The epg5 knockout zebrafish line: a model to study Vici syndrome

Abstract

The EPG5 protein is a RAB7A effector involved in fusion specificity between autophagosomes and late endosomes or lysosomes during macroautophagy/autophagy. Mutations in the human EPG5 gene cause a rare and severe multisystem disorder called Vici syndrome. In this work, we show that zebrafish epg5-/mutants from both heterozygous and incrossed homozygous matings are viable and can develop to the age of sexual maturity without conspicuous defects in external appearance. In agreement with the dysfunctional autophagy of Vici syndrome, western blot revealed higher levels of the Lc3-II autophagy marker in epg5-/mutants with respect to wild type controls. Moreover, starvation elicited higher accumulation of Lc3-II in epg5-/than in wild type larvae, together with a significant reduction of skeletal muscle birefringence. Accordingly, muscle ultrastructural analysis revealed accumulation of degradationdefective autolysosomes in starved epg5-/mutants. By aging, epg5-/mutants showed impaired motility and muscle thinning, together with accumulation of non-degradative autophagic vacuoles. Furthermore, epg5-/adults displayed morphological alterations in gonads and heart. These findings point at the zebrafish epg5 mutant as a valuable model for EPG5-related disorders, thus providing a new tool for dissecting the contribution of EPG5 on the onset and progression of Vici syndrome as well as for the screening of autophagy-stimulating drugs.

Autophagy in Neuronal Development and Plasticity

“…It is possible that the human mutations result in aberrant protein function that is not phenocopied in null mutant model organisms. Indeed, zebrafish epg5-deficient CRISPR/Cas9 models show no overt physical defects or neuronal deficits, despite showing accumulation of non-degradative autophagic vesicles [27]. Interestingly, the clinical neurological features more commonly associated with genetic mutations in autophagy genes are developmental delay, cognitive decline and functional deficits, rather than structural defects in brain development (see Table 1).…”

epg5 knockout leads to the impairment of reproductive success and courtship behaviour in a zebrafish model of autophagy-related diseases

“…Interestingly, in another epg5 null model, the medaka fish, defective autophagy was related to an impairment of spermatogenesis, due to the inability to clear mitochondria and germplasm during sperm differentiation, which led to failure to reproduce [ 5 ]. A reduction of the reproductive capabilities was reported also in a epg5 null mutant zebrafish line [ 6 ] that showed a clear impairment of the autophagic flux, as demonstrated by Western blot and ultrastructural analysis of both larvae and adults, delay in the intestinal development and alterations in the heart and gonads morphology.…”

“…Altogether these recent findings suggest a possible direct link between disruption/impairment of the autophagic processes and reproduction at both the physiological and the behavioural level. The previously developed KO epg5 zebrafish line, in which some preliminary indications on the impairment of reproductive capabilities have been found [ 6 ], served, in the present study, as a suitable model to deepen our understanding of this possible multilevel link. To this aim we applied a multifaceted analysis of the effects of the epg5 KO on the male reproductive performances, with particular attention to i) breeding success, ii) fertility rate, iii) offspring survival, iv) ejaculate quality, v) sperm and testes morphology, vi) courtship behaviour.…”

Natterin-like depletion by CRISPR/Cas9 impairs zebrafish (Danio rerio) embryonic development

“…The detection of expressed mRNAs through WISH is a robust tool for assessing the spatial distribution of gene transcripts [ 48 ]. The zebrafish is especially suitable for this analysis and there are many described gene expression patterns available online at the Zebrafish Information Network (ZFIN, https://zfin.org/action/expression/search/ ) [ 47 , 49 , 50 ].…”