Human immunodeficiency virus-associated depression: contributions of immuno-inflammatory, monoaminergic, neurodegenerative, and neurotrophic pathways.

In the era of greatly improved pharmacological treatment of HIV infection through highly active antiretroviral therapy (HAART), HIV patients experience reduced viral loads, reduced opportunistic infections, increased CD4+ T cell count, and greater life expectancy. Although life expectancy is increased, patients often develop neurological disturbances that may persist for long periods, seriously jeopardizing quality of life and adherence to the medication protocols of HAART. For these reasons, HIV-associated neurological disorders have gained importance in both clinical and basic investigations of HIV infection. Depression is the most prevalent neuropsychiatric disorder among people living with HIV. In this review, we discuss how HIV can predispose infected individuals to depression by several interrelated mechanisms. These include inducing chronic elevation of cytokines through activation of microglia and astrocytes; decreasing monoaminergic function; inducing neurotoxicity, especially in dopaminergic neurons; and reducing brain-derived neurotrophic factor. These viral pathways interact with psychosocial factors to create the depressive state. HIV depression has a great impact on quality of life and implementation of antiretroviral therapy, and thus, recognition of these modes of action is significant for understanding HIV neuropathology and for selecting modalities for pharmacologic treatment.

A nerve growth factor-regulated messenger RNA encodes a new intermediate filament protein.

Differential screening of a cDNA library from the PC12 rat pheochromocytoma cell line previously revealed a clone, clone 73, whose corresponding mRNA is induced by nerve growth factor (NGF). Induction parallels NGF-stimulated PC12 differentiation from a chromaffinlike phenotype to a sympathetic neuronlike phenotype. We report that DNA sequence analysis reveals that clone 73 mRNA encodes an intermediate filament (IF) protein whose predicted amino acid sequence is distinct from the known sequences of other members of the IF protein family. The sequence has highest homology with desmin and vimentin and includes the highly conserved central alpha-helical rod domain with the characteristic heptad repeat of hydrophobic residues, but has lower homology in the amino-terminal head and carboxyl-terminal tail domains. The head domain contains a large number of serine residues which are potential phosphorylation sites. The expression of clone 73 in vivo in the nervous system of the adult rat was investigated by in situ hybridization of clone 73 probes to tissue sections. The mRNA is expressed at high levels in ganglia of the peripheral nervous system, including the superior cervical ganglion (sympathetic), ciliary ganglion (parasympathetic), and dorsal root ganglion (sensory). In the central nervous system, motor nuclei of cranial nerves III, IV, V, VI, VII, X, and XII as well as ventral horn motor neurons and a restricted set of other central nervous system nuclei express the clone 73 mRNA. Tissues apart from those of the nervous system did not in general express the mRNA, with only very low levels detected in adrenal gland. We discuss the implications of these results for the mechanism of NGF-induced PC12 cell differentiation, the pathways of neuronal development in vivo, and the possible function of the clone 73 IF protein and its relationship to other IF proteins.

Methylation-sensitive sequence-specific DNA binding by the c-Myc basic region.

The function of the c-Myc oncoprotein and its role in cell growth control is unclear. A basic region of c-Myc is structurally related to the basic motifs of helix-loop-helix (HLH) and leucine zipper proteins, which provide sequence-specific DNA binding function. The c-Myc basic region was tested for its ability to bind DNA by attaching it to the HLH dimerization interface of the E12 enhancer binding factor. Dimers of the chimeric protein, termed E6, specifically bound an E box element (GGCCACGTGACC) recognized by other HLH proteins in a manner dependent on the integrity of the c-Myc basic motif. Methylation of the core CpG in the E box recognition site specifically inhibited binding by E6, but not by two other HLH proteins. Expression of E6 (but not an E6 DNA binding mutant) suppressed the ability of c-myc to cooperate with H-ras in a rat embryo fibroblast transformation assay, suggesting that the DNA recognition specificity of E6 is related to that of c-Myc in vivo.

Stimulation of neuronal acetylcholine receptors induces rapid gene transcription.

Cholinergic agonists rapidly and transiently induced transcription of the c-fos protooncogene and one or more actin genes in neuronally differentiated PC12 cells. Transcription was activated within minutes after stimulation of the nicotinic acetylcholine receptor and required an influx of extracellular Ca2+ ions through voltage-sensitive calcium channels. Nicotine activation proceeded by a different pathway from activation by nerve growth factor, whose stimulation of these genes is independent of extracellular Ca2+ ions. These findings suggest that neurotransmitters may rapidly activate specific gene transcription in nondividing neuronally differentiated cells. They also suggest a functional role for neurotransmitter induction of c-fos and actin expression in the nervous system.

Structure of the gene encoding peripherin, an NGF-regulated neuronal-specific type III intermediate filament protein.

We have cloned the rat gene encoding peripherin, a neuronal-specific intermediate filament protein that is NGF-regulated. Determination of the complete sequence, including 821 nucleotides of the 5'-flanking region, allows us to make conclusions about the evolutionary origin of the peripherin gene, its homology with other intermediate filament proteins, and possible mechanisms of regulation of peripherin expression in neurons. The positions of the eight peripherin gene introns correspond to the intron patterns of desmin, vimentin, and GFAP, with one example of intron sliding. Together with protein sequence homologies, this conclusively demonstrates that peripherin is a type III intermediate filament protein. The peripherin promoter contains sequences homologous to regions of other NGF-regulated promoters, which may function in peripherin induction by NGF.

Differential spatial and temporal expression of two type III intermediate filament proteins in olfactory receptor neurons.

Olfactory receptor neurons (ORNs) do not express the typical neuronal intermediate filament proteins (IFPs), the neurofilament triplet proteins. Immunocytochemical evidence shows that ORNs coexpress vimentin and peripherin but distribute them differently. Specifically, ORNs contain vimentin in dendrites, cell bodies, and axons, but not in terminals in glomeruli; peripherin is present in axons, but excluded from dendrites, cell bodies, and terminal glomeruli. In adult rats, ORN axon fascicles are variably stained with antisera for peripherin; in juvenile rats, staining of fascicles is uniform. Staining with antibody to vimentin is uniform in both adult and juvenile ORN axon fascicles. The unusual pattern of IFP expression and intracellular sorting may have implications for the unique plastic and regenerative capacities of these neurons.

Mutagenesis reveals a role for ABP/GRIP binding to GluR2 in synaptic surface accumulation of the AMPA receptor.

We studied the role of PDZ proteins GRIP, ABP, and PICK1 in GluR2 AMPA receptor trafficking. An epitope-tagged MycGluR2 subunit, when expressed in hippocampal cultured neurons, was specifically targeted to the synaptic surface. With the mutant MycGluR2delta1-10, which lacks the PDZ binding site, the overall dendritic intracellular transport and the synaptic surface targeting were not affected. However, over time, Myc-GluR2delta1-10 accumulated at synapses significantly less than MycGluR2. Notably, a single residue substitution, S880A, which blocks binding to ABP/GRIP but not to PICK1, reduced synaptic accumulation to the same extent as the PDZ site truncation. We conclude that the association of GluR2 with ABP and/or GRIP but not PICK1 is essential for maintaining the synaptic surface accumulation of the receptor, possibly by limiting its endocytotic rate.

The AMPA receptor GluR2 C terminus can mediate a reversible, ATP-dependent interaction with NSF and alpha- and beta-SNAPs.

In this study, we demonstrate specific interaction of the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit C-terminal peptide with an ATPase N-ethylmaleimide-sensitive fusion protein (NSF) and alpha- and beta-soluble NSF attachment proteins (SNAPs), as well as dendritic colocalization of these proteins. The assembly of the GluR2-NSF-SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the SNAP receptor (SNARE) membrane fusion apparatus. We provide evidence that the molar ratio of NSF to SNAP in the GluR2-NSF-SNAP complex is similar to that of the t-SNARE syntaxin-NSF-SNAP complex. NSF is known to disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis. We propose a model in which NSF functions as a chaperone in the molecular processing of the AMPA receptor.

Novel anchorage of GluR2/3 to the postsynaptic density by the AMPA receptor-binding protein ABP.

We report the cloning of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor-binding protein (ABP), a postsynaptic density (PSD) protein related to glutamate receptor-interacting protein (GRIP) with two sets of three PDZ domains, which binds the GluR2/3 AMPA receptor subunits. ABP exhibits widespread CNS expression and is found at the postsynaptic membrane. We show that the protein interactions of the ABP/GRIP family differ from the PSD-95 family, which binds N-methyl-D-aspartate (NMDA) receptors. ABP binds to the GluR2/3 C-terminal VKI-COOH motif via class II hydrophobic PDZ interactions, distinct from the class I PSD-95-NMDA receptor interaction. ABP and GRIP also form homo- and heteromultimers through PDZ-PDZ interactions but do not bind PSD-95. We suggest that the ABP/GRIP and PSD-95 families form distinct scaffolds that anchor, respectively, AMPA and NMDA receptors.

A new bind for Myc.

Recent studies centered on the c-Myc basic/helix-loop-helix/leucine zipper (B/HLH/LZ) motifs have led to the identification of a DNA recognition sequence for c-Myc and the isolation of a novel protein that forms a DNA-binding complex with c-Myc in vitro. These advances may make it possible to address directly the long-standing question of c-Myc function in vivo.

Altered transcriptional activity of c-fos promoter plasmids in v-raf-transformed NIH 3T3 cells.

In cells transformed by v-raf, an oncogenic counterpart of the serine/threonine kinase Raf-1, regulatory elements of the c-fos promoter were active under conditions of cell growth or stimulation for which they were inactive in untransformed control cells. This suggests that v-raf transforms by deregulating transcription of early response genes.

Functional analysis of an isolated fos promoter element with AP-1 site homology reveals cell type-specific transcriptional properties.

A DNA element located at positions -295 to -289 of the c-fos promoter (FAP site) is highly homologous to a consensus 12-O-tetradecanoyl phorbol-13-acetate-responsive element (TRE) and to a cyclic AMP (cAMP)-responsive element (CRE). We found that an oligonucleotide containing the FAP element was a transcription regulator which was distinct from both the TRE and CRE. When cloned in multiple copies in front of a reporter gene in HeLa cells, the FAP oligonucleotide was a powerful constitutive activator sequence. Conversely, in the same cells, reporter plasmids containing multiple copies of the TRE of the human metallothionein gene required phorbol esters for their induction. In PC12 cells, the FAP oligonucleotide was cAMP responsive. Its activity was mediated through a cAMP-dependent protein kinase II and did not rely on ongoing protein synthesis for activation. Adenovirus E1a proteins activated viral promoters through ATF (activation transcription factor) consensus binding sequences identical to the CRE. However, E1a repressed the FAP oligonucleotide-associated transcriptional activity in HeLa cells. In PC12 cells, E1a neither transactivated nor transrepressed the basal and cAMP-stimulated FAP activity. In contrast, the CRE of the human c-fos promoter located at -60 was weakly induced by cAMP and E1a in both HeLa and PC12 cells. We suggest that the FAP oligonucleotide acts through a factor(s) distinct from those employed by the TRE and CRE and that the FAP-associated protein factor(s) may differ in HeLa and PC12 cells in expression or posttranslational regulation.

The amino-terminal region of the adenovirus serotype 5 E1a protein performs two separate functions when expressed in primary baby rat kidney cells.

Adenovirus serotype 5 E1a proteins immortalize primary cells and in cooperation with products of a second oncogene, such as adenovirus serotype 5 E1b or EJ ras, produce full transformation. E1a also activates transcription of specific viral and cellular promoters, represses enhancer-dependent genes, and induces cellular DNA synthesis in quiescent cells. Comparison of different adenovirus serotypes has identified three conserved regions in the E1a protein sequence. We have analyzed E1a mutants with deletions-linker insertions in or preceding the first conserved region, region 1 (amino acids 40 through 77 of adenovirus serotype 5 E1a). E1a mutants which have in-frame deletions-substitutions in region 1 or pre-region 1 sequences were reconstructed into adenovirus to yield a total of 14 mutant viruses. All the mutant viruses showed wild-type growth in HeLa cells, confirming that region 1 is nonessential in these cells. However, we show that region 1 provides two distinct functions in infected primary rodent cells. One function is essential for induction of cell DNA synthesis, and the other is essential for focus formation. In addition, our results are consistent with a requirement for the DNA induction function in focus formation.

Repression of insulin gene expression by adenovirus type 5 E1a proteins.

Insulin gene transcription relies on enhancer and promoter elements which are active in pancreatic beta cells. We showed that adenovirus type 5 infection of HIT T-15 cells, a transformed hamster beta cell line, represses insulin gene transcription and mRNA levels. Using expression plasmids transiently introduced into HIT T-15 cells, we showed that adenovirus type 5 E1a transcription regulatory proteins repress insulin enhancer-promoter element activity as assayed with a surrogate xanthine-guanine phosphoribosyltransferase gene. We relate E1a repression of the insulin gene to other examples of repression of enhancer-dependent genes by E1a and discuss the possible relationship of this repression to insulin gene regulation.

The nerve growth factor-responsive PC12 cell line does not express the Myc dimerization partner Max.

Heterodimerization of Max with the nuclear oncoprotein Myc and the differentiation-associated proteins Mad and Mxi1 enables these factors to bind E-box sites in DNA and control genes implicated in cell proliferation and differentiation. We show that in the PC12 pheochromocytoma tumor cell line, functional Max protein is not expressed because of the synthesis of a mutant max transcript. This transcript encodes a protein incapable of homo- or heterodimerization. Furthermore, the mutant Max protein, unlike wild-type Max, is incapable of repressing transcription from an E-box element. Synthesis of mutant max transcripts appears to be due to a homozygous chromosomal alteration within the max gene. Reintroduction of max into PC12 cells results in repression of E-box-dependent transcription and a reduction in growth rate, which may explain the loss of Max expression either during the growth of the pheochromocytoma or in subsequent passage of the PC12 cell line in vitro. Finally, the ability of these cells to divide, differentiate, and apoptose in the absence of Max demonstrates for the first time that these processes can occur via Max- and possibly Myc-independent mechanisms.

HeLa cell beta-tubulin gene transcription is stimulated by adenovirus 5 in parallel with viral early genes by an E1a-dependent mechanism.

We report that the rate of transcription of cellular beta-tubulin genes increases during the early phase of adenovirus infection of HeLa cells, with kinetics very similar to those routinely found for viral genes. This activation depends upon adenovirus early region E1a, which encodes products that activate early virus transcription. To compare the responses of viral and cellular genes to E1a, we infected HeLa cells with dl312, a transcriptionally inactive deletion mutant that lacks a functional E1a gene. We then superinfected the cells with a helper virus, dl327, which encodes active E1a products, and measured changes in the rates of transcription of various cell and viral genes. Early region E3 of dl312 was activated 0 to 6 h postinfection and then repressed at 8 h postinfection, thus reproducing the two-step kinetics characteristic of a wild-type infection. Synthesis of beta-tubulin nuclear RNA was also transiently induced two- to six-fold, rising and falling in a manner similar to E3 transcription. An increase in helper virus multiplicity gave an increase in beta-tubulin stimulation, but dl312 alone, even at a high multiplicity of infection, gave no induction, confirming the requirement for E1a. beta-Actin nuclear RNA was actively synthesized before infection, but it was not further stimulated by the virus. Cellular beta-globin gene transcription was not stimulated by the virus, although transcription of a transfected beta-globin plasmid was induced by the virus or from a cotransfected E1a expression plasmid. We conclude that adenovirus 5 can stimulate beta-tubulin gene transcription. We discuss the significance for the viral life cycle of viral stimulation of cell genes and consider possible mechanisms in the light of the results obtained with beta-actin and beta-tubulin.

Selective inhibition of adenovirus type 2 early region II and III transcription by an anisomycin block of protein synthesis.

The transcription of adenovirus type 2 genes proceeds through a broad three-phase program. From 1 to 4 h postinfection six early transcription units (EIa, EIb, EII, EIII, EIV, and the promoter-proximal segment of the late transcription unit) are activated. From 4 to 6 h postinfection transcription of the early genes is depressed. After the onset of viral DNA replication at approximately 6 h postinfection, the transcript from the late promoter is antiterminated, and this transcript dominates viral RNA synthesis. The early activation period also proceeds through a series of stages; early regions EIa and EIV are activated first, followed by early region EII. We show that in the presence of anisomycin, a stringent inhibitor of protein synthesis, nuclear RNA and cytoplasmic polyadenylated RNA from regions EIa and EIV accumulate at normal rates, whereas RNAs from regions EII and EIII do not accumulate. We also show that failure to accumulate RNAs from regions EII and EIII is due to reduction of the rate of transcription by greater than 90%. We conclude that the regulation of the promoters for early regions EII and EIII is distinct from the mechanism that operates on the other early transcription units. The promoters for early regions EII and EIII diverge and lie approximately 500 nucleotides apart. We examined the structure of viral chromatin in this region early during infection by mild DNase I digestion in isolated nuclei and indirect end labeling with a DNA probe near these promoters. In control, drug-free cells where EII and EIII are transcribed and in anisomycin-treated cells where EII and EIII are not transcribed we detect the same regular DNase I pattern. We suggest that regulation of EII and EIII is not mediated through a change in gross chromatin structure, but rather by a viral effector, possibly a product of region EIa.

Identification and characterization of mRNAs regulated by nerve growth factor in PC12 cells.

Differential screening of cDNA libraries was used to detect and prepare probes for mRNAs that are regulated in PC12 rat pheochromocytoma cells by long-term (2-week) treatment with nerve growth factor (NGF). In response to NGF, PC12 cells change from a chromaffin cell-like to a sympathetic-neuron-like phenotype. Thus, one aim of this study was to identify NGF-regulated mRNAs that may be associated with the attainment of neuronal properties. Eight NGF-regulated mRNAs are described. Five of these increase 3- to 10-fold and three decrease 2- to 10-fold after long-term NGF exposure. Each mRNA was characterized with respect to the time course of the NGF response, regulation by agents other than NGF, and rat tissue distribution. Partial sequences of the cDNAs were used to search for homologies to known sequences. Homology analysis revealed that one mRNA (increased 10-fold) encodes the peptide thymosin beta 4 and a second mRNA (decreased 2-fold) encodes tyrosine hydroxylase. Another of the increased mRNAs was very abundant in sympathetic ganglia, barely detectable in brain and adrenals, and undetectable in all other tissues surveyed. One of the decreased mRNAs, by contrast, was very abundant in the adrenals and nearly absent in the sympathetic ganglia. With the exception of fibroblast growth factor, which is the only other agent known to mimic the differentiating effects of NGF on PC12 cells, none of the treatments tested (epidermal growth factor, insulin, dibutyryl cyclic AMP, dexamethasone, phorbol ester, and depolarization) reproduced the regulation observed with NGF. These and additional findings suggest that the NGF-regulated mRNAs may play roles in the establishment of the neuronal phenotype and that the probes described here will be useful to study the mechanism of action of NGF and the development and differentiation of neurons.

Effect of protein synthesis inhibitors on growth factor activation of c-fos, c-myc, and actin gene transcription.

Stimulation of quiescent 3T3 cells with purified growth factors or of the pheochromocytoma cell line PC12 with nerve growth factor results in the rapid transient induction of c-fos, c-myc, and actin gene transcription (M.E. Greenberg and E.B. Ziff, Nature [London] 312:711-716; M.E. Greenberg, L.A. Greene, and E.B. Ziff, J. Biol. Chem. 26:14101-14110). We used protein synthesis inhibitors to investigate whether synthesis of new proteins plays a role in the rapid induction and subsequent repression of the transcription of these genes. Pretreatment of quiescent 3T3 cells with the inhibitor anisomycin before growth factor stimulation caused a superinduction of c-fos and c-myc mRNA levels upon growth factor addition. Nuclear runoff transcription analyses of 3T3 cells indicated that anisomycin potentiated c-fos, c-myc, and also actin expression at the transcriptional level, possibly by inhibiting transcriptional repression. Somewhat different results were obtained when PC12 cells were incubated with either anisomycin or cycloheximide. In PC12 cells protein synthesis inhibitors superinduced nerve growth factor activation of c-fos mRNA production but completely abolished the activation of c-myc. The results suggest that in PC12 cells c-fos transcription is activated by a protein-synthesis-independent mechanism, whereas c-myc stimulation requires new protein synthesis. The difference in the effect of anisomycin on growth factor activation of c-myc expression in 3T3 versus PC12 cells may be due to differential stringency of protein synthesis inhibition in the two cells or could reflect cell type differences in c-myc regulation.

Mutation of the c-fos gene dyad symmetry element inhibits serum inducibility of transcription in vivo and the nuclear regulatory factor binding in vitro.

In vitro mutagenesis of a 61-base-pair DNA sequence element that is necessary for induction of the c-fos proto-oncogene by growth factors revealed that a small region of dyad symmetry within the sequence element is critical for c-fos transcriptional activation. The same c-fos dyad symmetry element was found to bind a nuclear protein in vitro, causing a specific mobility shift of this c-fos regulatory sequence. An analysis of insertion and deletion mutants established a strict correlation between the ability of the dyad symmetry element to promote serum activation of c-fos transcription and in vitro nuclear protein binding. These experiments suggest that the DNA mobility shift assay detects a nuclear protein that mediates growth factor stimulation of c-fos expression. In vitro competition experiments indicate that the c-fos regulatory factor also binds to sequences within another growth factor-inducible gene, the beta-actin gene.