Michael J. Zuscik

Overexpression of the α 1B -adrenergic receptor causes apoptotic neurodegeneration: Multiple system atrophy

Progress toward elucidating the function of α1B-adrenergic receptors (α1BARs) in the central nervous system has been constrained by a lack of agonists and antagonists with adequate α1B-specificity. We have obviated this constraint by generating transgenic mice engineered to overexpress either wild-type or constitutively active α1BARs in tissues that normally express the receptor, including the brain. All transgenic lines showed granulovacular neurodegeneration, beginning in α1B-expressing domains of the brain and progressing with age to encompass all areas. The degeneration was apoptotic and did not occur in non-transgenic mice. Correspondingly, transgenic mice showed an age-progressive hindlimb disorder that was parkinsonian-like, as demonstrated by rescue of the dysfunction by 3, 4-dihydroxyphenylalanine and considerable dopaminergic-neuronal degeneration in the substantia nigra. Transgenic mice also had a grand mal seizure disorder accompanied by a corresponding dysplasia and neurodegeneration of the cerebral cortex. Both behavioral phenotypes (locomotor impairment and seizure) could be partially rescued with the α1AR antagonist terazosin, indicating that α1AR signaling participated directly in the pathology. Our results indicate that overstimulation of α1BAR leads to apoptotic neurodegeneration with a corresponding multiple system atrophy indicative of Shy-Drager syndrome, a disease whose etiology is unknown.

Phe-308 and Phe-312 in Transmembrane Domain 7 Are Major Sites of α1-Adrenergic Receptor Antagonist Binding IMIDAZOLINE AGONISTS BIND LIKE ANTAGONISTS

Although agonist binding in adrenergic receptors is fairly well understood and involves residues located in transmembrane domains 3 through 6, there are few residues reported that are involved in antagonist binding. In fact, a major docking site for antagonists has never been reported in any G-protein coupled receptor. It has been speculated that antagonist binding is quite diverse depending upon the chemical structure of the antagonist, which can be quite different from agonists. We now report the identification of two phenylalanine residues in transmembrane domain 7 of the α1a-adrenergic receptor (Phe-312 and Phe-308) that are a major site of antagonist affinity. Mutation of either Phe-308 or Phe-312 resulted in significant losses of affinity (4–1200-fold) for the antagonists prazosin, WB4101, BMY7378, (+) niguldipine, and 5-methylurapidil, with no changes in affinity for phenethylamine-type agonists such as epinephrine, methoxamine, or phenylephrine. Interestingly, both residues are involved in the binding of all imidazoline-type agonists such as oxymetazoline, cirazoline, and clonidine, confirming previous evidence that this class of ligand binds differently than phenethylamine-type agonists and may be more antagonist-like, which may explain their partial agonist properties. In modeling these interactions with previous mutagenesis studies and using the current backbone structure of rhodopsin, we conclude that antagonist binding is docked higher in the pocket closer to the extracellular surface than agonist binding and appears skewed toward transmembrane domain 7.

Mice expressing the α1B‐adrenergic receptor induces a synucleinopathy with excessive tyrosine nitration but decreased phosphorylation

We had previously reported that systemic overexpression of the α1B‐adrenergic receptor (AR) in a transgenic mouse induced a neurodegenerative disease that resembled the parkinsonian‐like syndrome called multiple system atrophy (MSA). We now report that our mouse model has cytoplasmic inclusion bodies that colocalize with oligodendrocytes and neurons, are positive for α‐synuclein and ubiquitin, and therefore may be classified as a synucleinopathy. α‐Synuclein monomers as well as multimers were present in brain extracts from both normal and transgenic mice. However, similar to human MSA and other synucleinopathies, transgenic mice showed an increase in abnormal aggregated forms of α‐synuclein, which also increased its nitrated content with age. However, the same extracts displayed decreased phosphorylation of α‐synuclein. Other traits particular to MSA such as Purkinje cell loss in the cerebellum and degeneration of the intermediolateral cell columns of the spinal cord also exist in our mouse model but differences still exist between them. Interestingly, long‐term therapy with the α1‐AR antagonist, terazosin, resulted in protection against the symptomatic as well as the neurodegeneration and α‐synuclein inclusion body formation, suggesting that signaling of the α1B‐AR is the cause of the pathology. We conclude that overexpression of the α1B‐AR can cause a synucleinopathy similar to other parkinsonian syndromes.

Gene expression profiling of α1b-adrenergic receptor-induced cardiac hypertrophy by oligonucleotide arrays

OBJECTIVEnCardiac hypertrophy is closely associated with the development of cardiomyopathies that lead to heart failure. The alpha(1B) adrenergic receptor (alpha(1)-AR) is an important regulator of the hypertrophic process. Cardiac hypertrophy induced by systemic overexpression of the alpha(1b)-AR in a mouse model does not progress to heart failure. We wanted to explore potential gene expression differences that characterize this type of hypertrophy that may identify genes that prevent progression to heart failure.nnnMETHODSnTransgenic and normal mice (B6CBA) representing two time points were compared; one at 2-3 months of age before disease manifests and the other at 12 months when the hypertrophy is significant. Age-matched hearts were removed, cRNA prepared and biotinylated. Aliquots of the cRNA was subjected to hybridization with Affymetrix chips representing 12,656 murine genes. Gene expression profiles were compared with normal age-matched controls as the baseline and confirmed by Northern and Western analysis.nnnRESULTSnThe non-EST genes could be grouped into five functional classifications: embryonic, proliferative, inflammatory, cardiac-related, and apoptotic. Growth response genes involved primarily Src-related receptors and signaling pathways. Transgenic hearts also had a 60% higher Src protein content. There was an inflammatory response that was verified by an increase in IgG and kappa-chained immunoglobulins by western analysis. Apoptosis may be regulated by cell cycle arrest through a p53-dependent mechanism. Cardiac gene expression was decreased for common hypertrophy-inducing proteins such as actin, collagen and GP130 pathways.nnnCONCLUSIONSnOur results suggest a profile of gene expression in a case of atypical cardiac hypertrophy that does not progress to heart failure. Since many of these altered gene expressions have not been linked to heart failure models, our findings may provide a novel insight into the particular role that the alpha(1B)AR plays in its overall progression or regression.

Systemic overexpression of the α1B-adrenergic receptor in mice: An animal model of epilepsy

Summary: u2002Purpose: A lack of selective α1‐adrenergic receptor (α1‐ARs) agonists and antagonists has made it difficult to clarify the precise function of these receptors in the CNS. We recently generated transgenic mice that overexpress either wild‐type or a constitutively active mutant α1B‐AR in tissues that normally express the receptor. Both wild‐type and mutant mice showed an age‐progressive neurodegeneration with locomotor impairment and probable stress‐induced motor events, which can be partially reversed by α1‐AR antagonists. We hypothesized that the wild‐type and mutant mice may exhibit spontaneous epileptogenicity as compared with normal (nontransgenic) mice.