RABV travels faster and is more directed when transported with p75NTR.

<p>(<b>A–C</b>) Multi-channel live imaging of EGFP-RABV 2 hours after addition to distal axon compartment of DRG explant previously treated with a fluorescent antibody against p75NTR. Arrowheads: p75NTR-positive RABV puncta, scale bar = 10 µm. (<b>D,E</b>) Kymographs of and P75NTR extracted from time lapse depicted in (A–C). (<b>F</b>) RABV-only tracks (green) are less directed than RABV-p75NTR tracks (yellow), as shown when overlaying corresponding kymographs. Vertical scale bar = 5 µm, horizontal scale bar = 40 seconds. (<b>G–O</b>) Characterization of directed RABV puncta, transported with and without p75NTR, n = 184 and n = 122, respectively. (G) RABV presents higher speeds when transported with p75NTR, due to less frequent (H) and shorter pauses (I). Overall RABV-p75NTR spent less time paused on average (J), Diameter and intensity measurements revealed that RABV puncta positive for p75NTR were larger (<b>K</b>) and had higher intensity levels (<b>L</b>) than p75NTR-negative puncta. (<b>M–O</b>) p75NTR positive puncta (blue) are faster, more directed and present higher displacements over time, compared to p75NTR negative puncta (red), illustrated by distribution of instantaneous velocities in (M) (RABV+p75: n = 8051 events; RABV-p75: n = 7423 events) displacement plotted over time (N) and mean square displacement (O). Data is pulled from two separate experiments, error bars represent SEM. *p<0.05.</p

A microfluidic system for tracking retrograde transport in sensory axons.

<p>(<b>A</b>) A Polydimethylsiloxane (PDMS) microfluidic chamber used for explant culture. (<b>B,C</b>) 40 µl interval towards the proximal compartment (top) prevents fluorescent dye from diffusing to the proximal compartment, allowing several hours of compartmental separation. Bright field (<b>B</b>) and fluorescent images (<b>C</b>) taken 7.5 hours after addition of Sulforhodamine B fluorescent dye to the distal compartment (bottom). (<b>D</b>) DRG explants are healthy and extend axons through micro-grooves to distal compartment after 2–3 days in vitro. One microgroove typically contains 2–5 axons. Mosaic of 10× images of Calcein-stained DRG explant taken after 5 DIV. Scale bar = 50 µm.</p

Suggested model.

<p>In order to arrive at the cell body and subsequently the CNS, rabies virus hijacks a fast route using the p75NTR endosomal pathway. In a p75NTR dependent path, RABV manipulates the axonal transport machinery to migrate faster to the cell body. An alternative, slower path, may involve alternative RABV receptors.</p

Rabies virus retrograde transport in DRG is faster and more directed than that of NGF.

<p>Retrograde transport of (<b>A</b>) EGFP-RABV and (<b>B</b>) Quantum-dot conjugated NGF in DRG explants, roughly 2 hours after addition to distal axon compartment. Arrows and arrowheads pointing at transported particles. Scale bars = 10 µm. (<b>C,D</b>) Kymograph for EGFP-RABV and NGF respectively. Horizontal scale bars = 5 µm, vertical scale bars = 40 seconds. (<b>E–J</b>) Characterization of manually tracked directed particles of RABV (n = 209) and NGF (n = 149). (<b>E</b>) RABV is transported faster than NGF, as seen by average speed. (<b>F</b>) RABV is more directed and pauses less than NGF (<b>G</b>) Average pause duration was not significantly different. (<b>H</b>) RABV spent a smaller fraction of its run paused (<b>I</b>) Instantaneous velocities of RABV particles (n = 9885 events) are higher than those of NGF particles (n = 8973 events). Positive and negative values represent retrograde and anterograde velocities, respectively. (<b>J</b>) RABV particles travel larger net distances than NGF's, as seen by mean squared displacements. Data was pulled from two separate experiments. Error bars represent SEM, *p<0.0001.</p

RABV and NGF present similar internalization kinetics at the axon tip.

<p>Live TIRF microscopy was used to track RABV and NGF internalization in DRG neuron tips. (<b>A</b>) EGFP-RABV (dashed circles) is detected on the surface of a neuron tip (white line). Lower panels present a particle (circled in black) disappearing gradually into the cell over a course of ∼9 seconds. (<b>B</b>) Qdot labeled NGF undergoes gradual internalization at the tip of the DRG neuron. (<b>C</b>) Average internalization time of RABV and NGF (n = 6 and 8, respectively), from onset of gradual signal reduction to its disappearance, do not differ significantly [p = 0.148]. (<b>D</b>) Representative time course of individual RABV (blue) and NGF (red) particles intensity profile, from detection on the cell surface to their internalization. Arrows represent direction of the neuron soma. Scale bars = 5 µm.</p

RABV binds and internalizes with p75NTR in DRG neuron tips.

<p>Co-localization of EGFP-RABV with p75NTR is shown by live TIRF imaging and sub-pixel localization algorithms. (<b>A</b>) RABV-p75 particles shift from the periphery to the center of the growth cone, where they are internalized into the cell. Lower panels zoom in on dashed square, showing co-localized puncta (left) shifting towards the center of the growth cone (middle) until finally internalized (right). (<b>B</b>) Presentation of six separate events of RABV and p75NTR binding and internalization on the surface of the growth cone shown in (A). Colored trajectories denote displacement from point of detection to point of disappearance. (<b>C</b>) RABV and p75NTR are internalized together, illustrated by corresponding plots of puncta intensity over time (normalized to background), calculated for co-localized particles shown in lower panels of (A). Scale bars = 5 µm. (<b>D</b>) Zoom-in on colocalized RABV and p75 spot, taken from panel (A), scale bar = 1 µm. (<b>E</b>) Overlay of 1D-Gaussian fits of p75 and RABV intensity profiles at the x-axis of the image in panel (D). (<b>F</b>) Representative overlay of radial symmetry fits of the x-y intensity profiles of p75 and RABV spots. σ is the standard deviation of each fitting function; distance between the two spot centers is 51.3 nm. (<b>G</b>) Knockdown of p75NTR decreases rabies virus infection for shorts time incubation. DRGs embryonic cells infected with lentiviral vectors (LV) containing 4 different EGFP-tagged shRNA's against p75NTR or LV-EGFP, were transfected with RABV for 30 or 120 minutes. Low levels of infected neurons were found in shRNA-p75-EGFP cells Average RABV infection rates were normalized to LV-EGFP controls (n = 4 experiments, error bars = SEM, *p<0.005, **p<0.0005).</p

Co-transport of RABV and NGF.

<p>(<b>A–C</b>) EGFP-RABV and Qdot-NGF were simultaneously added to the distal compartment of a DRG explant at 3DIV. Dual-channel live imaging revealed multiple events of RABV co-transported with NGF, illustrated by corresponding images from either channel and overlay. Arrowheads: co-localized puncta trafficked over time. Scale bar = 10 µm. (<b>D–F</b>) Kymographs of EGFP-RABV and Qdot-NGF show mutual transport of RABV and NGF. (<b>G</b>) Two populations of RABV-NGF particles were identified by distribution of average track speeds, as seen by fitted curve (red, minima point represented by dashed line, n = 46). (<b>H–J</b>) “Slow” (n = 38) and “fast” (n = 8) populations according to the minima (1.01 µm/sec) in (G). “Slow” tracks paused more than “fast” tracks (<b>H</b>), and though stop duration was not significantly different (<b>I</b>), spent a larger fraction of their travel paused (<b>J</b>). Data is pulled from two experiments. Error bars represent SEM, *p<0.02.</p

Phosphatidylserine Ameliorates Neurodegenerative Symptoms and Enhances Axonal Transport in a Mouse Model of Familial Dysautonomia.

Familial Dysautonomia (FD) is a neurodegenerative disease in which aberrant tissue-specific splicing of IKBKAP exon 20 leads to reduction of IKAP protein levels in neuronal tissues. Here we generated a conditional knockout (CKO) mouse in which exon 20 of IKBKAP is deleted in the nervous system. The CKO FD mice exhibit developmental delays, sensory abnormalities, and less organized dorsal root ganglia (DRGs) with attenuated axons compared to wild-type mice. Furthermore, the CKO FD DRGs show elevated HDAC6 levels, reduced acetylated α-tubulin, unstable microtubules, and impairment of axonal retrograde transport of nerve growth factor (NGF). These abnormalities in DRG properties underlie neuronal degeneration and FD symptoms. Phosphatidylserine treatment decreased HDAC6 levels and thus increased acetylation of α-tubulin. Further PS treatment resulted in recovery of axonal outgrowth and enhanced retrograde axonal transport by decreasing histone deacetylase 6 (HDAC6) levels and thus increasing acetylation of α-tubulin levels. Thus, we have identified the molecular pathway that leads to neurodegeneration in FD and have demonstrated that phosphatidylserine treatment has the potential to slow progression of neurodegeneration

Generation of <i>Tyrp2-Cre;IKBKAP</i>^{FDloxP/FDloxP} (CKO^<i>Tyrp2</i> FD) mice.

<p>(<b>A</b>) Two <i>loxP</i> sequences were inserted in the introns flanking exon 20 of the <i>IKBKAP</i> gene (<i>IKBAKP</i>^{FDloxP/FDloxP} mouse, the mouse shown on the left). <i>IKBAKP</i>^{FDloxP/FDloxP} mice were mated with <i>Tyrp2-Cre</i> mice (the mouse on the right). The lower panel shows a schematic representation of the <i>IKBKAP</i>^{<i>FDloxP/FDloxP</i>} construct. <i>Cre</i> activation leads to exon 20 deletion in targeted tissues. (<b>B-E</b>) Whole-mount immunostaining using Cre (red) and Tuj-1 (green) antibodies of (B) control and (C-E) CKO^<i>Tyrp2</i> FD mice; enlargements are shown in D and E. (<b>F</b>) DNA from the indicated organs was extracted and analyzed to examine exon 20 deletion. Green arrowhead indicates removal of <i>IKBKAP</i> exon 20. (<b>G</b>) Western blot of IKAP in the lungs, DRGs, cerebellums (Cere.), and forebrains (FB) of control and CKO^<i>Tyrp2</i> FD mice. (<b>H</b>) Left panel: Photographs of CKO^<i>Tyrp2</i> FD and control littermates 10 days after birth (P10). Middle panel: Weights of CKO^<i>Tyrp2</i> FD and control mice (n = 40 per group, ***p<0.001). Right panel: Weights of brains from CKO^<i>Tyrp2</i> FD and control littermates (n = 10 per group, **p<0.01). (<b>I</b>) CKO^<i>Tyrp2</i> FD mice have brownish and swollen intestines (indicated by arrow). (<b>J</b>) Tail hanging test of three-month old CKO^<i>Tyrp2</i> FD and control littermates and plots of hindpaw gaps during tail hanging (n = 5 per group, ***p<0.001). (<b>K</b>) Hot-plate analgesia evaluation of thermal sensation and peripheral sensory nerve function of CKO^<i>Tyrp2</i> FD and control mice (n = 20 per group, *p<0.05). (<b>L</b>) qRT-PCR analysis of genes, known to be abnormally regulated in FD patients in brains (n = 3 of each group) and DRGs (pool of four mice for each group) of CKO^<i>Tyrp2</i> FD and control mice. Error bars represent ± SEM and for DRGs error bars represent technical standard deviation of three repeats.</p