Zln005

Example 5

Ppargc1a activator ZLN005 (25 mg/kg, Sigma) was administered orally once a day starting 30 minutes after MPTP administration on Day 1 for 7 consecutive days in 0.5% methylcellulose (Sigma). For protein expression studies, animals were sacrificed on Day 8, 24 hours after the 7th oral dosage of ZLN005, and paraformaldehyde-perfused brain tissues were processed for immunohistochemical analysis of dopaminergic neurons in the substantia nigra.

One representative picture of each of Veh (n=3), MPTP-Ctrl (n=5), and MPTP-ZLN (n=5) animals is shown in FIG. 5. The brown staining represents tyrosine hydroxylase expression in dopaminergic neurons of the substantia nigra. The results show that MPTP administration led to a depletion of these neurons, which was reversed by treatment with ZLN005.

Example 9

Ppargc1a, which is an activator of mitochondrial biogenesis, is widely expressed in cells throughout the body. Ppargc1a activator ZLN005 (25 mg/kg, Sigma) was administered orally once on Day 1 in 0.5% methylcellulose (Sigma) immediately before the first dose of STZ. Treatment with ZLN005 was continued on a daily schedule until Day 4.

For gene expression studies, animals were sacrificed on Day 4, and PBS-perfused brain tissues were processed for RNA isolation, cDNA synthesis and real-time quantitative PCR (Invitrogen).

The results are summarized in FIG. 9. The results show that Ppargc1a activator ZLN005 increased expression of genes involved in Ppargc1a signaling and antioxidant defense (Tfam, Cytc), and decreased the expression of genes involved in β-amyloid generation (App and Psen1), in the brains of STZ-treated-animals (n=10 animal per condition). Unpaired t-tests were used for statistical analyses.

Example 2

Ppargc1a, an inducer of mitochondrial biogenesis, is widely expressed in cells throughout the body. Ppargc1a activator ZLN005 (25 mg/kg, Sigma) was administered orally once a day starting 30 minutes after MPTP administration on Day 1 (when animals exhibited PD-like symptoms) for 3 consecutive days in 0.5% methylcellulose (Sigma).

For gene expression studies, animals were sacrificed on Day 4, 24 hours after the 3rd oral dosage of ZLN005, and PBS-perfused brain tissues were processed for RNA isolation, cDNA synthesis, and real-time quantitative PCR (Invitrogen). The results are summarized in FIG. 2. The results show that the Ppargc1a activator, ZLN005, increases expression of genes involved in Ppargc1a signaling (Pgc1a), mitochondrial genes (Tfam, Nrf2, Ucp3-downstream targets of Ppargc1a), and anti-oxidative stress genes (Ant, Sod1, Sod2) in the brains of MPTP-treated animals. There was also a 15% upregulation of tyrosine hydroxylase (Th), the enzyme that is critical for dopamine synthesis in the brain. These results indicate that ZLN005 penetrated the blood-brain barrier and activated the Ppargc1a pathway in the brain. Unpaired t-tests were used for statistical analyses.

Example 18

ALS transgenic mice were orally treated 3 times a week with 0.5% methylcellulose or ZLN005 (Sigma) at 25 mg/kg in 0.5% methylcellulose, starting at 5, 10 and 15 weeks of age.

Survival of ALS-Ctrl (n=18) and ALS-ZLN mice (n=8) were monitored until all animals succumbed. The results (FIG. 18) demonstrate that ALS-ZLN at 5 or 10 weeks of age significantly increased survival (mean survival of 131-132 days) in comparison to ALS-Ctrl (mean survival of 119 days); p-values <0.05. Log-rank test was used for statistical analysis.

Example 2

Ppargc1a, an inducer of mitochondrial biogenesis, is widely expressed in cells throughout the body. Ppargc1a activator ZLN005 (25 mg/kg, Sigma) was administered orally once a day starting 30 minutes after MPTP administration on Day 1 (when animals exhibited PD-like symptoms) for 3 consecutive days in 0.5% methylcellulose (Sigma).

For gene expression studies, animals were sacrificed on Day 4, 24 hours after the 3rd oral dosage of ZLN005, and PBS-perfused brain tissues were processed for RNA isolation, cDNA synthesis, and real-time quantitative PCR (Invitrogen). The results are summarized in FIG. 2. The results show that the Ppargc1a activator, ZLN005, increases expression of genes involved in Ppargc1a signaling (Pgc1a), mitochondrial genes (Tfam, Nrf2, Ucp3-downstream targets of Ppargc1a), and anti-oxidative stress genes (Ant, Sod1, Sod2) in the brains of MPTP-treated animals. There was also a 15% upregulation of tyrosine hydroxylase (Th), the enzyme that is critical for dopamine synthesis in the brain. These results indicate that ZLN005 penetrated the blood-brain barrier and activated the Ppargc1a pathway in the brain. Unpaired t-tests were used for statistical analyses.

Example 10

Ppargc1a activator ZLN005 (25 mg/kg, Sigma) was administered orally once in 0.5% methylcellulose (Sigma) immediately before the first dose of STZ on Day1. Treatment with ZLN005 was continued on a daily schedule until Day 7.

For microglia analysis, animals were sacrificed on Day 7, and PBS-perfused brain tissues were digested with Collagenase IV and processed for flow cytometry. Microglia were phenotyped with antibodies directed against mouse TNF-α (Biolegend) and metabolic dyes ThioltrackerViolet, and MitotrackerRed (Invitrogen) for flow cytometric acquisition (LSRII, BD) and analysis (FlowJo). The results are summarized in FIG. 10.

The results show that ZLN005 suppressed TNF-α production in microglia isolated from STZ-Ctrl (n=8) when compared with Veh animals (n=8) (A), which indicates that neuroinflammation was inhibited. The results also show that ZLN005 enhanced (B) the production of glutathione, an antioxidant with neuroprotective properties, and (C) mitochondrial potential, a marker of functional integrity of mitochondria, in microglia isolated from STZ-treated animals (n=4). These results indicate that ZLN005 reverses metabolic dysfunction in microglia induced by STZ. ANOVA was used for statistical analyses.

Example 9

Ppargc1a, which is an activator of mitochondrial biogenesis, is widely expressed in cells throughout the body. Ppargc1a activator ZLN005 (25 mg/kg, Sigma) was administered orally once on Day 1 in 0.5% methylcellulose (Sigma) immediately before the first dose of STZ. Treatment with ZLN005 was continued on a daily schedule until Day 4.

For gene expression studies, animals were sacrificed on Day 4, and PBS-perfused brain tissues were processed for RNA isolation, cDNA synthesis and real-time quantitative PCR (Invitrogen).

The results are summarized in FIG. 9. The results show that Ppargc1a activator ZLN005 increased expression of genes involved in Ppargc1a signaling and antioxidant defense (Tfam, Cytc), and decreased the expression of genes involved in β-amyloid generation (App and Psen1), in the brains of STZ-treated-animals (n=10 animal per condition). Unpaired t-tests were used for statistical analyses.