*6.1. VEGF and FLK-1 Expression in the Oculomotor System*

A high level of VEGF expression has been broadly related to neuronal survival. The expression of VEGF and Flk-1 is high during the embryonic stages but decreases during the adult state, being restricted to some areas of the adult CNS [145]. A higher basal level of VEGF and its receptor Flk-1 has been detected in ocular motoneurons compared to other brainstem motoneurons that are more vulnerable to neurodegeneration [143]. Thus, these oculomotor neurons could form one of these discrete CNS regions that retain the ability to express VEGF and Flk-1 after development.

Likewise, VEGF decreases the levels of pro-apoptotic proteins caspase-3, caspase-9, and Bax, and induces an increased expression of the anti-apoptotic protein Bcl-2 [146]. Therefore, the higher expression of VEGF observed in oculomotor neurons could yield a greater expression of anti-apoptotic proteins, which may be one of the reasons why these neurons show resistance against neurodegeneration. Furthermore, it has been observed that an increased expression of VEGF leads to a greater expression of its Flk-1 receptor [147], which correlates with the fact that a higher expression of Flk-1 was observed in those motoneurons that in turn expressed more VEGF [143].

Motoneurons are known to be especially susceptible to changes in Flk-1 expression, with a linear relationship between a lower expression of this RTK and a more significant loss of motoneurons. This occurs as a consequence of the blockade of the neuroprotective effect of VEGF, preventing activation of the PI3-K/Akt pathway, phospholipase C, and the p38MAPK protein [38]. These claims are supported by other studies where it was shown that the degeneration of spinal motoneurons could be delayed in transgenic SOD1 mice that overexpressed the Flk-1 receptor, thanks to the survival signals generated after VEGF binding [67]. All these findings give the Flk-1 receptor a key role in selective resistance that specific populations of motoneurons show against excitotoxic processes and neurodegeneration.

#### *6.2. VEGF Sources to Ocular Motoneurons*

The high level of VEGF found in the soma of extraocular motoneurons compared to the observed in other brainstem motoneurons [143] could be one more of the reasons for the lower vulnerability of this population to neurodegeneration. But, which is the origin of that higher VEGF content? Several possibilities could be considered: (i) VEGF could be synthesized by the motoneurons themselves and act as an autocrine source for extraocular motoneurons; (ii) it could reach motoneurons from surrounding cells, such as glial cells and endothelial cells on the blood vessels; (iii) VEGF could also come from the target muscles via retrograde to the innervating motoneurons (Figure 2).

**Figure 2.** In this scheme, the different pathways of VEGF supply for motoneurons are illustrated. (i) autocrine: selfproduction of VEGF by the motoneurons themselves; (ii) paracrine: VEGF arriving at the motoneurons from the surrounding cells and blood vessels; and (iii) retrograde: VEGF can also reach the soma of motoneurons from their target muscles.

#### 6.2.1. Via Autocrine

Motoneurons are known to synthesize trophic factors, including neurotrophins such as BDNF, NGF, and NT-3 [148,149]. It is well-known that they also express their receptors, allowing these motoneurons to receive and use the trophic factors as an autocrine source [141,149]. Their production has been shown to vary in response to diverse insults [120,150].

As aforementioned, ocular motoneurons can synthesize VEGF both in control situation and after injury [151], and they express VEGF receptors on their surface [143,151]. Therefore, it is possible that those motoneurons are also acting as an autocrine source of VEGF. Thus, the increased VEGF highlights the autocrine functions of the VEGF, as previously described in the CNS [22,152], this pathway being one of the essential vias of VEGF supply to ocular motoneurons [151].

### 6.2.2. Via Paracrine

The high level of VEGF located in extraocular motoneurons could also indicate that VEGF is acting as a paracrine factor for the adjacent neurons. The fact that the presence of the Flk-1 receptor is increased in this pool of motoneurons allows them to receive higher amounts of the trophic factor from the neighboring cells. Thus, the upregulation of Flk-1 in the ocular motoneurons highlights the paracrine functions of VEGF [151,152].

Astrocytes are involved in almost all physiological processes that ensure the wellbeing of neurons [153,154]. Likewise, astrocytes also play a role in neurodegenerative processes since the selective deterioration of the glutamate transporter EAAT2 causes the extracellular accumulation of excitotoxic levels of this amino acid and an increase in the entry of Ca2+ in neurons [155]. ROS are believed to induce this oxidative disruption of glutamate transport and promote the spread of this damage, affecting motoneurons. Consequently, glutamate levels increase further, inducing more ROS in motoneurons

and triggering a progressive cascade of selective motoneuronal injury, with consequent astrocytic and microglial activation [156]. On the other hand, the cells of the microglia are the specialized macrophages of the CNS [157]. They are an essential component of the inflammatory response to lesions and pathogens [158]. After an injury to the CNS they are the first glial cells to respond, producing pro-inflammatory mediators [159] and promoting the reaction of neurotoxic astrocytes [160].

However, the expression of VEGF driven by the glial cells surrounding brainstem motoneurons is low under control circumstances [151]. A low expression of both mRNA and VEGF protein in glial cells in a control situation was also previously described [161,162], ruling out the possible role of these neural cells as a paracrine source of VEGF to motoneurons at basal conditions. Therefore, astrocytes and microglia do not seem to be contributing to the differential expression of VEGF detected between oculomotor, facial, and hypoglossal motoneurons in a control situation. Nevertheless, glial cells have been reported to modify their VEGF expression under adverse conditions [25,163].

It is well-known that VEGF also acts as a growth factor for vascular endothelial cells forming the blood vessels [2], promoting vascular proliferation and permeability and therefore providing oxygen and nutrients to neurons, which contribute to their wellness. Administration of exogenous VEGF in the brainstem is not accompanied by either angiogenesis or a significantly increased vascular permeability around treated motoneurons [164]. Therefore, it could be assumed that the action of this factor on motoneuron survival was likely due to a direct effect on the motoneurons instead of an indirect effect due to increased blood perfusion. Besides, no differences were observed in the vascularization of these motor nuclei, neither in control nor after an injury [151,164].

Therefore, paracrine actions of VEGF do not seem to be crucial for the differences observed in resistance to degeneration between diverse pools of brainstem motoneurons.

#### 6.2.3. Retrograde Via

Three pairs of extraocular muscles (EOMs) are inserted around the eye, functioning as antagonistic to each other. These are: (i) the medial rectus and lateral rectus muscles, producing eye movements in the horizontal plane; (ii) the superior and inferior rectus muscles, in charge of vertical movements; and (iii) the superior and inferior oblique muscles, which mediate oblique movements [165].

EOMs are anatomically and functionally quite different from other muscles (reviewed in [166]). Most skeletal muscles exclusively have single innervation fibers (SIF), with a single axon forming part of the NMJs and constituting the motor unit, in which a motoneuron innervates 300–2000 muscle fibers (ratio 1: 300–2000). Furthermore, these SIF fibers have a high content of mitochondria and oxidative enzymes, which results in faster contractions. On the other hand, EOMs present a high percentage (20%) of fibers with multiple innervations (MIF), characterized by forming smaller motor units (1:5 ratio), with lower mitochondrial content and fewer oxidative enzymes, a relatively slow, graduated contraction [166,167]. This constitution favors a fine and precise muscular control, modulating the ocular movement and resulting in a more stable vision [168].

It is important to highlight that fast motor units degenerate before slow ones, due, at least in part, to the fact that the motoneurons that supply the slow contraction muscles can compensate the death of its neighboring motoneurons temporarily by generating compensatory axonal branches and reinnervation of the denervated muscle [166]. In contrast, motor-neuronal populations that exclusively present fibers with SIF innervation lose contact with their target muscles much earlier and, consequently, are more vulnerable to neurodegeneration. This resistance has been demonstrated in SOD1 mouse models, where the EOMs remain fully innervated in stages in which the limb muscles show deep denervation [108,169,170]. Therefore, the EOMs can maintain NMJs for a longer time, which leads to a greater retrograde trophic support from the EOMs to the projecting motoneurons.

EOMs have been shown to express VEGF and, therefore, are good candidates to intervene in trophic supply towards motoneurons [151]. Previous studies described the anterograde and retrograde transport of VEGF to neurons, the latter being crucial for maintaining the integrity and functionality of NMJs [82,171]. The importance of trophic supply to ocular motoneurons is also emphasized by the higher expression of BDNF, NGF, and NT-3 found in the EOMs, compared to the buccinator and tongue muscles, target muscles for facial and hypoglossal motoneurons, respectively [172], emphasizing the role of the retrograde pathway as a source of trophic factors.

Although the level of VEGF expression is similar in buccinator, tongue, and EOMs, there is a higher density of Flk-1 receptors in the pre-synaptic terminal of the EOMs compared to the muscles innervated by facial and hypoglossal motoneurons [151]. Previous studies have also shown the presence of the Flk-1 receptor at NMJs level of the abducens motoneurons, projecting by the abducens nerve towards the lateral rectus muscle [173]. These data support the idea that, although extraocular, facial, and hypoglossal muscle fibers were found to be positive for VEGF, not all target muscles appear to be acting to the same extent as the retrograde source of this factor towards the motoneurons that innervate them [151]. In this sense, the VEGF reaching the motoneurons of the ocular motor system through the retrograde pathway may have a more significant influence than the VEGF that comes to the facial or hypoglossal motoneurons.

All this evidence suggests that the retrograde function of VEGF is important and determinant for the survival of brainstem motoneurons.
