Light Directs Zebrafish period2 Expression via Conserved D and E Boxes

A highly conserved promoter module in a vertebrate clock gene confers light-regulated gene expression

Nano-scale architecture of blood-brain barrier tight-junctions

Tight junctions (TJs) between blood-brain barrier (BBB) endothelial cells construct a robust physical barrier, whose damage underlies BBB dysfunctions related to several neurodegenerative diseases. What makes these highly specialized BBB-TJs extremely restrictive remains unknown. Here, we use super-resolution microscopy (dSTORM) to uncover new structural and functional properties of BBB TJs. Focusing on three major components, Nano-scale resolution revealed sparse (occludin) vs. clustered (ZO1/claudin-5) molecular architecture. In mouse development, permeable TJs become first restrictive to large molecules, and only later to small molecules, with claudin-5 proteins arrangement compacting during this maturation process. Mechanistically, we reveal that ZO1 clustering is independent of claudin-5 in vivo. In contrast to accepted knowledge, we found that in the developmental context, total levels of claudin-5 inversely correlate with TJ functionality. Our super-resolution studies provide a unique perspective of BBB TJs and open new directions for understanding TJ functionality in biological barriers, ultimately enabling restoration in disease or modulation for drug delivery

Huntington’s Disease iPSC-Derived Brain Microvascular Endothelial Cells Reveal WNT-Mediated Angiogenic and Blood-Brain Barrier Deficits

Brain microvascular endothelial cells (BMECs) are an essential component of the blood-brain barrier (BBB) that shields the brain against toxins and immune cells. While BBB dysfunction exists in neurological disorders, including Huntington's disease (HD), it is not known if BMECs themselves are functionally compromised to promote BBB dysfunction. Further, the underlying mechanisms of BBB dysfunction remain elusive given limitations with mouse models and post-mortem tissue to identify primary deficits. We undertook a transcriptome and functional analysis of human induced pluripotent stem cell (iPSC)-derived BMECs (iBMEC) from HD patients or unaffected controls. We demonstrate that HD iBMECs have intrinsic abnormalities in angiogenesis and barrier properties, as well as in signaling pathways governing these processes. Thus, our findings provide an iPSC-derived BBB model for a neurodegenerative disease and demonstrate autonomous neurovascular deficits that may underlie HD pathology with implications for therapeutics and drug delivery.American Heart Association (12PRE10410000)American Heart Association (CIRMTG2-01152)National Institutes of Health (U.S.) (NIHNS089076

PLEKHM2 Loss of Function Impairs the Activity of iPSC-Derived Neurons via Regulation of Autophagic Flux

Pleckstrin Homology And RUN Domain Containing M2 (PLEKHM2) [delAG] mutation causes dilated cardiomyopathy with left ventricular non-compaction (DCM-LVNC), resulting in a premature death of PLEKHM2[delAG] individuals due to heart failure. PLEKHM2 is a factor involved in autophagy, a master regulator of cellular homeostasis, decomposing pathogens, proteins and other cellular components. Autophagy is mainly carried out by the lysosome, containing degradation enzymes, and by the autophagosome, which engulfs substances marked for decomposition. PLEKHM2 promotes lysosomal movement toward the cell periphery. Autophagic dysregulation is associated with neurodegenerative diseases’ pathogenesis. Thus, modulation of autophagy holds considerable potential as a therapeutic target for such disorders. We hypothesized that PLEKHM2 is involved in neuronal development and function, and that mutated PLEKHM2 (PLEKHM2[delAG]) neurons will present impaired functions. Here, we studied PLEKHM2-related abnormalities in induced pluripotent stem cell (iPSC)-derived motor neurons (iMNs) as a neuronal model. PLEKHM2[delAG] iMN cultures had healthy control-like differentiation potential but exhibited reduced autophagic activity. Electrophysiological measurements revealed that PLEKHM2[delAG] iMN cultures displayed delayed functional maturation and more frequent and unsynchronized activity. This was associated with increased size and a more perinuclear lysosome cellular distribution. Thus, our results suggest that PLEKHM2 is involved in the functional development of neurons through the regulation of autophagic flux

Altered Behavioral Performance and Live Imaging of Circuit-Specific Neural Deficiencies in a Zebrafish Model for Psychomotor Retardation

<div><p>The mechanisms and treatment of psychomotor retardation, which includes motor and cognitive impairment, are indefinite. The Allan-Herndon-Dudley syndrome (AHDS) is an X-linked psychomotor retardation characterized by delayed development, severe intellectual disability, muscle hypotonia, and spastic paraplegia, in combination with disturbed thyroid hormone (TH) parameters. AHDS has been associated with mutations in the monocarboxylate transporter 8 (<i>mct8</i>/<i>slc16a2</i>) gene, which is a TH transporter. In order to determine the pathophysiological mechanisms of AHDS, MCT8 knockout mice were intensively studied. Although these mice faithfully replicated the abnormal serum TH levels, they failed to exhibit the neurological and behavioral symptoms of AHDS patients. Here, we generated an <i>mct8</i> mutant (<i>mct8</i>−/−) zebrafish using zinc-finger nuclease (ZFN)-mediated targeted gene editing system. The elimination of MCT8 decreased the expression levels of TH receptors; however, it did not affect the expression of other TH-related genes. Similar to human patients, <i>mct8</i>−/− larvae exhibited neurological and behavioral deficiencies. High-throughput behavioral assays demonstrated that <i>mct8</i>−/− larvae exhibited reduced locomotor activity, altered response to external light and dark transitions and an increase in sleep time. These deficiencies in behavioral performance were associated with altered expression of myelin-related genes and neuron-specific deficiencies in circuit formation. Time-lapse imaging of single-axon arbors and synapses in live <i>mct8</i>−/− larvae revealed a reduction in filopodia dynamics and axon branching in sensory neurons and decreased synaptic density in motor neurons. These phenotypes enable assessment of the therapeutic potential of three TH analogs that can enter the cells in the absence of MCT8. The TH analogs restored the myelin and axon outgrowth deficiencies in <i>mct8</i>−/− larvae. These findings suggest a mechanism by which MCT8 regulates neural circuit assembly, ultimately mediating sensory and motor control of behavioral performance. We also propose that the administration of TH analogs early during embryo development can specifically reduce neurological damage in AHDS patients.</p></div

Loss of MCT8 reduces synaptic density in axonal arbors of the motor neuron.

<p><b>A–C</b>. Confocal imaging of a 2 dpf live <i>tg</i>(<i>mct8:EGFP</i>) embryo co-injected with <i>huc:GAL4</i> and <i>uas:tRFP</i> constructs revealed co-localization of <i>mct8</i> (green) and the <i>huc</i> pan-neural marker (red) in a motor neuron. <b>D</b>. Schematic illustration of an axonal arbor in a motor neuron. Each color represents a single branch that was subjected to ImageJ software analysis. <b>E</b>. Lateral view of a 3 dpf <i>tg(huc:GAL4Xuas:memYFP)</i> embryo. memYFP expression driven by the <i>huc</i> promoter is observed in the spinal cord (SC) and in descending motor neurons. The dashed frame marks a single motor neuron that was selected for further comparative studies. High magnification of the framed area is shown in the trunk of 6 dpf <i>tg(huc:GAL4Xuas:memYFP)/mct8+/−</i> and <i>tg(huc:GAL4Xuas:memYFP)/mct8−/−</i> representative larvae (<b>F</b> and <b>G</b>, respectively). <b>H</b>. Lateral view of a 30 hpf <i>tg(mct8:GAL4Xuas:SYP-EGFP)</i> embryo. SYP-EGFP expression driven by the <i>mct8</i> promoter is observed in the spinal cord (SC) and in descending motor neurons. In order to compare the number of synapses in <i>mct8+/−</i> and <i>mct8−/−</i> larvae, single motor-neuron arbors were selected (dashed frame). High magnification of the dashed frame is shown in 6 dpf <i>tg(mct8:GAL4)/(uas:SYP-EGFP)/mct8+/−</i> and <i>tg(mct8:GAL4)/(uas:SYP-EGFP)/mct8−/−</i> representative larvae (<b>I</b> and <b>J</b>, respectively). The total arbor length (<b>K</b>) and the number of branches (<b>L</b>) were measured in 3 and 6 dpf <i>mct8+/−</i> larvae and in 3 and 6 dpf <i>mct8−/−</i> larvae. <b>M</b>. Synapse density in the axons of the motor-neurons was measured along the last 50 µm of a single branch in 3 and 6 dpf <i>mct8+/−</i> larvae and in 3 and 6 dpf <i>mct8−/−</i> larvae. <b>N</b>. The total number of synapses was measured in the motor-neuron arbor of 6 dpf <i>mct8+/−</i> and <i>mct8−/−</i> larvae. Scale bar = 30 µm. Values represented as means±SEM (standard error of the mean). Statistical significance determined by <i>t</i>-test: Two-sample assuming unequal variances followed by one-sample Kolmogorov-Smirnov test, to assume normal distribution (*<i>p<0.05</i>).</p

MCT8 regulates axon branching in the Rohon-Beard sensory neurons.

<p><b>A</b>. A representative scheme of the Rohon-Beard (RB) sensory neuron location in zebrafish larvae. <b>B–D</b>. Double fluorescent ISH in 33 hpf embryos revealed co-localization of <i>p2rx3.1</i> (green) and <i>mct8</i> (red) in RB cell bodies. <b>E–F</b>. Whole mount ISH showed the spatial expression of <i>p2rx3.1</i> in the dorsal spinal cord of 2 dpf WT-sibling (<b>E</b>) and <i>mct8−/−</i> larvae (<b>F</b>). <b>G–I</b>. Whole-mount ISH and immunofluorescence revealed co-localization of EGFP (green) and <i>p2rx3.1</i> (red) in the cell body of an RB neuron in 2 dpf <i>huc:GAL4+uas:memYFP</i>-injected embryos. <b>J</b>. The percentages of embryos that express <i>memYFP</i> in single arborized RB neurons in the tail (black bars), are shown in 2 dpf WT-sibling, <i>mct8−/−</i> and <i>mct8</i> mRNA-injected <i>mct8−/−</i> embryos. Statistical significance was determined by the Chi square test. Different letters indicate significant difference. <b>K</b>. The percentages of embryos that express <i>memYFP</i> in single arborized RB neurons in the tail (black bars), are shown in 2 dpf WT-sibling, <i>mct8−/−</i>, WT-sibling treated with 0.5 nM TRIAC and <i>mct8−/−</i> treated with 0.5 nM TRIAC. Statistical significance was determined by the Chi square test. Different letters indicate significant difference. <b>L, M</b>. Lateral view of arborized RB-neuron that projects toward the tail in 2 dpf live <i>mct8−/−</i> and WT-sibling embryos, which are transiently expressed <i>huc:GAL4</i> and <i>uas:memYFP</i> constructs. <b>N</b>. Schematic illustration of arborized RB sensory neuron. Each color represents a single branch that was subjected to ImageJ software analysis. Filopodia are colored in black. The total length (<b>O</b>), average length (<b>P</b>), and number of branches (<b>Q</b>) measured in <i>mct8−/−</i> and WT-sibling embryos. Scale bar = 30 µm. Values represented as means±SEM (standard error of the mean). Statistical significance determined by <i>t</i>-test: Two-sample assuming unequal variances followed by one-sample Kolmogorov-Smirnov test, to assume normal distribution (*<i>p<0.05</i>).</p

Sleep architecture of <i>mct8−/−</i> larvae.

<p><b>A–C</b>. Recording of sleep was performed in 6 dpf <i>mct8−/−</i> and WT-sibling larvae during 24 h under a 14 h light/10 h dark cycle. Total sleep time (<b>A</b>), the number of sleep/wake transitions (<b>B</b>) and sleep-bout length (<b>C</b>) monitored in <i>mct8−/−</i> and WT-sibling larvae. Values are represented as means±SEM (standard error of the mean). Statistical significance was determined by <i>t</i>-test: two-sample assuming unequal variances (** <i>p<0.001</i>).</p

MCT8 mutant exhibits reduced locomotor activity and altered responses to light/dark transitions.

<p>Locomotor activity recording was performed in 6 dpf <i>mct8−/−</i> larvae and their WT siblings throughout a daily cycle under a 14 h light/10 h dark cycle (<b>A–C</b>), or during 3 h of 30 min light/30 min dark intervals (<b>D–F</b>). White and black horizontal boxes represent light and dark periods, respectively. Average total activity of each genotype was measured as the average distance movement in 1 min (<b>A</b> and <b>D</b>). Dotted boxes represent 1 h and 5 min (in <b>A</b> and <b>D</b>, respectively) before and after the light-to-dark and dark-to-light transitions. The average total activity of each genotype was measured during day and night as well as during short light and dark periods (<b>B</b> and <b>E</b>, respectively). Differences in the average total activity of each genotype were calculated by comparing 1 h after and 1 h before the day-to-night and night-to-day transitions, as well as by comparing 5 min after and 5 min before light-to-dark and dark-to-light transitions (<b>C</b> and <b>F</b>, respectively). Values are represented as means±SEM (standard error of the mean). Statistical significance determined by <i>t</i>-test: two-sample assuming unequal variances (*<i>p<0.05</i>, ** <i>p<0.001</i>).</p