An acetylation-phosphorylation switch that regulates tau aggregation propensity and function.

The aberrant accumulation of tau protein is a pathological hallmark of a class of neurodegenerative diseases known as tauopathies, including Alzheimer's disease and related dementias. On the basis of previous observations that tau is a direct substrate of histone deacetylase 6 (HDAC6), we sought to map all HDAC6-responsive sites in tau and determine how acetylation in a site-specific manner affects tau's biophysical properties in vitro Our findings indicate that several acetylation sites in tau are responsive to HDAC6 and that acetylation on Lys-321 (within a KCGS motif) is both essential for acetylation-mediated inhibition of tau aggregation in vitro and a molecular tactic for preventing phosphorylation on the downstream Ser-324 residue. To determine the functional consequence of this HDAC6-regulated phosphorylation event, we examined tau's ability to promote microtubule assembly and found that phosphorylation of Ser-324 interferes with the normal microtubule-stabilizing function of tau. Tau phosphorylation of Ser-324 (pSer-324) has not previously been evaluated in the context of tauopathy, and here we observed increased deposition of pSer-324-positive tau both in mouse models of tauopathy and in patients with Alzheimer's disease. These findings uncover a novel acetylation-phosphorylation switch at Lys-321/Ser-324 that coordinately regulates tau polymerization and function. Because the disease relevance of this finding is evident, additional studies are needed to examine the role of pSer-324 in tau pathobiology and to determine whether therapeutically modulating this acetylation-phosphorylation switch affects disease progression in vivo.

Acetylation of the KXGS motifs in tau is a critical determinant in modulation of tau aggregation and clearance.

The accumulation of hyperphosphorylated tau in neurofibrillary tangles (NFTs) is a neuropathological hallmark of tauopathies, including Alzheimer's disease (AD) and chronic traumatic encephalopathy, but effective therapies directly targeting the tau protein are currently lacking. Herein, we describe a novel mechanism in which the acetylation of tau on KXGS motifs inhibits phosphorylation on this same motif, and also prevents tau aggregation. Using a site-specific antibody to detect acetylation of KXGS motifs, we demonstrate that these sites are hypoacetylated in patients with AD, as well as a mouse model of tauopathy, suggesting that loss of acetylation on KXGS motifs renders tau vulnerable to pathogenic insults. Furthermore, we identify histone deacetylase 6 (HDAC6) as the enzyme responsible for the deacetylation of these residues, and provide proof of concept that acute treatment with a selective and blood-brain barrier-permeable HDAC6 inhibitor enhances acetylation and decreases phosphorylation on tau's KXGS motifs in vivo. As such, we have uncovered a novel therapeutic pathway that can be manipulated to block the formation of pathogenic tau species in disease.

Loss of HDAC6, a novel CHIP substrate, alleviates abnormal tau accumulation.

The abnormal accumulation of the microtubule-binding protein tau is associated with a number of neurodegenerative conditions, and correlates with cognitive decline in Alzheimer's disease. The ubiquitin ligase carboxy terminus of Hsp70-interacting protein (CHIP) and the molecular chaperone Hsp90 are implicated in protein triage decisions involving tau, and have consequently been targeted for therapeutic approaches aimed at decreasing tau burden. Here, we present evidence that CHIP binds, ubiquitinates and regulates expression of histone deacetylase 6 (HDAC6). As the deacetylase for Hsp90, HDAC6 modulates Hsp90 function and determines the favorability of refolding versus degradation of Hsp90 client proteins. Moreover, we demonstrate that HDAC6 levels positively correlate with tau burden, while a decrease in HDAC6 activity or expression promotes tau clearance. Consistent with previous research on Hsp90 clients in cancer, we provide evidence that a loss of HDAC6 activity augments the efficacy of an Hsp90 inhibitor and drives client degradation, in this case tau. Therefore, our current findings not only identify HDAC6 as a critical factor for the regulation of tau levels, but also indicate that a multi-faceted treatment approach could more effectively arrest tau accumulation in disease.

Mice deficient in fractalkine are less susceptible to cerebral ischemia-reperfusion injury.

Fractalkine (FKN), also known as neurotactin, is a CX(3)C chemokine that exists in both secreted and neuronal membrane-bound forms and is upregulated during brain inflammation. There is accumulating evidence that FKN induces chemotaxis by binding to its receptor CX(3)CR1 on leukocytes and microglia. We generated FKN-deficient mice to study the role of FKN in postischemic brain injury. After transient focal cerebral ischemia, FKN-deficient mice had a 28% reduction in infarction size and lower mortality rate, when compared to wild-type littermates. The findings of this study indicate a possible role for FKN in augmenting postischemic injury and mortality after transient focal cerebral ischemia.

Casein kinase II induced polymerization of soluble TDP-43 into filaments is inhibited by heat shock proteins.

BACKGROUND: Trans-activation Response DNA-binding Protein-43 (TDP-43) lesions are observed in Amyotrophic Lateral Sclerosis (ALS), Frontotemporal Lobar Degeneration with ubiquitin inclusions (FTLD-TDP) and 25-50% of Alzheimer's Disease (AD) cases. These abnormal protein inclusions are composed of either amorphous TDP-43 aggregates or highly ordered filaments. The filamentous TDP-43 accumulations typically contain clean 10-12 nm filaments though wider 18-20 nm coated filaments may be observed. The TDP-43 present within these lesions is phosphorylated, truncated and ubiquitinated, and these modifications appear to be abnormal as they are linked to both a cellular heat shock response and microglial activation. The mechanisms associated with this abnormal TDP-43 accumulation are believed to result in a loss of TDP-43 function, perhaps due to the post-translational modifications or resulting from physical sequestration of the TDP-43. The formation of TDP-43 inclusions involves cellular translocation and conversion of TDP-43 into fibrillogenic forms, but the ability of these accumulations to sequester normal TDP-43 and propagate this behavior between neurons pathologically is mostly inferred. The lack of methodology to produce soluble full length TDP-43 and recapitulate this polymerization into filaments as observed in disease has limited our understanding of these pathogenic cascades. RESULTS: The protocols described here generate soluble, full-length and untagged TDP-43 allowing for a direct assessment of the impact of various posttranslational modifications on TDP-43 function. We demonstrate that Casein Kinase II (CKII) promotes the polymerization of this soluble TDP-43 into 10 nm diameter filaments that resemble the most common TDP-43 structures observed in disease. Furthermore, these filaments are recognized as abnormal by Heat Shock Proteins (HSPs) which can inhibit TDP-43 polymerization or directly promote TDP-43 filament depolymerization. CONCLUSION: These findings demonstrate CKII induces polymerization of soluble TDP-43 into filaments and Hsp90 promotes TDP-43 filament depolymerization. These findings provide rational for potential therapeutic intervention at these points in TDP-43 proteinopathies.

Severe amygdala dysfunction in a MAPT transgenic mouse model of frontotemporal dementia.

Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is a neurodegenerative tauopathy caused by mutations in the tau gene (MAPT). Individuals with FTDP-17 have deficits in learning, memory, and language, in addition to personality and behavioral changes that are often characterized by a lack of social inhibition. Several transgenic mouse models expressing tau mutations have been tested extensively for memory or motor impairments, though reports of amygdala-dependent behaviors are lacking. To this end, we tested the rTg4510 mouse model on a behavioral battery that included amygdala-dependent tasks of exploration. As expected, rTg4510 mice exhibit profound impairments in hippocampal-dependent learning and memory tests, including contextual fear conditioning. However, rTg4510 mice also display an abnormal hyperexploratory phenotype in the open-field assay, elevated plus maze, light-dark exploration, and cued fear conditioning, indicative of amygdala dysfunction. Furthermore, significant tau burden is detected in the amygdala of both rTg4510 mice and human FTDP-17 patients, suggesting that the rTg4510 mouse model recapitulates the behavioral disturbances and neurodegeneration of the amygdala characteristic of FTDP-17.

A uterine decidual cell cytokine ensures pregnancy-dependent adaptations to a physiological stressor.

In the mouse, decidual cells differentiate from uterine stromal cells in response to steroid hormones and signals arising from the embryo. Decidual cells are crucially involved in creating the intrauterine environment conducive to embryonic development. Among their many functions is the production of cytokines related to prolactin (PRL), including decidual prolactin-related protein (DPRP). DPRP is a heparin-binding cytokine, which is abundantly expressed in uterine decidua. In this investigation, we have isolated the mouse Dprp gene, characterized its structure and evaluated its biological role. Dprp-null mice were made by replacing exons 2 to 6 of the Dprp gene with an in-frame enhanced green fluorescent protein (EGFP) gene and a neomycin (neo) resistance cassette. Heterozygous intercross breeding of the mutant mice yielded the expected mendelian ratio. Pregnant heterozygote females expressed EGFP within decidual tissue in locations identical to endogenous Dprp mRNA and protein expression. Homozygous Dprp-null mutant male and female mice were viable, exhibited normal postnatal growth rates, were fertile and produced normal litter sizes. A prominent phenotype was observed when pregnant Dprp-null mice were exposed to a physiological stressor. DPRP deficiency interfered with pregnancy-dependent adaptations to hypoxia resulting in pregnancy failure. Termination of pregnancy was associated with aberrations in mesometrial decidual cells, mesometrial vascular integrity, and disruptions in chorioallantoic placenta morphogenesis. The observations suggest that DPRP participates in pregnancy-dependent adaptations to a physiological stressor.

Integration of embryonic stem cells in metanephric kidney organ culture.

Many stages of nephrogenesis can be studied using cultured embryonic kidneys, but there is no efficient technique available to readily knockdown or overexpress transgenes for rapid evaluation of resulting phenotypes. Embryonic stem (ES) cells have unlimited developmental potential and can be manipulated at the molecular genetic level by a variety of methods. The aim of this study was to determine if ES cells could respond to developmental signals within the mouse embryonic day 12 to embryonic day 13 (E12 to E13) kidney microenvironment and incorporate into kidney structures. ROSA26 ES cells were shown to express beta-galactosidase ubiquitously when cultured in the presence of leukemia inhibitory factor to suppress differentiation. When these cells were microinjected into E12 to E13 metanephroi and then placed in transwell organ culture, ES cell-derived, beta-galactosidase-positive cells were identified in epithelial structures resembling tubules. On rare occasions, individual ES cells were observed in structures resembling glomerular tufts. Electron microscopy showed that the ES cell-derived tubules were surrounded by basement membrane and had apical microvilli and junctional complexes. Marker analysis revealed that a subset of these epithelial tubules bound Lotus tetragonolobus and expressed alpha(1) Na(+)/K(+) ATPase. ES cells were infected before injection with a cytomegalovirus promoter-green fluorescence protein (GFP) adenovirus and GFP expression was found as early as 18 h, persisting for up to 48 h in cultured kidneys. This ES cell technology may achieve the objective of obtaining a versatile cell culture system in which molecular interventions can be used in vitro and consequences of these perturbations on the normal kidney development program in vivo can be studied.

A prolactin family paralog regulates reproductive adaptations to a physiological stressor.

Successful species develop strategies to optimize their reproductive performance. This optimization likely includes the evolution of genes that specifically permit reproduction in physiologically challenging conditions. The prolactin (PRL) family gene cluster is one of 25 mouse-specific gene clusters, the majority of which are associated with reproduction. A prevailing theme characterizing the PRL family is its connection with pregnancy and mechanisms controlling viviparity. PRL-like protein A (PLP-A) is one of 26 genes located within the PRL family locus. It is a nonclassical member of the PRL family (e.g., PLP-A does not use the PRL receptor) produced by trophoblast cells of the chorioallantoic placenta and acts on uterine natural killer cells. In this report, the biology of PLP-A has been investigated by generating mice with a PLP-A null mutation. Under standardized animal husbandry conditions, PLP-A possesses modest effects on reproductive performance. However, this same gene is critical for reproduction when mice are exposed to a physiological stressor. Wild-type mice exposed to hypobaric hypoxia during gestation readily adapt and maintain their pregnancies, whereas PLP-A null mutant mice fail to adapt, resulting in pregnancy failure. PLP-A contributes to hypoxia-induced adaptations critical to hemochorial placentation and thus nutrient flow to extraembryonic and embryonic tissues. The findings provide insights into species-specific reproductive adaptations.