John W. Cave

Dopamine Systems in the Forebrain

The brain contains a number of distinct regions that share expression ofdopamine (DA) and its requisite biosynthetic machinery, but otherwise encompass a diverse array of features and functions. Across the vertebrate family, the olfactory bulb (OB) contains the major DA system in the forebrain. OB DA cells are primarily periglomerular interneurons that define the glomerular structures in which they receive innervation from olfactory receptor neurons as well as mitral and tufted cells, the primary OB output neurons. The OB DA cells are necessary for both discrimination and the dynamic range over which odorant sensory information can be detected. In the embryo, OB DA neurons are derived from the ventricular area of the evaginating telencephalon, the dorsal lateral ganglionic eminence and the septum. However, most OB DA interneurons are generated postnatally and continue to be produced throughout adult life from neural stem cells in the subventricular zone of the lateral ventricle and rostral migratory stream. Adult born OB DA neurons are capable of integrating into existing circuits and do not appear to degenerate in Parkinsons disease. Several genes have been identified that regulate the differentiation of OB DA interneurons from neural stem cells. These include transcription factors that modify the expression of tyrosine hydroxylase, the first enzyme in the DA biosynthetic pathway and a reliable marker of the DA phenotype. Elucidation of the molecular genetic pathways of OB DA differentiation may advance the development of strategies to treat neurological disease.

ER81 and CaMKIV identify anatomically and phenotypically defined subsets of mouse olfactory bulb interneurons

The mechanisms underlying dopamine (DA) phenotypic differentiation in the olfactory bulb (OB) have not yet been fully elucidated and are the subject of some controversy. OB DA interneurons destined for the glomerular layer were shown to originate in the subventricular zone (SVZ) and in the rostral migratory stream (RMS). The current study investigated whether calcium/calmodulin‐dependent protein kinase IV (CaMKIV) either alone or together with the Ets transcription factor ER81 was necessary for phenotypic determination during migration of progenitors. In most brain areas, including the OB, CaMKIV and ER81 displayed a reciprocal distribution. In the SVZ, only ER81 could be demonstrated. In the RMS, a subpopulation of progenitors contained ER81, but few, if any, contained CaMKIV. In OB, CaMKIV expression, restricted to deep granule cells, showed limited overlap with ER81. ER81 expression was weak in deep granule cells. Strong labeling occurred in the mitral and glomerular layers, where ER81 colabeled dopaminergic periglomerular cells that expressed either tyrosine hydroxylase (TH) or green fluorescent protein, the latter reporter gene under control of 9‐kb of 5′ TH promoter. Odor deprivation resulted in a significant 5.2‐fold decline in TH immunoreactivity, but ER81 exhibited a relatively small 1.7‐fold decline in immunoreactivity. TH expression as well as brain and bulb size were unchanged in CaMKIV knockout mice. These data suggest that ER81 may be required but is not sufficient for DA neuron differentiation and that CaMKIV is not directly involved in TH gene regulation. J. Comp. Neurol. 502:485–496, 2007.

Differential Regulation of Dopaminergic Gene Expression by Er81

A recent study proposed that differentiation of dopaminergic neurons requires a conserved “dopamine motif” (DA-motif) that functions as a binding site for ETS DNA binding domain transcription factors. In the mammalian olfactory bulb (OB), the expression of a set of five genes [including tyrosine hydroxylase (Th)] that are necessary for differentiation of dopaminergic neurons was suggested to be regulated by the ETS-domain transcription factor ER81 via the DA-motif. To investigate this putative regulatory role of ER81, expression levels of these five genes were compared in both olfactory bulbs of adult wild-type mice subjected to unilateral naris closure and the olfactory bulbs of neonatal Er81 wild-type and mutant mice. These studies found that ER81 was necessary only for Th expression and not the other cassette genes. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assays (EMSA) experiments showed that ER81 bound directly to a consensus binding site/DA-motif in the rodent Th proximal promoter. However, the ER81 binding site/DA-motif in the Th proximal promoter is poorly conserved in other mammals. Both ChIP assays with canine OB tissue and EMSA experiments with the human Th proximal promoter did not detect ER81 binding to the Th DA-motif from these species. These results suggest that regulation of Th expression by the direct binding of ER81 to the Th promoter is a species-specific mechanism. These findings indicate that ER81 is not necessary for expression of the OB dopaminergic gene cassette and that the DA-motif is not involved in differentiation of the mammalian OB dopaminergic phenotype.

Adult subventricular zone neural stem cells as a potential source of dopaminergic replacement neurons.

Clinical trials engrafting human fetal ventral mesencephalic tissue have demonstrated, in principle, that cell replacement therapy provides substantial long-lasting improvement of motor impairments generated by Parkinsons Disease (PD). The use of fetal tissue is not practical for widespread clinical implementation of this therapy, but stem cells are a promising alternative source for obtaining replacement cells. The ideal stem cell source has yet to be established and, in this review, we discuss the potential of neural stem cells in the adult subventricular zone (SVZ) as an autologous source of replacement cells. We identify three key challenges for further developing this potential source of replacement cells: (1) improving survival of transplanted cells, (2) suppressing glial progenitor proliferation and survival, and (3) developing methods to efficiently produce dopaminergic neurons. Subventricular neural stem cells naturally produce a dopaminergic interneuron phenotype that has an apparent lack of vulnerability to PD-mediated degeneration. We also discuss whether olfactory bulb dopaminergic neurons derived from adult SVZ neural stem cells are a suitable source for cell replacement strategies.

Therapeutic targeting of oxygen-sensing prolyl hydroxylases abrogates ATF4-dependent neuronal death and improves outcomes after brain hemorrhage in several rodent models.

Blocking oxygen-sensing prolyl hydroxylases in the rodent CNS enhances functional recovery after brain hemorrhage. Beating back damage from brain bleeding Brain bleeding is associated with stroke, anticoagulant use, amyloid angiopathy, and brain trauma. Blood in the brain leads to the deposition of toxic iron, and as expected, chelators of iron can enhance functional recovery after stroke. Here, Karuppagounder et al. show that iron chelators protect from a bleeding stroke not by binding all iron but rather by targeting a small family of iron-containing enzymes, the hypoxia-inducible factor prolyl hydroxylases. The target enzymes are oxygen sensors that, when inhibited, engage a broad homeostatic response to low oxygen and oxidative stress. The authors characterize and validate a selective, brain-penetrant inhibitor of brain oxygen sensors, which they call adaptaquin, as a new candidate treatment for brain bleeding in several rodent models. Protective doses of adaptaquin were used in combination with unbiased RNA profiling to identify an unexpected hypoxia-inducible factor–independent pathway mediated by the prodeath transcription factor ATF4. Disability or death due to intracerebral hemorrhage (ICH) is attributed to blood lysis, liberation of iron, and consequent oxidative stress. Iron chelators bind to free iron and prevent neuronal death induced by oxidative stress and disability due to ICH, but the mechanisms for this effect remain unclear. We show that the hypoxia-inducible factor prolyl hydroxylase domain (HIF-PHD) family of iron-dependent, oxygen-sensing enzymes are effectors of iron chelation. Molecular reduction of the three HIF-PHD enzyme isoforms in the mouse striatum improved functional recovery after ICH. A low-molecular-weight hydroxyquinoline inhibitor of the HIF-PHD enzymes, adaptaquin, reduced neuronal death and behavioral deficits after ICH in several rodent models without affecting total iron or zinc distribution in the brain. Unexpectedly, protection from oxidative death in vitro or from ICH in vivo by adaptaquin was associated with suppression of activity of the prodeath factor ATF4 rather than activation of an HIF-dependent prosurvival pathway. Together, these findings demonstrate that brain-specific inactivation of the HIF-PHD metalloenzymes with the blood-brain barrier–permeable inhibitor adaptaquin can improve functional outcomes after ICH in several rodent models.

Epigenetic control of neurotransmitter expression in olfactory bulb interneurons

Defining the molecular mechanisms that underlie development and maintenance of neuronal phenotypic diversity in the CNS is a fundamental challenge in developmental neurobiology. The vast majority of olfactory bulb (OB) interneurons are GABAergic and this neurotransmitter phenotype is specified in migrating neuroblasts by transcription of either or both glutamic acid decarboxylase 1 (Gad1) and Gad2. A subset of OB interneurons also co‐express dopamine, but transcriptional repression of tyrosine hydroxylase (Th) suppresses the dopaminergic phenotype until these neurons terminally differentiate. In mature OB interneurons, GABA and dopamine levels are modulated by odorant‐induced synaptic activity‐dependent regulation of Gad1 and Th transcription. The molecular mechanisms that specify and maintain the GABAergic and dopaminergic phenotypes in the OB are not clearly delineated. In this report, we review previous studies and present novel findings that provide insight into the contribution of epigenetic regulatory mechanisms for controlling expression of these neurotransmitter phenotypes in the OB. We show that HDAC enzymes suppress the dopaminergic phenotype in migrating neuroblasts by repressing Th transcription. In the mature interneurons, both Th and Gad1 transcription levels are modulated by synaptic activity‐dependent recruitment of acetylated Histone H3 on both the Th and Gad1 proximal promoters. We also show that HDAC2 has the opposite transcriptional response to odorant‐induced synaptic activity when compared to Th and Gad1. These findings suggest that HDAC2 mediates, in part, the activity‐dependent chromatin remodeling of the Th and Gad1 proximal promoters in mature OB interneurons.

γ-Aminobutyric acid-mediated regulation of the activity-dependent olfactory bulb dopaminergic phenotype

γ‐Aminobutyric acid (GABA) regulates the proliferation and migration of olfactory bulb (OB) interneuron progenitors derived from the subventricular zone (SVZ), but the role of GABA in the differentiation of these progenitors has been largely unexplored. This study examines the role of GABA in the differentiation of OB dopaminergic interneurons using neonatal forebrain organotypic slice cultures prepared from transgenic mice expressing green fluorescent protein (GFP) under the control of the tyrosine hydroxylase (Th) gene promoter (ThGFP). KCl‐mediated depolarization of the slices induced ThGFP expression. The addition of GABA to the depolarized slices further increased GFP fluorescence by inducing ThGFP expression in an additional set of periglomerular cells. These findings show that GABA promoted differentiation of SVZ‐derived OB dopaminergic interneurons and suggest that GABA indirectly regulated Th expression and OB dopaminergic neuron differentiation through an acceleration of the maturation rate for the dopaminergic progenitors. Additional studies revealed that the effect of GABA on ThGFP expression required activation of L‐ and P/Q‐type Ca2+ channels as well as GABAA and GABAB receptors. These voltage‐gated Ca2+ channels and GABA receptors have previously been shown to be required for the coexpressed GABAergic phenotype in the OB interneurons. Together, these findings suggest that Th expression and the differentiation of OB dopaminergic interneurons are coupled to the coexpressed GABAergic phenotype and demonstrate a novel role for GABA in neurogenesis.

Regulation of tyrosine hydroxylase transcription by hnRNP K and DNA secondary structure

Regulation of tyrosine hydroxylase gene (Th) transcription is critical for specifying and maintaining the dopaminergic neuronal phenotype. Here we define a molecular regulatory mechanism for Th transcription conserved in tetrapod vertebrates. We show that heterogeneous nuclear ribonucleoprotein (hnRNP) K is a transactivator of Th transcription. It binds to previously unreported and evolutionarily conserved G:C-rich regions in the Th proximal promoter. hnRNP K directly binds C-rich single DNA strands within these conserved regions and also associates with double-stranded sequences when proteins, such as CREB, are bound to an adjacent cis-regulatory element. The single DNA strands within the conserved G:C-rich regions adopt either G-quadruplex or i-motif secondary structures. We also show that small molecule-mediated stabilization of these secondary structures represses Th promoter activity. These data suggest that these secondary structures are targets for pharmacological modulation of the dopaminergic phenotype.

Histone deacetylase inhibitors de-repress tyrosine hydroxylase expression in the olfactory bulb and rostral migratory stream

Most olfactory bulb (OB) interneurons are derived from neural stem cells in the subventricular zone (SVZ) and migrate to the OB via the rostral migratory stream (RMS). Mature dopaminergic interneurons in the OB glomerular layer are readily identified by their synaptic activity-dependent expression of tyrosine hydroxylase (TH). Paradoxically, TH is not expressed in neural progenitors migrating in the RMS, even though ambient GABA and glutamate depolarize these progenitors. In forebrain slice cultures prepared from transgenic mice containing a GFP reporter gene under the control of the Th 9kb upstream regulatory region, treatment with histone deacetylase (HDAC) inhibitors (either sodium butyrate, Trichostatin A or Scriptaid) induced Th-GFP expression specifically in the RMS independently of depolarizing conditions in the culture media. Th-GFP expression in the glomerular layer was also increased in slices treated with Trichostatin A, but this increased expression was dependent on depolarizing concentrations of KCl in the culture media. Th-GFP expression was also induced in the RMS in vivo by intra-peritoneal injections with either sodium butyrate or valproic acid. Quantitative RT-PCR analysis of neurosphere cultures confirmed that HDAC inhibitors de-repressed Th expression in SVZ-derived neural progenitors. Together, these findings suggest that HDAC function is critical for regulating Th expression levels in both neural progenitors and mature OB dopaminergic neurons. However, the differential responses to the combinatorial exposure of HDAC inhibitors and depolarizing culture conditions indicate that Th expression in mature OB neurons and neural progenitors in the RMS are regulated by distinct HDAC-mediated mechanisms.

Solution nuclear magnetic resonance structure of a protein disulfide oxidoreductase from Methanococcus jannaschii

The solution structure of the protein disulfide oxidoreductase Mj0307 in the reduced form has been solved by nuclear magnetic resonance. The secondary and tertiary structure of this protein from the archaebacterium Methanococcus jannaschii is similar to the structures that have been solved for the glutaredoxin proteins from Escherichia coli, although Mj0307 also shows features that are characteristic of thioredoxin proteins. Some aspects of Mj0307s unique behavior can be explained by comparing structure‐based sequence alignments with mesophilic bacterial and eukaryotic glutaredoxin and thioredoxin proteins. It is proposed that Mj0307, and similar archaebacterial proteins, may be most closely related to the mesophilic bacterial NrdH proteins. Together these proteins may form a unique subgroup within the family of protein disulfide oxidoreductases.