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Abstract Background PTEN, a syndromic autism spectrum disorder (ASD) risk gene, is mutated in approximately 10% of macrocephalic ASD cases. Despite the described genetic association between PTEN and ASD and ensuing studies, we continue to have a limited understanding of how PTEN disruption drives ASD pathogenesis and maintenance. Methods We derived neural stem cells (NSCs) from the dentate gyrus (DG) of Pten m3m4 mice, a model that recapitulates PTEN-ASD phenotypes. We subsequently characterized the expression of stemness factors, proliferation, and differentiation of neurons and glia in Pten m3m4 NSCs using immunofluorescent and immunoblotting approaches. We also measured Creb phosphorylation by Western blot analysis and expression of Creb-regulated genes with qRT-PCR. Results The m3m4 mutation decreases Pten localization to the nucleus and its global expression over time. Pten m3m4 NSCs exhibit persistent stemness characteristics associated with increased proliferation and a resistance to neuronal maturation during differentiation. Given the increased proliferation of Pten m3m4 NSCs, a significant increase in the population of immature neurons relative to mature neurons occurs, an approximately tenfold decrease in the ratio between the homozygous mutant and wildtype. There is an opposite pattern of differentiation in some Pten m3m4 glia, specifically an increase in astrocytes. These aberrant differentiation patterns associate with changes in Creb activation in Pten m3m4/m3m4 NSCs. We specifically observed loss of Creb phosphorylation at S133 in Pten m3m4/m3m4 NSCs and a subsequent decrease in expression of Creb-regulated genes important to neuronal function (i.e., Bdnf). Interestingly, Bdnf treatment is able to partially rescue the stunted neuronal maturation phenotype in Pten m3m4/m3m4 NSCs. Conclusions Constitutional disruption of Pten nuclear localization with subsequent global decrease in Pten expression generates abnormal patterns of differentiation, a stunting of neuronal maturation. The propensity of Pten disruption to restrain neurons to a more progenitor-like state may be an important feature contributing to PTEN-ASD pathogenesis. Graphical abstract

Aggregation of the normal cellular prion protein, PrP, is important in the pathogenesis of prion disease. PrP binds glycosaminoglycan (GAG) and divalent cations, such as Cu2+ and Zn2+. Here, we report our findings that GAG and Cu2+ promote the aggregation of recombinant human PrP (rPrP). The normal cellular prion protein has five octapeptide repeats. In the presence of either GAG or Cu2+, mutant rPrPs with eight or ten octapeptide repeats are more aggregation prone, exhibit faster kinetics and form larger aggregates than wild-type PrP. When the GAG-binding motif, KKRPK, is deleted the effect of GAG but not that of Cu2+ is abolished. By contrast, when the Cu2+-binding motif, the octapeptide-repeat region, is deleted, neither GAG nor Cu2+ is able to promote aggregation. Therefore, the octapeptide-repeat region is critical in the aggregation of rPrP, irrespective of the promoting ligand. Furthermore, aggregation of rPrP in the presence of GAG is blocked with anti-PrP mAbs, whereas none of the tested anti-PrP mAbs block Cu2+-promoted aggregation. However, a mAb that is specific for an epitope at the N-terminus enhances aggregation in the presence of either GAG or Cu2+. Therefore, although binding of either GAG or Cu2+ promotes the aggregation of rPrP, their aggregation processes are different, suggesting multiple pathways of rPrP aggregation. [ABSTRACT FROM AUTHOR]

Overwhelming evidence supports the amyloid hypothesis of Alzheimer’s disease that stipulates that the relative level of the 42 amino acid β-amyloid peptide (Aβ42) in relationship to Aβ40 is critical to the pathogenesis of the disease. While it is clear that the multi-subunit gamma secretase is responsible for cleavage of the amyloid precursor protein (APP) into Aβ42 and Aβ40, the exact molecular mechanisms regulating the production of the various Aβ species remain elusive. To elucidate the underlying mechanisms, we replaced individual amino acid residues from positions 43 to 52 of Aβ with phenylalanine to examine the effects on the production of Aβ40 and Aβ42. All mutants, except for V50F, resulted in a decrease in total Aβ with a more prominent reduction in Aβ for residues 45, 48, and 51, following an every three residue repetition pattern. In addition, the mutations with the strongest reductions in total Aβ had the largest increases in the ratio of Aβ42/Aβ40. Curiously, the T43F, V44F, and T48F mutations caused a striking decrease in the accumulation of membrane bound Aβ46, albeit by a different mechanism. Our data suggest that initial cleavage of APP at the ε site is crucial in the generation of Aβ. The implicated sequential cleavage and an α-helical model may lead to a better understanding of the γ-secretase-mediated APP processing and may also provide useful information for therapy and drug design aimed at altering Aβ production. [ABSTRACT FROM AUTHOR]

The normal PrPC (cellular prion protein) contains sLeX [sialyl-LeX (Lewis X)] and LeX. sLeX is a ligand of selectins. To examine whether PrPC is a ligand of selectins, we generated three human PrPC–Ig fusion proteins: one with LeX, one with sLeX, and the other with neither LeX nor sLeX. Only LeX-PrPC–Ig binds E-, L- and P-selectins. Binding is Ca2+-dependent and occurs with nanomolar affinity. Removal of sialic acid on sLeX-PrPC–Ig enables the fusion protein to bind all selectins. These findings were confirmed with brain-derived PrPC. The selectins precipitated PrPC in human brain in a Ca2+-dependent manner. Treatment of brain homogenates with neuraminidase increased the amounts of PrPC precipitated. Therefore the presence of sialic acid prevents the binding of PrPC in human brain to selectins. Hence, human brain PrPC interacts with selectins in a manner that is distinct from interactions in peripheral tissues. Alternations in these interactions may have pathological consequences. [ABSTRACT FROM AUTHOR]

Mutation in the prion gene PRNP accounts for 10–15% of human prion diseases. However, little is known about the mechanisms by which mutant prion proteins (PrPs) cause disease. Here we investigated the effects of 10 different pathogenic mutations on the conformation and ligand-binding activity of recombinant human PrP (rPrP). We found that mutant rPrPs react more strongly with N terminus-specific antibodies, indicative of a more exposed N terminus. The N terminus of PrP contains a glycosaminoglycan (GAG)-binding motif. Binding of GAG is important in prion disease. Accordingly, all mutant rPrPs bind more GAG, and GAG promotes the aggregation of mutant rPrPs more efficiently than wild-type recombinant normal cellular PrP (rPrpC). Furthermore, point mutations in PRNP also cause conformational changes in the region between residues 109 and 136, resulting in the exposure of a second, normally buried, GAG-binding motif. Importantly, brain-derived PrP from transgenic mice, which express a pathogenic mutant with nine extra octapeptide repeats, also binds more strongly to GAG than wild-type PrPC. Thus, several rPrPs with distinct pathogenic mutations have common conformational changes, which enhance binding to GAG. These changes may contribute to the pathogenesis of inherited prion diseases. [ABSTRACT FROM AUTHOR]

Mutation in the prion gene, PRNP, accounts for approx. 10–15% of human prion diseases. However, little is known about the mechanisms by which a mutant prion protein (PrP) causes disease. We compared the biochemical properties of a wild-type human prion protein, rPrPC (recombinant wild-type PrP), which has five octapeptide-repeats, with two recombinant human prion proteins with insertion mutations, one with three more octapeptide repeats, rPrP8OR, and the other with five more octapeptide repeats, rPrP10OR. We found that the insertion mutant proteins are more prone to aggregate, and the degree and kinetics of aggregation are proportional to the number of inserts. The octapeptide-repeat and α-helix 1 regions are important in aggregate formation, because aggregation is inhibited with monoclonal antibodies that are specific for epitopes in these regions. We also showed that a small amount of mutant protein could enhance the formation of mixed aggregates that are composed of mutant protein and wild-type rPrPC. Accordingly, rPrP10OR is also more efficient in promoting the aggregation of rPrPC than rPrP8OR. These findings provide a biochemical explanation for the clinical observations that the severity of the disease in patients with insertion mutations is proportional to the number of inserts, and thus have implications for the pathogenesis of inherited human prion disease. [ABSTRACT FROM AUTHOR]

We compared the biochemical properties of a wild type recombinant normal human cellular prion protein, rPrPc, with a recombinant mutant human prion protein that has three additional octapeptide repeats, rPrPSOR. Monoclonal antibodies that are specific for the N terminus of rPrPc react much better with rPrPSOR than rPrPc, suggesting that the N terminus of rPrPSOR is more exposed and hence more available for antibody binding. The N terminus of PrPc contains a glycosaminoglycan binding motif. Accordingly, rPrPSOR also binds more glycosaminoglycan than rPrPc. In addition, the divalent cation copper modulates the conformations of rPrPc and rPrPSOR differently. When compared with rPrPc, rPrPSOR is also more susceptible to oxidative damage. Furthermore, the abnormalities associated with rPrPSOR are recapitulated, but even more profoundly, in another insertion mutant, which has five extra octapeptide repeats, rPrP10OR. Therefore, insertion mutants appear to share common features, and the degree of abnormality is proportional to the number of insertions. Any of these anomalies may contribute to the pathogenesis of inherited human prion disease. [ABSTRACT FROM AUTHOR]

The conversion of the normal cellular prion protein, PrPC, into the protease-resistant, scrapie PrPSc aggregate is the cause of prion diseases. We developed a novel enzyme-linked immunosorbent assay (ELISA) that is specific for PrP aggregate by screening 30 anti-PrP monoclonal antibodies (MAbs) for their ability to react with recombinant mouse, ovine, bovine, or human PrP dimers. One MAb that reacts with all four recombinant PrP dimers also reacts with PrPSc aggregates in ME7-, 139A-, or 22L-infected mouse brains. The PrPSc aggregate is proteinase K resistant, has a mass of 2,000 kDa or more, and is present at a time when no protease-resistant PrP is detectable. This simple and sensitive assay provides the basis for the development of a diagnostic test for prion diseases in other species. Finally, the principle of the aggregate-specific ELISA we have developed may be applicable to other diseases caused by abnormal protein aggregation, such as Alzheimer's disease or Parkinson's disease. [ABSTRACT FROM AUTHOR]

Infection with any one of three strains of mouse scrapie prion (PrPSc), 139A, ME7, or 22L, results in the accumulation of two underglycosylated, full-length PrP species and an N-terminally truncated PrP species that are not detectable in uninfected animals. The levels of the N-terminally truncated PrP species vary depending on PrPSc strain. Furthermore, 22L-infected brains consistently have the highest levels of proteinase K (PK)resistant PrP species, followed by ME7- and 139A-infected brains. The three strains of PrPSc are equally susceptible to PK and proteases papain and chymotrypsin. Their protease resistance patterns are also similar. In sucrose gradient velocity sedimentation, the aberrant PrP species partition with PrPSc aggregates, indicating that they are physically associated with PrPSc. In ME7-infected animals, one of the underglycosylated, full-length PrP species is detected much earlier than the other, before both the onset of clinical disease and the detection of PK-resistant PrP species. In contrast, the appearance of the N-terminally truncated PrP species coincides with the presence of PK-resistant species and the manifestation of clinical symptoms. Therefore, accumulation of the underglycosylated, full-length PrP species is an early biochemical fingerprint of PrPSc infection. Accumulation of the underglycosylated, full-length PrP species and the aberrant N-terminally truncated PrP species may be important in the pathogenesis of prion disease. [ABSTRACT FROM AUTHOR]