Detection of Intranasally Delivered Bone Marrow-Derived Mesenchymal Stromal Cells in the Lesioned Mouse Brain: A Cautionary Report

Bone marrow-derived mesenchymal stromal cells (MSCs) hold promise for autologous treatment of neuropathologies. Intranasal delivery is relatively noninvasive and has recently been reported to result in transport of MSCs to the brain. However, the ability of MSCs to migrate from nasal passages to sites of neuropathology and ultimately survive has not been fully examined. In this paper, we harvested MSCs from transgenic mice expressing enhanced green fluorescent protein (cells hereafter referred to as MSC-EGFP) and delivered them intranasally to wild-type mice sustaining mechanical lesions in the striatum. Using fluorescent, colorimetric, and ultrastructural detection methods, GFP-expressing cells were undetectable in the brain from 3 hours to 2 months after MSC delivery. However, bright autofluorescence that strongly resembled emission from GFP was observed in the olfactory bulb and striatum of lesioned control and MSC-EGFP-treated mice. In a control experiment, we directly implanted MSC-EGFPs into the mouse striatum and detected robust GFP expression 1 and 7 days after implantation. These findings suggest that—under our conditions—intranasally delivered MSC-EGFPs do not survive or migrate in the brain. Furthermore, our observations highlight the necessity of including appropriate controls when working with GFP as a cellular marker

Myosin Va binding to neurofilaments is essential for correct myosin Va distribution and transport and neurofilament density

The identification of molecular motors that modulate the neuronal cytoskeleton has been elusive. Here, we show that a molecular motor protein, myosin Va, is present in high proportions in the cytoskeleton of mouse CNS and peripheral nerves. Immunoelectron microscopy, coimmunoprecipitation, and blot overlay analyses demonstrate that myosin Va in axons associates with neurofilaments, and that the NF-L subunit is its major ligand. A physiological association is indicated by observations that the level of myosin Va is reduced in axons of NF-L–null mice lacking neurofilaments and increased in mice overexpressing NF-L, but unchanged in NF-H–null mice. In vivo pulse-labeled myosin Va advances along axons at slow transport rates overlapping with those of neurofilament proteins and actin, both of which coimmunoprecipitate with myosin Va. Eliminating neurofilaments from mice selectively accelerates myosin Va translocation and redistributes myosin Va to the actin-rich subaxolemma and membranous organelles. Finally, peripheral axons of dilute-lethal mice, lacking functional myosin Va, display selectively increased neurofilament number and levels of neurofilament proteins without altering axon caliber. These results identify myosin Va as a neurofilament-associated protein, and show that this association is essential to establish the normal distribution, axonal transport, and content of myosin Va, and the proper numbers of neurofilaments in axons

Dissociation of Axonal Neurofilament Content from Its Transport Rate

The axonal cytoskeleton of neurofilament (NF) is a long-lived network of fibrous elements believed to be a stationary structure maintained by a small pool of transported cytoskeletal precursors. Accordingly, it may be predicted that NF content in axons can vary independently from the transport rate of NF. In the present report, we confirm this prediction by showing that human NFH transgenic mice and transgenic mice expressing human NFL Ser55 (Asp) develop nearly identical abnormal patterns of NF accumulation and distribution in association with opposite changes in NF slow transport rates. We also show that the rate of NF transport in wild-type mice remains constant along a length of the optic axon where NF content varies 3-fold. Moreover, knockout mice lacking NFH develop even more extreme (6-fold) proximal to distal variation in NF number, which is associated with a normal wild-type rate of NF transport. The independence of regional NF content and NF transport is consistent with previous evidence suggesting that the rate of incorporation of transported NF precursors into a metabolically stable stationary cytoskeletal network is the major determinant of axonal NF content, enabling the generation of the striking local variations in NF number seen along axons

NFM phosphorylation increases along the optic pathway in a proximal-to-distal manner.

<p>Coomassie blue-stained 2D gels of Triton-insoluble fractions from consecutive 1 mm segments of the optic nerve and tract demonstrate that phosphorylated NFM (indicated by arrows, pI 5.0) shows proximal to distal increase (<b>A</b>). The ratios of phosphorylated NFM over non-phosphorylated NFM (indicated by arrowheads, pI 5.5) increase up to about 300% along optic pathway (Mean ± SD, n = 2, <b>B</b>).</p

Relationship of the number of NFs to the cross-sectional area at two levels (50 μm and 700μm) along the optic nerve in WT (A, C) and hNFH tg mice (B, D).

<p>Region-specific shifts in inter-neurofilament spacing in hNFH tg mice (F, H, J) compared with WT controls (E, G, I) at the 50 μm, 700 μm and 7000 μm levels of the optic nerve. The nearest neighbor distance was calculated for every NF in axons of calibers representative of those in the total axonal population at the 50 μm, 700 μm and 7000 mm levels of the optic pathway.</p

NF transport rates differ in mice exhibiting the same pattern of abnormal NF accumulation along axons.

<p>NF transport rate was determined by intravitreal injection of radiolabeled 35S-methionine into wild-type controls (<b>A-B, G-H</b>), hNFH tg (<b>C-D, I-J</b>) and hNFL Ser55 tg mice (<b>E-F, K-L</b>). At 7 and 16 days after pulse labeling, optic pathways were cut into consecutive 1.1mm segments and fractionated into cytoskeleton and soluble fractions with a Triton X-100–containing buffer. Fractionated proteins were separated on 5–15% SDS-polyacrylamide gels, transferred to nitrocellulose, and visualized by x-ray film and phosphorimaging. (<b>B, D, F, H, J, L</b>) Distributions of NFM were quantified despite nearly identical NF distribution (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133848#pone.0133848.g001" target="_blank">Fig 1D</a></b>). NFM transport was significantly slowed in hNFH tg mice (<b>D</b>, <b>J</b>) but not in hNFL Ser 55 tg mice (<b>F</b>, <b>L</b>). Despite marked differences in NF distribution between hNFL Ser 55 tg and WT mice, NF transport in the tg model is unchanged or slightly faster (<b>F</b>, <b>L</b>). NFM peaks are denoted by dashed lines.</p

NFM phosphorylation increases along the optic pathway in a proximal-to-distal manner.

<p>Coomassie blue-stained 2D gels of Triton-insoluble fractions from consecutive 1 mm segments of the optic nerve and tract demonstrate that phosphorylated NFM (indicated by arrows, pI 5.0) shows proximal to distal increase (<b>A</b>). The ratios of phosphorylated NFM over non-phosphorylated NFM (indicated by arrowheads, pI 5.5) increase up to about 300% along optic pathway (Mean ± SD, n = 2, <b>B</b>).</p

NFM transport rate remains constant along optic axons except the first segment.

<p>Contradicting a critical assumption of the Li et al. mathematical model, NF transport rates determined at 7 (<b>A</b>), 14 (<b>B</b>), 21 (<b>C</b>), 42 days (<b>D</b>) post injection as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133848#pone.0133848.g004" target="_blank">Fig 4</a>, show that NFM peak advance is constant along the optic axons [for example, 0.14 ± 0.02 mm/day (Mean ± SEM, n = 19) at 7 days; 0.14 ± 0.01 mm/day (Mean ± SEM, n = 14) at 14 days] (<b>E</b>) until the wave exits the optic window (<b>A</b>-<b>E</b>). (<b>F</b>) Percentage of NFM radioactivity (in dpm) in optic nerve (segments 1–4) relative to total NFM dmp in optic pathway (segments 1–8) in long-term labeling studies from two independent analyses [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133848#pone.0133848.ref007" target="_blank">7</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133848#pone.0133848.ref009" target="_blank">9</a>]. The constant percentage in optic nerve at 75-to-192 days indicates no net movement of NF containing labeled NFM. Total number of mice analyzed in both studies equals 262–302. Gray bar, Nixon and Logvinenko, 1986; black bar, Yuan et al. 2009.</p

Two genetic mouse models exhibit similar patterns of abnormal axonal NF accumulation.

<p>(<b>A</b>) shows Coomassie Blue-stained gels of NFL, NFM and NFH distribution from 8 segments of optic pathway of WT, hNFH tg and hNFL Ser55 tg mice and also the distributions of hNFH and hNFL recognized by anti-human NFH specific antibody in hNFH tg and anti-human NFL specific antibody in hNFL Ser55 tg, respectively. Cross-sections of axons at the 50μm level of the optic nerve shows increased NF number in hNFH tg and hNFL Ser55 tg mice (<b>B</b>) and higher magnification of marked areas in B (C). Scale bar, 100nm. Average NF density measured in optic axons at levels 50, 700, 4000, and 7000 μm from the retinal excavation in 4 month old hNFH tg, hNFL Ser55 tg and WT mice (<b>D</b>). In each genotype of mice, 996 to 1377 axons in a size distribution matching that of the entire caliber distribution in the optic nerve axons were analyzed.</p

Model describing fates of newly assembled NF proteins in transport and formation of a non-uniform stationary axonal cytoskeletal network.

<p><b>(A)</b> The radiolabeled sub-population of newly synthesized NF and protofilaments (red dotted lines and dots) is transported along axons within the slow wave of axonal transport at a broad range of rates averaging 0.14 ± 0.02 mm per day, and reflecting brief periods of rapid movement and longer periods of pausing (<b>E</b>). As they are transported, radiolabeled NFs are continually incorporated into the stationary NF network containing preexisting unlabeled NF (indicated as black filaments) (<b>A</b>) which turn over at a very slow rate (half-life > 83 days). The greater level of incorporation into the stationary network at distal axonal levels (Nixon and Logvinenko 1986) generates the proximal-to-distal gradient of increasing NF content along optic axons at steady state in mature neurons (<b>B</b>-<b>D</b>).</p