Bidirectional molecular communication between the motoneuron and the muscle is vital for neuromuscular junction (NMJ) formation and maintenance. The molecular mechanisms underlying such communication are of keen interest and could provide new targets for intervention in motoneuron disease. Here, we developed a microfluidic platform with motoneuron cell bodies on one side and muscle cells on the other, connected by motor axons extending through microgrooves to form functional NMJs. Using this system, we were able to differentiate between the proximal and distal effects of oxidative stress and glial-derived neurotrophic factor (GDNF), demonstrating a dying-back degeneration and retrograde transmission of pro-survival signaling, respectively. Furthermore, we show that GDNF acts differently on motoneuron axons versus soma, promoting axonal growth and innervation only when applied locally to axons. Finally, we track for the first time the retrograde transport of secreted GDNF from muscle to neuron. Thus, our data suggests spatially distinct effects of GDNF – facilitating growth and muscle innervation at axon terminals and survival pathways in the soma.

Rabies virus (RABV) is a neurotropic virus that depends on long distance axonal transport in order to reach the central nervous system (CNS). The strategy RABV uses to hijack the cellular transport machinery is still not clear. It is thought that RABV interacts with membrane receptors in order to internalize and exploit the endosomal trafficking pathway, yet this has never been demonstrated directly. The p75 Nerve Growth Factor (NGF) receptor (p75NTR) binds RABV Glycoprotein (RABV-G) with high affinity. However, as p75NTR is not essential for RABV infection, the specific role of this interaction remains in question. Here we used live cell imaging to track RABV entry at nerve terminals and studied its retrograde transport along the axon with and without the p75NTR receptor. First, we found that NGF, an endogenous p75NTR ligand, and RABV, are localized in corresponding domains along nerve tips. RABV and NGF were internalized at similar time frames, suggesting comparable entry machineries. Next, we demonstrated that RABV could internalize together with p75NTR. Characterizing RABV retrograde movement along the axon, we showed the virus is transported in acidic compartments, mostly with p75NTR. Interestingly, RABV is transported faster than NGF, suggesting that RABV not only hijacks the transport machinery but can also manipulate it. Co-transport of RABV and NGF identified two modes of transport, slow and fast, that may represent a differential control of the trafficking machinery by RABV. Finally, we determined that p75NTR-dependent transport of RABV is faster and more directed than p75NTR-independent RABV transport. This fast route to the neuronal cell body is characterized by both an increase in instantaneous velocities and fewer, shorter stops en route. Hence, RABV may employ p75NTR-dependent transport as a fast mechanism to facilitate movement to the CNS.

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.

Rabies virus (RABV) spread is widely accepted to occur only by retrograde axonal transport. However, examples of anterograde RABV spread in peripheral neurons such as dorsal root ganglion (DRG) neurons indicated a possible bidirectional transport by an uncharacterized mechanism. Here, we analyzed the axonal transport of fluorescence-labeled RABV in DRG neurons by livecell microscopy. Both entry-related retrograde transport of RABV after infection at axon endings and postreplicative transport of newly formed virus were visualized in compartmentalized DRG neuron cultures. Whereas entry-related transport at 1.5 m/s occurred only retrogradely, after 2 days of infection, multiple particles were observed in axons moving in both the anterograde and retrograde directions. The dynamics of postreplicative retrograde transport (1.6 m/s) were similar to those of entry-related retrograde transport. In contrast, anterograde particle transport at 3.4 m/s was faster, indicating active particle transport. Interestingly, RABV missing the glycoproteins did not move anterogradely within the axon. Thus, anterograde RABV particle transport depended on the RABV glycoprotein. Moreover, colocalization of green fluorescent protein (GFP)-labeled ribonucleoproteins (RNPs) and glycoprotein in distal axonal regions as well as cotransport of labeled RNPs with membrane-anchored mCherry reporter confirmed that either complete enveloped virus particles or vesicle associated RNPs were transported. Our data show that anterograde RABV movement in peripheral DRG neurons occurs by active motor protein-dependent transport. We propose two models for postreplicative long-distance transport in peripheral neurons: either transport of complete virus particles or cotransport of RNPs and G-containing vesicles through axons to release virus at distal sites of infected DRG neurons. IMPORTANCERabies virus retrograde axonal transport by dynein motors supports virus spread over long distances and lethal infection of the central nervous system. Though active rabies virus transport has been widely accepted to be unidirectional, evidence for anterograde spread in peripheral neurons supports the hypothesis that in some neurons RABV also enters the anterograde pathway by so-far unknown mechanisms. By live microscopy we visualized fast anterograde axonal transport of rabies virus. The velocities exceeded those of retrograde movements, suggesting that active, most likely kinesin-dependent transport machineries are involved. Dependency of anterograde transport on the expression of virus glycoprotein G and cotransport with vesicles further suggest that complete enveloped virus particles or cotransport of virus ribonucleoprotein and G-containing vesicles occurred. These data provide the first insight in the mechanism of anterograde rabies virus transport and substantially contribute to the understanding of RABV replication and spread of newly formed virus in peripheral neurons.

During development, neurons undergo apoptosis if they do not receive adequate trophic support from tissues they innervate or when detrimental factors activate the p75 neurotrophin receptor (p75NTR) at their axon ends. Trophic factor deprivation (TFD) or activation of p75NTR in distal axons results in a retrograde degenerative signal. However, the nature of this signal and the regulation of its transport are poorly understood. Here, we identify p75NTR intracellular domain (ICD) and histone deacetylase 1 (HDAC1) as part of a retrograde pro-apoptotic signal generated in response to TFD or ligand binding to p75NTR in sympathetic neurons. We report an unconventional function of HDAC1 in retrograde transport of a degenerative signal and its constitutive presence in sympathetic axons. HDAC1 deacetylates dynactin subunit p150, which enhances its interaction with dynein. These findings define p75NTR ICD as a retrograde degenerative signal and reveal p150 deacetylation as a unique mechanism regulating axonal transport.