Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) has been shown to activate the eIF2α kinase PERK to directly regulate translation initiation. Tight control of PERK-eIF2α signaling has been shown to be necessary for normal long-lasting synaptic plasticity and cognitive function, including memory. In contrast, chronic activation of PERK-eIF2α signaling has been shown to contribute to pathophysiology, including memory impairments, associated with multiple neurological diseases, making this pathway an attractive therapeutic target. Herein, using multiple genetic approaches we show that selective deletion of the PERK in mouse midbrain dopaminergic (DA) neurons results in multiple cognitive and motor phenotypes. Conditional expression of phospho-mutant eIF2α in DA neurons recapitulated the phenotypes caused by deletion of PERK, consistent with a causal role of decreased eIF2α phosphorylation for these phenotypes. In addition, deletion of PERK in DA neurons resulted in altered de novo translation, as well as changes in axonal DA release and uptake in the striatum that mirror the pattern of motor changes observed. Taken together, our findings show that proper regulation of PERK-eIF2α signaling in DA neurons is required for normal cognitive and motor function in a non-pathological state, and also provide new insight concerning the onset of neuropsychiatric disorders that accompany UPR failure.
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...
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