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        The Cytoskeleton as a Pharmacological Target in Neurodegenerative Diseases

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        The cytoskeleton plays a key role in maintaining the highly asymmetrical shape and structural polarity of neurons that are essential for neuritogenesis and neuronal physiology. In neurodegenerative diseases, the cytoskeleton is abnormally assembled and impairment of neurotransmission occurs. Cumulative evidence suggests that neurodegenerative diseases and psychiatric illnesses are associated with cytoskeletal alterations in neurons that, in turn, lose synaptic connectivity and the ability to transmit incoming axonal information to the somatodendritic domain. Major components of the neuronal cytoskeleton are the microtubules (MT). MTs, composed of the tubulin heterodimer backbone and decorated/regulated by MT-associated proteins (MAPs), provide a dynamic skeleton for cellular structures as well as trails for motor protein movement of important cargo.
        The following suggested set of selected assays are intended to evaluate MT changes and effects by candidate drugs. These methods assess different characteristics of MTs in versatile assays as follows. (1) MT polymerization assays. (2) Quantifying polymerized vs. soluble tubulin by fractionation of tubulin pools and evaluating percent of polymerized tubulin as well as MAPs, by size fractionations immunoassays. (3) Tubulin isoform expression, for example, beta 3 tubulin expression at the level of RNA and protein as a marker for neuronal differentiation and neurite outgrowth using quantitative reverse transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunofluorescence. (4) The microtubule-associated protein 2, MAP2 expression as a marker for neurite outgrowth and neuronal survival. (5) The microtubule-associated protein tau expression, phosphorylation, and aggregation as a marker for tau pathology (tauopathy), a major hallmark of Alzheimer’s disease (AD). (6) MT dynamics by distinguishing stable and dynamic microtubules through their content of posttranslationally modified α-tubulin. MTs that are rich in tyrosinated/detyrosinated tubulin are visualized using specific antibodies and immunofluorescence. (7) Measurements of cellular/axoplasmic transport.
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