Image_3_Overexpression of the autism candidate gene Cyfip1 pathologically enhances olivo-cerebellar signaling in mice.tif

Cyfip1, the gene encoding cytoplasmic FMR1 interacting protein 1, has been of interest as an autism candidate gene for years. A potential role in autism spectrum disorder (ASD) is suggested by its location on human chromosome 15q11-13, an instable region that gives rise to a variety of copy number variations associated with syndromic autism. In addition, the CYFIP1 protein acts as a binding partner to Fragile X Messenger Ribonucleoprotein (FMRP) in the regulation of translation initiation. Mutation of FMR1, the gene encoding FMRP, causes Fragile X syndrome, another form of syndromic autism. Here, in mice overexpressing CYFIP1, we study response properties of cerebellar Purkinje cells to activity of the climbing fiber input that originates from the inferior olive and provides an instructive signal in sensorimotor input analysis and plasticity. We find that CYFIP1 overexpression results in enhanced localization of the synaptic organizer neurexin 1 (NRXN1) at climbing fiber synaptic input sites on Purkinje cell primary dendrites and concomitant enhanced climbing fiber synaptic transmission (CF-EPSCs) measured using whole-cell patch-clamp recordings from Purkinje cells in vitro. Moreover, using two-photon measurements of GCaMP6f-encoded climbing fiber signals in Purkinje cells of intact mice, we observe enhanced responses to air puff stimuli applied to the whisker field. These findings resemble our previous phenotypic observations in a mouse model for the human 15q11-13 duplication, which does not extend to the Cyfip1 locus. Thus, our study demonstrates that CYFIP1 overexpression shares a limited set of olivo-cerebellar phenotypes as those resulting from an increased number of copies of non-overlapping genes located on chromosome 15q11-13.</p

Image_1_Overexpression of the autism candidate gene Cyfip1 pathologically enhances olivo-cerebellar signaling in mice.tif

Cyfip1, the gene encoding cytoplasmic FMR1 interacting protein 1, has been of interest as an autism candidate gene for years. A potential role in autism spectrum disorder (ASD) is suggested by its location on human chromosome 15q11-13, an instable region that gives rise to a variety of copy number variations associated with syndromic autism. In addition, the CYFIP1 protein acts as a binding partner to Fragile X Messenger Ribonucleoprotein (FMRP) in the regulation of translation initiation. Mutation of FMR1, the gene encoding FMRP, causes Fragile X syndrome, another form of syndromic autism. Here, in mice overexpressing CYFIP1, we study response properties of cerebellar Purkinje cells to activity of the climbing fiber input that originates from the inferior olive and provides an instructive signal in sensorimotor input analysis and plasticity. We find that CYFIP1 overexpression results in enhanced localization of the synaptic organizer neurexin 1 (NRXN1) at climbing fiber synaptic input sites on Purkinje cell primary dendrites and concomitant enhanced climbing fiber synaptic transmission (CF-EPSCs) measured using whole-cell patch-clamp recordings from Purkinje cells in vitro. Moreover, using two-photon measurements of GCaMP6f-encoded climbing fiber signals in Purkinje cells of intact mice, we observe enhanced responses to air puff stimuli applied to the whisker field. These findings resemble our previous phenotypic observations in a mouse model for the human 15q11-13 duplication, which does not extend to the Cyfip1 locus. Thus, our study demonstrates that CYFIP1 overexpression shares a limited set of olivo-cerebellar phenotypes as those resulting from an increased number of copies of non-overlapping genes located on chromosome 15q11-13.</p

Image_4_Overexpression of the autism candidate gene Cyfip1 pathologically enhances olivo-cerebellar signaling in mice.TIF

Cyfip1, the gene encoding cytoplasmic FMR1 interacting protein 1, has been of interest as an autism candidate gene for years. A potential role in autism spectrum disorder (ASD) is suggested by its location on human chromosome 15q11-13, an instable region that gives rise to a variety of copy number variations associated with syndromic autism. In addition, the CYFIP1 protein acts as a binding partner to Fragile X Messenger Ribonucleoprotein (FMRP) in the regulation of translation initiation. Mutation of FMR1, the gene encoding FMRP, causes Fragile X syndrome, another form of syndromic autism. Here, in mice overexpressing CYFIP1, we study response properties of cerebellar Purkinje cells to activity of the climbing fiber input that originates from the inferior olive and provides an instructive signal in sensorimotor input analysis and plasticity. We find that CYFIP1 overexpression results in enhanced localization of the synaptic organizer neurexin 1 (NRXN1) at climbing fiber synaptic input sites on Purkinje cell primary dendrites and concomitant enhanced climbing fiber synaptic transmission (CF-EPSCs) measured using whole-cell patch-clamp recordings from Purkinje cells in vitro. Moreover, using two-photon measurements of GCaMP6f-encoded climbing fiber signals in Purkinje cells of intact mice, we observe enhanced responses to air puff stimuli applied to the whisker field. These findings resemble our previous phenotypic observations in a mouse model for the human 15q11-13 duplication, which does not extend to the Cyfip1 locus. Thus, our study demonstrates that CYFIP1 overexpression shares a limited set of olivo-cerebellar phenotypes as those resulting from an increased number of copies of non-overlapping genes located on chromosome 15q11-13.</p

Atg7 and Pten double deficiency synergistically increases axon terminal size in midbrain DA neurons.

<p>(<b>A</b>) Schema of mouse mating to obtain Atg7 and Pten double deficient mice. <i>Dat^Cre/+</i> background animals were used for the analyses. Animals lacking <i>Cre</i> (<i>Pten^flox/*Atg7^flox/*</i>) were not used in this study. Asterisk indicates ‘+’ or ‘flox’. (<b>B</b>) Neurodegeneration in <i>Atg7</i> cKO mice was rescued by secondary deletion of Pten. Secondary Pten deletion (<i>Atg7/Pten</i> double cKO) suppressed the loss of TH-positive DA neurons in the substantia nigra of 2-month-old <i>Atg7</i> cKO mice. Representative TH-stained midbrain sections are presented. Bar, 250 µm. (right) Quantification of TH-positive DA neuron number in the substantia nigra of <i>Atg7/Pten</i> double cKO mice. n = 4 per genotype. **, <i>p</i><0.01. (<b>C</b>) The enlarged axon terminal phenotype of <i>Atg7</i> cKO mice was greatly enhanced in <i>Atg7/Pten</i> double cKO mice, whereas Pten deficiency alone (<i>Pten</i> cKO) did not significantly change the axon terminal size. (left) Giant (arrowheads, >9.8 µm²) and moderately enlarged (arrows, 4.4∼9.8 µm²) axon terminals were seen in the striatum of <i>Atg7/Pten</i> double cKO mice, whereas only moderately enlarged axon terminals (arrows) were seen in <i>Atg7</i> cKO mice and no enlarged axon terminals were seen in <i>Pten</i> cKO mice. Bars, 20 µm. (right) Quantification of enlarged axon terminal distribution. Black bar, giant terminals (>9.8 µm²); white bar, moderately enlarged terminals (4.4∼9.8 µm²). n = 6 per genotype. **, <i>p</i><0.01. (<b>D</b>) The soma of TH-positive DA neurons in <i>Atg7/Pten</i> double cKO mice were dramatically enlarged (79% increase versus control cWT) relative to <i>Atg7</i> cKO mice (15% increase versus control cWT) and <i>Pten</i> cKO mice (32% increase versus control cWT). (left) Representative sections stained with anti-TH antibody. Bars, 10 µm. (right) Quantification of the average cell size, presented as a fraction of DA neuron soma size in control cWT mice. n = 290∼417 TH-positive DA neurons per genotype. **, <i>p</i><0.01.</p

Biochemical analyses of the protein extracts from the striatal synaptosomes of <i>Atg7</i> cKO mice.

<p>(<b>A</b>) Evidence for reduced macroautophagy activity in axon terminals of <i>Atg7</i> cKO mice. Conversions of LC3-I and GABARAPL1-I into modified lipidated forms associated with autophagosome formation – termed LC3-II and GABARAPL1-II, respectively – were significantly decreased in striatal synaptosomal preparations from <i>Atg7</i> cKO mice (relative to cWT mice); the incomplete reduction likely reflects the presence of non-dopaminergic axon terminals. White, <i>Atg7</i> cWT; Black, <i>Atg7</i> cKO. n = 5 per group. **, <i>p</i><0.01. (<b>B</b>) Moderately reduced accumulation of early endosome markers in striatal synaptosomal preparations from <i>Atg7</i> cKO mice. Both EEA1 and Rab5 are significantly decreased in striatal synaptosomal preparations from <i>Atg7</i> cKO mice (relative to cWT mice), whereas late endosomal and lysosomal markers, Rab7 and Cathepsin B, unchanged. pro, procathepsin B; act, active mature Cathepsin B. Internal control Actin is same as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003845#pgen-1003845-g003" target="_blank">Figure 3A</a>. White, <i>Atg7</i> cWT; Black, <i>Atg7</i> cKO. n = 5 per group. *, <i>p</i><0.05. (<b>C</b>) Selectively increased accumulation of Synaptobrevin II in striatal synaptosomal preparations from <i>Atg7</i> cKO mice. Other synaptic markers were not significantly altered. Internal control Actin is same as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003845#pgen-1003845-g003" target="_blank">Figure 3A</a>. White, <i>Atg7</i> cWT; Black, <i>Atg7</i> cKO. n = 5 per group. *, <i>p</i><0.05. (<b>D</b>) Non-canonical alterations of PI3K/mTOR pathway signaling in synaptosomal preparations from <i>Atg7</i> cKO mice. Phosphorylation of AKT at Ser 473 (S473) was decreased in <i>Atg7</i> cKO mice, whereas phosphorylation at Thr 308 (T308) unchanged. Phosphorylations of mTOR at Ser 2448 (S2448) or Ser 2481 (S2481) were unchanged in <i>Atg7</i> cKO mice. Phosphorylations of 4EBP1 at Ser 65 (S65) and Thr 70 (T70) were increased in the striatal synaptosomes of <i>Atg7</i> cKO mice, whereas phosphorylations of S6 (T389) and S6K (S235/236) were unchanged. Internal control Actin is same as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003845#pgen-1003845-g003" target="_blank">Figure 3A</a>. n = 5 per group. **, <i>p</i><0.01.</p

Characterization of enlarged TH-positive axon terminals of <i>Atg7</i> cKO mice.

<p>(<b>A</b>) Enlarged axon terminals in the striatum of 2-month-old <i>Atg7</i> cKO mice were positive for the axon terminal proteins of midbrain DA neurons. Enlarged axon terminals (arrows in green) in <i>Atg7</i> cKO mice were stained with VMAT2 (arrows in red). Bars, 10 µm. (<b>B</b>, <b>C</b>) Enlarged axon terminals in 2-month-old <i>Atg7</i> cKO mice are Ub- and p62-negative. Enlarged axon terminals (arrows in green) in the striatum of <i>Atg7</i> cKO mice were not stained with the markers for protein inclusions such as Ub (red in ‘B’) and p62 (red in ‘C’), suggesting that they are distinct from the inclusions seen in the cell somas of <i>Atg7</i> cKO mice (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003845#pgen-1003845-g001" target="_blank">Figure 1D</a>). Bars, 10 µm. (<b>D</b>) Ultrastructural analysis of dopaminergic axon terminals in the striatum of 3-month-old <i>Atg7</i> cWT or <i>Atg7</i> cKO mice by immunoelectron microscopy with an antibody to TH. Red circles indicate the gold particle-conjugated anti-TH antibody. Bars, 200 nm. (right) Quantification of the size distribution of axon terminals in striatal sections. Each dot (•) represents approximately 1.6% of the total axon terminal number. n = 611 terminals for cWT and 592 terminals for cKO sections.</p

Enlarged soma size in TH-positive DA neurons of <i>Atg7</i> cKO mice.

<p>(<b>A</b>) Enlarged cell soma of TH-positive DA neurons in <i>Atg7</i> cKO mice. The soma area (brown in pictures) of nigral TH-positive DA neurons in <i>Atg7</i> cKO mice (black in graph) was approximately 15% larger than that in <i>Atg7</i> cWT mice (white in graph), as quantified using Image-J software (Image J, Bethesda, MD) and presented relative to the area of the <i>Atg7</i> cWT group. Bars, 10 µm. (<i>Atg7</i> cWT mice = 1.0); n = 194 to 377 TH-positive DA neurons per group. **, <i>p</i><0.01. (<b>B</b>) The distribution of cell soma size of TH-positive DA neurons in <i>Atg7</i> cWT and <i>Atg7</i> cKO mice. The soma size of TH-positive DA neurons in <i>Atg7</i> cKO mice (black circle) was on average approximately 15% larger than that in <i>Atg7</i> cWT mice (white circle).</p

Atg7 regulates morphological plasticity of mature dopaminergic axon terminals.

<p>(<b>A</b>) Scheme of AAV2-Cre/GFP viral transduction of adult <i>Atg7^flox/flox</i> substantia nigra. Eight-week-old <i>Atg7^flox/+</i> and <i>Atg7^flox/flox</i> mice were stereotactically injected with AAV2-Cre/GFP viral solution, and sacrificed 4- or 8-weeks later. (<b>B</b>) AAV2-Cre/GFP viral transduction of substantia nigra led to prominent GFP fluorescence (green) in a majority of TH-positive DA neurons (red) at 4- or 8-weeks after the injection; no GFP fluorescence was seen in untransduced animals (data not shown). (<b>C</b>) Transduction of Cre/GFP virus into adult <i>Atg7^flox/flox</i> substantia nigra reproduced the enlarged axon terminal phenotype seen in <i>Atg7</i> cKO mice. At 8-weeks after injection, enlarged TH-positive axon terminals (arrows) were seen in the striatum of <i>Atg7^flox/flox</i> mice injected with AAV2-Cre/GFP viral solution. No enlarged axon terminals were seen in the striatum of <i>Atg7^flox/+</i> mice with AAV2-Cre/GFP virus or <i>Atg7^flox/flox</i> mice with control AAV2-GFP virus lacking Cre. Bars, 20 µm. (right) Quantification of the density of enlarged axon terminals in mice injected with AAV2-Cre/GFP virus. TH-positive axon terminal enlargement in <i>Atg7^flox/flox</i> mouse striatum was seen at 8-weeks after the AAV2-Cre/GFP virus injection. White, <i>Atg7^flox/+</i>; Black, <i>Atg7^flox/flox</i>. n = 3 per group. **, <i>p</i><0.01.</p

Characterization of PI3K/mTOR pathways in TH-positive DA neurons of <i>Atg7</i> and/or <i>Pten</i> cKO mice.

<p>PI3K/mTOR pathway signaling, in terms of accumulation of phospho-AKT (S473), phospho-TOR (S2448), and phospho-S6 (S235/236) as indicated (in red) were unchanged in the TH-positive (green) midbrain DA neurons of <i>Atg7</i> cKO mice (C), whereas these markers were increased in TH-positive DA neurons of <i>Pten</i> cKO and <i>Atg7/Pten</i> double cKO mice (arrows in B and D). (<b>A</b>) Control cWT mice, (<b>B</b>) <i>Pten</i> cKO mice, (<b>C</b>) <i>Atg7</i> cKO mice, and (<b>D</b>) <i>Atg7/Pten</i> double cKO mice. Scale bars, 10 µm.</p

Image_2_Overexpression of the autism candidate gene Cyfip1 pathologically enhances olivo-cerebellar signaling in mice.tif

Cyfip1, the gene encoding cytoplasmic FMR1 interacting protein 1, has been of interest as an autism candidate gene for years. A potential role in autism spectrum disorder (ASD) is suggested by its location on human chromosome 15q11-13, an instable region that gives rise to a variety of copy number variations associated with syndromic autism. In addition, the CYFIP1 protein acts as a binding partner to Fragile X Messenger Ribonucleoprotein (FMRP) in the regulation of translation initiation. Mutation of FMR1, the gene encoding FMRP, causes Fragile X syndrome, another form of syndromic autism. Here, in mice overexpressing CYFIP1, we study response properties of cerebellar Purkinje cells to activity of the climbing fiber input that originates from the inferior olive and provides an instructive signal in sensorimotor input analysis and plasticity. We find that CYFIP1 overexpression results in enhanced localization of the synaptic organizer neurexin 1 (NRXN1) at climbing fiber synaptic input sites on Purkinje cell primary dendrites and concomitant enhanced climbing fiber synaptic transmission (CF-EPSCs) measured using whole-cell patch-clamp recordings from Purkinje cells in vitro. Moreover, using two-photon measurements of GCaMP6f-encoded climbing fiber signals in Purkinje cells of intact mice, we observe enhanced responses to air puff stimuli applied to the whisker field. These findings resemble our previous phenotypic observations in a mouse model for the human 15q11-13 duplication, which does not extend to the Cyfip1 locus. Thus, our study demonstrates that CYFIP1 overexpression shares a limited set of olivo-cerebellar phenotypes as those resulting from an increased number of copies of non-overlapping genes located on chromosome 15q11-13.</p