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Sodium valproate: an old drug with new roles
Opinion
Sodium valproate: an old drug with new roles Natalia N. Nalivaeva1,2, Nikolai D. Belyaev1 and Anthony J. Turner1 1 2
Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, RAS, Saint Petersburg, 194223, Russia
Sodium valproate, or Epilim, has been widely used as a broad spectrum, anticonvulsant drug for over 40 years and exhibits a good safety profile. Some of the actions of valproate arise from its more recently described histone deacetylase (HDAC) inhibitory properties and hence it can specifically modulate gene expression. There is now accumulating evidence that HDAC inhibitors may have potential in the treatment of CNS disorders and, in this context, valproate has much potential as a brainpenetrant, clinically available and well tested drug. This article reviews the pharmacology of this remarkable molecule, focusing on its actions as a neuroprotectant and hence with new potential in the treatment of neurodegenerative diseases. Introduction Valproic acid was originally identified serendipitously as an anti-convulsant drug in 1963 by Pierre Eymard [1] and rapidly came into regular clinical usage in France from the late 1960s, being introduced into the U.K. in 1972 and the U.S. from 1978. Sodium valproate (Epilim1, Depakine1) is unusual among anti-convulsants in that it has broad activity against both generalized and partial seizures, acts as a mood stabilizer in bipolar disorder and is effective in migraine treatment [2]. It is relatively free of side-effects compared to other anticonvulsants and is routinely used in epileptic patients, in some cases successfully for decades [3]. It is also a drug of choice in the treatment of childhood epilepsies. Severe reactions to valproate, e.g. liver toxicity, are rare. Despite its long-standing usage, the mechanism of the anti-convulsant activity of valproate is still controversial. Mechanistic studies originally focused on its ability to dampen neuronal hyperexcitability by potentiation of inhibitory neurotransmission. This was believed to be through an effect on g-aminobutyric acid (GABA) metabolism, particularly since valproic acid partly resembles GABA chemically in being a small, aliphatic acid (2-propylpentanoic acid; n-dipropylacetic acid) (Figure 1). Valproate did indeed increase brain levels of GABA, initially suspected to be through inhibition of GABA aminotransferase [4]. However, other GABA metabolising enzymes have been suggested as more plausible targets in the GABAergic system [5]. More recent studies have shown new activities for valproate including effects on voltage-gated sodium channels [6] which may be direct or act by disrupting the membrane environment of the Corresponding author: Turner, A.J. ([email protected])
channels. Valproate also appears to modulate NMDA receptor-mediated actions further enhancing neuronal inhibition [7] explaining its anti-epileptic activity. Nevertheless, what is undisputed is that valproate is a multi-faceted drug due to its diverse actions, exhaustively tested clinically, and which may have new potential as a therapeutic in some other central nervous system disorders ranging from schizophrenia (reviewed in [8]) to neurodegeneration. This is largely mediated by its more recently discovered activity as a broad acting, histone deacetylase (HDAC) inhibitor [9] modifying chromatin structure and neuronal gene expression. Indeed, the chronic antiepileptic effects of valproate may be due in part to HDAC-mediated regulation of the GABA synthetic enzyme, glutamate decarboxylase [10]. Our opinion is that valproate may specifically have efficacy in neurological disease, particularly through its newly discovered effects on amyloid precursor protein (APP) and amyloid metabolism [11,12]. We speculate that the development of more selective HDAC inhibitors could form a new class of therapeutics in neurodegenerative and psychiatric disorders. HDAC inhibitors and chromatin remodelling The dynamics of chromatin provides the major regulatory factor underlying gene expression and the major epigenetic modifications of chromatin involve the reversible acetylation and methylation of core histones on key lysine residues and DNA methylation at CpG islands. Both these modifications have been implicated in a range of neurodegenerative and neuropsychiatric disorders [8,13]. The reversible acetylation of histone lysine residues by histone acetyltransferases (HAT) is hence a key regulator of gene expression with acetylation leading to an open chromatin structure facilitating RNA polymerase binding and transcription. HDACs have multiple functions, in particular they are major components of repressive complexes and act to compact the chromatin and repress transcription (Figure 2). This has promoted an intensive search for inhibitors of HDACs as drugs to modulate gene transcription through their effects on the chromatin modifications [14]. Mammalian HDACs total 18 in number, are subdivided into four classes (I, IIa/b, III and IV) and are diverse mechanistically. Class III HDACs are also known as sirtuins and are NAD+-dependent enzymes which have been implicated in stress resistance and as mediators of longevity. The others are zinc-dependent enzymes. Valproate primarily inhibits Class I HDACs (HDAC 1, 2, 3 and 8), which are constitutively located in the nucleus [9].
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Box 1. The metabolism of the amyloid precursor protein (APP) leading to formation of Alzheimer senile plaques
Figure 1. Chemical formulae of (a), the anticonvulsant and histone deacetylase (HDAC) inhibitor, valproic acid (VA); (b), the inhibitory neurotransmitter, gaminobutyric acid (GABA), and (c), the clinically approved HDAC inhibitor, vorinostat (suberoylanilide hydroxamic acid, SAHA).
Epigenetic changes in gene expression are particularly implicated in tumour growth [14] and HDAC inhibition can lead to re-expression of pro-apoptotic genes while inhibiting the cell cycle and cell division. Following the approval in 2006 of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat) (Figure 1) by the U.S. Food and Drug Administration (FDA) in the treatment of cutaneous T cell lymphoma, a number of HDAC inhibitors are currently in phase II or III clinical trials for cancer treatments and have other therapeutic potential ranging from anti-infective agents to treatments for b-haemoglobinopathies [15]. Valproate itself has recently proved successful in combination therapy for thyroid carcinoma [16].
APP is a constitutive transmembrane protein which undergoes proteolytic cleavage by b- and g- secretases (shown as pale blue and red arrows in Scheme 1) producing a soluble ectodomain referred to as sAPPb, the neurotoxic amyloid b peptide (Ab) and the APP intracellular domain (AICD). Ab has the property to form neurotoxic oligomers and then to aggregate further, ultimately forming the socalled senile plaques, which are one of the pathological hallmarks of Alzheimer’s disease. This APP processing pathway is considered amyloidogenic in contrast with the cleavage of APP by a-secretase within the Ab domain, which prevents formation of Ab. This nonamyloidogenic processing leads to formation of the soluble ectodomain sAPPa, a short peptide p3 and AICD. AICD binds to the neprilysin (NEP) promoter and activates neprilysin expression (NEP). This binding requires AICD stabilisation by Fe65 and also involves the histone-acetyl transferase, Tip60. AICD also controls expression of some other genes. If the NEP gene is silenced through histone deacetylation by HDACs, expression of NEP is inhibited. However, incubation of cells with VA, and hence inhibition of HDACs, allows AICD to bind to the NEP promoter and re-activate NEP expression [12]. NEP, a cell-surface ectopeptidase, in turn, degrades Ab and reverses amyloid plaque accumulation. VA also inhibits g-secretase activity and hence Ab production [11].
APP metabolism and Alzheimer’s disease (AD) AD is characterized pathologically by the accumulation of extracellular amyloid plaques composed mainly of the amyloid-b peptide (Ab) and intracellular neurofibrillary tangles of the hyperphosphorylated form of the tau protein. Ab is primarily a 40-42 amino acid peptide that is generated from the transmembrane APP through successive proteolysis by two membrane-bound aspartic proteinases: b- and g-secretases [17]. Alternatively, APP can be metabolised through a non-amyloidogenic pathway mediated by the metalloproteinase a-secretases [18] (Box 1 and Scheme 1). Traditionally, the insidious accumulation of Ab has been regarded as an irreversible event. However,
Scheme 1. Schematic representation of the proteolytic processing of the Alzheimer’s amyloid precursor protein (APP) ultimately to form oligomers and then the senile plaques (shown as brown spots) as well as AICD, regulation by AICD of NEP gene expression and degradation of Ab peptide by neprilysin.
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Figure 2. Epigenetic modifications of genes at the chromatin level. DNA methylation and histone acetylation and methylation are involved in gene activation and silencing. Acetylation of histone tails by histone acetyl transferases (HAT) leads to opening of the nucleosome chain which allows transcription factors (TF) to bind gene promoter regions on DNA and activate gene expression. In contrast, DNA methylation by DNA methylases causes gene silencing. Histone deacetylases (HDACs) remove histone acetyl groups while valproic acid (VA), and other HDAC inhibitors, inhibit this process allowing histone acetylation and gene activation.
Ab probably has a normal physiological role as a regulatory peptide [19] and its steady state concentration is tightly regulated by perivascular mechanisms [20] as well as by proteolytic cleavage [21]. Ever since the proposal of the amyloid cascade hypothesis of AD [22], inhibition of Ab formation has been seen as a primary therapeutic target. However, while much AD research has focused on severe, familial forms of the human disease, or equivalent animal models, the much more prevalent, late-onset forms of the disease may primarily be due to deficiencies in Ab clearance rather than its formation [21]. Consecutive processing of APP by b- and gsecretases generates Ab, as well as an APP IntraCellular Domain (AICD) (Box 1 and Scheme 1), which appears to serve as a transcriptional activator regulating expression of a small group of neuronal genes. Among these is the amyloid-degrading enzyme, neprilysin (NEP) [23], which is a zinc metallopeptidase and ectoenzyme that also serves to inactivate synaptic neuropeptides, e.g. enkephalins and tachykinins, as well as Ab [21]. Other genes associated with neurodegeneration and reported to be regulated by AICD include glycogen synthase kinase-3b [24], the epidermal growth factor receptor (EGFR) [25] and the tumour suppressor protein, p53 [26] (and hence indirectly the prion protein [27], which causes the transmissible spongiform encephalopathies such as ‘‘mad cow disease’’). The exact mechanism of action of AICD remains unclear and is controversial but the consensus view is that it functions in combination with the APP-tail binding protein Fe65 which stabilises AICD against proteolytic degradation, and the chromatin-associated HAT, Tip60 [11,28]. Chromatin immunoprecipitation (ChIP) studies have established that AICD can directly bind to target gene promoters enhancing (NEP) [12] or negatively regulating EGFR promoter activity [25]. Hence, regulation of the amyloid-degrading enzyme NEP by AICD (Box 1) represents an elegant feedback mechanism controlling Ab levels as originally proposed [23]. AICD occupancy of the NEP promoter is correlated with enhanced promoter-
associated histone acetylation and HDAC inhibitors such as valproate, or trichostatin A, can enhance transcription of the NEP gene resulting in increased enzyme activity and amyloid-degrading potential [12]. Further emphasizing the potential of valproate, it has recently been demonstrated that a one month treatment with the drug could inhibit formation of the neuritic Ab plaques in transgenic AD model mice [11]. This effect appeared to be due, at least in part, to an indirect inhibition of the g-secretase pathway which generates Ab. Consequently, Ab42 levels were decreased to one-third of control levels. Hence, both groups independently proposed that valproate could provide a new therapeutic option in AD [11,12]. HDAC inhibitors, amyloid metabolism and learning and memory (Box 2) A general alteration of chromatin structure could provide the backdrop for the changes in gene expression seen during the aging process and in neurodegeneration. Such chromatin remodelling, specifically through increased histone acetylation is associated with the recovery of learning and memory in a transgenic mouse model and the remodelling can be induced by exposing the mice to enriched housing conditions (environmental enrichment), which specifically increased acetylation and methylation of histones H3 and H4 in hippocampus and cortex [29]. Consistent with these observations, injection of the HDAC inhibitors sodium butyrate or trichostatin A increased learning capacity in wild-type mice and reversed learning deficits in transgenic mice showing synaptic loss [29]. These effects were subsequently attributed to HDAC2 as a negative regulator of memory formation rather than HDAC1 through the use of mice over-expressing or lacking these enzymes [30]. The related HDAC inhibitor, phenylbutyrate, was independently shown to reverse spatial learning and memory deficits in an AD mouse model suggesting that this drug may have some efficacy in AD [31]. In an entirely separate study, environmental enrichment has also been shown to up-regulate expression of the 511
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amyloid-degrading enzyme, NEP, and to reduce amyloid deposition in transgenic mice, although this was not directly correlated with any specific chromatin changes [32]. In the same study that demonstrated an effect of valproate in vivo on reduction of amyloid plaques in rodent brain [11], it was also shown that the chronic valproate treatment improved memory and behavioural deficits in these mice as assessed by the Morris water maze test. The effects on chromatin modification and amyloid-degrading activity were not, however, examined in these studies although such treatment should enhance brain histone acetylation levels and neuronal gene expression. If HDAC2 is indeed a key player in inhibiting memory formation and synaptic plasticity [30], then the additional ability of valproate to induce the selective proteasomal degradation of HDAC2 [33] may enhance even further its efficacy. In summary, the combined effects of valproate in reducing Ab formation and enhancing Ab clearance should be synergistic in contributing to behavioural improvements, although whether this translates to the human situation needs exploring in detail. Although a consistent body of information is beginning to accumulate in relation to HDAC inhibitors and neurodegeneration, there are, however, potential pitfalls in chronic treatment with such compounds.
huntingtin protein was known to cause a dysregulation of transcription. Subsequently, an HDAC inhibitor was shown to have efficacy in a Huntington’s disease mouse model both in ameliorating the disease phenotype and reversing the transcriptional abnormalities [34]. Friedreich’s ataxia, another triplet expansion neurodegenerative disorder (of the gene frataxin), is also accompanied by specific gene silencing and reduced histone acetylation, which can be reversed by similar HDAC inhibitors. In a mouse model of the disease such compounds were able to normalize frataxin levels and the associated transcriptional down-regulation [35]. The neuroprotective role of HDAC inhibitors in neurodegenerative diseases appears to relate to conditions where oxidative stress, inflammatory mechanisms or neuronal apoptosis may be involved. For example, valproate, as well as other HDAC inhibitors appear to attenuate the dopaminergic neurotoxicity which is induced by lipopolysaccharide, in part through inducing apoptosis of activated microglia [36] suggesting their potential in the treatment of Parkinson’s disease. Furthermore, valproate and other HDAC inhibitors can protect dopaminergic neurons through upregulating the transcription of astrocyte neurotrophic factors (GDNF, BDNF) by astrocytes [37] and phenylbutyrate appears to provide protection in a rodent model of Parkinson’s disease [38]. The potential of HDAC inhibitors in the treatment of demyelinating disorders is more debatable but the underlying rationale for use of HDAC inhibitors as both neuroprotectants and anti-inflammatories in such conditions has been outlined [39]. The remyelination programme, which declines with aging, is clearly under epigenetic control and a recent study has shown, using the cuprizone model of demyelination, that elevated levels of acetylated histones can impede the repair process of remyelination [40]. Nevertheless, some studies have shown a beneficial effect of HDAC inhibitors in the mouse model of multiple sclerosis, experimental allergic encephalomyelitis, which is ascribed to increased expression of protective genes and apoptosis of inflammatory cells [41]. Motor neuron diseases may also be responsive to the application of HDAC inhibitors, which have given critical insight into underlying disease mechanisms, and clinical trials of such compounds in amyotrophic lateral sclerosis and spinal muscular atrophy are currently in progress [42]. Even in stroke, where postinsult treatment is currently limited to tissue plasminogen activator to enhance recovery, HDAC inhibitors have considerable potential and certainly seem to improve clinical outcome in ischaemic models [43]. A more comprehensive survey of other potential applications of HDAC inhibitors in CNS disorders is to be found in [44].
Valproate and other neurodegenerative conditions Intuitively, given their proapoptotic effects, HDAC inhibitors might not seem likely to be efficacious in neurodegenerative disorders and perhaps even exacerbate the conditions; anti-cancer drugs are not obvious candidates for preventing neuronal cell death. However, evidence is accumulating that there may well be benefits of these compounds, valproate included, in a number of quite distinct neurological conditions. The first example to be reported was in Huntington’s disease where the mutant
Future prospects Pre-clinical and clinical trials of HDAC inhibitors in neurodegenerative diseases are in various stages of progress. To date there is no substantive data regarding anticonvulsant drugs and AD and those trials that have taken place have been small scale focusing on late stage disease and behavioural changes such as agitation and aggression (reviewed in [45]). A phase 3 two year clinical trial of low dose (10 mg/kg/d) valproate in 300 AD patients (the ‘‘valproate in dementia’’ or VALID study: http://
Box 2. Mechanisms of learning and memory The neurological basis of learning and memory is considered a dynamic process at the molecular, cellular, and brain network levels which involves changes in the properties of neuronal cells and their functions in response to various stimuli (both external and internal). These changes may occur via rapid post-translational modification of the existing proteins (short-term memory) or via alterations of gene expression (memory consolidation). Modification of the properties of the neuronal cells subsequently leads to changes in their interactions with other cells in the neuronal networks hence encoding an experience of this particular stimulus. Although these changes are usually sufficient for short-term memory, the lasting memory or establishment of a new reflex requires repetition of this network activation. In this reinforcement process cells use the same molecular mechanisms which were activated by the original stimuli including alterations in gene expression. Since activation of gene expression requires modification of chromatin structure and inhibition of histone deacetylases, application of HDAC inhibitors can change the reaction of cells to various stimuli and as such facilitate memory formation and reinforcement. Ab (and in particular its soluble oligomers) can disrupt synaptic plasticity and memory at extremely low concentrations via various signalling mechanisms, including stress-activated kinases and oxidative/nitrosative stress mediators. Learning difficulties and memory loss, particularly of recent memories, are early signs of dementias such as Alzheimer’s disease. Evaluating potential therapeutics in animal models of the disease (e.g. transgenic rodent models) usually involves testing for recovery of behavioural and cognitive deficits using standard tests such as the Morris water maze.
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Opinion www.adcs.org/Studies/VN.aspx) has not reported any efficacy although its primary aim was to assess the efficacy of valproate on agitation and other early personality changes in dementia but secondarily on whether valproate slows disease progression in AD and improves memory. Whatever the outcome of such trials it seems there is still much potential in the well established drug valproate, particularly but not exclusively through its HDAC inhibitory actions and a detailed exploration of its effects on ADrelated genes would be valuable. The multiplicity of actions of valproate on neuronal systems (neurotransmitters, receptors, ion channels, chromatin remodelling) does make interpetation of data on its actions complex. Furthermore, non-specific HDAC inhibitors like valproate can modify some 2% of genes, potentially causing both beneficial and harmful effects limiting potential efficacy. The next generation of HDAC inhibitors, selective for individual sub-classes and isoforms of the deacetylases, may prove to be even more effective not only in neurology and psychiatry but also in other major classes of disease. Valproate has the distinction, however, of having paved the way for drug development in some of the major human neurological diseases. Acknowledgements We thank the Medical Research Council of Great Britain and Russian Academy of Sciences Programme ‘‘Fundamental Sciences to Medicine’’ for financial support.
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15 Rotili, D. et al. (2009) Non-cancer uses of histone deacetylase inhibitors: effects on infectious diseases and b-hemoglobinopathies. Curr. Top. Med. Chem. 9, 272–291 16 Noguchi, H. et al. (2009) Successful treatment of anaplastic thyroid carcinoma with a combination of oral valproic acid, chemotherapy, radiation and surgery. Endocr. J. 56, 245–249 17 Hunt, C.E. and Turner, A.J. (2009) Cell biology, regulation and inhibition of b-secretase (BACE-1). FEBS J. 276, 1845–1859 18 Allinson, T.M. et al. (2003) ADAMs family members as amyloid precursor protein a-secretases. J. Neurosci. Res. 74, 342–352 19 Pearson, H.A. and Peers, C. (2006) Physiological roles for amyloid b peptides. J. Physiol. 575, 5–10 20 Weller, R.O. et al. (2008) Perivascular drainage of amyloid-b peptides from the brain and its failure in cerebral amyloid angiopathy and Alzheimer’s disease. Brain Pathol. 18, 253–266 21 Nalivaeva, N.N. et al. Amyloid-degrading enzymes as therapeutic targets in Alzheimer’s disease. Curr. Alzheimer Res. 5, 212-224 22 Hardy, J.A. and Higgins, G.A. (1992) Alzheimer’s disease: the amyloid cascade hypothesis. Science 256, 184–185 23 Pardossi-Piquard, R. et al. (2005) Presenilin-dependent transcriptional control of the Ab-degrading enzyme neprilysin by intracellular domains of bAPP and APLP. Neuron 46, 541–554 24 Ryan, K.A. and Pimplikar, S.W. (2005) Activation of GSK-3 and phosphorylation of CRMP2 in transgenic mice expressing APP intracellular domain. J. Cell Biol. 171, 327–335 25 Zhang, Y.W. et al. (2007) Presenilin/g-secretase-dependent processing of b-amyloid precursor protein regulates EGF receptor expression. Proc. Natl. Acad. Sci. USA 104, 10613–10618 26 Alves da Costa, C. et al. (2006) Presenilin-dependent g-secretasemediated control of p53-associated cell death in Alzheimer’s disease. J. Neurosci. 26, 6377–6385 27 Vincent, B. et al. (2009) p53-Dependent transcriptional control of cellular prion by presenilins. J. Neurosci. 29, 6752–6760 28 Cao, X. and Su¨dhof, T.C. (2001) A transcriptionally active complex of APP with Fe65 and histone acetyltransferase Tip60. Science 293, 115– 120 29 Fischer, A. et al. (2007) Recovery of learning and memory is associated with chromatin remodelling. Nature 447, 178–182 30 Guan, J-S. et al. (2009) HDAC2 negatively regulates memory formation and synaptic plasticity. Nature 459, 55–60 31 Ricobaraza, A. et al. (2009) Phenylbutyrate ameliorates cognitive deficit and reduces tau pathology in an Alzheimer’s disease mouse model. Neuropsychopharmacology 34, 1721–1732 32 Lazarov, O. et al. (2005) Environmental enrichment reduces Ab levels and amyloid deposition in transgenic mice. Cell 120, 701–713 33 Kra¨mer, O.H. et al. (2003) The histone deacetylase inhibitor valproic acid selectively induces proteasomal degradation of HDAC2. EMBO J. 22, 3411–3420 34 Thomas, E.A. et al. (2008) The HDAC inhibitor 4b ameliorates the disease phenotype and transcriptional abnormalities in Huntington’s disease transgenic mice. Proc. Natl. Acad. Sci. USA 105, 15564– 15569 35 Rai, M. et al. (2008) HDAC inhibitors correct frataxin deficiency in a Friedreich ataxia mouse model. PLoS ONE 3, e1958 36 Chen, P.S. et al. (2007) Valproic acid and other histone deacetylase inhibitors induce microglial apoptosis and attenuate lipopolysaccharide-induced dopaminergic neurotoxicity. Neuroscience 149, 203–212 37 Wu, X. et al. (2008) Histone deacetylase inhibitors up-regulate astrocyte GDNF and BDNF gene transcription and protect dopaminergic neurons. Int. J. Neuropsychopharmacology 11, 1123– 1134 38 Gardian, G. et al. (2008) Neuroprotective effects of phenylbutyrate against MPTP neurotoxicity. Neuromolecular Med. 5, 235–241 39 Gray, S.G. and Dangond, F. (2006) Rationale for the use of histone deacetylase inhibitors as a dual therapeutic modality in multiple sclerosis. Epigenetics 1, 67–75 40 Shen, S. et al. (2008) Age-dependent epigenetic control of differentiation inhibitors is critical for remyelination efficiency. Nat. Neurosci. 11, 1024–1034 41 Camelo, S. et al. (2005) Transcriptional therapy with the histone deacetylase inhibitor trichostatin A ameliorates experimental autoimmune encephalomyelitis. J. Neuroimmunol. 164, 10–21
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Opinion 42 Echaniz-Laguna, A. et al. (2008) Histone deacetylase inhibitors: therapeutic agents and research tools for deciphering motor neuron diseases. Curr. Med. Chem. 15, 1263–1273 43 Langley, B. et al. (2009) Targeting histone deacetylases as a multifaceted approach to treat the diverse outcomes of stroke. Stroke 40, 2899–2905
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Sodium valproate: an old drug with new roles Natalia N. Nalivaeva1,2, Nikolai D. Belyaev1 and Anthony J. Turner1 1 2
Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK I.M. Sechenov Institute of Evolutionary Physiology and Biochemistry, RAS, Saint Petersburg, 194223, Russia
Sodium valproate, or Epilim, has been widely used as a broad spectrum, anticonvulsant drug for over 40 years and exhibits a good safety profile. Some of the actions of valproate arise from its more recently described histone deacetylase (HDAC) inhibitory properties and hence it can specifically modulate gene expression. There is now accumulating evidence that HDAC inhibitors may have potential in the treatment of CNS disorders and, in this context, valproate has much potential as a brainpenetrant, clinically available and well tested drug. This article reviews the pharmacology of this remarkable molecule, focusing on its actions as a neuroprotectant and hence with new potential in the treatment of neurodegenerative diseases. Introduction Valproic acid was originally identified serendipitously as an anti-convulsant drug in 1963 by Pierre Eymard [1] and rapidly came into regular clinical usage in France from the late 1960s, being introduced into the U.K. in 1972 and the U.S. from 1978. Sodium valproate (Epilim1, Depakine1) is unusual among anti-convulsants in that it has broad activity against both generalized and partial seizures, acts as a mood stabilizer in bipolar disorder and is effective in migraine treatment [2]. It is relatively free of side-effects compared to other anticonvulsants and is routinely used in epileptic patients, in some cases successfully for decades [3]. It is also a drug of choice in the treatment of childhood epilepsies. Severe reactions to valproate, e.g. liver toxicity, are rare. Despite its long-standing usage, the mechanism of the anti-convulsant activity of valproate is still controversial. Mechanistic studies originally focused on its ability to dampen neuronal hyperexcitability by potentiation of inhibitory neurotransmission. This was believed to be through an effect on g-aminobutyric acid (GABA) metabolism, particularly since valproic acid partly resembles GABA chemically in being a small, aliphatic acid (2-propylpentanoic acid; n-dipropylacetic acid) (Figure 1). Valproate did indeed increase brain levels of GABA, initially suspected to be through inhibition of GABA aminotransferase [4]. However, other GABA metabolising enzymes have been suggested as more plausible targets in the GABAergic system [5]. More recent studies have shown new activities for valproate including effects on voltage-gated sodium channels [6] which may be direct or act by disrupting the membrane environment of the Corresponding author: Turner, A.J. ([email protected])
channels. Valproate also appears to modulate NMDA receptor-mediated actions further enhancing neuronal inhibition [7] explaining its anti-epileptic activity. Nevertheless, what is undisputed is that valproate is a multi-faceted drug due to its diverse actions, exhaustively tested clinically, and which may have new potential as a therapeutic in some other central nervous system disorders ranging from schizophrenia (reviewed in [8]) to neurodegeneration. This is largely mediated by its more recently discovered activity as a broad acting, histone deacetylase (HDAC) inhibitor [9] modifying chromatin structure and neuronal gene expression. Indeed, the chronic antiepileptic effects of valproate may be due in part to HDAC-mediated regulation of the GABA synthetic enzyme, glutamate decarboxylase [10]. Our opinion is that valproate may specifically have efficacy in neurological disease, particularly through its newly discovered effects on amyloid precursor protein (APP) and amyloid metabolism [11,12]. We speculate that the development of more selective HDAC inhibitors could form a new class of therapeutics in neurodegenerative and psychiatric disorders. HDAC inhibitors and chromatin remodelling The dynamics of chromatin provides the major regulatory factor underlying gene expression and the major epigenetic modifications of chromatin involve the reversible acetylation and methylation of core histones on key lysine residues and DNA methylation at CpG islands. Both these modifications have been implicated in a range of neurodegenerative and neuropsychiatric disorders [8,13]. The reversible acetylation of histone lysine residues by histone acetyltransferases (HAT) is hence a key regulator of gene expression with acetylation leading to an open chromatin structure facilitating RNA polymerase binding and transcription. HDACs have multiple functions, in particular they are major components of repressive complexes and act to compact the chromatin and repress transcription (Figure 2). This has promoted an intensive search for inhibitors of HDACs as drugs to modulate gene transcription through their effects on the chromatin modifications [14]. Mammalian HDACs total 18 in number, are subdivided into four classes (I, IIa/b, III and IV) and are diverse mechanistically. Class III HDACs are also known as sirtuins and are NAD+-dependent enzymes which have been implicated in stress resistance and as mediators of longevity. The others are zinc-dependent enzymes. Valproate primarily inhibits Class I HDACs (HDAC 1, 2, 3 and 8), which are constitutively located in the nucleus [9].
0165-6147/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tips.2009.07.002
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Trends in Pharmacological Sciences Vol.30 No.10
Box 1. The metabolism of the amyloid precursor protein (APP) leading to formation of Alzheimer senile plaques
Figure 1. Chemical formulae of (a), the anticonvulsant and histone deacetylase (HDAC) inhibitor, valproic acid (VA); (b), the inhibitory neurotransmitter, gaminobutyric acid (GABA), and (c), the clinically approved HDAC inhibitor, vorinostat (suberoylanilide hydroxamic acid, SAHA).
Epigenetic changes in gene expression are particularly implicated in tumour growth [14] and HDAC inhibition can lead to re-expression of pro-apoptotic genes while inhibiting the cell cycle and cell division. Following the approval in 2006 of the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA; vorinostat) (Figure 1) by the U.S. Food and Drug Administration (FDA) in the treatment of cutaneous T cell lymphoma, a number of HDAC inhibitors are currently in phase II or III clinical trials for cancer treatments and have other therapeutic potential ranging from anti-infective agents to treatments for b-haemoglobinopathies [15]. Valproate itself has recently proved successful in combination therapy for thyroid carcinoma [16].
APP is a constitutive transmembrane protein which undergoes proteolytic cleavage by b- and g- secretases (shown as pale blue and red arrows in Scheme 1) producing a soluble ectodomain referred to as sAPPb, the neurotoxic amyloid b peptide (Ab) and the APP intracellular domain (AICD). Ab has the property to form neurotoxic oligomers and then to aggregate further, ultimately forming the socalled senile plaques, which are one of the pathological hallmarks of Alzheimer’s disease. This APP processing pathway is considered amyloidogenic in contrast with the cleavage of APP by a-secretase within the Ab domain, which prevents formation of Ab. This nonamyloidogenic processing leads to formation of the soluble ectodomain sAPPa, a short peptide p3 and AICD. AICD binds to the neprilysin (NEP) promoter and activates neprilysin expression (NEP). This binding requires AICD stabilisation by Fe65 and also involves the histone-acetyl transferase, Tip60. AICD also controls expression of some other genes. If the NEP gene is silenced through histone deacetylation by HDACs, expression of NEP is inhibited. However, incubation of cells with VA, and hence inhibition of HDACs, allows AICD to bind to the NEP promoter and re-activate NEP expression [12]. NEP, a cell-surface ectopeptidase, in turn, degrades Ab and reverses amyloid plaque accumulation. VA also inhibits g-secretase activity and hence Ab production [11].
APP metabolism and Alzheimer’s disease (AD) AD is characterized pathologically by the accumulation of extracellular amyloid plaques composed mainly of the amyloid-b peptide (Ab) and intracellular neurofibrillary tangles of the hyperphosphorylated form of the tau protein. Ab is primarily a 40-42 amino acid peptide that is generated from the transmembrane APP through successive proteolysis by two membrane-bound aspartic proteinases: b- and g-secretases [17]. Alternatively, APP can be metabolised through a non-amyloidogenic pathway mediated by the metalloproteinase a-secretases [18] (Box 1 and Scheme 1). Traditionally, the insidious accumulation of Ab has been regarded as an irreversible event. However,
Scheme 1. Schematic representation of the proteolytic processing of the Alzheimer’s amyloid precursor protein (APP) ultimately to form oligomers and then the senile plaques (shown as brown spots) as well as AICD, regulation by AICD of NEP gene expression and degradation of Ab peptide by neprilysin.
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Figure 2. Epigenetic modifications of genes at the chromatin level. DNA methylation and histone acetylation and methylation are involved in gene activation and silencing. Acetylation of histone tails by histone acetyl transferases (HAT) leads to opening of the nucleosome chain which allows transcription factors (TF) to bind gene promoter regions on DNA and activate gene expression. In contrast, DNA methylation by DNA methylases causes gene silencing. Histone deacetylases (HDACs) remove histone acetyl groups while valproic acid (VA), and other HDAC inhibitors, inhibit this process allowing histone acetylation and gene activation.
Ab probably has a normal physiological role as a regulatory peptide [19] and its steady state concentration is tightly regulated by perivascular mechanisms [20] as well as by proteolytic cleavage [21]. Ever since the proposal of the amyloid cascade hypothesis of AD [22], inhibition of Ab formation has been seen as a primary therapeutic target. However, while much AD research has focused on severe, familial forms of the human disease, or equivalent animal models, the much more prevalent, late-onset forms of the disease may primarily be due to deficiencies in Ab clearance rather than its formation [21]. Consecutive processing of APP by b- and gsecretases generates Ab, as well as an APP IntraCellular Domain (AICD) (Box 1 and Scheme 1), which appears to serve as a transcriptional activator regulating expression of a small group of neuronal genes. Among these is the amyloid-degrading enzyme, neprilysin (NEP) [23], which is a zinc metallopeptidase and ectoenzyme that also serves to inactivate synaptic neuropeptides, e.g. enkephalins and tachykinins, as well as Ab [21]. Other genes associated with neurodegeneration and reported to be regulated by AICD include glycogen synthase kinase-3b [24], the epidermal growth factor receptor (EGFR) [25] and the tumour suppressor protein, p53 [26] (and hence indirectly the prion protein [27], which causes the transmissible spongiform encephalopathies such as ‘‘mad cow disease’’). The exact mechanism of action of AICD remains unclear and is controversial but the consensus view is that it functions in combination with the APP-tail binding protein Fe65 which stabilises AICD against proteolytic degradation, and the chromatin-associated HAT, Tip60 [11,28]. Chromatin immunoprecipitation (ChIP) studies have established that AICD can directly bind to target gene promoters enhancing (NEP) [12] or negatively regulating EGFR promoter activity [25]. Hence, regulation of the amyloid-degrading enzyme NEP by AICD (Box 1) represents an elegant feedback mechanism controlling Ab levels as originally proposed [23]. AICD occupancy of the NEP promoter is correlated with enhanced promoter-
associated histone acetylation and HDAC inhibitors such as valproate, or trichostatin A, can enhance transcription of the NEP gene resulting in increased enzyme activity and amyloid-degrading potential [12]. Further emphasizing the potential of valproate, it has recently been demonstrated that a one month treatment with the drug could inhibit formation of the neuritic Ab plaques in transgenic AD model mice [11]. This effect appeared to be due, at least in part, to an indirect inhibition of the g-secretase pathway which generates Ab. Consequently, Ab42 levels were decreased to one-third of control levels. Hence, both groups independently proposed that valproate could provide a new therapeutic option in AD [11,12]. HDAC inhibitors, amyloid metabolism and learning and memory (Box 2) A general alteration of chromatin structure could provide the backdrop for the changes in gene expression seen during the aging process and in neurodegeneration. Such chromatin remodelling, specifically through increased histone acetylation is associated with the recovery of learning and memory in a transgenic mouse model and the remodelling can be induced by exposing the mice to enriched housing conditions (environmental enrichment), which specifically increased acetylation and methylation of histones H3 and H4 in hippocampus and cortex [29]. Consistent with these observations, injection of the HDAC inhibitors sodium butyrate or trichostatin A increased learning capacity in wild-type mice and reversed learning deficits in transgenic mice showing synaptic loss [29]. These effects were subsequently attributed to HDAC2 as a negative regulator of memory formation rather than HDAC1 through the use of mice over-expressing or lacking these enzymes [30]. The related HDAC inhibitor, phenylbutyrate, was independently shown to reverse spatial learning and memory deficits in an AD mouse model suggesting that this drug may have some efficacy in AD [31]. In an entirely separate study, environmental enrichment has also been shown to up-regulate expression of the 511
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amyloid-degrading enzyme, NEP, and to reduce amyloid deposition in transgenic mice, although this was not directly correlated with any specific chromatin changes [32]. In the same study that demonstrated an effect of valproate in vivo on reduction of amyloid plaques in rodent brain [11], it was also shown that the chronic valproate treatment improved memory and behavioural deficits in these mice as assessed by the Morris water maze test. The effects on chromatin modification and amyloid-degrading activity were not, however, examined in these studies although such treatment should enhance brain histone acetylation levels and neuronal gene expression. If HDAC2 is indeed a key player in inhibiting memory formation and synaptic plasticity [30], then the additional ability of valproate to induce the selective proteasomal degradation of HDAC2 [33] may enhance even further its efficacy. In summary, the combined effects of valproate in reducing Ab formation and enhancing Ab clearance should be synergistic in contributing to behavioural improvements, although whether this translates to the human situation needs exploring in detail. Although a consistent body of information is beginning to accumulate in relation to HDAC inhibitors and neurodegeneration, there are, however, potential pitfalls in chronic treatment with such compounds.
huntingtin protein was known to cause a dysregulation of transcription. Subsequently, an HDAC inhibitor was shown to have efficacy in a Huntington’s disease mouse model both in ameliorating the disease phenotype and reversing the transcriptional abnormalities [34]. Friedreich’s ataxia, another triplet expansion neurodegenerative disorder (of the gene frataxin), is also accompanied by specific gene silencing and reduced histone acetylation, which can be reversed by similar HDAC inhibitors. In a mouse model of the disease such compounds were able to normalize frataxin levels and the associated transcriptional down-regulation [35]. The neuroprotective role of HDAC inhibitors in neurodegenerative diseases appears to relate to conditions where oxidative stress, inflammatory mechanisms or neuronal apoptosis may be involved. For example, valproate, as well as other HDAC inhibitors appear to attenuate the dopaminergic neurotoxicity which is induced by lipopolysaccharide, in part through inducing apoptosis of activated microglia [36] suggesting their potential in the treatment of Parkinson’s disease. Furthermore, valproate and other HDAC inhibitors can protect dopaminergic neurons through upregulating the transcription of astrocyte neurotrophic factors (GDNF, BDNF) by astrocytes [37] and phenylbutyrate appears to provide protection in a rodent model of Parkinson’s disease [38]. The potential of HDAC inhibitors in the treatment of demyelinating disorders is more debatable but the underlying rationale for use of HDAC inhibitors as both neuroprotectants and anti-inflammatories in such conditions has been outlined [39]. The remyelination programme, which declines with aging, is clearly under epigenetic control and a recent study has shown, using the cuprizone model of demyelination, that elevated levels of acetylated histones can impede the repair process of remyelination [40]. Nevertheless, some studies have shown a beneficial effect of HDAC inhibitors in the mouse model of multiple sclerosis, experimental allergic encephalomyelitis, which is ascribed to increased expression of protective genes and apoptosis of inflammatory cells [41]. Motor neuron diseases may also be responsive to the application of HDAC inhibitors, which have given critical insight into underlying disease mechanisms, and clinical trials of such compounds in amyotrophic lateral sclerosis and spinal muscular atrophy are currently in progress [42]. Even in stroke, where postinsult treatment is currently limited to tissue plasminogen activator to enhance recovery, HDAC inhibitors have considerable potential and certainly seem to improve clinical outcome in ischaemic models [43]. A more comprehensive survey of other potential applications of HDAC inhibitors in CNS disorders is to be found in [44].
Valproate and other neurodegenerative conditions Intuitively, given their proapoptotic effects, HDAC inhibitors might not seem likely to be efficacious in neurodegenerative disorders and perhaps even exacerbate the conditions; anti-cancer drugs are not obvious candidates for preventing neuronal cell death. However, evidence is accumulating that there may well be benefits of these compounds, valproate included, in a number of quite distinct neurological conditions. The first example to be reported was in Huntington’s disease where the mutant
Future prospects Pre-clinical and clinical trials of HDAC inhibitors in neurodegenerative diseases are in various stages of progress. To date there is no substantive data regarding anticonvulsant drugs and AD and those trials that have taken place have been small scale focusing on late stage disease and behavioural changes such as agitation and aggression (reviewed in [45]). A phase 3 two year clinical trial of low dose (10 mg/kg/d) valproate in 300 AD patients (the ‘‘valproate in dementia’’ or VALID study: http://
Box 2. Mechanisms of learning and memory The neurological basis of learning and memory is considered a dynamic process at the molecular, cellular, and brain network levels which involves changes in the properties of neuronal cells and their functions in response to various stimuli (both external and internal). These changes may occur via rapid post-translational modification of the existing proteins (short-term memory) or via alterations of gene expression (memory consolidation). Modification of the properties of the neuronal cells subsequently leads to changes in their interactions with other cells in the neuronal networks hence encoding an experience of this particular stimulus. Although these changes are usually sufficient for short-term memory, the lasting memory or establishment of a new reflex requires repetition of this network activation. In this reinforcement process cells use the same molecular mechanisms which were activated by the original stimuli including alterations in gene expression. Since activation of gene expression requires modification of chromatin structure and inhibition of histone deacetylases, application of HDAC inhibitors can change the reaction of cells to various stimuli and as such facilitate memory formation and reinforcement. Ab (and in particular its soluble oligomers) can disrupt synaptic plasticity and memory at extremely low concentrations via various signalling mechanisms, including stress-activated kinases and oxidative/nitrosative stress mediators. Learning difficulties and memory loss, particularly of recent memories, are early signs of dementias such as Alzheimer’s disease. Evaluating potential therapeutics in animal models of the disease (e.g. transgenic rodent models) usually involves testing for recovery of behavioural and cognitive deficits using standard tests such as the Morris water maze.
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Opinion www.adcs.org/Studies/VN.aspx) has not reported any efficacy although its primary aim was to assess the efficacy of valproate on agitation and other early personality changes in dementia but secondarily on whether valproate slows disease progression in AD and improves memory. Whatever the outcome of such trials it seems there is still much potential in the well established drug valproate, particularly but not exclusively through its HDAC inhibitory actions and a detailed exploration of its effects on ADrelated genes would be valuable. The multiplicity of actions of valproate on neuronal systems (neurotransmitters, receptors, ion channels, chromatin remodelling) does make interpetation of data on its actions complex. Furthermore, non-specific HDAC inhibitors like valproate can modify some 2% of genes, potentially causing both beneficial and harmful effects limiting potential efficacy. The next generation of HDAC inhibitors, selective for individual sub-classes and isoforms of the deacetylases, may prove to be even more effective not only in neurology and psychiatry but also in other major classes of disease. Valproate has the distinction, however, of having paved the way for drug development in some of the major human neurological diseases. Acknowledgements We thank the Medical Research Council of Great Britain and Russian Academy of Sciences Programme ‘‘Fundamental Sciences to Medicine’’ for financial support.
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