A marked reduction in NF number and density at 50 µm area of NF-(H/M)^tailΔ optic axons.

<p>Distribution of NFs and MTs in WT and NF-(H/M)^tailΔ optic axons at 50 µm (A). Bar: 100 nm. WT and NF-(H/M)^tailΔ optic nerves at 50 µm area analyzed for NF and MT number (B, C) and density (D, E). NF (B) and MT numbers (C) at 50 µm in WT and NF-(H/M)^tailΔ mice. NF (D) and MT density (E) at 50 µm area. Data is from WT-1000, and NF-(H/M)^tailΔ - 1000 axons. Error bars represent SEM in all experiments.</p

NF-M/H tails regulate NF integration, number, density and establishment of stationary NF networks in optic axons.

<p>Distribution of NFs and MTs in WT and NF-(H/M)^tailΔ optic axons at 2 mm region of optic nerve (A). Bar: 100 nm. WT and NF-(H/M)^tailΔ optic nerves at 2 mm area analyzed for NF and MT number (B, D) and density (C, E). NF (B) and MT numbers (C) at 2 mm in WT and NF-(H/M)^tailΔ mice. NF (D) and MT density (E) at 2 mm area of WT and NF-(H/M)^tailΔ optic axons. Data is from WT-1000, and NF-(H/M)^tailΔ - 1000 axons. (F). Integration of NFs along the optic axonal segments is reduced in NF-(H/M)^tailΔ optic axons. Calnexin staining is used as a loading control. Error bars represent SEM in all experiments.</p

Absence of NF-H/M tails does not influence the rate of transport of NFs in optic axons.

<p>The speed and composition of slow axonal transport was determined by intra-vitreal injection of radiolabeled [³⁵S]-methionine of 3–4-month-old WT (A, C) and NF-(H/M)^tailΔ (B, D) mice for 3, 7 and 14 days. Optic nerve proteins were fractionated into cytoskeleton (A, B) and soluble fractions (C, D) with a Triton X-100 containing buffer. Fractionated proteins were separated on 5–15% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, and visualized by x-ray film and phosphorimaging. <i>(E–P)</i> Quantification of NF-subunits, tubulin and actin transport in optic nerves. (E, H&K)-NF-H&NF-H^tailΔ; (F, I&L)-NF-M&NF-M^tailΔ; (G, J&M)-NF-L; (N&P)-tubulin; and (O)-actin. (E-G)-3day; (H–J)-7 day; (A–D; K–P)-14 day transport.</p

Increased turnover and degradation of NF-L in NF-(H/M)^tailΔ optic axons.

<p>(A) Unaltered NF-L synthesis in NF-(H/M)^tailΔ optic axons. (B). Decreased ratio of retained NF-L in NF-(H/M)^tailΔ optic axons indicate increased turnover of NF-L while the ratios of fodrin (C), p-20 (D), and tubulin (E) were not significantly altered. Increased NF-L degradation in optic nerve (F), sciatic nerve (G) and spinal cord (H) of NF-(H/M)^tailΔ mice as evidenced by generation of proteolytic fragments detected by immunoblots probed with NR-4 antibody (see the arrows in panels F–H). Error bars represent SEM in all experiments.</p

Loss of both the tails of NF-H/M subunits results in reduced neurofilament content in axons.

<p>Equal amounts of optic (lanes 1–4) and sciatic nerve (lanes 5–8) extracts were fractionated on 7% SDS-polyacrylamide gels, immunoblotted with NF-H (Fig. A), NF-M (Fig. B), NF-L (Fig. C), NF triplets (Fig. D), tail deleted NF-H and NF-M (Fig. E), α-tubulin (Fig. F), β-tubulin (Fig. G), and β_III-tubulin (H) antibodies. Quantification of NF-L in optic (Fig. I), and sciatic nerves (Fig. J); NF-H^tailΔ (Fig. K); NF-M^tailΔ (Fig. L); full length NF-H (Fig. M), and full length NF-M (Fig. N). NF preparation from WT spinal cords indicates the position of full length NF subunits on immunoblots (lanes 9&10). Error bars represent SEM in all experiments.</p

Autophagy is a novel pathway for neurofilament protein degradation <i>in vivo</i>

How macroautophagy/autophagy influences neurofilament (NF) proteins in neurons, a frequent target in neurodegenerative diseases and injury, is not known. NFs in axons have exceptionally long half-lives in vivo enabling formation of large stable supporting networks, but they can be rapidly degraded during Wallerian degeneration initiated by a limited calpain cleavage. Here, we identify autophagy as a previously unrecognized pathway for NF subunit protein degradation that modulates constitutive and inducible NF turnover in vivo. Levels of NEFL/NF-L, NEFM/NF-M, and NEFH/NF-H subunits rise substantially in neuroblastoma (N2a) cells after blocking autophagy either with the phosphatidylinositol 3-kinase (PtdIns3K) inhibitor 3-methyladenine (3-MA), by depleting ATG5 expression with shRNA, or by using both treatments. In contrast, activating autophagy with rapamycin significantly lowers NF levels in N2a cells. In the mouse brain, NF subunit levels increase in vivo after intracerebroventricular infusion of 3-MA. Furthermore, using tomographic confocal microscopy, immunoelectron microscopy, and biochemical fractionation, we demonstrate the presence of NF proteins intra-lumenally within autophagosomes (APs), autolysosomes (ALs), and lysosomes (LYs). Our findings establish a prominent role for autophagy in NF proteolysis. Autophagy may regulate axon cytoskeleton size and responses of the NF cytoskeleton to injury and disease.</p