Gary G. Cadd

Distinct patterns of cAMP-dependent protein kinase gene expression in mouse brain

In situ hybridization was used to localize cAMP-dependent protein kinase (PKA) mRNAs in the adult mouse CNS. The PKA holoenzyme contains two catalytic (C) subunits and a regulatory (R) subunit dimer. Our studies demonstrate expression of two isoforms of C (C alpha and C beta) and four isoforms of R (RI alpha, RI beta, RII alpha, and RII beta) in the CNS. mRNAs for C alpha, RI alpha, and RI beta preferentially localize in the neocortex, caudate-putamen, hypothalamus, thalamus, and hippocampus. Hybridization with C beta and RII beta probes is clearly distinguished from the C alpha-like pattern by a reduced level of hybridization in the thalamus and by a relative increase in expression in the dentate gyrus compared with cell layers CA1-3 in the hippocampus. RII alpha transcripts are very specifically localized in the medial habenula. The differential expression of PKA subunit genes suggests that functional differences in cAMP responses within neural tissues may be mediated by the biochemical properties of specific PKA isoforms.

Galanin transgenic mice display cognitive and neurochemical deficits characteristic of Alzheimer's disease

Galanin is a neuropeptide with multiple inhibitory actions on neurotransmission and memory. In Alzheimers disease (AD), increased galanin-containing fibers hyperinnervate cholinergic neurons within the basal forebrain in association with a decline in cognition. We generated transgenic mice (GAL-tg) that overexpress galanin under the control of the dopamine β-hydroxylase promoter to study the neurochemical and behavioral sequelae of a mouse model of galanin overexpression in AD. Overexpression of galanin was associated with a reduction in the number of identifiable neurons producing acetylcholine in the horizontal limb of the diagonal band. Behavioral phenotyping indicated that GAL-tgs displayed normal general health and sensory and motor abilities; however, GAL-tg mice showed selective performance deficits on the Morris spatial navigational task and the social transmission of food preference olfactory memory test. These results suggest that elevated expression of galanin contributes to the neurochemical and cognitive impairments characteristic of AD.

Analysis of the cAMP-Dependent Protein Kinase System Using Molecular Genetic Approaches

Publisher Summary This chapter focuses on the new approaches to the study of the cyclic adenosine monophosphate (cAMP)-dependent protein kinase system with the utilization of the techniques of molecular genetics. The chapter describes the characterization of two catalytic and four regulatory subunit genes in mammalian systems. Three of these subunits—RIα, RIIα, and Cα—are constitutively expressed in most tissues although the levels of messenger RNA (mRNA) can vary significantly, most notably in male germ cells during spermatogenesis. The other three subunits—Cβ, RIβ, and RIIβ—are highly expressed in brain and are either absent or at very low levels in many other tissues. In particular, the RIIβ gene is highly inducible in several differentiating systems. The chapter further discusses the differences in functional properties of the Cα and Cβ subunits. Future work with specific antibodies should help resolve the potential for specific interactions between subtypes of R and C subunits.

A unique mRNA species for a regulatory subunit of cAMP-dependent protein kinase is specifically induced in haploid germ cells.

Cyclic AMP (cAMP) and its action by way of cAMP‐dependent protein kinase is important for sperm motility. Previous studies on germ cells have demonstrated a selective decrease in the amount of type I cAMP‐dependent protein kinase during spermatid development, and that type II was the major form present in elongating spermatids and in mature sperm. This would indicate activation of a gene in haploid germ cells, encoding a regulatory subunit of type II protein kinase. However, haploid expression of such a gene has so far not been shown. In the present study we demonstrate high‐levelled expression of a unique mRNA species for a specific regulatory subunit of type II cAMP‐dependent protein kinase at late stages of spermatogenesis, i.e. during spermatid elongation.

A C/EBP-binding site in the transferrin promoter is essential for expression in the liver but not the brain of transgenic mice

The gene for the iron-binding protein transferrin is transcribed at a high level in liver hepatocytes but is also active in several other cell types, including oligodendrocytes in the brain. Enhancer elements between bp -560 and -44 of the transferrin gene promoter specifically activated transcription from a heterologous promoter in transgenic mouse liver and brain. Within this region, a potent cis-acting element between bp -98 and -83 was found to be essential for gene activity in both cultured hepatocytes and transgenic mouse liver. The -98 to -83 element contains a CCAAT sequence and is specifically bound by a nuclear factor from mouse liver that is homologous to rat liver C/EBP (CAAT enhancer-binding protein). Point mutations within this binding site inhibit factor binding and abolish transcription in transfected hepatoma cells. When placed in the context of the 3,000-bp transferrin promoter, the C/EBP binding site mutation causes a complete loss of transcription in transgenic mouse liver; however, transgene expression in the brain of the same animals was unaffected. These results suggest a modular structure for the transferrin promoter and demonstrate that deletions or specific point mutations can be used to generate transgene promoters with an activity more restricted than that of their endogenous counterparts.

Application of Molecular Genetic Techniques to the cAMP-Dependent Protein Kinase System

Publisher Summary This chapter describes modifications of the standard techniques used for either protein kinase characterization or gene regulation studies with emphasis on the unique possibilities and pitfalls that are observed when manipulating a multisubunit enzyme system by gene transfer techniques. In utilizing the techniques, it is important to fully appreciate the complex regulatory network in which the cAMP-dependent protein kinases function. For example, the overexpression of exogenous C subunit has led to a compensatory increase in regulatory subunits in several cell types. The chapter also describe four basic parts of eukaryotic expression vectors: (1) a transcriptional promoter sequence, (2) the structural gene coding for the protein which is to be expressed, (3) a eukaryotic poly(A) addition signal sequence, and (4) a prokaryotic replication/antibiotic resistance region for growth of large amounts of the expression vector in bacteria. Expression vectors may also contain intron sequences that must be removed by RNA splicing to produce a functional mRNA but these are not essential in most cases and are not included in the protein kinase vectors.