Differential Effects of Phenylalanine on Rac1, Cdc42, and RhoA Expression and Activity in Cultured Cortical Neurons

Abstract

Phenylketonuria (PKU) is characterized by a high concentration of phenylalanine, which can lead to mental retardation. One of the characteristic pathologic changes in untreated phenylketonuria patients is a reduction in the number of axons, dendrites, and synapses in the brain. This is thought to be due to the toxic effects of phenylalanine and/or its metabolites, however, the underlying mechanism remains unclear. In this study, we observed that phenylalanine reduced the number of dendrites and dendritic spines in cultured neurons. We further demonstrated that phenylalanine down-regulated Rac1, Cdc42, and RhoA mRNA and protein expression. Pull-down assays indicated that phenylalanine caused a decrease in Rac1/Cdc42 activity but increased RhoA activity. Expression of a dominant negative RhoA or treatment with a Rho-associated kinase specific inhibitor, Y-27632, partly inhibited the phenylalanine-induced decrease in dendrite numbers. In conclusion, we have demonstrated that phenylalanine affects the expression and activity of Rac1, Cdc42, and RhoA. Furthermore, RhoA signaling is involved in the inhibitory effect of phenylalanine on dendritic branching. These results may provide an important insight into the molecular mechanism underlying phenylalanine-induced abnormalities of dendrites, specifically in phenylketonuria neuronal injury. (Pediatr Res 62: 8-13, 2007) P henylketonuria (PKU) is one of the most frequent inborn disorders of amino acid metabolism. It is caused by a deficiency in phenylalanine hydroxylase (PAH), resulting in an accumulation of its upstream metabolite phenylalanine, mainly in brain tissue and the cerebrospinal fluid of PKU patients. Phenylalanine interferes with brain development and results in severe MR, microcephaly, epilepsy, and behavioral problems. These symptoms appear to be the direct consequence of neuronal cell loss, white matter abnormalities, dendritic simplification, and synaptic density reduction (1-5). Previous studies demonstrated that phenylalanine reduced Naϩ, Kϩ-ATPase and creatine kinase activity in rat brain (6 -8). Moreover, some neurotransmitters such as serotonin and dopamine were deficient in PKU animal models (9). Glushakov et al. (10) found that a high concentration of phenylalanine attenuated NMDA-activated currents in cultured hippocampal neurons. In vivo 31 P-MRS demonstrated abnormalities of cerebral energy metabolism in PKU, indicating a link between phenylalanine neurotoxicity and imbalances of cerebral energy metabolism (11). Lately, Horster et al. (5) revealed that phenylalanine reduced synaptic density and simultaneously inhibited pyruvate kinase in mixed cortical cultures. Our laboratory reported that phenylalanine and its metabolites caused neuronal cell apoptosis in vitro (12,13). Although these observations demonstrate that phenylalanine is probably the main causative agent of brain injury in PKU, the pathophysiological mechanisms of its actions in brain injury are multiple and not fully understood.As dendrites are the site of most synaptic c...

The Behavioral Consequence of Phenylketonuria in Mice Depends on the Genetic Background

“…How these PKU-related changes can result in a different functional outcome is not clear. Concerning raised Phe concentrations, in in vitro models of PKU, increased Phe concentrations seems to affect post- and presynaptic markers, proteins involved in cytoskeleton organization, and neuronal morphology (Zhang and Gu, 2005; Hörster et al, 2006; Zhang et al, 2007; Li et al, 2010; Horling et al, 2015; Schlegel et al, 2016). In vivo , although both strains show changes in different markers related to synaptic functioning, both BTBR PKU and B6 PKU show affected synaptic plasticity and overall neuronal functioning (Andolina et al, 2011; Liang et al, 2011; Horling et al, 2015; Bruinenberg et al, 2016).…”

Role of peptidylarginine deiminase 2 (PAD2) in mammary carcinoma cell migration

“…Real-time quantitative PCR analysis was performed using the following primers [39, 40]: PAD2 ( PADI2 ) Forward (5′-TCTCAGGCCTGGTCTCCA-3′) and Reverse (5′- AAGATGGGAGTCAGGGGAAT-3′); E-cadherin ( CDH1 ) Forward (5′- TGGAGGAATTCTTGCTTTGC-3′) and Reverse (5′- CGCTCTCCTCCGAAGAAAC-3′); β-actin ( ACTB ) Forward (5′- CCAACCGCGAGAAGATGA-3′) and Reverse (5′- CCAGAGGCGTACAGGGATAG-3′); RhoA ( RHOA ) Forward (5′-CCAAATGTGCCCATCATCCTAGTTG-3′) and Reverse (5′-TCCGTCTTTGGTCTTTGCTGAACAC-3′); Rac1 ( RAC1 ) Forward (5′-CAATGCGTTCCCTGGAGAGTACA-3′) and Reverse (5′-ACGTCTGTTTGCGGGTAGGAGAG-3′); Cdc42 ( CDC42 ) Forward (5′-TAACTCACCACTGTCCAAAGACTCC-3′) and Reverse (5′-CCTCATCAAACACATTCTTCAGACC-3′) and Power SYBR Green PCR Master Mix (Applied Bioystems). The samples were normalized to β-actin and three biological replicates were performed.…”

Metabolomics analysis reveals perturbations of cerebrocortical metabolic pathways in the Pahenu2 mouse model of phenylketonuria

“…Li and colleagues discovered that phenylalanine suppresses neurite outgrowth and induces rat cortical neuronal death in vitro by decreasing the expression of the brain‐derived neurotrophic factor at both mRNA and protein levels. Moreover, Zhang and Schlegel demonstrated decreased dendritic arborization of cortical neurons in cultures, reduced dendritic length, and loss of synapses in hippocampal slice cultures after phenylalanine treatment. Despite long‐term numerous clinical and experimental studies, the exact pathogenetic mechanisms underlying phenylalanine‐evoked impairment of brain function in this neurometabolic disorder remain elusive; in particular, few studies have focused on its metabolic mechanisms.…”