The pathologic hallmarks of Alzheimer’s disease (AD) are extracellular amyloid-β (Aβ) protein-containing neuritic plaques and intracellular hyperphosphorylated tau-containing neurofibrillary tangles. Early-onset AD is associated with mutations in three genes involved in Aβ proteolysis displaying autosomal-dominant inheritance patterns in humans: amyloid-β precursor protein (APP), Presenilin 1 (PSEN1), and Presenilin 2 (PSEN2). Late-onset AD is linked to a number of genetic risk factors including the apolipoprotein E (ApoE), which is a cholesterol transport protein, and the neuronal sortilin-related receptor (SORL1), which acts as a sorting receptor for APP.
In humans, APP undergoes post-translational processing. If APP is cleaved by α-secretase, a benign Aβ peptide is produced. Alternatively, APP can undergo two sequential cleavages by β-secretase and γ-secretase to generate the pathogenic Aβ peptide pushing it toward the late endosomal pathway. Depending on where the γ-secretase cleaves the Aβ C-terminal, either Aβ42 or Aβ40 will be generated. Aβ42 is the longer form of Aβ, which is more cytotoxic than the shorter Aβ40 peptide.
According to the amyloid hypothesis, deposits of Aβ outside the neuron are the underlying cause of AD. The competing tau hypothesis states that the hyperphosphorylated tau protein, which forms neurofibrillary tangles inside neurons, is the catalyst for AD disease progression. This leads to the disassembly of microtubules essential for neuronal transport, disrupting neurotransmitter communication between neurons, and ultimately resulting in cell death.
Zebrafish as a Model for AD
Several of the human genes encoding the enzymes required for the post-translational modifications of APP have been found with a high percent of amino acid similarity in zebrafish. Zebrafish have two genes similar to human APP: appa
]. During gastrulation, both app genes are expressed in the entire embryo, whereas at 24 hpf, the appa
paralogs are expressed in the telencephalon, the vDC, the trigeminal ganglia, and the posterior lateral line ganglia. Orthologs of the β-secretase and γ-secretase complexes are found in zebrafish and are expressed in the CNS [48
]. Despite conservation of the Aβ domain and of the secretases between zebrafish and humans, a zebrafish Aβ peptide has yet to be found and it is not known if the above post-translational modifications that occur in human APP processing also occur in zebrafish.
Loss of zebrafish Appa and Appb function by MO knockdown resulted in reduced body length and defective convergent-extension movements during gastrulation [50
]. Interestingly, these defects are rescued by wild-type human APP mRNA, but not by the Swedish mutant APP, known to cause familial AD. Both zebrafish psen1
are expressed ubiquitously during embryogenesis; however, psen2
is more restricted to the CNS, eye, and spinal cord at 1 dpf [48
]. One ortholog of the human β-secretase enzyme has been annotated by Ensembl (Zv9) in zebrafish: BACE1 on chromosome 15. Further characterization of this gene in zebrafish is required to determine if it functions similarly in zebrafish as it does in humans. Another candidate gene that requires more in-depth characterization is the ApoE ε4 susceptibility gene, whose function in zebrafish is not well understood. ApoE is expressed in the zebrafish eyes, and some cells of the mesencephalic, telencephalic, and rhombencephalic brain areas, suggesting that it may play a significant function in the CNS [51
To understand how the microtubule-associated protein tau (MAPT) contributes to tau pathology and how tau redistributes from neuronal axons to neuronal soma forming pathogenic neurofibrillary tangles, a zebrafish model transiently expressing mutant human tau has been reported [52
]. In this study, human tau carrying mutations at sites associated with hereditary dementias was fused to GFP while under the control of the zebrafish pan-neural–specific GATA-2 promoter. GFP-positive neurons were found in the brain, retina, and spinal cord. In the brain, there was evidence of hallmark AD-associated cytoskeletal pathology, including disruption of tau trafficking and cytoskeletal filaments in the axon, accumulation of tau in the cell body near the axon, accumulation of fibrillar tau throughout the cell body, and presence of hyperphosphorylated tau. Despite showing that mutant human tau-GFP is hyperphosphorylated and could be used to monitor the formation of tangles, this strategy failed to generate a transgenic line stably expressing tau-GFP.
Bai et al. [53
] established a stable transgenic zebrafish line expressing either GFP or mutant human 4-repeat tau under the control of the zebrafish enolase-2 promoter, Tg(eno2:GFP
) or Tg(eno2:Tau
), respectively. Zebrafish enolase-2 was chosen because its expression in differentiated neuronal axons starts after the early stages of zebrafish development. This prevents pathogenic tau from disrupting the development of neuronal precursors and from causing defects that are not attributable to late-onset neurodegeneration. In Tg(eno2:Tau
) zebrafish, tau was expressed throughout the CNS and accumulations were present in the retina, spine, axons, and neuronal soma throughout the brain. Stable expression of tau into adulthood will facilitate the examination of pathologic tau in the age-related progression of AD.
Stable transgenic zebrafish expressing human tau with a mutation (TAU-P301L) found in frontotemporal dementia has been generated [54
••]. TAU-P301L is expressed in zebrafish neurons using the Gal4-UAS system, where DsRed and TAU-P301L are expressed concomitantly in early neuronal cells (under the control of the zebrafish HuC promoter), permitting the observation of red tau-expressing cell in embryos. TAU-P301L expression showed pathologic features of tauopathies, including human tau hyperphosphorylation, tangle formation, and neurodegeneration in the spinal cord. Moreover, tau-expressing embryos have behavioral deficits in escape response after touch stimulus. This study also reported testing of inhibitors of the tau kinase GSK3β, which lead to reduced tau phosphorylation in vivo. Thus, this model serves as a valuable tool to study AD and related tauopathies in vivo, especially because tau hyperphosphorylation developed within only 32 h.
It has been shown that APP can be diverted away from the late endosomal pathway by a SORL1-dependent switch, which sequesters APP into recycling endosomes, preventing the formation of Aβ [55
•]. Reduced expression of SORL1 is seen in AD brain tissue and is associated with an increase in Aβ production [56
]. This genetic association between AD and SORL1 expression is a result of single nucleotide polymorphisms (SNPs) found within the SORL1
•]. It has been demonstrated that SORL1 binds directly to APP and differentially regulates the sorting of APP into the late endosomal pathway leading to the production of cytotoxic Aβ or into the retromer recycling pathway, sequestering APP from the β- and γ-secretases. The overexpression of SORL1 in HEK cells reduces Aβ production by 82%, likely by diverting APP into the retromer recycling pathway. Controversy does exist over whether or not there is an association between AD and SORL1
. Currently, more research into the link between SORL1
and late-onset AD risk is necessary. Of potential benefit in the elucidation of a link between SORL1 and AD, a gene coding for a Sorl1-like protein is located on zebrafish chromosome 15, according to the latest Ensembl version of the zebrafish genome (Zv9). Expression and function of zebrafish sorl1
have yet to be investigated.