mTOR pathway inhibition prevents neuroinflammation and neuronal death in a mouse model of cerebral palsy

Highlights

• In mice, hypoxia, ischemia and lipopolysaccharide (HIL) models cerebral palsy.

• Rapamycin treatment prevents brain injury at 24 h and 1 month induced by HIL.

• Rapamycin reduces neuroinflammation at 24 h and 1 month induced by HIL.

• Rapamycin may inhibit neuronal death by inducing autophagy.

• mTOR inhibition may provide a new preventative treatment option for cerebral palsy.

Abstract

Background and purpose

Mammalian target of rapamycin (mTOR) pathway signaling governs cellular responses to hypoxia and inflammation including induction of autophagy and cell survival. Cerebral palsy (CP) is a neurodevelopmental disorder linked to hypoxic and inflammatory brain injury however, a role for mTOR modulation in CP has not been investigated. We hypothesized that mTOR pathway inhibition would diminish inflammation and prevent neuronal death in a mouse model of CP.

Methods

Mouse pups (P6) were subjected to hypoxia–ischemia and lipopolysaccharide-induced inflammation (HIL), a model of CP causing neuronal injury within the hippocampus, periventricular white matter, and neocortex. mTOR pathway inhibition was achieved with rapamycin (an mTOR inhibitor; 5 mg/kg) or PF-4708671 (an inhibitor of the downstream p70S6kinase, S6K, 75 mg/kg) immediately following HIL, and then for 3 subsequent days. Phospho-activation of the mTOR effectors p70S6kinase and ribosomal S6 protein and expression of hypoxia inducible factor 1 (HIF-1α) were assayed. Neuronal cell death was defined with Fluoro-Jade C (FJC) and autophagy was measured using Beclin-1 and LC3II expression. Iba-1 labeled, activated microglia were quantified.

Results

Neuronal death, enhanced HIF-1α expression, and numerous Iba-1 labeled, activated microglia were evident at 24 and 48 h following HIL. Basal mTOR signaling, as evidenced by phosphorylated-S6 and -S6K levels, was unchanged by HIL. Rapamycin or PF-4,708,671 treatment significantly reduced mTOR signaling, neuronal death, HIF-1α expression, and microglial activation, coincident with enhanced expression of Beclin-1 and LC3II, markers of autophagy induction.

Conclusions

mTOR pathway inhibition prevented neuronal death and diminished neuroinflammation in this model of CP. Persistent mTOR signaling following HIL suggests a failure of autophagy induction, which may contribute to neuronal death in CP. These results suggest that mTOR signaling may be a novel therapeutic target to reduce neuronal cell death in CP.

Introduction

Cerebral palsy (CP) is among the most common neurodevelopmental disorders, affecting 2–3 out of every 1000 live births (Kirby et al., 2011). CP is characterized by a heterogeneous phenotype including impaired motor function, intellectual disability, blindness, and epilepsy in 25–50% of affected children (Bax et al., 2005, Pakula et al., 2009). Clear risk factors for CP include prematurity, low birth weight, multiple gestations, coagulation disorders, intraventricular hemorrhage, placental pathology, and especially, hypoxic–ischemic brain injury (Keogh and Badawi, 2006). Of particular relevance, inflammation resulting from maternal or fetal infection dramatically increases the risk for CP, especially if superimposed on prenatal or perinatal hypoxia–ischemia (Nelson and Grether, 1998, Wheater and Rennie, 2000). Retrospective case–control analysis illustrates that preterm infants with CP and white matter injury had increased culture-positive infections of the blood, cerebrospinal fluid, and trachea during the neonatal period (Graham et al., 2004). Moreover, the activation of inflammatory pathways during fetal life may sensitize the brain to the effects of hypoxia–ischemia (Fleiss and Gressens, 2012). Indeed, the most widely accepted animal models of CP combine hypoxia–ischemia plus lipopolysaccharide-induced inflammation (HIL) exposure during development, resulting in greater cellular injury and more substantial motor and behavioral deficits than either insult alone (Eklind et al., 2004, Shen et al., 2010, Shen et al., 2012, Girard et al., 2009, Hu et al., 2013). The patterns of neuronal injury in established HIL mouse models serve to model one subtype of CP (Shen et al., 2010, Shen et al., 2012) and include selective cell death in the hippocampus, cortex, thalamus, and the periventricular white matter, all neuropathological hallmarks of CP (Krägeloh-Mann et al., 2002, Bax et al., 2006, Krägeloh-Mann and Horber, 2007).

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that regulates cellular growth and proliferation in response to various environmental stimuli, including nutrients, oxygen, energy, and growth factors (Hall, 2008). mTOR signaling, altered independently by hypoxia–ischemia and inflammation, plays a critical role in regulating cell death following environmental stress. For example, lipopolysaccharide (LPS) induces an mTOR-dependent release of cytokines and pro-inflammatory mediators, such as IL-1β, 6, 8, and TNFα (Kusaba et al., 2005, Weichhart et al., 2008), which have been implicated in neuronal and white matter damage (Allan and Rothwell, 2001, Kadhim et al., 2001). Interestingly, enhanced mTOR activity, by suppression of the upstream mTOR inhibitors Tsc1 or Tsc2, increases vulnerability of neurons to hypoxic–ischemic injury (Ng et al., 2011, Papadakis et al., 2013), whereas, decreasing mTOR activity via Tsc1 overexpression fosters resistance to ischemia-induced damage (Papadakis et al., 2013). mTOR inhibition with rapamycin treatment prior to injury reduces neuronal death and increases autophagy in an animal model of neonatal stroke (Carloni et al., 2008, Chen et al., 2012). Autophagy, a regulated intracellular degradation process, may play a role in cell survival during bioenergetic stress (Levine and Klionsky, 2004, Kiffin et al., 2006, Wu et al., 2009) and thus, rapamycin-induced autophagy is a potential mechanism of mTOR-dependent neuroprotection (Carloni et al., 2008, Carloni et al., 2010).

While the mTOR signaling cascade has been linked to several pediatric neurological disorders (Curatolo et al., 2001, Goorden et al., 2007, Sharma et al., 2010, Talos et al., 2012, Zeng et al., 2008), manipulation of the mTOR signaling cascade as a pre-clinical therapeutic strategy in CP has not been investigated. While previous studies in neonatal stroke models have used mTOR inhibitors prior to hypoxic–ischemic injury conditions, in a clinically relevant paradigm, we hypothesize that mTOR pathway inhibition with rapamycin following HIL may reduce neuronal death and neuroinflammation in a mouse model of CP. If successful, our approach could provide a completely new cell signaling cascade to investigate for therapeutic development in a subset of infants at high risk for CP.