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Title: Investigating the role of mitogen activated protein kinase phosphatase-2 on CNS function
Author: Rahman, Nor Zaihana Abdul
ISNI:       0000 0004 5347 4568
Awarding Body: University of Strathclyde
Current Institution: University of Strathclyde
Date of Award: 2015
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Mitogen-activated protein kinase phosphatases (MKPs) belong to the dual-specificity phosphatase (DUSP) family and are negative regulators of mitogen-activated protein kinases (MAPKs). Recently, MKP-2 has been shown to play a novel role in development, the immune system and cancer. However, our knowledge in relation to its function in the central nervous system (CNS) is limited. Hence, we have utilised novel MKP-2 knockout mice to investigate the role of this phosphatase in CNS function. The effect of MKP-2 deletion on CNS was first investigated using primary hippocampal culture by examining astrocyte proliferation and neurite length using immunocytochemistry techniques performed on cultures 1-7 days in vitro (DIV). To investigate the functional consequence of MKP-2 deletion on CNS at 3, 7 and 11 DIV, intracellular calcium concentrations were determined by fluorescence imaging using Fluo-4 dye. Na+, K+ current, synaptic transmission was also investigated using patch clamp electrophysiology. To examine the effect MKP-2 deletion in whole tissue, standard western blotting techniques were utilised to examine the consequence of MKP-2 deletion on ERK activity in the hippocampus of 3 week old mice. The functional effect of MKP-2 deletion on basal synaptic transmission and paired pulse facilitation (PPF) on acute hippocampal slices, field excitatory postsynaptic potentials (fEPSPs) were recorded from acute hippocampal slices of 3 week old mice. Astrocytes number and neurite length were reduced in 1-3 DIV in MKP-2-/- compared to MKP-2+/+. However, there is no difference in astrocyte proliferation and neurite length in 4-7 DIV. Further investigation into the reduction of neurite length, it was due to impairment in astrocyte function and not account of reduction in astrocyte proliferation. Investigating further into the functional consequences, astrocytic intracellular calcium was reduced at 7 DIV however neuronal intracellular calcium was increased at 11 DIV in MKP-2-/- primary hippocampal culture. Furthermore, Na+ and K+ current were also reduced at 7 DIV. However, spontaneous excitatory postsynaptic current (sEPSC) and synapse number has been shown to increase at 7 and 11 DIV. The mechanism underlying this seems to be not related to ERK phosphorylation as no difference in ERK activation was evident when compared between MKP-2+/+ and MKP-2-/-. In further experiments in acute hippocampal slices, MKP-2 deletion leads to a reduction in ERK activity within the hippocampus. In contrast, increased ERK activity was observed in the heart and liver. Basal synaptic transmission was enhanced in MKP-2-/- mice at high stimulus intensities compared to MKP-2+/+ but PPF was unaltered at all in inter-stimulus times tested (10-500 ms). In summary, in this thesis I demonstrate that even though MKP-2 deletion reduced astrocyte growth and neurite length at 3 DIV, it doesn't affect their functional properties at early development. However, alterations of functional activity at 7 and 11 DIV and acute hippocampal culture suggest that MKP-2 deletion might play a role in functional activity when the neuron is fully developed. This data suggest a novel physiological role for MKP-2 in the brain and might reveal valuable insight for the drug development.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available