A protease-resistant 61-residue prion peptide causes neurodegeneration in transgenic mice.

An abridged prion protein (PrP) molecule of 106 amino acids, designated PrP106, is capable of forming infectious miniprions in transgenic mice (S. Supattapone, P. Bosque, T. Muramoto, H. Wille, C. Aagaard, D. Peretz, H.-O. B. Nguyen, C. Heinrich, M. Torchia, J. Safar, F. E. Cohen, S. J. DeArmond, S. B. Prusiner, and M. Scott, Cell 96:869-878, 1999). We removed additional sequences from PrP106 and identified a 61-residue peptide, designated PrP61, that spontaneously adopted a protease-resistant conformation in neuroblastoma cells. Synthetic PrP61 bearing a carboxy-terminal lipid moiety polymerized into protease-resistant, beta-sheet-enriched amyloid fibrils at a physiological salt concentration. Transgenic mice expressing low levels of PrP61 died spontaneously with ataxia. Neuropathological examination revealed accumulation of protease-resistant PrP61 within neuronal dendrites and cell bodies, apparently causing apoptosis. PrP61 may be a useful model for deciphering the mechanism by which PrP molecules acquire protease resistance and become neurotoxic.

Branched polyamines cure prion-infected neuroblastoma cells.

Branched polyamines, including polyamidoamine and polypropyleneimine (PPI) dendrimers, are able to purge PrP(Sc), the disease-causing isoform of the prion protein, from scrapie-infected neuroblastoma (ScN2a) cells in culture (S. Supattapone, H.-O. B. Nguyen, F. E. Cohen, S. B. Prusiner, and M. R. Scott, Proc. Natl. Acad. Sci. USA 96:14529-14534, 1999). We now demonstrate that exposure of ScN2a cells to 3 microg of PPI generation 4.0/ml for 4 weeks not only reduced PrP(Sc) to a level undetectable by Western blot but also eradicated prion infectivity as determined by a bioassay in mice. Exposure of purified RML prions to branched polyamines in vitro disaggregated the prion rods, reduced the beta-sheet content of PrP 27-30, and rendered PrP 27-30 susceptible to proteolysis. The susceptibility of PrP(Sc) to proteolytic digestion induced by branched polyamines in vitro was strain dependent. Notably, PrP(Sc) from bovine spongiform encephalopathy-infected brain was susceptible to PPI-mediated denaturation in vitro, whereas PrP(Sc) from natural sheep scrapie-infected brain was resistant. Fluorescein-labeled PPI accumulated specifically in lysosomes, suggesting that branched polyamines act within this acidic compartment to mediate PrP(Sc) clearance. Branched polyamines are the first class of compounds shown to cure prion infection in living cells and may prove useful as therapeutic, disinfecting, and strain-typing reagents for prion diseases.

Identification of two prion protein regions that modify scrapie incubation time.

A series of prion transmission experiments was performed in transgenic (Tg) mice expressing either wild-type, chimeric, or truncated prion protein (PrP) molecules. Following inoculation with Rocky Mountain Laboratory (RML) murine prions, scrapie incubation times for Tg(MoPrP)4053, Tg(MHM2)294/Prnp(0/0), and Tg(MoPrP, Delta23-88)9949/Prnp(0/0) mice were approximately 50, 120, and 160 days, respectively. Similar scrapie incubation times were obtained after inoculation of these lines of Tg mice with either MHM2(MHM2(RML)) or MoPrP(Delta23-88)(RML) prions, excluding the possibility that sequence-dependent transmission barriers could account for the observed differences. Tg(MHM2)294/Prnp(0/0) mice displayed prolonged scrapie incubation times with four different strains of murine prions. These data provide evidence that the N terminus of MoPrP and the chimeric region of MHM2 PrP (residues 108 through 111) both influence the inherent efficiency of prion propagation.

Affinity-tagged miniprion derivatives spontaneously adopt protease-resistant conformations.

An abridged PrP molecule of 106 amino acids designated PrP106 can form infectious miniprions in transgenic (Tg) mice (29). Addition of six-histidine (His(6)) affinity tags to selective sites within PrP106 resulted unexpectedly in new PrP proteins that spontaneously adopted protease-resistant conformations when expressed in neuroblastoma cells and Tg mice. Acquisition of protease resistance depended on the length, charge, and placement of the affinity tag. Introduction of the disease-linked mutation E200K into the sequence of PrP106(140/6His) increased the recovery of protease-resistant PrP fivefold, whereas introduction of the mutations C213A and Delta214-220 did not affect the recovery of protease-resistant PrP. Treatment of cultured cells expressing affinity-tagged PrP106 mutants with polypropyleneimine dendrimer rendered these proteins sensitive to protease digestion in a manner similar to wild-type PrP(Sc). We conclude that certain affinity-tagged PrP106 proteins spontaneously fold into conformations partially resembling, yet distinct from, wild-type PrP(Sc). These proteins might be useful tools in the identification of new disease-causing mutations as well as for screening compounds for therapeutic efficacy.

Transgenic models of prion disease.

There is growing concern that bovine spongiform encephalopathy (BSE) may have passed from cattle to humans, resulting in approximately 70 cases of an atypical, variant CJD (vCJD) in teenagers and young adults. We report here that transgenic (Tg) mice expressing full-length bovine (Bo) PrP serially propagate BSE prions and that there is no species barrier for transmission from cattle to Tg(BoPrP) mice. Surprisingly, these same mice were also highly susceptible to vCJD and natural sheep scrapie. The incubation times (approximately 250 d), neuropathology, and PrP(Sc) isoforms in Tg(BoPrP) mice inoculated with vCJD and BSE brain extracts were indistinguishable and differed dramatically from those seen in these mice injected with natural scrapie. In efforts to identify PrP sequences required for prion formation, we found that a redacted prion protein of only 106 amino acids (PrP106) containing two large deletions supported prion propagation. In Tg(PrP106) mice, an artificial transmission barrier for the passage of full-length mouse prions was diminished by the coexpression of full-length wt MoPrP(C), suggesting that wt MoPrP acts in trans to accelerate the replication of "miniprions" containing PrP(Sc)106. Following a single passage (approximately 300 d) in Tg(PrP106) mice, the miniprions efficiently transmitted disease to Tg(PrP106) mice after only approximately 66 days. Our findings with Tg(BoPrP) mice provide compelling evidence that prions from cattle with BSE have infected humans and caused fatal neurodegeneration, the unique features of miniprions offer new insights into the mechanism of prion replication, and the trans-acting effects of full-length PrP coexpression suggest a new approach to the development of even more efficient animal models for prion diseases.

Elimination of prions by branched polyamines and implications for therapeutics.

We report that branched polyamines, including polyamidoamide dendimers, polypropyleneimine, and polyethyleneimine, are able to purge PrP(Sc), the protease-resistant isoform of the prion protein, from scrapie-infected neuroblastoma (ScN2a) cells in culture. The removal of PrP(Sc) by these compounds depends on both the concentration of branched polymer and the duration of exposure. Chronic exposure of ScN2a cells to low noncytotoxic concentrations of branched polyamines for 1 wk reduced PrP(Sc) to an undetectable level, a condition that persisted at least 3 wk after removal of the compound. Structure-activity analysis revealed that a high surface density of primary amino groups is required for polyamines to eliminate PrP(Sc) effectively from cells. The removal of PrP(Sc) by branched polyamines is attenuated by chloroquine in living cells, and exposure of scrapie-infected brain extracts with branched polyamines at acidic pH rendered the PrP(Sc) susceptible to protease in vitro, suggesting that endosomes or lysozomes may be the site of action. Our studies suggest that branched polyamines might be useful therapeutic agents for treatment of prion diseases and perhaps a variety of other degenerative disorders.

Prion protein of 106 residues creates an artifical transmission barrier for prion replication in transgenic mice.

A redacted prion protein (PrP) of 106 amino acids with two large deletions was expressed in transgenic (Tg) mice deficient for wild-type (wt) PrP (Prnp0/0) and supported prion propagation. RML prions containing full-length PrP(Sc)produced disease in Tg(PrP106)Prnp0/0 mice after approximately 300 days, while transmission of RML106 prions containing PrP(Sc)106 created disease in Tg(PrP106) Prnp0/0 mice after only approximately 66 days on repeated passage. This artificial transmission barrier for the passage of RML prions was diminished by the coexpression of wt MoPrPc in Tg(PrP106)Prnp+/0 mice that developed scrapie in approximately 165 days, suggesting that wt MoPrP acts in trans to accelerate replication of RML106 prions. Purified PrP(Sc)106 was protease resistant, formed filaments, and was insoluble in nondenaturing detergents. The unique features of RML106 prions offer insights into the mechanism of prion replication, and the small size of PrP(Sc)106 should facilitate structural analysis.

Purification and characterization of the inositol 1,4,5- trisphosphate receptor protein from rat vas deferens.

Among rat peripheral tissues examined, Ins(1,4,5)P(3) receptor binding is highest in the vas deferens, with levels about 25% of those of the cerebellum. We have purified the InsP(3) receptor binding protein from rat vas deferens membranes 600-fold. The purified protein displays a single 260 kDa band on SDS/PAGE, and the native protein has an apparent molecular mass of 1000 kDa, the same as in cerebellum. The inositol phosphate specificity, pH-dependence and influence of various reagents are the same for purified vas deferens and cerebellar receptors. Whereas particulate InsP(3) binding in cerebellum is potently inhibited by Ca(2+), particulate and purified vas deferens receptor binding of InsP(3) is not influenced by Ca(2+). Vas deferens appears to lack calmedin activity, but the InsP(3) receptor is sensitive to Ca(2+) inhibition conferred by brain calmedin. The vas deferens may prove to be a valuable tissue for characterizing functional aspects of InsP(3) receptors.

Rat brain endoplasmic reticulum calcium pools are anatomically and functionally segregated.

Autoradiographic imaging can localize 45Ca2+ selectively accumulated via the Ca2+, Mg2(+)-ATPase into endoplasmic reticulum stores in rat brain slices. 45Ca2+ accumulation is markedly stimulated by oxalate and displays a heterogeneous distribution which resembles the mRNA distribution for a sarcoendoplasmic reticulum Ca2+, Mg2(+)-ATPase. Inositol 1,4,5-triphosphate [I(1,4,5)P3] inhibits 45Ca2+ accumulation selectively into regions corresponding to those enriched in I(1,4,5)P3 receptor-binding sites and in Ca2+, -Mg2(+)-ATPase mRNA. Thus rat brain endoplasmic reticulum calcium stores are anatomically and functionally differentiated.

The inositol 1,4,5,-trisphosphate receptor in cerebellar Purkinje cells: quantitative immunogold labeling reveals concentration in an ER subcompartment.

The Ca2+ mobilization effect of inositol 1,4,5-trisphosphate, the second messenger generated via receptor-stimulated hydrolysis of phosphatidylinositol 4,5-bisphosphate, is mediated by binding to intracellular receptors, which are expressed in high concentration in cerebellar Purkinje cells. Partially conflicting previous reports localized the receptor to various subcellular structures: elements of ER, both rough and smooth-surfaced, the nuclear envelope, and even the plasma membrane. We have now reinvestigated the problem quantitatively by using cryosections of rat cerebellar tissue immunolabeled with polyclonal monospecific antibodies against the inositol 1,4,5-trisphosphate receptor. By immunofluorescence the receptor was detected only in Purkinje cells, whereas the other cells of the cerebellar cortex remained negative. In immunogold-decorated ultrathin cryosections of the Purkinje cell body, the receptor was concentrated in cisternal stacks (piles of up to 12 parallel cisternae separated by regularly spaced bridges, located both in the deep cytoplasm and beneath the plasma membrane; average density, greater than 5 particles/micron of membrane profile); in cisternal singlets and doublets adjacent to the plasma membrane (average density, approximately 2.5 particles/micron); and in other apparently smooth-surfaced vesicular and tubular profiles. Additional smooth-surfaced elements were unlabeled. Perinuclear and rough-surfaced ER cisternae were labeled much less by themselves (approximately 0.5 particles/micron, two- to threefold the background), but were often in direct membrane continuity with heavily labeled, smooth-surfaced tubules and cisternal stacks. Finally, mitochondria, Golgi cisternae, multivesicular bodies, and the plasma membrane were unlabeled. In dendrites, approximately half of the nonmitochondrial, membrane-bound structures (cisternae, tubules, and vesicles), as well as small cisternal stacks, were labeled. Dendritic spines always contained immunolabeled cisternae and vesicles. The dendritic plasma membrane, of both shaft and spines, was consistently unlabeled. These results identify a large, smooth-surfaced ER subcompartment that appears equipped to play a key role in the control of Ca2+ homeostasis: in particular, in the generation of [Ca2+]i transients triggered by activation of specific receptors, such as the quisqualate-preferring trans(+/-)-1-amino-1,3-cyclopentamedicarboxylic acid glutamatergic receptors, which are largely expressed by Purkinje cells.

Solubilization and separation of inositol 1,3,4,5-tetrakisphosphate- and inositol 1,4,5-trisphosphate-binding proteins and metabolizing enzymes in rat brain.

The two inositol phosphate-binding proteins, the Ins(1,4,5)P3 (InsP3) and Ins(1,3,4,5)P4 (InsP4) receptors, and the two particulate InsP3-metabolizing enzymes, InsP3 5-phosphatase and InsP3 3-kinase, were solubilized with detergent from rat cerebellar membranes. These four activities are shown to be distinct molecular species by separation using a variety of protein chromatographic steps. The pharmacology of the partially purified InsP4-binding site indicates that the binding has a high affinity and selectivity for InsP4 over InsP3. These results suggest the existence of a distinct specific InsP4-binding protein which may represent the receptor for this putative second messenger.

Free calcium rise and mitogenesis in glial cells caused by endothelin.

The peptide endothelin causes a biphasic rise in intracellular free calcium levels in cultured Type-1 astrocytes and C6 glioma cells, suggesting that glial cells may be the physiological target of endothelin in the brain. Endothelin also causes a calcium-dependent increase [3H]thymidine incorporation in primary cultures of rat cerebellum, indicating that, among other possible roles, this peptide may mediate mitogenesis in brain.

Purified inositol 1,4,5-trisphosphate receptor mediates calcium flux in reconstituted lipid vesicles.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), a second messenger molecule involved in actions of neurotransmitters, hormones and growth factors, releases calcium from vesicular non-mitochondrial intracellular stores. An Ins(1,4,5)P3 binding protein, purified from brain membranes, has been shown to be phosphorylated by cyclic-AMP-dependent protein kinase and localized by immunohistochemical techniques to intracellular particles associated with the endoplasmic reticulum. Although the specificity of the Ins(1,4,5)P3 binding protein for inositol phosphates and the high affinity of the protein for Ins(1,4,5)P3 indicate that it is a physiological Ins(1,4,5)P3 receptor mediating calcium release, direct evidence for this has been difficult to obtain. Also, it is unclear whether a single protein mediates both the recognition of Ins(1,4,5)P3 and calcium transport or whether these two functions involve two or more distinct proteins. In the present study we report reconstitution of the purified Ins(1,4,5)P3 binding protein into lipid vesicles. We show that Ins(1,4,5)P3 and other inositol phosphates stimulate calcium flux in the reconstituted vesicles with potencies and specificities that match the calcium releasing actions of Ins(1,4,5)P3. These results indicate that the purified Ins(1,4,5)P3 binding protein is a physiological receptor responsible for calcium release.

Isolation and functional characterization of an inositol trisphosphate receptor from brain.

We have identified an IP3 receptor protein in brain membranes through the binding of radiolabelled IP3. Autoradiographic studies localize the receptor to various areas of the brain with highest densities in Purkinje cells of the cerebellum. IP3 binding is inhibited by physiologic intracellular concentrations of calcium. Purification of the IP3 receptor to homogeneity reveals it to be comprised of four identical subunits of 260 kD each. Antisera to the purified receptor protein have been employed for immunohistochemical studies which, at the electron microscopic level, localize the IP3 receptor to a subdivision of the rough endoplasmic reticulum occurring in synaptic areas and in close association with the nuclear membrane. The IP3 receptor protein is selectively phosphorylated by cyclic AMP (cAMP) dependent protein kinase. This phosphorylation decreases 10-fold the potency of IP3 in releasing calcium from brain membranes.

Inositol 1,4,5-trisphosphate receptor localized to endoplasmic reticulum in cerebellar Purkinje neurons.

Inositol 1,4,5-trisphosphate (InsP3) mediates the effects of several neurotransmitters, hormones and growth factors by mobilizing Ca2+ from a vesicular, non-mitochondrial intracellular store. Many studies have indirectly suggested the endoplasmic reticulum (ER) to be the site of InsP3 action, though some have implicated the plasma membrane or a newly described smooth surfaced structure, termed the calciosome. Using antibodies directed against a purified InsP3-receptor glycoprotein, of relative molecular mass 260,000, in electron microscope immunocytochemical studies of rat cerebellar Purkinje cells, we have now localized the InsP3 receptor to ER, including portions of the rough endoplasmic reticulum, a population of smooth-membrane-bound organelles (smooth ER), a portion of subplasmalemmal cisternae and the nuclear membrane, but not to mitochondria or the cell membrane. These results suggest that in cerebellar Purkinje cells, InsP3-induced intracellular calcium release is not the property of a single organelle, but is effected by specialized portions of both rough and smooth ER, and possibly by other smooth surfaced structures. The present findings are the first immunocytochemical demonstration of an InsP3 receptor within a cell.

Inositolphospholipid-linked glutamate receptors mediate cerebellar parallel-fiber-Purkinje-cell synaptic transmission.

In slices of adult rat cerebellum inositolphospholipid turnover is stimulated markedly by glutamate and its rigid analogues quisqualate and ibotenate. The drug and amino acid specificity of the response reflects a quisqualate-preferring excitatory amino acid receptor. The absence of glutamate-enhanced inositolphospholipid turnover in mice with Purkinje-cell degeneration indicates that the inositolphospholipid-linked quisqualate receptor mediates parallel fiber-Purkinje cell synaptic transmission. The quantitative prominence of this synapse accounts for the massive enrichment of elements of the inositolphospholipid system in cerebellar Purkinje cells.

Cyclic AMP-dependent phosphorylation of a brain inositol trisphosphate receptor decreases its release of calcium.

We report the stoichiometric phosphorylation of an inositol 1,4,5-trisphosphate receptor-binding protein from rat brain by the cAMP-dependent protein kinase but not by protein kinase C or Ca2+/calmodulin-dependent protein kinase. This phosphorylation event does not markedly alter [3H]inositol 1,4,5-trisphosphate-binding characteristics. However, inositol 1,4,5-trisphosphate is only 10% as potent in releasing 45Ca2+ from phosphorylated, as compared with native, cerebellar microsomes. Phosphorylation of the inositol 1,4,5-trisphosphate-binding protein by the cAMP-dependent protein kinase may provide a biochemical substrate for second-messenger cross talk.