4. Huntington’s Disease Models
Huntington’s disease (HD) is an autosomal dominant, incurable, and fatal neurodegenerative disorder. Initially, HD patients display excessive movements of the limbs and face, and then gradually progress to exaggerated body movements described as chorea. Patients exhibit progressive symptoms, such as psychiatric, cognitive, and motor dysfunction, and this disease is usually lethal 10–20 years after the onset
[87][88][89]. HD is caused by an expansion of the polyglutamine-coding region in the N-terminus of the huntingtin protein (HTT)
[90]. HTT is a 350 kDa protein that is ubiquitously expressed, evolutionarily conserved, and likely to be involved in many cellular processes
[91][92][93]. However, the precise mechanisms underlying the functions of the HTT gene remain incompletely understood.
The zebrafish HTT protein consists of 3121 amino acids and shares 70% identity with the human HTT orthologue
[94]. Compared with the HTT-null mutation mice
[92], HTT-null mutation zebrafish are viable, so the zebrafish is believed to be a suitable model to study the mechanisms of HD. To investigate the roles of HTT, several previous zebrafish HD models used MO to observe the effects of HTT deficiency in the early zebrafish development
[95][96][97]. One study revealed that HTT-deficient zebrafish had hypochromic blood because of the decrease in hemoglobin production, despite the presence of iron within blood cells, and speculated that the disturbance of HTT’s normal function in the iron pathway leads to HD pathology and especially to its neuronal specificity
[95]. By use of the same HTT-deficient model, Henshall et al. reported the effects of the loss-of-function of HTT on the developing nervous system and found obvious defects in the morphology of olfactory placode, neuromasts, and branchial arches, which led them to postulate that HTT may have a specific function that enables the formation of telencephalic progenitor cells and preplacodal cells in the forebrain
[96]. Another study of the morpholino-based HTT loss-of-function zebrafish observed massive apoptosis of neuronal cells, accompanied by impaired neuronal development, small eyes and heads, and the enlargement of brain ventricles. Interestingly, it was observed that the expression of brain-derived neurotrophic factor (BDNF) was reduced. Notably, treatment of HTT-MO zebrafish embryos with exogenous BDNF rescued these defects, which suggests that increasing the BDNF expression might be a useful strategy for HD treatment
[97].
In addition, some scientists established HD zebrafish models through the transgenic technology
[98][99][100]. Schiffer et al. transiently expressed 102 polyglutamine repeats in the N-terminal fragment of the HTT protein fused with GFP (Q102-GFP) in zebrafish and found an accumulation of this mutant protein in large SDS-insoluble inclusions in the zebrafish embryos, thus reproducing an important feature of the HD pathology. The expression of the mutant HTT protein resulted in an increase in abnormal morphology and the occurrence of apoptosis in zebrafish embryos. A further study found that soluble mutant HTT protein forms are responsible for toxicity and aberrant polyglutamine aggregates in zebrafish
[98]. The same study also found that its toxicity can be suppressed by the heat-shock proteins Hsp40 and Hsp70. Importantly, by the use of this HD transgenic model, two inhibitors of the Q102-GFP aggregation in vivo were identified, both of which are compounds of the
N′-benzylidene-benzohydrazide class (293G02 and 306H03). In another study, a stable transgenic zebrafish line, which expressed a Q71-GFP fusion protein under the control of the rhodopsin promoter, was constructed to screen FDA-approved drugs to identify novel autophagy-inducing pathways. Three drugs (L-type Ca
2+ channel antagonists, the K
+ATP channel opener minoxidil, and the G
i signalling activator clonidine), which participate in a cyclical mTOR-independent pathway that regulates autophagy, were detected. This pathway has lots of candidate points to induce autophagy and reduce aggregates
[99].
Cre-
loxP system was also sometimes used to generate conditionally inducible transgenic zebrafish to study HD. For example, Veldman et al. created an inducible zebrafish HD model, in which the N-terminal 17 amino acids (N17) in the context of the exon 1 fragment of HTT were deleted, coupled with 97Q expansion (mHTT-ΔN17-exon1). That study found that, compared with the mHTT-ΔN17-exon1 line, fish with intact N17 and 97Q expansion (mHTT-exon1) had more delayed-onset movement deficits with slower progression. This model confirmed that the deletion of N17 terminal amino acids of the HTT will lead to an accelerated HD-like phenotype in zebrafish
[100]. Recently, a separate study treated a transgenic HD zebrafish model with a phosphodiesterase 5 (PDE5) inhibitor and found an obvious decrease in the mutant HTT protein levels, cell death, and morphological abnormalities
[101].
5. Other Neurodegenerative Disease Models
In addition to the above studies, zebrafish and medaka were also used in the investigation of some other rare neurodegenerative disorders. Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by the motor neuron loss, and thus progressive muscle weakness and eventual death, primarily due to respiratory failure. The most prevalent genetic cause of ALS and frontotemporal dementia (FTD) is a hexanucleotide repeat expansion (HRE) within the first intron of the C9orf72 gene
[102][103]. Shaw et al. generated two zebrafish lines to express C9orf72 HREs. This model recapitulates the motor deficits, cognitive impairment, muscle atrophy, motor neuron loss, and mortality in early adulthood that was observed in human C9orf72-ALS/FTD patients. Moreover, this stable transgenic model represents a powerful potential for the screening of therapeutic compounds
[104]. In another study, several transgenic C9orf72-associated repeat zebrafish lines were generated by TOL2-mediated transposition. These models confirm the poly-GA toxicity in zebrafish. The reduction of poly-GA protein rescues toxicity, indicating its potential therapeutic value to treat C9orf72 repeat expansion carriers
[105]. Conversely, Yeh et al. constructed two transient loss-of-function zebrafish larvae (C9orf72
u-DENN, C9orf72
c-DENN) using a morpholino injection. These models facilitate advances in the understanding of the functions of C9orf72 and provide potential mechanisms to elucidate the pathogenesis of ALS-FTD
[106]. Mutations in the superoxide dismutase 1 (SOD1) gene were identified as another cause of ALS. In a previous study, by outcrossing the G93Ros10-SH1 line with the wildtype AB zebrafish strain, a mutant SOD1 zebrafish model was generated and used for high throughput screening to identify neuroprotective compounds
[107].
Spinocerebellar ataxias (SCAs) are global neurodegenerative diseases leading to motor discoordination, which is always caused by the affected cerebellar Purkinje cells (PCs). A previous study generated a transgenic SCA type 13 (SCA13) model, which mimics a human pathological SCA13
R420H mutation. This model exhibited neuropathological and behavioural changes similar to those manifested by SCA-affected patients
[108]. Based on the same model, Namikawa et al. reported an SCA13-triggered cell-autonomous PC degeneration, which results in eye movement deficits
[109]. In a previous study in our lab, we constructed an
NPC1 knock-out zebrafish model using the CRISPR/Cas9-mediated technology
[110]. This model developed symptoms similar to those observed in human patients of Niemann-Pick type C disease (NPC). We observed the loss of Purkinje cells in the cerebella of the
NPC1−/− homozygous fish
[110] and the aberrant motor behaviour, i.e., ataxias, a typical pathological character of human NPC1 patients (unpublished data), indicating its potential value for investigating the molecular mechanisms of NPC1.
In addition, a previous study generated a Gaucher disease (GD) model in medaka by the use of a high-resolution melting assay in the TILLING library for the
glucocerebrosidase (
GBA) gene
[111]. In this study, it was observed that the
GBAW337X/W337X (
GBA−/−) medaka displayed complete deficiency in GCase activity, and it showed similar pathological phenotypes with human neuronopathic GD. Importantly, compared with the perinatal death in humans and mice lacking the GCase activity, the
GBA−/− medaka survived for months, enabling the investigation of the pathological progression
[111].