A new therapy prevents intellectual disability in mouse with phenylketonuria.

Untreated phenylketonuria (PKU) results in severe neurodevelopmental disorders, which can be partially prevented by an early and rigorous limitation of phenylalanine (Phe) intake. Enzyme substitution therapy with recombinant Anabaena variabilis Phe Ammonia Lyase (rAvPAL) proved to be effective in reducing blood Phe levels in preclinical and clinical studies of adults with PKU. Aims of present study were: a) to gather proofs of clinical efficacy of rAvPAL treatment in preventing neurological impairment in an early treated murine model of PKU; b) to test the advantages of an alternative delivering system for rAvPAL such as autologous erythrocytes. BTBR-Pahenu2-/- mice were treated from 15 to 64 post-natal days with weekly infusions of erythrocytes loaded with rAvPAL. Behavioral, neurochemical, and brain histological markers denoting untreated PKU were examined in early treated adult mice in comparison with untreated and wild type animals. rAvPAL therapy normalized blood and brain Phe; prevented cognitive developmental failure, brain depletion of serotonin, dendritic spine abnormalities, and myelin basic protein reduction. No adverse events or inactivating immune reaction were observed. In conclusion present study testifies the clinical efficacy of rAvPAL treatment in a preclinical model of PKU and the advantages of erythrocytes as carrier of the enzyme in term of frequency of the administrations and prevention of immunological reactions.

Unbalance between Excitation and Inhibition in Phenylketonuria, a Genetic Metabolic Disease Associated with Autism.

Phenylketonuria (PKU) is the most common genetic metabolic disease with a well-documented association with autism spectrum disorders. It is characterized by the deficiency of the phenylalanine hydroxylase activity, causing plasmatic hyperphenylalaninemia and variable neurological and cognitive impairments. Among the potential pathophysiological mechanisms implicated in autism spectrum disorders is the excitation/inhibition (E/I) imbalance which might result from alterations in excitatory/inhibitory synapse development, synaptic transmission and plasticity, downstream signalling pathways, and intrinsic neuronal excitability. Here, we investigated functional and molecular alterations in the prefrontal cortex (pFC) of BTBR-Pahenu2 (ENU2) mice, the animal model of PKU. Our data show higher frequency of inhibitory transmissions and significant reduced frequency of excitatory transmissions in the PKU-affected mice in comparison to wild type. Moreover, in the pFC of ENU2 mice, we reported higher levels of the post-synaptic cell-adhesion proteins neuroligin1 and 2. Altogether, our data point toward an imbalance in the E/I neurotransmission favouring inhibition in the pFC of ENU2 mice, along with alterations of the molecular components involved in the organization of cortical synapse. In addition to being the first evidence of E/I imbalance within cortical areas of a mouse model of PKU, our study provides further evidence of E/I imbalance in animal models of pathology associated with autism spectrum disorders.

P-cresol Alters Brain Dopamine Metabolism and Exacerbates Autism-Like Behaviors in the BTBR Mouse.

Background: Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social interaction/communication, stereotypic behaviors, restricted interests, and abnormal sensory-processing. Several studies have reported significantly elevated urinary and foecal levels of p-cresol in ASD children, an aromatic compound either of environmental origin or produced by specific gut bacterial strains. Methods: Since p-cresol is a known uremic toxin, able to negatively affect multiple brain functions, the present study was undertaken to assess the effects of a single acute injection of low- or high-dose (1 or 10 mg/kg i.v. respectively) of p-cresol in behavioral and neurochemical phenotypes of BTBR mice, a reliable animal model of human ASD. Results: P-cresol significantly increased anxiety-like behaviors and hyperactivity in the open field, in addition to producing stereotypic behaviors and loss of social preference in BTBR mice. Tissue levels of monoaminergic neurotransmitters and their metabolites unveiled significantly activated dopamine turnover in amygdala as well as in dorsal and ventral striatum after p-cresol administration; no effect was recorded in medial-prefrontal cortex and hippocampus. Conclusion: Our study supports a gene x environment interaction model, whereby p-cresol, acting upon a susceptible genetic background, can acutely induce autism-like behaviors and produce abnormal dopamine metabolism in the reward circuitry.

Targeting mGlu5 Metabotropic Glutamate Receptors in the Treatment of Cognitive Dysfunction in a Mouse Model of Phenylketonuria.

We studied group-I metabotropic glutamate (mGlu) receptors in Pahenu2 (ENU2) mice, which mimic the genetics and neurobiology of human phenylketonuria (PKU), a metabolic disorder characterized, if untreated, by autism, and intellectual disability (ID). Male ENU2 mice showed increased mGlu5 receptor protein levels in the hippocampus and corpus striatum (but not in the prefrontal cortex) whereas the transcript of the mGlu5 receptor was unchanged. No changes in mGlu1 receptor mRNA and protein levels were found in any of the three brain regions of ENU2 mice. We extended the analysis to Homer proteins, which act as scaffolds by linking mGlu1 and mGlu5 receptors to effector proteins. Expression of the long isoforms of Homer was significantly reduced in the hippocampus of ENU2 mice, whereas levels of the short Homer isoform (Homer 1a) were unchanged. mGlu5 receptors were less associated to immunoprecipitated Homer in the hippocampus of ENU2 mice. The lack of mGlu5 receptor-mediated long-term depression (LTD) in wild-type mice (of BTBR strain) precluded the analysis of hippocampal synaptic plasticity in ENU2 mice. We therefore performed a behavioral analysis to examine whether pharmacological blockade of mGlu5 receptors could correct behavioral abnormalities in ENU2 mice. Using the same apparatus we sequentially assessed locomotor activity, object exploration, and spatial object recognition (spatial novelty test) after displacing some of the objects from their original position in the arena. Systemic treatment with the mGlu5 receptor antagonist, MPEP (20 mg/kg, i.p.), had a striking effect in the spatial novelty test by substantially increasing the time spent in exploring the displaced objects in ENU2 mice (but not in wild-type mice). These suggest a role for mGlu5 receptors in the pathophysiology of ID in PKU and suggest that, also in adult untreated animals, cognitive dysfunction may be improved by targeting these receptors with an appropriate therapy.

Early-onset behavioral and neurochemical deficits in the genetic mouse model of phenylketonuria.

Phenylketonuria (PKU) is one of the most common human inborn errors of metabolism, caused by phenylalanine hydroxylase deficiency, leading to high phenylalanine and low tyrosine levels in blood and brain causing profound cognitive disability, if untreated. Since 1960, population is screened for hyperphenylalaninemia shortly after birth and submitted to early treatment in order to prevent the major manifestations of the disease. However, the dietetic regimen (phenylalanine free diet) is difficult to maintain, and despite the recommendation to a strict and lifelong compliance, up to 60% of adolescents partially or totally abandons the treatment. The development and the study of new treatments continue to be sought, taking advantage of preclinical models, the most used of which is the PAHenu2 (BTBR ENU2), the genetic murine model of PKU. To date, adult behavioral and neurochemical alterations have been mainly investigated in ENU2 mice, whereas there are no clear indications about the onset of these deficiencies. Here we investigated and report, for the first time, a comprehensive behavioral and neurochemical assay of the developing ENU2 mice. Overall, our findings demonstrate that ENU2 mice are significantly smaller than WT until pnd 24, present a significant delay in the acquisition of tested developmental reflexes, impaired communicative, motor and social skills, and have early reduced biogenic amine levels in several brain areas. Our results extend the understanding of behavioral and cerebral abnormalities in PKU mice, providing instruments to an early preclinical evaluation of the effects of new treatments.