ZNF804a Regulates Expression of the Schizophrenia-Associated Genes PRSS16, COMT, PDE4B, and DRD2

ZNF804a was identified by a genome-wide association study (GWAS) in which a single nucleotide polymorphism (SNP rs1344706) in ZNF804a reached genome-wide statistical significance for association with a combined diagnosis of schizophrenia (SZ) and bipolar disorder. Although the molecular function of ZNF804a is unknown, the amino acid sequence is predicted to contain a C2H2-type zinc-finger domain and suggests ZNF804a plays a role in DNA binding and transcription. Here, we confirm that ZNF804a directly contributes to transcriptional control by regulating the expression of several SZ associated genes and directly interacts with chromatin proximal to the promoter regions of PRSS16 and COMT, the two genes we find upregulated by ZNF804a. Using immunochemistry we establish that ZNF804a is localized to the nucleus of rat neural progenitor cells in culture and in vivo. We demonstrate that expression of ZNF804a results in a significant increase in transcript levels of PRSS16 and COMT, relative to GFP transfected controls, and a statistically significant decrease in transcript levels of PDE4B and DRD2. Furthermore, we show using chromatin immunoprecipitation assays (ChIP) that both epitope-tagged and endogenous ZNF804a directly interacts with the promoter regions of PRSS16 and COMT, suggesting a direct upregulation of transcription by ZNF804a on the expression of these genes. These results are the first to confirm that ZNF804a regulates transcription levels of four SZ associated genes, and binds to chromatin proximal to promoters of two SZ genes. These results suggest a model where ZNF804a may modulate a transcriptional network of SZ associated genes

DYX1C1 is required for axonemal dynein assembly and ciliary motility

Dyx1c1 has been associated with dyslexia and neuronal migration in the developing neocortex. Unexpectedly, we found that deletion of Dyx1c1 exons 2–4 in mice caused a phenotype resembling primary ciliary dyskinesia (PCD), a genetically heterogeneous disorder characterized by chronic airway disease, laterality defects, and male infertility. This phenotype was confirmed independently in mice with a Dyx1c1c.T2A start codon mutation recovered from an ENU mutagenesis screen. Morpholinos targeting dyx1c1 in zebrafish also created laterality and ciliary motility defects. In humans, recessive loss-of-function DYX1C1 mutations were identified in twelve PCD individuals. Ultrastructural and immunofluorescence analyses of DYX1C1-mutant motile cilia in mice and humans revealed disruptions of outer and inner dynein arms (ODA/IDA). DYX1C1 localizes to the cytoplasm of respiratory epithelial cells, its interactome is enriched for molecular chaperones, and it interacts with the cytoplasmic ODA/IDA assembly factor DNAAF2/KTU. Thus, we propose that DYX1C1 is a newly identified dynein axonemal assembly factor (DNAAF4)

GABAergic regulation of cerebellar NG2 cell development is altered in perinatal white matter injury.

Diffuse white matter injury (DWMI), a leading cause of neurodevelopmental disabilities in preterm infants, is characterized by reduced oligodendrocyte formation. NG2-expressing oligodendrocyte precursor cells (NG2 cells) are exposed to various extrinsic regulatory signals, including the neurotransmitter GABA. We investigated GABAergic signaling to cerebellar white matter NG2 cells in a mouse model of DWMI (chronic neonatal hypoxia). We found that hypoxia caused a loss of GABAA receptor-mediated synaptic input to NG2 cells, extensive proliferation of these cells and delayed oligodendrocyte maturation, leading to dysmyelination. Treatment of control mice with a GABAA receptor antagonist or deletion of the chloride-accumulating transporter NKCC1 mimicked the effects of hypoxia. Conversely, blockade of GABA catabolism or GABA uptake reduced NG2 cell numbers and increased the formation of mature oligodendrocytes both in control and hypoxic mice. Our results indicate that GABAergic signaling regulates NG2 cell differentiation and proliferation in vivo, and suggest that its perturbation is a key factor in DWMI

DYX1C1 is required for axonemal dynein assembly and ciliary motility

DYX1C1 has been associated with dyslexia and neuronal migration in the developing neocortex. Unexpectedly, we found that deleting exons 2–4 of Dyx1c1 in mice caused a phenotype resembling primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease, laterality defects and male infertility. This phenotype was confirmed independently in mice with a Dyx1c1 c.T2A start-codon mutation recovered from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Morpholinos targeting dyx1c1 in zebrafish also caused laterality and ciliary motility defects. In humans, we identified recessive loss-of-function DYX1C1 mutations in 12 individuals with PCD. Ultrastructural and immunofluorescence analyses of DYX1C1-mutant motile cilia in mice and humans showed disruptions of outer and inner dynein arms (ODAs and IDAs, respectively). DYX1C1 localizes to the cytoplasm of respiratory epithelial cells, its interactome is enriched for molecular chaperones, and it interacts with the cytoplasmic ODA and IDA assembly factor DNAAF2 (KTU). Thus, we propose that DYX1C1 is a newly identified dynein axonemal assembly factor (DNAAF4)

Disrupted-in-Schizophrenia (DISC1) Functions Presynaptically at Glutamatergic Synapses

The pathophysiology of schizophrenia is believed to involve defects in synaptic transmission, and the function of many schizophrenia-associated genes, including DISC1, have been linked to synaptic function at glutamatergic synapses. Here we develop a rodent model via in utero electroporation to assay the presynaptic function of DISC1 at glutamatergic synapses. We used a combination of mosaic transgene expression, RNAi knockdown and optogenetics to restrict both genetic manipulation and synaptic stimulation of glutamatergic neurons presynaptic to other layer 2/3 neocortical pyramidal neurons that were then targeted for whole-cell patch-clamp recording. We show that expression of the DISC1 c-terminal truncation variant that is associated with Schizophrenia alters the frequency of mEPSCs and the kinetics of evoked glutamate release. In addition, we show that expression level of DISC1 is correlated with the probability of glutamate release such that increased DISC1 expression results in paired-pulse depression and RNAi knockdown of DISC1 produces pairedpulse facilitation. Overall, our results support a direct presynaptic function for the schizophrenia-associated gene, DISC1

Presynaptic transmitter release is regulated by the expression levels of DISC1.

<p>A) Representative EPSCs recorded from an untransfected neuron and evoked from presynaptic neurons expressing ChR2 and either dsRed (control) or wtDISC1. B) Summary graph showing the mean±SEM PPR recorded from control and wtDISC1 conditions. C) Summary plot comparing the CV values vs PPR for each condition. D) Representative EPSCs evoked from neurons expressing DISC1 RNAi (D1 RNAi) and D1 RNAi+DISC1-GFP (rescue). E) Summary graph showing the mean±SEM PPR recorded from D1 RNAi and rescue conditions. F) Summary plot comparing CV values vs PPR for each condition. All EPSC traces are normalized to the first EPSC peak amplitude.</p

Presynaptic expression of DISC1ΔC alters the kinetics of evoked glutamate release and inhibits synchronous glutamate release.

<p>A) Normalized EPSCs recorded from an untransfected neuron and evoked from presynaptic terminals expressing ChR2 and either dsRed (control), DISC1ΔC, wtDISC1 or D1 RNAi. B) Summary graphs showing the mean ± SEM of four different kinetic measures of EPSCs evoked from presynaptic terminals expressing ChR2 and either dsRed (control), DISC1ΔC, wtDISC1 or D1 RNAi. C) Two representative recordings from a neuron in control or DISC1ΔC conditions showing 10 consecutive traces that are normalized and overlaid (top traces). The average trial-to-trial variance for the traces above is displayed over the duration of the waveform (bottom trace). D) A histogram depicting the average trial-to-trial variance for all neurons in each condition (bin = 0.001 A²). E) The ratio of the average trial-to-trial variance for each condition (black trace) is displayed over the duration of the EPSC waveform. The dotted blue line indicates the threshold for statistical significance (p<0.05; F-test). A representative DISC1ΔC EPSC (red trace) is overlaid to emphasize the aspects of the EPSC waveform that produce the most variance.</p

Postnatal expression of DISC1ΔC enhances the frequency of mEPSCs.

<p>A) Expression of DISC1ΔC is induced by postnatal administration of 4-OHT as seen by expression of GFP fused to DISC1ΔC in this P28 brain slice. No GFP expression is observed in vehicle treated animals (- 4-OHT). Scale bar equals 100 µm. B) DISC1ΔC-GFP is localized to the soma, dendrite (arrow), and axons (arrowhead) of layer 2/3 pyramidal cells. Expression is also observed in axon terminal field of layer 5 and axon tracts of the corpus callosum. Scale bar equals 20 µm. C) Example mEPSC sweeps from transfected layer 2/3 neurons expressing either GFP (control), DISC1ΔC, wtDISC1 or D1 RNAi. Traces shown above are the average of all the captured mEPSC from every recording for each condition. D) Summary graph showing the frequency of mEPSCs are nearly doubled by DISC1ΔC expression. This increase in synaptic activity was present to the same extent in both transfected cells expressing DISC1ΔC and neighboring non-transfected cells. All recordings performed in the presence of gabazine (5 µM) and TTX (1 µM).</p

Advantages and limitations of the use of optogenetic approach in studying fast-scale spike encoding.

Understanding single-neuron computations and encoding performed by spike-generation mechanisms of cortical neurons is one of the central challenges for cell electrophysiology and computational neuroscience. An established paradigm to study spike encoding in controlled conditions in vitro uses intracellular injection of a mixture of signals with fluctuating currents that mimic in vivo-like background activity. However this technique has two serious limitations: it uses current injection, while synaptic activation leads to changes of conductance, and current injection is technically most feasible in the soma, while the vast majority of synaptic inputs are located on the dendrites. Recent progress in optogenetics provides an opportunity to circumvent these limitations. Transgenic expression of light-activated ionic channels, such as Channelrhodopsin2 (ChR2), allows induction of controlled conductance changes even in thin distant dendrites. Here we show that photostimulation provides a useful extension of the tools to study neuronal encoding, but it has its own limitations. Optically induced fluctuating currents have a low cutoff (~70 Hz), thus limiting the dynamic range of frequency response of cortical neurons. This leads to severe underestimation of the ability of neurons to phase-lock their firing to high frequency components of the input. This limitation could be worked around by using short (2 ms) light stimuli which produce membrane potential responses resembling EPSPs by their fast onset and prolonged decay kinetics. We show that combining application of short light stimuli to different parts of dendritic tree for mimicking distant EPSCs with somatic injection of fluctuating current that mimics fluctuations of membrane potential in vivo, allowed us to study fast encoding of artificial EPSPs photoinduced at different distances from the soma. We conclude that dendritic photostimulation of ChR2 with short light pulses provides a powerful tool to investigate population encoding of simulated synaptic potentials generated in dendrites at different distances from the soma