Caveolin isoform switching as a molecular, structural, and metabolic regulator of microglia.

Microglia are ramified cells that serve as central nervous system (CNS) guardians, capable of proliferation, migration, and generation of inflammatory cytokines. In non-pathological states, these cells exhibit ramified morphology with processes intermingling with neurons and astrocytes. Under pathological conditions, they acquire a rounded amoeboid morphology and proliferative and migratory capabilities. Such morphological changes require cytoskeleton rearrangements. The molecular control points for polymerization states of microtubules and actin are still under investigation. Caveolins (Cavs), membrane/lipid raft proteins, are expressed in inflammatory cells, yet the role of caveolin isoforms in microglia physiology is debatable. We propose that caveolins provide a necessary control point in the regulation of cytoskeletal dynamics, and thus investigated a role for caveolins in microglia biology. We detected mRNA and protein for both Cav-1 and Cav-3. Cav-1 protein was significantly less and localized to plasmalemma (PM) and cytoplasmic vesicles (CVs) in the microglial inactive state, while the active (amoeboid-shaped) microglia exhibited increased Cav-1 expression. In contrast, Cav-3 was highly expressed in the inactive state and localized with cellular processes and perinuclear regions and was detected in active amoeboid microglia. Pharmacological manipulation of the cytoskeleton in the active or non-active state altered caveolin expression. Additionally, increased Cav-1 expression also increased mitochondrial respiration, suggesting possible regulatory roles in cell metabolism necessary to facilitate the morphological changes. The present findings strongly suggest that regulation of microglial morphology and activity are in part due to caveolin isoforms, providing promising novel therapeutic targets in CNS injury or disease.

Neurokinin B causes acute GnRH secretion and repression of GnRH transcription in GT1-7 GnRH neurons.

Genetic studies in human patients with idiopathic hypogonadotropic hypogonadism (IHH) identified mutations in the genes that encode neurokinin B (NKB) and the neurokinin 3 receptor (NK3R). However, determining the mechanism whereby NKB regulates gonadotropin secretion has been difficult because of conflicting results from in vivo studies investigating the luteinizing hormone (LH) response to senktide, a NK3R agonist. NK3R is expressed in a subset of GnRH neurons and in kisspeptin neurons that are known to regulate GnRH secretion. Thus, one potential source of inconsistency is that NKB could produce opposing direct and indirect effects on GnRH secretion. Here, we employ the GT1-7 cell model to elucidate the direct effects of NKB on GnRH neuron function. We find that GT1-7 cells express NK3R and respond to acute senktide treatment with c-Fos induction and increased GnRH secretion. In contrast, long-term senktide treatment decreased GnRH secretion. Next, we focus on the examination of the mechanism underlying the long-term decrease in secretion and determine that senktide treatment represses transcription of GnRH. We further show that this repression of GnRH transcription may involve enhanced c-Fos protein binding at novel activator protein-1 (AP-1) half-sites identified in enhancer 1 and the promoter, as well as chromatin remodeling at the promoter of the GnRH gene. These data indicate that NKB could directly regulate secretion from NK3R-expressing GnRH neurons. Furthermore, whether the response is inhibitory or stimulatory toward GnRH secretion could depend on the history or length of exposure to NKB because of a repressive effect on GnRH transcription.

Nasal embryonic LHRH factor (NELF) mutations in patients with normosmic hypogonadotropic hypogonadism and Kallmann syndrome.

OBJECTIVE: To determine if mutations in NELF, a gene isolated from migratory GnRH neurons, cause normosmic idiopathic hypogonadotropic hypogonadism (IHH) and Kallmann syndrome (KS). DESIGN: Molecular analysis correlated with phenotype. SETTING: Academic medical center. PATIENT(S): A total of 168 IHH/KS patients as well as unrelated control subjects were studied for NELF mutations. INTERVENTION(S): NELF coding regions/splice junctions were subjected to polymerase chain reaction (PCR)-based DNA sequencing. Eleven additional IHH/KS genes were sequenced in three patients with NELF mutations. MAIN OUTCOME MEASURE(S): Mutations were confirmed by sorting intolerant from tolerant, reverse-transcription (RT)-PCR, and Western blot analysis. RESULT(S): Three novel NELF mutations absent in 372 ethnically matched control subjects were identified in 3/168 (1.8%) IHH/KS patients. One IHH patient had compound heterozygous NELF mutations (c.629-21G>C and c.629-23C>G), and he did not have mutations in 11 other known IHH/KS genes. Two unrelated KS patients had heterozygous NELF mutations and mutation in a second gene: NELF/KAL1 (c.757G>A; p.Ala253Thr of NELF and c.488_490delGTT; p.Cys163del of KAL1) and NELF/TACR3 (c.1160-13C>T of NELF and c.824G>A; p.Trp275X of TACR3). In vitro evidence of these NELF mutations included reduced protein expression and splicing defects. CONCLUSION(S): Our findings suggest that NELF is associated with normosmic IHH and KS, either singly or in combination with a mutation in another gene.