Becky C. Carlyle

DISC1, PDE4B, and NDE1 at the centrosome and synapse

Disrupted-In-Schizophrenia 1 (DISC1) is a risk factor for schizophrenia and other major mental illnesses. Its protein binding partners include the Nuclear Distribution Factor E Homologs (NDE1 and NDEL1), LIS1, and phosphodiesterases 4B and 4D (PDE4B and PDE4D). We demonstrate that NDE1, NDEL1 and LIS1, together with their binding partner dynein, associate with DISC1, PDE4B and PDE4D within the cell, and provide evidence that this complex is present at the centrosome. LIS1 and NDEL1 have been previously suggested to be synaptic, and we now demonstrate localisation of DISC1, NDE1, and PDE4B at synapses in cultured neurons. NDE1 is phosphorylated by cAMP-dependant Protein Kinase A (PKA), whose activity is, in turn, regulated by the cAMP hydrolysis activity of phosphodiesterases, including PDE4. We propose that DISC1 acts as an assembly scaffold for all of these proteins and that the NDE1/NDEL1/LIS1/dynein complex is modulated by cAMP levels via PKA and PDE4.

DISC1: Structure, Function, and Therapeutic Potential for Major Mental Illness

Disrupted in schizophrenia 1 (DISC1) is well established as a genetic risk factor across a spectrum of psychiatric disorders, a role supported by a growing body of biological studies, making the DISC1 protein interaction network an attractive therapeutic target. By contrast, there is a relative deficit of structural information to relate to the myriad biological functions of DISC1. Here, we critically appraise the available bioinformatics and biochemical analyses on DISC1 and key interacting proteins, and integrate this with the genetic and biological data. We review, analyze, and make predictions regarding the secondary structure and propensity for disordered regions within DISC1, its protein-interaction domains, subcellular localization motifs, and the structural and functional implications of common and ultrarare DISC1 variants associated with major mental illness. We discuss signaling pathways of high pharmacological potential wherein DISC1 participates, including those involving phosphodiesterase 4 (PDE4) and glycogen synthase kinase 3 (GSK3). These predictions and priority areas can inform future research in the translational and potentially guide the therapeutic processes.

PKA Phosphorylation of NDE1 Is DISC1/PDE4 Dependent and Modulates Its Interaction with LIS1 and NDEL1

Nuclear distribution factor E-homolog 1 (NDE1), Lissencephaly 1 (LIS1), and NDE-like 1 (NDEL1) together participate in essential neurodevelopmental processes, including neuronal precursor proliferation and differentiation, neuronal migration, and neurite outgrowth. NDE1/LIS1/NDEL1 interacts with Disrupted in Schizophrenia 1 (DISC1) and the cAMP-hydrolyzing enzyme phosphodiesterase 4 (PDE4). DISC1, PDE4, NDE1, and NDEL1 have each been implicated as genetic risk factors for major mental illness. Here, we demonstrate that DISC1 and PDE4 modulate NDE1 phosphorylation by cAMP-dependent protein kinase A (PKA) and identify a novel PKA substrate site on NDE1 at threonine-131 (T131). Homology modeling predicts that phosphorylation at T131 modulates NDE1–LIS1 and NDE1–NDEL1 interactions, which we confirm experimentally. DISC1–PDE4 interaction thus modulates organization of the NDE1/NDEL1/LIS1 complex. T131-phosphorylated NDE1 is present at the postsynaptic density, in proximal axons, within the nucleus, and at the centrosome where it becomes substantially enriched during mitosis. Mutation of the NDE1 T131 site to mimic PKA phosphorylation inhibits neurite outgrowth. Thus PKA-dependent phosphorylation of the NDE1/LIS1/NDEL1 complex is DISC1–PDE4 modulated and likely to regulate its neural functions.

NDE1 and NDEL1: Multimerisation, alternate splicing and DISC1 interaction

Nuclear Distribution Factor E Homolog 1 (NDE1) and NDE-Like 1 (NDEL1) are highly homologous mammalian proteins. However, whereas NDEL1 is well studied, there is remarkably little known about NDE1. We demonstrate the presence of multiple isoforms of both NDE1 and NDEL1 in the brain, showing that NDE1 binds directly to multiple isoforms of Disrupted in Schizophrenia 1 (DISC1), and to itself. We also show that NDE1 can complex with NDEL1. Together these results predict a high degree of complexity of DISC1-mediated regulation of neuronal activity.

Maternal separation with early weaning: A rodent model providing novel insights into neglect associated developmental deficits

Child neglect is the most prevalent form of child maltreatment in the United States, and poses a serious public health concern. Children who survive such episodes go on to experience long-lasting psychological and behavioral problems, including higher rates of post-traumatic stress disorder symptoms, depression, alcohol and drug abuse, attention-deficit/hyperactivity disorder, and cognitive deficits. To date, most research into the causes of these life-long problems has focused on well-established targets such as stress responsive systems, including the hypothalamus-pituitary-adrenal axis. Using the maternal separation and early weaning model, we have attempted to provide comprehensive molecular profiling of a model of early-life neglect in an organism amenable to genomic manipulation: the mouse. In this article, we report new findings generated with this model using chromatin immunoprecipitation sequencing, diffuse tensor magnetic resonance imaging, and behavioral analyses. We also review the validity of the maternal separation and early weaning model, which reflects behavioral deficits observed in neglected humans including hyperactivity, anxiety, and attentional deficits. Finally, we summarize the molecular characterization of these animals, including RNA profiling and label-free proteomics, which highlight protein translation and myelination as novel pathways of interest.

Functional Genomic and Proteomic Analysis Reveals Disruption of Myelin-Related Genes and Translation in a Mouse Model of Early Life Neglect

Early life neglect is an important public health problem which can lead to lasting psychological dysfunction. Good animal models are necessary to understand the mechanisms responsible for the behavioral and anatomical pathology that results. We recently described a novel model of early life neglect, maternal separation with early weaning (MSEW), that produces behavioral changes in the mouse that persist into adulthood. To begin to understand the mechanism by which MSEW leads to these changes we applied cDNA microarray, next-generation RNA-sequencing (RNA-seq), label-free proteomics, multiple reaction monitoring (MRM) proteomics, and methylation analysis to tissue samples obtained from medial prefrontal cortex to determine the molecular changes induced by MSEW that persist into adulthood. The results show that MSEW leads to dysregulation of markers of mature oligodendrocytes and genes involved in protein translation and other categories, an apparent downward biasing of translation, and methylation changes in the promoter regions of selected dysregulated genes. These findings are likely to prove useful in understanding the mechanism by which early life neglect affects brain structure, cognition, and behavior.

cAMP-PKA phosphorylation of tau confers risk for degeneration in aging association cortex

Significance This study of the monkey cerebral cortex revealed age-related changes that help to answer key questions about Alzheimer’s disease (AD): (i) why advancing age is the highest risk factor for AD and (ii) why neurofibrillary tangles (NFTs) in AD selectively target extensively interconnected pyramidal neurons in the highly evolved association cortex, but do not afflict the primary sensory cortex. NFTs arise from phosphorylation of tau protein. In aged prefrontal association cortex, we found increased cAMP-dependent protein kinase (PKA) phosphorylation of tau and corresponding loss of phosphodiesterase 4A, an enzyme that regulates cAMP-PKA signaling in dendritic spines where pyramidal neurons interconnect. In contrast, phosphorylated tau was not observed in aged primary visual cortex. These data provide new strategies for early intervention in AD. The pattern of neurodegeneration in Alzheimer’s disease (AD) is very distinctive: neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau selectively affect pyramidal neurons of the aging association cortex that interconnect extensively through glutamate synapses on dendritic spines. In contrast, primary sensory cortices have few NFTs, even in late-stage disease. Understanding this selective vulnerability, and why advancing age is such a high risk factor for the degenerative process, may help to reveal disease etiology and provide targets for intervention. Our study has revealed age-related increase in cAMP-dependent protein kinase (PKA) phosphorylation of tau at serine 214 (pS214-tau) in monkey dorsolateral prefrontal association cortex (dlPFC), which specifically targets spine synapses and the Ca2+-storing spine apparatus. This increase is mirrored by loss of phosphodiesterase 4A from the spine apparatus, consistent with increase in cAMP-Ca2+ signaling in aging spines. Phosphorylated tau was not detected in primary visual cortex, similar to the pattern observed in AD. We also report electron microscopic evidence of previously unidentified vesicular trafficking of phosphorylated tau in normal association cortex—in axons in young dlPFC vs. in spines in aged dlPFC—consistent with the transneuronal lesion spread reported in genetic rodent models. pS214-Tau was not observed in normal aged mice, suggesting that it arises with the evolutionary expansion of corticocortical connections in primates, crossing the threshold into NFTs and degeneration in humans. Thus, the cAMP-Ca2+ signaling mechanisms, needed for flexibly modulating network strength in young association cortex, confer vulnerability to degeneration when dysregulated with advancing age.

Parallel declines in cognition, motivation, and locomotion in aging mice: association with immune gene upregulation in the medial prefrontal cortex

Aging in humans is associated with parallel changes in cognition, motivation, and motoric performance. Based on the human aging literature, we hypothesized that this constellation of age-related changes is mediated by the medial prefrontal cortex and that it would be observed in aging mice. Toward this end, we performed detailed assessments of cognition, motivation, and motoric behavior in aging mice. We assessed behavioral and cognitive performance in C57Bl/6 mice aged 6, 18, and 24 months, and followed this with microarray analysis of tissue from the medial prefrontal cortex and analysis of serum cytokine levels. Multivariate modeling of these data suggested that the age-related changes in cognition, motivation, motor performance, and prefrontal immune gene expression were highly correlated. Peripheral cytokine levels were also correlated with these variables, but less strongly than measures of prefrontal immune gene upregulation. To determine whether the observed immune gene expression changes were due to prefrontal microglial cells, we isolated CD11b-positive cells from the prefrontal cortex and subject them to next-generation RNA sequencing. Many of the immune changes present in whole medial prefrontal cortex were enriched in this cell population. These data suggest that, as in humans, cognition, motivation, and motoric performance in the mouse change together with age and are strongly associated with CNS immune gene upregulation.

Co-ordinated action of DISC1, PDE4B and GSK3|[beta]| in modulation of cAMP signalling

Disrupted-in-Schizophrenia-1 (DISC1) is a risk factor for major mental illness, including schizophrenia and depression.1 DISC1 interacts with multiple proteins involved in intracellular signalling pathways, including two enzymes cAMP phosphodiesterase-4 (PDE4)2 and glycogen synthase kinase-3β (GSK3β),3 that are inhibited by the prototypic antidepressant/antipsychotic Rolipram4 and the mood stabiliser Lithium (LiCl),5 respectively. PDE4 itself has been directly implicated as a risk factor for mental illness,1, 2, 6 whereas the likely involvement of GSK3β in molecular pathways underlying mental illness is long standing and now linked to DISC1.7 DISC1 inhibits GSK3β,3 and we have previously suggested that DISC1 similarly modulates PDE4.2 These enzymes have overlapping binding sites on DISC1,3, 8 suggesting that their function within the DISC1 complex may be related. Moreover, it has recently been demonstrated that GSK3β phosphorylates PDE4D.9 Accordingly, we now provide evidence that pharmacological inhibition of GSK3β decreases PDE4 activity, and demonstrate that the stimulatory effect of Forskolin upon PDE4 activity is modulated by the co-ordinated action of DISC1 and GSK3β.

Neuroanatomical changes in a mouse model of early life neglect

Using a novel mouse model of early life neglect and abuse (ENA) based on maternal separation with early weaning, George et al. (BMC Neurosci 11:123, 2010) demonstrated behavioral abnormalities in adult mice, and Bordner et al. (Front Psychiatry 2(18):1–18, 2011) described concomitant changes in mRNA and protein expression. Using the same model, here we report neuroanatomical changes that include smaller brain size and abnormal inter-hemispheric asymmetry, decreases in cortical thickness, abnormalities in subcortical structures, and white matter disorganization and atrophy most severely affecting the left hemisphere. Because of the similarities between the neuroanatomical changes observed in our mouse model and those described in human survivors of ENA, this novel animal model is potentially useful for studies of human ENA too costly or cumbersome to be carried out in primates. Moreover, our current knowledge of the mouse genome makes this model particularly suited for targeted anatomical, molecular, and pharmacological experimentation not yet possible in other species.