p62 Pathology Model in the Rat Substantia Nigra with Filamentous Inclusions and Progressive Neurodegeneration

Abstract

One of the proteins most frequently found in neuropathological lesions is the ubiquitin binding protein p62 (sequestosome 1). Post-mortem analysis of p62 is a defining diagnostic marker in several neurodegenerative diseases including amyotrophic lateral sclerosis and inclusion body myositis. Since p62 functions in protein degradation pathways including autophagy, the build-up of p62-positive inclusions suggests defects in protein clearance. p62 was expressed unilaterally in the rat substantia nigra with an adeno-associated virus vector (AAV9) in order to study p62 neuropathology. Inclusions formed within neurons from several days to several weeks after gene transfer. By electron microscopy, the inclusions were found to contain packed 10 nm thick filaments, and mitochondria cristae structure was disrupted, resulting in the formation of empty spaces. In corollary cell culture transfections, p62 clearly impaired mitochondrial function. To probe for potential effects on macroautophagy, we co-expressed p62 with a double fluorescent tagged reporter for the autophagosome protein LC3 in the rat. p62 induced a dramatic and specific dissociation of the two tags. By 12 weeks, a rotational behavior phenotype manifested, consistent with a significant loss of dopaminergic neurons analyzed post-mortem. p62 overexpression resulted in a progressive and robust pathology model with neuronal inclusions and neurodegeneration. p62 gene transfer could be a novel methodological probe to disrupt mitochondrial function or autophagy in the brain and other tissues in vivo.

Axonal Protection by Ripasudil, a Rho Kinase Inhibitor, via Modulating Autophagy in TNF-Induced Optic Nerve Degeneration

“…8 One recent study has reported that p62 transfection resulted in mitochondrial dysfunction and progressive neurodegeneration in the rat brain. 9 Consistent with our previous results, 12 the current study also confirmed upregulated p62 levels in optic nerve degeneration, which was prevented by ripasudil. p62 may correlate with autophagic flux, and decreases in its level indicate autophagy induction while increases indicate autophagy inhibition.…”

“…[5][6][7] Some recent studies have suggested a detrimental role of p62 (SQSTM1) with regulation of autophagy in certain conditions in neurodegeneration. [8][9][10] We previously reported upregulated p62 in the optic nerve in two different models such as a hypertensive glaucoma model 11 and TNF-induced axon damage model. 12 Microtubule-associated protein light chain 3 (LC3) is distributed in autophagosomes 13 and LC3-II correlates with autophagosomes numbers.…”

Cre‐dependent AAV vectors for highly targeted expression of disease‐related proteins and neurodegeneration in the substantia nigra

“…We conclude that a subtoxic level of a-synuclein was expressed in this study compared with previous studies in which AAV a-synuclein gene transfer did induce a lesion to the nigrostriatal system (4,5). Importantly, there were no untoward effects of expressing YFP in the Cre-targeted manner detected under these conditions because high levels of a control fluorescent protein in neurons can become toxic, as with green fluorescent protein (31,32).…”

“…Immunofluorescent or immunoperoxidase staining procedures followed previously described methods (4,31). The primary antibodies and their dilutions were as follows: anti-tyrosine hydroxylase from Pel-Freez (Rogers, AR, USA) at 1:2000; anti-a-synuclein at 1:2000 (NACP-98, kindly provided by M. Farrer, University of British Columbia, Vancouver, BC, Canada); anti-tau at 1:2000 (Tau-5, kindly provided by L. Petrucelli, Mayo Clinic, Rochester, MN, USA); and anti-TDP-43 from Abnova (Taipei, Taiwan) at 1:2000 (human TDP-43 specific).…”

Filamentous Aggregation of Sequestosome-1/p62 in Brain Neurons and Neuroepithelial Cells upon Tyr-Cre-Mediated Deletion of the Autophagy Gene Atg7

“…Previous studies have suggested that protein aggregates containing endogenous p62 have a filamentous organization, e.g., in dendrites of dopaminergic neurons of mice that carry a tyrosine hydroxylase (TH) cell-specific deletion of Atg7 ( Atg7 f/f ;TH-IRES-Cre ) [ 43 ]. However, only recently evidence from studies involving recombinant p62 in vitro [ 55 , 56 ] and recombinant p62 expressed by an adeno-associated virus vector injected into the rat substantia nigra [ 57 ] have suggested that p62 itself is sufficient to form filaments. The structure and the dimensions of the p62 filaments in neurons and neuroepithelial cells of Atg7 f/f Tyr-Cre mice is similar or identical to those reported for pure p62 [ 55 ] indicating that endogenous p62 is the main, if not the only, component of these filaments.…”