Synaptic Polarity Depends on Phosphatidylinositol Signaling Regulated by myo-Inositol Monophosphatase in Caenorhabditis elegans

Although neurons are highly polarized, how neuronal polarity is generated remains poorly understood. An evolutionarily conserved inositol-producing enzyme myo-inositol monophosphatase (IMPase) is essential for polarized localization of synaptic molecules in Caenorhabditis elegans and can be inhibited by lithium, a drug for bipolar disorder. The synaptic defect of IMPase mutants causes defects in sensory behaviors including thermotaxis. Here we show that the abnormalities of IMPase mutants can be suppressed by mutations in two enzymes, phospholipase Cβ or synaptojanin, which presumably reduce the level of membrane phosphatidylinositol 4,5-bisphosphate (PIP2). We also found that mutations in phospholipase Cβ conferred resistance to lithium treatment. Our results suggest that reduction of PIP2 on plasma membrane is a major cause of abnormal synaptic polarity in IMPase mutants and provide the first in vivo evidence that lithium impairs neuronal PIP2 synthesis through inhibition of IMPase. We propose that the PIP2 signaling regulated by IMPase plays a novel and fundamental role in the synaptic polarity

<em>dnc-1/dynactin 1</em> Knockdown Disrupts Transport of Autophagosomes and Induces Motor Neuron Degeneration

<div><p>Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. We previously showed that the expression of dynactin 1, an axon motor protein regulating retrograde transport, is markedly reduced in spinal motor neurons of sporadic ALS patients, although the mechanisms by which decreased dynactin 1 levels cause neurodegeneration have yet to be elucidated. The accumulation of autophagosomes in degenerated motor neurons is another key pathological feature of sporadic ALS. Since autophagosomes are cargo of dynein/dynactin complexes and play a crucial role in the turnover of several organelles and proteins, we hypothesized that the quantitative loss of dynactin 1 disrupts the transport of autophagosomes and induces the degeneration of motor neuron. In the present study, we generated a <em>Caenorhabditis elegans</em> model in which the expression of DNC-1, the homolog of dynactin 1, is specifically knocked down in motor neurons. This model exhibited severe motor defects together with axonal and neuronal degeneration. We also observed impaired movement and increased number of autophagosomes in the degenerated neurons. Furthermore, the combination of rapamycin, an activator of autophagy, and trichostatin which facilitates axonal transport dramatically ameliorated the motor phenotype and axonal degeneration of this model. Thus, our results suggest that decreased expression of dynactin 1 induces motor neuron degeneration and that the transport of autophagosomes is a novel and substantial therapeutic target for motor neuron degeneration.</p> </div

Morphological changes in ventral motor neurons.

<p>(<i>A</i>) Representative view of fluorescent GFP microscopic images of the ventral nerve cord in a <i>control(RNAi) C. elegans</i>. All of the motor neurons (white asterisks) were located in the ventral side of the worm. Axons from the motor neurons project within the ventral nerve cord or toward the dorsal side. (<i>B–E</i>) Representative view of the ventral nerve cord in the <i>control(RNAi)</i> worms (<i>B, C</i>) and <i>dnc-1(RNAi)</i> worms (<i>D, E</i>). The degenerated axons were defasciculated (arrows in <i>D, E</i>) and formed spheroids (arrowheads in <i>D, E</i>) in the <i>dnc-1(RNAi)</i> worms. Mild defasciculation was observed occasionally in the <i>control(RNAi)</i> worms (arrow in <i>C</i>). While the cell bodies of the motor neurons were regular and round in <i>control(RNAi)</i> and young adult <i>dnc-1(RNAi)</i> worms (white asterisks in <i>B–D</i>), abnormally shaped cell bodies (yellow asterisks in <i>E</i>) were observed only in the worms with severe axonal changes. (<i>F</i>) Semi-quantification of the abnormal morphological changes in the <i>control(RNAi)</i> and <i>dnc-1(RNAi)</i> worms. The percentage of worms with axonal defasciculation, axonal spheroids, or cell body degeneration on days 4, 7, and 10. (<i>G</i>) Population of <i>dnc-1(RNAi)</i> worms with and without cell body degeneration (black and gray boxes, respectively) on day 4. (<i>H</i>) Correlation between the axonal defasciculation index and locomotor function in the <i>dnc-1(RNAi)</i> worms. The axonal defasciculation index represents the degree of axonal defasciculation (its details are described in the Materials and Methods). Scale bars = 20 μm. The statistical analysis in <i>F</i> was performed using Fisher's exact probability test (*p<0.05, **p<0.001, and ***p<0.0001) and Pearson's correlation coefficient in <i>H</i>.</p

Accumulation of autophagosomes and motor neuron degeneration in the <i>dnc-1</i><i>(</i><i>RNAi</i><i>)</i> worms.

<p>(<i>A, B</i>) Representative kymographs of Lgg1::DsRed in the ventral nerve cord from the <i>control(RNAi</i> (<i>A</i>) and <i>dnc-1(RNAi)</i> (<i>B</i>) worms derived from time-lapse images. Vertical lines represent stationary/docked Lgg1 puncta, while the oblique lines (labeled with arrowheads) represent the tracks of moving Lgg1 puncta. The slope of this track is an indicator of velocity. (<i>C</i>) The number of Lgg1 puncta within a single kymograph image. (<i>D, E</i>) Quantitative analyses of the mobility of puncta. The number of puncta that moved more than 2 μm was counted (<i>D</i>). The ratio of moving puncta was calculated by dividing the number of moving puncta by the total number of puncta (<i>E</i>). (<i>F</i>) The mean velocities of Lgg1 puncta. A total of 20 time-lapse images were analyzed for each strain in <i>C–F</i>. (<i>G</i>) The number of Lgg1 puncta was increased in the <i>dnc-1(RNAi)</i> worms compared with the <i>control(RNAi)</i> worms (n = 15 for each group). (<i>H, I</i>) Accumulation of autophagosomes in the <i>dnc-1(RNAi)</i> worms was correlated with the severity of axonal defasciculation (<i>H</i>) and locomotor function (<i>I</i>) (n = 20 for each analysis). (<i>J–L</i>) Ultrastructural images of ventral motor neurons from the <i>dnc-1(RNAi)</i> worms. Aberrant membranous vesicles including degenerated mitochondria were observed in the cytoplasm (<i>J</i>) and axons (<i>K</i>) (arrows). Autophagosome-like, double membrane vesicles (asterisk in <i>L</i>) were also found in the axons of the <i>dnc-1(RNAi)</i> worms (<i>L</i>). Scale bar = 500 nm (A–C) or 10 μm (D). Statistical analyses were performed using Student's t test (*p<0.05 and **p<0.0001) and Pearson's correlation coefficient in <i>H</i> and <i>I</i>. The error bars are S.E.M.</p

Impaired transport and abnormal accumulation of autophagosomes in the axons of <i>dnc-1</i><i>(</i><i>RNAi</i><i>)</i> motor neurons.

<p>(<i>A, B</i>) Representative time-lapse images of autophagosome (DsRed-tagged Lgg1) transport in an axon (GFP-tagged shRNA; green) of a primary cultured motor neuron from the <i>control(RNAi)</i> (<i>A</i>) and <i>dnc-1(RNAi)</i> (<i>B</i>) worms. The autophagosomes were transported smoothly along the axon (arrows) of the <i>control(RNAi)</i> motor neuron (<i>A</i>). The autophagosome (arrows) was transported anterogradely, but was trapped where the axon was slightly narrowed (arrowhead) (<i>B</i>). There were also autophagosomes that accumulated in the distal part of the axon (<i>B</i>, bar). (<i>C</i>) Histograms of Lgg1::DsRed velocity in the retrograde (white bars) and anterograde (black bars) directions in neurons from the <i>control(RNAi)</i> and <i>dnc-1(RNAi)</i> worms. (<i>D</i>) Histograms of Lgg1::DsRed run-length in the <i>control(RNAi)</i> and <i>dnc-1(RNAi)</i> neurons. (<i>E, F</i>) Mean velocity (<i>E</i>) and run-length (<i>F</i>) of autophagosomes (n = 70 vesicles for each strain) in <i>control(RNAi)</i> and <i>dnc-1(RNAi)</i> neurons. Scale bar = 5 μm (A and B). The statistical analyses in <i>E</i> and <i>F</i> were performed using the Mann-Whitney U test (*p<0.05 and **p<0.0001). The error bars are S.E.M.</p

Ultrastructure of degenerating motor neurons.

<p>Electron microscopy of transverse sections of ventral motor neurons from the <i>control(RNAi)</i> (<i>A, B</i>) and <i>dnc-1(RNAi)</i> (<i>C–F</i>) worms. The dashed lines in <i>B</i>, <i>D</i>, and <i>F</i> denote the boundaries of the main bundle of axons. Each round-shaped component inside the dashed line is an axon. In the <i>dnc-1(RNAi)</i> worms, whorl-like inclusions (W) and vacuoles (V) were observed (<i>D–F</i>). In the worms with mild axonal degeneration (<i>D</i>), few morphological changes were observed in the cytoplasm (<i>C</i>); however, in the later stage with severe axonal degeneration (<i>F</i>), the cell bodies were also affected (<i>E</i>). Scale bars = 20 μm.</p

Defective axonal transport of synaptobrevin-1 in <i>dnc-1</i><i>(</i><i>RNAi</i><i>) </i><i>C. elegans</i>.

<p>(<i>A, B</i>) Expression patterns of DsRed-tagged synaptobrevin-1 (SNB-1) in the dorsal nerve cord. In the <i>control(RNAi)</i> worms, SNB-1 puncta (arrowheads) are regularly spaced with a uniform shape. In the <i>dnc-1(RNAi)</i> worms (B), they are irregularly spaced and abnormally accumulated (white bars) with occasional clumps. (<i>C, D</i>) Histograms of the distances between neighboring SNB-1 puncta. The average distance between puncta in the <i>control(RNAi)</i> (3.240±1.716 μm, n = 139) and <i>dnc-1(RNAi)</i> (3.855±2.764 μm, n = 104) worms was not significantly different (p = 0.996 by Student's t test), but the peak of the control histogram was higher than that of the <i>dnc-1(RNAi)</i> histogram, proving that the localization of SNB1 was irregular. (<i>E, F</i>) Representative kymographs of SNB-1::DsRed in the ventral nerve cord from the <i>control(RNAi)</i> (<i>E</i>) and <i>dnc-1(RNAi)</i> (<i>F</i>) worms derived from time-lapse imaging. Vertical lines represent stationary/docked SNB-1 puncta and oblique lines (labeled with yellow arrowheads) represent the tracks of moving SNB-1 puncta. The slope of this track is an indicator of velocity. (<i>G</i>) The number of SNB-1 puncta within a single image of kymograph was not different between the <i>control(RNAi)</i> and the <i>dnc-1(RNAi)</i> worms. (<i>H</i>) The mean velocities of SNB-1 puncta. (<i>I, J</i>) The quantitative analysis of mobile puncta. The number of puncta which moved more than 2 μm was counted (<i>I</i>). The ratio of moving puncta was calculated by dividing the number of moving puncta by the total number of SNB-1 puncta (<i>J</i>). A total of 20 time laps images were analyzed from each strains in <i>G</i>–<i>J</i>. Scale bar (black)  = 10 μm (<i>B</i>). Statistical analyses were performed using Student's t test (*p<0.05, **p<0.001, ***p<0.0001). Error bars are S.E.M.</p

Creation of the motor neuron-specific <i>dnc-1-</i>KD <i>C. elegans</i> model.

<p>(<i>A</i>) Fluorescent visualization of ventral cholinergic motor neurons and their neurites in transgenic <i>C. elegans</i> worms expressing acr2p::<i>shRNA</i>::<i>gfp</i>. (<i>B</i>) Representative immunohistochemical staining of GFP and <i>in situ</i> hybridization against <i>dnc-1</i> in ventral cholinergic motor neurons and their neurites in the <i>control(RNAi)</i> and <i>dnc-1(RNAi)</i> worms. (<i>C</i>) The number of GFP-positive motor neurons (white arrows in <i>B</i>) was not significantly different between the <i>control(RNAi)</i> and <i>dnc-1(RNAi)</i> worms (n = 20 animals for each strain). (<i>D</i>) Conversely, the number of <i>dnc-1</i> mRNA-positive neurons (black arrows in <i>B</i>) was remarkably decreased in the <i>dnc-1(RNAi)</i> worms (n = 20 animals for each strain). (<i>E</i>) Representative images of <i>in situ</i> hybridization for <i>dnc-1</i> in the head neurons. Scale bars = 100 μm (<i>A</i>), 10 μm (<i>B</i>), and 20 μm (<i>E</i>). Statistical analyses were performed using Student's t test (*p<0.0001). The error bars are S.E.M.</p

Dysfunction of autophagy causes axonal degeneration.

<p>(<i>A</i>) Treatment with 3-MA decreased the number of autophagosomes in the ventral nerve cord in a dose dependent manner (n = 15 for each group). (<i>B–E</i>) The effects of 3-MA on the locomotor function (<i>C</i>) and axonal morphology (<i>B, D,</i> and <i>E</i>) of the <i>control(RNAi)</i> worms. Treatment with 3-MA increased axonal defasciculation (arrows in <i>B</i> and the graph in <i>D</i>) and the number of axonal spheroids (arrowheads in <i>B</i> and the graph in <i>E</i>) (n = 15 for each group). (<i>F–H</i>) The effects of 3-MA on the locomotor function (<i>F</i>) and axonal morphology (<i>G, H</i>) of the <i>dnc-1(RNAi)</i> worms (n = 15 for each group). Scale bar = 10 μm. Statistical analyses were performed using Dunnett's post hoc test (<i>A</i>) and Student's t test (B, <i>D</i>, and <i>E</i>) (*p<0.05, **p<0.001, and ***p<0.0001). The error bars are S.E.M.</p

Dysregulated expression of dynactin 1 and the accumulation of autophagosomes in SALS patients.

<p>(<i>A</i>) Representative <i>in situ</i> hybridization for <i>DCTN1</i> in the spinal cords of control and ALS patients. (<i>B, C</i>) Representative immunohistochemistry for dynactin 1 and microtubule-associated protein 1 light chain 3 alpha (LC3) on consecutive spinal cord (<i>B</i>) and cerebellar (<i>C</i>) sections from control and ALS patients. (<i>D</i>) Quantification of the signal intensity of LC3 in anterior horn neurons of the spinal cord (n = 20 sections from 4 patients for each group). (<i>E</i>) Correlation between LC3 intensity and the expression of <i>DCTN1</i> in individual motor neurons from SALS patients (n = 12 consecutive sections from 3 SALS patients). (<i>F</i>) Correlation between the intensity of LC3 immunoreactivity and the size of motor neurons in SALS patients (n = 20 sections from 4 patients). (<i>G–L</i>) Electron microscopy images of spinal motor neurons. Representative lower magnification image of a motor neuron from a control patient (<i>G</i>) and lower (<i>H</i>) and higher magnification images (<i>I–L</i>) from SALS patients. The open arrowheads indicate lipofuscin. There were abundant autophagic vacuoles, e.g., multi-lamellar bodies (arrowheads in <i>I, K</i>), autophagosome-like double membrane vesicles (arrows in <i>K</i>, <i>J</i>), and autolysosomes (asterisks in <i>L</i>) in the motor neurons of SALS patients, but not of the control. Scale bar = 50 μm (<i>A–C</i>), 2 μm (<i>G, H</i>), or 1μm (<i>I–L</i>). Statistical analyses were performed using Student's t test (*p<0.0001) and Pearson's correlation coefficient in <i>E</i> and <i>F</i>. The error bars are S.E.M.</p