Gemfibrozil, food and drug administration-approved lipid-lowering drug, increases longevity in mouse model of late infantile neuronal ceroid lipofuscinosis

Abstract

Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL) is a rare neurodegenerative disease caused by mutations in the Cln2 gene that leads to deficiency or loss of function of the tripeptidyl peptidase 1 (TPP1) enzyme. TPP1 deficiency is known to cause the accumulation of autofluoroscent lipid-protein pigments in brain. Similar to other neurodegenerative disorders, LINCL is also associated with neuroinflammation and neuronal damage. Despite investigations, no effective therapy is currently available for LINCL. Therefore, we administered gemfibrozil (gem), an food and drug administration (FDA)-approved lipid-lowering drug, which has been shown to stimulate lysosomal biogenesis and induce anti-inflammation, orally, at a dose of 7.5 mg/kg body wt/day to Cln2^(-/-) mice. We observed that gem-fed Cln2^(-/-) mice lived longer by more than 10 weeks and had better motor activity compared to vehicle (0.1% Methyl cellulose) treatment. Gem treatment lowered the burden of storage materials, increased anti-inflammatory factors like SOCS3 and IL-1Ra, up-regulated anti-apoptotic molecule like phospho-Bad, and reduced neuronal apoptosis in the brain of Cln2^(-/-) mice. Collectively, this study reinforces a neuroprotective role of gem that may be of therapeutic interest in improving the quality of life in LINCL patients.

Keywords: anti-inflammation; apoptosis; batten disease; gemfibrozil; longevity; mouse model.

Figure 1. Gemfibrozil (gem) prolongs the life span of Cln2 ^(−/−) mice

Cln2 ^(−/−) animals were treated with gem (dissolved in 0.1% MeC) orally at a dose of 7.5 mg/kg body weight/day. Treatment started from 4 weeks of age for all groups. One group of animals received only MeC as vehicle. A) Percentage of survival is shown by Kaplan–Meier plot. B) Mean survival days are shown for all three groups. Forty mice (n=40) containing 20 males and 20 female were used in each group.

Figure 2. Gem treatment delays the loss of motor activity in Cln2 ^(−/−) mice

Cln2 ^(−/−) animals (4 weeks old) were treated with gem (dissolved in 0.1% MeC) orally at a dose of 7.5 mg/kg body weight/day. After 8 weeks of treatment, mice were monitored for horizontal activity (A), movement time (B), number of movement (C), total distance travelled (D), stereotypy counts (E), and rest time (F). Cln2 ^(−/−) mice receiving only vehicle and background-matched wild type (WT) mice were also run for comparison. Results represent mean ± SEM of twelve mice (n=12) per group. Male and female mice were kept in each group in equal ratio.

Figure 3. Gem treatment reduces storage materials in vivo in the motor cortex of Cln2 ^(−/−) mice

Cln2 ^(−/−) animals (4 weeks old) were treated with gem (dissolved in 0.1% MeC) orally at a dose of 7.5 mg/kg body weight/day. After 8 weeks of treatment, storage pigments were observed in cortical sections by immunofluorescence analysis of subunit C (A). DAPI was used to visualize nucleus. B) Subunit C positive immunofluorescence was quantified in two different sections (two images per section) of each of six different mice (n=6) per group using NIH Image J software as described under Methods section. ^ap < 0.001 vs WT-control; ^bp < 0.05 vs Cln2 ^(−/−).

Figure 4. Gem treatment attenuates apoptosis in vivo in the motor cortex of Cln2 ^(−/−) mice

Cln2 ^(−/−) animals (4 weeks old) were treated with gem (dissolved in 0.1% MeC) orally at a dose of 7.5 mg/kg body weight/day. After 8 weeks of treatment, cortical sections (immediately dorsal to CA1 hippocampal region) were double-labeled for NeuN and TUNEL (A). NeuN⁺TUNEL⁺ cells were counted (B) in two different cortical sections of each of six different mice (n=6) per group. C) Cortical homogenates were immunoblotted for phospho-BAD. Actin was run as loading control. D) Bands were scanned and values (P-BAD/Actin) are presented as relative to WT control. Results are mean ± SEM of four mice per group.

Figure 5. Gem treatment inhibits apoptosis in vivo in the striatum of Cln2 ^(−/−) mice

Cln2 ^(−/−) animals (4 weeks old) were treated with gem (dissolved in 0.1% MeC) orally at a dose of 7.5 mg/kg body weight/day. After 8 weeks of treatment, striatal sections were double-labeled for NeuN and TUNEL (A). NeuN⁺TUNEL⁺ cells were counted (B) in two different sections of each of six different mice (n=6) per group in an Olympus IX81 fluorescence microscope using the MicroSuite imaging software. C) Striatal homogenates were immunoblotted for phospho-BAD. Actin was run as loading control. D) Bands were scanned and values (P-BAD/Actin) are presented as relative to WT control. Results are mean ± SEM of four mice per group.

Figure 6. Gem treatment upregulates the expression of anti-inflammatory molecules in vivo in the CNS of Cln2 ^(−/−) mice

Cln2 ^(−/−) animals (4 weeks old) were treated with gem (dissolved in 0.1% MeC) orally at a dose of 7.5 mg/kg body weight/day. After 8 weeks of treatment, the expression of SOCS3 and IL-1Ra was monitored in motor cortex (A–C) and striatum (D–F) extracts by Western blot. Actin was run as loading control. Bands were scanned and values (SOCS3/Actin, B & E; IL-1Ra/Actin, C & F) are presented as relative to WT control for cortex (B & C) and striatum (E & F). Results are mean ± SEM of four mice per group.

Figure 7. Gem treatment upregulates SOCS3 in vivo in the striatum of Cln2 ^(−/−) mice

Cln2 ^(−/−) animals (4 weeks old) were treated with gem (dissolved in 0.1% MeC) orally at a dose of 7.5 mg/kg body weight/day. After 8 weeks of treatment, striatal sections were double-labeled for GFAP & SOCS3 (A) and Iba1 & SOCS3 (B). Total SOCS3⁺ (C), GFAP⁺SOCS3⁺ (D) and Iba1⁺SOCS3⁺ (E) cells were counted in two different sections of six different mice per group in an Olympus IX81 fluorescence microscope using the MicroSuite imaging software. Results are mean ± SEM of six mice per group.

Figure 8. Gem treatment upregulates IL-1Ra in vivo in the striatum of Cln2 ^(−/−) mice

Cln2 ^(−/−) animals (4 weeks old) were treated with gem (dissolved in 0.1% MeC) orally at a dose of 7.5 mg/kg body weight/day. After 8 weeks of treatment, striatal sections were double-labeled for GFAP & IL-1Ra (A) and Iba1 & IL-1Ra (B). Total IL-1Ra⁺ (C), GFAP⁺IL-1Ra⁺ (D) and Iba1⁺IL-1Ra⁺ (E) cells were counted in two different sections of six different mice per group in an Olympus IX81 fluorescence microscope using the MicroSuite imaging software. Results are mean ± SEM of six mice per group.