Nervous System Of Transgenic Mice Research Articles

Activation of the unfolded protein response promotes axonal regeneration after peripheral nerve injury.

Although protein-folding stress at the endoplasmic reticulum (ER) is emerging as a driver of neuronal dysfunction in models of spinal cord injury and neurodegeneration, the contribution of this pathway to peripheral nerve damage remains poorly explored. Here we targeted the unfolded protein response (UPR), an adaptive reaction against ER stress, in mouse models of sciatic nerve injury and found that ablation of the transcription factor XBP1, but not ATF4, significantly delay locomotor recovery. XBP1 deficiency led to decreased macrophage recruitment, a reduction in myelin removal and axonal regeneration. Conversely, overexpression of XBP1s in the nervous system in transgenic mice enhanced locomotor recovery after sciatic nerve crush, associated to an improvement in key pro-regenerative events. To assess the therapeutic potential of UPR manipulation to axonal regeneration, we locally delivered XBP1s or an shRNA targeting this transcription factor to sensory neurons of the dorsal root ganglia using a gene therapy approach and found an enhancement or reduction of axonal regeneration in vivo, respectively. Our results demonstrate a functional role of specific components of the ER proteostasis network in the cellular changes associated to regeneration and functional recovery after peripheral nerve injury.

Myotonic dystrophy CTG expansion affects synaptic vesicle proteins, neurotransmission and mouse behaviour

Myotonic dystrophy type 1 is a complex multisystemic inherited disorder, which displays multiple debilitating neurological manifestations. Despite recent progress in the understanding of the molecular pathogenesis of myotonic dystrophy type 1 in skeletal muscle and heart, the pathways affected in the central nervous system are largely unknown. To address this question, we studied the only transgenic mouse line expressing CTG trinucleotide repeats in the central nervous system. These mice recreate molecular features of RNA toxicity, such as RNA foci accumulation and missplicing. They exhibit relevant behavioural and cognitive phenotypes, deficits in short-term synaptic plasticity, as well as changes in neurochemical levels. In the search for disease intermediates affected by disease mutation, a global proteomics approach revealed RAB3A upregulation and synapsin I hyperphosphorylation in the central nervous system of transgenic mice, transfected cells and post-mortem brains of patients with myotonic dystrophy type 1. These protein defects were associated with electrophysiological and behavioural deficits in mice and altered spontaneous neurosecretion in cell culture. Taking advantage of a relevant transgenic mouse of a complex human disease, we found a novel connection between physiological phenotypes and synaptic protein dysregulation, indicative of synaptic dysfunction in myotonic dystrophy type 1 brain pathology.

Development and Characterization of Thy*IκBα-SI Transgenic Mice

We developed and characterized a new transgenic model where NF-κB activity is inhibited only in mature neurons. Transgenic mouse strain Thy*IκBα-SI was created using trans-dominant super inhibitor NF-κB (IκBα-SI), which is a multimutant form of IκBα inhibitory protein cloned into specific neutral Thy-1.2 cassette. Detailed molecular analysis showed that the transgene and its product (IκBα-SI protein) are expressed in the nervous system of transgenic mice. In situ hybridization showed that Thy*IκBα-SI in the nervous system is expressed exclusively in neurons. The developed model provides wide opportunities for studying functional role of NF-κB in mature neurons in the central and peripheral nervous system in vivo.

Interferon-γ Prevents Death of Bystander Neurons during CD8 T Cell Responses in the Brain

T cells restricted to neurotropic viruses are potentially harmful as their activity may result in the destruction of neurons. In the Borna disease virus (BDV) model, antiviral CD8 T cells entering the brain of infected mice cause neurological disease but no substantial loss of neurons unless the animals lack interferon-gamma (IFN-gamma). We show here that glutamate receptor antagonists failed to prevent BDV-induced neuronal loss in IFN-gamma-deficient mice, suggesting that excitotoxicity resulting from glutamate receptor overstimulation is an unlikely explanation for the neuronal damage. Experiments with IFN-gamma-deficient mice lacking eosinophils indicated that these cells, which specifically accumulate in the infected brains of IFN-gamma-deficient mice, are not responsible for CA1 neuronal death. Interestingly, BDV-induced damage of CA1 neurons was reduced significantly in IFN-gamma-deficient mice lacking perforin, suggesting a key role for CD8 T cells in this pathological process. Specific death of hippocampal CA1 neurons could be triggered by adoptive transfer of BDV-specific CD8 T cells from IFN-gamma-deficient mice into uninfected mice that express transgene-encoded BDV antigen at high level in astrocytes. These results indicate that attack by CD8 T cells that cause the death of CA1 neurons might be directed toward regional astrocytes and that IFN-gamma protects vulnerable CA1 neurons from collateral damage resulting from exposure to potentially toxic substances generated as a result of CD8 T cell-mediated impairment of astrocyte function.

γ-Synucleinopathy: neurodegeneration associated with overexpression of the mouse protein

The role of α-synuclein in pathogenesis of familial and idiopathic forms of Parkinson’s disease, and other human disorders known as α-synucleinopathies, is well established. In contrast, the involvement of two other members of the synuclein family, β-synuclein and γ-synuclein, in the development and progression of neurodegeneration is poorly studied. However, there is a growing body of evidence that α-synuclein and β-synuclein have opposite neuropathophysiological effects. Unlike α-synuclein, overexpressed β-synuclein does not cause pathological changes in the nervous system of transgenic mice and even ameliorates the pathology caused by overexpressed α-synuclein. To assess the consequences of excess expression of the third family member, γ-synuclein, on the nervous system we generated transgenic mice expressing high levels of mouse γ-synuclein under control of Thy-1 promoter. These animals develop severe age- and transgene dose-dependent neuropathology, motor deficits and die prematurely. Histopathological changes include aggregation of γ-synuclein, accumulation of various inclusions in neuronal cell bodies and processes, and astrogliosis. These changes are seen throughout the nervous system but are most prominent in the spinal cord where they lead to loss of spinal motor neurons. Our data suggest that down-regulation of small heat shock protein HSPB1 and disintegration of neurofilament network play a role in motor neurons dysfunction and death. These findings demonstrate that γ-synuclein can be involved in neuropathophysiological changes and the death of susceptible neurons suggesting the necessity of further investigations of the potential role of this synuclein in disease.

Lysyl-tRNA Synthetase Is a Target for Mutant SOD1 Toxicity in Mitochondria

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting the motor neurons. The majority of familial forms of ALS are caused by mutations in the Cu,Zn-superoxide dismutase (SOD1). In mutant SOD1 spinal cord motor neurons, mitochondria develop abnormal morphology, bioenergetic defects, and degeneration. However, the mechanisms of mitochondrial toxicity are still unclear. One possibility is that mutant SOD1 establishes aberrant interactions with nuclear-encoded mitochondrial proteins, which can interfere with their normal trafficking from the cytosol to mitochondria. Lysyl-tRNA synthetase (KARS), an enzyme required for protein translation that was shown to interact with mutant SOD1 in yeast, is a good candidate as a target for interaction with mutant SOD1 at the mitochondrion in mammals because of its dual cytosolic and mitochondrial localization. Here, we show that in mammalian cells mutant SOD1 interacts preferentially with the mitochondrial form of KARS (mitoKARS). KARS-SOD1 interactions occur also in the mitochondria of the nervous system in transgenic mice. In the presence of mutant SOD1, mitoKARS displays a high propensity to misfold and aggregate prior to its import into mitochondria, becoming a target for proteasome degradation. Impaired mitoKARS import correlates with decreased mitochondrial protein synthesis. Ultimately, the abnormal interactions between mutant SOD1 and mitoKARS result in mitochondrial morphological abnormalities and cell toxicity. mitoKARS is the first described member of a group of mitochondrial proteins whose interaction with mutant SOD1 contributes to mitochondrial dysfunction in ALS.

Structures of the G85R Variant of SOD1 in Familial Amyotrophic Lateral Sclerosis

Mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause a dominant form of the progressive neurodegenerative disease amyotrophic lateral sclerosis. Transgenic mice expressing the human G85R SOD1 variant develop paralytic symptoms concomitant with the appearance of SOD1-enriched proteinaceous inclusions in their neural tissues. The process(es) through which misfolding or aggregation of G85R SOD1 induces motor neuron toxicity is not understood. Here we present structures of the human G85R SOD1 variant determined by single crystal x-ray diffraction. Alterations in structure of the metal-binding loop elements relative to the wild type enzyme suggest a molecular basis for the metal ion deficiency of the G85R SOD1 protein observed in the central nervous system of transgenic mice and in purified recombinant G85R SOD1. These findings support the notion that metal-deficient and/or disulfide-reduced mutant SOD1 species contribute to toxicity in SOD1-linked amyotrophic lateral sclerosis.

Immortalized hypothalamic gonadotropin-releasing hormone neurons.

The neuroendocrine hypothalamus has been intensively studied using whole animals and tissue slices. However, it has been difficult to approach questions at the molecular and cellular level. By targeting expression of the oncogene product, simian virus 40 T antigen, in transgenic mice using the regulatory domain of the rat gonadotropin-releasing hormone (GnRH) gene, we have produced specific hypothalamic tumours. These tumours have been cultured to produce clonal cell lines (GT-1 cells) that express T antigen, GnRH and many other neuronal markers, but do not express other hypothalamic hormones. These immortal cell lines have a distinctive neuronal phenotype, process the GnRH peptide accurately and secrete GnRH in a pulsatile pattern. Thus, by targeting oncogenesis to a defined population of neurons using the regulatory region of a gene that is expressed late in differentiation of that cell lineage, we have succeeded in immortalizing hypothalamic GnRH neurons. The GT-1 cell lines are an excellent model for future molecular, cell biological, physiological and biochemical investigations into the mechanisms involved in regulation of GnRH and the characteristics of an isolated central nervous system neuron. Their derivation demonstrates the utility of targeting tumorigenesis to specific differentiated neurons of the central nervous system in transgenic mice.

Age-related Changes in Tau Expression in Transgenic Mouse Model of Amyotrophic Lateral Sclerosis

The work is a continuation of studies on tau expression and alternative splicing in the central nervous system of transgenic mice harboring human SOD1 with G93A amyotrophic lateral sclerosis (ALS)-associated mutation. Since age is an important risk factor for ALS, we expanded the studies into younger animals (age 5 and 25 days). We also included cerebellum, a structure not studied in the context of neurodegeneration in ALS. We found decreased total tau-mRNA expression in hippocampus but not in cortex and spinal cord of young transgenics, and a lack of exon 10 in 5-day-old mice. In cerebellum, the total tau-mRNA expression was increased in transgenic animals during the whole period of life, however at the symptomatic stage of ALS (age 120 days) the level of protein was decreased. It can be concluded that the SOD1 G93A mutation causes early alterations of tau expression in cns, which are not exclusively restricted to the upper and lower motor neuron.

Expression of reef coral fluorescent proteins in the central nervous system of transgenic mice

Reef coral fluorescent proteins (RCFPs) are bright fluorescent proteins (FPs) covering a wide spectral range. We used various RCFP genes to transgenically color different cell populations in the brain. The mouse Thy1.2 promoter was used to target expression of HcRed1 in neurons, the human glial fibrillary acidic protein (GFAP) promoter to label astrocytes with AmCyan1, AsRed2 and mRFP1 as well as the mouse proteolipid protein promoter to mark oligodendrocytes with DsRed1. In brain sections of transgenic mice, RCFP expression was found to be highly specific using immunohistochemistry and fluorescence microscopy. In contrast to transgenic mice with expression of jellyfish FP variants, RCFPs formed numerous fluorescent precipitates. These aggregates were primarily found in cell somata and also in cell processes. Older mice were more affected than younger ones. Despite these fluorescent deposits, physiological properties of RCFP expressing brain cells such as whole-cell membrane currents or glutamate-evoked calcium signaling seemed to be unaffected.While brightness and spectral variation of RCFPs are optimal for expression in transgenic animals used in physiological experiments, the formation of fluorescent precipitates in various cell types limits their use for morphological cell analysis in situ.

Expanded dynamic range of fluorescent indicators for Ca(2+) by circularly permuted yellow fluorescent proteins.

Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Although most indicators have cyan- and yellow-emitting fluorescent proteins (CFP and YFP) as FRET donor and acceptor, their poor dynamic range often prevents detection of subtle but significant signals. Here, we optimized the relative orientation of the two chromophores in the Ca(2+) indicator, yellow cameleon (YC), by fusing YFP at different angles. We generated circularly permuted YFPs (cpYFPs) that showed efficient maturation and acid stability. One of the cpYFPs incorporated in YC absorbs a great amount of excited energy from CFP in its Ca(2+)-saturated form, thereby increasing the Ca(2+)-dependent change in the ratio of YFP/CFP by nearly 600%. Both in cultured cells and in the nervous system of transgenic mice, the new YC enables visualization of subcellular Ca(2+) dynamics with better spatial and temporal resolution than before. Our study provides an important guide for the development and improvement of indicators using GFP-based FRET.

Disruption of EphA/ephrin-A signaling in the nigrostriatal system reduces dopaminergic innervation and dissociates behavioral responses to amphetamine and cocaine

We have investigated functional roles of EphA/ephrin-A signaling in the development and function of the nigrostriatal system by overexpressing a soluble, broad-range EphA receptor antagonist in the central nervous system of transgenic mice. Adult transgenic mice showed a 30–40% reduction in the total volume of the substantia nigra (SN) without detectable differences in the number of dopaminergic neurons. Using fluorogold retrograde tracing from the striatum, we detected a 40–50% reduction in the number of dopaminergic neurons that could be traced from this structure in transgenic mice, suggesting that, a lower proportion of these cells were able to reach the striatum after disruption of EphA/ephrin-A signaling. In spite of this, total dopamine content in the striatum of transgenic mice was comparable to wild type. Analysis of locomotor activity and its regulation by pharmacological treatments that stimulate dopaminergic transmission revealed an unexpected dissociation of the behavioral responses to amphetamine and cocaine. In particular, transgenic mice were relatively insensitive to amphetamine while retaining normal responsiveness to cocaine, which, to the best of our knowledge, represents the first report of a dissociation of the behavioral responses to these two psychostimulants. Together, these results reveal an unexpected role for EphA/ephrin-A signaling in the normal connectivity and function of midbrain dopaminergic neurons.

Antisense peptide nucleic acid-mediated knockdown of the p75 neurotrophin receptor delays motor neuron disease in mutant SOD1 transgenic mice.

Re-expression of the death-signalling p75 neurotrophin receptor (p75NTR) is associated with injury and neurodegeneration in the adult nervous system. The induction of p75NTR expression in mature degenerating spinal motor neurons of humans and transgenic mice with amyotrophic lateral sclerosis (ALS) suggests a role of p75NTR in the progression of motor neuron disease (MND). In this study, we designed, synthesized and evaluated novel antisense peptide nucleic acid (PNA) constructs targeting p75NTR as a potential gene knockdown therapeutic strategy for ALS. An 11-mer antisense PNA directed at the initiation codon, but not downstream gene sequences, dose-dependently inhibited p75NTR expression and death-signalling by nerve growth factor (NGF) in Schwann cell cultures. Antisense phosphorothioate oligonucleotide (PS-ODN) sequences used for comparison failed to confer such inhibitory activity. Systemic intraperitoneal administration of this antisense PNA to mutant superoxide dismutase 1 (SOD1G93A) transgenic mice significantly delayed locomotor impairment and mortality compared with mice injected with nonsense or scrambled PNA sequences. Reductions in p75NTR expression and subsequent caspase-3 activation in spinal cords were consistent with increased survival in antisense PNA-treated mice. The uptake of fluorescent-labelled antisense PNA in the nervous system of transgenic mice was also confirmed. This study suggests that p75NTR may be a promising antisense target in the treatment of ALS.

Immunohistochemical study on the distribution of MnSOD in the central nervous system of the transgenic mice expressing a human Cu/Zn SOD mutation

In the present study, we used the SOD1 G93A mutant transgenic mice as an animal model of amyotrophic lateral sclerosis (ALS) and performed immunohistochemical studies to investigate the changes of MnSOD in the central nervous system of transgenic mice at the age of 8, 13, and 18 weeks. In the spinal cord of wild-type SOD1 (wtSOD1) and SOD1 G93A transgenic mice, MnSOD-immunoreactive neurons were distributed mainly in the anterior horn, although they were also observed in the posterior horn. The staining intensity of MnSOD was significantly increased in the spinal cord of SOD1 G93A transgenic mice at presymptomatic and symptomatic stage. In the brainstem of symptomatic SOD1 G93A transgenic mice, significantly increased immunoreactivity for MnSOD was observed in abducens nucleus, facial nucleus, dorsal motor nucleus of vagus, hypoglossal nucleus, medullary and pontine reticular formation, superior and inferior olivary nucleus, and cochlear nucleus. The present study provides the first evidence that MnSOD immunoreactivity was increased in the central nervous system of SOD G93A transgenic mice, suggesting that mitochondria may play an important role in the pathogenesis and progress of ALS. The mechanisms underlying the increased immunoreactivity for MnSOD, and the functional implications of these increases, require elucidation.

Math1-driven GFP expression in the developing nervous system of transgenic mice

Math1 is a bHLH transcription factor expressed in neural progenitor cells in multiple regions of the nervous system. Previously we identified a Math1 enhancer that directs expression of reporter genes in a Math1 specific pattern [Development 127 (2000) 1185]. We have used a portion of this enhancer to drive expression of a nuclear GFP reporter in the Math1 lineage in transgenic mice. In this transgenic mouse strain, GFP is expressed in Math1 domains in the (1) developing spinal cord in progenitors to dI1 dorsal interneurons, (2) granule-cell progenitors in the developing cerebellum, (3) Merkel cells in the skin, and (4) hair cells in the developing vestibular and auditory systems. Furthermore, non-Math1 related expression is detected that is likely due to the absence of inhibitory regulatory sequences from the transgene. These expression domains include (1) the apical ectodermal ridge in developing limbs, (2) post-mitotic cells in the developing cortex and spinal cord, (3) the dentate gyrus, (4) retina, and (5) olfactory epithelium. Because GFP marks specific neuronal cell types in living tissue, this transgenic strain is a powerful tool for future studies on the development and electrophysiological properties of distinct cell types in the central nervous system and in sensory systems.

Tetracycline-dependent regulation of formamidopyrimidine DNA glycosylase in transgenic mice conditionally reduces oxidative DNA damage in vivo.

8-Oxo-deoxyguanosine (8-oxo-dG) is a pervasive oxidative DNA lesion formed by endogenous oxidative stress and enhanced by drugs and environmental chemicals. This lesion results in transcriptional errors and mutations and is linked to neurodegeneration, teratogenesis, cancer, and other pathologies. We demonstrate that the neonatal central nervous system of transgenic mice carrying the tetracycline-regulable DNA repair gene formamidopyrimidine DNA glycosylase (fpg) has a 50% reduction in 8-oxo-dG levels. This enhanced DNA repair is suppressed by treatment with doxycycline. For the first time, this murine model permits the level of a specific DNA oxidation product to be regulated in a temporally and spatially specific manner, allowing its role as a primary or secondary factor in neurodegenerative disease to be determined in vivo.

Reactive astrocytes express cAMP-response-element-binding protein (CREB) binding protein (CBP) in the central nervous system of transgenic mice expressing a human Cu/Zn superoxide dismutase mutation

In the present study, we used the superoxide dismutase 1(SODI) G93A transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS) and performed immunohistochemical studies to investigate the changes of cAMP-response-element-binding protein (CREB) binding protein (CBP) in the central nervous system of transgenic mice. The distribution of CBP-immunoreactive neurons was not different between control and transgenic mice, whereas CBP-immunoreactive astrocytes were found only in transgenic mice. CBP-immunoreactive astrocytes were detected in the spinal cord, brainstem, midbrain and cerebellar nuclei of transgenic mice. The present study provides the first evidence that CBP immunoreactive astrocytes were observed in the central nervous system of transgenic mice, suggesting that reactive astrocytes may play an important role in the pathogenesis and progress of ALS.

Immunohistochemical study on the distribution of alpha-synuclein in the central nervous system of transgenic mice expressing a human Cu/Zn superoxide dismutase mutation

We used the SOD1 G93A transgenic mice as an in vivo model of amyotrophic lateral sclerosis (ALS) and performed immunohistochemical studies to investigate whether α-synuclein is involved in the pathogenesis of ALS. In the spinal cord of transgenic mice, immunohistochemistry showed intense staining of α-synuclein mainly in the anterior horn. In the hippocampus of transgenic mice, differential increases in the staining density of α-synuclein were observed. In the cerebellar cortex of transgenic mice, the prominent immunostaining of α-synuclein was found in the molecular and granular layers. The present study provides the first in vivo evidence that α-synuclein immunoreactivity was increased in the central nervous system of SOD G93A transgenic mice, suggesting that α-synuclein might play an important role in the pathogenesis of ALS. However, the functional implications of these increases require elucidation.

Immunohistochemical study on the distribution of homocysteine in the central nervous system of transgenic mice expressing a human Cu/Zn SOD mutation

In the present study, we used the transgenic mice expressing a human Cu/Zn SOD mutation (SOD1 G93A) as an in vivo model of ALS and performed immunohistochemical studies to investigate the changes of homocysteine in the central nervous system of symptomatic transgenic mice. In control and presymptomatic transgenic mice, homocysteine-immunoreactive astrocytes were not detected in any region. In symptomatic transgenic mice, homocysteine-immunoreactive astrocytes were distributed in the spinal cord, brainstem and cerebellar nuclei of transgenic mice. In the hippocampal formation of transgenic mice, pyramidal cells in the CA1-3 regions and granule cells in the dentate gyrus showed homocysteine immunoreactivity. The present study provides the first in vivo evidence that homocysteine immunoreactive astrocytes were found in the central nervous system of symptomatic SOD G93A transgenic mice, suggesting that reactive astrocytes may play an important role in the pathogenesis and progress of ALS. This study also suggests that increased expression of homocysteine in the hippocampal neurons might reflect a role of homocysteine in an abnormality of hippocampal function of ALS.

Neuropathology in Mice Expressing Human α-Synuclein

The presynaptic protein alpha-synuclein is a prime suspect for contributing to Lewy pathology and clinical aspects of diseases, including Parkinson's disease, dementia with Lewy bodies, and a Lewy body variant of Alzheimer's disease. alpha-Synuclein accumulates in Lewy bodies and Lewy neurites, and two missense mutations (A53T and A30P) in the alpha-synuclein gene are genetically linked to rare familial forms of Parkinson's disease. Under control of mouse Thy1 regulatory sequences, expression of A53T mutant human alpha-synuclein in the nervous system of transgenic mice generated animals with neuronal alpha-synucleinopathy, features strikingly similar to those observed in human brains with Lewy pathology, neuronal degeneration, and motor defects, despite a lack of transgene expression in dopaminergic neurons of the substantia nigra pars compacta. Neurons in brainstem and motor neurons appeared particularly vulnerable. Motor neuron pathology included axonal damage and denervation of neuromuscular junctions in several muscles examined, suggesting that alpha-synuclein interfered with a universal mechanism of synapse maintenance. Thy1 transgene expression of wild-type human alpha-synuclein resulted in similar pathological changes, thus supporting a central role for mutant and wild-type alpha-synuclein in familial and idiotypic forms of diseases with neuronal alpha-synucleinopathy and Lewy pathology. These mouse models provide a means to address fundamental aspects of alpha-synucleinopathy and test therapeutic strategies.