Increased CDK5 Expression in HIV Encephalitis Contributes to Neurodegeneration via Tau Phosphorylation and Is Reversed with Roscovitine
2011; Elsevier BV; Volume: 178; Issue: 4 Linguagem: Inglês
10.1016/j.ajpath.2010.12.033
ISSN1525-2191
AutoresChristina Patrick, Leslie Crews, Paula Desplats, Wilmar Dumaop, Edward Rockenstein, Cristian L. Achim, Ian Everall, Eliezer Masliah,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoRecent treatments with highly active antiretroviral therapy (HAART) regimens have been shown to improve general clinical status in patients with human immunodeficiency virus (HIV) infection; however, the prevalence of cognitive alterations and neurodegeneration has remained the same or has increased. These deficits are more pronounced in the subset of HIV patients with the inflammatory condition known as HIV encephalitis (HIVE). Activation of signaling pathways such as GSK3β and CDK5 has been implicated in the mechanisms of HIV neurotoxicity; however, the downstream mediators of these effects are unclear. The present study investigated the involvement of CDK5 and tau phosphorylation in the mechanisms of neurodegeneration in HIVE. In the frontal cortex of patients with HIVE, increased levels of CDK5 and p35 expression were associated with abnormal tau phosphorylation. Similarly, transgenic mice engineered to express the HIV protein gp120 exhibited increased brain levels of CDK5 and p35, alterations in tau phosphorylation, and dendritic degeneration. In contrast, genetic knockdown of CDK5 or treatment with the CDK5 inhibitor roscovitine improved behavioral performance in the water maze test and reduced neurodegeneration, abnormal tau phosphorylation, and astrogliosis in gp120 transgenic mice. These findings indicate that abnormal CDK5 activation contributes to the neurodegenerative process in HIVE via abnormal tau phosphorylation; thus, reducing CDK5 might ameliorate the cognitive impairments associated with HIVE. Recent treatments with highly active antiretroviral therapy (HAART) regimens have been shown to improve general clinical status in patients with human immunodeficiency virus (HIV) infection; however, the prevalence of cognitive alterations and neurodegeneration has remained the same or has increased. These deficits are more pronounced in the subset of HIV patients with the inflammatory condition known as HIV encephalitis (HIVE). Activation of signaling pathways such as GSK3β and CDK5 has been implicated in the mechanisms of HIV neurotoxicity; however, the downstream mediators of these effects are unclear. The present study investigated the involvement of CDK5 and tau phosphorylation in the mechanisms of neurodegeneration in HIVE. In the frontal cortex of patients with HIVE, increased levels of CDK5 and p35 expression were associated with abnormal tau phosphorylation. Similarly, transgenic mice engineered to express the HIV protein gp120 exhibited increased brain levels of CDK5 and p35, alterations in tau phosphorylation, and dendritic degeneration. In contrast, genetic knockdown of CDK5 or treatment with the CDK5 inhibitor roscovitine improved behavioral performance in the water maze test and reduced neurodegeneration, abnormal tau phosphorylation, and astrogliosis in gp120 transgenic mice. These findings indicate that abnormal CDK5 activation contributes to the neurodegenerative process in HIVE via abnormal tau phosphorylation; thus, reducing CDK5 might ameliorate the cognitive impairments associated with HIVE. The human immunodeficiency virus (HIV) enters the central nervous system (CNS) early in the progression of the disease, resulting in a spectrum of behavioral and motor alterations that range from mild cognitive deficits to dementia.1Gendelman H.E. Persidsky Y. Ghorpade A. Limoges J. Stins M. Fiala M. Morrisett R. The neuropathogenesis of the AIDS dementia complex.AIDS. 1997; 11: S35-S45PubMed Google Scholar, 2McArthur J. Neurological manifestations of AIDS.Medicine. 1987; 66: 407-437Crossref PubMed Scopus (370) Google Scholar, 3McArthur J.C. Haughey N. Gartner S. Conant K. Pardo C. Nath A. Sacktor N. Human immunodeficiency virus-associated dementia: an evolving disease.J Neurovirol. 2003; 9: 205-221Crossref PubMed Scopus (335) Google Scholar, 4González-Scarano F. Martín-García J. The neuropathogenesis of AIDS.Nat Rev Immunol. 2005; 5: 69-81Crossref PubMed Scopus (891) Google Scholar Despite the notable efficacy of highly active antiretroviral therapy (HAART) in lowering HIV levels in plasma, recent studies have shown that HIV can persist in the CNS for a long period of time.5Bell J.E. An update on the neuropathology of HIV in the HAART era.Histopathology. 2004; 45: 549-559Crossref PubMed Scopus (109) Google Scholar, 6Gray F. Chrétien F. Vallat-Decouvelaere A.V. Scaravilli F. The changing pattern of HIV neuropathology in the HAART era.J Neuropathol Exp Neurol. 2003; 62: 429-440PubMed Google Scholar Chronic presence of HIV in the brain is associated with neurodegenerative changes that contribute to cognitive alterations.4González-Scarano F. Martín-García J. The neuropathogenesis of AIDS.Nat Rev Immunol. 2005; 5: 69-81Crossref PubMed Scopus (891) Google Scholar, 7Crews L. Patrick C. Achim C.L. Everall I.P. Masliah E. Molecular pathology of neuro-AIDS (CNS-HIV).Int J Mol Sci. 2009; 10: 1045-1063Crossref PubMed Scopus (60) Google Scholar, 8Everall I.P. Hansen L.A. Masliah E. The shifting patterns of HIV encephalitis neuropathology.Neurotox Res. 2005; 8: 51-61Crossref PubMed Scopus (99) Google Scholar, 9Lawrence D.M. Major E.O. HIV-1 and the brain: connections between HIV-1-associated dementia, neuropathology and neuroimmunology.Microbes Infect. 2002; 4: 301-308Crossref PubMed Scopus (81) Google Scholar, 10Diesing T.S. Swindells S. Gelbard H. Gendelman H.E. HIV-1-associated dementia: a basic science and clinical perspective.AIDS Read. 2002; 12: 358-368PubMed Google Scholar Neuronal damage resulting from the neuroinflammatory process associated with HIV has been shown to lead to cognitive impairment.11Masliah E. Heaton R.K. Marcotte T.D. Ellis R.J. Wiley C.A. Mallory M. Achim C.L. McCutchan J.A. Nelson J.A. Atkinson J.H. Grant I. Dendritic injury is a pathological substrate for human immunodeficiency virus-related cognitive disorders HNRC Group The HIV Neurobehavioral Research Center.Ann Neurol. 1997; 42: 963-972Crossref PubMed Scopus (424) Google Scholar, 12Masliah E. Ge N. Achim C.L. DeTeresa R. Wiley C.A. Patterns of neurodegeneration in HIV encephalitis.J NeuroAIDS. 1996; 1: 161-173PubMed Google Scholar, 13Masliah E. Ge N. Mucke L. Pathogenesis of HIV-1 associated neurodegeneration.Crit Rev Neurobiol. 1996; 10: 57-67Crossref PubMed Scopus (103) Google Scholar, 14Wiley C.A. Achim C.L. Human immunodeficiency virus encephalitis and dementia.Ann Neurol. 1995; 38: 559-560Crossref PubMed Scopus (22) Google Scholar Increased life span of HIV patients and an increase in resistant strains of HIV has drawn attention to the prevalence of cognitive alterations in this population,8Everall I.P. Hansen L.A. Masliah E. The shifting patterns of HIV encephalitis neuropathology.Neurotox Res. 2005; 8: 51-61Crossref PubMed Scopus (99) Google Scholar, 15Wendel K.A. McArthur J.C. Acute meningoencephalitis in chronic human immunodeficiency virus (HIV) infection: putative central nervous system escape of HIV replication.Clin Infect Dis. 2003; 37: 1107-1111Crossref PubMed Scopus (44) Google Scholar and these deficits represent an important morbidity factor in HIV.3McArthur J.C. Haughey N. Gartner S. Conant K. Pardo C. Nath A. Sacktor N. Human immunodeficiency virus-associated dementia: an evolving disease.J Neurovirol. 2003; 9: 205-221Crossref PubMed Scopus (335) Google Scholar, 6Gray F. Chrétien F. Vallat-Decouvelaere A.V. Scaravilli F. The changing pattern of HIV neuropathology in the HAART era.J Neuropathol Exp Neurol. 2003; 62: 429-440PubMed Google Scholar, 16Sacktor N. McDermott M.P. Marder K. Schifitto G. Selnes O.A. McArthur J.C. Stern Y. Albert S. Palumbo D. Kieburtz K. De Marcaida J.A. Cohen B. Epstein L. HIV-associated cognitive impairment before and after the advent of combination therapy.J Neurovirol. 2002; 8: 136-142Crossref PubMed Scopus (472) Google Scholar, 17Maschke M. Kastrup O. Esser S. Ross B. Hengge U. Hufnagel A. Incidence and prevalence of neurological disorders associated with HIV since the introduction of highly active antiretroviral therapy (HAART).J Neurol Neurosurg Psychiatry. 2000; 69: 376-380Crossref PubMed Scopus (248) Google Scholar, 18Grant I. Heaton R. Atkinson J. Group H. Neurocognitive disorder in HIV-1 infection. HIV and dementia: proceedings of the NIMH-sponsored conference “Pathogenesis of HIV infection of the brain: impact on function and behavior”.in: Oldstone M.B.A. Vitkovic L. Springer-Verlag, Heidelberg1995: 9-30Google Scholar HIV encephalitis (HIVE) is an inflammatory condition characterized by the presence of HIV-infected macrophages and microglial cells, astrogliosis, white matter damage, and neurodegeneration characterized by dendritic and synaptic damage in the neocortex and hippocampus.11Masliah E. Heaton R.K. Marcotte T.D. Ellis R.J. Wiley C.A. Mallory M. Achim C.L. McCutchan J.A. Nelson J.A. Atkinson J.H. Grant I. Dendritic injury is a pathological substrate for human immunodeficiency virus-related cognitive disorders HNRC Group The HIV Neurobehavioral Research Center.Ann Neurol. 1997; 42: 963-972Crossref PubMed Scopus (424) Google Scholar Viral proteins and cytokines produced by macrophages and microglia have been shown to induce neuronal dysfunction, neuron damage, and loss of nerve cells.19Nath A. Human immunodeficiency virus (HIV) proteins in neuropathogenesis of HIV dementia.J Infect Dis. 2002; 186: S193-S198Crossref PubMed Scopus (279) Google Scholar, 20Kaul M. Lipton S.A. Chemokines and activated macrophages in HIV gp120-induced neuronal apoptosis.Proc Natl Acad Sci USA. 1999; 96: 8212-8216Crossref PubMed Scopus (457) Google Scholar, 21Meucci O. Fatatis A. Simen A.A. Bushell T.J. Gray P.W. Miller R.J. Chemokines regulate hippocampal neuronal signaling and gp120 neurotoxicity.Proc Natl Acad Sci USA. 1998; 95: 14500-14505Crossref PubMed Scopus (614) Google Scholar, 22Sanders V.J. Pittman C.A. White M.G. Wang G. Wiley C.A. Achim C.L. Chemokines and receptors in HIV encephalitis.AIDS. 1998; 12: 1021-1026Crossref PubMed Scopus (225) Google Scholar, 23Giulian D. Vaca K. Noonan C. Secretion of neurotoxins by mononuclear phagocytes infected with HIV-1.Science. 1990; 250: 1593-1596Crossref PubMed Scopus (566) Google Scholar, 24Pulliam L. Zhou M. Stubblebine M. Bitler C.M. Differential modulation of cell death proteins in human brain cells by tumor necrosis factor alpha and platelet activating factor.J Neurosci Res. 1998; 54: 530-538Crossref PubMed Scopus (42) Google Scholar, 25Pulliam L. Clarke J.A. McGuire D. McGrath M.S. Investigation of HIV-infected macrophage neurotoxin production from patients with AIDS dementia.Adv Neuroimmunol. 1994; 4: 195-198Abstract Full Text PDF PubMed Scopus (27) Google Scholar, 26Wang Z. Trillo-Pazos G. Kim S.Y. Canki M. Morgello S. Sharer L.R. Gelbard H.A. Su Z.Z. Kang D.C. Brooks A.I. Fisher P.B. Volsky D.J. Effects of human immunodeficiency virus type 1 on astrocyte gene expression and function: potential role in neuropathogenesis.J Neurovirol. 2004; 10: 25-32Crossref PubMed Scopus (113) Google Scholar, 27Brandimarti R. Khan M.Z. Fatatis A. Meucci O. Regulation of cell cycle proteins by chemokine receptors: a novel pathway in human immunodeficiency virus neuropathogenesis?.J Neurovirol. 2004; 10: 108-112Crossref PubMed Scopus (22) Google Scholar, 28Martín-García J. Kolson D.L. González-Scarano F. Chemokine receptors in the brain: their role in HIV infection and pathogenesis.AIDS. 2002; 16: 1709-1730Crossref PubMed Scopus (42) Google Scholar HIV infection in the CNS has also been shown to promote neuronal calcium dysregulation, mitochondrial damage, oxidative stress,29Ozdener H. Molecular mechanisms of HIV-1 associated neurodegeneration.J Biosci. 2005; 30: 391-405Crossref PubMed Scopus (55) Google Scholar, 30Lipton S. HIV-related neuronal injury Potential therapeutic intervention with calcium channel antagonists and NMDA antagonists.Mol Neurobiol. 1994; 8: 181-196Crossref PubMed Scopus (95) Google Scholar and caspase 3-dependent apoptosis.31Garden G.A. Budd S.L. Tsai E. Hanson L. Kaul M. D'Emilia D.M. Friedlander R.M. Yuan J. Masliah E. Lipton S.A. Caspase cascades in human immunodeficiency virus-associated neurodegeneration.J Neurosci. 2002; 22: 4015-4024PubMed Google Scholar In addition, activation of calcium-dependent signaling pathways might also contribute to neurodegeneration. Among these, activation of mitogen-activated protein kinase 10 (MAPK10; also known as c-Jun N-terminal kinase 3, or JNK3), double-stranded RNA-activated protein kinase (PKR),32Alirezaei M. Watry D.D. Flynn C.F. Kiosses W.B. Masliah E. Williams B.R. Kaul M. Lipton S.A. Fox H.S. Human immunodeficiency virus-1/surface glycoprotein 120 induces apoptosis through RNA-activated protein kinase signaling in neurons.J Neurosci. 2007; 27: 11047-11055Crossref PubMed Scopus (56) Google Scholar glycogen synthase kinase-3β (GSK3β),7Crews L. Patrick C. Achim C.L. Everall I.P. Masliah E. Molecular pathology of neuro-AIDS (CNS-HIV).Int J Mol Sci. 2009; 10: 1045-1063Crossref PubMed Scopus (60) Google Scholar, 33Hashimoto M. Sagara Y. Everall I.P. Mallory M. Everson A. Langford D. Masliah E. Fibroblast growth factor 1 regulates signaling via the glycogen synthase kinase-3beta pathway Implications for neuroprotection.J Biol Chem. 2002; 277: 32985-32991Crossref PubMed Scopus (111) Google Scholar, 34Maggirwar S.B. Tong N. Ramirez S. Gelbard H.A. Dewhurst S. HIV-1 Tat-mediated activation of glycogen synthase kinase-3beta contributes to Tat-mediated neurotoxicity.J Neurochem. 1999; 73: 578-586Crossref PubMed Scopus (149) Google Scholar, 35Dewhurst S. Maggirwar S.B. Schifitto G. Gendelman H.E. Gelbard H.A. Glycogen synthase kinase 3 beta (GSK-3 beta) as a therapeutic target in neuroAIDS.J Neuroimmune Pharmacol. 2007; 2: 93-96Crossref PubMed Scopus (38) Google Scholar and cyclin-dependent kinase 5 (CDK5)7Crews L. Patrick C. Achim C.L. Everall I.P. Masliah E. Molecular pathology of neuro-AIDS (CNS-HIV).Int J Mol Sci. 2009; 10: 1045-1063Crossref PubMed Scopus (60) Google Scholar, 36Wang Y. White M.G. Akay C. Chodroff R.A. Robinson J. Lindl K.A. Dichter M.A. Qian Y. Mao Z. Kolson D.L. Jordan-Sciutto K.L. Activation of cyclin-dependent kinase 5 by calpains contributes to human immunodeficiency virus-induced neurotoxicity.J Neurochem. 2007; 103: 439-455Crossref PubMed Scopus (48) Google Scholar have been shown to play a role. Recent studies have shown that the abnormal activation of CDK5 might play a role in Alzheimer's disease (AD),37Baumann K. Mandelkow E.M. Biernat J. Piwnica-Worms H. Mandelkow E. Abnormal Alzheimer-like phosphorylation of tau-protein by cyclin-dependent kinases cdk2 and cdk5.FEBS Lett. 1993; 336: 417-424Abstract Full Text PDF PubMed Scopus (419) Google Scholar, 38Lee M.S. Kwon Y.T. Li M. Peng J. Friedlander R.M. Tsai L.H. Neurotoxicity induces cleavage of p35 to p25 by calpain.Nature. 2000; 405: 360-364Crossref PubMed Scopus (904) Google Scholar Parkinson's disease39Qu D. Rashidian J. Mount M.P. Aleyasin H. Parsanejad M. Lira A. Haque E. Zhang Y. Callaghan S. Daigle M. Rousseaux M.W. Slack R.S. Albert P.R. Vincent I. Woulfe J.M. Park D.S. Role of Cdk5-mediated phosphorylation of Prx2 in MPTP toxicity and Parkinson's disease.Neuron. 2007; 55: 37-52Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar and Huntington's disease.40Anne S.L. Saudou F. Humbert S. Phosphorylation of huntingtin by cyclin-dependent kinase 5 is induced by DNA damage and regulates wild-type and mutant huntingtin toxicity in neurons.J Neurosci. 2007; 27: 7318-7328Crossref PubMed Scopus (106) Google Scholar Under physiological conditions, CDK5 is activated by the neuron-specific proteins p35/p39; under pathological conditions, elevated intracellular calcium activates calpain-1, which in turns abnormally cleaves p35 into p25.38Lee M.S. Kwon Y.T. Li M. Peng J. Friedlander R.M. Tsai L.H. Neurotoxicity induces cleavage of p35 to p25 by calpain.Nature. 2000; 405: 360-364Crossref PubMed Scopus (904) Google Scholar The p25 fragment is more stable than p35 and constitutively activates CDK5,38Lee M.S. Kwon Y.T. Li M. Peng J. Friedlander R.M. Tsai L.H. Neurotoxicity induces cleavage of p35 to p25 by calpain.Nature. 2000; 405: 360-364Crossref PubMed Scopus (904) Google Scholar resulting in aberrant phosphorylation of neuronal substrates41Ahlijanian M.K. Barrezueta N.X. Williams R.D. Jakowski A. Kowsz K.P. McCarthy S. Coskran T. Carlo A. Seymour P.A. Burkhardt J.E. Nelson R.B. McNeish J.D. Hyperphosphorylated tau and neurofilament and cytoskeletal disruptions in mice overexpressing human p25, an activator of cdk5.Proc Natl Acad Sci USA. 2000; 97: 2910-2915Crossref PubMed Scopus (305) Google Scholar and in neuronal cell death. In patients with AD, the constitutively active p25/CDK5 complex results in the hyperphosphorylation of the microtubule-associated protein tau, which is abundantly present in the neurofibrillary tangles and may contribute to neuronal cell death.37Baumann K. Mandelkow E.M. Biernat J. Piwnica-Worms H. Mandelkow E. Abnormal Alzheimer-like phosphorylation of tau-protein by cyclin-dependent kinases cdk2 and cdk5.FEBS Lett. 1993; 336: 417-424Abstract Full Text PDF PubMed Scopus (419) Google Scholar, 38Lee M.S. Kwon Y.T. Li M. Peng J. Friedlander R.M. Tsai L.H. Neurotoxicity induces cleavage of p35 to p25 by calpain.Nature. 2000; 405: 360-364Crossref PubMed Scopus (904) Google Scholar, 41Ahlijanian M.K. Barrezueta N.X. Williams R.D. Jakowski A. Kowsz K.P. McCarthy S. Coskran T. Carlo A. Seymour P.A. Burkhardt J.E. Nelson R.B. McNeish J.D. Hyperphosphorylated tau and neurofilament and cytoskeletal disruptions in mice overexpressing human p25, an activator of cdk5.Proc Natl Acad Sci USA. 2000; 97: 2910-2915Crossref PubMed Scopus (305) Google Scholar Recent studies suggest that CDK5 might also play a role in the mechanisms of neurotoxicity in patients with HIV-associated cognitive alterations. For example, in a gene array study, we found that expression levels of CDK5 and related family members are dysregulated in the frontal cortex of patients with HIVE.42Masliah E. Roberts E.S. Langford D. Everall I. Crews L. Adame A. Rockenstein E. Fox H.S. Patterns of gene dysregulation in the frontal cortex of patients with HIV encephalitis [Erratum appeared in J Neuroimmunol 2005, 162:197].J Neuroimmunol. 2004; 157: 163-175Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar Moreover, a recent study showed that in the brains of patients with HIV, calpain activity and the subsequent calpain-mediated generation of p25 from p35 were increased, leading to activation of the CDK5 pathway; however, the downstream targets involved in mediating the neurotoxic effects of HIV via abnormal CDK5 activation remain unclear.36Wang Y. White M.G. Akay C. Chodroff R.A. Robinson J. Lindl K.A. Dichter M.A. Qian Y. Mao Z. Kolson D.L. Jordan-Sciutto K.L. Activation of cyclin-dependent kinase 5 by calpains contributes to human immunodeficiency virus-induced neurotoxicity.J Neurochem. 2007; 103: 439-455Crossref PubMed Scopus (48) Google Scholar Thus, given the role of tau in CDK5-mediated neurotoxicity in AD, this protein is an important candidate to consider in the pathogenesis of HIVE.37Baumann K. Mandelkow E.M. Biernat J. Piwnica-Worms H. Mandelkow E. Abnormal Alzheimer-like phosphorylation of tau-protein by cyclin-dependent kinases cdk2 and cdk5.FEBS Lett. 1993; 336: 417-424Abstract Full Text PDF PubMed Scopus (419) Google Scholar, 38Lee M.S. Kwon Y.T. Li M. Peng J. Friedlander R.M. Tsai L.H. Neurotoxicity induces cleavage of p35 to p25 by calpain.Nature. 2000; 405: 360-364Crossref PubMed Scopus (904) Google Scholar The primary objective of our study was to investigate in vivo the involvement of abnormal CDK5 activation and tau phosphorylation in the mechanisms of neurodegeneration in human cases of HIVE and in gp120 transgenic (Tg) mice. We found that abnormal activation of CDK5 resulted in aberrant tau phosphorylation, which in turn might contribute to dendritic damage and neurodegeneration. In contrast, partial genetic ablation of CDK5, or pharmacological manipulation with the CDK5 inhibitor roscovitine, reversed the tau pathology and neurodegenerative phenotype in the gp120 Tg mice. For the present study, HIV-positive cases with and without encephalitis were selected from a cohort of 43 HIV-positive cases from the HIV Neurobehavioral Research Center and the California NeuroAIDS Tissue Network at the University of California, San Diego (UCSD). Subjects were excluded if they had a history of CNS opportunistic infections or non-HIV-related developmental, neurological, psychiatric, or metabolic conditions that might affect CNS functioning (eg, loss of consciousness exceeding 30 minutes, psychosis, substance dependence). For inclusion in the present study, a total of 16 age-matched cases were identified with and without encephalitis (n = 8 per group), and without other complications (Table 1). All cases had neuromedical and neuropsychological examinations within a median of 12 months before death. Most patients died as a result of acute bronchopneumonia or septicemia, and autopsy was performed within 24 hours of death. Autopsy findings were consistent with AIDS, and the associated pathology was most frequently due to systemic CMV, Kaposi sarcoma, and liver disease. In all cases, neuropathological assessment was performed in paraffin sections from the frontal, parietal, and temporal cortices, and the hippocampus, basal ganglia, and brainstem stained with H&E, or immunolabeled with antibodies against p24 and glial fibrillary acidic protein (GFAP, a marker of astrogliosis).43Achim C.L. Heyes M.P. Wiley C.A. Quantitation of human immunodeficiency virus, immune activation factors, and quinolinic acid in AIDS brains.J Clin Invest. 1993; 91: 2769-2775Crossref PubMed Scopus (182) Google Scholar, 44Masliah E. Achim C. Ge N. DeTeresa R. Terry R. Wiley C. Spectrum of human immunodeficiency virus-associated neocortical damage.Ann Neurol. 1992; 32: 321-329Crossref PubMed Scopus (327) Google Scholar The diagnosis of HIVE was based on the presence of microglial nodules, astrogliosis, HIV-p24 positive cells, and myelin pallor. Additional analysis was performed with a subset of five age-matched (non-HIV) cases from the UCSD-Medical Center Autopsy Service.Table 1Demographic and Clinico-Pathological Characteristics of HIV+ Cases with and without HIV EncephalitisNeuropathology diagnosisAgeSexPM timeBrain weightCause of deathNeurocognitive diagnosisCD4 countsPlasma HIV RNA loadCSF HIV RNA loadHistory of stimulant dependenceNormal52F31300Diffuse alveolar damageNormal31500YesNormal53M141410BronchopneumoniaNPI420410019NoNormal47M121200BronchopneumoniaNormal48404896181YesNormal45M51220BronchopneumoniaNPI78500YesNormal43M141210SepticemiaNormal32000NoNormal46M181450BronchopneumoniaMCMD61661323688NoNormal56M61350BronchopneumoniaNormal176056NoNormal45M141410Congestive heart failureNormal51000NoHIV encephalitis31M81220SepticemiaHAD219535215677YesHIV encephalitis39M121370SepticemiaNPI86851152567NoHIV encephalitis40M101200BronchopneumoniaHAD141615047272277NoHIV encephalitis46M61125SepticemiaMCMD17815001116413NoHIV encephalitis43M61350SepticemiaHAD325900047100NoHIV encephalitis37M121300BronchopneumoniaHAD02797479644YesHIV encephalitis27M81190SepticemiaNPI5270951592YesHIV encephalitis40F121040BronchopneumoniaMCMD883696229YesPM, post-mortem; F, female; M, male; NPI, neuropsychological impairment; MCMD, minor cognitive/motor disorder; HAD, HIV-associated dementia. Open table in a new tab PM, post-mortem; F, female; M, male; NPI, neuropsychological impairment; MCMD, minor cognitive/motor disorder; HAD, HIV-associated dementia. For studies of CDK5 activation in an animal model of HIV-protein mediated neurotoxicity, Tg mice expressing high levels of gp120 under the control of the GFAP promoter were used.45Toggas S.M. Masliah E. Rockenstein E.M. Rall G.F. Abraham C.R. Mucke L. Central nervous system damage produced by expression of the HIV-1 coat protein gp120 in transgenic mice.Nature. 1994; 367: 188-193Crossref PubMed Scopus (605) Google Scholar These mice develop neurodegeneration accompanied by astrogliosis, microgliosis,45Toggas S.M. Masliah E. Rockenstein E.M. Rall G.F. Abraham C.R. Mucke L. Central nervous system damage produced by expression of the HIV-1 coat protein gp120 in transgenic mice.Nature. 1994; 367: 188-193Crossref PubMed Scopus (605) Google Scholar and memory deficits evinced in the water maze test.46D'Hooge R. Franck F. Mucke L. De Deyn P.P. Age-related behavioural deficits in transgenic mice expressing the HIV-1 coat protein gp120.Eur J Neurosci. 1999; 11: 4398-4402Crossref PubMed Scopus (62) Google Scholar To study the effects of genetic CDK5 inhibition in vivo, CDK5 heterozygous deficient mice (Cdk5+/−) were crossed47Ohshima T. Ward J.M. Huh C.G. Longenecker G. Veeranna Pant H.C. Brady R.O. Martin L.J. Kulkarni A.B. Targeted disruption of the cyclin-dependent kinase 5 gene results in abnormal corticogenesis, neuronal pathology and perinatal death.Proc Natl Acad Sci USA. 1996; 93: 11173-11178Crossref PubMed Scopus (808) Google Scholar with the GFAP-gp120 Tg mice. Full ablation of both copies of CDK5 (Cdk5−/−)causes severe neurodevelopmental alterations, so to study CDK5 knockdown in the adult mouse brain, the Cdk5+/− animals were used as a model of reduced CDK5 activity. Cdk5+/− mice were kindly provided by the laboratory of Dr. Joseph Gleeson (UCSD). For in vivo studies, 12-month-old wild-type non-Tg, Cdk5+/−, gp120 Tg, or gp120 Tg/Cdk5+/− crossed mice (n = 8 mice per group, 12 months old) were tested in the water maze to assess learning and memory. The mice were sacrificed within one week of behavioral testing, and brains were removed for biochemical analyses of frozen or fixed brain tissues. For pharmacological treatments, an additional 24 mice (12-month-old non-Tg and gp120 Tg animals) received intracerebral infusions of vehicle (saline) alone or roscovitine (Calbiochem, San Diego, CA) at a concentration of 20 mg/kg into the lateral ventricle. Six mice per group received each treatment, as follows: n = 6 non-Tg with saline, n = 6 non-Tg with roscovitine, n = 6 gp120 Tg with saline, n = 6 gp120 Tg with roscovitine. For this purpose, mice were anesthetized, and under sterile conditions a 26-gauge stainless steel cannula was implanted stereotaxically into the lateral ventricle using the bregma as a reference (Franklin and Paxinos coordinates: bregma 0.5 mm; 1.1 mm lateral; depth 3 mm) and secured to the cranium using superglue. The cannula was connected via a 5-mm coil of V3 Biolab vinyl to a model 1007D osmotic minipump (Alzet, Cupertino, CA) surgically placed subcutaneously beneath the shoulder. The solutions were delivered at a flow rate of 0.5 μL/hour for 2 weeks. The pump was left for an additional 2 weeks and mice were tested in the water maze at 1 month after initiation of the infusions. All procedures were completed under the specifications set forth by the Institutional Animal Care and Use Committee (UCSD). In brief, as previously described,48Rockenstein E. Adame A. Mante M. Moessler H. Windisch M. Masliah E. The neuroprotective effects of Cerebrolysin in a transgenic model of Alzheimer's disease are associated with improved behavioral performance.J Neural Transm. 2003; 110: 1313-1327Crossref PubMed Scopus (71) Google Scholar to evaluate learning and memory in the roscovitine-infused mice and in the gp120 Tg/Cdk5+/− mice, the animals were trained in the water maze, beginning with a 3-day exposure to the visible platform (days 1 to 3). Then mice were tested for learning and memory with the hidden platform for 4 days (days 4 to 7), followed by one probe test and one final visible test trial on day 8. In the memory portion of the water maze behavioral test (probe test), the platform was removed to evaluate the time spent and distance traveled swimming in the target quadrant where the platform had been located. All experiments were approved by the UCSD Animal Subjects committee and were performed according to NIH recommendations for animal use. In accordance with NIH guidelines for the humane treatment of animals, mice were anesthetized with chloral hydrate and were sacrificed by transcardial flush-perfusion with 0.9% saline. Brains were removed and divided sagittally. One hemibrain was postfixed in phosphate-buffered 4% paraformaldehyde at 4°C for 48 hours and then was sectioned at 40 μm with a microtome (Vibratome 2000; Leica Microinstruments, Bannockburn, IL); the other hemibrain was snap-frozen and stored at −70°C for protein analysis. All experiments described were approved by the UCSD Animal Subjects committee and were performed according to NIH recommendations for animal use. Frontal cortex tissues from human and mouse brains were homogenized and fractionated using a buffer that facilitates separation of the membrane and cytosolic fractions (1.0 mmol/L HEPES, 5.0 mmol/L benzamidine, 2.0 mmol/L 2-mercaptoethanol, 3.0 mmol/L EDTA, 0.5 mmol/L magnesium sulfate, 0.05% sodium azide; final pH 8.8).49Pham E. Crews L. Ubhi K. Hansen L. Adame A. Cartier A. Salmon D. Galasko D. Michael S. Savas J.N. Yates J.R. Glabe C. Masliah E. Progressive accumulation of amyloid-beta oligomers in Alzheimer's disease and in amyloid precursor protein transgenic mice is accompanied by selective alterations in synaptic scaffold proteins.FEBS J. 2010; 277: 3051-3067Crossref PubMed Scopus (168) Google Scholar In brief, as previously described,50Cole G. Dobkins K. Hansen L. Terry R. Saitoh T. Decreased levels of protein kinase C in Alzheimer brain [Erratum appeared in Brain Res 1991, 558:177].Brain Res. 1988; 452: 165-170Crossref PubMed Scopus (225) Google Scholar, 51Masliah E. Rockenstein E. Veinbergs I. Mallory M. Hashimoto M. Takeda A. Sagara Sisk A. Mucke L. Dopaminergic loss and inclusion body formation in alpha-synuclein mice: implications for neuro
Referência(s)