Binggong Chang

A novel method for preclinical detection of PrPSc in blood.

In this study, we demonstrate that a moderate amount of protein misfolding cyclic amplification (PMCA) coupled to a novel surround optical fibre immunoassay (SOFIA) detection scheme can be used to detect the disease-associated form of the prion protein (PrP(Sc)) in protease-untreated plasma from preclinical and clinical scrapie sheep, and white-tailed deer with chronic wasting disease, following natural and experimental infection. PrP(Sc), resulting from a conformational change of the normal (cellular) form of prion protein (PrP(C)), is considered central to neuropathogenesis and serves as the only reliable molecular marker for prion disease diagnosis. While the highest levels of PrP(Sc) are present in the central nervous system, the development of a reasonable diagnostic assay requires the use of body fluids that characteristically contain exceedingly low levels of PrP(Sc). PrP(Sc) has been detected in the blood of sick animals by means of PMCA technology. However, repeated cycling over several days, which is necessary for PMCA of blood material, has been reported to result in decreased specificity (false positives). To generate an assay for PrP(Sc) in blood that is both highly sensitive and specific, we have utilized limited serial PMCA (sPMCA) with SOFIA. We did not find any enhancement of sPMCA with the addition of polyadenylic acid nor was it necessary to match the genotypes of the PrP(C) and PrP(Sc) sources for efficient amplification.

Prion Disease Detection, PMCA Kinetics, and IgG in Urine from Sheep Naturally/Experimentally Infected with Scrapie and Deer with Preclinical/Clinical Chronic Wasting Disease

ABSTRACT Prion diseases, also known as transmissible spongiform encephalopathies, are fatal neurodegenerative disorders. Low levels of infectious agent and limited, infrequent success of disease transmissibility and PrPSc detection have been reported with urine from experimentally infected clinical cervids and rodents. We report the detection of prion disease-associated seeding activity (PASA) in urine from naturally and orally infected sheep with clinical scrapie agent and orally infected preclinical and infected white-tailed deer with clinical chronic wasting disease (CWD). This is the first report on PASA detection of PrPSc from the urine of naturally or preclinical prion-diseased ovine or cervids. Detection was achieved by using the surround optical fiber immunoassay (SOFIA) to measure the products of limited serial protein misfolding cyclic amplification (sPMCA). Conversion of PrPC to PrPSc was not influenced by the presence of poly(A) during sPMCA or by the homogeneity of the PrP genotypes between the PrPC source and urine donor animals. Analysis of the sPMCA-SOFIA data resembled a linear, rather than an exponential, course. Compared to uninfected animals, there was a 2- to 4-log increase of proteinase K-sensitive, light chain immunoglobulin G (IgG) fragments in scrapie-infected sheep but not in infected CWD-infected deer. The higher-than-normal range of IgG levels found in the naturally and experimentally infected clinical scrapie-infected sheep were independent of their genotypes. Although analysis of urine samples throughout the course of infection would be necessary to determine the usefulness of altered IgG levels as a disease biomarker, detection of PrPSc from PASA in urine points to its potential value for antemortem diagnosis of prion diseases.

Surround optical fiber immunoassay (SOFIA): an ultra-sensitive assay for prion protein detection.

We describe the development of a new technology (SOFIA) and demonstrate its utility by establishing a sensitive and specific assay for PrP(Sc). SOFIA is a surround optical fiber immunoassay which is comprised of a set of specific monoclonal antibodies and comprehensive capture of high energy fluorescence emission. In its current format, this system is capable of detecting less than 10 attogram (ag) of hamster, sheep and deer recombinant PrP. Approximately 10 ag of PrP(Sc) from 263 K-infected hamster brains can be detected with similar lower limits of PrP(Sc) detection from the brains of scrapie-infected sheep and deer infected with chronic wasting disease. These detection limits allow protease treated and untreated material to be diluted beyond the point where PrP(C), non-specific proteins or other extraneous material may interfere with PrP(Sc) signal detection and/or specificity. This not only eliminates the issue of specificity of PrP(Sc) detection but also increases sensitivity since the possibility of partial PrP(Sc) proteolysis is no longer a concern. SOFIA will likely lead to early antemortem detection of transmissible encephalopathies and is also amenable for use with additional target amplification protocols. SOFIA represents a sensitive means for detecting specific proteins involved in disease pathogenesis and/or diagnosis that extends beyond the scope of the transmissible spongiform encephalopathies.

A novel, ultrasensitive assay for tau: potential for assessing traumatic brain injury in tissues and biofluids.

Traumatic brain injury (TBI) is a cause of death and disability and can lead to tauopathy-related dementia at an early age. Pathologically, TBI results in axonal injury that is coupled to tau hyperphosphorylation, leading to microtubule instability and tau-mediated neurodegeneration. This suggests that the forms of this protein might serve as neuroinjury-related biomarkers for diagnosis of injury severity and prognosis of the neurological damage prior to clinical expression. We initially determined whether we could detect tau in body fluids using a highly sensitive assay. We used a novel immunoassay, enhanced immunoassay using multi-arrayed fiberoptics (EIMAF) either alone or in combination with rolling circle amplification (a-EIMAF) for the detection of total (T) and phosphorylated (P) tau proteins from brains and biofluids (blood, CSF) of rodents following controlled cortical impact (CCI) and human patients post severe TBI (sTBI). This assay technology for tau is the most sensitive to date with a detection limit of approximately 100 ag/mL for either T-tau and P-tau. In the rodent models, T-tau and P-tau levels in brain and blood increased following CCI during the acute phase and remained high during the chronic phase (30 d). In human CSF samples, T-tau and P-tau increased during the sampling period (5-6 d). T-tau and P-tau in human serum rose during the acute phase and decreased during the chronic stage but was still detectable beyond six months post sTBI. Thus, EIMAF has the potential for assessing both the severity of the proximal injury and the prognosis using easily accessible samples.

Re-Assessment of PrPSc Distribution in Sporadic and Variant CJD

Human prion diseases are fatal neurodegenerative disorders associated with an accumulation of PrPSc in the central nervous system (CNS). Of the human prion diseases, sporadic Creutzfeldt-Jakob disease (sCJD), which has no known origin, is the most common form while variant CJD (vCJD) is an acquired human prion disease reported to differ from other human prion diseases in its neurological, neuropathological, and biochemical phenotype. Peripheral tissue involvement in prion disease, as judged by PrPSc accumulation in the tonsil, spleen, and lymph node has been reported in vCJD as well as several animal models of prion diseases. However, this distribution of PrPSc has not been consistently reported for sCJD. We reexamined CNS and non-CNS tissue distribution and levels of PrPSc in both sCJD and vCJD. Using a sensitive immunoassay, termed SOFIA, we also assessed PrPSc levels in human body fluids from sCJD as well as in vCJD-infected humanized transgenic mice (Tg666). Unexpectedly, the levels of PrPSc in non-CNS human tissues (spleens, lymph nodes, tonsils) from both sCJD and vCJD did not differ significantly and, as expected, were several logs lower than in the brain. Using protein misfolding cyclic amplification (PMCA) followed by SOFIA, PrPSc was detected in cerebrospinal fluid (CSF), but not in urine or blood, in sCJD patients. In addition, using PMCA and SOFIA, we demonstrated that blood from vCJD-infected Tg666 mice showing clinical disease contained prion disease-associated seeding activity although the data was not statistically significant likely due to the limited number of samples examined. These studies provide a comparison of PrPSc in sCJD vs. vCJD as well as analysis of body fluids. Further, these studies also provide circumstantial evidence that in human prion diseases, as in the animal prion diseases, a direct comparison and intraspecies correlation cannot be made between the levels of PrPSc and infectivity.

Temporal MRI characterization, neurobiochemical and neurobehavioral changes in a mouse repetitive concussive head injury model.

Single and repeated sports-related mild traumatic brain injury (mTBI), also referred to as concussion, can result in chronic post-concussive syndrome (PCS), neuropsychological and cognitive deficits, or chronic traumatic encephalopathy (CTE). However PCS is often difficult to diagnose using routine clinical, neuroimaging or laboratory evaluations, while CTE currently only can be definitively diagnosed postmortem. We sought to develop an animal model to simulate human repetitive concussive head injury for systematic study. In this study, mice received single or multiple head impacts by a stereotaxic impact device with a custom-made rubber tip-fitted impactor. Dynamic changes in MRI, neurobiochemical markers (Tau hyperphosphorylation and glia activation in brain tissues) and neurobehavioral functions such as anxiety, depression, motor function and cognitive function at various acute/subacute (1-7 day post-injury) and chronic (14-60 days post-injury) time points were examined. To explore the potential biomarkers of rCHI, serum levels of total Tau (T-Tau) and phosphorylated Tau (P-Tau) were also monitored at various time points. Our results show temporal dynamics of MRI consistent with structural perturbation in the acute phase and neurobiochemical changes (P-Tau and GFAP induction) in the subacute and chronic phase as well as development of chronic neurobehavioral changes, which resemble those observed in mTBI patients.

Comparing Plasma Phospho Tau, Total Tau, and Phospho Tau–Total Tau Ratio as Acute and Chronic Traumatic Brain Injury Biomarkers

Importance Annually in the United States, at least 3.5 million people seek medical attention for traumatic brain injury (TBI). The development of therapies for TBI is limited by the absence of diagnostic and prognostic biomarkers. Microtubule-associated protein tau is an axonal phosphoprotein. To date, the presence of the hypophosphorylated tau protein (P-tau) in plasma from patients with acute TBI and chronic TBI has not been investigated. Objective To examine the associations between plasma P-tau and total-tau (T-tau) levels and injury presence, severity, type of pathoanatomic lesion (neuroimaging), and patient outcomes in acute and chronic TBI. Design, Setting, and Participants In the TRACK-TBI Pilot study, plasma was collected at a single time point from 196 patients with acute TBI admitted to 3 level I trauma centers (<24 hours after injury) and 21 patients with TBI admitted to inpatient rehabilitation units (mean [SD], 176.4 [44.5] days after injury). Control samples were purchased from a commercial vendor. The TRACK-TBI Pilot study was conducted from April 1, 2010, to June 30, 2012. Data analysis for the current investigation was performed from August 1, 2015, to March 13, 2017. Main Outcomes and Measures Plasma samples were assayed for P-tau (using an antibody that specifically recognizes phosphothreonine-231) and T-tau using ultra-high sensitivity laser-based immunoassay multi-arrayed fiberoptics conjugated with rolling circle amplification. Results In the 217 patients with TBI, 161 (74.2%) were men; mean (SD) age was 42.5 (18.1) years. The P-tau and T-tau levels and P-tau–T-tau ratio in patients with acute TBI were higher than those in healthy controls. Receiver operating characteristic analysis for the 3 tau indices demonstrated accuracy with area under the curve (AUC) of 1.000, 0.916, and 1.000, respectively, for discriminating mild TBI (Glasgow Coma Scale [GCS] score, 13-15, n = 162) from healthy controls. The P-tau level and P-tau–T-tau ratio were higher in individuals with more severe TBI (GCS, ⩽12 vs 13-15). The P-tau level and P-tau–T-tau ratio outperformed the T-tau level in distinguishing cranial computed tomography–positive from -negative cases (AUC = 0.921, 0.923, and 0.646, respectively). Acute P-tau levels and P-tau–T-tau ratio weakly distinguished patients with TBI who had good outcomes (Glasgow Outcome Scale–Extended GOS-E, 7-8) (AUC = 0.663 and 0.658, respectively) and identified those with poor outcomes (GOS-E, ⩽4 vs >4) (AUC = 0.771 and 0.777, respectively). Plasma samples from patients with chronic TBI also showed elevated P-tau levels and a P-tau–T-tau ratio significantly higher than that of healthy controls, with both P-tau indices strongly discriminating patients with chronic TBI from healthy controls (AUC = 1.000 and 0.963, respectively). Conclusions and Relevance Plasma P-tau levels and P-tau–T-tau ratio outperformed T-tau level as diagnostic and prognostic biomarkers for acute TBI. Compared with T-tau levels alone, P-tau levels and P-tau–T-tau ratios show more robust and sustained elevations among patients with chronic TBI.

PrP antibody binding-induced epitope modulation evokes immunocooperativity

We have characterized the antibody-antigen binding events of the prion protein (PrP) utilizing three new PrP-specific monoclonal antibodies (Mabs). The degree of immunoreactivity was dependent on the denaturation treatment with the combination of heat and SDS resulting in the highest levels of epitope accessibility and antibody binding. Interestingly however, this harsh denaturation treatment was not sufficient to completely and irreversibly abolish protein conformation. The Mabs differed in their PrP epitopes with Mab 08-1/11F12 binding in the region of PrP(93-122), Mab 08-1/8E9 reacting to PrP(155-200) and Mab 08-1/5D6 directed to an undefined conformational epitope. Using normal and infected brains from hamsters, sheep and deer, we demonstrate that the binding of PrP to one Mab triggers PrP epitope unmasking, which enhances the binding of a second Mab. This phenomenon, termed positive immunocooperativity, is specific regarding epitope and the sequence of binding events. Positive immunocooperativity will likely increase immunoassay sensitivity since assay conditions for PrP(Sc) detection does not require protease digestion.

T-Tau and P-Tau in Brain and Blood from Natural and Experimental Prion Diseases

Synaptic abnormalities are prominent in prion disease pathogenesis and are responsible for functional deficits. The microtubule associated protein, Tau, binds to and stabilizes microtubules in axons ensuring axonal transport of synaptic components. Tau phosphorylation reduces its affinity for microtubules leading to their instability and resulting in disrupted axonal transport and synaptic dysfunction. We report on the levels of total Tau (T-Tau) and phosphorylated Tau (P-Tau), measured by highly sensitive laser-based immunoassays, in the central nervous system and biofluids from experimentally transmitted prion disease in mice and natural cases of sporadic Creutzfeldt-Jakob Disease (sCJD) in humans. We found that, in contrast to sCJD where only the levels of T-Tau in brain are increased, both T-Tau and P-Tau are increased in the brains of symptomatic mice experimentally infected with the ME7, 139A and 22L mouse-adapted scrapie strains. The increased levels of T-Tau in sCJD brain, compared to control samples, were also observed in patient plasma. In contrast, there was no detectable increase in T-Tau and P-Tau in plasma from symptomatic experimentally infected mice. Furthermore, our data suggests that in mice showing clinical signs of prion disease the levels and/or ratios of T-Tau and P-Tau are only a useful parameter for differentiating the mouse-adapted scrapie strains that differ in the extent of disease. We conclude that the neuropathogenesis associated with P-Tau and synaptic dysfunction is similar for at least two of the mouse-adapted scrapie strains tested but may differ between sporadic and experimentally transmitted prion diseases.

Influence of Mabs on PrPSc Formation Using In Vitro and Cell-Free Systems

PrPSc is believed to serve as a template for the conversion of PrPC to the abnormal isoform. This process requires contact between the two proteins and implies that there may be critical contact sites that are important for conversion. We hypothesized that antibodies binding to either PrPcor PrPSc would hinder or prevent the formation of the PrPC–PrPSc complex and thus slow down or prevent the conversion process. Two systems were used to analyze the effect of different antibodies on PrPSc formation: (i) neuroblastoma cells persistently infected with the 22L mouse-adapted scrapie stain, and (ii) protein misfolding cyclic amplification (PMCA), which uses PrPSc as a template or seed, and a series of incubations and sonications, to convert PrPC to PrPSc. The two systems yielded similar results, in most cases, and demonstrate that PrP-specific monoclonal antibodies (Mabs) vary in their ability to inhibit the PrPC–PrPSc conversion process. Based on the numerous and varied Mabs analyzed, the inhibitory effect does not appear to be epitope specific, related to PrPC conformation, or to cell membrane localization, but is influenced by the targeted PrP region (amino vs carboxy).