Atg9a deficiency causes axon-specific lesions including neuronal circuit dysgenesis

Conditional knockout mice for Atg9a, specifically in brain tissue, were generated to understand the roles of ATG9A in the neural tissue cells. The mice were born normally, but half of them died within one wk, and none lived beyond 4 wk of age. SQSTM1/p62 and NBR1, receptor proteins for selective autophagy, together with ubiquitin, accumulated in Atg9a-deficient neurosoma at postnatal d 15 (P15), indicating an inhibition of autophagy, whereas these proteins were significantly decreased at P28, as evidenced by immunohistochemistry, electron microscopy and western blot. Conversely, degenerative changes such as spongiosis of nerve fiber tracts proceeded in axons and their terminals that were occupied with aberrant membrane structures and amorphous materials at P28, although no clear-cut degenerative change was detected in neuronal cell bodies. Different from autophagy, diffusion tensor magnetic resonance imaging and histological observations revealed Atg9a-deficiency-induced dysgenesis of the corpus callosum and anterior commissure. As for the neurite extensions of primary cultured neurons, the neurite outgrowth after 3 d culturing was significantly impaired in primary neurons from atg9a-KO mouse brains, but not in those from atg7-KO and atg16l1-KO brains. Moreover, this tendency was also confirmed in Atg9a-knockdown neurons under an atg7-KO background, indicating the role of ATG9A in the regulation of neurite outgrowth that is independent of autophagy. These results suggest that Atg9a deficiency causes progressive degeneration in the axons and their terminals, but not in neuronal cell bodies, where the degradations of SQSTM1/p62 and NBR1 were insufficiently suppressed. Moreover, the deletion of Atg9a impaired nerve fiber tract formation

Enrichment of GABARAP Relative to LC3 in the Axonal Initial Segments of Neurons

<div><p>GABA_A receptor-associated protein (GABARAP) was initially identified as a protein that interacts with GABA_A receptor. Although LC3 (microtubule-associated protein 1 light chain 3), a GABARAP homolog, has been localized in the dendrites and cell bodies of neurons under normal conditions, the subcellular distribution of GABARAP in neurons remains unclear. Subcellular fractionation indicated that endogenous GABARAP was localized to the microsome-enriched and synaptic vesicle-enriched fractions of mouse brain as GABARAP-I, an unlipidated form. To investigate the distribution of GABARAP in neurons, we generated GFP-GABARAP transgenic mice. Immunohistochemistry in these transgenic mice showed that positive signals for GFP-GABARAP were widely distributed in neurons in various brain regions, including the hippocampus and cerebellum. Interestingly, intense diffuse and/or fibrillary expression of GFP-GABARAP was detected along the axonal initial segments (AIS) of hippocampal pyramidal neurons and cerebellar Purkinje cells, in addition to the cell bodies and dendrites of these neurons. In contrast, only slight amounts of LC3 were detected along the AIS of these neurons, while diffuse and/or fibrillary staining for LC3 was mainly detected in their cell bodies and dendrites. These results indicated that, compared with LC3, GABARAP is enriched in the AIS, in addition to the cell bodies and dendrites, of these hippocampal pyramidal neurons and cerebellar Purkinje cells.</p> </div

Most endogenous GABARAP in the brain is GABARAP-I.

<p>A crude synaptosomal fraction (P2') was prepared from mouse brain, and the residual supernatant containing small cell fragments such as microsomes and soluble proteins was centrifuged at 100,000×g to yield P3 pellet (microsome-enriched fraction) and S2 supernatant (soluble protein-enriched fraction). After hypotonic lysis of the P2' fraction, the lysate was centrifuged at 33,000×g to yield the lysate-pellet (LP1) and the lysate-supernatant (LS1). LS1 was further centrifuged at 260,000×g for 2 h. After discarding the supernatant, the pellet (LP2) was collected as a synaptic vesicle-enriched fraction. Note that little GABARAP-PL was present in any fraction, while LC3-II was present in both the microsome-enriched and synaptic vesicle-enriched fractions.</p

Starvation conditions have little effect on GFP-GABARAP expression in the heart, liver and skeletal muscle.

<p>(A) Expression of GFP-GABARAP in tissues of GFP-GABARAP transgenic mice. Cell lysates were immunoblotted with anti-GFP antibody to detect GFP-GABARAP. Endogenous GABARAP and LC3 were detected with anti-GABARAP and anti-LC3 antibodies, respectively. Arrow-heads indicate GFP-GABARAP; asterisks indicate non-specific bands in the skeletal muscle. CON, lysate from control mouse tissue; tg, lysate from GFP-GABARAP transgenic mouse tissue; SK muscle, skeletal muscle. (<b>B</b>–<b>I</b>) Confocal fluorescence images of GFP-GABARAP in the <b>heart</b> (<b>B</b> and <b>C</b>), <b>liver</b> (<b>E</b> and <b>F</b>), and <b>SK muscle</b> (<b>H</b> and <b>I</b>). Fluorescence of GFP-GABARAP was observed under confocal laser-scanning microscopy (FV1000: Olympus), and GFP-GABARAP dots were counted using an ImageJ program (<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a>) with a TopHat plugin (<a href="http://rsb.info.nih.gov/ij/plugins/lipschitz/" target="_blank">http://rsb.info.nih.gov/ij/plugins/lipschitz/</a>). (<b>D</b>, <b>G</b>, and <b>J</b>) Relative ratios of GFP-GABARAP dots per unit area in the heart (<b>D</b>), liver (<b>E</b>), and skeletal muscle (<b>J</b>), using at least 10 images from each tissue in four mice. <b>Fed</b>, tissues under fed conditions; <b>Starvation</b>, tissues under starvation conditions for 48 h. Bars indicate 10 mm.</p

GFP-GABARAP, but little LC3, is enriched in the axonal initial segments of neurons.

<p>(A–L) Distribution patterns of GFP-GABARAP (green) and endogenous LC3 (red) in the pyramidal neurons of the CA1 region of the hippocampus (A–D) and cerebellar Purkinje cells (E–L) of GFP-GABARAP transgenic mice. Boxed areas in E–H are enlarged and shown in I-L. Nuclei were stained with DAPI (blue). Immunopositive signals for GFP and LC3 co-localized in the dendrites of hippocampal pyramidal and cerebellar Purkinje cells located in the stratum radiatum (sr) and the molecular layer (m), respectively (arrowheads). Intense immunopositivity for GFP, but not for LC3, was present along the axonal initial segments of these neurons (arrows). (M–P) Fluorescence intensity of GFP (M, O) and LC3 (N, P) immunoreactivity in the somata (S), dendrites (D) and axon initial segments (A) of pyramidal neurons in the hippocampus (Hp) (M, N) and Purkinje cells in the cerebellum (Cb) (O, P). Intensities are normalized relative to those of somata. Vertical bars represent means ± SEMs (n = 10 and 7 for hippocampal neurons and cerebellar Purkinje cells, respectively). Both in the hippocampal neurons and cerebellar Purkinje cells, the intensity of GFP immunoreactivity was highest in the axonal initial segments, whereas the highest immunoreactivity for LC3 was detected in the dendrites (*<i>P</i><0.01, one-way ANOVA followed by Tukey's <i>post hoc</i> test). The intensity of LC3 immunoreactivity in the axon initial segments was almost negligible. Abbreviations: so, stratum oriens; sp, stratum pyramidale; p, Purkinje cell layer; g, granular cell layer. Bars indicate 30 µm in (A–H) and 60 µm in (I–L).</p

GFP-GABARAP colocalizes with ankyrin-G and MAP2.

<p>(A–H) The pyramidal neurons of the CA1 region of the hippocampus (A–D) and cerebellar Purkinje cells (E–H) of GFP-GABARAP transgenic mice were stained with antibodies to GFP (green), ankyrin-G (red) and MAP2 (blue). GFP and MAP2 co-localized in the dendrites of hippocampal pyramidal and cerebellar Purkinje cells in the stratum radiatum (sr) and molecular layer (m), respectively (arrowheads). GFP was also detected in the ankyrin-G-positive axonal initial segments of these neurons (arrows). (I–L) Calbindin-positive axons of Purkinje cells of GFP-GABARAP transgenic mice (an asterisk), specifically axonal segments positive for ankyrin-G (blue) and calbindin (red) (arrow), were strongly positive for GFP signal (green), whereas distal axons devoid of ankyrin-G immunoreactivity (arrows) were not. (M–Q) Cultured cortical neurons from GFP-GABARAP transgenic mice immunostained for MAP2 (white), GFP (green), ankyrin-G (red) and tau-1 (blue). The asterisk indicates the cell body of a GFP-GABARAP-positive neuron. Weak immunoreactivity for GFP was detected in MAP2-positive dendrites (open arrowheads) (M, N). (N–Q) Strong GFP-GABARAP signals detected in the cell body and axon initial segment, which were positive for ankyrin-G and tau-1. In contrast, little GFP signal could be detected in tau-1-positive distal axons devoid of ankyrin-G immunoreactivity (arrows). Abbreviations: so, stratum oriens; sp stratum pyramidale; p, Purkinje cell layer; g, granular cell layer. Bars indicate 30 µm.</p

Sodium orthovanadate inhibits p53-mediated apoptosis

We recently reported a novel suppressive effect of sodium orthovanadate (vanadate) on the DNA-binding activity of p53. Here, we showed that vanadate had a more potent antiapoptotic activity than three other chemical p53 inhibitors, including pifithrin-alpha. Although the other agents inhibited p53\u27s transcriptional activity, they did not suppress p53-dependent apoptosis in irradiated MOLT-4 cells. To investigate the cause for the different effects of vanadate and the other inhibitors, we chose PFT-alpha and PFT-mu (an inhibitor of the p53-mediated transcription-independent apoptotic pathway), as references, and determined their and vanadate\u27s effect on p53-mediated apoptosis, with a focus on the transcription-independent pathway. We found that vanadate suppressed the p53-associated apoptotic events at the mitochondria, including the loss of mitochondrial membrane potential, the conformational change of Bax and Bak, the mitochondrial translocation of p53, and p53\u27s interaction with Bcl-2. Vanadate also suppressed the apoptosis-inducing activity of a mitochondrially targeted temperature-sensitive p53 in stable transfectants of the SaOS-2 cell line. Finally, we tested vanadate\u27s potential as a radioprotector. Vanadate completely protected mice from a sublethal dose of 8 Gy and partially from a lethal dose of 12 Gy. Our data demonstrate that vanadate can suppress both the transcription-dependent and the transcription-independent p53 pathways, and suggest that both pathways must be inhibited to completely block p53-mediated apoptosis

<i>Atg9a</i> deficiency causes axon-specific lesions including neuronal circuit dysgenesis

<p>Conditional knockout mice for <i>Atg9a</i>, specifically in brain tissue, were generated to understand the roles of ATG9A in the neural tissue cells. The mice were born normally, but half of them died within one wk, and none lived beyond 4 wk of age. SQSTM1/p62 and NBR1, receptor proteins for selective autophagy, together with ubiquitin, accumulated in <i>Atg9a</i>-deficient neurosoma at postnatal d 15 (P15), indicating an inhibition of autophagy, whereas these proteins were significantly decreased at P28, as evidenced by immunohistochemistry, electron microscopy and western blot. Conversely, degenerative changes such as spongiosis of nerve fiber tracts proceeded in axons and their terminals that were occupied with aberrant membrane structures and amorphous materials at P28, although no clear-cut degenerative change was detected in neuronal cell bodies. Different from autophagy, diffusion tensor magnetic resonance imaging and histological observations revealed <i>Atg9a</i>-deficiency-induced dysgenesis of the corpus callosum and anterior commissure. As for the neurite extensions of primary cultured neurons, the neurite outgrowth after 3 d culturing was significantly impaired in primary neurons from <i>atg9a</i>-KO mouse brains, but not in those from <i>atg7</i>-KO and <i>atg16l1</i>-KO brains. Moreover, this tendency was also confirmed in <i>Atg9a</i>-knockdown neurons under an <i>atg7</i>-KO background, indicating the role of ATG9A in the regulation of neurite outgrowth that is independent of autophagy. These results suggest that <i>Atg9a</i> deficiency causes progressive degeneration in the axons and their terminals, but not in neuronal cell bodies, where the degradations of SQSTM1/p62 and NBR1 were insufficiently suppressed. Moreover, the deletion of <i>Atg9a</i> impaired nerve fiber tract formation.</p