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First Symposium on Pediatric Neurotransmitter Diseases

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First Symposium on Pediatric Neurotransmitter Diseases

Co-Hosted by National Institute of Neurological Disorders and Stroke, NIH
Stanford University
Washington, D.C.
May 17 - 19, 2002

Meeting Summary

Session I: Neurochemistry Associated with the PNDs; Darryl C. De Vivo, M.D., Chair

Session I included three presentations focusing on basic neurochemical features of GABA, catecholamines and gamma-hydroxybutyric acid (GHB). The objective was to provide the neurochemical fundamentals applicable to the diseases discussed in future sessions. Dr. O. C. Snead presented first on functional aspects of GABA neurotransmission.

Brain function is dependent upon the capacity for neurons within interconnecting neuronal circuitry to excite or inhibit one another. Excitation and inhibition are achieved through synaptic transmission, which in turn is mediated by chemical messengers called neurotransmitters. Neurotransmission may be thought of as translation of an electrical signal to a chemical signal (mediated by the neurotransmitter), back to an electrical signal. For a neurochemical in the brain to be considered a neurotransmitter, several criteria must be met: 1) there must be neuronal synthesis; 2) the neurochemical must be found in the presynaptic terminal with release in amounts sufficient to exert a defined action on the postsynaptic neuron; 3) when administered exogenously, it must mimic the actions of the endogenously released transmitter; and 4) there must be a defined biochemical sequence for removal of the neurochemical from the synaptic cleft. In mammalian brain, the primary excitatory neurotransmitter meeting these criteria is glutamic acid, while GABA (gamma-aminobutyric acid) fulfills these criteria as the primary inhibitory neurotransmitter.

Once released into the synaptic cleft, GABA may interact at the postsynaptic membrane to induce both fast and slow inhibitory neurotransmission, or it may act on the presynaptic neuron from where it was released to inhibit further release of GABA, so-called pre-synaptic inhibition. GABA may be removed from the synaptic cleft by reuptake both into the presynaptic neuron and into surrounding glia by GABA transporter proteins. Fast postsynaptic inhibition is mediated by the GABAA receptor (GABAAR), a ligand-gated chloride ion channel composed of five different subunits. Slow inhibitory neurotransmission is mediated by the GABAB receptor (GABABR), a metabotropic receptor coupled to various effector systems (G proteins, inwardly-rectifying potassium channels, etc).

The term metabotropic refers to the fact that activation of the GABABR does not induce an electrical change in the postsynaptic membrane directly, but rather leads to a biochemical cascade which then results in postsynaptic membrane hyperpolarization. Depending upon synaptic localization, activation of GABAB receptors can produce either inhibition or disinhibition of synaptic transmission. Presynaptically, GABAB autoreceptors (located on GABAergic neurons) and heteroreceptors (located on other neurotransmitter releasing neurons) may inhibit neurotransmitter release via inhibition of calcium channels. Postsynaptically, GABAB receptor activation produces increased potassium conductance, leading to slow, long-lasting GABAergic inhibition.

Any alteration in GABA metabolism has the potential to result in seizures, and therefore the most common disorder in which GABA is targetted as treatment is epilepsy. However, other disorders, including psychiatric disease, spasticity, stiff man syndrome, and succinic semialdehyde dehydrogenase deficiency, may also be related to disordered GABAergic function in brain.

Dr. Teodoro Bottiglieri next presented a concise overview on metabolism and regulation of biogenic amines. Biogenic amines include the catecholamines dopamine (DA), norepinephrine (NE) and epinephrine (EP), in addition to the indoleamines serotonin (5-HT) and melatonin. Alterations in 5-HT, NE and DA levels have been implicated in diverse neurologic disorders including depression, dementia, schizophrenia, Parkinson's disease, epilepsy, Huntington's disease, Segawa's disease and autism. Serotonin is derived from the physiologic amino acid tryptophan via the combined actions of tryptophan hydroxylase and aromatic amino acid decarboxylase. The major metabolite used to track serotonin turnover, 5-hydroxyindole acetic acid (5-HIAA), is derived from serotonin by the combined actions of monamine oxidase and aldehyde dehydrogenase. In the pineal gland, serotonin is metabolized to melatonin with S-adenosylmethionine as cofactor.

Production of DA begins with the conversion of tyrosine to L-DOPA, catalyzed by tetrahydrobiopterin-dependent tyrosine hydroxylase. L-DOPA is then metabolized to DA by DOPA decarboxylase, a pyridoxine-dependent enzyme. Further metabolism of DA results in production of NE and EP, also requiring S-adenosylmethionine as methyl donor. Drugs which inhibit DA and 5-HT reuptake or breakdown (pargyline, selegeline, imipramine, amphetamine, etc) have demonstrated utility in a variety of neurologic disorders, most likely achieving their therapeutic effect by increasing DA and 5-HT levels within the synaptic cleft. Dr. Bottiglieri presented evidence showing that biogenic amines are further regulated by various receptors, which are stimulated or inhibited by DA, NE or 5HT, and are located either on the pre-synpatic or post-synpatic neuron. Transgenic mice unable to express such receptors have been useful in understanding the role of these biogenic amines in behavior and neurological disorders.

The final presentation focused on gamma-hydroxybutyric acid, once again presented by Dr. Snead. GHB is an ubiquitous short chain fatty acid whose primary precursor is GABA. The concentration of GHB in brain is < 1% of its parent compound, GABA. GHB may be a neurotransmitter, but this has been contested in the literature. GHB synthetic enzymes correlate in location with GHB high-affinity binding sites, and GHB is released by neuronal depolarization in a calcium-dependent fashion. Further, mammalian brain possesses a sodium-dependent GHB uptake system, and GHB is capable of stimulating second messenger systems (cyclic GMP). These observations suggest that GHB is a neurotransmitter.

The pharmacologic action of GHB is mediated via low- and high-affinity GHB receptors (G protein coupled) as well as through the GABABR, for which GHB is a weak agonist. Pharmacologically, GHB inhibits DA release from the presynaptic receptor. GHB has been the subject of a voluminous literature in the last 30 years, and today research is further expanding. GHB is an emerging drug of abuse (most likely linked to its dopaminergic effects), leading the Department of Justice to categorize it as a Class I controlled substance in 1999. Simultaneously, GHB is utilized clinically to treat cataplexy, alcohol- and opiate-withdrawal syndromes, and to induce anaesthesia. Its ability to induce profound EEG abnormalities and behavioral changes has led to its use in the induction of absence seizures in rodent models, and there is speculation that GHB is involved in the pathogenesis of pediatric absence epilepsy. GHB is markedly increased in the inborn error of human metabolism succinic semialdehyde dehydrogenase deficiency, and likely contributes to the neurologic abnormalities seen in patients with this disease.

Session II: Clinical overview of the PNDs; Andrea L. Gropman, M.D., Chair

The disorders known collectively as "pediatric neurotransmitter disorders" consist of several possibly under-recognized, recently identified errors of metabolism that affect the production of neurotransmitters. Neurotransmitters have vast CNS effects, controlling aspects of memory and cognition, temperature regulation, pain control, and motor function to name a few. The diseases within the category of PNDs include aromatic L-amino acid decarboxylase deficiency, tyrosine hydroxylase deficiency, GTP cyclohydrolase deficiency (Segawa disease), and succinic semialdehyde dehydrogenase deficiency. Many of the presenting features are non-specific, or overlap with features seen in other disorders, thus delaying or preventing diagnosis. Appropriate diagnosis can be achieved by specialized testing of cerebrospinal fluid or urine. The mechanism of inheritance for these disorders is known, and for many of the PNDs, appropriate therapeutic options are available. Thus, it is critical to establish the correct diagnoses in these patients, not only to benefit those affected, but also to offer appropriate genetic counseling to at risk individuals.

This session, held on Saturday, May 18th presented an overview of the clinical features of pediatric neurotransmitter disorders, including their classification, presentation, metabolic features, diagnosis, and aspects of medical treatment and management.

Dr. Kathryn Swoboda spoke of her experience with the aromatic l-amino acid decarboxylase deficiency disorders (ALAAD). The major metabolic defect in this category of PNDs is deficiency of central and peripheral catecholamines and serotonin owing to a deficiency of the enzyme aromatic L- amino acid decarboxylase deficiency. The disorder is transmitted in an autosomal recessive manner. Major clinical features include generalized hypotonia, paroxysmal movement disorders ( ie torticollis, limb dystonia, flexor spasms, myoclonic jerks, limb tremor, athetosis, blepharospasm, oral facial dystonia), oculogyric crisis, temperature instability, sleep disturbance, irritability, and developmental delay. In the newborn period, infants may present with hypothermia, lethargy, poor suck, ptosis and hypotension. Originally, the patients may be thought to have cerebral palsy, seizure disorders, mitochondrial disorders, myasthenia gravis, or hyperekplexia, A number of automic features are present including abnormalities of sweating, GERD, increased salivation, apnea and cardiorespiratory arrest. Dr. Swoboda showed a videotape demonstrating some of the major clinical features. Autonomic testing in two patients showed normal to high vagal tone and abnormal sympathetic responses to alterations of heart rate or blood pressure. Neuroimaging studies show no defining features. A PET study in one patient revealed complete absence of dopamine uptake. She presented data from David Goldstein and Keith Hyland, regarding patient plasma catecholamine and serotonin levels , plasma AADC activity/CSF neurotransmitter metabolites, which forms the basis for clinical diagnosis. With regard to medication use, her preliminary studies have revealed some success in reducing frequency and severity of spells and improved voluntary movement with dopamine receptor agonists (pergolide, pramipexole, ropinirole, bromocriptine), although small numbers of patients were used and many experienced dose related irritability or dyskinesias. The next group of agents showing benefit were the anticholinergics (artane, tranylcypromine), antiepileptics (topirimate, klonopin) and MAO inhibitors (selegiline). Also, the serotonergic agents showed some benefit in decreasing irritability (fluoxetine, ergotamine, buspirone, zolmitriptan). Phenylephrine reversed ptosis in about half of patients who were tried on this agent.

In general, the outcome for this group of patients appears to be non-satisfactory, with the majority of patients remaining non-verbal and nonambulatory. A few patients in this series achieved assisted or independent ambulation. Lastly, Dr. Swoboda presented the possibility that there may be increased psychiatric disease in the family histories of patients with ALAAD. Whether this is related to the carrier status of AADC mutation remains to be investigated.

Dr. Georg Hoffman spoke of his experience with Tyrosine hydroxylase deficiency. He presented several slides of patients demonstrating the cardinal clinical features of this disorder: oculogyric crises, parkinsonian symptoms, tremor, hypokinesia, truncal hypotonia, irritability, and alterations in tone with hypotonia on one end of the spectrum to opisthotonos and spasticity at the other extreme. The metabolic defect in tyrosine hydroxylase results in decreased CNS catecholamine levels (including HVA and MHPG), while serotonin metabolism is unaffected (5-HIAA). The tetrahydrobiopterin and neopterin levels are normal, which allows for distinguishing THD from forms of GTPCH deficiency (see next). The disorder is transmitted in an autosomal recessive manner and three disease-associated mutations (missense) have been identified. The features of dopamine deficiency include the tremors, oculogyric crises, akinesia, rigidity, and dystonia. The manifestations of norepinephrine deficiency include ptosis, miosis, increased oculopharyngeal secretions, and postural hypotension. In these patients, only some have responded to dopamine and those unresponsive to dopamine may respond to selegiline.

Dr. Masaya Segawa presented his experience with hereditary progressive dystonia with marked diurnal fluctuation/dopa responsive dystonia, dominant GTP cyclohydrolase I deficiency. First described in 1971, this disorder is a hereditary basal ganglia disease with diurnal fluctuation, inherited in an autosomal dominant manner. It is differentiated form other dopa responsive basal ganglia disorders by its early age of onset (typically 5-6 years of age), diurnal fluctuation (ie worse in the evening, improved in the morning) postural dystonia as a constant feature, later developing tremor, and the absence of cognitive or autonomic features. Characteristically it also demonstrates sustained response to L-dopa (exquisite sensitivity) without any side effects. Early development may be normal or patients may have hypotonia and difficulty in crawling, delayed language, or dystonia in early childhood.

GTP cyclohydrolase is the first enzyme required for the synthesis of tetrahydrobiopterin. Metabolic confirmation can be obtained by measuring biogenic amine metabolites and pterins in CSF. Tetrahydrobiopterin and neopterin concentrations in CSF will be low, along with reduced levels of HVA. Definitive diagnosis is made by mutation analysis of the GTPCH gene (chromosome 14q22.1-22.2). There are no common mutations, and in many cases, a mutation may not be found.

Patients who have undergone PET studies show normal or subnormal FDA PET. Histopathologic studies show absence of degenerative changes in the substantia nigra and basal ganglia. There is reduction of tyrosine hydroxylase in the substantia nigra and reduced dopamine in the ventral caudate nuclease where the D1 receptors are predominantly affected. In addition, there is a reduction of neopterin and biopterin in the striatum. Dopamine transporter activities in the striatum are normal.

Patients with heterogeneous mutations of the GTPCH gene show dominant inheritance with low penetrance, selective impairment of dopamine neurotransmission, preferential involvement of the D1 direct pathway, diurnal fluctuation, and female predominance. Patients with phenotypic variations include those with focal dystonias (ie writer's cramp), paroxysmal dystonia, action dystonia, oculogyric crisis, and muscle hypotonia and developmental delays seen in patients who are compound heterozygotes. Patients with recessive inheritance of mutations in GTPCH gene have hyperphenylalaninemia with neopterin, biopterin, dopamine, and serotonin deficiency (tetrahydrobiopterin is a cofactor not only for tryptophan and tyrosine hydroxylases, but also phenylalanine hydroxylase in the liver) along with onset in infancy, epilepsy and mental retardation. Treatment with dopamine is effective and long lasting.

Lastly, Dr. Philip Pearl presented his experience with a small series of patients with Succinic semialdehyde dehydrogenase deficiency (SSADH). SSADH is a rare autosomal recessive disorder affecting the breakdown of GABA. Due to enzyme deficiency, GABA is not broken down to succinic acid (which then enters the Kreb cycle), but accumulates as gamma hydroxybutyrate. It is unclear whether decreased GABA, and/or elevations of GHB account for the phenotype. The gene has been identified to chromosome 6p22, where greater than 47 disease causing mutations have been identified (leading to absence of functional protein--slice site, missense, frameshift). The presenting features are non-specific and include mental retardation, seizures, hypotonia, nonprogressive ataxia, disproportionate language impairment, autistic features, aggression, anxiety, and hallucinations. Patients may be identified by excessive urinary excretion of gamma hydroxybutyrate (GHB) measured by specific ion monitoring on GCMS. Dr. Pearl then discussed imaging findings in the seven patients he studied: 5/5 demonstrated increased T2 weighted signal in the globus pallidi with normal 3H-MR spectroscopy. One patient showed cerebellar hypoplasia. Reviewing the literature of published cases of SSADH, he found evidence of T2 hyperintensities in globus pallidus, white matter, dentate nucleus, brainstem, as well as reports of delayed myelination, cerebral atrophy, and cerebellar atrophy. EEG results in one patient showed diffuse background slowing, sleep spindle asynchrony, sleep activated spike wave complexes, and central/temporal focal spike discharges. Literature review demonstrated similar findings with the addition of one patient with lack of REM stage sleep.

Of the seven patients he reported, about half have epilepsy, which is concordant with a seizure frequency of about 50% in the literature. All of his patients had generalized tonic clonic seizures, one with absence seizures, and two with history of convulsive status epilepticus. He had limited success with vigabatrin treatment (GABA transaminase inhibitor--ie should lead to improved GABA levels due to blocking breakdown), and found the benzodiazepines helpful for anxiety. He concluded that SSADH may be under-recognized and that cases of autism be screened for urinary excretion of GHB.

Session III: Diagnoses and Therapeutic Intervention for PNDs; Keith Hyland, Ph.D., Chair

The session was designed to present and allow discussion of the clinical signs and symptoms and the analytical methods required to arrive at a diagnosis of a PND. In addition, treatment options were presented for the various conditions and the PND patient was discussed in terms of special nursing considerations and the long-term impact on patient and family well being.

Dr. Georg Hoffmann gave an overview of the approach the clinician takes when considering the possibility of a pediatric neurotransmitter disease. He emphasized the need to exclude other diagnoses via blood or urine testing prior to resorting to cerebrospinal fluid collection and that the measurement of serum prolactin was useful as a peripheral marker of abnormal central dopamine metabolism. Symptoms associated with PND's can include tremor, other movement disorders, occular gyric crises, unstable temperature regulation and other autonomic signs. However, tyrosine hydroxylase deficiency can present as a severe encephalopathy and dopa responsive dystonia due to GTP cyclohydrolase deficiency has an extremely variable phenotype. The possibility of a PND should always be considered in a child who has been given the label of cerebral palsy where there is no obvious cause for the condition.

Dr. Keith Hyland outlined the importance of lumbar cerebrospinal fluid analysis for the diagnosis of the disorders of serotonin and catecholamine metabolism. Emphasis was placed on the critical importance of correct sample collection, handling and storage if meaningful results are to be obtained. Methodology was presented and example chromatograms were provided that indicated the possibility for a deficiency of GTP cyclohydrolase, sepiapterin reductase, tyrosine hydroxylase or aromatic L-amino acid decarboxylase. Problems with diagnoses were discussed and an overview of disorders yet to be discovered was presented. These included deficiencies of tryptophan hydroxylase, catechol-O-methyltransferase, hydroxyindole methyltransferase the vescicular amine transporter, the pre-synaptic amine transporters, post synaptic receptors, GABA defects and other receptor disorders.

Dr. Blair Ford provided an overview of the biochemistry, clinical features and treatment of dopa responsive dystonia (dominantly inherited GTP cyclohydrolase deficiency), tyrosine hydroxylase deficiency and aromatic L-amino acid decarboxylase deficiency. Emphasis was placed on the use of levodopa or dopamine agonists in the treatment of dopa responsive dystonia and tyrosine hydroxylase deficiency with the understanding that there may be receptor supersensitivity to dopamine agonists with accompanying drug-induced dyskinesias. Treatment of aromatic L-amino acid decarboxylase deficiency requires the use of dopamine agonists in conjunction with monoamine oxidase inhibitors. In addition, as the enzyme requires vitamin B6, trials with this cofactor should also be tried. Dr Ford summarized that the therapy for the defects of biogenic amine disorders is not optimum, that earlier diagnosis and initiation of therapy may be beneficial but in the long run gene replacement may be the optimal treatment approach.

Dr Andrea Gropman provided an overview of the biochemistry, clinical features and treatment of succinic semialdehyde dehydrogenase (SSADH) deficiency. Current therapeutic intervention has been limited to Vigabatrin that aims to prevent GABA breakdown and decrease succinic semialdehyde and gamma hydroxybutyrate (GHB) levels. Efficacy of Vigabatrin has been limited possibly due to remaining high levels of GHB in the brain. Emphasis was placed on the SSADH-mouse model for exploring disease mechanisms, pathology and for the investigation of other potential therapeutic agents. Vigabatrin in the animal model elevated brain GABA but did not affect brain GHB. Using survival as an outcome marker, taurine and the GABAB receptor antagonist CGP 35348 increased survival and these pharmacological agents may prove beneficial to the human situation.

Catherine Ascher RN, provided a poignant reminder that the long term care of a patient with a PND may be an arduous task both for health care providers and the affected families. Emphasis was placed on family and patient needs following diagnosis. These included the need for access to support groups and the necessity for referral to a pediatrician knowledgeable in this area. Physical, occupational, speech, feeding and behavioral therapies are required in many patients on a continuing basis and the problems and treatment issues relating to the profuse sweating, oculogyric crises, movement disorders, autonomic disregulation and gastrointestinal problems were all discussed.

Session IV: Animal Models of the PNDs; Kathryn J. Swoboda, M.D., Chair

A number of animal models are currently available which provide us with additional insights into the underlying pathophysiology of the various neurotransmitter deficiency disorders. Topics in this session included an overview of mouse models of catecholamine defects, by Xiaoxi Zhuang; a summary of new therapeutic insights relating to the SSADH knockout mouse, by K. Michael Gibson; and an overview of insights gained from the hph-1 mouse, a model of GTP cyclohydrolase deficiency, by Keith Hyland.

Dr. Zhuang reviewed the strategies involved in the creation of gene deletion (knockout) animal models and gene addition (transgenic) models. Both strategies have been used in the creation of animal models of catecholamine defects, involving numerous components of the dopaminergic synapse. These include TH (tyrosine hydroxylase), VMAT2 and DAT (dopamine transporters), D1 - D5 dopamine receptors, and Golf. The TH knockout animal proved to be an embryonic lethal. However, rescue of TH activity in norepinephrine neurons allowed the generation of dopamine deficient mice who were hypoactive and aphagic, with early postnatal lethality. The mice showed long-term survival with L-DOPA injection with an enhanced response to D1 or D2 dopamine receptor agonists manifest by hyperactivity and stereotyped movements. The DAT knockout mouse, in which dopamine reuptake into the presynaptic dopaminergic terminal is impaired, is hyperactive, with anterior pituitary hypoplasia and dwarfism. Evaluation of a number of psychostimulants in this model, including methylphenidate, amphetamine, cocaine, and nisoxetine, induce a paradoxical calming effect, measured by decreased horizontal activity in cm per unit time. A DAT "knockdown" mouse model has also been created via insertion of additional sequences within the DAT gene construct. No growth retardation is seen in the dopamine transporter "knockdown" mice. Compensatory changes observed in the DAT knockdown mouse include increased extracellular dopamine concentrations but decreased total tissue dopamine concentration. Marked slowing of uptake of dopamine is balanced in part by a significant decrease in dopamine release. Increased dopamine receptor sensitivity has also been observed in these mice in response to quinpirole and SKF-81297. In summary, the DAT knockdown mice have reduced DAT level compared to controls, a 30% reduction in dopamine clearance, approximately 50% tissue dopamine levels, a 200% increase in extracellular dopamine levels, and a 50% reduction in tyrosine hydroxylase. They demonstrate normal postsynaptic receptor expression density for D1 and D2 receptors, but approximately 50% reduction in the expression of presynaptic D2 receptors. However, the postsynaptic activity of the D1 and D2 receptors is reduced. In terms of behavioral analysis, they demonstrate increased locomotor activity and decreased habituation in an open field test. Additional testing on these mice evaluating prepulse inhibition of acoustic startle revealed a trend towards increased inhibition at lower prepulse intensities, and decreased inhibition at increased prepulse intensities. Working memory, as measured in a radial arm maze test, revealed no significant differences from wild-type animals. Impulsivity, as measured in a differential-reinforcement-of-low-rate paradigm, revealed a significant increase in the first 15 second epoch, followed by a decrease in the second 15 second epoch. Dr. Zhang also reviewed strategies available for creating spatial and temporal resolutions in gene knockout paradigms. Knocking out a gene at a specific developmental stage is termed an inducible knockout, while knocking out a gene in specific organs and tissues is termed a tissue specific knockout. Creating a tetracycline inducible system requires several steps. In the specific paradigm that Dr. Zhang provides for this model, the first step is to knock in a neo-stop-tetO site just after the promoter of the gene of interest. This creates a knockout paradigm by preventing transcription. A brain specific promoter is then attached to a tet-transactivator, allowing rescue of the phenotype. Addition of doxycycline to this system aborts the rescue, creating a knockout of the gene at a specific point during development. He also outlined the steps involved in creation of a tissue specific knockout. These include making a tissue-specific Cre recombinase mouse, tagging the gene of interest with Cre recognition targets, and deleting the gene of interest by Cre recombinase. In this way, dopamine and serotonin neuron-specific Cre mice can be created. In summary, it is possible to manipulate mouse models in a variety of ways to better understand the pathophysiology of the catecholamine defects. There are a variety of methods available to analyze the resulting compensatory changes in the system. It is possible to do fairly sophisticated behavioral analyses on mice, and to turn on or turn off a gene of interest in specific organs or tissues at various times in development to explore questions regarding the pathophysiology of these disorders.

Dr. Gibson provided an overview of a murine model of succinic semialdehyde dehydrogenase deficiency (SSADH) deficiency. The first clinical identification of patients with SSADH deficiency was in 1983, followed by the development of an isotope dilution assay for GHB in 1990, an enzyme assay in 1991, the first prenatal diagnosis in 1994, and the cloning of the human gene in 1995. In 2000, the first knockout mouse was created, and ongoing efforts over the past two years have explored various therapeutic interventions in the mouse model. SSADH is involved in the catabolism of succinic semialdehyde, which is derived from GABA, an inhibitory neurotransmitter. Succinic semialdehyde is catabolized via SSADH into succinic acid, which enters the Kreb's cycle. Alternatively it is converted to 4-hydroxybutyric acid. This is the compound that accumulates in the setting of SSADH deficiency, resulting in at least some of the clinical manifestations of the disorder. Clinical features are variable and include psychomotor retardation, delayed or absent speech, hypotonia, ataxia, behavioral problems, seizures and EEG abnormalities. Metabolic abnormalities include marked elevations in urine, plasma and cerebrospinal fluid (CSF) GHB, as well as modest elevations in CSF GABA. Dr. Gibson reviewed the mechanism of action of Vigabatrin, an irreversible inhibitor of GABA transaminase, which should theoretically result in lower levels of succinic semialdehyde and its metabolites. Vigabatrin is typically used as an anticonvulsant. A pilot trial in patients with SSADH deficiency, at a dosage of 25-250 mg/kg/day, resulted in no obvious change in urinary GHB output and only modest improvements in ataxia, hypotonia and speech. It was discontinued in most patients due to lack of apparent benefit. It is unclear at present why patients failed to demonstrate benefit, and why a reduction in urine GHB was not noted. The mouse mutant shows significant growth retardation compared to its wild type counterpart. SSADH activities in tissue homogenates were reduced to essentially zero in the brain and heart, with less than 1% residual activity in the liver and kidney. GABA and GHB levels in urine, as well as tissue GHB concentrations, were markedly elevated. Brain total GABA was significantly elevated, and was specifically elevated in hippocampus, frontal cortex, parietal cortes and cerebellum. A number of abnormalities of specific amino acid concentrations were noted in mutant vs wild type animals. Glutamine levels in brain were significantly reduced, and were specifically reduced in hippocampus, cerebellum, frontal and parietal cortex. Arginine, aspartate and ornithine were significantly increased in frontal and parietal cortex. Serine was substantially elevated in hippocampus, frontal cortex and cerebellum, and glycine was elevated in frontal and parietal cortex. Cystathionine and alanine were elevated in hippocampus, frontal and parietal cortex, and glutamate was elevated in hippocampus. It is known that GHB acts primarily by inhibiting presynaptic dopamine release in vivo. Potential therapies for SSADH deficiency include naltrexone, an opioid receptor antagonist;NCS-382, a selective GHB receptor antagonist which blocks striatal dopamine release following GHB administration; and taurine, an essential amino acid which is found in high quantities in breast milk. Survival curves in mutant mice given various agents at the time of weaning were reviewed. Administration of Vigabatrin, taurine or CGP 35348 increase the percent of animals surviving to age 50 days compared to untreated animals. The backbone of the compound NCS-382 strongly resembles that of GHB. NCS-382 reveals the highest percent survival to date of any of the compounds tested. NCS-382 appears to work via antagonism of GHB for its high affinity reaction with the GHB receptor. CGP 35348 interferes with the lower affinity reaction of GHB with the GABA B receptor. The poor clinical response with treatment with Vigabatrin in humans may be explained in part by the animal model. Although Vigabatrin increases brain GABA levels by 50%, GHB is not lowered. An adenoviral mediated liver gene therapy paradigm has been used in an attempt to rescue the phenotype in the mouse model by administering the construct at day of life 10, prior to weaning. The percent survival in the treated animals significantly exceeds that of the untreated animals after 20 days of life. This data is preliminary and additional studies are needed with regard to these therapeutic interventions. However, therapeutic interventions appear feasible based on the above data. Additional questions remain regarding whether or not SSADH deficiency is associated with oxidative damage in light of the reduction of the Krebs cycle intermediate succinic semialdehyde, and whether or not rescue of liver function alone will have a significant impact on the neurologic phenotype.

Dr. Hyland provided an overview of the hph-1 mouse, a model of autosomal dominant GTP cyclohydrolase deficiency (Segawa's disease). He reviewed what is known about the pattern of biochemical abnormalities in the human disorder, in which GTPCH deficiency results in decreased neopterin, tetrahydrobiopterin (BH4), homovanillic acid, and 5-hydroxyindoleacetic acid. He pointed out the important role of BH4 as a cofactor in the function of the hydroxylases phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH), and tryptophan hydroxylase (TRYPH), critical in the generation of the neurotransmitter precursors L-DOPA and 5-HTP. A number of unanswered questions remain regarding Segawa's disease, including the observed diurnal variation in symptoms, increased penetrance in females, variability of the phenotype, and mechanism of decreased amounts of TH in the striatum. As the underlying defect in the mouse model is unknown (no mutation in the GTP cyclohydrolase gene has been identified as yet), which raises questions regarding the validity of the mouse model in understanding the human condition. From a biochemical standpoint, however, it appears to be an excellent model, as manifest by decreased BH4 in striatum, as well as decreased brain amines and amine metabolites. The mechanism of decreased neurotransmitter metabolites had been initially presumed to be due to the decreased amount of available cofactor, BH4, resulting in sub-saturating concentrations for the hydroxylases. In vivo activities of TH and TRYPH, and in vitro striatal TH activities in the animal model were reduced, while in vitro AADC activity was preserved. In vitro liver PAH activity was also significantly decreased, with preserved AADC activity. This raised the question of whether the mechanism of decreased neurotransmitter metabolism is a general phenomenon relating to BH4 requiring enzymes. The mechanism of decreased hydroxylase activities could theoretically be due to an effect of BH4 on stability or synthesis of the proteins. To address this question, correction of peripheral and central BH4 levels was attained via intraperitoneal injection of high dose (200 mg/kg) BH4. BH4 brain levels remained elevated for at least 10 hours above controls. Following correction of BH4 levels, striatal TH activity showed a progressive increase in the hours post injection. Western blots of striatal TH and liver PAH before and after BH4 injection revealed a substantial increase in measurable protein levels. Liver PAH activity also demonstrated a substantial increase in the hours post injection. TH and PAH gene expression, as assessed by TH and PAH cDNA levels, also substantially increased in the hours post injection. This data suggests that BH4 controls the steady state concentration of TH and PAH by a mechanism involving activation of gene expression, and that the decrease in striatal TH occurs because of decreased gene expression. Given the similarities between the mouse model and human disease, Dr. Hyland concludes that the hph-1 mouse is a good model to study pathophysiological mechanisms in Segawa disease.

Session V: Future Directions for Research and Treatment of PNDs; Georg Hoffmann, M.D., Chair

Session V focused on future interventions for PNDs, those still in development and currently being implemented in animal models, including hepatocyte and liver repopulation techniques, the use of neural stem cells, and gene therapy approaches. Most of these techniques were described using animal model systems other than PNDs.

Dr. J. Roy Chowdhury presented an overview of hepatocyte transplantation and liver repopulation for treatment of inherited diseases. In animal models, transplantation of primary hepatocytes has ameliorated several liver-based metabolic disorders, including Crigler-Najjar syndrome type 1 (CN-1; hyperbilirubinemia), analbuminemia, familial hypercholesterolemia and Wilson's disease. Hepatocyte repopulation has been used clinically in CN-1, in which repopulation of 5% of the liver mass with transplanted hepatocytes resulted in a 50% reduction of serum bilirubin. While it has become abundantly clear that adult hepatocytes have remarkable regenerative capacity, there is a need to identify a strong proliferative stimulus for transplanted hepatocytes that is not recognized by host hepatocytes.

One approach to this problem has been the use of preparative irradiation, used widely in bone marrow transplantation. Transplantation of normal hepatocytes from congenic donor rats into rats subjected to partial hepatectomy and hepatic irradiation has resulted in almost total replacement of host hepatocytes by progeny derived from transplanted non-irradiated cells. This technique, applied to the rodent CN-1 model, led to complete normalization of serum bilirubin levels. This promising methodology of hepatocyte repopulation, however, remains limited by the shortage of donor livers as source of hepatocytes. To overcome this obstacle, investigators are studying the utility of conditionally immortalized hepatocytes, which can be expanded in vitro with induction of quiescence following transplantation. Studies with immortalized hepatocytes are underway in the rodent model of CN-1.

In the second presentation, Dr. Clive Svendsen of the Waisman Center, University of Wisconsin, Madison, spoke on the topic of genetic modification of human neuronal stem cells, and their potential implications for brain repair. Stem cells represent specialized precursor cells with the capacity for indefinite expansion. By definition, pluripotent stem cells have the capacity to form most tissues, whereas multipotent stem cells (i.e., blood cells) have a more limited capacity. Totipotent stem cells have the capacity to form all tissues (i.e., fertilized egg). Totipotent stem cells form the hollow sphere known as the blastocyst. Pluripotent stem cells hold great promise in a variety of settings, including cell and tissue therapy, drug screening and other applications. However, we currently lack essential knowledge as to those factors responsible for cell specialization.

In their laboratories at the Waisman Center, Dr. Svendsen and colleagues have developed a novel method for production of large-scale quantities of human neural precursor cells. In this context, precursor cells could be stimulated to produce monolayers of both astrocytes and neurons, without production of oligodendrocytes. Dr. Svendsen showed serial rodent brain sections with clear evidence of astrocyte migration following transplantation of human neural stem cells (neurospheres). These neurospheres can be infected with wild-type vectors (i.e., tyrosine hydroxylase (TH), an enzyme critical for dopamine production), and studies have documented long-term expression of TH activity. Loss of dopaminergic neurons is a hallmark of Parkinson's disease. However, TH production alone does not necessarily imply a functional dopaminergic neuron. An additional approach of deriving dopaminergic neurons has been to explore transfection of human neurospheres with Nurr 1, an orphan nuclear receptor which is a transcriptional activator of endogenous TH in neural progenitor cells. Dr. Svendsen showed data indicating that neurospheres could be efficiently infected with a Nurr 1 lentiviral construct.

Dr. Svendsen discussed the potential utility of overexpressed growth factors for the potential treatment of PNDs. Important growth factors in neurological disease with potential utility include nerve growth factor (NGF) in Alzheimer's disease, ciliary neurotrophic factor (CNTF) for Huntington's disease, brain derived neurotrophic factor (BDNF) and insulin-like growth factor (IGF) for amyotrophic lateral sclerosis (ALS) and stroke, and GDNF for ALS and Parkinson's disease. Dr. Svendsen posited the question as to what GDNF application might achieve in PNDs (in relation to the capacity to induce DA release)? Since GDNF and other growth factors are exceed size limitations imposed by the blood-brain barrier, direct infusion of the growth factor would be necessary, or direct infusion of stem cell grafts containing the appropriate growth factor. In closing, Dr. Svendsen noted that neural stem cells can now be produced in large numbers in the laboratory, and these cells can produce neurons with the capacity for transplantation into injured rodent brains. In the future, these neurons may have the capacity to function as "mini-pumps" for enzyme replacement within the brain of PND patients.

In the final presentation, Dr. Un Jung Kang described the potential for gene therapy in catecholamine deficiency disorders, using Parkinson's disease (PD) as the model system. Dopamine (DA) is depleted in PD brain, and therapeutic trials have focused on DA repletion using L-DOPA (3,4-dihydroxyphenylalanine) and DA agonists. Production of DA requires the concerted actions of tyrosine hydroxylase (TH; converting L-tyrosine to L-DOPA) and aromatic L-amino acid decarboxylase (AADC; converting L-DOPA to DA). TH is a tetrahydrobiopterin requiring enzyme, and the production of this cofactor is dependent upon the action of GTP cyclohydrolase (GC) on GTP. Thus, three enzyme activities are critically important in the production of DA in brain.

In rodents, Parkinsonian lesions may be induced by application of 6-hydroxydopamine, which results in moderate to severe forelimb akinesia. Primary fibroblasts transfected with retroviral constructs containing TH and GC can be effectively grafted into dopamine-denervated rat striatum, and the levels of L-DOPA assessed by in vivo microdialysis. Using this system, increased and sustained amounts of L-DOPA could be obtained. However, DA production requires AADC activity as well. Retroviral co-transfection of grafts containing all three genes, TH, GC and AADC resulted in substantial in vivo DA synthesis. Unfortunately, a drawback to this approach is the capacity of DA to feedback inhibit TH activity. Thus, to avoid this feedback inhibition, the vesicular monamine transporter (VMAT) gene was introduced to the genetically modified cells in order to store DA. VMAT gene therapy led to the highest sustained levels of DA release determined in vivo. These studies have direct relevance to PNDs, many of which have significantly altered catecholamine levels. Understanding the interactions and roles of genes involved in DA synthesis and processing could help us with designing gene therapy most appropriate for the specific genetic deficits present in PNDs.



Organizing Committee

Symposium Medical & Scientific Organizing Committee

Cathrine Ascher, R.N. MSN, Schneider Children's Hospital, New Hyde Park , NY
Darryl C. De Vivo, M.D., Columbia-Presbyterian Medical Center, New York, NY
K. Michael Gibson, Ph.D., FACMG, Oregon Health Sciences University, Portland, OR
Andrea Gropman, M.D., National Institutes of Health, Bethesda, MD
Keith Hyland, Ph.D, Baylor University Medical Center, Dallas, TX
Phillip L. Pearl, M.D. Children's National Medical Center, Washington, DC
Giovanna Spinella, M.D., National Institutes of Health, Bethesda, MD
Kathryn J. Swoboda, M.D. Primary Children's Medical Center, Salt Lake City, UT


Medical & Scientific Organizing Committee
Teodoro Bottiglieri, Ph.D., Baylor University Medical Center, Dallas TX
Jayanta Roy Chowdhury, M.D. Marion Bessin Liver Research Center, Bronx, NY
Blair Ford, M.D., Columbia Presbyterian Medical Center, New York, NY
Georg F. Hoffmann, M.D., University of Heidelberg, Germany
Un Jung Kang, M.D. The University of Chicago, Il
Masaya Segawa, M.D., Ph.D., Segawa Neurological Clinic for Children, Tokyo, Japan
O.C. Snead, M.D., Hospital for Sick Children, Toronto, CA
Clive Svendsen, Ph.D., The Waisman Center, Madison, WI

PND Board Members/Family Disease Representatives

Charlie Cargill - GTP I Disease Representative
Stacey Cargill - GTP I Disease Representative
Kelly Heger - Aromatic L- Amino Acid Decarboxylase Deficiency Disease Representative
Brad Hoffman- VP PND, Succinic Semialdehyde Dehyrodgenase Deficiency Disease Representative
Carolyn Hoffman -VP. PND, Succinic Semialdehyde Dehyrodgenase Deficiency Disease Representative
Liz McKinnon- Sec. PND, Tyrosine Hydroxylase Deficiency Disease Representative
Nancy Speller - Pres. PND, Unknown Biogenic Amine Defects Disease Representative
John Speller - Tres. PND, Unknown Biogenic Amine Defects Disease Representative




Friday , May 17th 2002
3:00-7:00 pm Arrival and Registration
Second Floor Lobby
7:30-10:00 pm Welcoming Reception (Open to all)
Congressional/Old Georgetown Room Second Floor
Saturday, May 18th 2002
7:00-8:30 am Registration and Continental Breakfast
Second Floor Lobby
8:30-8:45 am Welcome and Opening Remarks
Cabinet/Judiciary Room Second floor
Nancy B. Speller, President/Founder PND Association
Stephen C. Groft, Pharm.D.,Director National Institute of Health Office Of Rare Diseases
8:45-10:05 am Session I-Neurochemistry Associated with the PNDs, Darryl C. De Vivo, MD-Chair
8:45-9:10 am Functional Aspects of GABA Neurotransmission - Snead
9:10-9:40 am Biogenic Amines-Metabolism and Regulation - Bottiglieri
9:40-10:05 am GHB: A Potential Neurotransmitter in Mammalian CNS - Snead
10:05-10:20 am Roundtable Discussion
10:20-10:35 am Coffee Break
10:35-12:15 pm Session II -Clinical Overview of the PNDs, Andrea Gropman, MD-Chair
10:35-11:00 am Aromatic L-Amino Acid Decarboxylase Deficiency- Swoboda
11:00-11:25 am Tyrosine Hydroxylase Deficiency- Hoffmann
11:25-11:50 pm Hereditary Progressive Dystonia with Marked Diurnal Fluctuation (Dominantly Inherited GTP Cyclohydrolase 1 Deficiency)- Segawa
11:50-12:15 pm Succinic Semialdehyde Dehydrogenase (SSADH) Deficiency (4-Hydroxybutyric Aciduria) in Children and Adults- Pearl
12:15-12:30 pm Roundtable Discussion - Disease Databases
12:30-1:30 pm Lunch
Old Georgetown/Congressional Second Floor
1:30-4:15 pm Session III - Diagnoses and Therapeutic Intervention for PND's, Keith Hyland, PhD-Chair
1:30-2:00 pm The Clinician's Approach to the Diagnosis of the Patient with a Suspected PND- Hoffmann
2:00-2:30 pm The Lumbar Puncture for Diagnosis of the PND's- Hyland
2:30-2:45 pm Roundtable Discussion
2:45-3:00 pm Coffee Break
3:00-3:30 pm Therapeutic Intervention for Disorders of Biogenic Amine Metabolism-Ford
3:30-3:55 pm Vigabatrin and Newer Interventions in SSADH Deficiency- Gropman
3:55-4:15 pm Nursing Considerations for the Child with a PND - Ascher
4:15-5:15 pm PND Association Family Disease Representatives , Medical & Scientific Organizing Committee. "Everything you Ever Wanted to Ask about PND's" Question and Answer Chaired by Phillip L. Pearl MD,
5:15 pm Adjourn
Sunday, May 19, 2002
7:00-8:00 am Continental Breakfast
Second Floor Lobby
8:00-9:10 am Session IV-Animal Models of PNDs, Kathryn Swoboda, MD- Chair
Cabinet/Judiciary Room Second Floor
8:00- 8:25 am Animal Models of Catecholamine Defects-Zhuang
8:25-8:50 am New Therapeutic Insights from a Murine Model of SSADH Deficiency- Gibson
8:50-9:10 am The hph-1 Mouse, a Model of GTP Cyclohydrolase Deficiency- Hyland
9:10-9:50 am Roundtable Discussion- "Research Recommendations- Impediments, Challenges and Opportunities".
9:50-10:05 am Coffee Break
10:05-11:05 am Session V-Future Directions for Research and Treatment of PND's , G. Hoffmann, MD-Chair
10:05-10:35 am Liver Cell Transplantation and Liver Repopulation for Inherited Diseases-Chowdhury
10:35-11:05 am Genetic Modification of Human Neuronal Stem Cells; Implications for Brain Repair - Svendson
11:05-11:30 am Gene Therapy for Catecholamines Deficiencies: Lessons from Parkinson Models-Kang
11:30-11:40 am Roundtable Discussion
11:40-12:30 pm Summary Discussion - Defining and Developing a Strategy Plan to Achieve Future Goals and Objectives of the PND Association. Chaired by the Scientific Organization Committee and Invited Speakers (open to all participants)
12:30-12:45 pm Giovanna Spinella, MD and Darryl C. De Vivo, MD-Concluding Remarks and Closing
12:30 pm Adjourn



Last updated April 8, 2011