Corticospinal neurons exhibit a novel pattern of cytoskeletal gene expression after injury

Abstract

We examined changes in the expression of major cytoskeletal protein mRNAs in adult hamster corticospinal neurons after axotomy. While a number of studies had determined that peripheral neurons exhibit major alterations in cytoskeletal gene expression after axotomy, no previous studies had addressed the question of whether or not intrinsic mammalian CNS neurons, which do not have the ability to successfully regenerate axons after injury, alter their expression of tubulin and neurofilament genes after injury. In the present study we used in situ hybridization methods to examine this issue. 35S-labeled cDNA probes for the low molecular weight neurofilament protein (NF-L) mRNA and an alpha-tubulin mRNA species (M alpha 1) were used for in situ hybridizations of sections of the sensorimotor cortex obtained 2, 7, and 14 days after unilateral axotomy of the corticospinal tract in the caudal medulla. Film as well as emulsion autoradiography showed dramatic decreases in both alpha-tubulin and NF-L mRNA levels within axotomized neurons in layer Vb of the sensorimotor cortex. Tubulin mRNA levels were decreased as early as 2 days after injury whereas NF-L mRNA levels were not decreased until later times. Ribosomal RNA (rRNA) levels in axotomized corticospinal neurons were also examined using in situ hybridization with a 35S-labeled rDNA probe. These studies showed only a slight decrease in rRNA levels in corticospinal neurons at 14 days after axotomy. Immunoblotting experiments of total protein from corticospinal axons in the medulla were performed to assess whether the axonal composition immediately proximal to the injury site reflected changes in cell body gene expression. Both alpha-tubulin and NF-L levels were found to decrease in corticospinal axons by 28 days after injury. These findings, to our knowledge, are the first to demonstrate that a class of mammalian CNS neurons have an intrinsically different cytoskeletal response to axonal injury than do PNS neurons. The failure to upregulate tubulin gene expression following injury may contribute to the ineffective regenerative response of these long-tract CNS neurons.

Recovery of Neurofilament Expression Selectively in Regenerating Reticulospinal Neurons

“…In goldfish, the expression of mRNA for the NF ON-1 increased during optic nerve regeneration (Tesser et al, 1986;Glasgow et al, 1994). On the other hand, NF mRNA, protein synthesis, and transport are reduced in mammalian peripheral (Hoffman et al, 1985;Goldstein et al, 1988;Oblinger and Lasek, 1988) and central neurons (Mikucki and Oblinger, 1991;Tetzlaff et al, 1991;Hoffman et al, 1993;McKerracher et al, 1993a) after axotomy. The changes are reversed in peripheral neurons only after regenerating axons reach their targets (Hoffman et al, 1985;Hoffman and Cleveland, 1988;Muma et al, 1990;Wong and Oblinger, 1990b), and they persist if regeneration is prevented (Tetzlaff et al, 1988;Jiang et al, 1994).…”

“…The changes are reversed in peripheral neurons only after regenerating axons reach their targets (Hoffman et al, 1985;Hoffman and Cleveland, 1988;Muma et al, 1990;Wong and Oblinger, 1990b), and they persist if regeneration is prevented (Tetzlaff et al, 1988;Jiang et al, 1994). In mammalian central neurons, which normally do not regenerate, NF message remains permanently depressed (Mikucki and Oblinger, 1991;Tetzlaff et al, 1991). These observations have led to the widespread assumption that NFs play only a passive role in axonal regeneration.…”

Heterogeneous Nuclear Ribonucleoprotein K, an RNA-Binding Protein, Is Required for Optic Axon Regeneration inXenopus laevis

“…Meanwhile, removing neurofilaments reduces rates of axon elongation in both developing and regenerating axons (Zhu et al, 1997;Walker et al, 2001). Injury-induced changes in expression of these two genes, although very similar among anamniotes exhibiting successful CNS axon regeneration, are quite different in the amniotes (Mikucki and Oblinger, 1991;Bates and Meyer, 1993;Schaden et al, 1994;Zhao and Szaro, 1995;Rodger et al, 2001;Kaneda et al, 2008). Thus, their expression is tightly coupled with gene regulatory mechanisms controlling the success of CNS axon regeneration.…”

Increased expression of multiple neurofilament mRNAs during regeneration of vertebrate central nervous system axons

“…Peripherinand ␣-internexin-like nIFs also increase during goldfish optic nerve regeneration (Glasgow et al, 1992;, but comparable changes in nIF expression fail to occur in injured mammalian CNS (Mikucki and Oblinger, 1991), arguing that they are linked to successful recovery from injury. Moreover, the relative magnitudes of changes in the expression of individual subunits must be highly regulated, since substantially altering nIF stoichiometries can cause NFs to aggregate and axons to degenerate (Beaulieu et al, , 2000.…”