Coordinate Regulation of Mature Dopaminergic Axon Morphology by Macroautophagy and the PTEN Signaling Pathway

Macroautophagy is a conserved mechanism for the bulk degradation of proteins and organelles. Pathological studies have implicated defective macroautophagy in neurodegeneration, but physiological functions of macroautophagy in adult neurons remain unclear. Here we show that Atg7, an essential macroautophagy component, regulates dopaminergic axon terminal morphology. Mature Atg7-deficient midbrain dopamine (DA) neurons harbored selectively enlarged axonal terminals. This contrasted with the phenotype of DA neurons deficient in Pten – a key negative regulator of the mTOR kinase signaling pathway and neuron size – that displayed enlarged soma but unaltered axon terminals. Surprisingly, concomitant deficiency of both Atg7 and Pten led to a dramatic enhancement of axon terminal enlargement relative to Atg7 deletion alone. Similar genetic interactions between Atg7 and Pten were observed in the context of DA turnover and DA-dependent locomotor behaviors. These data suggest a model for morphological regulation of mature dopaminergic axon terminals whereby the impact of mTOR pathway is suppressed by macroautophagy

Macroautophagy deficiency mediates age-dependent neurodegeneration through a phospho-tau pathway

Background: Macroautophagy is an evolutionarily conserved mechanism for bulk intracellular degradation of proteins and organelles. Pathological studies have implicated macroautophagy defects in human neurodegenerative disorders of aging including Alzheimer’s disease and tauopathies. Neuronal deficiency of macroautophagy throughout mouse embryonic development results in neurodevelopmental defects and early postnatal mortality. However, the role of macroautophagy in mature CNS neurons, and the relationship with human disease neuropathology, remains unclear. Here we describe mice deficient in an essential macroautophagy component, Atg7, specifically within postnatal CNS neurons. Results: Postnatal forebrain-specific Atg7 conditional knockout (cKO) mice displayed age-dependent neurodegeneration and ubiquitin- and p62-positive inclusions. Phosphorylated tau was significantly accumulated in Atg7 cKO brains, but neurofibrillary tangles that typify end-stage human tauopathy were not apparent. A major tau kinase, glycogen synthase kinase 3β (GSK3β), was also accumulated in Atg7 cKO brains. Chronic pharmacological inhibition of tau phosphorylation, or genetic deletion of tau, significantly rescued Atg7-deficiency-mediated neurodegeneration, but did not suppress inclusion formation. Conclusions: These data elucidate a role for macroautophagy in the long-term survival and physiological function of adult CNS neurons. Neurodegeneration in the context of macroautophagy deficiency is mediated through a phospho-tau pathway

Clinical and Diffusion Tensor MRI Findings in Congenital Homonymous Hemianopia

A 29-year-old woman underwent brain MRI for increasing frequency of migraine headaches. The MRI report recommended further evaluation for septo-optic dysplasia. The patient reported no visual deficits. Her examination showed normal visual acuities, color vision, pupillary function, and a normal optic disc in the right eye with mild temporal disc pallor in the left eye. Automated perimetry revealed an incongruous left homonymous hemianopia (Fig. 1). Optical coherence tomography (OCT) showed thinning of the retinal nerve fiber layer to 60 µm in the right eye and 54 µm in the left eye. There was ganglion cell layer thinning in a hemianopic pattern with temporal thinning in the right eye and nasal thinning in the left eye. This corresponded with the left homonymous hemianopia

Free induction decay navigator motion metrics for prediction of diagnostic image quality in pediatric MRI

Purpose To investigate the ability of free induction decay navigator (FIDnav)-based motion monitoring to predict diagnostic utility and reduce the time and cost associated with acquiring diagnostically useful images in a pediatric patient cohort.Methods A study was carried out in 102 pediatric patients (aged 0-18 years) at 3T using a 32-channel head coil array. Subjects were scanned with an FID-navigated MPRAGE sequence and images were graded by two radiologists using a five-point scale to evaluate the impact of motion artifacts on diagnostic image quality. The correlation between image quality and four integrated FIDnav motion metrics was investigated, as well as the sensitivity and specificity of each FIDnav-based metric to detect different levels of motion corruption in the images. Potential time and cost savings were also assessed by retrospectively applying an optimal detection threshold to FIDnav motion scores.Results A total of 12% of images were rated as non-diagnostic, while a further 12% had compromised diagnostic value due to motion artifacts. FID-navigated metrics exhibited a moderately strong correlation with image grade (Spearman's rho >= 0.56). Integrating the cross-correlation between FIDnav signal vectors achieved the highest sensitivity and specificity for detecting non-diagnostic images, yielding total time savings of 7% across all scans. This corresponded to a financial benefit of $2080 in this study.Conclusions Our results indicate that integrated motion metrics from FIDnavs embedded in structural MRI are a useful predictor of diagnostic image quality, which translates to substantial time and cost savings when applied to pediatric MRI examinations

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

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

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