Palmitoylation Regulates Epidermal Homeostasis and Hair Follicle Differentiation

Palmitoylation is a key post-translational modification mediated by a family of DHHC-containing palmitoyl acyl-transferases (PATs). Unlike other lipid modifications, palmitoylation is reversible and thus often regulates dynamic protein interactions. We find that the mouse hair loss mutant, depilated, (dep) is due to a single amino acid deletion in the PAT, Zdhhc21, resulting in protein mislocalization and loss of palmitoylation activity. We examined expression of Zdhhc21 protein in skin and find it restricted to specific hair lineages. Loss of Zdhhc21 function results in delayed hair shaft differentiation, at the site of expression of the gene, but also leads to hyperplasia of the interfollicular epidermis (IFE) and sebaceous glands, distant from the expression site. The specific delay in follicle differentiation is associated with attenuated anagen propagation and is reflected by decreased levels of Lef1, nuclear β-catenin, and Foxn1 in hair shaft progenitors. In the thickened basal compartment of mutant IFE, phospho-ERK and cell proliferation are increased, suggesting increased signaling through EGFR or integrin-related receptors, with a parallel reduction in expression of the key differentiation factor Gata3. We show that the Src-family kinase, Fyn, involved in keratinocyte differentiation, is a direct palmitoylation target of Zdhhc21 and is mislocalized in mutant follicles. This study is the first to demonstrate a key role for palmitoylation in regulating developmental signals in mammalian tissue homeostasis

Nr4a1-eGFP Is a Marker of Striosome-Matrix Architecture, Development and Activity in the Extended Striatum

Transgenic mice expressing eGFP under population specific promoters are widely used in neuroscience to identify specific subsets of neurons in situ and as sensors of neuronal activity in vivo. Mice expressing eGFP from a bacterial artificial chromosome under the Nr4a1 promoter have high expression within the basal ganglia, particularly within the striosome compartments and striatal-like regions of the extended amygdala (bed nucleus of the stria terminalis, striatal fundus, central amygdaloid nucleus and intercalated cells). Grossly, eGFP expression is inverse to the matrix marker calbindin 28K and overlaps with mu-opioid receptor immunoreactivity in the striatum. This pattern of expression is similar to Drd1, but not Drd2, dopamine receptor driven eGFP expression in structures targeted by medium spiny neuron afferents. Striosomal expression is strong developmentally where Nr4a1-eGFP expression overlaps with Drd1, TrkB, tyrosine hydroxylase and phospho-ERK, but not phospho-CREB, immunoreactivity in “dopamine islands”. Exposure of adolescent mice to methylphenidate resulted in an increase in eGFP in both compartments in the dorsolateral striatum but eGFP expression remained brighter in the striosomes. To address the role of activity in Nr4a1-eGFP expression, primary striatal cultures were prepared from neonatal mice and treated with forskolin, BDNF, SKF-83822 or high extracellular potassium and eGFP was measured fluorometrically in lysates. eGFP was induced in both neurons and contaminating glia in response to forskolin but SKF-83822, brain derived neurotrophic factor and depolarization increased eGFP in neuronal-like cells selectively. High levels of eGFP were primarily associated with Drd1+ neurons in vitro detected by immunofluorescence; however ∼15% of the brightly expressing cells contained punctate met-enkephalin immunoreactivity. The Nr4a1-GFP mouse strain will be a useful model for examining the connectivity, physiology, activity and development of the striosome system

Generational Association Studies of Dopaminergic Genes in Reward Deficiency Syndrome (RDS) Subjects: Selecting Appropriate Phenotypes for Reward Dependence Behaviors

Abnormal behaviors involving dopaminergic gene polymorphisms often reflect an insufficiency of usual feelings of satisfaction, or Reward Deficiency Syndrome (RDS). RDS results from a dysfunction in the “brain reward cascade,” a complex interaction among neurotransmitters (primarily dopaminergic and opioidergic). Individuals with a family history of alcoholism or other addictions may be born with a deficiency in the ability to produce or use these neurotransmitters. Exposure to prolonged periods of stress and alcohol or other substances also can lead to a corruption of the brain reward cascade function. We evaluated the potential association of four variants of dopaminergic candidate genes in RDS (dopamine D1 receptor gene [DRD1]; dopamine D2 receptor gene [DRD2]; dopamine transporter gene [DAT1]; dopamine beta-hydroxylase gene [DBH]). Methodology: We genotyped an experimental group of 55 subjects derived from up to five generations of two independent multiple-affected families compared to rigorously screened control subjects (e.g., N = 30 super controls for DRD2 gene polymorphisms). Data related to RDS behaviors were collected on these subjects plus 13 deceased family members. Results: Among the genotyped family members, the DRD2 Taq1 and the DAT1 10/10 alleles were significantly (at least p < 0.015) more often found in the RDS families vs. controls. The TaqA1 allele occurred in 100% of Family A individuals (N = 32) and 47.8% of Family B subjects (11 of 23). No significant differences were found between the experimental and control positive rates for the other variants. Conclusions: Although our sample size was limited, and linkage analysis is necessary, the results support the putative role of dopaminergic polymorphisms in RDS behaviors. This study shows the importance of a nonspecific RDS phenotype and informs an understanding of how evaluating single subset behaviors of RDS may lead to spurious results. Utilization of a nonspecific “reward” phenotype may be a paradigm shift in future association and linkage studies involving dopaminergic polymorphisms and other neurotransmitter gene candidates

Factors Associated with Revision Surgery after Internal Fixation of Hip Fractures

Background: Femoral neck fractures are associated with high rates of revision surgery after management with internal fixation. Using data from the Fixation using Alternative Implants for the Treatment of Hip fractures (FAITH) trial evaluating methods of internal fixation in patients with femoral neck fractures, we investigated associations between baseline and surgical factors and the need for revision surgery to promote healing, relieve pain, treat infection or improve function over 24 months postsurgery. Additionally, we investigated factors associated with (1) hardware removal and (2) implant exchange from cancellous screws (CS) or sliding hip screw (SHS) to total hip arthroplasty, hemiarthroplasty, or another internal fixation device. Methods: We identified 15 potential factors a priori that may be associated with revision surgery, 7 with hardware removal, and 14 with implant exchange. We used multivariable Cox proportional hazards analyses in our investigation. Results: Factors associated with increased risk of revision surgery included: female sex, [hazard ratio (HR) 1.79, 95% confidence interval (CI) 1.25-2.50; P = 0.001], higher body mass index (fo

hVGAT-mCherry: A novel molecular tool for analysis of GABAergic neurons derived from human pluripotent stem cells

BACKGROUND: GABAergic synaptic transmission is known to play a critical role in the assembly of neuronal circuits during development and is responsible for maintaining the balance between excitatory and inhibitory signaling in the brain during maturation into adulthood. Importantly, defects in GABAergic neuronal function and signaling have been linked to a number of neurological diseases, including autism spectrum disorders, schizophrenia, and epilepsy. With patient-specific induced pluripotent stem cell (iPSC)-based models of neurological disease, it is now possible to investigate the disease mechanisms that underlie deficits in GABAergic function in affected human neurons. To that end, tools that enable the labeling and purification of viable GABAergic neurons from human pluripotent stem cells would be of great value. RESULTS: To address the need for tools that facilitate the identification and isolation of viable GABAergic neurons from the in vitro differentiation of iPSC lines, a cell type-specific promoter-driven fluorescent reporter construct was developed that utilizes the human vesicular GABA transporter (hVGAT) promoter to drive the expression of mCherry specifically in VGAT-expressing neurons. The transduction of iPSC-derived forebrain neuronal cultures with the hVGAT promoter-mCherry lentiviral reporter construct specifically labeled GABAergic neurons. Immunocytochemical analysis of hVGAT-mCherry expression cells showed significant co-labelling with the GABAergic neuronal markers for endogenous VGAT, GABA, and GAD67. Expression of mCherry from the VGAT promoter showed expression in several cortical interneuron subtypes to similar levels. In addition, an effective and reproducible protocol was developed to facilitate the fluorescent activated cell sorting (FACS)-mediated purification of high yields of viable VGAT-positive cells. CONCLUSIONS: These studies demonstrate the utility of the hVGAT-mCherry reporter construct as an effective tool for studying GABAergic neurons differentiated in vitro from human pluripotent stem cells. This approach could provide a means of obtaining large quantities of viable GABAergic neurons derived from disease-specific hiPSCs that could be used for functional assays or high-throughput screening of small molecule libraries