Neurological Disorders: Molecules, Mechanisms and Therapeutics
The neuronal pathology of schizophrenia: molecules and mechanisms G.P. Reynolds and M.K. Harte1 Division of Psychiatry and Neuroscience, Queen’s University Belfast, Belfast BT9 7BL, Northern Ireland, U.K.
Abstract There is an accumulation of evidence for abnormalities in schizophrenia of both the major neurotransmitter systems of the brain – those of GABA (γ -aminobutyric acid) and glutamate. Initial studies have found deficits in the putative neuronal marker, N-acetylaspartate, in a number of brain regions in schizophrenia. The animal models have provided some interesting correlates and discrepancies with these findings. The deficit in inhibitory interneurons within structures implicated in schizophrenic symptomatology may well have direct functional relevance, and can be induced by animal models of the disease such as subchronic phencyclidine administration or social isolation. Their association with these animal models suggests an environmental involvement. A loss of glutamatergic function in schizophrenia is supported by decreases in markers for the neuronal glutamate transporter in striatal structures that receive cortical glutamatergic projections. Deficits in the VGluT1 (vesicular glutamate transporter-1) in both striatal and hippocampal regions support this observation, and the association of VGluT1 density with a genetic risk factor for schizophrenia points to genetic influences on these glutamatergic deficits. Further studies differentiating neuronal loss from diminished activity and improved models allowing us to determine the temporal and causal relationships between GABAergic and glutamatergic deficits will lead to a better understanding of the processes underlying the neuronal pathology of schizophrenia.
Introduction From the middle of the last century, research into the brain in schizophrenia has attempted to implicate in the disorder many different neurochemicals, particularly neurotransmitters and other compounds with direct effects on neuronal activity. Few of these theories have lasted long, although for over 30 years the dopamine hypothesis of schizophrenia has been an important stimulus to research. This was first based on the ability of amphetamine and dopamine agonists to induce a schizophreniform psychosis, and was strengthened by the finding that almost all antipsychotic drugs are effective antagonists of the dopamine D2 receptor subtype. Subsequently, the higher density of dopamine D2 receptors found in post-mortem brain from schizophrenic patients led to the formulation of a modified dopamine hypothesis in which elevated D2 receptors were proposed to underlie the positive symptoms of schizophrenia. However, an up-regulation of D2 receptors is seen in animals after chronic administration of antipsychotic drugs, a treatment that most schizophrenic patients will inevitably have received. It seemed likely, therefore, that the elevation seen was a consequence of drug treatment and unrelated to the disease process. Despite some remaining inconsistencies, most post-mortem and PET Key words: γ -aminobutyric acid (GABA), glutamate, isolation rearing, N-acetylaspartate, phencyclidine, schizophrenia. Abbreviations used: CB, calbindin; CBP, calcium-binding protein; CR, calretinin; GABA, γ aminobutyric acid; MRS, magnetic resonance spectroscopy; NAA, N-acetylaspartate; NAAG, N-acetylaspartylglutamate; NMDA, N-methyl-D-aspartate; PCP, phencyclidine; PET, positron emission tomography; PV, parvalbumin; VGluT1, vesicular glutamate transporter 1. 1 To whom correspondence should be addressed (email
[email protected]).
(positron emission tomography) studies of D2 receptors conclude that there is no elevation in drug-free schizophrenic patients. Nevertheless, dopamine dysfunction is still implicated in schizophrenia, although with no evidence that it might represent a primary pathology in the disease. Post-mortem studies show elevations of dopamine concentration in the amygdala [1], and PET has demonstrated increases in stimulated dopamine release in the striatum [2] in patients with schizophrenia. These findings are likely, however, to be secondary changes reflecting dysfunction and/or deficits in other neuronal systems. Certainly there is substantial evidence for neuronal deficits in schizophrenia. Neuroimaging has identified increases in ventricular size, reflecting volume decreases in brain regions, particularly in the structures of the neocortex and medial temporal lobe. Many functional imaging studies, including PET determination of energy metabolism, have also implicated these structures in the various symptoms of schizophrenia. These macroscopic findings are reflected at the neuronal level by substantial evidence for neuronal deficits and dysfunction. This short review will concentrate on some evidence from our own studies demonstrating changes in neuronal systems containing the major inhibitory and excitatory neurotransmitters of the human brain: respectively GABA (γ -aminobutyric acid) and glutamate. We shall also draw on similarities and discrepancies with two animal models of the disease, which provide some clues as to the aetiological origins of the abnormalities observed in schizophrenia. C 2007
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GABAergic deficits Post-mortem studies have provided evidence for abnormalities of the GABAergic system in schizophrenia. Deficits in GABA-containing neurons are consistently reported, particularly in the frontal cortex and hippocampus. Benes et al. [3] found reduced density of interneurons in CA2 and CA3 regions in the hippocampus of brains of schizophrenia patients, including drug-free patients, suggesting that deficits in interneurons may play a contributory role in the pathophysiology of schizophrenia. Consistent with this interpretation, neurochemical studies have demonstrated a deficit of GABAergic uptake sites in the hippocampus [4], while other reports showed a widespread compensatory up-regulation of postsynaptic-specific GABAA receptor-binding activity throughout most subfields of the hippocampal formation of schizophrenia patients [5]. However, our understanding of the GABA system in schizophrenia must take into account the fact that there are several different types of non-pyramidal neuron that utilize GABA as a neurotransmitter, and that each subset of neurons may show a unique pattern of change. The CBPs (calciumbinding proteins) PV (parvalbumin), CB (calbindin) and CR (calretinin) can be used as markers for specific subpopulations of GABAergic interneurons in the brain. It is now generally accepted that the major neuronal role of these CBPs is the buffering and transport of calcium ions and the regulation of various enzyme systems. As cellular degeneration is often accompanied by impaired calcium homoeostasis, a protective role for CBPs has been postulated [6]. PV is particularly interesting in that it shows a late onset of expression, occurring after GABAergic neurons have been formed [7], imparting a period during which these cells may be particularly susceptible to neurotoxic insult. We have previously reported deficits of PV-immunoreactive cells both in the frontal cortex [8,9] and, more profoundly, in the hippocampus in schizophrenia [10]. These GABAergic deficits in schizophrenia could well be an initial deficit, perhaps of neurodevelopmental origin, that subsequently results in further progressive neuronal dysfunction and the development of schizophrenia in the second or third decade of life. These GABAergic deficits are unrelated to antipsychotic drug treatment, age or duration of illness [10,11]. Deficits in CB have also been reported in schizophrenia [8,12]. In contrast, most of the studies using human postmortem tissue have reported that CR immunoreactive neurons are unaffected in schizophrenia [10,12,13]. Although current animal models of schizophrenia are inevitably limited, behavioural and pharmacological studies concentrate on two approaches with substantial validity. These may be pharmacological [subchronic administration of an NMDA (N-methyl-D-aspartate) antagonist such as PCP (phencyclidine)] or non-pharmacological (isolation rearing from weaning) animal models. PCP has been shown to induce a psychosis in humans that closely resembles schizophrenia in both positive and negative symptoms and also exacerbates the symptoms in C 2007
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stabilized patients [14]. Thus it has been proposed that NMDA receptor hypofunction may be viewed as a model for the disease mechanism, which could explain the symptoms and course of schizophrenia [15]. In reviewing the neuropsychopharmacology of PCP, Jentsch and Roth [16] have concluded that long-term repeated administration of PCP to both humans and animals models the symptoms of schizophrenia better than acute treatment. In animals long-term repeated administration of PCP has been shown to mimic certain aspects of the disorder. Utilizing a subchronic or chronic intermittent treatment regime, our laboratory and others have found that, compared with vehicle-treated controls, animals receiving PCP exhibit reductions in PV-immunoreactive cells in the frontal cortex and hippocampus [17,18]. Isolation rearing of rats is a non-lesion manipulation that leads to deficits in PPI (prepulse inhibition) of the startle reflex and other behavioural and neurochemical alterations reminiscent of schizophrenia [19]. Compared with socially housed rats, isolated rats exhibited reductions in PV- and CB-immunoreactive cells in the hippocampus, with no significant change in CR [20]. These findings demonstrate selective abnormalities of subpopulations of GABAergic interneurons in the hippocampus of isolation reared rats, which resemble the neuronal deficits seen in this region in schizophrenia. In conclusion, we find a deficit of PV-immunoreactive hippocampal neurons in animals receiving subchronic PCP [18] as well as in those reared in isolation [20]. These findings mimic the pathology of the disease in their specificity; no deficits of CR-containing neurons are observed in these animal models.
NAA (N-acetylaspartate) and glutamatergic deficits NAA is an amino acid that is present in high concentrations in the CNS (central nervous system), with intraneuronal concentrations between 10 and 14 mM. NAA is synthesized in neuronal mitochondria from acetyl-coA and aspartate by the enzyme NAA transferase. In the adult rat brain, NAA is found almost exclusively in neurons, being absent from mature glial cells [21]. Although it is 50 years since NAA was first discovered [22] there is still no consensus on its principal metabolic or neurochemical function. It has been shown to be involved in myelin synthesis [23], osmoregulation [24], and a recent study suggests a possible role as an anti-inflammatory [25]. NAA is also a precursor for the biosynthesis of NAAG (N-acetylaspartylglutamate), a neuropeptide with actions at NMDA and metabotropic glutamate receptors [26]. NAA correlates with neuronal density and as such it has been widely used as a neuronal marker, with deficits in NAA thought to reflect neuronal loss. However, previous studies demonstrating the reversibility of NAA following acute brain injury [27,28] indicate that NAA may be a marker of both neuronal loss and cellular dysfunction [29]. The current focus on NAA dysfunction in neuropsychiatric disorders stems from the prominence of the NAA signal in MRS (magnetic resonance spectroscopy). This has led to
Neurological Disorders: Molecules, Mechanisms and Therapeutics
Table 1 Deficits in NAA in schizophrenic post-mortem tissue,
ing a glutamatergic hypofunction [40]. More recently, we have found that striatal deficits in glutamatergic innervation are reflected in losses of immunoreactivity of the neuronal Isolation Schizophrenia PCP model rearing model excitatory amino acid transporter 3. We have gone on to investigate a more specific and selective marker for glutamatergic terminals in the brain, namely VGluT1 (vesicular glutaFrontal cortex – – – mate transporter 1). VGluTs are glutamate transporters localTemporal cortex ↓ ↓ ↓ ized on the membrane of synaptic vesicles in the presynaptic Striatum ↓ – – neuron. VGluT1, in particular, is exclusive to glutamatergic Hippocampus ↓ – – terminals and undetectable in other cell types and neuronal compartments [41–43], and as such provides a very selective a wide number of MRS studies looking at NAA in brain re- marker for presynaptic glutamatergic terminals in the brain. gions in neurodegenerative and psychiatric diseases, notably We find deficits in VGluT1 in both striatal and hippocampal schizophrenia. Neuronal dysfunction, determined by MRS regions in schizophrenia [44]. Preliminary studies demonstrated an association between measurement of NAA deficits in vivo, is seen in cortical and medial temporal structures in schizophrenia [30]. We and VGluT1 density and a genetic risk factor for schizophrenia others have shown that these deficits are unlikely to be due to [45]. A single polymorphism in neuregulin1, identified as treatment with antipsychotic medication [31–33] and may in a risk factor for the disease by Stefansson et al. [46], was associated with VGluT1 in human post-mortem striatal and fact be related to the disease process. In our laboratory we use a sensitive HPLC method to hippocampal tissue, with the at-risk allele predicting lower determine levels of NAA in post-mortem tissue. In the first densities of the glutamatergic marker. These findings provide of these studies we demonstrated a significant deficit in NAA a link between the genotype of a genetic risk factor for schizoin the temporal cortex in schizophrenia, with no change in phrenia and glutamatergic innervation. The results provide the frontal cortex [34]. Interestingly, we also find selective further evidence that the glutamatergic deficits that we see in deficits in NAA in the temporal cortex both in the PCP animal our human studies may, in part, be genetically determined, model [35] and in the isolation rearing model [36]. There was and may indicate why these deficits are not seen in the no change in NAA in any other region examined in these environmental animal models we studied. Further studies differentiating neuronal loss from diminmodels, namely the frontal cortex, striatum or hippocampus ished activity, and improved models allowing us to determine (Table 1). Specific temporal cortex deficits in NAA can be induced the temporal and causal relationships between GABAergic by both pharmacological and non-pharmacological effects in and glutamatergic deficits, as well as identifying the role animals. This raises the question whether temporal cortical of specific genetic and environmental factors, will lead to a NAA deficits might reflect an environmental aetiology of better understanding of the processes underlying the neuronal pathology of schizophrenia. schizophrenia. In our recent studies, we have found that in schizophrenia the NAA deficits extend to regions of the medial temporal lobe, the striatum, the hippocampus and amygdala (L.M. References 1 Reynolds, G.P. (1983) Nature 305, 527–529 Reynolds, personal communication). It is of interest to note 2 Laruelle, M. and Abi-Dargham, A. (1999) J. Psychopharmacol. 13, 358–371 that the NAA deficits that we see in the striatum and 3 Benes, F.M., Kwok, E.W., Vincent, S.L. and Todtenkopf, M.S. (1998) hippocampus in the human post-mortem tissue are not seen in Biol. Psychiatry 44, 88–97 either of the environmental animal models mentioned above 4 Reynolds, G.P., Czudek, C. and Andrews, H.B. (1990) Biol. Psychiatry 27, 1038–1044 (Table 1). Thus, in contrast with the findings in temporal 5 Benes, F.M., Khan, Y., Vincent, S.L. and Wickramasinghe, R. (1996) cortex, this leads us to speculate whether hippocampal (and Synapse 22, 338–349 subcortical) NAA deficits reflect a non-environmental, per6 Heizmann, C.W. (1992) Trends Neurosci. 15, 259–264 7 Solbach, S. and Celio, M.R. (1991) Anat. Embryol. 184, 103–124 haps genetic, aetiology of schizophrenia. 8 Beasley, C.L., Zhang, Z.J., Patten, I. and Reynolds, G.P. (2002) NAA has been suggested to reflect glutamate concenBiol. Psychiatry 52, 708–715 trations and thus may provide a marker of glutamatergic 9 Beasley, C.L. and Reynolds, G.P. (1997) Schizophr. Res. 24, 349–355 neuronal function [37]. In support of this are several observ- 10 Zhang, Z.J. and Reynolds, G.P. (2002) Schizophr. Res. 55, 1–10 ations implicating abnormalities of glutamate in the brain 11 Cahir, M., Costello, I., King, D.J. and Reynolds, G.P. (2005) Neurosci. Lett. 373, 57–60 in schizophrenia. We have found a deficit in the number of 12 Cotter, D., Landau, S., Beasley, C., Stevenson, R., Chana, G., MacMillan, L. and Everall, I. (2002) Biol. Psychiatry 57, 377–386 glutamate uptake sites in the caudate nucleus, putamen and nucleus accumbens in schizophrenia [38], which we interpret 13 Reynolds, G.P. and Beasley, C.L. (2001) J. Chem. Neuroanat. 22, 95–100 14 Javitt, D.C. and Zukin, S.R. (1991) Am. J. Psychiatry 148, 1301–1308 as a deficit of cortico-striatal glutamatergic innervation. 15 Olney, J.W., Newcomer, J.W. and Farber, N.B. (1999) J. Psychiatry Res. 33, 523–533 Consistent with this is the increase in the density of NMDA receptors in the putamen from schizophrenic patients when 16 Jentsch, J.D. and Roth, R.H. (1999) Neuropsychopharmacology 20, 201–225 compared with controls [39]. Such increases in glutamate 17 Cochran, S.M., Kennedy, M., McKerchar, C.E., Steward, L.J., Pratt, J.A. and Morris, B.J. (2003) Neuropsychopharmacology 28, 265–275 receptors have been interpreted as compensatory and reflect-
correlates and discrepancies with two animal models
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34 Nudmamud, S., Reynolds, L.M. and Reynolds, G.P. (2003) Biol. Psychiatry 53, 1138–1141 35 Reynolds, L.M., Cochran, S.M., Morris, B.J., Pratt, J.A. and Reynolds, G.P. (2005) Schizophr. Res. 73, 147–152 36 Harte, M.K., Powell, S.B., Reynolds, L.M., Swerdlow, N.R., Geyer, M.A. and Reynolds, G.P. (2004) Biol. Psychiatry 56, 296–299 37 Petroff, O.A., Errante, L.D., Rothman, D.L., Kim, J.H. and Spencer, D.D. (2002) Ann. Neurol. 52, 635–642 38 Aparicio-Legarza, M.I., Cutts, A.J., Davis, B. and Reynolds, G.P. (1997) Neurosci. Lett. 232, 13–16 39 Aparicio-Legarza, M.I., Davis, B., Hutson, P.H. and Reynolds, G.P. (1998) Neurosci. Lett. 241, 143–146 40 Deakin, J.F.W., Slater, P., Simpson, M.D.C., Gilchrist, A.C., Skon, W.J., Royston, M.C., Reynolds, G.P. and Cross, C.J. (1989) J. Neurochem. 52, 1781–1786 41 Bellocchio, E.E., Hu, H., Pohorille, A., Chan, J., Pickel, V.M. and Edwards, R.H. (1998) J. Neurosci. 18, 8648–8459 42 Bellocchio, E.E., Reimer, R.J., Fremeau, Jr, R.T. and Edwards, R.H. (2000) Science 289, 957–960 43 Fremeau, Jr, R.T., Troyer, M.D., Pahner, I., Nygaard, G.O., Tran, C.H., Reimer, R.J., Bellocchio, E.E., Fortin, D., Storm-Mathisen, J. and Edwards, R.H. (2001) Neuron 31, 247–260 44 Piyabhan, P. and Reynolds, G.P. (2006) J. Psychopharm. 20, MB26 45 Reynolds, G.P., Piyabhan, P., Nudmamud-Thanoi, S., Dempster, E. and Collier, D.A. (2006) Biol. Psychiatry 59, 123S 46 Stefansson, H., Sigurdsson, E., Inthorsdottir, V., Bjornsdottir, S., Sigmundsson, T., Ghosh, S., Brynjolfsson, J., Gunnarsdottir, S., Ivarsson, O., Chou, T.T. et al. (2002) Am. J. Hum. Genet. 71, 877–892
Received 31 August 2006
