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1.
Mol Psychiatry ; 22(7): 981-989, 2017 07.
Article in English | MEDLINE | ID: mdl-27777419

ABSTRACT

Recent data suggest that intraneuronal accumulation of metabolites of the amyloid-ß-precursor protein (APP) is neurotoxic. We observed that transgenic mice overexpressing in neurons a human APP gene harboring the APPE693Q (Dutch) mutation have intraneuronal lysosomal accumulation of APP carboxylterminal fragments (APP-CTFs) and oligomeric amyloid ß (oAß) but no histological evidence of amyloid deposition. Morphometric quantification using the lysosomal marker protein 2 (LAMP-2) immunolabeling showed higher neuronal lysosomal counts in brain of 12-months-old APPE693Q as compared with age-matched non-transgenic littermates, and western blots showed increased lysosomal proteins including LAMP-2, cathepsin D and LC3. At 24 months of age, these mice also exhibited an accumulation of α-synuclein in the brain, along with increased conversion of LC3-I to LC3-II, an autophagosomal/autolysosomal marker. In addition to lysosomal changes at 12 months of age, these mice developed cholinergic neuronal loss in the basal forebrain, GABAergic neuronal loss in the cortex, hippocampus and basal forebrain and gliosis and microgliosis in the hippocampus. These findings suggest a role for the intraneuronal accumulation of oAß and APP-CTFs and resultant lysosomal pathology at early stages of Alzheimer's disease-related pathology.


Subject(s)
Amyloid beta-Peptides/metabolism , Lysosomal-Associated Membrane Protein 2/metabolism , Proteins/metabolism , Alzheimer Disease/genetics , Amyloid beta-Protein Precursor/genetics , Animals , Brain/metabolism , Cerebral Cortex/metabolism , Disease Models, Animal , Hippocampus/metabolism , Humans , Lysosomal-Associated Membrane Protein 2/genetics , Mice , Mice, Transgenic , Neurons/metabolism , Peptide Fragments/metabolism , alpha-Synuclein/metabolism
3.
Mol Psychiatry ; 21(8): 1099-111, 2016 08.
Article in English | MEDLINE | ID: mdl-26552589

ABSTRACT

Identification and characterization of molecular mechanisms that connect genetic risk factors to initiation and evolution of disease pathophysiology represent major goals and opportunities for improving therapeutic and diagnostic outcomes in Alzheimer's disease (AD). Integrative genomic analysis of the human AD brain transcriptome holds potential for revealing novel mechanisms of dysfunction that underlie the onset and/or progression of the disease. We performed an integrative genomic analysis of brain tissue-derived transcriptomes measured from two lines of mice expressing distinct mutant AD-related proteins. The first line expresses oligomerogenic mutant APP(E693Q) inside neurons, leading to the accumulation of amyloid beta (Aß) oligomers and behavioral impairment, but never develops parenchymal fibrillar amyloid deposits. The second line expresses APP(KM670/671NL)/PSEN1(Δexon9) in neurons and accumulates fibrillar Aß amyloid and amyloid plaques accompanied by neuritic dystrophy and behavioral impairment. We performed RNA sequencing analyses of the dentate gyrus and entorhinal cortex from each line and from wild-type mice. We then performed an integrative genomic analysis to identify dysregulated molecules and pathways, comparing transgenic mice with wild-type controls as well as to each other. We also compared these results with datasets derived from human AD brain. Differential gene and exon expression analysis revealed pervasive alterations in APP/Aß metabolism, epigenetic control of neurogenesis, cytoskeletal organization and extracellular matrix (ECM) regulation. Comparative molecular analysis converged on FMR1 (Fragile X Mental Retardation 1), an important negative regulator of APP translation and oligomerogenesis in the post-synaptic space. Integration of these transcriptomic results with human postmortem AD gene networks, differential expression and differential splicing signatures identified significant similarities in pathway dysregulation, including ECM regulation and neurogenesis, as well as strong overlap with AD-associated co-expression network structures. The strong overlap in molecular systems features supports the relevance of these findings from the AD mouse models to human AD.


Subject(s)
Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Presenilin-1/genetics , Alzheimer Disease/genetics , Amyloid beta-Peptides/metabolism , Animals , Brain/metabolism , Disease Models, Animal , Fibrillar Collagens , Fragile X Mental Retardation Protein/metabolism , Humans , Mice , Mice, Transgenic , Mutation , Neurogenesis , Neurons/metabolism , Plaque, Amyloid/pathology , Risk Factors , Transcriptome/genetics
4.
Mol Psychiatry ; 20(1): 109-17, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25349165

ABSTRACT

Certain mutant Alzheimer's amyloid-ß (Aß) peptides (that is, Dutch mutant APP(E693Q)) form complexes with gangliosides (GAß). These mutant Aß peptides may also undergo accelerated aggregation and accumulation upon exposure to GM2 and GM3. We hypothesized that increasing ß-hexosaminidase (ß-hex) activity would lead to a reduction in GM2 levels, which in turn, would cause a reduction in Aß aggregation and accumulation. The small molecule OT1001 is a ß-hex-targeted pharmacological chaperone with good bioavailability, blood-brain barrier penetration, high selectivity for ß-hex and low cytotoxicity. Dutch APP(E693Q) transgenic mice accumulate oligomeric Aß as they age, as well as Aß oligomer-dose-dependent anxiety and impaired novel object recognition (NOR). Treatment of Dutch APP(E693Q) mice with OT1001 caused a dose-dependent increase in brain ß-hex levels up to threefold over those observed at baseline. OT1001 treatment was associated with reduced anxiety, improved learning behavior in the NOR task and dramatically reduced GAß accumulation in the subiculum and perirhinal cortex, both of which are brain regions required for normal NOR. Pharmacological chaperones that increase ß-hex activity may be useful in reducing accumulation of certain mutant species of Aß and in preventing the associated behavioral pathology.


Subject(s)
Alzheimer Disease/complications , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Antipsychotic Agents/therapeutic use , Cognition Disorders , Gangliosides/metabolism , beta-N-Acetylhexosaminidases/metabolism , Alzheimer Disease/genetics , Animals , Blood-Testis Barrier/drug effects , Cells, Cultured , Cognition Disorders/drug therapy , Cognition Disorders/etiology , Cognition Disorders/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Fibroblasts/drug effects , Fibroblasts/metabolism , Gangliosides/therapeutic use , Humans , Maze Learning/drug effects , Mice , Mice, Inbred C57BL , Mutation/genetics , Recognition, Psychology/drug effects , Time Factors
5.
Mol Psychiatry ; 19(11): 1235-42, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25113378

ABSTRACT

Proneurogenic compounds have recently shown promise in some mouse models of Alzheimer's pathology. Antagonists at Group II metabotropic glutamate receptors (Group II mGluR: mGlu2, mGlu3) are reported to stimulate neurogenesis. Agonists at those receptors trigger γ-secretase-inhibitor-sensitive biogenesis of Aß42 peptides from isolated synaptic terminals, which is selectively suppressed by antagonist pretreatment. We have assessed the therapeutic potential of chronic pharmacological inhibition of Group II mGluR in Dutch APP (Alzheimer's amyloid precursor protein E693Q) transgenic mice that accumulate Dutch amyloid-ß (Aß) oligomers but never develop Aß plaques. BCI-838 is a clinically well-tolerated, orally bioavailable, investigational prodrug that delivers to the brain BCI-632, the active Group II mGluR antagonist metabolite. Dutch Aß-oligomer-forming APP transgenic mice (APP E693Q) were dosed with BCI-838 for 3 months. Chronic treatment with BCI-838 was associated with reversal of transgene-related amnestic behavior, reduction in anxiety, reduction in levels of brain Aß monomers and oligomers, and stimulation of hippocampal neurogenesis. Group II mGluR inhibition may offer a unique package of relevant properties as an Alzheimer's disease therapeutic or prophylactic by providing both attenuation of neuropathology and stimulation of repair.


Subject(s)
Alzheimer Disease/drug therapy , Alzheimer Disease/psychology , Anxiety/drug therapy , Learning/drug effects , Psychotropic Drugs/pharmacology , Receptors, Metabotropic Glutamate/antagonists & inhibitors , Alzheimer Disease/physiopathology , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Anxiety/physiopathology , Disease Models, Animal , Hippocampus/drug effects , Hippocampus/physiopathology , Humans , Learning/physiology , Neurogenesis/drug effects , Neurogenesis/physiology , Psychotropic Drugs/chemistry , Receptors, Metabotropic Glutamate/metabolism
6.
Mol Psychiatry ; 18(8): 882-8, 2013 Aug.
Article in English | MEDLINE | ID: mdl-22869031

ABSTRACT

Latrepirdine (Dimebon; dimebolin) is a neuroactive compound that was associated with enhanced cognition, neuroprotection and neurogenesis in laboratory animals, and has entered phase II clinical trials for both Alzheimer's disease and Huntington's disease (HD). Based on recent indications that latrepirdine protects cells against cytotoxicity associated with expression of aggregatable neurodegeneration-related proteins, including Aß42 and γ-synuclein, we sought to determine whether latrepirdine offers protection to Saccharomyces cerevisiae. We utilized separate and parallel expression in yeast of several neurodegeneration-related proteins, including α-synuclein (α-syn), the amyotrophic lateral sclerosis-associated genes TDP43 and FUS, and the HD-associated protein huntingtin with a 103 copy-polyglutamine expansion (HTT gene; htt-103Q). Latrepirdine effects on α-syn clearance and toxicity were also measured following treatment of SH-SY5Y cells or chronic treatment of wild-type mice. Latrepirdine only protected yeast against the cytotoxicity associated with α-syn, and this appeared to occur via induction of autophagy. We further report that latrepirdine stimulated the degradation of α-syn in differentiated SH-SY5Y neurons, and in mouse brain following chronic administration, in parallel with elevation of the levels of markers of autophagic activity. Ongoing experiments will determine the utility of latrepirdine to abrogate α-syn accumulation in transgenic mouse models of α-syn neuropathology. We propose that latrepirdine may represent a novel scaffold for discovery of robust pro-autophagic/anti-neurodegeneration compounds, which might yield clinical benefit for synucleinopathies including Parkinson's disease, Lewy body dementia, rapid eye movement (REM) sleep disorder and/or multiple system atrophy, following optimization of its pro-autophagic and pro-neurogenic activities.


Subject(s)
Autophagy/drug effects , Indoles/pharmacology , Neuroprotective Agents/pharmacology , alpha-Synuclein/antagonists & inhibitors , Amyloid beta-Peptides , Animals , Brain/drug effects , Brain/metabolism , Cell Death/drug effects , Cells, Cultured , Humans , Male , Mice , Neuroprotective Agents/therapeutic use , Peptide Fragments , Saccharomyces cerevisiae , alpha-Synuclein/metabolism , alpha-Synuclein/toxicity
7.
Mol Psychiatry ; 18(8): 889-97, 2013 Aug.
Article in English | MEDLINE | ID: mdl-22850627

ABSTRACT

Latrepirdine (Dimebon) is a pro-neurogenic, antihistaminic compound that has yielded mixed results in clinical trials of mild to moderate Alzheimer's disease, with a dramatically positive outcome in a Russian clinical trial that was unconfirmed in a replication trial in the United States. We sought to determine whether latrepirdine (LAT)-stimulated amyloid precursor protein (APP) catabolism is at least partially attributable to regulation of macroautophagy, a highly conserved protein catabolism pathway that is known to be impaired in brains of patients with Alzheimer's disease (AD). We utilized several mammalian cellular models to determine whether LAT regulates mammalian target of rapamycin (mTOR) and Atg5-dependent autophagy. Male TgCRND8 mice were chronically administered LAT prior to behavior analysis in the cued and contextual fear conditioning paradigm, as well as immunohistological and biochemical analysis of AD-related neuropathology. Treatment of cultured mammalian cells with LAT led to enhanced mTOR- and Atg5-dependent autophagy. Latrepirdine treatment of TgCRND8 transgenic mice was associated with improved learning behavior and with a reduction in accumulation of Aß42 and α-synuclein. We conclude that LAT possesses pro-autophagic properties in addition to the previously reported pro-neurogenic properties, both of which are potentially relevant to the treatment and/or prevention of neurodegenerative diseases. We suggest that elucidation of the molecular mechanism(s) underlying LAT effects on neurogenesis, autophagy and behavior might warranty the further study of LAT as a potentially viable lead compound that might yield more consistent clinical benefit following the optimization of its pro-neurogenic, pro-autophagic and/or pro-cognitive activities.


Subject(s)
Alzheimer Disease/drug therapy , Autophagy/drug effects , Cognition/drug effects , Indoles/pharmacology , Neuroprotective Agents/pharmacology , Alzheimer Disease/pathology , Alzheimer Disease/psychology , Amyloid beta-Peptides/metabolism , Animals , Autophagy-Related Protein 5 , Brain/drug effects , Brain/metabolism , Cells, Cultured , Dose-Response Relationship, Drug , Indoles/therapeutic use , Mice , Mice, Transgenic , Microtubule-Associated Proteins/metabolism , Neuroprotective Agents/therapeutic use , Peptide Fragments/metabolism , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/metabolism , alpha-Synuclein/metabolism
8.
Gene Ther ; 18(7): 666-73, 2011 Jul.
Article in English | MEDLINE | ID: mdl-21368900

ABSTRACT

Therapeutic RNA interference (RNAi) has emerged as a promising approach for the treatment of many incurable diseases, including cancer, infectious disease or neurodegenerative disorders. Demonstration of efficacy and safety in animal models is necessary before planning human application. Our group and others have previously shown the potential of this approach for the dominantly inherited neurological disease DYT1 dystonia by achieving potent short-hairpin RNA (shRNA)-mediated silencing of the disease protein, torsinA, in cultured cells. To establish the feasibility of this approach in vivo, we pursued viral delivery of shRNA in two different mouse models. Surprisingly, intrastriatal injections of adeno-associated virus serotype 2/1 (AAV2/1) vectors expressing different shRNAs, whether targeting torsinA expression or mismatched controls, resulted in significant toxicity with progressive weight loss, motor dysfunction and animal demise. Histological analysis showed shRNA-induced neurodegeneration. Toxicity was not observed in animals that received control AAV2/1 encoding no shRNA, and was independent of genotype, occurring in both DYT1 and wild-type animals. Interestingly, the different genetic background of both mouse models influenced toxicity, being earlier and more severe in 129/SvEv than in C57BL/6 mice. In conclusion, our studies demonstrate that expression of shRNA in the mammalian brain can lead to lethal toxicity. Furthermore, the genetic background of rodents modifies their sensitivity to this form of toxicity, a factor that should be taken into consideration in the design of preclinical therapeutic RNAi trials.


Subject(s)
Corpus Striatum/metabolism , Dystonia/therapy , Genetic Therapy/methods , Molecular Chaperones/genetics , RNA Interference , RNA, Small Interfering/metabolism , RNA, Small Interfering/toxicity , Animals , Dependovirus/genetics , Dystonia/genetics , Dystonia/mortality , Feasibility Studies , Genetic Therapy/adverse effects , Genetic Vectors , Mice , Mice, Transgenic
9.
J Neurochem ; 79(5): 1027-32, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11739615

ABSTRACT

Brain-derived neurotrophic factor (BDNF) regulates several properties of striatal dopaminoceptive medium-sized spiny neurons (MSNs) in vivo and in vitro, including expression levels of DARPP-32 (dopamine and cyclic adenosine 3',5'-monophosphate-regulated phosphoprotein, 32 kDa). DARPP-32 is expressed in 96% of the MSNs, and is a key modulator of dopamine actions. We investigated the intracellular signal transduction pathways activated by BDNF in MSNs and via which BDNF induces DARPP-32 expression. We found that phosphorylation of the cyclic AMP response element binding protein (CREB) is only transiently increased following stimulation of MSNs by BDNF, whereas increased phosphorylation of the extracellular signal regulated kinases 1 and 2 (Erk1/2) and Akt is sustained for longer than 4 h. Treatment of cultures with inhibitors of mitogen-activated protein kinase kinase (MEK) or phosphatidylinositide 3-kinase (PI3K) showed that the majority of the BDNF-induced increase in DARPP-32 requires the PI3K pathway. We also found that inhibition of PI3K reduces BDNF-induced Erk phosphorylation, indicating that cross-talk between these pathways may play a prominent role in MSNs.


Subject(s)
Brain Chemistry/drug effects , Brain-Derived Neurotrophic Factor/pharmacology , Nerve Tissue Proteins , Neurons/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphoproteins/biosynthesis , Animals , Blotting, Western , Brain-Derived Neurotrophic Factor/antagonists & inhibitors , Cell Size , Chromones/pharmacology , Cyclic AMP Response Element-Binding Protein/metabolism , Dopamine and cAMP-Regulated Phosphoprotein 32 , Enzyme Inhibitors/pharmacology , Female , Mice , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinases/metabolism , Morpholines/pharmacology , Neurons/drug effects , Neurons/ultrastructure , Phosphoinositide-3 Kinase Inhibitors , Phosphorylation , Pregnancy , Signal Transduction/drug effects
10.
Exp Neurol ; 167(2): 215-26, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11161610

ABSTRACT

The ST14A cell line was previously derived from embryonic day 14 rat striatal primordia by retroviral transduction of the temperature-sensitive SV40 large T antigen. We showed that cell division and expression of nestin persists at 33 degrees C, the permissive temperature, whereas cell division ceases, nestin expression decreases, and MAP2 expression increases at the nonpermissive temperature of 39 degrees C. In this study, we further characterized the cells and found that they express other general and subtype-specific neuronal characteristics. ST14A cells express enolase and beta III-tubulin. Furthermore, they express the striatal marker DARPP-32, which is up-regulated upon differentiation of the cells by growth in serum-free medium. Stimulation with dopamine, the D2-dopamine receptor agonist quinpirole, or the D1-dopamine receptor agonist SKF82958 results in phosphorylation of CREB. Treatment of the cells with a mixture of reagents which stimulate the MAPK and adenylyl cyclase pathways radically changes the morphology of the ST14A cells. The cells develop numerous neurite-like appearing processes which stain with beta III-tubulin. Moreover, under these conditions, intracellular injection of rectangular depolarizing current stimuli elicits overshooting action potentials with a relatively fast depolarization rate when starting from a strongly hyperpolarized membrane potential. Taken together, these data imply that the ST14A cell line displays some of the characteristics of a medium-size spiny neuron subtype and provides a new tool to elucidate the pathways and molecules involved in medium-size spiny neuron differentiation and disease.


Subject(s)
Nerve Tissue Proteins , Neurons/classification , Neurons/cytology , Action Potentials/physiology , Adenylyl Cyclases/metabolism , Animals , Antigens, Differentiation/biosynthesis , Cell Division/physiology , Cell Line , Corpus Striatum/cytology , Corpus Striatum/embryology , Cyclic AMP Response Element-Binding Protein/metabolism , Dopamine Agonists/pharmacology , Dopamine and cAMP-Regulated Phosphoprotein 32 , Electric Stimulation , Intermediate Filament Proteins/biosynthesis , MAP Kinase Signaling System/drug effects , Microtubule-Associated Proteins/biosynthesis , Nestin , Neurites/drug effects , Neurons/drug effects , Neurons/metabolism , Phosphoproteins/biosynthesis , Phosphopyruvate Hydratase/biosynthesis , Phosphorylation/drug effects , Rats , Temperature , Tubulin/biosynthesis
11.
J Neurosci ; 19(13): 5409-19, 1999 Jul 01.
Article in English | MEDLINE | ID: mdl-10377350

ABSTRACT

The medium spiny neuron (MSN) is the major output neuron of the caudate nucleus and uses GABA as its primary neurotransmitter. A majority of MSNs coexpress DARPP-32 and ARPP-21, two dopamine and cyclic AMP-regulated phosphoproteins, and most of the matrix neurons express calbindin. DARPP-32 is the most commonly used MSN marker, but previous attempts to express this gene in vitro have failed. In this study we found that DARPP-32 is expressed in <12% of E13- or E17-derived striatal neurons when they are grown in defined media at high or low density in serum, dopamine, or Neurobasal/N2 (Life Technologies), and ARPP-21 is expressed in <1%. The percentage increases to 25% for DARPP-32 and 10% for ARPP-21 when the same cells are grown in Neurobasal/B27 (Life Technologies) for 7 d. After growth in Neurobasal/B27 plus brain-derived neurotrophic factor (BDNF) for 7 d, E13-derived MSNs are 53.7% DARPP-32-positive and 29. 0% ARPP-21-positive; E17-derived MSNs are 66.8% DARPP-32-positive and 51.5% ARPP-21-positive. The percentage of calbindin-positive neurons also is increased under these conditions. Finally, ARPP-21 expression is reduced in mice with a targeted deletion of the BDNF gene. We conclude that BDNF is required for the maturation of a large subset of patch and matrix MSNs in vivo and in vitro. In addition, we introduce a culture system in which highly differentiated MSNs may be generated, maintained, and studied.


Subject(s)
Brain-Derived Neurotrophic Factor/pharmacology , Caudate Nucleus/cytology , Gene Expression Regulation/drug effects , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Phosphoproteins/metabolism , gamma-Aminobutyric Acid/physiology , Animals , Brain-Derived Neurotrophic Factor/genetics , Brain-Derived Neurotrophic Factor/physiology , Calbindins , Cell Count/drug effects , Cell Culture Techniques/methods , Cell Differentiation/drug effects , Cell Survival/drug effects , Cells, Cultured , Culture Media , Dopamine/pharmacology , Dopamine and cAMP-Regulated Phosphoprotein 32 , Female , Gene Deletion , Mice , Nerve Growth Factors/pharmacology , Neurons/cytology , Neurons/drug effects , S100 Calcium Binding Protein G/metabolism
12.
Development ; 125(15): 2847-55, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9655807

ABSTRACT

Transplantation of immature CNS-derived cells into the developing brain is a powerful approach to investigate the factors that regulate neuronal position and phenotype. CNS progenitor cells dissociated from the embryonic striatum and implanted into the brain of embryos of the same species generate cells that reaggregate to form easily recognizable structures that we previously called clusters and cells that disperse and integrate as single cells into the host brain. We sought to determine if the neurons in the clusters differentiate according to their final location or acquire a striatal phenotype in heterotopic positions. We transplanted dissociated cells from the E14 rat medial and lateral ganglionic eminences, either combined or in isolation, into the E16 embryonic rat brain. At all time points, we found clusters of BrdU- and DiI-labelled donor cells located in the forebrain and hindbrain, without any apparent preference for striatum. Immunocytochemical analyses revealed that cells in the clusters expressed DARPP-32 and ARPP-21, two antigens typically co-expressed in striatal medium-sized spiny neurons. In agreement with observations previously noted by several groups, isolated cells integrated into heterologous host areas do not express basal ganglia phenotypes. These data imply that immature striatal neuronal progenitors exert a community effect on each other that is permissive and/or instructive for development of a striatal phenotype in heterotopic locations.


Subject(s)
Basal Ganglia/embryology , Brain/embryology , Corpus Striatum/embryology , Animals , Antigens, Differentiation , Astrocytes , Basal Ganglia/cytology , Basal Ganglia/transplantation , Cell Aggregation , Corpus Striatum/cytology , Dopamine and cAMP-Regulated Phosphoprotein 32 , Nerve Tissue Proteins/isolation & purification , Phenotype , Phosphoproteins/isolation & purification , Rats , Stem Cell Transplantation , Stem Cells/cytology , Transplantation, Heterotopic
13.
Neuroscience ; 79(2): 509-16, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9200733

ABSTRACT

The medium spiny neuron is the predominant striatal neuronal subtype. The striatum, a participant in motor and cognitive functions, is a site of pathophysiology in prevalent neuropsychiatric diseases and is the target of many currently utilized pharmacologic agents. DARPP-32, a dopamine and cyclic AMP-regulated phosphoprotein, is a widely-used marker of mature striatal medium-sized neurons, but the molecules regulating DARPP-32 transcription have not been identified. We show that a null mutation in the mouse brain-derived neurotrophic factor gene leads to decreased DARPP-32 immunoreactivity in striatal medium spiny neurons at birth and postnatal day 10. Striatal DARPP-32 messenger RNA and protein are decreased relative to wild-type littermate controls. In densely plated (1 x 10(6) cells/cm2) primary cultures derived from the ganglionic eminences, addition of brain-derived neurotrophic factor (100 ng/ml) to defined media results in a greater than 3-fold increase in the number of DARPP-32-immunopositive cells after 12 h and greater than 4-fold (P<0.005) after 24 h. The increase in DARPP-32-immunopositivity is abolished by the addition of 2 microg/ml actinomycin D without a significant effect on cell viability. These data suggest that brain-derived neurotrophic factor directly or indirectly regulates DARPP-32 transcription in medium spiny neurons. This is the first demonstration of transcriptional regulation of DARPP-32, and the first evidence of a forebrain abnormality in a newborn neurotrophin "knockout" mouse.


Subject(s)
Brain-Derived Neurotrophic Factor/pharmacology , Corpus Striatum/drug effects , Enzyme Inhibitors/metabolism , Nerve Tissue Proteins/metabolism , Neurons/drug effects , Phosphoproteins , Animals , Dopamine and cAMP-Regulated Phosphoprotein 32 , Immunohistochemistry , In Vitro Techniques , Mice , Mice, Mutant Strains , Phenotype
14.
Brain Res Mol Brain Res ; 52(1): 62-70, 1997 Dec 01.
Article in English | MEDLINE | ID: mdl-9450678

ABSTRACT

Repeated lesions of rat cerebral cortex result in transient peaks in the level of the c-fos transcript, but after the second lesion, this peak is substantially diminished. Using this lesion paradigm, we have analyzed the participation of the c-fos promoter elements SRE and DSE in the regulation of c-fos transcription. Following a single lesion, SRE/DSE binding activity peaked at 2 h, subsequent to the maximal levels of c-fos mRNA and parallel to the peak of c-Fos protein. After a second lesion (reinduction), 4 h following the initial lesion, SRE/DSE binding activity peaked after only 30 min and was significantly higher than following the first lesion. Once again, this peak occurred after the peak of c-fos mRNA expression and parallel with the second peak of c-Fos protein expression. These results suggested that the SRE and DSE promoter elements participated in the induction and down-regulation of c-fos transcription in vivo and suggested the possible involvement of Fos protein in its own regulation. The ability of Fos/Fra proteins to participate in a transcriptional complex was confirmed in gel-shift experiments with an AP-1 element, and the biphasic trend of binding activity was observed. Supershift experiments were performed to directly determine whether Fos protein was participating in SRE and/or DSE transcriptional complexes. No alterations in the position or intensity of the shifted band were observed using Fos/Fra antiserum suggesting that Fos/Fra proteins could be involved in c-fos down-regulation through mechanisms other than direct participation in the SRE/DSE transcription complex.


Subject(s)
Brain/metabolism , Cerebral Cortex/physiology , Nerve Tissue Proteins/genetics , Promoter Regions, Genetic , Proto-Oncogene Proteins c-fos/genetics , RNA, Messenger/biosynthesis , Animals , Down-Regulation , Male , Protein Binding , Rats
15.
Brain Res ; 709(1): 10-6, 1996 Feb 12.
Article in English | MEDLINE | ID: mdl-8869551

ABSTRACT

ARPP-21 (cAMP-regulated phosphoprotein, Mr = 21,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis) is a phosphoprotein highly enriched in concentration in the neurons of the limbic striatum. It is likely a third messenger in the intracellular cascade of events following neuronal stimulation by first-messenger activators of the adenylate cyclase system, including dopamine via the D1 receptor. ARPP-21 expression is restricted to telencephalic post-mitotic, post-migrational neurons, and its precise pattern of temporal and spatial expression makes it an attractive candidate for the study of transcriptional regulation of neuronal maturation. To define genomic regions likely to contain functional promoter elements, we isolated the murine ARPP-21 gene. Primer extension and T2 RNase protection analyses identified multiple transcription start sites, but 1.3 kb of 5'-flanking DNA revealed few consensus transcription factor binding sequences. A series of transient transfection assays in clonal cell lines which do not express ARPP-21 identified a basal promoter active in both neuronal and non-neuronal lines. Expression in all lines was decreased by the inclusion of regions further upstream, and extinguished by the inclusion of the first intron. Further analyses are likely to reveal cell specific regulatory sequences.


Subject(s)
Genes , Limbic System/metabolism , Mice/genetics , Neurons/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Promoter Regions, Genetic , Animals , Base Sequence , Cell Line , Limbic System/cytology , Molecular Probes/genetics , Molecular Sequence Data , Rats
16.
Am J Physiol ; 269(4 Pt 2): F564-70, 1995 Oct.
Article in English | MEDLINE | ID: mdl-7485543

ABSTRACT

DARPP-32, a dopamine- and adenosine 3',5'-cyclic monophosphate (cAMP)-regulated inhibitor of protein phosphatase-1, is highly colocalized with neuronal and nonneuronal D1-type receptors. DARPP-32 concentration is enriched in the renal outer medulla and in the medium-size spiny neurons of the brain. In the ascending limb of the loop of Henle, DARPP-32 is phosphorylated following stimulation by dopamine and other first messengers, and in this form inhibits the activity of the Na(+)-K(+)-adenosinetriphosphatase pump. For functional analysis of the DARPP-32 promoter in the kidney, we characterized the murine gene. There are two groups of transcription start sites utilized in the brain, but the proximal set appears to be preferentially used in the kidney. In four of four lines of mice carrying a DARPP-32/lacZ transgene with 2.1 kb of 5'-flanking DNA, adult kidney lacZ transgene expression mimicked that of endogenous DARPP-32. There was no ectopic expression in peripheral organs. We conclude that the sequences necessary for direction of DARPP-32 expression to the medullary thick ascending limb are contained within this 2.1-kb fragment.


Subject(s)
Gene Expression , Loop of Henle/physiology , Nerve Tissue Proteins/genetics , Promoter Regions, Genetic , Transgenes , Animals , Base Sequence , Cloning, Molecular , Dopamine and cAMP-Regulated Phosphoprotein 32 , Genes , Lac Operon , Mice , Mice, Transgenic , Molecular Sequence Data , Oligonucleotide Probes/genetics , Phosphoproteins/genetics
17.
Biochem Biophys Res Commun ; 206(2): 652-8, 1995 Jan 17.
Article in English | MEDLINE | ID: mdl-7826384

ABSTRACT

The mechanism of inhibition of protein phosphatase-1 catalytic subunit (PP-1c) by recombinant DARPP-32 and synthetic peptides was studied. DARPP-32 was expressed in Escherichia coli as a non-fusion protein using a pEt-3a plasmid, purified to homogeneity and shown to have physicochemical properties similar to those of the protein purified from bovine brain. Recombinant DARPP-32 phosphorylated on threonine-34 by cAMP-dependent protein kinase inhibited PP-1c with an IC50 approximately 0.5 nM, comparable to that obtained with bovine DARPP-32. Non-phosphorylated DARPP-32, and mutated forms in which threonine-34 was replaced by an alanine or a glutamic acid, inhibited PP-1c with an IC50 approximately 1 microM. Surface plasmon resonance analysis showed binding of PP-1c to nonphospho- and phospho-DARPP-32-(8-38) synthetic peptides with apparent Kd values of 1.2 and 0.3 microM, respectively, supporting the existence of an interaction between non-phosphorylated DARPP-32 and PP-1c that is increased by phosphorylation of DARPP-32 at threonine-34. These results suggest a model in which DARPP-32 interacts with PP-1c by at least two low affinity sites, the combination of which is responsible for the high affinity (nM) inhibition.


Subject(s)
Nerve Tissue Proteins/pharmacology , Peptide Fragments/pharmacology , Phosphopeptides/pharmacology , Phosphoprotein Phosphatases/antagonists & inhibitors , Amino Acid Sequence , Animals , Base Sequence , Cattle , Cell Line , Cyclic AMP-Dependent Protein Kinases/metabolism , Dopamine and cAMP-Regulated Phosphoprotein 32 , Escherichia coli , Kinetics , Molecular Sequence Data , Muscle, Skeletal/enzymology , Mutagenesis, Site-Directed , Myocardium/enzymology , Oligodeoxyribonucleotides , Peptide Fragments/chemical synthesis , Phosphopeptides/chemical synthesis , Phosphoproteins/pharmacology , Phosphorylation , Protein Binding , Protein Phosphatase 1 , Rabbits , Recombinant Proteins/pharmacology , Spodoptera
18.
J Neurosci ; 15(1 Pt 2): 676-88, 1995 Jan.
Article in English | MEDLINE | ID: mdl-7823171

ABSTRACT

Immortalized hybrid cells were generated by somatic cell fusion of 18-d-old embryonic corpus striatum of the mouse strain C57BL/6J with the N18TG2 neuroblastoma. One of the cell populations obtained was treated with a combination of 1 mM n-butyric acid and 10 microM SKF 38393 (a specific D1 agonist), and a surviving cell population (E1X) was subcloned. Twenty-seven monoclonal cell lines were obtained and screened for the expression of striatal-specific characteristics including gamma-aminobutyric acid (GABA), choline acetyltransferase (ChAT), acetylcholine (ACh), mRNA for specific dopamine receptors, and dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein, M(r) 32,000 (DARPP-32), and functional D1 and D2 dopamine receptors. Neither the parent hybrid cell population (E1X) nor any of the monoclonal cell lines examined expressed GABA levels significantly different than that of the N18TG2 parent neuroblastoma cells (1.36 +/- 0.07 micrograms/mg protein). The range of ChAT activity in the monoclonal hybrid cell lines was 5.5 +/- 0.3 to 921.3 +/- 97.4 pmol/min/mg protein. Two of the cell lines expressing ChAT activity (X52 and X58) contained ACh (49.64 +/- 4.23 and 1.78 +/- 0.07 ng/mg protein, respectively). The neuronal origin of four of the monoclonal hybrid lines was shown by their immunoreactivity, following differentiation with 10 microM forskolin, to neurofilament protein, a neuron-specific marker. The monoclonal hybrid cell lines, but not the N18TG2 neuroblastoma, were shown to express an array of D1, D2, and D5 receptor mRNA as well as DARPP-32 mRNA. Two monoclonal cell lines expressed D1 receptor binding sites (X57, 29.2 +/- 4.5 fmol/mg protein and X62, 43.8 +/- 6.8 fmol/mg protein) which mediated the stimulation of adenylate cyclase activity. One cell line, X58, expressed only D2 dopamine receptors (80.9 +/- 9.8 fmol/mg protein) which were negatively coupled to adenylate cyclase activity. These findings suggest that the immortalized monoclonal hybrid cell lines are of neuronal origin and have incorporated elements of the medium spiny and cholinergic neurons of the developing striatum.


Subject(s)
Acetylcholine/metabolism , Acetylcholinesterase/metabolism , Corpus Striatum/cytology , Corpus Striatum/metabolism , Receptors, Dopamine/metabolism , Adenylyl Cyclases/metabolism , Animals , Base Sequence , Binding Sites , Biomarkers , Cell Line , Cytoskeleton/metabolism , Dopamine and cAMP-Regulated Phosphoprotein 32 , Hybrid Cells , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Nerve Tissue Proteins/genetics , Phosphoproteins/genetics , RNA, Messenger/metabolism , Receptors, Dopamine/genetics
19.
Dev Biol ; 163(2): 480-90, 1994 Jun.
Article in English | MEDLINE | ID: mdl-8200483

ABSTRACT

Adrenal chromaffin cells and neurons of the sympathetic ganglia are derived from common precursors in the neural crest. The phenotype of the sympathoadrenal progenitor cell is unknown, but adult chromaffin cells are distinguished by the expression of phenylethanolamine-N-methyltransferase (PNMT) and the lack of neurofilament (NF) and neuritic processes. Mature neurons have processes and express NF, but are PNMT-. We hypothesize that embryonic adrenal cells are multipotential. This implies that the cells can coactivate all the traits characteristic of mature sympathetic neurons and chromaffin cells and then selectively extinguish expression of either the chromaffin or the neuronal traits, depending on the environment. We further asked whether this repression is plastic and can be environmentally modified in adult chromaffin cells. We demonstrate that, in vivo, embryonic (e-) rat adrenal cells coexpress PNMT and the intermediate- and high-molecular-weight neurofilaments at e-15.5 (21%), e-16.5 (40%), and e-20.5 (23%). When cultured in complete or glucocorticoid-depleted media for 5 to 14 days, 20% of adult bovine chromaffin cells which remain PNMT+ reexpress NF and extend NF+ and PNMT+ processes. Both the expression of NF and the extension of neurites are inhibited by the addition of 10(-7) M dexamethasone to complete media. We conclude that the embryonic adrenal medullary cells simultaneously express traits of mature chromaffin cells and neurons and that the phenotypes remain labile in the adult mammalian chromaffin cell. In addition, coexpression of PNMT, NF, and neurite extension are not mutually exclusive in either the embryonic or adult adrenal chromaffin cell.


Subject(s)
Adrenal Medulla/cytology , Adrenal Medulla/enzymology , Neurofilament Proteins/metabolism , Neurons/cytology , Phenylethanolamine N-Methyltransferase/metabolism , Animals , Cattle , Cells, Cultured , Epinephrine/biosynthesis , Female , Glucocorticoids/pharmacology , Male , Neural Crest/cytology , Rats
20.
Neuroscience ; 51(1): 65-75, 1992 Nov.
Article in English | MEDLINE | ID: mdl-1465187

ABSTRACT

The regional and cellular ontogeny of the mRNA encoding the dopamine- and cAMP-regulated phosphoprotein, DARPP-32, has been studied in rat striatum by quantitative in situ hybridization histochemistry. The mRNA for DARPP-32 exhibited a characteristic developmental profile. The hybridization signal was first visible on the day of birth, at which time DARPP-32 mRNA was concentrated in patches in the caudate-putamen. By the end of the first postnatal week, the majority of neurons in the caudate-putamen expressed the DARPP-32 message. Levels of mRNA per cell increased markedly during the second postnatal week, and peaked around the beginning of the third week. The adult level of DARPP-32 mRNA was lower than that observed at the apex of mRNA expression, on a per cell basis, while the proportion of neurons expressing detectable levels of message remained relatively constant. In the nucleus accumbens and olfactory tubercle, DARPP-32 mRNA development lagged somewhat behind that observed in the caudate-putamen, but was similar in other respects. A non-quantitative study employing an oligonucleotide probe complementary to the mRNA encoding another cAMP-regulated phosphoprotein, ARPP-21, revealed a similar developmental sequence to DARPP-32. The present results suggest that for DARPP-32 mRNA, genetic and, possibly, environmental factors play a role in determining the developmental patterns observed.


Subject(s)
Aging/physiology , Caudate Nucleus/metabolism , Gene Expression Regulation , Nerve Tissue Proteins/genetics , Phosphoproteins/genetics , Putamen/metabolism , RNA, Messenger/metabolism , Animals , Autoradiography , Base Sequence , Brain/metabolism , Caudate Nucleus/embryology , Caudate Nucleus/growth & development , DNA/genetics , Dopamine and cAMP-Regulated Phosphoprotein 32 , Embryonic and Fetal Development , Gestational Age , In Situ Hybridization , Molecular Sequence Data , Nerve Tissue Proteins/biosynthesis , Oligodeoxyribonucleotides , Organ Specificity , Phosphoproteins/biosynthesis , Putamen/embryology , Putamen/growth & development , RNA, Messenger/analysis , Rats , Rats, Sprague-Dawley , Sulfur Radioisotopes
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