VEGF expression disparities in brainstem motor neurons of the SOD1G93A ALS model: Correlations with neuronal vulnerability.

Amyotrophic lateral sclerosis (ALS) is a rare neuromuscular disease characterized by severe muscle weakness mainly due to degeneration and death of motor neurons. A peculiarity of the neurodegenerative processes is the variable susceptibility among distinct neuronal populations, exemplified by the contrasting resilience of motor neurons innervating the ocular motor system and the more vulnerable facial and hypoglossal motor neurons. The crucial role of vascular endothelial growth factor (VEGF) as a neuroprotective factor in the nervous system is well-established since a deficit of VEGF has been related to motoneuronal degeneration. In this study, we investigated the survival of ocular, facial, and hypoglossal motor neurons utilizing the murine SOD1G93A ALS model at various stages of the disease. Our primary objective was to determine whether the survival of the different brainstem motor neurons was linked to disparate VEGF expression levels in resilient and susceptible motor neurons throughout neurodegeneration. Our findings revealed a selective loss of motor neurons exclusively within the vulnerable nuclei. Furthermore, a significantly higher level of VEGF was detected in the more resistant motor neurons, the extraocular ones. We also examined whether TDP-43 dynamics in the brainstem motor neuron of SOD mice was altered. Our data suggests that the increased VEGF levels observed in extraocular motor neurons may potentially underlie their resistance during the neurodegenerative processes in ALS in a TDP-43-independent manner. Our work might help to better understand the underlying mechanisms of selective vulnerability of motor neurons in ALS.

Substance P-Botulinum Mediates Long-term Silencing of Pain Pathways that can be Re-instated with a Second Injection of the Construct in Mice.

Chronic pain presents an enormous personal and economic burden and there is an urgent need for effective treatments. In a mouse model of chronic neuropathic pain, selective silencing of key neurons in spinal pain signalling networks with botulinum constructs resulted in a reduction of pain behaviours associated with the peripheral nerve. However, to establish clinical relevance it was important to know how long this silencing period lasted. Now, we show that neuronal silencing and the concomitant reduction of neuropathic mechanical and thermal hypersensitivity lasts for up to 120d following a single injection of botulinum construct. Crucially, we show that silencing and analgesia can then be reinstated with a second injection of the botulinum conjugate. Here we demonstrate that single doses of botulinum-toxin conjugates are a powerful new way of providing long-term neuronal silencing and pain relief. PERSPECTIVE: This research demonstrates that botulinum-toxin conjugates are a powerful new way of providing long-term neuronal silencing without toxicity following a single injection of the conjugate and have the potential for repeated dosing when silencing reverses.

Neuroprotective Effect of Vascular Endothelial Growth Factor on Motoneurons of the Oculomotor System.

Vascular endothelial growth factor (VEGF) was initially characterized as a potent angiogenic factor based on its activity on the vascular system. However, it is now well established that VEGF also plays a crucial role as a neuroprotective factor in the nervous system. A deficit of VEGF has been related to motoneuronal degeneration, such as that occurring in amyotrophic lateral sclerosis (ALS). Strikingly, motoneurons of the oculomotor system show lesser vulnerability to neurodegeneration in ALS compared to other motoneurons. These motoneurons presented higher amounts of VEGF and its receptor Flk-1 than other brainstem pools. That higher VEGF level could be due to an enhanced retrograde input from their target muscles, but it can also be produced by the motoneurons themselves and act in an autocrine way. By contrast, VEGF's paracrine supply from the vicinity cells, such as glial cells, seems to represent a minor source of VEGF for brainstem motoneurons. In addition, ocular motoneurons experiment an increase in VEGF and Flk-1 level in response to axotomy, not observed in facial or hypoglossal motoneurons. Therefore, in this review, we summarize the differences in VEGF availability that could contribute to the higher resistance of extraocular motoneurons to injury and neurodegenerative diseases.

Sources and lesion-induced changes of VEGF expression in brainstem motoneurons.

Motoneurons of the oculomotor system show lesser vulnerability to neurodegeneration compared to other cranial motoneurons, as seen in amyotrophic lateral sclerosis (ALS). The overexpression of vascular endothelial growth factor (VEGF) is involved in motoneuronal protection. As previously shown, motoneurons innervating extraocular muscles present a higher amount of VEGF and its receptor Flk-1 compared to facial or hypoglossal motoneurons. Therefore, we aimed to study the possible sources of VEGF to brainstem motoneurons, such as glial cells and target muscles. We also studied the regulation of VEGF in response to axotomy in ocular, facial, and hypoglossal motor nuclei. Basal VEGF expression in astrocytes and microglial cells of the cranial motor nuclei was low. Although the presence of VEGF in the different target muscles for brainstem motoneurons was similar, the presynaptic element of the ocular neuromuscular junction showed higher amounts of Flk-1, which could result in greater efficiency in the capture of the factor by oculomotor neurons. Seven days after axotomy, a clear glial reaction was observed in all the brainstem nuclei, but the levels of the neurotrophic factor remained low in glial cells. Only the injured motoneurons of the oculomotor system showed an increase in VEGF and Flk-1, but such an increase was not detected in axotomized facial or hypoglossal motoneurons. Taken together, our findings suggest that the ocular motoneurons themselves upregulate VEGF expression in response to lesion. In conclusion, the low VEGF expression observed in glial cells suggests that these cells are not the main source of VEGF for brainstem motoneurons. Therefore, the higher VEGF expression observed in motoneurons innervating extraocular muscles is likely due either to the fact that this factor is more avidly taken up from the target muscles, in basal conditions, or is produced by these motoneurons themselves, and acts in an autocrine manner after axotomy.

Vascular Endothelial Growth Factor (VEGF) Prevents the Downregulation of the Cholinergic Phenotype in Axotomized Motoneurons of the Adult Rat.

Vascular endothelial growth factor (VEGF) was initially characterized by its activity on the vascular system. However, there is growing evidence indicating that VEGF also acts as a neuroprotective factor, and that its administration to neurons suffering from trauma or disease is able to rescue them from cell death. We questioned whether VEGF could also maintain damaged neurons in a neurotransmissive mode by evaluating the synthesis of their neurotransmitter, and whether its action would be direct or through its well-known angiogenic activity. Adult rat extraocular motoneurons were chosen as the experimental model. Lesion was performed by monocular enucleation and immediately a gelatine sponge soaked in VEGF was implanted intraorbitally. After 7 days, abducens, trochlear, and oculomotor nuclei were examined by immunohistochemistry against choline acetyltransferase (ChAT), the biosynthetic enzyme of the motoneuronal neurotransmitter acetylcholine. Lesioned motoneurons exhibited a noticeable ChAT downregulation which was prevented by VEGF administration. To explore whether this action was mediated via an increase in blood vessels or in their permeability, we performed immunohistochemistry against laminin, glucose transporter-1 and the plasmatic protein albumin. The quantification of the immunolabeling intensity against these three proteins showed no significant differences between VEGF-treated, axotomized and control animals. Therefore, the present data indicate that VEGF is able to sustain the cholinergic phenotype in damaged motoneurons, which is a first step for adequate neuromuscular neurotransmission, and that this action seems to be mediated directly on neurons since no sign of angiogenic activity was evident. These data reinforces the therapeutical potential of VEGF in motoneuronal diseases.

Extraocular Motor System Exhibits a Higher Expression of Neurotrophins When Compared with Other Brainstem Motor Systems.

Extraocular motoneurons resist degeneration in diseases such as amyotrophic lateral sclerosis. The main objective of the present work was to characterize the presence of neurotrophins in extraocular motoneurons and muscles of the adult rat. We also compared these results with those obtained from other cranial motor systems, such as facial and hypoglossal, which indeed suffer neurodegeneration. Immunocytochemical analysis was used to describe the expression of nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 in oculomotor, trochlear, abducens, facial, and hypoglossal nuclei of adult rats, and Western blots were used to describe the presence of neurotrophins in extraocular, facial (buccinator), and tongue muscles, which are innervated by the above-mentioned motoneurons. In brainstem samples, brain-derived neurotrophic factor was present both in extraocular and facial motoneuron somata, and to a lesser degree, in hypoglossal motoneurons. Neurotrophin-3 was present in extraocular motor nuclei, while facial and hypoglossal motoneurons were almost devoid of this protein. Finally, nerve growth factor was not present in the soma of any group of motoneurons, although it was present in dendrites of motoneurons located in the neuropil. Neuropil optical density levels were higher in extraocular motoneuron nuclei when compared with facial and hypoglossal nuclei. Neurotrophins could be originated in target muscles, since Western blot analyses revealed the presence of the three molecules in all sampled muscles, to a larger extent in extraocular muscles when compared with facial and tongue muscles. We suggest that the different neurotrophin availability could be related to the particular resistance of extraocular motoneurons to neurodegeneration.

Extraocular motoneurons of the adult rat show higher levels of vascular endothelial growth factor and its receptor Flk-1 than other cranial motoneurons.

Recent studies show a relationship between the deficit of vascular endothelial growth factor (VEGF) and motoneuronal degeneration, such as that occurring in amyotrophic lateral sclerosis (ALS). VEGF delivery protects motoneurons from cell death and delayed neurodegeneration in animal models of ALS. Strikingly, extraocular motoneurons show lesser vulnerability to neurodegeneration in ALS compared to other cranial or spinal motoneurons. Therefore, the present study investigates possible differences in VEGF and its main receptor VEGFR-2 or Flk-1 between extraocular and non-extraocular brainstem motoneurons. We performed immunohistochemistry and Western blot to determine the presence of VEGF and Flk-1 in rat motoneurons located in the three extraocular motor nuclei (abducens, trochlear and oculomotor) and to compare it to that observed in two other brainstem nuclei (hypoglossal and facial) that are vulnerable to degeneration. Extraocular motoneurons presented higher amounts of VEGF and its receptor Flk-1 than other brainstem motoneurons, and thus these molecules could be participating in their higher resistance to neurodegeneration. In conclusion, we hypothesize that differences in VEGF availability and signaling could be a contributing factor to the different susceptibility of extraocular motoneurons, when compared with other motoneurons, in neurodegenerative diseases.