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Schizophrenia is a debilitating neurodegenerative disease that affects 1 of the worlds population. Its non-phenomenal symptoms were treated with anti-psychotic drugs. The drugs helped in reducing the psychotic symptoms but failed to deal with the core symptom of the disease, cognitive degeneration. It was not until the late 1980, after the advent of modern neuroimaging tools, that we have been able to map the effects of schizophrenia. Advances in genetic tools have also enabled us begin mapping the genetic linkage of the disease. Schizophrenia involves a complex relationship between genetics and the environment and clusters of genes are switched on and off at various stages of development. It is due to the complexity of the disease that we have had to use Model systems, such as the laboratory mice, to study the disease extensively. It was only in late 2000s that creation of Knockout mice, to study genetic factors of schizophrenia, became economically and technologically viable. The field has expanded with pioneering research done in labs around the world. This dissertation deals with the key mutant mice that have been created to study the disease and how they can be used to create the first generation of anti-schizophrenia drugs.
Schizophrenia is a complex psychiatric disorder caused due to neurocognitive degeneration. Diverse positive and negative symptoms are exhibited by schizophrenics which are compounded by cognitive defects. These cognitive defects were known to exist but were only started to be mapped from 1980s. Enlargement of Lateral ventricles, loss of density of dendritic spines , loss of cortical volume and low latent inhibition were some of the common effects schizophrenia. Schizophrenia has a heritability factor of 0.8 but the genetic linkage of various factors involved is still being researched. Mice, due to their homology and other factors, are ideal candidates to study the genetic basis of Schizophrenia. The economic and technological viability of creating mutant and transgenic mice has helped advance such research. Mutations are induced randomly and sometimes specifically using chemicals such as N-nitroso-N-ethylurea and gene inserts using specific promoters. This dissertation would look at the various different mutant phenotypic mice and their mutations and how accurately these mutations mirror the effects of schizophrenia, as seen in humans. Creation of pharmacological and genetic models are essential for the creation of a new generation of drugs/therapies that act on the core symptoms(cognitive defects) of Schizophrenia.
Key Words : Schizophrenia, Transgene(tg), DISC1, NRG1, Dominant Negative(DN), Cognitive, Lateral Ventricles
Schizophrenia, or dementia praecox, affects 0.3-0.7% of people at some point in their lives(van Os and Kapur 2009).It is a mental illness characterized by a breakdown in cognitive and emotional thought processes. Genetics, epigenetics and neurobiology are important factors behind the disease. Schizophrenia was first delineated as a disease by Emil Kraepeilin in 1893. At the time, he named it dementia praecox with the illness being characterized by a loss in cognitive function(dementia) and an early onset of symptoms(praecox)(Kraepelin 1893). The terminology he used is extremely relevant because he considered the disease to be primarily characterized by cognitive decline and not by the psychosis symptoms. Schizophrenia has positive and negative symptoms. Positive symptoms characterized by symptoms that the patient possess and a normal person does not, for e.g. delusion, hallucinations and psychosis. Negative symptoms are characterized by deficiency in emotional responses and/or thought processes when compared to a normal person. These include anhedonia, asociality and avolition(A 2003).
A combination of environmental and genetic factors plays a key role in the development of schizophrenia. Genetic heritability is complex as the disease involves many clusters of genes working in an epigenetic fashion as well as environmental factors affecting the expression of these genes(Picchioni and Murray 2007). These genes are still being mapped and the relationship between genetic factors and environmental effects, like pre natal stressor and drug use, are still being studied.
From the 1950s, since the serendipitous discovery of anti-psychotic drugs(Ban 2006), schizophrenia has been a key focus for drugs companies. However, till the advent of modern neuroimaging tools and genetic sequencing in the 21st century, the drugs have merely 'treated' the secondary psychotic symptoms of schizophrenia. Psychosis is merely a nonspecific phenomenon of the illness. The anti-psychotic drugs primarily worked on the dopaminergic and serotonergic receptors and recently drugs, that target cholinergic and glutamatergic receptors, have undergone clinical trials (Patil, Zhang et al. 2007). Drugs being used today still fail to act on the key effect of schizophrenia, progressively degenerative cognitive decline.
Data collected by the World Health Organization is used here to give a statistical overview of the spread of Schizophrenia. The spread of the disease does not show any clear correlation between ethnical or regional boundaries, with the disease showing an uniform spread throughout the world(WHO 2011). Males are 1.5x more likely to develop the disease when compared to females(Picchioni and Murray 2007). The peak age for onset of the disease was between 20-28 years for Males and 26-32 for females. It was estimated that one in 10000 people between the age 12-60 are diagnosed each year with schizophrenia(WHO 2011). Estrogen has been suggested to be playing a protective factor for women against schizophrenia(Kulkarni, Riedel et al. 2001), thus showing lower prevalence when compared to men.
Till the advent of modern neuroimaging tools such as Computer Assisted Tomography and MRI scanning, schizophrenia's neurological effects remained unknown. The first MRI study O was done by Smith, R.C. et al in 1984(Smith, Calderon et al. 1984). After which hundreds of different studies have been conducted to map neurological abnormalities and impact of schizophrenia(Shenton, Dickey et al. 2001). There are subtle differences in brain structure that seem to compound and manifest itself as schizophrenia. These differences include abnormalities in medial temporal lobe structure, ventricular enlargement, frontal lobe enlargement etc.(Shenton, Dickey et al. 2001) . The emergence of these abnormalities has been linked to maldevelopment in early brain development and adolescence(Keshavan 1999, Fatemi and Folsom 2009). Genetics and the Environment clearly show a direct linkage in the development of such abnormalities(Fatemi and Folsom 2009).
Mice as a Model Organism
Mice share 99% of genes with humans, thus making them an ideal candidate to study human diseases. Manipulation of the mice genome scales extensively to the human genome, manipulations that otherwise cannot be done in humans. The fact that mice have been used as a model system to study human diseases has already created thousands of different reference mice mutant models. These mutants are used by laboratories around the world to develop new pharmacological models and Knockout/Knock-In mutants. Mice also provide a novel platform to study a disease as complex as Schizophrenia as the mice's cognitive, social and emotional effects can be observed. Factors which cannot be observed in other model like the Zebrafish(Rosenthal and Brown 2007).
Epigenetical interaction of both genes and the environment can be observed in mice and the homology of the mice genome to the human genome makes scaling possible(Rosenthal and Brown 2007). Schizophrenia is a very complex human disorder involving many clusters of genes and various neuronal and neurochemical pathways. More extensive future research has to be conducted to be able to map these pathways precisely(Geyer, Olivier et al. 2012). From research that has been conducted various gene Knockout mutants have been able to replicate the symptoms (positive, negative and cognitive) and abnormalities of schizophrenia. This dissertation will deal with these specific mutants and how these mutant are being used to create a new generation of anti-schizophrenia drugs.
Behavioural Assays and Traits
Schizophrenia is a very complex human disorder due to which several isolated traits, related to the symptoms of schizophrenia, have to be used for correlation in animal models. Some of these assays are as follows
Pre-Pulse inhibition(PPI), is used to characterize defective working memory. It involves measuring the startling response to high intensity (mostly auditory) stimuli which is preceded by similar lower intensity stimuli. Typically low in schizophrenics
Latent Inhibition(LI), specifically low LI is characteristic of patients with Schizophrenia(Cohen, Sereni et al. 2004). It involves forming association of repeated stimuli (pre-exposed) with novel stimuli.
Paired Click Gating or P50 gating, similar to PPI in which a click followed by another click (with 50ms) produces a reduced response. Reduction is 70-80% in normal with 10-20% reduction in schizophrenics. This assay is used to asses sensorimotor gating.
Social Withdrawal, Modeled in animals by socially isolating them
Locomotor Defects - repetitive behavior associated with schizophrenics. Related to defects in Frontotemporal lobe and easily observed in animal models
Cortical Volume, reduced cortical volume(MRI scans) is an anatomical indicator of schizophrenia in both animals and humans
NMDA receptor/gene expression and Dopamine Release, neuroimaging studies are used to model animal drug models as well as mutant models
The model of transmission of schizophrenia still remain elusive but has been shown to be complex and non-mendelian(Sullivan PF 2006). It is clear that there are many different mutations that cause the disease but no single mutation has been found to be the cause in all cases. In Table 1 the risk genes for schizophrenia have been listed
Disruption in Schizophrenia
Table 1. Data adapted from (Sullivan PF 2006, Le-Niculescu, Balaraman et al. 2007, Wedenoja, Loukola et al. 2007). Shows genes that have clear linkage to risk of schizophrenia.
Disrupted-In-Schizophrenia 1 was one of the first genes to show a clear linkage to the development of schizophrenia. It was first described in a Scottish family with a history of Schizophrenia and other psychological disorders. The disruption is caused due to a translocation on chromosome 1(Millar, James et al. 2004, Pletnikov, Ayhan et al. 2007). DISC1 encodes for a synaptic protein that has various isoforms found in different quantities at various compartments within the cell. It is also expressed in essential neuronal areas, involved in schizophrenia, such as the cerebral cortex, amygdala, hippocampus and the cerebellum.DISC1 was also found to have crucial roles in pre and post natal neuronal development, particularly involved in synaptogenesis and also in enlargement of lateral ventricles(Figure 1). Transgenic mice were created primarily using Tet-Off system, inducing both loss of function and partial loss of function of DISC 1 gene(Jaaro-Peled 2009). Seven transgenic lines were created with different mutations in the gene and each of the exhibiting different symptoms related with schizophrenia
MRI Scan of DN-DISC1 Tg Mutant Mice Brain.
(Hikida, Jaaro-Peled et al. 2007)
Figure 1 . Shows MRI scan results of WT to tg(DN-DISC1) mutant mice. In 1(a) the histograms show that the Lateral Ventricular(LV) is higher for tg over WT over a period of six weeks and also in 3months and six weeks. 1(b) shows an increase in the ratio of LV and RV(right ventricular) volume. The picture in top panel of 1(c) shows a 2 dimensional representation of LV and the bottom panel in (c) shows a 3dimernsional representation.(Hikida, Jaaro-Peled et al. 2007)
Δ25 bp mutant
A frameshift mutation that results in the formation of stop codon in exon 7 of DISC1 was induced. This involved a 25bp deletion in exon 6. The deletion did not affect mRNA levels but was found to produce the symptoms in line with schizophrenia. Abnormalities were found in the working memory of Δ25 mutants. These abnormalities were found by using the T-Maze test(Koike, Arguello et al. 2006). The abnormalities observed in spatial working memory show correlation between DISC1 mutation and prefrontal dysfuncton, a key symptom of Schizophrenia.
Q31L and L100P Mutants
N-nitroso-N-ethylurea(ENU) is commonly used to induce random point mutation. Q31L and L100P are missense mutations induced in exon 2 by ENU. Both mutations were found to exhibit anatomical, behavioral and neurological abnormalities with L100P mutation exhibiting schizophrenia-like symptoms(Jaaro-Peled 2009). These symptoms were characterized by defects in Pre-Pulse Inhibition(PPI) and latent inhibition. Pre Pulse inhibition tests the response to a startle inducing stimuli which is preceded by a lower intensity stimuli. It was found that Homozygous recessive (100P/100P) and heterozygous (100P/+) had lower PPI when compared to the wild-type(+/+), suggesting a deficit in information processing(Clapcote, Lipina et al. 2007). Latent Inhibiton(LI) tests also showed similar results. In the T-Maze test, the homozygous recessive(100P/100p) mutant was found to have defects in spatial working memory.(Clapcote, Lipina et al. 2007)
αCaMKII-ΔC DISC1 and BAC-ΔC DISC1 transgenic mice
Transgenic Mice expressing a C-terminal truncated DISC1, under control of α-calmodulin kinase II, in dominant negative form were used for the experiment(Koike, Arguello et al. 2006). The abnormalities found in the Double Negative DISC1(DN-DISC1) mutant were similar to abnormailities observed in patient with schizophrenia. These include enlargement of lateral ventricles, deficiency in sensorimotor gating and deficiencies in subset of interneurons within the cortex(Koike, Arguello et al. 2006).
Using bacterial artificial chromosome(BAC) truncated DISC1 gene was overexpressed causing effects similar to other mutations, with abnormalities in spatial learning and working memory(Shen, Lang et al. 2008, Jaaro-Peled 2009).
DISC1 mutants show enlarged ventricles(Figure 1) and brain volume decrease, similar to that of schizophrenia patients. Cellular, Biochemical and Behavioral Abnormailites have been found in DISC1 mutants that are found in schizophrenia patients(Jaaro-Peled 2009). However, emotional or social response characteristics of schizophrenia were not found to have any relation to the mutation. Therefore drugs targeting DISC1 mutation along with anti-psychotic drugs could help in addressing the core symptoms of Schizophrenia.
Dystrobrevin-binding protein 1(Dysbindin)is a synaptic protein involved is exocytotic regulation, receptor trafficking in neurons and vesicle biogenesis(Karlsgodt, Robleto et al. 2011). The gene for dysbindin is located on chromosome 6 at p24-22. Mutations in this gene show strong susceptibility to schizophrenia and defects in working memory. Dysbindin has also shown, in post-mortem studies, to be reduced in the hippocampal area of schizophrenics(Weickert, Rothmond et al. 2008).. A naturally occurring mouse mutant known as 'sandy'(sdy) was found with a homozygous deletion of exon 2 of the gene. This mutation results in the loss dysbindin protein. Since dysbindin is involved in both pre-synpatic and post-synaptic neurons, the mutants showed many symptoms unique to schizophrenia. These symptoms include larger vesicular size, lower release of vesicles from hippocampal CA 1 and defective synaptic structure (Weickert, Rothmond et al. 2008)
Sdy mutants exhibited various abnormal behaviors .
Novel Object recognition, sdy mice exhibited less novel object preference, suggesting loss of dysbindin leads to long delay recognition memory
Social Withdrawal, lack of dysbindin causes one of the negative symptoms of schizophrenia.
Alteration of neurotransmitter release leads to the effects seen in dysbindin null mice(sdy)
(Weickert, Rothmond et al. 2008)
Neuregulin1 and ErbB4
Neuregulin1(NRG1) and its receptor ErbB4 have been studied extensively and has been shown to have a strong association with schizophrenia. NRG1 plays crucial roles during the pre-natal development . It aids in the radial migration of neurons to the cortex and cerebellum and also in tangential migration. NRG1 is essential for axon guidance, a key factor behind schizophrenia(Jaaro-Peled, Hayashi-Takagi et al. 2009)
NRG1 AND ErbB4 interaction controls spine regulation and dendritic spine density in post-natal stages. NRG1 KO mice thus show reduced dentritic spine density, a key feature of schizophrenia(Jaaro-Peled, Hayashi-Takagi et al. 2009). NRG1KO mice also showed enlarged lateral ventricles(similar to DN-DISC1 mutant). Mice lacking ErbB4 also show reduced dendritic spine density in the cortex and hippocampus. The compounded effect of decreased spine density is increased aggression, hyperactivity(T-Maze) and PPI deficit, all classic signs of schizophrenia(Jaaro-Peled, Hayashi-Takagi et al. 2009). Social behavior was also found to be defective in various tg NRG1 types(Kato, Kasai et al. 2010).
NRG1 and ErbB4 could possibly be working in a connected pathway to DISC1. Defects in these pathways would be manifested as the key positive, negative and cognitive symptoms of Schizophrenia .More research involving Knockout/partial loss of function of both these pathways are needed before a clinical model can be formed.
Comparative figures of Dendritic spine density in (A) Patients with Schizophrenia and (B) NRG1 KO mice.
Reduction in dendritic spine density. In 2(A) SZ panel shows spine density of a patient suffering from schizophrenia. In 2(B) NRG1KO panel shows the reduction of dendritic spine density in the KO mouse. (Chen, Johnson et al. 2008)
Reelin is a protein that plays a crucial role in synaptic genesis and plasticity within the CNS(Cassidy, Mulvany et al. 2010). In patients with schizophrenia, it has been found that reelin mRNA and protein content are reduced in the cerebellum ,the hippocampus and the frontal cortex. Mutants homozygous for reelin mutation were found to have behavioral symptoms more severe than schizophrenia(Bellon, Le Pen et al. 2009). However, heterozygote mutant mice showed few relevant characteristic symptoms of schizophrenia. There were subtle deficits in learning acquisition and and social interaction. But for more positive symptoms, like hyperactivity and spatial reference memory, the mutant mice did not exhibit any consistent defects(Arguello and Gogos 2010, Jones, Watson et al. 2011). Mixed results were also present in PPI and LI tests with the protocol followed, type and duration of tests affecting results(Jones, Watson et al. 2011). As reelin clearly shows linkage in schizophrenia , it is imperative that reelin plays an important role in schizophrenia. However more research has to be carried out to precisely map reelin's effects and how it works with genetic cascades of DISC1 and NRG1.
Apomorphine Susceptible and Unsusceptible Rats
Apomorphine is a dopamine D1/D2 receptor agonist. In high doses, it induces key positive symptoms of schizophrenia like locomotive hyperactivity. To study the effects at a fundamentally genetic level, Wistar mice(mice with an increased response to apomorphine) were bred to create Apomorphine Susceptible(APO-SUS) and Apomorphine Unsusceptiple(APO-UNSUS) mice(Ellenbroek and Cools 2002). As expected both mice showed exact opposite responses to various different tests(Ellenbroek and Cools 2002). APO-SUS mice showed reduced PPI and LI, classic symptoms of schizophrenia, and APO-UNSUS showed no such effects unless at high doses of the drug amphetamine(Le-Niculescu, Balaraman et al. 2007). APO-SUS mice also showed an increase in Dopamine D2 receptors in the nucleus accumbens(nAcc)(Le-Niculescu, Balaraman et al. 2007), part of the pleasure center of the brain, which is similar to a feature found during post mortem of schizophrenics. Pups of APO-SUS mice were also found to have relatively retarded somatic development when compared to normal mice.
As creation of Knockout mutants and next generation sequencing becomes more affordable, the more links that can be found in Schizophrenia. It is a very complex disorder, seemingly unique to humans, which affects anywhere between 30-70million people worldwide (van Os and Kapur 2009). For more than 50 years, this disorder was treated with anti-psychotic drugs that had effect on the actual disease. These drugs merely masked the psychotic effects of the disease and did not address the core symptom of neurocognitive degeneration. It has been shown in this dissertation that a newly emerging field of mice mutants could hold the key to the development of anti-schizophrenia models. The interlinked pathways connecting the ever complex neuronal pathways are the key to finding the various causes of schizophrenia. Mapping of these pathways, using mutants and the mice genome as a model system, provides researchers with information that can be used in the next few decades to develop a viable pharmacological/therapeutic model to combat schizophrenia. But as it stands today, further research is required in all viable pathways involved in Schizophrenia and more focus should be placed by drug manufacturers on combating the core symptoms of the disease.