Disruption of Type IV Intermediate Filament Network in Mice Lacking the Neurofilament Medium and Heavy Subunits He ´le `ne Jacomy, Qinzang Zhu, Se ´bastien Couillard-Despre ´s, Jean-Martin Beaulieu, and Jean-Pierre Julien Montreal General Hospital Research Institute, Montreal, Que ´bec, Canada Abstract: To clarify the role of the neurofilament (NF) medium (NF-M) and heavy (NF-H) subunits, we gener- ated mice with targeted disruption of both NF-M and NF-H genes. The absence of the NF-M subunit resulted in a two- to threefold reduction in the caliber of large my- elinated axons, whereas the lack of NF-H subunits had little effect on the radial growth of motor axons. In NF- M-/- mice, the velocity of axonal transport of NF light (NF-L) and NF-H proteins was increased by about two- fold, whereas the steady-state levels of assembled NF-L were reduced. Although the NF-M or NF-H subunits are each dispensable for the formation of intermediate fila- ments, the absence of both subunits in double NF-M; NF-H knockout mice led to a scarcity of intermediate filament structures in axons and to a marked approxi- mately twofold increase in the number of microtubules. Protein analysis indicated that the levels of NF-L and -internexin proteins were reduced dramatically through- out the nervous system. Immunohistochemistry of spinal cord from the NF-M-/-;NF-H-/- mice revealed en- hanced NF-L staining in the perikaryon of motor neurons but a weak NF-L staining in axons. In addition, axonal transport studies carried out by the injection of [ 35 S]me- thionine into spinal cord revealed after 30 days very low levels of newly synthesized NF-L proteins in the sciatic nerve of NF-M-/-;NF-H-/- mice. The combined re- sults demonstrate a requirement of the high-molecular- weight subunits for the assembly of type IV intermediate filament proteins and for the efficient translocation of NF-L proteins into the axonal compartment. Key Words: Neurofilaments—Knockout mice —Axonal transport— Axonal caliber—-Internexin. J. Neurochem. 73, 972–984 (1999). The adult neuronal cytoskeleton is composed of three interconnected filaments: the actin microfilaments, mi- crotubules, and intermediate filaments (IFs). On the basis of sequence homology and the exon–intron gene struc- ture, the IF proteins have been classified into five distinct groups. Only type III and IV IF proteins are expressed in mature neurons. Neurofilaments (NFs), which are made up by the copolymerization of three polypeptides desig- nated as the NF light (NF-L; 61 kDa), medium (NF-M; 90 kDa), and heavy (NF-H; 115 kDa) subunits (Lee et al., 1993), belong to the type IV IFs together with -in- ternexin. During embryonic neurogenesis, the NF-L and NF-M proteins are coexpressed, whereas the activation of NF-H expression is delayed to the postnatal period (Shaw and Weber, 1982; Julien et al., 1986). -Intern- exin is also expressed in the developing nervous system (Fliegner et al., 1994). At adult age, -internexin is expressed at much lower levels than the three NF sub- units in large motor neurons of the spinal cord and the cranial nerve ganglia (Fliegner et al., 1994), but it per- sists as the dominant IF system in small-caliber axons. Peripherin, a type III IF protein, is abundant in neurons of the peripheral nervous system and in some central nervous system neurons with peripheral projections. The expression of peripherin overlaps with that of NF pro- teins, in many axons during development and in subsets of adult neurons (Troy et al., 1990). In large myelinated axons, NFs constitute the most abundant cytoskeletal element, outnumbering microtu- bules. Transfection studies in cultured cells provided evidence that NFs are obligate heteropolymers, requiring the NF-L subunit together with NF-M or NF-H to poly- merize into extended 10-nm filaments (Ching and Liem, 1993; Lee et al., 1993). However, conflicting data have been published on whether the NF-L protein is able to self-assemble into IFs in vivo. Some laboratories found that the NF-L is unable to form homopolymers (Gibb et al., 1996; Carter et al., 1997; our unpublished observa- tion), whereas Carter et al. (1998) recently reported that the human NF-L protein is able to self-assemble into filaments in transfected cells. Following their synthesis in the perikarya, the NF subunits are converted to insoluble structures (Black et Received March 24, 1999; revised manuscript April 27, 1999; ac- cepted April 28, 1999. Address correspondence and reprint requests to Dr. J.-P. Julien at Montreal General Hospital Research Institute, 1650 Cedar Avenue, Montreal, Que ´bec, Canada H3G 1A4. Abbreviations used: DRG, dorsal root ganglia; IF, intermediate filament; NF, neurofilament; NF-H, NF-L, and NF-M, neurofilament heavy, light, and medium subunit, respectively; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; TBS, Tris-buffered saline. 972 Journal of Neurochemistry Lippincott Williams & Wilkins, Inc., Philadelphia © 1999 International Society for Neurochemistry