Binding studies of muscarinic cholinergic receptors in cultured adult skin fibroblasts

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FOURTH INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE

The central nervous system is a recognized target of steroid hormone action. Alteratkms in gluccccmicoid hormones are known to affect the development of brain snd behaviour. We investigated the effects of long-term adrenalectomy on hippocampal neurons and spatial memory tasks in male Sprague-Dawley rats.?he damage was assessed throughout the hippocampus by counting cells in Nissl stained sections from admnalectomized tats with attenuated weight gain (ADX) and normal weight gain (ADXNW) and compared to those in sections from sham operated and naive controls. Admnal&omized rats, sham operated and naive controls were tested in the Morris water maze 4 months after surgery. The ADX

dentate gyms M2 has more binding in the granular layer whereas Ml receptor density is more in the molecular layer. In the present investigation iti vitro autoradiography was used to visualize M2 bindlne sites in the hiuoocamnus of adrenalectomized rats. Four‘;nonths after &ena&tomy the animals were sacrificed and 10 pm coronal sections from adrenalectomized rats, sham operated and naive rats wen incubated with 3H-AFDX-384 (10 nM, specific activity 90.4 Ci/mMol) to study the distribution of M2 muscarinic receptor. Quantitative measuerment of the receptors were done in different subfields of the hippocampus using a computer assisted analyzing syste.m. Our preliminary results indicate a significant inCAl, CA3, CA4 and dentate gyms of decmase in M2 binding sites adrenalectomized rats cunpated to sham and control groups.

rats had significantly reduced number of dentate granule cells. Moreover, significant reductions in the number of pyramidal cells were. observed in the CAl, CA2, CA3 and CA4 regions of the hippocampus. Analysis of body weight and serum corticostemne levels after adnmalcctomy suggested that the individual

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differences in hippocatnpal neuron degeneration appear to be due to incomplete

COS CELLS OVEREXPRESSING THE LDL-RECEPTORTAKE UP APO E FROM FRESH CEREBROSPINAL FLUID. T.G. Jensen, F. Heath and A.L. Jorgensen. Inst. Hum. Genet. University of Aarhus, Dept. Med. University Hospital Aarhus Amtssygehus, Aarhus, Denmark.

adrcnalcctomies or mmainiig ectopic tissue. ADX rats were signilicandy slower in learning the Morris mare memory task compared to ADXNW, sham operated and naive contmls. These data indicate that hlppocampal damage following longterm adrenslectomy causes learning impairment depending on the dcgrcc of neuronal damage.Thus the adrenal glands play a role ln maintaining the structural integrity of the normal adult hippocampus.

64 ANIMAL MODEL OF ALZHEIMER’S DISEASE: CHARACTERISTIC OF AVOIDANCE LEARNING OF RAT WITH ENTORHINAL CORTEX LESION. A.Ueki, C.Miua and K.Miyoshi. Dept. of Neuropsychiatry, l-l, Mukogawa-cho, Nishinomiya, Hyogo College of Medicine, Hyogo 663, Japan. The initial stage of Alzheimer’s disease is characterized by neuropathological alteration in the entorhinal cortex. Because of the anatomical relationship to the hippocampus, the entorhinal cortex may play a crucial role in memory formation. The purpose of the present study was to determine if there is any relationship between entorhinal cortex and learning behavior, to compare entorhinal cortex lesion with hippocampal and to model one aspect of the lesion in avoidance learning, neurodegeneration observed in Alzheimer’s disease. Ibotenic acid (20nmol in 1~1 of artificial cerebrospinal fluid) was infused into the entorhinal cortex or hippocampus bilaterally. Male Wistar rats (ZBO-31Og) were randamly assigned to 4 groups: bilateral entorhinal cortex-lesioned rats (n=4), bilateral hippocampal lesioned rats (n-4) and sham-operated controls for entorhinal cortex lesion (n=4) and hippocampal lesion (n=4). Entorhinal cortex-lesioned animals did not reveal any sensorimotor disturbances. But increased locomotor activity and sensitivity were found in rats with hippocampal lesion. Ibotenic acid-induced lesion of all rats were located within the confines of the entorhinal cortex or hippocampus. Three weeks after the operation, the entorhinal cortex-lesioned rats showed impaired acquisition of passive and active avoidance responses. The hippocampal lesioned rats exhibited an impairment in acquisition no significant deficits in of passive avoidance. However, acquisition of active avoidance responses were found in rats with hippocampal lesion. The discrepancy between passive and active avoidance learning of hippocampal lesioned rats may be These findings confirm the due to sensorimotor disturbances. importance of entorhinal cortex in memory acquisition and indicate that rats with partial neuronal loss in the entorhinal cortex may be a useful Alzheimer’s disease.

model

for

the

memory

disturbance

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MUSCARINIC CHOLINERGIC RECEF’TOR CHANGES IN THE HIPFGCAMFVS OF ADRENALECIDMIZED RATS. A. Islam, N. Bogdanovlc, B. Winblad and A. A&m. Department of Gerlatrlc Medic&, Karolloska Institute, Huddinge University Hospital, Huddinge, Sweden. Adrenalcctomy in adult rats nsults in loss of dentate granule cells. In addition to the dentate gyrus, adrenalectomy also affects other hippocampal fields. ‘llu. hlppocampus is rich in muscarinic reaptors. It has been shown that Ml tors are mainly localized to the saatumoriensoftheCA*andCPQFegions.MZreceptrnsanmostly localized in the pyramidal layer of hippocampal formation. In the

of

Late onset familial and sporadic Alzheimer disease (AD) are associated with inheritance of the E4 allele at the Apo E locus on chromosome 19 (Corder et al. (1993) Science 261, 921923, Saunders et al. (1993) Neurology 43, 1467-1472). In vitro experiments have shown isoform specific binding of Apor /3-amyloid and to tau, the two proteins which are currently implicated in the pathogenesis of Alzheimer neurodegeneration (Strittmatteret al. (1993) PNAS USA 90, 8098-8102, Strittmatter et al. (1994) PNAS USA in press). Using Cos cells overexpressing the LDL-receptor we studied binding and internalizationof Apo E via the LDL-R. Transfected Cos cells were incubated in fresh cerebrospinalfluid (CSF) at 37°C. InternalizedApo E was detected using anti-APO E antibodies (BoehringerMannheim) and a secondary fluorochromeconjugated antibody. The !oca!izationof immunofluorescence indicates that Apo E is present.in the cytoplasm of the transfected Cos cells after incubation in CSF. No such fluorescence was detected when mock-transfected Cos cells were used or when transfected Cos cells were incubated in growth medium (10% fetal calf serum). To clearly establish cytoplasmic localization and to investigate intracellulartrafficking of Apo E we have initiated electron microscopic examination of Apo E containing Cos cells. Acknowledgement:We thank J. Jacobsen Dept. Neurology for providing us with the CSF samples.

67 BINDING STUDIES OF MUSCARINIC CHOLINERGIC RECEPTORS IN CULTURED ADULT SKIN FIBROBLASTS M. VESTLING, N. VENIZELOS, J. JOHNSTON, R. COWBURN, L. LANNFBLT, B. WINBLAD AND A. ADEM Department

of Geriatric Medicine, Karollnaka Institute, Novum KFC, 141% Huddiige, Sweden

The presence of muacarlnlc cholinergic receptors was studied in cultured skin fibroblasts obtained from adult controls. Homogenate radioligand binding studies showed that human skin fibroblasts possess specific binding sites for the non-selective muscarinic antagonists [aH]quinuclidinyl benzilate @H]QNB) and [SHYJmethyl-scopolamine, as well as for the M2-selective antagonist [SHIAF-DX 3s4. The specificity of [%-IlQNB binding to fibroblast cell membranes was characterlsed using a range of concentrations (IO-2 - IO-11 M) of different cholinergic antagonists, including atroplne, pirenzipine (Ml-selective), methoctramine (M2-selective) and p-fluore hexahydro-alla-dlfenldol (pF-HSSID; Msselective). These antagonists gave a rank order of potency of atropine > methoctratrdne > pF-HSSID > pirenzipine when tested against the specific binding of 0.2 nM PHIQNB. These results indicate that the major muscarinic receptor on human skin flbroblaata is the M2 subtype. These cells would appear to have only low levels of the Ml and M3 subtypes. We are currently studying the levels of different muscarinic

FOURTH INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE

receptor subtypes in fibroblast cell lines from controls and mutation

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bearing members of the Swedish family with the APP 670/671 mutation, in order to assess their relevance as a potential peripheral marker for the choline+ changes seen inAlzheimer'sdisease.

CADMIUM CHLORIDE INDUCES OVEREXPRESSION AND ALTERED PROCESSING OF DAPP IN MAMMALIAN CELL LINES. R. B. Denman, M. Smedman, W. Ju, R. Rubenstein, A. Potempska and D. L Miller. NYS Institute for Basic Research, 1050 Forest Hill Road, Staten Island, NY 10314 USA. Mammalian expression vectors bearing the human metallothionein IIA (Mt IIA) promoter are commonly used to overexpress cloned genes. Induction is accomplished by addition of trace levels of heavy metal ions such as Cd+s, Cu+s and Zn+* to the media. However, even at micromolar doses Cd+’ can have varied effects upon cells. We have examined the effect of Cd+* in three transfected COS-7 cell lines. Each line contained the episomal vector pMEP4 which contains a metallothionein IIA promoter. In two of the lines ribozymes targeted lo DAPP mRNA were inserted behind this promoter; the other line contained the vector-alone. Previous analysis revealed that BAPP mRNA and protein levels were reduced in riboyme-containing cell lines compared to vector-alone cells upon addition of glucocorticoids. However, following a 36 hr. induction by Cd+2(1O PM), amyloid peptide precursor (BAPP) and DAPP mRNA steady-state levels increased in all three transfected cell lines. In addition, all three Cd+*-induced cell lines exhibited altered BAPP processing. Similar changes were also observed in untransfected COS7 and PC12 cells treated with Cd+*. Altered BAPP processing was found lo correlate with the expression of the inducible form of the heat shock protein, HSP74 but not with other heat shock proteins or metallothionein. Heat shock itself (42q, 30 min.) however, did not significantly perturb either BAPP mRNA or BAPP protein steady-state levels or alter processing, although both HSP70 mRNA and protein were significantly overexpressed. These data suggest (1) effects observed from overexpression from Mt IIA promoter-based vectors induced by Cd+* be interpreted cautiously, and (2) alteration and stimulation of IlAPP expression by heavy metal ions may bc an appropriate model to test hypotheses concerning the role of BAPP in the etiology of Alzheimer’s disease.

68 OVEREXPRESSION OF HUMAN TAUd,, IN PC12 AND CHO CELLS. N. Haque, R.B. Denman, 1. Grundke-Iqbal and KIqbal. New York State Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314 USA. Tau is a neuronal microtubule-associated protein that promotes the assembly and stability of microtubules. In its hyperphosphorylated forms, tau is the major subunit of paired helical filaments which form the neurofibrillary tangles in the brain of patients with Alzheimer disease (Grundke-Iqbal, et al. Proc. Nat]. Acad. Sci. USA, 83:4913-4917, 1986; Iqbal, et al., ibid, 865646-5650, 1989). The objective of the present study is to generate a cellular model of abnormally phosphorylated human tau. Towards this objective, a full length human tau cDNA insert coding for tau,,, (the four-repeat longest isoform of tau) was prepared by polymerase chain reaction (PCR) using pRK172 tau plasmid DNA (Gc-edert et a/, EMBO .I. 9:4225-4230, 1990) as a template. The resulting 1.4 kb tau insert was purified and subcloned into the vector PCRTM, downstream of the T7 RNA polymerase promoter in both the sense [Clone TA-TlO) and the Bacterial expression of tau,, was antisense (Clone TA-T8) orientation. induced in TA-TlO transfected E. ColiBE21 cells by 4mM isopropyl-B-Dthiogalactoside (IPTG). Recombinant, rIauqql, in total cell homogenates was confirmed by Western blots developed with mAb Tau-1. b’amHI/Xbol fragments were excised from the TA-TlO and TA-T8 clones, gel purified downstream and cloned into the eukaryotic expression vector pcDNAl/Neo of the CMV promoter. The resulting sense and antisense plasmid constructs were used to generate stably-transfected cell lines in both PC12 and CHO cells. Preliminary analysis has shown that CHO cells containing the rtau,,,-sense consrruct were morphologically distinct from both the antisense-tau,,, and vector-alone containing CHO cells. (Supported in part by NIH grants AG 08076, AG 05892, AG 04220, NS 18105, and a Zenith Award (to K.I.) from the Alzheimer’s Disease Assoc.).

69 DERIVATION OF A TEMPERATURE-SENSITIVE HIPPOCAMPAL PRECURSOR CELL LINE AND ~mlA;PLANTATiON INTO THE NEWBORN MOUSE L. E. Sabbey. K. R. Sales, P. T. Keith, C. F. Hohmann’ and R. F. Santerre Lilly Research Laboratories, Eli Lilly and Company, Indianapolis. IN 46265 ‘Kennedy Kreiger Institute and Morgan State University, Baltimore. MD A hippccampal precursor cell line that can be maintained in continuous culture or differentiated to express astrocytic and neuronal properties was isolated from the H-2Kb-tsA56 transgenic mouse (Immortomouse) (Jat et al., PNAS 865096, 1991). Mixed brain cultures were established from the hippocampus of E15-16 mouse embryos. Clonal cell lines expressing temperature-sensitive SV4OTAg (tsA56) were derived from primary cultures maintained at WC in the presence of 25 w/ml a-interferon and 25 @ml D-interferon. Cultures were differentiated at 39°C in the absence of interferons. Cell division ceased in the differentiated cultures and the cells developed neuronal and astrocyte-like morphologies. By immunocytochemistry differentiated cells were negative for SV40TAg and positive for glial fibrillary acidic protein (GFAP) and neurofilament protein (NF). Non-differentiated. fluorescent-tagged cells were injected into the hippocampal region of 2- day old mouse pups. At two and four weeks post-transplantation small numbers of trens anted cells were immunocytochemically positive for GFA I+ and NF. This temperature-sensitive hippocampal precursor cell line offers a unique cell system for in vitro genetic manipulation, transplantation and in viva differentiation to model diseases of the central nervous system.

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71 EXPRESSION OF THE ALZI-IEIMER -ID-PROMOTING FAClORS a,-ANTICHYMOTRYFSIN AND AFOLIPOF’ROTEIN E IS INDUCED IN ASTROCYTES BY IL,-1. S.Das, L. Geller, M. Nietbanuner, and H. Potter. Department of Neurobiology, Harvard Medical School, Boston MA 02115. The amyloid deposits of Alzheimer’s disease contain, in addition to the P-protein (AP), lesser amounts of the protease inhibitor a,-antichymotrypsin (ACT) and the lipid carrier protein apolipoprotein E (apoE). We have recently shown that these proteins act as pathological chaperones, binding to the bprotein and strongly promoting its polymerization into amyloid filaments in vitro (Ma et. al. this Conference). The data of the present report show that ACT and apoE synthesis is induced in human astrocytes in culture by IL-l, a lymphokine whose expression is upregulated in Alzheiiner’s disease brain. Furthermore, ACT and apoE are constitutirely expressed in confluent glial cultures prepared from human cortex, an area of the brain prone to Alzheimer amyloid formation. This constitutive expression of ACT and apoE in cortical cultures can be blocked by IL-l receptor antibodies or by the removal of the microglial cells, which express ILl. Confluent cultures prepared from human cerebellum or brain stem, or from rat brain, tissues which do not accumulate mature amyloid deposits, express apoE, but not ACT. These results indicate that the IL-l-induced expression of these two amyloid-promoting proteins, particularly ACT, may help direct the regional and temporal production of mature amyloid filaments inAlzheimer'sdisease brain.

72 REDUCTION OF APP LEVELS IN PC12 CELLS TREATED WITH ANTI-SENSE OLIGONUCLEOTIDE. R.E. Majocha, Sudhir Agrawal'., J.Y. Tang', E. Humke, J. Newton and C.A. Marotta. Depts. Psychiatry & Human Behavior and Neuroscience, Brown University and Miriam Hospital,