Gene Expression-Targeted Isoflavone Therapy: Facts, Questions and Further Possibilities

Transcription

1 Gene Expression-Targeted Isoflavone Therapy: Facts, Questions and Further Possibilities Grzegorz Wegrzyn Department of Molecular Biology University of Gdansk Gdansk, Poland

2 Lysosomal storage diseases (LSD) A group of over 50 diseases Each disease is caused by a deficiency in: (i) a specific lysosmal hydrolase (leading to an inability to degrade particular macromolecules, for example: sphingolipids, glycoproteins, glycosaminoglycans) (ii) a protein involved in transport of particular compounds through lysosomal membranes (iii) an enzyme that modifies lysosomal proteins, ensuring their proper localization and function (iv) an activator of particular lysosomal enzyme

3 lysosomes nucleus Normal fibroblast LSD fibroblast

4 The deposition of undegraded substrates in tissues throughout the body leads to a multisystemic disease Growth deficiency (dwarfism) Central nervous system and sensory organs affetced Bones and joints problems (Disostosis multiplex) Visceral organs dysfunctions

5 Pathogenic cascades in LSD

6 Mucopolysaccharidoses (MPS): a group of lysosomal storage diseases Glycosaminoglycans (GAGs) are accumulated in MPS Different kinds of GAGs, whose degradation is inhibited at different steps, are accumulated in various MPS types.

7 Degradation of dermatan sulfate I2S enzyme α-l-iduronidase N-acetylgalactosamine 4-sulfatase β-hexosaminidase A, B, S β-glucuronidase I2S = iduronate-2-sulfatase NAc = N-acetylgalactosamine H 2 COH COOH H 2 COH O COOH S O O O O O O O O etc O S NA C NA C 1 Neufeld & Muenzer. In: The Metabolic and Molecular Bases of Inherited Disease. 2001:3421.

8 MPS are chronic, progressive and lifethreatening diseases Clinical features include: - organomegaly (e.g. liver, spleen, tongue) - dysostosis multiplex - obstructive airway disease - impaired cardiovascular functions - impaired hearing and vision - joint stiffness - hernias - spinal cord compression - hydrocephalus - mental retardation (in some cases) - SINGNIFICANY SHORTENED LIFE SPAN (death usually within a childhood)

9 Potential therapies for LSD: - Enzyme replacement therapy (ERT) - Bone marrow (or stem cell) transplantation - Gene therapy - Stop codon read-through - Small chaperones - Substrate optimization therapy - Substrate reduction therapy

10 Potential therapies for LSD: - Enzyme replacement therapy (ERT) - Bone marrow (or stem cell) transplantation - Gene therapy - Stop codon read-through - Small chaperones - Substrate optimization therapy - Substrate reduction therapy

11 The idea of substrate reduction therapy Precursors (for synthesis) SYNTHESIS DEGRADATION Substrate (for degradation) NORMAL (Synthesis = Degradation)

12 The idea of substrate reduction therapy Precursors (for synthesis) SYNTHESIS DEGRADATION Substrate (for degradation) Inhibited due to mutations LSD (Synthesis > Degradation)

13 The idea of substrate reduction therapy Inhibited by therapeutics Precursors (for synthesis) SYNTHESIS DEGRADATION Substrate (for degradation) Inhibited due to mutations SRT for LSD (Synthesis = Degradation)

14 Facts

15 Pediatric Research (2006) 60, Inhibition of glycosaminoglycan synthesis using rhodamine B in a mouse model of mucopolysaccharidosis type IIIA Roberts A.L., Thomas B.J., Wilkinson A.S., Fletcher J.M., Byers S.

16 Rodamine B is an inhibitor of GAG synthesis but the mechanism of its action in unknown

17 Reduction of GAG accumulation in MPS fibroblasts treated with rhodamine B for 14 days

18 MPS IIIA mouse treated with rhodamine B (weekly infusions of 1 mg/kg) for 6 months Untreated MPS IIIA mouse Control (a healthy mouse)

19 European Journal of Human Genetics (2006) 14, Genistein-mediated inhibition of glycosaminoglycan synthesis as a basis for gene expression-targeted isoflavone therapy for mucopolysaccharidoses Ewa Piotrowska, Joanna Jakobkiewicz-Banecka, Sylwia Baranska, Anna Tylki-Szymanska, Barbara Czartoryska, Alicja Wegrzyn and Grzegorz Wegrzyn

20 5, 7-dihydroxy-3- (4-hydroxyphenyl)-4H-1-benzopyran-4-one 4', 5, 7-trihydroxyisoflavone Genistein

21 MPS IIIA cell MPS I cells Genistein Inhibition of glycosaminoglycan (GAG) synthesis by genistein Piotrowska et al. (2006) Eur. J. Hum. Genet. 14:

22 Inhibition of EGF receptor phosphorylation (long term experiment - 24 h)

23 EGF EGF receptor Genistein Signal transduction (kinases cascade) including action of protein tyrosine kinases Activation of transcription factors Stimulation of gene expression (transcription) Jakobkiewicz-Banecka et al. (2009) J. Biomed. Sci. 16: 26

24 MPS IIIB mouse model

25 Total GAGs[ug]/DNA[ug] Short-term experiment AIM: To establish the most effective and non-toxic dose of genistein which can significantly reduce GAG storage in MPS IIIB mice and be used as substrate reduction therapy for Sanfilippo syndrome Dose [mg/kg/day] 0 Wt [GAGs/DNA] Total GAGs Genistein[mg/kg/day] HO O 5 Histology OH O Genistein OH MPS IIIB 160 Hair morphology Malinowska et al. (2009) Mol. Genet. Metabol.

26 Reduction of GAG storage in liver of MPS IIIB mice Wild-type MPS IIIB untreated MPS IIIB genistein (160 mg/kg for 8 weeks)

27 Electron Microscopy Score Score MPS IIIB WT 500x 1000x 500x 1000x 500x 1000x 5,50 5,00 4,50 4,00 3,50 3,00 2,50 2,00 1,50 1,00 0,50 0,00 Before treatment 160 mg/kg genistein Carrier control Hair morphology males Genistein [mg/kg/day] 5,50 5,00 4,50 4,00 3,50 3,00 2,50 2,00 1,50 1,00 0,50 0,00 Hair morphology females Genistein [mg/kg/day]

28 Total GAGs[ug]/DNA[ug] Long-term experiment MPS IIIB Wt Weight Blood Soy free diet Urine Every 2 months Hair Histology Biochemistry MPS IIIB MassSpec Genistein[mg/kg/day] End Point Wt Diet with genistein 160 mg/kg TopScan Circadian rythm Bar crossing Inverted Screen 6 and 8 months Malinowska et al. (2010) PLoS ONE

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31 Behavioral tests Inverted screen test Bar crossing test Open field test Home cage behavior

32 Behavioral tests

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34 Mice: Sgsh / (IIIa) Sgsh / Ext1 +/ (IIIaE1) Sgsh / Ext1 +/ Ext2 +/ (IIIaE1E2)

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36 gsrt ameliorates lysosomal storage in MPS IIIa mice Lamanna W C et al. J. Biol. Chem. 2012;287:

37 gsrt reduces hepatomegaly in MPS IIIa mice Lamanna W C et al. J. Biol. Chem. 2012;287:

38 gsrt ameliorates markers of neuropathology in MPS IIIa mice Lamanna W C et al. J. Biol. Chem. 2012;287:

39 Ext1/Ext2 heterozygosity improves the efficacy of ERT in MPS IIIa cells and mice Lamanna W C et al. J. Biol. Chem. 2012;287:

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49 Microarray analyses using HumanHT-12 Expression BeadChip Transcriptomes of human fibroblasts treated with genistien Gene expression modulation Modulation targets No. of genes Genistein concentration [ M] Time of exposure [h] up-regulated genes whole genome GAG metabolism GSL metabolism down-regulated genes whole genome GAG metabolism GSL metabolism

50 Expression of genes coding for enzymes involved in GAG synthesis and degradation is response to genistein, as assessed by microarray analyses and qrt-pcr A Relative to GAPDH as a control GAG biosynthesis GAG degradation B GAG biosynthesis GAG degradation Relative totbp as a control

51 GAG synthesis pathways GAG degradation pathways A Stimulation by genistein Inhibition by genistein No strong effect of genistein B

52 Expression of genes coding for enzymes involved in glycosphingolipids (GSL) synthesis and degradation is response to genistein, as assessed by microarray analyses and qrt-pcr A Relative to GAPDH as a control GSL biosynthesis GSL degradation B GSL biosynthesis GSL degradation Relative totbp as a control

53 Expression TFEB, a gene coding for the master positive regulator of lysosomal biogenesis, is stimulated by genistein mrna level Protein level

54 Genistein stimulates lysosomal 24 h 48h biogenesis in HDFa cells Control no genistein 30 µm genistein 60 µm genistein 100 µm genistein

55 Genistein stimulates lysosomal 24 h 48h biogenesis in MPS I cells Control no genistein 30 µm genistein 60 µm genistein 100 µm genistein

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65 Genistein decreases levels of total GAGs and heparan sulfate in MPS III patients

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68 Glycosaminoglycans (GAGs) DEGRADATION SYNTHESIS Precursors Primary GAG storage Secondary storage Inflammation Oxidative stress P-tau accumulation Apoptosis Genistein Synapse disappearance and dysfunction Dysfunctions of cells, tissues and organs Symptoms Wegrzyn (2012) IUBMB Life 64:

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76 Microarray analyses using HumanHT-12 Expression BeadChip Transcriptomes of human fibroblasts treated with genistien, kaempferol, daidzein, and their combinations upregulated genes downregulated genes Gene expression modulation Modulation targets whole genome GAG metabolism GSL metabolism whole genome GAG metabolism GSL metaboli sm No. of genes Genistein [ M] Kaempferol [ M] Genistein + Kaempfer ol [ M] Daidzein [ M] Genistein + Daidzein [ M] Time of exposure [h]

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