Dr. Francesc Palau - 'Neuropatías periféricas hereditarias'
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Los días 11 y 12 de diciembre de 2014, la Fundación Ramón Areces celebró el Simposio Internacional 'Neuropatías periféricas hereditarias. Desde la biología a la terapéutica' en colaboración con CIBERER-ISCIII y el Centro de Investigación Príncipe Felipe. El tipo más común de estas patologías es la enfermedad de Charcot-Marie-Tooth, un trastorno neuromuscular hereditario con una prevalencia estimada de 17-40 afectados por 100.000 habitantes. Durante estos dos días, investigadores mostraron sus avances en la mejora del diagnóstico y el tratamiento y, por ende, de la aproximación clínica y la calidad de vida de las personas afectadas por estas patologías.
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Dr. Francesc Palau - 'Neuropatías periféricas hereditarias'
- 1. MOUSE MODELS FORMOUSE MODELS FOR CHARCOT-MARIE-TOOTH NEUROPATHYCHARCOT-MARIE-TOOTH NEUROPATHY Francesc Palau, MD, PhDFrancesc Palau, MD, PhD UGMM Genetics and Molecular Medicine UnitGenetics and Molecular Medicine Unit Centro de Investigación Príncipe FelipeCentro de Investigación Príncipe Felipe CIBER on Rare Diseases (CIBERER)CIBER on Rare Diseases (CIBERER) ValenciaValencia http://www.cipf.es/web/portada/enfermedades-raras http://www.ciberer.es
- 2. 1. Mouse as a model of human genetic disease 2. The biological and therapeutical aspects a. Genetic heterogeneity of CMT b. Clinical and pathophysiological diversity of hereditary peripheral neuropathy 3. Mouse and rat models of Charcot-Marie-Tooth disease 4. The case of mitochondrial CMT mice: Gdap1 and Mfn2 MAIN POINTS
- 3. 1.Mouse as a model of human genetic disease
- 4. Are mouse gene phenotypes mimicking human genetic disease? The mouse is a common model used in research as a way of studying human biology and diseases, but a new study questions whether this is an accurate approach considering some stark differences - aside from many similarities - between the two species Read more: http://www.natureworldnews.com/articles/10525/20141120/should-the-mouse-model-be-used-to- study-human-diseases.htm#ixzz3LDhVQoY6
- 6. Programme Genomic region(s) Website MGU (MRC, U.K.) Genome-wide/Chromosome 13 36H deletion www.mgu.har.mrc.ac.uk GSF (Germany) Genome-wide www0.gsf.de/ieg/groups/genome /enu.html Jackson Laboratory (U.S.A.) Genome-wide nmf.jax.org/index.html Northwestern University (U.S.A.) Genome-wide genome.northwestern.edu/neuro Oak Ridge National Laboratory (U.S.A.) Chromosomes 7, 10, 15 and X www.tnmouse.org/neuromutagen esis RIKEN Genomic Sciences Centre (Japan) Genome-wide www.gsc.riken.go.jp Novartis Research (U.S.A.) Genome-wide web.gnf.org/scientific/mouse.htm l University of Toronto (Canada) Genome-wide www.phenogenomics.ca/databas es/enu_P.htm Major mouse mutagenesis projects incorporating neurological-based screens
- 7. 2a. Genetic heterogeneity of CMT
- 8. Number of genes found per year since the identification of the CMT1A duplication in 1991 Timmerman V, Strickland AV, Züchner S. Genes 2014 CMT – the complexity of a Mendelian disease
- 9. 80 currently known genes (orange symbols) and their corresponding chromosomal loci (vertical bars). The corresponding phenotypes are indicated by blue symbols and are according to the disease nomenclature. Timmerman V, Strickland AV, Züchner S. Genes 2014 CMT genetic heterogeneity – Gene Map
- 10. Functional categories of genes involved in CMT and related neuropathies Timmerman V, Strickland AV, Züchner S. Genes 2014 CMT Biological Diversity
- 11. 2b. Clinical and pathophysiological diversity of hereditary peripheral neuropathy
- 12. Multiple functional proteins in myelin and axon are associated with CMT and related neuropathies CMT – Demyelinating and Axonal Neuropathies
- 14. Onion bulb formation observed in semi-thin sections (A) and by electron microscopy (B). https://www.bcm.edu/departments/neurology/n euromuscular/?pmid=13873 Hereditary neuropathy with liability to pressure palsies. (A) Note nerve fibers with "thickened" myelin sheath as is seen in semi-thin sections (arrow). In a single teased nerve fiber preparation (B), the "thickened" area appears as sausage-like structure (tomaculous neuropathy). HNPP 17p11.2 deletion CMT1A 17p11.2 duplication AR-CMT2K: GDAP1 Q163X/Q163X CMT2A: MFN2 +/M376I Sevilla et al., Brain 2003 Pareyson et al., Lancet Neurol 2013 AXONOPATHIESMYELINOPATHIES
- 15. 3. Mouse and rat models of Charcot-Marie-Tooth disease
- 16. Interindividual variability within the same mutant strain Mildly affected ratsSeverely affected rats Transgenic animals that performed poorly in the bar test showed neurogenic muscle atrophy in lower limb, with numerous atrophic fibers (red arrowheads), presumably secondary to denervation. Right, mildly affected rats have more normal histology. CMT rats show interindividual variation in Pmp22 expression Sereda et al. Nature Med 2003 CMT1A dup rat
- 17. (A, B) Light and (C, D) electron microscopy of ventral spinal roots of cx32def/RAG‐1+/? (A, C) and cx32def/RAG‐1–/– (B, D) mice. Large myelinated fibre density was fairly well preserved in 16‐ year old patient with a Cx32 mutation (Ser26Leu) (‐ F), while they were markedly diminished and axonal sprouts were abundant (G) in a 61 year old patient with a Cx32 mutation‐ ‐ (Phe69Leu). Hattori N. et al. Brain 2003 Patient’s disease versus Mouse model – CMTX1 CMTX1: GBJ1 and connexin 32 Mouse ventral spinal rootsPatients’ sural biopsies
- 18. Arnaud et al. PNAS 2009 Patient’s disease versus Mouse model – CMT4C CMT4C: SH3TC2 Patient’s biopsy Mouse Sh3tc2 KO
- 19. 4. The case of mitochondrial CMT mice: Gdap1 and Mfn2
- 20. CMT2A and Mitofusin 2 transgenic mice – Mfn2R94Q Cartoni et al. Brain 2010 Pareyson et al., Lancet Neurol 2013
- 21. Gdap1 knockout mice (Gdap1-/- ) – AR-CMT2K and CMT4AAR-CMT2K, Gdap1Q163X/Q163X Sevilla et al. 2003 CMT2K, Gdap1+/R120W Sivera et al. 2010 Niemann et al. Brain 2014 Hypomyelinating neuropathy: increased g‐ratio Barneo‐Muñoz et al. (under review) – 5 month mice Axonal neuropathy: reduced CMAPs
- 22. Mouse model Gdap1-/- – phenotype
- 24. 5-moth-old mice Gdap1-/-WT DistalProximal Increased number of mitochondria: increased fission or enlarged mitochondria (fusion)? No loss of the number of axons in the sciatic nerve
- 25. Gdap1-null mice motor neurons •Dilated endoplasmatic reticulum cisternae •Dilated perinuclear space (PS) •Tubular mitochondria with swollen cristae •Increase number of phagolysosomes (*) and autophagosomes (AV) •Disperse mitochondrial distribution Alterations in the motor neurons ultrastructureWTGdap1-/- WT mice motor neurons •Polarized mitochondrial distribution •Round mitochondria with organized cristae •Compacted endoplasmatic reticulum cistarnae ( )
- 26. WTGdap1-/-- Acetylated α-tub β-III tub merge Deacetylated α-tubulin Acetylated α-tubulin Hinckelmann et al; 2013Trends in Cell Biology. Pla-Martin et al; 2013 Neurobiology of Disease. Instability of the microtubules network ***
- 27. Alterations in mitochondrial axonal transport Gdap1-/- 5µm WT Anterograde 5µm Microfluidics Chambers Time (300sec) 5µm 5µm Time (300sec)
- 28. HADHA PK-M2 LDHA β-F1 SDHB CORE 2 COX IV GAPDH SOD2 CATALASE MFN I MFN II COX I COX II NDUFS3 β-ACTIN A Relative expression Glycolysis OXPHOS Mitochondrial dynamics β-Oxidation Oxidative stress Relative expression Relative expression Relative expression Relative expression SKELETAL MUSCLE HADHA PK-M2 LDHA β-F1 SDHB CORE 2 COX IV GAPDH SOD2 CATALASE MFN1 MFN2 COX I COX II NDUFS3 β-ACTIN Relative expression Glycolysis OXPHOS Mitochondrial dynamics β-Oxidation Relative expression Relative expression Relative expression Relative expression CEREBELLUM B Oxidative stress Relative expression HADHA PK-M2 LDHA β-F1 SDHB CORE 2 COX IV GAPDH SOD2 CATALASE MFN I MFN II COX I COX II NDUFS3 β-ACTIN Relative expression Glycolysis OXPHOS Mitochondrial dynamics β-Oxidation Relative expression Relative expression Relative expression PERIPHERAL NERVE Oxidative stress C
- 29. Ca2+ homeostasis ER M Ca2+ homeostasis Store-Operated Ca2+ Entry (SOCE) Mechanism Plasmatic Membrane Cytosol Endoplasmic Reticulum Mitochondria Mitochondria + M RE LD RE Gdap1-/- Motor neuron
- 30. Depletion of Ca2+ and decrease in SOCE activity Treated motor neurons with Thapsigargin Treated motor neurons with Ionomycin GDAP1 null situation Pla-Martin et al Neurobiol Dis 2013. Physiological situation Barneo-Muñoz et al. 2014 (under review)
- 31. Genetics & Molecular Medicine pital Universitari La Fe and IIS La Fe – Teresa Sevilla and Juan Vílchez versitat de València – Josema Torres and Federico Pallardó Groups MSO – Jorgina Satrústegui and José M. Cuezva Groups versité de Laussanne – Roman Chrast Group versidad Católica de Valencia – Jerónimo Forteza Group
Editor's Notes
- Figure 1. Metabolic protein profiles in tissues of GDAP1-KO mice. One µl tissue extracts from liver and muscle (A), brain and cerebellum (B) and spinal cord and peripheral nerves (C) of ten control (white columns) and ten GDAP1-KO (grey columns) mice were spotted onto RPPM and processed as indicated. Representative RPPM are shown. Histograms show the expression level of the markers (mean ± SEM) in muscle (A), cerebellum (B) and peripheral nerves (C) of control (gray bars) and GDAP1-KO (closed bars) mice. For printing details see Supplemental Fig. S1B. β-actin was used as loading control. The expression level of the different proteins was expressed relative to that found in HCT116 colon cancer cells. *, P<0.05 when compared to control by Student’s t test