Measuring Nonapoptotic Caspase Activity with a Transgenic Reporter in Mice.

The protease caspase-3 is a key mediator of apoptotic programmed cell death. But weak or transient caspase activity can contribute to neuronal differentiation, axonal pathfinding, and synaptic long-term depression. Despite the importance of sublethal, or nonapoptotic, caspase activity in neurodevelopment and neural plasticity, there has been no simple method for mapping and quantifying nonapoptotic caspase activity (NACA) in rodent brains.

Increased levels of Stress-inducible phosphoprotein-1 accelerates amyloid-β deposition in a mouse model of Alzheimer's disease.

Molecular chaperones and co-chaperones, which are part of the protein quality control machinery, have been shown to regulate distinct aspects of Alzheimer's Disease (AD) pathology in multiple ways. Notably, the co-chaperone STI1, which presents increased levels in AD, can protect mammalian neurons from amyloid-β toxicity in vitro and reduced STI1 levels worsen Aβ toxicity in C. elegans.

Chronic hM3Dq signaling in microglia ameliorates neuroinflammation in male mice.

Microglia express muscarinic G protein-coupled receptors (GPCRs) that sense cholinergic activity and are activated by acetylcholine to potentially regulate microglial functions. Knowledge about how distinct types of muscarinic GPCR signaling regulate microglia function in vivo is still poor, partly due to the fact that some of these receptors are also present in astrocytes and neurons. We generated mice expressing the hM3Dq Designer Receptor Exclusively Activated by Designer Drugs (DREADD) selectively in microglia to investigate the role of muscarinic M3Gq-linked signaling.

Brain tumor acidification using drugs simultaneously targeting multiple pH regulatory mechanisms.

Introduction: Non-invasively distinguishing aggressive from non-aggressive brain tumors is an important clinical challenge. Intracellular pH (pH) regulation is essential for normal cell function and is normally maintained within a narrow range. Cancer cells are characterized by a reversed intracellular to extracellular pH gradient, compared to healthy cells, that is maintained by several distinct mechanisms.

Detection of Active Caspase-3 in Mouse Models of Stroke and Alzheimer's Disease with a Novel Dual Positron Emission Tomography/Fluorescent Tracer [Ga]Ga-TC3-OGDOTA.

Apoptosis is a feature of stroke and Alzheimer's disease (AD), yet there is no accepted method to detect or follow apoptosis in the brain in vivo. We developed a bifunctional tracer [Ga]Ga-TC3-OGDOTA containing a cell-penetrating peptide separated from fluorescent Oregon Green and Ga-bound labels by the caspase-3 recognition peptide DEVD. We hypothesized that this design would allow [Ga]Ga-TC3-OGDOTA to accumulate in apoptotic cells.

Mechanisms of neuroprotection against ischemic insult by stress-inducible phosphoprotein-1/prion protein complex.

Stress-inducible phosphoprotein 1 (STI1) acts as a neuroprotective factor in the ischemic brain and its levels are increased following ischemia. Previous work has suggested that some of these STI1 actions in a stroke model depend on the recruitment of bone marrow-derived stem cells to improve outcomes after ischemic insult. However, STI1 can directly increase neuroprotective signaling in neurons by engaging with the cellular prion protein (PrP ) and activating α7 nicotinic acetylcholine receptors (α7nAChR).

The Hsp70/Hsp90 Chaperone Machinery in Neurodegenerative Diseases.

The accumulation of misfolded proteins in the human brain is one of the critical features of many neurodegenerative diseases, including Alzheimer's disease (AD). Assembles of beta-amyloid (Aβ) peptide-either soluble (oligomers) or insoluble (plaques) and of tau protein, which form neurofibrillary tangles, are the major hallmarks of AD. Chaperones and co-chaperones regulate protein folding and client maturation, but they also target misfolded or aggregated proteins for refolding or for degradation, mostly by the proteasome.

Regulation of Amyloid β Oligomer Binding to Neurons and Neurotoxicity by the Prion Protein-mGluR5 Complex.

The prion protein (PrP) has been suggested to operate as a scaffold/receptor protein in neurons, participating in both physiological and pathological associated events. PrP, laminin, and metabotropic glutamate receptor 5 (mGluR5) form a protein complex on the plasma membrane that can trigger signaling pathways involved in neuronal differentiation. PrP and mGluR5 are co-receptors also for β-amyloid oligomers (AβOs) and have been shown to modulate toxicity and neuronal death in Alzheimer's disease.

Cholinergic Regulation of hnRNPA2/B1 Translation by M1 Muscarinic Receptors.

Unlabelled: Cholinergic vulnerability, characterized by loss of acetylcholine (ACh), is one of the hallmarks of Alzheimer's disease (AD). Previous work has suggested that decreased ACh activity in AD may contribute to pathological changes through global alterations in alternative splicing. This occurs, at least partially, via the regulation of the expression of a critical protein family in RNA processing, heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins.

Domains of STIP1 responsible for regulating PrPC-dependent amyloid-β oligomer toxicity.

Soluble oligomers of amyloid-beta peptide (AβO) transmit neurotoxic signals through the cellular prion protein (PrP(C)) in Alzheimer's disease (AD). Secreted stress-inducible phosphoprotein 1 (STIP1), an Hsp70 and Hsp90 cochaperone, inhibits AβO binding to PrP(C) and protects neurons from AβO-induced cell death. Here, we investigated the molecular interactions between AβO and STIP1 binding to PrP(C) and their effect on neuronal cell death.

The Transient Receptor Potential Melastatin 2 (TRPM2) Channel Contributes to β-Amyloid Oligomer-Related Neurotoxicity and Memory Impairment.

Unlabelled: In Alzheimer's disease, accumulation of soluble oligomers of β-amyloid peptide is known to be highly toxic, causing disturbances in synaptic activity and neuronal death. Multiple studies relate these effects to increased oxidative stress and aberrant activity of calcium-permeable cation channels leading to calcium imbalance. The transient receptor potential melastatin 2 (TRPM2) channel, a Ca(2+)-permeable nonselective cation channel activated by oxidative stress, has been implicated in neurodegenerative diseases, and more recently in amyloid-induced toxicity.

The prion protein ligand, stress-inducible phosphoprotein 1, regulates amyloid-β oligomer toxicity.

In Alzheimer's disease (AD), soluble amyloid-β oligomers (AβOs) trigger neurotoxic signaling, at least partially, via the cellular prion protein (PrP(C)). However, it is unknown whether other ligands of PrP(C) can regulate this potentially toxic interaction. Stress-inducible phosphoprotein 1 (STI1), an Hsp90 cochaperone secreted by astrocytes, binds to PrP(C) in the vicinity of the AβO binding site to protect neurons against toxic stimuli.

Regulation of stress-inducible phosphoprotein 1 nuclear retention by protein inhibitor of activated STAT PIAS1.

Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation.

Increased prion protein processing and expression of metabotropic glutamate receptor 1 in a mouse model of Alzheimer's disease.

Prion protein (PrP(C) ), a glycosylphosphatidylinositol-anchored protein corrupted in prion diseases, has been shown recently to interact with group I metabotropic glutamate receptors (mGluRs). Moreover, both PrP(C) and mGluRs were proposed to function as putative receptors for β-amyloid in Alzheimer's disease. PrP(C) can be processed in neurons via α or β-cleavage to produce PrP(C) fragments that are neuroprotective or toxic, respectively.

Laminin-γ1 chain and stress inducible protein 1 synergistically mediate PrPC-dependent axonal growth via Ca2+ mobilization in dorsal root ganglia neurons.

Prion protein (PrP(C)) is a cell surface glycoprotein that is abundantly expressed in nervous system. The elucidation of the PrP(C) interactome network and its significance on neural physiology is crucial to understanding neurodegenerative events associated with prion and Alzheimer's diseases. PrP(C) co-opts stress inducible protein 1/alpha7 nicotinic acetylcholine receptor (STI1/α7nAChR) or laminin/Type I metabotropic glutamate receptors (mGluR1/5) to modulate hippocampal neuronal survival and differentiation.

A new mechanism for transmissible prion diseases.

The transmissible agent of prion disease consists of prion protein (PrP) in β-sheet-rich state (PrP(Sc)) that can replicate its conformation according to a template-assisted mechanism. This mechanism postulates that the folding pattern of a newly recruited polypeptide accurately reproduces that of the PrP(Sc) template. Here, three conformationally distinct amyloid states were prepared in vitro using Syrian hamster recombinant PrP (rPrP) in the absence of cellular cofactors.

Dissecting structural basis of the unique substrate selectivity of human enteropeptidase catalytic subunit.

Enteropeptidase is a key enzyme in the digestion system of higher animals. It initiates enzymatic cascade cleaving trypsinogen activation peptide after a unique sequence DDDDK. Recently, we have found specific activity of human enteropeptidase catalytic subunit (L-HEP) being significantly higher than that of its bovine ortholog (L-BEP).

Atomic force fluorescence microscopy in the characterization of amyloid fibril assembly and oligomeric intermediates.

Atomic force microscopy (AFM) has become a conventional tool for elucidation of the molecular mechanisms of protein aggregation and, specifically, for analysis of assembly pathways, architecture, aggregation state, and heterogeneity of oligomeric intermediates or mature fibrils. AFM imaging provides useful information about particle dimensions, shape, and substructure with nanometer resolution. Conventional AFM methods have been very helpful in the analysis of polymorphic assemblies formed in vitro from homogeneous proteins or peptides.

Molecular structure of amyloid fibrils controls the relationship between fibrillar size and toxicity.

Background: According to the prevailing view, soluble oligomers or small fibrillar fragments are considered to be the most toxic species in prion diseases. To test this hypothesis, two conformationally different amyloid states were produced from the same highly pure recombinant full-length prion protein (rPrP). The cytotoxic potential of intact fibrils and fibrillar fragments generated by sonication from these two states was tested using cultured cells.

Amyloid features and neuronal toxicity of mature prion fibrils are highly sensitive to high pressure.

Prion proteins (PrP) can aggregate into toxic and possibly infectious amyloid fibrils. This particular macrostructure confers on them an extreme and still unexplained stability. To provide mechanistic insights into this self-assembly process, we used high pressure as a thermodynamic tool for perturbing the structure of mature amyloid fibrils that were prepared from recombinant full-length mouse PrP.

Highly efficient protein misfolding cyclic amplification.

Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrP(C) into PrP(Sc) in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrP(C) may be available for conversion.

The α-helical C-terminal domain of full-length recombinant PrP converts to an in-register parallel β-sheet structure in PrP fibrils: evidence from solid state nuclear magnetic resonance.

We report the results of solid state nuclear magnetic resonance (NMR) measurements on amyloid fibrils formed by the full-length prion protein PrP (residues 23−231, Syrian hamster sequence). Measurements of intermolecular 13C−13C dipole−dipole couplings in selectively carbonyl-labeled samples indicate that β-sheets in these fibrils have an in-register parallel structure, as previously observed in amyloid fibrils associated with Alzheimer’s disease and type 2 diabetes and in yeast prion fibrils. Two-dimensional 13C−13C and 15N−13C solid state NMR spectra of a uniformly 15N- and 13C-labeled sample indicate that a relatively small fraction of the full sequence, localized to the C-terminal end, forms the structurally ordered, immobilized core.

Two amyloid States of the prion protein display significantly different folding patterns.

It has been well established that a single amino acid sequence can give rise to several conformationally distinct amyloid states. The extent to which amyloid structures formed within the same sequence are different, however, remains unclear. To address this question, we studied two amyloid states (referred to as R- and S-fibrils) produced in vitro from highly purified full-length recombinant prion protein.

Conformational switching within individual amyloid fibrils.

A key structural component of amyloid fibrils is a highly ordered, crystalline-like cross-beta-sheet core. Conformationally different amyloid structures can be formed within the same amino acid sequence. It is generally assumed that individual fibrils consist of conformationally uniform cross-beta-structures.

The polybasic N-terminal region of the prion protein controls the physical properties of both the cellular and fibrillar forms of PrP.

Individual variations in structure and morphology of amyloid fibrils produced from a single polypeptide are likely to underlie the molecular origin of prion strains and control the efficiency of the species barrier in the transmission of prions. Previously, we observed that the shape of amyloid fibrils produced from full-length prion protein (PrP 23-231) varied substantially for different batches of purified recombinant PrP. Variations in fibril morphology were also observed for different fractions that corresponded to the highly pure PrP peak collected at the last step of purification.