VEGF and Neuronal Survival

1 1 2 3 VEGF and Neuronal Survival 4 5 6 Paula M. Calvo, Rosendo G. Hernández, 7 Angel M. Pastor* and Rosa R. de la Cruz 8 9 Departamento de Fisiología, Facultad de Biología, Universidad de Sevilla, 10 41012-Sevilla, Spain. 11 12 13 14 15 16 * Corresponding Author: 17 Angel M. Pastor 18 Departamento de Fisiología 19 Facultad de Biología 20 Universidad de Sevilla 21 Avda. Reina Mercedes, 6 22 41012-Sevilla 23 Spain 24 25 Email: [email protected] 26 27 2 Abstract 28 Vascular endothelial growth factor (VEGF) is well known by its angiogenic activity, however, 29 recent evidence has revealed a neuroprotective action of this factor on injured or diseased 30 neurons. In the present review, we summarize the most relevant findings that have contributed 31 to establish a link between VEGF deficiency and neuronal degeneration. It should be 32 emphasized that the design of the mutant mice VEGFδ/δ has been crucial to establish the 33 neuroprotective role of VEGF. These mice, which develop over time reduced levels of VEGF, 34 show adult-onset muscle weakness and motoneuron degeneration that resemble amyotrophic 35 lateral sclerosis (ALS). VEGF administration through different technical approaches to animal 36 models of ALS has been demonstrated to ameliorate motoneuronal degeneration and improve 37 motor performance. Altogether, the results presented in this review highlight VEGF as an 38 essential motoneuron neurotrophic factor endowed with promising therapeutic potential for the 39 treatment of motoneuron disorders. 40 41 Keywords 42 Neurotrophic factors, neuroprotection, ALS, axotomy, extraocular motoneurons 43 44 3 Introduction 45 The vascular endothelial growth factor (VEGF) was identified as the result of two parallel lines 46 of research. In 1983, Senger and others carried out the partial purification of a protein derived 47 from a guinea pig tumor cell line, which they named “vascular permeability factor” (VPF), due 48 to its ability of increasing blood vessel permeability in skin. In 1989, Conolly and others 49 isolated and sequenced, from U937 tumor cells, the human VPF molecule. In that same year, 50 Ferrara and Henzel reported the isolation of a diffusible endothelial cell-specific mitogen from 51 medium conditioned by bovine pituitary follicular cells, which they named “vascular 52 endothelial growth factor” (VEGF) to reflect the restricted target-cell specificity of this 53 molecule. The sequence of purified VEGF proved that this protein did not match any known 54 protein in available databases (Ferrara and Henzel 1989). cDNA cloning of VEGF (Leung and 55 others 1989) and VPF (Keck and others 1989), both reported also in 1989, demonstrated that 56 VEGF and VPF were the same molecule. All these findings revealed that VEGF is a potent, 57 diffusible, and specific factor for vascular endothelial cells and led to the hypothesis that this 58 molecule could play a role in the regulation of physiological and pathological growth of blood 59 vessels (Ferrara and Henzel 1989; Ferrara and others 1991; Leung and others 1989). Therefore, 60 VEGF was historically first associated with its potent actions on endothelial cells, inducing 61 vasculogenesis, angiogenesis and increased vascular permeability (Apte and others 2019; 62 Ferrara 1999, 2004; Yancopoulus and others 2000). 63 Subsequently, different evidence indicated that, in addition to its vascular biological 64 activity, VEGF also acts as a neuroprotective factor. Numerous works have demonstrated that 65 the administration of VEGF exerts trophic effects after different types of lesion and neuronal 66 types. For instance, VEGF protects against seizure-induced neuronal loss in hippocampus 67 (Nicoletti and others 2008) and attenuates status-epilepticus behavioral impairments (Nicoletti 68 and others 2010; Ureña-Guerrero and others 2020). VEGF acts as a neurotrophic factor 69 4 improving the damaged hypoxic/ischemic brain (Guo and others 2016; Sun and others 2003; 70 Yang and others 2018), and promotes the anatomical and functional recovery of injured 71 peripheral nerves in the avascular cornea (Pan and others 2013) and after olfactory nerve 72 bulbectomy (Beecher and others 2019). It also rescues hippocampal neurons from glutamate- 73 induced excitotoxicity (Matsuzaki and others 2001). Interestingly, VEGF exerts neurorescue 74 effects in animal models of neurodegenerative diseases, such as Alzheimer and Parkinson, in 75 cerebral cortex and hippocampus (Guo and others 2019; Ureña-Guerrero and others 2020) and 76 in the substantia nigra and striatum (Yasuhara and others 2005). 77 Strikingly, although VEGF was discovered by its angiogenic activity, this factor has an 78 evolutionarily ancient role as a neurotrophic factor controlling neural cell morphogenesis in 79 invertebrates. Thus, the nematode worm Caenorhabditis elegans lacks a vascular system, but 80 VEGF is present as a trophic molecule necessary for the development of its nervous system. In 81 insects, such as Drosophila melanogaster, which have a rudimentary vasculature, VEGF 82 controls nervous system development. In vertebrates, which have an elaborated network of 83 vessels, VEGF controls both nervous and vascular development, suggesting that vessels co- 84 opted VEGF from nerves to regulate their development (Zacchigna and others 2008). 85 86 VEGF family and receptors 87 The VEGF family members are dimeric glycoproteins of approximately 40 kDa. In mammals, 88 the VEGF family consists of five members with a high degree of homology, which comprises 89 VEGF-A (the founding molecule of this family, also known as VEGF), VEGF-B, VEGF-C, 90 VEGF-D and the placental growth factor (PlGF). In addition, proteins that are structurally 91 related to the VEGFs are present in parapoxvirus (VEGF-E) and in snake venom (VEGF-F) 92 (Olsson and others 2006). VEGF family members have been implicated in angiogenesis, 93 5 lymphangiogenesis, and may play different roles in the nervous system (e.g., neuronal wiring 94 in the CNS, peripheral nerve development and vascularization, neural migration, neurogenesis, 95 neurite outgrowth, neuronal survival, synaptic plasticity, neuroprotection and 96 neuroregeneration) (Lladó and others 2013; Ruiz de Almodovar and others 2009), and has been 97 implicated in the etiology and treatment of various neurological diseases (Lange and others 98 2016). VEGF-B is a weak angiogenic stimulator, and appears to have a relatively restricted 99 angiogenic activity in the ischemic heart (Li and others 2008). VEGF-B also acts as 100 neuroprotective against neurodegeneration and physiological alterations (Dhondt and others 101 2011; Poesen and others 2008) The molecules VEGF-C and VEGF-D act on the lymphatic 102 endothelium promoting the process of lymphangiogenesis (Lohela and others 2009). PlGF 103 stimulates angiogenesis selectively under pathological conditions (Carmeliet and others 2001). 104 The signaling of VEGFs is carried out through binding to three cell surface membrane 105 receptors, which are endowed with tyrosin kinase intracellular activity. Each VEGF factor binds 106 with different affinities and selectivities to these three receptors: VEGFR-1 (Flt-1), VEGFR-2 107 (KDR/Flk-1) and VEGFR-3 (Flt-4). VEGFRs are formed by two monomers with several 108 extracellular immunoglobulin domains each, which mediate ligand binding, followed by a 109 transmembrane chain and an intracellular effector comprising two regions with tyrosine kinase 110 activity. VEGFRs form homodimers, but also heterodimers of unknown function (Ferrara 2004; 111 Ruiz de Almodovar and others 2009). VEGF binds to VEGFR-1 and VEGFR-2, VEGF-B and 112 PlGF binds to VEGFR-1, and VEGF-C and VEGF-D interact with VEGFR-3 and VEGFR-2 113 (Lange and others 2016; Fig. 1). 114 VEGFR-2 is the best characterized signaling receptor, promoting angiogenesis in health 115 and disease; it stimulates endothelial cell proliferation, survival, migration and vascular 116 permeability. VEGFR-2 also signals numerous processes in various neural types (Carmeliet and 117 Ruiz de Almodovar 2013; Ruiz de Almodovar and others 2009). VEGFR-2 has a high tyrosine 118 6 kinase activity (Takahashi and Shibuya 2005). Although VEGFR-1 was discovered before 119 VEGFR-2, its role is still unclear. VEGFR-1 has a weak tyrosine kinase activity but binds 120 VEGF with at least 10-fold higher affinity than VEGFR-2 (Takahashi and Shibuya 2005). 121 Consequently, signaling through VEGFR-2 promotes the phosphorylation of a larger number 122 of intracellular effector proteins due to its strong tyrosine kinase activity, and thus VEGFR-2 is 123 considered the principal mediator of VEGF actions. On the other hand, due to the higher affinity 124 of VEGFR1 for VEGF, it has been proposed that VEGFR-1 could act as a “decoy” receptor 125 that traps VEGF and thus prevents excessive activation of VEGFR-2 (Ferrara and Davis-Smyth 126 1997; Koch and Claesson-Welsh 2012). That is, VEGFR-1 would negatively regulate the 127 activity of VEGFR-2. The third tyrosine kinase VEGFR, VEGFR-3, is present in the endothelial 128 cells of lymphatic vessels, and binds the ligands VEGF-C and VEGF-D (Takahashi and 129 Shibuya 2005). 130 The members of the VEGF family can also bind selectively two co-receptors named 131 neuropilins (NRP): neuropilin-1 (NRP-1) and neuropilin-2 (NRP-2) (Fig. 1). They were initially 132 identified as semaphorin receptor, proteins involved in axonal guidance. NRPs are cell surface 133 glycoproteins whose intracellular domains are short and lack tyrosine kinase activity. 134 Nevertheless, when coexpress with VEFGRs, NRPs enhance the binding to VEGFRs and 135 increase the effectiveness of VEGFR-mediated signaling transduction (Ferrara and others 2003; 136 Takahashi and Shibuya 2005). 137 The specific ligands for VEGFRs and NRPs are illustrated in Fig. 1, and are as follows: 138 VEGF (i.e., VEGF-A) binds to VEGFR-1, VEGFR-2, NRP-1 and NRP-2; VEGF-B binds to 139 VEGFR-1 and NRP-1; PlGF binds to VEGFR-1, NRP-1 and NRP-2; and VEGF-C and VEGF- 140 D interact with VEGFR-3, VEGFR-2, NRP-1 and NRP-2 (Ferrara and others 2003; Lange and 141 others 2016; Takahashi and Shibuya 2005). 142 7 143 Regulation of VEGF by hypoxia 144 Hypoxia is the principal stimulus for VEGF expression. When local oxygen tension decreases, 145 VEGF levels rise promoting angiogenesis, i.e., the formation of new blood vessels from pre- 146 existing ones, which prevents the oxygen deficit in the tissue. Thus, the regulation of VEGF by 147 hypoxia plays an adaptive, homeostatic role in the maintenance of a proper delivery of oxygen 148 and nutrients to areas with low vascular perfusion (Pronto-Laborinho and others 2014). Some 149 studies have shown that the hypoxia-regulated mechanism of the VEGF gene and the 150 erythropoietin gene shares similarities (Pronto-Laborinho and others 2014). 151 The increase in VEGF protein levels under hypoxic conditions is due to control at both 152 the transcriptional and translational level. At the transcriptional level, the gene sequence 153 involved in hypoxia regulation is termed “hypoxia-response element” (HRE) and has been 154 located in the 5´ promoter of the human and murine VEGF gene (Ferrara 2004). Hypoxia- 155 inducible transcription factors (HIF) bind HRE and thereby upregulate VEGF transcription 156 under hypoxic situations. HIF is a heterodimeric protein formed by two constitutively expressed 157 subunits HIF-1α (120 kDa) and HIF-1β (92 kDa) (Oosthuyse and others 2001; Storkebaum and 158 others 2004; Wang and others 1995). 159 Under normoxia (Fig. 2), HIF-1α is hydroxylated at two proline residues by the 160 enzymatic action of proline hydroxylase proteins (PHD), which require molecular oxygen to 161 carry out this action. This step is necessary for the interaction with the von Hippel-Lindau 162 (VHL) protein, a component of the E3 ubiquitin ligase complex, tagging HIF-1α for degradation 163 (Pronto-Laborinho and others 2014; Pugh and Ratcliffe 2003; Salceda and Caro 1997). The 164 proteasome degradation of HIF-1α preclude VEGF transcription in normoxia (Fig. 2). 165 However, in hypoxia, PHDs are inactive due to lack of O2, and consequently HIF-1α are not 166 8 hydroxylated and thus not ubiquitinated for proteasome degradation. Therefore, HIF-1α protein 167 levels increase and HIF-1α translocate into the nucleus and associates with HIF-1β and the 168 coactivators P300/CBP. This complex binds to HRE in the VEGF gene promoter, activating 169 VEGF transcription (Semenza 2001, 2010). In this way, VEGF levels increase in response to 170 hypoxia (Fig. 2). HIF-1α can also increase in response to excitotoxicity (Vazquez-Valls and 171 others 2011) and the levels of VEGF consequently rise under this neurotoxic condition 172 (Castañeda-Cabral and others 2017). 173 VEGF can also be regulated at the translational level. Hypoxia promotes the 174 stabilization of VEGF mRNA through proteins that binds to sequences located in the region 175 UTR (3´untranslated region) of the mRNA such as HuR (Levy and others 1998; Pronto- 176 Laborinho and others 2014; Storkebaum and others 2004). It should be mentioned that VEGF 177 receptors are also increased by hypoxia, as reported in endothelial cells of lung, heart, brain, 178 kidney and liver following systemic hypoxia (Marti and Risau, 1998), and in skeletal muscle in 179 response to chronic ischemia (Milkiewicz and others 2003). The response of receptors to 180 hypoxia enhances VEGF signaling, favoring increased vascular perfusion in ischemic tissues. 181 182 VEGF and ALS: evidences form transgenic animals 183 184 ALS is a devastating neurodegenerative disease characterized by rapidly progressive 185 degeneration of motoneurons in the spinal cord, brainstem, and premotor neurons in the motor 186 cortex, leading to severe weakness and muscle atrophy. The mean survival of ALS patients is 187 3-5 years after symptom onset. Most ALS cases are sporadic, but approximately 10% are 188 familial, one fifth of which are caused by mutations in the gene encoding the antioxidant Cu/Zn 189 superoxide dismutase (SOD1). The pathogenesis of ALS appears to be a complex interplay of 190 9 several processes, such as oxidative stress, excitotoxicity, protein aggregation, mitochondrial 191 dysfunction, deleterious effects derived from glial cells, abnormal axonal transport, deficit in 192 neurotrophic factors, ultimately leading to motoneuron death. However, the pathophysiological 193 precise mechanisms of motoneuron degeneration in ALS are still unknown. The only drug 194 currently approved worldwide in ALS is riluzole, which acts by decreasing glutamate activity 195 in the CNS, although its impact on survival is modest and ALS is still an incurable disease 196 (Bruijn and others 2004; Dhasmana and others 2022; Kaur and others 2016; Robberech and 197 Philips 2013; Valko and Ciesla 2019). Mice and rats, expressing a mutant SOD1 transgene, 198 develop ALS with clinical, anatomical and pathological features that are highly reminiscent of 199 those found in human ALS patients. SOD1 transgenic mice and rats are a classic animal model 200 for the study of ALS and have been widely used in numerous ALS-related investigations 201 (Tovar-y-Romo and others 2009). 202 The link between VEGF and ALS was established in a fortuitous way. Attempts were 203 made to generate knockout mice that failed to express VEGF, or either of its two principal 204 receptors (VEGFR-1 or VEGFR-2), in studies aimed at investigating the relevance of hypoxic 205 regulation of VEGF-dependent angiogenesis. However, these knockout mice were not viable 206 and they died at embryonic stage due to lack of development of the vascular system (Greenberg 207 and Jin 2004). To overcome this obstacle, Oosthuyse and others (2001) generated mutant mice 208 in which the HRE (i.e., hypoxia-response element) sequence in the VEGF gene promoter was 209 deleted (VEGFδ/δ mice). These genetically engineered mice resulted in an unexpected 210 phenotype, unveiling a novel role of VEGF in the survival of adult motoneurons. 211 VEGFδ/δ mice exhibited impaired upregulation of VEGF by hypoxia in neural tissue, so 212 that spinal VEGF levels were reduced by 75%. Strikingly, these VEGFδ/δ mice developed 213 symptoms of motoneuron disease beyond five months of age. They became progressively less 214 mobile and showed signs of severe muscle weakness and limb paresis. Behavioral tests revealed 215 16 activity (Fig. 4). Oosthuyse and others (2001) studied the possible VEGF neuroprotective effect 359 on cultured primary motoneurons of VEGF+/+ embryos, which lack vascular irrigation. The 360 addition of VEGF to the culture increased survival of motoneurons by 20% in baseline 361 conditions and by 50% after serum deprivation. VEGF also protected the mouse motoneuron 362 NSC-34 cells against apoptosis induced by tumor necrosis factor-α, hypoxia, oxidative stress 363 (H2O2), or serum deprivation. The survival effect of VEGF required the activation of both 364 VEGFR-2 and NRP-1, since the percentage of survival partially dropped by adding either anti- 365 NRP-1 antibody or anti-VEGFR-2 antibody to the culture medium. A combination of anti- 366 VEGFR-2 and anti-NRP-1 antibodies was required to completely neutralize the VEGF- 367 dependent motoneuron survival activity (Oosthuyse and others 2001). Altogether, it is possible 368 that motoneurons in VEGFδ/δ mice degenerate because of insufficient VEGF-dependent 369 neuroprotection. Since VEGF is strongly and rapidly induced by hypoxia and other insults (e.g., 370 acidosis), VEGF may represent a neuroprotective factor for motoneurons exposed to stress 371 conditions. Therefore, motoneuronal VEGF-mediated neuroprotection may be relevant not only 372 during hypoxia but also under other conditions that can represent a damage to motoneurons. 373 For instance, excitotoxicity due to an excess of glutamate has been reported as one of the 374 possible causes of ALS (Dhasmana and others 2022; Valko and Ciesla 2019) and VEGF 375 administered in vivo protects motoneurons against excitotoxicity (Tovar-y-Romo and others 376 2007; Tovar-y-Romo and Tapia 2010). 377 Proof of direct effect of VEGF in neuroprotection is the lack of angiogenesis caused by 378 VEGF delivery (Azzouz and others 2004). Moreover, the intracerebrospinal administration of 379 VEGF in SOD1 rats is also beneficial without inducing changes to blood vessel density or 380 permeability, suggestive also of a direct neurotrophic effect of VEGF (Storkebaum and others 381 2005). In another work, VEGF was administered using osmotic minipumps in a rat model of 382 spinal cord excitotoxicity, and the factor was demonstrated to rescue motoneurons from 383 17 excitotoxic cell death with no changes in the vascular architecture of the spinal cord (Tovar-y- 384 Romo and others 2007). Interestingly, a dose-dependent study of the effects of VEGF 385 administration on ischemic rat brains demonstrated that doses not inducing angiogenesis were 386 the only neuroprotective. On the contrary, the high dose of VEGF induced angiogenesis and 387 showed absence of neuroprotection perhaps due to the deleterious effects of brain edema and 388 inflammation (Manoonkitiwongsa and others 2004). 389 390 Neurotrophic action of VEGF in extraocular motoneurons 391 Extraocular motoneurons are involved in the generation of eye movements and lay in three 392 distinct brainstem nuclei: the oculomotor, the trochlear and the abducens nuclei. The 393 physiological actions of VEGF have been studied in detail in the abducens motoneurons of the 394 cat following lesion and exogenous VEGF administration, as well as after anti-VEGF antibody 395 treatment in undamaged motoneurons. The abducens nucleus offers several advantages for the 396 study of lesion-induced plasticity: the discharge pattern of abducens motoneurons is well 397 characterized, and both their afferents and the signals they carry have been described in detail 398 (Büttner-Ennever 2006; Davis-López de Carrizosa and others 2011; Delgado-García and others 399 1986; Escudero and Delgado-García 1988; Escudero and others 1992; Horn and Straka 2021). 400 In addition, the firing activity of abducens motoneurons can be recorded using the alert chronic 401 animal preparation, which allows a direct correlation of motoneuronal firing with eye 402 movements under different experimental conditions (e.g., lesion, VEGF or anti-VEGF antibody 403 treatment). 404 Extraocular motoneurons are less vulnerable than other cranial or spinal motoneurons 405 to degeneration in ALS (Haenggeli and Kato 2002; Reiner and others 1995). However, it is 406 important to note that they also exhibit some signs of degeneration but at very late stages of the 407 18 disease (Kimura and others 2014; Sharma and others 2011; Takahashi and others 1993; Tjust 408 and others 2017). 409 410 Peripheral administration of VEGF to axotomized abducens motoneurons 411 Extraocular eye muscles contain VEGF (Calvo and others 2018; Silva-Hucha and others 2020) 412 and thus the axotomy of abducens motoneurons leaves these cells deprived of their retrograde 413 source of VEGF. The recording of the discharge of these motoneurons in alert cats has revealed 414 that (control) abducens motoneurons show a typical tonic-phasic firing pattern that correlates 415 with eye position and eye velocity, respectively, during the different types of eye movements 416 (Delgado-García and others 1986). When these motoneurons are axotomized, they exhibit an 417 overall decrease in firing rate and a significant reduction in neuronal eye position and velocity 418 sensitivities due, at least in part, to the loss of synaptic inputs (a process known as “synaptic 419 stripping”, reviewed in Alvarez and others 2020), as first described by Delgado-García and 420 others (1988) and later confirmed by other authors (Calvo and others, 2018, 2020; Davis-López 421 de Carrizosa and others, 2009, 2010). 422 The chronic administration of VEGF to the proximal stump of the transected VIth 423 (abducens) nerve prevents and recovers the synaptic loss and the firing alterations induced by 424 axotomy. These findings were obtained after using two different protocols of administration. In 425 the immediate administration protocol, VEGF first dose was applied just immediately after 426 lesion, preventing the appearance of axotomy-induced alterations. In the delayed administration 427 protocol (VEGF first dose 20 days after lesion), VEGF recovered the synaptic and physiological 428 changes already present in severed motoneurons (Calvo and others 2018). It was also 429 demonstrated that the tyrosine kinase receptors of VEGF, i.e., VEGFR-1 and VEGFR-2, 430 mediate different roles in the maintenance of synaptic afferents to abducens motoneurons. It 431 19 should be mentioned that, in contrast to VEGF, the administration to axotomized abducens 432 motoneurons of the neurotrophins BDNF, NT-3 or NGF induces only partial recovery of 433 synaptic inputs and discharge activity (Davis-López and others 2009, 2010). 434 Interestingly, the administration of VEGF-B, using the immediate administration 435 protocol, also prevents the modifications induced by injury in abducens motoneurons (Calvo 436 and others 2018). Previous results have also reported a neuroprotective role for VEGF-B in 437 spinal motoneurons. Thus, VEGF-B protects cultured primary motoneurons against 438 degeneration. Moreover, mice lacking VEGF-B also develop a more severe form of 439 motoneuron degeneration when intercrossed with mutant SOD1 mice. When delivered 440 intracerebroventricularly, VEGF-B prolongs the survival of mutant SOD1 rats (Poesen and 441 others 2008). Altogether, these results may have an important therapeutic value, because 442 VEGF-B is virtually non-angiogenic and, therefore, its administration in the case of 443 motoneuronal disorders could produce beneficial effects without the side-effects of a high dose 444 of VEGF, such as tissue edema and inflammation due to excessive angiogenesis and vascular 445 permeability (Calvo and others 2018; Manoonkitiwongsa and others 2004; Poesen and others 446 2008). 447 Several types of brainstem and spinal motoneurons downregulate the expression of 448 choline acetyltransferase when axotomized (reviewed by Navarro and others 2007; see also 449 Morcuende and others 2005, 2013). Thus, peripheral administration of VEGF to axotomized 450 extraocular motoneurons in adult rats also prevents the loss of their cholinergic phenotype. 451 (Acosta and others 2018). The immunostaining, enzymatic activity, and mRNA levels of 452 choline acetyltransferase are also reduced in spinal motoneurons of ALS patients (Nagata and 453 others 1982; Oda and others 1995; Virgo and others 1992). All these findings suggest that 454 VEGF administered to diseased motoneurons not only rescues them from cell death (as 455 20 described above) but also maintains their neurotransmissive phenotype, synaptic inputs, and 456 discharge pattern (Calvo and others 2018; Acosta and others 2018). 457 458 Intraventricular delivery of a single dose of VEGF to axotomized abducens motoneurons 459 The administration of VEGF into the IVth ventricle, located dorsal to the abducens nucleus, 460 just after the section of the VIth nerve, also prevents the axotomy-induced alterations in the 461 discharge pattern and synaptic inputs of damaged abducens motoneurons, as demonstrated by 462 single-unit extracellular recordings and immunocytochemistry (Calvo and others 2020). 463 In addition, the study of the vasculature after VEGF intraventricular delivery revealed 464 absence of angiogenesis in the abducens nucleus as well as in other brainstem nuclei (Calvo 465 and others 2020). Absence of angiogenesis and vascular leakage has also been shown after the 466 peripheral administration of VEGF in the three extraoculomotor nuclei after axotomizing all 467 extraocular motoneurons in adult rats (Acosta and others 2018). These findings indicate that 468 the beneficial effects obtained with VEGF result likely from a direct action of the factor on the 469 motoneurons themselves rather than by an indirect action on the vascular system (Acosta and 470 others 2018; Calvo and others 2020). A recent study using an anti-VEGF-antibody, to reduce 471 the retrograde delivery and transport of this factor, has shown that control, treated extraocular 472 motoneurons turn into a lesioned state, both in terms of firing and synaptic coverage (Calvo and 473 others 2022) 474 A schematic drawing is illustrated in Fig. 5 to compare the changes in synaptic inputs 475 and firing of abducens motoneurons in the three conditions, i.e., control (Fig. 5A), axotomy 476 (Fig. 5B), and anti-VEGF antibody treatment (Fig. 5C). The data obtained in extraocular 477 motoneurons demonstrating the neurotrophic effects of VEGF (Acosta and others 2018; Calvo 478 and others 2018, 2020, 2022) are consistent with all the evidence summarized in the present 479 21 review supporting the neuroprotective effects of VEGF studied mainly in spinal motoneurons 480 from animal models of motoneuron disease. 481 482 Conclusions 483 Vascular endothelial growth factor (VEGF) was discovered by its angiogenic activity, but 484 strikingly it appears first in evolution as a neurotrophic factor, necessary for the appropriate 485 development of the nervous system in invertebrates lacking a vascular system or having a 486 rudimentary vasculature. Neuroscience interest in VEGF has been growing remarkably in 487 recent years due to numerous evidence showing that VEGF is neuroprotective. Mutant mice 488 characterized by low levels of VEGF (VEGFδ/δ) suffer severe adult-onset muscle weakness and 489 neurodegeneration, symptoms that resemble ALS. Experiments of crossbreeding between 490 transgenic mice have reinforced the role of VEGF as an essential factor for motoneuron 491 survival. Thus, when SOD1 mice (a classical animal model of ALS) are crossed with mice 492 overexpressing VEGF (VEGF+/+), the double transgenic animals show reduced motoneuron 493 degeneration and a longer life expectancy. The opposite occurs when VEGFδ/δ and SOD1 mice 494 are crossed, as the double mutants show earlier and more severe motoneuron degeneration and 495 muscle weakness. 496 Recent results in extraocular motoneurons also point to a role of VEGF in 497 neuroprotection since the administration to the proximal stump of the sectioned nerve or 498 intraventricularly prevents and recovers all structural and functional changes induced by lesion, 499 returning axotomized motoneurons into a normal operation mode. A striking result has recently 500 been shown in this model consisting in the administration of VEGF neutralizing antibody to 501 intact, undamaged extraocular motoneurons. Results have shown that antibody treated 502 22 motoneurons resemble injured motoneurons in that they discharge abnormally at low 503 frequencies and also experience synaptic loss. 504 All the findings summarized in the present review indicate that VEGF is an essential 505 neurotrophic factor for the survival and physiology of motoneurons and suggest that this factor 506 might have a high potential therapeutic value in the treatment of motor neuron disorders. 507 508 Declaration of Conflicting Interests 509 The authors declare no potential conflicts of interest with respect to the research, authorship, 510 and/or publication of this article. 511 512 Funding 513 The authors disclosed receipt of the following financial support for the research, authorship, 514 and/or publication of this article. This publication is part of the I+D+i project P20_00529 515 Consejería de Transformación Económica Industria y Conocimiento, Junta de Andalucía- 516 FEDER. Research materials were also supported by project PGC2018-094654-B-100 funded 517 by MCIN/AEI/10.13039/501100011033, FEDER “A way of making Europe”. P.M.C. was a 518 scholar of Ministerio de Educación y Ciencia (BES-2016-077912) in Spain and now a 519 “Margarita Salas” postdoctoral fellow. R.G.H. is a postdoctoral fellow from PAIDI-2019, 520 "Talento Doctores" Junta de Andalucía in Spain. Confocal images were performed in the 521 Central Research Services of Universidad de Sevilla. 522 523 ORCID iDs 524 Angel M. Pastor: https://orcid.org/0000-0002-6213-7454 525 23 Rosa R de la Cruz: https://orcid.org/0000-0001-5301-5108 526 Paula Martín Calvo https://orcid.org/0000-0001-9817-8728 527 Rosendo García Hernández https://orcid.org/0000-0001-7098-8630 528 529 530 24 References 531 - Acosta L, Morcuende S, Silva-Hucha S, Pastor AM, de la Cruz RR. 2018. 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Biochem Biophys 859 Res Commun 363(4):989-93. 860 861 39 Figure legends 862 Figure 1. VEGF family and receptors. The figure is a representation of the members of 863 the vascular endothelial growth factor (VEGF) family and the specific binding of each member 864 to the different VEGF receptors. In addition to VEGF-A (also known as VEGF), the other 865 isoforms of the VEGF family in mammals are VEGF-B, VEGF-C, VEGF-D, and placental 866 growth factor (PlGF). VEGF receptors include the tyrosine kinases VEGFR-1, VEGFR-2 and 867 VEGFR-3 and the co-receptors neuropilin-1 (NRP-1) and NRP-2. 868 Figure 2. Regulation of VEGF by hypoxia. Under normoxia, HIF-1α is hydroxylated 869 by proline (P) hydroxylase domain-containing proteins (PHD), which requires O2 for their 870 enzymatic activity. Hydroxylation of HIF-1α allows its interaction with the von Hippel-Lindau 871 (VHL) protein, a component of the E3 ubiquitin ligase complex. This interaction results in 872 ubiquitination (Ub) and degradation by the proteasome pathway of HIF-1α and, consequently, 873 prevents VEGF transcription in normoxia. Under hypoxic conditions, PHDs are inactive due to 874 lack of O2. Therefore, HIF-1α cannot be hydroxylated and thus not ubiquitinated for proteasome 875 degradation. This allows HIF-1α to enter the nucleus, dimerizes with HIF-1β and, in association 876 with the p300/CBP coactivators, this complex binds to HRE in the VEGF gene promoter 877 activating VEGF transcription. 878 Figure 3. Motoneuronal degeneration in VEGFδ/δ mice. (A, B) Images of sciatic nerve 879 semi-thin sections from wild type (A) and VEGFδ/δ mice (B). In contrast to the wild type (A), 880 the sciatic nerve of the VEGFδ/δ mice shows signs of axonal loss and Wallerian degeneration 881 (B). (C, D) Transmission electron microscopy images of spinal cord motoneurons from wild 882 type (C) and VEGFδ/δ mice (D). The motoneuron in C shows typical ultrastructural 883 characteristics of this cell type such as a large round nucleus, with homogeneous chromatic and 884 a conspicuous nucleolus, and a cytoplasm containing a prominent rough endoplasmic reticulum 885 40 and mitochondria. The ultrastructure of the VEGFδ/δ motoneuron (D) shows signs of clear 886 degeneration. Note cell shrinkage, an irregular nucleus with aggregates of peripheral chromatin, 887 and a reduced cytoplasm with vacuolized mitochondria and endoplasmic reticulum. Reprinted 888 from Carmeliet P, Storkebaum E. 2002. Vascular and neuronal effects of VEGF in the nervous 889 system: implications for neurological disorders. Semin Cell Dev Biol 13(1):39-53. Copyright 890 (2002), with permission from Elsevier. 891 Figure 4. Proposed mechanisms of action of VEGF in ALS motoneurons. (A) In healthy 892 conditions, VEGF is adequately synthesized and acts on motoneurons by two likely 893 mechanisms. First, due to its effects on the vasculature, motoneurons are provided by 894 appropriate levels of oxygen and nutrients. Second, VEGF acts also as a neurotrophic factor for 895 motoneurons. (B) In ALS, the synthesis of VEGF is reduced. The effects of low levels of VEGF 896 on blood vessels produces a deficit in oxygen and nutrient supply to motoneurons. In addition, 897 there is insufficient VEGF neurotrophic support. These two processes leads to motoneuron 898 degeneration. (C) The potential therapeutic value of VEGF can be used as a strategy to promote 899 motoneuron recovery. VEGF delivery (through different routes and methods) reestablishes 900 normal levels of VEGF that would act on diseased motoneurons enhancing the supply of oxygen 901 and nutrients via the restored vascular function and by providing the required neurotrophic 902 support and thus preventing motoneurons from degeneration. 903 Figure 5. Schematic representation summarizing the effects of the administration of 904 anti-VEGF antibody to abducens motoneurons on their discharge activity and synaptic inputs. 905 (A) Control motoneurons receive abundant synaptic boutons contacting their cell body, of both 906 excitatory (in red) and inhibitory (in blue) nature. They exhibit a typical tonic-phasic discharge 907 pattern of action potentials (AP) in correlation with eye movements (EP, eye position). (B) 908 After axotomy, the motoneuron experiences a significant loss of afferent synaptic boutons 909 (excitatory and inhibitory), a phenomenon known as synaptic stripping. In congruence, the 910 41 discharge pattern of axotomized motoneurons is markedly reduced. (C) Uninjured motoneurons 911 treated with the neutralizing antibody against VEGF (VEGF ab) applied in the muscle lack their 912 retrograde source of VEGF. Treated motoneurons also show synaptic stripping and a reduced 913 firing pattern in correlation with eye movements, resembling the axotomy situation. 914 915