RESUMEN
Precise regulation of protein phosphorylation is critical for many cellular processes, and dysfunction in this process has been linked to various neurological disorders and diseases. Protein phosphatase 1 (PP1) is a ubiquitously expressed serine/threonine phosphatase with three major isoforms, (α, ß, γ) and hundreds of known substrates. Previously, we reported that PP1α and PP1γ are essential for the known role of PP1 in synaptic physiology and learning/memory, while PP1ß displayed a surprising opposing function. De novo mutations in PP1ß cause neurodevelopmental disorders in humans, but the mechanisms involved are currently unknown. A Cre-Lox system was used to delete PP1ß specifically in neurons in order to study its effects on developing mice. These animals fail to survive to 3 postnatal weeks, and exhibit deficits in cortical myelination and glutamate release. There was defective compound action potential (CAP) propagation in the optic nerve of the null mice, which was traced to a deficit in the formation of nodes of Ranvier. Finally, it was found that phosphorylation of the PP1ß-specific substrate, myosin light chain 2 (MLC2), is significantly enhanced in PP1ß null optic nerves. Several novel important in vivo roles of PP1ß in neurons were discovered, and these data will aid future investigations in delineating the mechanisms by which de novo mutations in PP1ß lead to intellectual and developmental delays in patients.
RESUMEN
Protein phosphatase 1 (PP1) regulates synaptic plasticity and has been described as a molecular constraint on learning and memory. There are three neuronal isoforms, PP1α, PP1ß, and PP1γ, but little is known about their individual functions. PP1α and PP1γ are assumed to mediate the effects of PP1 on learning and memory based on their enrichment at dendritic spines and their preferential binding to neurabin and spinophilin, major PP1 synaptic scaffolding proteins. However, it was recently discovered that human de novo PP1ß mutations cause intellectual disability, suggesting an important but ill-defined role for PP1ß. In this study, we investigated the functions of each PP1 isoform in hippocampal synaptic physiology using conditional CA1-specific knockout mice. In stark contrast to classic PP1 function, we found that PP1ß promotes synaptic plasticity as well as spatial memory. These changes in synaptic plasticity and memory are accompanied by changes in GluA1 phosphorylation, GluN2A levels, and dendritic spine density and morphology, including silent synapse number. These functions of PP1ß reveal a previously unidentified signaling pathway regulating spine maturation and plasticity, broadening our understanding of the complex role of PP1 in synaptic physiology.
RESUMEN
Reversible phosphorylation is a fundamental regulatory mechanism required for many biological processes and is coordinated by the opposing actions of protein kinases and phosphatases. Protein phosphatase 1 (PP1) is a major protein phosphatase that plays an important role in many fundamental physiological processes including synaptic transmission and memory formation. Here we investigate the regulation of PP1 by prominent signaling proteins and synaptic scaffolds including GSK3ß, inhibitor-2 (I-2), neurabin (Nrb), and actin. While GSK3ß is known to regulate PP1 via phosphorylation of the PP1-binding protein I-2, we found that GSK3ß directly regulates PP1 via inhibitory phosphorylation in neurons. Additionally, using bioluminescence resonance energy transfer (BRET), we found that GSK3ß alters PP1-I-2 interaction in living cells. The effect of GSK3ß on PP1-I-2 interaction is independent of the PP1 C-terminal tail, contrary to predictions based on previous findings from purified proteins. I-2 has been shown to form a trimeric complex with PP1 and Nrb, a major synaptic scaffold for promoting PP1 localization to the actin cytoskeleton. Utilizing BRET, we found that Nrb promotes PP1-actin interaction, however no BRET was detected between I-2 and F-actin. Finally, we found that stabilizing F-actin promotes Nrb-PP1 binding and may also lead to conformational changes between Nrb-I-2 and Nrb-F-actin complexes. Overall, our findings elaborate the dynamic regulation of PP1 complexes by GSK3ß, targeting proteins, and actin polymerization.
Asunto(s)
Citoesqueleto de Actina , Actinas , Proteína Fosfatasa 1/metabolismo , Actinas/metabolismo , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Citoesqueleto de Actina/metabolismo , FosforilaciónRESUMEN
Inhibitor-2 (I-2) is a prototypic inhibitor of protein phosphatase-1 (PP1), a major serine-threonine phosphatase that regulates synaptic plasticity and learning and memory. Although I-2 is a potent inhibitor of PP1 in vitro, our previous work has elucidated that, in vivo, I-2 may act as a positive regulator of PP1. Here we show that I-2 and PP1γ, but not PP1α, positively regulate synaptic transmission in hippocampal neurons. Moreover, we demonstrated that I-2 enhanced PP1γ interaction with its major synaptic scaffold, neurabin, by Förster resonance energy transfer (FRET)/Fluorescence lifetime imaging microscopy (FLIM) studies, while having a limited effect on PP1 auto-inhibitory phosphorylation. Furthermore, our study indicates that the effect of I-2 on PP1 activity in vivo is dictated by I-2 threonine-72 phosphorylation. Our work thus demonstrates a molecular mechanism by which I-2 positively regulates PP1 function in synaptic transmission.
RESUMEN
Nuclear inhibitor of protein phosphatase 1 (NIPP1) is a known regulator of gene expression and plays roles in many physiological or pathological processes such as stem cell proliferation and skin inflammation. While NIPP1 has many regulatory roles in proliferating cells, its function in the central nervous system (CNS) has not been directly investigated. In the present study, we examined NIPP1 CNS function using a conditional knockout (cKO) mouse model in which the Nipp1 gene is excised from neural precursor cells. These mice exhibited severe developmental impairments that led to premature lethality. To delineate the neurological changes occurring in these animals, we first assessed microtubule-associated protein tau, a known target of NIPP1 activity. We found that phosphorylation of tau is significantly enhanced in NIPP1 cKO mice. Consistent with this, we found altered AKT and PP1 activity in NIPP1 cKO mice, suggesting that increased tau phosphorylation likely results from a shift in kinase/phosphatase activity. Secondly, we observed tremors in the NIPP1 cKO mice which prompted us to explore the integrity of the myelin sheath, an integral structure for CNS function. We demonstrated that in NIPP1 cKO mice, there is a significant decrease in MBP protein expression in the cortex, along with deficits in both the conduction of compound action potentials (CAP) and the percentage of myelinated axons in the optic nerve. Our study suggests that NIPP1 in neural precursor cells regulates phosphorylation of tau and CNS myelination and may represent a novel therapeutic target for neurodegenerative diseases.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular , Células-Madre Neurales , Ratones , Animales , Proteína Fosfatasa 1/metabolismo , Fosforilación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células-Madre Neurales/metabolismo , Sistema Nervioso Central/metabolismo , Vaina de Mielina/metabolismoRESUMEN
Arsenic is a well-established carcinogen known to increase mortality, but its effects on the central nervous system are less well understood. Epidemiological studies suggest that early life exposure is associated with learning deficits and behavioral changes. Studies in arsenic-exposed rodents have begun to shed light on potential mechanistic underpinnings, including changes in synaptic transmission and plasticity. However, previous studies relied on extended exposure into adulthood, and little is known about the effect of arsenic exposure in early development. Here, we studied the effects of early developmental arsenic exposure in juvenile mice on synaptic transmission and plasticity in the hippocampus. C57BL/6J females were exposed to arsenic (0, 50 ppb, 36 ppm) via drinking water two weeks prior to mating, with continued exposure throughout gestation and parturition. Electrophysiological recordings were then performed on juvenile offspring prior to weaning. In this paradigm, the offspring are exposed to arsenic indirectly, via the mother. We found that high (36 ppm) and relatively low (50 ppb) arsenic exposure both decreased basal synaptic transmission. A compensatory increase in pre-synaptic vesicular release was only observed in the high-exposure group. These results suggest that indirect, ecologically relevant arsenic exposure in early development impacts hippocampal synaptic transmission and plasticity that could underlie learning deficits reported in epidemiological studies.
RESUMEN
Protein phosphatases, by counteracting protein kinases, regulate the reversible phosphorylation of many substrates involved in synaptic plasticity, a cellular model for learning and memory. A prominent phosphatase regulating synaptic plasticity and neurologic disorders is the serine/threonine protein phosphatase 1 (PP1). PP1 has three isoforms (α, ß, and γ, encoded by three different genes), which are regulated by a vast number of interacting subunits that define their enzymatic substrate specificity. In this review, we discuss evidence showing that PP1 regulates synaptic transmission and plasticity, as well as presenting novel models of PP1 regulation suggested by recent experimental evidence. We also outline the required targeting of PP1 by neurabin and spinophilin to achieve substrate specificity at the synapse to regulate AMPAR and NMDAR function. We then highlight the role of inhibitor-2 in regulating PP1 function in plasticity, including its positive regulation of PP1 function in vivo in memory formation. We also discuss the distinct function of the three PP1 isoforms in synaptic plasticity and brain function, as well as briefly discuss the role of inhibitory phosphorylation of PP1, which has received recent emphasis in the regulation of PP1 activity in neurons.
Asunto(s)
Plasticidad Neuronal/fisiología , Proteína Fosfatasa 1/fisiología , Transmisión Sináptica/fisiología , Animales , Humanos , Proteína Fosfatasa 1/química , Estructura Terciaria de Proteína , Receptores de N-Metil-D-Aspartato/fisiología , Transducción de Señal/fisiologíaRESUMEN
Factors contributing to the high rate of discard among deceased donor kidneys remain poorly understood and the influence of resource limitations of weekends on kidney transplantation is unknown. To quantify this we used data from the Scientific Registry of Transplant Recipients and assembled a retrospective cohort of 181,799 deceased donor kidneys recovered for transplantation from 2000-2013. We identified the impact of the day of the week on the procurement and subsequent utilization or discard of deceased donor kidneys in the United States, as well as report the geographic variation of the impact of weekends on transplantation. Compared with weekday kidneys, organs procured on weekends were significantly more likely to be discarded than transplanted (odds ratio: 1.16; 95% confidence interval: 1.13-1.19), even after adjusting for organ quality (adjusted odds ratio: 1.13; 95% confidence interval: 1.10-1.17). Weekend discards were of a significantly higher quality than weekday discards (Kidney Donor Profile Index: 76.5% vs. 77.3%). Considerable geographic variation was noted in the proportion of transplants that occurred over the weekend. Kidneys available for transplant over the weekend were significantly more likely to be used at larger transplant centers, be shared without payback, and experienced shorter cold ischemia times. Thus, factors other than kidney quality are contributing to the discard of deceased donor kidneys, particularly during weekends. Policy prescriptions, administrative or organizational solutions within transplant programs may potentially mitigate against the recent increase in kidney discards.