Richard J. Smeyne

Targeted disruption of the trkB neurotrophin receptor gene results in nervous system lesions and neonatal death

We have generated mice carrying a germline mutation in the tyrosine kinase catalytic domain of the trkB gene. This mutation eliminates expression of gp145trkB, a protein-tyrosine kinase that serves as the signaling receptor for two members of the nerve growth factor family of neurotrophins, brain-derived neurotrophic factor and neurotrophin-4. Mice homozygous for this mutation, trkBTK(-/-), develop to birth. However, these animals do not display feeding activity, and most die by P1. Neuroanatomical examination of trkBTK (-/-) mice revealed neuronal deficiencies in the central (facial motor nucleus and spinal cord) and peripheral (trigeminal and dorsal root ganglia) nervous systems. These findings illustrate the role of the gp145trkB protein-tyrosine kinase receptor in the ontogeny of the mammalian nervous system.

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.

Recessive resistance to thyroid hormone in mice lacking thyroid hormone receptor beta: evidence for tissue-specific modulation of receptor function.

The diverse functions of thyroid hormone (T3) are presumed to be mediated by two genes encoding the related receptors, TRalpha and TRbeta. However, the in vivo functions of TRalpha and TRbeta are undefined. Here, we report that targeted inactivation of the mouse TRbeta gene results in goitre and elevated levels of thyroid hormone. Also, thyroid‐stimulating hormone (TSH), which is released by pituitary thyrotropes and which is normally suppressed by increased levels of thyroid hormone, was present at elevated levels in homozygous mutant (Thrb−/−) mice. These findings suggest a unique role for TRbeta that cannot be substituted by TRalpha in the T3‐dependent feedback regulation of TSH transcription. Thrb−/− mice provide a recessive model for the human syndrome of resistance to thyroid hormone (RTH) that exhibits a similar endocrine disorder but which is typically caused by dominant TRbeta mutants that are transcriptional inhibitors. It is unknown whether TRalpha, TRbeta or other receptors are targets for inhibition in dominant RTH; however, the analysis of Thrb−/− mice suggests that antagonism of TRbeta‐mediated pathways underlies the disorder of the pituitary‐thyroid axis. Interestingly, in the brain, the absence of TRbeta may not mimic the defects often associated with dominant RTH, since no overt behavioural or neuroanatomical abnormalities were detected in Thrb−/− mice. These data define in vivo functions for TRbeta and indicate that specificity in T3 signalling is conferred by distinct receptor genes.

Nrf2-mediated neuroprotection in the MPTP mouse model of Parkinson's disease: Critical role for the astrocyte

Oxidative stress has been implicated in the etiology of Parkinsons disease (PD) and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of PD. It is known that under conditions of oxidative stress, the transcription factor NF-E2-related factor (Nrf2) binds to antioxidant response element (ARE) to induce antioxidant and phase II detoxification enzymes. To investigate the role of Nrf2 in the process of MPTP-induced toxicity, mice expressing the human placental alkaline phosphatase (hPAP) gene driven by a promoter containing a core ARE sequence (ARE-hPAP) were used. ARE-hPAP mice were injected (30 mg/kg) once per day for 5 days and killed 7 days after the last MPTP injection. In response to this design, ARE-dependent gene expression was decreased in striatum whereas it was increased in substantia nigra. The same MPTP protocol was applied in Nrf2+/+ and Nrf2−/− mice; Nrf2 deficiency increases MPTP sensitivity. Furthermore, we evaluated the potential for astrocytic Nrf2 overexpression to protect from MPTP toxicity. Transgenic mice with Nrf2 under control of the astrocyte-specific promoter for the glial fribillary acidic protein (GFAP-Nrf2) on both a Nrf2+/+ and Nrf2−/− background were administered MPTP. In the latter case, only the astrocytes expressed Nrf2. Independent of background, MPTP-mediated toxicity was abolished in GFAP-Nrf2 mice. These striking results indicate that Nrf2 expression restricted to astrocytes is sufficient to protect against MPTP and astrocytic modulation of the Nrf2-ARE pathway is a promising target for therapeutics aimed at reducing or preventing neuronal death in PD.

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.

Fos-IacZ transgenic mice: Mapping sites of gene induction in the central nervous system

Abstract A transgenic mouse line containing a fos-lacZ fusion gene was derived in which β-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.

Highly pathogenic H5N1 influenza virus can enter the central nervous system and induce neuroinflammation and neurodegeneration

One of the greatest influenza pandemic threats at this time is posed by the highly pathogenic H5N1 avian influenza viruses. To date, 61% of the 433 known human cases of H5N1 infection have proved fatal. Animals infected by H5N1 viruses have demonstrated acute neurological signs ranging from mild encephalitis to motor disturbances to coma. However, no studies have examined the longer-term neurologic consequences of H5N1 infection among surviving hosts. Using the C57BL/6J mouse, a mouse strain that can be infected by the A/Vietnam/1203/04 H5N1 virus without adaptation, we show that this virus travels from the peripheral nervous system into the CNS to higher levels of the neuroaxis. In regions infected by H5N1 virus, we observe activation of microglia and alpha-synuclein phosphorylation and aggregation that persists long after resolution of the infection. We also observe a significant loss of dopaminergic neurons in the substantia nigra pars compacta 60 days after infection. Our results suggest that a pandemic H5N1 pathogen, or other neurotropic influenza virus, could initiate CNS disorders of protein aggregation including Parkinsons and Alzheimers diseases.

Local Control of Granule Cell Generation by Cerebellar Purkinje Cells

Cerebellar Purkinje cells were ablated by the specific expression of diphtheria toxin in these cells in transgenic mice. Purkinje cell degeneration during early postnatal development shows a zonally restricted pattern which has been exploited in order to look for local secondary effects. The most obvious early effect is the alignment of gaps in the Purkinje cell layer with dramatically thinned zones in the overlying EGL, the germinal layer from which granule cells are generated. Within these EGL zones in the transgenic mutant, markers that distinguish matrix from mantle cells demonstrate a preferential loss of the proliferative cells. Comparison of BrdU incorporation in the mutant vs wild-type confirms the reduction in proliferation. In the mutant, in situ labeling of DNA fragmentation associated with apoptotic cell death shows abundant labeling of granule cells that have exited the EGL, but not of progenitor cells in the EGL. Thus, although a trophic role for Purkinje cells has been well documented, these observations further suggest a mitogenic role which can be exerted locally.

A Golgi-Cox morphological analysis of neuronal changes induced by environmental enrichment

Exposure to an enriched environment (EE), consisting of a combination of increased exercise, social interactions and learning, has been shown to produce many positive effects in the CNS. In this study, we use a Golgi-Cox analysis to examine and dissect the role of various components of the enriched environment on two measures of neuronal growth: total cell volume and total dendritic length in four regions of the brain. In the hippocampus, CA1 and dentate gyrus cells, animals raised in an enriched environment demonstrate significant morphological change. These changes were not observed in layer V pyramidal neurons of the cerebral cortex or spiny neurons located in the striatum. To determine if one or more of the individual components of the EE were responsible for the changes in neuronal morphology, we examined mice raised with free access to exercise wheels. In these mice, no morphological changes were observed. These results suggest that changes in the CA1 and dentate gyrus morphology were a result of alterations in the animals environment and not an increase in motor activity.

Glutathione metabolism and Parkinson's disease

It has been established that oxidative stress, defined as the condition in which the sum of free radicals in a cell exceeds the antioxidant capacity of the cell, contributes to the pathogenesis of Parkinson disease. Glutathione is a ubiquitous thiol tripeptide that acts alone or in concert with enzymes within cells to reduce superoxide radicals, hydroxyl radicals, and peroxynitrites. In this review, we examine the synthesis, metabolism, and functional interactions of glutathione and discuss how these relate to the protection of dopaminergic neurons from oxidative damage and its therapeutic potential in Parkinson disease.