Generalized tetracycline induced Cre recombinase expression through the ROSA26 locus of recombinant mice.

Inducible Cre recombinase systems have been developed to bypass initial lethal phenotypes and to provide access to later embryonic or adult phenotypes. Here we describe the generation of a recombinant mouse that combines a tetracycline dependent switch with generalized Cre recombinase expression by targeting the ubiquitously expressed ROSA26 locus. This transgenic strain was developed using a simplified gene delivery system integrating both elements, the reverse tetracycline controlled trans-activator (rtTA) and rtTA inducible promoter into a single vector. In this transgenic strain, the endogenous ROSA26 promoter drives rtTA expression through a splice acceptor site. The tetracycline inducible promoter, cloned in opposite orientation to the ROSA26 locus and separated from the rtTA element by a 5 kb human p53 intron, drives Cre recombinase expression. Crossing these mice with a Cre reporter strain showed that Cre DNA-mediated recombination was ubiquitously and effectively induced during various prenatal developmental windows. Background Cre recombinase expression levels were observed in some tissues in the absence of the inducer, mostly during late embryonic developmental stages and in adult animals. Background recombination levels were low during development and most prominent in nervous tissue. Cre recombinase expression could not be effectively induced in adult animals. While rtTA mRNA levels were high in developmental and adult tissues, Cre recombinase mRNA levels remained low after doxycycline treatment. The mouse strain described here provides a valuable tool to further analyze the function of genes during specific developmental windows, by allowing the effective inactivation of their function throughout defined stages of embryonic development.

Gene expression patterns for GDNF and its receptors in the human putamen affected by Parkinson's disease: a real-time PCR study.

Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-beta superfamily, is a potent trophic factor for dopaminergic neurons of the ventral midbrain, which are known to degenerate during Parkinson's disease (PD). The neuroprotective, neurorestorative, and stimulatory properties of GDNF has prompted numerous suggestions that this trophic factor may be a potential therapeutic tool to treat PD, and it has also been widely speculated that altered GDNF expression levels may be involved in the pathophysiology of the disease. In this study, we have investigated if mRNA expression levels for GDNF and/or its receptors are altered during PD in the human putamen, a target area for dopamine neurons of the substantia nigra compacta. Expression levels were analyzed with quantitative real-time reverse transcriptase polymerase reaction (RT qPCR) in post-mortem tissues from PD patients and aged matched controls. Primer pairs specific for GDNF (isoforms I and II), and its receptor molecules, GFRalpha1 and cRET were utilized. GDNF, cRET and GFRalpha1 mRNA expression was clearly detected in the putamen of control and Parkinson's disease patients. A modest but significant upregulation of GDNF mRNA levels (Isoform I) was observed in the putamen of Parkinson's disease patients with a marked loss of nigral neurons. No significant changes were observed for the expression of cRet and GFRa1. These data suggest that the extensive loss of dopaminergic neurons in the substantia nigra, and concomitant loss of striatal dopamine, may induce compensatory changes in the expression of target derived GDNF, but not its receptor system.

Tetracycline-inducible expression systems for the generation of transgenic animals: a comparison of various inducible systems carried in a single vector.

The most often used tetracycline-regulated transgenic mice system requires the generation of two transgenic strains, one carrying an inducible promoter and the other a transactivator. In this study, we report the design of a universal and simplified regulatory gene delivery vector to facilitate the generation of conditional transgenic animals that integrate both the tetracycline regulatory and response elements in a single vector. The newly developed tetracycline reversed transactivator rtTA-M2 was used in all our constructs, based on its highly improved properties with respect to specificity, stability and inducibility. To minimize interference between the different tetracycline-inducible promoters used in this study (tetracycline-responsive element (TRE), TRE-tight, or Tk-tetO) and the rtTA-M2 transactivator, both elements were cloned in opposite directions and separated by a 5 kb human p53 intron. The functionality of this system was confirmed after in vitro transfection in a mammalian cell line. Overall induction by the tetracycline-responsive element promoter was significantly higher than that induced by the newly developed TRE-tight promoter. However, the TRE-tight promoter showed a significantly tighter expression with minimal background, and still maintained high induction levels. The minimal Tk-tetO promoter showed a very weak induction capacity. Our study demonstrates that this combination of elements, placed in a single vector is sufficient for delivering a functional tetracycline-inducible system to a mammalian cell line. Moreover, additional modifications to this regulatory gene delivery system, such as the introduction of specific cloning sites and selection markers, have been designed with the idea of creating a simplified and universal inducible system to facilitate the generation of conditional transgenic, knock-out, and knock-in animals.

Short interfering RNAs (siRNAs) for reducing dopaminergic phenotypic markers.

Short interfering RNAs (siRNAs) are double-stranded RNAs of approximately 19-29 nucleotides designed to suppress the expression of homologous genes by a process known as RNA interference (RNAi). In this study we have characterized several short interfering RNAs to reduce (knockdown) the expression of genes related to the dopaminergic system of the ventral mesencephalon. We report here effective suppression of all targeted genes, tyrosine hydroxylase (TH), the transcription factor Nr4a2 (Nurr1) and the GDNF receptor moiety cRet, by co-transfection of plasmids expressing gene specific siRNAs under control of the human U6 promoter with a reporter plasmid coding for firefly luciferase and fused in tandem to the cDNA for the gene of interest. The effective suppression of Nr4a2, cRet and tyrosine hydroxylase suggest that the U6 expression cassette could be used to deliver siRNA to mammalian cells in vivo and actively suppress the expression of dopamine related mammalian genes. The characterization of highly effective siRNAs for DA phenotypic markers may open new avenues for loss-of-function phenotypic analysis, and may lead to new approaches for gene therapy.

Minimally invasive presacral retrieval of a failed AxiaLIF rod implant: technical note and illustrative cases.

BACKGROUND CONTEXT: There are few reported cases of failed axial lumbar interbody fusion (AxiaLIF) in the existing neurosurgical literature, and an anecdotal case of open paramedian retroperitoneal approach to L5-S1 level for retrieval of AxiaLIF rod has been published. PURPOSE: The object of this study is to illustrate a minimally invasive presacral rod retrieval technique in cases with failed AxiaLIF causing lumbosacral instability. STUDY DESIGN/SETTING: Retrospective case series. METHODS: A retrospective analysis of the initial 26 cases of AxiaLIF done at our institution was performed; two cases of failed AxiaLIF that required rod removal were identified for detailed study. Available literature on the minimally invasive presacral techniques for rod retrieval was researched, and the use of a novel rod retrieval device with an expanding hex tip is discussed. RESULTS: Using a minimally invasive presacral approach through the previous surgical corridor, the authors were able to retrieve the AxiaLIF rod implant and then proceed with an alternative fusion technique. Both patients improved clinically and radiographically after revision. Removal of the presacral rod was not associated with vascular or bowel complications and required minimal operating room time with minimal blood loss. CONCLUSIONS: To the authors' knowledge, this is the first report demonstrating the safety and efficacy of minimally invasive presacral approach for removal of AxiaLIF rods in patients with failed AxiaLIF. As the AxiaLIF procedure is rapidly gaining acceptance among spine surgeons, we can expect to see increasing numbers of failed procedures as well. Understanding options for revision strategies is important for surgeons considering the use of this technique.

NMDA receptors on non-dopaminergic neurons in the VTA support cocaine sensitization.

BACKGROUND: The initiation of behavioral sensitization to cocaine and other psychomotor stimulants is thought to reflect N-methyl-D-aspartate receptor (NMDAR)-mediated synaptic plasticity in the mesolimbic dopamine (DA) circuitry. The importance of drug induced NMDAR mediated adaptations in ventral tegmental area (VTA) DA neurons, and its association with drug seeking behaviors, has recently been evaluated in Cre-loxp mice lacking functional NMDARs in DA neurons expressing Cre recombinase under the control of the endogenous dopamine transporter gene (NR1(DATCre) mice). METHODOLOGY AND PRINCIPAL FINDINGS: Using an additional NR1(DATCre) mouse transgenic model, we demonstrate that while the selective inactivation of NMDARs in DA neurons eliminates the induction of molecular changes leading to synaptic strengthening, behavioral measures such as cocaine induced locomotor sensitization and conditioned place preference remain intact in NR1(DATCre) mice. Since VTA DA neurons projecting to the prefrontal cortex and amygdala express little or no detectable levels of the dopamine transporter, it has been speculated that NMDA receptors in DA neurons projecting to these brain areas may have been spared in NR1(DATCre) mice. Here we demonstrate that the NMDA receptor gene is ablated in the majority of VTA DA neurons, including those exhibiting undetectable DAT expression levels in our NR1(DATCre) transgenic model, and that application of an NMDAR antagonist within the VTA of NR1(DATCre) animals still blocks sensitization to cocaine. CONCLUSIONS/SIGNIFICANCE: These results eliminate the possibility of NMDAR mediated neuroplasticity in the different DA neuronal subpopulations in our NR1(DATCre) mouse model and therefore suggest that NMDARs on non-DA neurons within the VTA must play a major role in cocaine-related addictive behavior.

Selective deletion of PTEN in dopamine neurons leads to trophic effects and adaptation of striatal medium spiny projecting neurons.

The widespread distribution of the tumor suppressor PTEN in the nervous system suggests a role in a broad range of brain functions. PTEN negatively regulates the signaling pathways initiated by protein kinase B (Akt) thereby regulating signals for growth, proliferation and cell survival. Pten deletion in the mouse brain has revealed its role in controlling cell size and number. In this study, we used Cre-loxP technology to specifically inactivate Pten in dopamine (DA) neurons (Pten KO mice). The resulting mutant mice showed neuronal hypertrophy, and an increased number of dopaminergic neurons and fibers in the ventral mesencephalon. Interestingly, quantitative microdialysis studies in Pten KO mice revealed no alterations in basal DA extracellular levels or evoked DA release in the dorsal striatum, despite a significant increase in total DA tissue levels. Striatal dopamine receptor D1 (DRD1) and prodynorphin (PDyn) mRNA levels were significantly elevated in KO animals, suggesting an enhancement in neuronal activity associated with the striatonigral projection pathway, while dopamine receptor D2 (DRD2) and preproenkephalin (PPE) mRNA levels remained unchanged. In addition, PTEN inactivation protected DA neurons and significantly enhanced DA-dependent behavioral functions in KO mice after a progressive 6OHDA lesion. These results provide further evidence about the role of PTEN in the brain and suggest that manipulation of the PTEN/Akt signaling pathway during development may alter the basal state of dopaminergic neurotransmission and could provide a therapeutic strategy for the treatment of Parkinson's disease, and other neurodegenerative disorders.

Alterations in prodynorphin, proenkephalin, and GAD67 mRNA levels in the aged human putamen: correlation with Parkinson's disease.

A real-time quantitative PCR approach was used to quantify mRNA levels corresponding to the neuropeptides enkephalin, dynorphin, and the 67-kDa isoform of glutamic acid decarboxylase (GAD67) in the human putamen from young and aged individuals as well as from aged patients affected by Parkinson's disease (PD). cDNA-specific primers were designed to amplify GAD67, proenkephalin (pENK), prodynorphin (pDYN), and the housekeeping genes glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and guanine nucleotide binding protein, beta-peptide 2-like I (GNB2LI). GAPDH and GNB2LI mRNA levels were similarly expressed among the groups and were therefore used as endogenous reference genes. Normalized data showed that mRNA levels for both pENK and pDYN were reduced in the putamen of aged controls and aged individuals affected by PD, compared with young controls. In addition, we showed that GAD67 mRNA levels did not change during aging and PD. Further analyses showed no differences in mRNA levels, for pENK, pDYN, or GAD67 mRNA, between PD patients and aged matched controls. These findings contrast with animal models of parkinsonism, for which expression of pDYN, pENK, and GAD67 mRNA has been reported to change after striatal dopamine denervation. Compensatory mechanisms and regional differences within the human putamen as well as the severity index of the disease, clinical diagnosis, and response to phalmacological therapy are possible reasons for these results. The present study suggests that alteration of neuropeptide pathways in the human putamen may be involved in the functional deterioration of parts of the extrapyramidal system during aging.

Characterization of a mouse strain expressing Cre recombinase from the 3' untranslated region of the dopamine transporter locus.

Dopamine (DA) neurotransmission has been implicated in several neurological and psychiatric disorders. The dopamine transporter (DAT) is highly expressed in dopaminergic neurons of the ventral mesencephalon and regulates neurotransmission by transporting DA back into the presynaptic terminals. To mediate restricted DNA recombination events into DA neurons using the Cre/loxP technology, we have generated a knockin mouse expressing Cre recombinase under the transcriptional control of the endogenous DAT promoter. To minimize interference with DAT function by preservation of both DAT alleles, Cre recombinase expression was driven from the 3' untranslated region (3'UTR) of the endogenous DAT gene by means of an internal ribosomal entry sequence. Crossing this murine line with a LacZ reporter showed colocalization of DAT immunocytochemistry and beta-galactosidase staining in all regions analyzed. This knockin mouse can be used for generating tissue specific knockouts in mice carrying genes flanked by loxP sites, and will facilitate the analysis of gene function in dopaminergic neurons.

Effects of cerebral ischemia in mice deficient in Persephin.

Persephin (Pspn), a recently cloned member of the transforming growth factor-beta superfamily (TGF-beta) and glial cell line-derived neurotrophic factor (GDNF) subfamily, is distributed throughout the nervous system at extremely low levels and is thought to function as a survival factor for midbrain dopaminergic and spinal motor neurons in vivo. Here, we report that mice lacking Pspn by homologous recombination show normal development and behavior, but are hypersensitive to cerebral ischemia. A 300% increase in infarction volume was observed after middle cerebral artery occlusion. We find that glutamate-induced Ca(2+) influx, thought to be a major component of ischemic neuronal cell death, can be regulated directly by the Persephin protein (PSP) and that PSP can reduce hypoxia/reperfusion cell death in vitro. Neuronal cell death can be prevented or markedly attenuated by administration of recombinant human PSP in vivo before ischemia in both mouse and rat models. Taken together, these data indicate that PSP is a potent modulator of excitotoxicity in the central nervous system with pronounced neuroprotective activity. Our findings support the view that PSP signaling can exert an important control function in the context of stroke and glutamate-mediated neurotoxicity, and also suggest that future therapeutic approaches may involve this novel trophic protein.