Preclinical Studies in VCP Disease Using Mouse Models Virginia - PowerPoint PPT Presentation

Preclinical Studies in VCP Disease Using Mouse Models Virginia Kimonis, MD., MRCP Professor, Dept. of Pediatrics, Division of Genetics and Genomic Medicine Univ. of California, Irvine vkimonis@uci.edu ENMC Conference Heemskerk, The Netherlands

Animal Models of VCP mutations • Deinhardt et al 2004, Mol Neuropath Lab, Cancer Research, UK – No homozygous p97-/- pups - early embryonic lethal – Heterozygous KO mouse normal size and fertility with normal p97/VCP expression • Weihl C et al. Hum Mol Genet. 2007 – Transgenic mice expressing p97/VCP-WT or the most common IBMPFD mutant, p97/VCP R155H, under a muscle specific promoter – R155H developed age dependant muscle weakness starting at 6 mo, abnormal muscle architecture, autophagocytic vacuoles, and Ub containing protein inclusions.

Transgenic expression of inclusion body myopathy associated mutant p97/VCP causes weakness and ubiquitinated protein inclusions in mice Weihl C, Miller SE, Hanson PI, Pestronk A. Dual immunofluorescence of 3-month-old quadriceps muscle from (B) control, (C) TgVCP- WT and (D) TgVCP-RH animals using anti- p97/VCP (red) and anti-collagen IV (green) antibodies showing increase in sarcolemma VCP Diaphragm muscle from ( A , D , F ) TgVCP-WT or ( B , C , E , G , H ) TgVCP-RH (B, I , J ) at 6 months, (C–E) 10 months or (F and G) 12 months of age (H) Congo red in the sarcoplasm and myonuclei of 15-month-old TgVCP-RH12 animal skeletal muscle. Panels I and J are quadriceps skeletal muscle from 6-month-old TgVCP-WT (I) and TgVCP-RH animals immunostained for ubiquitinated proteins with FK2 antibody.

Custer S, Neumann M, Lu H, Wright A and Taylor J. Transgenic mice expressing mutant forms VCP/p97 recapitulate the full spectrum of IBMPFD including degeneration in muscle, brain and bone. Hum Mol Genet. 2010 May 1;19(9):1741-55. 9 mo mice- centralized nuclei irregular fiber size and inflammatory infiltrates (arrows).

Custer S et al. 2010 Marked degeneration of the normal sarcomeric structure in both VCP-R155H and VCP-A232E muscle, and also the accumulation of degenerated mitochondria

Giles Watts &Kimonis designed Knock-in mouse model for IBMPFD. I KI

Badadani M, Nalbandian A, Watts GD, Vesa J, Kitazawa M, Su H, Tanaja J, Dec E, Wallace DC, Mukherjee J, Caiozzo V, Warman M, Kimonis VE. VCP Associated Inclusion Body Myopathy and Paget Disease of Bone Knock-In Mouse Model Exhibits Tissue Pathology Typical of Human Disease. 2010. PLoS ONE 5(10). Nalbandian A, Llewellyn KJ, Badadani M, Yin HZ, Nguyen C, Katheria V, Watts G, Mukherjee J, Vesa J, Caiozzo V, Mozaffar T, Weiss JH and Kimonis VE. A Progressive Translational Mouse Model of Human VCP Disease: The VCP R155H/+ Mouse. Muscle Nerve. 2012 Jul 12.

Progression of muscle weakness in knock-in Neo+ R155H mice. Grip Strength Rotarod testing

Histological analysis of the VCP R155H/+ mouse quadriceps muscles.

Bone micro CT imaging, histology and giant osteoclast formation of VCPR155H/+ and wild-type mice.

Histological analyses of spinal cord ventral horn astrocytes in 15 mo. WT and Het. VCP R155H mouse demonstrates neuronal atrophy and astrocyte proliferation Yin HZ, Nalbandian A, Hsu C-I, Li S, Llewellyn K, Mozaffar T, Kimonis VE, Weiss J. A mutant valosin-containing protein (VCP) gene knockin mouse model of ALS .Cell Death Dis. 2012.

THE HOMOZYGOTE VCPR155H/R155H MOUSE MODEL EXHIBITS ACCELERATED HUMAN VCP- ASSOCIATED DISEASE PATHOLOGY Nalbandian A , et a al. (2012) The Homozygote VCP(R155H/R155H) Mouse Model Exhibits Accelerated Human VCP-Associated Disease Pathology. PLoS oS O ONE 7(9):e46308.

Survival Curve of Homozygote VCP R155H/R155H VCP IBMPFD Mouse Model. Animals do not survive after 21 days

Phenotype and histological analyses of the VCP R155H/R155H homozygote knock-in mice

MicroCT and hind limb bone imaging in VCPR155H/R155H and WT mice.

Summary of findings in Neo- Heterozygote & Homozygote R155H VCP KI mice Heterozygotes • The 15- and 24-mo mice have muscle weakness • Muscle :variation in fiber size, centrally located nuclei and rare vacuoles. • Muscle and brain: cytoplasmic accumulation of TDP-43, ubiquitin-positive inclusion bodies and increased autophagocytic activity. • Spinal cords neurodegenerative changes and TDP-43 pathology of the motor neurons cells. EMG-ALS like • MicroCT and bone histology: Paget-like lesions Homozygotes • Lethality 21 days • Muscle and brain pathology • Useful model also for myopathy, ALS, and PDB

Translational Studies with the R155H mouse

Nalbandian A, Nguyen C, Katheria V, Llewellyn KJ, Badadani M, Caiozzo V, Kimonis VE. Exercise Training Reverses Skeletal Muscle Atrophy in an Experimental Model of VCP Disease. PLoS One. 2013 Oct 9;8(10) WT mice. Weights, muscle strength measurements and histological analyses of sedentary and exercised VCPR155H/+ and

Targeted Excision of VCP R155H mutation by Cre- LoxP Technology as a Promising Therapeutic Strategy for Inclusion Body Myopathy Nalbandian A, Llewellyn K, Nguyen C, Monuki E, Kimonis V. Hum Gene Ther Methods. 2014 Dec 29. a b M M VCP 1000 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 700 500 200 R155H LoxP LoxP Exon 3 Exon 2 Exon 6 Exon 4 Exon 5 c Recombination No recombination 550bp 2000bp 2 3 2 3 6 6 4 5

Targeted Excision of VCP R155H mutation by Cre- LoxP Technology improves myopathy b a * * Corn c Oil VCP R155H/+ Wild Type Tamoxifen R155H/+ CRE-ER TM -VCP Type Wild 100 µM

Nuclear Envelope Ubiquitin Reconstruction Dependent Protein Degradation Apoptosis VALOSIN B MUT1 Membrane CONTAINING Fusion PROTEIN (NSFL1C) Autophagy Endoplasmic Reticulum Associated Transcription Cell Cycle Control Degradation Activation

Nalbandian A, Llewellyn KJ, Nguyen C, Yazdi PG Kimonis VE. Rapamycin and Chloroquine: the in vitro and in vivo effects of autophagy-modifying drugs show unexpected results in valosin containing protein multisystem proteinopathy. PLoS One. 2015 Apr 17;10(4)

Rapamycin and Chloroquine: the in in v vit itro and in in v viv ivo effects of autophagy-modifying drugs show unexpected results in valosin containing protein multisystem proteinopathy

Rapamycin-induced autophagy aggravates pathology and weakness in a mouse model of VCP-associated myopathy. Ching JK, Weihl CC. Autophagy. 2013 May; 9(5):799-800. • Mice treated every other day for 21 days • Mice developed weakness, increase CPK, increase in vacuolated and atrophic fibers • Inhibition of mTOR with rapamycin resulting in autophagosome biogenesis worsens muscle degeneration

Llewellyn KJ, Nalbandian A, Jung KM, Nguyen C, Avanesian A, Mozaffar T, Piomelli D, Kimonis VE. Lipid-enriched diet slows down progression in a murine model of VCP-associated disease. Hum Mol Genet. 2013 Oct 24.

Survival curve for the untreated and treated homozygous VCP R155H/R155H IBMPFD mouse model. VCP R155H/R155H animals fed an increased fat diet several survive >3 months Percentage difference 2020x Fatty acids (normal) VS 2019 (higher-fat) diet C16:0 Palmitic 0.3% increase C18:0 Stearic 0.1% increase C18:1ω9 Oleic 0.6% increase C18:2ω6 Linoleic 1.3% increase C18:3ω3 Linolenic 0.1% increase 9 % Diet Total saturated 0.4% increase Total monounsaturated 0.6% increase Total polysaturated 1.5% increase 6 % Diet Amino Acids Phenylalanine 0.1% increase Isoleucine 0.1% increase Proline 0.1% decrease

Measurements, and histological analyses of WT and VCP R155H/R155H mice on normal and HFDs. Wild Type VCP R155H/R155H D A 3 weeks ND E 3 weeks LED B F 4 months LED C G 9 months LED 200 μ m

Lipid analyses of quadriceps and livers from VCP R155H/R155H and WT animals on normal and lipid-enriched diets

Summary • VCP inclusion body myopathy is an important heterogeneous and under-recognized disorder • VCP R155H mice are excellent models of human disease for translational studies • Uphill exercise improved grip strength/Rotarod • Cre excision of VCP improved pathology. Exon skipping or allele silencing may be a promising strategy in patients • ?Autophagy modifying drugs as therapy. • High fat diet improves survival and pathology in homozygotes with translational potential

ACKNOWLEDGEMENTS Patients Clinical Projects Abhilasha Surampalli MBBS Samantha Reiter Avkahan Claudia Shambaugh Laboratory Angele Nalbandian, PhD Katrina Llewellyn,PhD Arianna Gomez Naomi Walker Collaborators Daniele Piomelli Kwang Jung John H. Weiss FUNDING NIH NIAMS Vince Caiozzo MDA Hong Yin NICHD-RDCRN Tahseen Mozaffar CHOC-IRVINE COLLABORATIVE GRANT Annabel Wang CODY’s QUEST Masashi Kitazawa ICTS Phil Schwartz