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
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
5.
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
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
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
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
Notas del editor
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