Molecular cloning of m1-receptor-regulated genes (Folgeprojekt)

Projektdetails:

Thematik: Grundlagenforschung
Förderstatus: abgeschlossen
Art der Förderung: Standard Projekt
Institution: Universität Hamburg, Zentrum für Molekularbiologie
Projektleiter: Prof. Dr. Roger M. Nitsch
Laufzeit: 01. November 1996 - 31. Oktober 1998
Fördersumme: 76.694,00 Euro
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Projektbeschreibung

It is likely that many of the clinical signs of Alzheimer’s disease are caused by the loss of cholinergic synapses. Post-synaptic receptors for acetylcholine exist in a variety of subtypes. The ml subtype is the predominant brain-specific receptor in hippocampus and cortex, two major areas involved in memory processing. These are the two areas that are particularly affected by the degeneration of the cholinergic neurotransmitter system in Alzheimer’s disease.

Learning and memory require long term changes of neuronal circuits. Such changes are known as neuronal plasticity, and they are typically mediated by reshaping of the branches and the surfaces of neurons. In particular, the ‘strength’ of synapses increases with frequent uses, whereas synapses that are not used are reduced, and sometimes, eliminated. Such use-dependent dynamic alterations of the structure and the function of neuronal circuits are mediated by changes in the expression of activity-dependent neuronal genes.

In recent years, some activity-dependent neuronal genes were identified, but genes whose expression is regulated by m1 receptors are unknown. This project is designed to identify genes that are regulated by m1 receptors, to clone and to characterise them.

In preliminary studies, Nitsch already found 3 genes, sting1, maz, and gos, which were activated by m1 receptors. During this funding period, he has further characterised these genes.

Abschlussbericht

Alzheimer's disease (AD) is the most frequent of all degenerative brain diseases. Clinical signs begin with impaired memory, progress to dementia, and inevitably lead to failure of all but the most primitive brain functions. AD is the fourth most common cause of death. At autopsy, severe shrinkage and loss of neurons, a long with such abnormal protein structures as amyloid plaques and neurofibrally tangles are found in brain tissue.

The clinical symptoms of AD are most closely related to the massive loss of synapses, the connections that connect neurons for communication with chemical neurotransmitters. There are several such neurotransmitter systems in brain, and most of them are damaged to a certain degree in AD brains. Among the most heavily damaged systems in the cholinergic system which plays a major role in learning and memory. In addition, this system seems to be involved in the regulations of the cleavage of the amyloid precursor protein, and thus, in amyloid formation. It is likely that many of the clinical signs of AD are caused by the loss of cholinergic synapses. Normally, cholinergic cells stimulate subsequent neurons by releasing the neurotransmitter acetylcholine in to the synapse that connects both cells, and by stimulating a neurotransmitter receptor in the membrane of the postsynaptic cell. Binding of acetylcholine to the receptor initiates a signal cascade that transduces the stimulating signal into the interior of the cell, and ultimately into the cell nucleus where DNA is located that carries copies of all human genes. The molecules that transmit the receptor-initiated signal into the cell nucleus are called transcription factors. They can bind to particular parts in the DNA called gene promoters and activate specific target genes. An activated gene is transcribed in to messenger RNA that is transported out of dependent neuronal functions as learning and memory.

During the initial 18 months of AFI funding, we discovered, cloned and sequenced several such neurotransmitter receptor inducible genes that encode proteins with important functions. H-cyr61, for instance, is a secretory protein that promotes the interaction between neuron and that strengthens the binding of neurons to their environment. Thus, receptor mediated induction of this gene may be expected to increase neurites outgrowth and to strengthen synaptic connections. Another example is gig-2, a gene that encodes a protein with a possible function in the cellular signal transduction. Additional genes that we identified with these experiments include the entire family of egr transcription factors. These genes encode important proteins that function in the regulation of a yet additional set of target genes, including the acetylcholinesterase gene that encodes AChE, an enzyme that is important for the degradation of the neurotransmitter acetylcholine.

These studies have three major consequences fo r our understanding of Alzheimer's disease and its therapy. First, we expect that the above neurotransmitter-induced genes are expressed at lower than normal levels in brains of Alzheimer's disease patients, because of the decrease in acetylcholine-mediated neurotransmission in this disease. To test this hypothesis, we are analyzing levels of expression of genes in brains from Alzheimer's disease patients in the current remaining AFI funding period. Second, our results generate a therapeutic opportunity for recovering expression levels of these genes by using pharmacological compounds designed to replace lost neurotransmitters.

Drugs designed to replace acetylcholine are currently tested in clinical trials for their efficacy to treat Alzheimer's disease. These drugs include the AChE inhibitors Tacrine (Parke-Davis/Warner-Lambert), Exelon (Novartis), Aricept (Pfizer/Eisai) and Metrifonate (Bayer), as well as several muscarinic agonists including Talsaclidine (Boehringer Ingelheim), LU25M (Lundbeck) and Xanomeline (Lilly). A common result of current efficacy studies is that beneficial effects appear to be restricted to the prevention of further decline for several weeks to months, after which time the rates of decline tend to parallel that of the placebo-treated groups. Our data suggest hat this rather limited efficacy may be caused by the potential role of all these compounds to increase synaptic levels of AChE - via the above inducible egr transcription factors - resulting in yet accelerated degradation of the already depleted brain concentration of ACh. This possibility underscores the crucial importance to identify and study genes that are regulated by these treatments. We therefore initiated a program that is designed to discover ultimately all genes that are under the control of muscarinic neurotransmission.

During the initial AFI funding period, we identified and characterized 7 such genes with an intriguing pattern of functions that include transcriptional regulation, signal transduction, as well as cell adhesion and growth. Data generated during the initial period of AFI funding strongly suggest that expression of these genes is clearly influenced by drugs that are currently used for the treatment of Alzheimer's disease. Detailed understanding of the pattern of genes that are regulated by these drugs will enable us to design novel, finely tuned treatments that combine the positive effects of cholinergic stimulation with major reductions in unwanted cellular responses.

Wissenschaftliche Publikationen auf Basis des geförderten Projekts

Albrecht,C., von der Kammer, H., Mayhaus, M., Klaudiny, J., Schweizer, M. and Nitsch, R.M. (2000). Muscarinic Acetylcholine Receptors Induce the Expression of the Immediate Early Growth Regulatory Gene CYR61. J. Biol. Chem., 275: 28929-28936.

Von der Kammer, H. Albrecht, C., Mayhaus, M.,  Hofmann, B., Stanke, G. and Nitsch, R.M. (1999). Identification of genes regulated by muscarinic acetylcholine receptors: application of an improved and statistically comprehensive mRNA differential display technique. Nucleic Acids Res., 27(10):2211-8.

Von der Kammer, H., Mayhaus, M., Albrecht, C., Enderich, J.,Wegener, M., and Nitsch, R.M. (1998). Muscarinic acetylcholine receptors activate expression of the Egr gene family of transcription factors. J. Biol. Chem., 273:14538-14544.

Foto: Krisztian Juhasz


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