Nationwide Children’s Hospital August 25, 2015

Nuclear Envelope and Muscular Dystrophy

Howard J. Worman, M.D. Columbia University

Columbia University Medical Center The

By D. W. Fawcett The Nuclear Envelope:

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 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 (“”) 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 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 Mutations

EDM – 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

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 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 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 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

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