Karen L. O’Malley

Identification of cell type-specific promoter elements associated with the rat tyrosine hydroxylase gene using transgenic founder analysis

Transcriptional regulatory elements capable of directing transgene expression to individual cells are powerful tools for manipulating a given CNS circuit. Delineating these elements via traditional transgenic analysis is both costly and labor intensive. Here we have used the rat tyrosine hydroxylase (TH) promoter as a model to describe and validate the use of founder animals for systematic promoter studies. No significant differences were found when data obtained from founder animals expressing a 6.0 kb TH promoter directing LacZ were compared with animals derived from an analogous transgenic line. Subsequent studies with founder animals expressing beta-galactosidase directed by various lengths of rat TH promoter revealed different patterns of expression. Specifically, a locus coeruleus regulatory domain was localized between 3.4 and 6.0 kb of the rat TH promoter, a hypothalamic regulatory domain between 2.5 and 3.4 kb and a brainstem regulatory domain between 0.8 and 6.0 kb. At least one element of a midbrain specific regulatory domain was within 2.5 kb of the transcriptional start site. Olfactory bulb specific elements however appeared to reside outside of the sequences tested. Specific patterns of ectopic gene expression were also observed suggesting the presence of negative regulatory elements. Thus, TH appears to be regulated in a complex modular fashion by both positive and negative regulatory elements. Taken together, this study demonstrates the feasibility and reliability of founder analysis for promoter studies of genes expressed in complex spatial and temporal patterns.

Dynamic Changes in Striatal Dopamine D2 and D3 Receptor Protein and mRNA in Response to 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine (MPTP) Denervation in Baboons

Loss of nigrostriatal neurons leads to striatal dopamine deficiency and subsequent development of parkinsonism. The effects of this denervation on D2-like receptors in striatum remain unclear. Most studies have demonstrated increases in striatal dopamine D2-like receptors in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-mediated denervation, but others have found either decreases or no change in binding. To clarify the response to denervation, we have investigated the time-dependent changes in dopamine D2, D3, and D4 receptor protein and mRNA levels in unilaterally MPTP-lesioned baboons. MPTP (0.4 mg/kg) was infused into one internal carotid artery, producing a contralateral hemi-parkinsonian syndrome. After MPTP treatment, the animals were maintained for 17–480 d and then euthanized. MPTP decreased ipsilateral dopamine content by >90%, which did not change with time. Ipsilateral D2-like receptor binding in caudate and putamen initially decreased then increased two- to sevenfold over the first 100 d and returned to near baseline levels by 480 d. Relative levels of D2mRNA were essentially unchanged over this period. D4 mRNA was not detected. In contrast, D3 mRNA increased sixfold by 2 weeks and then decreased. At the peak period of increase in binding sites, all D2-like receptors were in a micromolar affinity agonist-binding state, implying an increase in uncoupled D2but not D3 receptor protein. Taken together, these data suggest that MPTP-induced changes in D2-like dopamine receptors are complex and include translational or post-translational mechanisms.

A microdevice platform for visualizing mitochondrial transport in aligned dopaminergic axons.

Experimental evidence points to the importance of mitochondrial transport defects in contributing to major neurodegenerative diseases, such as Parkinsons disease (PD). Studies of mitochondrial transport along single axons are difficult with traditional dissociated culture systems and the fragility of the midbrain dopaminergic cultures precludes their survival in previously developed microfluidic devices with an enclosed architecture. Using soft lithography, we generated a microdevice from polydimethylsiloxane (PDMS) for the purpose of studying the transport of mitochondria along single dopaminergic axons. The device comprises two large open culture chambers connected by a parallel array of microchannels that achieves fluidic separation of axons from the soma and allows the tracking of mitochondrial movement along oriented axons. Dopaminergic neurons from midbrain cultures were successfully cultured within the microdevices for up to 4 weeks and extended their axons across the microchannels. Axonal mitochondria within the microchannels were labeled by transduction with a mitochondrial-targeted DsRed2 lentiviral vector or with the mitochondria-specific dye, Mitotracker Deep Red and were visually tracked with conventional confocal microscopy. The methodology and device that we have described here will allow further study of the role of mitochondrial transport defects play in major neurodegenerative diseases.

Nuclear localization of functional metabotropic glutamate receptor mGlu1 in HEK293 cells and cortical neurons: role in nuclear calcium mobilization and development.

The Group I metabotropic glutamate receptor (mGlu1) plays an important role in neuromodulation, development, and synaptic plasticity. Using immunocytochemistry, subcellular fractionation, and western blot analysis, the present study shows that mGlu1a receptors are present on nuclear membranes in stably transfected human embryonic kidney 293 (HEK293) cells as well as being endogenously expressed on rat cortical nuclei. Both glutamate and the group I agonist, quisqualate, directly activate nuclear mGlu1 receptors leading to a characteristic oscillatory pattern of calcium flux in isolated HEK nuclei and a slow rise to plateau in isolated cortical nuclei. In either case calcium responses could be terminated upon application of the mGlu1‐selective antagonist, 7‐(hydroxyamino)cyclopropa[b]chromen‐1a‐carboxylate ethyl ester. Responses could also be blocked by ryanodine and inositol 1,4,5‐triphosphate receptor inhibitors, demonstrating the involvement of these calcium channels. Agonist activation of intracellular receptors was driven by Na+‐dependent and ‐independent processes in nuclei isolated from either HEK or cortical neurons. Finally, mGlu1 nuclear receptors were dramatically up‐regulated in the course of post‐natal development. Therefore, like the other Group I receptor, mGlu5, mGlu1 can function as an intracellular receptor, suggesting a more encompassing role for nuclear G protein‐coupled receptors and downstream signaling elements in the regulation of nuclear events.

Intracellular GPCRs Play Key Roles in Synaptic Plasticity

The trillions of synaptic connections within the human brain are shaped by experience and neuronal activity, both of which underlie synaptic plasticity and ultimately learning and memory. G protein-coupled receptors (GPCRs) play key roles in synaptic plasticity by strengthening or weakening synapses and/or shaping dendritic spines. While most studies of synaptic plasticity have focused on cell surface receptors and their downstream signaling partners, emerging data point to a critical new role for the very same receptors to signal from inside the cell. Intracellular receptors have been localized to the nucleus, endoplasmic reticulum, lysosome, and mitochondria. From these intracellular positions, such receptors may couple to different signaling systems, display unique desensitization patterns, and/or show distinct patterns of subcellular distribution. Intracellular GPCRs can be activated at the cell surface, endocytosed, and transported to an intracellular site or simply activated in situ by de novo ligand synthesis, diffusion of permeable ligands, or active transport of non-permeable ligands. Current findings reinforce the notion that intracellular GPCRs play a dynamic role in synaptic plasticity and learning and memory. As new intracellular GPCR roles are defined, the need to selectively tailor agonists and/or antagonists to both intracellular and cell surface receptors may lead to the development of more effective therapeutic tools.

Does axonopathy play a role in Parkinson's disease?

Background Impaired axonal transport may play an early, pivotal role in a variety of neurodegenerative disorders, including Parkinson’s disease (PD). For example, postmortem studies on PD patients show widespread axonal pathology preceding the loss of cell bodies, animal models of PD-linked mutations such as the R1441G LRRK2 mutation exhibit decreased dopamine (DA) terminal fields together with increased dystrophic processes and abnormal axonal swellings, reduced axonal transport is also seen with a-synuclein mutants, and the PD-like toxin, MPP, de-polymerizes microtubules leading to axon fragmentation as well as directly inhibiting axon transport in the isolated squid axoplasm. Thus, PD-linked environmental and genetic factors support the hypothesis that axon transport failure plays an early, critical role in this disorder.

Functional G Protein-Coupled Receptors on Nuclei from Brain and Primary Cultured Neurons

A growing number of G protein-coupled receptors (GPCRs) have been identified on nuclear membranes. In many cases, it is unknown how the intracellular GPCR is activated, how it is trafficked to nuclear membranes, and what long-term signaling consequences follow nuclear receptor activation. Here we describe how to isolate nuclei that are free from plasma membrane and cytoplasmic contamination yet still exhibit physiological properties following receptor activation.

Protein Oxidation Triggers the Unfolded Protein Response and Neuronal Injury in Chemically Induced Parkinson Disease

The recent identification of genetic mutations linked to Parkinson’s disease (PD), such as α-synuclein, parkin, and LRRK2, has highlighted the role of aberrant protein handling and degradation in this disorder. Moreover, a growing body of data suggests that environmental toxins that mimic PD also exhibit faulty protein handling, providing a mechanistic link between toxicity and the identified PD mutations. In particular, toxin-mediated cell stress and/or some PD mutations can trigger unfolded protein response, a cell-protective mechanism intended for surviving short-term cellular perturbations. If this process cannot overcome the insult, it is thought that apoptosis is rapidly activated. Although the toxicity of several parkinsonian mimetics is thought to stem from the production of reactive oxygen species, whether oxidative stress and other forms of cell stress are subsequent or parallel events is not well established. Emerging data collected using molecular, biochemical, and cellular techniques suggest that oxidative stress precedes the appearance of unfolded protein response which, in turn, precedes apoptosis. Knowledge of the signaling pathways utilized by parkinsonian mimetics as well as their temporal induction may aid in designing more effective interventions in models of PD and ultimately to treat PD in humans.