**2.1 Histopathological studies in ALS demonstrating intraretinal protein inclusions**

(**Table 1**, **Figure 1**) Accumulated and altered proteins can interfere with neuronal traffic or can abduct proteins that are essential for proper neuronal functioning causing neurotoxicity [62]. The ubiquitin proteasome system plays an important role in ALS, with reactive ubiquitin inclusions being characteristic of this pathology [5, 63]. Among them, TDP-43 and p62 proteins are specifically indicative of ALS. These inclusions, which are positive for P62 and negative for TDP-43, have been demonstrated in the brain, hippocampus, and cerebellum in ALS patients [64, 65].

There are scarce studies that have focused on the histopathology of retinal tissue in both ALS patients and animal models of mammals with ALS. Actually, the first histopathological analysis in the retina was performed in 2014 on a patient with the C9orf72 mutation. In this study, protein intracytoplasmic p62-positive and pTDP43 negative perinuclear aggregates, typical of ALS/frontotemporal dementia (FTD), were observed in the inner nuclear layer (INL) of the retina [66]. Both the poly-(GA) n dipeptide repeats and ubiquitin in the retina were positively stained for p62, similar to the perinuclear inclusions localized in the brains, specifically in the dentate gyrus, of patients with this mutation [66]. The authors suggested that most of the p62-positive inclusions found were likely placed within the cones of bipolar cells (OFF bipolar cells) and between amacrine and horizontal cells, because they were also stained with GLT-1 and recoverin; in addition, these retinal deposits could be related to the contrast sensitivity impairment manifested by the patient [66]. Moreover, Volpe et al. [67] analyzed two retinas from ALS patients with C9orf72 mutations and demonstrated (i) specific p62 inclusions mostly in the INL (94.9%) and in a smaller proportion in the retinal ganglion cell layer (GCL) (5.1%) in one patient, and (ii) ganglion cell axonal atrophy specifically in the papillomacular bundle in the second patient. On the other hand, abundant positive ubiquilin 2-positive inclusions were also shown in a transgenic mice experimental model with mutant UBQLN2, mostly in the inner plexiform layer (IPL), with a smaller amount in the outer plexiform layer (OPL) and a scarce amount in the GCL. This ubiquilin 2 aggregation in the layers of the retina with more synapses is associated to the ubiquilin 2 accumulation in the dendritic spines of the hippocampus, and it may also be related to the dementia observed in this experimental model. Furthermore, few ubiquilin 2-positive aggregates were detected between the neurosensorial retina and the retinal pigment epithelium, whose appearance was analogous to that of drusen [67]. Similarly, in patients with FTD and progranulin deficiency, lipofuscin deposits were found, sometimes associated with subretinal drusen-like aggregates [68]. Retinal thinning in these patients was detected by OCT before symptoms, suggesting that the eye is affected in progranulin-deficient frontotemporal dementia disease [69].

Eye degeneration was reported in an ALS *Drosophila* model that expressed C9orf72 repeat expansion. The expansion of a noncoding GGGGCC hexanucleotide repeat of the C9orf72 gene on chromosome 9p21 is the most common point mutation in familiar ALS, which generates dipeptide repeat proteins that aggregate in the brain. It is noteworthy that some synthesized compounds revealed a significant biological effect by blocking the neurodegeneration of fly retina at different efficacy levels and upgrading



*ALS: amyotrophic lateral sclerosis; FTD: frontotemporal dementia; INL: inner nuclear layer; UBQLN2P497H: dysfunctional ubiquilin 2; TG: transgenic; IPL: inner plexiform layer; OPL: outer plexiform layer; ONL: outer nuclear layer; INL: inner nuclear layer; GCL: ganglion cell layer; sub-RPE: subretinal pigment epithelium; hSOD1: human superoxide dismutase 1; Ranbp2: RAN-binding protein 2; RGCs: retinal ganglion cells; PAS: periodic acid Schiff; P-NF: phosphorylated form of neurofilament; NP-NF: non-phosphorylated form of neurofilament; RNFL: retinal nerve fiber layer; pRNFL: peripapillary retinal nerve fiber layer; OCT: optic coherence tomography; ALSFRS-R: ALS Functional Rating Scale—Revised.*
