Genetic reduction of PERK-eIF2α signaling in dopaminergic neurons drives cognitive and age-dependent motor dysfunction

Abstract

2An array of phenotypes in animal models of neurodegenerative disease have been shown to 3 be reversed by neuronal inhibition of PERK, an eIF2α kinase that modulates the unfolded 4 protein response (UPR). This suggests that targeting PERK therapeutically could be beneficial 5 for treatment of human disease. Herein, using multiple genetic approaches we show that 6 selective deletion of the PERK in mouse midbrain dopaminergic (DA) neurons results in 7 multiple cognitive and age-dependent motor phenotypes. Conditional expression of phospho-8 mutant eIF2 in DA neurons recapitulated the phenotypes caused by deletion of PERK, 9 consistent with a causal role of decreased eIF2 phosphorylation. In addition, deletion of 10 PERK in DA neurons resulted in altered de novo translation, as well as age-dependent 11 changes in axonal DA release and uptake in the striatum that mirror the pattern of motor 12 changes observed. Taken together, our findings show that proper regulation of PERK-eIF2α 13 signaling in DA neurons is required for normal cognitive and motor function across lifespan, 14 and also highlight the need for caution in the proposed use of sustained PERK inhibition in 15 neurons as a therapeutic strategy in the treatment of neurodegenerative disorders.In order to evaluate whether proper cell type-specific regulation of PERK-eIF2 105 signaling in DA neurons is required for normal cognitive and motor function, we generated 106 mice containing a DA transporter (DAT) promoter-driven Cre transgene (DAT-Cre; Jackson 107 Laboratory, stock number: 006660) 21 and a conditional allele of Perk (Perk loxP ; termed PERK f/f ; 108 Fig. 1a) 22 . The expression of the Cre transgene and the Perk loxP allele was determined using 109 PCR-specific primers (Fig. 1b). The resulting conditional knockout mice (PERK f/f DAT-Cre), 110 which lack PERK in DA neurons of both the ventral tegmental area (VTA) and the substantia 111 nigra pars compacta (SNc) represent the primary experimental mouse line used here, along 112 with their littermate control mice (WT DAT-Cre). 114Cell-specific deletion of PERK in DA neurons was first verified at the protein expression 115 level by treating coronal midbrain slices containing the VTA and SNc with thapsigargin, which 116 inhibits ER Ca 2+ sequestration and is a potent inducer of ER stress and eIF2 phosphorylation. 117Immunostaining for PERK after thapsigargin exposure was clearly seen in tyrosine-118 hydroxylase positive (TH+) DA neurons in both SNc and VTA in WT DAT-Cre mice, whereas 119 PERK staining was not detected in SNc or VTA DA neurons of PERK f/f DAT-Cre mice, 120 although PERK expression in non-DA (TH-) cells remained intact ( Fig. 1c-d). Consistent with 121 these results, downstream UPR targets of PERK such as p-eIF2 ( Supplementary Fig. 1a-d) 122 and ATF4 ( Supplementary Fig. 1e-f) were significantly reduced in both SNc and VTA DA 123 neurons of PERK f/f DAT-Cre mice compared to thapsigargin-treated controls. Taken together, 124 these results demonstrate a reduced UPR after thapsigargin...

Mastering organismal aging through the endoplasmic reticulum proteostasis network

“…Three studies available in the BioRxiv repository might provide the first direct evidence for an involvement of the UPR in normal brain aging (Cabral-Miranda et al, 2020;Krukowski et al, 2020;Longo et al, 2020). Genetic depletion of PERK in dopaminergic neurons was shown to reduce dopamine release in the striatum on an age-dependent manner, associated with progressive motor dysfunction (Longo et al, 2020).…”

“…It has been reported that the genetic or pharmacological reduction of eIF2α phosphorylation or ATF4/PERK expression improves synaptic plasticity and the performance of animals in memory‐related tasks (reviewed in (Martinez et al, 2018)). Three studies available in the BioRxiv repository might provide the first direct evidence for an involvement of the UPR in normal brain aging (Cabral‐Miranda et al, 2020; Krukowski et al, 2020; Longo et al, 2020). Genetic depletion of PERK in dopaminergic neurons was shown to reduce dopamine release in the striatum on an age‐dependent manner, associated with progressive motor dysfunction (Longo et al, 2020).…”

“…Three studies available in the BioRxiv repository might provide the first direct evidence for an involvement of the UPR in normal brain aging (Cabral‐Miranda et al, 2020; Krukowski et al, 2020; Longo et al, 2020). Genetic depletion of PERK in dopaminergic neurons was shown to reduce dopamine release in the striatum on an age‐dependent manner, associated with progressive motor dysfunction (Longo et al, 2020). In contrast, treatment of aged animals with ISRIB, a small molecule that blocks the consequences of eIF2α phosphorylation, reverses the natural decay of cognitive function in mice, improving behavioral, and electrophysiological parameters (Krukowski et al, 2020).…”

Chromosome 22q11.2 deletion causes PERK-dependent vulnerability in dopaminergic neurons

“… [48] Moreover, a recent study shows that a specific-deletion of PERK in mouse midbrain dopaminergic neurons drives the cognitive and motor dysfunction. [49] We should certainly validate this hypothesis with in vivo analysis in the future. In addition, we believe that PERK dysfunction is at least partially responsible for the defect in F-actin dynamics based on our pharmacological manipulations and gene-editing analyses; however, we do not exclude the possibility of other genes in the 22q11.2 region directly affecting the F-actin dynamics.…”