Ronnie Folkesson

Alzheimer's disease: Molecular genetics and transgenic animal models

Disease-causing mutations in the amyloid precursor protein (APP) gene have been found on chromosome 21 during the last 2 years in some early onset Alzheimers disease (AD) families. Genetic evidence shows that other genes than the APP are also involved in the aetiology of AD. Linkage to a loci on chromosome 14 has been found in early onset disease. The identification of APP mutation has led to the realization that APP mismetabolism is a central event in the aetiology and pathogenesis of the disease. Experiments to test this in transgenic mice have so far met with little success. There are many possible explanations for the problems to generate transgenic mice. These include the possibilities that mice are incapable of developing AD for reasons dependent on their APP sequence; and that appropriate regulation of APP gene is required for pathology to develop. Current attempts that seem promising to model the disease pathology are the use of homologous recombination to insert the pathogenic mutation and transfection of YACs into transgenic animals.

Cloning and expression of the human N-methyl-d-aspartate receptor subunit NR3A

Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding to a 93% homology at the amino acid level. Based on available data from human genome databases, we localized the gene to chromosome 9. The transcript could be detected by in situ hybridization in human fetal spinal cord and forebrain. Two splice variants of NR3A have been reported in rat brain, the longer of the two containing a 60 bp insert in the intracellular domain. We were unable to detect this 60 bp insert in fetal or adult human brain, suggesting that only the short variant is expressed in humans.

Expression of the cholecystokinin gene in a human (small-cell) lung carcinoma cell-line.

Expression of the cholecystokinin (CCK), gastrin and enkephalin A genes were studied by Northern blot analysis and a library of sequence‐specific radioimmunoassays in human cell lines. The human small‐cell lung carcinoma line (SCLC) U‐1690 expressed moderate levels of CCK mRNA as compared to the human neuroepithelioma cell line SK‐N‐MC. Neither gastrin nor (pro)enkephalin A mRNAs were detectable in the U‐1690 cell line. In contrast, the SCLC‐line H‐69 expressed Enk A but no CCK mRNA. The radioimmunoassays showed that the CCK mRNA transcript in the SCLC line U‐1690 also is translated, and that preproCCK is processed into bioactive, carboxyamidated CCK peptides. Thus, the human small cell carcinoma cell line U‐1690 is a useful model for studies of cell‐specific CCK gene expression.

Environmental influence on somatostatin levels and gene expression in the rat brain

In the present study we have quantified preprosomatostatin-mRNA and somatostatin levels in rat brain following environmental stimulation. Animals were housed for 30 days in an enriched or impoverished environment prior to analysis. After 30 days of housing half of the rats from each environment were behaviourally tested for 3 days. Housing in enriched environment improved performance in a spatial learning situation. The open-field behaviour of these animals was characterized by initially higher rearing scores and a more rapid habituation to novel environment as measured by spontaneous locomotor activity. We found significantly elevated somatostatin levels in the cortex following enriched environment, compared with impoverished environment. Exposure to behavioural testing of impoverished animals led to increased cortical somatostatin levels. Hypothalamic somatostatin levels increased significantly after housing in enriched environment, while the testing procedure had no influence. Our data shows that the somatostatin system in the rat brain was activated in association with cognitive changes, that were induced by housing in an enriched environment.

Cognitive impairment in the Tg6590 transgenic rat model of Alzheimer’s disease

Recently, interest in the rat as an animal model of Alzheimer’s disease (AD) has been growing. We have previously described the Tg6590 transgenic rat line expressing the amyloid precursor protein containing the Swedish AD mutation (K670M/N671L) that shows early stages of Aβ deposition, predominantly in cerebrovascular blood vessels, after 15 months of age. Here we show that by the age of 9 months, that is long before the appearance of Aβ deposits, the Tg6590 rats exhibit deficits in the Morris water maze spatial navigation task and altered spontaneous behaviour in the open‐field test. The levels of soluble Aβ were elevated both in the hippocampus and cortex of transgenic animals. Magnetic resonance imaging showed no major changes in the brains of transgenic animals, although they tended to have enlarged lateral ventricles when compared to control animals. The Tg6590 transgenic rat line should prove a suitable model of early AD for advanced studies including serial cerebrospinal fluid sampling, electrophysiology, neuroimaging or complex behavioural testing.

Cypermethrin alters Glial Fibrillary Acidic Protein levels in the rat brain.

Pyrethroids, widely used insecticides, are biologically active in neurons. Whether they act on the non-neuronal brain cells remains an open question. Thus, the aim of this study was to examine whether Cypermethrin intoxication affects astroglial cells in the rat brain. The levels of Glial Fibrillary Acidic Protein (GFAP) in different brain regions were measured by ELISA following oral treatment with 5 or 10% of LD(50) of Cypermethrin per day for 6 days. A significant decrease of GFAP was observed in different brain regions of treated animals. The cerebral cortex showed the most pronounced effect with GFAP levels reduced to 81% of the controls 2 days after treatment and 77% 21 days after treatment. Although we did not find profound changes in the morphology of astrocytes in Cypermethrin treated animals, the decrease in GFAP suggests that astrocytes were affected by low doses of pyrethroids. The possible consequences were discussed.

Phorbol 12-myristate-13-acetate (PMA) stimulates a differential expression of cholecystokinin (CCK) and c-fos MRNA in a human neuroblastoma cell line

Regulation of cholecystokinin (CCK) and the proto‐oncogene c‐fos mRNA expression was studied in the human neuroblastoma cell line SK‐N‐MC. Cells were treated either with the tumor promoting phorbolester phorbol‐12‐myristate‐13‐acetate (PMA), the phosphodiesterase inhibitor isobutyl‐methylxanthine (IBMX), which results in an elevated intracellular cyclic AMP (cAMP) level, or with a combination of PMA and IBMX. The level of CCK and c‐fos mRNA was determined by Northern‐blot analysis with CCK and c‐fos specific antisense RNA probes after 4–24 h of drug treatment. Treatment with PMA and IBMX for 4–24 hours transiently raised the CCK MRNA level ∼1.5–3.5 times compared to the controls, and the combination PMA and IBMX had an additive effect and elevated CCK MRNA abundance 1.5–6.5 times. Under the same experimental conditions, both PMA and IBMX elevated the c‐fos MRNA level ∼3–5.5 times. The drug combination showed a pronounced synergistic effect and raised the c‐fos mRNA level ∼3–20 times as compared to controls. Apparently, CCK and c‐fos mRNA expression appears to be regulated by similar protein kinase C (PKC) and cAMP‐dependent mechanisms in SK‐N‐MC cells.

Impact of a deletion of the full-length and short isoform of p75NTR on cholinergic innervation and the population of postmitotic doublecortin positive cells in the dentate gyrus

Analyses of mice carrying a deletion of the pan-neurotrophin receptor p75NTR have allowed identifying p75NTR as an important structural regulator of the hippocampus. Most of the previous analyses were done using p75NTRExIII knockout mice which still express the short isoform of p75NTR. To scrutinize the role of p75NTR in the hippocampus, we analyzed adult and aged p75NTRExIV knockout mice, in which both, the short and the full-length isoform are deleted. Deletion of these isoforms induced morphological alterations in the adult dentate gyrus (DG), leading to an increase in the thickness of the molecular and granular layer. Based on these observations, we next determined the morphological substrates that might contribute to this phenotype. The cholinergic innervation of the molecular and granular layer of the DG was found to be significantly increased in the knockout mice. Furthermore, adult neurogenesis in the DG was found to be significantly altered with increased numbers of doublecortin (DCX) positive cells and reduced numbers of apoptotic cells in p75NTRExIV knockout mice. However, cell proliferation as measured by phosphohiston H3 (PH3) positive cell numbers was not affected. These morphological alterations (number of DCX-positive cells and increased cholinergic fiber densities) as well as reduced cell death in the DG are likely to contribute to the observed thickening of the granular layer in p75NTRExIV knockout mice. In addition, Sholl-analysis of DCX-positive neurons revealed a higher dendritic complexity and could thus be a possible morphological correlate for the increased thickness of the molecular layer in p75NTR deficient animals. Our data clearly demonstrate that deletion of both, the short and the full-length isoform of p75NTR affects DG morphology, due to alterations of the cholinergic system and an imbalance between neurogenesis and programmed cell death within the subgranular zone.

Alzheimer transgenic models

Purpose of review A number of Alzheimer transgenic mouse models have been generated using a variety of different constructs containing the amyloid precursor protein. Some of the models develop features of Alzheimers disease, including amyloid pathology, cholinergic deficits and cognitive impairment. Recent publications have demonstrated the benefits of using transgenic animals as models of Alzheimers disease. Recent findings Genetic crosses with animals expressing other transgenes relevant to Alzheimers disease have enhanced our understanding of the underlying pathogenic mechanisms. In the past year the Alzheimer transgenic models have increasingly been used in the development of anti-Alzheimer drugs. Summary Although the current transgenic animals are still not complete models of Alzheimers disease, they are useful for studies on pathogenic mechanisms and the evaluation of new anti-amyloid treatment strategies.

SOMATOSTATINERGIC PHENOTYPE MARKERS IN THE HUMAN NEUROBLASTOMA CELL-LINE LA-N-2

We have characterized somatostatinergic phenotype markers of the human neuroblastoma, LA‐N‐2. A single mRNA‐transcript (∼850bp) and two cellular somatostatin immunoreactivity forms, a high molecular weight form (M r 15,000) and a fragment corresponding to somatostatin‐28 was found, while the somatostatin‐14 peptide was absent. Saturation binding experiments demonstrated a single class of high‐affinity somatostatin receptors with K d and B max of 0.27 ± 0.03 nM and 45 ± 1 fmol/mg protein. Partial G‐protein uncoupling (30%) was demonstrated, using GTPγS, with an affinity of 9.7 nM. The LA‐N‐2 cell line, previously shown to be cholinergic, may serve as a simplified system to elucidate heterologous neurotransmittor interactions. Such studies are of interest since dysfunctions of the cholinergic basal forebrain neurons and somatostatin immunoreactive interneurons have been consistently observed in Alzheimers disease.