First evidence of overlaps between HIV-Associated Dementia (HAD) and non-viral neurodegenerative diseases: proteomic analysis of the frontal cortex from HIV+ patients with and without dementia
© Zhou et al; licensee BioMed Central Ltd. 2010
Received: 26 December 2009
Accepted: 24 June 2010
Published: 24 June 2010
The pathogenesis of HIV-associated dementia (HAD) is poorly understood. To date, detailed proteomic fingerprinting directly from autopsied brain tissues of HAD and HIV non-dementia patients has not been performed.
Here, we have analyzed total proteins from the frontal cortex of 9 HAD and 5 HIV non-dementia patients. Using 2-Dimensional differential in-gel electrophoresis (2-DIGE) to analyze the brain tissue proteome, 76 differentially expressed proteins (p < 0.05; fold change>1.25) were identified between HAD and HIV non-dementia patients, of which 36 protein spots (based on 3D appearance of spots on the images) were chosen for the mass spectrometry analysis. The large majority of identified proteins were represented in the energy metabolic (mitochondria) and signal transduction pathways. Furthermore, over 90% of the protein candidates are common to both HAD and other non-viral neurodegenerative disease, such as Alzheimer's disease. The data was further validated using specific antibodies to 4 proteins (CA2, GS, CKMT and CRMP2) by western blot (WB) in the same samples used for 2D-DIGE, with additional confirmation by immunohistochemitsry (IHC) using frontal lobe tissue from different HAD and HIV+ non-dementia patients. The validation for all 4 antibodies by WB and IHC was in concordance with the DIGE results, lending further credence to the current findings.
These results suggest not only convergent pathogenetic pathways for the two diseases but also the possibility of increased Alzheimer's disease (AD) susceptibility in HAD patients whose life expectancy has been significantly increased by highly active antiretroviral therapy.
HIV-1 associated dementia (HAD) is a common complication of HIV disease with a prevalence of at least 20% in advanced HIV infection in the pre-highly active antiretroviral therapy (HAART) era . Even in patients taking HAART, milder forms of cognitive impairment remain common and functionally significant . The reasons for the continued presence and development of HAD and its milder forms, despite effective HAART are not clear. Furthermore, due to the longevity of HIV patients after the advent of HAART, the prevalence of HAD has increased . It has been hypothesized that Alzheimer's disease will significantly increase among elderly HIV-infected individuals . Thus, there is the possibility of HIV initiating or facilitating a neurodegenerative process.
Various arrays and bioinformatic approaches have been utilized to explore the pathogenesis of HAD [5–9]. Based on the differentially expressed genes, many cellular processes, including T-cell receptor-mediated signaling, sub-cellular trafficking, transcriptional regulation, and a variety of cellular metabolic pathways have been identified. However, there are two issues with these studies. First, the transcriptomic gene expression has not been validated at the protein level. Second, several HAD pathogenesis proteome-based studies are only confined to cultured cells and cerebrospinal fluid (CSF) [10–13]. To date, protein changes directly in the native HIV-infected brain tissue have not been reported.
Therefore, in the present study, we employed 2D-DIGE, coupled with mass spectrometry, on the total protein extracts from the autopsied human frontal cortex tissue of HAD and HIV non-dementia patients to identify differentially expressed protein candidates between these two groups and to define the pathways and processes, which might be involved in the pathogenesis of HAD, along with any overlapping proteomic features between HAD and other neurodegenerative diseases, such as AD. Our study is unique in using the native brain tissues obtained from HIV+ individuals at autopsy for a detailed proteomic analysis.
Results and Discussion
Significant alteration of protein profiles in HAD brains as opposed to HIV non-demented brains
Identified proteins and related pathways and neurological diseases summary
Related Neurological disease
Fructose-bisphosphate aldolase C [Homo sapiens]
Schizophrenia, bipolar disorder, and depression 
L-lactate dehydrogenase A chain [Homo sapiens]
L-lactate dehydrogenase B chain (LDH) [Homo sapiens]
Alcohol dehydrogenase [NADP+] [Homo sapiens]
Triosephosphate isomerase [Homo sapiens]
Phosphoglycerate mutase 1 (Brain) variant (Fragment) [Homo sapiens]
Oxidative phosphorylation pathway
cDNA FLJ30970 fis, clone HEART2000444, highly similar to Homo sapiens phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP), mRNA [Homo sapiens]
V-type proton ATPase subunit E 1 [Homo sapiens]
(ATP5H)Isoform 2 of O75947. [Homo sapiens]
Cytochrome b-c1 complex subunit Rieske, mitochondrial [Homo sapiens]
NADH dehydrogenase (ubiquinone) 1 beta subcomplex subunit 10 [Homo sapiens]
Nitrogen metabolism pathway
Glutamine synthetase [Homo sapiens]
Carbonic anhydrase 2 [Homo sapiens]
Arachidonic acid metabolism pathway
Carbonyl r Carbonyl reductase [NADPH] 1 [Homo sapiens]
Purine metabolism pathway
Nucleoside diphosphate kinase B [Homo sapiens]
DS, AD 
Arginine and proline metabolism pathway
Creatine kinase, ubiquitous mitochondrial [Homo sapiens]
Alzheimer's and Pick's Disease 
Glutathione metabolism pathway
Glutathione S-transferase P [Homo sapiens]
MAPK signalling pathway
Stathmin [Homo sapiens]
Growth factor receptor-bound protein 2 [Homo sapiens]
Calcium signalling pathway
cDNA FLJ60120, highly similar to Voltage-dependent anion-selective channel protein 2 [Homo sapiens]*
Guanine nucleotide-binding Protein G(o) subunit alpha [Homo sapiens]
Familial Alzheimer's disease 
Axon guidance pathway
Dihydropyrimidinase-related protein 2 [Homo sapiens]
Parkinson's disease pathway
H5 [Homo sapiens]*
Autosomal-recessive juvenile parkinsonism 
Antigen processing and presentation pathway
Heat shock cognate 71 kDa protein [Homo sapiens]
(ACOT7)Isoform 6 of O00154. [Homo sapiens]
Aspartate aminotransferase [Homo sapiens]
Elongation factor Tu, mitochondrial [Homo sapiens]
Infantile Encephalopathy 
Neurocalcin-delta [Homo sapiens]*
cDNA FLJ54102, highly similar to Beta-soluble NSF attachment protein [Homo sapiens]
Guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 [Homo sapiens]
Putative uncharacterized protein PRDX5 [Homo sapiens]
It is interesting to note that more than 90% of the proteins identified in the current study have been reported previously in relation to AD or other neurological diseases (Table 1), thereby lending further credence to our observations. Among them, 9 of 31 proteins have already been reported to interact with HIV directly or indirectly. According to their molecular functions, these 9 proteins can be categorized into 4 groups: proteins involved in metabolic pathways/processes; proteins involved in signal transduction pathways/processes; and antigen presenting protein. In the following section, we will discuss these nine proteins by their functions and three other functionally similar proteins, which have neither been reported previously in HIV infection nor in the context of other neurological diseases.
a. Proteins involved in metabolic pathways/processes
In the current study, we have found both creatine kinase (CK; EC 18.104.22.168) and glutamine synthetase (GS; EC 22.214.171.124) significantly increased in HAD brains in comparison to HIV non-demented brains. They are involved in energy related metabolism (arginine and proline metabolism and nitrogen metabolism, respectively). Furthermore, they are two of the three major specifically oxidized proteins in AD brains . CK plays an important role in facilitating energy transfer within cells with high energy flux or requirements by catalyzing the reversible transfer of a phosphoryl group between adenosine-5'-triphosphate (ATP) and creatine. Cytoplasmic brain CK (BB form) and ubiquitous mitochondrial CK (uMtCK), among the four isoforms of CK, have been reported co-expressed  at various levels throughout the entire brain and serve as an efficient energy buffering and shuttle system in the brain . For uMtCK, the change in AD brains is not significant although a sharp decrease of its activity has been reported . Our results on the HAD brain proteome suggest that HIV can manipulate the energy production or transfer for its own use by altering uMtCK expression since HIV-1 Tat peptide can fuse with human brain CK, and this fusion can increase CK activity after being transduced into PC12 cells . GS is a ubiquitous enzyme, which plays an important role in recycling the glutamate. It is mainly localized in astroglial cells. It has been used as a biomarker of oxidative stress  and potential diagnostic marker of AD . Its presence is enhanced in neurological diseases associated with reactive astrogliosis . Previous in vitro studies have also reported a positive correlation of GS with HIV replication  or the concentration of HIV glycoprotein 120 . Our findings provide in vivo evidence that HIV infection can alter GS expression in human brain, thereby impairing the glutamine/glutamate cycle.
Further, in this context, it is important to mention that we identified another protein, elongation factor Tu, mitochondrial (EF-Tu), which plays a vital role in energy-related cellular metabolic processes and can interact with HIV. It is involved in the mitochondrial protein translation and its mutations are associated with combined oxidative phosphorylation deficiency, which can lead to fatal encephalopathy . Previous studies have shown that EF1a, one of its family members, can interact with the entire HIV-1 Gag polyprotein . Therefore, the increase of EF-Tu in HAD brains, observed in the current study, raises the possibility that HIV-host interaction might partly contribute to the abnormal oxidative phosphorylation. Thus, HIV might be able to trigger oxidative stress in the HAD brains, which is a vital step in the development of neurodegeneration. Our findings also concur with a previous in vitro study that HIV-1 infection does induce the generation of reactive oxygen species (ROS) . Further in vivo studies on enzyme activity and protein modifications are needed in the context of neurological manifestations of HIV disease.
b. Proteins involved in signal transduction pathways/processes
Two important proteins, stathmin (STMN1) and growth factor receptor-bound protein2 (GRB2), identified in our study, are involved in mitogen activated protein kinase (MAPK) signalling pathways, which are known to play multiple roles in HIV disease and NK effector functions [27, 28]. Up-regulation of STMN1 in HAD brains was found in our study, whereas a decrease of STMN1 had been previously reported in AD and DS brains . STMN1 belongs to a class of regulatory proteins related to microtubule dynamics  and is vital for cellular processes, including intracellular transport, maintenance of cell shape and cell polarity . It is widely distributed in the neuronal cell body, dendrites, axons, and growth cones  and is involved in the destabilization of microtubule, which is essential for axon and dendrite differentiation, growth, and maintenance and even the generation of neuronal size, shape, and compartmentalization . In addition, Nishi et al.  have demonstrated that STMN1 expression can inhibit enhancement of HIV-1 particle production by suppressing cytokine signaling-1. Our results indicate that alteration of STMN1 might be one of the positive host responses upon HIV infection at the protein level, which can inhibit the virion assembly and production.
GRB2 is an adaptor protein involved in signal transduction/cell communication and T-cell activation . GRB2 coupled to Son-of-sevenless-1 (SOS-1), which can activate the RAS/MAPK pathway , and form one of the primary components of signal transduction cascade . In neurodegenerative processes it directly interacts with amyloid precursor protein (APP) and presenilin (PS1), which play an important role in the onset of AD . Moreover, increased levels of Grb3-3, an isoform of Grb2, were seen in PBMCs from HIV-1 infected subjects . In the current study, a mild increase of Grb-2 protein was seen in HAD brains, which complements previous studies on AD brains. Its increase in HAD brains is probably triggered by HIV-1 since Grb3-3 can be induced by HIV Tat and Nef proteins independently . In the context of HAD, it might be involved in the MAPK activation and possibly tau hyper-phosphoylation, which needs to be elucidated in future.
The guanine nucleotide-binding proteins (G proteins) families also play a crucial role in signal transduction. They function as molecular switches between extracellular events and intracellular effectors. Through the coupling between their receptors and several heterotrimeric G-proteins in NK cells, the C, CC, CXC and CX3C chemokines can activate NK cells and induce intracellular signalling pathways in the NK cells [41, 42]. In the present study, we found a significant up-regulation in both Go subunit alpha and beta-2. Our results are consistent with previous in vitro array studies . It might indicate a stronger host antiviral response by activating the NK cells and then death of the infected host cells. In this context, it is important to reiterate that the CC chemokine RANTES, MIP-1b and the CXC chemokine SDF-1a, which signal through M-tropic HIV-1 co-receptors CCR5 and CCR3 and T-tropic HIV-1 co-receptor CXCR4 respectively, are activated by G proteins [43, 44]. In non-viral neuro-degeneration, G-protein can also couple with two of the three causative gene products of familial Alzheimer's disease, APP  and PS1 . Data (reviewed by Cowburn ) have suggested that the neurochemical pathology of AD includes severe disruption of the neurotransmitter receptor/G-protein mediated phosphatidylinositol hydrolysis and adenylyl cyclase signal transduction pathways. β2 subunit and β γ complex of G-proteins have been reported to participate in chemokine-induced NK cell chemotaxis  and mediate apoptosis , respectively. In the context of HIV, further studies are needed to explore the mechanism of how HIV interferes with G proteins expression and utilizes G proteins to manipulate the signalling pathways for its survival.
Another important protein interacting with HIV identified in this study is neurocalcin, which is a Ca2+-binding protein distributed abundantly in the central nervous system and has been reported to play a role in neuronal signalling . So far, at least 6 isoforms of neurocalcin have been identified. Neurocalcin δ is expressed mainly in glial cells . It has been reported that one of the target proteins of neurocalcin δ is S100β , which has been shown to be up-regulated in HIV infection and very important to HAD neuropathogenesis . To the author's knowledge, neurocalcin δ has been only reported to be site down-regulated in the temporal lobe of AD brains [53, 54] without comparison to control brain. In our study, we found an up-regulation of neurocalcin in the frontal cortex of HAD brains and our results suggest that neurocalcin δ and S100β complex might participate in the pathogenesis of HAD by inducing gliosis, growth of dystrophic neurites, and calcium-mediated neuronal cell loss.
c. Antigen presenting and other proteins
Heat shock cognate 71 kDa protein (HSC-71) was mildly but significantly increased in the HAD brains. HSC-71 is the constitutively expressed member of the heat shock protein 70 (Hsp70) family and has 85% homology with Hsp70. Hsp70 protein level can increase due to HIV infection or oxidative stress [55, 56]. Furthermore, Hsp70 can trigger an increased immune response by incorporating into the membrane of HIV virions and prevent HIV induced astrocytes apoptosis . In the context of neurodegeneration, HSC71 plays more important roles. For instance, it can interact with the cytoplasmic domain of APP in the presence of proteasome inhibitors. This indicates that HSC-71 might participate in proteasome structural maintenance and mis-folded protein conformational recognition . Although HSC-71 has been reported to have an insignificant down-regulation in the AD brains , it appears more oxidatively modified and possibly glycosylated in the AD brains [14, 60]. Taking these observations in the context of our study, the increase in the expression of HSC-71 could be a consequence of HIV infection or increased oxidative stress seen during HIV infection .
Worthy of noting is that we have found three functionally similar proteins (ACOT7, FLJ54102 and FLJ60120), which have neither been reported previously in HIV infection nor in the context of other neurological diseases. ACOT7 or Acyl-CoA thioesterases (EC 126.96.36.199.) are enzymes that catalyze the hydrolysis of CoA esters of various molecules to the free acid plus coenzyme A (CoA) , which differentiates them from long-chain acyl-CoA synthetases because long-chain acyl-CoA synthetases ligate fatty acids to CoA, to produce the CoA ester . They are implicated in the regulation of intracellular levels of CoA esters, the corresponding free acid, CoASH and cellular processes involving these compounds. ACOT8 under recently revised nomenclature was identified as hACTEIII  and hTE , which can interact with and activate the HIV-1 Nef protein. So far, only long chain acyl-CoA synthetases have been reported to be related to neurodegenerative disease . Interestingly, we found up-regulation of the ACOT7 protein uniquely in the HAD brains implying its likely interaction with HIV.
The second protein is cDNA FLJ54102, a highly similar protein to beta-soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein. We observed an up-regulation of FLJ54102 in the HAD brains. Soluble NSF attachment proteins (SNAPs) are highly conserved proteins, which are involved in intracellular membrane fusion and vesicular trafficking. There are three individual isoforms of SNAPs: α, β, and γ . Among them, β-SNAP is brain specific and it has been reported that maximal levels of its expression are in the hippocampus . Schiavo  showed that β-SNAP, NSF, SNAP receptor, and the calcium-binding protein synaptotagmin (SYT) assemble cooperatively to form a docking and fusion complex. HIV Tat has been reported to be able to fuse with NSF and inhibit the extrocytosis , while SNAP receptor has been reported to be involved in fusion events of endosomal Gag-RNA complexes with the plasma membrane to generate virions through an endosome-dependent route . The expression of SNAPs has also been shown to be significantly reduced in the AD and DS brains .
The third is cDNA FLJ60120, highly similar to voltage-dependent anion-selective channel protein (VDAC) 2. VDAC is a mitochondrial outer-membrane protein, which plays an important role in the pore formation and cytochrome c release . It can regulate cell death by binding to B-cell CLL/lymphoma 2 (Bcl-2) family pro- or anti- apoptotic protein [74, 75]. It can also regulate the mitochondrial function by controlling metabolite fluxes through the mitochondrial membrane [76, 77]. It can impact on glucose metabolism by directly binding to glycolytic enzymes . More importantly, it has been reported to be involved in neurodegenerative disorders and mental retardation [78–81]. By binding VDAC, HIV Vpr can induce apoptosis through a direct effect on the mitochondrial permeability transition pore complex (PTPC) . Three of human VDACs have been cloned and termed, VDAC1, VDAC2 and VDAC3. VDAC2 has been shown to over-express in the AD brains . Our findings on HAD brains suggest possible association with apoptosis and the synaptic loss in HAD brains due to the impairment of energy pathways during HIV infection.
Apart from these proteins discussed at above, other identified proteins in the current study so far haven't been reported to be related to HIV infections. However, some proteins, especially carbonic anhydrase 2 (CA2) and carbonyl reductase [NADPH] 1, changed dramatically in the HAD brains (Table 1). Moreover, both of them have been reported to change in AD brains and are involved in AD pathogenesis. Thus, our results might indicate that HIV is able to alter those candidate proteins that directly or indirectly affect neurological functions. This sheds light on some novel candidate host proteins that possibly interact with HIV and modulate neuropathogenesis.
Pathway and network analysis
For annotation and pathway analysis of these identified proteins, Metacore and DAVID were used. The 31 identified differentially expressed proteins were transferred into the official gene symbol ID code based on their corresponding Swiss-prot database accession numbers. There were 19 statistically significant locations (FDR<0.05, p < 0.01), the details of which are shown in the Additional file 1. Among them, the mitochondrion, cytoplasm, cytoplasmic part, cytosol and mitochondrial inner membrane rank the top 5 according to the statistical significance. Functionally, most of them are enzymes, while several transporting proteins and generic binding protein were also observed (see Additional file 2).
It is broadly accepted that metabolic pathways/processes are closely related to neurodegenerative diseases. It has been shown that the impairment of energy metabolism can exacerbate synaptic dysfunction, neuronal injury, which together may lead to neurodegenerative disorders . The oxidative phosphorylation system (OXPHOS), using glucose as critical substrates, normally provides more than 95% of ATP used for cellular energy. Inefficient glycolysis plays a crucial role in the pathogenesis of AD . Further, the mitochondrial genes in complex I of OXPHOS have been demonstrated to be significantly down-regulated in AD and Parkinson's brains [85, 86]. In the context of HIV, it has also been shown that HIV-1 targets the energy generating system of the host cells by affecting mitochondrial DNA and protein . HIV envelope glycoprotein gp120 can impair glucose metabolism in vitro and in vivo [88, 89]. Furthermore, HIV-1 matrix protein, p17, can stimulate gluconeogenesis by inducing the expression of Fructose 1, 6 bisphosphatase, which can convert fructose-1, 6-bisphosphate to fructose 6-phosphate and produce NADPH . Also, it has been reported that HIV can reduce the activity of complex I by down-regulating the expression of NDUFA6 at protein level .
Supporting these arguments, our pathway/process analysis has shown that most of the identified proteins in the current study were involved in metabolic pathways or processes. Figure 2 and Figure 3 shows the Glycolysis and Oxidative phosphorylation KEGG pathways. We have observed a significant decrease in the levels of two key enzymes of Glycolysis pathway (TPI and PGAM1) and complex I of Oxidative phosphorylation pathway (NDUFB10), while complex III and complex IV were up-regulated. These findings overlap with AD brains as well . It is probably due to a great demand on energy production. In addition, it has been shown that HIV-1 infection does induce ROS, thereby leading to T cell death . Our results provide a further explanation that HIV might influence the production of ROS through regulating the Oxidative phosphorylation pathway. Figure 6 shows the metabolic network, which includes identified proteins in the current study (marked with a circle), and the proteins within the metabolic network and closely interact with the identified proteins. All edges are supported by at least 1 reference from the literature. Collectively, this complex alteration of proteins provides strong support for abnormalities in metabolic pathways/processes during HAD in humans.
The overlap between HAD and non-viral dementia
It is interesting that more than 90% of the proteins identified in the current study share an overlap with proteins reported from other neurodegenerative brains (Table 1). Although the alteration of these pathways/processes and proteins are possibly more generalized in neurodegenerative disorders rather than specifically abnormal in individual diseases, the critical overlap between HAD and non-viral neurodegenerative disorders, such as AD, is worthy of attention. Previous studies have shown that brain infections, such as herpes simplex, are related to the occurrence and development of AD process [92–94]. In addition, Brosseau et al. , have shown the first case of HIV-associated dementia with characteristics of Alzheimer's disease in a patient with AIDS, which supports the functional and neurophysiological relevance of the data shown in this study. Our findings at the proteomic level further raise the possibility that HIV might initiate or facilitate a neurodegenerative process. Our findings are consistent with previous reports at the genetic level . However, until detailed proteomic fingerprinting is available from various virus-mediated neurodegenerative diseases (such as HSV encephalitis), it is difficult to rule out whether these overlaps at the protein level exist only between HAD and non-viral neurodegenerative diseases or human brain responds to different pathogens similarly. Nonetheless, our study is the first to provide this tantalizing evidence in favor of this major proteomic overlap between HAD and non-viral neurodegeneration, which in the future may clarify the involvement of any pathogenic etiology in non-viral neurodegenerative processes.
In contrast, the GS, CKMT, and CRMP2 were up-regulated proteins, which changed from mild (FC<1.5) to moderate (1.5<FC<2). Apart from their direct relevance in neurological disease, they were also chosen for validation because of their increase in fold-change in 2D-DIGE data. They were recognized by their specific antibodies at sizes 42, 47 and 63 kDa, respectively (Figures 7B, C and 7D). The western blot results of these three proteins followed the trend of 2D-DIGE data (p < 0.05) but not in numeracy (Figure 7E), with the exception of two samples where they were not recognized by the antibodies (sample #2434 and #H0011db with GS and CKMT). This is possible because the low sensitivity of western blot techniques/antibodies compared to 2D-DIGE/CyDye. Although some noticeable sample-to-sample variation within the same group was observed, no statistical relationship with dementia stage was found apart from some visual differences in relationship. Figure 7 shows the 3D DeCyder interpretation of all 4 proteins together with their corresponding western blot results.
Our study is the first demonstration of evidence showing overlapping proteins between viral (HIV) and non-viral neurodegenerative diseases. Although the majority of proteins identified in this study have been previously reported in relation to other neurodegenerative or psychiatric diseases, these findings do provide a strong foundation not only for understanding possible mechanisms of HAD, but also provide a much needed foundation for clarifying the possible involvement of a pathogenic etiology in non-viral neurodegenerative diseases. Furthermore, we also observed a significant involvement of Glycolysis and Oxidative phosphorylation, the two important energy related metabolic pathways, in the HAD brains, which has been fully demonstrated in the current study at the pathway level. Interestingly, the involvement of these pathways in HAD also coincides with their involvement in other neurodegenerative diseases, such as AD and PD.
Materials and methods
Patient details and brain tissue collection
Brain tissue samples were obtained from HIV-1-infected patients with or without HAD through the National Neuro-AIDS Tissue Consortium (NNTC, Request #R203) and the Westmead Hospital, Sydney, Australia (Reference No: 5465). Samples were collected at post-mortem, shipped frozen on dry ice and stored frozen at -70°C until use. Frontal cortex of male patients 9 with HAD and 5 without were used for this study due to its importance to motor impairment and involvement in AD [97, 98]. The average age for HAD and non-HAD patients was 43.57 ± 14.77 and 50.2 ± 11.88, respectively, (P = 0.41). Clinical profiles of all patients are shown in Additional file 4. This study was conducted according to the principles expressed in the Declaration of Helsinki. Use of samples in this study was approved by Institutional Review Board and the Ethics Committee of the NNTC Allocations, the University of Sydney and the Westmead Hospital individually. The family members of the patients have given written, informed consent for the use of autopsied brain tissue. For the diagnostic criteria for HAD, the criteria defined by the American Academy of Neurology 1991 were used (American Academy of Neurology. Nomenclature and research case definitions for neurological manifestations of HIV type 1 infection 1: Report of a working group of AAN of neurology and AIDS task force, 1991).
In each case, 30 mg of frozen brain tissue was measured on dry ice and then transferred into a 2 mL Eppendorf tube. The tissues were lysed in 1 mL of 4°C lysis buffer (7 M urea, 2 M thiourea, 30 mM Tris, 4% CHAPS, 2% ASB, 1% Sigma protease inhibitor cocktail), vortexed for 30 seconds and cooled for 1 minute on ice (repeat 25 cycles), sonicated for 1 minute at 4°C and cooled for 1 minute on ice (repeat 3 cycles), and centrifuged for 50 minutes at 14000 rpm at 4°C. Protein concentrations were determined using the 2-D Quant kit (GE Healthcare), as per manufacturer's instructions.
Protein labeling with CyDyes
Protein samples were labelled with CyDyes (GE Healthcare), as per manufacturer's instructions. 25 μg of total protein from each sample was mixed in an Eppendorf tube (Eppendorf, Düsseldorf, Germany) and labelled with Cy2 minimal dye, and 50 μg protein was taken from the mix and used as an internal standard on each gel for the subsequent 2D electrophoresis and image analysis. In parallel, 50 μg proteins of each sample were labelled with either Cy3 or Cy5, and the dyes scrambled within each group to avoid possible dye bias. The sample volumes were adjusted to 18 μL with labelling buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris), followed by addition of 1 μL dye (working solution) to each individual sample. The samples were left on ice for 30 minutes in the dark, followed by adding 1 μL of 10 mmol/L lysine to stop the reaction.
2D Electrophoresis and image analysis
One sample from each of the CyDye groups was mixed together and adjusted to final concentrations of 1% DTT, 1% IPG buffer at a total volume of 350 μL with lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 0.04% bromophenol blue) and was used to rehydrate 17 cm IPG strips (pH 3-10, non-linear; Bio-Rad) overnight. First dimension isoelectric focusing (IEF) was carried out with IPGphor II (GE Healthcare). The strips were focused at a constant temperature of 20°C with approximately 55 kVh (150 V for 3 hours, gradient to 300 V for 1 minute, 300 V for 5 hours, gradient to 8000 V for 4 hours, 8000 V for 4 hours, gradient to 400 V for 30 minutes, 400 V for 4 hours). The strips then were incubated with equilibration buffer (50 mM Tris-HCL pH 8.8, 6 M urea, 30% glycerol, 2% sodium dodecyl sulfate, 0.02% bromophenol blue) containing 65 mM DTT for 15 minutes, followed by 130 mM iodoacetamide in equilibration buffer for the next 15 minutes. The second dimension SDS-PAGE was performed by mounting the IPG strips onto 8% to 18% polyacrylamide gradient gel (Jule Precast Gels, USA) and running the gels in the Protean II multi cell electrophoresis system (Bio-Rad) at 16 mA/gel for the initial hour and 25 mA/gel at 10°C constantly until bromophenol blue reached the bottom of the gel. Following this, the gels were scanned on a Typhoon Trio variable mode imager (GE Healthcare) at 100 microns resolution to produce a Cy2, Cy3 and Cy5 image for each gel according to the manufacturer's protocol. The images were cropped with ImageQuant software (GE Healthcare) and analyzed by automated Difference In-gel Analysis (DIA) and Biological Variation Analysis (BVA) using Decyder software version 6.5 (GE Healthcare). After the gels were scanned, they were removed from the glass plates fixed with 10% methanol, 7% acetic acid overnight, stained with Coomassie Brilliant Blue G250 (Coomassie Brilliant Blue G250 0.5 g, Ammonium sulphate 50 g, 85% Phosphoric Acid 6 mL in 500 mL water and add 125 mL methanol before use) overnight and de-stained with 1% acetic acid overnight.
Mass Spectrometry and Protein identification
Spots selected for protein identification were manually excised using a scalpel, de-stained in 60% (v/v) 50 mM ammonium bicarbonate (pH 7.8), 40% (v/v) acetonitrile and then dehydrated in 100% acetonitrile for 1 minute before being dried in a vacuum centrifuge. Gel pieces were rehydrated in 10 μL trypsin solution (12 ng/μl porcine modified sequencing grade trypsin in 50 mM ammonium bicarbonate, pH 7.8) at 4°C for 1 hour. Excess trypsin solution was removed and 15 μL of 50 mM ammonium bicarbonate (pH 7.8) was added prior to incubating overnight at 37°C. For MALDI-TOF MS analysis, 1 μL of peptide was spotted onto a target plate with an equal volume of matrix solution (10 mg/mL α-cyano-4-hydroxycinnamic acid in 70% (v/v) acetonitrile, 1% (v/v) TFA). Mass spectra were acquired in the mass:charge range of 875-3500 m/z on a QSTAR XL mass spectrometer equipped with an oMALDI source (Applied Biosystems Inc., Foster City, CA, USA). The monoisotopic peak masses were subjected to database searching against the MSDB comprehensive non-redundant database using MASCOT vr 2.0 (Matrix Science, London, UK). Parameters for protein identification included searching with a mass error tolerance of 50 ppm per peptide, 1 missed tryptic cleavage, and allowing oxidation of methionine as an optional modification. Confident matches were defined by the MASCOT score and statistical significance (p < 0.05), the number of matching peptides and the percentage of total amino acid sequence covered by those matching peptides. Peptide mixtures that provided poor initial mass spectra were concentrated and desalted using C18 PerfectPure reverse-phase micro-columns (Eppendorf, Düsseldorf, Germany) according to the manufacturer's instructions and eluted in matrix solution directly onto the target plate. MALDI-TOF MS was then performed as described above.
Functional analysis of protein findings
Once identified with mass spectrometry, the gene encoding counterpart of each individual protein was searched in the Swiss-prot database http://www.expasy.org based on their Swiss-prot database accession numbers. Afterwards, the gene symbol and the fold change of each protein were loaded to Metacore http://portal.genego.com and DAVID http://david.abcc.ncifcrf.gov/ to determine the pathways and biological processes associated with each individual protein. The analysis was performed using the algorithm within the software. The pathways and processes statistically significant to the data set were represented by maps in the database and network, respectively.
Functional Validation of proteins using Western blotting and Immuno-histochemistry
Western blot and immunohistochemistry were employed to validate the 2D-Dige data. A subset of the same samples for the 2D-Dige study was used for the western blot. 40 ug of proteins were separated by 12% SDS-PAGE and then transferred to PVDF membranes (Millipore, USA) or nitrocellulose membranes (Amersham, USA) using Bio-Rad apparatus (Bio-Rad, USA). Membranes were blocked in 5% skim milk powder or 5% BSA in Tris-buffered saline (TBS) (20 mM Tris and 0.9% NaCl, pH 7.4) for 1 hour at room temperature. Following that, they were incubated for 2 hours at room temperature with each of the following primary antibodies: rabbit anti-Carbonic Anhydrase II (1:10000), Creatine kinase MT (1:75), Glutamine Synthetase (1:2000) (Abcam, US) and with another primary antibody rabbit anti-CRMP2 (1:4000) (Abcam, US) incubated overnight at 4°C. Mouse anti-Actin (1:6000, DAKO, USA) was used as control antibody. Membranes were washed four times with TTBS (TBS with 0.05% Tween20) and then incubated for 1 hour with anti-rabbit HRP-conjugated secondary antibody (Dako, USA; 1:6000) followed by chemiluminescence ECL detection (GE, USA) and exposure to autoradiography film (Kodak, France). Films were scanned with HP scanjet8200 (HP, USA) and the images were collected and analysed using Imagel software http://rsbweb.nih.gov/ij/. Statistically significant differences between patients were estimated with the Mann-Whitney test (P-value < 0.05).
Different samples were used for immuno-histochemistry. The serial sections of frontal lobes from 1 HAD versus 1 HIV non-dementia patients were used for the experiment. Patient details were well documented previously (Patient A and D in ). Tissues were fixed in 20% formalin, followed by paraffin embedding. Sections were cut into 6 μm thick for immuno-histochemical staining. Heat antigen retrieval was performed using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0). 3% H2O2, NH4Cl, Glycine, goat serum and Bovine Serum Albumin (BSA) (Aurion) blocking steps were used. Slides then were incubated over night at 4°C or at room temperature with the same primary antibodies used for western blot: rabbit anti-Carbonic Anhydrase II (1:800), Creatine kinase MT (1:10), Glutamine Synthetase (1:100) and rabbit anti-CRMP2 (1:100) (Abcam, US), washed in PBS and then incubated for one hour with the swine anti-rabit HRP-conjugated secondary antibody (1:100; Dako, USA). The detection were made using DAB kit (Dako, USA). The images were captured using a Leica microscope and analysed neuropathologically.
Authors thank NNTC for providing brain samples used for this study and Dr. Ashley Schoell (Los Angeles) for facilitating that. Authors are thankful to Ms. Mary Simonian and Virginia James for technical assistance with immunohistochemistry. Authors are also thankful to Mrs. April Davis for technical advice with western blot. In addition, this publication was made possible from NIH funding through the NIMH and NINDS Institutes by the following grants: Manhattan HIV Brain Bank: U01MH083501, R24MH59724; Texas NeuroAIDS Research Center U01MH083507, R24 NS45491; National Neurological AIDS Bank 5U01MH083500, NS 38841; California NeuroAIDS Tissue Network U01MH083506, R24MH59745; and Statistics and Data Coordinating Center U01MH083545, N01MH32002. This study was funded by the World AIDS Foundation grant to NKS. HR is a NSW Cancer Institute Research Fellow.
- Cysique LA, Maruff P, Brew BJ: Prevalence and pattern of neuropsychological impairment in human immunodeficiency virus-infected/acquired immunodeficiency syndrome (HIV/AIDS) patients across pre- and post-highly active antiretroviral therapy eras: a combined study of two cohorts. Journal of neurovirology. 2004, 10: 350-357. 10.1080/13550280490521078.PubMedGoogle Scholar
- Joseph J, Clifford D, Douglas SD, Fox H, Gendelman HE, Gonzalez-Scarano F, Grant I, Major E, McArthur J: Planning Future Strategies for Domestic and International NeuroAIDS Research, July 24-25, 2008. J Neuroimmune Pharmacol. 2009, 4: 283-297. 10.1007/s11481-009-9159-1.PubMedPubMed CentralGoogle Scholar
- Dore GJ, McDonald A, Li Y, Kaldor JM, Brew BJ: Marked improvement in survival following AIDS dementia complex in the era of highly active antiretroviral therapy. AIDS (London, England). 2003, 17: 1539-1545.Google Scholar
- Alisky JM: The coming problem of HIV-associated Alzheimer's disease. Medical hypotheses. 2007, 69: 1140-1143. 10.1016/j.mehy.2007.02.030.PubMedGoogle Scholar
- Sui Y, Potula R, Pinson D, Adany I, Li Z, Day J, Buch E, Segebrecht J, Villinger F, Liu Z, Huang M, Narayan O, Buch S: Microarray analysis of cytokine and chemokine genes in the brains of macaques with SHIV-encephalitis. Journal of medical primatology. 2003, 32: 229-239. 10.1034/j.1600-0684.2003.00030.x.PubMedGoogle Scholar
- Geiss GK, Bumgarner RE, An MC, Agy MB, van 't Wout AB, Hammersmark E, Carter VS, Upchurch D, Mullins JI, Katze MG: Large-scale monitoring of host cell gene expression during HIV-1 infection using cDNA microarrays. Virology. 2000, 266: 8-16. 10.1006/viro.1999.0044.PubMedGoogle Scholar
- Galey D, Becker K, Haughey N, Kalehua A, Taub D, Woodward J, Mattson MP, Nath A: Differential transcriptional regulation by human immunodeficiency virus type 1 and gp120 in human astrocytes. Journal of neurovirology. 2003, 9: 358-371.PubMedGoogle Scholar
- Vahey MT, Nau ME, Taubman M, Yalley-Ogunro J, Silvera P, Lewis MG: Patterns of gene expression in peripheral blood mononuclear cells of rhesus macaques infected with SIVmac251 and exhibiting differential rates of disease progression. AIDS research and human retroviruses. 2003, 19: 369-387. 10.1089/088922203765551728.PubMedGoogle Scholar
- Roberts ES, Zandonatti MA, Watry DD, Madden LJ, Henriksen SJ, Taffe MA, Fox HS: Induction of pathogenic sets of genes in macrophages and neurons in NeuroAIDS. The American journal of pathology. 2003, 162: 2041-2057.PubMedPubMed CentralGoogle Scholar
- Ricardo-Dukelow M, Kadiu I, Rozek W, Schlautman J, Persidsky Y, Ciborowski P, Kanmogne GD, Gendelman HE: HIV-1 infected monocyte-derived macrophages affect the human brain microvascular endothelial cell proteome: new insights into blood-brain barrier dysfunction for HIV-1-associated dementia. J Neuroimmunol. 2007, 185: 37-46. 10.1016/j.jneuroim.2007.01.004.PubMedPubMed CentralGoogle Scholar
- Glanzer JG, Enose Y, Wang T, Kadiu I, Gong N, Rozek W, Liu J, Schlautman JD, Ciborowski PS, Thomas MP, Gendelman HE: Genomic and proteomic microglial profiling: pathways for neuroprotective inflammatory responses following nerve fragment clearance and activation. Journal of neurochemistry. 2007, 102: 627-645. 10.1111/j.1471-4159.2007.04568.x.PubMedGoogle Scholar
- Rozek W, Ricardo-Dukelow M, Holloway S, Gendelman HE, Wojna V, Melendez LM, Ciborowski P: Cerebrospinal fluid proteomic profiling of HIV-1-infected patients with cognitive impairment. Journal of proteome research. 2007, 6: 4189-4199. 10.1021/pr070220c.PubMedGoogle Scholar
- Laspiur JP, Anderson ER, Ciborowski P, Wojna V, Rozek W, Duan F, Mayo R, Rodriguez E, Plaud-Valentin M, Rodriguez-Orengo J, Gendelman HE, Melendez LM: CSF proteomic fingerprints for HIV-associated cognitive impairment. J Neuroimmunol. 2007, 192: 157-170. 10.1016/j.jneuroim.2007.08.004.PubMedPubMed CentralGoogle Scholar
- Castegna A, Aksenov M, Aksenova M, Thongboonkerd V, Klein JB, Pierce WM, Booze R, Markesbery WR, Butterfield DA: Proteomic identification of oxidatively modified proteins in Alzheimer's disease brain. Part I: creatine kinase BB, glutamine synthase, and ubiquitin carboxy-terminal hydrolase L-1. Free radical biology & medicine. 2002, 33: 562-571.Google Scholar
- Eppenberger HM, Dawson DM, Kaplan NO: The comparative enzymology of creatine kinases. I. Isolation and characterization from chicken and rabbit tissues. The Journal of biological chemistry. 1967, 242: 204-209.PubMedGoogle Scholar
- Wallimann T, Hemmer W: Creatine kinase in non-muscle tissues and cells. Molecular and cellular biochemistry. 1994, 133-134: 193-220. 10.1007/BF01267955.PubMedGoogle Scholar
- Aksenov MY, Aksenova MV, Payne RM, Smith CD, Markesbery WR, Carney JM: The expression of creatine kinase isoenzymes in neocortex of patients with neurodegenerative disorders: Alzheimer's and Pick's disease. Experimental neurology. 1997, 146: 458-465. 10.1006/exnr.1997.6550.PubMedGoogle Scholar
- Jeong MS, Kim DW, Lee MJ, Lee YP, Kim SY, Lee SH, Jang SH, Lee KS, Park J, Kang TC, Cho SW, Kwon OS, Eum WS, Choi SY: HIV-1 Tat-mediated protein transduction of human brain creatine kinase into PC12 cells. BMB reports. 2008, 41: 537-541.PubMedGoogle Scholar
- Smith CD, Carney JM, Starke-Reed PE, Oliver CN, Stadtman ER, Floyd RA, Markesbery WR: Excess brain protein oxidation and enzyme dysfunction in normal aging and in Alzheimer disease. Proceedings of the National Academy of Sciences of the United States of America. 1991, 88: 10540-10543. 10.1073/pnas.88.23.10540.PubMedPubMed CentralGoogle Scholar
- Gunnersen D, Haley B: Detection of glutamine synthetase in the cerebrospinal fluid of Alzheimer diseased patients: a potential diagnostic biochemical marker. Proceedings of the National Academy of Sciences of the United States of America. 1992, 89: 11949-11953. 10.1073/pnas.89.24.11949.PubMedPubMed CentralGoogle Scholar
- Stringaris AK, Bruck W, Tumani H, Schmidt H, Nau R: Increased glutamine synthetase immunoreactivity in experimental pneumococcal meningitis. Acta neuropathologica. 1997, 93: 215-218. 10.1007/s004010050606.PubMedGoogle Scholar
- Porcheray F, Leone C, Samah B, Rimaniol AC, Dereuddre-Bosquet N, Gras G: Glutamate metabolism in HIV-infected macrophages: implications for the CNS. American journal of physiology. 2006, 291: C618-626. 10.1152/ajpcell.00021.2006.PubMedGoogle Scholar
- Visalli V, Muscoli C, Sacco I, Sculco F, Palma E, Costa N, Colica C, Rotiroti D, Mollace V: N-acetylcysteine prevents HIV gp 120-related damage of human cultured astrocytes: correlation with glutamine synthase dysfunction. BMC neuroscience. 2007, 8: 106-10.1186/1471-2202-8-106.PubMedPubMed CentralGoogle Scholar
- Valente L, Tiranti V, Marsano RM, Malfatti E, Fernandez-Vizarra E, Donnini C, Mereghetti P, De Gioia L, Burlina A, Castellan C, Comi GP, Savasta S, Ferrero I, Zeviani M: Infantile encephalopathy and defective mitochondrial DNA translation in patients with mutations of mitochondrial elongation factors EFG1 and EFTu. American journal of human genetics. 2007, 80: 44-58. 10.1086/510559.PubMedPubMed CentralGoogle Scholar
- Cimarelli A, Luban J: Translation elongation factor 1-alpha interacts specifically with the human immunodeficiency virus type 1 Gag polyprotein. Journal of virology. 1999, 73: 5388-5401.PubMedPubMed CentralGoogle Scholar
- Macho A, Castedo M, Marchetti P, Aguilar JJ, Decaudin D, Zamzami N, Girard PM, Uriel J, Kroemer G: Mitochondrial dysfunctions in circulating T lymphocytes from human immunodeficiency virus-1 carriers. Blood. 1995, 86: 2481-2487.PubMedGoogle Scholar
- Berg NN, Puente LG, Dawicki W, Ostergaard HL: Sustained TCR signaling is required for mitogen-activated protein kinase activation and degranulation by cytotoxic T lymphocytes. J Immunol. 1998, 161: 2919-2924.PubMedGoogle Scholar
- Toschi E, Bacigalupo I, Strippoli R, Chiozzini C, Cereseto A, Falchi M, Nappi F, Sgadari C, Barillari G, Mainiero F, Ensoli B: HIV-1 Tat regulates endothelial cell cycle progression via activation of the Ras/ERK MAPK signaling pathway. Molecular biology of the cell. 2006, 17: 1985-1994. 10.1091/mbc.E05-08-0717.PubMedPubMed CentralGoogle Scholar
- Cheon MS, Fountoulakis M, Cairns NJ, Dierssen M, Herkner K, Lubec G: Decreased protein levels of stathmin in adult brains with Down syndrome and Alzheimer's disease. Journal of neural transmission. 2001, 281-288.Google Scholar
- Jourdain L, Curmi P, Sobel A, Pantaloni D, Carlier MF: Stathmin: a tubulin-sequestering protein which forms a ternary T2 S complex with two tubulin molecules. Biochemistry. 1997, 36: 10817-10821. 10.1021/bi971491b.PubMedGoogle Scholar
- Belmont LD, Mitchison TJ: Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of microtubules. Cell. 1996, 84: 623-631. 10.1016/S0092-8674(00)81037-5.PubMedGoogle Scholar
- Gavet O, El Messari S, Ozon S, Sobel A: Regulation and subcellular localization of the microtubule-destabilizing stathmin family phosphoproteins in cortical neurons. Journal of neuroscience research. 2002, 68: 535-550. 10.1002/jnr.10234.PubMedGoogle Scholar
- Walczak CE: Microtubule dynamics and tubulin interacting proteins. Current opinion in cell biology. 2000, 12: 52-56. 10.1016/S0955-0674(99)00056-3.PubMedGoogle Scholar
- Nishi M, Ryo A, Tsurutani N, Ohba K, Sawasaki T, Morishita R, Perrem K, Aoki I, Morikawa Y, Yamamoto N: Requirement for microtubule integrity in the SOCS1-mediated intracellular dynamics of HIV-1 Gag. FEBS letters. 2009, 583: 1243-1250. 10.1016/j.febslet.2009.03.041.PubMedGoogle Scholar
- Downward J: The GRB2/Sem-5 adaptor protein. FEBS letters. 1994, 338: 113-117. 10.1016/0014-5793(94)80346-3.PubMedGoogle Scholar
- Aktas H, Cai H, Cooper GM: Ras links growth factor signaling to the cell cycle machinery via regulation of cyclin D1 and the Cdk inhibitor p27KIP1. Molecular and cellular biology. 1997, 17: 3850-3857.PubMedPubMed CentralGoogle Scholar
- Simon MA, Bowtell DD, Dodson GS, Laverty TR, Rubin GM: Ras1 and a putative guanine nucleotide exchange factor perform crucial steps in signaling by the sevenless protein tyrosine kinase. Cell. 1991, 67: 701-716. 10.1016/0092-8674(91)90065-7.PubMedGoogle Scholar
- Nizzari M, Venezia V, Repetto E, Caorsi V, Magrassi R, Gagliani MC, Carlo P, Florio T, Schettini G, Tacchetti C, Russo T, Diaspro A, Russo C: Amyloid precursor protein and Presenilin1 interact with the adaptor GRB2 and modulate ERK 1,2 signaling. The Journal of biological chemistry. 2007, 282: 13833-13844. 10.1074/jbc.M610146200.PubMedGoogle Scholar
- Li X, Multon MC, Henin Y, Schweighoffer F, Venot C, Josef J, Zhou C, LaVecchio J, Stuckert P, Raab M, Mhashilkar A, Tocque B, Marasco WA: Grb3-3 is up-regulated in HIV-1-infected T-cells and can potentiate cell activation through NFATc. The Journal of biological chemistry. 2000, 275: 30925-30933. 10.1074/jbc.M005535200.PubMedGoogle Scholar
- Li X, Multon MC, Henin Y, Schweighoffer F, Venot C, LaVecchio J, Josef J, Stuckert P, Mhashilkar A, Tocque B, Marasco WA: Upregulation of the apoptosis-associated protein Grb3-3 in HIV-1-infected human CD4(+) lymphocytes. Biochemical and biophysical research communications. 2000, 276: 362-370. 10.1006/bbrc.2000.3415.PubMedGoogle Scholar
- Maghazachi AA: Intracellular signalling pathways induced by chemokines in natural killer cells. Cellular signalling. 1999, 11: 385-390. 10.1016/S0898-6568(99)00008-X.PubMedGoogle Scholar
- al-Aoukaty A, Schall TJ, Maghazachi AA: Differential coupling of CC chemokine receptors to multiple heterotrimeric G proteins in human interleukin-2-activated natural killer cells. Blood. 1996, 87: 4255-4260.PubMedGoogle Scholar
- Dikic I, Dikic I, Schlessinger J: Identification of a new Pyk2 isoform implicated in chemokine and antigen receptor signaling. The Journal of biological chemistry. 1998, 273: 14301-14308. 10.1074/jbc.273.23.14301.PubMedGoogle Scholar
- Ganju RK, Dutt P, Wu L, Newman W, Avraham H, Avraham S, Groopman JE: Beta-chemokine receptor CCR5 signals via the novel tyrosine kinase RAFTK. Blood. 1998, 91: 791-797.PubMedGoogle Scholar
- Nishimoto I, Okamoto T, Matsuura Y, Takahashi S, Okamoto T, Murayama Y, Ogata E: Alzheimer amyloid protein precursor complexes with brain GTP-binding protein G(o). Nature. 1993, 362: 75-79. 10.1038/362075a0.PubMedGoogle Scholar
- Smine A, Xu X, Nishiyama K, Katada T, Gambetti P, Yadav SP, Wu X, Shi YC, Yasuhara S, Homburger V, Okamoto T: Regulation of brain G-protein go by Alzheimer's disease gene presenilin-1. The Journal of biological chemistry. 1998, 273: 16281-16288. 10.1074/jbc.273.26.16281.PubMedGoogle Scholar
- Cowburn RF, Fowler CJ, O'Neill C: Neurotransmitters, signal transduction and second-messengers in Alzheimer's disease. Acta neurologica Scandinavica. 1996, 165: 25-32.PubMedGoogle Scholar
- Maghazachi AA, Skalhegg BS, Rolstad B, Al-Aoukaty A: Interferon-inducible protein-10 and lymphotactin induce the chemotaxis and mobilization of intracellular calcium in natural killer cells through pertussis toxin-sensitive and -insensitive heterotrimeric G-proteins. Faseb J. 1997, 11: 765-774.PubMedGoogle Scholar
- Giambarella U, Yamatsuji T, Okamoto T, Matsui T, Ikezu T, Murayama Y, Levine MA, Katz A, Gautam N, Nishimoto I: G protein betagamma complex-mediated apoptosis by familial Alzheimer's disease mutant of APP. The EMBO journal. 1997, 16: 4897-4907. 10.1093/emboj/16.16.4897.PubMedPubMed CentralGoogle Scholar
- Vijay-Kumar S, Kumar VD: Neurocalcin. Role in neuronal signaling. Methods in molecular biology (Clifton, NJ). 2002, 172: 261-279.Google Scholar
- Okazaki K, Iino S, Inoue S, Kobayashi S, Hidaka H: Differential distribution of neurocalcin isoforms in rat spinal cord, dorsal root ganglia and muscle spindle. Biochimica et biophysica acta. 1994, 1223: 311-317.PubMedGoogle Scholar
- Stanley LC, Mrak RE, Woody RC, Perrot LJ, Zhang S, Marshak DR, Nelson SJ, Griffin WS: Glial cytokines as neuropathogenic factors in HIV infection: pathogenic similarities to Alzheimer's disease. Journal of neuropathology and experimental neurology. 1994, 53: 231-238. 10.1097/00005072-199405000-00003.PubMedGoogle Scholar
- Shimohama S, Chachin M, Taniguchi T, Hidaka H, Kimura J: Changes of neurocalcin, a calcium-binding protein, in the brain of patients with Alzheimer's disease. Brain Res. 1996, 716: 233-236. 10.1016/0006-8993(96)00070-4.PubMedGoogle Scholar
- Yokota T, Mishra M, Akatsu H, Tani Y, Miyauchi T, Yamamoto T, Kosaka K, Nagai Y, Sawada T, Heese K: Brain site-specific gene expression analysis in Alzheimer's disease patients. European journal of clinical investigation. 2006, 36: 820-830. 10.1111/j.1365-2362.2006.01722.x.PubMedGoogle Scholar
- Wainberg Z, Oliveira M, Lerner S, Tao Y, Brenner BG: Modulation of stress protein (hsp27 and hsp70) expression in CD4+ lymphocytic cells following acute infection with human immunodeficiency virus type-1. Virology. 1997, 233: 364-373. 10.1006/viro.1997.8618.PubMedGoogle Scholar
- Peng J, Jones GL, Watson K: Stress proteins as biomarkers of oxidative stress: effects of antioxidant supplements. Free radical biology & medicine. 2000, 28: 1598-1606.Google Scholar
- Pocernich CB, Boyd-Kimball D, Poon HF, Thongboonkerd V, Lynn BC, Klein JB, Calebrese V, Nath A, Butterfield DA: Proteomics analysis of human astrocytes expressing the HIV protein Tat. Brain research. 2005, 133: 307-316. 10.1016/j.molbrainres.2004.10.023.PubMedGoogle Scholar
- Kouchi Z, Sorimachi H, Suzuki K, Ishiura S: Proteasome inhibitors induce the association of Alzheimer's amyloid precursor protein with Hsc73. Biochemical and biophysical research communications. 1999, 254: 804-810. 10.1006/bbrc.1998.9977.PubMedGoogle Scholar
- Yoo BC, Kim SH, Cairns N, Fountoulakis M, Lubec G: Deranged expression of molecular chaperones in brains of patients with Alzheimer's disease. Biochemical and biophysical research communications. 2001, 280: 249-258. 10.1006/bbrc.2000.4109.PubMedGoogle Scholar
- Kanninen K, Goldsteins G, Auriola S, Alafuzoff I, Koistinaho J: Glycosylation changes in Alzheimer's disease as revealed by a proteomic approach. Neuroscience letters. 2004, 367: 235-240. 10.1016/j.neulet.2004.06.013.PubMedGoogle Scholar
- Wu JQ, Dwyer DE, Dyer WB, Yang YH, Wang B, Saksena NK: Transcriptional profiles in CD8+ T cells from HIV+ progressors on HAART are characterized by coordinated up-regulation of oxidative phosphorylation enzymes and interferon responses. Virology. 2008, 380: 124-135. 10.1016/j.virol.2008.06.039.PubMedGoogle Scholar
- Hunt MC, Alexson SE: The role Acyl-CoA thioesterases play in mediating intracellular lipid metabolism. Progress in lipid research. 2002, 41: 99-130. 10.1016/S0163-7827(01)00017-0.PubMedGoogle Scholar
- Mashek DG, Bornfeldt KE, Coleman RA, Berger J, Bernlohr DA, Black P, DiRusso CC, Farber SA, Guo W, Hashimoto N, Khodiyar V, Kuypers FA, Maltais LJ, Nebert DW, Renieri A, Schaffer JE, Stahl A, Watkins PA, Vasiliou V, Yamamoto TT: Revised nomenclature for the mammalian long-chain acyl-CoA synthetase gene family. Journal of lipid research. 2004, 45: 1958-1961. 10.1194/jlr.E400002-JLR200.PubMedGoogle Scholar
- Watanabe H, Shiratori T, Shoji H, Miyatake S, Okazaki Y, Ikuta K, Sato T, Saito T: A novel acyl-CoA thioesterase enhances its enzymatic activity by direct binding with HIV Nef. Biochemical and biophysical research communications. 1997, 238: 234-239. 10.1006/bbrc.1997.7217.PubMedGoogle Scholar
- Liu LX, Margottin F, Le Gall S, Schwartz O, Selig L, Benarous R, Benichou S: Binding of HIV-1 Nef to a novel thioesterase enzyme correlates with Nef-mediated CD4 down-regulation. The Journal of biological chemistry. 1997, 272: 13779-13785. 10.1074/jbc.272.21.13779.PubMedGoogle Scholar
- Periquet M, Corti O, Jacquier S, Brice A: Proteomic analysis of parkin knockout mice: alterations in energy metabolism, protein handling and synaptic function. Journal of neurochemistry. 2005, 95: 1259-1276. 10.1111/j.1471-4159.2005.03442.x.PubMedGoogle Scholar
- Clary DO, Griff IC, Rothman JE: SNAPs, a family of NSF attachment proteins involved in intracellular membrane fusion in animals and yeast. Cell. 1990, 61: 709-721. 10.1016/0092-8674(90)90482-T.PubMedGoogle Scholar
- Puschel AW, O'Connor V, Betz H: The N-ethylmaleimide-sensitive fusion protein (NSF) is preferentially expressed in the nervous system. FEBS letters. 1994, 347: 55-58. 10.1016/0014-5793(94)00505-2.PubMedGoogle Scholar
- Schiavo G, Gmachl MJ, Stenbeck G, Sollner TH, Rothman JE: A possible docking and fusion particle for synaptic transmission. Nature. 1995, 378: 733-736. 10.1038/378733a0.PubMedGoogle Scholar
- Matsushita K, Morrell CN, Lowenstein CJ: A novel class of fusion polypeptides inhibits exocytosis. Molecular pharmacology. 2005, 67: 1137-1144. 10.1124/mol.104.004275.PubMedGoogle Scholar
- Molle D, Segura-Morales C, Camus G, Berlioz-Torrent C, Kjems J, Basyuk E, Bertrand E: Endosomal trafficking of HIV-1 gag and genomic RNAs regulates viral egress. The Journal of biological chemistry. 2009, 284: 19727-19743. 10.1074/jbc.M109.019844.PubMedPubMed CentralGoogle Scholar
- Yoo BC, Cairns N, Fountoulakis M, Lubec G: Synaptosomal proteins, beta-soluble N-ethylmaleimide-sensitive factor attachment protein (beta-SNAP), gamma-SNAP and synaptotagmin I in brain of patients with Down syndrome and Alzheimer's disease. Dementia and geriatric cognitive disorders. 2001, 12: 219-225. 10.1159/000051261.PubMedGoogle Scholar
- Tsujimoto Y, Shimizu S: VDAC regulation by the Bcl-2 family of proteins. Cell death and differentiation. 2000, 7: 1174-1181. 10.1038/sj.cdd.4400780.PubMedGoogle Scholar
- Yoo BC, Fountoulakis M, Cairns N, Lubec G: Changes of voltage-dependent anion-selective channel proteins VDAC1 and VDAC2 brain levels in patients with Alzheimer's disease and Down syndrome. Electrophoresis. 2001, 22: 172-179. 10.1002/1522-2683(200101)22:1<172::AID-ELPS172>3.0.CO;2-P.PubMedGoogle Scholar
- Adams JM, Cory S: The Bcl-2 protein family: arbiters of cell survival. Science (New York, NY). 1998, 281: 1322-1326.Google Scholar
- Hodge T, Colombini M: Regulation of metabolite flux through voltage-gating of VDAC channels. The Journal of membrane biology. 1997, 157: 271-279. 10.1007/s002329900235.PubMedGoogle Scholar
- Liu MY, Colombini M: Regulation of mitochondrial respiration by controlling the permeability of the outer membrane through the mitochondrial channel, VDAC. Biochimica et biophysica acta. 1992, 1098: 255-260. 10.1016/S0005-2728(05)80344-5.PubMedGoogle Scholar
- Colleaux L, May M, Belougne J, Lepaslier D, Schwartz C, Fontes M: Localisation of two candidate genes for mental retardation using a YAC physical map of the Xq21.1-21.2 subbands. Journal of medical genetics. 1996, 33: 353-357. 10.1136/jmg.33.5.353.PubMedPubMed CentralGoogle Scholar
- Greber S, Lubec G, Cairns N, Fountoulakis M: Decreased levels of synaptosomal associated protein 25 in the brain of patients with Down syndrome and Alzheimer's disease. Electrophoresis. 1999, 20: 928-934. 10.1002/(SICI)1522-2683(19990101)20:4/5<928::AID-ELPS928>3.0.CO;2-Z.PubMedGoogle Scholar
- Krapfenbauer K, Yoo BC, Cairns N, Lubec G: Differential display reveals deteriorated mRNA levels of NADH3 (complex I) in cerebellum of patients with Down syndrome. Journal of neural transmission. 1999, 57: 211-220.PubMedGoogle Scholar
- Nagy Z, Esiri MM, LeGris M, Matthews PM: Mitochondrial enzyme expression in the hippocampus in relation to Alzheimer-type pathology. Acta neuropathologica. 1999, 97: 346-354. 10.1007/s004010050997.PubMedGoogle Scholar
- Jacotot E, Ravagnan L, Loeffler M, Ferri KF, Vieira HL, Zamzami N, Costantini P, Druillennec S, Hoebeke J, Briand JP, Irinopoulou T, Daugas E, Susin SA, Cointe D, Xie ZH, Reed JC, Roques BP, Kroemer G: The HIV-1 viral protein R induces apoptosis via a direct effect on the mitochondrial permeability transition pore. The Journal of experimental medicine. 2000, 191: 33-46. 10.1084/jem.191.1.33.PubMedPubMed CentralGoogle Scholar
- Ockner RK: Integration of Metabolism, Energetics, and Signal Transduction. Springer. 2004, chapter 13:Google Scholar
- Hoyer S, Oesterreich K, Wagner O: Glucose metabolism as the site of the primary abnormality in early-onset dementia of Alzheimer type?. J Neurol. 1988, 235: 143-148. 10.1007/BF00314304.PubMedGoogle Scholar
- Manczak M, Park BS, Jung Y, Reddy PH: Differential expression of oxidative phosphorylation genes in patients with Alzheimer's disease: implications for early mitochondrial dysfunction and oxidative damage. Neuromolecular medicine. 2004, 5: 147-162. 10.1385/NMM:5:2:147.PubMedGoogle Scholar
- Wallace DC, Shoffner JM, Watts RL, Juncos JL, Torroni A: Mitochondrial oxidative phosphorylation defects in Parkinson's disease. Annals of neurology. 1992, 32: 113-114. 10.1002/ana.410320123.PubMedGoogle Scholar
- Cossarizza A, Troiano L, Mussini C: Mitochondria and HIV infection: the first decade. Journal of biological regulators and homeostatic agents. 2002, 16: 18-24.PubMedGoogle Scholar
- Vignoli AL, Martini I, Haglid KG, Silvestroni L, Augusti-Tocco G, Biagioni S: Neuronal glycolytic pathway impairment induced by HIV envelope glycoprotein gp120. Molecular and cellular biochemistry. 2000, 215: 73-80. 10.1023/A:1026590916661.PubMedGoogle Scholar
- Kimes AS, London ED, Szabo G, Raymon L, Tabakoff B: Reduction of cerebral glucose utilization by the HIV envelope glycoprotein Gp-120. Experimental neurology. 1991, 112: 224-228. 10.1016/0014-4886(91)90073-L.PubMedGoogle Scholar
- Besancon F, Just J, Bourgeade MF, Van Weyenbergh J, Solomon D, Guillozo H, Wietzerbin J, Cayre YE: HIV-1 p17 and IFN-gamma both induce fructose 1,6-bisphosphatase. J Interferon Cytokine Res. 1997, 17: 461-467. 10.1089/jir.1997.17.461.PubMedGoogle Scholar
- Ladha JS, Tripathy MK, Mitra D: Mitochondrial complex I activity is impaired during HIV-1-induced T-cell apoptosis. Cell death and differentiation. 2005, 12: 1417-1428. 10.1038/sj.cdd.4401668.PubMedGoogle Scholar
- Honjo K, van Reekum R, Verhoeff NP: Alzheimer's disease and infection: do infectious agents contribute to progression of Alzheimer's disease?. Alzheimers Dement. 2009, 5: 348-360. 10.1016/j.jalz.2008.12.001.PubMedGoogle Scholar
- Itzhaki RF, Wozniak MA: Herpes simplex virus type 1 in Alzheimer's disease: the enemy within. J Alzheimers Dis. 2008, 13: 393-405.PubMedGoogle Scholar
- Kinoshita J: Pathogens as a cause of Alzheimer's disease. Neurobiology of aging. 2004, 25: 639-640. 10.1016/j.neurobiolaging.2004.02.007.PubMedGoogle Scholar
- Brousseau KM, Filley CM, Kaye K, Kiser JJ, Adler LE, Connick E: Dementia with features of Alzheimer's disease and HIV-associated dementia in an elderly man with AIDS. AIDS (London, England). 2009, 23: 1029-1031.Google Scholar
- Shapshak P, Rodriguez HE, Kayathri R, Levine A, Chiappelli F, Minagar A: Alzheimer's disease and HIV associated dementia related genes: I. location and function. Bioinformation. 2008, 2: 348-357.PubMedPubMed CentralGoogle Scholar
- Burbaeva G, Boksha IS, Tereshkina EB, Savushkina OK, Starodubtseva LI, Turishcheva MS: Glutamate metabolizing enzymes in prefrontal cortex of Alzheimer's disease patients. Neurochemical research. 2005, 30: 1443-1451. 10.1007/s11064-005-8654-x.PubMedGoogle Scholar
- Passingham RE, Bengtsson SL, Lau HC: Medial frontal cortex: from self-generated action to reflection on one's own performance. Trends in cognitive sciences. 2010, 14: 16-21. 10.1016/j.tics.2009.11.001.PubMedPubMed CentralGoogle Scholar
- Zhou L, Rua R, Ng T, Vongrad V, Ho YS, Geczy C, Hsu K, Brew BJ, Saksena NK: Evidence for predilection of macrophage infiltration patterns in the deeper midline and mesial temporal structures of the brain uniquely in patients with HIV-associated dementia. BMC infectious diseases. 2009, 9: 192-10.1186/1471-2334-9-192.PubMedPubMed CentralGoogle Scholar
- Johnston-Wilson NL, Sims CD, Hofmann JP, Anderson L, Shore AD, Torrey EF, Yolken RH: Disease-specific alterations in frontal cortex brain proteins in schizophrenia, bipolar disorder, and major depressive disorder. The Stanley Neuropathology Consortium. Mol Psychiatry. 2000, 5: 142-149. 10.1038/sj.mp.4000696.PubMedGoogle Scholar
- Ovadi J, Orosz F, Hollan S: Functional aspects of cellular microcompartmentation in the development of neurodegeneration: mutation induced aberrant protein-protein associations. Molecular and cellular biochemistry. 2004, 256-257: 83-93. 10.1023/B:MCBI.0000009860.86969.72.PubMedGoogle Scholar
- Sultana R, Boyd-Kimball D, Cai J, Pierce WM, Klein JB, Merchant M, Butterfield DA: Proteomics analysis of the Alzheimer's disease hippocampal proteome. J Alzheimers Dis. 2007, 11: 153-164.PubMedGoogle Scholar
- Sly WS, Hewett-Emmett D, Whyte MP, Yu YS, Tashian RE: Carbonic anhydrase II deficiency identified as the primary defect in the autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification. Proceedings of the National Academy of Sciences of the United States of America. 1983, 80: 2752-2756. 10.1073/pnas.80.9.2752.PubMedPubMed CentralGoogle Scholar
- Sultana R, Boyd-Kimball D, Poon HF, Cai J, Pierce WM, Klein JB, Merchant M, Markesbery WR, Butterfield DA: Redox proteomics identification of oxidized proteins in Alzheimer's disease hippocampus and cerebellum: an approach to understand pathological and biochemical alterations in AD. Neurobiology of aging. 2006, 27: 1564-1576. 10.1016/j.neurobiolaging.2005.09.021.PubMedGoogle Scholar
- Balcz B, Kirchner L, Cairns N, Fountoulakis M, Lubec G: Increased brain protein levels of carbonyl reductase and alcohol dehydrogenase in Down syndrome and Alzheimer's disease. Journal of neural transmission. 2001, 193-201.Google Scholar
- Kim SH, Fountoulakis M, Cairns NJ, Lubec G: Human brain nucleoside diphosphate kinase activity is decreased in Alzheimer's disease and Down syndrome. Biochemical and biophysical research communications. 2002, 296: 970-975. 10.1016/S0006-291X(02)02035-1.PubMedGoogle Scholar
- Lovell MA, Xie C, Markesbery WR: Decreased glutathione transferase activity in brain and ventricular fluid in Alzheimer's disease. Neurology. 1998, 51: 1562-1566.PubMedGoogle Scholar
- Menegon A, Board PG, Blackburn AC, Mellick GD, Le Couteur DG: Parkinson's disease, pesticides, and glutathione transferase polymorphisms. Lancet. 1998, 352: 1344-1346. 10.1016/S0140-6736(98)03453-9.PubMedGoogle Scholar
- Jin LW, Masliah E, Iimoto D, Deteresa R, Mallory M, Sundsmo M, Mori N, Sobel A, Saitoh T: Neurofibrillary tangle-associated alteration of stathmin in Alzheimer's disease. Neurobiology of aging. 1996, 17: 331-341. 10.1016/0197-4580(96)00021-8.PubMedGoogle Scholar
- Zhou D, Noviello C, D'Ambrosio C, Scaloni A, D'Adamio L: Growth factor receptor-bound protein 2 interaction with the tyrosine-phosphorylated tail of amyloid beta precursor protein is mediated by its Src homology 2 domain. The Journal of biological chemistry. 2004, 279: 25374-25380. 10.1074/jbc.M400488200.PubMedGoogle Scholar
- Shimura H, Hattori N, Kubo S, Mizuno Y, Asakawa S, Minoshima S, Shimizu N, Iwai K, Chiba T, Tanaka K, Suzuki T: Familial Parkinson disease gene product, parkin, is a ubiquitin-protein ligase. Nature genetics. 2000, 25: 302-305. 10.1038/77060.PubMedGoogle Scholar
- Sowell RA, Owen JB, Butterfield DA: Proteomics in animal models of Alzheimer's and Parkinson's diseases. Ageing research reviews. 2009, 8: 1-17. 10.1016/j.arr.2008.07.003.PubMedPubMed CentralGoogle Scholar
- Lovell MA, Xie C, Gabbita SP, Markesbery WR: Decreased thioredoxin and increased thioredoxin reductase levels in Alzheimer's disease brain. Free radical biology & medicine. 2000, 28: 418-427.Google Scholar
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