Complete and long-term rescue of lesioned adult motoneurons by lentiviral-mediated expression of glial cell line-derived neurotrophic factor in the facial nucleus.

To date, delivery of neurotrophic factors has only allowed to transiently protect axotomized facial motoneurons against cell death. In the present report, long-term protection of these neurons was evaluated by continuously expressing the neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) within the facial nucleus using a lentiviral vector system. The viral vector was injected unilaterally into the facial nucleus of 4-month-old Balb/C mice. In contrast to axotomy in other adult rodents, facial nerve lesion in these animals leads to a progressive and sustained loss and/or atrophy of >50% of the motoneurons. This model thus represents an attractive model to evaluate potential protective effects of neurotrophic factors for adult-onset motoneuron diseases, such as amyotrophic lateral sclerosis. One month after unilateral lentiviral vector injection, the facial nerve was sectioned, and the animals were killed 3 months later. Viral delivery of the GDNF gene led to long-term expression and extensive diffusion of GDNF within the brainstem. In addition, axotomized motoneurons were completely protected against cell death, because 95% of the motoneurons were present as demonstrated by both Nissl staining and choline acetyltransferase immunoreactivity. Furthermore, GDNF prevented lesion-induced neuronal atrophy and maintained proximal motoneuron axons, despite the absence of target cell reinnervation. This is the first evidence that viral-mediated delivery of GDNF close to the motoneuron cell bodies of the facial nucleus of adult mice can lead to complete and long-term protection against lesion-induced cell death.     

Motoneurons crave glial cell line-derived neurotrophic factor

This is a commentary on the developmental and therapeutic relevance of recent studies in the glial fibrillary acid protein (GFAP)-glial cell line-derived neurotrophic factor (GDNF) transgenic mouse reported by Zhao et al. (2004). This interesting study demonstrated that increased expression of GDNF in astrocytes increases the number of neighboring motoneurons of certain motoneuron subpopulations by diminishing programmed cell death during development. In addition, astrocyte-derived GDNF was shown to protect facial motoneurons from injury-induced cell death. Since this is the first direct demonstration that secretion of GDNF from astrocytes in the CNS can affect motoneuron development in utero and motoneuron survival after axotomy, novel approaches for motor neuron disease are suggested. The known target neurons that respond to GDNF are reviewed, as are studies using GDNF gene delivery in animal models of amyotrophic lateral sclerosis (ALS). It is postulated that GDNF is a factor to which many motoneurons respond along their whole extent from soma to axon to terminal.     

Cellular changes in motoneurons in a transgenic mouse model for amyotrophic lateral sclerosis as revealed by monoclonal antibody Py.

Transgenic mice (G93A) carrying the human amyotrophic lateral sclerosis (ALS) linked superoxide dismutase 1 (SOD1) mutations develop a motoneuron disease resembling human ALS. The affected motoneurons are characterized by the presence of cellular alterations. The antigen recognized by the monoclonal antibody Py is suggested to be associated with the neurofilamentous and microtubular elements of the cytoskeleton of specific neuron populations including the spinal motoneurons. The aim of the present study was to measure changes in the relative Py-immunoreactivity per identified Choline-Acetyl-Transferase (ChAT)-immunoreactive motoneuron during the disease progression. The relative Py-immunoreactivity of identified spinal motoneurons was measured on double stained (Py and ChAT) motoneurons using a digital imaging system coupled to an inverse microscope. A significant decrease of Py-immunoreactivity was already noted in the pre-symptomatic stages of the disease even before the onset of massive motoneuron degeneration. It is concluded that the Py-antibody detects early intracellular abnormalities related to neurodegenerative changes in spinal motoneurons of transgenic SOD1-(G93A) mice.     

The role of p75NTR in modulating neurotrophin survival effects in developing motoneurons

Neurotrophins exert their biological functions on neuronal cells through two types of receptors, the trk tyrosine kinases and the low-affinity neurotrophin receptor (p75NTR), which can bind all neurotrophins with similar affinity. The p75NTR is highly expressed in developing motoneurons and in adult motoneurons after axotomy, suggestive of a physiological role in mediating neurotrophin responses under such conditions. In order to characterize this specific function of p75NTR, we have tested the effects of nerve growth factor (NGF) on embryonic motoneurons from control and p75NTR-deficient mice. NGF antagonizes brain-derived neurotrophic factor (BDNF)- and neurotrophin-3 (NT-3)-mediated survival in control but not p75NTR-deficient motoneurons. Survival of cultured motoneurons in the presence of 0.5 ng/mL of either ciliary neurotrophic factor (CNTF) or glial-derived neurotrophic factor (GDNF) was not reduced by 20 ng/mL NGF. Dose-response investigations revealed that five times higher concentrations of BDNF are required for half-maximal survival of p75NTR-deficient motoneurons in comparison to motoneurons from wild-type controls. After facial nerve lesion in newborn wild-type mice, local administration of NGF reduced survival of corresponding motoneurons to less than 2% compared to the unlesioned control side. In p75NTR-deficient mice, the same treatment did not enhance facial motoneuron death on the lesioned side. In the facial nucleus of 1-week-old p75NTR -/- mice, a significant reduction of motoneurons was observed at the unlesioned side in comparison to p75NTR +/+ mice. The observation that motoneuron cell numbers are reduced in the facial nucleus of newborn p75NTR-deficient mice suggests that p75NTR might not function as a physiological cell death receptor in developing motoneurons.     

Oxidative damage to mitochondrial DNA in spinal motoneurons of transgenic ALS mice

In order to clarify a possible role of oxidative stress in motoneuron death in amyotrophic lateral sclerosis (ALS), we examined a presence of 8-hydroxy-2-deoxyguanosine (8-OHdG), one of the best markers of the oxidative DNA damage, in the spinal cord of transgenic mice harboring a mutant Cu/Zn superoxide dismutase (SOD1) gene. Immunocytochemistry showed a progressive accumulation of 8-OHdG in ventral horn neurons from early and presymptomatic stage (25 weeks) before significant loss of ventral horn neurons, while no detectable 8-OHdG in non-transgenic control mice. At the late and symptomatic stage (35 weeks), the 8-OHdG-like immunoreactivity spread over the posterior horn of spinal cord in Tg mice. The immunoreactivity for 8-OHdG was not localized in the nucleus but in cytoplasm with small granular pattern. These data suggest that an oxidative damage to mitochondrial DNA is happening in spinal motoneurons of the Tg mice from very early stage of the disease, and may be involved in the mechanism of the subsequent motoneuron death in this model.     

Reduced calreticulin levels link endoplasmic reticulum stress and Fas-triggered cell death in motoneurons vulnerable to ALS

Cellular responses to protein misfolding are thought to play key roles in triggering neurodegeneration. In the mutant superoxide dismutase (mSOD1) model of amyotrophic lateral sclerosis (ALS), subsets of motoneurons are selectively vulnerable to degeneration. Fast fatigable motoneurons selectively activate an endoplasmic reticulum (ER) stress response that drives their early degeneration while a subset of mSOD1 motoneurons show exacerbated sensitivity to activation of the motoneuron-specific Fas/NO pathway. However, the links between the two mechanisms and the molecular basis of their cellular specificity remained unclear. We show that Fas activation leads, specifically in mSOD1 motoneurons, to reductions in levels of calreticulin (CRT), a calcium-binding ER chaperone. Decreased expression of CRT is both necessary and sufficient to trigger SOD1(G93A) motoneuron death through the Fas/NO pathway. In SOD1(G93A) mice in vivo, reductions in CRT precede muscle denervation and are restricted to vulnerable motor pools. In vitro, both reduced CRT and Fas activation trigger an ER stress response that is restricted to, and required for death of, vulnerable SOD1(G93A) motoneurons. Our data reveal CRT as a critical link between a motoneuron-specific death pathway and the ER stress response and point to a role of CRT levels in modulating motoneuron vulnerability to ALS.     

Changes in the expression of P2X1 and P2X2 purinergic receptors in facial motoneurons after nerve lesions in rodents and correlation with motoneuron degeneration

Involvement of P2X1 and P2X2 purinergic receptors in motoneuron response to injury was investigated with Western blotting and immunohistochemistry and correlated with motoneuron loss, Bcl-2 expression, nitric oxide synthase induction and glial activation. P2X1 was highly induced in rat facial motoneurons after nerve resection, which causes slowly occurring neurodegeneration. P2X1 induction was lower and less persistent after nerve crush, permissive for fiber regeneration. P2X2 expression was found in nuclei of rat facial motoneurons, with nuclear export in the cytoplasm after nerve resection. P2X1 induction in axotomized facial motoneurons was impaired in superoxide dismutase (SOD)1-G93A-mutant mice, a model of motoneuron disease. The data in rats point to a correlation of P2X1 induction with motoneuron degeneration, which also involves P2X2 intracellular changes, rather than with axon regeneration effort. The data in mice show that the SOD1 mutation interferes with injury-elicited P2X1 induction, suggesting alterations of ATP release from mutant motoneurons after damage.     

Specific Expression of Glial-Derived Neurotrophic Factor in Muscles as Gene Therapy Strategy for Amyotrophic Lateral Sclerosis

Glial cell line–derived neurotrophic factor (GDNF) is a powerful neuroprotective growth factor. However, systemic or intrathecal administration of GDNF is associated with side effects. Here, we aimed to avoid this by restricting the transgene expression to the skeletal muscle by gene therapy. To specifically target most skeletal muscles in the mouse model of amyotrophic lateral sclerosis (ALS), SOD1^G93A transgenic mice were intravenously injected with adeno-associated vectors coding for GDNF under the control of the desmin promoter. Treated and control SOD1^G93A mice were evaluated by rotarod and nerve conduction tests from 8 to 20 weeks of age, and then histological and molecular analyses were performed. Muscle-specific GDNF expression delayed the progression of the disease in SOD1^G93A female and male mice by preserving the neuromuscular function; increasing the number of innervated neuromuscular junctions, the survival of spinal motoneurons; and reducing glial reactivity in treated SOD1^G93A mice. These beneficial actions are attributed to a paracrine protective mechanism from the muscle to the motoneurons by GDNF. Importantly, no adverse secondary effects were detected. These results highlight the potential of muscle GDNF-targeted expression for ALS therapy.Supplementary InformationThe online version contains supplementary material available at 10.1007/s13311-021-01025-6.Key Words: GDNF, Amyotrophic lateral sclerosis, Motoneuron, Gene therapy, AAV, Neuromuscular junction     

Increase in presynaptic territory of C-terminals on lumbar motoneurons of G93A SOD1 mice during disease progression

Compensatory synaptic plasticity is reported in muscle and the central nervous system of motor neuron disease patients, and transgenic SOD1 mice, but direct ultrastructural evidence for spinal motoneurons is lacking. Prompted by our observation in spinal cords from autopsied patients suggesting selective enlargement of the ultrastructurally distinctive C‐type terminal synapsing with spinal motoneurons, we examined the ultrastructural synaptology of lumbar motoneurons during disease progression in age‐ and sex‐matched wild‐type mice, transgenic G93A SOD1 mice, and mice overexpressing normal human SOD1 (Wt_SOD1). Prescribed criteria classified presynaptic terminals of motoneurons into five ultrastructural classes (S, F, T, M, and C). Computerized morphometry on electronmicrographs was used to measure their appositional lengths, coverage of the motoneuron membrane, and sizes of postsynaptic structures. No terminal degeneration occurred in wild‐type or Wt_SOD1 mice. In transgenic mice, degeneration of motoneurons and S‐terminals and F‐terminals commenced presymptomatically (10 weeks), and continued into the symptomatic stage (18 weeks). However, C‐terminals were preserved. Morphometry confirmed significant reductions in frequency and membrane coverage for S‐terminals and F‐terminals between 10 and 18 weeks, but a maintained frequency of C‐terminals coupled with increased appositional length and coverage of the motoneuron membrane. Increased C‐terminal size was matched by growth of its characterizing postsynaptic cistern and Nissl body. The results reveal selective preservation and increased presynaptic territory of the C‐type terminal. As C‐terminals derive from cholinergic intrasegmental propriospinal interneurons and may modulate motoneuron excitability, their increased presynaptic territory on surviving motoneurons of transgenic SOD1 mice may represent a means of maintaining excitability, compensating for the loss of overall presynaptic input.     

Adenoviral gene transfer of GDNF,BDNF and TGF beta 2, but not CNTF,cardiotrophin-1 or IGF1, protects injured adult motoneurons after facial nerve avulsion

We examined neuroprotective effects of recombinant adenoviral vectors encoding glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT1), insulin-like growth factor-1 (IGF1), and transforming growth factor-beta2 (TGFbeta2) on lesioned adult rat facial motoneurons. The right facial nerves of adult Fischer 344 male rats were avulsed and removed from the stylomastoid foramen, and adenoviral vectors were injected into the facial canal. Animals avulsed and treated with adenovirus encoding GDNF, BDNF, CNTF, CT1, IGF1 and TGFbeta2 showed intense immunolabeling for these factors in lesioned facial motoneurons, respectively, indicating adenoviral induction of the neurotrophic factors in these neurons. The treatment with adenovirus encoding GDNF, BDNF, or TGFbeta2 after avulsion significantly prevented the loss of lesioned facial motoneurons, improved choline acetyltransferase immunoreactivity and prevented the induction of nitric oxide synthase activity in these neurons. The treatment with adenovirus encoding CNTF, CT1 or IGF1, however, failed to protect these neurons after avulsion. These results indicate that the gene transfer of GDNF and BDNF and TGFbeta2 but not CNTF, CT1 or IGF1 may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.     

Mechano-growth factor, an IGF-I splice variant, rescues motoneurons and improves muscle function in SOD1 mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motoneuron degeneration. Although viral delivery of IGF-I has shown therapeutic efficacy in the SOD1^G93A mouse model of ALS, clinical trials of IGF-I in ALS patients have led to conflicting results. Here we examine the effects of an IGF-I splice variant, mechano-growth factor (MGF) which has previously been shown to have greater neuroprotective effects than IGF-I in a number of models of neurodegeneration. A mammalian expression plasmid containing either MGF or, for comparison, the IGF-I cDNA sequence was delivered to the hindlimb muscles of SOD1^G93A mice at 70 days of age, at symptom onset. Treatment with either IGF-I or MGF resulted in a significant improvement in hindlimb muscle strength, and an increase in motor unit and motoneuron survival. Significantly more motoneurons survived in MGF treated mice.     

Prolonged survival and milder impairment of motor function in the SOD1 ALS mouse model devoid of fibroblast growth factor 2

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective motoneuron loss in brain and spinal cord. Mutations in the superoxide dismutase (SOD) 1 gene account for 10-20% of familial ALS patients. The ALS-mouse model over-expressing a mutant human SOD1 (G93A) gene closely mimics human ALS disease. The cause for the selective death of motoneurons is still unclear, but among several pathomechanisms discussed, loss of neurotrophic factors is one possibility. Basic fibroblast growth factor 2 (FGF-2) plays a prominent role in the motor system. In order to evaluate a role of FGF-2 in ALS pathogenesis, double mouse mutants transgenic for the human SOD1 mutation and lacking the endogenous FGF-2 gene were generated. Both heterozygous and homozygous FGF-2 deficient mutant SOD1 mice showed a significant delay in disease onset and less impaired motor performance in comparison to mutant SOD1 mice with normal FGF-2 levels. Survival of the double mouse mutants was significantly prolonged for two weeks. Motoneuron numbers were significantly higher in the double mutants and astrocytosis was diminished at disease endstage. While one would initially have expected that FGF-2 deficiency deteriorates the phenotype of mutant SOD1 animals, our results revealed a protective effect of FGF-2 reduction. In search of the underlying mechanisms, we could show up-regulation of other neurotrophic factors with proven protective effects in the ALS mouse model, ciliary neurotrophic factor (CNTF) and glial derived neurotrophic factor (GDNF) in muscle and spinal cord tissue of double mutant animals.     

Adenoviral expression of TDP‐43 and FUS genes and shRNAs for protein degradation pathways in rodent motoneurons in vitro and in vivo

Formation of cytoplasmic aggregates in neuronal and glial cells is one of the pathological hallmarks of amyotrophic lateral sclerosis (ALS). Mutations in two genes encoding transactivation response (TAR) DNA‐binding protein 43 (TDP‐43) and fused in sarcoma (FUS), both of which are main constituents of cytoplasmic aggregates, have been identified in patients with familial and sporadic ALS. Impairment of protein degradation machineries has also been recognized to participate in motoneuron degeneration in ALS. In the present study, we produced recombinant adenovirus vectors encoding wild type and mutant TDP‐43 and FUS, and those encoding short hairpin RNAs (shRNAs) for proteasome (PSMC1), autophagy (ATG5), and endosome (VPS24) systems to investigate whether the coupled gene transductions in motoneurons by these adenoviruses elicit ALS pathology. Cultured neurons, astrocytes and oligodendrocytes differentiated from adult rat neural stem cells and motoneurons derived from mouse embryonic stem cells were successfully infected with these adenoviruses showing cytoplasmic aggregate formation. When these adenoviruses were injected into the facial nerves of adult rats, exogenous TDP‐43 and FUS proteins were strongly expressed in facial motoneurons by a retrograde axonal transport of the adenoviruses. Co‐infections of adenovirus encoding shRNA for PSMC1, ATG5 or VPS24 with TDP‐43 or FUS adenovirus enhanced cytoplasmic aggregate formation in facial motoneurons, suggesting that impairment of protein degradation pathways accelerates formation of TDP‐43 and FUS‐positive aggregates in ALS.     

Human Cu/Zn Superoxide Dismutase (SOD1) Overexpression in Mice Causes Mitochondrial Vacuolization, Axonal Degeneration, and Premature Motoneuron Death and Accelerates Motoneuron Disease in Mice Expressing a Familial Amyotrophic Lateral Sclerosis Mutant SOD1

Cytosolic Cu/Zn superoxide dismutase (SOD1) is a ubiquitous small cytosolic metalloenzyme that catalyzes the conversion of superoxide anion to hydrogen peroxide (H₂O₂). Mutations in the SOD1 gene cause a familial form of amyotrophic lateral sclerosis (fALS). The mechanism by which mutant SOD1s causes ALS is not understood. Transgenic mice expressing multiple copies of fALS-mutant SOD1s develop an ALS-like motoneuron disease resembling ALS. Here we report that transgenic mice expressing a high concentration of wild-type human SOD1 (hSOD1_WT) develop an array of neurodegenerative changes consisting of (1) swelling and vacuolization of mitochondria, predominantly in axons in the spinal cord, brain stem, and subiculum; (2) axonal degeneration in a number of long fiber tracts, predominantly the spinocerebellar tracts; and (3) at 2 years of age, a moderate loss of spinal motoneurons. Parallel to the development of neurodegenerative changes, hSOD1_WT mice also develop mild motor abnormalities. Interestingly, mitochondrial vacuolization was associated with accumulation of hSOD1 immunoreactivity, suggesting that the development of mitochondrial pathology is associated with disturbed SOD1 turnover. In this study we also crossed hSOD1_WT mice with a line of fALS-mutant SOD1 mice (hSOD1_G93A) to generate “double” transgenic mice that express high levels of both wild-type and G93A mutant hSOD1. The “double” transgenic mice show accelerated motoneuron death, earlier onset of paresis, and earlier death as compared with hSOD1_G93A littermates. Thus in vivo expression of high levels of wild-type hSOD1 is not only harmful to neurons in itself, but also increases or facilitates the deleterious action of a fALS-mutant SOD1. Our data indicate that it is important for motoneurons to control the SOD1 concentration throughout their processes, and that events that lead to improper synthesis, transport, or breakdown of SOD1 causing its accumulation are potentially dangerous.     

Immunohistochemical study on the distribution of MnSOD in the central nervous system of the transgenic mice expressing a human Cu/Zn SOD mutation

In the present study, we used the SOD1^G93A mutant transgenic mice as an animal model of amyotrophic lateral sclerosis (ALS) and performed immunohistochemical studies to investigate the changes of MnSOD in the central nervous system of transgenic mice at the age of 8, 13, and 18 weeks. In the spinal cord of wild-type SOD1 (wtSOD1) and SOD1^G93A transgenic mice, MnSOD-immunoreactive neurons were distributed mainly in the anterior horn, although they were also observed in the posterior horn. The staining intensity of MnSOD was significantly increased in the spinal cord of SOD1^G93A transgenic mice at presymptomatic and symptomatic stage. In the brainstem of symptomatic SOD1^G93A transgenic mice, significantly increased immunoreactivity for MnSOD was observed in abducens nucleus, facial nucleus, dorsal motor nucleus of vagus, hypoglossal nucleus, medullary and pontine reticular formation, superior and inferior olivary nucleus, and cochlear nucleus. The present study provides the first evidence that MnSOD immunoreactivity was increased in the central nervous system of SOD^G93A transgenic mice, suggesting that mitochondria may play an important role in the pathogenesis and progress of ALS. The mechanisms underlying the increased immunoreactivity for MnSOD, and the functional implications of these increases, require elucidation.     

Differential gene expression in the axotomized facial motor nucleus of presymptomatic SOD1 mice

Previously, we compared molecular profiles of one population of wild-type (WT) mouse facial motoneurons (FMNs) surviving with FMNs undergoing significant cell death after axotomy. Regardless of their ultimate fate, injured FMNs respond with a vigorous pro-survival/regenerative molecular response. In contrast, the neuropil surrounding the two different injured FMN populations contained distinct molecular differences that support a causative role for glial and/or immune-derived molecules in directing contrasting responses of the same cell types to the same injury. In the current investigation, we utilized the facial nerve axotomy model and a presymptomatic amyotrophic lateral sclerosis (ALS) mouse (SOD1) model to experimentally mimic the axonal die-back process observed in ALS pathogenesis without the confounding variable of disease onset. Presymptomatic SOD1 mice had a significant decrease in FMN survival compared with WT, which suggests an increased susceptibility to axotomy. Laser microdissection was used to accurately collect uninjured and axotomized facial motor nuclei of WT and presymptomatic SOD1 mice for mRNA expression pattern analyses of pro-survival/pro-regeneration genes, neuropil-specific genes, and genes involved in or responsive to the interaction of FMNs and non-neuronal cells. Axotomized presymptomatic SOD1 FMNs displayed a dynamic pro-survival/regenerative response to axotomy, similar to WT, despite increased cell death. However, significant differences were revealed when the axotomy-induced gene expression response of presymptomatic SOD1 neuropil was compared with WT. We propose that the increased susceptibility of presymptomatic SOD1 FMNs to axotomy-induced cell death and, by extrapolation, disease progression, is not intrinsic to the motoneuron, but rather involves a dysregulated response by non-neuronal cells in the surrounding neuropil.     

Fas small interfering RNA reduces motoneuron death in amyotrophic lateral sclerosis mice

OBJECTIVE: Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease characterized by selective motoneuron death. Understanding of the molecular mechanisms that trigger and regulate motoneuron degeneration could be relevant to ALS and other motoneuron disorders. This study investigates the role of Fas-linked motoneuron death in the pathogenesis of ALS. METHODS: We performed in vitro and in vivo small interfering RNA-mediated interference, by silencing the Fas receptor on motoneurons that carry the superoxide dismutase-1 (SOD1)-G93A mutation. RESULTS: We observed a significant reduction in Fas expression at messenger RNA (p < 0.001) and protein levels. Treated motoneurons demonstrated an increase in survival and a reduction in cytochrome c release from mitochondria. In vivo, continuous intrathecal administration of Fas small interfering RNA by an osmotic minipump improved motor function and survival in SOD1-G93A mice (mean increase, 18 days; p < 0.0001). Treated mice showed a significant reduction in Fas and Fas mediators p38 mitogen-activated protein kinase, neuronal nitric oxide synthase, and caspase-8. INTERPRETATION: Fas silencing interferes with motoneuron-specific downstream death pathways and results in increased motoneuron survival and amelioration of the SOD1-G93A phenotype, suggesting new possible strategies for molecular therapy of ALS.     

Peripheral nerve avulsion injuries as experimental models for adult motoneuron degeneration

We have used adult rat peripheral nerve avulsion models to evaluate the effects of neuroprotective molecules on motoneuron degeneration. The right facial nerves of adult Fischer 344 male rats were avulsed and adenoviral vectors encoding glial cell line‐derived neurotrophic factor (GDNF), brain‐derived neurotrophic factor (BDNF), transforming growth factor‐β2 (TGFβ2), and growth inhibitory factor (GIF) were injected into the facial canal. The treatment with the vectors significantly prevented the loss of lesioned facial motoneurons, improved choline acetyltransferase (ChAT) immunoreactivity and suppressed the induction of nitric oxide synthase activity in these neurons. In separate experiments, animals were orally administered a solution of a neuroprotective compound T‐588 after avulsion. Both free oral administration and oral tube administration of T‐588 improved the survival of injured motoneurons and ameliorated their ChAT immunoreactivity. These results indicate that the gene transfer of GDNF, BDNF, TGFβ2, and GIF and oral administration of T‐588 may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.