Patent Application
20080254462
The present invention relates to the role of mutations that perturb expression of ZDHHC8 in increased susceptibility to schizophrenia. It is based, at least in part, on the discovery of a mechanism by which a high risk allele disrupts expression of functional ZDHHC8, and on the discovery that two proteins involved in synaptic pathways associated with schizophrenia interact with ZDHHC8, at least one of which is a substrate for that enzyme.
Microdeletions in chromosome region 22q11 are associated, with relatively high frequency, with severe mental illness. Between one out of three and one out of four subjects with a microdeletion in 22q11 develop schizophrenia or schizoaffective disorder (1-4, 13). A background 0.025% occurrence rate of these deletions in the general population (5, 13) increases to a rate of up to 2% of adult schizophrenic patients (6, 13) and up to 6% of cases with childhood-onset schizophrenia (7, 13). Accordingly, whereas a person in the general population has a risk of about 1% of developing schizophrenia, that risk may be 25-30 times higher in persons having a deletion in the 22q11 region (13). Additional studies have reported suggestive linkage results in the 22q11 region (8, 9, 13) and that schizophrenic patients carrying a 22q11 deletion bear the hallmark neuropsychological and neuroanatomical features of classical schizophrenia (10-12, 13). It therefore seems likely that the 22q11 region harbors genes that, alone or in combination, are causally implicated in schizophrenia in a subset of patients.
Although the majority (87%) of 22q11 deletions are 3 Mb in size, a “schizophrenia critical region” confined to 1.5 Mb has been identified; as the deletions are mediated by low copy repeat (“LCR”) sequences, this critical region has been denoted “LCR-A to -B” (6, 13, 14, 15). The majority of the genes in the region are known (http://genome.ucsc.edu), making this locus amenable to a molecular genetic analysis, and Liu et al. (16) performed linkage disequilibrium (LD) studies in family samples to evaluate whether nondeletion common variants of individual genes within the 22q11 region might play an even greater role than microdeletions in susceptibility to schizophrenia in the general population (13). The LD analysis delineated a subregion of 22q11 in which PRODH2 and DGCR6 are the only known genes, and identified a relatively common haplotype where the schizophrenia susceptibility variant(s) likely reside (16, 13). While the conclusions of these studies implicated PRODH2 as playing an important role, comparison of the increase in the morbid risk of schizophrenia associated with the identified PRODH2 variation to the risk associated with the 22q11 microdeletion could not exclude contribution from other genes in the region (16, 17, 13). Further LD studies (13) revealed a limited number of candidate genes, including two membrane-associated proteins, KIAA1292 (now known as ZDHHC8) and NOGO-R, which are highly expressed in brain regions implicated in schizophrenia.
The present invention relates to the role of mutations that perturb expression of ZDHHC8 in increased susceptibility to schizophrenia and related disorders. It is based, at least in part, on the discoveries that (i) a point mutation associated with increased risk of schizophrenia prevented proper splicing of ZDHHC8 mRNA; (ii) ZDHHC8 interacts with two homologous proteins (PSD95 and PSD93) which are key signaling molecules in glutamatergic synapses; (iii) ZDHHC8 palmitoylates and facilitates membrane translocation of PSD95; and (iv) palmitoylation of PSD95 by ZDHHC8 is necessary for maintaining a homeostatic balance between excitatory glutamatergic and inhibitory GABAergic synapses.
In a first set of embodiments, the present invention provides for methods of identifying a subject at risk of developing schizophrenia or a related disorder, comprising identifying, in the individual, a mutation that decreases or prevents expression of wild-type ZDHHC8 when compared to a subject not diagnosed with schizophrenia or a related disorder. In related embodiments, the invention may be used to aid in the diagnosis of schizophrenia or a related disorder in a subject.
In a second set of embodiments, the present invention provides for a transgenic non-human animal in which expression of at least one copy of endogenous ZDHHC8 is decreased or prevented. In preferred embodiments, expression of both copies of endogenous ZDHHC8 is decreased or prevented, and most preferably, detectable expression of the active protein is absent.
In a third set of embodiments, the present invention provides for methods and assay systems for identifying agents that may be used to treat schizophrenia or a related disorder in a subject carrying a mutation that decreases or prevents ZDHHC8 expression. As one non-limiting example, a test agent may be administered to a transgenic animal in which expression of one or both copies of ZDHHC8 is decreased or prevented, and the animal may be tested for behavior, such as spontaneous exploratory behavior, where an increase in such behavior bears a positive correlation with the ability of the test agent to treat schizophrenia or a related disorder. In another non-limiting example, a cell in which endogenous ZDHHC8 expression is decreased or prevented may be exposed to a test agent, and the level of PSD93 or PSD95 activity, palmitoylation, and/or membrane translocation may be measured, where an increase in the activity, palmitoylation, and/or membrane translocation of either PSD93 or PSD95, especially PSD95, bears a positive correlations with the ability of the test agent to treat schizophrenia or a related disorder. In another non-limiting example, a cell in which endogenous ZDHHC8 expression is decreased or prevented may be exposed to a test agent, and the ratio of excitatory glutamatergic to inhibitory GABAergic synapses may be measured, where an increase in the ratio of excitatory glutamatergic to inhibitory GABAergic synapses bears a positive correlations with the ability of the test agent to treat schizophrenia or a related disorder.
In a fourth set of embodiments, the present invention provides for a method of treating a subject suffering from schizophrenia or a related disorder comprising administering, to the subject, an effective amount of an isolated nucleic acid encoding human ZDHHC8, operably linked to a suitable promoter, optionally comprised in a suitable vector molecule.
For clarity, and not by way of limitation, the detailed description of the invention is divided into the following subsections:
(i) diagnostic methods based on ZDHHC8;
(ii) transgenic animals;
(iii) assay systems; and
(iv) methods of treatment.
The present invention provides for methods for identifying a subject at risk of developing, or suffering from, schizophrenia or a related disorder, comprising identifying, in the individual, a mutation that decreases or prevents expression of wild-type ZDHHC8.
“Schizophrenia” is defined herein as set forth in the Diagnostic and Statistical Manual of Mental Disorders (“DSM-IV”; reference (4)), set forth herein as TABLE 1 (below). Related disorders include schizoaffective disorder, schizophreniform disorder, schizotypal disorder, and schizoid disorder, all as defined in the DSMIV, which is incorporated by reference herein. “Related disorders” also encompass non-human disorders which are considered, in the veterinary art, to be analogous to the above-listed human disorders, and include any one or more of the following in a non-human subject: learning problem, hypoactivity, repetitive behavior, and/or anxious, fearful, or dependent personality.
A subject may be a human or non-human subject, such as a dog, cat, or horse. Preferably, the subject is a human, and may be an adult, infant, child, or fetus.
“A mutation that decreases or prevents expression of wild-type ZDHHC8” means a mutation which may be a deletion, substitution, insertion, or inversion, which leads to a decreased level of ZDHHC8 mRNA and/or ZDHHC8 protein and/or ZDHHC8 activity (e.g., palmitoyltransferase activity) in the patient. The wild-type nucleic acid sequence of ZDHHC8 is set forth in
The foregoing may be determined using a sample from the subject which may be any suitable clinical sample, including but not limited to a blood sample (e.g., collection of peripheral blood lymphocytes), a tissue sample (e.g., epithelial cells), fetal cells obtained through amniocentesis or chorionic villus sampling, sperm cells, etc. To measure PSD93 or PSD95 palmitoylation the sample may be a brain biopsy.
Thus, the present invention may be used to identify an individual at risk of developing schizophrenia. Subjects identified as exhibiting decreased or absent expression of ZDHHHC8; decreased activity of ZDHHC8 (e.g. palmitoyltransferase activity); decreased activity of PSD93 or PSD95 (e.g. mediating the effects of the neurotransmitter glutamine via the formation of protein scaffolding), especially PSD95; or decreased palmitoylation and/or membrane translocation of PSD93 or PSD95, especially PSD95, when compared to a subject not diagnosed with schizophrenia or a related disorder, may be encouraged to enter a pschiatric evaluation and/or treatment program that may result in prompter treatment of symptoms that may arise.
Further, the present invention may be used to aid in establishing a diagnosis of schizophrenia or a related disorder, where decreased or absent ZDHHC8 expression; decreased activity of ZDHHC8; decreased activity of PSD93 or PSD95, especially PSD95; or decreased palmitoylation and/or membrane translocation of PSD93 or PSD95, especially PSD95, when compared to a subject not diagnosed with schizophrenia or a related disorder, is supportive of a diagnosis of schizophrenia or a related condition. Such a diagnostic test may further be used to direct a subject, having decreased or absent ZDHHC8 expression; decreased activity of ZDHHC8; decreased activity of PSD93 or PSD95, especially PSD95; or decreased palmitoylation and/or membrane translocation of PSD93 or PSD95, especially PSD95, when compared to a subject not diagnosed with schizophrenia or a related disorder, to a treatment regimens which compensates for that defect, for example, to a treatment regimen which increases levels of ZDHHC8; increases the activity of ZDHHC8; increases the activity of PSD93 or PSD95, especially PSD95; reintroduces a ZDHHC8, gene, or promotes palmitoylation/membrane translocation of PSD93 or PSD95, especially PSD95.
The present invention provides for a transgenic non-human animal in which expression of at least one copy of endogenous ZDHHC8 is decreased or prevented. In preferred embodiments, expression of both copies of endogenous ZDHHC8 is decreased or prevented, and most preferably, detectable expression of the active protein is absent. In specific, non-limiting embodiments, the transgenic animal is a “knock-out” mouse which is homozygous or heterozygous for a knock-out mutation. Such a mouse may be prepared using standard laboratory techniques.
The present invention further envisions a “knock-out” transgenic animal, in which endogenous ZDHCC8 is expressed at very low or undetectable levels, into which a heterologous ZDHCC8-encoding transgene is introduced. The transgene may be from the same or a different species as its transgenic host. The ZDHCC8 may be wild-type or mutant gene. The transgene may be operably linked to a promoter element, which may be, for example and not by way of limitation, a constitutively active promoter, a neuron specific promoter, and/or an inducible promoter.
Such transgenic animals may be used as experimental models of schizophrenia and related disorders and may be used to identify agents useful in treating schizophrenia and/or related disorders (see below).
The present invention provides for methods and assay systems for identifying agents that may be used to treat schizophrenia or a related disorder in a subject carrying a mutation that decreases or prevents ZDHHC8 expression. As one non-limiting example, a test agent may be administered to a transgenic animal in which expression of one or both copies of ZDHHC8 is decreased or prevented, and the animal may be tested for behavior, such as spontaneous exploratory behavior, (e.g., total distance traveled, or (for rodents) number of vertical moves, or entry into centerfield), where an increase in such behavior bears a positive correlation with the ability of the test agent to treat schizophrenia or a related disorder. In an analogous test in rodents, a test agent which may have therapeutic benefit would increase prepulse inhibition scores (see Example 6, below, for an assay for prepulse inhibition of the acoustic startle response). In preferred, non-limiting embodiments, the transgenic animal is a female, as females tend to exhibit mutant phenotypes more than males.
In another non-limiting example, a cell in which endogenous ZDHHC8 expression is decreased or prevented, or activity is decreased, may be exposed to a test agent, and the level of PSD93 or PSD95 activity, palmitoylation, and/or membrane translocation may be measured, where an increase in the activity, palmitoylation, and/or membrane translocation of either PSD93 or PSD95, especially PS95, bears a positive correlations with the ability of the test agent to treat schizophrenia or a related disorder. The test cell may be a human or a non-human cell, and may be a neuronal or non-neuronal cell.
In a specific non-limiting embodiment of the invention, the cell is a human cell which is heterozygous or homozygous for a mutation that disrupts ZDHHC8 expression, into which a fusion protein comprising PSD95 linked to a fluorescent protein, such as GFP, YFP, BFP, RFP, or CFP. A test agent is administered to said cell, and the cell is evaluated for the effect of test agent on membrane translocation of PSD95, using immunocytochemistry and/or cellular fractionation. The ability of a test agent to increase membrane translocation of PSD95 relative to negative control (absence of test agent) correlates positively with the ability of the test agent to treat schizophrenia or a related disorder. A test agent having such a positive result may then optionally be subjected to further testing, for example, in a transgenic animal having a homozygous or heterozygous mutation in ZDHHC8 expression, where an increase in exploratory behavior or an increase in prepulse inhibition score further positively correlates with therapeutic efficacy.
In an alternative non-limiting embodiment of the invention, an assay may utilize a cell which is a human cell which is heterozygous or homozygous for a mutation that disrupts ZDHHC8 expression. A test agent may be administered to the cell, and the amount of PSD95 palmitoylation may be measured and compared to the amount in a negative control cell. The ability of the test agent to increase palmitoylation correlates positively with the ability of the test agent to treat schizophrenia or a related disorder. A test agent having such a positive result may then optionally be subjected to further testing, for example, in a transgenic animal having a homozygous or heterozygous mutation in ZDHHC8 expression, where an increase in exploratory behavior or an increase in prepulse inhibition score further positively correlates with therapeutic efficacy.
The present invention provides for a method of treating a subject suffering from schizophrenia or a related disorder comprising administering, to the subject, an effective amount of an isolated nucleic acid encoding human ZDHHC8, operably linked to a suitable promoter, optionally comprised in a suitable vector molecule. Suitable promoters include, but are not limited to, constitutively active promoters, neuron specific promoters (e.g., neuron specific enolase promoter), and inducible promoters (e.g., tetracycline inducible promoters). Suitable vectors include, but are not limited to, adenovirus, adeno-associated virus, retrovirus, vaccinia virus, etc. As a non-limiting example, ZDHHC8-encoding nucleic acids which may be used according to the invention include a human ZDHHC8 nucleic acid encoding a protein having a sequence as set forth in GenBank Acc. No. NM—013373 and
The present invention further provides for methods of treating a subject suffering from schizophrenia or a related disorder comprising administering, to the subject, an agent which increases the level of palmitoylated PSD93 and/or PSD95, such as Palmitoyl Coenzyme A, lecithin, or an isolated nucleic acid comprising a nucleic acid encoding human Zdhhc8, operably linked to a suitable promoter.
This working example was essentially published as Mukai J. et al. Evidence that the gene encoding ZDHHC8 contributes to the risk of schizophrenia. Nature Genetics 36, 725-731, published less than a year prior to the filing of this provisional application, and incorporated by reference in its entirety herein.
Construction and analysis of ZDHHC8 minigenes. Primer pair F6-ZDHHC8, R6-ZDHHC8 (TABLE 3, at the end of this section) was used for amplification of the genomic sequence surrounding the alternatively spliced exons 4 and 5 of ZDHHC8. Human genomic DNA homozygous for AA or GG at SNP rs175174 was used as PCR template. The forward primer was tailed with EcoRV, while the reverse primer was tailed with NotI to facilitate cloning. PCR product was purified using Qiaquick PCR Purification Kit (Qiagen). The purified PCR product was then double digested with EcoRV and NotI (New England Biolabs), purified using Qiaquick Gel Purification Kit and ligated into vector pcDNA3.1 (Invitrogen), which was pre-digested with EcoRV and NotI. The ligation product was then transformed into DH5α. Plasmids isolated from the transformants were sequenced to confirm the ZDHHC8 genomic sequence and the genotypes for SNP rs175174. Plasmids of ZDHHC8 minigene constructs were purified using Endofree Plasmid Maxi Kit (Qiagen) and transfected to 293 cells using Lipofectamine 2000 transfection reagent (Life Technologies). 293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine at 37° C. under humidified atmosphere containing 5% CO2. One day before the transfection, 5×105 cells were plated on a 35 mm×10 mm cell culture dish (Corning Incorporated) in the growth medium without antibiotics. On the day of the transfection, 4 μg DNA diluted in 250 μl DMEM was combined with 10 μl LipofectAmine-2000 (Invitrogen) diluted in 250 μl DMEM. The mixture was then incubated at room temperature for 20 min and added to the cell culture dish. The cells were further incubated at 37° C., 5% CO2 for 36-48 h. Total RNA from the transfected cells was purified using RNeasy Mini Purification kit according to the manufacturer's instructions (Qiagen).
Quantification of the ZDHHC8 minigene expression. For semi-quantitative polyacrylamide gel electrophoresis, PCR was performed using Herculase™Enhanced DNA Polymerase following the manufacturer's instructions (Stratagene), except that [α-32P]dCTP was used instead of dCTP to label the PCR product. A touchdown protocol was used for the amplifications, consisting of denaturation at 94° C.×3 min, 15 cycles of 94° C.×30 sec. 68° C. (minus 1° C./cycle)×45 sec, 72° C.×45 sec, 5 cycles of 94° C.×30 sec, 53° C.×45 sec, 72° C.×45 sec, and a final extension cycle of 72° C.×7 min. The PCR products were separated on 4-12% polyacrylamide gels (Invitrogen). The gels were then exposed to KODAK X-OMAT AR film and the band densities were quantified by using MCID-4.0 (Imaging Research Inc.). For real-time quantitative (Taqman) PCR assay, the total RNA was transcripted by using Taqman Reverse Transcription Reagents (Applied Biosystems). A random hexamer was used as the primer for reverse transcription. The transcripts from the minigene were amplified by using Taqman Universal PCR Master Mix following the manufacturer's instructions (Applied Biosystems). The quantitative PCR was performed in an ABI Prism 7700 Sequence Detection System (Applied Biosystems).
Cell culture and transfection. Primary cultures of hippocampal neurons were obtained from mice embryos at embryonic day 15-16. The entire hippocampus was isolated and dissociated with trypsin treatment and trituration, and cells were plated on poly-L-lysine-coated 12 mm glass coverslips at a cell density of 4×104. Neurons were maintained in Neurobasal media (Gibco) supplemented with B-27 (Gibco), 0.5 mM L-glutamine, penicillin and streptomycin. For transfection of ZDHHC8 and YFP-PSD-95, hippocampal cultures were transfected with 0.5 μg of DNA using LipofectAmine-2000 in Neurobasal media supplemented with B-27 and 0.5 mM L-glutamine. HeLa cells were obtained from the American Type Culture Collection and maintained in DMEM supplemented with 10% fetal bovine serum. For transfection, HeLa cells (2×104 per 12 mm cover slip) were transiently transfected with 0.5 μg of DNA using LipofectAmine-2000 in DMEM supplemented with 10% fetal bovine serum and cultured for 18 h. Expression vector for human ZDHHC8 was constructed by PCR amplification of ZDHHC8 coding sequence using the oligonucleotide pair: 5′-CGGAATTCGCCGCCACCATGCCCCGCAGCCCC (SEQ. ID. NO. 20) and 5′-CGGGATCCTATCACACCGAGATCTCGTAGGTGGT (SEQ. ID. NO. 21), digestion with EcoRI/BamHI, and ligation of the resulting fragment into EcoRI/BamHI-digested pcDNA3.1/Myc-His(−)C.
Immunocytochemistry. The 280GP antibody was obtained by immunization of guinea pigs with a synthetic peptide derived from the C-terminal sequence of human ZDHHC8 (KKVSGVGGTTYEISV) (SEQ. ID. NO.: 17). Pilot experiments indicated that 280GP recognizes only exogenously transfected protein with high specificity. For double-staining of ZDHHC8 and NMDA receptor, neurons were fixed with methanol for 10 min at −20° C., blocked with 1% BSA and 1% normal goat serum, and incubated with 280GP and mouse monoclonal antibody to NMDAR2B (BD Transduction Laboratories) at 1:200 dilutions for 1 h at room temperature. After several rinses with PBS, cells were incubated with the anti-guinea pig IgG-Cy3 (Jackson ImmunoResearch Laboratory; diluted 1:100 in blocking solution) and anti-mouse Cy5 (Jackson ImmunoResearch Laboratory; diluted 1:100 in blocking solution) at room temperature for 30 min. For HeLa cells, cells were fixed with 3.7% paraformaldehyde for 30 min at room temperature, and permeabilized with 0.1% sodium citrate containing 0.1% Triton X-100 for 20 min on ice. After blocking with 1% BSA and 1% normal goat serum for 1 h at room temperature, cells were incubated with anti-EEA 1 mouse monoclonal antibody (BD Transduction Laboratories) at 1:50 dilutions, anti-mannose 6-phosphate receptor mouse monoclonal antibody (Affinity Bioreagents) at 1:100 dilutions, anti-GM130 mouse monoclonal antibody (BD Transduction Laboratories) at 1:100 dilutions, anti-golgin-97 mouse monoclonal antibody (Molecular Probes) at 1:100 dilutions, and anti-GRP-78 rabbit polyclonal antibody (Stressgen) at 1:100 dilutions as primary antibodies. After several rinses with PBS, cells were incubated with the anti-guinea pig IgG-Cy3, and anti-mouse IgG-Alexa Fluor 488 (Molecular Probes; diluted 1:100 in blocking solution), or anti-rabbit IgG-Alexa Fluor 488 (Molecular Probes; diluted 1:100 in blocking solution) at room temperature for 30 min. To-Pro-3 iodide (Molecular Probes) was used to visualize the nucleus. Coverslips were mounted on Vectashield (Vector Laboratory). Fluorescent images of cells were captured and analyzed on LSM510 Meta Confocal laser-scanning microscope (Zeiss).
Gross brain morphology. Four Zdhhc8 null and four wild-type littermate mice, 9-15 weeks of age, were used. Brains were post-fixed in 4% paraformaldehyde and transferred to 30% sucrose at 4° C. until they sank. Using a sliding microtome, 40 mm coronal sections were obtained and one series of every four sections through the entire brain was Nissl-stained with cresyl violet (Sigma). Regions of interest were digitally photographed (Spot RT Camera, Diagnostic Instruments, Inc.) at 40× and 100× magnification (Nikon E800 microscope). The photographs were analyzed using NIH Image program (1.63).
Generation of the Zdhhc8 knockout mice. Homologous recombination in ES cells was performed as described previously (39) using standard homologous recombination methods (see
Assay for prepulse inhibition of the acoustic startle response. Adult (4-mo old) Zdhhc8 null and wild-type littermate mice were housed individually for 2 weeks prior to testing. Testing was conducted in a SR-Lab system (San Diego Instruments). Response amplitude was calculated as the maximum response level occurring during the 100 ms recording. Because animals can in principle habituate to the prepulse, as well as to the startle stimulus, the number of trials was kept to the essential minimum. Immediately after placement in the chamber, the animal was given a 5 min acclimation period during which background noise (67 dB) was continually present, and then received 8 sets of the following 4 trial types distributed pseudorandomly and separated by an average of 15 sec intertrial intervals: Trial 1: 40 ms, 120 dB noise burst alone; Trial 2-3: 120 dB startle stimuli preceded 100 ms by a 20 ms, 78 dB or 82 dB noise burst (prepulse); Trial 4: no-stimulus/background noise alone (67 dB). Data was analyzed using ANOVA repeated measures.
Open field assay. Animals were not pre-exposed to the chamber before testing. Activity was monitored in a clear acrylic chamber directly illuminated and equipped with infrared sensors for the automatic recording of horizontal and vertical activity (Colbourn Instruments). Each animal was placed initially in the periphery of the chamber, and recordings of the total distance traveled, the number of rearings (vertical moves) and the number of entries in the centerfield over the next 15 min were used as a measure of a locomotor response to novelty. 47 female mice (17 homozygous Zdhhc8 mutant, 17 heterozygous Zdhhc8 mutant and 13 wild-type littermate controls) and 45 male mice (13 homozygous Zdhhc8 mutant, 19 heterozygous Zdhhc8 mutant and 13 wild-type littermate controls) were tested.
Drug administration. MK801 was purchased from Sigma. Each experimental group consisted of 8-10 mice, which received the same number of injections, administered intraperitoneally, of either drug (0.4 mg per kg body weight) or vehicle. After 30 minutes of prehabituation in the open field, the mice were removed, injected and then left to rest for 10 minutes in their home cage before returning them to the open field arena, where they were monitored for an additional 30 minutes.
Fetal brain samples. Tissues were obtained from the tissue collection and distribution program at the University of Washington, Laboratory for the study of embryology. The tissues were from 90-120 day old specimens and were snap-frozen in liquid nitrogen.
Patient samples. Both our US and Afrikaner adult schizophrenia family samples have been described in detail elsewhere (19, 13). All methods were approved by Institutional Review Boards at participating sites and all participants signed appropriate informed consent. Allele transmission calculations were performed using the TDT method as described elsewhere (19, 13).
Association with schizophrenia in the distal part of the 22q11 locus implicated 5 neighboring SNPs distributed within a haplotypic block of 80 kb and having alleles with nominally significant association results (13;
Preliminary analysis of EST databases identified alternatively spliced variants of the ZDHHC8 gene involving exons 4 and 5 (13). Therefore, a potential effect of rs175174 genotype on splicing warranted further investigation. Total or polyA+ RNA was prepared from fresh (non-transformed) human lymphocytes and subjected to reverse transcription and PCR amplification using primers located in exons 1 and 8. The PCR products were separated on a 1.2% agarose gel, purified and sequenced. This analysis revealed a highly reproducible, complex pattern of alternative splicing around intron 4. Interestingly, the two major products of comparable abundance were identified in both the total (
It was determined whether the genotype at SNP rs175174 affects the rate of intron 4 retention and therefore the ratio of the intron 4-containing splice form over the fully spliced form. Specifically, we examined whether presence of the risk allele A results in the production of relatively higher levels of the unspliced form. Minigenes from the “risk” and “non-risk” alleles containing exon 4, intron 4, exon 5, as well as flanking genomic sequences were constructed and transfected into 293 cells. Both minigenes were sequenced in their entirety and no variation beyond the rs175174 A/G was present. Initial semi-quantitative PCR amplification analysis revealed that the pattern of splicing observed in lymphocytes was recapitulated in total RNA prepared from 293 cells transiently transfected with the minigene (
ZDHHC8 is predicted to have four transmembrane (TM) domains and a cysteine-rich domain that includes a DHHC motif and a Cys4 zinc-finger-like (Z) metal binding site (
Recent work has shown that palmitate reversibly modifies numerous classes of neuronal proteins, including proteins important for neuronal development, neurotransmitter receptors and synaptic scaffolding proteins (29). ZDHHC8 is widely expressed in the adult human brain (13). More detailed analysis of expression in the adult mouse brain demonstrated higher expression levels in the cortex and hippocampus (
Given the effects of ZDHHC8 deficit on behavior, it may be relevant that protein palmitoylation modulates several neurotransmitter systems (29), including activity-dependent plasticity at glutamatergic synapses (38). The effects of the psychomimetic dizocilpine (MK801), which is traditionally considered to be a NMDA-receptor blocker but probably also results in secondary activation of non-NMDA receptor glutamatergic neurotransmission by increasing glutamate efflux (41), in homozygous Zdhhc8 mutant mice and wild-type littermate controls. The ratio of activity used was during the half-hour after the injection to the activity during the half-hour before the injection as an index of the locomotor-activating effects of the drug. In terms of ratio index, homozygous Zdhhc8 mutant female (but not male) mice seemed to be somewhat less sensitive to MK801-induced stimulation of locomotor activity than wild-type littermate controls (P=0.05;
Experiments were performed to determine whether a similar sexual dimorphism exists between functional variants of the ZDHHC8 gene and schizophrenia in humans. SNP rs175174 was examined in an extended sample of 389 parent-proband triads from the US and the Afrikaner population of South Africa that includes the families described in the original study. Indeed, in the extended sample of nuclear families the presence of a sexually dimorphic effect was confirmed that was also initially observed in the discovery sample (13; TABLE 2, at the end of this section). Specifically, analysis of allele transmission by gender revealed a, striking sex-related heterogeneity of transmission (heterogeneity χ2=8.2, P=0.004). The effect of SNP rs175174 was found to be highly significant in females (allele A predisposing χ2=12.2, P=0.0005, T/U: 82/43). Males did not show any effect (χ2=0.02, P=0.88, T/U: 106/108). Interestingly, two recent studies involving large-scale collections of schizophrenic patients also identified striking female-specific effects for variants of two strong candidate schizophrenia susceptibility genes (32, 33), including the gene for COMT also located within the 22q11 microdeletion.
An emerging “genetic” picture is that the 22q11 microdeletion-associated schizophrenia may have the characteristics of a contiguous gene syndrome where more than one gene may contribute to the dramatic increase in disease risk (13). Systematic approaches as well as candidate gene studies designed to identify schizophrenia susceptibility genes from the 22q 11 region have implicated so far the genes encoding PRODH (20, 34, 35), COMT (32, 36) and ZDHHC8 (13 and herein). Recent studies in animal models (17) have started addressing potential interactions between these genes. These studies have shown that PRODH deficiency (that likely results in dysregulation of glutamate transmission (17, 36)) leads to a secondary dopaminergic hyperresponsivity and subsequent compensatory changes that include increase in the levels of COMT-mediated dopamine breakdown. Therefore, deficiency of the 22q11 schizophrenia susceptibility genes modulates the disease risk either by impairing synaptic function or by failing to compensate for such impairment. Such synergistic interaction could in principle lead to the unprecedented increase in schizophrenia risk associated with microdeletions of this locus. It is of considerable interest that protein palmitoylation can modulate several neurotransmitter systems (29, 37) and, most notably, as shown in recent studies, can regulate synaptic strength and activity-dependent plasticity at glutamatergic synapses (38).
a.F, forward (top-strand) primer; R, reverse (bottom-strand) primer.
b.Underlined sequences represent the restriction enzyme sites used for cloning.
Using the yeast two-hybrid approach as well as co-immunoprecipitation assays (
A series of ZDHHC8 mutants were prepared, either lacking the DHHC motif (“ΔDHHC”); carrying a mutation substituting cysteine for alanine at position 134, so that the DHHC motif become DHHA (“C134”); or lacking the C terminus (“230”). As shown in
The foregoing results are consistent with PSD95 being an authentic substrate of ZDHHC8. Activity-dependent palmitoylation of PSD95 has been known to affect plasticity at glutamatergic synapses. This is important because it is widely believed that impaired glutamate transmission underlies both the cognitive and negative symptoms of schizophrenia.
It is widely believed that impaired glutamate transmission underlies both the cognitive and negative symptoms of schizophrenia. Abnormal expression of Post-Synaptic Density (PSD)-proteins, which form a protein scaffold mediating the effects of the neurotransmitter glutamate, is thought to be involved in the pathophysiology of schizophrenia. Recently, it was shown that the level of one of these proteins (PSD95) was significantly decreased in the frontal cortex of individuals with schizophrenia (49).
We have shown that PSD95 is a substrate for ZDHHC8 palmitoyltransferase activity in vitro and in vivo. The palmitoylation of PSD-95 is critical for PSD95 clustering of AMPA receptors at excitatory glutamatergic synapses (38), and is dynamically regulated by synaptic activity, such that cycling of palmitate on PSD95 can contribute to aspects of synaptic plasticity (38). Accordingly, we analyzed the effects of Zdhhc8 deficiency on synaptic function in primary cultured hippocampal neurons. We found that the total protein expression of PSD95 and surface expression of GluR2 receptor were reduced in neurons from heterozygous as well as homozygous Zdhhc8-deficient mice, indicating that ZDHHC8 plays fundamental roles in controlling glutamate transmission, consistent with its role as a schizophrenia susceptibility gene.
The balance between excitatory and inhibitory synapses is a tightly regulated process that requires differential recruitment of proteins that dictate the specificity of newly formed contacts. Decreased expression of PSD95 induced changes in the number of excitatory versus inhibitory contacts and results in overall decrease in the ratio of excitatory (glutamatergic) to inhibitory (GABAergic) synaptic currents (50). We found that the reduction of PSD95 induced by Zdhhc8 deficiency resulted in a decrease in the ratio of excitatory/inhibitory synaptic number in neurons from heterozygous as well as homozygous Zdhhc8-deficient mice (
We also found that the deficiency in Zdhhc8 in hippocampal neurons affects dendritic tree morphogenesis (
Various publications are cited herein, the contents of which are hereby incorporated by reference in their entireties.
This application claims priority to U.S. Provisional Patent Application Ser. No. 60/686,178, filed Jun. 1, 2005, the contents of which is hereby incorporated by reference in its entirety herein.
The subject matter of this application was developed, at least in part, under a grant from the United States National Institute of Mental Health Grant No. RO1 MH067068, so that the United States Government holds certain rights herein.
| Number | Date | Country | |
|---|---|---|---|
| 60686178 | Jun 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US06/21446 | Jun 2006 | US |
| Child | 11947361 | US |
