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Derivation of a temperature-sensitive hippocampal precursor cell line and transplantation into the newborn mouse brain
FOURTH INTERNATIONAL CONFERENCE ON ALZHEIMER’S DISEASE
receptor subtypes in fibroblast cell lines from controls and mutation
70
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.
s17
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,
receptor subtypes in fibroblast cell lines from controls and mutation
70
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.
s17
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,