RESUMO
A spinal root avulsion is the most severe proximal peripheral nerve lesion possible. Avulsion of ventral root filaments disconnects spinal motoneurons from their target muscles, resulting in complete paralysis. In patients that undergo brachial plexus nerve repair, axonal regeneration is a slow process. It takes months or even years to bridge the distance from the lesion site to the distal targets located in the forearm. Following ventral root avulsion, without additional pharmacological or surgical treatments, progressive death of motoneurons occurs within 2 weeks (Koliatsos et al., 1994). Reimplantation of the avulsed ventral root or peripheral nerve graft can act as a conduit for regenerating axons and increases motoneuron survival (Chai et al., 2000). However, this beneficial effect is transient. Combined with protracted and poor long-distance axonal regeneration, this results in permanent function loss. To overcome motoneuron death and improve functional recovery, several promising intervention strategies are being developed. Here, we focus on GDNF gene-therapy. We first introduce the experimental ventral root avulsion model and discuss its value as a proxy to study clinical neurotmetic nerve lesions. Second, we discuss our recent studies showing that GDNF gene-therapy is a powerful strategy to promote long-term motoneuron survival and improve function when target muscle reinnervation occurs within a critical post-lesion period. Based upon these observations, we discuss the influence of timing of the intervention, and of the duration, concentration and location of GDNF delivery on functional outcome. Finally, we provide a perspective on future research directions to realize functional recovery using gene therapy.
RESUMO
Ventral root avulsion leads to severe motoneuron degeneration and prolonged distal nerve denervation. After a critical period, a state of chronic denervation develops as repair Schwann cells lose their pro-regenerative properties and inhibitory factors such as CSPGs accumulate in the denervated nerve. In rats with ventral root avulsion injuries, we combined timed GDNF gene therapy delivered to the proximal nerve roots with the digestion of inhibitory CSPGs in the distal denervated nerve using sustained lentiviral-mediated chondroitinase ABC (ChABC) enzyme expression. Following reimplantation of lumbar ventral roots, timed GDNF-gene therapy enhanced motoneuron survival up to 45 weeks and improved axonal outgrowth, electrophysiological recovery, and muscle reinnervation. Despite a timed GDNF expression period, a subset of animals displayed axonal coils. Lentiviral delivery of ChABC enabled digestion of inhibitory CSPGs for up to 45 weeks in the chronically denervated nerve. ChABC gene therapy alone did not enhance motoneuron survival, but led to improved muscle reinnervation and modest electrophysiological recovery during later stages of the regeneration process. Combining GDNF treatment with digestion of inhibitory CSPGs did not have a significant synergistic effect. This study suggests a delicate balance exists between treatment duration and concentration in order to achieve therapeutic effects.
Assuntos
Condroitina ABC Liase/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Regeneração Nervosa/genética , Raízes Nervosas Espinhais/fisiologia , Animais , Axônios/fisiologia , Linhagem Celular , Feminino , Terapia Genética/métodos , Células HEK293 , Humanos , Neurônios Motores/fisiologia , Regeneração Nervosa/fisiologia , Ratos , Ratos Wistar , Recuperação de Função Fisiológica/genética , Células de Schwann/fisiologiaRESUMO
Avulsion of spinal nerve roots is a severe proximal peripheral nerve lesion. Despite neurosurgical repair, recovery of function in human patients is disappointing, because spinal motor neurons degenerate progressively, axons grow slowly and the distal Schwann cells which are instrumental to supporting axon extension lose their pro-regenerative properties. We have recently shown that timed GDNF gene therapy (dox-i-GDNF) in a lumbar plexus injury model promotes axon regeneration and improves electrophysiological recovery but fails to stimulate voluntary hind paw function. Here we report that dox-i-GDNF treatment following avulsion and re-implantation of cervical ventral roots leads to sustained motoneuron survival and recovery of voluntary function. These improvements were associated with a twofold increase in motor axon regeneration and enhanced reinnervation of the hand musculature. In this cervical model the distal hand muscles are located 6,5â¯cm from the reimplantation site, whereas following a lumber lesion this distance is twice as long. Since the first signs of muscle reinnervation are observed 6â¯weeks after the lesion, this suggests that regenerating axons reached the hand musculature before a critical state of chronic denervation has developed. These results demonstrate that the beneficial effects of timed GDNF-gene therapy are more robust following spinal nerve avulsion lesions that allow reinnervation of target muscles within a relatively short time window after the lesion. This study is an important step in demonstrating the potential of timed GDNF-gene therapy to enhance axon regeneration after neurosurgical repair of a severe proximal nerve lesion.
Assuntos
Neuropatias do Plexo Braquial , Terapia Genética/métodos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/administração & dosagem , Regeneração Nervosa/fisiologia , Recuperação de Função Fisiológica , Animais , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Radiculopatia , Ratos , Ratos Wistar , Recuperação de Função Fisiológica/fisiologiaRESUMO
Neurosurgical repair in patients with proximal nerve lesions results in unsatisfactory recovery of function. Gene therapy for neurotrophic factors is a powerful strategy to promote axon regeneration. Glial cell line-derived neurotrophic factor (GDNF) gene therapy promotes motor neuron survival and axon outgrowth; however, uncontrolled delivery of GDNF results in axon entrapment. We report that time-restricted GDNF expression (1 month) using an immune-evasive doxycycline-inducible gene switch attenuated local axon entrapment in avulsed reimplanted ventral spinal roots, was sufficient to promote long-term motor neuron survival (24 weeks) and facilitated the recovery of compound muscle action potentials by 8 weeks. These improvements were associated with an increase in long-distance regeneration of motor axons. In contrast, persistent GDNF expression impaired axon regeneration by inducing axon entrapment. These findings demonstrate that timed expression can resolve the deleterious effect of uncontrolled growth factor delivery and shows that inducible growth factor gene therapy can be employed to enhance the efficacy of axon regeneration after neurosurgical repair of a proximal nerve lesion in rats. This preclinical study is an important step in the ongoing development of a neurotrophic factor gene therapy for patients with severe proximal nerve lesions.
Assuntos
Axônios/fisiologia , Genes de Troca/fisiologia , Terapia Genética/métodos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Evasão da Resposta Imune/fisiologia , Regeneração Nervosa/fisiologia , Animais , Axônios/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Feminino , Genes de Troca/efeitos dos fármacos , Fator Neurotrófico Derivado de Linhagem de Célula Glial/administração & dosagem , Evasão da Resposta Imune/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Ratos , Ratos Wistar , Células de Schwann/efeitos dos fármacos , Células de Schwann/fisiologia , Fatores de TempoRESUMO
Adeno-associated viral vectors have numerous applications in neuroscience, including the study of gene function in health and disease, targeting of light-sensitive proteins to anatomically distinct sets of neurons to manipulate neuronal activity (optogenetics), and the delivery of fluorescent protein to study anatomical connectivity in the brain. Moreover several phase I/II clinical trials for gene therapy of eye and brain diseases with adeno-associated viral vectors have shown that these vectors are well tolerated by human patients. In this chapter we describe a detailed protocol for the small scale production of recombinant adeno-associated viral vectors. This protocol can be executed by investigators with experience in cell culture and molecular biological techniques in any well-equipped molecular neurobiology laboratory. With this protocol we typically obtain research batches of 100-200 µL that range in titer from 5 × 1012 to 2 × 1013 genomic copies/mL.
Assuntos
Encefalopatias/terapia , Dependovirus/genética , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos , Encefalopatias/genética , Oftalmopatias/genética , Oftalmopatias/terapia , Células HEK293 , Humanos , Injeções Intraoculares/métodos , Sistema Nervoso/metabolismo , PlasmídeosRESUMO
Repulsive guidance molecule member a (RGMa) is a membrane-associated or released guidance molecule that is involved in axon guidance, cell patterning, and cell survival. In our previous work, we showed that RGMa is significantly upregulated in the substantia nigra of patients with Parkinson's disease. Here we demonstrate the expression of RGMa in midbrain human dopaminergic (DA) neurons. To investigate whether RGMa might model aspects of the neuropathology of Parkinson's disease in mouse, we targeted RGMa to adult midbrain dopaminergic neurons using adeno-associated viral vectors. Overexpression of RGMa resulted in a progressive movement disorder, including motor coordination and imbalance, which is typical for a loss of DA release in the striatum. In line with this, RGMa induced selective degeneration of dopaminergic neurons in the substantia nigra (SN) and affected the integrity of the nigrostriatal system. The degeneration of dopaminergic neurons was accompanied by a strong microglia and astrocyte activation. The behavioral, molecular, and anatomical changes induced by RGMa in mice are remarkably similar to the clinical and neuropathological hallmarks of Parkinson's disease. Our data indicate that dysregulation of RGMa plays an important role in the pathology of Parkinson's disease, and antibody-mediated functional interference with RGMa may be a disease modifying treatment option.SIGNIFICANCE STATEMENT Parkinson's disease (PD) is a neurodegenerative disease characterized by severe motor dysfunction due to progressive degeneration of mesencephalic dopaminergic (DA) neurons in the substantia nigra. To date, there is no regenerative treatment available. We previously showed that repulsive guidance molecule member a (RGMa) is upregulated in the substantia nigra of PD patients. Adeno-associated virus-mediated targeting of RGMa to mouse DA neurons showed that overexpression of this repulsive axon guidance and cell patterning cue models the behavioral and neuropathological characteristics of PD in a remarkable way. These findings have implications for therapy development as interfering with the function of this specific axon guidance cue may be beneficial to the survival of DA neurons.
Assuntos
Proteínas do Tecido Nervoso/genética , Doença de Parkinson/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Linhagem Celular Tumoral , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Feminino , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microglia/patologia , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/patologia , Doença de Parkinson/fisiopatologia , Substância Negra/metabolismo , Substância Negra/patologiaRESUMO
The dorsal column lesion model of spinal cord injury targets sensory fibres which originate from the dorsal root ganglia and ascend in the dorsal funiculus. It has the advantages that fibres can be specifically traced from the sciatic nerve, verifiably complete lesions can be performed of the labelled fibres, and it can be used to study sprouting in the central nervous system from the conditioning lesion effect. However, functional deficits from this type of lesion are mild, making assessment of experimental treatment-induced functional recovery difficult. Here, five functional tests were compared for their sensitivity to functional deficits, and hence their suitability to reliably measure recovery of function after dorsal column injury. We assessed the tape removal test, the rope crossing test, CatWalk gait analysis, and the horizontal ladder, and introduce a new test, the inclined rolling ladder. Animals with dorsal column injuries at C4 or T7 level were compared to sham-operated animals for a duration of eight weeks. As well as comparing groups at individual timepoints we also compared the longitudinal data over the whole time course with linear mixed models (LMMs), and for tests where steps are scored as success/error, using generalized LMMs for binomial data. Although, generally, function recovered to sham levels within 2-6 weeks, in most tests we were able to detect significant deficits with whole time-course comparisons. On the horizontal ladder deficits were detected until 5-6 weeks. With the new inclined rolling ladder functional deficits were somewhat more consistent over the testing period and appeared to last for 6-7 weeks. Of the CatWalk parameters base of support was sensitive to cervical and thoracic lesions while hind-paw print-width was affected by cervical lesion only. The inclined rolling ladder test in combination with the horizontal ladder and the CatWalk may prove useful to monitor functional recovery after experimental treatment in this lesion model.
Assuntos
Recuperação de Função Fisiológica , Traumatismos da Medula Espinal/fisiopatologia , Medula Espinal/fisiopatologia , Animais , Medula Cervical/lesões , Medula Cervical/fisiopatologia , Modelos Animais de Doenças , Feminino , Transtornos Neurológicos da Marcha/diagnóstico , Transtornos Neurológicos da Marcha/fisiopatologia , Gânglios Espinais/lesões , Gânglios Espinais/fisiopatologia , Regeneração Nervosa , Ratos , Ratos Endogâmicos F344 , Corno Dorsal da Medula Espinal/lesões , Corno Dorsal da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/diagnóstico , CaminhadaRESUMO
Root avulsions due to traction to the brachial plexus causes complete and permanent loss of function. Until fairly recent, such lesions were considered impossible to repair. Here we review clinical repair strategies and current progress in experimental ventral root avulsion lesions. The current gold standard in patients with a root avulsion is nerve transfer, whereas reimplantation of the avulsed root into the spinal cord has been performed in a limited number of cases. These neurosurgical repair strategies have significant benefit for the patient but functional recovery remains incomplete. Developing new ways to improve the functional outcome of neurosurgical repair is therefore essential. In the laboratory, the molecular and cellular changes following ventral root avulsion and the efficacy of intervention strategies have been studied at the level of spinal motoneurons, the ventral spinal root and peripheral nerve, and the skeletal muscle. We present an overview of cell-based pharmacological and neurotrophic factor treatment approaches that have been applied in combination with surgical reimplantation. These interventions all demonstrate neuroprotective effects on avulsed motoneurons, often accompanied with various degrees of axonal regeneration. However, effects on survival are usually transient and robust axon regeneration over long distances has as yet not been achieved. Key future areas of research include finding ways to further extend the post-lesion survival period of motoneurons, the identification of neuron-intrinsic factors which can promote persistent and long-distance axon regeneration, and finally prolonging the pro-regenerative state of Schwann cells in the distal nerve.
Assuntos
Regeneração Nervosa , Radiculopatia/terapia , Raízes Nervosas Espinhais/fisiopatologia , Animais , Terapia Genética/métodos , Humanos , Fármacos Neuroprotetores/uso terapêutico , Radiculopatia/patologia , Radiculopatia/fisiopatologia , Raízes Nervosas Espinhais/metabolismo , Transplante de Células-Tronco/métodosRESUMO
Axonal regeneration after injury requires the coordinated expression of genes in injured neurons. We previously showed that either reducing expression or blocking function of the transcriptional repressor NFIL3 activates transcription of regeneration-associated genes Arg1 and Gap43 and strongly promotes axon outgrowth in vitro. Here we tested whether genetic deletion or dominant-negative inhibition of NFIL3 could promote axon regeneration and functional recovery after peripheral nerve lesion in vivo. Contrary to our expectations, we observed no changes in the expression of regeneration-associated genes and a significant delay in functional recovery following genetic deletion of Nfil3. When NFIL3 function was inhibited specifically in dorsal root ganglia prior to sciatic nerve injury, we observed a decrease in regenerative axon growth into the distal nerve segment rather than an increase. Finally, we show that deletion of Nfil3 changes sciatic nerve lesion-induced expression in dorsal root ganglia of genes that are not typically involved in regeneration, including several olfactory receptors and developmental transcription factors. Together our findings show that removal of NFIL3 in vivo does not recapitulate the regeneration-promoting effects that were previously observed in vitro, indicating that in vivo transcriptional control of regeneration is probably more complex and more robust against perturbation than in vitro data may suggest.
Assuntos
Axônios/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Deleção de Genes , Regeneração Nervosa , Proteínas Repressoras/metabolismo , Animais , Células Cultivadas , Gânglios Espinais/metabolismo , Regulação da Expressão Gênica , Ontologia Genética , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Compressão Nervosa , Neurônios/metabolismo , Ratos Wistar , Recuperação de Função Fisiológica , Nervo Isquiático/lesões , Nervo Isquiático/patologia , Nervo Isquiático/fisiopatologiaRESUMO
Fibroblast growth factor 2 (FGF-2) is a trophic factor expressed by glial cells and different neuronal populations. Addition of FGF-2 to spinal cord and dorsal root ganglia (DRG) explants demonstrated that FGF-2 specifically increases motor neuron axonal growth. To further explore the potential capability of FGF-2 to promote axon regeneration, we produced a lentiviral vector (LV) to overexpress FGF-2 (LV-FGF2) in the injured rat peripheral nerve. Cultured Schwann cells transduced with FGF-2 and added to collagen matrix embedding spinal cord or DRG explants significantly increased motor but not sensory neurite outgrowth. LV-FGF2 was as effective as direct addition of the trophic factor to promote motor axon growth in vitro. Direct injection of LV-FGF2 into the rat sciatic nerve resulted in increased expression of FGF-2, which was localized in the basal lamina of Schwann cells. To investigate the in vivo effect of FGF-2 overexpression on axonal regeneration after nerve injury, Schwann cells transduced with LV-FGF2 were grafted in a silicone tube used to repair the resected rat sciatic nerve. Electrophysiological tests conducted for up to 2 months after injury revealed accelerated and more marked reinnervation of hindlimb muscles in the animals treated with LV-FGF2, with an increase in the number of motor and sensory neurons that reached the distal tibial nerve at the end of follow-up.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Neurônios Motores/fisiologia , Regeneração Nervosa , Células de Schwann/metabolismo , Células de Schwann/transplante , Nervo Isquiático/lesões , Animais , Axônios/fisiologia , Proliferação de Células/fisiologia , Células Cultivadas , Técnicas de Cocultura , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Gânglios Espinais/fisiopatologia , Vetores Genéticos , Células HEK293 , Membro Posterior/fisiopatologia , Humanos , Lentivirus/genética , Músculo Esquelético/fisiopatologia , Ratos Endogâmicos F344 , Nervo Isquiático/fisiopatologia , Células Receptoras Sensoriais/fisiologia , Medula Espinal/fisiopatologia , Nervo Tibial/fisiopatologia , Alicerces TeciduaisRESUMO
Although the peripheral nerve is capable of regeneration, only a small minority of patients regain normal function after surgical reconstruction of a major peripheral nerve lesion, resulting in a severe and lasting negative impact on the quality of life. Glial cell-line derived neurotrophic factor (GDNF) has potent survival- and outgrowth-promoting effects on motoneurons, but locally elevated levels of GDNF cause trapping of regenerating axons and the formation of nerve coils. This phenomenon has been called the "candy store" effect. In this study we created gradients of GDNF in the sciatic nerve after a ventral root avulsion. This approach also allowed us to study the effect of increasing concentrations of GDNF on Schwann cell proliferation and morphology in the injured peripheral nerve. We demonstrate that lentiviral vectors can be used to create a 4 cm long GDNF gradient in the intact and lesioned rat sciatic nerve. Nerve coils were formed throughout the gradient and the number and size of the nerve coils increased with increasing GDNF levels in the nerve. In the nerve coils, Schwann cell density is increased, their morphology is disrupted and myelination of axons is severely impaired. The total number of regenerated and surviving motoneurons is not enhanced after the distal application of a GDNF gradient, but increased sprouting does result in higher number of motor axon in the distal segment of the sciatic nerve. These results show that lentiviral vector mediated overexpression of GDNF exerts multiple effects on both Schwann cells and axons and that nerve coil formation already occurs at relatively low concentrations of exogenous GDNF. Controlled expression of GDNF, by using a viral vector with regulatable GDNF expression, may be required to avoid motor axon trapping and to prevent the effects on Schwann cell proliferation and myelination.
Assuntos
Vetores Genéticos/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Lentivirus/genética , Traumatismos dos Nervos Periféricos/genética , Traumatismos dos Nervos Periféricos/metabolismo , Animais , Axônios/metabolismo , Sobrevivência Celular , Feminino , Expressão Gênica , Vetores Genéticos/administração & dosagem , Neurônios Motores/metabolismo , Bainha de Mielina/metabolismo , Regeneração Nervosa , Ratos , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Fatores de Tempo , Transdução GenéticaRESUMO
Peripheral nerve injury in humans often leads to incomplete functional recovery. In this review we discuss the potential for gene therapy to be used as a strategy alongside surgical repair techniques for the study of peripheral nerve regeneration in rodent models and with a view to its eventual use for the promotion of successful regeneration in the clinic. Gene therapy can be defined as the introduction of a foreign, therapeutic gene into living cells in order to treat a disease. The first attempts to express a foreign gene in peripheral neurons date back more than 25 years. The vectors used at that time were imperfect-mainly because they contained viral genes that were expressed in the target cells and elicited an immunological response. Fortunately significant progress has been made: today adeno-associated viral vectors can be produced completely free of viral genes and Phase I and II clinical studies have shown that these vectors are well tolerated. The technology for gene delivery has reached a state of readiness for clinical translation in many fields of neurology, including peripheral nerve repair. The current range of potential therapeutic genes for the repair of the traumatized peripheral nerve has also grown over the years and now includes neurotrophic factors with specificities for various subtypes of peripheral neurons, cell adhesion and extracellular matrix molecules and transcription factors. This review for this Festschrift, published to celebrate the 70th birthday of Willem Hendrik Gispen, contains many "footprints" from the time the senior author (JV) worked with Willem Hendrik, first as a student intern, then as a Ph.D. student (1983-1987) and later as a postdoctoral fellow (1989-1993). The preface of this article highlights personal memories of a time that will never come back.
Assuntos
Terapia Genética/métodos , Regeneração Nervosa/genética , Traumatismos dos Nervos Periféricos/fisiopatologia , Traumatismos dos Nervos Periféricos/terapia , Animais , Dependovirus/genética , Humanos , Neurônios/metabolismo , Traumatismos dos Nervos Periféricos/genética , Traumatismos dos Nervos Periféricos/patologia , Células de Schwann/metabolismoRESUMO
Olfactory ensheathing cells (OECs) have neuro-restorative properties in animal models for spinal cord injury, stroke, and amyotrophic lateral sclerosis. Here we used a multistep screening approach to discover genes specifically contributing to the regeneration-promoting properties of OECs. Microarray screening of the injured olfactory pathway and of cultured OECs identified 102 genes that were subsequently functionally characterized in cocultures of OECs and primary dorsal root ganglion (DRG) neurons. Selective siRNA-mediated knockdown of 16 genes in OECs (ADAMTS1, BM385941, FZD1, GFRA1, LEPRE1, NCAM1, NID2, NRP1, MSLN, RND1, S100A9, SCARB2, SERPINI1, SERPINF1, TGFB2, and VAV1) significantly reduced outgrowth of cocultured DRG neurons, indicating that endogenous expression of these genes in OECs supports neurite extension of DRG neurons. In a gain-of-function screen for 18 genes, six (CX3CL1, FZD1, LEPRE1, S100A9, SCARB2, and SERPINI1) enhanced and one (TIMP2) inhibited neurite growth. The most potent hit in both the loss- and gain-of-function screens was SCARB2, a protein that promotes cholesterol secretion. Transplants of fibroblasts that were genetically modified to overexpress SCARB2 significantly increased the number of regenerating DRG axons that grew toward the center of a spinal cord lesion in rats. We conclude that expression of SCARB2 enhances regenerative sprouting and that SCARB2 contributes to OEC-mediated neuronal repair.
Assuntos
Axônios/fisiologia , Proteínas de Membrana Lisossomal/biossíntese , Impressão Molecular/métodos , Regeneração Nervosa/fisiologia , Mucosa Olfatória/fisiologia , Receptores Depuradores/biossíntese , Células Receptoras Sensoriais/fisiologia , Animais , Células Cultivadas , Feminino , Testes Genéticos/métodos , Células HEK293 , Humanos , Proteínas de Membrana Lisossomal/genética , Mesotelina , Bulbo Olfatório/fisiologia , Mucosa Olfatória/citologia , Gravidez , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , Receptores Depuradores/genética , Células Receptoras Sensoriais/citologiaRESUMO
PURPOSE: Parkinson's disease (PD) is a movement disorder mainly characterized by progressive neurodegeneration of dopaminergic (DAergic) neurons in the substantia nigra (SN). As yet, unknown molecular changes contribute to the development of PD leading to a great need for in vivo models that herald this disorder. Here we characterize an animal model presenting early PD pathology. METHODS: Young, adult C57/BL6 mice were treated for five weeks twice a week with 15 mg/kg 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) in combination with 250 mg/kg probenecid. During the treatment mice were tested on their dopamine dependent movement skills. The integrity of their nigrostriatal system was examined through immunohistochemical studies. RESULTS: During the treatment, mice developed dopamine-dependent movement deficits induced by loss of tyrosine hydroxylase (TH) positive nigrostriatal axon terminals. Immunohistochemical study identified astrogliosis and microgliosis in the SN but no decrease of TH immunostaining, demonstrating lack of DAergic neuron degeneration. We also observed formation of α-synuclein inclusion bodies in the SN. CONCLUSIONS: The combined features of this MPTP model appear to represent an early neurotoxic cellular stress to the SN neurons bearing a striking resemblance to the early stages of PD neuropathology. This model might prove very useful to investigate early neurodegenerative events in the nigrostriatal DAergic system and to study the effects of potential treatment strategies counteracting the early PD cellular changes.
Assuntos
Modelos Animais de Doenças , Intoxicação por MPTP/patologia , Movimento/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Substância Negra/efeitos dos fármacos , Animais , Intoxicação por MPTP/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Neurônios/metabolismo , Neurônios/patologia , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Substância Negra/metabolismo , Substância Negra/patologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
In this study, we assess the feasibility of bioluminescence imaging to monitor the survival of Schwann cells (SCs) and olfactory ensheathing glia cells (OECs) after implantation in the lesioned spinal cord of adult rats. To this end, purified SCs and OECs were genetically modified with lentiviral vectors encoding luciferase-2 and GFP and implanted in the lesioned dorsal column. The bioluminescent signal was monitored for over 3 months, and at 7 and 98 days postsurgery, the signal was compared to standard histological analysis of GFP expression in the spinal cords. The temporal profile of the bioluminescent signal showed three distinct phases for both cell types. (I) A relatively stable signal in the first week. (II) A progressive decline in signal strength in the second and third week. (III) After the third week, the average bioluminescent signal stabilized for both cell types. Interestingly, in the first week, the peak of the bioluminescent signal after luciferin injection was delayed when compared to later time points. Similar to in vitro, our data indicated a linear relationship between the in vivo bioluminescent signal and the GFP signal of the SCs and OECs in the spinal cords when the results of both the 7 and 98 day time points are combined. This is the first report of the use of in vivo bioluminescence to monitor cell survival in the lesioned rat spinal cord. Bioluminescence could be a potentially powerful, noninvasive strategy to examine the efficacy of treatments that aim to improve the survival of proregenerative cells transplanted in the injured rat spinal cord.
Assuntos
Neuroglia/transplante , Bulbo Olfatório/patologia , Células de Schwann/transplante , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/cirurgia , Animais , Modelos Animais de Doenças , Feminino , Proteínas de Fluorescência Verde/análise , Medições Luminescentes/métodos , Neuroglia/patologia , Bulbo Olfatório/transplante , Ratos , Células de Schwann/patologiaRESUMO
There are many indications that neurogenesis is impaired in Parkinson's disease, which might be due to a lack of dopamine in the subventricular zone. An impairment in neurogenesis may have negative consequences for the development of new therapeutic approaches in Parkinson's disease, as neural stem cells are a potential source for endogenous repair. In this study, we examined the subventricular zone of 10 patients with Parkinson's disease and 10 age- and sex-matched controls for proliferation and neural stem cell numbers. We also included five cases with incidental Lewy body disease, which showed Parkinson's disease pathology but no clinical symptoms and thus did not receive dopaminergic treatment. We quantified the neural stem cell number and proliferative capacity in the subventricular zone of these three donor groups. We found subventricular neural stem cells in each donor, with a high variation in number. We did not observe significant differences in neural stem cell number or in proliferation between the groups. Additionally, we were able to culture neural stem cells from post-mortem brain of several patients with Parkinson's disease, confirming the presence of viable neural stem cells in these brains. We have also examined the subventricular zone of a chronic, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease mouse model, and again found no effect of dopaminergic denervation on precursor proliferation. Lastly, we investigated the proliferation capacity of two different human neural stem cell lines in response to dopamine. Both cell lines did not respond with a change in proliferation to treatment with dopamine agonists and an antagonist. In summary, the adult neural stem cell pool in the subventricular zone was not clearly affected in the human parkinsonian brain or a Parkinson's disease mouse model. Furthermore, we did not find evidence that dopamine has a direct effect on human neural stem cell proliferation in vitro. Thus, we conclude that the number of adult neural stem cells is probably not diminished in the parkinsonian brain and that dopamine depletion most likely has no effect on human neural stem cells.
Assuntos
Encéfalo/patologia , Proliferação de Células , Ventrículos Cerebrais/patologia , Intoxicação por MPTP/patologia , Neurogênese/fisiologia , Doença de Parkinson/patologia , Idoso , Idoso de 80 Anos ou mais , Animais , Encéfalo/metabolismo , Encéfalo/fisiopatologia , Células Cultivadas , Ventrículos Cerebrais/metabolismo , Ventrículos Cerebrais/fisiopatologia , Feminino , Humanos , Intoxicação por MPTP/metabolismo , Intoxicação por MPTP/fisiopatologia , Masculino , Camundongos , Células-Tronco Neurais , Doença de Parkinson/metabolismo , Doença de Parkinson/fisiopatologiaRESUMO
All cellular processes are regulated by condition-specific and time-dependent interactions between transcription factors and their target genes. While in simple organisms, e.g. bacteria and yeast, a large amount of experimental data is available to support functional transcription regulatory interactions, in mammalian systems reconstruction of gene regulatory networks still heavily depends on the accurate prediction of transcription factor binding sites. Here, we present a new method, log-linear modeling of 3D contingency tables (LLM3D), to predict functional transcription factor binding sites. LLM3D combines gene expression data, gene ontology annotation and computationally predicted transcription factor binding sites in a single statistical analysis, and offers a methodological improvement over existing enrichment-based methods. We show that LLM3D successfully identifies novel transcriptional regulators of the yeast metabolic cycle, and correctly predicts key regulators of mouse embryonic stem cell self-renewal more accurately than existing enrichment-based methods. Moreover, in a clinically relevant in vivo injury model of mammalian neurons, LLM3D identified peroxisome proliferator-activated receptor γ (PPARγ) as a neuron-intrinsic transcriptional regulator of regenerative axon growth. In conclusion, LLM3D provides a significant improvement over existing methods in predicting functional transcription regulatory interactions in the absence of experimental transcription factor binding data.
Assuntos
Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Genoma , Modelos Lineares , Camundongos , Regeneração Nervosa/genética , Neurônios/metabolismo , PPAR gama/metabolismo , Ratos , Ratos Wistar , Leveduras/genética , Leveduras/metabolismoRESUMO
For many experiments in the study of the peripheral nervous system, it would be useful to genetically manipulate primary sensory neurons. We have compared vectors based on adeno-associated virus (AAV) serotypes 1, 2, 3, 4, 5, 6, and 8, and lentivirus (LV), all expressing green fluorescent protein (GFP), for efficiency of transduction of sensory neurons, expression level, cellular tropism, and persistence of transgene expression following direct injection into the dorsal root ganglia (DRG), using histological quantification and qPCR. Two weeks after injection, AAV1, AAV5, and AAV6 had transduced the most neurons. The time course of GFP expression from these three vectors was studied from 1 to 12 weeks after injection. AAV5 was the most effective serotype overall, followed by AAV1. Both these serotypes showed increasing neuronal transduction rates at later time points, with some injections of AAV5 yielding over 90% of DRG neurons GFP(+) at 12 weeks. AAV6 performed well initially, but transduction rates declined dramatically between 4 and 12 weeks. AAV1 and AAV5 both transduced large-diameter neurons, IB4(+) neurons, and CGRP(+) neurons. In conclusion, AAV5 is a highly effective gene therapy vector for primary sensory neurons following direct injection into the DRG.
Assuntos
Dependovirus/classificação , Gânglios Espinais , Terapia Genética , Vetores Genéticos , Animais , Dependovirus/genética , Feminino , Plasmídeos , Ratos , Ratos Wistar , Sorotipagem , Transdução GenéticaRESUMO
BACKGROUND: After a spinal cord lesion, axon regeneration is inhibited by the presence of a diversity of inhibitory molecules in the lesion environment. At and around the lesion site myelin-associated inhibitors, chondroitin sulfate proteoglycans (CSPGs) and several axon guidance molecules, including all members of the secreted (class 3) Semaphorins, are expressed. Interfering with multiple inhibitory signals could potentially enhance the previously reported beneficial effects of blocking single molecules. RNA interference (RNAi) is a tool that can be used to simultaneously silence expression of multiple genes. In this study we aimed to employ adeno-associated virus (AAV) mediated expression of short hairpin RNAs (shRNAs) to target all Semaphorin class 3 signaling by knocking down its receptors, Neuropilin 1 (Npn-1) and Neuropilin 2 (Npn-2). RESULTS: We have successfully generated shRNAs that knock down Npn-1 and Npn-2 in a neuronal cell line. We detected substantial knockdown of Npn-2 mRNA when AAV5 viral vector particles expressing Npn-2 specific shRNAs were injected in dorsal root ganglia (DRG) of the rat. Unexpectedly however, AAV1-mediated expression of Npn-2 shRNAs and a control shRNA in the red nucleus resulted in an adverse tissue response and neuronal degeneration. The observed toxicity was dose dependent and was not seen with control GFP expressing AAV vectors, implicating the shRNAs as the causative toxic agents. CONCLUSIONS: RNAi is a powerful tool to knock down Semaphorin receptor expression in neuronal cells in vitro and in vivo. However, when shRNAs are expressed at high levels in CNS neurons, they trigger an adverse tissue response leading to neuronal degradation.
Assuntos
Dependovirus/genética , Vetores Genéticos , Degeneração Neural/etiologia , Neurônios/fisiologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Animais , Linhagem Celular , Feminino , Gânglios Espinais/fisiologia , Técnicas de Silenciamento de Genes , Humanos , Lentivirus/genética , Neuropilina-1/genética , Neuropilina-1/metabolismo , Neuropilina-2/genética , Neuropilina-2/metabolismo , RNA Interferente Pequeno/genética , Ratos , Ratos Wistar , Núcleo Rubro/fisiologia , Semaforinas/metabolismoRESUMO
Reconstructive surgery of the peripheral nerve has undergone major technical improvements over the last decades, leading to a significant improvement in the clinical outcome of surgery. Nonetheless, functional recovery remains suboptimal in the majority of patients after nerve repair surgery. In this review, we first discuss the molecular mechanisms involved in peripheral nerve injury and regeneration, with a special emphasis on the role of neurotrophic factors. We then identify five major challenges that currently exist in the clinical practice of nerve repair and their molecular basis. The first challenge is the slow rate of axonal outgrowth after peripheral nerve repair. The second problem is that of scar formation at the site of nerve injury, which is detrimental to functional recovery. As a third issue, we discuss the difficulty in assessing the degree of injury in closed traction lesions without total loss of continuity of the involved nerve elements. The fourth challenge is the problem of misrouting of regenerating axons. As a fifth and final issue we discuss the potential drawbacks of using sensory nerve grafts to support the regeneration of motoneurons. For all these challenges, solutions are likely to emerge from (a) a better understanding of their molecular basis and (b) the ability to influence these processes at a molecular level, possibly with the aid of viral vectors. We discuss how lentiviral vectors have been applied in the peripheral nerve to express neurotrophic factors and summarize both the advantages and drawbacks of this approach. Finally, we discuss how lentiviral vectors can be used to provide new, molecular neurobiology-based, approaches to address the clinical challenges described above.