Identification of Host Proteins Predictive of Early Stage Mycobacterium tuberculosis Infection.

The objective of this study was to identify blood-based protein biomarkers of early stage Mycobacterium tuberculosis (Mtb) infection. We utilized plasma and serum specimens from TB patients and their contacts (age≥12) enrolled in a household contact study in Uganda. In the discovery phase cross-sectional samples from 104 HIV-uninfected persons classified as either active TB, latent Mtb infection (LTBI), tuberculin skin test (TST) converters, or persistent TST-negative were analyzed. Two hundred eighty-nine statistically significant (false discovery rate corrected p<0.05) differentially expressed proteins were identified across all comparisons. Proteins associated with cellular immunity and lipid metabolism were induced early after Mtb infection. One hundred and fifty-nine proteins were selected for a targeted mass spectrometry assay. A set of longitudinal samples from 52 TST-negative subjects who converted to TST-positive or remained TST-negative were analyzed, and multivariate logistic regression was used to identify unique protein panels able to predict TST conversion with cross-validated AUC>0.85. Panel performance was confirmed with an independent validation set of longitudinal samples from 16 subjects. These candidate protein biomarkers may allow for the identification of recently Mtb infected individuals at highest risk for developing active TB and most likely to benefit from preventive therapy.

Local inhibition of Rho signaling by cell-permeable recombinant protein BA-210 prevents secondary damage and promotes functional recovery following acute spinal cord injury.

Spinal cord injury (SCI) leads to robust Rho activation at the lesion site. Here, we demonstrate that BA-210, a cell-permeable fusion protein derived from C3 transferase, formulated in fibrin sealant and delivered topically onto the dura matter, diffuses into the spinal cord and inactivates Rho in a dose-dependent manner. Treatment with BA-210 in rats with thoracic spinal cord contusion increased tissue sparing around the lesion area and led to significant improvement of locomotor function. In mice, BA-210 improved functional outcome when treatment was either applied at the time of injury or delayed by 24 h. In both rats and mice, treatment with BA-210 was well tolerated. Rats gained body weight normally, and BA-210 treatment had no impact on the development of allodynia. Inactivating Rho with BA-210 holds promise for treating patients with SCI.

Genes contributing to prion pathogenesis.

Prion diseases are caused by conversion of a normally folded, non-pathogenic isoform of the prion protein (PrP(C)) to a misfolded, pathogenic isoform (PrP(Sc)). Prion inoculation experiments in mice expressing homologous PrP(C) molecules on different genetic backgrounds displayed different incubation times, indicating that the conversion reaction may be influenced by other gene products. To identify genes that contribute to prion pathogenesis, we analysed incubation times of prions in mice in which the gene product was inactivated, knocked out or overexpressed. We tested 20 candidate genes, because their products either colocalize with PrP, are associated with Alzheimer's disease, are elevated during prion disease, or function in PrP-mediated signalling, PrP glycosylation, or protein maintenance. Whereas some of the candidates tested may have a role in the normal function of PrP(C), our data show that many genes previously implicated in prion replication have no discernible effect on the pathogenesis of prion disease. While most genes tested did not significantly affect survival times, ablation of the amyloid beta (A4) precursor protein (App) or interleukin-1 receptor, type I (Il1r1), and transgenic overexpression of human superoxide dismutase 1 (SOD1) prolonged incubation times by 13, 16 and 19 %, respectively.

Developmental expression of PrP in the post-implantation embryo.

Since prion protein (PrP) mRNA and PrP(C) expression levels in transgenic (Tg) mice using the CosSHa.tet vector correlate well with the PrP transgene copy, we constructed Prnp-LacZ Tg animals expressing beta-galactosidase that was inserted into the CosSHa.tet vector. The CosSHa.tet vector was created from a large PrP cosmid clone in which the PrP open reading frame was deleted. In the developing nervous system, the beta-galactosidase marker was not expressed in the neural progenitors of the mitotically active ventricular zone. It is first expressed in cells that have ceased proliferating, migrated radially from the ventricular zone, and differentiated into neurons in the intermediate layer. At E11.5 p.c., motor neurons in the ventral neural tube clearly express the marker transgene. Expression in dorsal neural tube neurons is observed at later stages, after their differentiation. These results indicate that Prnp gene expression in the nervous system begins in post-mitotic neural cells that have undergone neuronal differentiation. This pattern of Prnp expression in the nervous system appears to persist throughout the adult life of mammals.

Targeting soluble Abeta peptide with Tramiprosate for the treatment of brain amyloidosis.

Amyloid beta-peptide (Abeta) is a major constituent of senile plaques in Alzheimer's disease (AD). Neurotoxicity results from the conformational transition of Abeta from random-coil to beta-sheet and its oligomerization. Among a series of ionic compounds able to interact with soluble Abeta, Tramiprosate (3-amino-1-propanesulfonic acid; 3APS; Alzhemedtrade mark) was found to maintain Abeta in a non-fibrillar form, to decrease Abeta(42)-induced cell death in neuronal cell cultures, and to inhibit amyloid deposition. Tramiprosate crosses the murine blood-brain barrier (BBB) to exert its activity. Treatment of TgCRND8 mice with Tramiprosate resulted in significant reduction (approximately 30%) in the brain amyloid plaque load and a significant decrease in the cerebral levels of soluble and insoluble Abeta(40) and Abeta(42) (approximately 20-30%). A dose-dependent reduction (up to 60%) of plasma Abeta levels was also observed, suggesting that Tramiprosate influences the central pool of Abeta, changing either its efflux or its metabolism in the brain. We propose that Tramiprosate, which targets soluble Abeta, represents a new and promising therapeutic class of drugs for the treatment of AD.

Deficiency in neuronal TGF-beta signaling promotes neurodegeneration and Alzheimer's pathology.

Alzheimer's disease (AD) is characterized by progressive neurodegeneration and cerebral accumulation of the beta-amyloid peptide (Abeta), but it is unknown what makes neurons susceptible to degeneration. We report that the TGF-beta type II receptor (TbetaRII) is mainly expressed by neurons, and that TbetaRII levels are reduced in human AD brain and correlate with pathological hallmarks of the disease. Reducing neuronal TGF-beta signaling in mice resulted in age-dependent neurodegeneration and promoted Abeta accumulation and dendritic loss in a mouse model of AD. In cultured cells, reduced TGF-beta signaling caused neuronal degeneration and resulted in increased levels of secreted Abeta and beta-secretase-cleaved soluble amyloid precursor protein. These results show that reduced neuronal TGF-beta signaling increases age-dependent neurodegeneration and AD-like disease in vivo. Increasing neuronal TGF-beta signaling may thus reduce neurodegeneration and be beneficial in AD.

Mapping genetic modulators of amyloid plaque deposition in TgCRND8 transgenic mice.

Alzheimer's disease (AD) is a complex disorder for which various in vivo models exist. The TgCRND8 mouse, transgenic for the human amyloid precursor protein, is an aggressive early onset model of brain amyloid deposition. Preliminary studies revealed that when the transgene is expressed on an A/J genetic background, these mice not only survive longer but also deposit less parenchymal amyloid-beta (Abeta) peptides as compared to those on a C57BL/6 background. We performed a genome-wide study of an F2 intercross between TgCRND8 on an A/J background and C57BL/6 mice, to identify genetic modulators of amyloid accumulation and deposition. We identified four highly significant QTLs that together account for 55% of the phenotypic variance in the number of plaques (Thioflavin S). QTLs were found on the distal part of chromosome 4 with an LOD score of 8.1 at D4Mit251, on chromosome 11 with an LOD score of 5.5 at D11Mit242, on chromosome 9 with an LOD score of 5.0 at D9Mit336 and on the proximal part of chromosome 8 with an LOD score of 4.5 at D8Mit223. A/J alleles at these loci are protective and all decreased the amount of Abeta deposition. Interestingly, the QTL on chromosome 11 is also significantly linked to the levels of brain Abeta(42) and Abeta(40). Although these QTLs do not control the levels of plasmatic Abeta, other regions on chromosomes 1 and 6 show significant linkage. Further characterization of these QTL regions may lead to the identification of genes involved in the pathogenesis of AD.

The cAMP-specific phosphodiesterase 4B mediates Abeta-induced microglial activation.

Microglial activation is a key player in the degenerative process that accompanies the deposition of amyloid-beta (Abeta) peptide into senile plaques in Alzheimer's disease (AD) patients. The goal of this study is to identify novel genes involved in microglial activation in response to Abeta peptide. Prompted by the fact that soluble Abeta(1-42) (sAbeta(1-42))-stimulated primary rat microglia produce more tumor necrosis factor-alpha (TNF-alpha) than fibrillar Abeta(1-42) (fAbeta(1-42))-stimulated microglia, we examined gene expression in these cells following stimulation using cDNA arrays. This analysis confirms the upregulation caused by both sAbeta(1-42) and fAbeta(1-42) of pro-inflammatory molecules such as TNF-alpha, interleukin-1beta and macrophage inflammatory protein-1alpha. In addition, other transcripts not previously described in the context of Abeta-induced microglial activation were identified. The modulation of some of these genes within microglial cells seems to be specific to sAbeta(1-42) as compared to fAbeta(1-42) suggesting that different forms of Abeta may activate distinct pathways during the progression of AD. Importantly, we demonstrate that Pde4B, a cAMP-specific phosphodiesterase, is upregulated by Abeta and results in an increased production of TNF-alpha. Inhibition of Pde4B reduces by up to 70% the release of TNF-alpha from sAbeta-stimulated microglial cells, implicating cAMP as an important mediator of Abeta-induced microglial activation.

Prion clearance in bigenic mice.

The clearance of prions from the brain was investigated in bigenic mice designated Tg(tTA : PrP(+/0))3, in which expression of the cellular prion protein (PrP(C)) was regulated by oral doxycycline administration. With suppression of PrP(C) expression, the incubation time for RML prions was prolonged almost threefold from approximately 150 to approximately 430 days. To determine the clearance rate of disease-causing PrP(Sc), bigenic mice were given oral doxycycline beginning 98 days after inoculation with RML prions and sacrificed at various time points over the subsequent 56 days. The half-life (t1/2) for PrP(Sc) was approximately 1.5 days in mouse brain, in reasonable agreement with the apparent t1/2 of 30 h that was determined in a separate study for scrapie-infected mouse neuroblastoma (ScN2a) cells in culture. Both protease-sensitive and -resistant conformers of PrP(Sc) were cleared at the same rate. The t1/2 value for PrP(C) clearance from brain was approximately 18 h, which was considerably longer than the t1/2 of 5 h found in ScN2a cells. The capability of the brain to clear prions raises the possibility that PrP(Sc) is normally made at low levels and continually cleared, and that PrP(Sc) may have a function in cellular metabolism. Moreover, these bigenic mice make it possible to determine both components of PrP(Sc) accumulation, i.e. the rates of formation and clearance, for various strains of prions exhibiting different incubation times.

Mouse syngenic in vitro blood-brain barrier model: a new tool to examine inflammatory events in cerebral endothelium.

Although cerebral endothelium disturbance is commonly observed in central nervous system (CNS) inflammatory pathologies, neither the cause of this phenomenon nor the effective participation of blood-brain barrier (BBB) in such diseases are well established. Observations were mostly made in vivo using mouse models of chronic inflammation. This paper presents a new mouse in vitro model suitable for the study of underlying mechanistic events touching BBB functions during CNS inflammatory disturbances. This model consists of a coculture with both primary cell types isolated from mice. Mouse brain capillary endothelial cell (MBCEC)s coming from brain capillaries are in culture with their in vivo partners and form differentiated monolayers that retain endothelial markers and numerous phenotypic properties of in vivo cerebral endothelium, such as: (1) peripheral distribution of tight junction proteins (occludin, claudin-5, claudin-3 and JAM-1); (2) high trans-endothelium electrical resistance value; (3) attenuated paracellular flux of sucrose and inulin; (4) P-gp expression; (5) no MECA-32 expression. Furthermore, this endothelium expresses cell adhesion molecules described in vivo and shows intracellular cell adhesion molecule-1 and vascular cell adhesion molecule-1 upregulation under lipopolysaccharide-treatment. Therefore, this well-differentiated model using autologous cells appears as a suitable support to reconstitute pathological in vitro BBB model.

Differences in the amyloid-beta-induced inflammatory response in microglia from C57BL/6 and A/J strains of mice.

The microglial inflammatory response to Abeta(1-42) stimulation with or without IFN-gamma priming was investigated in low and high responder strains of mice, A/J and C57BL/6, respectively. A/J microglia showed moderate morphological changes upon stimulation with IFN-gamma alone or with Abeta(1-42). Conversely, C57BL/6 microglia showed major changes in their cellular morphology, which were accompanied by a decrease in NO release and a marked increase in TNF-alpha production. These results indicate that the magnitude of the microglial inflammatory response to Abeta is strongly influenced by genetic factors. Individual differences in the regulation of the microglial response may be a key player in the rate of development of the neuropathology of AD.

Inflammation occurs early during the Abeta deposition process in TgCRND8 mice.

Alzheimer's disease (AD) is characterized by a progressive cognitive decline leading to dementia and involves the deposition of amyloid-beta (Abeta) peptides into senile plaques. Other neuropathological features that accompany progression of the disease include a decrease in synaptic density, neurofibrillary tangles, dystrophic neurites, inflammation, and neuronal cell loss. In this study, we report the early kinetics of brain amyloid deposition and its associated inflammation in an early onset transgenic mouse model of AD (TgCRND8) harboring the human amyloid precursor protein gene with the Indiana and Swedish mutations. Both diffuse and compact plaques were detected as early as 9-10 weeks of age. Abeta-immunoreactive (Abeta-IR) plaques (4G8-positive) appeared first in the neocortex and amygdala, then in the hippocampal formation, and lastly in the thalamus. Compact plaques (ThioS-positive) with an amyloid core were observed as early as diffuse plaques were detected, but in lower numbers. Amyloid deposition increased progressively with age. The formation of plaques was concurrent with the appearance of activated microglial cells and shortly followed by the clustering of activated astrocytes around plaques at 13-14 weeks of age. This TgCRND8 mouse model allows for a rapid, time-dependent study of the relationship between the fibrillogenic process and the inflammatory response during the brain amyloidogenic process.

Mutant PrPSc conformers induced by a synthetic peptide and several prion strains.

Gerstmann-Sträussler-Scheinker (GSS) disease is a dominantly inherited, human prion disease caused by a mutation in the prion protein (PrP) gene. One mutation causing GSS is P102L, denoted P101L in mouse PrP (MoPrP). In a line of transgenic mice denoted Tg2866, the P101L mutation in MoPrP produced neurodegeneration when expressed at high levels. MoPrP(Sc)(P101L) was detected both by the conformation-dependent immunoassay and after protease digestion at 4 degrees C. Transmission of prions from the brains of Tg2866 mice to those of Tg196 mice expressing low levels of MoPrP(P101L) was accompanied by accumulation of protease-resistant MoPrP(Sc)(P101L) that had previously escaped detection due to its low concentration. This conformer exhibited characteristics similar to those found in brain tissue from GSS patients. Earlier, we demonstrated that a synthetic peptide harboring the P101L mutation and folded into a beta-rich conformation initiates GSS in Tg196 mice (29). Here we report that this peptide-induced disease can be serially passaged in Tg196 mice and that the PrP conformers accompanying disease progression are conformationally indistinguishable from MoPrP(Sc)(P101L) found in Tg2866 mice developing spontaneous prion disease. In contrast to GSS prions, the 301V, RML, and 139A prion strains produced large amounts of protease-resistant PrP(Sc) in the brains of Tg196 mice. Our results argue that MoPrP(Sc)(P101L) may exist in at least several different conformations, each of which is biologically active. Such conformations occurred spontaneously in Tg2866 mice expressing high levels of MoPrP(C)(P101L) as well as in Tg196 mice expressing low levels of MoPrP(C)(P101L) that were inoculated with brain extracts from ill Tg2866 mice, with a synthetic peptide with the P101L mutation and folded into a beta-rich structure, or with prions recovered from sheep with scrapie or cattle with bovine spongiform encephalopathy.

Stereoselective interactions of peptide inhibitors with the beta-amyloid peptide.

Residues 16-20 of the beta-amyloid peptide (A beta) function as a self-recognition element during A beta assembly into fibers. Peptides containing this motif retain the ability to interact with A beta and, in some cases, potently inhibit its assembly. Replacing L- with D-amino acids could stabilize such peptides and permit their evaluation as therapeutic agents for Alzheimer's disease. Here we have assessed the effect that such a chiral reversal has on inhibitory potency. D-enantiomers of five peptides, KLVFFA, KKLVFFA, KFVFFA, KIVFFA, and KVVFFA, were unexpectedly more active as inhibitors in an in vitro fibrillogenesis assay. Circular dichroism showed that D-KLVFFA more effectively prevented A beta adopting the beta-sheet secondary structure correlated with fibrillogenesis. Electron microscopy showed that fiber formation was also more strongly inhibited by D-KLVFFA. Heterochiral inhibition was confirmed using D-A beta, on the principle that enantiomeric proteins exhibit reciprocal chiral biochemical interactions. With D-Abeta, L-KLVFFA was the more potent inhibitor, rather than d-KLVFFA. Most significantly, D-peptides were more potent at reducing the toxicity of both A beta1-40 and A beta 1-42 toward neuronal cells in culture. This unforeseen heterochiral stereoselectivity of A beta for D-peptide inhibitors should be considered during future design of peptide-based inhibitors of A beta neurotoxicity and fibrillogenesis.

Abbreviated incubation times for human prions in mice expressing a chimeric mouse-human prion protein transgene.

Transgenic (Tg) mouse lines that express chimeric mouse-human prion protein (PrP), designated MHu2M, are susceptible to prions from patients with sporadic Creutzfeldt-Jakob disease (sCJD). With the aim of decreasing the incubation time to fewer than 200 days, we constructed transgenes in which one or more of the nine human residues in MHu2M were changed to mouse. The construct with murine residues at positions 165 and 167 was expressed in Tg(MHu2M,M165V,E167Q) mice and resulted in shortening the incubation time to approximately 110 days for prions from sCJD patients. The construct with a murine residue at position 96 resulted in lengthening the incubation time to more than 280 days for sCJD prions. When murine residues 96, 165, and 167 were expressed, the abbreviated incubation times for sCJD prions were abolished. Variant CJD prions showed prolonged incubation times between 300 and 700 days in Tg(MHu2M) mice on first passage and incubation times of approximately 350 days in Tg(MHu2M,M165V,E167Q) mice. On second and third passages of variant CJD prions in Tg(MHu2M) mice, multiple strains of prions were detected based on incubation times and the sizes of the protease-resistant, deglycosylated PrP(Sc) fragments. Our discovery of a previously undescribed chimeric transgene with abbreviated incubation times for sCJD prions should facilitate studies on the prion species barrier and human prion diversity.

Dominant-negative inhibition of prion replication in transgenic mice.

Our discovery of dominant-negative inhibition of prion formation in cultured cells provided an explanation for the resistance of some sheep to scrapie and humans to Creutzfeldt-Jakob disease. To determine whether dominant-negative inhibition occurs in vivo, we produced transgenic (Tg) mice expressing prion protein (PrP) with either the Q167R or Q218K mutation alone or in combination with wild-type (wt) PrP. Tg(MoPrP,Q167R)Prnp(0/0) mice expressing mutant PrP at levels equal to non-Tg mice remained healthy for >550 days, indicating that inoculation with prions did not cause disease. Immunoblots of brain homogenates and histologic analysis did not reveal abnormalities. Tg(MoPrP,Q167R)Prnp(+/+) mice expressing both mutant and wt PrP did not exhibit neurologic dysfunction, but their brains revealed low levels of the PrP pathogenic isoform (PrP(Sc)), and sections showed numerous vacuoles and severe astrocytic gliosis at 300 days after inoculation. Both Tg(MoPrP,Q218K)Prnp(0/0) and Tg(MoPrP,Q218K)Prnp(+/+) mice expressing high levels of the transgene product remained healthy for >300 days after inoculation. Neither PrP(Sc) nor neuropathologic changes were found. Our studies demonstrate that although dominant-negative inhibition of wt PrP(Sc) formation occurs, expression of the dominant-negative PrP at the same level as wt PrP does not prevent prion formation completely. However, expression of dominant-negative PrP alone had no deleterious effects on the mice and did not support prion propagation.

Respiratory function in mice lacking or overexpressing the prion protein.

We investigated a possible involvement of the prion protein in ventilatory control in four groups of mice, those deficient for the prion protein (PrP(c)), those overexpressing the prion protein, and two groups of genetically and age-matched controls. Ventilatory patterns of unrestrained mice were measured in a whole-body plethysmograph. Between each genotype and its control, we compared ventilation at rest and the ventilatory response to moderate hypoxia (10-12% O2), hyperoxia and hyperoxic hypercapnia. Mice lacking or overexpressing PrP(c) and their respective controls showed similar ventilatory patterns at rest and similar chemosensory responses when awake and under urethane anesthesia. Our results do not support the view that PrP(c) may play any significant role in basal ventilation or in the chemosensory ventilatory control of adult mice.