Pten regulates neuronal soma size: a mouse model of Lhermitte-Duclos disease.

Somatic inactivation of PTEN occurs in different human tumors including glioblastoma, endometrial carcinoma and prostate carcinoma. Germline mutations in PTEN result in a range of phenotypic abnormalities that occur with variable penetrance, including neurological features such as macrocephaly, seizures, ataxia and Lhermitte-Duclos disease (also described as dysplastic gangliocytoma of the cerebellum). Homozygous deletion of Pten causes embryonic lethality in mice. To investigate function in the brain, we used Cre-loxP technology to selectively inactivate Pten in specific mouse neuronal populations. Loss of Pten resulted in progressive macrocephaly and seizures. Neurons lacking Pten expressed high levels of phosphorylated Akt and showed a progressive increase in soma size without evidence of abnormal proliferation. Cerebellar abnormalities closely resembled the histopathology of human Lhermitte-Duclos disease. These results indicate that Pten regulates neuronal size in vivo in a cell-autonomous manner and provide new insights into the etiology of Lhermitte-Duclos disease.

A promoter that drives transgene expression in cerebellar Purkinje and retinal bipolar neurons.

A genomic clone encoding the Purkinje cell-specific L7 protein has been isolated and utilized to drive the expression of beta-galactosidase in mice. Three independent transgenic lines, germ line transformed with an L7-beta-galactosidase fusion gene, exhibit beta-galactosidase expression in both cerebellar Purkinje cells and retinal bipolar neurons. This distribution is the same as that previously determined for the L7 protein by immunohistochemistry. The transgenic murine lines can be used to obtain populations of marked Purkinje and bipolar neurons. Similar L7 promoter constructs can be used to express other foreign genes specifically in these two classes of neurons.

Dynamic organization of developing Purkinje cells revealed by transgene expression.

The cerebellum has many properties that make it a useful model for investigating neural development. Purkinje cells, the major output neurons of the cerebellar cortex, have drawn special attention because of the availability of biochemical markers and mutants that affect their development. The spatial expression of L7, a protein specific for Purkinje cells, and L7 beta Gal, a gene expressed in transgenic mice that was constructed from the L7 promoter and the marker beta-galactosidase, delineated bands of Purkinje cells that increased in number during early postnatal development. Expression of the transgene in adult reeler mutant mice, which show inverted cortical lamination, and in primary culture showed that the initial expression of L7 is intrinsic to Purkinje cells and does not depend on extracellular signals. This may reflect an underlying developmental map in cerebellum.

Two distinct GUCY2C circuits with PMV (hypothalamic) and SN/VTA (midbrain) origin.

Guanylyl cyclase C (GUCY2C) is the afferent central receptor in the gut-brain endocrine axis regulated by the anorexigenic intestinal hormone uroguanylin. GUCY2C mRNA and protein are produced in the hypothalamus, a major center regulating appetite and metabolic homeostasis. Further, GUCY2C mRNA and protein are expressed in the ventral midbrain, a principal structure regulating hedonic reward from behaviors including eating. While GUCY2C is expressed in hypothalamus and midbrain, its precise neuroanatomical organization and relationship with circuits regulating satiety remain unknown. Here, we reveal that hypothalamic GUCY2C mRNA is confined to the ventral premammillary nucleus (PMV), while in midbrain it is produced by neurons in the ventral tegmental area (VTA) and substantia nigra (SN). GUCY2C in the PMV is produced by 46% of neurons expressing anorexigenic leptin receptors, while in the VTA/SN it is produced in most tyrosine hydroxylase-immunoreactive neurons. In contrast to mRNA, GUCY2C protein is widely distributed throughout the brain in canonical sites of PMV and VTA/SN axonal projections. Selective stereotaxic ablation of PMV or VTA/SN neurons eliminated GUCY2C only in their respective canonical projection sites. Conversely, specific anterograde tracer analyses of PMV or VTA/SN neurons confirmed distinct GUCY2C-immunoreactive axons projecting to those canonical locations. Together, these findings reveal two discrete neuronal circuits expressing GUCY2C originating in the PMV in the hypothalamus and in the VTA/SN in midbrain, which separately project to other sites throughout the brain. They suggest a structural basis for a role for the GUCY2C-uroguanylin gut-brain endocrine axis in regulating homeostatic and behavioral components contributing to satiety.

Regulation of c-fos expression in transgenic mice requires multiple interdependent transcription control elements.

Transcription control regions of eukaryotic genes contain multiple sequence elements proposed to function independently to regulate transcription. We developed transgenic mice carrying fos-lacZ fusion genes with clustered point mutations in each of several distinct regulatory sequences: the sis-inducible element, the serum response element, the fos AP-1 site, and the calcium/cAMP response element. Analysis of Fos-lacZ expression in the CNS and in cultured cells demonstrated that all of the regulatory elements tested were required in concert for tissue- and stimulus-specific regulation of the c-fos promoter. This implies that the regulation of c-fos expression requires the concerted action of multiple control elements that direct the assembly of an interdependent transcription complex.

Control of segment-like patterns of gene expression in the mouse cerebellum.

A Purkinje cell-specific transgene, L7-lacZ, is expressed in a series of parasagitally oriented stripes in the mouse cerebellum. This banding pattern can be perturbed by promoter mutation, showing that a combination of positive and negative control elements contributes to the temporal and spatial map of L7 gene expression. In addition to the parasagittal stripes, certain mutations reveal Purkinje cells organized into compartments oriented in the transverse plane of the cerebellum. Transcription factors of the POU or homeobox families appear to be involved in controlling L7 expression in the transverse orientation. Strikingly, some of the domains of gene expression revealed by the mutations appear to correspond to functional compartments of Purkinje cells, thereby suggesting an underlying genetic principle used to orchestrate functional organization in the nervous system.

fos-lacZ transgenic mice: mapping sites of gene induction in the central nervous system.

A transgenic mouse line containing a fos-lacZ fusion gene was derived in which beta-galactosidase activity identified cell populations expressing fos either constitutively or after stimulation. Seizures and light pulses induced nuclear lacZ activity in defined populations of neurons in vivo, and an array of neurotransmitters, including glutamate, induced the transgene in primary brain cultures. In unstimulated mice, the major sites of fos-lacZ expression were skin, hair follicle, and bone. fos-lacZ mice provide a new avenue for activity mapping studies based on gene expression.

Temporal mRNA profiles of inflammatory mediators in the murine 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrimidine model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). With the exception of a few rare familial forms of the disease, the precise molecular mechanisms underlying PD are unknown. Inflammation is a common finding in the PD brain, but due to the limitation of postmortem analysis its relationship to disease progression cannot be established. However, studies using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD have also identified inflammatory responses in the nigrostriatal pathway that precede neuronal degeneration in the SNpc. To assess the pathological relevance of these inflammatory responses and to identify candidate genes that might contribute to neuronal vulnerability, we used quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to measure mRNA levels of 11 cytokine and chemokine encoding genes in the striatum of MPTP-sensitive (C57BL/6J) and MPTP-insensitive (Swiss Webster, SWR) mice following administration of MPTP. The mRNA levels of all 11 genes changed following MPTP treatment, indicating the presence of inflammatory responses in both strains. Furthermore, of the 11 genes examined only 3, interleukin 6 (Il-6), macrophage inflammatory protein 1 alpha/CC chemokine ligand 3 (Mip-1alpha/Ccl3) and macrophage inflammatory protein 1 beta/CC chemokine ligand 4 (Mip-1beta/Ccl4), were differentially regulated between C57BL/6J and SWR mice. In both mouse strains, the level of monocyte chemoattractant protein 1/CC chemokine ligand 2 (Mcp-1/Ccl2) mRNA was the first to increase following MPTP administration, and might represent a key initiating component of the inflammatory response. Using Mcp-1/Ccl2 knockout mice backcrossed onto a C57BL/6J background we found that MPTP-stimulated Mip-1alpha/Ccl3 and Mip-1beta/Ccl4 mRNA expression was significantly lower in the knockout mice; suggesting that Mcp-1/Ccl2 contributes to MPTP-enhanced expression of Mip-1alpha/Ccl3 and Mip-1beta/Ccl4. However, stereological analysis of SNpc neuronal loss in Mcp-1/Ccl2 knockout and wild-type mice showed no differences. These findings suggest that it is the ability of dopaminergic SNpc neurons to survive an inflammatory insult, rather than genetically determined differences in the inflammatory response itself, that underlie the molecular basis of MPTP resistance.

Regulation of proto-oncogene expression in adult and developing lungs.

Activation of immediate-early gene expression has been associated with mitogenesis, differentiation, nerve cell depolarization, and recently, terminal differentiation processes and programmed cell death. Previous evidence also suggested that immediate-early genes play a role in the physiology of the lungs (J. I. Morgan, D. R. Cohen, J. L. Hempstead, and T. Curran, Science 237:192-197, 1987). Therefore, we analyzed c-fos expression in adult and developing lung tissues. Seizures elicited by chemoconvulsants induced expression of mRNA for c-fos, c-jun, and junB and Fos-like immunoreactivity in lung tissue. The use of pharmacological antagonists and adrenalectomy indicated that this increased expression was neurogenic. Interestingly, by using a fos-lacZ transgenic mouse, it was shown that Fos-LacZ expression in response to seizure occurred preferentially in clusters of epithelial cells at the poles of the bronchioles. This was the same location of Fos-LacZ expression detected during early lung development. These data imply that pharmacological induction of immediate-early gene expression in adult mice recapitulates an embryological program of gene expression.

Adult and in utero exposure to cocaine alters sensitivity to the Parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Cocaine abuse is a significant problem in the United States, including its use by approximately 1% of pregnant women. Cocaine acts as an indirect agonist of dopamine at the dopamine transporter, resulting in the presence of excess dopamine in the synapse. Since synaptic dopamine can rapidly oxidize to form free radicals, it was hypothesized that exposure to this drug might produce damage in dopaminergic systems such as the substantia nigra pars compacta, damage to which is a hallmark of Parkinson's disease. To test this hypothesis we exposed mice both in utero and as adults to cocaine and examined its effects on the nigrostriatal system. We found that exposure to cocaine both in utero or as adults did not affect substantia nigra cell number, but did make these neurons more susceptible to the parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. We also found long-lasting changes in D2 receptor mRNA levels as well as changes in the monoamine transport system and several growth factors. This work suggests that use of cocaine might be a predisposing factor for development of Parkinson's disease in both adults exposed chronically as well as in individuals exposed prenatally.

MPTP and SNpc DA neuronal vulnerability: role of dopamine, superoxide and nitric oxide in neurotoxicity. Minireview.

Parkinson disease (PD) is a common neurodegenerative disease of unknown origin that is characterized, mainly, by a significant reduction in the number of dopamine neurons in the substantia nigra pars compacta (SNpc) of the brain and a dramatic reduction in dopamine levels in the corpus striatum. For reasons that we do not know, the dopamine neuron seems to be more vulnerable to damage than any other neuron in the brain. Although hypotheses of damage to the dopamine neuron include oxidative stress, growth factor decline, excitotoxicity, inflammation in the SNpc and protein aggregation, oxidative stress in the nigrostriatal dopaminergic system garners a significant amount of attention. In the oxidative stress hypothesis of PD, superoxide, nitric oxide and dopamine all conspire to create an environment that can be detrimental to the dopamine neuron. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), the tool of choice for investigations into the mechanisms involved in the death of dopamine neurons in PD, has been used extensively in attempts to sort out what happens in and around the dopamine neuron. Herein, we review the roles of dopamine, superoxide and nitric oxide in the demise of the dopamine neuron in the MPTP model of PD as it relates to the death of the dopamine neuron noted in PD.

HIF1α is necessary for exercise-induced neuroprotection while HIF2α is needed for dopaminergic neuron survival in the substantia nigra pars compacta.

Exercise reduces the risk of developing a number of neurological disorders and increases the efficiency of cellular energy production. However, overly strenuous exercise produces oxidative stress. Proper oxygenation is crucial for the health of all tissues, and tight regulation of cellular oxygen is critical to balance O2 levels and redox homeostasis in the brain. Hypoxia Inducible Factor (HIF)1α and HIF2α are transcription factors regulated by cellular oxygen concentration that initiate gene regulation of vascular development, redox homeostasis, and cell cycle control. HIF1α and HIF2α contribute to important adaptive mechanisms that occur when oxygen and ROS homeostasis become unbalanced. It has been shown that preconditioning by exposure to a stressor prior to a hypoxic event reduces damage that would otherwise occur. Previously we reported that 3 months of exercise protects SNpc dopaminergic (DA) neurons from toxicity caused by Complex I inhibition. Here, we identify the cells in the SNpc that express HIF1α and HIF2α and show that running exercise produces hypoxia in SNpc DA neurons, and alters the expression of HIF1α and HIF2α. In mice carrying a conditional knockout of Hif1α in postnatal neurons we observe that exercise alone produces SNpc TH+ DA neuron loss. Loss of HIF1α also abolishes exercise-induced neuroprotection. In mice lacking Hif2α in postnatal neurons, the number of TH+ DA neurons in the adult SNpc is diminished, but 3months of exercise rescues this loss. We conclude that HIF1α is necessary for exercise-induced neuroprotection and both HIF1α and HIF2α are necessary for the survival and function of adult SNpc DA neurons.

Correspondence between L7-lacZ-expressing Purkinje cells and labeled olivocerebellar fibers during late embryogenesis in the mouse.

It has been suggested that Purkinje cells (PC) play a role in organizing topographic relationships of several cerebellar afferent systems, including olivocerebellar fibers. This hypothesis is based on the observation that PC in the rat express biochemical heterogeneities during the presumptive period of olivocerebellar fiber ingrowth to the cerebellum. Previous studies designed to investigate the organization of murine olivocerebellar fibers during embryogenesis have suggested that interactions with PC may play a role in segregating olivocerebellar fibers after they enter the cerebellum. To determine whether PC heterogeneities are related to olivocerebellar fiber organization, transgenic mice carrying a beta-galactosidase (beta-gal) reporter gene linked to the promoter from the PC-specific gene L7/pcp-2 were used in neuroanatomical tracing experiments. Expression of the transgene mirrors endogenous L7/pcp-2 expression, which is upregulated earliest in parasagittal strips of the vermal cortex. Studies were conducted in vitro by using brainstem-cerebellar explants from embryonic day 17/18 (E17/18) and 18/19 mice. Applications of neuroanatomical tracer (horseradish peroxidase or neurobiotin) were made in either the caudal medial accessory olive (cMAO) or the rostral olive. These studies indicate that groups of olivocerebellar fibers and clusters of L7/lacZ+ and L7/lacZ-Purkinje cells respect common distribution boundaries during late embryogenesis. The strong correspondence between the distribution patterns generated by these two markers suggests that expression of L7/pcp-2 and the topographic organization of olivocerebellar (OC) fibers are not interdependent, but may be regulated by a common event or interaction, of a presently unknown nature, which occurs earlier during cerebellar development.

Temporal and spatial expression of a fos-lacZ transgene in the developing nervous system.

A Fos-lacZ transgenic mouse has been described that accurately recapitulates both constitutive and inducible patterns of c-fos expression in adult mice. Here we describe the developmental expression of the transgene in the brain during the early postnatal period. On the day of birth, expression of the transgene is observed in several discrete regions of the CNS; including the olfactory bulb, hippocampus, retrosplenial cortex, parafascicular nucleus of the thalamus, and several cranial nerve nuclei. In these regions, expression declines to adult levels by three weeks. In other regions of the CNS, expression appeared transiently after P0.

Absence of neuroanatomical and behavioral deficits in L7/pcp-2-null mice.

L7/pcp-2 is expressed exclusively in cerebellar Purkinje and retinal bipolar neurons. While the function of L7/pcp-2 is unknown, its mRNA is trafficked into dendrites, suggesting a role in dendritic physiology. To elucidate its function, L7/pcp-2-null mice were generated. These mice are neurologically normal with no signs of cerebellar or retinal dysfunction. The mice are indistinguishable from wild-type littermates with regards to gross neuroanatomy and fine structure of Purkinje cell dendrites.

Caspase-3-dependent neuronal death in the hippocampus following kainic acid treatment.

In this study, we examined the levels of activated caspase-3 in the kainic acid (KA) model of hippocampal degeneration in both sensitive (FVB/N) and resistant (129/SvEMS) strains of mice. At 30 h, 2 and 4 days following KA administration, animals were sacrificed and brains examined for pyknosis, TUNEL labeling, and activated caspase-3 immunoreactivity. Catalytically active caspase-3 was first detected 30 h following KA treatment in the sensitive, FVB/N strain. This was 18 h before the appearance of pyknosis or TUNEL labeling. The expression of activated caspase-3 continues up to 4 days post-injection. No activated caspase-3 immunoreactivity was detected in the resistant, 129/SvEMS strain, neither was there evidence of pyknosis or TUNEL staining. This suggests that activation of caspase-3 is a necessary component of KA-induced cell death.

Developmental expression of the GIRK family of inward rectifying potassium channels: implications for abnormalities in the weaver mutant mouse.

G-protein-gated inward rectifying potassium channels (GIRKs) are a newly identified gene family. These gene products are thought to form functional channels through the assembly of heteromeric subunits. Recently, it has been demonstrated that a point mutation in the GIRK2 gene, one of the GIRK family members, is the cause of the neurological and reproductive defects observed in the weaver (wv) mutant mouse. The mechanism(s) by which a single amino acid substitution in GIRK2 protein leads to the severe phenotypes in the wv / wv mouse is not fully understood. However, it implicates the importance of GIRK channels in neuronal development. To characterize the mRNA expression patterns of GIRK1-3 during mouse brain development we have used in situ hybridization analyses. We found that the expression of all three genes showed developmental regulation. In most areas that showed expression, the levels of GIRK1-3 transcripts reached their peak at around postnatal day 10 (P10). In general, GIRK1 showed the least fluctuation in its levels of expression during development, while dynamic changes were found with the levels of GIRK2 and GIRK3 transcripts. GIRK3 becomes the predominant inward rectifying K+-channel in the brain at later postnatal ages. In the CNS regions affected in the wv / wv mouse, GIRK2 is the predominant inward rectifying channel that is expressed. This suggests that the presence of the other subtypes are able to compensate for the mutated GIRK2 channel in weaver neurons that survive.

Differential strain susceptibility following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration acts in an autosomal dominant fashion: quantitative analysis in seven strains of Mus musculus.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been used as a potent neurotoxin to approximate, in animals, the pathology that is observed in human Parkinson's disease. In this study, we examine the toxicity of MPTP in seven strains of mice, spanning a genetic continuum of Mus musculus as a prelude to uncovering complex traits associated with MPTP toxicity. Seven days following injection of 80 mg/kg MPTP (4x20 mg/kg every 2 h), we find that the individual mouse strains exhibit dramatic differences in SNpc neuron survival, ranging from 63% cell loss in C57BL/6J mice to 14% cell loss in Swiss-Webster (SW) mice. In order to determine if the susceptibility trait was dominant, additive or recessive, we crossed C57Bl/6J mice with either SWR/J or AKR/J mice and examined the effect of MPTP on F1 C57BL/6JxSWR/J or F1 C57BL/6JxAKR/J animals. We find that all of the F1 animals were phenotypically identical to the C57BL/6J animals. In addition, no gender differences were noted in any of the MPTP-treated inbred mice or in the F1 animals. These results suggest that susceptibility to cell loss following MPTP is autosomal dominant and this polymorphism is carried on the C57BL/6J allele.

Postnatal development of the wild-type and weaver cerebellum after embryonic administration of propylthiouracil (PTU).

In this study we used propylthiouracil (PTU), a thyroid hormone-inhibiting compound, to render +/+, wv/+, and wv/wv embryos hypothyroid in order to test if the appearance of external granule layer (EGL) cell death in the weaver cerebellum is affected by alteration of granule cell development. At birth, the number of EGL cells in the PTU-treated cerebellum was reduced, compared to control animals, by 50%. Also, the amount of cell death was reduced in the PTU-treated wv/wv cerebellum. As adults, no differences were seen between PTU-treated and untreated mutant or normal cerebella. If the hypothyroid treatment that results in a 50% decrease in EGL cell number is due to an extension of cell cycling time, then the expression of the weaver phenotype of cell death likely follows granule cell exit from the cell cycle.