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Research reporting from the Hebrew University of Jerusalem

Year 2015

 

http://huj-fondation-papa-2015The molecular basis of the laminopathic-based Charcot-Marie-Tooth disorder – from a mutation to a potential treatment

Research report submitted by Professor Yosef Gruenbaum, PhD, Department of

Genetics, The Hebrew University of Jerusalem

Introduction

The Charcot-Marie-Tooth (CMT) disorders, also known as “hereditary motor and

sensory neuropathies” (HMSNs) form the most common group of inherited

neuropathies, affecting 10–40/100,000 individuals. The CMT disorders can be

divided into two subtypes: demyelinating (i.e., type 1, or CMT1) and axonal (i.e., type

2, or CMT2) neuropathies. Genetically, CMT2 is a heterogeneous group of peripheral

neuropathies [1]. Seven genes and thirty loci have been reported for CMT1. In

contrast, only two specific genes responsible for autosomal dominant CMT2 (ADCMT2)

and one gene for autosomal recessive (AR) CMT2 (AR-CMT2) have been

identified. These include mutations in the type IV intermediate filaments (IF) gene

neurofilament-light (NF-L) gene, (MIM 162280), microtubule kinesin superfamily

member motor protein KIF1Bb (MIM 118210), and the type V nuclear intermediate

filament gene LMNA (MIM 150330), which encodes lamins A/C. Besides these

mutations, gene defects in MPZ (MIM 118200), which encodes myelin protein zero

causes both CMT1 and CMT2 phenotypes.

The mutations in the LMNA gene cause AR-CMT2, which is a rare and

severe condition. Clinically, the main symptoms in 90% of cases are early onset of

the disease, symmetrical muscle weakness and wasting (predominantly in the distal

lower limbs), foot deformities, and walking difficulties associated with reduced or

absent tendon reflexes. Confirmation of diagnosis relies essentially on

electrophysiology, which shows NCVs 138 m/s at the median nerve, and on

histopathology after nerve biopsy, which evidences a loss of myelinated fibers with

(for AD-CMT2) or without (for AR-CMT2) regenerative attempts.

The amino acid substitution R298C in LMNA encoding lamins A/C causes

AD-CMT2 [2]. Mutations in other residues in the LMNA gene cause 13 other

diseases, collectively termed laminopathies, including AR-Emery Dreifuss and limbgirdle

muscular dystrophies, heart-hand syndrome, dilated cardiomyopathy, partial

2

lipodystrophy, Seip syndrome, diabetes, Mandibuloacral Dysplasia, Hutchison-Gilford

and atypical Werner progeria syndromes and Restrictive Dermopathy [3, 4].

It is still unknown why mutations in specific residues in the lamin A isoforms

cause specific diseases and what are the relationship between a specific mutation

and the phenotypes that it causes. The structural model suggests that changes in

lamin filament assembly causes weakening in the scaffold of nuclei leading to cell

death. The gene expression model suggests that specific transcriptional regulators

interact with lamins A/C. For example, a specific mutation in lamins A/C causes a

loss or abnormal activity of a specific transcriptional regulator and cell death of

specific cells. The cell proliferation model suggests that lamins A/C mutations affect

the regulation of the cell cycle in specific type of cells [5].

Mammals have a highly complex nuclear lamina system comprising of 3 lamin

genes and probably hundreds of lamin-interacting proteins, which makes the

understanding of the molecular basis of CMT2 extremely difficult. In contrast, C.

elegans have a simple evolutionarily conserved nuclear lamina [6, 7]. In addition,

genetic analysis in C. elegans is relatively simple. Thus, studies of lamin in C.

elegans can address fundamental questions regarding: why mutations in lamins

cause death of nerve cells, which transcriptional regulators in the nerve cell require

R298 for its interaction, do nerve cell nuclei change their shape due to CMT2

mutations and how lamin assembly into filaments is affected by the R298C mutation.

The combined research in mammalian cells and in C. elegans should provide a

better understanding of the disease and will suggest ways for treating it.

Another complication to the story of laminopathic diseases is the fact that

mutations in lamin cause abnormal post-translational modifications of its carboxyl

terminus, leaving a fraction of farnesylated molecules (reviewed in [4].

Experiments performed during the past year

Further testing the mechanical model of laminopathies There are roughly 14

distinct heritable autosomal dominant diseases associated with mutations in lamins

A/C, including Emery-Dreifuss muscular dystrophy (EDMD). The mechanical model

proposes that the lamin mutations change the mechanical properties of muscle

nuclei, leading to cell death and tissue deterioration. Here, we developed an

experimental protocol that analyzes the effect of disease-linked lamin mutations on

the response of nuclei to mechanical strain in living C. elegans. We found that an

EDMD mutation, L535P, disrupts nuclear mechanical properties specifically in

muscle nuclei. Inhibiting lamin prenylation rescued the mechanical properties of the

EDMD nuclei, reversed the muscle phenotypes and led to normal motility. The LINC

3

complex and emerin were also required to regulate the mechanical response of C.

elegans nuclei. This study provides evidence to support the mechanical model and

offers a potential future therapeutic approach towards curing laminopathic diseases.

Figure 1: Experimental concept. A late larval L4 animal is glued at both ends to a

deformable silicone membrane.

The membrane is placed on the

stretching device under a

fluorescence microscope.

Images of GFP::lamin are taken

before stretching, at maximal

stretching and after relaxation

from a 1 min stretch. (A) Analysis

of nuclear mechanical response

to external strain application

(ESA) in intact C. elegans.

Normalized nuclear strain (NNS)

was measured as the longitudinal deformation of the nucleus during ESA (light red

arrow, right panel) along the stretch axis (directionality of light and dark red arrows)

compared to the initial length of the same nucleus (light red arrow, left panel) before

strain application, normalized to the longitudinal deformation of the surrounding

tissue (dark red arrows) along the stretch axis. (B).Analysis of nuclear recovery from

mechanical strain. Residual strain (RS) was measured as the length of the nucleus

(light red arrow, left panel) along the stretch axis (directionality of light and dark red

arrows) before the application of external strain (light red arrow, left panel) compared

to its length along the stretch axis after the strain has been eliminated (light red arrow,

right panel).

4

Figure 2: The

EDMD lamin

mutation L535P

causes

alterations in the

response of

muscle nuclei to

mechanical

strain. The

mechanical

response in

different living C.

elegans strains to

external strain

application (ESA)

was measured in

muscle (A) and

hypodermis (B)

nuclei.

Emerin::GFP was

used when lmn-1

was

downregulated. EV, a strain expressing emerin::GFP, was subjected to feeding with

an empty vector. The p-values for the different normalized nuclear strain (NNS)

experiments were 0.67, 2e-13, 0.76, 0.42, 0.78, 6.34e-6 and 7.08e-7 in (A) and 0.98,

0.0002, 0.41, 0.08, 0.72 and 0.74 in (B) for EV, lmn-1(RNAi), R460C, G472D, L229P,

Q159K and L535P, respectively. The residual strain (RS) in different living C.

elegans strains after relaxation from ESA was measured in muscle (C) and

hypodermis (D) nuclei. The p-values for the different residual strain (RS) were: 0.86,

0.0006, 2.5e-7 and 1.59e-10 in (C) and 0.75, 0.03, 9.44e-6 and 0.08 in (D) for R460C,

lmn-1 RNAi, Q159K and L535P, respectively. Error bars represent SEM. P values

are compared to wild-type lamin::GFP..

5

Figure 3: Downregulation of fdps-1 rescues both the nuclear mechanical

properties and disease

phenotypes of L535P animals. (A)

NNS values were measured in

response to fdps-1 (RNAi)

treatment in muscle (left panels)

and hypodermis (right panels)

nuclei. Significant changes were

observed in fdps-1 (RNAi) of wildtype

nuclei in both muscle and

hypodermis compared to untreated

nuclei (p=0.004 and 0.001

respectively). A significant change

was also observed in L535P

muscle nuclei subjected to fdps-1

(RNAi) (p= 8.2e-8) compared to untreated L535P nuclei. (B) Recovery from

mechanical strain was analyzed in muscle (left panel) and hypodermis (right panel)

nuclei. Significant changes were observed in fdps-1 (RNAi) of L535P muscle nuclei

(p= 1.2e-19) and hypodermis nuclei (p= 6.2e-6). Error bars represent SEM. (C)

Phalloidin staining in C. elegans subjected to empty vector (EV) (left panels) or fdps-

1 (RNAi) (right panels). Top panels – wild-type lamin; middle panels -animals

expressing the L535P lamin mutation; bottom panels – enlargement of a region taken

from the middle panels. Scale bar, 10 mm. (D) Electron microscopy analysis of the

effects of fdps-1 (RNAi) on muscle organization in C. elegans subjected to treatment

with EV (left panels) or fdps-1 (RNAi) (right panels). In animals expressing wild type

lamin (top panels), fdps-1 (RNAi) treatment did not cause a change in muscle

organization (black arrowheads). In contrast, fdps-1 (RNAi) rescued the aberrant

muscle morphology observed in animals expressing the L535P mutation (black

arrowheads in bottom panels). (E). Analysis of animal motility in wild-type and L535P

strains in response to fdps-1 (RNAi) treatment. A significant change was observed in

the L535P animals following fdps-1 RNAi treatment (p= 2.4e-13). Error bars represent

SEM. * p<0.05, **p<0.005, ***p<0.0005.

Figure 4: Downregulation of unc-84 affects both the nuclear mechanical

properties and motility of L535P animals. (A) NNS values were measured in

response to unc-84 (RNAi) treatment in muscle (left panels) and hypodermis (right

panels) nuclei. Significant changes were observed in L535P muscle nuclei (p=0.01

6

compared to untreated L535P nuclei) and in unc-84 (RNAi) wild-type hypodermal

nuclei (p= 0.03 compared to untreated wild-type hypodermal nuclei) (B) Recovery

from mechanical strain was analyzed in muscle (left panel) and hypodermis (right

panel) nuclei. Significant changes were observed in L535P unc-84 (RNAi) muscle

nuclei compared to untreated L535P (p=0.0002), in hypodermal nuclei subjected to

unc-84 (RNAi) compared to wild-type nuclei (p=0.0007) and in hypodermal L535P

nuclei subjected to unc-84 (RNAi) (p=8.8e-7 compared to untreated hypodermal

L535P nuclei). (C) Analysis of animal motility in wild-type and L535P strains in

response to unc-84 (RNAi) treatment. A significant change was observed in the

L535P animals following unc-84 RNAi treatment (p= 0.001). Error bars represent

SEM. * p<0.05, **p<0.005, ***p<0.0005.

Figure 5: emr-1 (RNAi) affects the nuclear mechanical properties but not the

motility of L535P animals. (A) NNS values were measured in response to emr-1

(RNAi) treatment in muscle (left panels) and hypodermis (right panels) nuclei.

Significant changes were only observed in L535P emr-1 (RNAi) hypodermal nuclei

(p=6.6e-5compared to untreated hypodermal L535P nuclei). (B) Recovery from

mechanical strain was analyzed in muscle (left panel) and hypodermis (right panel)

nuclei. Significant changes in both muscle and hypodermal nuclei were observed for

both strains (in wild-type lamin 0.01 and 0.0003 and in L535P animals 4.9e-6 and

0.006 respectively). (C) Analysis of animal motility in wild-type and L535P strains in

response to emr-1 (RNAi) treatment. Error bars represent SEM. * p<0.05, **p<0.005,

***p<0.0005.

References

1. Gemignani F, Marbini A (2001) Charcot–Marie–Tooth disease (CMT):

distinctive phenotypic

and genotypic features in CMT type 2. J Neuro Sci 184: 1-9

2. De Sandre-Giovannoli A et al (2002) Homozygous defects in LMNA,

encoding Lamin A/C nuclear-envelope proteins, cause autosomal recessive axonal

neuropathy in human (Charcot-Marie-Tooth Disorder Type 2) and mouse. Am J

Hum Genet 70: 726-736

3. Mattout A, Dechat T, Adam SA, Goldman RD, Gruenbaum Y (2006)

Nuclear lamins, diseases and aging. Curr Opin Cell Biol 18: 1-7

4. Prokocimer M, Davidovich M, Nissim-Rafinia M, Wiesel-Motiuk N, Bar D,

Barkan R, Meshorer E, Gruenbaum Y (2009) Nuclear lamins: key regulators

of nuclear structure and activities. J Cell Mol Med 13: 1059-1085

5. Gruenbaum Y, Margalit A, Goldman RD, Shumaker DK, Wilson KL

(2005) The nuclear lamina comes of age. Nat Rev Mol Cell Biol 6: 21-31

6. Cohen M, Lee KK, Wilson KL, Gruenbaum Y (2001) Transcriptional

repression, apoptosis, human disease and the functional evolution of the

nuclear lamina. Trends Bioc Sci 26: 41-47

7. Melcer S, Gruenbaum Y, Krohne G (2007) Invertebrate lamins. Exp Cell Res

313: 2157-2166

8. Grossman E, Dahan I, Stick R, Goldberg MW, Gruenbaum Y, Medalia O

(2012) Filaments assembly of ectopically expressed Caenorhabditis elegans

lamin within Xenopus oocytes. J Struct Biol 177: 113-118

9. Bengtsson L, Wilson KL (2006) Barrier-to-autointegration factor

phosphorylation on Ser-4 regulates emerin binding to lamin A in vitro and

emerin localization in vivo. Mol Biol Cell 17: 1154-1163

Fondation de bourse de recherche sur la sclérose amyotrophique latérale (maladie de charcot) à la mémoire de Jacques Kluger

2013-2014

 

Research report submitted by Professor Yosef Gruenbaum, PhD, Department of Genetics, The Hebrew University of Jerusalem

 

The Molecular Basis of the Laminopathic-Based Charcot-Marie-Tooth Disorder – From a Mutation to a Potential Treatment

Introduction

The Charcot-Marie-Tooth (CMT) disorders, also known as “hereditary motor and sensory neuropathies” (HMSNs) form the most common group of inherited neuropathies, affecting 10–40/100,000 individuals. The CMT disorders can be divided into two subtypes: demyelinating (i.e., type 1, or CMT1) and axonal (i.e., type 2, or CMT2) neuropathies. Genetically, CMT2 is a heterogeneous group of peripheral neuropathies [1]. Seven genes and thirty loci have been reported for CMT1. In contrast, only two specific genes responsible for autosomal dominant CMT2 (AD-CMT2) and one gene for authosomal recessive CMT2 (AR-CMT2) have been identified. These include mutations in the type IV intermediate filaments (IF) gene neurofilament-light (NF-L) gene, (MIM 162280), microtubule kinesin superfamily member motor protein KIF1Bb (MIM 118210), and the type V nuclear intermediate filament gene LMNA (MIM 150330), which encodes lamins A/C. Besides these mutations, gene defects in MPZ (MIM 118200), which encodes myelin protein zero causes both CMT1 and CMT2 phenotypes.

The mutations in the LMNA gene cause AR-CMT2, which is a rare and severe condition. Clinically, the main symptoms in 90% of cases are early onset of the disease, symmetrical muscle weakness and wasting (predominantly in the distal lower limbs), foot deformities, and walking difficulties associated with reduced or absent tendon reflexes. Confirmation of diagnosis relies essentially on electrophysiology, which shows NCVs 138 m/s at the median nerve, and on histopathology after nerve biopsy, which evidences a loss of myelinated fibers with (for AD-CMT2) or without (for AR-CMT2) regenerative attempts.

The amino acid substitution R298C in LMNA encoding lamins A/C causes AD-CMT2 [2]. Mutations in other residues in the LMNA gene cause 11 other diseases, collectively termed laminopathies, including AR-Emery Dreifuss and limb-girdle muscular dystrophies, heart-hand syndrome, dilated cardiomyopathy, partial lipodystrophy, Seip syndrome, diabetes, Mandibuloacral Dysplasia, Hutchison-Gilford and atypical Werner progeria syndromes and Restrictive Dermopathy [3, 4].

It is still unknown why mutations in specific residues in the lamin A isoforms cause specific diseases and what are the relationship between a specific mutation and the phenotypes that it causes. The structural model suggests that changes in lamin filament assembly causes weakening in the scaffold of nuclei leading to cell death. The gene expression model suggests that specific transcriptional regulators interact with lamins A/C. For example, a specific mutation in lamins A/C causes a loss or abnormal activity of a specific transcriptional regulator and cell death of specific cells. The cell proliferation model suggests that lamins A/C mutations affect the regulation of the cell cycle in specific type of cells [5].

Mammals have a highly complex nuclear lamina system comprising of 3 lamin genes and probably hundreds of lamin-interacting proteins, which makes the understanding of the molecular basis of CMT2 extremely difficult. In contrast, C. elegans have a simple evolutionarily conserved nuclear lamina [6, 7]. In addition, genetic analysis in C. elegans is relatively simple. Thus, studies of lamin in C. elegans can address fundamental questions regarding: why mutations in lamins cause death of nerve cells, which transcriptional regulators in the nerve cell require R298 for its interaction, do nerve cell nuclei change their shape due to CMT2 mutations and how lamin assembly into filaments is affected by the R298C mutation. The combined research in mammalian cells and in C. elegans should provide a better understanding of the disease and will suggest ways for treating it.

Another complication to the story of laminopathic diseases is the fact that mutations in lamin cause abnormal post-translational modifications of its carboxyl terminus, leaving a fraction of farnesylated molecules (reviewed in [4].

 

Experiments performed during the past year

Testing the mechanical model of laminopathies

There are more than 14 distinct heritable diseases associated with mutations in lamins including muscular dystrophies, lipodystrophies, Charcot-Marie-Tooth and accelerated aging. The mechanical model for these diseases suggests that lamin mutations increase nuclear fragility, resulting in cell death and progressive failure in tissues such as muscle and peripheral neurons that are exposed to repetitive mechanical stress. However, It remains unclear how mutations in ubiquitously expressed lamins can lead to often highly tissue-specific disorders. Many of the lamin residues that cause laminopathic diseases (including CMT), as well as most lamin functions and the effects of the disease-linked mutations, are conserved in Caenorhabditis elegans. Here, we tested the effects of disease-linked lamin mutations on the response of living C. elegans nuclei from different tissues to mechanical strain in the context of living animals. We are using the mechanical device show in in Figure 1 to test changes in the mechanical properties of nuclei.

Figure 1. The mechanical device that we use to measure nuclear deformation: A late larval L4 animal is glued at both ends to a deformable silicone membrane. The membrane is placed on the stretching device under a fluorescence microscope. Images of GFP::lamin are taken before stretching, at maximal stretching of 21.7% membrane strain and after relaxation,

 

 

We found that certain mutations cause a tissue specific response to the mechanical strain applied on the worm, and the affected tissues correspond to the type of disease (Figs 2&3).

 

Figure 2: Analysis of normalized nuclear strain in stretched nuclei.

Normalized nuclear strain (NNS – A) is measured as the deformation of the nucleus (thin red arrows) along the stretch axis (green arrows) in response to the membrane being stretched, normalized to the deformation of the membrane (blue arrows) along the stretch axis. B) Images taken of L4 worms (top panel, scale bar- 50 µm) and nuclei before (left panels) and during (right panels) membrane stretching. Stretch axis indicated in red. C) Normalized nuclear strain in muscle tissue. The progeria-linked mutation, Q159K (p=6.34e-6), and the EDMD-linked mutation L535P (p=7.08e-7), increased the rigidity of muscle nuclei in response to mechanical strain, as compared to control muscle nuclei of worms carrying WT lamin or lamin with the benign mutation R460C. In contrast, down-regulation of lamin caused muscle nuclei to be more flexible (p=2.01e-13), similar to an EDMD causing mutation T164P (2.66e-5). D) Normalized nuclear strain in hypodermal tissue. Similar to its effect in muscle cells, the progeria-linked mutation Q159K (p=0.000221) caused nuclei to be more rigid and lamin down-regulation caused the nuclei to be more flexible (p=9.57e-7). The T164P caused hypodermal nuclei to be more flexible, but the differences were not significant. Also, the L535P EDMD-linked mutation did not show a hypodermal phenotype. E) Normalized nuclear strain in neurons. Neuronal nuclei from all mutant strains showed no significant change in NNS from that of wild-type worms expressing lamin::GFP.

 

Figure 3: Analysis of the ability of nuclei to return to their original shape and dimensions. A) Induced nuclear strain (INS) is measured as the deformation of the nucleus (thin red arrows) along the stretch axis (green arrows) before and after the application of strain. B) Images taken of nuclei before (left panels) and after (right panels) membrane stretching. Stretch axis indicated in red. C) Induced nuclear strain in muscle tissue. The progeria-linked mutation, Q159K (p=2.5e-7), and the EDMD-linked mutation L535P (p=1.59e-10), demonstrated impaired ability to return to original nuclei dimensions and an inward collapse phenotype, as compared to control muscle nuclei of worms carrying WT lamin and a benign lamin mutation (R460C). Down-regulation of lamin caused the same phenotype, but to a lesser extent (p=0.0005). D) Induced nuclear strain in hypodermal tissue. Similar to the effect in muscle cells, the progeria-linked mutation Q159K (p=9.4e-6) demonstrated an inward collapse phenotype. Lamin down-regulation showed the same phenotype but to a much lesser extent (p=0.03). The T164P and L535P EDMD-linked mutations did not have a hypodermal phenotype. E) Induced nuclear strain in nerve tissue. Nerve nuclei from all mutant worms showed no significant change in INS from that of the worms carrying WT lamin.

In a related study we compared the amount of GFP::lamin in different tissues and found that the amount of peripheral lamin is similar in neurons and muscle nuclei and is 10-20% reduced in hypodermal cells (data not shown).

 

References

  1. Gemignani F, Marbini A (2001) Charcot–Marie–Tooth disease (CMT): distinctive phenotypic

and genotypic features in CMT type 2. J Neuro Sci 184: 1-9

  1. De Sandre-Giovannoli A et al (2002) Homozygous defects in LMNA, encoding Lamin A/C nuclear-envelope proteins, cause autosomal recessive axonal neuropathy in human (Charcot-Marie-Tooth Disorder Type 2) and mouse. Am J Hum Genet 70: 726-736
  2. Mattout A, Dechat T, Adam SA, Goldman RD, Gruenbaum Y (2006) Nuclear lamins, diseases and aging. Curr Opin Cell Biol 18: 1-7
  3. Prokocimer M, Davidovich M, Nissim-Rafinia M, Wiesel-Motiuk N, Bar D, Barkan R, Meshorer E, Gruenbaum Y (2009) Nuclear lamins: key regulators of nuclear structure and activities. J Cell Mol Med 13: 1059-1085
  4. Gruenbaum Y, Margalit A, Goldman RD, Shumaker DK, Wilson KL (2005) The nuclear lamina comes of age. Nat Rev Mol Cell Biol 6: 21-31
  5. Cohen M, Lee KK, Wilson KL, Gruenbaum Y (2001) Transcriptional repression, apoptosis, human disease and the functional evolution of the nuclear lamina. Trends Bioc Sci 26: 41-47
  6. Melcer S, Gruenbaum Y, Krohne G (2007) Invertebrate lamins. Exp Cell Res 313: 2157-2166
  7. Grossman E, Dahan I, Stick R, Goldberg MW, Gruenbaum Y, Medalia O (2012) Filaments assembly of ectopically expressed Caenorhabditis elegans lamin within Xenopus oocytes. J Struct Biol 177: 113-118
  8. Bengtsson L, Wilson KL (2006) Barrier-to-autointegration factor phosphorylation on Ser-4 regulates emerin binding to lamin A in vitro and emerin localization in vivo. Mol Biol Cell 17: 1154-1163




























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