Odorant Sensory Input Modulates DNA Secondary Structure Formation and Heterogeneous Ribonucleoprotein Recruitment on the Tyrosine Hydroxylase and Glutamic Acid Decarboxylase 1 Promoters in the Olfactory Bulb.

Adaptation of neural circuits to changes in sensory input can modify several cellular processes within neurons, including neurotransmitter biosynthesis levels. For a subset of olfactory bulb interneurons, activity-dependent changes in GABA are reflected by corresponding changes in Glutamate decarboxylase 1 (Gad1) expression levels. Mechanisms regulating Gad1 promoter activity are poorly understood, but here we show that a conserved G:C-rich region in the mouse Gad1 proximal promoter region both recruits heterogeneous nuclear ribonucleoproteins (hnRNPs) that facilitate transcription and forms single-stranded DNA secondary structures associated with transcriptional repression. This promoter architecture and function is shared with Tyrosine hydroxylase (Th), which is also modulated by odorant-dependent activity in the olfactory bulb. This study shows that the balance between DNA secondary structure formation and hnRNP binding on the mouse Th and Gad1 promoters in the olfactory bulb is responsive to changes in odorant-dependent sensory input. These findings reveal that Th and Gad1 share a novel transcription regulatory mechanism that facilitates sensory input-dependent regulation of dopamine and GABA expression.SIGNIFICANCE STATEMENT Adaptation of neural circuits to changes in sensory input can modify several cellular processes within neurons, including neurotransmitter biosynthesis levels. This study shows that transcription of genes encoding rate-limiting enzymes for GABA and dopamine biosynthesis (Gad1 and Th, respectively) in the mammalian olfactory bulb is regulated by G:C-rich regions that both recruit heterogeneous nuclear ribonucleoproteins (hnRNPs) to facilitate transcription and form single-stranded DNA secondary structures associated with repression. hnRNP binding and formation of DNA secondary structure on the Th and Gad1 promoters are mutually exclusive, and odorant sensory input levels regulate the balance between these regulatory features. These findings reveal that Th and Gad1 share a transcription regulatory mechanism that facilitates odorant-dependent regulation of dopamine and GABA expression levels.

Partial Conservation between Mice and Humans in Olfactory Bulb Interneuron Transcription Factor Codes.

The mammalian main olfactory bulb (OB) has a large population of GABAergic inhibitory interneurons that contains several subtypes defined by the co-expression other neurotransmitters and calcium binding proteins. The three most commonly studied OB interneuron subtypes co-express either Calretinin, Calbindin, or Tyrosine hydroxylase (Th). Combinations of transcription factors used to specify the phenotype of progenitors are referred to as transcription factor codes, and the current understanding of transcription factor codes that specify OB inhibitory neuron phenotypes are largely based on studies in mice. The conservation of these transcription factor codes in the human OB, however, has not been investigated. The aim of this study was to establish whether transcription factor codes in OB interneurons are conserved between mice and humans. This study compared the co-expression of Foxp2, Meis2, Pax6, and Sp8 transcription factors with Calretinin, Calbindin, or Th in human and mouse OB interneurons. This analysis found strong conservation of Calretinin co-expression with Sp8 and Meis2 as well as Th co-expression with Pax6 and Meis2. This analysis also showed that selective Foxp2 co-expression with Calbindin was conserved between mice and humans, which suggests Foxp2 is a novel determinant of the OB Calbindin interneuron phenotype. Together, the findings in this study provide insight into the conservation of transcription codes for OB interneuron phenotypes between humans and mice, as well as reveal some important differences between the species. This advance in our understanding of transcription factor codes in OB interneurons provides an important complement to the codes that have been established for other regions within the mammalian central nervous system, such as the cortex and spinal cord.

Cytoarchitectural changes in the olfactory bulb of Parkinson's disease patients.

Olfactory dysfunction is associated with nearly all the cases of Parkinson's disease (PD) and typically manifests years before motor symptoms are detected. The cellular mechanisms underlying this dysfunction, however, are not understood. In this study, olfactory bulbs (OBs) from male control and PD subjects were examined by histology for changes in cytoarchitecture. These studies found that the general OB laminar organization and the number of interneurons expressing tyrosine hydroxylase were unaltered. In contrast, the number of mitral/tufted projection neurons and interneurons expressing Calretinin were significantly decreased in PD subjects. This study reveals changes in OB cytoarchitecture mediated by PD and provides valuable insight into identifying specific OB neuronal populations vulnerable to PD-related neurodegeneration.

TMPyP4, a Stabilizer of Nucleic Acid Secondary Structure, Is a Novel Acetylcholinesterase Inhibitor.

The porphyrin compound, TMPyP4 (5,10,15,20-Tetrakis-(N-methyl-4-pyridyl)porphine), is widely used as a photosensitizer and a modulator of nucleic acid secondary structure stability. Our group recently showed in cultured cells and forebrain slice cultures that this compound can also down regulate expression of Tyrosine hydroxylase (Th), which encodes the rate-limiting enzyme in catecholamine biosynthesis, by stabilizing DNA secondary structures in the Th proximal promoter. The current study sought to establish whether treatment with TMPyP4 could modify mouse Th expression levels in vivo. Intraperitoneal administration of low TMPyP4 doses (10mg/kg), similar to those used for photosensitization, did not significantly reduce Th transcript levels in several catecholaminergic regions. Administration of a high dose (40 mg/kg), similar to those used for tumor xenograph reduction, unexpectedly induced flaccid paralysis in an age and sex-dependent manner. In vitro analyses revealed that TMPyP4, but not putative metabolites, inhibited Acetylcholinesterase activity and pre-treatment of TMPyP4 with Hemeoxygenase-2 (HO-2) rescued Acetylcholinesterase function. Age-dependent differences in HO-2 expression levels may account for some of the variable in vivo effects of high TMPyP4 doses. Together, these studies indicate that only low doses of TMPyP4, such as those typically used for photosensitization, are well tolerated in vivo. Thus, despite its widespread use in vitro, TMPyP4 is not ideal for modifying neuronal gene expression in vivo by manipulating nucleic acid secondary structure stability, which highlights the need to identify more clinically suitable compounds that can modulate nucleic acid secondary structure and gene expression.

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 to C-rich single-stranded DNA within these conserved regions and also associates with double-stranded sequences when proteins, such as CRE-binding protein, 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.