Conformation-dependent membrane permeabilization by neurotoxic PrP oligomers: The role of the H2H3 oligomerization domain.

The relationship between prion propagation and the generation of neurotoxic species and clinical onset remains unclear. Several converging lines of evidence suggest that interactions with lipids promote various precursors to form aggregation-prone states that are involved in amyloid fibrils. Here, we compared the cytotoxicities of different soluble isolated oligomeric constructs from murine full-length PrP and from the restricted helical H2H3 domain with their effects on lipid vesicles.

A novel and rapid method for obtaining high titre intact prion strains from mammalian brain.

Mammalian prions exist as multiple strains which produce characteristic and highly reproducible phenotypes in defined hosts. How this strain diversity is encoded by a protein-only agent remains one of the most interesting and challenging questions in biology with wide relevance to understanding other diseases involving the aggregation or polymerisation of misfolded host proteins. Progress in understanding mammalian prion strains has however been severely limited by the complexity and variability of the methods used for their isolation from infected tissue and no high resolution structures have yet been reported.

Endogenous prion protein conversion is required for prion-induced neuritic alterations and neuronal death.

Prions cause fatal neurodegenerative conditions and result from the conversion of host-encoded cellular prion protein (PrP(C)) into abnormally folded scrapie PrP (PrP(Sc)). Prions can propagate both in neurons and astrocytes, yet neurotoxicity mechanisms remain unclear. Recently, PrP(C) was proposed to mediate neurotoxic signaling of β-sheet-rich PrP and non-PrP conformers independently of conversion.

Isolation of proteinase K-sensitive prions using pronase E and phosphotungstic acid.

Disease-related prion protein, PrP(Sc), is classically distinguished from its normal cellular precursor, PrP(C), by its detergent insolubility and partial resistance to proteolysis. Molecular diagnosis of prion disease typically relies upon detection of protease-resistant fragments of PrP(Sc) using proteinase K, however it is now apparent that the majority of disease-related PrP and indeed prion infectivity may be destroyed by this treatment. Here we report that digestion of RML prion-infected mouse brain with pronase E, followed by precipitation with sodium phosphotungstic acid, eliminates the large majority of brain proteins, including PrP(C), while preserving >70% of infectious prion titre.

Endogenous proteolytic cleavage of disease-associated prion protein to produce C2 fragments is strongly cell- and tissue-dependent.

The abnormally folded form of the prion protein (PrP(Sc)) accumulating in nervous and lymphoid tissues of prion-infected individuals can be naturally cleaved to generate a N-terminal-truncated fragment called C2. Information about the identity of the cellular proteases involved in this process and its possible role in prion biology has remained limited and controversial. We investigated PrP(Sc) N-terminal trimming in different cell lines and primary cultured nerve cells, and in the brain and spleen tissue from transgenic mice infected by ovine and mouse prions.

Detection and characterization of proteinase K-sensitive disease-related prion protein with thermolysin.

Disease-related PrP(Sc) [pathogenic PrP (prion protein)] is classically distinguished from its normal cellular precursor, PrP(C)(cellular PrP) by its detergent insolubility and partial resistance to proteolysis. Although molecular diagnosis of prion disease has historically relied upon detection of protease-resistant fragments of PrP(Sc) using PK (proteinase K), it is now apparent that a substantial fraction of disease-related PrP is destroyed by this protease. Recently, thermolysin has been identified as a complementary tool to PK, permitting isolation of PrP(Sc) in its full-length form.

Conditional, genetic disruption of ciliary neurotrophic factor receptors reveals a role in adult motor neuron survival.

Indirect evidence suggests that endogenous ciliary neurotrophic factor (CNTF) receptor signaling can promote motor neuron (MN) survival in the adult. If so, proper targeting of this signaling may selectively counteract the effects of adult MN diseases. However, direct evidence for CNTF receptor involvement in adult MN survival is lacking, presumably because the unconditional blockade of the mouse CNTF receptor in vivo [through genetic disruption of the essential CNTF receptor alpha (CNTFRalpha) gene] leads to uniform perinatal death of the mice.

High-throughput single copy DNA amplification and cell analysis in engineered nanoliter droplets.

A high-throughput single copy genetic amplification (SCGA) process is developed that utilizes a microfabricated droplet generator (microDG) to rapidly encapsulate individual DNA molecules or cells together with primer functionalized microbeads in uniform PCR mix droplets. The nanoliter volume droplets uniquely enable quantitative high-yield amplification of DNA targets suitable for long-range sequencing and genetic analysis. A hybrid glass-polydimethylsiloxane (PDMS) microdevice assembly is used to integrate a micropump into the microDG that provides uniform droplet size, controlled generation frequency, and effective bead incorporation.

Kuru prions and sporadic Creutzfeldt-Jakob disease prions have equivalent transmission properties in transgenic and wild-type mice.

Kuru provides our principal experience of an epidemic human prion disease and primarily affected the Fore linguistic group of the Eastern Highlands of Papua New Guinea. Kuru was transmitted by the practice of consuming dead relatives as a mark of respect and mourning (transumption). To date, detailed information of the prion strain type propagated in kuru has been lacking.

Modulating the mechanical properties of self-assembled peptide hydrogels via native chemical ligation.

Hydrogels produced from self-assembling peptides and peptide derivatives are being investigated as synthetic extracellular matrices for defined cell culture substrates and scaffolds for regenerative medicine. In many cases, however, they are less stiff than the tissues and extracellular matrices they are intended to mimic, and they are prone to cohesive failure. We employed native chemical ligation to produce peptide bonds between the termini of fibrillized beta-sheet peptides to increase gel stiffness in a chemically specific manner while maintaining the morphology of the self-assembled fibrils.

Prion strain- and species-dependent effects of antiprion molecules in primary neuronal cultures.

Transmissible spongiform encephalopathies (TSE) arise as a consequence of infection of the central nervous system by prions and are incurable. To date, most antiprion compounds identified by in vitro screening failed to exhibit therapeutic activity in animals, thus calling for new assays that could more accurately predict their in vivo potency. Primary nerve cell cultures are routinely used to assess neurotoxicity of chemical compounds.

Inline injection microdevice for attomole-scale sanger DNA sequencing.

A new affinity-capture-based inline purification, concentration, and injection method is developed for microchip capillary electrophoresis (CE) and used to perform efficient attomole-scale Sanger DNA sequencing separations. The microdevice comprises three axial domains for nanoliter-scale sequencing sample containment, sample plug formation, and high-resolution capillary gel electrophoresis. Purified and concentrated inline sample plugs are formed by electrophoretically driving Sanger sequencing extension fragments into an affinity-capture polymer network positioned within a CE separation channel.

The S1P2 sphingosine 1-phosphate receptor is essential for auditory and vestibular function.

Sphingosine 1-phosphate (S1P) is an endogenous growth factor with potent effects on many different cell types. Most of these effects are produced by activation of one or more of a family of G-protein coupled receptors. The S1P2 receptor can mediate S1P-induced proliferation, differentiation and survival in a wide variety of cells in culture.

Activation of the JNK-c-Jun pathway during the early phase of neuronal apoptosis induced by PrP106-126 and prion infection.

Prion diseases are neurodegenerative pathologies characterized by apoptotic neuronal death. Although the late execution phase of neuronal apoptosis is beginning to be characterized, the sequence of events occurring during the early decision phase is not yet well known. In murine cortical neurons in primary culture, apoptosis was first induced by exposure to a synthetic peptide homologous to residues 106-126 of the human prion protein (PrP), PrP106-126.

Prions can infect primary cultured neurons and astrocytes and promote neuronal cell death.

Transmissible spongiform encephalopathies arise as a consequence of infection of the central nervous system by prions, where neurons and glial cells are regarded as primary targets. Neuronal loss and gliosis, associated with the accumulation of misfolded prion protein (PrP), are hallmarks of prion diseases; yet the mechanisms underlying such disorders remain unclear. Here we introduced a cell system based on primary cerebellar cultures established from transgenic mice expressing ovine PrP and then exposed to sheep scrapie agent.