Effects of Sigma-1 Receptor Ligands on Peripheral Nerve Regeneration

Peripheral nerve injuries lead to the loss of motor, sensory and autonomic functions in the territories supplied by the injured nerve. Currently, nerve injuries are managed by surgical repair procedures, and there are no effective drugs in the clinic for improving the capacity of axonal regeneration. Sigma-1 receptor (Sig-1R) is an endoplasmic reticulum chaperon protein involved in many functions, including neuroprotection and neuroplasticity. A few previous studies using Sig-1R ligands reported results that suggest this receptor as a putative target to enhance regeneration. The aim of this study was to evaluate the possible effects of Sig-1R ligands on axonal regeneration in a sciatic nerve section and repair model in mice. To this end, mice were treated either with the Sig-1R agonist PRE-084 or the antagonist BD1063, and a Sig-1R knock-out (KO) mice group was also studied. The electrophysiological and histological data showed that treatment with Sig-1R ligands, or the lack of this protein, did not markedly modify the process of axonal regeneration and target reinnervation after sciatic nerve injury. Nevertheless, the nociceptive tests provided results indicating a role of Sig-1R in sensory perception after nerve injury, and immunohistochemical labeling indicated a regulatory role in inflammatory cell infiltration in the injured nerve

Evaluation of neuroprotective effects of sigma 1 receptor ligands on motoneuron degeneration models

Les malalties de les motoneurones (MMN) són un conjunt ampli de trastorns neurològics esporàdics i hereditaris caracteritzats per la degeneració d'aquest tipus de neurones, les motoneurones (MN), amb afeccions molt debilitants i greus amb opcions terapèutiques limitades. L'esclerosi lateral amiotròfica (ELA), la MNN més freqüent en adults, és una malaltia neurodegenerativa fatal que cursa amb la degeneració de les MNs de la medul·la espinal i del cervell. Actualment, els tractaments disponibles tenen lleus millores sobre la supervivència del pacient allargant la vida uns mesos. Per tant, és urgent comprendre els mecanismes moleculars implicats en la degeneració de les MNs per identificar noves dianes terapèutiques i desenvolupar teràpies efectives que protegeixin i restaurin la integritat neuronal. Recentment, entre d'altres dianes terapèutiques proposades per la neuroprotecció, el receptor Sigma-1 (Sig-1R) ha guanyat interès. L'objectiu d'aquesta tesi era avaluar el potencial terapèutic de diversos lligands del Sig-1R per promoure la supervivència de les MNs i el manteniment de la funció neuromuscular. Amb aquest propòsit, primer vam emprar el model in vitro de cultiu organotípic de medul·la espinal sotmès a una excitotoxicitat crònica i, a continuació, dos models murins, la lesió del nervi espinal i els ratolins transgènics SOD1G93A. Els nostres resultats in vitro van revelar que els lligands del Sig-1R provats (PRE-084, BD1063, SA4503, EST79232 i EST79376) augmentaven la supervivència de les MNs sotmeses a una excitotoxicitat crònica. D'altra banda, els estudis in vivo van demostrar que els efectes neuroprotectors observats diferien entre els diversos lligands del Sig-1R testats. En el model murí SOD1G93A, el tractament amb els diversos lligands del Sig-1R va ser capaç de preservar la funció i les unions neuromusculars de les extremitats posteriors, augmentar el nombre de MNs supervivents i modular la reactivitat glial a la medul·la espinal. Aquests lligands del Sig-1R també van ser capaços d'augmentar la supervivència de les MNs i modular la reactivitat glial després d'una lesió del nervi espinal. En conjunt, els resultats d'aquesta tesi aporten evidències clares que el Sig-1R és una diana interessant pel tractament de les MMN.Las enfermedades de las motoneuronas (EMN) son un grupo amplio de trastornos neurológicos esporádicos y hereditarios caracterizados por la degeneración de este tipo de neuronas, las motoneuronas (MNs), con afecciones muy debilitantes y grave con unas opciones terapéuticas limitadas. La esclerosis lateral amiotrófica (ELA), la EMN más frecuente en adultos, es una enfermedad neurodegenerativa mortal caracterizada por la degeneración de las MNs de la médula espinal y el cerebro. Actualmente, los tratamientos disponibles tienen efectos modestos sobre la supervivencia del paciente alargando la vida unos meses. Por tanto, existe una necesidad urgente de comprender los mecanismos moleculares implicados en la degeneración de las MNs para identificar nuevas dianas terapéuticas y desarrollar terapias eficaces que protejan y restauren la integridad neuronal. Recientemente, entre otras dianas para la neuroprotecion, el receptor Sigma-1 (Sig-1R) ha ganado interés. El objetivo de esta tesis era evaluar el potencial terapéutico de varios ligandos del Sig-1R para promover la supervivencia de las MNs y el mantenimiento de la función neuromuscular. Para ello, primero utilizamos el modelo in vitro de cultivo organotípico de médula espinal sometido a una excitotoxicidad crónica, y luego, dos modelos murinos, la lesión del nervio espinal y los ratones transgénicos SOD1G93A. Nuestros resultados in vitro revelaron que los ligandos del Sig-1R probados (PRE-084, BD1063, SA4503, EST79232 y EST79376) aumentaban la supervivencia de las MNs sometidas a una excitotoxicidad crónica. Por otro lado, los estudios in vivo mostraron que los efectos neuroprotectores observados diferían entre los diversos ligandos Sig-1R probados. En el ratón SOD1G93A, el tratamiento con varios ligandos Sig-1R fue eficaz para preservar la función y las uniones neuromusculares de las extremidades posteriores, aumentar el número de MNs supervivientes y modular la reactividad glial en la médula espinal. Estos ligandos del Sig-1R también fueron capaces de aumentar la supervivencia de las MNs y modular la reactividad glial después de una lesión del nervio espinal. En conjunto, los resultados de esta tesis proporcionan una clara evidencia de que el Sig-1R es una diana interesante para el tratamiento de las EMN.Motor neuron diseases (MND) include a wide type of sporadic and hereditary neurological disorders characterized by degeneration of motor neurons (MNs), and are highly debilitating and severe conditions for which only limited therapeutic options are available. Amyotrophic lateral sclerosis (ALS), the most frequent MND in adults, is a fatal neurodegenerative disease with degeneration of MNs in the spinal cord and the brain. Currently, available treatments have modest effects on patient survival. Thus, there is an urgent need to understand the molecular mechanisms involved in the MN degeneration to discover new therapeutic targets and develop effective therapies protecting and restoring neuronal integrity. Among the putative targets for neuroprotection, recently the Sigma-1 receptor (Sig-1R) has gained attention. The aim of the present thesis was to assess the therapeutic potential of several Sig-1R ligands for promoting MN survival and maintenance of neuromuscular function. For this purpose, we first used an in vitro model of spinal cord organotypic culture subjected to chronic excitotoxicity, and then, two murine models, the spinal nerve injury and the transgenic SOD1G93A mice. Our in vitro results revealed that the Sig-1R ligands tested (PRE-084, BD1063, SA4503, EST79232 and EST79376) increased MN survival under chronic excitotoxicity. On the other hand, in vivo studies showed that the neuroprotective effects observed differed between the Sig-1R ligands tested. In the SOD1G93A mouse treatment with several Sig-1R ligands was effective to preserve neuromuscular function and junctions of the hindlimbs, to increase the number of surviving MNs and to modulate glial reactivity in the spinal cord. These Sig-1R ligands were also able to increase MN survival and modulate glial reactivity after a spinal nerve injury. Altogether, the results of this thesis provide clear evidence that Sig-1R is an interesting target for the treatment of MND

Evaluation of neuroprotective effects of sigma 1 receptor ligands on motoneuron degeneration models

Les malalties de les motoneurones (MMN) són un conjunt ampli de trastorns neurològics esporàdics i hereditaris caracteritzats per la degeneració d’aquest tipus de neurones, les motoneurones (MN), amb afeccions molt debilitants i greus amb opcions terapèutiques limitades. L’esclerosi lateral amiotròfica (ELA), la MNN més freqüent en adults, és una malaltia neurodegenerativa fatal que cursa amb la degeneració de les MNs de la medul·la espinal i del cervell. Actualment, els tractaments disponibles tenen lleus millores sobre la supervivència del pacient allargant la vida uns mesos. Per tant, és urgent comprendre els mecanismes moleculars implicats en la degeneració de les MNs per identificar noves dianes terapèutiques i desenvolupar teràpies efectives que protegeixin i restaurin la integritat neuronal. Recentment, entre d’altres dianes terapèutiques proposades per la neuroprotecció, el receptor Sigma-1 (Sig-1R) ha guanyat interès. L’objectiu d’aquesta tesi era avaluar el potencial terapèutic de diversos lligands del Sig-1R per promoure la supervivència de les MNs i el manteniment de la funció neuromuscular. Amb aquest propòsit, primer vam emprar el model in vitro de cultiu organotípic de medul·la espinal sotmès a una excitotoxicitat crònica i, a continuació, dos models murins, la lesió del nervi espinal i els ratolins transgènics SOD1G93A. Els nostres resultats in vitro van revelar que els lligands del Sig-1R provats (PRE-084, BD1063, SA4503, EST79232 i EST79376) augmentaven la supervivència de les MNs sotmeses a una excitotoxicitat crònica. D’altra banda, els estudis in vivo van demostrar que els efectes neuroprotectors observats diferien entre els diversos lligands del Sig-1R testats. En el model murí SOD1G93A, el tractament amb els diversos lligands del Sig-1R va ser capaç de preservar la funció i les unions neuromusculars de les extremitats posteriors, augmentar el nombre de MNs supervivents i modular la reactivitat glial a la medul·la espinal. Aquests lligands del Sig-1R també van ser capaços d’augmentar la supervivència de les MNs i modular la reactivitat glial després d’una lesió del nervi espinal. En conjunt, els resultats d’aquesta tesi aporten evidències clares que el Sig-1R és una diana interessant pel tractament de les MMN.Las enfermedades de las motoneuronas (EMN) son un grupo amplio de trastornos neurológicos esporádicos y hereditarios caracterizados por la degeneración de este tipo de neuronas, las motoneuronas (MNs), con afecciones muy debilitantes y grave con unas opciones terapéuticas limitadas. La esclerosis lateral amiotrófica (ELA), la EMN más frecuente en adultos, es una enfermedad neurodegenerativa mortal caracterizada por la degeneración de las MNs de la médula espinal y el cerebro. Actualmente, los tratamientos disponibles tienen efectos modestos sobre la supervivencia del paciente alargando la vida unos meses. Por tanto, existe una necesidad urgente de comprender los mecanismos moleculares implicados en la degeneración de las MNs para identificar nuevas dianas terapéuticas y desarrollar terapias eficaces que protejan y restauren la integridad neuronal. Recientemente, entre otras dianas para la neuroprotecion, el receptor Sigma-1 (Sig-1R) ha ganado interés. El objetivo de esta tesis era evaluar el potencial terapéutico de varios ligandos del Sig-1R para promover la supervivencia de las MNs y el mantenimiento de la función neuromuscular. Para ello, primero utilizamos el modelo in vitro de cultivo organotípico de médula espinal sometido a una excitotoxicidad crónica, y luego, dos modelos murinos, la lesión del nervio espinal y los ratones transgénicos SOD1G93A. Nuestros resultados in vitro revelaron que los ligandos del Sig-1R probados (PRE-084, BD1063, SA4503, EST79232 y EST79376) aumentaban la supervivencia de las MNs sometidas a una excitotoxicidad crónica. Por otro lado, los estudios in vivo mostraron que los efectos neuroprotectores observados diferían entre los diversos ligandos Sig-1R probados. En el ratón SOD1G93A, el tratamiento con varios ligandos Sig-1R fue eficaz para preservar la función y las uniones neuromusculares de las extremidades posteriores, aumentar el número de MNs supervivientes y modular la reactividad glial en la médula espinal. Estos ligandos del Sig-1R también fueron capaces de aumentar la supervivencia de las MNs y modular la reactividad glial después de una lesión del nervio espinal. En conjunto, los resultados de esta tesis proporcionan una clara evidencia de que el Sig-1R es una diana interesante para el tratamiento de las EMN.Motor neuron diseases (MND) include a wide type of sporadic and hereditary neurological disorders characterized by degeneration of motor neurons (MNs), and are highly debilitating and severe conditions for which only limited therapeutic options are available. Amyotrophic lateral sclerosis (ALS), the most frequent MND in adults, is a fatal neurodegenerative disease with degeneration of MNs in the spinal cord and the brain. Currently, available treatments have modest effects on patient survival. Thus, there is an urgent need to understand the molecular mechanisms involved in the MN degeneration to discover new therapeutic targets and develop effective therapies protecting and restoring neuronal integrity. Among the putative targets for neuroprotection, recently the Sigma-1 receptor (Sig-1R) has gained attention. The aim of the present thesis was to assess the therapeutic potential of several Sig-1R ligands for promoting MN survival and maintenance of neuromuscular function. For this purpose, we first used an in vitro model of spinal cord organotypic culture subjected to chronic excitotoxicity, and then, two murine models, the spinal nerve injury and the transgenic SOD1G93A mice. Our in vitro results revealed that the Sig-1R ligands tested (PRE-084, BD1063, SA4503, EST79232 and EST79376) increased MN survival under chronic excitotoxicity. On the other hand, in vivo studies showed that the neuroprotective effects observed differed between the Sig-1R ligands tested. In the SOD1G93A mouse treatment with several Sig-1R ligands was effective to preserve neuromuscular function and junctions of the hindlimbs, to increase the number of surviving MNs and to modulate glial reactivity in the spinal cord. These Sig-1R ligands were also able to increase MN survival and modulate glial reactivity after a spinal nerve injury. Altogether, the results of this thesis provide clear evidence that Sig-1R is an interesting target for the treatment of MND.Universitat Autònoma de Barcelona. Programa de Doctorat en Neurocièncie

EST79232 and EST79376, Two Novel Sigma-1 Receptor Ligands, Exert Neuroprotection on Models of Motoneuron Degeneration

Motor neuron diseases (MNDs) include sporadic and hereditary neurological disorders characterized by progressive degeneration of motor neurons (MNs). Sigma-1 receptor (Sig-1R) is a protein enriched in MNs, and mutations on its gene lead to various types of MND. Previous studies have suggested that Sig-1R is a target to prevent MN degeneration. In this study, two novel synthesized Sig-1R ligands, coded EST79232 and EST79376, from the same chemical series, with the same scaffold and similar physicochemical properties but opposite functionality on Sig-1R, were evaluated as neuroprotective compounds to prevent MN degeneration. We used an in vitro model of spinal cord organotypic cultures under chronic excitotoxicity and two in vivo models, the spinal nerve injury and the superoxide dismutase 1 (SOD1)G93A mice, to characterize the effects of these Sig-1R ligands on MN survival and modulation of glial reactivity. The antagonist EST79376 preserved MNs in vitro and after spinal nerve injury but was not able to improve MN death in SOD1G93A mice. In contrast, the agonist EST79232 significantly increased MN survival in the three models of MN degeneration evaluated and had a mild beneficial effect on motor function in SOD1G93A mice. In vivo, Sig-1R ligand EST79232 had a more potent effect on preventing MN degeneration than EST79376. These data further support the interest in Sig-1R as a therapeutic target for neurodegeneration

Effects of Sigma-1 Receptor Ligands on Peripheral Nerve Regeneration

Peripheral nerve injuries lead to the loss of motor, sensory and autonomic functions in the territories supplied by the injured nerve. Currently, nerve injuries are managed by surgical repair procedures, and there are no effective drugs in the clinic for improving the capacity of axonal regeneration. Sigma-1 receptor (Sig-1R) is an endoplasmic reticulum chaperon protein involved in many functions, including neuroprotection and neuroplasticity. A few previous studies using Sig-1R ligands reported results that suggest this receptor as a putative target to enhance regeneration. The aim of this study was to evaluate the possible effects of Sig-1R ligands on axonal regeneration in a sciatic nerve section and repair model in mice. To this end, mice were treated either with the Sig-1R agonist PRE-084 or the antagonist BD1063, and a Sig-1R knock-out (KO) mice group was also studied. The electrophysiological and histological data showed that treatment with Sig-1R ligands, or the lack of this protein, did not markedly modify the process of axonal regeneration and target reinnervation after sciatic nerve injury. Nevertheless, the nociceptive tests provided results indicating a role of Sig-1R in sensory perception after nerve injury, and immunohistochemical labeling indicated a regulatory role in inflammatory cell infiltration in the injured nerve

Neuroprotective Effects of Sigma 1 Receptor Ligands on Motoneuron Death after Spinal Root Injury in Mice

Loss of motor neurons (MNs) after spinal root injury is a drawback limiting the recovery after palliative surgery by nerve or muscle transfers. Research based on preventing MN death is a hallmark to improve the perspectives of recovery following severe nerve injuries. Sigma-1 receptor (Sig-1R) is a protein highly expressed in MNs, proposed as neuroprotective target for ameliorating MN degenerative conditions. Here, we used a model of L4–L5 rhizotomy in adult mice to induce MN degeneration and to evaluate the neuroprotective role of Sig-1R ligands (PRE-084, SA4503 and BD1063). Lumbar spinal cord was collected at 7, 14, 28 and 42 days post-injury (dpi) for immunohistochemistry, immunofluorescence and Western blot analyses. This proximal axotomy at the immediate postganglionic level resulted in significant death, up to 40% of spinal MNs at 42 days after injury and showed markedly increased glial reactivity. Sig-1R ligands PRE-084, SA4503 and BD1063 reduced MN loss by about 20%, associated to modulation of endoplasmic reticulum stress markers IRE1α and XBP1. These pathways are Sig-1R specific since they were not produced in Sig-1R knockout mice. These findings suggest that Sig-1R is a promising target for the treatment of MN cell death after neural injuries

EST79232 and EST79376, Two novel sigma-1 receptor ligands, exert neuroprotection on models of motoneuron degeneration

Motor neuron diseases (MNDs) include sporadic and hereditary neurological disorders characterized by progressive degeneration of motor neurons (MNs). Sigma-1 receptor (Sig-1R) is a protein enriched in MNs, and mutations on its gene lead to various types of MND. Previous studies have suggested that Sig-1R is a target to prevent MN degeneration. In this study, two novel synthesized Sig-1R ligands, coded EST79232 and EST79376, from the same chemical series, with the same scaffold and similar physicochemical properties but opposite functionality on Sig-1R, were evaluated as neuroprotective compounds to prevent MN degeneration. We used an in vitro model of spinal cord organotypic cultures under chronic excitotoxicity and two in vivo models, the spinal nerve injury and the superoxide dismutase 1 (SOD1)G93A mice, to characterize the effects of these Sig-1R ligands on MN survival and modulation of glial reactivity. The antagonist EST79376 preserved MNs in vitro and after spinal nerve injury but was not able to improve MN death in SOD1G93A mice. In contrast, the agonist EST79232 significantly increased MN survival in the three models of MN degeneration evaluated and had a mild beneficial effect on motor function in SOD1G93A mice. In vivo, Sig-1R ligand EST79232 had a more potent effect on preventing MN degeneration than EST79376. These data further support the interest in Sig-1R as a therapeutic target for neurodegeneration

Microglial cell loss after ischemic stroke favors brain neutrophil accumulation

Stroke attracts neutrophils to the injured brain tissue where they can damage the integrity of the blood-brain barrier and exacerbate the lesion. However, the mechanisms involved in neutrophil transmigration, location and accumulation in the ischemic brain are not fully elucidated. Neutrophils can reach the perivascular spaces of brain vessels after crossing the endothelial cell layer and endothelial basal lamina of post-capillary venules, or migrating from the leptomeninges following pial vessel extravasation and/or a suggested translocation from the skull bone marrow. Based on previous observations of microglia phagocytosing neutrophils recruited to the ischemic brain lesion, we hypothesized that microglial cells might control neutrophil accumulation in the injured brain. We studied a model of permanent occlusion of the middle cerebral artery in mice, including microglia- and neutrophil-reporter mice. Using various in vitro and in vivo strategies to impair microglial function or to eliminate microglia by targeting colony stimulating factor 1 receptor (CSF1R), this study demonstrates that microglial phagocytosis of neutrophils has fundamental consequences for the ischemic tissue. We found that reactive microglia engulf neutrophils at the periphery of the ischemic lesion, whereas local microglial cell loss and dystrophy occurring in the ischemic core are associated with the accumulation of neutrophils first in perivascular spaces and later in the parenchyma. Accordingly, microglia depletion by long-term treatment with a CSF1R inhibitor increased the numbers of neutrophils and enlarged the ischemic lesion. Hence, microglial phagocytic function sets a critical line of defense against the vascular and tissue damaging capacity of neutrophils in brain ischemia