Signaling through the vascular endothelial growth factor receptor VEGFR-2 protects hippocampal neurons from mitochondrial dysfunction and oxidative stress

Highlights

• Nutritional stress upregulates VEGF and its receptors in mature hippocampal neurons.

• Activated VEGFR-2 signals neuroprotection through the PI3K/Akt and ERK1/2 pathways.

• VEGFR-2 inactivation decreased viability with mitochondrial dysfunction and Oxidative stress.

• VEGF and VEGF-B signaling or NAC counteract the insult induced by VEGFR-2 inhibition.

Abstract

Vascular endothelial growth factor VEGF (VEGF-A or VEGF₁₆₅) is a potent angiogenic factor that also signals neuroprotection through activation of its cognate receptor VEGFR-2. In this capacity, VEGF signaling can rescue neurons from the damage induced by stressful stimuli many of which elicit oxidative stress. However, the regulatory role that VEGFR-2 plays in providing neuroprotection remains elusive. Therefore, we investigated the effects of VEGFR-2 inhibition on primary cultures of mature hippocampal neurons undergoing nutritional stress. We found that neurons cultured under nutritional stress had increased expression of VEGF and its receptors, VEGFR-1, VEGFR-2, and NP-1, as well as enhanced levels of VEGFR-2 phosphorylation. These neurons also showed increased activation of the prosurvival pathways for MEK/ERK1/2 and PI3K/Akt, enhanced phosphorylation (inactivation) of the proapoptotic BAD, and higher levels of the antiapoptotic protein Bcl-xL, all of which were augmented by treatments with exogenous VEGF and blocked by VEGFR-2 inhibition. The blockade of VEGFR-2 function also elicited a cytotoxicity that was accompanied by caspase-3 activation, induction of hemeoxygenase-1 (HO-1), oxidative stress, and a collapse in the mitochondrial membrane potential (ΔΨ_m). Knockdown of VEGFR-2 by siRNA generated a similar pattern of redox change and mitochondrial impairment. Pretreatments with VEGF, VEGF-B, or the antioxidant N-acetylcysteine (NAC) rescued SU1498 or siRNA-treated neurons from the mitochondrial dysfunction and oxidative stress induced by VEGFR-2 inhibition in a timely fashion. These findings suggested that VEGF or VEGF-B can provide neuroprotection by signaling through an alternate VEGF receptor. Together, our findings suggest that VEGF signaling through VEGFR-2 plays a critical regulatory role in protecting stressed hippocampal neurons from the damaging effects of an oxidative insult. These findings also implicate VEGFR-1 or NP-1 as compensatory receptors that mediate neuroprotection when VEGFR-2 function is blocked.

Introduction

Oxidative stress and mitochondrial dysfunction contribute to the pathogenesis of a number of neurodegenerative disorders including Alzheimer's and Parkinsonʼs disease [1]. One approach for protecting neurons from oxidative insults is to administer neurotrophic growth factors. One such factor is VEGF (VEGF-A or VEGF₁₆₅) which is a mitogen that stimulates angiogenesis and neuroprotection through autocrine or paracrine mechanisms [2], [3]. In addition, VEGF can stimulate axonal outgrowth, and rescue rat mesencephalic neurons or hippocampal cells from death induced by serum withdrawal, ischemia, hypoxia, and glutamate-induced toxicity [4], [5], [6] and elicit neuroprotection via angiogenesis and neurogenesis [7], [8], [9]. Delineating this neuroprotective mechanism is complex since VEGF can undergo cell-surface interactions with different cognate tyrosine kinase receptors such as VEGFR-1, VEGFR-2, and the non-tyrosine kinase members of the neuropilin family of class 3 semaphorin receptors Neuropilin-1 and -2 (NP-1, NP-2) receptors [10]. While VEGF mediates most biological effects through VEGFR-2, it can interact with NP-1 as a coreceptor that enhances VEGF signaling. Ligand binding to VEGFR-1 and VEGFR-2 results in receptor dimerization followed by autophosphorylation and activation of downstream signaling cascades [3]. In neurons, NP-1 is a cell-surface receptor for both VEGF and class 3 semaphorins and plays a functional role in axonal pathfinding, retraction, and collapse during development. While the function of VEGFR-1 remains unclear, it is implicated as a decoy receptor that sequesters VEGF from activating VEGFR-2. VEGF-B, which is a ligand for VEGFR-1 but not VEGFR-2, is poorly angiogenic in the brain but can protect against mitochondrial membrane permeabilization [11], proapoptotic gene expression [12], and is coexpressed with genes encoding mitochondrial proteins [13]. Thus, VEGF-B may protect against mitochondrial dysfunction independent of angiogenesis.

Oxidative insults are implicated as causative factors of neuronal damage in several different neurological disorders and increasing evidence from in vitro and in vivo studies shows that VEGF signaling protects neurons from insults which are known to induce oxidative stress [14], [15]. In this context, VEGF overexpression has been shown to delay the onset of neuronal death in an animal model of amyotrophic lateral sclerosis (ALS) where oxidative stress is a contributing factor [16]. VEGF has been shown to mediate neuroprotection under stress conditions through the downstream activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase MEK/ERK1/2 pathways [6], [17], [18], [19] and by suppressing caspase activation [20]. While several of these studies show that VEGF activates VEGFR-2 to mediate survival, the protective capacity of VEGFR-2 signaling remains unclear. Therefore, the aim of these studies is to determine the impact of VEGFR-2 signaling on the survival of mature hippocampal neurons by blocking its activation using pharmacological and gene silencing methods.

In previous studies we used a neuronal model of serum-deprived SK-N-SH cells to show that VEGF signaling through VEGFR-2 prevents a caspase-dependent cell death [21]. Therefore, we addressed whether VEGF signaling through VEGFR-2 protected rat hippocampal neurons from the harmful effects of nutritional stress. Depriving cultured neurons from vital nutrients provide a viable model to identify the molecular basis of neuronal insults that would occur under pathological conditions in vivo. Our findings show that a blockade of VEGFR-2 function in hippocampal neurons leads to a rapid loss in viability that is manifested by induction of markers of oxidative stress, mitochondrial dysfunction, and a loss in the activation of prosurvival pathways. Notably, the inclusion of exogenous VEGF or VEGF-B mediates a time-dependent rescue from this response, suggesting that a molecular switch to an alternate receptor can provide neuroprotection when VEGFR-2 activity is blocked. Our findings establish a link between VEGFR-2 signaling and mitochondrial function in differentiated rat neurons and provide insight on oxidative stress-related pathways that mediate neuronal damage and how exogenous VEGF or VEGF-B may counteract these events.