Journal Club Presentation 11/01

PHTX 639 Journal Club Presentation 11.01.19

 

siddiquiz

 

10 thoughts on “Journal Club Presentation 11/01

  1. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    GABAA alpha5 receptors are highly expressed in the hippocampus and have a negative effect on memory performance. Alpha5 is upregulated in the hippocampus of AD patients and alpha5 deficient mice demonstrate improved performance in the water maze test for spatial learning. The BZ site on GABA receptors offers a target for developing highly selective allosteric modulators. It was hypothesized that using a GABAA alpha5 NAM would improve cognitive impairment with respect to memory performance without anxiogenic or convulsant effects associated with GABA antagonists. Such a compound, ONO-8590580 (ONO), was identified via receptor binding and FLIPR functional assays and used in this study. In vitro binding data was obtained using HEK293 cells expressing alpha1, 2, 3, and 5 subunits in association with beta-3-gamma-2 subunits. Tetanus induced LTP in rat brain hippocampal slices was used to assess the effect of ONO on LTP. Alpha5 receptor occupancy was measured with a binding assay using ONO and tritium labeled Ro15-4513. Passive avoidance test in a light-dark box was used to measure anxiogenic effects and PTZ administered peripherally was used to measure convulsant effects. Eight-arm radial maze and elevated plus maze tests were employed to assess memory performance. Overall, ONO was found to augment tetanus-LTP, block MK-801 memory deficit in passive avoidance, and block scopolamine memory deficit in radial maze test all without anxiogenic effects in elevated plus maze test or proconvulsant effects in PTZ induced seizure test.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    The in vitro binding data was obtained using HEK293 cells which being human-derived, I feel are an appropriate expression system for the GABA receptors under study. In vitro binding and in vivo receptor occupancy were both measured against Ro15-4513 which is a partial inverse agonist structurally similar to flumazenil (antagonist). Ro15-4513 in this instance is the more appropriate ligand to use because flumazenil does not selectively bind alpha1 subunits. Tetanus induced LTP occurs via inhibition of GABA release and since ONO is a NAM at GABA receptors, it makes sense that tetanus was used to assess the effect of ONO on LTP. With respect to the behavioral components of this study, the passive avoidance test and elevated plus maze test are both commonly used to assess anxiogenic or anxiolytic compounds. The elevated plus maze measures anxiolytic behavior and the passive avoidance test involves a learning/memory component. I think these two behavioral tests are sufficient to demonstrate the anxiolytic and memory enhancing properties of ONO but there are other tests that can be used such as marble-burying. The radial eight arm maze test measures reference memory and working memory and was an appropriate method for assessing memory performance. ONO was shown to block memory deficits induced by MK-801 in passive avoidance test and MK-801 or scopolamine in the eight-arm radial maze test, which all together seems to show convincingly that negative modulation of GABA is beneficial to cognitive performance. The Morris water maze test could also have been used as it assess spatial memory though the eight-arm radial maze test is more suited for repeated measures and better at detecting steady-state reference and working memory. The morris water maze is also a fear-conditioned task and so I don’t it would be as translatable to studying memory deficits in humans such as that experienced by AD patients.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    The introduction does well to lay out the rationale behind the study, citing literature with regards to alzheimer’s disease (disease relvance), GABAA subunit structure features and general effects of GABAA modulation. The discussion corroborates the results obtained with ONO with findings on other GABAA ligands (comparing ONO to other GABAA NAMs). The theory behind the effects of these ligands (GABA NAM and memory performance/anxiolysis) is sound and I think the results do support the overall conclusion.

  2. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    This study investigated the efficacy of orally administered ONO-8590580, a GABAA a5 negative allosteric modulator for treatment of Alzheimer’s disease (AD) in the aging population. They proposed this research question based on the need to identify better efficacious treatments for AD due to the limited efficacy of current approved pharmacological treatments. They hypothesized that the GABAA a5–selective NAM ONO-8590580 should improve cognitive impairment without anxiogenic and proconvulsant effects. They then use several in vivo as well as in vitro methods to analyze the pharmacodynamic properties of the drug and its behavioral profile on cognition, anxiety and convulsant effects. They demonstrated that ONO-8590580 significantly enhanced LTP in rat hippocampal slices and improved cognitive deficits in rats without anxiogenic-like or proconvulsant effects; and that ONO-8590580 could be beneficial in preclinical setting for evaluating cognitive disorders like AD.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    They utilized methods such as: cell culturing for assessing their in vitro parameters like radioligand binding studies as well as current recordings to measure the in vitro efficacy of the drug. Long term potentiation was induced and measured in hippocampal slices using electrophysiological techniques. In vivo receptor occupancy was tested in rat hippocampus using the [3H]-Ro15-4513 binding assay. Lastly, their behavioral tests included the: passive avoidance test to evaluate cognitive function (learning and memory), the eight-arm radial maze test to assess spatial learning and memory, elevated plus maze assay to test for anxiety and PTZ proconvulsant test to assess proconvulsant effects of the drug.
    Fig 2: ONO-8590580 binds to GABAA a1, a2, and a3 subunits but with higher affinity to a5 (lower Ki than the rest).
    Fig 3: shows efficacy of ONO-8590580 on human recombinant GABAA receptors. It dose-dependently inhibited current induced by GABA.
    Fig 4: ONO-8590580 significantly induced LTP in the rat hippocampus after oral administration
    Fig 6: ONO dose-dependently increased the escape latency compared with control in the retention trial in the passive avoidance test.
    Fig 7: High dose of ONO significantly decreased the number of errors and the total latency compared with the control in the eight-arm radial maze test.
    Fig 8: Shows no anxiogenic-like effect in the elevated plus maze test and no proconvulsant effect in the mouse PTZ test.
    I think the methodological approaches used in this study were pertinent to the proposed research question to be answered. They used necessary techniques to validate their findings and support their analysis, making sure to include both in vitro as well as in vivo studies to validate and support their conclusions.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    I think the paper does a great job at explaining the rational and premise of the study. The introduction does a great job at giving adequate background and stating the problem state. They subsequently go on to express what their hypothesis is and to evaluate it using numerous approaches. The organization as well as the flow of the article in terms of the methodology and the progression of the results and discussion were thorough and the conclusions strongly supported. However, some concerns I had with the paper is their use of different rodent types as well as different strains in the behavioral tests they used. In the passive avoidance test and the elevated plus maze test they used Male Sprague Dawley rats but used male Wistar rats for the eight-arm radial maze test and male ICR mice for the PTZ proconvulsant test? A more pressing concern however is their use of male rodents in the study. Since Alzheimer’s disease is known to disproportionately affect more females than males why do they not assess sex differences or state that in their future directions?

  3. The purpose of this study was to study the potential of a GABA a5 negative allosteric modulator, ONO-8590580, in improving long-term potentiation and cognitive deficits as a means of treating dementia and Alzheimer’s disease. It also sought to assess the anxiogenic and proconvulsant effects of this drug due to other GABA subunits potentiating these effects. Receptor occupancy was studied by administering varying doses orally of ONO-8590580 prior to sacrifice and then receptor occupancy was measured post-sacrifice. ONO-8950580 was found to have a receptor occupancy of 68% at a dose of 10mg/kg. Transgenic rats were used to study behavioral effects of the NAM on AD. Cell cultures were done on cells expressing each of the GABA receptor subunits and then both in vitro radioligand binding studies and in vitro efficacy studies were performed. Long-term potentiation in rat brain slices was measured using a multielectrode array system and receptor occupancy was also studied in the brain slices. To study drug-dependent behavior, the rats performed in a passive avoidance test and an eight-arm radial maze test. They also performed an elevated plus maze to assess anxiolytic effects. Lastly mice were administered vehicle, ONO-8590580, or FG-7142 followed by PTZ (a convulsant) to test for potential proconvulsant properties.
    For the in vitro experiments, it was concluded that ONO-8590580 has a high affinity for alpha-5 subunits and low affinity for the other subunits. They also showed that ONO-8590580 concentration-dependently inhibited the current induced by the EC20 concentration of GABA in cells with alpha-5 subunit receptors and the drug had little to no effect on the other receptors. With regard to LTP, ONO-8590580 was shown to significantly enhance the slope of the bursts and the burst did not return to baseline after 1 hour like they did in the control. The receptor occupancy test showed competition with another GABA alpha-5 binder and the percent occupancy was dose dependent. In the passive avoidance test, ONO-8590580 significantly increased the escape latency compared with control in the retention trial. In the eight-arm maze test, ONO-8590580 reduced both latency and the number of errors made while, Donepezil, used as a positive control, only reduced latency but had no significant effect on errors. The elevated plus maze showed to effects of ONO-8590580 on anxiety compared to controls and the positive control FG-7142. Lastly, ONO-8590580 was found to have no pro-convulsant effects compared to controls and FG-7142. Overall, the methodology appears strong and the statistical analysis is sound and supports their conclusions. One criticism is that I didn’t see much as far as dose-time curves is concerned. They gave ONO-8590580 orally and waited about an hour before tests without any knowledge of whether the peak effect might be seen sooner or later. I did appreciate that in some of the experiments they not only used a control but also used a positive control to assess ONO-8590580’s performance. Another critique I have is why they did not test ONO-8590580 in reversing the memory-blocking effects of Etomidate? L655-708, another NAM, was shown to reverse these effects but they didn’t test ONO-8590580 specifically. I am also curious if more retention test trials would’ve proved beneficial in the passive avoidance test. They only had one retention trial after the acquisition trial. It might have been beneficial to see a few more retention trial carried out after repeated dose of ONO-8590580.
    Overall, the introduction was concise but provided adequate background information to understand the purpose, significance, and rationale for the study. They also ensured to provide proper references to previous related studies especially regarding those that proved the transgenic mice as an appropriate model for AD. An interesting future study would be to try and understand how large of a role NMDA dysfunction plays in dementia and in turn, how much of an effect ONO-8590580 can potentially have given that there are various other factors at play like beta-amyloid plaque build-up. In other words, what percent of regression back to baseline cognitive performance can we see on average in rats given ONO-8590580? This wasn’t mentioned in the paper.

  4. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    – Hypothesis: a GABAA α5-selective NAM should improve cognitive impairment without anxiogenic and proconvulsant effects
    – ONO-8590580 binds to GABAA α1, α2, α3, and α5, induced long term potentiation after a θ-burst in the CA1, increased the escape latency of rodents when compared with control in retention trials, and decreased the number of errors and latency in the eight-arm radial maze test
    – Donepezil decreased total latency of the eight-arm radial maze test, but did not decrease the number of errors
    – FG-7142 decreased time spent in open arms of an elevated plus maze and decreased the dose required in induce colonic seizure, while ONO did not affect time spent in open arms of the plus maze, nor had any effect on the threshold for PTZ-induced colonic seizures

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    – Even with so many experiments being conducted, the results were extremely short and brief, while the methods were much more elaborate
    – It felt like there was so much done, but very little data actually collected
    – I appreciated that they included a control, but I am still unsure why they included scopolamine and PTZ
    – These rodents were only a few months old at most; given that Alzheimer’s disease is a disease that affects the elderly, wouldn’t it have been useful to instead use older subjects?
    – While the statistical methods were mentioned, the only assignment of each test to a method was included in the figures; I would have appreciated if at the end of the statistical analysis section the method was included next to the test (eg: passive avoidance test (normal group: Wilcoxon rank-sum test; control group: Steel test))
    – the results didn’t include any statistical measures, only that certain results were “significant”, without showing any of their values

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    – The authors did bring their results back to Alzheimer’s disease, but spent most of the discussion focusing on that rather than focusing more on ONO-8590580 and GABAA α5 and how their results relate to the literature as a whole; it feels a bit like they were honing in too much on a single subject
    – For how much they speak on Alzheimer’s disease in the discussion, the authors spend very little time on the current findings on the disease in the introduction
    – They do spend plenty of time devoted to why they chose to focus on a GABAA α5 modulator and focus on the current literature on GABAA and the α subunits

  5. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    – The hypothesis is that GABA alpha 5 NAMs will enhance hippocampal memory function without side effects such as anxiety or convulsion risk.
    – Results: Figure 1 depicts the molecular structure of the novel GABA a5 NAM (ONO-8590580). Figure 2 shows the binding affinity for this compound at the a5 target as well as GABA a1-3. These results show that ONO is binding with greater selectivity for a5 but still targets the other three subtypes as well. Figure 3 shows the results of a patch-clamp study used to measure whether ONO would inhibit the current produced by GABA in HEK cells. These results show that the inhibition of the current by this NAM was a decrease of approximately 44%. Next, figure 4 shows that long-term potentiation was induced on rat hippocampus slices and the effects of ONO were tested. These results show that ONO significantly induced LTP and the duration to return to baseline was longer than the controls where fEPSP slopes were enhanced. Figure 5 shows the receptor occupancy of ONO in rat hippocampus collected after in vivo administration. These results show the receptor occupancy is dose-dependent and the drug has a ED50 value of approximately 2 mg/kg. Next, the authors conducted a passive avoidance test using MK-801 as a model for cognitive dysfunction related to dementia. ONO significantly increased escape latency in this model in a dose-dependent manner. In figure 7 the authors use a multi-drug combo to model cholinergic degeneration and hypofunction of glutamatergic transmission and tested rats in the radial maze test. ONO significantly decreased the number of errors and total latency in this model. Lastly, in figure 8A the authors show that ONO has no effect on rats in the elevated plus maze assay. They also administered PTZ to induce clonic seizures and showed that ONO had no effect on the threshold of PTZ required to induce convulsive activity.
    – In conclusion the authors results demonstrate that ONO acts as a functionally selective GABA a5 NAM and enhances LTP in rat hippocampus. Also, ONO improved cognitive deficits in behavioral models without producing anxiety-like or proconvulsant effects making it a candidate for further investigation to treat cognitive disorders.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
    – The methods are very thorough, and the authors provide a lot of detail in each methods section for all their experimental components. It is nice that they went into this much detail as it makes it more likely another group can replicate their study if desired.
    – The results section is also clear and easy to follow. It would have been nice if they added more statistical details (such as F-values and degrees of freedom), however this may be something not required or allowed by the journal. The indications of significant effects on all their figures are clear with the P-value and type of analysis used being described clearly.
    – The authors also did a good job with having the proper positive controls for all their experiments and therefore their results are substantiated by the data provided.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.
    – The introduction gives thorough rational for the premise of their study by giving an overview of the current pharmacological treatments for AD and the role of GABA in disease models.
    – The discussion is also thorough, and the authors give background on other relevant literature on how it supports their hypothesis and how the ONO compound is different than other previously described a5 selective compounds in the literature. Their conclusions also are in line with what we know about the pathology of AD and how their results provide strong rational why this novel GABA a5 NAM is a potential candidate for therapeutic use in AD.

    1. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
      H: The compound, ONO-8590580, acts as an efficacious GABAA α5 negative allosteric modulator for learning and memory related effects of GABA.
      R: Using a in-vitro binding assay, the efficacy of ONO-590580 was evaluated for multiple subunit types and showed highest efficacy for the α5 subunit. The authors further displayed that ONO-590580 was not as effective in α2,3 and 4 as for α5. LTP was then evaluated in rat hippocampal slices following injection of ONO-590580 and showed that the NAM induced LTP after a θ-burst in CA1. An in-vivo receptor occupancy assay showed that ONO-590580 had an ED50 of 1.9mg/kg. The cognitive impairment paradigm was used with MK801 and showed that ONO-590580 did not affect the acquisition but it did have an effect on the escape latency. In the 8-arm maze test, ONO-590580 decreased the number of mistakes and the latency of the task. Finally, ONO-590580 showed no effect of elevated plus maze or PTZ-induced clonic seizure.
      C: ONO-590580 negatively modulates the effect of GABAA α5 subunit activation and its effects of learning and memory but not anxiolytic effects.
      2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
      M: In the first experimental binding assay, the authors reported that different binding assays were used for α5 versus the other subunits tested; this left me skeptical of their efficacy results. They then explained that a control was used in the LTP assay but didn’t explain what the control was. The in-vivo receptor occupancy assay provided an ED50 for ONO-590580 but did not compare this is data to any other data to provide significance to the data shown. The authors did use proper drug controls for their in-vivo assays. Futhermore, they described α5 as being the most efficacious but the data for α3 seemed fairly similar in Fig. 2. Although the use of in-vitro methods was substantial; an in-vivo learning and memory assay such as the Morris Water Maze or objection recognition would have further strengthened the paper and provided a better insight to whether the findings are clinically translatable and similar to previous findings that were mentioned in their introduction.
      R: Data analysis, though some was provided, was not adequate for all the data shown. More statistics would have strengthened their results.

      3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.
      I: The authors did a great job outlining the overall need for novel memory related therapeutics, more specifically for Alzheimer’s disease. They then provided adequate rationale for exploring the GABAergic system in particular, such as genetic and pharmacological in-vitro and in-vivo manipulations that showed effects on learning and memory. However, towards the end of the introduction the authors were explaining the different subunit manipulations that had been previously studied and continued by saying that this was their rationale for investigating the GABAA α5 subunit though they mentioned nothing specifically about that subunit prior.
      C/D: Similar to prior results in the field, they concluded that their drug was a NAM for the GABAA α5 subunit and that it modulated the effects of GABA on learning and memory. They also concluded it had no effect on anxiety-like behaviors. They included an extensive amount of in-vitro and in-vivo data that supported this conclusion. One thing I think they missed was including a current AD drug as a comparison for their in-vivo work. I also would have liked to see this drug also tested in a paradigm such as drug discrimination compared to a benzodiazepine or a currently used AD drug. The evaluation of sex on these results would also be interesting to see.

  6. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    Hypothesis: The synthetic compound ONO-8590580 is a negative allosteric modulator (NAM) for the GABAa α5 subunit that can improve cognitive function via action on the hippocampus.
    Results: It was found that, in rodents, ONO-8590580 improved LTP in hippocampal slices and had a beneficial effect on cognition without producing convulsions or anxiogenesis. The authors therefore concluded that the compound could be worth investigating in preclinical trials for treatment of Alzheimer’s.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
    Methods: Techniques such as radioligand binding assays and electrophysiological assessments of long-term potentiation, as well as behavioral assays such as radial maze tests and elevated plus maze, were pertinent to the research question. In addition, experiments like the proconvulsant test were necessary to determine whether the test compound increased susceptibility to seizures, which would preclude the possibility of conduction additional preclinical studies with ONO. Controls appear to have been used. Data analysis (Student’s t test- differences between two groups; Wilcoxon rank-sum test, Dunnett’s) were in keeping with the experimental design of the studies.
    However, I was a little confused about why the authors didn’t use the same strain of animal throughout for the sake of consistency. For instance, why use Wistar rats for the eight-arm radial maze test when other behavioral tests, such as elevated plus maze, were run with Sprague-Dawley rats? In addition, it might have been advisable to test females in addition to males. It doesn’t look like the possibility of sex differences was addressed, which could potentially be an issue of interest.
    The data given in the article generally support the author’s purported results.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.
    Introduction: Authors relate their work to the clinic by pointing out the need for better treatment options for Alzheimer’s and then segueing into changes in expression of GABAa α5 in the disorder. Authors explain why agents that inhibit GABAa α5 would be expected to ameliorate the condition, thus providing a rationale for the present study. The authors also demonstrate that they consulted the literature and conducted the necessary screens to decide on the type of compound that would be best to test.
    Discussion: Contextualizes results by explaining how they are consistent with previous studies on GABAa α5. Authors explain how their rodent models imitate the cognitive and neurological deficits in Alzheimer’s. The authors also touch on possible future directions, such as studying whether ONO could be a viable treatment for neurodegenerative disorders other than Alzeheimer’s. Overall, the results presented support the authors’ conclusion.

  7. Donald Jessup
    1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.

    Hypothesis: The GABAa-alpha5 NAM ONO-8590580, will improve hippocampal learning and memory in a rodent model without proconvulsant or anxiogenic effects

    Results/Methods: 1.) In Vitro Binding of ONO-8590580 to GABAA Receptors, was demonstrated in by radiolabeled ligand binding experiments and showed ONO8590580 binds with high affinity to GABAA a5 2.) The efficacy of ONO-8590580 on the GABAA receptor subtypes was assessed using HEK293 cell current recordings and showed that ONO-8590580 concentration-dependently inhibited the current induced by the EC20 concentration of GABA in human GABAA a5b3g2 expressing cells by a maximum calculated inhibition of -44.4%. 3.) Changes in LTP in rat hippocampal slices were observed following theta-burst stimulation in the presence or absence of ONO-8590580. Results indicated that ONO-8590580 was able to enhance LTP and prevent return to baseline 1-hour after stimulation. 4.) In Vivo receptor occupancy in rat hippocampus was also shown by radioligand binding similar to the first experiments but was delivered through an oral route. Results indicated that the ratio of GABAA a5 occupancy values produced by ONO-8590580 at 1, 3, 10, and 20 mg/kg were 44%, 53%, 71%, and 89%, respectively and the ED50 value was established as 1.9mg/kg 5.) The study also evaluated the effect of ONO-8590580 on MK-801–induced cognitive deficit in the passive avoidance test and found that ONO-8590580 (3, 10, and 20 mg/kg) significantly increased the escape latency compared with control, but only in the retention (vs acquisition) trial. 6.) The effect of ONO-8590580 on the
    MK-801/scopolamine-induced cognitive deficit was evaluated in an eight-arm radial maze test. The results of which demonstrated that ONO-8590580 (20 mg/kg, p.o.) significantly decreased the number of errors
    and the total latency compared with the control Vs the positive control Donepezil, which decreased latencies in the trial but not errors. 7.) In the rat elevated plus maze test, FG-7142 significantly decreased the time spent on the open arms, whereas ONO-8590580 had no effect compared with vehicle-treated animals. Additionally, in the mouse PTZ test, FG-7142 decreased the dose of PTZ required to induce clonic seizure, but ONO-8590580 had no effect on PTZ-induced clonic seizure

    Conclusion: ONO-8590580, a functionally selective GABAa a5 NAM, significantly enhanced
    LTP in rat hippocampal slices and improved cognitive deficits in rats without anxiogenic-like or proconvulsant effects.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    The study encompassed many different experimental methodologies to investigate and characterize the molecular, physiological, and behavioral elements behind the ONO-8590580 NAM. The approach presented in the study would seem to suggest that they started with a very simple pharmacological question in trying to just establish the efficacy of the compound for GABAa before building up through the relevant pharmacology, physiology, and ultimately behavior. I felt that kept the authors experimental methodology constrained to answering the central hypothesis without chasing down the various secondary questions this studies data no doubt produced. Overall I felt that the authors analysis of their data was well controlled and didn’t deviate from the data that was being presented and while I would have liked to see some morphological data on the hippocampal pyramidal cells, such as dendritic density, spine maturity etc., I admit it would be mostly supplemental as the study seems well supported already.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    The introduction was very thorough with regards to establishing the relevant background literature particularly in laying out the clinical value in exploring this particularly selective NAM. Additionally, they did a fairly good job of guiding the reader through their rational for the study. By contrast, while I did thoroughly appreciate that the conclusion establishes the impact this publication has for this field as well as what knowledge gap specifically was being serviced with the data, I also felt it simply dragged on and while it wasn’t necessarily an issue, I would have much preferred a more detailed results section (which was succinct to say the least). Overall the authors conclusions were supported effectively by the reported data and the diverse set of experimental methods

  8. 1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    Kawaharada et al. hypothesized that ONO-8590580, a novel negative allosteric modulator (NAM) of the GABAA receptor subunit α5 would improve cognitive deficits without producing anxiogenic and proconvulsant like effects. The major result to support this is their finding that different doses of ONO-8590580 increased escape latency in the rat passive avoidance test as compared to control see fig. 6/7. Another major result they showed was that in two different assays, elevated plus maze test to test for anxiety and PTZ administered solution test to test for convulsion-like activity, ONO-8590580 had no significant effect on either pro-assay see figure 8. Overall, Kawaharada et al concluded that ONO-8590580 a selective GABAA receptor subunit α5 NAM enhanced long term potentiation (LTP) in hippocampus slices and improved cognitive deficits without producing anxiolytic or proconvulsive effects in a rodent model.
    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.
    Method/result 1: Kawaharada et al showed an in vitro assay of ONO-8590580 inhibiting [3H]-Ro15-4513 binding affinities (Ki) values to the GABAA receptor and its different subunits (α1, 2, 3, 5) using the scintillation proximity assay (SPA) on HEK293 cells. Fig. 2 shows their results with their special interest on the α5 subunit. The affinity of α5 was Ki = 7.9 nM and affinities for α1 Ki =140 nM, α2 Ki =32 nM, α3 are Ki =24 nM. They state in their methods that they utilized controls to calculate inhibition such as DMSO and flumazenil although that data is not shown, I’m not sure why? Although they show the affinities differently, they do not show direct comparison of them or a control, so I would’ve liked to see that being show.
    Methods/result 2: Kawaharada et al showed the relative efficacy of ONO-8590580 on GABAA receptors using patch-clam recordings performed using PatchXpress. They used HEK293 cells that expressed the above subunits described. The current responses were performed in the presence of DMSO as their control/baseline? to get an EC20 maximal concentration value to then test the different GABAA subunit/concentrations. Their main result lies in fig. 3 which shows ONO-8590580 inhibited only the current induced GABA α5 and they state that ONO-8590580 was not effective at inhibiting the other subunits. I am wondering why they used different concentrations to see the inhibition of different subunits with the lowest at α5 = 0.34 nM, is this telling us that the subunit is already super sensitive to this compound? Again, they could’ve showed control curves so that they could’ve ran statistics to better prove their conclusion.
    Method/result 3: Kawaharada et al showed the effect of ONO-8590580 to induce LTP in hippocampal slices in rat. They did this by the θ-burst protocol. Their conclusion based off of fig. 4 is that ONO-8590580 was able to significantly reduce LTP in the CA1. A time-dependent increase in fEPSP is seen even after the θ-burst although for the slope data shown they did not show statistical analysis done to compare to see if the effect is there, but they did run statistical analysis on the %baseline data shown.
    The lack of good controls used in some of the assays makes me question the viability/replicability of the data. Also, the fact that not much statistical conclusions are made. Also, since they describe location specificity of this ligand I was expecting to see some genetic manipulation work done. They also state they are interested in this to target Alzheimer’s Disease and go on to do elevated plus maze work and passive avoidance test so I am just wondering how those behavioral assays translate to targeting Alzheimer’s Disease. I can see why the passive avoidance test is done (it’s testing cognition but the EPM tests for anxiety).
    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.
    Their introduction describes Alzheimer’s Disease, its issues and main pharmacological barriers that exist. Kawaharada et al have a good amount of supporting citations from the literature that support the idea behind their rationale. I like that they talked about the binding site and the different subunits. The discussion does a better job at diving deeper into their findings than their results section did. In the end they bring it back to the importance of this in the clinical setting and they also mention future work that needs to be done to further support the idea of the GABAA receptor subunit α5 NAM.

  9. Student Aminatta Tejan-Kamara Instructor Dr. Shelton Date 10/27/19
    Please answer the following. Do not exceed 1 page for your answer; you may use outline/bullet points.
    1. Briefly summarize the hypothesis, major results with methods as needed, and overall conclusion.
    • Hypothesis- Authors hypothesized that ONO-8590580 (ONO) a GABAA α5-selective negative allosteric modulator i.e. an inhibitor of GABAA would improve cognitive deficits without the anxiogenic and proconvulsant effects.
    • Major results- ONO binds GABAA α5 with high affinity demonstrating ONO’s binding selectivity. ONO contributes to improving cognitive deficits induced by MK-801 (NMDA antagonist) and Scopolamine (muscarinic antagonist), methods used was the eight-arm radial maze test that served to assess how ONO would effect the number of errors the rats made after being administered the respective antagonists. It was found that ONO “significantly decreased the number of errors and the total latency compared with the control (donepezil/positive control), ONO also noted to effectively enhance LTP because of inhibition of GABAA α5 in the preclinical models.
    • Overall conclusion: ONO-8590580, a “novel GABAA α5 NAM,” improves cognitive impairment without the anxiogenic or proconvulsant side effects.

    2. Critically review the Methods and Results, including appropriateness of methods, use of controls, data analysis, and whether results are substantiated by the data provided.

    As stated previously, the overall conclusion states that ONO-8590580, a GABAA α5 selective NAM improves cognitive impairment without anxiogenic and proconvulsant side effects.
    • Experimenters opted to use SPA binding assay to assess binding affinity, which is appropriate given the assay’s flexibility (with respect to concentrations of components involved) and lack of separation step which leaves less room for error and subsequently a higher throughput (compared to more traditional binding assays), given the reported Z factor values in the binding assay for GABAA α1, GABAA α2, GABAA α3, and GABAA α5, GABAA α5 had the highest Z factor value of 0.54 implying the assay is robust; the results illustrated in Figure 2 of the paper further confirm the selectivity for GABAA α5. This method supports the selectivity of ONO.
    • Experiments implement a PTZ test in mouse and Elevated Plus maze test in rats, to assess epileptic activity and anxiety respectively, ONO-8590580 was shown to have no effect on threshold for PTZ induced seizure and no effect on anxiety. In terms of appropriateness of methods, the elevated maze test is considered an adequate behavioral assay to quantify anxiety-like behavior, additionally the PTZ test allows for proper assessment of potential seizure activity/lack thereof to be assessed with the PTZ induced seizure paradigm. The findings from these methods support the conclusion that ONO does not promote anxiogenic/proconvulsant behavior, however the experimenters decision to use mice for one behavioral assay and rats for another is unclear, it may be preferable to allow the cohort of rodents across their series of studies to be consistent. The rationale behind the different rodents is not explicitly understood and could be arguably weakening the strength of their conclusions due to the concern of biased methodology to yield desired results.
    Overall, given the various methods and the results from the noted figures, the conclusion is supported.

    3. Critically review the Introduction/Discussion/Conclusions. Points for critique include rationale for scientific premise, relationship of the findings to literature in the field, whether results support the overall conclusion.

    Authors offer thoughtful introduction that guided the direction of their studies, the significance of the aging societal population warrants the need for these presented studies, the potential contribution these studies would provide the field is nearly unequivocal. The rationale for the scientific premise is understood, without dispute. The results the experiments present, with emphasis to the ONO-8590580 selective GABAA α5 binding (Figure 2) and the potential cognitive enhancement in AD patients (figure 7) demonstrate that the conclusion is supported.

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