RESUMEN

Axonal damage is a common feature of traumatic injury and neurodegenerative disease. The capacity for axons to regenerate and to recover functionality after injury is a phenomenon that is seen readily in the peripheral nervous system, especially in rodent models, but human axonal regeneration is limited and does not lead to full functional recovery. Here we describe a system where dynamics of human axonal outgrowth and regeneration can be evaluated via live imaging of human-induced pluripotent stem cell (hiPSC)-derived neurons cultured in microfluidic systems, in which cell bodies are isolated from their axons. This system could aid in studying axonal outgrowth dynamics and could be useful for testing potential drugs that encourage regeneration and repair of the nervous system.

Asunto(s)

Axones , Células Madre Pluripotentes Inducidas , Neuronas Motoras , Regeneración Nerviosa , Humanos , Células Madre Pluripotentes Inducidas/citología , Axones/fisiología , Neuronas Motoras/fisiología , Neuronas Motoras/citología , Regeneración Nerviosa/fisiología , Microfluídica/métodos , Microfluídica/instrumentación , Diferenciación Celular , Células Cultivadas , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Técnicas de Cultivo de Célula/métodos

RESUMEN

Peripheral nerve injuries (PNIs) represent a significant clinical challenge, particularly in elderly populations where axonal remyelination and regeneration are impaired. Developing therapies to enhance these processes is crucial for improving PNI repair outcomes. Glutamate carboxypeptidase II (GCPII) is a neuropeptidase that plays a pivotal role in modulating glutamate signaling through its enzymatic cleavage of the abundant neuropeptide N-acetyl aspartyl glutamate (NAAG) to liberate glutamate. Within the PNS, GCPII is expressed in Schwann cells and activated macrophages, and its expression is amplified with aging. In this study, we explored the therapeutic potential of inhibiting GCPII activity following PNI. We report significant GCPII protein and activity upregulation following PNI, which was normalized by the potent and selective GCPII inhibitor 2-(phosphonomethyl)-pentanedioic acid (2-PMPA). In vitro, 2-PMPA robustly enhanced myelination in dorsal root ganglion (DRG) explants. In vivo, using a sciatic nerve crush injury model in aged mice, 2-PMPA accelerated remyelination, as evidenced by increased myelin sheath thickness and higher numbers of remyelinated axons. These findings suggest that GCPII inhibition may be a promising therapeutic strategy to enhance remyelination and potentially improve functional recovery after PNI, which is especially relevant in elderly PNI patients where this process is compromised.

Asunto(s)

Glutamato Carboxipeptidasa II , Traumatismos de los Nervios Periféricos , Remielinización , Animales , Ratones , Traumatismos de los Nervios Periféricos/tratamiento farmacológico , Traumatismos de los Nervios Periféricos/metabolismo , Remielinización/efectos de los fármacos , Glutamato Carboxipeptidasa II/antagonistas & inhibidores , Glutamato Carboxipeptidasa II/metabolismo , Vaina de Mielina/metabolismo , Vaina de Mielina/efectos de los fármacos , Envejecimiento/efectos de los fármacos , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Ratones Endogámicos C57BL , Regeneración Nerviosa/efectos de los fármacos , Nervio Ciático/lesiones , Nervio Ciático/efectos de los fármacos , Masculino , Axones/efectos de los fármacos , Axones/metabolismo

RESUMEN

Anti-ganglioside antibodies (anti-Gg Abs) have been linked to delayed/poor clinical recovery in both axonal and demyelinating forms of Guillain-Barrè Syndrome (GBS). In many instances, the incomplete recovery is attributed to the peripheral nervous system's failure to regenerate. The cross-linking of cell surface gangliosides by anti-Gg Abs triggers inhibition of nerve repair in both in vitro and in vivo axon regeneration paradigms. This mechanism involves the activation of the small GTPase RhoA, which negatively modulates the growth cone cytoskeleton. At present, the identity/es of the receptor/s responsible for transducing the signal that ultimately leads to RhoA activation remains poorly understood. The aim of this work was to identify the transducer molecule responsible for the inhibitory effect of anti-Gg Abs on nerve repair. Putative candidate molecules were identified through proteomic mass spectrometry of ganglioside affinity-captured proteins from rat cerebellar granule neurons (Prendergast et al., 2014). These candidates were evaluated using an in vitro model of neurite outgrowth with primary cultured dorsal root ganglion neurons (DRGn) and an in vivo model of axon regeneration. Using an shRNA-strategy to silence putative candidates on DRGn, we identified tumor necrosis factor receptor 1A protein (TNFR1A) as a transducer molecule for the inhibitory effect on neurite outgrowth from rat/mouse DRGn cultures of a well characterized mAb targeting the related gangliosides GD1a and GT1b. Interestingly, lack of TNFr1A expression on DRGn abolished the inhibitory effect on neurite outgrowth caused by anti-GD1a but not anti-GT1b specific mAbs, suggesting specificity of GD1a/transducer signaling. Similar results were obtained using primary DRGn cultures from TNFR1a-null mice, which did not activate RhoA after exposure to anti-GD1a mAbs. Generation of single point mutants at the stalk region of TNFR1A identified a critical amino acid for transducing GD1a signaling, suggesting a direct interaction. Finally, passive immunization with an anti-GD1a/GT1b mAb in an in vivo model of axon regeneration exhibited reduced inhibitory activity in TNFR1a-null mice compared to wild type mice. In conclusion, these findings identify TNFR1A as a novel transducer receptor for the inhibitory effect exerted by anti-GD1a Abs on nerve repair, representing a significant step forward toward understanding the factors contributing to poor clinical recovery in GBS associated with anti-Gg Abs.

Asunto(s)

Axones , Gangliósidos , Inmunoglobulina G , Regeneración Nerviosa , Receptores Tipo I de Factores de Necrosis Tumoral , Proteína de Unión al GTP rhoA , Animales , Ratones , Ratas , Axones/metabolismo , Axones/inmunología , Células Cultivadas , Gangliósidos/metabolismo , Gangliósidos/inmunología , Síndrome de Guillain-Barré/inmunología , Síndrome de Guillain-Barré/metabolismo , Síndrome de Guillain-Barré/patología , Inmunoglobulina G/inmunología , Inmunoglobulina G/metabolismo , Inmunoglobulina G/farmacología , Ratones Noqueados , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/inmunología , Proteína de Unión al GTP rhoA/metabolismo , Proteína de Unión al GTP rhoA/inmunología , Transducción de Señal

RESUMEN

OBJECTIVE: To evaluate the longitudinal correlations between sulfatide/lysosulfatide levels and central and peripheral nervous system function in children with metachromatic leukodystrophy (MLD) and to explore the impact of intravenous recombinant human arylsulfatase A (rhASA) treatment on myelin turnover. METHODS: A Phase 1/2 study of intravenous rhASA investigated cerebrospinal fluid (CSF) and sural nerve sulfatide levels, 88-item Gross Motor Function Measure (GMFM-88) total score, sensory and motor nerve conduction, brain N-acetylaspartate (NAA) levels, and sural nerve histology in 13 children with MLD. Myelinated and unmyelinated nerves from an untreated MLD mouse model were also analyzed. RESULTS: CSF sulfatide levels correlated with neither Z-scores for GMFM-88 nor brain NAA levels; however, CSF sulfatide levels correlated negatively with Z-scores of nerve conduction parameters, number of large (≥7 µm) myelinated fibers, and myelin/fiber diameter slope, and positively with nerve g-ratios and cortical latencies of somatosensory-evoked potentials. Quantity of endoneural litter positively correlated with sural nerve sulfatide/lysosulfatide levels. CSF sulfatide levels decreased with continuous high-dose treatment; this change correlated with improved nerve conduction. At 26 weeks after treatment, nerve g-ratio decreased by 2%, and inclusion bodies per Schwann cell unit increased by 55%. In mice, abnormal sulfatide storage was observed in non-myelinating Schwann cells in Remak bundles of sciatic nerves but not in unmyelinated urethral nerves. INTERPRETATION: Lower sulfatide levels in the CSF and peripheral nerves correlate with better peripheral nerve function in children with MLD; intravenous rhASA treatment may reduce CSF sulfatide levels and enhance sulfatide/lysosulfatide processing and remyelination in peripheral nerves.

Asunto(s)

Leucodistrofia Metacromática , Psicosina/análogos & derivados , Niño , Humanos , Ratones , Animales , Leucodistrofia Metacromática/tratamiento farmacológico , Sulfoglicoesfingolípidos/farmacología , Cerebrósido Sulfatasa , Nervio Ciático/patología

RESUMEN

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease, characterized by degeneration of upper and lower motor neurons that leads to muscle weakness, paralysis, and death, but the effects of disease-causing mutations on axonal outgrowth of neurons derived from human induced pluripotent stem cells (iPSC)-derived motor neurons (hiPSC-MN) are poorly understood. The use of hiPSC-MN is a promising tool to develop more relevant models for target identification and drug development in ALS research, but questions remain concerning the effects of distinct disease-causing mutations on axon regeneration. Mutations in superoxide dismutase 1 (SOD1) were the first to be discovered in ALS patients. Here, we investigated the effect of the SOD1A4V mutation on axonal regeneration of hiPSC-MNs, utilizing compartmentalized microfluidic devices, which are powerful tools for studying hiPSC-MN distal axons. Surprisingly, SOD1+/A4V hiPSC-MNs regenerated axons more quickly following axotomy than those expressing the native form of SOD1. Though initial axon regrowth was not significantly different following axotomy, enhanced regeneration was apparent at later time points, indicating an increased rate of outgrowth. This regeneration model could be used to identify factors that enhance the rate of human axon regeneration.

Asunto(s)

Esclerosis Amiotrófica Lateral , Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Humanos , Esclerosis Amiotrófica Lateral/genética , Superóxido Dismutasa-1/genética , Axones , Superóxido Dismutasa/genética , Regeneración Nerviosa , Neuronas Motoras/fisiología , Mutación

RESUMEN

Following peripheral nerve injury, multiple cell types, including axons, Schwann cells, and macrophages, coordinate to promote nerve regeneration. However, this capacity for repair is limited, particularly in older populations, and current treatments are insufficient. A critical component of the regeneration response is the network of cell-to-cell signaling in the injured nerve microenvironment. Sheddases are expressed in the peripheral nerve and play a role in the regulation if this cell-to-cell signaling through cleavage of transmembrane proteins, enabling the regulation of multiple pathways through cis- and trans-cellular regulatory mechanisms. Enhanced axonal regeneration has been observed in mice with deletion of the sheddase beta-secretase (BACE1), a transmembrane aspartyl protease that has been studied in the context of Alzheimer's disease. BACE1 knockout (KO) mice display enhanced macrophage recruitment and activity following nerve injury, although it is unclear whether this plays a role in driving the enhanced axonal regeneration. Further, it is unknown by what mechanism(s) BACE1 increases macrophage recruitment and activity. BACE1 has many substrates, several of which are known to have immunomodulatory activity. This review will discuss current knowledge of the role of BACE1 and other sheddases in peripheral nerve regeneration and outline known immunomodulatory BACE1 substrates and what potential roles they could play in peripheral nerve regeneration. Currently, the literature suggests that BACE1 and substrates that are expressed by neurons and Schwann cells are likely to be more important for this process than those expressed by macrophages. More broadly, BACE1 may play a role as an effector of immunomodulation beyond the peripheral nerve.

Asunto(s)

Secretasas de la Proteína Precursora del Amiloide/genética , Ácido Aspártico Endopeptidasas/genética , Macrófagos/patología , Regeneración Nerviosa/genética , Traumatismos de los Nervios Periféricos/genética , Traumatismos de los Nervios Periféricos/patología , Animales , Humanos , Nervios Periféricos/patología

RESUMEN

Nervous system disorders are prevalent health issues that will only continue to increase in frequency as the population ages. Dying-back axonopathy is a hallmark of many neurologic diseases and leads to axonal disconnection from their targets, which in turn leads to functional impairment. During the course of many of neurologic diseases, axons can regenerate or sprout in an attempt to reconnect with the target and restore synapse function. In amyotrophic lateral sclerosis (ALS), distal motor axons retract from neuromuscular junctions early in the disease-course before significant motor neuron death. There is evidence of compensatory motor axon sprouting and reinnervation of neuromuscular junctions in ALS that is usually quickly overtaken by the disease course. Potential drugs that enhance compensatory sprouting and encourage reinnervation may slow symptom progression and retain muscle function for a longer period of time in ALS and in other diseases that exhibit dying-back axonopathy. There remain many outstanding questions as to the impact of distinct disease-causing mutations on axonal outgrowth and regeneration, especially in regards to motor neurons derived from patient induced pluripotent stem cells. Compartmentalized microfluidic chambers are powerful tools for studying the distal axons of human induced pluripotent stem cells-derived motor neurons, and have recently been used to demonstrate striking regeneration defects in human motor neurons harboring ALS disease-causing mutations. Modeling the human neuromuscular circuit with human induced pluripotent stem cells-derived motor neurons will be critical for developing drugs that enhance axonal regeneration, sprouting, and reinnervation of neuromuscular junctions. In this review we will discuss compensatory axonal sprouting as a potential therapeutic target for ALS, and the use of compartmentalized microfluidic devices to find drugs that enhance regeneration and axonal sprouting of motor axons.

RESUMEN

Oligodendrocytes (OLs) are important for myelination and shuttling energy metabolites lactate and pyruvate toward axons through their expression of monocarboxylate transporter 1 (MCT1). Recent studies suggest that loss of OL MCT1 causes axonal degeneration. However, it is unknown how widespread and chronic loss of MCT1 in OLs specifically affects neuronal energy homeostasis with aging. To answer this, MCT1 conditional null mice were generated that allow for OL-specific MCT1 ablation. We observe that MCT1 loss from OL lineage cells is dispensable for normal myelination and axonal energy homeostasis early in life. By contrast, loss of OL lineage MCT1 expression with aging leads to significant axonal degeneration with concomitant hypomyelination. These data support the hypothesis that MCT1 is important for neuronal energy homeostasis in the aging central nervous system (CNS). The reduction in OL MCT1 that occurs with aging may enhance the risk for axonal degeneration and atrophy in neurodegenerative diseases.

Asunto(s)

Axones/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Vaina de Mielina/metabolismo , Degeneración Nerviosa/metabolismo , Oligodendroglía/metabolismo , Simportadores/metabolismo , Animales , Femenino , Masculino , Ratones , Ratones Transgénicos , Transportadores de Ácidos Monocarboxílicos/deficiencia , Vaina de Mielina/patología , Oligodendroglía/patología , Simportadores/deficiencia

RESUMEN

OBJECTIVE: Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease caused by deficient activity of arylsulfatase A (ASA), resulting in severe motor and cognitive dysfunction. This phase 1/2 study evaluated the safety and efficacy of intravenous (IV) recombinant human ASA (rhASA; HGT-1111, previously known as Metazym) in children with MLD. METHODS: Thirteen children with MLD (symptom onset < 4 years of age) were enrolled in an open-label, nonrandomized, dose-escalation trial and received IV rhASA at 50, 100, or 200 U/kg body weight every 14 (± 4) days for 52 weeks (NCT00418561; NCT00633139). Eleven children continued to receive rhASA at 100 or 200 U/kg during a 24-month extension period (NCT00681811). Outcome measures included safety observations, changes in motor and cognitive function, and changes in nerve conduction and morphometry. RESULTS: There were no serious adverse events considered related to IV rhASA. Motor function and developmental testing scores declined during the study in all dose groups; no significant differences were observed between groups. Nerve conduction studies and morphometric analysis indicated that peripheral nerve pathology did not worsen during the study in any dose group. INTERPRETATION: IV rhASA was generally well tolerated. There was no evidence of efficacy in preventing motor and cognitive deterioration, suggesting that IV rhASA may not cross the blood-brain barrier in therapeutic quantities. The relative stability of peripheral nerve function during the study indicates that rhASA may be beneficial if delivered to the appropriate target site and supports the development of rhASA for intrathecal administration in MLD.

Asunto(s)

Cerebrósido Sulfatasa/administración & dosificación , Leucodistrofia Metacromática/tratamiento farmacológico , Encéfalo/efectos de los fármacos , Cerebrósido Sulfatasa/farmacocinética , Preescolar , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Masculino , Conducción Nerviosa/efectos de los fármacos , Nervios Periféricos/efectos de los fármacos

RESUMEN

Following peripheral nerve injury, macrophages are recruited to the injury site from circulation to clear cellular debris. Injured ß-secretase 1 (BACE1) knockout mice have enhanced macrophage recruitment and debris clearance, which may be due to BACE1 activity in macrophages or the hypomyelination observed in BACE1 knockout mice. To assess if BACE1 expression by macrophages mediates enhanced macrophage recruitment we utilized mice with macrophage specific deletion of BACE1 and saw no increase in macrophage recruitment following injury. This study suggests that expression of BACE1 by macrophages may not be essential for increased recruitment observed previously in global BACE1 KO mice.

Asunto(s)

Secretasas de la Proteína Precursora del Amiloide/deficiencia , Ácido Aspártico Endopeptidasas/deficiencia , Macrófagos/metabolismo , Regeneración Nerviosa/fisiología , Traumatismos de los Nervios Periféricos/metabolismo , Animales , Células Cultivadas , Femenino , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Traumatismos de los Nervios Periféricos/patología

RESUMEN

While the peripheral nervous system is able to repair itself following injury and disease, recovery is often slow and incomplete, with no available treatments to enhance the effectiveness of regeneration. Using knock-out and transgenic overexpressor mice, we previously reported that BACE1, an aspartyl protease, as reported by Hemming et al. (PLoS One 4:12, 2009), negatively regulates peripheral nerve regeneration. Here, we investigated whether pharmacological inhibition of BACE may enhance peripheral nerve repair following traumatic nerve injury or neurodegenerative disease. BACE inhibitor-treated mice had increased numbers of regenerating axons and enhanced functional recovery after a sciatic nerve crush while inhibition increased axonal sprouting following a partial nerve injury. In the SOD1G93A ALS mouse model, BACE inhibition increased axonal regeneration with improved muscle re-innervation. CHL1, a BACE1 substrate, was elevated in treated mice and may mediate enhanced regeneration. Our data demonstrates that pharmacological BACE inhibition accelerates peripheral axon regeneration after varied nerve injuries and could be used as a potential therapy.

Asunto(s)

Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Esclerosis Amiotrófica Lateral/enzimología , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/metabolismo , Axones/fisiología , Regeneración Nerviosa/fisiología , Traumatismos de los Nervios Periféricos/enzimología , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/genética , Animales , Axones/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/uso terapéutico , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Regeneración Nerviosa/efectos de los fármacos , Traumatismos de los Nervios Periféricos/tratamiento farmacológico , Traumatismos de los Nervios Periféricos/genética , Superóxido Dismutasa/genética

RESUMEN

Notch signaling is a highly conserved intercellular pathway with tightly regulated and pleiotropic roles in normal tissue development and homeostasis. Dysregulated Notch signaling has also been implicated in human disease, including multiple forms of cancer, and represents an emerging therapeutic target. Successful development of such therapeutics requires a detailed understanding of potential on-target toxicities. Here, we identify autosomal dominant mutations of the canonical Notch ligand Jagged1 (or JAG1) as a cause of peripheral nerve disease in 2 unrelated families with the hereditary axonal neuropathy Charcot-Marie-Tooth disease type 2 (CMT2). Affected individuals in both families exhibited severe vocal fold paresis, a rare feature of peripheral nerve disease that can be life-threatening. Our studies of mutant protein posttranslational modification and localization indicated that the mutations (p.Ser577Arg, p.Ser650Pro) impair protein glycosylation and reduce JAG1 cell surface expression. Mice harboring heterozygous CMT2-associated mutations exhibited mild peripheral neuropathy, and homozygous expression resulted in embryonic lethality by midgestation. Together, our findings highlight a critical role for JAG1 in maintaining peripheral nerve integrity, particularly in the recurrent laryngeal nerve, and provide a basis for the evaluation of peripheral neuropathy as part of the clinical development of Notch pathway-modulating therapeutics.

Asunto(s)

Enfermedad de Charcot-Marie-Tooth , Genes Dominantes , Proteína Jagged-1 , Mutación Missense , Transducción de Señal/genética , Sustitución de Aminoácidos , Animales , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/metabolismo , Femenino , Glicosilación , Humanos , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Masculino , Ratones , Receptores Notch/genética , Receptores Notch/metabolismo

RESUMEN

Chemotherapy-Induced Peripheral Neurotoxicity (CIPN) is often dose-limiting and impacts life quality and survival of cancer patients. Ghrelin agonists have neuroprotectant effects and may have a role in treating or preventing CIPN. We evaluated the CNS-penetrant ghrelin agonist HM01 in three experimental models of CIPN at doses of 3-30 mg/kg p.o. daily monitoring orexigenic properties, nerve conduction, mechanical allodynia, and intra-epidermal nerve fiber density (IENFD). In a cisplatin-based study, rats were dosed daily for 3 days (0.5 mg/kg i.p.) + HM01. Cisplatin treatment induced mechanical hypersensitivity which was significantly reduced by HM01. In a second study, oxaliplatin was administered to mice (6 mg/kg i.p. 3 times/week for 4 weeks) resulting in significant digital nerve conduction velocity (NCV) deficits and reduction of IENFD. Concurrent HM01 dose dependently prevented the decline in NCV and attenuated the reduction in IENFD. Pharmacokinetic studies showed HM01 accumulation in the dorsal root ganglia and sciatic nerves which reached concentrations > 10 fold that of plasma. In a third model, HM01 was tested in preventive and therapeutic paradigms in a bortezomib-based rat model (0.2 mg/kg i.v., 3 times/week for 8 weeks). In the preventive setting, HM01 blocked bortezomib-induced hyperalgesia and IENFD reduction at all doses tested. In the therapeutic setting, significant effect was observed, but only at the highest dose. Altogether, the robust peripheral nervous system penetration of HM01 and its ability to improve multiple oxaliplatin-, cisplatin-, and bortezomib-induced neurotoxicities suggest that HM01 may be a useful neuroprotective adjuvant for CIPN.

Asunto(s)

Antineoplásicos/efectos adversos , Derivados del Benceno/farmacología , Ghrelina/agonistas , Sistema Nervioso/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Animales , Peso Corporal/efectos de los fármacos , Cisplatino/efectos adversos , Relación Dosis-Respuesta a Droga , Ingestión de Alimentos/efectos de los fármacos , Femenino , Masculino , Ratones , Conducción Nerviosa/efectos de los fármacos , Piperidinas , Ratas

RESUMEN

While the peripheral nervous system has the capacity to regenerate following a nerve injury, it is often at a slow rate and results in unsatisfactory recovery, leaving patients with reduced function. Many regeneration associated genes have been identified over the years, which may shed some insight into how we can manipulate this intrinsic regenerative ability to enhance repair following peripheral nerve injuries. Our lab has identified the membrane bound protease beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1), or beta secretase, as a potential negative regulator of peripheral nerve regeneration. When beta secretase activity levels are abolished via a null mutation in mice, peripheral regeneration is enhanced following a sciatic nerve crush injury. Conversely, when activity levels are greatly increased by overexpressing beta secretase in mice, nerve regeneration and functional recovery are impaired after a sciatic nerve crush injury. In addition to our work, many substrates of beta secretase have been found to be involved in regulating neurite outgrowth and some have even been identified as regeneration associated genes. In this review, we set out to discuss BACE1 and its substrates with respect to axonal regeneration and speculate on the possibility of utilizing BACE1 inhibitors to enhance regeneration following acute nerve injury and potential uses in peripheral neuropathies.

RESUMEN

Axons of the peripheral nervous system possess the capacity to regenerate following injury. Previously, we showed that genetically knocking out Beta-Site APP-Cleaving Enzyme 1 (BACE1) leads to increased nerve regeneration. Two cellular components, macrophages and neurons, contribute to enhanced nerve regeneration in BACE1 knockout mice. Here, we utilized a transgenic mouse model that overexpresses BACE1 in its neurons to investigate whether neuronal BACE1 has an inverse effect on regeneration following nerve injury. We performed a sciatic nerve crush in BACE1 transgenic mice and control wild-type littermates, and evaluated the extent of both morphological and physiological improvements over time. At the earliest time point of 3days, we observed a significant decrease in the length of axonal sprouts growing out from the crush site in BACE1 transgenic mice. At later times (10 and 15days post-crush), there were significant reductions in the number of myelinated axons in the sciatic nerve and the percentage of re-innervated neuromuscular junctions in the gastrocnemius muscle. Transgenic mice had a functional electrophysiological delay in the recovery up to 8weeks post-crush compared to controls. These results indicate that BACE1 activity levels have an inverse effect on peripheral nerve repair after injury. The results obtained in this study provide evidence that neuronal BACE1 activity levels impact peripheral nerve regeneration. This data has clinical relevance by highlighting a novel drug target to enhance peripheral nerve repair, an area which currently does not have any approved therapeutics.

Asunto(s)

Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Axones/enzimología , Regeneración Nerviosa/fisiología , Recuperación de la Función/fisiología , Nervio Ciático/enzimología , Nervio Ciático/lesiones , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Ácido Aspártico Endopeptidasas/genética , Axones/patología , Modelos Animales de Enfermedad , Femenino , Humanos , Macrófagos/enzimología , Macrófagos/patología , Masculino , Ratones Transgénicos , Músculo Esquelético/enzimología , Músculo Esquelético/inervación , Músculo Esquelético/patología , Fibras Nerviosas Mielínicas/enzimología , Fibras Nerviosas Mielínicas/patología , Unión Neuromuscular/enzimología , Unión Neuromuscular/patología , Distribución Aleatoria , Nervio Ciático/patología

RESUMEN

Sensory and autonomic neuropathy affects the majority of type II diabetic patients. Clinically, autonomic evaluation often focuses on sudomotor function yet this is rarely assessed in animal models. We undertook morphological and functional studies to assess large myelinated and small unmyelinated axons in the db/db type II diabetes mouse model. We observed that autonomic innervation of sweat glands in the footpads was significantly reduced in db/db mice compared to control db/+ mice and this deficit was greater compared to reductions in intraepidermal sensory innervation of adjacent epidermis. Additionally, db/db mice formed significantly fewer sweat droplets compared to controls as early as 6 weeks of age, a time when no statistical differences were observed electrophysiologically between db/db and db/+ mice studies of large myelinated sensory and motor nerves. The rate of sweat droplet formation was significantly slower and the sweat droplet size larger and more variable in db/db mice compared to controls. Whereas pilocarpine and glycopyrrolate increased and decreased sweating, respectively, in 6 month-old controls, db/db mice did not respond to pharmacologic manipulations. Our findings indicate autonomic neuropathy is an early and prominent deficit in the db/db model and have implications for the development of therapies for peripheral diabetic neuropathy.

Asunto(s)

Sistema Nervioso Autónomo/patología , Sistema Nervioso Autónomo/fisiopatología , Diabetes Mellitus Experimental/patología , Diabetes Mellitus Experimental/fisiopatología , Diabetes Mellitus Tipo 2/fisiopatología , Extremidades/patología , Extremidades/fisiopatología , Sensación , Animales , Diabetes Mellitus Tipo 2/patología , Modelos Animales de Enfermedad , Epidermis/patología , Masculino , Estimulación Física , Glándulas Sudoríparas/inervación , Temperatura

RESUMEN

Peripheral neuropathy is a serious, dose-limiting side effect of cancer treatment with microtubule-targeting drugs. Symptoms present in a "stocking-glove" distribution, with longest nerves affected most acutely, suggesting a length-dependent component to the toxicity. Axonal transport of ATP-producing mitochondria along neuronal microtubules from cell body to synapse is crucial to neuronal function. We compared the effects of the drugs paclitaxel and ixabepilone that bind along the lengths of microtubules and the drugs eribulin and vincristine that bind at microtubule ends, on mitochondrial trafficking in cultured human neuronal SK-N-SH cells and on axonal transport in mouse sciatic nerves. Antiproliferative concentrations of paclitaxel and ixabepilone significantly inhibited the anterograde transport velocity of mitochondria in neuronal cells, whereas eribulin and vincristine inhibited transport only at significantly higher concentrations. Confirming these observations, anterogradely transported amyloid precursor protein accumulated in ligated sciatic nerves of control and eribulin-treated mice, but not in paclitaxel-treated mice, indicating that paclitaxel inhibited anterograde axonal transport, whereas eribulin did not. Electron microscopy of sciatic nerves of paclitaxel-treated mice showed reduced organelle accumulation proximal to the ligation consistent with inhibition of anterograde (kinesin based) transport by paclitaxel. In contrast, none of the drugs significantly affected retrograde (dynein based) transport in neuronal cells or mouse nerves. Collectively, these results suggest that paclitaxel and ixabepilone, which bind along the lengths and stabilize microtubules, inhibit kinesin-based axonal transport, but not dynein-based transport, whereas the microtubule-destabilizing drugs, eribulin and vincristine, which bind preferentially to microtubule ends, have significantly less effect on all microtubule-based axonal transport. Cancer Res; 76(17); 5115-23. ©2016 AACR.

Asunto(s)

Antineoplásicos/toxicidad , Transporte Axonal/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Neuronas/efectos de los fármacos , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Animales , Epotilonas/toxicidad , Furanos/toxicidad , Humanos , Cetonas/toxicidad , Ratones , Mitocondrias/efectos de los fármacos , Paclitaxel/toxicidad , Nervio Ciático/efectos de los fármacos , Moduladores de Tubulina/toxicidad , Vincristina/toxicidad

RESUMEN

BACKGROUND: Peripheral neuropathy is a common and dose-limiting side effect of many cancer chemotherapies. The taxane agents, including paclitaxel (Taxol(®)), are effective chemotherapeutic drugs but cause degeneration of predominantly large myelinated afferent sensory fibers of the peripheral nervous system in humans and animal models. Dorsal root ganglia (DRG) neurons are sensory neurons that have unipolar axons each with two branches: peripheral and central. While taxane agents induce degeneration of peripheral axons, whether they also cause degeneration of central nervous system axons is not clear. Using a mouse model of paclitaxel-induced neuropathy, we investigated the effects of paclitaxel on the central branches of sensory axons. RESULTS: We observed that in the spinal cords of paclitaxel-intoxicated mice, degenerated axons were present in the dorsal columns, where the central branches of DRG axons ascend rostrally. In the peripheral nerves, degenerated myelinated fibers were present in significantly greater numbers in distal segments than in proximal segments indicating that this model exhibits the distal-to-proximal degeneration pattern generally observed in human peripheral nerve disorders. CONCLUSIONS: We conclude that paclitaxel causes degeneration of both the peripheral and central branches of DRG axons, a finding that has implications for the site and mode of action of chemotherapy agents on the nervous system.

Asunto(s)

Antineoplásicos/toxicidad , Axones/efectos de los fármacos , Ganglios Espinales/efectos de los fármacos , Degeneración Nerviosa/inducido químicamente , Paclitaxel/toxicidad , Animales , Axones/patología , Modelos Animales de Enfermedad , Femenino , Ganglios Espinales/patología , Masculino , Ratones Endogámicos C57BL , Microscopía Electrónica , Degeneración Nerviosa/patología , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/patología , Nervio Ciático/efectos de los fármacos , Nervio Ciático/patología , Médula Espinal/efectos de los fármacos , Médula Espinal/patología

RESUMEN

Hematogenous macrophages remove myelin debris from injured peripheral nerves to provide a micro-environment conducive to axonal regeneration. Previously, we observed that injured peripheral nerves from Beta-site APP Cleaving Enzyme 1 (BACE1) knockout (KO) mice displayed earlier influx of and enhanced phagocytosis by macrophages when compared to wild-type (WT) mice. These observations suggest that BACE1 might regulate macrophage influx into distal stumps of injured nerves. To determine through which pathway BACE1 influences macrophage influx, we used a mouse inflammation antibody array to assay the expression of inflammation-related proteins in injured nerves of BACE1 KO and WT mice. The most significant change was in expression of tumor necrosis factor receptor 1 (TNFR1) in the distal stump of injured BACE1 KO nerves. Western blotting of protein extracts confirmed increased expression of TNFR1 and its downstream transcriptional factor NFκB in the BACE1 KO distal stumps. Additionally, treatment of WT mice with a BACE1 inhibitor resulted in increased TNFR1 expression and signaling in the distal stump of injured nerves. Exogenous TNFα increased nuclear translocation of p65 NFκB in BACE1 KO tissue and cultured fibroblasts compared with control WT. BACE1 regulates TNFR1 expression at the level of gene expression and not through proteolytic processing. The accelerated macrophage influx in injured nerves of BACE1 KO mice correlates with increased expression and signaling via TNFR1, indicating a link between BACE1 activity and TNFR1 expression/signaling that might contribute to repair of the injured nervous system.

Asunto(s)

Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Vaina de Mielina/metabolismo , Traumatismos de los Nervios Periféricos/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal/fisiología , Animales , Macrófagos/metabolismo , Ratones Noqueados , Regeneración Nerviosa/fisiología , Nervios Periféricos/metabolismo , Fagocitosis/fisiología

RESUMEN

OBJECTIVE: Metachromatic leukodystrophy (MLD) is an autosomal recessive lysosomal storage disorder due to deficient activity of arylsulfatase A (ASA) that causes accumulation of sulfatide and lysosulfatide. The disorder is associated with demyelination and axonal loss in the central and peripheral nervous systems. The late infantile form has an early-onset, rapidly progressive course with severe sensorimotor dysfunction. The relationship between the degree of nerve damage and (lyso)sulfatide accumulation is, however, not established. METHODS: In 13 children aged 2-5 years with severe motor impairment, markedly elevated cerebrospinal fluid (CSF) and sural nerve sulfatide and lysosulfatide levels, genotype, ASA mRNA levels, residual ASA, and protein cross-reactive immunological material (CRIM) confirmed the diagnosis. We studied the relationship between (lyso)sulfatide levels and (1) the clinical deficit in gross motor function (GMFM-88), (2) median and peroneal nerve motor and median and sural nerve sensory conduction studies (NCS), (3) median and tibial nerve somatosensory evoked potentials (SSEPs), (4) sural nerve histopathology, and (5) brain MR spectroscopy. RESULTS: Eleven patients had a sensory-motor demyelinating neuropathy on electrophysiological testing, whereas two patients had normal studies. Sural nerve and CSF (lyso)sulfatide levels strongly correlated with abnormalities in electrophysiological parameters and large myelinated fiber loss in the sural nerve, but there were no associations between (lyso)sulfatide levels and measures of central nervous system (CNS) involvement (GMFM-88 score, SSEP, and MR spectroscopy). INTERPRETATION: Nerve and CSF sulfatide and lysosulfatide accumulation provides a marker of disease severity in the PNS only; it does not reflect the extent of CNS involvement by the disease process. The magnitude of the biochemical disturbance produces a continuously graded spectrum of impairments in neurophysiological function and sural nerve histopathology.