Our long-range goal is to elucidate the potential role of isoaspartyl (isoAsp) protein formation in brain aging and age-related neurological disease. Accumulation of isoAsp sites is a major form of protein damage that is normally kept in check by protein L-isoaspartyl methyltransferase (PIMT), a repair enzyme that is highly enriched in brain. PIMT knockout mice accumulate high levels of isoAsp-damaged proteins, especially in the brain and testes, and their phenotype is mainly neurological;increased brain size, abnormal neuronal physiology and metabolic signaling pathways, decreased cognitive function, and fatal epilepsy at 4-10 weeks after birth. IsoAsp formation can disrupt protein function, can elicit auto-immunity, and is often accompanied by formation of protein aggregates in vitro. We hypothesize that inefficiencies in the repair of isoAsp sites in neurons contributes significantly to the neurodegeneration that occurs in advanced age and in certain brain diseases. Our recent findings also suggest that isoAsp formation may underlie a novel form of protein aggregation that involves covalent cross-linking. We propose to explore these ideas via the following four specific aims.
Aim 1 will compare brain extracts of PIMT -/- mice with wild type littermates to see if complete loss of this key repair enzyme leads to (a) isoAsp accumulation in synuclein and tau, two well studied proteins involved in several forms of neurodegeneration that have been reported by others to be highly susceptible to isoAsp formation, (b) aggregation of synuclein, tau, and collapsin-response mediator protein 2 (CRMP2), and (c) hyperphosphorylation of proteins in general, and site-specific hyperphosphorylation of tau and CRMP2.
Aim 2 will compare PIMT mice (which express 50-55% of normal PIMT activity) vs. wild type littermates to see how a moderate reduction of PIMT activity in vivo alters isoAsp accumulation, protein aggregation, and protein hyperphosphorylation (as in Aim 1) as a function of age.
Aims 3 stems from our recent findings that recombinant mouse CRMP2 undergoes isoAsp formation concomitant with formation of SDS-insoluble aggregates when it is aged in vitro at pH 7.4 and 37degC. We will carry out a series of studies to determine if, as we suspect from recent data, this aggregation involves covalent cross-linking and is mechanistically related to isoAsp formation.
Aim 4 will compare PIMT -/- mice with wild type littermates to look for functional changes in neuronal enzymes that accumulate isoAsp in vivo. We will initially focus on creatine kinase B (brain specific isoform) and the 70 kDa heat shock cognate protein (HSC70) which are among the 22 proteins we have found that accumulate high levels of isoAsp in the PIMT -/- mouse brain. If the hypotheses that guide these 4 aims are proven to be correct, this would call for pharmacological or genetic interventions that could boost the PIMT repair system to help stave off the decline in human cognitive function and neurological status that occurs in advanced age.

Public Health Relevance

Abnormal protein structures play a major role in degeneration of the human nervous system that occurs in advanced age and in diseases such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). This research will explore the possibility that the severity and time course of age and disease-related cognitive decline is significantly influenced by the status a key metabolic pathway that repairs damaged proteins in nerve cells. The results of this research should help elucidate the molecular basis of dementias and thereby pave the way for new therapies that moderate or delay their onset.

National Institute of Health (NIH)
National Institute of Neurological Disorders and Stroke (NINDS)
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Special Emphasis Panel (ZRG1-CMND-A (02))
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Mamounas, Laura
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University of California Irvine
Schools of Arts and Sciences
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Pulido, Mario A; DerHartunian, Meleeneh Kazarian; Qin, Zhenxia et al. (2016) Isoaspartylation appears to trigger small cell lung cancer-associated autoimmunity against neuronal protein ELAVL4. J Neuroimmunol 299:70-78
Qin, Zhenxia; Zhu, Jeff X; Aswad, Dana W (2016) The D-isoAsp-25 variant of histone H2B is highly enriched in active chromatin: potential role in the regulation of gene expression? Amino Acids 48:599-603
Qin, Zhenxia; Dimitrijevic, Aleksandra; Aswad, Dana W (2015) Accelerated protein damage in brains of PIMT+/- mice; a possible model for the variability of cognitive decline in human aging. Neurobiol Aging 36:1029-36
Qin, Zhenxia; Yang, Jing; Klassen, Henry J et al. (2014) Isoaspartyl protein damage and repair in mouse retina. Invest Ophthalmol Vis Sci 55:1572-9
Dimitrijevic, Aleksandra; Qin, Zhenxia; Aswad, Dana W (2014) Isoaspartyl formation in creatine kinase B is associated with loss of enzymatic activity; implications for the linkage of isoaspartate accumulation and neurological dysfunction in the PIMT knockout mouse. PLoS One 9:e100622
Qin, Zhenxia; Kaufman, Rachel S; Khoury, Rana N et al. (2013) Isoaspartate accumulation in mouse brain is associated with altered patterns of protein phosphorylation and acetylation, some of which are highly sex-dependent. PLoS One 8:e80758
Doyle, Hester A; Aswad, Dana W; Mamula, Mark J (2013) Autoimmunity to isomerized histone H2B in systemic lupus erythematosus. Autoimmunity 46:6-13
Morrison, Gareth J; Ganesan, Ranjani; Qin, Zhenxia et al. (2012) Considerations in the identification of endogenous substrates for protein L-isoaspartyl methyltransferase: the case of synuclein. PLoS One 7:e43288
Khoury, Mitri K; Parker, Ian; Aswad, Dana W (2010) Acquisition of chemiluminescent signals from immunoblots with a digital single-lens reflex camera. Anal Biochem 397:129-31
Carter, Wayne G; Aswad, Dana W (2008) Formation, localization, and repair of L-isoaspartyl sites in histones H2A and H2B in nucleosomes from rat liver and chicken erythrocytes. Biochemistry 47:10757-64

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