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Loss-of-function phenotype of a PKC?T219A knockin mouse strain.

ABSTRACT: BACKGROUND:Protein kinase C ? has been established as an important signaling intermediate in T-effector-cell activation and survival pathways by controlling activity of the key transcription factors NF-?B and NFAT. Previous studies identified an activation-induced auto-phosphorylation site at Thr-219, located between the tandem C1 domains of the regulatory fragment in PKC?, as a structural requirement for its correct membrane translocation and the subsequent transactivation of downstream signals leading to IL-2 production in a human T cell line. METHODS:The present work aimed to define the role of this phosphorylation switch on PKC? in a physiological context through a homozygous T219A knockin mouse strain. T cell activation was analyzed by H3-thymidine uptake (proliferative response), qRT-PCR and luminex measurements (cytokine production). NFAT and NF-?B transactivation responses were estimated by Gel mobility shift and Alpha Screen assays. Frequencies of T cell subsets were analyzed by flow cytometry. RESULTS:Despite a normal T cell development, in vitro activated effector T cells clearly revealed a requirement of Thr-219 phosphorylation site on PKC? for a transactivation of NF-?B and NFAT transcription factors and, subsequently, robust IL-2 and IFN-? expression. CONCLUSION:This phenotype is reminiscent of the PKC? knockout T cells, physiologically validating that this (p) Thr-219 auto-phosphorylation site indeed critically regulates PKC? function in primary mouse T cells.

SUBMITTER: Thuille N

PROVIDER: S-EPMC6836476 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Loss-of-function phenotype of a PKCθ<sup>T219A</sup> knockin mouse strain.

Thuille Nikolaus N Siegmund Kerstin K Klepsch Victoria V Schörgenhuber Jacqueline J Danklmaier Sarah S Leitges Michael M Baier Gottfried G

Cell communication and signaling : CCS 20191106 1

<h4>Background</h4>Protein kinase C θ has been established as an important signaling intermediate in T-effector-cell activation and survival pathways by controlling activity of the key transcription factors NF-κB and NFAT. Previous studies identified an activation-induced auto-phosphorylation site at Thr-219, located between the tandem C1 domains of the regulatory fragment in PKCθ, as a structural requirement for its correct membrane translocation and the subsequent transactivation of downstream ...[more]

PMID: 31694643

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Project description:The E693Δ (Osaka) mutation in APP is linked to familial Alzheimer's disease. While this mutation accelerates amyloid β (Aβ) oligomerization, only patient homozygotes suffer from dementia, implying that this mutation is recessive and causes loss-of-function of amyloid precursor protein (APP). To investigate the recessive trait, we generated a new mouse model by knocking-in the Osaka mutation into endogenous mouse APP. The produced homozygous, heterozygous, and non-knockin littermates were compared for memory, neuropathology, and synaptic plasticity. Homozygotes showed memory impairment at 4 months, whereas heterozygotes did not, even at 8 months. Immunohistochemical and biochemical analyses revealed that only homozygotes displayed intraneuronal accumulation of Aβ oligomers at 8 months, followed by abnormal tau phosphorylation, synapse loss, glial activation, and neuron loss. These pathologies were not observed at younger ages, suggesting that a certain mechanism other than Aβ accumulation underlies the memory disturbance at 4 months. For the electrophysiology studies at 4 months, high-frequency stimulation evoked long-term potentiation in all mice in the presence of picrotoxin, but in the absence of picrotoxin, such potentiation was observed only in homozygotes, suggesting their GABAergic deficit. In support of this, the levels of GABA-related proteins and the number of dentate GABAergic interneurons were decreased in 4-month-old homozygotes. Since APP has been shown to play a role in dentate GABAergic synapse formation, the observed GABAergic depletion is likely associated with an impairment of the APP function presumably caused by the Osaka mutation. Oral administration of diazepam to homozygotes from 6 months improved memory at 8 months, and furthermore, prevented Aβ oligomer accumulation, indicating that GABAergic deficiency is a cause of memory impairment and also a driving force of Aβ accumulation. Our findings suggest that the Osaka mutation causes loss of APP function, leading to GABAergic depletion and memory disorder when wild-type APP is absent, providing a mechanism of the recessive heredity.

Dataset Information

Loss-of-function phenotype of a PKC?T219A knockin mouse strain.

Publications

Loss-of-function phenotype of a PKCθ<sup>T219A</sup> knockin mouse strain.

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