Somatic Gene Transfer Using a Recombinant Adenoviral Vector (rAAV9) Encoding Pseudophosphorylated Human Thr175 Tau in Adult Rat Hippocampus Induces Tau Pathology

Abstract

Aberrant phosphorylation of the microtubule associated protein tau (tau) is associated with multiple neurodegenerative diseases where it is a contributes to neurotoxicity. We have observed that phosphorylation at Thr175 tau (pThr175 tau) exerts toxicity when expressed as a pseudophosphorylated tau construct (Thr175Asp) in vitro. To determine whether pThr175 tau can induce tau pathology in vivo with an accompanying clinical phenotype, we used a recombinant adenoviral expression vector (rAAV9) to express a GFP-tagged Thr175Asp tau protein construct in adult female Sprague-Dawley rat hippocampus. Ten rats per group were injected with rAAV9 vectors encoding either GFP, wild type GFP-tagged tau protein, Thr175Ala tau or Thr175Asp tau. 12 months postinjection, all rats were investigated by immunohistochemistry for GFP (extent of vector expression), pThr231 tau protein, activated GSK3β, and caspase-3 cleavage. Vector expression was primarily localized to hippocampal CA2 subregion. Tau protein pathology restricted to the CA2 region in the form of axonal beading, fibrils, and neurofibrillary tangles was observed in Thr175Asp tau inoculated brains and included colocalization with pThr231 tau and caspase-3 cleavage in this group only. Although no behavioral or imaging phenotype was observed, our results demonstrate that pThr175 tau protein is capable of exerting neuronal toxicity in vivo.

Synergistic toxicity in an in vivo model of neurodegeneration through the co-expression of human TDP-43M337V and tauT175D protein

“…Genotyping was confirmed by PCR to detect both ChAT and TDP-43. Expression was confirmed by RT-PCR of RNA from animals as previously described [22] and copy number analyses performed to ensure that experimental animals had similar transgene loads [22]. Due to the fact that the lines had to be carried as hemizygous lines, and only females were used in these studies, only 12.5% of the resulting offspring from the crosses were experimental animals.…”

“…Somatic gene transfer technique was used to express a recombinant adeno-associated virus (rAAV9) vector, allowing for the specific expression of human tau constructs in the hippocampus [22, 25]. We selected the toxic pseudophosphorylated tau variant human 2N4R tau with amino acid Thr 175 mutated to Asp to mimic phosphorylation (tau T175D ) based on our previous studies in which we demonstrated the toxicity of this ALSci associated tau variant in cell culture and rodent models [9, 21, 22, 37]. The constructs were designed to produce an N-terminus GFP-fused tau protein, which was then packaged into rAAV9 virus and injected into the hippocampus of the rats as previously described [22, 25].…”

Tau protein phosphorylation at Thr175 initiates fibril formation via accessibility of the N‐terminal phosphatase‐activating domain

“…The precise mechanisms by which pTyr 216 GSK3β levels are modulated by pThr 175 tau remain to be determined as we have only demonstrated mechanistic dependence of modulation of the pSer 9 regulatory site. Intriguingly, our previous investigations have demonstrated that pTyr 216 GSK3β colocalizes within dense pThr 175 and pThr 231 tau protein aggregates in human CTE cases (Moszczynski, Gopaul, et al, ). Therefore, if regulation of GSK3β at pSer 9 results in the formation of tau fibrils and these fibrils contained aggregated GSK3β, then increases in GSK3β translation might occur.…”

“…Our previous investigations have utilized a rodent model of a single TBI to induce the expression of endogenous pThr 175 tau immunoreactive fibrils by 90 days post‐TBI in vivo (Moszczynski, Gopaul, et al, ). Having demonstrated that the exposure of the tau PAD is elevated in Thr 175 Asp tau and that this enhanced exposure is critical to the formation of tau fibrils in vitro, we sought to model the development of pThr 175 tau fibrils in relation to PAD exposure in vivo.…”

“…Traumatic brain injury (TBI) is a significant risk factor for the development of various neurodegenerative diseases, including chronic traumatic encephalopathy (CTE) (McKee et al, ; McKee, Stein, Kiernan, & Alvarez, ), Alzheimer's disease (AD) (Ramos‐Cejudo et al, ) and amyotrophic lateral sclerosis (ALS) with cognitive impairment (Chen, Richard, Sandler, Umbach, & Kamel, ). These conditions share the neuropathological hallmarks of cytoplasmic inclusions of tau protein immunoreactive for phospho‐Thr 175 (pThr 175 ) tau and an elevated neuronal expression of active glycogen synthase kinase 3β (GSK3β) Hooper, Killick, & Lovestone, ; Lei, Ayton, Bush, & Adlard, ; Beurel, Grieco, & Jope, ; Moszczynski, Gopaul, et al, ; Moszczynski, Hintermayer & Strong, ). We have shown that both of these features can be reproduced in a rodent model of TBI (Moszczynski, Strong, et al, ), thus providing a model for studying the initiation and propagation of pThr 175 tauopathy.…”