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Analysis of Conditional Knock-Out of Calpain Small Subunit, Capns1, in Central Nervous System Development and Function

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Analysis of Conditional Knock-Out of Calpain Small Subunit, Capns1, in Central Nervous System Development and Function Analysis of Conditional Knock-out of Calpain Small Subunit, capns1, in Central Nervous System Development and Function MANDANA AMINI Thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for Doctorate in Philosophy degree in Neuroscience Department of Cellular and Molecular Medicine Faculty of Medicine University of Ottawa © Mandana Amini, Ottawa, Canada, 2014 Abstract Calpains, a highly conserved family of calcium-dependent cysteine proteases, are divided in two groups; classical and non-conventional calpains. Calpain-1 and calpain-2, the classical ones, are ubiquitously expressed and abundant in the CNS. Findings through different experimental approaches, predominantly pharmacological calpain inhibitors, proposed the necessity of the proteases for the modulation of various biological events particularly in the CNS, or a functional link between calpain and neurodegeneration. Significant functions associated with calpain activity are neuronal proliferation/differentiation, signal transduction, apoptosis, and synaptic plasticity; or neuronal death in Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, and ischemic stroke. However, due to limited insights of the approaches taken, such as non- specificity of the inhibitors, the exact roles of calpains in the CNS and the key mechanisms underlying them remain controversial. Calpain-1/calpain-2 germline knock- out are embryonic lethal at a very early stage hindering the use of these lines as mouse models for CNS studies. Accordingly, this thesis research introduced a unique brain- specific calpain-1/calpain-2 knock-out and explored the role of the proteases in brain development/function and in neuronal death. The first set of analyses examined how the elimination of calpain-1/calpain-2 activities in mouse brain impacts CNS development in general and synaptic plasticity in CA1 neurons of hippocampus. CNS-specific elimination of CAPNS1, the common small subunit, abolished calpain-1/calpain-2 activities in mouse brain. In contrast to Calpain-1/calpain-2 germ line knock-outs, the brain-specific knock-outs are viable and the general development of mouse brain is normal. However, morphology of dendrites in pyramidal neurons of the hippocampal II CA1 region showed significantly decreased dendritic branching complexity and spine density. Consistent with dendrite morphological abnormalities, electrophysiological analyses revealed a significant decrease in field excitatory postsynaptic potentials, long term potentiation, and learning and memory in the hippocampal CA1 neurons of the mutants. In the second part of this research we investigated the direct role of the calpains in neuronal death and their potential downstream targets in in vitro models of PD and ischemic stroke. Our findings indicated that ablation of calpains activity improves survival of different types of neurons against mitochondrial toxin 1-methyl-4- phenylpyridinium (MPP+), glutamate, and hypoxia. Importantly, we demonstrated an increase in p35-cleavage to p25, a cyclin dependent kinase 5 (Cdk5) activator, and that restoration of p25 significantly suppresses the neuronal survival associated with calpain deficiency. Taken together, this work unequivocally establishes two central roles of calpain-1/calpain-2 in CNS function in plasticity and neuronal death. III Dedication For the most loving ones in my life My beloved parents, For the person that I am today My sisters Atoosa, Maryam and Mina, For their love and support through my life My love Adrian and my sweet angel Hannah Eliza, For their unconditional love and devotion IV Table of contents Abstract ............................................................................................................................. II Dedication ........................................................................................................................ IV Table of contents .............................................................................................................. V Acknowledgement ........................................................................................................ VIII List of table and figures ................................................................................................... X List of abbreviations ..................................................................................................... XII List of publications ....................................................................................................... XVI Thesis format .............................................................................................................. XVII Chapter 1 ........................................................................................................................... 1 General Introduction ........................................................................................................ 1 1.1. Calpain System ......................................................................................................... 2 1.2. Calpain Structure ...................................................................................................... 5 1.3. Calpain activity ........................................................................................................ 9 1.4. Calpain substrates ................................................................................................... 11 1.5. Calpain inhibitors ................................................................................................... 13 1.6. Physiological functions of calpain in the CNS ....................................................... 15 1.6.1. Cell proliferation ......................................................................................... 16 1.6.2. Apoptosis .................................................................................................... 17 1.6.3. Dendrite and spine development ................................................................. 20 1.6.4. Synaptic plasticity ....................................................................................... 25 1.6.4.1. Excitatory synaptic transmission ............................................. 30 1.6.4.2. Ionotropic glutamate receptors ................................................ 34 1.6.4.3. Postsynaptic structural and molecular modifications in LTP .. 37 1.6.4.4. Calpain and excitatory synapse and LTP ................................ 40 1.7. Pathological functions of calpain in the CNS ........................................................ 48 1.7.1. Parkinson’s disease (PD) ............................................................................ 48 1.7.1.1. Pathology and etiology of PD ................................................. 49 1.7.1.2. MPTP: Parkinson-related toxin ............................................... 51 1.7.1.3. Pathogenesis of PD .................................................................. 52 1.7.1.4. Cell death pathways in PD ...................................................... 55 V 1.7.1.5. Calpain in PD .......................................................................... 56 1.7.2. Stroke in the CNS ....................................................................................... 57 1.7.2.1. Ischemic stroke and associated experimental models ............. 58 1.7.2.2. Pathophysiology of ischemic stroke ........................................ 59 1.7.2.3. Calpain in ischemic stroke ...................................................... 62 1.7.3. Calpain and Cdk5/p35/p25 system ............................................................. 64 1.8. Statement of research problem, rational and objectives ......................................... 67 Chapter 2 ......................................................................................................................... 69 Conditional disruption of calpain in the CNS alters dendrite morphology and impairs LTP and learning and memory ....................................................................... 69 Statement of author contribution ................................................................................... 70 Abstract ......................................................................................................................... 73 Introduction ................................................................................................................... 74 Materials and methods .................................................................................................. 77 Results ........................................................................................................................... 86 Discussion ................................................................................................................... 124 Chapter 3 ....................................................................................................................... 128 Classical calpain promotes neuronal death by cleavage of p35, a cdk5 co-activator, to p25 in Parkinson’s and ischemic related injuries .................................................. 128 Statement of author contribution ................................................................................. 129 Abstract ....................................................................................................................... 131 Introduction ................................................................................................................
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