Manic Mice: A Model Of Bipolar Disorder


 

Introduction

Bipolar disorder falls into a category of psychopathological conditions known as affective disorders. These conditions manifest themselves with  inappropriate and disproportionate exaggerations in mood state. Bipolar disorder itself consists of the swinging between major depressive and manic episodes. The manic episodes of bipolar disorder are the distinguishing factor that enable diagnosis. In many individuals however, manic episodes occur infrequently making diagnosis challenging. As a result, many patients are first diagnosed with depression and this leads to problems with treatment.

Circadian Rhythms and Bipolar Disorder

Patients with bipolar disorder suffer from disturbances in their circadian rhythms. In depressive episodes insomnia or hypersomnia are common factors, and in manic episodes there is a decreased “need for sleep”. The rhythm of sleep is also altered, in particular REM and slow wave stages of sleep. The normal rhythms of activity, hormonal secretions and appetite are also affected. Indeed the normalization of the sleep/wake cycle and exogenous zeitgebers plays an important part in treating many individuals suffering with the illness (Plante 2008). Recently SNPs (signal nucleotide polymorphisms) have been identified in genes that encode crucial components of our endogenous pacemakers associated with mood disorders (Benedetti 2008).  In addition to this, lithium and valproate, the most common mood stabilizing treatments both have known targets and effects in circadian rhythm biology. Lithium for example has been shown in many organisms to lengthen the circadian day and valproate to alter the expression of several circadian proteins.

Animal model of mania

CLOCK is a protein that is thought to play an important part in the molecular feedback mechanisms that make up our endogenous pacemaker. Described in this publication is a mouse model in which CLOCK was mutated to inhibit its interaction with BMAL1, its transcription regulatory complex partner. In the original paper several tests were employed to demonstrate that the mice exhibited different diagnostic criteria of mania (Roybal 2007).  One symptom of mania in humans is feelings of extreme euphoria. In mice this was examined by assessing helplessness in forced swim experiments. Positively the results indicated less helplessness in the mutants. Anxiety tests were also utilized and results were indicative of this second symptom of mania. The mutant mice also show a greater preference for rewarding stimuli, similar to the manic states in bipolar. This was examined by assessing the level of intracranial self-stimulation to the medial forebrain bundle. Finally disrupted circadian rhythms, hyperactivity and decreased sleep were also observed in the mice. Perhaps the most intriguing part of this model is the lithium sensitivity. Treatment with lithium was shown to ameliorate the mood-related effects of the mutation to wild type levels (in the helplessness and anxiety tests).

Evaluation (My Thoughts)

One major weakness in this model is the identification of CLOCK null mutant mice that exhibit normal circadian functioning (Jason 2006). This suggests that the CLOCK 19 mice dysfunction may result from new functions of the mutant CLOCK or a desynchronization of function or parallel function of CLOCK, with the mutations failing to completely inhibit CLOCKs function.

There is no evidence that suggests that mutations in the circadian system are required for the development of bipolar disorder in humans and therefore it could be argued that this model lacks any relevance to the human condition.

Bipolar disorder is more complex that the behavior displayed by these mice, which exhibit no cycling between mood states. These mice are at best a model of constant mania but their utility in predicting therapeutic efficacy remains unclear. At worst this model has no biological relevance for bipolar disorder. The observed influence of lithium in alleviating the effects of the mutation may be attributed to its intrinsic polypharmacology. In addition, in this study lithium was not able to restore all behavioral phenomena to wild type levels.

Despite these reservations it must be acknowledged that circadian rhythm dysfunction is an important factor in bipolar disorder and models that can be used in the development of drugs to “normalize” this are useful.

 

Blog written by: James Noble

References 

  1.  Benedetti F, Serretti A, Colombo C, Barbini B, Lorenzi C, Campori E, 
Smeraldi E Am J Med Genet B Neuropsychiatr Genet, 2003123:23–26. 

  2. David T. Plante, John W. Winkelman, Sleep Disturbance in Bipolar Disorder: Therapeutic Implications Am J Psychiatry 2008; 165:830–843
  3.  Jason P. DeBruyne, Elizabeth Noton, Christopher M. Lambert, Elizabeth S. Maywood, David R. Weaver, Steven M. Reppert, A Clock Shock: Mouse CLOCK Is Not Required for Circadian Oscillator Function, Neuron, 2006, 50 (3), 465-477
  4. Kole Roybal, David Theobold, Ami Graham, Jennifer A. DiNieri, Scott J. Russo, Vaishnav Krishnan, Sumana Chakravarty, Joseph Peevey, Nathan Oehrlein, Shari Birnbaum, Martha H. Vitaterna, Paul Orsulak, Joseph S. Takahashi, Eric J. Nestler, William A. Carlezon, Jr, and Colleen A. McClung,Mania-like behavior induced by disruption of CLOCK, PNAS 2007, 104 (15), 6406-6411

 

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Insight into the Pharmacokinetics of CNS Drugs: the Species-Independent Brain Tissue Binding Phenomenon


Neuroscience is one of the field of expertise of the Sussex Drug Discovery Centre, and as a medicinal chemist novice to this area of drug discovery, I decided to extend my knowledge of drug delivery to the brain and pharmacokinetics for central nervous system (CNS) pharmacological agents. A significant amount of extremely interesting literature covers neuropharmacokinetics, and I rapidly came across a number of essential principals such as the importance of high blood brain barrier (BBB) permeability or the complexity of predicting pharmacokinetics/pharmacodynamics (PK/PD) relationships for CNS drugs. Among these dozens of publications, one scientific article1 from a group of DMPK scientists at Pfizer particularly attracted my attention, since it was reporting that brain tissue binding is species-independent, something that I would not have expected to be true. Indeed, I was familiar with the well-known phenomenon of interspecies differences in plasma protein binding and would have expected the same for brain tissue binding. As a result, I got extremely curious in finding out why the story was different with brain tissues and how the team at Pfizer conducted their study to draw this conclusion.

Before getting into the details of this study, let’s first set the scene to refresh one’s knowledge of pharmacokinetics and make sure that we are all on the same page. One of the most important, well accepted and widely applied concept in drug discovery and development, used to establish PK/PD models among other things, is the free drug hypothesis. This concept stipulates that in vivo efficacy is solely determined by the free (unbound) drug concentration at the site of action rather than total (bound and unbound) drug concentration, and that only the free drug is able to distribute from the systemic circulation across membranes to tissues. This notion which is key to understand the action of all drugs becomes even more essential in the context of CNS pharmacological agents. Indeed, the CNS is an extravascular compartment separated from the systemic circulation by the BBB and only free drug from the plasma can cross this biological barrier. The capacity of a drug to diffuse through the BBB is referred to as CNS permeability or CNS penetration, and is controlled and influenced by a number of physicochemical properties. Once in the CNS, the drug undergoes binding to the brain tissues and only a portion of the free drug in the plasma is free in the CNS to exert a pharmacological effect.

A number of techniques have been developed and used to determine the unbound drug concentration in the brain, such as in vivo microdialysis, cerebrospinal fluid sampling, and a combined measurement of brain tissue binding and brain distribution from plasma and brain concentration time courses. The latest approach is routinely used in pharmaceutical research since it requires less compound-specific optimisation and provides a direct measurement in the compartment of interest. In addition, it is a less challenging, more reproducible and higher throughput technique than the other methods. The fraction unbound (fu) of drugs in brain tissues can be obtained from two different biological systems: brain homogenates or brain slices. Although brain slices are considered more physiologically relevant, brain homogenates are more widely used, since they are easier to handle and store than slices, and can be readily obtain from commercial suppliers. Both systems afford good correlation with each other, indicating that brain tissue binding is primarily governed by nonspecific binding to lipophilic components rather than binding to intact structural elements.

Similarly to plasma protein binding, brain tissue binding has been routinely determined in multiple species to account for any potential species dependence. However from 2007, a few studies using a limited number of drugs or a limited number of species indicated that brain tissue binding might be species-independent. Pfizer decided to conduct the present study to confirm the species-independent phenomenon of brain tissue binding and determine if it was not limited to certain species, certain classes of pharmaceutical agents or a certain nature of brain binding. With this objective, a large number of structurally diverse compounds, covering a wide range of physicochemical properties (logD from -1.43 to 6.01, MWt from 151 to 823 g/mol, tPSA from 12 to 220 Å2) and brain binding characteristics (fu from 0.0005 to 0.5) in multiple animal species, was selected. The brain tissue binding of these compounds was assessed in seven species and strains (Wistar Han rat, Sprague-Dawley rat, CD-1 mouse, Hartley guinea pig, beagle dog, cynomolgus monkey and human), commonly used in neuroscience drug discovery.

Rigorous statistical orthogonal regression was used to analyse the concordance of the data between species, strain and within strain. The results unambiguously demonstrate that the drugs unbound fractions were strongly correlated (R2 ranging from 0.93 to 0.99) across the various species and strains tested. Importantly, the cross-species/strain correlations were extremely similar to the interassay correlation with the same species. Statistical analysis were performed and indicated that no correction was required for the extrapolation of fraction unbound from one species to the other species/strains. These results suggest that determination of brain tissue binding in a single species can replace multispecies determinations. The authors argue that the difference in composition of the brain relative to the plasma is a likely cause of the species-independent nature of brain tissue binding. Indeed, the brain has a much higher lipid contents (11% lipid and 7.9% protein versus 0.65% lipid and 18% protein in the plasma) which could account for the predominant non-specific binding of drugs to brain tissues. The absence of species-specific brain proteins selectively binding to drugs could be another potential explanation.

As stated earlier, the results from this study allowed the DMPK scientists at Pfizer to conclude that measuring brain tissue binding is essential for drugs intended to be used as CNS pharmacological agents, but that a single species measurement is sufficient. In addition, these results demonstrated that brain tissue binding variations can be ruled out to explain observed interspecies differences in the behaviour of CNS drugs.

I personally found the scientific paper1 describing the above study extremely enlightening and hope that this blog article will be of interest to other medicinal chemists or scientists working in the field of neuroscience.

Blog written by Tristan Reuillon

 

References:

  1. Drug Metabolism and Disposition 2011, 39, 1270-1277, Species Independence in Brain Tissue Binding Using Brain Homogenates (doi: 10.1124/dmd.111.038778)

 

Aptamers as positive modulators


What to do when you want to validate an assay for drug discovery, but there are little or no literature tools available?

Well according to a publication from a group at Astra Zeneca, the use of Aptamers could be one way to solve this problem. Aptamers are lengths of DNA or RNA, generally 20-100 bases, which could be used in the same manner as small molecule tools, binding to targets of interest.

Using the historically difficult target glycine receptor as a model system (GlyRα1), the authors  generated aptamer libraries of RNA using the SELEX methodology (systematic evolution of ligands via exponential enrichment). This process involves the exposure of an aptamer library to your target, which has been immobilised (here the team used a biotin/streptavidin interaction), any unbound material is washed away and bound material is collected and amplified by RT-PCR. This is repeated across a number of iterations, improving the overall success rate.

In this case the SELEX process was run with a variety of different sources of the glycine receptor.  The active molecules generated from this method were then further validated using a radioligand filter binding assay. This resulted in eight aptamers being selected for scale up and further profiling in a variety of glycine receptor assays.

Selective binding of the aptamers was shown using SPR using immobilized glycine receptor, this was further supported by immunofluorescence of fixed cells and live cell imaging experiments.  Functional profiling of the aptamers occurred using a membrane potential dye assay supported with patch clamp electrophysiology.

The SPR measurements revealed all eight aptamers had Kd values in the low nanomolar range.  Interestingly two of the eight aptameters had slow on and off rates of binding .The cellular locations from the imaging experiments showed that the majority of the aptamer was present in the cytosol, and to a lesser extent at the plasma membrane. The authors suggested the cytosol accumulation may be due to interactions with Golgi and endoplasmic reticulum.

The functional assays highlighted some interesting findings. Using the membrane potential dye assay, five of the aptamers gave results suggesting they were positive modulators of the glycine receptor.  When this was further explored with one of the aptamers (c2) in a single cell patch clamp it was shown to be a positive modulator.

Overall the publication uses a variety of different supporting techniques to identify a positive modulator aptamer of the glycine receptor.

Could these molecules have a brighter future than just tools and become an alternative to small molecule therapeutics?  The issue of stability and delivery of the treatment have to be solved in each case, but the answer is yes.  As the authors point out, the FDA has approved the aptamer Macugen used for the treatment of age related macular degeneration.  The significant drawback of this medication is the requirement that it has to be injected into the eye of the patient.  So for the moment, tools seem to the current use for aptamers, however other clinical uses will be developed.

fig5

Figure showing the positive modulation of glycine receptor using aptamer C2

Shalaly, N. D., Aneiros, E., Blank, M., Mueller, J., Nyman, E., Blind, M., Dabrowski, M. A., et al. (2015). Positive Modulation of the Glycine Receptor by Means of Glycine Receptor-Binding Aptamers. Journal of Biomolecular Screening, 20(9), 1112-1123. Retrieved from http://jbx.sagepub.com/cgi/doi/10.1177/1087057115590575

 

Blog written by Gareth Williams

Recent advances in optogenetic technology: shedding new light onto neuropathic pain?


Over the last ten years, optogenetics has opened up a whole new world of possibilities within neuroscience research, highlighting the use of ion channels as physiological tools in the process.

Optogenetics combines the focal use of light with the targeted expression of light-sensitive proteins to manipulate and monitor the function of defined populations of cells1. These proteins are currently divided into two groups: the ‘actuator’ proteins, which can transduce a light stimulus into a neuronal signal, and the ‘indicator’ proteins, which report neuronal signals by the emission of light. Here, the focus is on the former. In a recent article in Nature Biotechnology (Dec 2015)2, Park and colleagues used the prokaryotic channelrhodopsin protein (a cation channel responsive to blue light) to reversibly stimulate peripheral and spinal pain pathways in mice which expressed this protein in all, or specific sub-populations, of sensory neurons. By using pulses of blue light, they were able to alter the membrane potential of the cells in which channelrhodopsins were expressed, resulting in neuronal activation, with observable effects on the animals’ behaviour. This in principle is nothing new; what is novel, and possibly revolutionary within optogenetics, in the method developed by this research group to deliver and control the light stimulus.

An improved, reversible pain model that enables natural behaviour.

The in vivo use of optogenetics to target discrete cell populations has several important advantages over lesion-based or genetic knockout animal models: spatial and temporal control over the stimulation or inhibition of the cell population, and more importantly, reversibility of the stimulus. In order to study pain, animals no longer have to be subject to constant pain. However, as with all developing technologies, the earlier incarnations of this technique have been far from optimal. Focal light stimulation has been achieved using fibre-optic cables, which during the course of an experiment, requires an animal to be effectively tethered by their light-source. This impedes natural behaviour and raises a number of both experimental and welfare questions, especially when monitoring behavioural changes is often one of the key read-outs of the experiment.

The research published by Park et al describes an elegant solution to this problem. They have developed a soft, stretchable wireless implant, smaller than a fingertip, which contains radio-frequency-powered LEDs capable of activation 30 cm from transmitter source. Having overcome the problems of heat generation and long-term durability, the implants have been used for reliable, reversible nerve stimulation for up to 6 months without impeding the normal movement or behaviour of animals when not in use (figure 1, used from reference 2 with permission).

Sarah's Lilley 11-01-16 Pic 1.png

Using this technology, implanted either above the sciatic nerve or lumbar spine, coupled with cre-recombinase-based transgenic expression of channelrhodopsin in the nociceptive neurones in mice, the group were able to observe robust, reproducible nocifensive responses on activation of the implanted blue LEDs, which reverted to normal behaviour on deactivation and were absent in cre-negative littermates with implants. Whole-cell voltage- and current-clamp of isolated DRG neurones from these mice confirmed frequency-matched depolarisation and neuronal firing in response to blue light stimulation.

This refined pain model appears to effectively mimic the symptoms of neuropathic pain, and could become an invaluable tool for the investigation of neuropathic pain relief, and possibly other peripheral neurological disorders. As an alternative approach, using optogenetic ion channels to stabilize membrane potential in order to reduce neuronal activity is also possible1. Indeed, the authors conclude with a reference to gene therapy approaches delivering optogenetic channels to human cells already undergoing clinical trial. With these implants, perhaps controlling chronic pain conditions may become as simple as flicking on a light switch?

Blog written by Sarah Lilley.

Refs:

  • Fenno L, Yizhar O & Deisseroth K (2011). The Development and Application of Optogenetics. Annu. Rev. Neurosci. 2011. 34:389–412
  • Park S et al (2015). Soft, stretchable, fully implantable miniaturised optoelectronic systems for wireless optogenetics. Nature Biotechnology 33(12):1280-1286 (doi:10.1038/nbt for methods)

Figure used by permission from Macmillan Publishers Ltd: Nature Biotechnology (citation 2), copyright 2015.