aPKC phosphorylation of HDAC6 results in increased deacetylation activity.

The Class II histone deacetylase, HDAC6, has been shown to be involved in cell motility, aggresome formation and mitochondria transport. HDAC6 deacetylase activity regulates α-tubulin acetylation levels and thus plays a critical role in these processes. In turn, HDAC6 activity can be regulated by interaction with various proteins including multiple kinases. Kinase mediated phosphorylation of HDAC6 can lead to either increased or reduced activity. Our previous research has shown that sequestosome1/p62 (SQSTM1/p62) interacts with HDAC6 and regulates its activity. As SQSTM1/p62 is a scaffolding protein known to interact directly with the zeta isoform of Protein Kinase C (PKCζ), we sought to examine if HDAC6 could be a substrate for PKCζ phosphorylation and if so, how its activity might be regulated. Our data demonstrate that HDAC6 is not only present in a protein complex with PKCζ but can also be phosphorylated by PKCζ. We also show that specific phosphorylation of HDAC6 by PKCζ increases HDAC6 deacetylase activity resulting in reduced acetylated tubulin levels. Our findings provide novel insight into the molecular mechanism by which HDAC6, PKCζ and SQSTM1/p62 function together in protein aggregate clearance. These results also highlight a new research direction which may prove fruitful for understanding the underlying cause of several neurodegenerative diseases.

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.

Interaction of SQSTM1 with the motor protein dynein--SQSTM1 is required for normal dynein function and trafficking.

The dynein motor protein complex is required for retrograde transport of vesicular cargo and for transport of aggregated proteins along microtubules for processing and degradation at perinuclear aggresomes. Disruption of this process leads to dysfunctional endosome accumulation and increased protein aggregation in the cell cytoplasm, both pathological features of neurodegenerative diseases. However, the exact mechanism of dynein functionality in these pathways is still being elucidated. Here, we show that the scaffolding protein SQSTM1 directly interacts with dynein through a previously unidentified dynein-binding site. This interaction is independent of HDAC6, a known interacting protein of both SQSTM1 and dynein. However, knockdown of HDAC6 increases the interaction of SQSTM1 with dynein, indicating a possible competitive interaction. Using different dynein cargoes, we show that SQSTM1 is required for proper dynein motility and trafficking along microtubules. Based on our results, we propose a new model of competitive interaction between SQSTM1 and HDAC6 with dynein. In this model, SQSTM1 would not only affect the association of polyubiquitylated protein aggregates and endosomes with dynein, but would also be required for normal dynein function.

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.

Behavioral effects of SQSTM1/p62 overexpression in mice: support for a mitochondrial role in depression and anxiety.

Affective spectrum and anxiety disorders have come to be recognized as the most prevalently diagnosed psychiatric disorders. Among a suite of potential causes, changes in mitochondrial energy metabolism and function have been associated with such disorders. Thus, proteins that specifically change mitochondrial functionality could be identified as molecular targets for drugs related to treatment for affective spectrum disorders. Here, we report generation of transgenic mice overexpressing the scaffolding and mitophagy related protein Sequestosome1 (SQSTM1/p62) or a single point mutant (P392L) in the UBA domain of SQSTM1/p62. We show that overexpression of SQSTM1/p62 increases mitochondrial energy output and improves transcription factor import into the mitochondrial matrix. These elevated levels of mitochondrial functionality correlate directly with discernible improvements in mouse behaviors related to affective spectrum and anxiety disorders. We also describe how overexpression of SQSTM1/p62 improves spatial learning and long term memory formation in these transgenic mice. These results suggest that SQSTM1/p62 provides an attractive target for therapeutic agents potentially suitable for the treatment of anxiety and affective spectrum disorders.

A role for sequestosome 1/p62 in mitochondrial dynamics, import and genome integrity.

As a signaling scaffold, p62/sequestosome (p62/SQSTM1) plays important roles in cell signaling and degradation of misfolded proteins. While localization of p62 to mitochondria has been reported, a description of its function once there, remains unclear. Here, we report that p62 is localized to mitochondria in non-stressed situations and demonstrate that deficiency in p62 exacerbates defects in mitochondrial membrane potential and energetics leading to mitochondrial dysfunction. We report on the relationship between mitochondrial protein import and p62. In a p62 null background, mitochondrial import of the mitochondrial transcription factor TFAM is disrupted. When p62 is returned, mitochondrial function is restored to more normal levels. We identify for the first time that p62 localization plays a role in regulating mitochondrial morphology, genome integrity and mitochondrial import of a key transcription factor. We present evidence that these responses to the presence of p62 extend beyond the protein's immediate influence on membrane potential.

Mining the TRAF6/p62 interactome for a selective ubiquitination motif.

A new approach is described here to predict ubiquitinated substrates of the E3 ubiquitin ligase, TRAF6, which takes into account its interaction with the scaffold protein SQSTM1/p62. A novel TRAF6 ubiquitination motif defined as [-(hydrophobic)-k-(hydrophobic)-x-x-(hydrophobic)- (polar)-(hydrophobic)-(polar)-(hydrophobic)] was identified and used to screen the TRAF6/p62 interactome composed of 155 proteins, that were either TRAF6 or p62 interactors, or a negative dataset, composed of 54 proteins with no known association to either TRAF6 or p62. NRIF (K19), TrkA (K485), TrkB (K811), TrkC (K602 and K815), NTRK2 (K828), NTRK3 (K829) and MBP (K169) were found to possess a perfect match for the amino acid consensus motif for TRAF6/p62 ubiquitination. Subsequent analyses revealed that this motif was biased to the C-terminal regions of the protein (nearly 50% the sites), and had preference for loops (~50%) and helices (~37%) over beta-strands (15% or less). In addition, the motif was observed to be in regions that were highly solvent accessible (nearly 90%). Our findings suggest that specific Lysines may be selected for ubiquitination based upon an embedded code defined by a specific amino acid motif with structural determinants. Collectively, our results reveal an unappreciated role for the scaffold protein in targeting ubiquitination. The findings described herein could be used to aid in identification of other E3/scaffold ubiquitination sites.

Contribution of the live-vertebrate trade toward taxonomic homogenization.

The process of taxonomic homogenization occurs through two mechanisms, extinctions and introductions, and leads to a reduction of global biodiversity. We used available U.S. trade data as a proxy for global trade in live vertebrates to assess the contribution of trade to the process of taxonomic homogenization. Data included all available U.S. importation and exportation records, estimation of extinction risk, and reports of establishment outside the native range for species within six vertebrate groups. Based on Monte Carlo sampling, the number of species traded, established outside of the native range, and threatened with extinction was not randomly distributed among vertebrate families. Twenty-eight percent of vertebrate families that were traded preferentially were also established or threatened with extinction, an unusually high percentage compared with the 7% of families that were not traded preferentially but that became established or threatened with extinction. The importance of trade in homogenization of vertebrates suggests that additional efforts should be made to prevent introductions and extinctions through this medium.

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 Alzheimer's disease (AD), Parkinson disease (PD), Huntington disease (HD), Frontotemporal dementia (FTD), and Pick's 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.

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.

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 3beta, 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.

Old mice, young islands and competing biogeographical hypotheses.

Naturally occurring variation within a small rodent species native to the southeastern USA, Peromyscus polionotus, has interested biologists for nearly a century. This species has contributed significantly to our understanding of geographical variation and has often been presented as an example of adaptive evolution. Much of the interest in this organism has been predicated on assumptions that the species is relatively young (<300 000 bp) and that coastal populations have a very recent history (<10 000 bp). To test these assumptions and the prevailing biogeographical hypothesis (Recurrent Invasion), we examined nucleotide sequence data from the cytochrome b and D-loop mitochondrial regions (2449 bp) for 79 samples of P. polionotus collected across the Gulf Coast region of Florida and Alabama. Samples representing Peromyscus maniculatus bairdii, P. m. sonoriensis, P. m. pallescens, and P. keeni were used as outgroups. The degree of cytochrome b divergence (approximately 4.4%) between P. maniculatus and P. polionotus was higher than expected. Analyses consistently indicated that three distinct groups are represented within P. polionotus from the Gulf Coast region. Among these, coastal populations (beach mice) form a monophyletic group and apparently represent a substantially older group (approximately 200 000 year. separation) than previously recognized. Our results were counter to the core assumptions of the existing biogeographical model but were consistent with an alternative hypothesis (Shore-line Tracking) which provides a more parsimonious explanation for the observed patterns. This research provides new insight into the evolutionary history of P. polionotus and highlights the importance of considering biogeographical history when evaluating extant patterns of natural variation.

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 Alzheimer's disease and other neurodegenerative diseases. Herein, we review p62's role in signaling, aggregation, and inclusion formation, and specifically as a possible contributor to Alzheimer's disease. The use of p62 as a potential target for the development of therapeutics and as a disease biomarker is also discussed.

Analysis of genetic variation in ribosomal DNA internal transcribed spacer and the NADH dehydrogenase subunit 4 mitochondrial genes of the onchocerciasis vector Simulium ochraceum.

Onchocerciasis is a serious disease vectored by black flies in the genus Simulium that are infected with the filarial parasite Onchocerca volvulus. In the Americas, black flies of the Simulium ochraceum s.l. species complex are important vectors of this parasite. Cytological studies have suggested that this species complex consists of at least three cytotypes that inhabit distinct habitats. In this study, the NADH dehydrogenase subunit four (ND4) and internal transcribed spacer (ITS) of the ribosomal RNA gene cluster were used to explore the degree of genetic diversity among S. ochraceum s.l. populations found in the three O. volvulus foci in Mexico. Both sequence regions were found to exhibit intra- and interpopulation variation. Four different ND4 alleles were found among the populations examined. Similarly, variation was noted in the ITS domain sequences within and among populations. Variation within the ITS sequence was primarily confined to a complex microsatellite locus. Four ITS length variants were observed, two of which were only seen in flies collected from the onchocerciasis focus in northern Chiapas. These data suggest that the ND4 and ITS sequences may prove to be useful markers for exploring interactions within and among the S. ochraceum s.l. populations in Mexico.

Effectiveness of a regional corridor in connecting two Florida black bear populations.

Corridors may mitigate the adverse effects of habitat fragmentation by restoring or maintaining connectivity between disjunct populations. The efficacy of corridors for large carnivores, however has rarely been evaluated objectively. We used noninvasive sampling, microsatellite analysis, and population assignment tests to evaluate the effectiveness of a regional corridor in connecting two Florida black bear (Ursus americanus floridanus) populations (Osceola and Ocala). Bear movement was predominantly unidirectional, with a limited mixing of individuals from the two populations in one area of the corridor We also documented bears in Osceola that were genetically assigned to Ocala and bears in Osceola that may be offspring from an Osceola-Ocala mating. Our results indicate that the Osceola-Ocala corridor is functional and provides a conduit for gene flow between these populations. Human development, however may hinder the use of the Osceola-Ocala corridor by bears. The noninvasive sampling and genetic methods we used provide a means of evaluating corridor effectiveness that can help identify linkages necessary for maintaining metapopulation structure and population viability.

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 (OTU's), representing a range of tree shapes, were used. Felsenstein's 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.