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Disrupted copper homeostasis: Pathogenic factor in autism spectrum disorder and side effect of valproic acid

  • Stephen I. Deutsch
    Correspondence
    Corresponding author at: Anne Armistead Robinson Endowed Chair in Psychiatry, Department of Psychiatry and Behavioral Sciences, Eastern Virginia Medical School, 825 Fairfax Avenue, Suite 710, Norfolk, VA 23507, United States.
    Affiliations
    Department of Psychiatry and Behavioral Sciences, Eastern Virginia Medical School, 825 Fairfax Avenue, Suite 710, Norfolk, VA 23507, United States
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  • David R. Spiegel
    Affiliations
    Department of Psychiatry and Behavioral Sciences, Eastern Virginia Medical School, 825 Fairfax Avenue, Suite 710, Norfolk, VA 23507, United States
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  • Jessica A. Burket
    Affiliations
    Department of Molecular Biology & Chemistry, Christopher Newport University, 1 Avenue of the Arts, Newport News, VA 23606, United States
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Open AccessPublished:August 29, 2021DOI:https://doi.org/10.1016/j.pmip.2021.100087

      Abstract

      A 23 year old male diagnosed with autism spectrum disorder and treated with valproic acid presented with acute onset of hepatic failure; he died less than two months later. Laboratory studies led to a diagnosis of Wilson Disease. The case raised questions about a possible pathogenic role of disrupted copper homeostasis in ASD, and exacerbation of this disruption as a result of treatment with valproic acid. Screening for abnormalities of copper homeostasis may stimulate strategies for therapeutic targeting of a contributing etiological factor and avoidance of contraindicated medications.

      Keywords

      1. Introduction

      A 23 year old male with a history of autism spectrum disorder (ASD), intellectual disability and a generalized tonic-clonic seizure developed acute onset of hepatic failure with hepatic encephalopathy that rapidly progressed to death within a timeframe of less than two months. Liver biopsy confirmed the presence of cirrhosis and laboratory studies of tissue and serum were consistent with a diagnosis of Wilson Disease. The patient’s medication regimen included Depakote. The case suggests that disturbed copper homeostasis may play a pathogenic role in at least some presentations of ASD, which could be exacerbated by promotion of biliary copper excretion by valproic acid.

      2. Case report

      The patient is a 23 year old white male diagnosed with ASD, comorbid intellectual disability, and a history of a generalized tonic-clonic seizure with brief loss of consciousness at age 18, who died secondary to fulminant hepatic failure attributed to the diagnosis of Wilson Disease that was made a few weeks prior to his death. The parents also think the patient may have had an earlier seizure episode because he was found having fallen in the bathroom at age 16, in addition to their description of “staring focal seizures”. The patient and his parents relocated to Coastal Virginia for the father’s employment and he was referred to our outpatient faculty practice by a community mental health center at age 20 because of “dangerousness”. Prior to his referral, the parents contacted local police officers with specialized training in responding to patients with histories of mental illness. The parents reported “we noticed a decline” after his witnessed seizure; for example, the patient “didn’t want to go out” with his mother to the movies, and no longer liked to leave the home. When first seen, the patient was not participating in a home schooling program. The mother reported “he needs reminds in the showering, step to step (i.e., reminders about the proper sequence of activities in order for him to shower)”. The patient was reported to be able to use the bathroom by himself, but this “started at age 12, really good in the last few years…will fecal smear”. Importantly, the patient’s biological mother, who served as the patient’s primary caregiver, was diagnosed with Neuromyelitis Optica (NMO), a demyelinating disorder and, as a result, had concerns about her ability to continue in this essential caregiving role. At the time that the patient was first seen, he was medicated by his primary care physician with quetiapine (300 mg twice a day), Depakote (500 mg at bedtime), and a bedtime dose of clonidine. Unfortunately, because of the patient’s large size (approximately 6 feet tall and weighing 270 lb) and uncooperativeness, blood could not be obtained for serum Depakote levels by either his primary care physician or us in the outpatient settings.
      The patient is the younger of two older neurotypical sisters. The mother reported that she became pregnant with the patient at age 35 and “it was a difficult pregnancy, I had to stay off my feet”. The mother denied alcohol, drugs and smoking during her pregnancy with the patient “just ibuprofen”, which she took for leg pain. The patient weighed 10 lb at birth and “he broke his collar bone when he was born (because of size)”; however, the mother denied a history of gestational diabetes. The mother reported that the patient developed normally until about 18–24 months of age, when she felt he regressed and “stopped” normally developing after his immunizations. The mother explained he “stopped turning when we called his name” “stopped smiling” and “he was starting to speak and that left”. At age 2, the patient was evaluated at a major academic health center and received a diagnosis of “PDD-autism spectrum”. At age two and a half years, the patient was enrolled in a three-day-per-week “infants and toddlers” program and, at age 3, he was enrolled full-time in “Child Find”, an early developmental program offered by the school. As a toddler, the patient displayed stereotypic movements, such as hand-flapping, twirling and tiptoe walking. The patient is reported to be a “picky” eater and is sensitive to the texture of his clothing “cuts all the tags out of everything, cuts them or pulls them out”. Language development was delayed, abnormal and “very strong in echolalia”, which he displayed during the initial and subsequent evaluations. Parents reported that the patient did not engage in imaginative play as a child nor did he socialize with age-matched peers. Patient also engaged in OCD-like behaviors, maintaining a meticulous filing system for a large collection of DVD’s and video-recorded movies. Moreover, at the time of his initial presentation to us, parents reported that the patient is “kind of in his own world” and concerned about possible auditory hallucinations. Family history was positive for prescription benzodiazepine abuse in the patient’s paternal grandmother and a paternal aunt diagnosed and hospitalized for bipolar disorder. A maternal history was positive for possible postpartum depression. At the time of the initial visit, in addition to sharing recommendations about local resources, a collaborative decision was made with the parents to increase his prescribed dosage of Depakote (to 250 mg in the morning and 500 mg at bedtime) and add risperidone as a prn medication for episodes of “dangerous” agitation. Over the course of three years, fluvoxamine was initiated for worsening of his OCD-like behaviors and the bedtime dose of quetiapine was significantly reduced.
      At age 23, parents were advised to seek hospitalization because of persistent hiccups with poor oral intake and appetite. On admission exam, patient was jaundiced with elevated levels of ammonia (169; reference range 28–80 mcg/dL) and clinically diagnosed with acute hepatic failure and hepatic encephalopathy. Depakote was discontinued in the hospital. Abdominal imaging was consistent with cirrhosis. The patient also had findings of portal hypertension and moderate splenomegaly. Patient was pancytopenic and experienced an acute seizure prior to a scheduled liver and bone marrow biopsy. His ammonia levels improved following several lactulose enemas. Interestingly, an ocular slit-lamp exam was negative for Kayser-Fleischer rings. The patient’s course of illness was one of rapid and progressive deterioration. He remained lethargic with poor appetite, occasionally distracted by his iPad, and regressed; he would be seen cuddling and sleeping with his father.
      The histological findings of the liver biopsy revealed a “mixed macro and microvesicular steatosis (approximately 60%)…evidence of end-stage fibrosis with nodular formation”. The copper content of a liver sample, measured in mcg/g dry weight, was 877 (reference value <50). Moreover, measurement of serum ceruloplasmin (12.2; reference range 16–31 mg/dL) and serum copper (56; reference range 63–121 µg/dL) were low, consistent with a diagnosis of Wilson Disease. The patient died within a month and a half of the initial hospitalization for this episode of care.

      4. Discussion

      Data implicate metal dyshomeostasis and abnormalities of the “metallome” (i.e., binding, distribution and metabolic handling of trace metals) in the pathogenesis of ASD [
      • Bjorklund G.
      The role of zinc and copper in autism spectrum disorders.
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      • Stanton J.E.
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      • McGourty K.
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      ,
      • Faber S.
      • Zinn G.M.
      • Kern II J.C.
      • Kingston H.M.S.
      The plasma zinc/serum copper ratio as a biomarker in children with autism spectrum disorders.
      ,
      • Crăciun E.C.
      • Bjørklund G.
      • Tinkov A.A.
      • Urbina M.A.
      • Skalny A.V.
      • Rad F.
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      Evaluation of whole blood zinc and copper levels in children with autism spectrum disorder.
      ,
      • Curtin P.
      • Austin C.
      • Curtin A.
      • Gennings C.
      • Arora M.
      • Tammimies K.
      • et al.
      Dynamical features in fetal and postnatal zinc-copper metabolic cycles predict the emergence of autism spectrum disorder.
      ,
      • Li S.
      • Wang J.
      • Bjørklund G.
      • Zhao W.
      • Yin C.
      Serum copper and zinc levels in individuals with autism spectrum disorders.
      ]. For example, elevated blood levels of copper and an increased ratio of copper to zinc have been reported in children with ASD and proposed as potential biomarkers of this disorder [
      • Bjorklund G.
      The role of zinc and copper in autism spectrum disorders.
      ,
      • Stanton J.E.
      • Malijauskaite S.
      • McGourty K.
      • Grabrucker A.M.
      The metallome as a link between the “omes” in autism spectrum disorders.
      ,
      • Faber S.
      • Zinn G.M.
      • Kern II J.C.
      • Kingston H.M.S.
      The plasma zinc/serum copper ratio as a biomarker in children with autism spectrum disorders.
      ,
      • Curtin P.
      • Austin C.
      • Curtin A.
      • Gennings C.
      • Arora M.
      • Tammimies K.
      • et al.
      Dynamical features in fetal and postnatal zinc-copper metabolic cycles predict the emergence of autism spectrum disorder.
      ,
      • Li S.
      • Wang J.
      • Bjørklund G.
      • Zhao W.
      • Yin C.
      Serum copper and zinc levels in individuals with autism spectrum disorders.
      ]. Copper is a redox-reactive metal that acts as a cofactor in many electron transfer reactions, whose free concentration must be maintained within a safe narrow range in order to prevent dysregulated oxidation of proteins and lipids due to elevated levels. Ordinarily, a steady state equilibrium exists between dietary absorption of copper from the stomach and duodenum into the portal circulation and its metabolic handling and biliary excretion by liver hepatocytes, an equilibrium that can be disrupted in disease states [
      • Schlief M.L.
      • Gitlin J.D.
      Copper homeostasis in the CNS: a novel link between the NMDA receptor and copper homeostasis in the hippocampus.
      ]. A large variety of proteins are involved in copper handling within and between cells and, thereby, assure maintenance of safe nontoxic concentrations. These proteins regulate directional transport of copper across plasma membranes of specialized cells, such as endothelial cells, hepatocytes, and neurons, and safely traffic copper to specific organelles and cellular pathways within cells by preventing toxic effects of excess free concentrations [
      • Schlief M.L.
      • Gitlin J.D.
      Copper homeostasis in the CNS: a novel link between the NMDA receptor and copper homeostasis in the hippocampus.
      ,
      • Gaier E.D.
      • Eipper B.A.
      • Mains R.E.
      Copper signaling in the mammalian nervous system: synaptic effects.
      ,
      • D’Ambrosi N.
      • Rossi L.
      Copper at synapse: release, binding and modulation of neurotransmission.
      ,
      • Ackerman C.M.
      • Chang C.J.
      Copper signaling in the brain and beyond.
      ].
      Liver hepatocytes also synthesize ceruloplasmin, an essential ferroxidase that incorporates six copper atoms in the late Golgi secretory pathway; ceruloplasmin is secreted by hepatocytes and safely transports about 95% of the copper in the circulation. Menkes disease and Wilson disease, which result from “loss of function” mutations of genes coding specific “P-type ATPases” (i.e., ATP7A and ATP7B, respectively), highlighted important roles for copper in CNS development and function; these “P-type ATPases” participate in directional transport of copper across specialized plasma membranes and its trafficking within cells [
      • Schlief M.L.
      • Gitlin J.D.
      Copper homeostasis in the CNS: a novel link between the NMDA receptor and copper homeostasis in the hippocampus.
      ,
      • Madsen E.
      • Gitlin J.D.
      Copper and iron disorders of the brain.
      ]. Menkes disease is an X-linked disorder that presents in early infancy with intractable seizures and failure to thrive and its pathogenesis results from consequences of impaired copper transport into the developing brain [
      • Schlief M.L.
      • Gitlin J.D.
      Copper homeostasis in the CNS: a novel link between the NMDA receptor and copper homeostasis in the hippocampus.
      ]. Neuropsychiatric complications of Wilson disease, an autosomal recessive disorder, most often present in the second to third decade of life with signs and symptoms referable to basal ganglia dysfunction, as well as disorders of mood, psychosis and cognition; the pathogenesis of Wilson disease is attributable to copper accumulation related to defective biliary excretion of copper [
      • Schlief M.L.
      • Gitlin J.D.
      Copper homeostasis in the CNS: a novel link between the NMDA receptor and copper homeostasis in the hippocampus.
      ]. Importantly, “downstream” consequences of impaired transport functions of these “P-type ATPases”, such as interference with synthesis and secretion of cuproenzymes, could play roles in pathogenic processes within the CNS. Further, data suggesting that synaptic release of copper modulates NMDA receptor-mediated neurotransmission, and perineuronal nets (PNNs), which are polyanionic, lattice-like embellishments of the extracellular matrix (ECM), bind redox-reactive metals, such as copper and iron, arouse additional interest in possible regulatory and pathogenic roles of copper in CNS [
      • Balogh G.T.
      • Illés J.
      • Székely Z.
      • Forrai E.
      • Gere A.
      Effect of different metal ions on the oxidative damage and antioxidant capacity of hyaluronic acid.
      ,
      • Burket J.A.
      • Urbano M.R.
      • Deutsch S.I.
      Sugarcoated perineuronal nets regulate “GABAergic” transmission: bittersweet hypothesis in autism spectrum disorder.
      ,
      • Wen T.H.
      • Binder D.K.
      • Ethell I.M.
      • Razak K.A.
      The perineuronal ‘safety’ net? Perineuronal net abnormalities in neurological disorders.
      ]. PNNs enwrap subpopulations of fast-spiking, parvalbumin-expressing (FS, PV+) GABAergic inhibitory basket cells and pyramidal cells in the neocortex, which are neural elements implicated in the pathogenesis of several major neuropsychiatric disorders, including ASD and schizophrenia [
      • Burket J.A.
      • Urbano M.R.
      • Deutsch S.I.
      Sugarcoated perineuronal nets regulate “GABAergic” transmission: bittersweet hypothesis in autism spectrum disorder.
      ,
      • Wen T.H.
      • Binder D.K.
      • Ethell I.M.
      • Razak K.A.
      The perineuronal ‘safety’ net? Perineuronal net abnormalities in neurological disorders.
      ,
      • Enwright J.F.
      • Sanapala S.
      • Foglio A.
      • Berry R.
      • Fish K.N.
      • Lewis D.A.
      Reduced labeling of parvalbumin neurons and perineuronal nets in the dorsolateral prefrontal cortex of subjects with schizophrenia.
      ,
      • Filice F.
      • Vörckel K.J.
      • Sungur A.Ö.
      • Wöhr M.
      • Schwaller B.
      Reduction in parvalbumin expression not loss of the parvalbumin-expressing GABA interneuron subpopulation in genetic parvalbumin and shank mouse models of autism.
      ,
      • Favuzzi E.
      • Marques-Smith A.
      • Deogracias R.
      • Winterflood C.M.
      • Sánchez-Aguilera A.
      • Mantoan L.
      • et al.
      Activity-dependent gating of parvalbumin interneuron function by the perineuronal net protein brevican.
      ].
      Incubating primary cultures of hippocampal neurons prepared from 18-day-old rat embryos in the presence of CuCl2 caused trafficking of ATP7A from the late Golgi to dendrites and axons; these data are consistent with dependence of the localization of the ATP7A on intraneuronal copper content [
      • Schlief M.L.
      • Craig A.M.
      • Gitlin J.D.
      NMDA receptor activation mediates copper homeostasis in hippocampal neurons.
      ]. Importantly, similar to the trafficking of the ATP7A observed in the presence of elevated copper, which is a key determinant of copper homeostasis, NMDA receptor-mediated depolarization of the cultured hippocampal neurons (achieved by incubation in the presence of glutamate and glycine in a Mg2+-free environment) also caused trafficking of the ATP7A to axons and dendrites. NMDA receptor-mediated translocation of the ATP7A to cytoplasmic vesicular compartments in axons and dendrites was blocked by 2-amino-phosphonovalerate (APV), an uncompetitive NMDA receptor channel blocker; thus, translocation is dependent on NMDA receptor-mediated Ca2+ flux. Moreover, the NMDA receptor-activated trafficking occurred independently of intracellular copper concentrations [
      • Schlief M.L.
      • Craig A.M.
      • Gitlin J.D.
      NMDA receptor activation mediates copper homeostasis in hippocampal neurons.
      ]. An important “downstream” consequence of NMDA receptor activated translocation of the ATP7A was shown to be copper efflux from these cultured hippocampal neurons [
      • Schlief M.L.
      • Craig A.M.
      • Gitlin J.D.
      NMDA receptor activation mediates copper homeostasis in hippocampal neurons.
      ]. Ultimately, this “downstream” effect of NMDA receptor activation causing efflux of a releasable pool of copper may have local “modulatory” or dampening effects on excessive NMDA receptor activation [
      • Schlief M.L.
      • Gitlin J.D.
      Copper homeostasis in the CNS: a novel link between the NMDA receptor and copper homeostasis in the hippocampus.
      ,
      • Gaier E.D.
      • Eipper B.A.
      • Mains R.E.
      Copper signaling in the mammalian nervous system: synaptic effects.
      ,
      • D’Ambrosi N.
      • Rossi L.
      Copper at synapse: release, binding and modulation of neurotransmission.
      ,
      • Ackerman C.M.
      • Chang C.J.
      Copper signaling in the brain and beyond.
      ,
      • Schlief M.L.
      • Craig A.M.
      • Gitlin J.D.
      NMDA receptor activation mediates copper homeostasis in hippocampal neurons.
      ]. Exogenous applications of micromolar concentrations of copper have been shown to noncompetitively antagonize NMDA receptor-activated currents in cultured hippocampal and cortical neurons. Conceivably, these noncompetitive antagonistic effects of copper could have functional consequences as low micromolar concentrations of copper interfered with induction of long-term potentiation in the CA1 region of rat hippocampus [
      • Schlief M.L.
      • Gitlin J.D.
      Copper homeostasis in the CNS: a novel link between the NMDA receptor and copper homeostasis in the hippocampus.
      ,
      • Gaier E.D.
      • Eipper B.A.
      • Mains R.E.
      Copper signaling in the mammalian nervous system: synaptic effects.
      ,
      • D’Ambrosi N.
      • Rossi L.
      Copper at synapse: release, binding and modulation of neurotransmission.
      ,
      • Ackerman C.M.
      • Chang C.J.
      Copper signaling in the brain and beyond.
      ,
      • Schlief M.L.
      • Craig A.M.
      • Gitlin J.D.
      NMDA receptor activation mediates copper homeostasis in hippocampal neurons.
      ].
      Copper’s probable role in synaptic transmission was supported by the depolarization-dependent release of 67Cu from preloaded synaptosomes and subsequent studies showing that both depolarization and NMDA-receptor activation resulted in trafficking of ATP7A to the plasma membrane of cultured hippocampal neurons, which could serve as a mechanism for creating this pool of releasable synaptic copper [
      • Schlief M.L.
      • Gitlin J.D.
      Copper homeostasis in the CNS: a novel link between the NMDA receptor and copper homeostasis in the hippocampus.
      ,
      • Gaier E.D.
      • Eipper B.A.
      • Mains R.E.
      Copper signaling in the mammalian nervous system: synaptic effects.
      ,
      • D’Ambrosi N.
      • Rossi L.
      Copper at synapse: release, binding and modulation of neurotransmission.
      ,
      • Ackerman C.M.
      • Chang C.J.
      Copper signaling in the brain and beyond.
      ]. Further, X-ray fluorescence microscopy and copper-sensing fluorescent dyes confirmed the existence of labile pools of copper that are dependent on depolarization and Ca2+-signaling; the labile releasable synaptic pool involves translocation and redistribution of copper from the cell body to dendrites. In addition to ATP7A, an adequately-sized releasable labile pool of neuronal copper depends on neuronal expression of CTR1, the “ion channel-like” copper import protein [
      • Schlief M.L.
      • Gitlin J.D.
      Copper homeostasis in the CNS: a novel link between the NMDA receptor and copper homeostasis in the hippocampus.
      ,
      • Gaier E.D.
      • Eipper B.A.
      • Mains R.E.
      Copper signaling in the mammalian nervous system: synaptic effects.
      ,
      • D’Ambrosi N.
      • Rossi L.
      Copper at synapse: release, binding and modulation of neurotransmission.
      ,
      • Ackerman C.M.
      • Chang C.J.
      Copper signaling in the brain and beyond.
      ]. As discussed, the releasable pool of copper may serve as a mechanism of intrasynaptic feedback inhibition, preventing too much NMDA receptor-activation. Further, longer-term intrasynaptic feedback inhibition by copper may be achieved by its stimulation of S-nitrosylation of the NMDA receptor, a post-translational modification of this tetrameric receptor protein.
      Synaptically-released copper also binds to and inhibits GABAA receptors, particularly α1 and α2-subunit containing GABAA receptors. These latter subunit-containing GABAA receptors are localized to subpopulations associated with parvalbumin-expressing (α1) and cholecystokinin/vasoactive intestinal peptide-expressing (α2) neurons [
      • Gaier E.D.
      • Eipper B.A.
      • Mains R.E.
      Copper signaling in the mammalian nervous system: synaptic effects.
      ]. Copper also has complex effects on the binding and currents generated by specific subtypes of P2X ionotropic purinergic receptors. Additionally, copper binds to several synaptic proteins implicated in the pathogenesis of neurodegenerative disorders, such as amyloid precursor protein (APP), prior protein (PrP) and α-synuclein (α-syn) [
      • Schlief M.L.
      • Gitlin J.D.
      Copper homeostasis in the CNS: a novel link between the NMDA receptor and copper homeostasis in the hippocampus.
      ,
      • Gaier E.D.
      • Eipper B.A.
      • Mains R.E.
      Copper signaling in the mammalian nervous system: synaptic effects.
      ,
      • D’Ambrosi N.
      • Rossi L.
      Copper at synapse: release, binding and modulation of neurotransmission.
      ,
      • Ackerman C.M.
      • Chang C.J.
      Copper signaling in the brain and beyond.
      ]. Thus, redox-reactive copper is an essential element required for normal CNS function and implicated in pathogenic mechanisms, including disruption of synaptic transmission.
      Increasingly, physicochemical studies of perineuronal nets (PNNs) and their isolated structural components inform possible functional roles of PNNs in the CNS, such as maintenance of the fast-spiking properties of a subset of parvalbumin-expressing GABAergic interneurons, and modulation of local temporal fluctuations in the binding of redox-reactive metals and their participation in scavenging of toxic free radical species [
      • Balogh G.T.
      • Illés J.
      • Székely Z.
      • Forrai E.
      • Gere A.
      Effect of different metal ions on the oxidative damage and antioxidant capacity of hyaluronic acid.
      ,
      • Burket J.A.
      • Urbano M.R.
      • Deutsch S.I.
      Sugarcoated perineuronal nets regulate “GABAergic” transmission: bittersweet hypothesis in autism spectrum disorder.
      ,
      • Morawski M.
      • Reinert T.
      • Meyer-Klaucke W.
      • Wagner F.E.
      • Tröger W.
      • Reinert A.
      • et al.
      Ion exchanger in the brain: Quantitative analysis of perineuronally fixed anionic binding sites suggests diffusion barriers with ion sorting properties.
      ]. The “Donnan-type distribution” of ions created by the “ionic sorting” property of PNNs surrounding fast-spiking parvalbumin-expressing GABAergic Basket cells contributes to generating high-frequency action potentials by pushing Cl- inward and pulling K+ outward [
      • Burket J.A.
      • Urbano M.R.
      • Deutsch S.I.
      Sugarcoated perineuronal nets regulate “GABAergic” transmission: bittersweet hypothesis in autism spectrum disorder.
      ,
      • Morawski M.
      • Reinert T.
      • Meyer-Klaucke W.
      • Wagner F.E.
      • Tröger W.
      • Reinert A.
      • et al.
      Ion exchanger in the brain: Quantitative analysis of perineuronally fixed anionic binding sites suggests diffusion barriers with ion sorting properties.
      ]. Data suggest that Cu2+ may protect hyaluronic acid (HA), a major component of the PNN with a long anionic, nonsulfated polysaccharide chain composed of repeating alternating disaccharide units of D-glucuronic acid and N-acetyl-glucosamine, by its ability to displace Fe2+ bound to HA [
      • Balogh G.T.
      • Illés J.
      • Székely Z.
      • Forrai E.
      • Gere A.
      Effect of different metal ions on the oxidative damage and antioxidant capacity of hyaluronic acid.
      ,
      • Burket J.A.
      • Urbano M.R.
      • Deutsch S.I.
      Sugarcoated perineuronal nets regulate “GABAergic” transmission: bittersweet hypothesis in autism spectrum disorder.
      ]. Binding of bivalent Fe2+ to the two carboxyl groups of each tetrasaccharide unit of HA leads to generation of hydroxyl radicals and peroxynitrite, which can cause oxidative degradation of HA. Moreover, independently of actual binding to HA, bivalent metal ions attracted to the PNNs by HA’s long polyanionic polysaccharide chain may provide protection by scavenging reactive free radicals in the local area of the neurons they enwrap. Because PNNs are critical structural components of neocortical circuits containing fast-spiking, parvalbumin-expressing GABAergic projections to assemblies of pyramidal neurons, there is growing interest in the physicochemical basis of their probable pathological roles in at least some presentations of ASD [
      • Burket J.A.
      • Urbano M.R.
      • Deutsch S.I.
      Sugarcoated perineuronal nets regulate “GABAergic” transmission: bittersweet hypothesis in autism spectrum disorder.
      ,
      • Wen T.H.
      • Binder D.K.
      • Ethell I.M.
      • Razak K.A.
      The perineuronal ‘safety’ net? Perineuronal net abnormalities in neurological disorders.
      ,
      • Enwright J.F.
      • Sanapala S.
      • Foglio A.
      • Berry R.
      • Fish K.N.
      • Lewis D.A.
      Reduced labeling of parvalbumin neurons and perineuronal nets in the dorsolateral prefrontal cortex of subjects with schizophrenia.
      ,
      • Filice F.
      • Vörckel K.J.
      • Sungur A.Ö.
      • Wöhr M.
      • Schwaller B.
      Reduction in parvalbumin expression not loss of the parvalbumin-expressing GABA interneuron subpopulation in genetic parvalbumin and shank mouse models of autism.
      ,
      • Favuzzi E.
      • Marques-Smith A.
      • Deogracias R.
      • Winterflood C.M.
      • Sánchez-Aguilera A.
      • Mantoan L.
      • et al.
      Activity-dependent gating of parvalbumin interneuron function by the perineuronal net protein brevican.
      ].
      Whereas valproic acid did not affect the absorption of copper from rat small intestine, which was measured using an in situ recirculation perfusion method, it did significantly increase bile flow and, thereby, increased biliary excretion of copper [
      • Kuzuya T.
      • Amioka K.
      • Nabeshima T.
      Valproic acid increases biliary copper excretion in the rat.
      ]. Serum concentrations of valproic acid of 31.1 ± 3.2 µg/ml measured at the end of the 2-h perfusion were associated with a total mean value for the increase in biliary copper excretion of 79% [
      • Kuzuya T.
      • Amioka K.
      • Nabeshima T.
      Valproic acid increases biliary copper excretion in the rat.
      ]. Since biliary excretion of copper is its major excretory route, therapeutic serum concentrations of valproic acid may be associated with disruptions of the homeostatic balance of copper in the body. Conceivably, valproic acid’s effect on bile flow and biliary copper excretion could result in pathogenic effects on CNS function, especially in patients at-risk for abnormalities of copper absorption, transport, distribution and metabolism [
      • Kuzuya T.
      • Amioka K.
      • Nabeshima T.
      Valproic acid increases biliary copper excretion in the rat.
      ].
      A clinical study of 101 adult outpatients treated with valproic acid as monotherapy (N = 75) or polytherapy (N = 26) for seizure disorders and healthy controls (N = 50) confirmed that increased biliary copper excretion can affect circulating levels of serum copper [
      • Lampón N.
      • Tutor J.C.
      Effect of valproic acid treatment on copper availability in adult epileptic patients.
      ]. ATP7B, a copper-transporting ATPase, has an important peripheral role in converting apoceruloplasmin, the inactive ferroxidase, into its enzymatically-active form by facilitating its incorporation of six copper atoms. Thus, ceruloplasmin oxidase assays measure the functionally active “holo” form of ceruloplasmin, serving as a biomarker of copper availability that is not confounded by age, gender, hormonal status, infection, inflammation, pregnancy or protein deficiency. Moreover, the ratio of serum copper to immunoreactive ceruloplasmin has been touted as a measure of copper status. These two serum measures were used to study effects of valproic acid on copper status in the human [
      • Lampón N.
      • Tutor J.C.
      Effect of valproic acid treatment on copper availability in adult epileptic patients.
      ]. Serum concentrations of valproic acid were 63.4 ± 2.2 mg/L and 49.2 ± 4.1 mg/L in the patients receiving monotherapy and polytherapy, respectively. The two measures of copper status were significantly correlated across the total number of subjects, controls and patients in the monotherapy and polytherapy groups [
      • Lampón N.
      • Tutor J.C.
      Effect of valproic acid treatment on copper availability in adult epileptic patients.
      ]. Importantly, the two measures of copper status were significantly lower in the two patient groups, compared to controls [
      • Lampón N.
      • Tutor J.C.
      Effect of valproic acid treatment on copper availability in adult epileptic patients.
      ]. Moreover, in the patients treated with valproic acid as monotherapy and polytherapy, specific oxidase activities of ceruloplasmin were lower than the lower limit of the reference range in 37.3% and 38.4% of each group, respectively. The data suggest that at least one-third of patients, whose serum valproic acid levels are within the therapeutic range, may have at least marginal copper deficiency; this cross-sectional study could not address possible effects of chronic exposure to valproic acid on copper status. Also, pathogenic effects of valproic acid on copper status may be exaggerated in patients with inborn errors that independently affect copper homeostasis.
      In this case, Depakote was initially prescribed by the neurologist for management of the patient’s seizure disorder, and was maintained and renewed by his primary care provider for this indication. Importantly, valproic acid can have a rare causative association with hyperammonemic encephalopathy, which is even more likely to emerge when co-administered with topiramate [
      • Deutsch S.I.
      • Burket J.A.
      • Rosse R.B.
      Valproate-induced hyperammonemic encephalopathy and normal liver functions: possible synergism with topiramate.
      ]. In psychiatric settings, because medical side effects of hyperammonemic encephalopathy include target symptoms prompting the off-label administration of Depakote in ASD, such as irritability and mood instability, recognition of hyperammonemic encephalopathy may be delayed. Thus, emergence of hyperammonemic encephalopathy caused by Depakote was an additional consideration independently of a possible etiopathogenic role of copper dyshomeostasis and enhanced biliary copper excretion in the presentation of this patient [
      • Deutsch S.I.
      • Burket J.A.
      • Rosse R.B.
      Valproate-induced hyperammonemic encephalopathy and normal liver functions: possible synergism with topiramate.
      ]; Depakote was discontinued in the hospital with the diagnosis of hepatic encephalopathy.
      Cuprizone, a copper chelator, can cause a demyelination syndrome in the mouse that models multiple sclerosis [
      • Herring N.R.
      • Konradi C.
      Myelin, copper, and the cuprizone model of schizophrenia.
      ]. The microscopic lesions caused by cuprizone show signs of edema, astrogliosis and demyelination. On a molecular and cellular level, cuprizone decreased expression of myelin-specific genes and interfered with the differentiation of oligodendrocytes in vitro [
      • Herring N.R.
      • Konradi C.
      Myelin, copper, and the cuprizone model of schizophrenia.
      ]. Thus, it may be of interest that the patient’s mother is diagnosed with “Neuromyelitis optica (NMO)”, which is a demyelinating syndrome [
      • Karussis D.
      The diagnosis of multiple sclerosis and the various related demyelinating syndromes: A critical review.
      ].
      In summary, levels of copper, a redox-reactive metal, within cells and biological fluids must be maintained within a narrow range: levels that are too low could adversely affect the activity of cuproenzymes, and levels that are too high could result in oxidative damage to lipids and proteins, among other consequences [
      • Schlief M.L.
      • Gitlin J.D.
      Copper homeostasis in the CNS: a novel link between the NMDA receptor and copper homeostasis in the hippocampus.
      ,
      • Gaier E.D.
      • Eipper B.A.
      • Mains R.E.
      Copper signaling in the mammalian nervous system: synaptic effects.
      ,
      • D’Ambrosi N.
      • Rossi L.
      Copper at synapse: release, binding and modulation of neurotransmission.
      ,
      • Ackerman C.M.
      • Chang C.J.
      Copper signaling in the brain and beyond.
      ]. Importantly, NMDA receptor, depolarization and Ca2+-dependent releasable pools of copper exist within neurons; these releasable copper pools have an inventory of effects on synaptic transmission, including possible intrasynaptic feedback inhibition of NMDA receptor-activation [
      • Schlief M.L.
      • Gitlin J.D.
      Copper homeostasis in the CNS: a novel link between the NMDA receptor and copper homeostasis in the hippocampus.
      ,
      • Gaier E.D.
      • Eipper B.A.
      • Mains R.E.
      Copper signaling in the mammalian nervous system: synaptic effects.
      ,
      • D’Ambrosi N.
      • Rossi L.
      Copper at synapse: release, binding and modulation of neurotransmission.
      ,
      • Ackerman C.M.
      • Chang C.J.
      Copper signaling in the brain and beyond.
      ]. Perineuronal nets (PNNs) play a variety of functional roles in the local environments of subpopulations of neurons they enwrap [
      • Burket J.A.
      • Urbano M.R.
      • Deutsch S.I.
      Sugarcoated perineuronal nets regulate “GABAergic” transmission: bittersweet hypothesis in autism spectrum disorder.
      ]. The polyanionic lattice-like structures bind copper, suggesting that they may protect against copper’s destructive oxidative effects when concentrations are too high, and serve as a local reservoir of copper when levels are too low. Finally, valproic acid, which, has a variety of approved and off-label indications in neuropsychiatric disorders, such as ASD, has been shown to increase biliary excretion of copper and thereby, affect copper homeostasis [
      • Lampón N.
      • Tutor J.C.
      Effect of valproic acid treatment on copper availability in adult epileptic patients.
      ]. Additionally, valproic acid is causally associated with hyperammonemic encephalopathy [
      • Deutsch S.I.
      • Burket J.A.
      • Rosse R.B.
      Valproate-induced hyperammonemic encephalopathy and normal liver functions: possible synergism with topiramate.
      ]. Many of these factors may have interacted to influence the clinical presentation throughout development of the patient and his end-of-life.

      Declaration of Competing Interest

      Drs. Deutsch, Spiegel and Burket have no conflicts of interest to disclose.

      Acknowledgement

      The authors acknowledge with gratitude and appreciation the opportunity given to us by the patient and his parents to participate in his care, teach our trainees, and learn about copper’s possible roles in CNS function and the pathogenesis of autism spectrum disorder.

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