In recent years, there has been a gradually growing awareness that the problems associated with abuse and addiction to psychoactive substances are very common in people with various mental disorders, including schizophrenia and bipolar disorder. But, although there is an increasing number of reports from the literature that these coexisting disorders are very common, as yet few therapeutic programs based on empirical data targeted specifically at this group of patients have been developed (Lewin & Hennesy, 1994). Based on available evidence, we can try to desribe the scale of the problem. The incidence of bipolar disorder and coexisting abuse of psychoactive substances has been described inter alia in the Epidemiological Catchment Area Study (Regier et al, 1990). This study was conducted among participants from five different municipalities in the U.S.. It showed that, in comparison with with patients with other psychiatric disorders, people with bipolar disorder had the highest comorbidity of disorders associated with alcohol use (46%) and drugs (41%). In addition, rates of disorders associated with the use of alcohol and drugs in people with bipolar disorder were much higher than the associated disorders in the general population (14% and 6%). It was also observed that in bipolar I there was a higher risk of coexistence of problems associated with substance abuse in comparison with bipolar II, wich were respectively 61% and 48% (Lewin & Hennesy, 1994). These results were confirmed by further epidemiological studies. In the National Comorbidity Study (NCS) focused on the respondents with a lifetime alcohol or drug use disorder, recruited for the evaluation, it was found that 51,4% of them also met criteria for at least one lifetime mental disorder. 50.9% of the NCS respondents with a lifetime mental disorder also had a history of alcohol or drug abuse or dependence (Kessler et al., 1996). As mentioned above, one of the most common example of dual diagnosic in the coexistence of schizophrenia and addiction to substances. In the studies of Helzer and Pryzbeck (1998) it was found out that four times more alcoholic than non-alcoholic subjects suffer from schizophrenia. The abuse of stimulants four times more common in schizophrenic than non-schizophrenic subjects (Leduc & Mittleman, 1995). In New York State among each 100 patients hospitalised for their first episode of schizophrenia, 35% of them were addicted to illicit drugs. During the relapses of schizophrenia 22% of the patients use psychoactive substances (DeLisi et al., 1991).

#### **3.1 Young age of patients with dual diagnosis**

Patients with dual diagnosis are often young people. De Millo (1989) observed the prevalence of various psychiatric disorders among adolescents addicted to drugs, which was much higher that a paralell incidence among adults, but he also emphasises the fact that there is a lack of standardisation of diagnostic tools for this population. According to Lysaker et al. (1994) among schizophrenic patients with concomitant use of cocaine, the age of first hospitalisation was earlier than in patients not biased. In turn, according to Menezes (1996) patients with dual diagnosis are mainly young men. These observations confirm the evaluations from the studies of Maynard and Cox (1998), who compared the demographic structure of patients hospitalised in psychiatric hospitals in the United States. They showed that among patients with dual diagnosis there was a larger number of younger ones (average age of 36-37 years), compared with those which had only a single psychiatric diagnosis (mean age of 42-43 years).

A study done in Swiss population, which was conducted in different groups of schizophrenic patients presented a higher average age of subjects without addiction, and also of those who were solely dependent on alcohol, in comparison with patients with a history of illicit drug abuse (Modestin et al., 2001).

Similarly, Kavanagh et al. (2004), examining patients with dual diagnosis in the population of Australia, found that the incidence of alcohol use in patients with dual diagnosis did not depend on the age group, whereas the use of other psychoactive substances was higher in younger groups. Epidemiological studies in England showed that the average age of patients with dual diagnosis is within the range of 34-38 years, but recent trends show a gradual decline of this age (Frisher et al., 2004).

#### **3.2 Sex differences among dual diagnosis patients**

430 Psychiatric Disorders – Trends and Developments

treatment systems. One of the most common examples of a dual diagnosis encountered in psychiatric clinical practice is comorbodity of schizophrenia or bipolar disorder and

In recent years, there has been a gradually growing awareness that the problems associated with abuse and addiction to psychoactive substances are very common in people with various mental disorders, including schizophrenia and bipolar disorder. But, although there is an increasing number of reports from the literature that these coexisting disorders are very common, as yet few therapeutic programs based on empirical data targeted specifically at this group of patients have been developed (Lewin & Hennesy, 1994). Based on available evidence, we can try to desribe the scale of the problem. The incidence of bipolar disorder and coexisting abuse of psychoactive substances has been described inter alia in the Epidemiological Catchment Area Study (Regier et al, 1990). This study was conducted among participants from five different municipalities in the U.S.. It showed that, in comparison with with patients with other psychiatric disorders, people with bipolar disorder had the highest comorbidity of disorders associated with alcohol use (46%) and drugs (41%). In addition, rates of disorders associated with the use of alcohol and drugs in people with bipolar disorder were much higher than the associated disorders in the general population (14% and 6%). It was also observed that in bipolar I there was a higher risk of coexistence of problems associated with substance abuse in comparison with bipolar II, wich were respectively 61% and 48% (Lewin & Hennesy, 1994). These results were confirmed by further epidemiological studies. In the National Comorbidity Study (NCS) focused on the respondents with a lifetime alcohol or drug use disorder, recruited for the evaluation, it was found that 51,4% of them also met criteria for at least one lifetime mental disorder. 50.9% of the NCS respondents with a lifetime mental disorder also had a history of alcohol or drug abuse or dependence (Kessler et al., 1996). As mentioned above, one of the most common example of dual diagnosic in the coexistence of schizophrenia and addiction to substances. In the studies of Helzer and Pryzbeck (1998) it was found out that four times more alcoholic than non-alcoholic subjects suffer from schizophrenia. The abuse of stimulants four times more common in schizophrenic than non-schizophrenic subjects (Leduc & Mittleman, 1995). In New York State among each 100 patients hospitalised for their first episode of schizophrenia, 35% of them were addicted to illicit drugs. During the relapses of

schizophrenia 22% of the patients use psychoactive substances (DeLisi et al., 1991).

Patients with dual diagnosis are often young people. De Millo (1989) observed the prevalence of various psychiatric disorders among adolescents addicted to drugs, which was much higher that a paralell incidence among adults, but he also emphasises the fact that there is a lack of standardisation of diagnostic tools for this population. According to Lysaker et al. (1994) among schizophrenic patients with concomitant use of cocaine, the age of first hospitalisation was earlier than in patients not biased. In turn, according to Menezes (1996) patients with dual diagnosis are mainly young men. These observations confirm the evaluations from the studies of Maynard and Cox (1998), who compared the demographic structure of patients hospitalised in psychiatric hospitals in the United States. They showed that among patients with dual diagnosis there was a larger number of younger ones

**3.1 Young age of patients with dual diagnosis**

psychoactive substance dependence.

**3. Epidemiology and demographics of dual diagnosis**

When discussing issues related to the comorbidity of a seriuos maental disorder and addiction to psychoactive substances, an important factor to be considered is the sex. Lewine (1981) in his review of the literature devoted to the differences between men and women suffering from schizophrenia emphasises that men are characterized by poorer premorbid social functioning, earlier age of first hospitalisation, more severe negative symptoms, more severe disease. Better premorbid functioning of women with schizophrenia is an element often emphasised in the literature. Women at the time of onset have a better social contacts, higher education, more permanent job, most of them have already left home, got married, or have a regular partner (Kalisz et al., 2001). Girls more often than boys have a secondary education (Krupka-Matuszczyk, 1998). Research on gender differences in schizophrenia have shown that women experience a milder form of the disease than men, which is associated with better premorbid functioning. Gearon and Bellack (2000) draw attention to the gender difference in patients with dual diagnosis. In the group of outpatients with coexisting schizophrenia and psychoactive substance dependence found that women, despite a better premorbid functioning and later age of onset, experience a deterioration, when they begin to use drugs. This may indicate that women are particularly susceptible to the adverse effects of psychoactive substances. The use of these substances and related social deficits, and reduced ability to process information in them may impair the ability to recognise hazards, such as the threat of violence or rape and protect themselves against them (Bellack & Gearon, 1998). Prevalence of drug use also has a correlation with gender. As noted Sieroslawski, in studies conducted in Poland and Europe in the early 90's, the prevalence among boys was more than twice higher than in girls, though he stressed that in some other countries, like Britain or the USA differentiation based on gender was not so clear (Sierosławski, 1998). Since the age of onset of schizophrenia in men is lower than in women, and drug use among young men is more prevalent, perhaps it may result in a fact that the population of patients with dual diagnosis is dominated by young men. Numerous studies show a predominance of males among people with dual diagnosis. In 1994 DeQuardo et al. evaluated the frequency of substance use among schizophrenic patients. In a population of addicts analysed by them, there were 48% of men and 20% of women. Maynard and Cox (1998) analyzed cross-sectionally a population of patients hospitalized in the U.S. due to various psychiatric disorders, and they found that among those with a dual diagnosis, the majority were males. Swartz et al. (2000) conducted

Comorbidity of a Serious Mental Illness with an Addiction to Psychoactive Substances 433

spectrum, such as cyclothymia, and addiction may be caused by the effects of consumption of psychoactive substances. This diagnostic error is less likely in patients with chronic, severe bipolar disorder or in those patients who clearly developed symptoms of bipolar disorder before they began using substances (Lewin & Hennesy, 2004). It is also sometimes difficult to make a differential diagnosis between schizophrenia and a drug related psychosis. Some helpful observations from the patient's behaviour may suggest the clinician the possibility of substance abuse by persons treated for various mental disorders. For example, patients can often ask the nurse for painkillers, and pharmaceuticals to treat different symptoms. This behaviour often distinguishes them from patients without substance abuse problems who are reluctant to take medications because of unpleasant experiences with side effect and because of an association with a stigma of a mental illness. Another clue suggesting addiction is a behaviour of the patient in the community of patients. Patients with a co-dependency often show a better functioning in a group of mentally ill persons. They show higher social skills and they are more adequate in the field of sexual behaviour. Patients with a co-dependence more often have a tendency to intimidate others, causing fear and respect among the staff and other patients (Solomon et

Why are disorders related to substance use so common in patients with serious mental illnesses? One of the attempts to explain this phenomenon is the self-medication hypothesis formulated by Khantzian (1985). In defining the self-medication, he draws attention to its two aspects. 1) There is a considerable degree of specificity in the choice of a psychoactive substance. 2) People use, abuse and become addicted to psychoactive substances because they reduce the feeling of psychological discomfort. This suggests that people with psychiatric disorders use specific substances (e.g. use heroin during a manic episode or stimulants during a depressive episode) in an attempt to prevent or "self-medicate" unpleasant symptoms, which ultimately leads to the re-use of these substances. The motivation to do it is often a result of subjective profits that the patients using them experience. The patients with schizophrenia use those substances in order to handle depression, to experience more profoundly different emotions, and to reduce the side-effects of the medication they are prescribed. The data show that illicit drugs are used to reduce depression (72%), and tension (64%) to increase pleasant emotions (62%), to enhance the ability to work and to learn (17%), to decrease the side-effects of the medication.(15%), to reduce hallucinations (11%) , suspicion (4%) and other symptoms (Dixon et al., 1991). The analysis of the causes of abuse of psychoactive substances by subjects with schizphrenia was made by a Canadian team, which attempted to balance gains and losses incurred by the patients reaching for psychoactive substances. In the patients' opinion marijuana and alcohol improved their social functioning. However besides the positive outcomes, the respondents emphasised that the reduction of depressive symptoms was often only their wish, because except for achieving such effects as relaxation, pleasure, being more active etc., the psychoactive substances may happent to be an uncontrolled and unexpected reason for an increase of depressive symptoms. In addition, in spite of achieving a feeling of increased satisfaction, the patients might experience an exacerbation of existing, or develop new positive symptoms of schizophrenia (Addington & Duchak, 1997). When evaluating the attractiveness of different substances for the patients suffering from schizophrenia it is very

**5. Reasons for substance abuse among mentally ill people**

al., 1993).

an epidemiological study including drug addicts in the U.S.. Among the people who developed a dual diagnosis, 70% were males, and most of them were in the age group of 30- 40 years. Salyers and Mueser (2001), studying a group of 404 patients with schizophrenia and schizoaffective disorder, found that in the subgroup that had used alcohol or drugs, women constituted a minority. Other sources of demographic and social analyses of patients with dual diagnosis indicate that the population of such patients consists mostly of men, who often do not work and are poorly educated (Drake & Mueser, 2000). Hambrecht and Hafner (2000) conducted research among a population of a billion participants from Germany, noting that among patients with the first episode of schizophrenia, male gender is a greater risk factor when it comes to cannabis abuse. Similar observations were made by Cantor-Graae in studies conducted in Sweden (2001) in which it was found that among patients with schizophrenia who use substances were far fewer women. French researchers analysed the demographics of patients with schizophrenia and the use of psychoactive substances. It turned out that in that group only 16.7% were women (Dervaux et al., 2003). Kavanagh et al. (2004) examining the incidence of substance use psychoactive drugs in patients with psychoses of various aetiologies noted a prevalence of males in the evaluated group.

#### **3.3 Level of education in the population of dual diagnosis patients**

As stated by Chouinard et al. (2003), the first episode of a serious mental illness usually appears at the beginning of adult life, and this is the age when people take important life decisions on, inter alia, the occupation. Suffering from psychiatric disorders is often associated with failure to achieve an adequate education, which may be a consequence of reduced social and economic status. In a Polish follow-up study conducted by Krupka-Matuszczyk (1998) on a group of adolescents including 142 persons, it was observed that after the first hospitalisation 30% of young people did not continue education interrupted by illness. Patients with schizophrenia or bipolar disorder often appear to be poorer, positioned (economically and socially) in worse groups and regions. Achieving a lower educational level may be also the result of the use of psychoactive substances. Swaim et al. (1997) repeatedly observed a higher proportion of drug users in those who interrupted their education prematurely. In a study conducted in Australia it was found that weekly marijuana use in adolescents increases the risk of disruption of school before completing it (Lynskey et al., 2003). Oboth and Anthony (2000) found that among young Americans intravenous drug use causes frequent interruptions of education in high school. In further studies, Kavanagh et al., (2004) observed that having a lower educational attainment was correlated with the use of cannabis, but there was no such correlation with other drugs. In another study, devoted to patients with dual diagnosis, it was found that patients with schizophrenia addicted to alcohol or drugs achieve worse education in comparison to patients with schizophrenia without concomitant dependence (Potvin et al., 2003). Thus, the coexistence of addiction and a serious mental ilness may imply in a summation of the associated adverse factors affecting the level of education of the patients.

#### **4. Diagnostic problems**

Conducting epidemiological studies is complicated by the fact that the described problem is associated with a number of diagnostic questions. For example it is possible that some of the observed elevated rates of coexistence of bipolar disorder, or disorders of the bipolar

an epidemiological study including drug addicts in the U.S.. Among the people who developed a dual diagnosis, 70% were males, and most of them were in the age group of 30- 40 years. Salyers and Mueser (2001), studying a group of 404 patients with schizophrenia and schizoaffective disorder, found that in the subgroup that had used alcohol or drugs, women constituted a minority. Other sources of demographic and social analyses of patients with dual diagnosis indicate that the population of such patients consists mostly of men, who often do not work and are poorly educated (Drake & Mueser, 2000). Hambrecht and Hafner (2000) conducted research among a population of a billion participants from Germany, noting that among patients with the first episode of schizophrenia, male gender is a greater risk factor when it comes to cannabis abuse. Similar observations were made by Cantor-Graae in studies conducted in Sweden (2001) in which it was found that among patients with schizophrenia who use substances were far fewer women. French researchers analysed the demographics of patients with schizophrenia and the use of psychoactive substances. It turned out that in that group only 16.7% were women (Dervaux et al., 2003). Kavanagh et al. (2004) examining the incidence of substance use psychoactive drugs in patients with psychoses of various aetiologies noted a prevalence of males in the evaluated

As stated by Chouinard et al. (2003), the first episode of a serious mental illness usually appears at the beginning of adult life, and this is the age when people take important life decisions on, inter alia, the occupation. Suffering from psychiatric disorders is often associated with failure to achieve an adequate education, which may be a consequence of reduced social and economic status. In a Polish follow-up study conducted by Krupka-Matuszczyk (1998) on a group of adolescents including 142 persons, it was observed that after the first hospitalisation 30% of young people did not continue education interrupted by illness. Patients with schizophrenia or bipolar disorder often appear to be poorer, positioned (economically and socially) in worse groups and regions. Achieving a lower educational level may be also the result of the use of psychoactive substances. Swaim et al. (1997) repeatedly observed a higher proportion of drug users in those who interrupted their education prematurely. In a study conducted in Australia it was found that weekly marijuana use in adolescents increases the risk of disruption of school before completing it (Lynskey et al., 2003). Oboth and Anthony (2000) found that among young Americans intravenous drug use causes frequent interruptions of education in high school. In further studies, Kavanagh et al., (2004) observed that having a lower educational attainment was correlated with the use of cannabis, but there was no such correlation with other drugs. In another study, devoted to patients with dual diagnosis, it was found that patients with schizophrenia addicted to alcohol or drugs achieve worse education in comparison to patients with schizophrenia without concomitant dependence (Potvin et al., 2003). Thus, the coexistence of addiction and a serious mental ilness may imply in a summation of the

**3.3 Level of education in the population of dual diagnosis patients**

associated adverse factors affecting the level of education of the patients.

Conducting epidemiological studies is complicated by the fact that the described problem is associated with a number of diagnostic questions. For example it is possible that some of the observed elevated rates of coexistence of bipolar disorder, or disorders of the bipolar

**4. Diagnostic problems**

group.

spectrum, such as cyclothymia, and addiction may be caused by the effects of consumption of psychoactive substances. This diagnostic error is less likely in patients with chronic, severe bipolar disorder or in those patients who clearly developed symptoms of bipolar disorder before they began using substances (Lewin & Hennesy, 2004). It is also sometimes difficult to make a differential diagnosis between schizophrenia and a drug related psychosis. Some helpful observations from the patient's behaviour may suggest the clinician the possibility of substance abuse by persons treated for various mental disorders. For example, patients can often ask the nurse for painkillers, and pharmaceuticals to treat different symptoms. This behaviour often distinguishes them from patients without substance abuse problems who are reluctant to take medications because of unpleasant experiences with side effect and because of an association with a stigma of a mental illness. Another clue suggesting addiction is a behaviour of the patient in the community of patients. Patients with a co-dependency often show a better functioning in a group of mentally ill persons. They show higher social skills and they are more adequate in the field of sexual behaviour. Patients with a co-dependence more often have a tendency to intimidate others, causing fear and respect among the staff and other patients (Solomon et al., 1993).

#### **5. Reasons for substance abuse among mentally ill people**

Why are disorders related to substance use so common in patients with serious mental illnesses? One of the attempts to explain this phenomenon is the self-medication hypothesis formulated by Khantzian (1985). In defining the self-medication, he draws attention to its two aspects. 1) There is a considerable degree of specificity in the choice of a psychoactive substance. 2) People use, abuse and become addicted to psychoactive substances because they reduce the feeling of psychological discomfort. This suggests that people with psychiatric disorders use specific substances (e.g. use heroin during a manic episode or stimulants during a depressive episode) in an attempt to prevent or "self-medicate" unpleasant symptoms, which ultimately leads to the re-use of these substances. The motivation to do it is often a result of subjective profits that the patients using them experience. The patients with schizophrenia use those substances in order to handle depression, to experience more profoundly different emotions, and to reduce the side-effects of the medication they are prescribed. The data show that illicit drugs are used to reduce depression (72%), and tension (64%) to increase pleasant emotions (62%), to enhance the ability to work and to learn (17%), to decrease the side-effects of the medication.(15%), to reduce hallucinations (11%) , suspicion (4%) and other symptoms (Dixon et al., 1991). The analysis of the causes of abuse of psychoactive substances by subjects with schizphrenia was made by a Canadian team, which attempted to balance gains and losses incurred by the patients reaching for psychoactive substances. In the patients' opinion marijuana and alcohol improved their social functioning. However besides the positive outcomes, the respondents emphasised that the reduction of depressive symptoms was often only their wish, because except for achieving such effects as relaxation, pleasure, being more active etc., the psychoactive substances may happent to be an uncontrolled and unexpected reason for an increase of depressive symptoms. In addition, in spite of achieving a feeling of increased satisfaction, the patients might experience an exacerbation of existing, or develop new positive symptoms of schizophrenia (Addington & Duchak, 1997). When evaluating the attractiveness of different substances for the patients suffering from schizophrenia it is very

Comorbidity of a Serious Mental Illness with an Addiction to Psychoactive Substances 435

PANSS than patients with schizophrenia alone (Margolese et al., 2003). Buhler et al (2002) examined populations of nonabused patients with schizophrenia and those addicted to alcohol and drugs (mainly cannabis). They found that patients with dual diagnosis experienced more severe positive symptoms. Similar results have also resulted in a study conducted by Green et al. (2002). In the work of Addington and Addington (1997) there was no overall significant difference in symptoms measured by PANSS scale between the group using and not using psychoactive substances. The exception was the subgroup of people who used cocaine, in which a higher severity of positive symptoms was observed. In a study conducted by Pencer and Addington (2003) analysing the cognitive functions in patients with first episode of psychosis, using psychoactive substances, an assessment of PANSS at the start, after one year and two years of observation was done. There was no difference in negative symptoms between the two groups, while those who did not use drugs had less severe positive symptoms. It must be remembered that the subjects throughout the period of observation have used psychoactive substances. Substance abuse can also have a negative impact on the course of the bipolar disorder. For example, it may be associated with earlier age of onset of the disorder and cause more difficulties in the therapy of the subtypes of the disease, such as rapid cycling, dysphoria and mixed states. Salloum et al. (2002) conducted a study covering 256 patients with acute manic episode, done in a municipal psychiatric ward, in which it was found that patients with severe symptoms of disease, abusing alcohol presented a significantly higher lability of mood, impulsivity and increased incidence of aggressive behaviour than patients with an acute manic episode without accompanying alcohol abuse. Furthermore, the coexistence of bipolar disorder and substance abuse is associated with an increased number of psychiatric hospitalisations and it is more difficult

to achieve remission of acute manic episodes.

**7.1 Pharmacological treatment**

**7. Treatment strategies for patients with dual diagnosis**

These observations lead to a conlusion that a specific approach for the treatment of comorbid mental ilness and disturbances associated with the use of psychoactive substances is necessary. Currently, clinicians must often rely on their own observations rather than on

In schizophrenic patients with comorbid substance abuse the treatment with secondgeneration antipsychotics may have beneficial effects on their symptoms. There are reports in the literature, which indicate the ability of these drugs to reduce other symptoms of the

In the studies of subjects with schizophrenia and schizoaffective disorder, previously diagnosed as drug resistant, a subgroup of patients with dual diagnosis was selected. When they were switch on clozapine from classical neuroleptics in within six months, the authors noted that patients with a coexistence and without a coexistence of drug dependence responsed equally to the treatment (Buckley et al., 1994). Similar observations were made Volavka (1999), who noted that during a 6-month treatment with clozapine it could not be determined whether the difference in the treatment response between patients with schizophrenia and schizoaffective disorder depends on the fact if they use or do not use psychoactive substances. In turn, Zimmet et al (2000) found that over 85% of patients who

disease, as well as reducing the amount of drug used by patients treated with them.

empirical data to determine which therapies are best for this group of patients.

important to distinguish between positive and negative symptoms of this disease. The positive symptoms usually implicate the abuse of tranquilisers, and the negative symptoms are most frequently a reason to develop a dependence than the positive symptoms. The incidence of negative symptoms is usually accompanied by increased suffering and the patient uses the substance to reduce it even if the relief is only transient. The study done in Australia (1995) was very important. 53 patients with comorbid diagnoses of schizophrenia and addiction to psychoactive substances were interviewed with the use of Brief Symptom Inventory and Schizophrenia/Substance Abuse Interview Schedule. Most of the patients reported that the use of substances was the reason for a development or exacerbation of their disease, 80% of them used drugs to handle their dysphoria and anxiety. The amphetamines caused their subjective improvement better than alcohol, however the choice of the substance depended mainly on what they could afford. Only cannabis exacerbated the positive symptoms and only amphetamines reduced negative symptoms (Baigent et al., 1995). In other studies it was proven that different substances have influence on different problems related to the disease. For example alcohol, cannabis and cocaine decrease depression, cannabis and alcohol decrease the level of anxiety and cocaine increases it (Coben & Levy, 1998). The analysis of subjective experiences of the patients shows that handling the positive symptoms is the most difficult for them as alcohol has here only a limited influence, cannabis increases these symptoms and the effect of cocaine may be diverse depending on the individual patients.

#### **6. Impact of substance use on the course of psychiatric disorders**

The observations of the clinicians treating the dual diagnosis patients concerning the influence of the substance abuse on the course of the mental illness are inconsistent. According to some of them the patients suffering from schizophrenia abusing substances present less positive and negative symptoms, according to others the substance abuse highly deteriorates the course of the disease. Especially the stimulants like cocaine have a negative impact on the intensity of psychiatric symptoms. According to data from literature the only result of substance abuse is the increase of frequency of hospitalizations (LeDuc & Mittleman, 1995), and according to some other studies the impact on different symptoms may be diverse, e.g. alcohol causes an increase of positive symptoms, and it is also responsible for a higher frequency of suicidal attempts (Soyka, 1994). We have available data from the literature on the relationship between substance use and severity of clinical symptoms. Lysaker et al., (1994), found that patients with schizophrenia who use cocaine have less intense negative symptoms than patients, who do not use cocaine. Serper et al. (1995) found in schizophrenic patients with a concomitant use of cocaine lower scores of negative symptoms and higher scores of depression and anxiety. Salyers and Mueser (2001) examining patients with schizophrenia who used alcohol and drugs, observed lower intensity of negative symptoms than in patients without concomitant dependence. However, Soyka et al., (2001) found that the coexistence of addiction, causes only minor differences in the scores of positive and negative symptoms. Clearer differentiation of severity was related only hallucinations among patients with dual diagnosis. Norwegian authors who analysed the relationship between substance use and severity of PANSS scores in psychotic patients who used and who did not use psychoactive substances, did not observe differences in positive negative and general symptoms (Moller & Linaker, 2004). In contrast, Canadian researchers found that patients with dual diagnosis had a higher score in PANSS than patients with schizophrenia alone (Margolese et al., 2003). Buhler et al (2002) examined populations of nonabused patients with schizophrenia and those addicted to alcohol and drugs (mainly cannabis). They found that patients with dual diagnosis experienced more severe positive symptoms. Similar results have also resulted in a study conducted by Green et al. (2002). In the work of Addington and Addington (1997) there was no overall significant difference in symptoms measured by PANSS scale between the group using and not using psychoactive substances. The exception was the subgroup of people who used cocaine, in which a higher severity of positive symptoms was observed. In a study conducted by Pencer and Addington (2003) analysing the cognitive functions in patients with first episode of psychosis, using psychoactive substances, an assessment of PANSS at the start, after one year and two years of observation was done. There was no difference in negative symptoms between the two groups, while those who did not use drugs had less severe positive symptoms. It must be remembered that the subjects throughout the period of observation have used psychoactive substances. Substance abuse can also have a negative impact on the course of the bipolar disorder. For example, it may be associated with earlier age of onset of the disorder and cause more difficulties in the therapy of the subtypes of the disease, such as rapid cycling, dysphoria and mixed states. Salloum et al. (2002) conducted a study covering 256 patients with acute manic episode, done in a municipal psychiatric ward, in which it was found that patients with severe symptoms of disease, abusing alcohol presented a significantly higher lability of mood, impulsivity and increased incidence of aggressive behaviour than patients with an acute manic episode without accompanying alcohol abuse. Furthermore, the coexistence of bipolar disorder and substance abuse is associated with an increased number of psychiatric hospitalisations and it is more difficult to achieve remission of acute manic episodes.

#### **7. Treatment strategies for patients with dual diagnosis**

These observations lead to a conlusion that a specific approach for the treatment of comorbid mental ilness and disturbances associated with the use of psychoactive substances is necessary. Currently, clinicians must often rely on their own observations rather than on empirical data to determine which therapies are best for this group of patients.

#### **7.1 Pharmacological treatment**

434 Psychiatric Disorders – Trends and Developments

important to distinguish between positive and negative symptoms of this disease. The positive symptoms usually implicate the abuse of tranquilisers, and the negative symptoms are most frequently a reason to develop a dependence than the positive symptoms. The incidence of negative symptoms is usually accompanied by increased suffering and the patient uses the substance to reduce it even if the relief is only transient. The study done in Australia (1995) was very important. 53 patients with comorbid diagnoses of schizophrenia and addiction to psychoactive substances were interviewed with the use of Brief Symptom Inventory and Schizophrenia/Substance Abuse Interview Schedule. Most of the patients reported that the use of substances was the reason for a development or exacerbation of their disease, 80% of them used drugs to handle their dysphoria and anxiety. The amphetamines caused their subjective improvement better than alcohol, however the choice of the substance depended mainly on what they could afford. Only cannabis exacerbated the positive symptoms and only amphetamines reduced negative symptoms (Baigent et al., 1995). In other studies it was proven that different substances have influence on different problems related to the disease. For example alcohol, cannabis and cocaine decrease depression, cannabis and alcohol decrease the level of anxiety and cocaine increases it (Coben & Levy, 1998). The analysis of subjective experiences of the patients shows that handling the positive symptoms is the most difficult for them as alcohol has here only a limited influence, cannabis increases these symptoms and the effect of cocaine may be

diverse depending on the individual patients.

**6. Impact of substance use on the course of psychiatric disorders**

The observations of the clinicians treating the dual diagnosis patients concerning the influence of the substance abuse on the course of the mental illness are inconsistent. According to some of them the patients suffering from schizophrenia abusing substances present less positive and negative symptoms, according to others the substance abuse highly deteriorates the course of the disease. Especially the stimulants like cocaine have a negative impact on the intensity of psychiatric symptoms. According to data from literature the only result of substance abuse is the increase of frequency of hospitalizations (LeDuc & Mittleman, 1995), and according to some other studies the impact on different symptoms may be diverse, e.g. alcohol causes an increase of positive symptoms, and it is also responsible for a higher frequency of suicidal attempts (Soyka, 1994). We have available data from the literature on the relationship between substance use and severity of clinical symptoms. Lysaker et al., (1994), found that patients with schizophrenia who use cocaine have less intense negative symptoms than patients, who do not use cocaine. Serper et al. (1995) found in schizophrenic patients with a concomitant use of cocaine lower scores of negative symptoms and higher scores of depression and anxiety. Salyers and Mueser (2001) examining patients with schizophrenia who used alcohol and drugs, observed lower intensity of negative symptoms than in patients without concomitant dependence. However, Soyka et al., (2001) found that the coexistence of addiction, causes only minor differences in the scores of positive and negative symptoms. Clearer differentiation of severity was related only hallucinations among patients with dual diagnosis. Norwegian authors who analysed the relationship between substance use and severity of PANSS scores in psychotic patients who used and who did not use psychoactive substances, did not observe differences in positive negative and general symptoms (Moller & Linaker, 2004). In contrast, Canadian researchers found that patients with dual diagnosis had a higher score in

In schizophrenic patients with comorbid substance abuse the treatment with secondgeneration antipsychotics may have beneficial effects on their symptoms. There are reports in the literature, which indicate the ability of these drugs to reduce other symptoms of the disease, as well as reducing the amount of drug used by patients treated with them.

In the studies of subjects with schizophrenia and schizoaffective disorder, previously diagnosed as drug resistant, a subgroup of patients with dual diagnosis was selected. When they were switch on clozapine from classical neuroleptics in within six months, the authors noted that patients with a coexistence and without a coexistence of drug dependence responsed equally to the treatment (Buckley et al., 1994). Similar observations were made Volavka (1999), who noted that during a 6-month treatment with clozapine it could not be determined whether the difference in the treatment response between patients with schizophrenia and schizoaffective disorder depends on the fact if they use or do not use psychoactive substances. In turn, Zimmet et al (2000) found that over 85% of patients who

Comorbidity of a Serious Mental Illness with an Addiction to Psychoactive Substances 437

diagnostic process. Depending on the patient's current condition, which for example may be acute or chronic, the patient may continue his or her treatment in a therapeutic community. The most important rules of the program are: 1) authority, 2) clear rules and principles. The fulfillment of these conditions is essential in building a therapeutic offer for the patients with an addiction to psychoactive substances and psychiatric disorders. These standards apply in therapeutic communities, where adherence to rules and principles is possible through the common responsibilty of all members of this community, both therapists and the patients. The most important factor in a succeful therapy are: receiving feedback from others, allowing to express emotions, a sense of belonging to a group, giving feedback to others, discovering that others have similar problems, receiving support from others, giving support to others, receiving tips and ideas from others. The participant of a therapeutic program is expected to make a gradual transition from the periphery of the therapeutic communitiy to a position, when he or she takes over certain responsibilities (May-Majewski, 2002). Training of social skills is also a very important part of rehabilitation of patients with dual diagnosis. It includes skills such as: communication, interpersonal problem solving, active participation in their pharmacotherapy, learning to take care of their own health (Sawicka, 2001). A very important element that should be included in these programs is also a cognitive skills training, because, according to the the literature, patients participating in cognitive skills training significantly improve their performance on neuropsychological tests, they increase their insight. Keshavan and Hogarty (1999) developed the concept of "social cognition", which is associated with the term "emotional intelligence". The so-called "social cognition" is the central element of pathophysiology of certainn mental disorders like schizophrenia. People predisposed to the development of schizophrenia may use immature (concrete as opposed to abstract) styles which are inadequate for the more complex and abstract cognitive requirements specific to adults. In this way, they do not understand the essence of the subtleties and nuances of social interaction (Lewis, 2004). It appears that cognitive impairment is associated with various aspects of social functioning and problems in vocational activities. Data from literature suggest that they are particularly important for social functioning, quality of life, social skills, ability to benefit from learning new skills

Understanding the causes of the problem described above is necessary for effective prevention of substance abuse by people with mental health problems. The results of available studies suggest that these patients have an increased risk of a relapse of both the disease and the addiction to substances. In addition to the symptoms they experience, one of the very important problem in the lives of these patients is their difficulty in social functioning. Effective therapy for these individuals should include: making it possible to create a protective environment, assisting these people in makin important changes in their lives, such as finding a good job, friends abstaining from drugs and alcohol, a group of people who can help the patient to find sense their lives. Patients directed to a therapeutic program must be motivated to make changes in their current life. This decision must be conscious and and be a free choice of the patient. It is also necessary to apply specific, individualied forms of therapy of, mental disorders and those associated with subsatnce

(Tamminga, 1998).

**8. Conclusion**

abuse.

used psychoactive substances during their treatment with clozapine reduced the quanities of abused psychoactive substances. To similar conclusions also led the studies by Green et al., (2002) and also by Drake et al., (2000) in relation to patients who use alcohol, as well as research of Buckley et al. (1998) embracing patients with dual diagnosis abusing alcohol, cigarettes and cocaine. As it turned out, the classical neuroleptic treatment did not reduce the amount of abused substances. Probably the positive effects of clozapine treatment is due to the fact that it acts on mesocortical and mesolimbic dopaminergic neurons that are associated the reward system (Green et al., 1999). According to Noordsy et al., (2003) in patients with schizophrenia the reward system does not function properly, which increases the susceptibility to substance use. This may also underly the poorer tolerability of conventional antipsychotics in patients with dual diagnosis and the effectiveness of clozapine and, to a lesser extent, of other atypical antipsychotics in the treatment of this group of patients. According to Stip et al., (2003) and Weisman (2003) in the treatment of patients with dual diagnosis the most promising results were obtained with clozapine, and slightly worse results with olanzapine and quetiapine. Other studies have shown a reduction in the frequency of alcohol use after the change of treatment from classical neuroleptics to risperidone (Huang, 1996).

As for the effect of various drugs used to treat bipolar disorder, a summary is made here Maremmani et al (2010). According to them, for example, carbamazepine is beneficial in preventing relapse in abusers of cocaine and has a positive effect on discomfort associated with fluctuations in mood, but it has no effect on the behaviour directed to obtain the substance. Selective response to lithium is achieved in alcoholics with a coexisting depression. Oxcarbazepine has a good effect of aggressive patients, while valproic acid generally is considered the drug effective in patients with coexisting bipolar disorder and addiction, but also in people with anxiety disorders and the accompanying dependence on psychoactive substances. The authors of that study conclude that some of these medicines are safe and effective agents that can be used in the treatment of addicted persons with mood disorders.

#### **7.2 Psychotherapy and rehabilitation**

Due to the above-described specific problems associated with diagnosing and treatiment of patients with dual diagnosis, specific treatment programs should be developed for them, which combine elements of both psychiatric treatment and addiction therapy. Understanding the causes of the problems described above is necessary for an effective prevention of drug abuse by people with mental illnesses. An example of therapeutic program for dual diagnosis patients with comorbid addiction to substances, with which the authors have a possibility to co-operate, is the Therapeutic Community "Famila" in Gliwice, Poland, which was founded by May-Majewski in the 80-ties. A comprehensive model of treating patients with dual diagnosis, offered by the Center for Addiction Treatment "Familia" allows them to move freely between the settings of two therapeutic models. The program is stationary, conducted in three separate buildings. They have registered 100 beds. The whole program employs three psychiatrists, 6 psychologists and 8 specialists in addiction therapy and nurses. The therapy takes place in a therapeutic community in which the average age is 23 years and in various small groups depending on the current needs of the patient. The treatment of the patient in the psychiatric ward usually follows a complex diagnostic process. Depending on the patient's current condition, which for example may be acute or chronic, the patient may continue his or her treatment in a therapeutic community. The most important rules of the program are: 1) authority, 2) clear rules and principles. The fulfillment of these conditions is essential in building a therapeutic offer for the patients with an addiction to psychoactive substances and psychiatric disorders. These standards apply in therapeutic communities, where adherence to rules and principles is possible through the common responsibilty of all members of this community, both therapists and the patients. The most important factor in a succeful therapy are: receiving feedback from others, allowing to express emotions, a sense of belonging to a group, giving feedback to others, discovering that others have similar problems, receiving support from others, giving support to others, receiving tips and ideas from others. The participant of a therapeutic program is expected to make a gradual transition from the periphery of the therapeutic communitiy to a position, when he or she takes over certain responsibilities (May-Majewski, 2002). Training of social skills is also a very important part of rehabilitation of patients with dual diagnosis. It includes skills such as: communication, interpersonal problem solving, active participation in their pharmacotherapy, learning to take care of their own health (Sawicka, 2001). A very important element that should be included in these programs is also a cognitive skills training, because, according to the the literature, patients participating in cognitive skills training significantly improve their performance on neuropsychological tests, they increase their insight. Keshavan and Hogarty (1999) developed the concept of "social cognition", which is associated with the term "emotional intelligence". The so-called "social cognition" is the central element of pathophysiology of certainn mental disorders like schizophrenia. People predisposed to the development of schizophrenia may use immature (concrete as opposed to abstract) styles which are inadequate for the more complex and abstract cognitive requirements specific to adults. In this way, they do not understand the essence of the subtleties and nuances of social interaction (Lewis, 2004). It appears that cognitive impairment is associated with various aspects of social functioning and problems in vocational activities. Data from literature suggest that they are particularly important for social functioning, quality of life, social skills, ability to benefit from learning new skills (Tamminga, 1998).

#### **8. Conclusion**

436 Psychiatric Disorders – Trends and Developments

used psychoactive substances during their treatment with clozapine reduced the quanities of abused psychoactive substances. To similar conclusions also led the studies by Green et al., (2002) and also by Drake et al., (2000) in relation to patients who use alcohol, as well as research of Buckley et al. (1998) embracing patients with dual diagnosis abusing alcohol, cigarettes and cocaine. As it turned out, the classical neuroleptic treatment did not reduce the amount of abused substances. Probably the positive effects of clozapine treatment is due to the fact that it acts on mesocortical and mesolimbic dopaminergic neurons that are associated the reward system (Green et al., 1999). According to Noordsy et al., (2003) in patients with schizophrenia the reward system does not function properly, which increases the susceptibility to substance use. This may also underly the poorer tolerability of conventional antipsychotics in patients with dual diagnosis and the effectiveness of clozapine and, to a lesser extent, of other atypical antipsychotics in the treatment of this group of patients. According to Stip et al., (2003) and Weisman (2003) in the treatment of patients with dual diagnosis the most promising results were obtained with clozapine, and slightly worse results with olanzapine and quetiapine. Other studies have shown a reduction in the frequency of alcohol use after the change of treatment from classical

As for the effect of various drugs used to treat bipolar disorder, a summary is made here Maremmani et al (2010). According to them, for example, carbamazepine is beneficial in preventing relapse in abusers of cocaine and has a positive effect on discomfort associated with fluctuations in mood, but it has no effect on the behaviour directed to obtain the substance. Selective response to lithium is achieved in alcoholics with a coexisting depression. Oxcarbazepine has a good effect of aggressive patients, while valproic acid generally is considered the drug effective in patients with coexisting bipolar disorder and addiction, but also in people with anxiety disorders and the accompanying dependence on psychoactive substances. The authors of that study conclude that some of these medicines are safe and effective agents that can be used in the treatment of addicted persons with

Due to the above-described specific problems associated with diagnosing and treatiment of patients with dual diagnosis, specific treatment programs should be developed for them, which combine elements of both psychiatric treatment and addiction therapy. Understanding the causes of the problems described above is necessary for an effective prevention of drug abuse by people with mental illnesses. An example of therapeutic program for dual diagnosis patients with comorbid addiction to substances, with which the authors have a possibility to co-operate, is the Therapeutic Community "Famila" in Gliwice, Poland, which was founded by May-Majewski in the 80-ties. A comprehensive model of treating patients with dual diagnosis, offered by the Center for Addiction Treatment "Familia" allows them to move freely between the settings of two therapeutic models. The program is stationary, conducted in three separate buildings. They have registered 100 beds. The whole program employs three psychiatrists, 6 psychologists and 8 specialists in addiction therapy and nurses. The therapy takes place in a therapeutic community in which the average age is 23 years and in various small groups depending on the current needs of the patient. The treatment of the patient in the psychiatric ward usually follows a complex

neuroleptics to risperidone (Huang, 1996).

**7.2 Psychotherapy and rehabilitation**

mood disorders.

Understanding the causes of the problem described above is necessary for effective prevention of substance abuse by people with mental health problems. The results of available studies suggest that these patients have an increased risk of a relapse of both the disease and the addiction to substances. In addition to the symptoms they experience, one of the very important problem in the lives of these patients is their difficulty in social functioning. Effective therapy for these individuals should include: making it possible to create a protective environment, assisting these people in makin important changes in their lives, such as finding a good job, friends abstaining from drugs and alcohol, a group of people who can help the patient to find sense their lives. Patients directed to a therapeutic program must be motivated to make changes in their current life. This decision must be conscious and and be a free choice of the patient. It is also necessary to apply specific, individualied forms of therapy of, mental disorders and those associated with subsatnce abuse.

Comorbidity of a Serious Mental Illness with an Addiction to Psychoactive Substances 439

Dixon, L.; Haas, G.; Weiden, P.J.; Sweeney, J. & Frances, A.J. (1991). Drug Abuse in

Drake, R.E. & Mueser, K.T. (2000). Psychosocial Approaches to Dual Diagnosis.

Drake, R.E.; Xie, H.; McHugo, G.J. & Green, A.I. (2000). The Effects of Clozapine on Alcohol

Frisher, M.; Collins, J.; Millson, D.; Crome, I. & Croft P. (2004). Prevalence of Comorbid

Gearon, J.S. & Bellack, A.S. (2000). Sex differences in illness presentation, course, and level of

Green, A.I., Salomon, M.S., Brenner, M.J., Rawlins, K. (2002). Treatment of schizophrenia

Green, A.I., Zimmet, S.V., Strous, R.D., Schildkraut, J.J. (1999). Clozapine for comorbid

*of psychiatry,* Vol.6, No.6, (March-April 1999) pp.287-296, ISSN: 1067-3229 Hambrecht, M. & Häfner, H. (2000). Cannabis, Vulnerability, and the Onset of

Helzer, J.E. & Pryzbeck, T.R. (1988). The Co-occurrence of Alcoholism with Other

Kalisz, A. & Cechnicki, A. (2001). Różnice w Czynnikach Rokowniczych Między

Keshavan, M.S. & Hogarty, G.E. (1999). Brain Maturational Processes and Delayed Onset in

Kessler, R.C.; Nelson C.B.; McGonagle, K.A.; Edlund, M.J .; Frank, R.G.; & Leaf P.L. (1996).

Vol.66, No.1, (January 1996), pp. 17–3, ISSN 0002-9432

Kavanagh, D.J.; Waghorn, G.; Jenner, L.; Chant, D.C.; Carr, V.; Evans, M.; Hemnan, H.; Jablensky, A. & McGrath, J.J. (2004). Demographic and Clinical Correlates of Comorbid

*of Psychiatry,* Vol.34, No.3 (June 2000), pp. 468-475, ISSN 0004-8674

*disorders,* Vol.1, No.2, (April 2002) pp.129-139, ISSN: 1568-007X

*Psychiatry,* Vol.148, No.2, pp. 224-230, ISSN 0002-953X

Vol.43, No1, pp. 65-70, (May 2000), ISSN 0920-9964

Vol.26, No.2, pp. 441-449, ISSN 0586-7614

1036-1041, ISSN 0143-005X

1937-1888

963, ISSN 0033-2674

115-124., ISSN 0920-9964

525-543, ISSN 0954-5794

*Schizophrenia Bulletin,* Vol.26, No.1, pp. 105-118, ISSN 0586-7614

Schizophrenic Patients: Clinical Correlates and Reasons for Use. *American Journal of*

and drug use disorders among patients with schizophrenia. *Schizophrenia Bulletin,*

Psychiatric Illness and Substance Misuse in Primary Care in England and Wales. *Journal of Epidemiology and Community Health,* Vol.58, No.12, (December 2004), pp.

Functioning in Substance-abusing Schizophrenia Patients. *Schizophrenia Research,*

and comorbid substance use disorder. *Current drug targets. CNS and neurological*

substance use disorder and schizophrenia: do patients with schizophrenia have a reward-deficiency syndrome that can be ameliorated by clozapine? *Harvard review*

Schizophrenia: an Epidemiological Perspective. *Australian and New Zealand Journal*

Psychiatric Ddisorders in the General Population and its Impact on Treatment. *Journal of Studies on Alcohol and Drugs,* Vol.49, No.3, (May 1988), pp. 219 –224, ISSN

Mężczyznami, i Kobietami Hospitalizowanymi po raz Pierwszy z Rozpoznaniem Schizofrenii. *Psychiatria Polska,* Vol.35, No.6, (November-December 2001), pp. 951-

Substance Use Disorders in Psychosis: Multivariate Analyses from an Epidemiological Sample. *Schizophrenia Research,* Vol.66, No.2-3, (February 2004), pp.

Schizophrenia. *Development and Psychopathology,* Vol.11, No.3, (Summer 1999), pp.

The Epidemiology of Co-occurring Addictive and Mental Disorders: Implications for Prevention and Service Utilization, *The American Journal of Orthopsychiatry,*

#### **9. References**

Addington, J. & Addington, D. (1997). Substance Abuse And Cognitive Functioning in

Addington J.; & Duchak V. (1997). Reasons For Substance Use in Schizophrenia. *Acta*

Baigent, M.; Holme, G. & Hafner, R.J. (1995). Self Reports of the Interaction Between

Bellack, A.S. & Gearon, J.S. (1998). Substance Abuse Treatment for People with

Buckley, P.F. (1998). Substance Abuse in Schizophrenia: a Review. *The Journal of Clinical*

Buckley, P.; Thompson, P.; Way, L. & Meltzer, H.Y. (1994). Substance Abuse Among

Bühler, B.; Hambrecht, M.; Löffler; W.; an der Heiden; W. & Häfner, H. (2002). Precipitation

Cantor-Graae, E.; Nordström, L.G. & McNeil, T.F. (2001). Substance Abuse in Schizophrenia:

Chouinard, S.; Stip, E.; Comtois, G.; Corbière, M.; Bolé, P.; Lamontagne, L.; Lecavalier, M. &

DeLisi, L.E.; Boccio, A.M.; Riordan, H.; Hoff, A.L.; Dorfman, A.; McClelland, J.; Kushner, M.;

DeMilio, L. (1989). Psychiatric Syndromes in Adolescent Substance Abusers. *American*

DeQuardo, J. R.; Carpenter, C. F. & Tandon, R. (1994). Patterns of Substance Abuse in

Dervaux, A.; Laqueille, X.; Bourdel, M.C.; Leborgne, M.H.; Olié, J.P.; Lôo, H. & Krebs, M.O.

*Research,* Vol.48, No.1, (March 2001), pp. 69-82, ISSN 0920-9964

Vol.28, No.2, (Autumn 2003), pp. 273-290, ISSN 0383-6320

Vol.38, No.1, (July, 1991), pp. 39-50, ISSN 0165-1781

No.3, (May-June 1994), pp. 267-275, ISSN 0022-3956

*L'Encéphale,* 2003, Vol.29, No.1, pp. 11-17, ISSN 0013-7006

*Psychiatry,* Vol.29, No.1 (March 1995), pp. 69-74, ISSN 0004-8674

*Psychiatry,* Vol.59, Suppl.3, pp. 26-30, ISSN 0160-6689

Schizophrenia, *Journal of Psychiatry and Neuroscience,* Vol.22, No.4, (March 1997), pp.

*Psychiatrica Scandinavica,* Vol.96, No.5 (December 1997), pp. 329-333, ISSN:0001 -

Substance Abuse and Schizophrenia, *Australian and New Zealand Journal of*

Schizophrenia. *Addictive Behaviors,* Vol.23, No.6 (November-December 1998),

Patients with Treatment-resistant Schizophrenia: Characteristics and Implications for Clozapine Therapy. *American Journal of Psychiatry*, Vol.151, No.3 (March 1994),

and Determination of the Onset and Course of Schizophrenia by Substance Abuse a Retrospective and Prospective Study of 232 Population-based First Illness Episodes. *Schizophrenia Research,* Vol.54, No.3, (April 2002), pp. 243-251, ISSN 0920-

a Review of the Literature and a Study of Correlates in Sweden. *Schizophrenia*

Beauregard, F. (2003). Young Patients with Severe Mental Illness Returning to School: Results of a Pilot Project Conducted in Montreal. *Santé mentale au Québec,*

Van Eyl, O. & Oden, N. (1991). Familial Thyroid Disease and Delayed Language Development in First Admission Patients with Schizophrenia. *Psychiatry Research,*

*Journal of Psychiatry,* Vol.146, No.9, (September 1989), pp. 1212-1214, ISSN 0002-

Schizophrenia: Nature and Significance. *Journal of Psychiatric Research,* 1994, Vol.28,

(2003) Cannabis and Schizophrenia: Demographic and Clinical Correlates.

**9. References**

690X

9964

953X

99-104, ISSN:1180-4882

pp.749-766, ISSN 0306-4603

pp. 385-389, ISSN 0002-953X

Comorbidity of a Serious Mental Illness with an Addiction to Psychoactive Substances 441

Salloum, I .M.; Cornelius, J. R.; Mezzich, J. E. & Kirisci, L. (2002). Impact of Concurrent

*Bipolar Disorders,* Vol.4, No.6, (December 2002), pp. 418-21, ISSN 1398-5647 Salyers M.P., Mueser K.T. (2001). Social functioning, psychopathology, and medication

Sawicka, M. (2005). Podobieństwa i Różnice w Stylu Radzenia Sobie ze Stresem Pomiędzy

Sciacca, K. (1987). Alcohol and Substance Abuse Programs at New York State

Sierosławski, J. (1998). Używanie Narkotyków przez Młodzież Polską na Tle Wybranych

Solomon J.; Zimberg, S. & E. Shollar (1993). *Dual Diagnosis: Evaluation, Treatment, Trainingand*

Soyka, M. (1994). Addiction and Schizophrenia. Nosological, Clinical and Therapeutic

Soyka, M.; Albus, M.; Immler, B.; Kathmann, N. & Hippius, H. (2001). Psychopathology

Stip, E.; Remington, G.J.; Dursun, S.M.; Reiss, J.P.; Rotstein, E.; MacEwan, G.W.; Chokka,

*Psychopharmacology,*Vol.23, No.4, (August 2003), pp 400-404, ISSN 0271-0749 Swaim, R.C.; Beauvais, F.; Chavez, E.L. & Oetting, E.R. (1997). The Effect of School Dropout

Regier, D.A.; Farmer, M.E.; Rae, D.S.; Locke, B.Z.; Keith, S.J.; Judd, L.L. & Goodwin, F.K.

Swartz, J.A.; Lurigio, A.J. & Goldstein, P. (2000). Severe Mental Illness and Substance Use

*Program Development,* Spinger, ISBN<PHONE_NUMBER>439, London, UK

*Psychiatrie,* Vol.62, No.6, (June 1994), pp. 186-196, ISSN 0720-4299

*Research,* Vol.48, No.1, (March 2001), pp.109-123, ISSN: 0920-9964

*Disabled,* Vol.9, No.2, (Winter 1987), pp. 1-3

pp. 25-40, ISSN 1233-9318

2001) pp. 232-238, ISSN 0940-1334

1990), pp. 2511–2518, ISSN 0098-7484

pp. 701-707, ISSN: 0003-990X

ISSN 0090-0036

0002-953X

Alcohol Misuse on Symptom Presentation of Acute Mania at Initial Evaluation.

sideeffects in relation to substance use and abuse in schizophrenia. *Schizophrenia*

Osobami Chorymi na Schizofrenię, Uzależnionymi a Osobami z Podwójnym Rozpoznaniem. *Psychiatria Polska,* Vol.29, No.6, pp. 1199-1210, ISSN 0033-2674 Serper; M.R., Alpert, M.; Richardson, N.A.; Dickson, S.; Allen, M.H. & Werner, A. (1995).

Clinical effects of recent cocaine use on patients with acute schizophrenia. *The American Journal of Psychiatry,* Vol.152, No.10, (October 1995), pp. 1464-1469, ISSN

PsychiatricCenters Develop and Expand. *AID Bulletin Addiction Intervention with the*

Krajów Europy. Wyniki badań ESPAD 1995. *Serwis Informacyjny Narkomania,* No.2,

Questions. 2. Substance Dependence and Schizophrenia. *Fortschritte der Neurologie-*

inDual Diagnosis and Non-addicted Schizophrenics - are There Differences? *European Archives of Psychiatry and Clinical Neuroscience,* Vol.,251, No.5, (October

P.R.; Jones, B. & Dickson, R.A.; Canadian Switch Study Group. (2003). A Canadian Multicenter Trial Assessing Memory and Executive Functions in Patients with Schizophrenia Spectrum Disorders Treated with Olanzapine. *Journal of Clinical*

Rates on Estimates of Adolescent Substance Use among Three Racial/Ethnic Groups. *American Journal of Public Health,* Vol.87, (January 1997), No.1, pp. 51-55,

(1990). Comorbidity of Mental Disorders with Alcohol and Other Drug Abuse. *JAMA: The Journal of the American Medical Association,* Vol.264, No.19, (November

Disorders Among Former Supplemental Security Income Beneficiaries For Drug Addiction and Alcoholism. *Archives of General Psychiatry,* Vo.57, No., (July 2000),

Khantzian, E.J. (1985). The Self-medication Hypothesis of Addictive Disorders: Focus on

Leduc, P. & G. Mittleman, G. (1995). Schizophrenia and Psychostimulant Abuse: a Review

Levin, F.R. & Hennessy, G. (2004). Bipolar Disorder and Substance Abuse. *Biological Psychiatry,* Vol.56, No.10, (November 2004), pp. 738–748, ISSN 0006-3223 Lewine, R.R. (1981). Sex Differences in Schizophrenia: Timing or Subtypes? *Psychological*

Lynskey, M.T., Coffey, C., Degenhardt, L., Carlin, J.B., Patton, G. (2003). A longitudinal

Lysaker, P.; Bell, M.; Beam-Goulet, J. & Milstein, R. (1994). Relationship of Positive and

*Mental Disease,* Vol.182, No.2, (February 1994), pp. 109-112, ISSN 0022-3018 May-Majewski, A. (2002). Normy i Zasady Leczenia Pacjentów z Podwójną Diagnozą.

Maynard, C. & Cox, G.B. (1998).Psychiatric hospitalization of persons with dual diagnoses:

Margolese, H.C.; Malchy, L.; Negrete, J.C.; Tempier, R. & Gill, K. (2004). Drug and Alcohol

Maremmani, I.; Pacini, M.; Lamanna, F.; Pani, P.P.; Perugi, G.; Deltito, J.; Salloum, I.M.&

Modestin, J.; Gladen, C.J. & Christen, S. (2001). A Comparative Study on Schizophrenic

Møller, T. & Linaker, O.M. (2004). Symptoms and Lifetime Treatment Experiences in

Noordsy, D.L., & Green, A.I. (2003). Pharmacotherapy for Schizophrenia and Co-occurring

Obot, I.S. & Anthony, J.C. (2000). School Dropout and Injecting Drug Use in a National

Pencer, A. & Addington, J. (2003). Substance Use and Cognition in Early Psychosis. *Journalof Psychiatry and Neuroscience,* Vol.28, No.1, (January 2003), pp. 48-54, ISSN 1180-4882 Potvin, S.; Stip, E. & Roy, J.Y. (2003). Schizophrenia and Addiction: An Evaluation of the

*Bulletin,* Vo.90, No.3, (November 1981), pp. 432-434, ISSN 0033-2909

1985), Vo.142, No.11, pp. 1259-1264, ISSN 002-953X

Vol.98, No.5, (May 2003), pp. 685-692, ISSN: 0965-2140

*Wiadomości Psychiatryczne,* Vol.5, No.1, ISSN 1505-7429

*CNS Spectrums,*Vol.15, No.2, pp. 95-109, ISSN 1092-8529

Vol.58, No.3, pp. 237-242, ISSN 0803-9488

1995), pp. 407–427, ISSN 0033-3158

pp. 1615-1617, ISSN 1075-2730

ISSN 0920-9964

ISSN 1055-0887

340-346, ISSN 1523-3812

203, ISSN 0013-7006

No.2, pp.145-155, ISSN 0047-2379

Heroin and Cocaine dependence. *The American Journal of Psychiatry,* (November

and Re-analysis of Clinical Evidence. *Psychopharmacology,* Vol.21, No.4, (October

study of the effects of adolescent cannabis use on high school completion. *Addiction,*

Negative Symptoms to Cocaine Abuse in Schizophrenia. *The Journal of Nervous and*

estimates from two national surveys. *Psychiatric Services,* No.49, (December 1998),

Use Among Patients with Schizophrenia and Related Psychoses: Levels and Consequences. *Schizophrenia Research,* Vol.67, No.2-3, (April 2004), pp. 157-166,

Akiskal, H. (2010). Mood Stabilizers in the Treatment of Substance Use Disorders.

Patients with Dual Diagnosis. *Journal of Addictive Diseases,* Vol.20, No.4, pp.41-51,

Psychotic Patients with and without Substance Abuse. *Nordic Journal of Psychiatry,*

substance use disorders. *Current Psychiatry Reports,*Vol.5, No.5, (October 2003), pp.

Sample of White Non-Hispanic American adults. *Journal of Drug Education,* Vol.30,

Self-medication Hypothesis. *L'Encéphale,* Vo.29, No.3 Pt 1, (May-June 2003), pp.193-

**Part 4**

**Biological Neuropsychiatry**

## **Part 4**

**Biological Neuropsychiatry**

442 Psychiatric Disorders – Trends and Developments

Tamminga, C.A.; Buchanan, R.W. & Gold, J.M. (1998). The Role of Negative Symptoms and

*Psychopharmacology,* Vo.13, Suppl.3, (March 1998), pp. 21-26, ISSN 0268-1315 Volavka, J. (1999). The Effects of Clozapine on Aggression and Substance Abuse

Weisman, R.L. (2003). Quetiapine in the Successful Treatment of Schizophrenia with

Zimmet, S.V., Strous, R.D., Burgess, E.S., Kohnstamm, S. & Green, A.I. (2000). Effects of

*Psychiatry and Medicine,* Vol.33, No.1, pp. 85-89, ISSN 0091-2174

No.1, (February 2000), pp.94-98, ISSN 0271-0749

46, ISSN 0160-6689

Cognitive Dysfunction in Schizophrenia Outcome. *International Clinical*

inSchizophrenic Patients. *The Journal of Clinical Psychiatry,* Vol.60, Suppl.12, pp. 43-

Comorbid Alcohol and Drug Dependence: a Case Report. *International Journal of*

Clozapine on Substance Use in Patients with Schizophrenia and Schizoaffective Disorder: a Retrospective Survey. *Journal of Clinical Psychopharmacology,* Vol.20,

**18**

*University, Japan*

**Molecular Mechanism of the Involvement**

**of the Susceptibility Genes,** *DISC1, PACAP,*

*3Department of Molecular Neuropharmacology, Graduate School of Medicine, Osaka*

No effective drugs are currently available for the treatment of mental diseases, primarily because the underlying mechanism of mental diseases have not been adequately explored at the molecular level. However, recent studies have examined several molecular cascades whose disturbances are associated with mental diseases such as schizophrenia, bipolar disease and major depression. The most common characteristics of these cascades us that they are all associated with neural circuit formation, suggesting that neurodevelopmental factors play a key role in the pathogenesis of mental diseases. The present review summarizes the available information on these molecular cascades and their association

**2. Molecular mechanism of PACAP-stathmin1 dependent psychiatric**

Pituitary adenylate cyclase polypeptide (PACAP) is involved in multiple brain function such as neurotransmission and neural plasticity (Hashimoto et al., 2001; Vaudry et al., 2000). It also has a neurotrophic effect via three heptahekical G protein coupled receptors, one of which is specific for PACAP (PAC1 receptor) and two others that are shared with vasoactive intestinal polypeptide (VPAC1 and VPAC2) (Hashimoto et al., 1993). Recently, mice that lack *Adcyap 1*, the gene encoding PACAP, (*Adcyap 1-/-* mice) were developed (Hashimoto et al., 2001, Shintani et al., 2002). *Adcyap 1-/-* mice display remarkable behavioral abnormalities providing evidence that PACAP plays a previously uncharacterized role in the regulation of

**1. Introduction**

with mental disease.

**disorders (Yamada et al., 2010)**

*TRAP1* **and** *Dysbindin* **in Major Psychiatric**

**Disorders Such as Schizophrenia,**

*1United Graduate School of Child Development, Osaka University, Kanazawa University and Hamamatsu University School of Medicine,*

**Depression and Bipolar Disease**

Taiichi Katayama1, Shinsuke Matsuzaki1,2, Tsuyosi Hattori3 and Masaya Tohyama1,2

*2Department of Anatomy and Neuroscience, Graduate school of Medicine, Osaka University*

### **Molecular Mechanism of the Involvement of the Susceptibility Genes,** *DISC1, PACAP, TRAP1* **and** *Dysbindin* **in Major Psychiatric Disorders Such as Schizophrenia, Depression and Bipolar Disease**

Taiichi Katayama1, Shinsuke Matsuzaki1,2, Tsuyosi Hattori3 and Masaya Tohyama1,2 *1United Graduate School of Child Development, Osaka University, Kanazawa University and Hamamatsu University School of Medicine, 2Department of Anatomy and Neuroscience, Graduate school of Medicine, Osaka University 3Department of Molecular Neuropharmacology, Graduate School of Medicine, Osaka University, Japan*

#### **1. Introduction**

No effective drugs are currently available for the treatment of mental diseases, primarily because the underlying mechanism of mental diseases have not been adequately explored at the molecular level. However, recent studies have examined several molecular cascades whose disturbances are associated with mental diseases such as schizophrenia, bipolar disease and major depression. The most common characteristics of these cascades us that they are all associated with neural circuit formation, suggesting that neurodevelopmental factors play a key role in the pathogenesis of mental diseases. The present review summarizes the available information on these molecular cascades and their association with mental disease.

#### **2. Molecular mechanism of PACAP-stathmin1 dependent psychiatric disorders (Yamada et al., 2010)**

Pituitary adenylate cyclase polypeptide (PACAP) is involved in multiple brain function such as neurotransmission and neural plasticity (Hashimoto et al., 2001; Vaudry et al., 2000). It also has a neurotrophic effect via three heptahekical G protein coupled receptors, one of which is specific for PACAP (PAC1 receptor) and two others that are shared with vasoactive intestinal polypeptide (VPAC1 and VPAC2) (Hashimoto et al., 1993). Recently, mice that lack *Adcyap 1*, the gene encoding PACAP, (*Adcyap 1-/-* mice) were developed (Hashimoto et al., 2001, Shintani et al., 2002). *Adcyap 1-/-* mice display remarkable behavioral abnormalities providing evidence that PACAP plays a previously uncharacterized role in the regulation of

Molecular Mechanism of the Involvement of the Susceptibility Genes, *DISC1, PACAP, TRAP1* and *Dysbindin* in Major Psychiatric Disorders Such as Schizophrenia, Depression and Bipolar Disease 447

A: PACAP regulated stathmin1 expression via PAC1 in PC12 cells. Alteration of stathmin1 mRNA levels 24 h after PACAP treatment, at the indicated concentrations were quantified by real-time PRC. Data are expressed as mean ±SEM relative to control values (n=3, PACAP

Stathmin1 over-expression in neurons caused abnormally pronounced arborization of axon fibers. Morphology of hippocampal primary neurons transfected with stathmin1. Overexpression of GFP (B) and GFP-stathmin1 (C). The neurons over-expressing stathmin1 have abnormally pronounced sprouting of axon fibers. D: Schematic drawing of the molecular

representation shows the pathway by which PACAP regulates the expression of stathmin1 by suppressing the function and expression of Ascl1 after increasing the expression of Hes1

**2.2.1 Stathmin1 gene promoter activity is regulated by basic helix loop helix (bHLH)**

A BLAST search identified the genomic sequence of rat stathmin1 in a chromosome 5 contig. PCR amplification of a 1885bp genomic DNA fragment that consisted of 1561 nt upstream of the stathmin1 transcription start site (+1), exon1, and part of intron 1 (+325) was then performed. This fragment was sequenced and subcloned into a pGL3 luciferase reporter vector. This fragment was also analyzed for transcription factor-binding sites using the DNAsis program. The 1.8kbp rat stathmin1 5' genomic sequence contained 12 (E1-E12) putative E boxes (CANNTG), which are potential binding sites for bHLH proteins, including neuronal transcription activators. To investigate the promoter activity of stathmin1, we constructed several expression plasmids for the luciferase assay after transient transfection in PC12 cells. Constructs containing E10 (such as STMN1-1, STMN1-2 and STMN 1-3) showed a high level of luciferase activity compared with control cells, but

1nM \*\*P=0.0044, 10nM \*\*P=0.0033, 100nM \*\*\*P=0.0003 compared with control. B,C;

pathway underlying PACAP regulation of stathmin1 expression. The schematic

Fig. 1. PACAP-stathmin1 cascade

and Id3 by activating ERK and p38.

**proteins via the E10 box**

psychomotor behavior. In addition, previous association study reported that several single nucleotide polymorphism (SNPs) in the vicinity of the PACAP gene locus were associated with schizophrenia (Hashimoto, R. et al., 2007). However, although nothing was known about the mechanism of PACAP deficiency-induced psychiatric illness, we have clarified these mechanisms.

#### **2.1 Down-regulation of PACAP expression induces up-regulation of stathmin1 expression in the dentate gyrus both** *in vivo* **and** *in vitro*

Real-time PCR showed that stathmin1 mRNA was markedly increased in the dentate gyrus of *Adcyap 1-/-* mice. An increased in stathmin1 protein levels in the dentate gyrus of *Adcyap 1- /-* mice was confirmed by western blot analysis. These findings were confirmed also i*n vitro* using PC12 cells. PACAP stimulation of PC12 cells caused a decrease in stathmin1 mRNA levels after 3 h, and expression continued to decrease over the next 24 h (Fig. 1A). Stathmin1 protein levels also decreased in response to PACAP, which caused a dose-dependent decrease of stathmin1 mRNA levels. The decrease of stathmin1 expression caused by PACAP stimulation was slightly, but statistically significantly, inhibited by pretreatment with a PAC1/VPAC2 receptor antagonist (PACAP6-38). In addition, pretreatment with a p38 antagonist (SB202190) or an ERK antagonist (PD98059) also inhibited the PACAP-induced decrease of stathmin1 expression. Co-administration of SB202190 and PD98059 strongly inhibited the effect of PACAP, reflecting the key roles played by p38 and ERK in the PACAP signaling pathway. On the other hand, VIP did not decrease stathmin1 expression. These results indicate that PACAP regulates stathmin1 expression via the PAC1 receptor in neurons of the dentate gyrus.

#### **2.2 Up-regulation of stathmin1 induces abnormal axonal arborization in neurons of the dentate gyrus subgranular zone**

In wild-type mice, cells expressing stathmin1 were preferentially located in the innermost part of the granular cell layer, the so-called subgranular zone (SGZ), where neurogenesis of granular cells occurs in adults. Two types of stathmin1 containing processes were found, namely; dendrites and axons. Immunoreactivity for stathmin1 was significantly increased in the SGZ neurons of *Adcyap 1-/-* mice, although the actual number of immunoreactive cells was similar in mutant and wild-type mice. . The number of dot-like immunoreactive fibers belonging to axons was significantly increased in the polymorphic layer of *Adcyap 1-/-* mice, compared with wild-type mice. In support of the *in vivo* datas, over-expression of stathmin1 in the hippocampal primary culture neurons caused dramatic changes of axon fibers. Arborization of axon fibers was markedly increased by stathmin1 over-expression compared with that in normal primary cultured neurons. The number of secondary neurites on axons was also increased following over-expression of stathmin1. These findings indicated that an increase in stathmin1 expression in SGZ neurons leads to abnormal axon arborization.

If PACAP directly regulates stathmin1 expression in vivo, SGZ neurons should express PAC1. In fact, strong expression of PAC1 mRNA was identified throughout the entire granular cell layer, including the SGZ. Furthermore, SGZ neurons expressed both stathmin1 protein and PAC1 mRNA. These results show that PACAP inhibits stathmin1 expression via the PAC1 receptor.

#### Fig. 1. PACAP-stathmin1 cascade

446 Psychiatric Disorders – Trends and Developments

psychomotor behavior. In addition, previous association study reported that several single nucleotide polymorphism (SNPs) in the vicinity of the PACAP gene locus were associated with schizophrenia (Hashimoto, R. et al., 2007). However, although nothing was known about the mechanism of PACAP deficiency-induced psychiatric illness, we have clarified

Real-time PCR showed that stathmin1 mRNA was markedly increased in the dentate gyrus of *Adcyap 1-/-* mice. An increased in stathmin1 protein levels in the dentate gyrus of *Adcyap 1- /-* mice was confirmed by western blot analysis. These findings were confirmed also i*n vitro* using PC12 cells. PACAP stimulation of PC12 cells caused a decrease in stathmin1 mRNA levels after 3 h, and expression continued to decrease over the next 24 h (Fig. 1A). Stathmin1 protein levels also decreased in response to PACAP, which caused a dose-dependent decrease of stathmin1 mRNA levels. The decrease of stathmin1 expression caused by PACAP stimulation was slightly, but statistically significantly, inhibited by pretreatment with a PAC1/VPAC2 receptor antagonist (PACAP6-38). In addition, pretreatment with a p38 antagonist (SB202190) or an ERK antagonist (PD98059) also inhibited the PACAP-induced decrease of stathmin1 expression. Co-administration of SB202190 and PD98059 strongly inhibited the effect of PACAP, reflecting the key roles played by p38 and ERK in the PACAP signaling pathway. On the other hand, VIP did not decrease stathmin1 expression. These results indicate that PACAP regulates stathmin1 expression via the PAC1 receptor in

**2.2 Up-regulation of stathmin1 induces abnormal axonal arborization in neurons of**

In wild-type mice, cells expressing stathmin1 were preferentially located in the innermost part of the granular cell layer, the so-called subgranular zone (SGZ), where neurogenesis of granular cells occurs in adults. Two types of stathmin1 containing processes were found, namely; dendrites and axons. Immunoreactivity for stathmin1 was significantly increased in the SGZ neurons of *Adcyap 1-/-* mice, although the actual number of immunoreactive cells was similar in mutant and wild-type mice. . The number of dot-like immunoreactive fibers belonging to axons was significantly increased in the polymorphic layer of *Adcyap 1-/-* mice, compared with wild-type mice. In support of the *in vivo* datas, over-expression of stathmin1 in the hippocampal primary culture neurons caused dramatic changes of axon fibers. Arborization of axon fibers was markedly increased by stathmin1 over-expression compared with that in normal primary cultured neurons. The number of secondary neurites on axons was also increased following over-expression of stathmin1. These findings indicated that an increase in stathmin1 expression in SGZ neurons leads to abnormal axon

If PACAP directly regulates stathmin1 expression in vivo, SGZ neurons should express PAC1. In fact, strong expression of PAC1 mRNA was identified throughout the entire granular cell layer, including the SGZ. Furthermore, SGZ neurons expressed both stathmin1 protein and PAC1 mRNA. These results show that PACAP inhibits stathmin1 expression via

**2.1 Down-regulation of PACAP expression induces up-regulation of stathmin1**

**expression in the dentate gyrus both** *in vivo* **and** *in vitro*

these mechanisms.

neurons of the dentate gyrus.

arborization.

the PAC1 receptor.

**the dentate gyrus subgranular zone**

A: PACAP regulated stathmin1 expression via PAC1 in PC12 cells. Alteration of stathmin1 mRNA levels 24 h after PACAP treatment, at the indicated concentrations were quantified by real-time PRC. Data are expressed as mean ±SEM relative to control values (n=3, PACAP 1nM \*\*P=0.0044, 10nM \*\*P=0.0033, 100nM \*\*\*P=0.0003 compared with control. B,C; Stathmin1 over-expression in neurons caused abnormally pronounced arborization of axon fibers. Morphology of hippocampal primary neurons transfected with stathmin1. Overexpression of GFP (B) and GFP-stathmin1 (C). The neurons over-expressing stathmin1 have abnormally pronounced sprouting of axon fibers. D: Schematic drawing of the molecular pathway underlying PACAP regulation of stathmin1 expression. The schematic representation shows the pathway by which PACAP regulates the expression of stathmin1 by suppressing the function and expression of Ascl1 after increasing the expression of Hes1 and Id3 by activating ERK and p38.

#### **2.2.1 Stathmin1 gene promoter activity is regulated by basic helix loop helix (bHLH) proteins via the E10 box**

A BLAST search identified the genomic sequence of rat stathmin1 in a chromosome 5 contig. PCR amplification of a 1885bp genomic DNA fragment that consisted of 1561 nt upstream of the stathmin1 transcription start site (+1), exon1, and part of intron 1 (+325) was then performed. This fragment was sequenced and subcloned into a pGL3 luciferase reporter vector. This fragment was also analyzed for transcription factor-binding sites using the DNAsis program. The 1.8kbp rat stathmin1 5' genomic sequence contained 12 (E1-E12) putative E boxes (CANNTG), which are potential binding sites for bHLH proteins, including neuronal transcription activators. To investigate the promoter activity of stathmin1, we constructed several expression plasmids for the luciferase assay after transient transfection in PC12 cells. Constructs containing E10 (such as STMN1-1, STMN1-2 and STMN 1-3) showed a high level of luciferase activity compared with control cells, but

Molecular Mechanism of the Involvement of the Susceptibility Genes, *DISC1, PACAP, TRAP1* and *Dysbindin* in Major Psychiatric Disorders Such as Schizophrenia, Depression and Bipolar Disease 449

and quantitatively regulated by Hes1, in response to PACAP signaling.

**2.3 Role of the PACAP-stathmin1 cascade in psychiatric disorders**

bipolar disorder.

transcription of Ascl1. In fact, the up-regulation of Hes1 in PC12 cells by PACAP stimulation led to inhibition of Ascl1 expression. In addition, a reduction of Hes1 expression also resulted in an elevation of Ascl1 expression to 1.2 fold the control level. These results indicate that Ascl1, which controls stathmin1 expression, was functionally regulated by Id3

As described above, PACAP inhibits stathmin1 expression. In addition, over-expression of stathmin1 causes abnormal axonal arborization (Fig. 1B,C), indicating that stathmin1 regulates the maturation of neurons and neural circuit formation. Furtehrmore, *Adcyap 1-/* mice are known to show behavioral abnormalities, some of which might have potential relevance to mental disorders such as schizophrenia (Hashimoto et al.,2001; Shintani et al., 2002), and several SNPs in the vicinity of the *PACAP* gene locus are associated with schizophrenia (Hashimoto et al., 2007). If so, stathmin1 expression should be altered in the brain of patients with schizophrenia. Our RT-PCR study showed that stathmin1 mRNA levels were significantly increased in schizophrenic patients compared with age-matched controls. In contrast, stathmin1 was not significantly increased in the brains of patients with

**3. Mechanism of PACAP-DBZ/DISC1 dependent psychiatric disorders**

DBZ (DISC1-binding zinc finger protein) was found as a DISC1(disrupted-in schizophrenia 1)-interacting molecules by yeast-2-hybrid screening of a complementary DNA (cDNA) library. Subsequent co-immunoprecipitation studies and yeast-2 hybrid assays showed that amino acids 348-597 of DISC1 act as the DBZ binding region, which indicates that the regions of DISC1 near the translocation breakpoint (amino acid 598) participate in the interaction with DBZ. DISC1 and DBZ co-localize diffusely in the cytoplasm and centrosome, and are involved in neurite extension. PACAP regulates the association

DBZ mRNA was expressed exclusively in the brain, but was not expressed in peripheral tissues. To examine the functional role of the DISC1-DBZ interaction, PC12 cells stably expressing DISC1-HA and mock-transfected cells were infected with Adv-DBZ-GFP or Adv-GFP for 24 h. Immunoprecipitation and western blot analysis confirmed the over-expression of DBZ-GFP and DISC1-HA as well as the association between these 2 molecules. Overexpression of both proteins in PC12 cells caused a significant reduction in the number of neurite bearing PC12 cells after PACAP stimulation. However, over-expression of either DBZ or DISC1 alone had no effect. The region of DBZ encompassing amino acids 152-301 interacts with DISC1. PC12 cells were transiently transfected with DBZ (152-301)-IRES-GFP or with GFP alone and treated with PACAP (100nM) for 48 h. The cells expressing DBZ (152-301) had a shorter neurite length than cells expressing GFP alone. Under these conditions, no significant change of apoptosis was detected and the number of transfected cells was similar. The effect of DBZ (152-301) on neurite growth was also confirmed in

**(Hattori et al.,2007; Katayama et al., 2009)**

primary cultured hippocampal neurons.

between DISC1 and DBZ (for details on DISC1, see the chapter 6).

**3.1 DISC1-DBZ interaction inhibits the neurite outgrowth (Fig.2)**

constructs containing a stathmin promoter lacking the E10 box (such as STMN1-4) did not show luciferase activity. Therefore, the E10 box was found to be a key motif that regulates stathmin1 expression through bHLH factors.

#### **2.2.2 An activating bHLH protein, Ascl1, activates the stathmin promoter**

Among activating bHLH proteins, Ascl1was found to activate the stathmin1 promoter. Cotransfection of PC12 cells with the stathmin1-promoter plasmid and the Ascl1 expression plasmid induced a dose-dependent increase in luciferase activity compared with cells transfected with the stathmin1 promoter plasmid alone. To examine whether endogenous Ascl1 protein could bind to the stathmin1 promoter sequence in PC12 cells, sheared chromatin was immunoprecipitated with an anti-Ascl1 antibody or with control IgG, followed by PCR amplification of the corresponding DNA regions using stathmin1 promoter specific primers. Analysis of amplified DNA showed that more sequences were amplified by primers flanking the E10 box than by primers flanking the E10-E11 boxes. In addition, co-localization of Ascl1 and stathmin1 in SGZ neurons was demonstrated by immunohistochemistry. These results established that endogenous Ascl1 protein binds to stathmin1 promoter and act as a major regulator of stathmin1 promoter activity.

#### **2.2.3 The inhibitory bHLH proteins, Hes1 and Id3, showed increased expression after PACAP stimulation**

As described above, PACAP inhibits stathmin1 expression. In addition, PACAP stimulation of PC12 cells caused an increase in the expression of the inhibitory HLH proteins, Hes1 and Id3 expression which belong to inhibitory HLH proteins. Inhibition of the PACAP signaling pathway, as through the inhibition of p38 and ERK, suppresses the effect of PACAP on stathmin1 expression. Moreover, the increase of Id3 mRNA levels in response to PACAP stimulation was inhibited by a p38 inhibitor (SB202190), but not by an ERK inhibitor (PD98059), while induction of Hes1 mRNA by PACAP stimulation was inhibited by both an ERK inhibitor and a p38 inhibitor. Co-administration of p38 and ERK inhibitors strongly inhibited thePACAP-induced induction of Hes1 mRNA. These findings showed that Hes1 expression is regulated by both the PACAP-ERK and PACAP-p38 pathways, whereas Id3 expression is mainly controlled by the PACAP-p38 pathway.

#### **2.2.4 Hes1 and Id3 suppress stathmin1 promoter activity via Ascl1 inhibition**

PC12 cells were co-transfected with a stathmin1 promoter plasmid and a Hes1 expression plasmid or an Id3 expression plasmid with or without an Ascl1 expression plasmid. Even without exogenous Ascl1 expression, a high level of luciferase activity was detected, owing to the action of the stathmin1 promoter. Expression of Hes1 or Id3 in these cells inhibited luciferase activity related to the stathmin1 promoter activity through endogenous Ascl1. In PC12 cells transfected with the stathmin1 promoter plasmid and the Ascl1 expression plasmid, luciferase activity was higher than that in PC12 cells without the Ascl1 expression plasmid. Id3 expression in these cells inhibited the up-regulation of stathmin1 promoter luciferase activity, while Hes1 expression failed to reduce the luciferase activity induced by exogenous Ascl1.These findings suggested that Id3 inhibits activation of the stathmin1 promoter by both exogenous and endogenous Ascl1, while Hes1 only blocked the effect of endogenous Ascl1. Thus, it is likely that Id3 regulates Ascl1 at the protein level, while Hes1 regulates Ascl1 transcription. If so, inhibition of Hes1 expression should increase the transcription of Ascl1. In fact, the up-regulation of Hes1 in PC12 cells by PACAP stimulation led to inhibition of Ascl1 expression. In addition, a reduction of Hes1 expression also resulted in an elevation of Ascl1 expression to 1.2 fold the control level. These results indicate that Ascl1, which controls stathmin1 expression, was functionally regulated by Id3 and quantitatively regulated by Hes1, in response to PACAP signaling.

#### **2.3 Role of the PACAP-stathmin1 cascade in psychiatric disorders**

448 Psychiatric Disorders – Trends and Developments

constructs containing a stathmin promoter lacking the E10 box (such as STMN1-4) did not show luciferase activity. Therefore, the E10 box was found to be a key motif that regulates

Among activating bHLH proteins, Ascl1was found to activate the stathmin1 promoter. Cotransfection of PC12 cells with the stathmin1-promoter plasmid and the Ascl1 expression plasmid induced a dose-dependent increase in luciferase activity compared with cells transfected with the stathmin1 promoter plasmid alone. To examine whether endogenous Ascl1 protein could bind to the stathmin1 promoter sequence in PC12 cells, sheared chromatin was immunoprecipitated with an anti-Ascl1 antibody or with control IgG, followed by PCR amplification of the corresponding DNA regions using stathmin1 promoter specific primers. Analysis of amplified DNA showed that more sequences were amplified by primers flanking the E10 box than by primers flanking the E10-E11 boxes. In addition, co-localization of Ascl1 and stathmin1 in SGZ neurons was demonstrated by immunohistochemistry. These results established that endogenous Ascl1 protein binds to

**2.2.2 An activating bHLH protein, Ascl1, activates the stathmin promoter**

stathmin1 promoter and act as a major regulator of stathmin1 promoter activity.

**2.2.4 Hes1 and Id3 suppress stathmin1 promoter activity via Ascl1 inhibition**

PC12 cells were co-transfected with a stathmin1 promoter plasmid and a Hes1 expression plasmid or an Id3 expression plasmid with or without an Ascl1 expression plasmid. Even without exogenous Ascl1 expression, a high level of luciferase activity was detected, owing to the action of the stathmin1 promoter. Expression of Hes1 or Id3 in these cells inhibited luciferase activity related to the stathmin1 promoter activity through endogenous Ascl1. In PC12 cells transfected with the stathmin1 promoter plasmid and the Ascl1 expression plasmid, luciferase activity was higher than that in PC12 cells without the Ascl1 expression plasmid. Id3 expression in these cells inhibited the up-regulation of stathmin1 promoter luciferase activity, while Hes1 expression failed to reduce the luciferase activity induced by exogenous Ascl1.These findings suggested that Id3 inhibits activation of the stathmin1 promoter by both exogenous and endogenous Ascl1, while Hes1 only blocked the effect of endogenous Ascl1. Thus, it is likely that Id3 regulates Ascl1 at the protein level, while Hes1 regulates Ascl1 transcription. If so, inhibition of Hes1 expression should increase the

expression is mainly controlled by the PACAP-p38 pathway.

**2.2.3 The inhibitory bHLH proteins, Hes1 and Id3, showed increased expression after**

As described above, PACAP inhibits stathmin1 expression. In addition, PACAP stimulation of PC12 cells caused an increase in the expression of the inhibitory HLH proteins, Hes1 and Id3 expression which belong to inhibitory HLH proteins. Inhibition of the PACAP signaling pathway, as through the inhibition of p38 and ERK, suppresses the effect of PACAP on stathmin1 expression. Moreover, the increase of Id3 mRNA levels in response to PACAP stimulation was inhibited by a p38 inhibitor (SB202190), but not by an ERK inhibitor (PD98059), while induction of Hes1 mRNA by PACAP stimulation was inhibited by both an ERK inhibitor and a p38 inhibitor. Co-administration of p38 and ERK inhibitors strongly inhibited thePACAP-induced induction of Hes1 mRNA. These findings showed that Hes1 expression is regulated by both the PACAP-ERK and PACAP-p38 pathways, whereas Id3

stathmin1 expression through bHLH factors.

**PACAP stimulation**

As described above, PACAP inhibits stathmin1 expression. In addition, over-expression of stathmin1 causes abnormal axonal arborization (Fig. 1B,C), indicating that stathmin1 regulates the maturation of neurons and neural circuit formation. Furtehrmore, *Adcyap 1-/* mice are known to show behavioral abnormalities, some of which might have potential relevance to mental disorders such as schizophrenia (Hashimoto et al.,2001; Shintani et al., 2002), and several SNPs in the vicinity of the *PACAP* gene locus are associated with schizophrenia (Hashimoto et al., 2007). If so, stathmin1 expression should be altered in the brain of patients with schizophrenia. Our RT-PCR study showed that stathmin1 mRNA levels were significantly increased in schizophrenic patients compared with age-matched controls. In contrast, stathmin1 was not significantly increased in the brains of patients with bipolar disorder.

#### **3. Mechanism of PACAP-DBZ/DISC1 dependent psychiatric disorders (Hattori et al.,2007; Katayama et al., 2009)**

DBZ (DISC1-binding zinc finger protein) was found as a DISC1(disrupted-in schizophrenia 1)-interacting molecules by yeast-2-hybrid screening of a complementary DNA (cDNA) library. Subsequent co-immunoprecipitation studies and yeast-2 hybrid assays showed that amino acids 348-597 of DISC1 act as the DBZ binding region, which indicates that the regions of DISC1 near the translocation breakpoint (amino acid 598) participate in the interaction with DBZ. DISC1 and DBZ co-localize diffusely in the cytoplasm and centrosome, and are involved in neurite extension. PACAP regulates the association between DISC1 and DBZ (for details on DISC1, see the chapter 6).

#### **3.1 DISC1-DBZ interaction inhibits the neurite outgrowth (Fig.2)**

DBZ mRNA was expressed exclusively in the brain, but was not expressed in peripheral tissues. To examine the functional role of the DISC1-DBZ interaction, PC12 cells stably expressing DISC1-HA and mock-transfected cells were infected with Adv-DBZ-GFP or Adv-GFP for 24 h. Immunoprecipitation and western blot analysis confirmed the over-expression of DBZ-GFP and DISC1-HA as well as the association between these 2 molecules. Overexpression of both proteins in PC12 cells caused a significant reduction in the number of neurite bearing PC12 cells after PACAP stimulation. However, over-expression of either DBZ or DISC1 alone had no effect. The region of DBZ encompassing amino acids 152-301 interacts with DISC1. PC12 cells were transiently transfected with DBZ (152-301)-IRES-GFP or with GFP alone and treated with PACAP (100nM) for 48 h. The cells expressing DBZ (152-301) had a shorter neurite length than cells expressing GFP alone. Under these conditions, no significant change of apoptosis was detected and the number of transfected cells was similar. The effect of DBZ (152-301) on neurite growth was also confirmed in primary cultured hippocampal neurons.

Molecular Mechanism of the Involvement of the Susceptibility Genes, *DISC1, PACAP, TRAP1* and *Dysbindin* in Major Psychiatric Disorders Such as Schizophrenia, Depression and Bipolar Disease 451

DISC1-DBZ binding through the ERK cascade but not through the cAMP cascade.

DISC1 from DBZ may not be induced, which results in an immature neural circuit.

**4. Molecular mechanism of Dysbindin-MARCKS cascade dependent**

decreased Dysbindin expression on vulnerability to schizophrenia remain unknown.

**4.1 Dysbindin- myristoylated alanin-rich protein kinase C substrate (MARCKS)**

Cell fractionation experiments using Dysbindin-FLAG-overexpressing HEK293 cells showed that Dysbindin exists mainly in the cytosol while a small amount is present in the nucleus. Immunohistochemical analysis also revealed that Dysbindin is localized mainly in the cytoplasm with a perinuclear high density region. However, a faint immunoreaction was seen within the nucleus. Furthermore, pretreatment with leptomycine-B(LPB), which inhibits the export of proteins from the nucleus to the cytoplasm, caused a slight increase of

**4.1.1 Dysbindin exists within the nucleus in addition to the cytoplasm**

Studies of postmortem brain tissue showed decreased Dysbindin (dystrobrevin binding protein 1; DTNBP1) protein and mRNA levels in patients with schizophrenia compared with controls (Bray et al., 2005; Talbot et al., 2004; Weickert et al., 2004). Chronic treatment of mice with antipsychotics did not affect the levels of Dysbindin protein and mRNA expression in their brains (Chiba et al., 2006; Talbot et al., 2004), suggesting the lower levels of Dysbindin protein and mRNA found in the postmortem brains of schizophrenia patients is not likely to be a simple artifact of antemortem drug treatment. In addition, several studies suggested that diverse high-risk SNPs and haplotypes could influence Dysbindin mRNA expression (Bray et al., 2005; Talbot et al., 2004; Weickert et al., 2004). These data indicate that the *Dysbindin* gene may confer susceptibility to schizophrenia through reduced Dysbindin expression. However, the molecular mechanisms underlying the effect of

**psychiatric disorders (Okuda et al., 2010)**

**cascade (Okuda et al., 2010)**

**psychiatric disorders (Fig.2)**

cyclase failed to influence the DISC1-DBZ interaction, showing that PACAP regulates

The findings described above show that the DISC1-DBZ interaction inhibits neurite out growth, and PACAP dissociates this interaction, resulting in neurite outgrowth. The involvement of the DISC1-DBZ interaction and PACAP in neurite outgrowth suggests that these molecules should be expressed in the early ontogenetical stages. In fact, a marked elevation of both DBZ and PAC1 (PACAP receptor) expression was observed during the perinatal stage (Hattori et al. in preparation). DISC1 shows a high level of expression in the developing cortex and hippocampus (Honda et al.,2004). In addition, down-regulation of DBZ caused a delay in the migration of cortical neurons (Sato et al., personal communication) and disturbance of cilia formation (Kumamoto et al., in preparation). The DISC1-DBZ interaction which is regulated by PACAP has therefore been shown to be involved in neurite out growth and the migration of neurons. In the brain of patients with PACAP-dependent psychiatric disorders, dissociation of the binding between DISC1 and DBZ does not occur and neurite extension may be inhibited. On the other hand, in brains in which translocation of DISC1 occurs, DBZ is unable bind to DISC1 and the dissociation of

**3.3 Role of the PACAP-DBZ cascade with regard to PACAP-DBZ/DISC1 dependent**

Fig. 2. A: Neurite outgrowth was inhibited by the DISC1-binding domain of DBZ [DBZ(152- 301)-IRES-GFP]. PC12 cells were transfected with DBZ (152-301)-IRES-GFP or GFP alone at 2 days after plating. After 24 h , cells were starved of serum for 4h and treated with 100nM PACAP for 48h. Phase-contrast and fluorescence microscopy images are shown. Diagrams display neurite length relative to cell body diameter of transfected PC12 cells. B: PACAPinduced transient inhibition of the endogenous DISC1-DBZ interaction in PC12 cells. Immunoprecipitation and western blot analysis (with anti-DISC1 or anti-DBZ antibodies) of PC12 cells lysates collected at the indicated times after stimulation with 100nM PACAP. Immunoprecipitaes obtained with an anti-DBZ antibody incubated with 5% of each lysates (5% input), were subjected to western blot analysis with the same antibody. Quantitation of relative band densities for DISC1 co-immunoprecipitated with DBZ, as well as for total DISC1 or DBZ protein, was performed by scanning densitometry. Data were expressed as the mean ±SEM. of at least 3 independent experiments. \*P<0.05 vs control (Student's ttest). C: A possible PACAP-DISC1/DBZ pathway . In the normal brain, PACAP leads to the temporal dissociation of the binding between DISC1 and DBZ , which in turn leads to normal neurite outgrowth. In the brain of schizophrenia patients, DBZ cannot bind to DISC1, and the PACAP pathway may proceed without inhibition, resulting in the formation of an abnormal neural circuit.

#### **3.2 PACAP regulates DISC1-DBZ binding (Fig. 2)**

Exposure to PACAP (100nM) increased the expression of endogenous DISC1 in PC12 cells by about 50% after 24 h, whereas it had no effect on DBZ expression. PACAP has a marked influence on the interaction between DBZ and DISC1. The co-immunoprecipitation of DISC1 and DBZ from PC12 cell lysates was reduced by approximately 80% 1h after treatment of cells with PACAP (100nM). However, this reduction was transient and there was a gradual return to control level by 24 h after treatment. Addition of an ERK inhibitor 24 h after PACAP treatment inhibited the rebinding of DBZ to DISC1, while an inhibitor of adenyl

Fig. 2. A: Neurite outgrowth was inhibited by the DISC1-binding domain of DBZ [DBZ(152- 301)-IRES-GFP]. PC12 cells were transfected with DBZ (152-301)-IRES-GFP or GFP alone at 2 days after plating. After 24 h , cells were starved of serum for 4h and treated with 100nM PACAP for 48h. Phase-contrast and fluorescence microscopy images are shown. Diagrams display neurite length relative to cell body diameter of transfected PC12 cells. B: PACAPinduced transient inhibition of the endogenous DISC1-DBZ interaction in PC12 cells.

Immunoprecipitation and western blot analysis (with anti-DISC1 or anti-DBZ antibodies) of PC12 cells lysates collected at the indicated times after stimulation with 100nM PACAP. Immunoprecipitaes obtained with an anti-DBZ antibody incubated with 5% of each lysates (5% input), were subjected to western blot analysis with the same antibody. Quantitation of relative band densities for DISC1 co-immunoprecipitated with DBZ, as well as for total DISC1 or DBZ protein, was performed by scanning densitometry. Data were expressed as the mean ±SEM. of at least 3 independent experiments. \*P<0.05 vs control (Student's ttest). C: A possible PACAP-DISC1/DBZ pathway . In the normal brain, PACAP leads to the temporal dissociation of the binding between DISC1 and DBZ , which in turn leads to normal neurite outgrowth. In the brain of schizophrenia patients, DBZ cannot bind to DISC1, and the PACAP pathway may proceed without inhibition, resulting in the formation

Exposure to PACAP (100nM) increased the expression of endogenous DISC1 in PC12 cells by about 50% after 24 h, whereas it had no effect on DBZ expression. PACAP has a marked influence on the interaction between DBZ and DISC1. The co-immunoprecipitation of DISC1 and DBZ from PC12 cell lysates was reduced by approximately 80% 1h after treatment of cells with PACAP (100nM). However, this reduction was transient and there was a gradual return to control level by 24 h after treatment. Addition of an ERK inhibitor 24 h after PACAP treatment inhibited the rebinding of DBZ to DISC1, while an inhibitor of adenyl

of an abnormal neural circuit.

**3.2 PACAP regulates DISC1-DBZ binding (Fig. 2)**

cyclase failed to influence the DISC1-DBZ interaction, showing that PACAP regulates DISC1-DBZ binding through the ERK cascade but not through the cAMP cascade.

#### **3.3 Role of the PACAP-DBZ cascade with regard to PACAP-DBZ/DISC1 dependent psychiatric disorders (Fig.2)**

The findings described above show that the DISC1-DBZ interaction inhibits neurite out growth, and PACAP dissociates this interaction, resulting in neurite outgrowth. The involvement of the DISC1-DBZ interaction and PACAP in neurite outgrowth suggests that these molecules should be expressed in the early ontogenetical stages. In fact, a marked elevation of both DBZ and PAC1 (PACAP receptor) expression was observed during the perinatal stage (Hattori et al. in preparation). DISC1 shows a high level of expression in the developing cortex and hippocampus (Honda et al.,2004). In addition, down-regulation of DBZ caused a delay in the migration of cortical neurons (Sato et al., personal communication) and disturbance of cilia formation (Kumamoto et al., in preparation). The DISC1-DBZ interaction which is regulated by PACAP has therefore been shown to be involved in neurite out growth and the migration of neurons. In the brain of patients with PACAP-dependent psychiatric disorders, dissociation of the binding between DISC1 and DBZ does not occur and neurite extension may be inhibited. On the other hand, in brains in which translocation of DISC1 occurs, DBZ is unable bind to DISC1 and the dissociation of DISC1 from DBZ may not be induced, which results in an immature neural circuit.

#### **4. Molecular mechanism of Dysbindin-MARCKS cascade dependent psychiatric disorders (Okuda et al., 2010)**

Studies of postmortem brain tissue showed decreased Dysbindin (dystrobrevin binding protein 1; DTNBP1) protein and mRNA levels in patients with schizophrenia compared with controls (Bray et al., 2005; Talbot et al., 2004; Weickert et al., 2004). Chronic treatment of mice with antipsychotics did not affect the levels of Dysbindin protein and mRNA expression in their brains (Chiba et al., 2006; Talbot et al., 2004), suggesting the lower levels of Dysbindin protein and mRNA found in the postmortem brains of schizophrenia patients is not likely to be a simple artifact of antemortem drug treatment. In addition, several studies suggested that diverse high-risk SNPs and haplotypes could influence Dysbindin mRNA expression (Bray et al., 2005; Talbot et al., 2004; Weickert et al., 2004). These data indicate that the *Dysbindin* gene may confer susceptibility to schizophrenia through reduced Dysbindin expression. However, the molecular mechanisms underlying the effect of decreased Dysbindin expression on vulnerability to schizophrenia remain unknown.

#### **4.1 Dysbindin- myristoylated alanin-rich protein kinase C substrate (MARCKS) cascade (Okuda et al., 2010)**

#### **4.1.1 Dysbindin exists within the nucleus in addition to the cytoplasm**

Cell fractionation experiments using Dysbindin-FLAG-overexpressing HEK293 cells showed that Dysbindin exists mainly in the cytosol while a small amount is present in the nucleus. Immunohistochemical analysis also revealed that Dysbindin is localized mainly in the cytoplasm with a perinuclear high density region. However, a faint immunoreaction was seen within the nucleus. Furthermore, pretreatment with leptomycine-B(LPB), which inhibits the export of proteins from the nucleus to the cytoplasm, caused a slight increase of

Molecular Mechanism of the Involvement of the Susceptibility Genes, *DISC1, PACAP, TRAP1* and *Dysbindin* in Major Psychiatric Disorders Such as Schizophrenia, Depression and Bipolar Disease 453

that the SP1 is indispensable for *MARCKS* transcription.

areindispensable for *MARCKS* transcription.

MARCKS expression (Fig. 3C).

(614)-Luc, and UTR (462)-Luc. The luciferase activity detected in SH-SY5Y cells expressing the UTR (1152)-Luc after retinoic acid stimulation was used as a baseline. In the cells transfected with UTR (953)-Luc containing both CCAAT sequence and UTR (700)-Luc containing CCAAT-1 sequence but lacking the CCAAT-2 sequence, luciferase activity remained at baseline level after stimulation with retinoic acid. However luciferase activity was markedly increased in cells expressing UTR (614)-Luc after retinoic acid stimulation. These results suggest that the CCAAT-2 motif plays an important role in the inhibition of MA*RCKS* transcription. Furthermore, SH-SY5Y cells transfected with UTR (462)-Luc lacking CCAAT-1, CCAAT-2 and SP1 region showed very low luciferase activity, indicating

To confirm that the CCAAT-2 region is important for the regulation of MARCKS transcription, several probes were designed for the luciferase assay (Fig. 3B), namely; D1- UTR (1152)-Luc which lacks the CCAAT-2 motif and its downstream region including Sp1 from UTR (1152)-Luc, D2-UTR (1152)-Luc which lacks the SP1 region and the downstream sequence from UTR (1152)-Luc, D3-UTR (1152)-Luc which lacks only the sequence downstream of the SP1 region, D4-UTR (1152)-Luc which lacks only the CCAAT motif from UTR (1152)-Luc, and M-UTR(1152)-Luc, which has a point mutation in the CCAAT-2 motif. Luciferase activity was detected in SH-SY5Y cells transfected with each probe, using the activity in cells transfected with UTR (1152)-Luc as the baseline value. Cells transfected with M-UTR (1152)-Luc and those transfected with D4-UTR (1152)-Luc exhibited marked increases in luciferase activity, showing that the CCAAT-2 motif plays a key role in the inhibition of *MARCKS* transcription. Furthermore, cells expressing D1-UTR (1152)-Luc, D2- UTR (1152)-Luc or D3-UTR (1152)-Luc exhibited no luciferase activity. These findings suggest that the sequence downstream of the Sp1 region, and the Sp1 region itself,

To confirm the involvement of Dysbindin in the altered MARCKS transcription levels via the CCAAT-2 motif, we compared the luciferase activity of UTR (1152)-Luc detected in Dysbindin knockdown cells with that of control cells. Knockdown of Dysbindin resulted in the up-regulation of luciferase activity in the UTR (1152)-Luc transfected cells. However, the effect of knockdown of Dysbindin knockdown on luciferase activity was not observed in D1-UTR (1152)-Luc transfected cells. These results suggest that Dysbindin regulates MARCKS transcription via the NF-YB binding motif CCAAT-2. On the other hand, the negligible levels of luciferase activity observed in cells transfected with probes lacking the sequence downstream of the Sp1 region, suggest that this this sequence is essential for *MARCKS* transcription. In fact, knockdown of Dysbindin caused the up-regulation of

**4.2 Role of the Dysbindin-MARCKS cascade in psychiatric disorders (Fig. 3D)**

cognitive functions (Burdick et al., 2006; Zinkstok et al., 2007).

SNPs in Dysbindin have been associated with intermediate cognitive phenotypes related to schizophrenia such as IQ and working and episodic memory, and a Dysbindin haplotype has been associated with higher educational attainment (Corvin et al., 2008; Donohoe et al., 2007). In addition, several papers show evidence of the involvement of Dysbindin in

Furthermore, accumulating evidence suggests the involvement of Dysbindin in neurotransmission. At the cellular level, Dysbindin is located at both pre- and post-synaptic terminals., and is thought to be involved in postsynaptic density function and the

Dysbindin and its nuclear localization. These findings show that the Dysbindin protein is shuttled between the nucleus and the cytoplasm.

#### **4.1.2 Dysbindin binds to the transcription factor NF-YB**

Using yeast 2-hybrid screening, several transcriptional factors including nuclear transcription factor Y beta (NF-YB), were identified as candidates for interaction with Dysbindin. NF-YB belong to a family of CCAAT-binding transcription factors that are important for the basal transcription of a class of regulatory genes and are involved in cellular reactions. HEK293T cells which express NF-YB endogenously were transfected with expression vectors for Dysbindin-V5 and subjected to immunoprecipitation to confirm the Dysbindin-NF-YB interaction. In addition, the Dysbindin-NF-YB interaction was shown in lysates from SH-SY5Y cells, which express both Dysbindin and NF-YB endogenously, as well as adult mouse brain lysates.

#### **4.1.3 Downregulation of Dysbindin causes up-regulation of the expression level of MARCKS**

The interaction between Dysbindin and NF-YB suggests that Dysbindin may be functionally involved in the transcription of genes regulated by NF-YB. We therefore screened for genes displaying altered expression by means of a DNA chip, using RNA extracts from the Dysbindin or NF-YB knockdown human neural cell line, SH-SY5Y. Among them identified, we focused on MARCKS, because protein kinase C has been involved in psychiatric diseases and because the promoter region of the *MARCKS* gene has the CCAAT binding motif specific for NF-YB.

The effect of Dysbindin on MARCKS *in vitro* was confirmed *in vivo* by examining the expression of the MARCKS protein product in the hippocampus of Dysbindin knockout mice with advanced age and comparing the levels with those of wild-type mice. In wildtype mice, a peak in MARCKS protein expression in the hippocampus was detected on postnatal days 15 and 20, and with a marked decrease in expression levels over time. However, this decrease was not observed in Dysbindin knockout mice, suggesting that down-regulation of Dysbindin may enhance the transcription of the MARCKS protein.

Chromatin immunoprecipitation analysis using SH-SY5Y cells over-expressing Dysbindin-Flag, was performed to explore the possibility that the Dysbindin-NF-YB complex could affect the transcription of *MARCKS via* interaction with the promoter region of *MARCKS*. PCR products from the chromatin immunoprecipitates suggested that Dysbindin and NF-YB simultaneously interact with the promoter region of *MARCKS*. These results indicate that the Dysbindin-NF-YB complex interacts with the promoter region of the *MARCKS* gene resulting in inhibition of MARCKS transcription.

#### **4.1.4 The transcriptional level of the** *MARCKS* **gene is regulated by Dysbindin via the NF-YB binding motif, CCAAT-2**

The 5'-UTR region of the *MARCKS* gene has 2 kinds of CCAAT sequences, namely; one CCAAT motif located between residues -1152 and -700 (CCAAT-1) and one located between UTR -700 and -614 (CCAAT-2). Because NF-YB binds to the CCAAT motif to regulate the transcription of target genes,the role of the CCAAT motifs in the regulation of *MARCKS* transcription was examined by luciferase assay with 5 vectors containing shorter RNA probes (Fig. 3A); These vectors were UTR(1152)-Luc, UTR (953)-Luc, UTR (700)-Luc, UTR

Dysbindin and its nuclear localization. These findings show that the Dysbindin protein is

Using yeast 2-hybrid screening, several transcriptional factors including nuclear transcription factor Y beta (NF-YB), were identified as candidates for interaction with Dysbindin. NF-YB belong to a family of CCAAT-binding transcription factors that are important for the basal transcription of a class of regulatory genes and are involved in cellular reactions. HEK293T cells which express NF-YB endogenously were transfected with expression vectors for Dysbindin-V5 and subjected to immunoprecipitation to confirm the Dysbindin-NF-YB interaction. In addition, the Dysbindin-NF-YB interaction was shown in lysates from SH-SY5Y cells, which express both Dysbindin and NF-YB endogenously, as

**4.1.3 Downregulation of Dysbindin causes up-regulation of the expression level of**

The interaction between Dysbindin and NF-YB suggests that Dysbindin may be functionally involved in the transcription of genes regulated by NF-YB. We therefore screened for genes displaying altered expression by means of a DNA chip, using RNA extracts from the Dysbindin or NF-YB knockdown human neural cell line, SH-SY5Y. Among them identified, we focused on MARCKS, because protein kinase C has been involved in psychiatric diseases and because the promoter region of the *MARCKS* gene has the CCAAT binding motif

The effect of Dysbindin on MARCKS *in vitro* was confirmed *in vivo* by examining the expression of the MARCKS protein product in the hippocampus of Dysbindin knockout mice with advanced age and comparing the levels with those of wild-type mice. In wildtype mice, a peak in MARCKS protein expression in the hippocampus was detected on postnatal days 15 and 20, and with a marked decrease in expression levels over time. However, this decrease was not observed in Dysbindin knockout mice, suggesting that down-regulation of Dysbindin may enhance the transcription of the MARCKS protein. Chromatin immunoprecipitation analysis using SH-SY5Y cells over-expressing Dysbindin-Flag, was performed to explore the possibility that the Dysbindin-NF-YB complex could affect the transcription of *MARCKS via* interaction with the promoter region of *MARCKS*. PCR products from the chromatin immunoprecipitates suggested that Dysbindin and NF-YB simultaneously interact with the promoter region of *MARCKS*. These results indicate that the Dysbindin-NF-YB complex interacts with the promoter region of the *MARCKS* gene

**4.1.4 The transcriptional level of the** *MARCKS* **gene is regulated by Dysbindin via the**

The 5'-UTR region of the *MARCKS* gene has 2 kinds of CCAAT sequences, namely; one CCAAT motif located between residues -1152 and -700 (CCAAT-1) and one located between UTR -700 and -614 (CCAAT-2). Because NF-YB binds to the CCAAT motif to regulate the transcription of target genes,the role of the CCAAT motifs in the regulation of *MARCKS* transcription was examined by luciferase assay with 5 vectors containing shorter RNA probes (Fig. 3A); These vectors were UTR(1152)-Luc, UTR (953)-Luc, UTR (700)-Luc, UTR

shuttled between the nucleus and the cytoplasm.

resulting in inhibition of MARCKS transcription.

**MARCKS**

specific for NF-YB.

**4.1.2 Dysbindin binds to the transcription factor NF-YB**

(614)-Luc, and UTR (462)-Luc. The luciferase activity detected in SH-SY5Y cells expressing the UTR (1152)-Luc after retinoic acid stimulation was used as a baseline. In the cells transfected with UTR (953)-Luc containing both CCAAT sequence and UTR (700)-Luc containing CCAAT-1 sequence but lacking the CCAAT-2 sequence, luciferase activity remained at baseline level after stimulation with retinoic acid. However luciferase activity was markedly increased in cells expressing UTR (614)-Luc after retinoic acid stimulation. These results suggest that the CCAAT-2 motif plays an important role in the inhibition of MA*RCKS* transcription. Furthermore, SH-SY5Y cells transfected with UTR (462)-Luc lacking CCAAT-1, CCAAT-2 and SP1 region showed very low luciferase activity, indicating that the SP1 is indispensable for *MARCKS* transcription.

To confirm that the CCAAT-2 region is important for the regulation of MARCKS transcription, several probes were designed for the luciferase assay (Fig. 3B), namely; D1- UTR (1152)-Luc which lacks the CCAAT-2 motif and its downstream region including Sp1 from UTR (1152)-Luc, D2-UTR (1152)-Luc which lacks the SP1 region and the downstream sequence from UTR (1152)-Luc, D3-UTR (1152)-Luc which lacks only the sequence downstream of the SP1 region, D4-UTR (1152)-Luc which lacks only the CCAAT motif from UTR (1152)-Luc, and M-UTR(1152)-Luc, which has a point mutation in the CCAAT-2 motif. Luciferase activity was detected in SH-SY5Y cells transfected with each probe, using the activity in cells transfected with UTR (1152)-Luc as the baseline value. Cells transfected with M-UTR (1152)-Luc and those transfected with D4-UTR (1152)-Luc exhibited marked increases in luciferase activity, showing that the CCAAT-2 motif plays a key role in the inhibition of *MARCKS* transcription. Furthermore, cells expressing D1-UTR (1152)-Luc, D2- UTR (1152)-Luc or D3-UTR (1152)-Luc exhibited no luciferase activity. These findings suggest that the sequence downstream of the Sp1 region, and the Sp1 region itself, areindispensable for *MARCKS* transcription.

To confirm the involvement of Dysbindin in the altered MARCKS transcription levels via the CCAAT-2 motif, we compared the luciferase activity of UTR (1152)-Luc detected in Dysbindin knockdown cells with that of control cells. Knockdown of Dysbindin resulted in the up-regulation of luciferase activity in the UTR (1152)-Luc transfected cells. However, the effect of knockdown of Dysbindin knockdown on luciferase activity was not observed in D1-UTR (1152)-Luc transfected cells. These results suggest that Dysbindin regulates MARCKS transcription via the NF-YB binding motif CCAAT-2. On the other hand, the negligible levels of luciferase activity observed in cells transfected with probes lacking the sequence downstream of the Sp1 region, suggest that this this sequence is essential for *MARCKS* transcription. In fact, knockdown of Dysbindin caused the up-regulation of MARCKS expression (Fig. 3C).

#### **4.2 Role of the Dysbindin-MARCKS cascade in psychiatric disorders (Fig. 3D)**

SNPs in Dysbindin have been associated with intermediate cognitive phenotypes related to schizophrenia such as IQ and working and episodic memory, and a Dysbindin haplotype has been associated with higher educational attainment (Corvin et al., 2008; Donohoe et al., 2007). In addition, several papers show evidence of the involvement of Dysbindin in cognitive functions (Burdick et al., 2006; Zinkstok et al., 2007).

Furthermore, accumulating evidence suggests the involvement of Dysbindin in neurotransmission. At the cellular level, Dysbindin is located at both pre- and post-synaptic terminals., and is thought to be involved in postsynaptic density function and the

Molecular Mechanism of the Involvement of the Susceptibility Genes, *DISC1, PACAP, TRAP1* and *Dysbindin* in Major Psychiatric Disorders Such as Schizophrenia, Depression and Bipolar Disease 455

regulation of SNAP25 expression (Kumamoto et al., 2006).

organization, which are required for neuronal development.

**5. Mechanism of tumor necrosis factor receptor (TNFR) associated protein 1 (TRAP1)-N-cadherin alteration-induced psychiatric disorders**