACi) that modulate epigenetic regulation and are approved for treating rare cancers have, in disease models, also been shown to mitigate neurological conditions, including chronic pain. They are of interest as non-opioid treatments, but achieving long-term efficacy with limited dosing has remained elusive. Here we utilize a triple combination formulation (TCF) comprised of a pan-HDACi vorinostat (Vo at its FDA-approved daily dose of 50mg/Kg), the caging agent 2-hydroxypropyl-ß-cyclodextrin (HPBCD) and polyethylene glycol (PEG) known to boost plasma and brain exposure and efficacy of Vo in mice and rats, of various ages, spared nerve injury (SNI) model of chronic neuropathic pain. Administration of the TCF (but not HPBCD and PEG) decreased mechanical allodynia for 4 weeks without antagonizing weight, anxiety, or mobility. This was achieved at less than 1% of the total dose of Vo approved for 4 weeks of tumor treatment and associated with decreased levels of major inflammatory markers and microglia in ipsilateral (but not contralateral) spinal cord regions. A single TCF injection was sufficient for 3-4 weeks of efficacy: this was mirrored in repeat injections, specific for the injured paw and not seen on sham treatment. Pharmacodynamics in an SNI mouse model suggested pain relief was sustained for days to weeks after Vo elimination. Doubling Vo in a single TCF injection proved effectiveness was limited to male rats, where the response amplitude tripled and remained effective for > 2 months, an efficacy that outperforms all currently available chronic pain pharmacotherapies. Together, these data suggest that through pharmacological modulation of Vo, the TCF enables single-dose effectiveness with extended action, reduces long-term HDACi dosage, and presents excellent potential to develop as a non-opioid treatment option for chronic pain.

Generation of three induced pluripotent stem cell lines from a patient with Kabuki syndrome carrying the KMT2D p.R4198X mutation.

Kabuki syndrome (KS) is a rare genetic disorder typically characterized by facial abnormalities, developmental delay, cognitive dysfunction, and organ impairment. In this report, fibroblast cells obtained from a KS patient containing a heterozygous KMT2D c.12592 C>T mutation (p.R4198X) were reprogrammed using non-integrative Sendai virus to generate three induced pluripotent stem cell (iPSC) clones. The iPSC lines retained the KS patient mutation, and displayed normal karyotypes, pluripotency marker expression, and the ability to differentiate into the three germ layers.

Genomic analyses of glycine decarboxylase neurogenic mutations yield a large-scale prediction model for prenatal disease.

Hundreds of mutations in a single gene result in rare diseases, but why mutations induce severe or attenuated states remains poorly understood. Defect in glycine decarboxylase (GLDC) causes Non-ketotic Hyperglycinemia (NKH), a neurological disease associated with elevation of plasma glycine. We unified a human multiparametric NKH mutation scale that separates severe from attenuated neurological disease with new in silico tools for murine and human genome level-analyses, gathered in vivo evidence from mice engineered with top-ranking attenuated and a highly pathogenic mutation, and integrated the data in a model of pre- and post-natal disease outcomes, relevant for over a hundred major and minor neurogenic mutations. Our findings suggest that highly severe neurogenic mutations predict fatal, prenatal disease that can be remedied by metabolic supplementation of dams, without amelioration of persistent plasma glycine. The work also provides a systems approach to identify functional consequences of mutations across hundreds of genetic diseases. Our studies provide a new framework for a large scale understanding of mutation functions and the prediction that severity of a neurogenic mutation is a direct measure of pre-natal disease in neurometabolic NKH mouse models. This framework can be extended to analyses of hundreds of monogenetic rare disorders where the underlying genes are known but understanding of the vast majority of mutations and why and how they cause disease, has yet to be realized.

Large scale analyses of genotype-phenotype relationships of glycine decarboxylase mutations and neurological disease severity.

Monogenetic diseases provide unique opportunity for studying complex, clinical states that underlie neurological severity. Loss of glycine decarboxylase (GLDC) can severely impact neurological development as seen in non-ketotic hyperglycinemia (NKH). NKH is a neuro-metabolic disorder lacking quantitative predictors of disease states. It is characterized by elevation of glycine, seizures and failure to thrive, but glycine reduction often fails to confer neurological benefit, suggesting need for alternate tools to distinguish severe from attenuated disease. A major challenge has been that there are 255 unique disease-causing missense mutations in GLDC, of which 206 remain entirely uncharacterized. Here we report a Multiparametric Mutation Score (MMS) developed by combining in silico predictions of stability, evolutionary conservation and protein interaction models and suitable to assess 251 of 255 mutations. In addition, we created a quantitative scale of clinical disease severity comprising of four major disease domains (seizure, cognitive failure, muscular and motor control and brain-malformation) to comprehensively score patient symptoms identified in 131 clinical reports published over the last 15 years. The resulting patient Clinical Outcomes Scores (COS) were used to optimize the MMS for biological and clinical relevance and yield a patient Weighted Multiparametric Mutation Score (WMMS) that separates severe from attenuated neurological disease (p = 1.2 e-5). Our study provides understanding for developing quantitative tools to predict clinical severity of neurological disease and a clinical scale that advances monitoring disease progression needed to evaluate new treatments for NKH.

Case report: a synonymous VHL mutation (c.414A > G, p.Pro138Pro) causes pathogenic familial hemangioblastoma through dysregulated splicing.

BACKGROUND: von Hippel-Lindau (VHL) disease is a familial neoplasia syndrome that results from the germline mutation of VHL. Pathogenic VHL mutations include deletion, frameshift, nonsense and missense mutations. Synonymous mutations are expected to be phenotypically silent and their role in VHL disease remains poorly understood. CASE PRESENTATION: We report a Caucasian male with a family history of pheochromocytoma and the synonymous VHL mutation c.414A > G (p.Pro138Pro). At 47-years, MRI revealed pheochromocytoma in the left adrenal gland and hemangioblastomas in the spine and brain. Pheochromocytoma was treated by adrenalectomy. Radiotherapy, followed by craniotomy and resection were needed to reduce hemangioblastomas to residual lesions. Two of three of the proband's children inherited the mutation and both presented with retinal hemangioblastomas without pheochromocytoma at age 7: one twin needed four laser treatments. Primary skin fibroblasts carrying the heterozygous mutation or wild type VHL were established from the family. Mutant fibroblasts downregulated full-length VHL mRNA and protein, and upregulated the short VHL mRNA isoform (a result of exon 2 skipping in splicing) at the mRNA level but not at the protein level. CONCLUSIONS: Our study shows that the synonymous VHL mutation c.414A > G can within 7 years induce pediatric retinal hemangioblastoma in absence of pheochromocytoma. This highlights the need to include splicing-altering synonymous mutations into the screening for VHL disease. This is also the first report on detecting and validating a synonymous VHL mutation using patient-derived fibroblasts. The mutation c.414A > G translates to p.Pro138Pro, yet it is not functionally silent, because it causes aberrant splicing by skipping exon 2. The reduced but not completely abolished pVHL protein in a loss-of-heterozygosity genetic backdrop may underlie the etiology of VHL disease.

Tolerance of chronic HDACi treatment for neurological, visceral and lung Niemann-Pick Type C disease in mice.

Histone deacetylase (HDAC) inhibitors are of significant interest as drugs. However, their use to treat neurological disorders has raised concern because HDACs are required for brain function. We have previously shown that a triple combination formulation (TCF) of the pan HDACi vorinostat (Vo), 2-hydroxypropyl-beta-cyclodextrin (HPBCD) and polyethylene glycol (PEG) 400 improves pharmacokinetic exposure and entry of Vo into the brain. TCF treatment significantly delayed both neurodegeneration and death in the Npc1 nmf164 murine model of Niemann-Pick Type C (NPC) disease. The TCF induces no metabolic toxicity, but its risk to normal brain functions and potential utility in treating lung disease, a major NPC clinical complication, remain unknown. Here we report that TCF administered in healthy mice for 8-10 months was not detrimental to the brain or neuromuscular functions based on quantitative analyses of Purkinje neurons, neuroinflammation, neurocognitive/muscular disease symptom progression, cerebellar/hippocampal nerve fiber-staining, and Hdac gene-expression. The TCF also improved delivery of Vo to lungs and reduced accumulation of foamy macrophages in Npc1 nmf164 mice, with no injury. Together, these data support feasibility of tolerable, chronic administration of an HDACi formulation that treats murine NPC neurological disease and lung pathology, a frequent cause of death in this and possibly additional disorders.

Pluronic based ß-cyclodextrin polyrotaxanes for treatment of Niemann-Pick Type C disease.

Niemann-Pick Type C disease (NPC) is a rare metabolic disorder characterized by disruption of normal cholesterol trafficking within the cells of the body. There are no FDA approved treatments available for NPC patients. Recently, the cycloheptaglucoside 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) has shown efficacy as a potential NPC therapeutic by extending lifetime in NPC mice, delaying neurodegeneration, and decreasing visceral and neurological cholesterol burden. Although promising, systemic HP-ß-CD treatment is limited by a pharmacokinetic profile characterized by rapid loss through renal filtration. To address these shortcomings, we sought to design a family of HP-ß-CD pro-drug delivery vehicles, known as polyrotaxanes (PR), capable of increasing the efficacy of a given injected dose by improving both pharmacokinetic profile and bioavailability of the HP-ß-CD agent. PR can effectively diminish the cholesterol pool within the liver, spleen, and kidney at molar concentrations 10-to-100-fold lower than monomeric HP-ß-CD. In addition to this proof-of-concept, use of PR scaffolds with differing physiochemical properties reveal structure-activity relationships in which PR characteristics, including hydrophobicity, threading efficiency and surface charge, were found to both decisively and subtly effect therapeutic efficacy. PR scaffolds exhibit absorption, pharmacokinetics, and biodistribution patterns that are significantly altered from monomeric HP-ß-CD. In all, PR scaffolds hold great promise as potential treatments for visceral disease in NPC patients.

Chronic administration of an HDAC inhibitor treats both neurological and systemic Niemann-Pick type C disease in a mouse model.

Histone deacetylase inhibitors (HDACi) are approved for treating rare cancers and are of interest as potential therapies for neurodegenerative disorders. We evaluated a triple combination formulation (TCF) comprising the pan-HDACi vorinostat, the caging agent 2-hydroxypropyl-ß-cyclodextrin (HPBCD), and polyethylene glycol (PEG) for treating a mouse model (the Npc1(nmf164) mouse) of Niemann-Pick type C (NPC) disease, a difficult-to-treat cerebellar disorder. Vorinostat alone showed activity in cultured primary cells derived from Npc1(nmf164) mice but did not improve animal survival. However, low-dose, once-weekly intraperitoneal injections of the TCF containing vorinostat increased histone acetylation in the mouse brain, preserved neurites and Purkinje cells, delayed symptoms of neurodegeneration, and extended mouse life span from 4 to almost 9 months. We demonstrate that the TCF boosted the ability of HDACi to cross the blood-brain barrier and was not toxic even when used long term. Further, the TCF enabled dose reduction, which has been a major challenge in HDACi therapy. TCF simultaneously treats neurodegenerative and systemic symptoms of Niemann-Pick type C disease in a mouse model.

Plasma signature of neurological disease in the monogenetic disorder Niemann-Pick Type C.

Early diagnosis of neurological disorders would greatly improve their management and treatment. A major hurdle is that inflammatory products of cerebral disease are not easily detected in blood. Inflammation in multiple organs and heterogeneity in disease present additional challenges in distinguishing the extent to which a blood-based marker reflects disease in brain or other afflicted organs. Murine models of the monogenetic disorder Niemann-Pick Type C present aggressive forms of cerebral and liver inflammatory disease. Microarray analyses previously revealed age-dependent changes in innate immunity transcripts in the mouse brain. We have now validated four putative secretory inflammatory markers that are also elevated in mouse liver. We include limited, first time analysis of human Niemann-Pick Type C liver and cerebellum. Furthermore, we utilized 2-hydroxypropyl-ß-cyclodextrin (HPßCD, an emerging therapeutic) administered intraperitoneally in mice, which abrogates inflammatory pathology in the liver but has limited effect on the brain. By analyzing the corresponding effects on inflammatory plasma proteins, we identified cathepsin S as a lead indicator of liver disease. In contrast, lysozyme was a marker of both brain and liver disease. 2-Hydroxypropyl-ß-cyclodextrin had no effect on transcripts of neuron-specific 24-hydroxylase, and its product 24(S)-hydroxycholesterol was not a useful indicator in mouse plasma. Our data suggest that dual analysis of levels of the inflammatory markers lysozyme and cathepsin S may enable detection of multiple distinct states of neurodegeneration in plasma.

Genomic expression analyses reveal lysosomal, innate immunity proteins, as disease correlates in murine models of a lysosomal storage disorder.

Niemann-Pick Type C (NPC) disease is a rare, genetic, lysosomal disorder with progressive neurodegeneration. Poor understanding of the pathophysiology and a lack of blood-based diagnostic markers are major hurdles in the treatment and management of NPC and several additional, neurological lysosomal disorders. To identify disease severity correlates, we undertook whole genome expression profiling of sentinel organs, brain, liver, and spleen of Balb/c Npc1(-/-) mice relative to Npc1(+/-) at an asymptomatic stage, as well as early- and late-symptomatic stages. Unexpectedly, we found prominent up regulation of innate immunity genes with age-dependent change in their expression, in all three organs. We shortlisted a set of 12 secretory genes whose expression steadily increased with age in both brain and liver, as potential plasma correlates of neurological and/or liver disease. Ten were innate immune genes with eight ascribed to lysosomes. Several are known to be elevated in diseased organs of murine models of other lysosomal diseases including Gaucher's disease, Sandhoff disease and MPSIIIB. We validated the top candidate lysozyme, in the plasma of Npc1(-/-) as well as Balb/c Npc1(nmf164) mice (bearing a point mutation closer to human disease mutants) and show its reduction in response to an emerging therapeutic. We further established elevation of innate immunity in Npc1(-/-) mice through multiple functional assays including inhibition of bacterial infection as well as cellular analysis and immunohistochemistry. These data revealed neutrophil elevation in the Npc1(-/-) spleen and liver (where large foci were detected proximal to damaged tissue). Together our results yield a set of lysosomal, secretory innate immunity genes that have potential to be developed as pan or specific plasma markers for neurological diseases associated with lysosomal storage and where diagnosis is a major problem. Further, the accumulation of neutrophils in diseased organs (hitherto not associated with NPC) suggests their role in pathophysiology and disease exacerbation.

WhiB2/Rv3260c, a cell division-associated protein of Mycobacterium tuberculosis H37Rv, has properties of a chaperone.

whiB-like genes have been found in all actinomycetes sequenced so far. The amino-acid sequences of WhiB proteins of Mycobacterium tuberculosis H37Rv are highly conserved and participate in several cellular functions. Unlike other WhiB proteins of M. tuberculosis that have properties of protein disulfide reductases, WhiB2 showed properties like a chaperone as it suppressed the aggregation of several model substrates (e.g. citrate synthase, rhodanese and luciferase). Suppression of aggregation of the model substrates did not require ATP. Four cysteine residues of WhiB2 form two intramolecular disulfide bonds; however, chaperone function was unaffected by the redox state of the cysteines. WhiB2 also restored the activity of chemically denatured citrate synthase and did not require either ATP or a co-chaperone for refolding. The results indicate that WhiB2, which has been shown to be associated with cell division in mycobacteria and streptomyces, has evolved independently of other WhiBs, although it retains basic properties of this group of proteins. This is the first report to show that a WhiB protein has chaperone-like function; therefore, this report will have major implications in attempts to understand the role of WhiB proteins in mycobacteria, particularly in cell division.

Redox biology of Mycobacterium tuberculosis H37Rv: protein-protein interaction between GlgB and WhiB1 involves exchange of thiol-disulfide.

BACKGROUND: Mycobacterium tuberculosis, an intracellular pathogen encounters redox stress throughout its life inside the host. In order to protect itself from the redox onslaughts of host immune system, M. tuberculosis appears to have developed accessory thioredoxin-like proteins which are represented by ORFs encoding WhiB-like proteins. We have earlier reported that WhiB1/Rv3219 is a thioredoxin like protein of M. tuberculosis and functions as a protein disulfide reductase. Generally thioredoxins have many substrate proteins. The current study aims to identify the substrate protein(s) of M. tuberculosis WhiB1. RESULTS: Using yeast two-hybrid screen, we identified alpha (1,4)-glucan branching enzyme (GlgB) of M. tuberculosis as a interaction partner of WhiB1. In vitro GST pull down assay confirmed the direct physical interaction between GlgB and WhiB1. Both mass spectrometry data of tryptic digests and in vitro labeling of cysteine residues with 4-acetamido-4' maleimidyl-stilbene-2, 2'-disulfonic acid showed that in GlgB, C95 and C658 are free but C193 and C617 form an intra-molecular disulfide bond. WhiB1 has a C37XXC40 motif thus a C40S mutation renders C37 to exist as a free thiol to form a hetero-disulfide bond with the cysteine residue of substrate protein. A disulfide mediated binary complex formation between GlgB and WhiB1C40S was shown by both in-solution protein-protein interaction and thioredoxin affinity chromatography. Finally, transfer of reducing equivalent from WhiB1 to GlgB disulfide was confirmed by 4-acetamido-4' maleimidyl-stilbene-2, 2'-disulfonic acid trapping by the reduced disulfide of GlgB. Two different thioredoxins, TrxB/Rv1471 and TrxC/Rv3914 of M. tuberculosis could not perform this reaction suggesting that the reduction of GlgB by WhiB1 is specific. CONCLUSION: We conclude that M. tuberculosis GlgB has one intra-molecular disulfide bond which is formed between C193 and C617. WhiB1, a thioredoxin like protein interacts with GlgB and transfers its electrons to the disulfide thus reduces the intra-molecular disulfide bond of GlgB. For the first time, we report that GlgB is one of the in vivo substrate of M. tuberculosis WhiB1.

Studies on structural and functional divergence among seven WhiB proteins of Mycobacterium tuberculosis H37Rv.

The whiB-like genes (1-7) of Mycobacterium tuberculosis are involved in cell division, nutrient starvation, pathogenesis, antibiotic resistance and stress sensing. Although the biochemical properties of WhiB1, WhiB3 and WhiB4 are known, there is no information about the other proteins. Here, we elucidate in detail the biochemical and biophysical properties of WhiB2, WhiB5, WhiB6 and WhiB7 of M. tuberculosis and present a comprehensive comparative study on the molecular properties of all WhiB proteins. UV-Vis spectroscopy has suggested the presence of a redox-sensitive [2Fe-2S] cluster in each of the WhiB proteins, which remains stably bound to the proteins in the presence of 8 m urea. The [2Fe-2S] cluster of each protein was oxidation labile but the rate of cluster loss decreased under reducing environments. The [2Fe-2S] cluster of each WhiB protein responded differently to the oxidative effect of air and oxidized glutathione. In all cases, disassembly of the [2Fe-2S] cluster was coupled with the oxidation of cysteine-thiols and the formation of two intramolecular disulfide bonds. Both CD and fluorescence spectroscopy revealed that WhiB proteins are structurally divergent members of the same family. Similar to WhiB1, WhiB3 and WhiB4, apo WhiB5, WhiB6 and WhiB7 also reduced the disulfide of insulin, a model substrate. However, the reduction efficiency varied significantly. Surprisingly, WhiB2 did not reduce the insulin disulfide, even though its basic properties were similar to those of others. The structural and functional divergence among WhiB proteins indicated that each WhiB protein is a distinguished member of the same family and together they may represent a novel redox system for M. tuberculosis.

Molecular function of WhiB4/Rv3681c of Mycobacterium tuberculosis H37Rv: a [4Fe-4S] cluster co-ordinating protein disulphide reductase.

The genome sequence of Mycobacterium tuberculosis H37Rv revealed the presence of seven whiB-like open reading frames. In spite of several genetic studies on whiB genes, the biochemical properties of WhiB proteins are poorly understood. All WhiB-like proteins have four conserved cysteine residues, out of which two are present in a CXXC motif. We report for the first time the detailed biochemical and biophysical properties of M. tuberculosis WhiB4/Rv3681c and demonstrate the functional relevance of four conserved cysteines and the CXXC motif. UV-visible absorption spectra of freshly purified mWhiB4 showed the presence of a [2Fe-2S] cluster, whereas the electron paramagnetic resonance (EPR) spectra of reconstituted protein showed the presence of a [4Fe-4S] cluster. The iron-sulphur cluster was redox sensitive but stably co-ordinated to the protein even in the presence of high concentration of chaotropic agents. Despite primary sequence divergence from thioredoxin family proteins, the apo mWhiB4 has properties similar to thioredoxins and functions as a protein disulphide reductase, whereas holo mWhiB4 is enzymatically inactive. Apart from the cysteine thiol of CXXC motif the distantly placed thiol pair also contributes equally to the enzymatic activity of mWhiB4. A functional model of mWhiB4 in redox signaling during oxidative stress in M. tuberculosis has been presented.

Expression and characterization of alpha-(1,4)-glucan branching enzyme Rv1326c of Mycobacterium tuberculosis H37Rv.

Glycogen branching enzyme (GlgB, EC 2.4.1.18) catalyzes the third step of glycogen biosynthesis by the cleavage of an alpha-(1,4)-glucosidic linkage and subsequent transfer of cleaved oligosaccharide to form a new alpha-(1,6)-branch. A single glgB gene Rv1326c is present in Mycobacterium tuberculosis. The predicted amino acid sequence of GlgB of M. tuberculosis has all the conserved regions of alpha-amylase family proteins. The overall amino acid identity to other GlgBs ranges from 48.5 to 99%. The glgB gene of M. tuberculosis was cloned and expressed in Escherichia coli. The recombinant protein was purified to homogeneity using metal affinity and ion exchange chromatography. The recombinant protein is a monomer as evidenced by gel filtration chromatography, is active as an enzyme, and uses amylose as the substrate. Enzyme activity was optimal at pH 7.0, 30 degrees C and divalent cations such as Zn2+ and Cu2+ inhibited activity. CD spectroscopy, proteolytic cleavage and mass spectroscopy analyses revealed that cysteine residues of GlgB form structural disulfide bond(s), which allow the protein to exist in two different redox-dependent conformational states. These conformations have different surface hydrophobicities as evidenced by ANS-fluorescence of oxidized and reduced GlgB. Although the conformational change did not affect the branching enzyme activity, the change in surface hydrophobicity could influence the interaction or dissociation of different cellular proteins with GlgB in response to different physiological states.