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.

Purinergic Receptor Blockade with Suramin Increases Survival of Postnatal Neural Progenitor Cells In Vitro.

Neural progenitor cells (NPCs) are self-renewing and multipotent cells that persist in the postnatal and adult brain in the subventricular zone and the hippocampus. NPCs can be expanded in vitro to be used in cell therapy. However, expansion is limited, since the survival and proliferation of adult NPCs decrease with serial passages. Many signaling pathways control NPC survival and renewal. Among these, purinergic receptor activation exerts differential effects on the biology of adult NPCs depending on the cellular context. In this study, we sought to analyze the effect of a general blockade of purinergic receptors with suramin on the proliferation and survival of NPCs isolated from the subventricular zone of postnatal rats, which are cultured as neurospheres. Treatment of neurospheres with suramin induced a significant increase in neurosphere diameter and in NPC number attributed to a decrease in apoptosis. Proliferation and multipotency were not affected. Suramin also induced an increase in the gap junction protein connexin43 and in vascular endothelial growth factor, which might be involved in the anti-apoptotic effect. Our results offer a valuable tool for increasing NPC survival before implantation in the lesioned brain and open the possibility of using this drug as adjunctive therapy to NPC transplantation.

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.

To Become or Not to Become Tumorigenic: Subventricular Zone Versus Hippocampal Neural Stem Cells.

Neural stem cells (NSCs) persist in the adult mammalian brain in two neurogenic regions: the subventricular zone lining the lateral ventricles and the dentate gyrus of the hippocampus. Compelling evidence suggests that NSCs of the subventricular zone could be the cell type of origin of glioblastoma, the most devastating brain tumor. Studies in glioblastoma patients revealed that NSCs of the tumor-free subventricular zone, harbor cancer-driver mutations that were found in the tumor cells but were not present in normal cortical tissue. Endogenous mutagenesis can also take place in hippocampal NSCs. However, to date, no conclusive studies have linked hippocampal mutations with glioblastoma development. In addition, glioblastoma cells often invade or are closely located to the subventricular zone, whereas they do not tend to infiltrate into the hippocampus. In this review we will analyze possible causes by which subventricular zone NSCs might be more susceptible to malignant transformation than their hippocampal counterparts. Cellular and molecular differences between the two neurogenic niches, as well as genotypic and phenotypic characteristics of their respective NSCs will be discussed regarding why the cell type originating glioblastoma brain tumors has been linked mainly to subventricular zone, but not to hippocampal NSCs.

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.

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.

Differential regulation of the expression of neurotrophin receptors in rat extraocular motoneurons after lesion.

Neurotrophins acting through high-affinity tyrosine kinase receptors (trkA, trkB, and trkC) play a crucial role in regulating survival and maintenance of specific neuronal functions after injury. Adult motoneurons supplying extraocular muscles survive after disconnection from the target, but suffer dramatic changes in morphological and physiological properties, due in part to the loss of their trophic support from the muscle. To investigate the dependence of the adult rat extraocular motoneurons on neurotrophins, we examined trkA, trkB, and trkC mRNA expression after axotomy by in situ hybridization. trkA mRNA expression was detectable at low levels in unlesioned motoneurons, and its expression was downregulated 1 and 3 days after injury. Expression of trkB and trkC mRNAs was stronger, and after axotomy a simultaneous, but inverse regulation of both receptors was observed. Thus, whereas a considerable increase in trkB expression was seen about 2 weeks after axotomy, the expression of trkC mRNA had decreased at the same post-lesion period. Injured extraocular motoneurons also experienced an initial induction in expression of calcitonin gene-related peptide and a transient downregulation of cholinergic characteristics, indicating a switch in the phenotype from a transmitter-specific to a regenerative state. These results suggest that specific neurotrophins may contribute differentially to the survival and regenerative responses of extraocular motoneurons after lesion.

Nerve growth factor regulates the firing patterns and synaptic composition of motoneurons.

Target-derived neurotrophins exert powerful synaptotrophic actions in the adult brain and are involved in the regulation of different forms of synaptic plasticity. Target disconnection produces a profound synaptic stripping due to the lack of trophic support. Consequently, target reinnervation leads to synaptic remodeling and restoration of cellular functions. Extraocular motoneurons are unique in that they normally express the TrkA neurotrophin receptor in the adult, a feature not seen in other cranial or spinal motoneurons, except after lesions such as axotomy or in neurodegenerative diseases like amyotrophic lateral sclerosis. We investigated the effects of nerve growth factor (NGF) by retrogradely delivering this neurotrophin to abducens motoneurons of adult cats. Axotomy reduced the density of somatic boutons and the overall tonic and phasic firing modulation. Treatment with NGF restored synaptic inputs and firing modulation in axotomized motoneurons. When K252a, a selective inhibitor of tyrosine kinase activity, was applied to specifically test TrkA effects, the NGF-mediated restoration of synapses and firing-related parameters was abolished. Discharge variability and recruitment threshold were, however, increased by NGF compared with control or axotomized motoneurons. Interestingly, these parameters returned to normal following application of REX, an antibody raised against neurotrophin receptor p75 (p75(NTR)). In conclusion, NGF, acting retrogradely through TrkA receptors, supports afferent boutons and regulates the burst and tonic signals correlated with eye movements. On the other hand, p75(NTR) activation regulates recruitment threshold, which impacts on firing regularity. To our knowledge, this is the first report showing powerful synaptotrophic effects of NGF on motoneurons in vivo.

Abducens internuclear neurons depend on their target motoneurons for survival during early postnatal development.

The highly specific projection of abducens internuclear neurons onto medial rectus motoneurons in the oculomotor nucleus is a good model to evaluate the dependence on target cells for survival during development and in the adult. Thus, the procedure we chose to selectively deprive abducens internuclear neurons of their natural target was the enucleation of postnatal day 1 rats to induce the death of medial rectus motoneurons. Two months later, we evaluated both the extent of reduction in target size, by immunocytochemistry against choline acetyltransferase (ChAT) and Nissl counting, and the percentage of abducens internuclear neurons surviving target loss, by calretinin immunostaining and horseradish peroxidase (HRP) retrograde tracing. Firstly, axotomized oculomotor motoneurons died in a high percentage ( approximately 80%) as visualized 2 months after lesion. In addition, we showed a transient (1 month) and reversible down-regulation of ChAT expression in extraocular motoneurons induced by injury. Secondly, 2 months after enucleation, 61.6% and 60.5% of the population of abducens internuclear neurons appeared stained by retrograde tracing and calretinin immunoreaction, respectively, indicating a significant extent of cell death after target loss (38.4% or 39.5%). By contrast, in the adult rat, neither extraocular motoneurons died in response to axotomy nor abducens internuclear neurons died due to the loss of their target motoneurons induced by the retrograde transport of toxic ricin injected in the medial rectus muscle. These results indicate that, during development, abducens internuclear neurons depend on their target motoneurons for survival, and that they lose this dependence with maturation.

Expression of Trk receptors in the oculomotor system of the adult cat.

We examined the expression of the three Trk receptors for neurotrophins (TrkA, TrkB, and TrkC) in the extraocular motor nuclei of the adult cat by using antibodies directed against the full-Trk proteins in combination with horseradish peroxidase retrograde tracing. The three receptors were present in all neuronal populations investigated, including abducens motoneurons and internuclear neurons, medial rectus motoneurons of the oculomotor nucleus, and trochlear motoneurons. They were also present in the vestibular and prepositus hypoglossi nuclei. TrkA, TrkB, and TrkC immunopositive cells were found in similar percentages in the oculomotor and in the trochlear nuclei. In the abducens nucleus, however, a significantly higher percentage of cells expressed TrkB than the other two receptors, among both motoneurons (81.8%) and internuclear neurons (88.4%). The percentages obtained for the three Trk receptors in identified neuronal populations pointed to the colocalization of two or three receptors in a large number of cells. We used confocal microscopy to elucidate the subcellular location of Trk receptors. In this case, abducens motoneurons and internuclear neurons were identified with antibodies against choline acetyltransferase and calretinin, respectively. We found a different pattern of staining for each neurotrophin receptor, suggesting the possibility that each receptor and its cognate ligand may use a different route for cellular signaling. Therefore, the expression of Trk receptors in oculomotor, trochlear, and abducens motoneurons, as well as abducens internuclear neurons, suggests that their associated neurotrophins may exert an influence on the normal operation of the oculomotor circuitry. The presence of multiple Trk receptors on individual cells indicates that they likely act in concert with each other to regulate distinct functions.

Increased neurogenesis and brain-derived neurotrophic factor in neurokinin-1 receptor gene knockout mice.

It has previously been shown that chronic treatment with antidepressant drugs increases neurogenesis and levels of brain-derived neurotrophic factor in the hippocampus. These changes have been correlated with changes in learning and long-term potentiation and may contribute to the therapeutic efficacy of antidepressant drug treatment. Recently, antagonists at the neurokinin-1 receptor, the preferred receptor for the neuropeptide substance P, have been shown to have antidepressant activity. Mice with disruption of the neurokinin-1 receptor gene are remarkably similar both behaviourally and neurochemically to mice maintained chronically on antidepressant drugs. We demonstrate here that there is a significant elevation of neurogenesis but not cell survival in the hippocampus of neurokinin-1 receptor knockout mice. Neurogenesis can be increased in wild-type but not neurokinin-1 receptor knockout mice by chronic treatment with antidepressant drugs which preferentially target noradrenergic and serotonergic pathways. Hippocampal levels of brain-derived neurotrophic factor are also two-fold higher in neurokinin-1 receptor knockout mice, whereas cortical levels are similar. Finally, we examined hippocampus-dependent learning and memory but found no clear enhancement in neurokinin-1 receptor knockout mice. These data argue against a simple correlation between increased levels of neurogenesis or brain-derived neurotrophic factor and mnemonic processes in the absence of increased cell survival. They support the hypothesis that increased neurogenesis, perhaps accompanied by higher levels of brain-derived neurotrophic factor, may contribute to the efficacy of antidepressant drug therapy.

Superficial NK1-expressing neurons control spinal excitability through activation of descending pathways.

The increase in pain sensitivity that follows injury is regulated by superficially located projection neurons in the dorsal horn of the spinal cord that express the neurokinin-1 (NK1) receptor. After selective ablation of these neurons in rats, we identified changes in receptive field size, mechanical and thermal coding and central sensitization of deeper dorsal horn neurons that are important for both pain sensations and reflexes. We were able to reproduce these changes by pharmacological block of descending serotonergic facilitatory pathways. Using Fos histochemistry, we found changes in the activation of serotonergic neurons in the brainstem as well as evidence for a loss of descending control of spinal excitability. We conclude that NK1-positive spinal projection neurons, activated by primary afferent input, project to higher brain areas that control spinal excitability--and therefore pain sensitivity--primarily through descending pathways from the brainstem.