Intrastriatal injection of an adenoviral vector expressing glial-cell-line-derived neurotrophic factor prevents dopaminergic neuron degeneration and behavioral impairment in a rat model of Parkinson disease

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor for adult nigral dopamine neurons in vivo. GDNF has both protective and restorative effects on the nigro-striatal dopaminergic (DA) system in animal models of Parkinson disease. Appropriate administration of this factor is essential for the success of its clinical application. Since it cannot cross the blood–brain barrier, a gene transfer method may be appropriate for delivery of the trophic factor to DA cells. We have constructed a recombinant adenovirus (Ad) encoding GDNF and injected it into rat striatum to make use of its ability to infect neurons and to be retrogradely transported by DA neurons. Ad-GDNF was found to drive production of large amounts of GDNF, as quantified by ELISA. The GDNF produced after gene transfer was biologically active: it increased the survival and differentiation of DA neurons in vitro. To test the efficacy of the Ad-mediated GDNF gene transfer in vivo, we used a progressive lesion model of Parkinson disease. Rats received injections unilaterally into their striatum first of Ad and then 6 days later of 6-hydroxydopamine. We found that mesencephalic nigral dopamine neurons of animals treated with the Ad-GDNF were protected, whereas those of animals treated with the Ad-β-galactosidase were not. This protection was associated with a difference in motor function: amphetamine-induced turning was much lower in animals that received the Ad-GDNF than in the animals that received Ad-β-galactosidase. This finding may have implications for the development of a treatment for Parkinson disease based on the use of neurotrophic factors.

Inactivation of the survival motor neuron gene, a candidate gene for human spinal muscular atrophy, leads to massive cell death in early mouse embryos

Proximal spinal muscular atrophy is an autosomal recessive human disease of spinal motor neurons leading to muscular weakness with onset predominantly in infancy and childhood. With an estimated heterozygote frequency of 1/40 it is the most common monogenic disorder lethal to infants; milder forms represent the second most common pediatric neuromuscular disorder. Two candidate genes—survival motor neuron (SMN) and neuronal apoptosis inhibitory protein have been identified on chromosome 5q13 by positional cloning. However, the functional impact of these genes and the mechanism leading to a degeneration of motor neurons remain to be defined. To analyze the role of the SMN gene product in vivo we generated SMN-deficient mice. In contrast to the human genome, which contains two copies, the mouse genome contains only one SMN gene. Mice with homozygous SMN disruption display massive cell death during early embryonic development, indicating that the SMN gene product is necessary for cellular survival and function.

Subplate neuron ablation alters neurotrophin expression and ocular dominance column formation

Ocular dominance column formation in visual cortex depends on both the presence of subplate neurons and the endogenous expression of neurotrophins. Here we show that deletion of subplate neurons, which supply glutamatergic inputs to visual cortex, leads to a paradoxical increase in brain-derived neurotrophic factor mRNA in the same region of visual cortex in which ocular dominance columns are absent. Subplate neuron ablation also increases glutamic acid decarboxylase-67 levels, indicating an alteration in cortical inhibition. These observations imply a role for this special class of neurons in modulating activity-dependent competition by regulating levels of neurotrophins and excitability within a developing cortical circuit.

Neural restrictive silencer factor recruits mSin3 and histone deacetylase complex to repress neuron-specific target genes

Accumulative evidence suggests that more than 20 neuron-specific genes are regulated by a transcriptional cis-regulatory element known as the neural restrictive silencer (NRS). A trans-acting repressor that binds the NRS, NRSF [also designated RE1-silencing transcription factor (REST)] has been cloned, but the mechanism by which it represses transcription is unknown. Here we show evidence that NRSF represses transcription of its target genes by recruiting mSin3 and histone deacetylase. Transfection experiments using a series of NRSF deletion constructs revealed the presence of two repression domains, RD-1 and RD-2, within the N- and C-terminal regions, respectively. A yeast two-hybrid screen using the RD-1 region as a bait identified a short form of mSin3B. In vitro pull-down assays and in vivo immunoprecipitation-Western analyses revealed a specific interaction between NRSF-RD1 and mSin3 PAH1-PAH2 domains. Furthermore, NRSF and mSin3 formed a complex with histone deacetylase 1, suggesting that NRSF-mediated repression involves histone deacetylation. When the deacetylation of histones was inhibited by tricostatin A in non-neuronal cells, mRNAs encoding several neuronal-specific genes such as SCG10, NMDAR1, and choline acetyltransferase became detectable. These results indicate that NRSF recruits mSin3 and histone deacetylase 1 to silence neural-specific genes and suggest further that repression of histone deacetylation is crucial for transcriptional activation of neural-specific genes during neuronal terminal differentiation.

X-ngnr-1 and Xath3 promote ectopic expression of sensory neuron markers in the neurula ectoderm and have distinct inducing properties in the retina

Xath3 encodes a Xenopus neuronal-specific basic helix–loop–helix transcription factor related to the Drosophila proneural factor atonal. We show here that Xath3 acts downstream of X-ngnr-1 during neuronal differentiation in the neural plate and retina and that its expression and activity are modulated by Notch signaling. X-ngnr-1 activates Xath3 and NeuroD by different mechanisms, and the latter two genes crossactivate each other. In the ectoderm, X-ngnr-1 and Xath3 have similar activities, inducing ectopic sensory neurons. Among the sensory-specific markers tested, only those that label cranial neurons were found to be ectopically activated. By contrast, in the retina, X-ngnr-1 and Xath3 overexpression promote the development of overlapping but distinct subtypes of retinal neurons. Together, these data suggest that X-ngnr-1 and Xath3 regulate successive stages of early neuronal differentiation and that, in addition to their general proneural properties, they may contribute, in a context-dependent manner, to some aspect of neuronal identity.

Effect of β-amyloid block of the fast-inactivating K+ channel on intracellular Ca2+ and excitability in a modeled neuron

β-Amyloid peptide (Aβ), one of the primary protein components of senile plaques found in Alzheimer disease, is believed to be toxic to neurons by a mechanism that may involve loss of intracellular calcium regulation. We have previously shown that Aβ blocks the fast-inactivating potassium (A) current. In this work, we show, through the use of a mathematical model, that the Aβ-mediated block of the A current could result in increased intracellular calcium levels and increased membrane excitability, both of which have been observed in vitro upon acute exposure to Aβ. Simulation results are compared with experimental data from the literature; the simulations quantitatively capture the observed concentration dependence of the neuronal response and the level of increase in intracellular calcium.

A relationship between behavior, neurotrophin expression, and new neuron survival

The high vocal center (HVC) controls song production in songbirds and sends a projection to the robust nucleus of the archistriatum (RA) of the descending vocal pathway. HVC receives new neurons in adulthood. Most of the new neurons project to RA and replace other neurons of the same kind. We show here that singing enhances mRNA and protein expression of brain-derived neurotrophic factor (BDNF) in the HVC of adult male canaries, Serinus canaria. The increased BDNF expression is proportional to the number of songs produced per unit time. Singing-induced BDNF expression in HVC occurs mainly in the RA-projecting neurons. Neuronal survival was compared among birds that did or did not sing during days 31–38 after BrdUrd injection. Survival of new HVC neurons is greater in the singing birds than in the nonsinging birds. A positive causal link between pathway use, neurotrophin expression, and new neuron survival may be common among systems that recruit new neurons in adulthood.

Survival motor neuron protein modulates neuron-specific apoptosis

Spinal muscular atrophy (SMA) is attributed to mutations in the SMN1 gene, leading to loss of spinal cord motor neurons. The neurotropic Sindbis virus vector system was used to investigate a role for the survival motor neuron (SMN) protein in regulating neuronal apoptosis. Here we show that SMN protects primary neurons and differentiated neuron-like stem cells, but not cultured cell lines from virus-induced apoptotic death. SMN also protects neurons in vivo and increases survival of virus-infected mice. SMN mutants (SMNΔ7 and SMN-Y272C) found in patients with SMA not only lack antiapoptotic activity but also are potently proapoptotic, causing increased neuronal apoptosis and animal mortality. Full-length SMN is proteolytically processed in brains undergoing apoptosis or after ischemic injury. Mutation of an Asp-252 of SMN abolished cleavage of SMN and increased the antiapoptotic function of full-length SMN in neurons. Taken together, deletions or mutations of the C terminus of SMN that result from proteolysis, splicing (SMNΔ7), or germ-line mutations (e.g., Y272C), produce a proapoptotic form of SMN that may contribute to neuronal death in SMA and perhaps other neurodegenerative disorders.

Heterogeneity of subcellular localization and electrophoretic mobility of survival motor neuron (SMN) protein in mammalian neural cells and tissues

Spinal muscular atrophy is caused by defects in the survival motor neuron (SMN) gene. To better understand the patterns of expression of SMN in neuronal cells and tissues, we raised a polyclonal antibody (abSMN) against a synthetic oligopeptide from SMN exon 2. AbSMN immunostaining in neuroblastoma cells and mouse and human central nervous system (CNS) showed intense labeling of nuclear “gems,” along with prominent nucleolar immunoreactivity in mouse and human CNS tissues. Strong cytoplasmic labeling was observed in the perikarya and proximal dendrites of human spinal motor neurons but not in their axons. Immunoblot analysis revealed a 34-kDa species in the insoluble protein fractions from human SY5Y neuroblastoma cells, embryonic mouse spinal cord cultures, and human CNS tissue. By contrast, a 38-kDa species was detected in the cytosolic fraction of SY5Y cells. We conclude that SMN protein is expressed prominently in both the cytoplasm and nucleus in multiple types of neurons in brain and spinal cord, a finding consistent with a role for SMN as a determinant of neuronal viability.

Mice transgenically overexpressing sulfonylurea receptor 1 in forebrain resist seizure induction and excitotoxic neuron death

The ability of the sulfonylurea receptor (SUR) 1 to suppress seizures and excitotoxic neuron damage was assessed in mice transgenically overexpressing this receptor. Fertilized eggs from FVB mice were injected with a construct containing SUR cDNA and a calcium-calmodulin kinase IIα promoter. The resulting mice showed normal gross anatomy, brain morphology and histology, and locomotor and cognitive behavior. However, they overexpressed the SUR1 transgene, yielding a 9- to 12-fold increase in the density of [3H]glibenclamide binding to the cortex, hippocampus, and striatum. These mice resisted kainic acid-induced seizures, showing a 36% decrease in average maximum seizure intensity and a 75% survival rate at a dose that killed 53% of the wild-type mice. Kainic acid-treated transgenic mice showed no significant loss of hippocampal pyramidal neurons or expression of heat shock protein 70, whereas wild-type mice lost 68–79% of pyramidal neurons in the CA1–3 subfields and expressed high levels of heat shock protein 70 after kainate administration. These results indicate that the transgenic overexpression of SUR1 alone in forebrain structures significantly protects mice from seizures and neuronal damage without interfering with locomotor or cognitive function.

Autoimmunity to βIV spectrin in paraneoplastic lower motor neuron syndrome

Paraneoplastic neurological disorders may result from autoimmunity directed against antigens shared by the affected neurons and the associated cancer cells. We have recently reported the case of a woman with breast cancer and paraneoplastic lower motor neuron syndrome whose serum contained autoantibodies directed against axon initial segments and nodes of Ranvier of myelinated axons, including the axons of motoneurons. Here, we show that major targets of the autoantibodies of this patient are βIVΣ1 spectrin and βIV spectrin 140, two isoforms of the novel βIV spectrin gene, as well as a neuronal surface epitope yet to be identified. Partial improvement of the neurological symptoms following cancer removal was associated with a drastic reduction in the titer of the autoantibodies against βIV spectrin and nodal antigens in general, consistent with the autoimmune pathogenesis of the paraneoplastic lower motor neuron syndrome. The identification of βIV spectrin isoforms and surface nodal antigens as novel autoimmune targets in lower motor neuron syndrome provide new insights into the pathogenesis of this severe neurological disease.

Simple neural networks for the amplification and utilization of small changes in neuron firing rates

I describe physiologically plausible “voter-coincidence” neural networks such that secondary “coincidence” neurons fire on the simultaneous receipt of sufficiently large sets of input pulses from primary sets of neurons. The networks operate such that the firing rate of the secondary, output neurons increases (or decreases) sharply when the mean firing rate of primary neurons increases (or decreases) to a much smaller degree. In certain sensory systems, signals that are generally smaller than the noise levels of individual primary detectors, are manifest in very small increases in the firing rates of sets of afferent neurons. For such systems, this kind of network can act to generate relatively large changes in the firing rate of secondary “coincidence” neurons. These differential amplification systems can be cascaded to generate sharp, “yes–no” spike signals that can direct behavioral responses.

Colony-forming progenitors from mouse olfactory epithelium: evidence for feedback regulation of neuron production.

The mammalian olfactory epithelium (OE) supports continual neurogenesis throughout life, suggesting that a neuronal stem cell exists in this system. In tissue culture, however, the capacity of the OE for neurogenesis ceases after a few days. In an attempt to identify conditions that support the survival of neuronal stem cells, a population of neuronal progenitors was isolated from embryonic mouse OE and cultured in defined serum-free medium. The vast majority of cells rapidly gave rise to neurons, which died shortly thereafter. However, when purified progenitors were co-cultured with cells derived from the stroma underlying the OE, a small subpopulation (0.07-0.1%) gave rise to proliferative colonies. A morphologically identifiable subset of these colonies generated new neurons as late as 7 days in vitro. Interestingly, development of these neuronal colonies was specifically inhibited when purified progenitors were plated onto stromal feeder cells in the presence of a large excess of differentiated OE neurons. These results indicate that a rare cell type, with the potential to undergo prolonged neurogenesis, can be isolated from mammalian OE and that stroma-derived factors are important in supporting neurogenesis by this cell. The data further suggest that differentiated neurons provide a signal that feeds back to inhibit production of new neurons by their own progenitors.

Identification of potential target genes for the neuron-restrictive silencer factor.

The neuron-restrictive silencer factor (NRSF) represses transcription of several neuronal genes in nonneuronal cells by binding to a 21-bp element called the neuron-restrictive silencer element (NRSE). We have performed data base searches with a composite NRSE to identify additional candidate NRSF target genes. Twenty-two more genes, 17 of which are expressed mainly in neurons, were found to contain NRSE-like sequences. Many of these putative NRSEs bound NRSF in vitro and repressed transcription in vivo. Most of the neuronal genes identified contribute to the basic structural or functional properties of neurons. However, two neuronal transcription factor genes contain NRSEs, suggesting that NRSF may repress neuronal differentiation both directly and indirectly. Functional NRSEs were also found in several nonneuronal genes, implying that NRSF may play a broader role than originally anticipated.