*VEGF and ALS*

ALS is characterized by being an adult neurodegenerative disease that causes progressive degeneration of motoneurons in the lower spinal cord, brainstem, and cortex. Consequently, it triggers astrogliosis, progressive atrophy of the skeletal musculature, and a reduction in voluntary movements, including those of the extremities and respiratory movements [72,73].

The disease affects five out of every 100,000 people worldwide, is progressive, and is generally fatal within 5 years after the onset of symptoms. 95% of cases are sporadic, and only 5% of patients have a family history, with a fifth of these caused by mutations in the SOD1 gene, located on chromosome 21 [15]. There are currently around 180 known mutations in SOD1 which are related to the pathogenesis of the disease [74], with the SOD1G93A mutant mouse model being the most widely used, studied, and well-characterized showing symptoms similar to the disease [75], and to which we refer in this review as the SOD1 model.

It has been shown that the expression of VEGF and its Flk-1 receptor undergo significant downregulation in the motoneurons of the spinal cord of SOD1 mice [76]. These findings correlate with other studies where SOD1 mice were crossed with transgenic mice that overexpressed Flk-1, resulting in a delayed onset of motor impairment and degeneration of motoneurons, and prolonged survival [67]. Moreover, as indicated before, the coincidence of both SOD1 mutation and VEGFδ/<sup>δ</sup> alteration produced an earlier and more severe motoneuronal degeneration [17].

Although the predominant hypothesis is that ALS is a disease of neural origin, some studies indicate that the disease involves a distal axonopathy and denervation of the neuromuscular junctions (NMJs) in the muscles of the extremities in the presymptomatic stage, that is, much before the loss of the motoneuron at the level of the spinal cord [77–79]. Several studies have shown that both the anterograde transport of VEGF and the intracerebroventricular infusion of this factor help protect and preserve the NMJs in a rat model SOD1 [67,80,81]. Likewise, experiments such as gene therapy of VEGF mediated by lentiviral vectors, or transplantation of stem cells that overexpress VEGF, have managed to significantly slow the progression of neurodegeneration, improving motor function and significantly prolonging the survival of motoneurons in the brainstem and the cervical and lumbar spinal cord [82,83]. All these findings give VEGF and its Flk-1 receptor an essential role in the treatment of motoneuronal diseases.

#### **4. Neuroprotective Effect of VEGF**

Among the possible pathogenic mechanisms linked to the degenerative neuronal process are oxidative stress, glutamate excitotoxicity, inflammation, mitochondrial and neurofilament dysfunction, protein aggregation, axonal transport abnormalities, and, ultimately, the activation of pathways that trigger apoptosis [84]. Numerous studies show the decisive neuroprotective role that VEGF plays in the CNS. These include experiments where an intracerebroventricular administration of VEGF stimulates neurogenesis in the adult hippocampus, promotes neurites growth, or provides greater protection to motoneurons [85,86]. Furthermore, it has also been shown that the retrograde transport of VEGF, after its intramuscular administration with lentiviral vectors, favors the survival of motoneurons [17,82]. At the same time, the supply of VEGF at the site of a spinal cord injury decreases lesion size, apoptosis levels, and retards neurodegeneration [36,87].

Two main hypotheses have been postulated to explain all of these VEGF effects. The first affirms that this factor promotes the vascular niche necessary for motoneurons to survive, and the second, that the binding of VEGF to Flk-1 promotes cell survival by blocking the process of apoptosis.

Another protective effect of VEGF is also due to the induction of the expression of the GluA2 subunit in AMPA receptors [64], which leads to a reduction in the entry of Ca2+ in neurons, which is a relevant mechanism involved in motoneuronal degeneration. All this makes VEGF and its Flk-1 receptor attractive candidates for evaluating its therapeutic potential in neurodegenerative disorders.

### *4.1. Anti-Apoptotic Effects of VEGF*

One of the mechanisms by which the binding of the VEGF-A isoform to the Flk-1 receptor improves and promotes cell survival is by blocking the process of apoptosis through the expression of anti-apoptotic proteins, such as the members of the Bcl-2 family and the generation of neuronal progenitors in the nervous system [16,88,89].

It is well known that the binding of VEGF with Flk-1 directly activates the PI3-K/Akt intracellular signaling pathway. This activation consequently causes an inhibition of the phosphorylation of p38MAPK, which is an essential factor in the cell death pathway [38,64], and an increase of the expression of the anti-apoptotic proteins Bcl-2 and A1, conceding greater protection to motoneurons against excitotoxicity. That effect has been described in diverse models of neurodegeneration, including ALS [38,70].

#### *4.2. Role of Excitotoxicity in Neurodegeneration and VEGF Protection*

Glutamate is the major excitatory neurotransmitter in the mammalian CNS and is involved in many aspects of normal brain function. However, an excess in the synaptic transmission of glutamate leads to an over-activation of the different types of receptors for this amino acid, which causes a massive entry of Ca2+ in the neurons and triggers the uncontrolled activation of damaging processes that, eventually, produce the destruction of the membrane, neurodegeneration and cell death [90]. Indeed, glutamate-mediated excitotoxicity is considered the primary mechanism leading to a degeneration of motoneurons in various neurodegenerative disorders, including Parkinson's disease, Alzheimer's disease, and ALS [91–93].

Two broad categories of glutamate receptors are known: (i) ionotropic receptors, which are ligand-activated ion channels, and comprise *N*-methyl-D-aspartate (NMDA), αamino-3-hydroxy-5-isoxazole propionate (AMPA), and kainate receptors; (ii) metabotropic receptors, which are associated to G proteins and coupled to the production of intracellular secondary messengers [94,95]. AMPA ionotropic receptors are heteromeric complexes composed of four subunits, GluA1-GluA4 (formerly GluR1-GluR4), with different combinations. The presence of the GluA2 subunit is known to decrease the permeability of these receptors to Ca2+ [95]. Therefore, the hyperactivation of AMPA receptors that lack this GluA2 subunit involves a massive entry of Ca2+ into the cell, which produces the activation of phospholipases, proteases, and endonucleases, inducing apoptotic or necrotic cell death, the production of reactive oxygen species (ROS) and a deficiency of mitochondrial function, with the consequent interruption of energy metabolism [90,96]. All of this generates neuronal degeneration, suggesting that the absence of the GluA2 subunit at AMPA receptors is a critical factor for the selective vulnerability of neurons to excitotoxicity.

Motoneurons are particularly susceptible to excitotoxicity due to their low expression of the GluA2 subunit [97,98]. Deficiency of this subunit exacerbates motoneuron degeneration in SOD1 mouse models, whose mutation is implicated in the accumulation of oxidative damage [99]. On top of that, many ALS patients have shown elevated glutamate levels in the cerebrospinal fluid, supporting the excitotoxic hypothesis of degeneration of the motoneurons [100]. Furthermore, glial cells that overexpress the SOD1 mutation are known to adversely affect the viability of spinal motoneurons by producing elevated levels of extracellular glutamate, leading to increased motoneuron degeneration and progressive paralysis [101].

Interestingly, the administration of exogenous recombinant VEGF is capable of preventing both excitotoxic neuronal death, induced by overactivation of AMPA receptors, and the consequent motor disorders [36]. That reduction in glutamate toxicity is mediated by the action of VEGF on PI3-K/Akt and MEK/ERK pathways [102]. Moreover, this neurotrophic factor has been shown, both in vitro and in vivo, to induce an increase in the expression of the GluA2 subunit in AMPA receptors [103], reducing the permeability to Ca2+ and, therefore, granting protection to motoneurons against excitotoxicity. Those experiments highlight the relevant role that VEGF plays in reducing excitotoxicity, making this factor an essential piece for the survival of motoneurons.

#### **5. Selective Vulnerability of Motoneurons to Neurodegeneration**

As stated above, motoneurons are particularly sensitive to excitotoxic neurodegeneration due, mainly, to their reduced capacity to blockade Ca2+ influx. However, a peculiarity of neurodegenerative processes is that specific neuronal populations show superior resistance to degeneration compared to other motor groups. In diseases such as ALS, motoneurons of some motor nuclei offer selective resistance and persist until the last stages of the disease, compared to other motoneurons that degenerate earlier [104]. Motoneurons of the oculomotor system are among those resistant populations [104,105], while motoneurons of the facial, hypoglossal, or trigeminal motor systems are vulnerable populations in the brainstem [106].

Likewise, between the possible differences that mark the selective vulnerability of the different motoneuronal groups is the differential expression of specific proteins. Several studies have shown that ocular motoneurons have a distinct transcriptional profile from other motoneurons in the expression of proteins related to synaptic transmission, including several glutamate and GABA receptor subunits, Ca2+-binding, ubiquitin-dependent proteolysis, mitochondrial function, or immune system processes [107–109].

Moreover, a greater expression of laminins, synaptophysin, and p75 receptor has been detected in muscle fibers of resistant motoneurons [110,111]. Thus, the differential expression of specific proteins and neurotrophic factors on the target muscles seems to influence the selective resistance of the different motor units against neurodegeneration and the deterioration of the NMJs, granting a vital role to the retrograde trophic contribution in motoneuronal survival.

#### **6. Properties of Ocular Motoneurons**

The ocular motoneurons present a series of morphological and functional characteristics that differentiate them from the rest of the motoneuronal populations. Several hypotheses have been proposed to explain the greater resistance of these cell populations to neurodegeneration.

Motoneurons of the ocular system show an extensive buffering capability of intracellular Ca2+ due to a greater expression of cytosolic Ca2+ binding proteins [112–114]. Overexpression of Ca2+ binding cytosolic proteins, such as calbindin D-28K (CaBP), calretinin (CR), and parvalbumin (PV), seem to give high protection to motoneurons [115–118]. Likewise, it has been observed that 85–100% of the motoneurons of the primate ocular motor nuclei contain PV, while only 20–30% of the neurons of the trigeminal, facial, and hypoglossal nuclei present it [109]. This PV distribution pattern coincides with the selective vulnerability between the brainstem motor nuclei [104]. Furthermore, additional experiments in SOD1 mice revealed that PV levels were significantly higher in ocular motoneurons compared to hypoglossal motoneurons [108].

Additionally, other studies in ALS models have related a low expression of the neuropeptide calcitonin gene-related peptide (CGRP), with higher resistance of motoneurons. Thus, CGRP could be a factor that promotes neuronal degeneration. Accordingly, motoneurons of the rat oculomotor system show lower expression of CGRP compared to other vulnerable motoneurons, such as facial or spinal ones, which would support these results [119,120]. However, these results have only been demonstrated in rats since a constitutive expression of CGRP has been observed in cats [121].

Notably, the extraocular motoneurons and the EOMs also express a higher proportion of the insulin-like growth factor 2 (IGF-2), which acts as a survival factor for motoneurons, and of its receptor IGF-1R, which mediates its survival effect [122,123]. Moreover, IGF-2 delivery to muscles preserved motoneurons and extended life-span in SOD mice [122]. It has also been shown that receptor α1 of the inhibitory neurotransmitter GABA-A (Gabra1) is preferably present in resistant motoneurons, such as ocular motoneurons, of symptomatic SOD1 mice and patients with end-stage ALS [107,108]. In contrast, vulnerable motoneurons show higher levels of GABA-A receptors α2 (Gabra2), dynein, and peripherin (intermediate neurofilament), which are involved in excitability and retrograde transport, which put these motoneurons at a higher risk [108]. Indeed, dysregulation in the dynein-dynactin or peripherin complex is well known to cause degeneration of the spinal motor neuron in mice due to faulty axonal transport [124,125], which is corroborated by the low levels of dynactin shown by spinal motoneurons in ALS patients [126]. Thus, the lower expression of dynein and peripherin is correlated with a lower vulnerability during neurodegenerative processes since retrograde transport is not affected [127]. Therefore, ocular motoneurons could continue receiving a correct trophic contribution from their target muscles, favoring the maintenance of their NMJs.

Motoneurons present heterogenic neurotrophic dependence [128–130]. It is known that these nerve cells receive NGF, BDNF, and NT-3 from the muscle [131] and that the need for neurotrophic contribution, as well as the expression of the different RTKs, both in a control situation and after inducing a lesion, vary among the diverse populations of motoneurons [120,132–135]. Indeed, the adult rat spinal and cranial motoneurons are known to express the TrkB and TrkC receptors but lack the TrkA receptor [132,136–139]. However, another peculiarity of ocular motoneurons is that they express TrkA both in control and after axotomy in the adult [120,140]. This gives them greater efficiency in their response to NGF [120,141], which acts as a potent survival factor for axotomized neonatal motoneurons [142] and plays an essential synaptotrophic and functional role in axotomized motoneurons of the abducens nuclei [140].

Remarkably, recently it has been shown that motoneurons of the oculomotor nuclei present higher expression of VEGF and Flk-1 in the motoneuronal soma compared to other more vulnerable groups of motoneurons, such as the facial and hypoglossal [143]. Previous studies have also shown weak immunoreactivity for VEGF in hypoglossal and facial motoneurons in control rats [144]. The neuroprotective role of VEGF has been extensively exposed above and, therefore, could also contribute to the extended survival of ocular motoneurons in neurodegenerative diseases.
