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1.
Neuroscience ; 359: 224-247, 2017 09 17.
Article in English | MEDLINE | ID: mdl-28754312

ABSTRACT

Many clinical studies have reported on the benefits of exercise therapy in patients with Parkinson's disease (PD). Exercise cannot stop the progression of PD or facilitate the recovery of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc) (Bega et al., 2014). To tease apart this paradox, we utilized a progressive MPTP (1-methyl-4-phenyl-1,2,3,6-tetra-hydropyridine) mouse model in which we initiated 4weeks of treadmill exercise after the completion of toxin administration (i.e., restoration). We found in our MPTP/exercise (MPTP+EX) group several measures of gait function that recovered compared to the MPTP only group. Although there was a small recovery of tyrosine hydroxylase (TH) positive DA neurons in the SNpc and terminals in the striatum, this increase was not statistically significant. These small changes in TH could not explain the improvement of motor function. The MPTP group had a significant 170% increase in the glycosylated/non-glycosylated dopamine transporter (DAT) and a 200% increase in microglial marker, IBA-1, in the striatum. The MPTP+EX group showed a nearly full recovery of these markers back to the vehicle levels. There was an increase in GLT-1 levels in the striatum due to exercise, with no change in striatal BDNF protein expression. Our data suggest that motor recovery was not prompted by any significant restoration of DA neurons or terminals, but rather the recovery of DAT and dampening the inflammatory response. Although exercise does not promote recovery of nigrostriatal DA, it should be used in conjunction with pharmaceutical methods for controlling PD symptoms.


Subject(s)
Corpus Striatum/physiopathology , Motor Activity , Parkinson Disease/physiopathology , Physical Conditioning, Animal , Recovery of Function , Substantia Nigra/physiopathology , Animals , Calcium-Binding Proteins/metabolism , Cell Count , Corpus Striatum/metabolism , Corpus Striatum/pathology , Disease Models, Animal , Dopamine Plasma Membrane Transport Proteins/metabolism , Dopaminergic Neurons/metabolism , Excitatory Amino Acid Transporter 2/metabolism , Male , Mice, Inbred C57BL , Microfilament Proteins/metabolism , Neural Pathways/metabolism , Neural Pathways/physiopathology , Parkinson Disease/pathology , Parkinson Disease/prevention & control , Parkinsonian Disorders/pathology , Parkinsonian Disorders/physiopathology , Parkinsonian Disorders/prevention & control , Substantia Nigra/metabolism , Substantia Nigra/pathology , Tyrosine 3-Monooxygenase/metabolism
2.
Mol Psychiatry ; 21(4): 574-84, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26303660

ABSTRACT

Latency-reversing agents (LRAs), including histone deacetylase inhibitors (HDACi), are being investigated as a strategy to eliminate latency in HIV-infected patients on suppressive antiretroviral therapy. The effectiveness of LRAs in activating latent infection in HIV strains derived from the central nervous system (CNS) is unknown. Here we show that CNS-derived HIV-1 strains possess polymorphisms within and surrounding the Sp transcription factor motifs in the long terminal repeat (LTR). These polymorphisms result in decreased ability of the transcription factor specificity protein 1 to bind CNS-derived LTRs, reducing the transcriptional activity of CNS-derived viruses. These mutations result in CNS-derived viruses being less responsive to activation by the HDACi panobinostat and romidepsin compared with lymphoid-derived viruses from the same subjects. Our findings suggest that HIV-1 strains residing in the CNS have unique transcriptional regulatory mechanisms, which impact the regulation of latency, the consideration of which is essential for the development of HIV-1 eradication strategies.


Subject(s)
Brain/virology , HIV Infections/virology , HIV-1/physiology , Histone Deacetylase Inhibitors/therapeutic use , Adult , Brain/metabolism , CD4-Positive T-Lymphocytes , Central Nervous System/metabolism , Cohort Studies , Depsipeptides/pharmacology , HIV Infections/drug therapy , HIV-1/genetics , Humans , Hydroxamic Acids/pharmacology , Indoles/pharmacology , Jurkat Cells , Male , Middle Aged , Panobinostat , Polymorphism, Genetic , Terminal Repeat Sequences , Transcriptional Activation , Virus Latency/drug effects
3.
Neuroscience ; 299: 156-74, 2015 Jul 23.
Article in English | MEDLINE | ID: mdl-25943481

ABSTRACT

Many studies have investigated exercise therapy in Parkinson's disease (PD) and have shown benefits in improving motor deficits. However, exercise does not slow down the progression of the disease or induce the revival of lost nigrostriatal neurons. To examine the dichotomy of behavioral improvement without the slowing or recovery of dopaminergic cell or terminal loss, we tested exercise therapy in an intervention paradigm where voluntary running wheels were installed half-way through our progressive PD mouse model. In our model, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is administered over 4 weeks with increased doses each week (8, 16, 24, 32-kg/mg). We found that after 4 weeks of MPTP treatment, mice that volunteered to exercise had behavioral recovery in several measures despite the loss of 73% and 53% tyrosine hydroxylase (TH) within the dorsolateral (DL) striatum and the substantia nigra (SN), respectively which was equivalent to the loss seen in the mice that did not exercise but were also administered MPTP for 4 weeks. Mice treated with 4 weeks of MPTP showed a 41% loss of vesicular monoamine transporter II (VMAT2), a 71% increase in the ratio of glycosylated/non-glycosylated dopamine transporter (DAT), and significant increases in glutamate transporters including VGLUT1, GLT-1, and excitatory amino acid carrier 1. MPTP mice that exercised showed recovery of all these biomarkers back to the levels seen in the vehicle group and showed less inflammation compared to the mice treated with MPTP for 4 weeks. Even though we did not measure tissue dopamine (DA) concentration, our data suggest that exercise does not alleviate motor deficits by sparing nigrostriatal neurons, but perhaps by stabilizing the extraneuronal neurotransmitters, as evident by a recovery of DA and glutamate transporters. However, suppressing inflammation could be another mechanism of this locomotor recovery. Although exercise will not be a successful treatment alone, it could supplement other pharmaceutical approaches to PD therapy.


Subject(s)
Corpus Striatum/metabolism , Neurons/metabolism , Parkinsonian Disorders/metabolism , Parkinsonian Disorders/physiopathology , Physical Conditioning, Animal , Substantia Nigra/metabolism , 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine/administration & dosage , Animals , Corpus Striatum/drug effects , Disease Models, Animal , Dopamine Plasma Membrane Transport Proteins/metabolism , Excitatory Amino Acid Transporter 2/metabolism , Excitatory Amino Acid Transporter 3/metabolism , Gait/drug effects , Hand Strength , Inflammation/metabolism , Male , Mice , Mice, Inbred C57BL , Neurons/drug effects , Parkinsonian Disorders/complications , Recovery of Function , Substantia Nigra/drug effects , Tyrosine 3-Monooxygenase/metabolism , Vesicular Glutamate Transport Protein 1/metabolism , Vesicular Monoamine Transport Proteins/metabolism
4.
Neuroscience ; 290: 454-71, 2015 Apr 02.
Article in English | MEDLINE | ID: mdl-25655214

ABSTRACT

Parkinson's disease (PD) is a progressive neurological disorder and current therapies help alleviate symptoms, but are not disease modifying. In the flavonoid class of compounds, 7,8-dihydroxyflavone (7,8-DHF) has been reported to elicit tyrosine kinase receptor B (TrkB) dimerization and autophosphorylation that further stimulates signaling cascades to promote cell survival/growth, differentiation, and plasticity. In this study we investigated if 7,8-DHF could prevent further loss of dopaminergic cells and terminals if introduced at the midpoint (i.e. intervention) of our progressive mouse model of PD. In our model, 1-methyl-4phenyl-1,2,3,6-tetrahyrdopyridine (MPTP) is administered with increased doses each week (8, 16, 24, 32-kg/mg) over a 4-week period. We found that despite 4 weeks of MPTP treatment, animals administered 7,8-DHF starting at the 2-week time period maintained 54% of the tyrosine hydroxylase (TH) levels within the dorsolateral (DL) striatum compared to the vehicle group, which was comparable to animals treated with MPTP for 2 weeks and was significantly greater compared to animals treated with MPTP for the full 4 weeks. Animals treated with MPTP and 7,8-DHF also demonstrated increased levels of, a sprouting-associated protein, superior cervical ganglion-10 (SCG10), phosphorylated TrkB (pTrkB), and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) within the DL striatum and substantia nigra (SN) compared to the 4-week MPTP-treated animals. In addition, motor deficits seen in the 2- and 4-week MPTP-treated animals were restored following administration of 7,8-DHF. We are reporting here for the first time that intervention with 7,8-DHF blocks further loss of dopaminergic terminals and restores motor deficits in our progressive MPTP mouse model. Our data suggest that 7,8-DHF has the potential to be a translational therapy in PD.


Subject(s)
Antiparkinson Agents/pharmacology , Corpus Striatum/drug effects , Flavones/pharmacology , MPTP Poisoning/drug therapy , Motor Activity/drug effects , Animals , Calcium-Binding Proteins , Corpus Striatum/pathology , Corpus Striatum/physiopathology , Forelimb/drug effects , Forelimb/physiopathology , Intracellular Signaling Peptides and Proteins/metabolism , MPTP Poisoning/pathology , MPTP Poisoning/physiopathology , Male , Mice, Inbred C57BL , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Motor Activity/physiology , Phosphorylation/drug effects , Random Allocation , Receptor, trkB/metabolism , Stathmin , Substantia Nigra/drug effects , Substantia Nigra/pathology , Substantia Nigra/physiopathology , Tyrosine 3-Monooxygenase/metabolism
5.
Neurology ; 70(9): 666-76, 2008 Feb 26.
Article in English | MEDLINE | ID: mdl-17687115

ABSTRACT

OBJECTIVE: The GRIN3B gene encodes NR3B, a motoneuron-specific member of the NMDA type of ionotropic glutamate receptors. NR3B reduces the Ca(2+)-permeability as well as the overall current of the receptor response and may thereby protect motoneurons against glutamate-mediated excitotoxicity. We tested whether genetic dysfunction of GRIN3B is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). METHODS: We searched for mutations in the GRIN3B coding region (3.1 kb) in 117 individuals with familial ALS and in 46 individuals with sporadic ALS. We genotyped the newly identified GRIN3B null allele and four "tag single nucleotide polymorphisms (SNPs)" at the GRIN3B locus in 342 individuals with sporadic ALS and in 374 matched controls. The GRIN3B null allele frequency was determined in 2,128 individuals from a worldwide panel of 42 populations. We furthermore compared the GRIN3B coding sequence in primates (human-macaque) and rodents (rat-mouse) to evaluate the molecular evolution of GRIN3B. RESULTS: Thirty-two SNPs, including 16 previously unreported SNPs, one 27-bp deletion, a polymorphic CAG repeat, and a 4-bp insertion (insCGTT), were identified. Mutational and case-control studies did not reveal variants that cause or modify disease in ALS. Intriguing is an insCGTT variant that truncates the protein at its amino terminus and results in a GRIN3B null allele. We demonstrated a global distribution of the null allele with allele frequencies ranging between 0 and 0.38, and we delineated a null allele specific haplotype of 9.89 kb. Comparative genomic analysis across four taxa demonstrated accelerated evolution of NR3B in primates. CONCLUSIONS: Our study supports the conclusions that 1) GRIN3B does not seem to be associated with familial or sporadic ALS, 2) the GRIN3B null allele is a common polymorphism, 3) the GRIN3B null allele has arisen once and early in human evolution, and 4) the GRIN3B gene belongs to a group of nervous system-related genes that have been subjected to faster evolution during evolution.


Subject(s)
Alleles , Motor Neuron Disease/genetics , Motor Neurons/physiology , Receptors, N-Methyl-D-Aspartate/genetics , Case-Control Studies , DNA Mutational Analysis , Gene Frequency , Genetic Predisposition to Disease/genetics , Genetics, Population , Genotype , Haplotypes , Humans , Linkage Disequilibrium , Motor Neuron Disease/diagnosis , Motor Neuron Disease/physiopathology , Polymorphism, Single Nucleotide/genetics , Superoxide Dismutase/genetics , Superoxide Dismutase-1
6.
AIDS Res Hum Retroviruses ; 17(16): 1481-8, 2001 Nov 01.
Article in English | MEDLINE | ID: mdl-11709092

ABSTRACT

c-Myb is expressed in proliferating T cells. Fifteen c-Myb-binding sites can be identified in the HIV-1 long terminal repeat (LTR), suggesting that c-Myb may regulate HIV-1 gene expression and virus replication. Increasing the cellular levels of c-Myb by transient transfection of CEM cells resulted in a 10- to 20-fold activation of HIV-1 LTR-driven gene expression and mutation of one high-affinity Myb-binding site within the LTR reduced this activation by 60 to 70%. Conversely, inhibition of c-Myb expression in MT-2 cells by treatment with c-myb antisense oligonucleotides decreased HIV-1 replication by 85%, as measured by reverse transcriptase activity and cytopathic effects. The effect of c-myb antisense oligonucleotides on HIV-1 gene expression and virus particle production appeared to be independent of cell proliferation, but dependent on the presence of c-Myb activity mediated through the HIV-1 LTR. These data show that c-myb expression affects HIV-1 replication in CD4(+) T cells.


Subject(s)
Gene Expression Regulation, Viral , HIV-1/genetics , Proto-Oncogene Proteins c-myb/pharmacology , Transcriptional Activation , Virus Replication/drug effects , CD4-Positive T-Lymphocytes/virology , Cell Line , DNA, Viral/metabolism , HIV Long Terminal Repeat/genetics , HIV Long Terminal Repeat/physiology , HIV-1/physiology , Humans , Proto-Oncogene Proteins c-myb/metabolism
7.
Nucleic Acids Res ; 28(3): 663-8, 2000 Feb 01.
Article in English | MEDLINE | ID: mdl-10637316

ABSTRACT

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) represents a model promoter system and the identification and characterisation of cellular proteins that interact with this region has provided a basic understanding about both general eukaryotic and HIV-1 proviral transcriptional regulation. To date a large number of sequence-specific DNA-protein interactions have been described for the HIV-1 LTR. The aim of this report is to provide a comprehensive, updated listing of these HIV-1 LTR interactions. It is intended as a reference point to facilitate on-going studies characterising the identity of cellular proteins interacting with the HIV-1 LTR and the functional role(s) of specific regions of the LTR for HIV-1 replication.


Subject(s)
DNA-Binding Proteins/metabolism , HIV Long Terminal Repeat/genetics , HIV-1/genetics , Promoter Regions, Genetic/genetics , Transcription Factors/metabolism , Enhancer Elements, Genetic/genetics , Gene Expression Regulation, Viral/genetics , Humans , Response Elements/genetics
8.
J Virol ; 73(1): 352-61, 1999 Jan.
Article in English | MEDLINE | ID: mdl-9847339

ABSTRACT

Astrocytes infected with human immunodeficiency virus type 1 (HIV-1) produce only minimal quantities of virus. The molecular events that limit acute-phase HIV-1 infection of astrocytes were examined after inducing acute-phase replication by transfection with the pNL4-3 proviral plasmid. The levels of HIV-1 mRNA were similarly high in both astrocytes and HeLa cells, but astrocytes produced approximately 50-fold less supernatant p24 than HeLa cells. We found that diminished HIV-1 production in astrocytes resulted from inefficient translation of gag, env, and nef mRNAs that were efficiently transported to the cytoplasm. Tat- or Rev-dependent reporter constructs showed no defect in Tat or Rev function in astrocytes compared with HeLa cells. HIV-1 mRNAs were correctly spliced, but only Rev and Tat proteins were efficiently translated from their native mRNAs. Pulse-chase labelling and immunoblot experiments revealed no defect in protein processing, but levels of Gag, Env, or Nef protein expressed were dramatically reduced in astrocytes compared to HeLa cells. These results demonstrate that inefficient translation of HIV-1 structural proteins underlies the restricted infection of astrocytes. The efficient expression of functional Tat and Rev by astrocytes may contribute to HIV-1 neuropathogenesis.


Subject(s)
Astrocytes/virology , Gene Products, rev/biosynthesis , Gene Products, tat/biosynthesis , HIV-1/physiology , Protein Biosynthesis , RNA, Messenger/analysis , 5' Untranslated Regions , Gene Products, env/biosynthesis , Gene Products, gag/biosynthesis , Gene Products, nef/biosynthesis , HIV Core Protein p24/biosynthesis , Humans , Tumor Cells, Cultured , nef Gene Products, Human Immunodeficiency Virus , rev Gene Products, Human Immunodeficiency Virus , tat Gene Products, Human Immunodeficiency Virus
9.
J Virol ; 70(9): 5786-90, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8709194

ABSTRACT

The human immunodeficiency virus type 1 (HIV-1) Rev protein mediates the accumulation of unspliced and singly spliced viral transcripts within the cytoplasm of infected cells, late in the infection cycle, leading to the expression of the viral structural proteins, Gag, Pol, and Env. Rev binds to a complex RNA structure, the Rev-responsive element (RRE), present in all Rev-responsive viral transcripts, relieving their nuclear sequestration. The precise mechanism by which RRE-containing transcripts are retained within the nucleus in the absence of Rev protein is not well understood. We previously demonstrated that the RRE alone plays a crucial role in the nuclear retention of RRE-containing env transcripts in stably transfected Drosophila cells. Here we extend our previous observations and demonstrate that the RRE is a principal determinant of nuclear retention for envelope transcripts in primate cells and, in particular, human CD4+ T cells.


Subject(s)
Gene Products, env/biosynthesis , Gene Products, rev/metabolism , Genes, env , HIV-1/metabolism , Regulatory Sequences, Nucleic Acid , Transcription, Genetic , Animals , Binding Sites , Blotting, Western , Cell Line , Chlorocebus aethiops , Drosophila , Genome, Viral , HIV-1/genetics , HeLa Cells , Humans , RNA, Messenger/biosynthesis , Recombinant Proteins/biosynthesis , Restriction Mapping , Transfection , rev Gene Products, Human Immunodeficiency Virus
10.
Biochemistry ; 33(13): 3807-18, 1994 Apr 05.
Article in English | MEDLINE | ID: mdl-8142383

ABSTRACT

In the oxidized "ES" state of cytochrome c peroxidase, Trp-191 is reversibly oxidized to a stable cation free radical by the hypervalent heme. To explore the potential for engineering a binding site for heterocyclic compounds at this site, the mutant W191G was constructed. Two independent crystal structures of W191G at 2.1- and 2.3-A resolution show that W191G contains a well-defined, approximately 180-A3 cavity at the Trp-191 site. The cavity is occupied by five ordered water molecules which participate in an extensive hydrogen-bonding network with each other, with polar main-chain atoms, and with the carboxylate of Asp-235. After a number of heterocyclic compounds were screened, evidence was obtained that substituted imidazoles bind to the cavity of W191G. Titration of W191G with imidazole resulted in a perturbation of the Soret absorption band that was not observed for W191H, W191F, or the native enzyme. The dissociation constants for binding of benzimidazole, imidazole, 2-ethylimidazole, 1-methylimidazole, 2-methylimidazole, and 1,2-dimethylimidazole to W191G were respectively 2.58, 0.70, 0.36, 0.057, 0.047, and 0.027 mM at pH 6.0. The highest binding affinity was exhibited by 1,2-dimethylimidazole, indicating that steric interactions and the efficiency of filling the cavity are important determinants for specificity. The Kd for imidazole binding increased from 0.7 mM at pH 6 to 3.0 mM at pH 8 and could be fit to a single proton ionization curve with a pKa of 7.4, demonstrating the preferential binding by the imidazolium ion (pKa = 7.3). The binding of a number of substituted imidazoles to the cavity of W191G was verified by X-ray crystallographic analysis. The most clearly defined density was observed for W191G crystals soaked in 1 mM 1,2-dimethylimidazole and was consistent with an oriented occupation in which the unsubstituted nitrogen forms a hydrogen bond or ion pair interaction with Asp-235. Thus, enhanced binding of positively charged molecules may be the result of interactions with this carboxylate. An analogous interaction may stabilize the developing positive charge on the Trp-191 radical of the wild-type enzyme. While the oxidation of imidazoles by the ferryl intermediate of W191G was neither expected nor observed, this study has defined the structural determinants for small molecule binding to an artificially created cavity near a heme center which is capable of generating oxidized species at a potential of over 1 V, and these results will guide future attempts for novel substrate oxidation by CCP.


Subject(s)
Cytochrome-c Peroxidase/metabolism , Amino Acid Sequence , Binding Sites , Crystallography, X-Ray , Cytochrome-c Peroxidase/chemistry , Hydrogen-Ion Concentration , Imidazoles/chemistry , Kinetics , Ligands , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Denaturation , Saccharomyces cerevisiae/enzymology , Structure-Activity Relationship
11.
Gene ; 94(2): 223-8, 1990 Oct 15.
Article in English | MEDLINE | ID: mdl-2258054

ABSTRACT

We have previously described a colorimetric test, designated an amplified DNA assay (ADA), for specific segments of DNA amplified by polymerase chain reactions (PCRs), suited to diagnostic applications. This relied on binding the amplified DNA via a sequence in one oligodeoxyribonucleotide (oligo) to the DNA-binding protein GCN4 coated on the wells of a microtiter dish. Avidin-peroxidase was then bound to biotin at the 5' end of the other oligo and detected colorimetrically. Two successive PCRs with nested oligos were utilized. We describe here several modifications that greatly simplify the ADA. First, we bind the DNA to a glutathione S-transferase-GCN4 fused polypeptide (GST-GCN4) and avidin-peroxidase simultaneously, rather than successively. Second, we carry out the two successive PCRs in the one reaction mixture, using the thermal stabilities of oligos of differing lengths to separate the two reactions. Third, PCRs can be performed in the wells of a microtiter dish and the amplified DNA captured and detected via GST-GCN4 immobilized on beads attached to the lid of the microtiter dish. Hence it is only necessary to pipette the DNA sample once, and up to 96 samples can then be handled simultaneously.


Subject(s)
Acquired Immunodeficiency Syndrome/genetics , Colorimetry/methods , HIV/genetics , Protein Kinases , Saccharomyces cerevisiae Proteins , Cells, Cultured , DNA-Binding Proteins , Enzyme-Linked Immunosorbent Assay , Fungal Proteins , Glutathione Transferase , Humans , In Vitro Techniques , Polymerase Chain Reaction , Transcription Factors
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