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

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

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 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

MCT8 reduces filopodia dynamics in the axons of RB neurons.

<p><b>A–B</b>. High magnification views of the dotted area shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004615#pgen-1004615-g007" target="_blank">Fig. 7L and 7M</a>, respectively. Arrows mark branches that contain filopodia and arrowheads mark branches that lack filopodia. <b>C</b>. Number of filopodian branches in <i>mct8−/−</i> and WT-sibling embryos. <b>D</b>. Time-lapse live imaging of axon arbor of RB sensory neuron (15 min intervals during 135 min). A representative series of images that were taken every 15 min in live <i>mct8−/−</i> and WT-sibling embryos is shown. Filopodia dynamics is defined as the number of new (green arrows) and lost (red arrows) filopodia per branch over time. <b>E</b>. Filopodia dynamics per branch during 150 min. <b>F–H</b>. Live imaging of synapses in the axons of the RB sensory neurons. <b>F</b>. Lateral view of axons and synapses marked with tRFP and SYP-EGFP, respectively. The dotted frame marks the area shown in high magnification in <b>G</b> and <b>H</b>. <b>I</b>. Synapse density in the RB-neuron arbor of <i>mct8−/−</i> and WT-sibling embryos measured along the last 50 µm of a single branch. 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>, ** <i>p<0.001</i>).</p

The expression of TH-induced and HPT-axis genes in <i>mct8</i>−/− embryos.

<p><b>A</b>. The expression pattern of <i>klf9</i> in the forebrain (FB), midbrain (MB), and hindbrain (HB) of 6 dpf larvae (lateral view), as detected by whole-mount ISH. <b>B</b>. <i>nrgna</i> is predominantly expressed in the dorsal forebrain (DFB) and HB in 3 dpf embryo (dorsal view), as detected by whole-mount ISH. <b>C</b>. Relative mRNA expression levels of <i>klf9</i> and <i>nrgna</i> in untreated and T3-treated WT embryos. <b>D</b>. Relative mRNA expression of <i>klf9</i> and <i>nrgna</i> in 3 dpf <i>mct8−/−</i> and their WT-sibling embryos. <b>E</b>. Relative mRNA expression levels of <i>tsh</i>, <i>trh</i>, <i>dio1</i>, <i>dio2</i>, <i>dio3</i>, <i>mct10</i>, <i>oatp1c1</i>, <i>thraa</i>, <i>thrab</i> and <i>thrb in</i> 3 dpf <i>mct8−/−</i> and their WT-sibling embryos. 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.</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

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

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