Enhanced Cortical Metabolic Activity in Females and Males of a Slow Progressing Mouse Model of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with selective degeneration of motor neurons in the central nervous system. The pathophysiology of ALS is not well understood. We have used ¹H-[¹³C]-NMR spectroscopy together with an administration of [1,6-¹³C₂]glucose and [2-¹³C]acetate in female and male SOD1^G37R mice to assess neuronal and astroglial metabolic activity, respectively, in the central nervous system in ALS condition. The female (p?=?0.0008) and male (p?<?0.0001) SOD1^G37R mice exhibited decreased forelimb strength when compared with wild-type mice. There was a reduction in N-acetylaspartylglutamate level, and elevation in myo-inositol in the spinal cord of female and male SOD1^G37R mice. The transgenic male mice exhibited increased acetate oxidation in the spinal cord (p?=?0.05) and cerebral cortex (p?=?0.03), while females showed an increase in the spinal cord (p?=?0.02) only. As acetate is transported and preferentially metabolized in the astrocytes, the finding of increased rate of acetate oxidation in the transgenic mice is suggestive of astrocytic involvement in the pathogenesis of ALS. The rates of glucose oxidation in glutamatergic (p?=?0.0004) and GABAergic neurons (p?=?0.0052) were increased in the cerebral cortex of male SOD1^G37R mice when compared with the controls. The female mice showed an increase in glutamatergic (p?=?0.039) neurometabolic activity only. The neurometabolic activity was unperturbed in the spinal cord of either sex. These data suggest differential changes in neurometabolic activity across the central nervous system in SOD1^G37R mice.

     

Alteration of neurotransmitter phenotype in noradrenergic neurons of transgenic mice.

The normal complement of neurotransmitters in noradrenergic neurons was altered by expressing the structural gene for the enzyme phenylethanolamine-N-methyltransferase (PNMT) under the control of the dopamine-beta-hydroxylase gene promoter in transgenic mice. This resulted in accumulation of large amounts of epinephrine in neurons of the sympathetic nervous system (SNS) and central nervous system (CNS) but did not reduce norepinephrine levels. Adrenalectomy reduced PNMT levels in the SNS and CNS, suggesting that the transgene is positively regulated by adrenal steroids. Epinephrine levels were unaffected by this treatment in the CNS, suggesting that PNMT is not rate limiting for epinephrine synthesis. However, catecholamines were elevated in a sympathetic ganglion and a target tissue of the SNS, perhaps due to up-regulation of tyrosine hydroxylase in response to adrenalectomy. These transgenic mice also reveal a marked difference in the ability of chromaffin cells and neurons to synthesize epinephrine.     

Passive immunization against beta-amyloid peptide protects central nervous system (CNS) neurons from increased vulnerability associated with an Alzheimer's disease-causing mutation

To characterize the effects of the familial Alzheimer's disease-causing Swedish mutations of amyloid precursor protein (SwAPP) on the vulnerability of central nervous system neurons, we induced epileptic seizures in transgenic mice expressing SwAPP. The transgene expression did not change the seizure threshold, but consistently more neurons degenerated in brains of SwAPP mice as compared with wild-type littermates. The degenerating neurons were stained both by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and by Gallyas silver impregnation. A susceptible population of neurons accumulated intracellular Abeta and immunoreacted with antibodies against activated caspase-3. To demonstrate that increased Abeta levels mediated the increased vulnerability, we infused antibodies against Abeta and found a significant reduction in neuronal loss that was paralleled by decreased brain levels of Abeta. Because the SwAPP mice exhibited no amyloid plaques at the age of these experiments, transgenic overproduction of Abeta in brain rendered neurons susceptible to damage much earlier than the onset of amyloid plaque formation. Our data underscore the possibility that Abeta is toxic, that it increases the vulnerability of neurons to excitotoxic events produced by seizures, and that lowering Abeta by passive immunization can protect neurons from Abeta-related toxicity.     

Mutant Cu/Zn-superoxide dismutase proteins have altered solubility and interact with heat shock/stress proteins in models of amyotrophic lateral sclerosis

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a familial form of amyotrophic lateral sclerosis. In humans and experimental models, death of motor neurons is preceded by formation of cytoplasmic aggregates containing mutant SOD-1 protein. In our previous studies, heat shock protein 70 (HSP70) prolonged viability of cultured motor neurons expressing mutant human SOD-1 and reduced formation of aggregates. In this paper, we report that mutant SOD-1 proteins have altered solubility in cells relative to wild-type SOD-1 and can form a direct association with HSP70 and other stress proteins. Whereas wild-type human and endogenous mouse SOD-1 were detergent-soluble, a portion of mutant SOD-1 was detergent-insoluble in protein extracts of NIH3T3 transfected with SOD-1 gene constructs, spinal cord cultures established from G93A SOD-1 transgenic mouse embryos, and lumbar spinal cord from adult G93A transgenic mice. A direct association of HSP70, HSP40, and alphaB-crystallin with mutant SOD-1 (G93A or G41S), but not wild-type or endogenous mouse SOD-1, was demonstrated by coimmunoprecipitation. Mutant SOD-1.HSP70 complexes were predominantly in the detergent-insoluble fraction. However, only a small percentage of total cellular mutant SOD-1 was detergent-insoluble, suggesting that mutation-induced alteration of protein conformation may not in itself be sufficient for direct interaction with heat shock proteins.     

Human poliovirus receptor gene expression and poliovirus tissue tropism in transgenic mice.

Expression of the human poliovirus receptor (PVR) in transgenic mice results in susceptibility to poliovirus infection. In the primate host, poliovirus infection is characterized by restricted tissue tropism. To determine the pattern of poliovirus tissue tropism in PVR transgenic mice, PVR gene expression and susceptibility to poliovirus infection were examined by in situ hybridization. PVR RNA is expressed in transgenic mice at high levels in neurons of the central and peripheral nervous system, developing T lymphocytes in the thymus, epithelial cells of Bowman's capsule and tubules in the kidney, alveolar cells in the lung, and endocrine cells in the adrenal cortex, and it is expressed at low levels in intestine, spleen, and skeletal muscle. After infection, poliovirus replication was detected only in neurons of the brain and spinal cord and in skeletal muscle. These results demonstrated that poliovirus tissue tropism is not governed solely by expression of the PVR gene nor by accessibility of cells to virus. Although transgenic mouse kidney tissue expressed poliovirus binding sites and was not a site of poliovirus replication, when cultivated in vitro, kidney cells developed susceptibility to infection. Identification of the changes in cultured kidney cells that permit poliovirus infection may provide information on the mechanism of poliovirus tissue tropism.     

Generation of the tamoxifen-inducible DBH-Cre transgenic mouse line DBH-CT

We generated transgenic mice bearing a tamoxifen-dependent Cre recombinase expressed under the control of the dopamine-β-hydroxylase promoter. By crossing to the ROSA26 reporter mice we show that tamoxifen-induced Cre recombinase in adult mice specifically activates β-galactosidase expression in differentiated noradrenergic neurons of the central and peripheral nervous system. Tamoxifen application in adult mice did not induce β-galactosidase activity in parasympathetic neurons that transiently express DBH during development. Thus, this transgenic mouse line represents a valuable tool to study gene function in mature noradrenergic neurons by conditional inactivation.     

Dopaminergic neuronal loss, reduced neurite complexity and autophagic abnormalities in transgenic mice expressing G2019S mutant LRRK2

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s) through which familial mutations precipitate neuronal degeneration and PD.     

Evidence for a cell-specific action of Reelin in the spinal cord

Reelin, the extracellular matrix protein missing in reeler mice, plays an important role in neuronal migration in the central nervous system. We examined the migratory pathways of phenotypically identified spinal cord neurons to determine whether their positions were altered in reeler mutants. Interneurons and projection neurons containing choline acetyltransferase and/or NADPH diaphorase were studied in E12.5-E17.5 reeler and wild-type embryos, and their final locations were assessed postnatally. While three groups of dorsal horn interneurons migrated and differentiated normally in reeler mice, the migrations of both sympathetic (SPNs) and parasympathetic preganglionic neurons (PPNs) were aberrant in the mutants. Initially reeler and wild-type SPNs were detected laterally near somatic motor neurons, but by E13.5, many reeler SPNs had mismigrated medially. Postnatally, 79% of wild-type SPNs were found laterally, whereas in reeler, 92% of these neurons were positioned medially. At E13.5, both reeler and wild-type PPNs were found laterally, but by E14.5, reeler PPNs were scattered across the intermediate spinal cord while wild-type neurons correctly maintained their lateral location. By postnatal day 16, 97% of PPNs were positioned laterally in wild-type mice; in contrast, only 62% of PPNs were found laterally in mutant mice. In E12.5-E14.5 wild-type mice, Reelin-secreting cells were localized along the dorsal and medial borders of both groups of preganglionic neurons, but did not form a solid barrier. In contrast, Dab1, the intracellular adaptor protein thought to function in Reelin signaling, was expressed in cells having positions consistent with their identification as SPNs and PPNs. In combination, these findings suggest that, in the absence of Reelin, both groups of autonomic motor neurons migrate medially past their normal locations, while somatic motor neurons and cholinergic interneurons in thoracic and sacral segments are positioned normally. These results suggest that Reelin acts in a cell-specific manner on the migration of cholinergic spinal cord neurons.     

Specific expression of the chicken delta-crystallin gene in the lens and the pyramidal neurons of the piriform cortex in transgenic mice

Two transgenic mice, 5-8 and 7-5, carrying the chicken delta-crystallin gene were produced by microinjecting cloned genes into male pronuclei. The mice were analyzed at 8 weeks of age with respect to gene integration and expression by means of blotting techniques and immunohistochemistry. Southern blot analysis indicated that both mice carried, on average, 50 copies of intact delta-crystallin gene per cell. Histological analysis of the mice using DNA-DNA in situ hybridization indicated that mouse 5-8 carried the delta-crystallin gene in every cell while mouse 7-5 was mosaic, with 20-40% of the cells of various tissues carrying the gene. Western blot analysis indicated that in both mice delta-crystallin is expressed in the lens and the cerebrum, but not in any other tissue examined. Immunohistological analysis revealed that, in the cerebrum of the mice, delta-crystallin was expressed specifically in pyramidal neurons located in layer IIb of the anterior piriform cortex. Thus, our results with transgenic mice not only demonstrate the primary specificity of delta-crystallin gene expression in authentic lens tissue, but reveal the unexpected specificity of this chicken gene in the central nervous system of the mouse.     

Overexpression of the human NFM subunit in transgenic mice modifies the level of endogenous NFL and the phosphorylation state of NFH subunits

Neurofilaments (NFs), the major intermediate filaments of central nervous system (CNS) and peripheral nervous system (PNS) neurons, are heteropolymers formed from the high (NFH), middle (NFM), and low (NFL) molecular weight NF subunits. To gain insights into how the expression of NF subunit proteins is regulated in vivo, two transgenes harboring coding sequences for human NFM (hNFM) with or without the hNFM multiphosphorylation repeat domain were introduced into mice. Expression of both hNFM constructs was driven by the hNFM promoter and resulted in increased levels of hNFM subunits concomitant with an elevation in the levels of mouse NFL (mNFL) proteins in the CNS of both lines of transgenic mice. The increased levels of mNFL appear specific to NFM because previous studies of transgenic mice overexpressing either NFL or NFH did not result in increased expression of either of the other two NF subunits. Further, levels of the most heavily phosphorylated isoforms of mouse NFH (mNFH) were reduced in the brains of these transgenic mice, and electron microscopic studies showed a higher packing density of NFs in large-diameter CNS axons of transgenic versus wild-type mice. Thus, reduced phosphorylation of the mNFH carboxy terminal domain may be a compensatory response of CNS neurons to the increase in NFs, and reduced negative charges on mNFH sidearms may allow axons to accommodate more NFs by increasing their packing density. Taken together, these studies imply that NFM may play a dominant role in the in vivo regulation of the levels of NFL protein, the stoichiometry of NF subunits, and the phosphorylation state of NFH. NFM and NFH proteins may assume similar functions in regulation of NF packing density in vivo.     

Crystal structure of neuroserpin: a neuronal serpin involved in a conformational disease

The protease inhibitor neuroserpin regulates the development of the nervous system and its plasticity in the adult. Neuroserpins carrying the Ser53Pro or Ser56Arg mutation form polymers in neuronal cells. We describe here the structure of wild-type neuroserpin in a cleaved form. The structure provides a basis to understand the role of the mutations in the polymerization process. We propose that these mutations could delay the insertion of the reactive center loop into the central beta-sheet A, an essential step in the inhibition and possibly in the polymerization of neuroserpin.     

Transgenic mice expressing the <Emphasis Type="Italic">Peripherin-EGFP</Emphasis> genomic reporter display intrinsic peripheral nervous system fluorescence

The development of homologous recombination methods for the precise modification of bacterial artificial chromosomes has allowed the introduction of disease causing mutations or fluorescent reporter genes into human loci for functional studies. We have introduced the EGFP gene into the human PRPH-1 locus to create the Peripherin-EGFP (hPRPH1-G) genomic reporter construct. The hPRPH1-G reporter was used to create transgenic mice with an intrinsically fluorescent peripheral nervous system (PNS). During development, hPRPH1-G expression was concomitant with the acquisition of neuronal cell fate and growing axons could be observed in whole embryo mounts. In the adult, sensory neurons were labeled in both the PNS and central nervous system, while motor neurons in the spinal cord had more limited expression. The fusion protein labeled long neuronal processes, highlighting the peripheral circuitry of hPRPH1-G transgenic mice to provide a useful resource for a range of neurobiological applications.     

Expression of human immunodeficiency virus type 1 in the nervous system of transgenic mice leads to neurological disease.

Patients infected with the human immunodeficiency virus type 1 (HIV-1) frequently develop central and peripheral nervous system complications, some of which may reflect the effect of the virus itself. In order to elucidate the pathogenic mechanisms of HIV in neurological disease in a small animal model, we generated transgenic mice expressing the entire HIV genome under control of the promoter for the human neurofilament NF-L gene. The transgene was predominantly expressed in anterior thalamic and spinal motor neurons. Animals developed a neurological syndrome characterized by hypoactivity and weakness and by axonal degeneration in peripheral nerves. These results provide evidence for a role of HIV in affecting both the central and peripheral nervous systems. This animal model may also facilitate the development of therapeutic agents against the human disease.     

The visna virus long terminal repeat directs expression of a reporter gene in activated macrophages, lymphocytes, and the central nervous systems of transgenic mice.

Visna virus is a lentivirus which causes a slow progressive disease involving the immune system and the central nervous system. To determine the role of the viral long terminal repeat (LTR) in targeting the virus to specific host cells and tissues, transgenic mice were constructed which contained the visna virus LTR directing expression of the bacterial gene encoding chloramphenicol acetyltransferase (CAT). Analysis of the transgenic mouse tissues for CAT activity revealed that the viral LTR was responsible, in part, for the tropism of visna virus for macrophages and the central nervous system. Expression of the LTR required the macrophage to be in an activated state both in vivo and in vitro. Thioglycolate activation of peritoneal macrophages in vivo and 12-O-tetradecanoylphorbol 13-acetate treatment in vitro induced expression of the visna virus LTR. Lymphocytes from the spleens of the transgenic mice expressed CAT activity, suggesting that visna virus was able to replicate in lymphocytes, as did human immunodeficiency virus and simian immunodeficiency virus. These studies demonstrated that the lentivirus LTR was responsible, in part, for cell and tissue tropism in vivo.     

Temporal regulation of Cre-recombinase activity in Scl-positive neurons of the central nervous system

The Cre/LoxP system provides a powerful tool to investigate gene function in vivo. This system requires Cre-recombinase expressing mouse lines that permit control of gene recombination in a tissue-specific and time-dependent manner. To allow spatio-temporal gene deletion in specific central nervous system (CNS) neuronal populations, we generated mice with a tamoxifen-inducible Cre (Cre-ER(T)) transgene under control of the Scl/Tal1 neural promoter/enhancer -0.9E3 (-0.9E3CreER(T) transgenic mice). Using Cre-reporter mice we have shown that tamoxifen-mediated Cre-ER(T) recombination in -0.9E3CreER(T) mice recapitulated the anticipated expression pattern of Scl in the caudal thalamus, midbrain, hindbrain, and spinal cord. Cre-mediated recombination was also effectively induced during embryogenesis and marked the same population of neurons as observed in the adult. Additionally, we identified a tamoxifen-independent constitutively active -0.9E3CreER(T) mouse line that will be useful for gene deletion during early neurogenesis. These -0.9E3CreER(T) mice will provide tools to investigate the role of neuronal genes in the developing and mature CNS. CNS.     

Increased inferior olivary neuron and cerebellar granule cell numbers in transgenic mice overexpressing the human Bcl-2 gene

Neuron-target interactions during development are critical for determining the final numbers of neurons in the nervous system. To investigate the role of Purkinje cells and programmed cell death in the regulation of afferent neuron numbers, we have counted olivary neurons and granule cells in two lines of transgenic mice (NSE73a and NSE71) that overexpress a human gene for bcl-2 (Hu-bcl-2) in Purkinje cells and olivary neurons, but not in granule cells. Bcl-2 overexpression in vivo reduces naturally occurring neuronal cell death and cell death following axotomy, target removal, or ischemia. Olivary neuron numbers in NSE73a and NSE71 transgenic mice are significantly increased compared to controls by 28% and 27%, respectively, while granule cell numbers are only increased in NSE73a mice (29% above controls). We have previously shown that Purkinje cell number is increased by 43% in NSE73a transgenics and by 23% in NSE71 transgenics. The ratio of Purkinje cells to olivary neurons is not significantly different between the control and transgenic mice, while the ratio of granule cells to Purkinje cells is significantly decreased in the NSE71 transgenic mice compared to controls and NSE73a transgenics. The increased numbers of olivary neurons suggest that bcl-2 overexpression rescues these neurons from programmed cell death. The increase in granule cell number in only one transgenic line is discussed with respect to hypotheses that Purkinje cells regulate both granule cell progenitor proliferation and the survival of differentiated granule cells. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 502–516, 1997     

Expression of the protein chaperone, clusterin, in spinal cord cells constitutively and following cellular stress, and upregulation by treatment with Hsp90 inhibitor

Clusterin, a protein chaperone found at high levels in physiological fluids, is expressed in nervous tissue and upregulated in several neurological diseases. To assess relevance to amyotrophic lateral sclerosis (ALS) and other motor neuron disorders, clusterin expression was evaluated using long-term dissociated cultures of murine spinal cord and SOD1^G93A transgenic mice, a model of familial ALS. Motor neurons and astrocytes constitutively expressed nuclear and cytoplasmic forms of clusterin, and secreted clusterin accumulated in culture media. Although clusterin can be stress inducible, heat shock failed to increase levels in these neural cell compartments despite robust upregulation of stress-inducible Hsp70 (HspA1) in non-neuronal cells. In common with HSPs, clusterin was upregulated by treatment with the Hsp90 inhibitor, geldanamycin, and thus could contribute to the neuroprotection previously identified for such compounds in disease models. Clusterin expression was not altered in cultured motor neurons expressing SOD1^G93A by gene transfer or in presymptomatic SOD1^G93A transgenic mice; however, clusterin immunolabeling was weakly increased in lumbar spinal cord of overtly symptomatic mice. More striking, mutant SOD1 inclusions, a pathological hallmark, were strongly labeled by anti-clusterin. Since secreted, as well as intracellular, mutant SOD1 contributes to toxicity, the extracellular chaperoning property of clusterin could be important for folding and clearance of SOD1 and other misfolded proteins in the extracellular space. Evaluation of chaperone-based therapies should include evaluation of clusterin as well as HSPs, using experimental models that replicate the control mechanisms operant in the cells and tissue of interest.     

HuD binds to three AU-rich sequences in the 3'-UTR of neuroserpin mRNA and promotes the accumulation of neuroserpin mRNA and protein

Neuroserpin is an axonally secreted serine protease inhibitor expressed in the nervous system that protects neurons from ischemia-induced apoptosis. Mutant neuroserpin forms have been found polymerized in inclusion bodies in a familial autosomal encephalopathy causing dementia, or associated with epilepsy. Regulation of neuroserpin expression is mostly unknown. Here we demonstrate that neuroserpin mRNA and the RNA-binding protein HuD are co-expressed in the rat central nervous system, and that HuD binds neuroserpin mRNA in vitro with high affinity. Gel-shift, supershift and T1 RNase assays revealed three HuD-binding sequences in the 3′-untranslated region (3′-UTR) of neuroserpin mRNA. They are AU-rich and 20, 51 and 19 nt in length. HuD binding to neuroserpin mRNA was also demonstrated in extracts of PC12 pheochromocytoma cells. Additionally, ectopic expression of increasing amounts of HuD in these cells results in the accumulation of neuroserpin 3′-UTR mRNA. Furthermore, stably transfected PC12 cells over-expressing HuD contain increased levels of both neuroserpin mRNAs (3.0 and 1.6 kb) and protein. Our results indicate that HuD stabilizes neuroserpin mRNA by binding to specific AU-rich sequences in its 3′-UTR, which prolongs the mRNA lifetime and increases protein level.     

Activity-dependent shedding of heparin-binding EGF-like growth factor in brain neurons

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is initially produced as a membrane-anchored precursor (pro-HB-EGF) and subsequently liberated from the cell membrane through ectodomain shedding. Here, we characterized the molecular regulation of pro-HB-EGF shedding in the central nervous system. Cultured neocortical or hippocampal neurons were transfected with the alkaline-phosphatase-tagged pro-HB-EGF gene and stimulated with various neurotransmitters. Both kainate and N-methyl-D-aspartate, but not agonists for metabotropic glutamate receptors, promoted pro-HB-EGF shedding and HB-EGF release, which were attenuated by an exocytosis blocker and metalloproteinase inhibitors. In the brain of transgenic mice over-expressing human pro-HB-EGF, kainate-induced seizure activity decreased content of pro-HB-EGF-like immunoreactivity and conversely increased levels of soluble HB-EGF. There was concomitant phosphorylation of EGF receptors (ErbB1) following seizures, suggesting that seizure activities liberated HB-EGF and activated neighboring ErbB1 receptors. Therefore, we propose that glutamatergic neurotransmission in the central nervous system plays a crucial role in regulating ectodomain shedding of pro-HB-EGF.