A Fine Balance of Dietary Lipids Improves Pathology of a Murine Model of VCP-Associated Multisystem Proteinopathy

Abstract

The discovery of effective therapies and of disease mechanisms underlying valosin containing protein (VCP)-associated myopathies and neurodegenerative disorders remains elusive. VCP disease, caused by mutations in the VCP gene, are a clinically and genetically heterogeneous group of disorders with manifestations varying from hereditary inclusion body myopathy, Paget's disease of bone, frontotemporal dementia (IBMPFD), and amyotrophic lateral sclerosis (ALS). In the present study, we examined the effects of higher dietary lipid percentages on VCPR155H/R155H, VCPR155H/+ and Wild Type (WT) mice from birth until 15 months of age by immunohistochemical and biochemical assays. Findings illustrated improvement in the muscle strength, histology, and autophagy signaling pathway in the heterozygote mice when fed 9% lipid-enriched diets (LED). However, increasing the LED by 12%, 30%, and 48% showed no improvement in homozygote and heterozygote survival, muscle pathology, lipid accumulation or the autophagy cascade. These findings suggest that a balanced lipid supplementation may have a therapeutic strategy for patients with VCP-associated multisystem proteinopathies.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Grip strength measurements and histological analyses of WT and VCPR155H/+ mice on normal and 9% LED.

(A) Grip strength measurements for the WT (blue) and VCPR155H/+ (red) mice placed on the 6% ND from birth and VCPR155H/+ mice placed on the 9% LED from birth (purple) and at 7 months of age onwards (green). H&E staining on WT and VCPR155H/+ on (B) ND (pathology shown by white arrows, right panel), (C) 9% LED from birth, and (D) 9% LED beginning at 7 months of age onwards (pathology shown by white arrows, right panel). (E) Western blot analyses of WT and VCPR155H/+ animals fed either ND or 9% LED. (F) Densitometry analyses relative to loading control (Beta-actin). Statistical significance is denoted by *P ≤ 0.05.

Fig 2. Survival curves of WT, VCPR155H/+, and VCPR155H/R155H mice on normal and varying lipid-enriched diets.

Kaplan-Meier survival probability (%) analysis for the WT, VCPR155H/+, and VCPR155H/R155H pregnant dams placed on the (A) 6% normal diet, (B) 9% LED, (C) 12% LED, (D) 30% LED, and (E) 48% LED. (F) Combined survival curve of all the LED regimens for the WT, VCPR155H/+, and VCPR155H/R155H. The number of mice analyzed per experiment is 35–40.

Fig 3. Strength measurements and histological stainings of quadriceps muscle of WT and VCPR155H/+ mice on normal and different lipid-enriched diets.

(A,B) Grip strength and Rotarod measurements analyses at 15-months of age, (C,D) H&E and (E,F) Oil Red O staining of quadriceps muscle from WT and VCPR155H/+ animals placed on the 9%, 12%, 30%, and 48% LED from birth. The number of mice analyzed per experiment is 8–10. Insets show magnified pathology, including rimmed vacuoles (orange arrows), atrophy (blue arrows), centralized nuclei (green arrows), and increased lipid droplets (white arrows).

Fig 4. Autophagy influx in the quadriceps of WT and VCPR155H/+ mice on normal and different lipid-enriched diets.

Immunohistochemical histological analyses comparing WT and heterozygote VCPR155H/+ knock-in animals on varying 12%, 30%, and 48% lipid-enriched diets stained with (A) TDP-43 (white arrows), (B) p62/SQSTM1, and (C) LC3-I/II. (D) Western blot of WT and heterozygote VCPR155H/+ knock-in animals on varying 12%, 30%, and 48% lipid-enriched diets analyzed for ubiquitin, optineurin, p62/SQSTM1, TDP-43, LC3-I/II, and Mucolipin (TRPML1). Magnified insets and white arrows indicate increased p62/SQSTM1 autophagic puncta, LC3 and TDP-43 protein expression translocated from the nucleus to the cytoplasm. (E) Densitometry analyses relative to loading control (Beta-actin). Statistical significance is denoted by *P ≤ 0.05. The number of mice analyzed per experiment is 8–10.

Fig 5. Effects of LED on the expression of lysosomal enzymes in the quadriceps of WT and VCPR155H/+ mice on normal and different lipid-enriched diets.

(A) Western blot analyses of protein expression levels of acid phosphatase, lysosomal acid lipase in WT and VCPR155H/+ knock-in animals on varying ND (6%), 9%, 12%, 30%, and 48% lipid-enriched diets (LED). (B,C) Densitometry analyses of acid phosphatase and LAL from various diets relative to loading control (Beta-actin). Statistical significance is denoted by *P ≤ 0.05. The number of mice analyzed per experiment is 8–10.