Beat Thöny

Influence of Early Stress on Social Abilities and Serotonergic Functions across Generations in Mice

Exposure to adverse environments during early development is a known risk factor for several psychiatric conditions including antisocial behavior and personality disorders. Here, we induced social anxiety and altered social recognition memory in adult mice using unpredictable maternal separation and maternal stress during early postnatal life. We show that these social defects are not only pronounced in the animals directly subjected to stress, but are also transmitted to their offspring across two generations. The defects are associated with impaired serotonergic signaling, in particular, reduced 5HT1A receptor expression in the dorsal raphe nucleus, and increased serotonin level in a dorsal raphe projection area. These findings underscore the susceptibility of social behaviors and serotonergic pathways to early stress, and the persistence of their perturbation across generations.

Identification of pharmacological chaperones as potential therapeutic agents to treat phenylketonuria

Phenylketonuria (PKU) is an inborn error of metabolism caused by mutations in phenylalanine hydroxylase (PAH). Over 500 disease-causing mutations have been identified in humans, most of which result in PAH protein misfolding and increased turnover in vivo. The use of pharmacological chaperones to stabilize or promote correct folding of mutant proteins represents a promising new direction in the treatment of misfolding diseases. We performed a high-throughput ligand screen of over 1,000 pharmacological agents and identified 4 compounds (I-IV) that enhanced the thermal stability of PAH and did not show substantial inhibition of PAH activity. In further studies, compounds III (3-amino-2-benzyl-7-nitro-4-(2-quinolyl)-1,2-dihydroisoquinolin-1-one) and IV (5,6-dimethyl-3-(4-methyl-2-pyridinyl)-2-thioxo-2,3-dihydrothieno[2,3- d]pyrimidin-4(1H)-one) stabilized the functional tetrameric conformation of recombinant WT-PAH and PKU mutants. These compounds also significantly increased activity and steady-state PAH protein levels in cells transiently transfected with either WT-PAH or PKU mutants. Furthermore, PAH activity in mouse liver increased after a 12-day oral administration of low doses of compounds III and IV. Thus, we have identified 2 small molecules that may represent promising alternatives in the treatment of PKU.

Cerebrospinal fluid pterins and folates in Aicardi-Goutières syndrome: A new phenotype

Objective: To describe three unrelated children with a distinctive variant of Aicardi-Goutières syndrome (AGS) characterized by microcephaly, severe mental and motor retardation, dyskinesia or spasticity, and occasional seizures. Results: Neuroimaging showed bilateral calcification of basal ganglia and white matter. CSF glucose, protein, cell count, and interferon alpha were normal. Abnormal CSF findings included extremely high neopterin (293 to 814 nmol/L; normal 12 to 30 nmol/L) and biopterin (226 to 416 nmol/L; normal 15 to 40 nmol/L) combined with lowered 5-methyltetrahydrofolate (23 to 48 nmol/L; normal 64 to 182 nmol/L) concentrations in two patients. The absence of pleocytosis and normal CSF interferon alpha was a characteristic finding compared to the classic AGS syndrome. Genetic and enzymatic tests excluded disorders of tetrahydrobiopterin metabolism, including mutation analysis of GTP cyclohydrolase feed-back regulatory protein. CSF investigations in three patients with classic AGS also showed increased pterins and partially lowered folate levels. Conclusions: Intrathecal overproduction of pterins is the first biochemical abnormality identified in patients with AGS variants. Long-term substitution with folinic acid (2-4 mg/kg/day) resulted in substantial clinical recovery with normalization of CSF folates and pterins in one patient and clinical improvement in another. The underlying defect remains unknown.

Mutations in the GTP Cyclohydrolase I and 6-Pyruvoyl-Tetrahydropterin Synthase Genes

Tetrahydrobiopterin deficiencies are highly heterogeneous disorders, with more than 30 molecular lesions identified in the past 2 years in the GTP cyclohydrolase I and 6‐pyruvoyl‐tetrahydropterin synthase genes. The spectrum of mutations causing a reduction of these two biosynthetic enzymes is reviewed. Only three mutations, two present homozygously, are reported in the GTP cyclohydrolase I gene to cause the rare autosomal recessively inherited form of hyperphenylalaninemia. Most of the other mutations, which are scattered over the entire coding region for the six exon‐containing GTP cyclohydrolase I gene, are observed in a heterozygous state with the wild‐type allele and are associated with the dominant DOPA‐responsive dystonia. Compound heterozygous or homozygous mutations spread over all six exons encoding the 6‐pyruvoyl‐tetrahydropterin synthase cause an autosomal recessively inherited variant of hyperphenylalaninemia, mostly accompanied by a deficiency of dopamine and serotonin. Hum Mutat 10:11–20, 1997.

Tetrahydrobiopterin Deficiency: From Phenotype to Genotype

Summary As a result of the selective screening worldwide during the last 18 years, approximately 250 patients with tetrahydrobiopterin deficiency were discovered. Most patients suffer from 6-pyruvoyl tetrahydropterin synthase deficiency (58%), followed by dihydropteridine reductase deficiency (35%), GTP cyclohydrolase I deficiency (3%), and “primapterinuria” (4%). The patients can be treated with neurotransmitter precursors, as well as with tetrahydrobiopterin. However, data on long term treatment are still scarce and it is therefore of great value to investigate all newborns with even mild hyperphenylalaninemia. Cloning of the enzymes involved in the biosynthesis and regeneration of tetrahydrobiopterin makes them to be easily accessible for biochemical and biological studies. So far, all proteins expressed heterologous are active in E. coli. Cloning of the wild type gene and mutant analysis of patients allow the rapid identification of the defective gene on the molecular level.

Tetrahydrobiopterin protects phenylalanine hydroxylase activity in vivo: Implications for tetrahydrobiopterin-responsive hyperphenylalaninemia

The natural cofactor of phenylalanine hydroxylase (PAH), tetrahydrobiopterin (BH4), regulates the enzyme activity as well as being essential in catalysis. BH4‐responsive PAH deficiency is a variant of hyperphenylalaninemia or phenylketonuria (PKU) caused by mutations in the human PAH gene that respond to oral BH4 loading by stimulating enzyme activity and therefore lowering serum phenylalanine. Here, we showed in a coupled transcription–translation in vitro assay that upon expression in the presence of BH4, wild‐type PAH enzyme activity was enhanced. We then investigated the effect of BH4 on PAH activity in transgenic mice that had a complete or partial deficiency in the endogenous cofactor biosynthesis. The rate of hepatic PAH enzyme activity increased significantly with BH4 content without affecting gene expression or Pah‐mRNA stability. These results indicate that BH4 has a chaperon‐like effect on PAH synthesis and/or is a protecting cofactor against enzyme auto‐inactivation and degradation also in vivo. Our findings thus contribute to the understanding of the regulation of PAH by its cofactor BH4 on an additional level and provide a molecular explanation for cofactor‐responsive PKU.

Correction of Murine PKU Following AAV-mediated Intramuscular Expression of a Complete Phenylalanine Hydroxylating System

Phenylketonuria (PKU) caused by phenylalanine hydroxylase (PAH) deficiency leads to toxic accumulation of phenylalanine (Phe). PAH is predominantly expressed in liver and its activity requires a supply of tetrahydrobiopterin (BH(4)) cofactor, but we propose that expression of a complete Phe hydroxylating system (PAH plus BH(4) synthetic enzymes) in skeletal muscle will lead to therapeutic reduction of blood Phe levels in Pah(enu2) mice, a model of human PKU. In order to test this hypothesis, we first developed transgenic Pah(enu2) mice that lack liver PAH activity but coexpress, in their skeletal muscle, PAH and guanosine triphosphate cyclohydrolase I (GTPCH). The latter is responsible for the committing enzymatic step in BH(4) biosynthesis. Despite sufficient muscle enzyme expression, these mice remained hyperphenylalaninemic, thereby suggesting that expression of additional BH(4) synthetic enzymes would be necessary. A recombinant triple-cistronic adeno-associated virus-2 (AAV2) pseudotype 1 vector expressing PAH along with GTPCH and 6-pyruvoyltetrahydrobiopterin synthase (PTPS), the next step in BH(4) synthesis, was generated. Injection of this vector into the gastrocnemius muscles of Pah(enu2) mice led to stable and long-term reduction of blood Phe and reversal of PKU-associated coat hypopigmentation. We propose that muscle-directed gene therapy will be a viable alternative treatment approach to PKU and other inborn errors of metabolism.

Computationally designed liver-specific transcriptional modules and hyperactive factor IX improve hepatic gene therapy.

The development of the next-generation gene therapy vectors for hemophilia requires using lower and thus potentially safer vector doses and augmenting their therapeutic efficacy. We have identified hepatocyte-specific transcriptional cis-regulatory modules (CRMs) by using a computational strategy that increased factor IX (FIX) levels 11- to 15-fold. Vector efficacy could be enhanced by combining these hepatocyte-specific CRMs with a synthetic codon-optimized hyperfunctional FIX-R338L Padua transgene. This Padua mutation boosted FIX activity up to sevenfold, with no apparent increase in thrombotic risk. We then validated this combination approach using self-complementary adenoassociated virus serotype 9 (scAAV9) vectors in hemophilia B mice. This resulted in sustained supraphysiologic FIX activity (400%), correction of the bleeding diathesis at clinically relevant, low vector doses (5 × 10(10) vector genomes [vg]/kg) that are considered safe in patients undergoing gene therapy. Moreover, immune tolerance could be induced that precluded induction of inhibitory antibodies to FIX upon immunization with recombinant FIX protein.

Tetrahydrobiopterin shows chaperone activity for tyrosine hydroxylase

Tyrosine hydroxylase (TH) is the rate‐limiting enzyme in the synthesis of catecholamine neurotransmitters. Primary inherited defects in TH have been associated with l‐DOPA responsive and non‐responsive dystonia and infantile parkinsonism. In this study, we show that both the cofactor (6R)‐l‐erythro‐5,6,7,8‐tetrahydrobiopterin (BH4) and the feedback inhibitor and catecholamine product dopamine increase the kinetic stability of human TH isoform 1 in vitro. Activity measurements and synthesis of the enzyme by in vitro transcription–translation revealed a complex regulation by the cofactor including both enzyme inactivation and conformational stabilization. Oral BH4 supplementation to mice increased TH activity and protein levels in brain extracts, while the Th‐mRNA level was not affected. All together our results indicate that the molecular mechanisms for the stabilization are a primary folding‐aid effect of BH4 and a secondary effect by increased synthesis and binding of catecholamine ligands. Our results also establish that orally administered BH4 crosses the blood–brain barrier and therapeutic regimes based on BH4 supplementation should thus consider the effect on TH. Furthermore, BH4 supplementation arises as a putative therapeutic agent in the treatment of brain disorders associated with TH misfolding, such as for the human TH isoform 1 mutation L205P.

Hyperphenylalaninemia with high levels of 7-biopterin is associated with mutations in the PCBD gene encoding the bifunctional protein pterin-4a-carbinolamine dehydratase and transcriptional coactivator (DCoH)

Pterin-4a-carbinolamine dehydratase (PCD) is required for efficient tetrahydrobiopterin regeneration after phenylalanine hydroxylase activity. This catalytic function was proposed to be specifically defective in newborns with a mild form of hyperphenylalaninemia (HPA) and persistent high urinary levels of primapterin (7-biopterin). A second regulatory task of the same protein is DCoH, a coactivation of transcription by hepatocyte nuclear factor 1alpha (HNF-1alpha), a function that is apparently not impaired in these HPA individuals. It has been shown elsewhere that the human PCD/DCoH bifunctional protein is encoded by a single 4-exon-containing gene, PCBD, located on chromosome 10q22. We have now examined the PCBD gene for mutations at the genomic level in six such HPA patients from four different families. By the use of new intron-specific primers, we detected, in all six patients, single, homozygous nucleotide alterations, in exon 4, that were inherited from their parents. These homozygous alterations predicted mutant PCD/DCoH with a single amino acid exchange, in two cases (alleles T78I), or premature stop codons, in the other four patients (alleles E86X and Q97X). Recombinant expression in Escherichia coli revealed that the mutant proteins-T78I, E86X, and Q97X-are almost entirely in the insoluble fraction, in contrast to wild type, which is expressed as a soluble protein. These data support the proposal that HPA in combination with urinary primapterin may be due to autosomal recessive inheritance of mutations in the PCBD gene specifically affecting the dehydratase activity.