Memantine Upregulates BDNF and Prevents Dopamine Deficits in SIV-Infected Macaques: a Novel Pharmacological Action of Memantine
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Neuropsychopharmacology (2008) 33, 2228–2236 & 2008 Nature Publishing Group All rights reserved 0893-133X/08 $30.00 www.neuropsychopharmacology.org Memantine Upregulates BDNF and Prevents Dopamine Deficits in SIV-Infected Macaques: A Novel Pharmacological Action of Memantine 1 1 2 3 4 5 Falko Meisner , Carsten Scheller , Susanne Kneitz , Sieghart Sopper , Eva Neuen-Jacob , Peter Riederer , 1 ,1,5 Volker ter Meulen and Eleni Koutsilieri* , for the German Competence Network HIV/AIDS 1 2 Institute of Virology and Immunobiology, University of Wurzburg, Germany; IZKF (Interdisciplinary Center for Clinical Research), University of 3 4 5 Wurzburg, Germany; German Primate Center, Go¨ttingen, Germany; Institute of Neuropathology, University of Duesseldorf, Germany; Clinical Neurochemistry (National Parkinson Foundation Center of Excellence Research Laboratory), Psychiatry and Psychotherapy, University of Wurzburg, Germany N-methyl-D-aspartate (NMDA) receptor activation is involved in the pathogenetic cascades of neurodegenerative disorders including human immunodeficiency virus (HIV) dementia. Memantine, an uncompetitive NMDA receptor antagonist, which has been recently approved for the treatment of Alzheimer’s disease, is being discussed as a potential adjunctive therapeutic substance for HIV dementia. We used simian immunodeficiency virus-infected rhesus macaques to assess the effects of memantine on brain dysfunction and brain pathology within 3–5 months after initial infection during early asymptomatic stage of disease. We had shown previously that within this time frame, marked changes were evident in the dopaminergic systems. Memantine was administered two weeks post infection, at peak viremia, in order to prevent early NMDA receptor activation due to immune mediators. We found that memantine prevented onset of dopamine deficits in the brains of SIV-infected macaques, without affecting early brain pathology or peripheral course of infection. Memantine specifically upregulated mRNA and protein expression of the neurotrophic factor brain-derived neurotrophic factor (BDNF), suggesting that the protective effect of memantine on dopamine function may be mechanistically remote from NMDA receptor antagonism. This novel pharmacological action of memantine may also be relevant for other neurodegenerative disorders and supports the involvement of neurotrophic factors in adult brain neuroprotection. Neuropsychopharmacology (2008) 33, 2228–2236; doi:10.1038/sj.npp.1301615; published online 31 October 2007 Keywords: memantine; dopamine; BDNF; dementia; HIV; SIV INTRODUCTION the evidence that neurons may die due to overactivation of NMDA receptors in HIV dementia comes from in vitro Infection of the central nervous system (CNS) by human studies on neurotoxic actions of HIV proteins (Mattson immunodeficiency virus (HIV) type-1 results in neuro- et al, 2005) and indirectly from observations of increased psychiatric disorders collectively known as HIV dementia. levels of products with NMDA-agonistic potential in CSF of Although the highly active antiretroviral therapy reduced HIV-infected patients (Heyes et al, 2001) and simian both the incidence and severity of HIV dementia (McArthur, immunodeficiency virus (SIV)-infected rhesus macaques 2004), the prevalence increased with the patients’ life span (Koutsilieri et al, 1999). Additionally, glutamate is increased getting longer and neuropsychological function did not in brains of asymptomatic SIV-infected animals without improve in all treated patients (Cysique et al, 2006). being accompanied by neuronal dysfunction (Bossuet et al, It is currently accepted that macrophages/microglia play a 2004), indicating intact compensatory mechanisms that key role in HIV-neurotoxicity through production of viral limit the glutamate-induced neurotoxic cascades during neurotoxins and/or immune activation with subsequent asymptomatic stage of disease. However, the combined release of neurotoxic factors acting at the N-methyl- mechanisms of increased pools of glutamate with dysregu- D-aspartate (NMDA) receptors (Kaul et al, 2001). Most of lation of NMDA receptor function due to immune activa- tion can result in neuronal calcium overload and cellular *Correspondence: PD Dr Eleni Koutsilieri, Clinical Neurochemistry, dysfunction and death (Mattson et al, 2005). It is therefore Department of Psychiatry and Psychotherapy, University of Wurzburg, Fu¨chsleinstr. 15, Wurzburg 97080, Germany, Tel: + + 49 931 crucial to stop neurochemical alterations before accumula- 20149928, Fax: + + 49 931 20149553, tion of toxic products and inflammation are widespread, E-mail: [email protected] namely, in early asymptomatic phase of infection. Received 26 June 2007; revised 18 September 2007; accepted 1 The NMDA antagonist memantine is an uncompetitive October 2007 NMDA receptor antagonist with improved voltage dependency, Pharmacological action of memantine F Meisner et al 2229 kinetics, and affinity compared to endogenous Mg2+ different time points after infection ranging from 11.9–20.1 (Danysz and Parsons, 2003) and is currently being used wpi. Brains were thoroughly perfused with 2 l of RPMI 1640 for treatment of senile dementias. Memantine has been medium (Gibco, Eggenstein, Germany) containing 3% fetal tested in a placebo-controlled study for the treatment of calf serum. The brains were quickly removed, partly sensory neuropathy associated with HIV without beneficial immersion-fixed (5% neutral buffered formaldehyde for outcome (Schifitto et al, 2006). However, memantine has light microscopy), and partly frozen at À701C for biochemi- been shown to exert protective effects on cell toxicity and cal analysis. Prior to neurochemical investigations, putamen synaptic functions. Memantine inhibits the HIV gp120- was dissected on a Teflon plate at À201C according to a evoked calcium changes in neurons and astrocytes and stereotactic atlas using standardized procedures. protects neurons from gp120-induced cell death (Lipton, 1992; Nath et al, 2000). Moreover, memantine is beneficial in transgenic mice constitutively expressing gp120 (Toggas BDNF Gene Expression et al, 1996) and it improves synaptic function in murine HIV encephalitis (Anderson et al, 2004), indicating that this Total RNA was extracted from putamen of rhesus macaques substance may be a good candidate for an adjunctive using the RNeasy Midi Kit (Qiagen GmbH, Hilden, therapy for HIV-infected humans. Germany). The samples were spectrophotometrically We found previously extensive impairment in the scanned from 220 to 320 nm; the A260/A280 of total RNA dopaminergic systems in brains of SIV-infected rhesus was typically 41.9. RNA quality was tested on the macaques during early infection (Jenuwein et al, 2004; RNAStdsenseChip using the Experion electrophoresis Scheller et al, 2005). In this study we hypothesized that early (BioRad, Germany). For all total RNAs extracted, 28S/18S dopamine deficits in brains of SIV-infected macaques may ratios were 41.5. Total RNAs were subjected to DNase-I be due to NMDA receptor activation due to inflammation (Qiagen GmbH) digestion to remove genomic DNA residues induced by invasion of virus and immune cells in the brain and subsequently cleaned with the RNeasy Midi Kit (Qiagen during initial peak brain viremia and we treated monkeys at GmbH). No significant differences between the spectral the peak viremia two weeks after infection with the NMDA purity, rate of degradation, molecular size, yield, or tissue antagonist memantine. Our central aim was to assess the pH between the groups were noted. protective potential of memantine on the observed early brain dysfunction. Gene Chip Arrays METHODS For preparation of labeled cRNA, the procedures described in detail in the Affymetrix Gene Chip Expression Analysis Animals Manual (Affymetrix Inc., Santa Clara, CA, USA) were followed. Confirmation of total RNA quality was conducted Juvenile rhesus monkeys (Macaca mulatta) of Chinese by hybridizing a small portion of labeled cRNA target to origin were housed individually in indoor facilities on a Affymetrix Test3 Array (Affymetrix 900341). Labeled cRNA 12/12 light/dark schedule at the German Primate Center was hybridized to GeneChip Rhesus Macaque Genome (Go¨ttingen, Germany). Dry food with fresh fruits (bananas Array (Affymetrix 900656) in an Affymetrix Fluidics Station and apples) as a dietary supplement was provided twice a 400. Hybridized arrays were stained with 10 mg/ml strepta- day and water was available ad lib. Prior to inoculation, vidin-R phycoerythrin (Molecular Probes, Eugene, OR, animals were demonstrated to be seronegative for STLV-1, USA), followed by staining with 3 mg/ml biotinylated goat SRV-1 (type D retrovirus), herpes B virus, and SIV. Eleven anti-streptavidin antibody (Sigma Chemical, St. Louis, MO, monkeys were infected under ketamine anesthesia (10 mg/ USA). Arrays were then stained once again with strepta- kg) with 100 MID50 (50% monkey infective dose) of vidin-R phycoerythrin and scanned using a GeneChip SIVmac251MPBMC. Four SIV-infected animals were treated Scanner 3000. The Gene Chip used contained several probe with memantine (10 mg/kg, per os) daily. The treatment sets specific for rhesus macaque house-keeping genes such started at 2 weeks post infection (wpi) and concurrent with as b-actin, elongation factor 1, and GAPDH which served as peak viremia. All infected animals were killed at predeter- positive controls. Several murine and yeast probe sets on mined time points without signs of simian