Michael C. Wooten

Genetic inactivation of p62 leads to accumulation of hyperphosphorylated tau and neurodegeneration.

The signaling adapter p62 plays a coordinating role in mediating phosphorylation and ubiquitin‐dependent trafficking of interacting proteins. However, there is little known about the physiologic role of this protein in brain. Here, we report age‐dependent constitutive activation of glycogen synthase kinase 3β, protein kinase B, mitogen‐activated protein kinase, and c‐Jun‐N‐terminal kinase in adult p62−/− mice resulting in hyperphosphorylated tau, neurofibrillary tangles, and neurodegeneration. Biochemical fractionation of p62−/− brain led to recovery of aggregated K63‐ubiquitinated tau. Loss of p62 was manifested by increased anxiety, depression, loss of working memory, and reduced serum brain‐derived neurotrophic factor levels. Our findings reveal a novel role for p62 as a chaperone that regulates tau solubility thereby preventing tau aggregation. This study provides a clear demonstration of an Alzheimer‐like phenotype in a mouse model in the absence of expression of human genes carrying mutations in amyloid‐beta protein precursor, presenilin, or tau. Thus, these findings provide new insight into manifestation of sporadic Alzheimer disease and the impact of obesity.

Signaling, Polyubiquitination, Trafficking, and Inclusions: Sequestosome 1/p62's Role in Neurodegenerative Disease

Aggregated misfolded proteins are hallmarks of most neurodegenerative diseases. In a chronic disease state, including pathologic situations of oxidative stress, these proteins are sequestered into inclusions. Accumulation of aggregated proteins can be prevented by chaperones, or by targeting their degradation to the UPS. If the accumulation of these proteins exceeds their degradation, they may impair the function of the proteasome. Alternatively, the function of the proteasome may be preserved by directing aggregated proteins to the autophagy-lysosome pathway for degradation. Sequestosome 1/p62 has recently been shown to interact with polyubiquitinated proteins through its UBA domain and may direct proteins to either the UPS or autophagosome. P62 is present in neuronal inclusions of individuals with Alzheimers disease and other neurodegenerative diseases. Herein, we review p62s role in signaling, aggregation, and inclusion formation, and specifically as a possible contributor to Alzheimers disease. The use of p62 as a potential target for the development of therapeutics and as a disease biomarker is also discussed.

Genetic variability and systematics of Gambusia in the southeastern United States.

Mosquitofish were sampled from 76 locations in 19 drainages in the southeastern United States. Thirteen polymorphic loci were resolved for fish from each location. Populations from eastern drainages had significantly higher levels of heterozygosity (H = 0.113) than those in western drainages (H = 0.055). Abrupt changes in allele frequencies for several loci occurred in the area of Mobile Bay, which corresponds with discontinuities previously reported for chromosomal and morphological data. Based on geographic patterns of allele frequencies, abrupt differentiation in local genetic constituency (DR = 0.443 between eastern Gambusia affinis holbrooki and G. a. afinis) and previously reported data on the existence of a reproductive barrier between the forms, the original taxonomic designations recognizing G. holbrooki and G. affinis as separate species are preferred. Populations in drainages west of Mobile Bay should be considered G. affinis, with those east of this divide being G. holbrooki.

Large mammals are genetically less variable

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Age-associated oxidative damage to the p62 promoter: implications for Alzheimer disease.

The absence of the p62 gene in mouse brain leads to biochemical and cognitive deficits that resemble Alzheimer disease (AD). In this context, the objective of this study was to examine the relationship between age-induced oxidative damage to the p62 promoter and AD. Increased 8-OHdG staining, a marker of oxidative stress, was observed in brain sections from mice deficient in the p62 gene compared to control. Treatment of MEF cells deficient in p62 with H(2)O(2) resulted in decreased cell survival and an absence of Nrf2 nuclear translocation. The mouse p62 promoter exhibited elevated oxidative damage with increasing age, and the degree of p62 promoter damage was also age-correlated in human brain samples. In human subjects, the expression of p62 was decreased in AD brain relative to age-matched controls, and likewise decreased p62 expression correlated with oxidative damage to the promoter. Treatment of HEK cells with H(2)O(2) resulted in decreased p62 expression concomitant with increased promoter damage. Consistent with these findings, a transgenic AD mouse model also exhibited increased p62 promoter damage and reduced p62 levels in brain. Altogether, our results reveal that oxidative damage to the p62 promoter correlates with decreased expression of p62 and may contribute to age-associated neurodegenerative disease such as AD and others.

Oxidative damage to the promoter region of SQSTM1/p62 is common to neurodegenerative disease

Recently we reported that declined SQSTM1/p62 expression in Alzheimer disease brain was age-correlated with oxidative damage to the p62 promoter. The objective of this study was to examine whether oxidative damage to the p62 promoter is common to DNA recovered from brain of individuals with neurodegenerative disease. Increased 8-OHdG staining was observed in brain sections from Alzheimers disease (AD), Parkinson disease (PD), Huntington disease (HD), Frontotemporal dementia (FTD), and Picks disease compared to control subjects. In parallel, the p62 promoter exhibited elevated oxidative damage in samples from various diseases compared to normal brain, and damage was negatively correlated with p62 expression in FTD samples. Oxidative damage to the p62 promoter induced by H2O2 treatment decreased its transcriptional activity. In keeping with this observation, the transcriptional activity of a Sp-1 element deletion mutant displayed reduced stimulus-induced activity. These findings reveal that oxidative damage to the p62 promoter decreased its transcriptional activity and might therefore account for decreased expression of p62. Altogether these results suggest that pharmacological means to increase p62 expression may be beneficial in delaying the onset of neurodegeneration.

Transport of protein kinase C isoforms to the nucleus of PC12 cells by nerve growth factor: association of atypical zeta-PKC with the nuclear matrix.

In an effort to understand the role of protein kinase C (PKC) in nerve growth factor‐induced differentiation, we studied the expression of PKC using isoform‐specific antibodies. Western blot analysis on whole cell lysates showed that α,β∥,γ,δ,ϵ,ζ, ı/λ and μ were expressed in PC12 cells, except for θ which was absent. In nuclei obtained from control PC12 cells, small amounts of δ, ϵ, ı/λ and ζ were detected. A computer‐assisted search algorithm was used to search for the presence of bipartite nuclear targeting motifs. In classical PKC isoforms α,β∥,γ, two bipartite motifs were present, while atypical ı/λ and ζ‐PKC displayed one motif, whereas novel PKC isoforms did not exhibit any bipartite motif structure. Treatment of cells with differentiating doses of nerve growth factor (NGF) resulted in changes of differential magnitude for all of the nuclear PKC isoforms in response to NGF. However, little change in γ‐PKC was observed in response to NGF. This analysis indicated that other factors may contribute to transport of PKC into the nucleus, in addition to the bipartite motif itself. Atypical ζ‐PKC is required for NGF‐induced neurite outgrowth of PC12 cells (Coleman and Wooten: J Mol Neurosci 5:39–57, 1994). Increases in nuclear ζ‐PKC were NGF dose‐dependant with a concomitant decrease in cytoplasmic immunoreactivity. The localization of ζ‐PKC was investigated by means of immunoelectron microscopy which revealed the localization of this isoform within the inner nuclear matrix bound to chromatin. Taken together, these findings suggest that ζ‐PKC may be involved in the regulation of nuclear processes. J. Neurosci. Res. 49:393–403, 1997.

SQSTM1/p62 interacts with HDAC6 and regulates deacetylase activity.

Protein aggregates can form in the cytoplasm of the cell and are accumulated at aggresomes localized to the microtubule organizing center (MTOC) where they are subsequently degraded by autophagy. In this process, aggregates are engulfed into autophagosomes which subsequently fuse with lysosomes for protein degradation. A member of the class II histone deacetylase family, histone deacetylase 6(HDAC6) has been shown to be involved in both aggresome formation and the fusion of autophagosomes with lysosomes making it an attractive target to regulate protein aggregation. The scaffolding protein sequestosome 1(SQSTM1)/p62 has also been shown to regulate accumulation and autophagic clearance of protein aggregates. Recent studies have revealed colocalization of HDAC6 and p62 to ubiquitinated mitochondria, as well as, ubiquitinated protein aggregates associated with the E3 ubiquitin ligase TRIM50. HDAC6 deacetylase activity is required for aggresome formation and can be regulated by protein interaction with HDAC6. Due to their colocalization at ubiquitinated protein aggregates, we sought to examine if p62 specifically interacted with HDAC6 and if so, if this interaction had any effect on HDAC6 activity and/or the physiological function of cortactin-F-actin assembly. We succeeded in identifying and mapping the direct interaction between HDAC6 and p62. We further show that this interaction regulates HDAC6 deacetylase activity. Data are presented demonstrating that the absence of p62 results in hyperactivation of HDAC6 and deacetylation of α-tubulin and cortactin. Further, upon induction of protein misfolding we show that p62 is required for perinuclear co-localization of cortactin-F-actin assemblies. Thus, our findings indicate that p62 plays a key role in regulating the recruitment of F-actin network assemblies to the MTOC, a critical cellular function that is required for successful autophagic clearance of protein aggregates.

Evaluation of the restricted maximum-likelihood method for estimating phylogenetic trees using simulated allele-frequency data

Comparisons are made of the accuracy of the restricted maximum‐likelihood, Wagner parsimony, and UPGMA (unweighted pair‐group method using arithmetic averages) clustering methods to estimate phylogenetic trees. Data matrices were generated by constructing simulated stochastic evolution in a multidimensional gene‐frequency space using a simple genetic‐drift model (Brownian‐motion, random‐walk) with constant rates of divergence in all lineages. Ten differentphylogenetic tree topologies of 20 operational taxonomic units (OTUs), representing a range of tree shapes, were used. Felsensteins restricted maximum‐likelihood method, Wagner parsimony, and UPGMA clustering were used to construct trees from the resulting data matrices. The computations for the restricted maximum‐likelihood method were performed on a Cray‐1 supercomputer since the required calculations (especially when optimized for the vector hardware) are performed substantially faster than on more conventional computing systems. The overall level of accuracy of tree reconstruction depends on the topology of the true phylogenetic tree. The UPGMA clustering method, especially when genetic‐distance coefficients are used, gives the most accurate estimates of the true phylogeny (for our model with constant evolutionary rates). For large numbers of loci, all methods give similar results, but trends in the results imply that the restricted maximum‐likelihood method would produce the most accurate trees if sample sizes were large enough.

Use of the Open Field Maze to Measure Locomotor and Anxiety-like Behavior in Mice

Animal models have proven to be invaluable to researchers trying to answer questions regarding the mechanisms of behavior. The Open Field Maze is one of the most commonly used platforms to measure behaviors in animal models. It is a fast and relatively easy test that provides a variety of behavioral information ranging from general ambulatory ability to data regarding the emotionality of the subject animal. As it relates to rodent models, the procedure allows the study of different strains of mice or rats both laboratory bred and wild-captured. The technique also readily lends itself to the investigation of different pharmacological compounds for anxiolytic or anxiogenic effects. Here, a protocol for use of the open field maze to describe mouse behaviors is detailed and a simple analysis of general locomotor ability and anxiety-related emotional behaviors between two strains of C57BL/6 mice is performed. Briefly, using the described protocol we show Wild Type mice exhibited significantly less anxiety related behaviors than did age-matched Knock Out mice while both strains exhibited similar ambulatory ability.