The PINK1/Parkin pathway of mitophagy exerts a protective effect during prion disease.

The PINK1/Parkin pathway of mitophagy has been implicated in the pathogenesis of Parkinson's disease. In prion diseases, a transmissible neurodegenerative disease caused by the misfolded and infectious prion protein (PrPSc), expression of both PINK1 and Parkin are elevated, suggesting that PINK1/Parkin mediated mitophagy may also play a role in prion pathogenesis. Using mice in which expression of either PINK1 (PINK1KO) or Parkin (ParkinKO) has been ablated, we analyzed the potential role of PINK1 and Parkin in prion pathogenesis. Prion infected PINK1KO and ParkinKO mice succumbed to disease more rapidly (153 and 150 days, respectively) than wild-type control C57Bl/6 mice (161 days). Faster incubation times in PINK1KO and ParkinKO mice did not correlate with altered prion pathology in the brain, altered expression of proteins associated with mitochondrial dynamics, or prion-related changes in mitochondrial respiration. However, the expression level of mitochondrial respiration Complex I, a major site for the formation of reactive oxygen species (ROS), was higher in prion infected PINK1KO and ParkinKO mice when compared to prion infected control mice. Our results demonstrate a protective role for PINK1/Parkin mitophagy during prion disease, likely by helping to minimize ROS formation via Complex I, leading to slower prion disease progression.

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes.

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes.

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

Lack of the immune adaptor molecule SARM1 accelerates disease in prion infected mice and is associated with increased mitochondrial respiration and decreased expression of NRF2.

Prion diseases are a group of fatal, transmissible neurodegenerative diseases of mammals. In the brain, axonal loss and neuronal death are prominent in prion infection, but the mechanisms remain poorly understood. Sterile alpha and heat/Armadillo motif 1 (SARM1) is a protein expressed in neurons of the brain that plays a critical role in axonal degeneration. Following damage to axons, it acquires an NADase activity that helps to regulate mitochondrial health by breaking down NAD+, a molecule critical for mitochondrial respiration. SARM1 has been proposed to have a protective effect in prion disease, and we hypothesized that it its role in regulating mitochondrial energetics may be involved. We therefore analyzed mitochondrial respiration in SARM1 knockout mice (SARM1KO) and wild-type mice inoculated either with prions or normal brain homogenate. Pathologically, disease was similar in both strains of mice, suggesting that SARM1 mediated axonal degradation is not the sole mechanism of axonal loss during prion disease. However, mitochondrial respiration was significantly increased and disease incubation time accelerated in prion infected SARM1KO mice when compared to wild-type mice. Increased levels of mitochondrial complexes II and IV and decreased levels of NRF2, a potent regulator of reactive oxygen species, were also apparent in the brains of SARM1KO mice when compared to wild-type mice. Our data suggest that SARM1 slows prion disease progression, likely by regulating mitochondrial respiration, which may help to mitigate oxidative stress via NRF2.

The effects of practicing a novel countermeasure on both the semantic and episodic memory-based complex trial protocols.

This study applied a countermeasure-resistant version of the Concealed Information Test - the Complex Trial Protocol (CTP) - in an information recognition scenario. We replicated and extended the effects of a novel countermeasure developed by Lukács et al., (2016) on both Semantic and Episodic CTPs. We measured participants' response time and P300 event-related potential to rare, crime-relevant probe stimuli, or frequent, non-crime-relevant irrelevant stimuli in two ways: 1) probe vs the average of all irrelevants (PvIall), and 2) probe vs the maximum irrelevant (PvImax). We hypothesized that countermeasure use would only impair information recognition (as indexed by P300) when participants had practiced the countermeasure beforehand. We further hypothesized that recognition of less salient, Episodic information (i.e., jewelry items from a mock crime) would be impaired by countermeasure use more than the recognition of highly salient, Semantic information (i.e., birthdates). Individual diagnostics based on the area under the receiver operating characteristic curve (Semantic CTP: practice n = 22, non-practice n = 23; Episodic CTP: practice n = 19, non-practice n = 18) revealed that the Semantic CTP was affected by the novel countermeasure, but both PvIall and PvImax analyses remained diagnostically useful. The Episodic CTP's performance, however, was reduced to chance, regardless of practice or analysis type. These results are important for both the field of deception detection and the CTP literature. Research on improvements to the Episodic CTP is required.

Transmission characteristics of heterozygous cases of Creutzfeldt-Jakob disease with variable abnormal prion protein allotypes.

In the human prion disease Creutzfeldt-Jakob disease (CJD), different CJD neuropathological subtypes are defined by the presence in normal prion protein (PrPC) of a methionine or valine at residue 129, by the molecular mass of the infectious prion protein PrPSc, by the pattern of PrPSc deposition, and by the distribution of spongiform change in the brain. Heterozygous cases of CJD potentially add another layer of complexity to defining CJD subtypes since PrPSc can have either a methionine (PrPSc-M129) or valine (PrPSc-V129) at residue 129. We have recently demonstrated that the relative amount of PrPSc-M129 versus PrPSc-V129, i.e. the PrPSc allotype ratio, varies between heterozygous CJD cases. In order to determine if differences in PrPSc allotype correlated with different disease phenotypes, we have inoculated 10 cases of heterozygous CJD (7 sporadic and 3 iatrogenic) into two transgenic mouse lines overexpressing PrPC with a methionine at codon 129. In one case, brain-region specific differences in PrPSc allotype appeared to correlate with differences in prion disease transmission and phenotype. In the other 9 cases inoculated, the presence of PrPSc-V129 was associated with plaque formation but differences in PrPSc allotype did not consistently correlate with disease incubation time or neuropathology. Thus, while the PrPSc allotype ratio may contribute to diverse prion phenotypes within a single brain, it does not appear to be a primary determinative factor of disease phenotype.

The enhancing effect of incongruent verbal priming stimuli on the CIT effect with pictorial probes in the P300-based complex trial protocol.

Previous research (Herring et al., 2011) indicated that certain types of incongruent verbal priming enhance responding to the subsequent (primed) stimuli. By priming participants in a P300-based Concealed Information Test (CIT), we examined the possible enhancement effects of priming stimuli in the P300 based Complex Trial Protocol (CTP) for face recognition. Participants were divided into two groups: one group with priming and one control group without. The probe (Pr) and irrelevants (Iall) of the two groups were faces, namely, pictures of the actor Tom Cruise (Pr) and of other unknown faces (Iall). One group had priming before Pr/Iall and one control group had no priming. The priming group was called the non-identical priming (NIP) group in which the verbal priming item (the name, "Bill Smith") is identical with neither Pr nor any of the Ialls. The group without priming is the control group which is called the non-priming group (NP) that simply experiences the basic Complex Trial Protocol. Results were that non-identical priming produced larger CIT effects than the control group, which is consistent with earlier findings. Also, the amplitude of the probe of the NIP group is larger than that of the NP group, while their irrelevants didn't show any significant difference. This means that the incongruent verbal priming did enhance the P300 CIT effect for the probe, which could further improve the accuracy of CTP for the concealed information test.

The effect of retroactive memory interference on the P300-based Complex Trial Protocol (CTP).

In a P300-based concealed information test (CIT), an increased response to a crime-related "probe" item of interest suggests concealed knowledge. Because the CIT's detection ability is based on knowledgeable parties recognizing the key item, weakening the crime memory might decrease probe identifiability and reduce diagnostic power. Research on retroactive memory interference (RI) has shown that acquiring new information after encoding a memory can degrade the original memory, which suggests that RI might pose a threat to CITs. To test this, Gronau et al. (2015) had participants complete a mock-crime, followed by either a control task or a RI manipulation task, intended to impair the crime memory. Both the simple guilty control and RI groups were subdivided into three time delay conditions: 1/3 of participants immediately completed the task and CIT, another 1/3 completed the task and returned a week later for the CIT, and the remaining participants completed both the task and CIT a week later. Results showed that RI reduced memory of crime details and skin conductance responses, while respiration line length was unaffected. Here, we extend Gronau et al. (2015), using the Complex Trial Protocol (CTP) version of the P300-based CIT, to investigate the influence of RI on recognition. The CIT effect was obvious in all six group × time delay subconditions, as evidenced by their significantly larger probe vs. irrelevant amplitudes, high percentage of bootstrapped iterations where probe > irrelevants, bootstrapped mean amplitude differences, and satisfactory hit rates. However, these indices of the CIT effect did not differ based on group or time delay, as was the case for target response error rates and P300 latencies. The only outcome of interest to vary by group or time delay was behavioral response times: both probe and combined irrelevant responses were delayed in the control (simple guilty) group. Thus, the evidence suggests that the RI manipulation used here does not threaten the P300-based CTP's accuracy. Results are considered as they compare to previous work, and limitations and possible explanations for our results are discussed.

Altered distribution, aggregation, and protease resistance of cellular prion protein following intracranial inoculation.

Prion protein (PrPC) is a protease-sensitive and soluble cell surface glycoprotein expressed in almost all mammalian cell types. PrPSc, a protease-resistant and insoluble form of PrPC, is the causative agent of prion diseases, fatal and transmissible neurogenerative diseases of mammals. Prion infection is initiated via either ingestion or inoculation of PrPSc or when host PrPC stochastically refolds into PrPSc. In either instance, the early events that occur during prion infection remain poorly understood. We have used transgenic mice expressing mouse PrPC tagged with a unique antibody epitope to monitor the response of host PrPC to prion inoculation. Following intracranial inoculation of either prion-infected or uninfected brain homogenate, we show that host PrPC can accumulate both intra-axonally and within the myelin membrane of axons suggesting that it may play a role in axonal loss following brain injury. Moreover, in response to the inoculation host PrPC exhibits an increased insolubility and protease resistance similar to that of PrPSc, even in the absence of infectious prions. Thus, our results raise the possibility that damage to the brain may be one trigger by which PrPC stochastically refolds into pathogenic PrPSc leading to productive prion infection.

Financial Incentive Does Not Affect P300 in the Complex Trial Protocol (CTP) Version of the Concealed Information Test (CIT) in Malingering Detection. II. Uninstructed Subjects.

Well-known research showed that the skin conductance response (SCR) of the Autonomic Nervous System (ANS) in the Concealed Information Test (CIT) is usually augmented in participants who are financially and motivationally incentivized to beat the CIT. This is not what happens with Reaction Time (RT)-based CITs, P300 CITs based on the 3-stimulus protocol, nor on the P300-based complex trial protocol for detection of malingering (however these tests differ from forensic CITs). The present report follows up the Rosenfeld et al. (1, 2) study of motivated malingerers instructed how to beat the test, with uninstructed motivated (paid and unpaid) and unmotivated ("simple malingering") subjects, using episodic and semantic memory probes. The Test of Memory Malingering (TOMM) validated behavioral differences among groups. The "CIT effect" (probe-minus-irrelevant P300 differences) did not differ among incentive groups, although as previously, semantic memory-evoked P300s exceeded episodic memory evoked P300s. An effect of specific test-beating instructions was found to enhance the CIT effect for semantic information.

Processing of high-titer prions for mass spectrometry inactivates prion infectivity.

Prions represent a class of universally fatal and transmissible neurodegenerative disorders that affect humans and other mammals. The prion agent contains a pathologically aggregated form of the host prion protein that can transmit infectivity without any bacterial or viral component and is thus difficult to inactivate using disinfection protocols designed for infectious microorganisms. Methods for prion inactivation include treatment with acids, bases, detergents, bleach, prolonged autoclaving and incineration. During these procedures, the sample is often either destroyed or damaged such that further analysis for research purposes is compromised. In this study we show that a straightforward denaturation and in-gel protease digestion protocol used to prepare prion-infected samples for mass spectroscopy leads to the loss of at least 7 logs of prion infectivity, yielding a final product that fails to transmit prion disease in vivo. We further show that the resultant sample remains suitable for mass spectrometry-based protein identifications. Thus, the procedure described can be used to prepare prion-infected samples for mass spectrometry analysis with greatly reduced biosafety concerns.

Moderate financial incentive does not appear to influence the P300 Concealed Information Test (CIT) effect in the Complex Trial Protocol (CTP) version of the CIT in a forensic scenario, while affecting P300 peak latencies and behavior.

Previous research indicated that the skin conductance response (SCR) of the Autonomic Nervous System (ANS) in the Concealed Information Test (CIT) is typically increased in subjects who are financially and otherwise incentivized to defeat the CIT (the paradoxical "motivational impairment" effect). This is not the case for RT-based CITs, nor for P300 tests based on the 3-stimulus protocol or Complex Trial Protocol for detection of cognitive malingering (although these are not the same as forensic CITs). The present report extends earlier studies of malingerers by running five groups of subjects (15-16 per group yielding 78 total) in a mock crime (forensic) scenario: paid (to beat the test) and unpaid, instructed and uninstructed, and simply guilty. There was no evidence that the "CIT effect" (probe-minus-irrelevant P300 differences) differed among groups, although behavioral differences among groups were seen.

Mitochondrial Respiration Is Impaired during Late-Stage Hamster Prion Infection.

Mitochondria are crucial to proper neuronal function and overall brain health. Mitochondrial dysfunction within the brain has been observed in many neurodegenerative diseases, including prion disease. Several markers of decreased mitochondrial activity during prion infection have been reported, yet the bioenergetic respiratory status of mitochondria from prion-infected animals is unknown. Here we show that clinically ill transgenic mice overexpressing hamster prion protein (Tg7) infected with the hamster prion strain 263K suffer from a severe deficit in mitochondrial oxygen consumption in response to the respiratory complex II substrate succinate. Characterization of the mitochondrial proteome of purified brain mitochondria from infected and uninfected Tg7 mice showed significant differences in the relative abundance of key mitochondrial electron transport proteins in 263K-infected animals relative to that in controls. Our results suggest that at clinical stages of prion infection, dysregulation of respiratory chain proteins may lead to impairment of mitochondrial respiration in the brain.IMPORTANCE Mitochondrial dysfunction is present in most major neurodegenerative diseases, and some studies have suggested that mitochondrial processes may be altered during prion disease. Here we show that hamster prion-infected transgenic mice overexpressing the hamster prion protein (Tg7 mice) suffer from mitochondrial respiratory deficits. Tg7 mice infected with the 263K hamster prion strain have little or no signs of mitochondrial dysfunction at the disease midpoint but suffer from a severe deficit in mitochondrial respiration at the clinical phase of disease. A proteomic analysis of the isolated brain mitochondria from clinically affected animals showed that several proteins involved in electron transport, mitochondrial dynamics, and mitochondrial protein synthesis were dysregulated. These results suggest that mitochondrial dysfunction, possibly exacerbated by prion protein overexpression, occurs at late stages during 263K prion disease and that this dysfunction may be the result of dysregulation of mitochondrial proteins.

A cardiovascular prescreening protocol for unmonitored in-office laryngology procedures.

OBJECTIVES: Currently, there are no cardiovascular (CV) preprocedure screening parameters for patients undergoing in-office laryngeal procedures (IOLP). Studies have shown significant changes in CV measures for IOLP. The aim was to develop and evaluate a pre-IOLP CV screening protocol. METHODS: Review of IOLP literature and consultation with an anesthesiologist and cardiologist led to the development of CV parameters and questions related to four metabolic equivalents (METS) of work as a patient-screening tool before IOLP. A separate cohort was screened with only a modified CV protocol. All patients were screened for heart rate (HR) and blood pressure (BP) elevation prior to the procedure. Need for further CV evaluation was characterized as systolic blood pressure BP >160, diastolic BP >100, and/or HR >110 beats/minute. Patients whose BP/HR exceeded these values were referred to their primary care physician (PCP) before re-screening. If parameters were exceeded again at the second screen, then the procedure was done under monitored anesthesia care. RESULTS: The first study phase included 56 patients. The fail rate was 40% largely related to four METS of work. The second study phase included 440 patients. The screen fail rate was 15 patients of 572 (2.6%). Of these, 12 patients of 132 (9.1%) failed the initial screen and were sent to their PCP for further evaluation, and five of 440 (1.4%) patients failed on the day of the procedure. Overall, five of 440 (1.5%) patients would qualify to have their site of service changed for their laryngology procedure from an unmonitored to a monitored setting due to the prescreening criteria. CONCLUSION: Few patients needed further workup based upon the in-office CV parameters set in this study. Patients with CV risk factors were identified by the screening protocol. Having established hemodynamic parameters in place may improve the safety of IOLP with a very low physician burden. LEVEL OF EVIDENCE: 2b Laryngoscope, 127:1845-1849, 2017.

Cellular prion protein is present in mitochondria of healthy mice.

Cellular prion protein (PrPC) is a mammalian glycoprotein which is usually found anchored to the plasma membrane via a glycophosphatidylinositol (GPI) anchor. PrPC misfolds to a pathogenic isoform PrPSc, the causative agent of neurodegenerative prion diseases. The precise function of PrPC remains elusive but may depend upon its cellular localization. Here we show that PrPC is present in brain mitochondria from 6-12 week old wild-type and transgenic mice in the absence of disease. Mitochondrial PrPC was fully processed with mature N-linked glycans and did not require the GPI anchor for localization. Protease treatment of purified mitochondria suggested that mitochondrial PrPC exists as a transmembrane isoform with the C-terminus facing the mitochondrial matrix and the N-terminus facing the intermembrane space. Taken together, our data suggest that PrPC can be found in mitochondria in the absence of disease, old age, mutation, or overexpression and that PrPC may affect mitochondrial function.

Attempts to Suppress Episodic Memories Fail but do Produce Demand: Evidence from the P300-Based Complex Trial Protocol and an Implicit Memory Test.

Instructions to voluntarily suppress memories of a mock crime have been reported to result in decreased P300 amplitude during a P300-based concealed information test (CIT) and reduced autobiographical Implicit Association Test (aIAT) D scores, supporting successful suppression. However, one such study, (Hu et al., Psychological science 26(7):1098-1106, 2015) used the P300-based Complex Trial Protocol with a 50-50 target to nontarget ratio, which could impose much response switching and thereby drain cognitive resources, also resulting in reduced P300. The present study replicated Hu et al. (Psychological science 26(7):1098-1106, 2015) with one major variation-a less intrusive 20-80 target to nontarget ratio that required less response switching. Detection rates were high using both the brainwave-based CIT (90% accuracy) and the aIAT (87% accuracy). However we found no significant differences between the suppression and simple guilty groups on the major indices of concealed information detection, which compare probe and irrelevant P300 responses. While we did find that overall P300 amplitude was reduced in the suppression group, this reduction was not specific to probe responses. Additionally, while there were group differences in aIAT hit rates, there were no differences in aIAT D scores. Taken together, these findings suggest that the previously demonstrated reductions in P300 are a reflection of task demand rather than of effective voluntary memory suppression.

Financial incentive does not affect P300 (in response to certain episodic and semantic probe stimuli) in the Complex Trial Protocol (CTP) version of the Concealed Information Test (CIT) in detection of malingering.

Previous research indicated that the skin conductance response of the autonomic nervous system in the Concealed Information Test (CIT) is typically increased in subjects who are financially and otherwise incentivized to defeat the CIT (the paradoxical "motivational impairment" effect). This is not the case for RT-based CITs, nor P300 tests based on the three-stimulus protocol for detection of cognitive malingering (although these are not the same as CITs). The present report is the first attempt to study the effect of financial motivation on the P300-based Complex Trial Protocol using both episodic and semantic memory probe and irrelevant stimuli. The Test of Memory Malingering (TOMM) was used to validate behavioral differences between the two groups we created by offering one (paid) group but not another (unpaid) group a financial reward for beating our tests. Group behavioral differences on the TOMM did confirm group manipulations. Probe-minus-irrelevant P300 differences did not differ between groups, although as previously, semantic memory-evoked P300s were larger than episodic memory-evoked P300s.

P300 amplitude at Pz and N200/N300 latency at F3 differ between participants simulating suspect versus witness roles in a mock crime.

Based on the autonomic nervous system (ANS) study by klein Selle, Verschuere, Kindt, Meijer, & Ben Shakhar (2016), 15 participants pretended to perform a crime shown on a video, which 16 other participants pretended to witness. Both groups then experienced a P300-based Concealed Information Test (CIT) protocol called the complex trial protocol. Both groups showed CIT effects, with a larger probe than irrelevant P300s at Pz. However, this effect was significantly larger in the suspect group. In contrast, only the suspect group showed delayed N200/N300 responses at F3-putative inhibitory signs. This supports the klein Selle et al. (2016) ANS study in that the suspect versus witness role-playing manipulation differentially affected inhibitory (vs. orienting) aspects of the CIT situation. Our results are also consistent with Ambach, Stark, Peper, & Vaitl (2008), who saw the same autonomic response fractionation as klein Selle et al., but using Furedy's differentiation of deception method (Furedy, Davis, & Gurevich, 1988). These similarities are discussed.

Instructions to suppress semantic memory enhances or has no effect on P300 in a concealed information test (CIT).

The present study investigated the extent to which people can suppress semantic memory as indexed with the P300 ERP and the autobiographical implicit association test (aIAT). In EXP 1, participants (22) were run in a counterbalanced repeated measures study in both simply knowledgeable (SK) and knowledgeable with suppression (SP) conditions. A P300-based, concealed information test ("Complex Trial Protocol"; CTP) with a 50/50 Target/Nontarget (T/NT) ratio was given both with and without instructions to suppress semantic memories. The results showed increased P300s to probe name stimuli, reduced (but still high positive) aIAT d-scores, and increased simple reaction times to all stimuli used in ERP tests in the SP condition. EXP 2 was similar, but with SP and SK in two separate groups, and a 20/80 T/NT ratio. Again, ERP and aIAT results failed to show a suppression effect for semantic memory. The behavioral data suggest some task demand effects under suppression instructions, and that EXP 1 was more demanding than EXP 2.

Bootstrapping the P300 in diagnostic psychophysiology: How many iterations are needed?

In psychophysiological research, bootstrapping procedures are often used to classify individual participants. How many iterations are required for a reliable bootstrap test is not universally agreed upon. To investigate the number of iterations needed for a stable bootstrap estimate, we reanalyzed P300 data collected in concealed information test paradigms. We also distinguished between the bootstrap and permutations approaches. We compared results in several studies using 100 versus 1,000 versus 10,000 iterations in the bootstrap, and we concluded that 100 iterations were adequate as results from all three iteration numbers correlated highly.