Michael F. Miles

The stress protein response in cultured neurons: characterization and evidence for a protective role in excitotoxicity.

We used purified cultures of cerebellar granule cells to investigate the possible protective role of stress proteins in an in vitro model of excitotoxicity. Initial experiments used one- and two-dimensional polyacrylamide gel electrophoresis to confirm the induction of typical stress protein size classes by heat shock, sodium arsenite, and the calcium ionophore A23187. Immunoblot analysis and immunocytochemistry verified the expression of the highly inducible 72 kd heat shock protein (HSP72). Granule cell cultures exposed to glutamate showed evidence of cellular injury that was prevented by the noncompetitive NMDA antagonist MK-801, yet glutamate did not induce a detectable stress protein response. Nonetheless, preinduction of heat shock proteins was associated with protection from toxic concentrations of glutamate. These results imply that the HSP72 expression observed in in vivo models of excitotoxicity may not be directly related to the effects of excitatory amino acids. However, the ability of stress protein induction to protect against injury from glutamate may offer a novel approach toward ameliorating damage from excitotoxins.

Up regulation of calbindin-D28K mRNA in the rat hippocampus following focal stimulation of the perforant path

Calbindin-D28K is a constitutive Ca2(+)-binding protein expressed in hippocampal neurons that are resistant to various forms of excitotoxic injury. However, the local factors controlling calbindin-D28K expression within the central nervous system are unknown. We report that neuronal excitation via the perforant path leads to an increased expression of calbindin-D28K mRNA within dentate granule cells. This response is related specifically to stimulation that induces prolonged periods of bursting afterdischarges and precedes cellular injury. The up regulation of calbindin-D28K mRNA occurs during the type of neuronal activation associated with elevated cytosolic Ca2+ and suggests that the maintenance of Ca2+ homeostasis includes a system of feedback control at the level of gene expression.

Interaction of Ethanol with Inducers of Glucose-regulated Stress Proteins ETHANOL POTENTIATES INDUCERS OF grp78 TRANSCRIPTION

GRP78, a molecular chaperone expressed in the endoplasmic reticulum, is a “glucose-regulated protein” induced by stress responses that deplete glucose or intracisternal calcium or otherwise disrupt glycoprotein trafficking. Previously we showed that chronic ethanol exposure increases the expression of GRP78. To further understand the mechanism underlying ethanol regulation of GRP78 expression, we studied the interaction between ethanol and classical modulators of GRP78 expression in NG108-15 neuroblastoma × glioma cells. We found that, in addition to increasing basal levels of GRP78 mRNA (“induction”), ethanol produced greater than additive increases in the induction of GRP78 mRNA by the “classical” GRP inducers A23187, brefeldin A, and thapsigargin (“potentiation”). Both the ethanol induction and potentiation responses modulated grp78 gene transcription as determined by stable transfection analyses with the rat grp78 promoter. Ethanol potentiated the action of all classical inducers of grp78 transcription that were studied. In contrast, co-treatment with the classical GRP inducers thapsigargin and tunicamycin produced only simple additive increases in grp78 promoter activity. Transient transfection studies with deletion mutants of the rat grp78 promoter showed that cis-acting promoter sequences required for ethanol induction differ from those mediating responses to classical GRP inducers. Furthermore, linker-scanning mutations of the grp78 promoter suggested that the ethanol potentiation response required a cis-acting promoter element different from those involved in induction by ethanol or classical inducing agents. While the ethanol induction response required 16-24 h to be detectable, ethanol potentiation of thapsigargin occurred within 6 h. The potentiation response also decayed rapidly after ethanol removal. In addition, the protein kinase A inhibitor R-cAMPS and protein phosphatase inhibitor okadaic acid both increased ethanol potentiation of thapsigargin while S-cAMPS, an activator of protein kinase A, decreased ethanol potentiation. Taken together, our findings suggest two mechanisms by which ethanol regulates grp78 transcription, both differing from the action of classical GRP inducers such as thapsigargin. One mechanism (potentiation) involves a protein phosphorylation cascade and potentiates the action of classical GRP inducers. In contrast, GRP78 induction by ethanol involves promoter sequences and a mechanistic pathway separate from that of the ethanol potentiation response or classical GRP78 inducers. These studies show that ethanol produces a novel and complex regulation of grp78 transcription which could be of particular importance during neuronal exposure to GRP-inducing stressors as might occur with central nervous system injury.

Phosducin-like protein (PhLP), a regulator of Gβγ function, interacts with the proteasomal protein SUG1

Phosducin-like protein (PhLP) and phosducin are highly homologous proteins that interact with the beta gamma subunits of guanine nucleotide binding proteins. While phosducin has a well-characterized role in retinal signal transduction, PhLP function remains unclear. To further understand the function of PhLP, we have examined other potential protein:protein interactions with PhLP using the yeast two-hybrid system. PhLP was found to interact with a mouse homologue of the yeast SUG1, a subunit of the 26S proteasome which may also indirectly modulate transcription. This interaction was further confirmed by an in vitro binding assay and co-immunoprecipitation of the two proteins in overexpression studies. Inhibition of proteasome function by lactacystin led to accumulation of high molecular weight, ubiquitin-immunoreactive protein precipitated by PhLP antiserum. We suggest that PhLP/SUG1 interaction may target PhLP for proteasomal degradation.

Cloning and characterization of the rat and human phosducin-like protein genes: structure, expression and chromosomal localization.

We isolated and characterized the rat gene encoding phosducin-like protein (PhLP), a putative heterotrimeric G protein modulator. The transcription start site was mapped by primer extension. The putative promoter region lacked a TATA sequence but contained a potential initiator element. Two splice variants were identified by RT-PCR of rat brain RNA, potentially generating either the full length or an amino-truncated protein. Only the full-length protein was immunodetected in all mouse tissues surveyed. Comparison of the conceptual translation product of the rat PhLP gene with those from human and Drosophila clones shows a striking conservation in the amino-terminal region of PhLP from these species. This contrasts with the relatively low degree of homology between PhLP and phosducin in this region, suggesting a functional role for this portion of the PhLP protein. Finally, we mapped the human PhLP gene by PCR analysis of somatic cell hybrids and the Stanford G3 radiation hybrid panel. The human PhLP gene (PDCL) is located on chromosome 9, linked to the polymorphic markers D9S1876 and D9S1674 (66-71 cM).

DNA arrays and functional genomics in neurobiology

Publisher Summary This chapter provides a background discussion of the approaches and techniques underlying DNA arrays and the application of this form of study. DNA arrays are used for purposes other than expression profiling, for example, for DNA sequencing or single nucleotide polymorphism detection. This chapter focuses on the use of these arrays in expression analysis. The chapter highlights early work suggesting that DNA arrays may offer significant advantages to the study of development and function of the central nervous system (CNS). The rapid development of new methodologies for RNA isolation, array preparation, and data analysis/ retrieval is highly likely, given the exponential growth in array studies. Given some of the special issues regarding expression profiling in neurobioiogy, it seems likely that a subdiscipline of functional neurogenomics may eventually develop. Perhaps the most exciting aspect of DNA array studies is the tremendous integrative and collaborative nature of such work. This approach is highly suited to neuroscience, a discipline long versed in the value of integrative science.

Regulation of Neuronal Gene Transcription by Ethanol

Much of the pathology caused by alcoholism can be attributed to the continued use of large quantities of ethanol. Alcoholics can expose themselves to extraordinarily large concentrations of ethanol due to an acquired resistance to the drug, termed tolerance. Resistance to ethanol can occur as dispositional (pharmacokinetic) or functional (pharmacodynamic) tolerance (Jaffe, 1985). Dispositional tolerance refers to an alteration in the rate of metabolism, absorption, distribution, or secretion. Functional tolerance refers to adaptive changes that occur within the central nervous system upon chronic ethanol exposure.