What's wrong with my genetically engineered animal?" is a common yet often difficult to answer qu... more What's wrong with my genetically engineered animal?" is a common yet often difficult to answer question in behavioral phenotyping. We present here a method termed Pattern Array for mining movement patterns and isolating those that best capture an effect of a genetic manipulation. We demonstrate the method by searching for early motor symptoms in the open-field behavior of SOD1 mutant rats, an animal model of amyotrophic lateral sclerosis. Pattern Array was able to identify a unique motor pattern that differentiated the SOD1 mutants from the wild-type controls 2 months before disease onset. This pattern included heavy braking while moving near the arena wall but turning away from it. SOD1 mutants performed this pattern significantly less than wild-type controls in 2 independent data sets. At such early age the SOD1 mutants could not be differentiated from the controls by standard behavioral measures or by subjective observation. The early discovered symptom may enable investigators to test therapies aimed for intervention rather than remediation. Our results demonstrate the feasibility and potential of detecting subtle behavioral effects using data mining strategies.
Basic fibroblast growth factor and interleukin-1 beta are known to regulate the expression of oth... more Basic fibroblast growth factor and interleukin-1 beta are known to regulate the expression of other trophic factors and to stimulate reactive gliosis in vivo. S100 beta is a glial-specific putative neurotrophic factor and has been considered a marker of the reactive status of astrocytes. Therefore, we tested the hypothesis that basic fibroblast growth factor-2 and interleukin-1 beta achieve their effects by altering S100 beta gene expression in cultured rat astrocytes using an RNase protection assay. Short-term treatment with basic fibroblast growth factor-2 produced a transient decrease in S100 beta messenger RNA which was followed by an increase after longer term treatment. In contrast, both short- and long-term treatment with interleukin-1 beta suppressed S100 beta messenger RNA. We measured levels of S100 beta nuclear primary transcript to assess whether alterations in transcriptional rate explain the changes in messenger RNA. Our results indicate that changes in transcription account for changes in steady state levels of messenger RNA since basic fibroblast growth factor-2-induced changes in S100 beta primary transcript temporally preceded changes in messenger RNA. We further measured intracellular S100 beta protein levels by enzyme-linked immunosorbent assay to determine whether changes in gene expression were translated into parallel changes in protein. Our results clearly demonstrate that basic fibroblast growth factor-2 and interleukin-1 beta influence the expression of the S100 beta gene, that this regulation appears to occur at the level of transcription, and that alterations in messenger RNA are sometimes, but not always, reflected in changes at the level of protein. These observations suggest that basic fibroblast growth factor-2 may amplify its trophic effects, in part, by influencing the expression of another trophic factor.
2011 IEEE First International Conference on Healthcare Informatics, Imaging and Systems Biology, 2011
ABSTRACT This manuscript focuses on distinguishing the locomotion of 6-Hydroxydopamine (6-OHDA) r... more ABSTRACT This manuscript focuses on distinguishing the locomotion of 6-Hydroxydopamine (6-OHDA) rats from untreated control rats and the efficient design of the experiment. A 6-OHDA rat is a model of Parkinson's disease and the locomotion is determined by measuring ground reaction forces. The hypothesis that changes in measured locomotion parameters can noninvasively detect Parkinson's disease in rats is tested. A gait analysis system that measures locomotion parameters (LP) of each individual limb along with a logistic regression model is used to assess the locomotion impairments of Sprague-Dawley rats before and after injecting 6-OHDA into their right medial forebrain bundle (MFB). We observed changes in the rats' locomotion as soon as they walked again through the system after recuperating from the surgery. Before the surgery the locomotion of the rats was normal, whereas after surgery the six rats demonstrated impaired locomotion. The statistical model used 7 LPs that characterize non vertical ground reaction forces and associated time parameters. The sensitivity and specificity of the 7-LP model is 92% and 95%, respectively. This gait analysis system can be used to test the efficacy of new pharmaceutical treatments for Parkinson's disease and other neurological ailments modeled in rats or mice. A system for noninvasively diagnosing neurological ailments that affect locomotion in humans can be developed using similar methodologies.
Micromolar concentrations of glutamate, acting at non-NMDA, kainate-preferring receptors, increas... more Micromolar concentrations of glutamate, acting at non-NMDA, kainate-preferring receptors, increased the survival of cultured mouse hippocampal neurons maintained in serum-free, chemically-defined medium. Glutamate in excess of 20 μM was excitotoxic. Thus, the survival versus glutamate dose response relation was bell-shaped with an optimal glutamate concentration near 1 μM. Hippocampal neurons from mice with the genetic defect, trisomy 16 (Ts16), died 2–3 times faster than normal (euploid) neurons. Moreover, glutamate, at all concentrations tested, failed to increase survival of Ts16 neurons. Ts16 is a naturally-occurring mouse genetic abnormality, the human analog of which (Down syndrome) leads to altered brain development and Alzheimer’s disease. These results demonstrate that the Ts16 genotype confers a defect in the glutamate-mediated survival response of hippocampal neurons and that this defect can account for their accelerated death.
Proceedings of the National Academy of Sciences, 1994
S100 beta is a calcium-binding protein that is expressed at high levels in brain primarily by ast... more S100 beta is a calcium-binding protein that is expressed at high levels in brain primarily by astrocytes. Addition of the disulfide-bonded dimeric form of S100 beta to primary neuronal and glial cultures and established cell lines induces axonal extension and alterations in astrocyte proliferation and phenotype, but evidence that S100 beta exerts the same effects in vivo has not been presented. An 8.9-kb murine S100b genomic clone was used to produce two lines of transgenic mice in which S100 beta RNA is increased in a dose-related manner to 2-fold and 7-fold above normal. These lines show concomitant increased S100 beta protein throughout the brain. Expression in both lines is cell type- and tissue-appropriate, and expression levels are correlated with the transgene copy number, demonstrating that sequences necessary for normal regulation of the gene are included within the cloned segment. In the hippocampus of adult transgenic mice, Western blotting detects elevated levels of glia...
Identification of targets and delivery platforms for gene therapy of neurodegenerative disorders ... more Identification of targets and delivery platforms for gene therapy of neurodegenerative disorders is a clinical challenge. We describe a novel paradigm in which the neuroprotective gene is the herpes simplex virus type 2 (HSV-2) antiapoptotic gene ICP10PK and the vector is the growth-compromised HSV-2 mutant DRR. DRR is delivered intranasally. It is not toxic in rats and mice. ICP10PK is expressed in the hippocampus of the DRR-treated animals for at least 42 days in the absence of virus replication and late virus gene expression. Its expression is regulated by an AP-1 amplification loop. Intranasally delivered DRR prevents kainic acid-induced seizures, neuronal loss, and inflammation, in both rats and mice. The data suggest that DRR is a promising therapeutic platform for neurodegenerative diseases.
We have previously shown that intrastriatal injection of DRR, the growth-compromised herpes simpl... more We have previously shown that intrastriatal injection of DRR, the growth-compromised herpes simplex virus type 2 (HSV-2) vector for the antiapoptotic protein ICP10PK, prevents apoptosis caused by the excitotoxin N-methyl-D-aspartate (NMDA) in a mouse model of glutamatergic neuronal cell death (Golembewski et al. [2007] Exp. Neurol. 203:381-393). Because apoptosis regulation is stimulus and cell type specific, our studies were designed to examine the mechanism of DRR-mediated neuroprotection in striatal neurons. Organotypic striatal cultures (OSC) that retain much of the synaptic circuitry of the intact striatum were infected with DRR or a growthcompromised HSV-2 vector that lacks ICP10PK (DPK) and examined for neuroprotection-associated signaling. The mutated ICP10 proteins (p175 and p95) were expressed in 70-80% of neurons from DRRand DPKinfected cultures, respectively, as determined by doubleimmunofluorescent staining with antibodies to ICP10 and NeuN or GAD65. DRRbut not DPK-treated OSC were protected from NMDA-induced apoptosis, as verified by ethidium homodimer staining, TUNEL, caspase-3 activation, and poly(AD-ribose) polymerase (PARP) cleavage. Neuroprotection was through ICP10PK-mediated activation of the survival pathways MEK/ERK and PI3-K/Akt, up-regulation of the antiapoptotic proteins Bag-1 and Bcl-2, and phosphorylation (inactivation) of the proapoptotic protein Bad. It was blocked by the MEK inhibitor U0126 or the PI3-K inhibitor LY294002, suggesting that either pathway can prevent NMDA-induced apoptosis. The data indicate that DRR-delivered ICP10PK stimulates redundant survival pathways that override proapoptotic cascades. DRR is a promising gene therapy platform against glutamatergic cell death. V
Expression of developmentally regulated antigens was used to characterize glial cells in cultures... more Expression of developmentally regulated antigens was used to characterize glial cells in cultures from embryonic mouse cerebral cortex. Over 90% of the cells had a flat morphology, and about 50% of these flat cells also expressed the ganglioside GD3. Up to 40% of all the GD3 expressing cells also expressed A2B5 antigen. Flat cells expressing either glial fibrillary acidic protein (GFAP), or GD3 or both were present at all times in vitro. These three populations of flat cells could not be further distinguished on the basis of NG2 or fibronectin expression, or with respect to their responses to the mitogens FGF-2, PDGF, or EGF. The glial cultures also contain a small number (approximately 5%) of process bearing cells with the morphological and immunocytochemical characteristics of oligodendrocyte precursors. The expression of GD3 by flat cells changed with time in culture as the fraction of flat cells expressing only GD3 declined and the fraction of cells expressing GFAP (with or without GD3) increased. The data are consistent with those flat cells expressing only GD3 being astrocyte precursors. Furthermore, between 1 and 3 weeks in vitro GD3/GFAP cells lose GD3 while retaining GFAP. Cells expressing only GFAP could be induced to express GD3 and A2B5 by treatment with FGF-2. The widespread and regulated expression of GD3 and A2B5 by murine glia is different from the restricted pattern of expression previously reported for these antigens in rat brain cell cultures. These results demonstrate that expression of GD3 and A2B5 by murine astrocytes depends on both culture age and extracellular signals and that these gangliosides are not markers for cell lineage in the mouse.
Genes from the Down syndrome (DS) critical region of human chromosome 21, which contribute to the... more Genes from the Down syndrome (DS) critical region of human chromosome 21, which contribute to the pathology of DS, are also found on mouse chromosome 16. Several animal models of DS with triplication of genes from the DS critical region have been generated, including mouse trisomy 16 (Ts16) and a partial trisomic mouse, Ts65Dn. Using computer-assisted imaging of fura-2 fluorescence, we found an elevation of intracellular cytoplasmic calcium in cortical astrocytes from neonatal Ts65Dn mouse brain, similar to that observed previously in embryonic Ts16 astrocytes. Furthermore, astrocytes from both Ts65Dn and Ts16 cortex fail to respond to the anti-proliferative actions of glutamate. These results suggest that defective regulation of cell proliferation and cellular calcium can result from triplication of DS critical region genes.
A locomotion analysis system for laboratory rats is presented. The system produces locomotion par... more A locomotion analysis system for laboratory rats is presented. The system produces locomotion parameters (LPs) in 4 different domains: force, space, time and frequency. Video images of the walking rats are used to associate the system signals with individual limbs. Numerous LPs can be derived for every test run when the rat walks through the system on the way to sweets and a personal toy placed at the exit. This manuscript demonstrates that in order to differentiate SOD1-G93A mutant rat, a model of amyotrophic lateral sclerosis (ALS), from a Sprague Dawley (SD) control rat at a pre-symptomatic stage, one has only to use 8 key parameters. These 8 parameters are the bio-markers of ALS. The spline-based transformed values of these parameters are used as explanatory variables of a logistic regression model. This model predicts the probability that the examined rat belongs to the SOD1-G93A group. The model differentiates faultlessly between the SOD1 and control groups from the very first time the rats walked through the system at 51 days old. This system provides a new paradigm for ALS diagnosis, and it can have a significant impact on the development of new therapeutic procedures for ALS. The methodology presented in this manuscript can further address the development and validation of therapeutic procedures for other neurological diseases that affect locomotion.
The regulation of intracellular Ca2+ was investigated in cultured astrocytes from the trisomy 16 ... more The regulation of intracellular Ca2+ was investigated in cultured astrocytes from the trisomy 16 (Ts16) mouse, an animal model for Down syndrome and Alzheimer's disease (AD). The cytoplasmic ionized Ca2+ concentration ([Ca2+]cyt) was determined using digital imaging of fura-2-loaded cells. The relative Ca2+ content of internal endoplasmic reticulum (ER) stores was estimated from the magnitude of the transient increase in [Ca2+]cyt induced by cyclopiazonic acid (CPA), an inhibitor of Ca2+ sequestration into ER stores. At rest, the average [Ca2+]cyt was 140 nM in euploid (normal) astrocytes, but over twice as high, 320 nM, in Ts16 cells. In the absence of extracellular Ca2+, CPA induced a transient increase in [Ca2+]cyt to over 1200 nM in Ts16 astrocytes as compared to only 500 nM in euploid cells, indicating an increased amount of Ca2+ in the Ts16 astrocyte ER. In contrast to euploid astrocytes, both resting [Ca2+]cyt and the amount of Ca2+ in the ER stores varied widely among individual Ts16 astrocytes. These results show that Ts16 produces a dysregulation of Ca2+ homeostasis leading to increased cytoplasmic and stored Ca2+. Since increases in [Ca2+]cyt have been implicated in the etiology of neurodegenerative diseases, including AD, this finding of abnormal Ca2+ homeostasis in a genetic model of human neurological disorders suggests that Ca2+ dysregulation may be a common feature underlying neurodegenerative processes.
Mechanisms that regulate the amount of releasable Ca2+ in intracellular stores of cultured mouse ... more Mechanisms that regulate the amount of releasable Ca2+ in intracellular stores of cultured mouse astrocytes were investigated using digital imaging of fura-2 loaded cells. At rest, the cytoplasmic Ca2+ concentration, [Ca2+]cyt, was about 110 nM. In the absence of extracellular Ca2+, cyclopiazonic acid (CPA), an inhibitor of the endoplasmic reticulum (ER) Ca(2+)-ATPase, induced a transient, four-fold increase in [Ca2+]cyt due to the release of Ca2+ from inositol triphosphate (IP3) sensitive stores. Caffeine (CAF), which releases Ca2+ from Ca(2+)-sensitive stores, induced a two-fold increase in [Ca2+]cyt. The CPA- and CAF-sensitive stores could be released independently. Changes in the amplitudes of the Ca2+ transients were taken as a measure of changes in store content. Removal of extracellular Na+ or addition of ouabain, which inhibit Ca2+ extrusion and promote Ca2+ entry across the plasmalemma via the Na/Ca exchanger, caused minimal increases in resting [Ca2+]cyt but greatly potentiated both CPA- and CAF-induced Ca2+ transients. The amount of Ca2+ releasable from the IP3(CPA) sensitive store was directly proportional to cytosolic Na+ concentration (i.e., inversely proportional to the transmembrane Na+ electrochemical gradient). Under these reduced Na+ gradient conditions, little, if any, Ca2+ destined for the ER stores enters the cells through voltage-dependent Ca2+ channels. These results demonstrate that mouse astrocytes contain two distinct ER Ca2+ stores, the larger, IP3- (CPA-) sensitive, and the smaller, Ca(2+)- (CAF-) sensitive. The Ca2+ content of both ER stores can be regulated by the Na/Ca exchanger. Thus, the magnitude of cellular responses to signals that are mediated by Ca2+ release induced by the two second messengers, IP3 and Ca2+, can be modulated by factors that affect the net transport of Ca2+ across the plasmalemma.
The Ts65Dn mouse, an adult model of Down syndrome displays behavioral deficits consistent with a ... more The Ts65Dn mouse, an adult model of Down syndrome displays behavioral deficits consistent with a dysfunctional hippocampus, similar to that seen with DS. In looking for mechanisms underlying these performance deficits, we have assessed adult neurogenesis in the dentate gyrus of Ts65Dn. Under untreated conditions, Ts65Dn mice (2-5 months old) showed markedly fewer BrdU-labeled cells than euploid animals. Chronic antidepressant treatment for over 3 weeks with the serotonin selective reuptake inhibitor, fluoxetine, increased neurogenesis in the Ts65Dn to comparable levels seen in the euploid by augmenting both proliferation and survival of BrdU-labeled cells in the subgranular layer and granule cell layer of the hippocampus, respectively.
During embryonic development of the mammalian cerebral cortex, the generation of the marginal zon... more During embryonic development of the mammalian cerebral cortex, the generation of the marginal zone (MZ) and subplate (SP) precedes that of the cortical plate (CP). MZ and SP neurons are believed to play a ‘pioneering’ role in directing the organization of the CP and the specificity of connections between the CP and other brain regions. Here we report that this sequential order of neurogenesis is disrupted in the trisomy 16 (Ts16) mouse, a potential animal model of Down syndrome. Bromodeoxyuridine labeling was used to establish the date of generation of postmitotic SP and CP neurons in the somatosensory cortex. As has been previously reported, most SP neurons in euploid (control) cortex were generated on embryonic day 12.5 (E12.5), and production of CP neurons began a day later. In contrast, in the Ts16 cortex, few SP neurons were born on E12.5 and most were generated on E13.5 and E14.5 when CP neurons were also being produced. Thus, in the Ts16 cortex, many CP neurons are born and a...
Spatial memory was assessed in the segmental trisomic 16 mouse (Ts65Dn), a potential model for Do... more Spatial memory was assessed in the segmental trisomic 16 mouse (Ts65Dn), a potential model for Down syndrome (DS), using the 1Zarm radial maze (RAM). Ts65Dn mice have a portion of mouse chromosome 16 syntenic to the distal end of human chromosome 21 triplicated. On each of 8 daily trials of the RAM, Ts65Dn mice made fewer correct choices than control mice and performed at or near chance levels, indicating a deficit in spatial working memory. On trials 9 and 10, Ts65Dn mice performed as well as control mice on the initial 12 choices, but required a greater number of choices to complete the RAM. The improved performance of Ts65Dn mice on trials 9 and 10 was lost when the animals were retested after a 50-day retention period, suggesting that long-term memory is also defective. These results are not likely explained by differences in either response bias or perceptual discrimination. Ts65Dn and control mice displayed comparable levels of performance in spontaneous alternation in a T-maze, demonstrating that simple spatial memory was not impaired. In the elevated plus maze, Ts65Dn mice did not display higher anxiety levels which could affect their performance in the RAM. In fact, Ts65Dn mice visited open arms on the elevated plus maze more frequently and spent more time on open arms than did control mice. Taken together, these results provide evidence for short-and long-term spatial memory deficits in Ts65Dn mice.
What's wrong with my genetically engineered animal?" is a common yet often difficult to answer qu... more What's wrong with my genetically engineered animal?" is a common yet often difficult to answer question in behavioral phenotyping. We present here a method termed Pattern Array for mining movement patterns and isolating those that best capture an effect of a genetic manipulation. We demonstrate the method by searching for early motor symptoms in the open-field behavior of SOD1 mutant rats, an animal model of amyotrophic lateral sclerosis. Pattern Array was able to identify a unique motor pattern that differentiated the SOD1 mutants from the wild-type controls 2 months before disease onset. This pattern included heavy braking while moving near the arena wall but turning away from it. SOD1 mutants performed this pattern significantly less than wild-type controls in 2 independent data sets. At such early age the SOD1 mutants could not be differentiated from the controls by standard behavioral measures or by subjective observation. The early discovered symptom may enable investigators to test therapies aimed for intervention rather than remediation. Our results demonstrate the feasibility and potential of detecting subtle behavioral effects using data mining strategies.
Basic fibroblast growth factor and interleukin-1 beta are known to regulate the expression of oth... more Basic fibroblast growth factor and interleukin-1 beta are known to regulate the expression of other trophic factors and to stimulate reactive gliosis in vivo. S100 beta is a glial-specific putative neurotrophic factor and has been considered a marker of the reactive status of astrocytes. Therefore, we tested the hypothesis that basic fibroblast growth factor-2 and interleukin-1 beta achieve their effects by altering S100 beta gene expression in cultured rat astrocytes using an RNase protection assay. Short-term treatment with basic fibroblast growth factor-2 produced a transient decrease in S100 beta messenger RNA which was followed by an increase after longer term treatment. In contrast, both short- and long-term treatment with interleukin-1 beta suppressed S100 beta messenger RNA. We measured levels of S100 beta nuclear primary transcript to assess whether alterations in transcriptional rate explain the changes in messenger RNA. Our results indicate that changes in transcription account for changes in steady state levels of messenger RNA since basic fibroblast growth factor-2-induced changes in S100 beta primary transcript temporally preceded changes in messenger RNA. We further measured intracellular S100 beta protein levels by enzyme-linked immunosorbent assay to determine whether changes in gene expression were translated into parallel changes in protein. Our results clearly demonstrate that basic fibroblast growth factor-2 and interleukin-1 beta influence the expression of the S100 beta gene, that this regulation appears to occur at the level of transcription, and that alterations in messenger RNA are sometimes, but not always, reflected in changes at the level of protein. These observations suggest that basic fibroblast growth factor-2 may amplify its trophic effects, in part, by influencing the expression of another trophic factor.
2011 IEEE First International Conference on Healthcare Informatics, Imaging and Systems Biology, 2011
ABSTRACT This manuscript focuses on distinguishing the locomotion of 6-Hydroxydopamine (6-OHDA) r... more ABSTRACT This manuscript focuses on distinguishing the locomotion of 6-Hydroxydopamine (6-OHDA) rats from untreated control rats and the efficient design of the experiment. A 6-OHDA rat is a model of Parkinson's disease and the locomotion is determined by measuring ground reaction forces. The hypothesis that changes in measured locomotion parameters can noninvasively detect Parkinson's disease in rats is tested. A gait analysis system that measures locomotion parameters (LP) of each individual limb along with a logistic regression model is used to assess the locomotion impairments of Sprague-Dawley rats before and after injecting 6-OHDA into their right medial forebrain bundle (MFB). We observed changes in the rats' locomotion as soon as they walked again through the system after recuperating from the surgery. Before the surgery the locomotion of the rats was normal, whereas after surgery the six rats demonstrated impaired locomotion. The statistical model used 7 LPs that characterize non vertical ground reaction forces and associated time parameters. The sensitivity and specificity of the 7-LP model is 92% and 95%, respectively. This gait analysis system can be used to test the efficacy of new pharmaceutical treatments for Parkinson's disease and other neurological ailments modeled in rats or mice. A system for noninvasively diagnosing neurological ailments that affect locomotion in humans can be developed using similar methodologies.
Micromolar concentrations of glutamate, acting at non-NMDA, kainate-preferring receptors, increas... more Micromolar concentrations of glutamate, acting at non-NMDA, kainate-preferring receptors, increased the survival of cultured mouse hippocampal neurons maintained in serum-free, chemically-defined medium. Glutamate in excess of 20 μM was excitotoxic. Thus, the survival versus glutamate dose response relation was bell-shaped with an optimal glutamate concentration near 1 μM. Hippocampal neurons from mice with the genetic defect, trisomy 16 (Ts16), died 2–3 times faster than normal (euploid) neurons. Moreover, glutamate, at all concentrations tested, failed to increase survival of Ts16 neurons. Ts16 is a naturally-occurring mouse genetic abnormality, the human analog of which (Down syndrome) leads to altered brain development and Alzheimer’s disease. These results demonstrate that the Ts16 genotype confers a defect in the glutamate-mediated survival response of hippocampal neurons and that this defect can account for their accelerated death.
Proceedings of the National Academy of Sciences, 1994
S100 beta is a calcium-binding protein that is expressed at high levels in brain primarily by ast... more S100 beta is a calcium-binding protein that is expressed at high levels in brain primarily by astrocytes. Addition of the disulfide-bonded dimeric form of S100 beta to primary neuronal and glial cultures and established cell lines induces axonal extension and alterations in astrocyte proliferation and phenotype, but evidence that S100 beta exerts the same effects in vivo has not been presented. An 8.9-kb murine S100b genomic clone was used to produce two lines of transgenic mice in which S100 beta RNA is increased in a dose-related manner to 2-fold and 7-fold above normal. These lines show concomitant increased S100 beta protein throughout the brain. Expression in both lines is cell type- and tissue-appropriate, and expression levels are correlated with the transgene copy number, demonstrating that sequences necessary for normal regulation of the gene are included within the cloned segment. In the hippocampus of adult transgenic mice, Western blotting detects elevated levels of glia...
Identification of targets and delivery platforms for gene therapy of neurodegenerative disorders ... more Identification of targets and delivery platforms for gene therapy of neurodegenerative disorders is a clinical challenge. We describe a novel paradigm in which the neuroprotective gene is the herpes simplex virus type 2 (HSV-2) antiapoptotic gene ICP10PK and the vector is the growth-compromised HSV-2 mutant DRR. DRR is delivered intranasally. It is not toxic in rats and mice. ICP10PK is expressed in the hippocampus of the DRR-treated animals for at least 42 days in the absence of virus replication and late virus gene expression. Its expression is regulated by an AP-1 amplification loop. Intranasally delivered DRR prevents kainic acid-induced seizures, neuronal loss, and inflammation, in both rats and mice. The data suggest that DRR is a promising therapeutic platform for neurodegenerative diseases.
We have previously shown that intrastriatal injection of DRR, the growth-compromised herpes simpl... more We have previously shown that intrastriatal injection of DRR, the growth-compromised herpes simplex virus type 2 (HSV-2) vector for the antiapoptotic protein ICP10PK, prevents apoptosis caused by the excitotoxin N-methyl-D-aspartate (NMDA) in a mouse model of glutamatergic neuronal cell death (Golembewski et al. [2007] Exp. Neurol. 203:381-393). Because apoptosis regulation is stimulus and cell type specific, our studies were designed to examine the mechanism of DRR-mediated neuroprotection in striatal neurons. Organotypic striatal cultures (OSC) that retain much of the synaptic circuitry of the intact striatum were infected with DRR or a growthcompromised HSV-2 vector that lacks ICP10PK (DPK) and examined for neuroprotection-associated signaling. The mutated ICP10 proteins (p175 and p95) were expressed in 70-80% of neurons from DRRand DPKinfected cultures, respectively, as determined by doubleimmunofluorescent staining with antibodies to ICP10 and NeuN or GAD65. DRRbut not DPK-treated OSC were protected from NMDA-induced apoptosis, as verified by ethidium homodimer staining, TUNEL, caspase-3 activation, and poly(AD-ribose) polymerase (PARP) cleavage. Neuroprotection was through ICP10PK-mediated activation of the survival pathways MEK/ERK and PI3-K/Akt, up-regulation of the antiapoptotic proteins Bag-1 and Bcl-2, and phosphorylation (inactivation) of the proapoptotic protein Bad. It was blocked by the MEK inhibitor U0126 or the PI3-K inhibitor LY294002, suggesting that either pathway can prevent NMDA-induced apoptosis. The data indicate that DRR-delivered ICP10PK stimulates redundant survival pathways that override proapoptotic cascades. DRR is a promising gene therapy platform against glutamatergic cell death. V
Expression of developmentally regulated antigens was used to characterize glial cells in cultures... more Expression of developmentally regulated antigens was used to characterize glial cells in cultures from embryonic mouse cerebral cortex. Over 90% of the cells had a flat morphology, and about 50% of these flat cells also expressed the ganglioside GD3. Up to 40% of all the GD3 expressing cells also expressed A2B5 antigen. Flat cells expressing either glial fibrillary acidic protein (GFAP), or GD3 or both were present at all times in vitro. These three populations of flat cells could not be further distinguished on the basis of NG2 or fibronectin expression, or with respect to their responses to the mitogens FGF-2, PDGF, or EGF. The glial cultures also contain a small number (approximately 5%) of process bearing cells with the morphological and immunocytochemical characteristics of oligodendrocyte precursors. The expression of GD3 by flat cells changed with time in culture as the fraction of flat cells expressing only GD3 declined and the fraction of cells expressing GFAP (with or without GD3) increased. The data are consistent with those flat cells expressing only GD3 being astrocyte precursors. Furthermore, between 1 and 3 weeks in vitro GD3/GFAP cells lose GD3 while retaining GFAP. Cells expressing only GFAP could be induced to express GD3 and A2B5 by treatment with FGF-2. The widespread and regulated expression of GD3 and A2B5 by murine glia is different from the restricted pattern of expression previously reported for these antigens in rat brain cell cultures. These results demonstrate that expression of GD3 and A2B5 by murine astrocytes depends on both culture age and extracellular signals and that these gangliosides are not markers for cell lineage in the mouse.
Genes from the Down syndrome (DS) critical region of human chromosome 21, which contribute to the... more Genes from the Down syndrome (DS) critical region of human chromosome 21, which contribute to the pathology of DS, are also found on mouse chromosome 16. Several animal models of DS with triplication of genes from the DS critical region have been generated, including mouse trisomy 16 (Ts16) and a partial trisomic mouse, Ts65Dn. Using computer-assisted imaging of fura-2 fluorescence, we found an elevation of intracellular cytoplasmic calcium in cortical astrocytes from neonatal Ts65Dn mouse brain, similar to that observed previously in embryonic Ts16 astrocytes. Furthermore, astrocytes from both Ts65Dn and Ts16 cortex fail to respond to the anti-proliferative actions of glutamate. These results suggest that defective regulation of cell proliferation and cellular calcium can result from triplication of DS critical region genes.
A locomotion analysis system for laboratory rats is presented. The system produces locomotion par... more A locomotion analysis system for laboratory rats is presented. The system produces locomotion parameters (LPs) in 4 different domains: force, space, time and frequency. Video images of the walking rats are used to associate the system signals with individual limbs. Numerous LPs can be derived for every test run when the rat walks through the system on the way to sweets and a personal toy placed at the exit. This manuscript demonstrates that in order to differentiate SOD1-G93A mutant rat, a model of amyotrophic lateral sclerosis (ALS), from a Sprague Dawley (SD) control rat at a pre-symptomatic stage, one has only to use 8 key parameters. These 8 parameters are the bio-markers of ALS. The spline-based transformed values of these parameters are used as explanatory variables of a logistic regression model. This model predicts the probability that the examined rat belongs to the SOD1-G93A group. The model differentiates faultlessly between the SOD1 and control groups from the very first time the rats walked through the system at 51 days old. This system provides a new paradigm for ALS diagnosis, and it can have a significant impact on the development of new therapeutic procedures for ALS. The methodology presented in this manuscript can further address the development and validation of therapeutic procedures for other neurological diseases that affect locomotion.
The regulation of intracellular Ca2+ was investigated in cultured astrocytes from the trisomy 16 ... more The regulation of intracellular Ca2+ was investigated in cultured astrocytes from the trisomy 16 (Ts16) mouse, an animal model for Down syndrome and Alzheimer's disease (AD). The cytoplasmic ionized Ca2+ concentration ([Ca2+]cyt) was determined using digital imaging of fura-2-loaded cells. The relative Ca2+ content of internal endoplasmic reticulum (ER) stores was estimated from the magnitude of the transient increase in [Ca2+]cyt induced by cyclopiazonic acid (CPA), an inhibitor of Ca2+ sequestration into ER stores. At rest, the average [Ca2+]cyt was 140 nM in euploid (normal) astrocytes, but over twice as high, 320 nM, in Ts16 cells. In the absence of extracellular Ca2+, CPA induced a transient increase in [Ca2+]cyt to over 1200 nM in Ts16 astrocytes as compared to only 500 nM in euploid cells, indicating an increased amount of Ca2+ in the Ts16 astrocyte ER. In contrast to euploid astrocytes, both resting [Ca2+]cyt and the amount of Ca2+ in the ER stores varied widely among individual Ts16 astrocytes. These results show that Ts16 produces a dysregulation of Ca2+ homeostasis leading to increased cytoplasmic and stored Ca2+. Since increases in [Ca2+]cyt have been implicated in the etiology of neurodegenerative diseases, including AD, this finding of abnormal Ca2+ homeostasis in a genetic model of human neurological disorders suggests that Ca2+ dysregulation may be a common feature underlying neurodegenerative processes.
Mechanisms that regulate the amount of releasable Ca2+ in intracellular stores of cultured mouse ... more Mechanisms that regulate the amount of releasable Ca2+ in intracellular stores of cultured mouse astrocytes were investigated using digital imaging of fura-2 loaded cells. At rest, the cytoplasmic Ca2+ concentration, [Ca2+]cyt, was about 110 nM. In the absence of extracellular Ca2+, cyclopiazonic acid (CPA), an inhibitor of the endoplasmic reticulum (ER) Ca(2+)-ATPase, induced a transient, four-fold increase in [Ca2+]cyt due to the release of Ca2+ from inositol triphosphate (IP3) sensitive stores. Caffeine (CAF), which releases Ca2+ from Ca(2+)-sensitive stores, induced a two-fold increase in [Ca2+]cyt. The CPA- and CAF-sensitive stores could be released independently. Changes in the amplitudes of the Ca2+ transients were taken as a measure of changes in store content. Removal of extracellular Na+ or addition of ouabain, which inhibit Ca2+ extrusion and promote Ca2+ entry across the plasmalemma via the Na/Ca exchanger, caused minimal increases in resting [Ca2+]cyt but greatly potentiated both CPA- and CAF-induced Ca2+ transients. The amount of Ca2+ releasable from the IP3(CPA) sensitive store was directly proportional to cytosolic Na+ concentration (i.e., inversely proportional to the transmembrane Na+ electrochemical gradient). Under these reduced Na+ gradient conditions, little, if any, Ca2+ destined for the ER stores enters the cells through voltage-dependent Ca2+ channels. These results demonstrate that mouse astrocytes contain two distinct ER Ca2+ stores, the larger, IP3- (CPA-) sensitive, and the smaller, Ca(2+)- (CAF-) sensitive. The Ca2+ content of both ER stores can be regulated by the Na/Ca exchanger. Thus, the magnitude of cellular responses to signals that are mediated by Ca2+ release induced by the two second messengers, IP3 and Ca2+, can be modulated by factors that affect the net transport of Ca2+ across the plasmalemma.
The Ts65Dn mouse, an adult model of Down syndrome displays behavioral deficits consistent with a ... more The Ts65Dn mouse, an adult model of Down syndrome displays behavioral deficits consistent with a dysfunctional hippocampus, similar to that seen with DS. In looking for mechanisms underlying these performance deficits, we have assessed adult neurogenesis in the dentate gyrus of Ts65Dn. Under untreated conditions, Ts65Dn mice (2-5 months old) showed markedly fewer BrdU-labeled cells than euploid animals. Chronic antidepressant treatment for over 3 weeks with the serotonin selective reuptake inhibitor, fluoxetine, increased neurogenesis in the Ts65Dn to comparable levels seen in the euploid by augmenting both proliferation and survival of BrdU-labeled cells in the subgranular layer and granule cell layer of the hippocampus, respectively.
During embryonic development of the mammalian cerebral cortex, the generation of the marginal zon... more During embryonic development of the mammalian cerebral cortex, the generation of the marginal zone (MZ) and subplate (SP) precedes that of the cortical plate (CP). MZ and SP neurons are believed to play a ‘pioneering’ role in directing the organization of the CP and the specificity of connections between the CP and other brain regions. Here we report that this sequential order of neurogenesis is disrupted in the trisomy 16 (Ts16) mouse, a potential animal model of Down syndrome. Bromodeoxyuridine labeling was used to establish the date of generation of postmitotic SP and CP neurons in the somatosensory cortex. As has been previously reported, most SP neurons in euploid (control) cortex were generated on embryonic day 12.5 (E12.5), and production of CP neurons began a day later. In contrast, in the Ts16 cortex, few SP neurons were born on E12.5 and most were generated on E13.5 and E14.5 when CP neurons were also being produced. Thus, in the Ts16 cortex, many CP neurons are born and a...
Spatial memory was assessed in the segmental trisomic 16 mouse (Ts65Dn), a potential model for Do... more Spatial memory was assessed in the segmental trisomic 16 mouse (Ts65Dn), a potential model for Down syndrome (DS), using the 1Zarm radial maze (RAM). Ts65Dn mice have a portion of mouse chromosome 16 syntenic to the distal end of human chromosome 21 triplicated. On each of 8 daily trials of the RAM, Ts65Dn mice made fewer correct choices than control mice and performed at or near chance levels, indicating a deficit in spatial working memory. On trials 9 and 10, Ts65Dn mice performed as well as control mice on the initial 12 choices, but required a greater number of choices to complete the RAM. The improved performance of Ts65Dn mice on trials 9 and 10 was lost when the animals were retested after a 50-day retention period, suggesting that long-term memory is also defective. These results are not likely explained by differences in either response bias or perceptual discrimination. Ts65Dn and control mice displayed comparable levels of performance in spontaneous alternation in a T-maze, demonstrating that simple spatial memory was not impaired. In the elevated plus maze, Ts65Dn mice did not display higher anxiety levels which could affect their performance in the RAM. In fact, Ts65Dn mice visited open arms on the elevated plus maze more frequently and spent more time on open arms than did control mice. Taken together, these results provide evidence for short-and long-term spatial memory deficits in Ts65Dn mice.
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Papers by Paul Yarowsky