Gene Therapy – Preservation of forelimb function by UPF1 …

Gene Therapy (2015) 22, 2028; doi:10.1038/gt.2014.101; published online 6 November 2014

K LJackson1, R DDayton1, E AOrchard2, SJu3, DRinge4, G APetsko4,5, L EMaquat6,7 and R LKlein1

Amyotrophic lateral sclerosis (ALS) is a deadly neurodegenerative disease involving progressive paralysis. There are no highly efficacious strategies to treat ALS despite great effort by doctors and scientists. Successful treatments in mouse models, most of which are based on rare familial mutations in the ALS gene SOD1, have so far had little impact on modifying the disease in humans. Novel models based on transactive response DNA-binding protein 43kDa (TDP-43) may offer a more predictive test system given that the vast majority of ALS cases harbor TDP-43 pathology in their neurons and glia.1, 2, 3 Abnormal TDP-43 aggregates are also prominent in the class of diseases known as frontotemporal lobar degeneration (FTLD-TDP).4 TDP-43 is an RNA-binding protein that is normally found predominantly in the nucleus. In FTLD-TDP and the majority of ALS, abnormal TDP-43 accumulation occurs in the cytoplasm in the form of hyperphosphorylated and ubiquitinated pathological protein aggregates, and thus serves as a post-mortem diagnostic marker. 1, 2, 3, 4 One of the ways by which TDP-43 has been studied in animals is by gene delivery, which has proven to be sufficiently reproducible to allow the discrimination of genotypephenotype differences among TDP-43 isoforms in our previous work.5, 6, 7 This reproducibility and the ability to experimentally control the onset and severity of the disease state offer advantages for modeling, given that TDP-43 overexpression is highly toxic to cells.8 Here we use TDP-43 gene transfer to induce motor paralysis in rats to study limb symptomatology that is germane to ALS as a platform for gene therapy. Overexpression of TDP-43 causes progressive paresis to paralysis of the limbs in a highly reproducible manner,6, 7 offering an assay for therapeutic efficacy such as gene therapy. Gene therapy is worth considering for this disease given that ALS is fatal and irreversible. In this report, recombinant TDP-43 expression was titrated to a low level for a partial disease state retaining restorative capacity.

Refinement of TDP-43 animal models continues to be an important goal in the field.9 Reports of experimental treatments that slow or block TDP-43-mediated toxicity are beginning to emerge, either by genetic or pharmacological interventions in several TDP-43 models.10, 11, 12, 13, 14 Here we report behavioral outcomes from testing an empirically chosen therapeutic target, cDNA coding for human upframeshift protein 1 (UPF1), in a rat model of ALS-like paralysis based on TDP-43.

UPF1 is best known for its role in nonsense-mediated mRNA decay (NMD), a surveillance mechanism that degrades mRNAs containing a premature termination codon, which can be generated, for example, through alternative splicing. NMD prevents the production of truncated proteins that could harm the cell. NMD is also involved in the regulation of the expression of ~10% of normal physiologic transcripts in the cell, and is essential in mice.15, 16, 17 We pursued the possibility that UPF1 could ameliorate ALS-like symptoms based on the work carried out in yeast and neuronal cultures.18, 19 In a genetic screen of several thousands of proteins, a yeast homolog of hUPF1, and then the human gene itself, was found to prevent FUS- and TDP-43-mediated toxicity in yeast, 19 Ju et al., unpublished. Barmada et al.18 have advanced this approach, demonstrating that UPF1 protects primary neuronal cultures from TDP-43 cytotoxicity, possibly by upregulating NMD, as inhibitors of NMD blocked the protective effect. 18 The fact that expressing UPF1 blocks the toxic actions of TDP-43 in yeast cells and cultured neurons is consistent with the hypothesis that TDP-43-induced toxicity involves inhibition of UPF1 function, because TDP-43 toxicity can be suppressed by adding back UPF1 to restore NMD.

The main purpose of this study was to evaluate the expression of human mycUPF1 (i.e. human UPF1 with an N-terminal myc epitope tag) as a protection against TDP-43-induced limb paralysis in rats. MycUPF1 was tested in parallel with several different types of control treatments, all confirming that mycUPF1 elicits a specific therapeutic effect. We also evaluated whether the expression of recombinant TDP-43 or mycUPF1 would affect either each others recombinant gene expression or the expression of endogenous rat TDP-43 or UPF1. The data demonstrate that augmenting the cellular abundance of UPF1 provides a useful means of abrogating the devastating paralysis induced by TDP-43 overexpression.

Exogenous TDP-43 and green fluorescent protein (GFP) expression levels were purposefully set relatively low compared with the previous studies to test a rat model with a partial lesion and restorative capacity. This titration was advantageous to observe a therapeutic effect, but the low expression levels rendered detection of the transgene products inefficient. Nevertheless, previous work demonstrated that intravenous adenoassociated virus vector (AAV9) TDP-43 gene transfer specifically induces hindlimb paralysis even when the resulting level of exogenous TDP-43 is only faintly detectable.6 We chose the intravenous AAV9 method because it produces widespread central nervous system (CNS) expression, leading to marked expression in spinal motor neurons, dorsal root ganglia (DRG) neurons and cerebellar Purkinje neurons,6, 20 with only a small fold overexpression of the encoded protein, for example, less than twofold overexpression relative to the corresponding endogenous protein as estimated in the spinal cord in Dayton et al.7

For studying the effect of mycUPF1 expression, we harvested DRG neurons because this tissue provides a relatively high percentage of transduced cells in the nervous system, allowing for detection of transgene product. By comparison, the spinal cord and cerebellar samples include a greater percentage of non-transduced cells. We used antibodies for total TDP-43 or total UPF1 that detect both the endogenous rat plus exogenous human TDP-43 or UPF1. In DRG, the increase in total TDP-43 expression in AAV9 TDP-43/Empty vs uninjected animals was 2.4-fold (t-test, P<0.02, N=3 per group), whereas for total UPF1, we estimated the increase to be 1.6-fold in AAV9 mycUPF1 vs uninjected subjects ( Figure 1). The fold increases were relatively lower in the spinal cord (Figure 1) and cerebellum (not shown), as expected: the estimated ratio in the spinal cord and cerebellum was 1.4- and 1.2-fold for AAV9 TDP-43/Empty vs uninjected subjects and 1.1- and 1.1-fold for AAV9 mycUPF1 vs uninjected subjects (N=3 per group). Although fold overexpression levels were small, recombinant mycUPF1 could be specifically visualized using myc antibody, which detected recombinant mycUPF1 only in subjects receiving AAV9 mycUPF1 only or AAV9 TDP-43/mycUPF1 ( Figure 2).

Overexpression of TDP-43 or UPF1 in the rat CNS. Protein from dissected DRG and lumbar spinal cord was analyzed by western blotting 12 weeks after intravenous injection of AAV9 expression vectors. Three animals are shown for each condition. The level of total TDP-43 (endogenous rat plus recombinant human TDP-43) was significantly increased in the DRG of the AAV9 TDP-43/Empty group compared with uninjected subjects (t-test, P<0.02, N=3), but less so in the spinal cord or cerebellum (not shown). The expression level of human mycUPF1 compared with endogenous rat UPF1 was relatively small in all the three regions. The bands were normalized to GAPDH. See Results for details.

Selective detection of only recombinant human TDP-43 or mycUPF1. (a) A human-specific TDP-43 antibody detected exogenous human but not endogenous rat TDP-43 in DRGs. The level of TDP-43 expression was indistinguishable with or without mycUPF1 coexpression. (b) The level of exogenous mycUPF1 was detected with a myc antibody and only observed in rats that received AAV9 mycUPF1. In contrast to (a), MycUPF1 expression levels were reduced when AAV9 TDP-43 was coexpressed (t-test, P<0.05, N=3 for DRG). The bands were normalized to GAPDH.