FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Jan 18, 2015
Epidermal growth factor receptor (EGFR) plays pivotal roles in cell proliferation, differentiatio... more Epidermal growth factor receptor (EGFR) plays pivotal roles in cell proliferation, differentiation, and tissue development, while EGFs protect neurons from toxic insults by binding EGFR and stimulating survival signaling. Furthermore, recent evidence implicates this receptor in neurometabolic disorders like Alzheimer disease and aging. Here we show that absence of presenilin 1 (PS1) results in dramatic decrease (>95%) of neuronal EGFR and that PS1-null (PS1(-/-)) brains have reduced amounts of this receptor. PS1(-/-) cortical neurons contain little EGFR and show no epidermal growth factor-induced survival signaling or protection against excitotoxicity, but exogenous EGFR rescues both functions even in absence of PS1. EGFR mRNA is greatly reduced (>95%) in PS1(-/-) neurons, and PS1(-/-) brains contain decreased amounts of this mRNA, although PS1 affects the stability of neither EGFR nor its mRNA. Exogenous PS1 increases neuronal EGFR mRNA, while down-regulation of PS1 decreases...
Haploinsufficiency of the Shank3 gene, which encodes a scaffolding protein at glutamatergic synap... more Haploinsufficiency of the Shank3 gene, which encodes a scaffolding protein at glutamatergic synapses, is a highly prevalent and penetrant risk factor for autism. Using combined behavioral, electrophysiological, biochemical, imaging, and molecular approaches, we find that Shank3-deficient mice exhibit autism-like social deficits and repetitive behaviors, as well as the significantly diminished NMDA receptor (NMDAR) synaptic function and synaptic distribution in prefrontal cortex. Concomitantly, Shank3-deficient mice have a marked loss of cortical actin filaments, which is associated with the reduced Rac1/PAK activity and increased activity of cofilin, the major actin depolymerizing factor. The social deficits and NMDAR hypofunction are rescued by inhibiting cofilin or activating Rac1 in Shank3-deficient mice and are induced by inhibiting PAK or Rac1 in wild-type mice. These results indicate that the aberrant regulation of synaptic actin filaments and loss of synaptic NMDARs contribut...
PNPLA6 mutations, known to be associated with the development of motor neuron phenotypes, have re... more PNPLA6 mutations, known to be associated with the development of motor neuron phenotypes, have recently been identified in families with Boucher-Neuhäuser syndrome. Boucher-Neuhäuser is a rare autosomal recessive syndrome characterized by the co-occurrence of cerebellar ataxia, hypogonadotropic hypogonadism, and chorioretinal dystrophy. Gait ataxia in Boucher-Neuhäuser usually manifests before early adulthood, although onset in the third or fourth decade has also been reported. However, given the recent identification of PNPLA6 mutations as the cause of this condition, the determining factors of age of symptom onset still need to be established. Here, we have identified a sporadic Boucher-Neuhäuser case with lateonset gait ataxia and relatively milder retinal changes due to compound heterozygous PNPLA6 mutations. Compound heterozygosity was confirmed by cloning and sequencing the patient's genomic DNA from coding exons 26-29. Furthermore, both mutations (one novel and one known) fell in the phospholipase esterase domain, where most pathogenic mutations seem to cluster. Taken together, we herein confirm PNPLA6 mutations as the leading cause of Boucher-Neuhäuser syndrome and suggest inquiring about a history of hypogonadism or visual changes in patients presenting with late-onset gait ataxia. We also advocate for neuroophthalmologic evaluation in suspected cases.
... Abstract/OtherAbstract: A new circadian variant was isolated by screening the intercross offs... more ... Abstract/OtherAbstract: A new circadian variant was isolated by screening the intercross offspring of wild-caught mice (Mus musculus castaneus). ... Tsuyoshi Watanabe; Tohru Suzuki; Akira Ishikawa; Yuki Yokota; Hiroki R Ueda; Rikuhiro G Yamada; Hajime Tei; Saki Imai; Shigeru ...
Obsessive compulsive disorder (OCD) is a syndrome characterized by recurrent and intrusive though... more Obsessive compulsive disorder (OCD) is a syndrome characterized by recurrent and intrusive thoughts and ritualistic behaviors or mental acts that a person feels compelled to perform. Twin studies, family studies, and segregation analyses provide compelling evidence that OCD has a strong genetic component. The SLITRK1 gene encodes a developmentally regulated stimulator of neurite outgrowth and previous studies have implicated rare variants in this gene in disorders in the OC spectrum, specifically Tourette syndrome (TS) and trichotillomania (TTM). The objective of the current study was to evaluate rare genetic variation in SLITRK1 in risk for OCD and to functionally characterize associated coding variants. We sequenced SLITRK1 coding exons in 381 individuals with OCD as well as in 356 control samples and identified three novel variants in seven individuals. We found that the combined mutation load in OCD relative to controls was significant (p = 0.036). We identified a missense N400I change in an individual with OCD, which was not found in more than 1000 control samples (P,0.05). In addition, we showed the the N400I variant failed to enhance neurite outgrowth in primary neuronal cultures, in contrast to wildtype SLITRK1, which enhanced neurite outgrowth in this assay. These important functional differences in the N400I variant, as compared to the wildtype SLITRK1 sequence, may contribute to OCD and OC spectrum symptoms. A synonymous L63L change identified in an individual with OCD and an additional missense change, T418S, was found in four individuals with OCD and in one individual without an OCD spectrum disorder. Examination of additional samples will help assess the role of rare SLITRK1 variation in OCD and in related psychiatric illness.
The Alzheimer disease (AD) amyloid protein precursor (APP) can bind the FE65 adaptor protein and ... more The Alzheimer disease (AD) amyloid protein precursor (APP) can bind the FE65 adaptor protein and this complex can regulate gene expression. We carried out yeast two-hybrid studies with a PTB domain of FE65, focusing on those genes that might be involved in nuclear signaling, and identified and validated Teashirt proteins as FE65 interacting proteins in neurons. Using reporter systems, we observed that FE65 could simultaneously recruit SET, a component of the inhibitor of acetyl transferase, and Teashirt, which in turn recruited histone deacetylases, to produce a powerful gene-silencing complex. We screened stable cell lines with a macroarray focusing on AD-related genes and identified CASP4, encoding caspase-4, as a target of this silencing complex. Chromatin immunoprecipitation showed a direct interaction of FE65 and Teashirt3 with the promoter region of CASP4. Expression studies in postmortem samples demonstrated decreasing expression of Teashirt and increasing expression of caspase-4 with progressive cognitive decline. Importantly, there were significant increases in caspase-4 expression associated with even the earliest neuritic plaque changes in AD. We evaluated a case-control cohort and observed evidence for a genetic association between the Teashirt genes TSHZ1 and TSHZ3 and AD, with the TSHZ3 SNP genotype correlating with expression of Teashirt3. The results were consistent with a model in which reduced expression of Teashirt3, mediated by genetic or other causes, increases caspase-4 expression, leading to progression of AD. Thus the cell biological, gene expression and genetic data support a role for Teashirt/caspase-4 in AD biology. As caspase-4 shows evidence of being a primate-specific gene, current models of AD and other neurodegenerative conditions may be incomplete because of the absence of this gene in the murine genome.
Neuregulin and the neuregulin receptor ERBB4 have been genetically and functionally implicated in... more Neuregulin and the neuregulin receptor ERBB4 have been genetically and functionally implicated in schizophrenia. In this study, we used the yeast two-hybrid system to identify proteins that interact with ERBB4, to identify genes and pathways that might contribute to schizophrenia susceptibility. We identified the MAGI scaffolding proteins as ERBB4-binding proteins. After validating the interaction of MAGI proteins with ERBB4 in mammalian cells, we demonstrated that ERBB4 expression, alone or in combination with ERBB2 or ERBB3, led to the tyrosine phosphorylation of MAGI proteins, and that this could be further enhanced with receptor activation by neuregulin. As MAGI proteins were previously shown to interact with receptor phosphotyrosine phosphatase b/f (RPTPb), we postulated that simultaneous binding of MAGI proteins to RPTPb and ERBB4 forms a phosphotyrosine kinase/phosphotyrosine phosphatase complex. Studies in cultured cells confirmed both a spatial and functional association between ERBB4, MAGI and RPTPb. Given the evidence for this functional association, we examined the genes coding for MAGI and RPTPb for genetic association with schizophrenia in a Caucasian United Kingdom case-control cohort (n = B1400). PTPRZ1, which codes for RPTPb, showed significant, gene-wide and hypothesis-wide association with schizophrenia in our study (best individual single-nucleotide polymorphism allelic P = 0.0003; gene-wide P = 0.0064; hypothesiswide P = 0.026). The data provide evidence for a role of PTPRZ1, and for RPTPb signaling abnormalities, in the etiology of schizophrenia. Furthermore, the data indicate a role for RPTPb in the modulation of ERBB4 signaling that may in turn provide further support for an important role of neuregulin/ERBB4 signaling in the molecular basis of schizophrenia.
The low-density lipoprotein receptor related protein 1 (LRP1) has been implicated in Alzheimer's ... more The low-density lipoprotein receptor related protein 1 (LRP1) has been implicated in Alzheimer's disease (AD) but its signalling has not been fully evaluated. There is good evidence that the cytoplasmic domain of LRP1 is involved in protein-protein interactions, important in the cell biology of LRP1.
Calsenilin/potassium channel-interacting protein (KChIP)3/ downstream regulatory element sequence... more Calsenilin/potassium channel-interacting protein (KChIP)3/ downstream regulatory element sequence antagonist modulator (DREAM) is a neuronal calcium-binding protein that has been shown to have multiple functions in the cell, including the regulation of presenilin processing, repression of transcription and modulation of A-type potassium channels. To gain a better understanding of the precise role of calsenilin in specific cellular compartments, an interactor hunt for proteins that bind to the N-terminal domain of calsenilin was carried out. Using a yeast two-hybrid system and co-immunoprecipitation studies, we have identified the transcriptional co-repressor C-terminal binding protein (CtBP)2 as an interactor for calsenilin and have shown that the two proteins can interact in vivo. In co-immunoprecipitation studies, calsenilin also interacted with CtBP1, a CtBP2 homolog. Our data also showed a calsenilindependent increase in c-fos protein levels in CtBP knockout fibroblasts, suggesting that CtBP may modulate the transcriptional repression of c-fos by calsenilin. Furthermore, the finding that histone deacetylase protein and activity were associated with the calsenilin-CtBP immunocomplex suggests a mechanism by which calsenilin-CtBP may act to repress transcription. Finally, we demonstrated that calsenilin and CtBP are present in synaptic vesicles and can interact in vivo.
The hnRNP C protein tetramer cooperatively binds 230 nt increments of pre-mRNA in vitro in a salt... more The hnRNP C protein tetramer cooperatively binds 230 nt increments of pre-mRNA in vitro in a salt-resistant manner and is located along the length of vertebrate transcripts in vivo. Based on these and other ®ndings it has been suggested that hnRNP C functions as a chaperonin to maintain long lengths of RNA topologically single-stranded and accessible to splicing factors. We report here that human C protein is lethal when expressed in the yeast Saccharomyces cerevisiae. Through a series of¯uorescent immunolocalization studies, lethality was observed to be associated with the rapid nuclear accumulation of both C protein and yeast pre-mRNA. Studies using various protein constructs and the two hybrid assay reveal that these events are dependent on the basic 40 residue high-af®nity RNA binding domain and its contiguous leucine zipper-like motif (the bZLM, residues 140-214). Additionally, equilibrium binding studies have shown that the bZLM is the determinant of C protein's saltresistant RNA binding mode. Taken together, these ®ndings further distinguish the bZIP-like domain as the major determinant of C protein's high-af®nity interaction with RNA, oligomerization, and its highly cooperative RNA binding activity. Finally, these ®ndings indicate that yeast and vertebrates may possess a conserved mechanism for general import of RNP although a true homolog to vertebrate C protein appears not to exist in yeast. Lethality is likely due to the absence in yeast of speci®c mechanisms for the removal of human C protein from nascent transcripts.
There is strong evidence for rare, highly penetrant genetic variants playing an etiological role ... more There is strong evidence for rare, highly penetrant genetic variants playing an etiological role in multiple neurodevelopmental disabilities, including autism spectrum disorders. The rate of discovery of such rare variants is increasing with the advent of larger sample collections, chromosome microarray analyses, and high-throughput sequencing. As the variants that are being discovered can be highly penetrant, they lead immediately to model systems with construct validity, critical for understanding the underlying neurobiology of these conditions, which in turn can provide leads for novel therapeutic targets. Moreover, these discoveries can benefit families with information about recurrence risk, resolve concerns about etiology, provide information about associated medical issues, and engender directed advocacy for specific genetic conditions. For these reasons, diagnostic laboratories are taking advantage of research data as they are produced. In the current report, we present our molecular analysis of a child with a purported disruptive mutation in SHANK3 identified by a commercial genetic testing laboratory and we provide evidence that this was not an etiological variant. The variant was a 1-bp insertion in exon 11 of the RefSeq gene, which we then determined was inherited from a healthy mother and found in~1% of controls. Since the variant would be predicted to disrupt the reference gene, and the penetrance of SHANK3 mutations is very high, we did follow up molecular and bioinformatic analyses and concluded that the presumptive exon containing the variant is not likely to be present in most or all SHANK3 transcripts. The results highlight difficulties that can arise with rapid translation of research findings to clinical practice. Researchers are in a unique position to generate resources with collated and curated information that can inform research, genetic testing, clinicians, and families about the best practices as pertains to rare genetic variants in neurodevelopmental disabilities. Of immediate importance would be a B R A I N R E S E A R C H 1 3 8 0 ( 2 0 1 1 ) 9 8 -1 0 5 ⁎ Corresponding author. Mount a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m w w w . e l s e v i e r . c o m / l o c a t e / b r a i n r e s well-curated database of gene variation identified in large numbers of typically developing individuals and in individuals affected with neurodevelopmental disabilities. Such a database would reduce false-positive results in clinical settings, would be helpful in structure-function analyses, and would direct translational research to pathways most likely to benefit families.
Background: Rare genetic variants of SLITRK1 have been previously associated with Tourette syndro... more Background: Rare genetic variants of SLITRK1 have been previously associated with Tourette syndrome (TS), attention-deficit/hyperactivity disorder (ADHD), and obsessive-compulsive disorder (OCD) symptoms.
Copy number variation (CNV) is an important determinant of human diversity and plays important ro... more Copy number variation (CNV) is an important determinant of human diversity and plays important roles in susceptibility to disease. Most studies of CNV carried out to date have made use of chromosome microarray and have had a lower size limit for detection of about 30 kilobases (kb). With the emergence of whole-exome sequencing studies, we asked whether such data could be used to reliably call rare exonic CNV in the size range of 1-30 kilobases (kb), making use of the eXome Hidden Markov Model (XHMM) program. By using both transmission information and validation by molecular methods, we confirmed that small CNV encompassing as few as three exons can be reliably called from whole-exome data. We applied this approach to an autism case-control sample (n ¼ 811, mean per-target read depth ¼ 161) and observed a significant increase in the burden of rare (MAF %1%) 1-30 kb CNV, 1-30 kb deletions, and 1-10 kb deletions in ASD. CNV in the 1-30 kb range frequently hit just a single gene, and we were therefore able to carry out enrichment and pathway analyses, where we observed enrichment for disruption of genes in cytoskeletal and autophagy pathways in ASD. In summary, our results showed that XHMM provided an effective means to assess small exonic CNV from whole-exome data, indicated that rare 1-30 kb exonic deletions could contribute to risk in up to 7% of individuals with ASD, and implicated a candidate pathway in developmental delay syndromes.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Jan 18, 2015
Epidermal growth factor receptor (EGFR) plays pivotal roles in cell proliferation, differentiatio... more Epidermal growth factor receptor (EGFR) plays pivotal roles in cell proliferation, differentiation, and tissue development, while EGFs protect neurons from toxic insults by binding EGFR and stimulating survival signaling. Furthermore, recent evidence implicates this receptor in neurometabolic disorders like Alzheimer disease and aging. Here we show that absence of presenilin 1 (PS1) results in dramatic decrease (>95%) of neuronal EGFR and that PS1-null (PS1(-/-)) brains have reduced amounts of this receptor. PS1(-/-) cortical neurons contain little EGFR and show no epidermal growth factor-induced survival signaling or protection against excitotoxicity, but exogenous EGFR rescues both functions even in absence of PS1. EGFR mRNA is greatly reduced (>95%) in PS1(-/-) neurons, and PS1(-/-) brains contain decreased amounts of this mRNA, although PS1 affects the stability of neither EGFR nor its mRNA. Exogenous PS1 increases neuronal EGFR mRNA, while down-regulation of PS1 decreases...
Haploinsufficiency of the Shank3 gene, which encodes a scaffolding protein at glutamatergic synap... more Haploinsufficiency of the Shank3 gene, which encodes a scaffolding protein at glutamatergic synapses, is a highly prevalent and penetrant risk factor for autism. Using combined behavioral, electrophysiological, biochemical, imaging, and molecular approaches, we find that Shank3-deficient mice exhibit autism-like social deficits and repetitive behaviors, as well as the significantly diminished NMDA receptor (NMDAR) synaptic function and synaptic distribution in prefrontal cortex. Concomitantly, Shank3-deficient mice have a marked loss of cortical actin filaments, which is associated with the reduced Rac1/PAK activity and increased activity of cofilin, the major actin depolymerizing factor. The social deficits and NMDAR hypofunction are rescued by inhibiting cofilin or activating Rac1 in Shank3-deficient mice and are induced by inhibiting PAK or Rac1 in wild-type mice. These results indicate that the aberrant regulation of synaptic actin filaments and loss of synaptic NMDARs contribut...
PNPLA6 mutations, known to be associated with the development of motor neuron phenotypes, have re... more PNPLA6 mutations, known to be associated with the development of motor neuron phenotypes, have recently been identified in families with Boucher-Neuhäuser syndrome. Boucher-Neuhäuser is a rare autosomal recessive syndrome characterized by the co-occurrence of cerebellar ataxia, hypogonadotropic hypogonadism, and chorioretinal dystrophy. Gait ataxia in Boucher-Neuhäuser usually manifests before early adulthood, although onset in the third or fourth decade has also been reported. However, given the recent identification of PNPLA6 mutations as the cause of this condition, the determining factors of age of symptom onset still need to be established. Here, we have identified a sporadic Boucher-Neuhäuser case with lateonset gait ataxia and relatively milder retinal changes due to compound heterozygous PNPLA6 mutations. Compound heterozygosity was confirmed by cloning and sequencing the patient's genomic DNA from coding exons 26-29. Furthermore, both mutations (one novel and one known) fell in the phospholipase esterase domain, where most pathogenic mutations seem to cluster. Taken together, we herein confirm PNPLA6 mutations as the leading cause of Boucher-Neuhäuser syndrome and suggest inquiring about a history of hypogonadism or visual changes in patients presenting with late-onset gait ataxia. We also advocate for neuroophthalmologic evaluation in suspected cases.
... Abstract/OtherAbstract: A new circadian variant was isolated by screening the intercross offs... more ... Abstract/OtherAbstract: A new circadian variant was isolated by screening the intercross offspring of wild-caught mice (Mus musculus castaneus). ... Tsuyoshi Watanabe; Tohru Suzuki; Akira Ishikawa; Yuki Yokota; Hiroki R Ueda; Rikuhiro G Yamada; Hajime Tei; Saki Imai; Shigeru ...
Obsessive compulsive disorder (OCD) is a syndrome characterized by recurrent and intrusive though... more Obsessive compulsive disorder (OCD) is a syndrome characterized by recurrent and intrusive thoughts and ritualistic behaviors or mental acts that a person feels compelled to perform. Twin studies, family studies, and segregation analyses provide compelling evidence that OCD has a strong genetic component. The SLITRK1 gene encodes a developmentally regulated stimulator of neurite outgrowth and previous studies have implicated rare variants in this gene in disorders in the OC spectrum, specifically Tourette syndrome (TS) and trichotillomania (TTM). The objective of the current study was to evaluate rare genetic variation in SLITRK1 in risk for OCD and to functionally characterize associated coding variants. We sequenced SLITRK1 coding exons in 381 individuals with OCD as well as in 356 control samples and identified three novel variants in seven individuals. We found that the combined mutation load in OCD relative to controls was significant (p = 0.036). We identified a missense N400I change in an individual with OCD, which was not found in more than 1000 control samples (P,0.05). In addition, we showed the the N400I variant failed to enhance neurite outgrowth in primary neuronal cultures, in contrast to wildtype SLITRK1, which enhanced neurite outgrowth in this assay. These important functional differences in the N400I variant, as compared to the wildtype SLITRK1 sequence, may contribute to OCD and OC spectrum symptoms. A synonymous L63L change identified in an individual with OCD and an additional missense change, T418S, was found in four individuals with OCD and in one individual without an OCD spectrum disorder. Examination of additional samples will help assess the role of rare SLITRK1 variation in OCD and in related psychiatric illness.
The Alzheimer disease (AD) amyloid protein precursor (APP) can bind the FE65 adaptor protein and ... more The Alzheimer disease (AD) amyloid protein precursor (APP) can bind the FE65 adaptor protein and this complex can regulate gene expression. We carried out yeast two-hybrid studies with a PTB domain of FE65, focusing on those genes that might be involved in nuclear signaling, and identified and validated Teashirt proteins as FE65 interacting proteins in neurons. Using reporter systems, we observed that FE65 could simultaneously recruit SET, a component of the inhibitor of acetyl transferase, and Teashirt, which in turn recruited histone deacetylases, to produce a powerful gene-silencing complex. We screened stable cell lines with a macroarray focusing on AD-related genes and identified CASP4, encoding caspase-4, as a target of this silencing complex. Chromatin immunoprecipitation showed a direct interaction of FE65 and Teashirt3 with the promoter region of CASP4. Expression studies in postmortem samples demonstrated decreasing expression of Teashirt and increasing expression of caspase-4 with progressive cognitive decline. Importantly, there were significant increases in caspase-4 expression associated with even the earliest neuritic plaque changes in AD. We evaluated a case-control cohort and observed evidence for a genetic association between the Teashirt genes TSHZ1 and TSHZ3 and AD, with the TSHZ3 SNP genotype correlating with expression of Teashirt3. The results were consistent with a model in which reduced expression of Teashirt3, mediated by genetic or other causes, increases caspase-4 expression, leading to progression of AD. Thus the cell biological, gene expression and genetic data support a role for Teashirt/caspase-4 in AD biology. As caspase-4 shows evidence of being a primate-specific gene, current models of AD and other neurodegenerative conditions may be incomplete because of the absence of this gene in the murine genome.
Neuregulin and the neuregulin receptor ERBB4 have been genetically and functionally implicated in... more Neuregulin and the neuregulin receptor ERBB4 have been genetically and functionally implicated in schizophrenia. In this study, we used the yeast two-hybrid system to identify proteins that interact with ERBB4, to identify genes and pathways that might contribute to schizophrenia susceptibility. We identified the MAGI scaffolding proteins as ERBB4-binding proteins. After validating the interaction of MAGI proteins with ERBB4 in mammalian cells, we demonstrated that ERBB4 expression, alone or in combination with ERBB2 or ERBB3, led to the tyrosine phosphorylation of MAGI proteins, and that this could be further enhanced with receptor activation by neuregulin. As MAGI proteins were previously shown to interact with receptor phosphotyrosine phosphatase b/f (RPTPb), we postulated that simultaneous binding of MAGI proteins to RPTPb and ERBB4 forms a phosphotyrosine kinase/phosphotyrosine phosphatase complex. Studies in cultured cells confirmed both a spatial and functional association between ERBB4, MAGI and RPTPb. Given the evidence for this functional association, we examined the genes coding for MAGI and RPTPb for genetic association with schizophrenia in a Caucasian United Kingdom case-control cohort (n = B1400). PTPRZ1, which codes for RPTPb, showed significant, gene-wide and hypothesis-wide association with schizophrenia in our study (best individual single-nucleotide polymorphism allelic P = 0.0003; gene-wide P = 0.0064; hypothesiswide P = 0.026). The data provide evidence for a role of PTPRZ1, and for RPTPb signaling abnormalities, in the etiology of schizophrenia. Furthermore, the data indicate a role for RPTPb in the modulation of ERBB4 signaling that may in turn provide further support for an important role of neuregulin/ERBB4 signaling in the molecular basis of schizophrenia.
The low-density lipoprotein receptor related protein 1 (LRP1) has been implicated in Alzheimer's ... more The low-density lipoprotein receptor related protein 1 (LRP1) has been implicated in Alzheimer's disease (AD) but its signalling has not been fully evaluated. There is good evidence that the cytoplasmic domain of LRP1 is involved in protein-protein interactions, important in the cell biology of LRP1.
Calsenilin/potassium channel-interacting protein (KChIP)3/ downstream regulatory element sequence... more Calsenilin/potassium channel-interacting protein (KChIP)3/ downstream regulatory element sequence antagonist modulator (DREAM) is a neuronal calcium-binding protein that has been shown to have multiple functions in the cell, including the regulation of presenilin processing, repression of transcription and modulation of A-type potassium channels. To gain a better understanding of the precise role of calsenilin in specific cellular compartments, an interactor hunt for proteins that bind to the N-terminal domain of calsenilin was carried out. Using a yeast two-hybrid system and co-immunoprecipitation studies, we have identified the transcriptional co-repressor C-terminal binding protein (CtBP)2 as an interactor for calsenilin and have shown that the two proteins can interact in vivo. In co-immunoprecipitation studies, calsenilin also interacted with CtBP1, a CtBP2 homolog. Our data also showed a calsenilindependent increase in c-fos protein levels in CtBP knockout fibroblasts, suggesting that CtBP may modulate the transcriptional repression of c-fos by calsenilin. Furthermore, the finding that histone deacetylase protein and activity were associated with the calsenilin-CtBP immunocomplex suggests a mechanism by which calsenilin-CtBP may act to repress transcription. Finally, we demonstrated that calsenilin and CtBP are present in synaptic vesicles and can interact in vivo.
The hnRNP C protein tetramer cooperatively binds 230 nt increments of pre-mRNA in vitro in a salt... more The hnRNP C protein tetramer cooperatively binds 230 nt increments of pre-mRNA in vitro in a salt-resistant manner and is located along the length of vertebrate transcripts in vivo. Based on these and other ®ndings it has been suggested that hnRNP C functions as a chaperonin to maintain long lengths of RNA topologically single-stranded and accessible to splicing factors. We report here that human C protein is lethal when expressed in the yeast Saccharomyces cerevisiae. Through a series of¯uorescent immunolocalization studies, lethality was observed to be associated with the rapid nuclear accumulation of both C protein and yeast pre-mRNA. Studies using various protein constructs and the two hybrid assay reveal that these events are dependent on the basic 40 residue high-af®nity RNA binding domain and its contiguous leucine zipper-like motif (the bZLM, residues 140-214). Additionally, equilibrium binding studies have shown that the bZLM is the determinant of C protein's saltresistant RNA binding mode. Taken together, these ®ndings further distinguish the bZIP-like domain as the major determinant of C protein's high-af®nity interaction with RNA, oligomerization, and its highly cooperative RNA binding activity. Finally, these ®ndings indicate that yeast and vertebrates may possess a conserved mechanism for general import of RNP although a true homolog to vertebrate C protein appears not to exist in yeast. Lethality is likely due to the absence in yeast of speci®c mechanisms for the removal of human C protein from nascent transcripts.
There is strong evidence for rare, highly penetrant genetic variants playing an etiological role ... more There is strong evidence for rare, highly penetrant genetic variants playing an etiological role in multiple neurodevelopmental disabilities, including autism spectrum disorders. The rate of discovery of such rare variants is increasing with the advent of larger sample collections, chromosome microarray analyses, and high-throughput sequencing. As the variants that are being discovered can be highly penetrant, they lead immediately to model systems with construct validity, critical for understanding the underlying neurobiology of these conditions, which in turn can provide leads for novel therapeutic targets. Moreover, these discoveries can benefit families with information about recurrence risk, resolve concerns about etiology, provide information about associated medical issues, and engender directed advocacy for specific genetic conditions. For these reasons, diagnostic laboratories are taking advantage of research data as they are produced. In the current report, we present our molecular analysis of a child with a purported disruptive mutation in SHANK3 identified by a commercial genetic testing laboratory and we provide evidence that this was not an etiological variant. The variant was a 1-bp insertion in exon 11 of the RefSeq gene, which we then determined was inherited from a healthy mother and found in~1% of controls. Since the variant would be predicted to disrupt the reference gene, and the penetrance of SHANK3 mutations is very high, we did follow up molecular and bioinformatic analyses and concluded that the presumptive exon containing the variant is not likely to be present in most or all SHANK3 transcripts. The results highlight difficulties that can arise with rapid translation of research findings to clinical practice. Researchers are in a unique position to generate resources with collated and curated information that can inform research, genetic testing, clinicians, and families about the best practices as pertains to rare genetic variants in neurodevelopmental disabilities. Of immediate importance would be a B R A I N R E S E A R C H 1 3 8 0 ( 2 0 1 1 ) 9 8 -1 0 5 ⁎ Corresponding author. Mount a v a i l a b l e a t w w w . s c i e n c e d i r e c t . c o m w w w . e l s e v i e r . c o m / l o c a t e / b r a i n r e s well-curated database of gene variation identified in large numbers of typically developing individuals and in individuals affected with neurodevelopmental disabilities. Such a database would reduce false-positive results in clinical settings, would be helpful in structure-function analyses, and would direct translational research to pathways most likely to benefit families.
Background: Rare genetic variants of SLITRK1 have been previously associated with Tourette syndro... more Background: Rare genetic variants of SLITRK1 have been previously associated with Tourette syndrome (TS), attention-deficit/hyperactivity disorder (ADHD), and obsessive-compulsive disorder (OCD) symptoms.
Copy number variation (CNV) is an important determinant of human diversity and plays important ro... more Copy number variation (CNV) is an important determinant of human diversity and plays important roles in susceptibility to disease. Most studies of CNV carried out to date have made use of chromosome microarray and have had a lower size limit for detection of about 30 kilobases (kb). With the emergence of whole-exome sequencing studies, we asked whether such data could be used to reliably call rare exonic CNV in the size range of 1-30 kilobases (kb), making use of the eXome Hidden Markov Model (XHMM) program. By using both transmission information and validation by molecular methods, we confirmed that small CNV encompassing as few as three exons can be reliably called from whole-exome data. We applied this approach to an autism case-control sample (n ¼ 811, mean per-target read depth ¼ 161) and observed a significant increase in the burden of rare (MAF %1%) 1-30 kb CNV, 1-30 kb deletions, and 1-10 kb deletions in ASD. CNV in the 1-30 kb range frequently hit just a single gene, and we were therefore able to carry out enrichment and pathway analyses, where we observed enrichment for disruption of genes in cytoskeletal and autophagy pathways in ASD. In summary, our results showed that XHMM provided an effective means to assess small exonic CNV from whole-exome data, indicated that rare 1-30 kb exonic deletions could contribute to risk in up to 7% of individuals with ASD, and implicated a candidate pathway in developmental delay syndromes.
Uploads
Papers by Yuji Kajiwara