Nationwide Children’s Hospital August 25, 2015
Nuclear Envelope and Muscular Dystrophy
Howard J. Worman, M.D. Columbia University
Columbia University Medical Center The Nuclear Envelope
By D. W. Fawcett The Nuclear Envelope: Nuclear Lamina
Dauer & Worman, 2009 Nuclear Lamins: A Brief “Ancient” History
Dwyer& Blobel (1975)
Aebi et al. (1986)
Aebi et al. (1986) Gerace, Blum & Blobel (1978) Goldman et al. (1986) McKeon et al. (1986) Fisher et al. (1986) HUMAN NUCLEAR LAMINS
LOCUS CHROMOSOME PROTEINS CELL-TYPES EXPRESSED
LMNA 1q21.2-21.3 Lamin A Differentiated Somatic
Lamin C Differentiated Somatic
Lamin A D10 Differentiated Somatic
Lamin C2 Germ
LMNB1 5q23.2-31.1 Lamin B1 Apparently All Somatic
LMNB2 19p13.3 Lamin B2 All or Most Somatic
Lamin B3 Germ LMNA Encoding A-type Lamins
Lin and Worman, JBC, 1993
Localization to chromosome 1q21.2–q21.3
Wynder et al., Genomics, 1995 Mutations in LMNA Cause Diseases (“Laminopathies”) with Four Major Tissue-Selective Phenotypes
Dauer and Worman Dev. Cell (2009) LMNA Genotype- Phenotype Correlations Dominant mutations causing muscle diseases are missense, splicing and small deletions and even haploinsufficiency
Recessive missense mutation peripheral neuropathy
Dominant mutations cluster in a small region encoded of exon 8 in partial lipodystrophy
Recessive missense in MAD
Dominant G608G or S608G in HGPF Worman and Courvalin (2005) Hutchinson-Gilford Progeria Syndrome (HGPS)
Normally, lamin A starts as a precursor prelamin A, which is fanesylated. The farnesylated precursor is recognized by ZMPSTE24 endoprotease and cleaved; mature lamin A is not farnesylated.
From Worman and Courvalin. (2005)
farnesyl
The LMNA mutation causing HGPS leads to expression of Paradisi et al. (2005) a truncated prelamin A (“progerin”), which remains farnesylated.
Modified from Eriksson et al. Nature (2003) Evidence that Abnormal Farnesylation of Progerin is Involved in Pathogenesis of HGPS
•Treatment of cultured fibroblasts from model mice and affected human with protein prenylation inhibitors reverse abnormal nuclear shape characteristic of cells expressing progerin •Treatment of mice with a targeted HGPS mutation with farnesyltransferase inhibitors improve phenotype •Based on above, clinical trials in affected children have been conducted but unfortunately uncontrolled ones •There are some LMNA mutations that cause progeroid phenotypes that apparently do not alter prenylation LMNA Mutations Causing Striated Muscle Disease Versus LMNA Mutations Causing Lipodystrophy
Dunnigan-type Familial Partial Lipodystrphy Emery-Dreifuss Muscular Dystrophy and Variant Disorders The vast majority of LMNA mutations in Dunnigan-type partial lipodsytrophy change the surface charge of an Ig fold in the lamin A/C tail domain
Cter Nter Cter Nter
W520
Y481 R453 R527
R482
G465 V442, V452, I469, N456 I497, T528, L530 K486
Krimm et al. Stucture (2001) Recessive LMNA Mutations
Charcot-Marie-Tooth type 2B1 Mandibuloacryl dysplasia
Tazir et al. (2004) Novelli et al. (2002)
LMNA R298C LMNA R2527H Integral Inner Nuclear Membrane Protein Gene Mutations
EDM – emerin Emery-Dreifuss Muscular Dystrophy (X-linked) and variants
LBR – lamin B receptor Pelger-Huët Anomaly (Heterozygous) HEM/Greenberg Skeletal Dysplasia (Homozygous)
MAN1/LEMD3 Osteopoikilosis, Buschke-Ollendorff Syndrome Outer Nuclear Membrane Protein/Perinuclear Space Gene Mutations SYNE1 – nesprin-1 Autosomal Recessive Cerebellar Ataxia Arthrogryposis Myopathies
SYNE2 – nesprin-2 Myopathies
SYNE4 – nesprin-4 High Frequency Hearing Loss
TOR1A – torsinA DYT1 Dystonia
Gundersen and Worman Cell (2013) LMNA Cardiomypathy and Muscular Dystrophy LMNA Linked Myopathies
Emery-Dreifuss Limb-girdle Type 1B Dilated Cardiomyopathy 1A
All three clinical phenotypes can occur in members of the same family with the same mutation and there is overlap between phenotypes: LMNA dilated cardiomyopathy with variable skeletal muscle involvement. LMNA Cardiomyopathy
•Approximately 6% to 9% of all dilated cardiomyopathies •Up to 30% of dilated cardiomyopathies with AV block and skeletal muscle involvement
van Berlo et al. J. Mol. Med. 2004; 83:79-83 Taylor et al. J. Am. Coll. Cardiol. 2003;41: 771-780 LMNAH222P/H222P Mouse Model of Emery-Dreifuss Muscular Dystrophy (Arimura et al. 2005)
Heart disease progression from 8 to 20 weeks of age in male mice
Heart structure and function Significant LV dilatation and decreased fractional shortening by echocardiography Abnormal Signaling in Hearts of LmnaH222P/H222 Mice
HEART at 10 weeks
AFFYMETRIX 430 2.0
MAP Kinase AKT/mTOR Early Activation of ERK1/2 in Hearts of LmnaH222P/H222P Mice
> ERK1/2 and downstream genes are abnormally activated prior to (4 weeks and 7 weeks) and concurrent with (10 weeks) the onset of cardiomyopathy in hearts of LmnaH222P/H222P mice
4 weeks
Choi et al. Sci Transl Med 2012 Muchir et al. J Clin Invest 2007 MEK1/2
MEK1/2 Inhibitor Protocol P ERK PD98059 Selumetinib P CIP-137401 Drug Administration ERK
1 6 w e e k s 2 0 w e e k s
E n d p o i n t s
-Echocardiography -Biochemical Markers -Cardiac Fibrosis
MEK 1 / 2 I n h i b i t o r T r e a t m e n t
Target Inhibition – decreased phosphorylated ERK1/2 (pERK1/2) in heart after selumetinib treatment MEK 1 / 2 I n h i b i t o r T r e a t m e n t
Muchir et al. 2012 MEK 1 / 2 I n h i b i t o r T r e a t m e n t
Fibrosis – decreased fibrosis tissue and collagen gene expression
20% 12% MEK 1 / 2 INHIBITION
Survival – prolonged median survival (proprietary MEK1/2 inhibitor) MEK1/2 Inhibitor Treatment Blocks AKT/mTOR in Hearts of LmnaH222P/H222P Mice
Chhoi et al. Sci Transl Med (2012) Increased AKT/mTOR Activity Correlates with Decreased Autophagy
Lmna Mice
Human Subjects
Chhoi et al. Sci Transl Med (2012) Temsirolimus Blocks mTOR Activity Improving Heart Function and Enhancing Autophagy
Chhoi et al. Sci Transl Med (2012) One Model of LMNA Cardiomyopathy
Normal Heart
Outstanding Theoretical Question: Outstanding Practical Question: How do LMNA mutation Can use of these drugs be CIP-137401 activate these pathways? optimized to treat human subjects?
Dilated Heart Emerin and LAP1 and Muscular Dystrophy Mutations in EMD Encoding Emerin Phenocopy Myopathies Caused by LMNA Mutations
X-linked Emery-Dreifuss Muscular Dystrophy (and related myopathies)
Emerin binds to A-type lamins In most cases, emerin is lacking from the nuclear envelope
Muscle Frozen Sections Anti-emerin Ab
Normal X-EDMD X-EDMD
Nagano et al. Nat Genet. 1996;12:254-259
Modified from A.E.H. Emery Emerin as a Candidate LAP1 Binding Protein
Lamina-associated polypeptide 1 (LAP1) integral inner nuclear membrane protein of unclear function
In an unbiased proteomics screen for interacting proteins in collaboration with Bill Dauer, emerin was found to be top candidate Emerin Binds to LAP1
Biochemical Assays
FRET
Shin et al. Dev Cell (2013) Loss of Emerin: of Mice and Men
Pacemaker
Apparently Normal Mouse
Melcon et al. Hum Mol Genet 2006;15:637-651 Emery-Dreifuss Muscular Ozawa et al. Am J Pathol 2006;168:907-917 Dystrophy A.E. H. Emery (2000) Why Don’t Emerin Null Mice Get Sick?
Mouse skeletal muscle has diminished emerin compared to human. Conversely, LAP1 is significantly higher in mouse than human striated muscle.
Shin et al. Dev Cell (2013) Therefore, We Depleted LAP1 from Mouse Skeletal Muscle
Shin et al. Dev Cell (2013) Mice Lacking LAP1 in Skeletal Muscle
Shin et al. Dev Cell (2013) LAP1 Depletion Causes Muscular Dystrophy
Shin et al. Dev Cell (2013) Depletion of LAP1 Striated Muscle on Emerin Null Background
Shin et al. Dev Cell (2013) Control: Deplete LAP1 from Hepatocytes
Shin et al. Dev Cell (2013) Deplete LAP1 from Hepatocytes – Minimal Pathology
Shin et al. Dev Cell (2013) Depletion of LAP1 from Heart
Shin et al. Nucleus (2014) Depletion of LAP1 from Heart Decreases Left Ventricular Fractional Shortening
Shin et al. Nucleus (2014) Disruption of Emerin-Lamin A/C-LAP1 Complex: Cardiomyoapthy and Muscular Dystrophy
Disruption Emerin-Lamin A/C-LAP1 Complex
ERK1/2 and AKT/mTOR Striated Muscle Disease
Columbia Past & Present Collaborators Elsewhere Revekka Boguslavsky Takuro Arimura Jason Choi Gisèle Bonne Gregg Gundersen Jean-Claude Courvalin Alan Herron Bill Dauer Stephen Lehnart Yukiko Hayashi Caroline LeDour Feng Lin Micheline Paulin-Levasseur Angelika Lüdtke Sophie Zinn-Justin Ivan Méndez-López Antoine Muchir Cecilia Östlund Paul Pavlides Jian Shan Ji-Yeon Shin Bruno Soullam Yuexia Wang Wei Wu Quan Ye Funding
BioAccelerate NYC Prize
Los Angeles Thoracic and Cardiovascular Foundation