Inhibition of p70 Ribosomal S6 Kinase (S6K1) Reduces Cortical Blood Flow in a Rat Model of Autism-Tuberous Sclerosis

Neurology Life Sciences Immunology Medicine Cell Biology
p70 ribosomal S6 kinase cerebral blood flow tuberous sclerosis complex autism spectrum disorders pf-4708671

Inhibition of p70 ribosomal S6 kinase (S6K1) Reduces Cerebral Cortical Blood Flow in Autism-Tuberous Sclerosis Rats: Research Report

Background

Tuberous Sclerosis Complex (TSC) is a genetic disorder caused by mutations in the TSC1 or TSC2 genes, primarily characterized by multiple benign tumors, including symptoms such as epilepsy, autism spectrum disorder (ASD), and intellectual disability. Previous studies have shown that TSC is associated with changes in cerebral blood flow and metabolic dysfunction. We have previously reported significantly increased cerebral blood flow in Eker rats (an animal model of TSC and autism). These studies suggest that treatment with rapamycin (inhibiting mTOR) can restore normal oxygen consumption and cerebral blood flow. This study aims to explore whether similar effects can be produced by inhibiting a key component of the mTOR signaling pathway, namely p70 ribosomal S6 kinase (S6K1).

Paper Source

This research was conducted by Oak Z. Chi, Xia Liu, Harvey Fortus, Guy Werlen, Estela Jacinto, and Harvey R. Weiss from various departments at Rutgers Robert Wood Johnson Medical School. The paper was published in 2024 in the journal “Neuromolecular Medicine” with DOI 10.1007/s12017-024-08780-7.

Research Process

Experimental Design and Procedures

The study used two rat models: Long-Evans rats (as control group) and Eker rats (TSC animal model). Each type of rat was divided into two groups: one receiving vehicle treatment (control group) and the other receiving PF-4708671 (S6K1 inhibitor) injection (dose 75 mg/kg, lasting 1 hour). The experimental procedure mainly included the following steps:

  1. Animal Preparation:

    • Rats underwent surgery under 2% isoflurane anesthesia, including femoral artery and vein catheterization. The venous catheter was used for radioactive tracer injection, and the arterial catheter was connected to a STATHAM P23DB transducer and IWORX data acquisition system to monitor heart rate and blood pressure. Blood samples were analyzed for hemoglobin, blood gases, and pH.
    • After surgery, isoflurane concentration was reduced to 1.4%. The vehicle group was injected with saline, while the PF-4708671 group received the S6K1 inhibitor.

  2. Regional Cerebral Blood Flow Measurement:

    • 20 μCi of ^(14)C-iodoantipyrine was injected as a tracer through the venous catheter. 10μl arterial blood samples were collected every 3 seconds for 60 seconds from the femoral artery.
    • Rat brains were partially thawed and cut into four regions: cortex, hippocampus, cerebellum, and pons, for slicing and autoradiographic analysis to determine ^(14)C-iodoantipyrine concentration in brain tissue.

  3. Western Blot Analysis:

    • Total proteins were extracted from rat cerebral cortex, separated by SDS-PAGE, transferred to PVDF membranes, and specific proteins and their phosphorylation states were detected by immunoblotting.

  4. Data Analysis and Statistical Processing:

    • ANOVA and Tukey post-hoc tests were used to analyze differences between treatment groups, with statistical significance set at P < 0.05.

Main Research Findings

  1. Cerebral Blood Flow Measurement Results:

    • Under basal conditions, Eker rats showed significantly higher blood flow in the cortex and hippocampus compared to Long-Evans rats (32% and 15% higher, respectively).
    • PF-4708671 treatment significantly reduced blood flow in the cortex and hippocampus of Eker rats but had no significant effect on the cerebellum and pons. No significant blood flow changes were observed in the control Long-Evans rats.

  2. Protein Phosphorylation Levels:

    • Phosphorylation levels of S6K1 target sites (S6 Ser240/244) were increased in the cortex of Eker rats but did not reach statistical significance. After PF-4708671 treatment, S6-S240/244 phosphorylation levels decreased in Eker rats but again did not reach statistical significance.
    • In contrast, phosphorylation levels of Akt at Ser473 were lower in Eker rats compared to Long-Evans rats, and PF-4708671 treatment had a moderate and statistically significant negative effect on this phosphorylation level.

Research Conclusions

The study found that the S6K1 inhibitor PF-4708671 effectively reduced cerebral blood flow in the cortex and hippocampus of autism-tuberous sclerosis rats (Eker rats). This effect depends on moderate inhibition of the mTOR signaling pathway. Although the inhibition of S6 phosphorylation did not reach statistical significance, the significant decrease in Akt phosphorylation may be one of its mechanisms.

Research Value and Significance

This study demonstrates that moderate inhibition of S6K1 can to some extent restore normal cerebral blood flow in TSC animal models, providing new insights for potential treatments of tuberous sclerosis and autism spectrum disorders. Unlike the broad inhibitory effects of rapamycin, S6K1-specific inhibitors may have lower side effects, thus having greater potential for clinical application.

Research Highlights

  • First demonstration that a specific S6K1 inhibitor (PF-4708671) can significantly reduce cerebral blood flow in the cortex and hippocampus of TSC animal models.
  • Proposed a treatment strategy that may have fewer side effects, with potential to alleviate TSC-related neuropathological symptoms such as epilepsy and ASD.
  • Provided new mechanistic insights, suggesting that moderate inhibition of the mTOR signaling pathway may play an important role in regulating cerebral blood flow and metabolic function.

This research provides valuable data support for future clinical applications of S6K1 inhibitors in the treatment of tuberous sclerosis and autism spectrum disorders.