FSH blockade improves cognition in mice with Alzheimer's disease.

Alzheimer's disease has a higher incidence in older women, with a spike in cognitive decline that tracks with visceral adiposity, dysregulated energy homeostasis and bone loss during the menopausal transition1,2. Inhibiting the action of follicle-stimulating hormone (FSH) reduces body fat, enhances thermogenesis, increases bone mass and lowers serum cholesterol in mice3-7. Here we show that FSH acts directly on hippocampal and cortical neurons to accelerate amyloid-ß and Tau deposition and impair cognition in mice displaying features of Alzheimer's disease. Blocking FSH action in these mice abrogates the Alzheimer's disease-like phenotype by inhibiting the neuronal C/EBPß-δ-secretase pathway. These data not only suggest a causal role for rising serum FSH levels in the exaggerated Alzheimer's disease pathophysiology during menopause, but also reveal an opportunity for treating Alzheimer's disease, obesity, osteoporosis and dyslipidaemia with a single FSH-blocking agent.

Angiotensin II type 2 receptor regulates ROMK-like K⁺ channel activity in the renal cortical collecting duct during high dietary K⁺ adaptation.

The kidney adjusts K⁺ excretion to match intake in part by regulation of the activity of apical K⁺ secretory channels, including renal outer medullary K⁺ (ROMK)-like K⁺ channels, in the cortical collecting duct (CCD). ANG II inhibits ROMK channels via the ANG II type 1 receptor (AT1R) during dietary K⁺ restriction. Because AT1Rs and ANG II type 2 receptors (AT2Rs) generally function in an antagonistic manner, we sought to characterize the regulation of ROMK channels by the AT2R. Patch-clamp experiments revealed that ANG II increased ROMK channel activity in CCDs isolated from high-K⁺ (HK)-fed but not normal K⁺ (NK)-fed rats. This response was blocked by PD-123319, an AT2R antagonist, but not by losartan, an AT1R antagonist, and was mimicked by the AT2R agonist CGP-42112. Nitric oxide (NO) synthase is present in CCD cells that express ROMK channels. Blockade of NO synthase with N-nitro-l-arginine methyl ester and free NO with 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt completely abolished ANG II-stimulated ROMK channel activity. NO enhances the synthesis of cGMP, which inhibits phosphodiesterases (PDEs) that normally degrade cAMP; cAMP increases ROMK channel activity. Pretreatment of CCDs with IBMX, a broad-spectrum PDE inhibitor, or cilostamide, a PDE3 inhibitor, abolished the stimulatory effect of ANG II on ROMK channels. Furthermore, PKA inhibitor peptide, but not an activator of the exchange protein directly activated by cAMP (Epac), also prevented the stimulatory effect of ANG II. We conclude that ANG II acts at the AT2R to stimulate ROMK channel activity in CCDs from HK-fed rats, a response opposite to that mediated by the AT1R in dietary K⁺-restricted animals, via a NO/cGMP pathway linked to a cAMP-PKA pathway.

Identification of a serotonin/glutamate receptor complex implicated in psychosis.

The psychosis associated with schizophrenia is characterized by alterations in sensory processing and perception. Some antipsychotic drugs were identified by their high affinity for serotonin 5-HT2A receptors (2AR). Drugs that interact with metabotropic glutamate receptors (mGluR) also have potential for the treatment of schizophrenia. The effects of hallucinogenic drugs, such as psilocybin and lysergic acid diethylamide, require the 2AR and resemble some of the core symptoms of schizophrenia. Here we show that the mGluR2 interacts through specific transmembrane helix domains with the 2AR, a member of an unrelated G-protein-coupled receptor family, to form functional complexes in brain cortex. The 2AR-mGluR2 complex triggers unique cellular responses when targeted by hallucinogenic drugs, and activation of mGluR2 abolishes hallucinogen-specific signalling and behavioural responses. In post-mortem human brain from untreated schizophrenic subjects, the 2AR is upregulated and the mGluR2 is downregulated, a pattern that could predispose to psychosis. These regulatory changes indicate that the 2AR-mGluR2 complex may be involved in the altered cortical processes of schizophrenia, and this complex is therefore a promising new target for the treatment of psychosis.

Brain atlas for glycoprotein hormone receptors at single-transcript level.

There is increasing evidence that anterior pituitary hormones, traditionally thought to have unitary functions in regulating single endocrine targets, act on multiple somatic tissues, such as bone, fat, and liver. There is also emerging evidence for anterior pituitary hormone action on brain receptors in mediating central neural and peripheral somatic functions. Here, we have created the most comprehensive neuroanatomical atlas on the expression of TSHR, LHCGR, and FSHR. We have used RNAscope, a technology that allows the detection of mRNA at single-transcript level, together with protein level validation, to document Tshr expression in 173 and Fshr expression in 353 brain regions, nuclei and subnuclei identified using the Atlas for the Mouse Brain in Stereotaxic Coordinates. We also identified Lhcgr transcripts in 401 brain regions, nuclei and subnuclei. Complementarily, we used ViewRNA, another single-transcript detection technology, to establish the expression of FSHR in human brain samples, where transcripts were co-localized in MALAT1-positive neurons. In addition, we show high expression for all three receptors in the ventricular region-with yet unknown functions. Intriguingly, Tshr and Fshr expression in the ependymal layer of the third ventricle was similar to that of the thyroid follicular cells and testicular Sertoli cells, respectively. In contrast, Fshr was localized to NeuN-positive neurons in the granular layer of the dentate gyrus in murine and human brain-both are Alzheimer's disease-vulnerable regions. Our atlas thus provides a vital resource for scientists to explore the link between the stimulation or inactivation of brain glycoprotein hormone receptors on somatic function. New actionable pathways for human disease may be unmasked through further studies.

Hallucinogens recruit specific cortical 5-HT(2A) receptor-mediated signaling pathways to affect behavior.

Hallucinogens, including mescaline, psilocybin, and lysergic acid diethylamide (LSD), profoundly affect perception, cognition, and mood. All known drugs of this class are 5-HT(2A) receptor (2AR) agonists, yet closely related 2AR agonists such as lisuride lack comparable psychoactive properties. Why only certain 2AR agonists are hallucinogens and which neural circuits mediate their effects are poorly understood. By genetically expressing 2AR only in cortex, we show that 2AR-regulated pathways on cortical neurons are sufficient to mediate the signaling pattern and behavioral response to hallucinogens. Hallucinogenic and nonhallucinogenic 2AR agonists both regulate signaling in the same 2AR-expressing cortical neurons. However, the signaling and behavioral responses to the hallucinogens are distinct. While lisuride and LSD both act at 2AR expressed by cortex neurons to regulate phospholipase C, LSD responses also involve pertussis toxin-sensitive heterotrimeric G(i/o) proteins and Src. These studies identify the long-elusive neural and signaling mechanisms responsible for the unique effects of hallucinogens.

The human herpesvirus 8 chemokine receptor vGPCR triggers autonomous proliferation of endothelial cells.

We have used a novel conditional transgenic system to study the mechanisms of angioproliferation induced by viral G protein-coupled receptor (vGPCR), the constitutively active chemokine receptor encoded by human herpesvirus 8 (HHV8, also known as Kaposi sarcoma herpesvirus). Using this system, we were able to control temporal expression of vGPCR and to monitor its expression in situ via the use of the surrogate marker LacZ. Upon treatment with doxycycline (DOX), cells expressing vGPCR and LacZ (vGPCR/LacZ(+) cells) progressively accumulated in areas where angioproliferation was observed. Sorted vGPCR/LacZ(+) cells from angiogenic lesions expressed markers characteristic of endothelial progenitor cells, produced angiogenic factors, and proliferated in vitro. Prolonged treatment of transgenic mice with DOX led to development of tumors in the skin of ears, tail, nose, and paws. vGPCR/LacZ(+) cells were frequent in early lesions but scarce within these tumors. Finally, transfer of vGPCR/LacZ(+) cells into Rag1(-/-) mice treated with DOX led to angioproliferation and, with time, to development of tumors containing both vGPCR/LacZ(+) and vGPCR/LacZ(-) cells. Taken together, these results indicate that vGPCR triggers angioproliferation directly and suggest a novel role for this molecule in the pathogenesis of Kaposi sarcoma.

Photoacoustic imaging of synovial tissue hypoxia in experimental post-traumatic osteoarthritis.

OBJECTIVES: This pilot study aimed to investigate the feasibility of non-invasively assessing synovial tissue hypoxia in vivo using photoacoustic (PA) imaging in a post-traumatic osteoarthritis model and explore its correlation with OA severity. METHODS: The three-dimensional vasculature structure and oxygenation level of synovial tissues of destabilization of the medial meniscus (DMM)-induced osteoarthritis (OA) mice were longitudinally monitored using PA imaging. Vascular volume/tissue volume (%) and tissue oxygen saturation (sO2) were validated against results obtained by established Power Doppler (PD) imaging and dynamic changes of inhaled O2 concentration respectively. PA changes were correlated with the histological grading of cartilage damages. RESULTS: PA-measurements of vascularity and sO2 demonstrated a strong correlation with localized blood flow detected by PD imaging (r = 0.506, p < 0.001) and inhaled O2 concentration. DMM knees exhibited much more vascularity in synovial tissue at 4 months after surgery (median 11.3%, IQR: 10.7-15.5%) than the intact knees at time zero (median:5.1%, IQR:3.8-6.8%, p < 0.001) as well as the sham-operated knees (median: 4%, IQR: 3.75-5.45%, p = 0.017). Paradoxically, synovial tissue sO2 was significantly lower in DDM knees (median: 37.7%, IQR: 36.4-40.6%) than both the intact (47.1%, IQR: 41.9-49.8% p = 0.001) and sham-operated knees (45.1% IQR: 45.1-52.4%, p = 0.017). The PA-detected synovial tissue hypoxia correlated with the severity of cartilage loss in DMM mice (rho = -0.597, p = 0.031). CONCLUSION: Here, we demonstrated PA imaging can be implemented for non-invasive imaging of the synovial tissue. Under PA imaging, synovitis in OA was characterized by increased angiogenesis and synovial tissue hypoxia; the latter was associated with the severity of OA.

Method for multiplex cellular detection of mRNAs using quantum dot fluorescent in situ hybridization.

The photostability and narrow emission spectra of non-organic quantum dot fluorophores (QDs) make them desirable candidates for fluorescent in situ hybridization (FISH) to study the expression of specific mRNA transcripts. We developed a novel method for direct QD labeling of modified oligonucleotide probes through streptavidin and biotin interactions, as well as protocols for their use in multiple-label FISH. We validated this technique in mouse brainstem sections. The subcellular localization of the vesicular monoamine transporter (Vmat2) mRNA corresponds when using probes labeled with two different QDs in the same hybridization. We developed protocols for combined direct QD FISH and QD immunohistochemical labeling within the same neurons as well as for simultaneous study of the subcellular distribution of multiple mRNA targets. We demonstrated increased sensitivity of FISH using QDs in comparison with organic fluorophores. These techniques gave excellent histological results both for multiplex FISH and combined FISH and immunohistochemistry. This approach can facilitate the ultrasensitive simultaneous study of multiple mRNA and protein markers in tissue culture and histological section.

Local protein synthesis mediates a rapid increase in dendritic elongation factor 1A after induction of late long-term potentiation.

The maintenance of long-term potentiation (LTP) requires a brief period of accelerated protein synthesis soon after synaptic stimulation, suggesting that an early phase of enhanced translation contributes to stable LTP. The mechanism regulating protein synthesis and the location and identities of mRNAs translated are not well understood. Here, we show in acute brain slices that the induction of protein synthesis-dependent hippocampal LTP increases the expression of elongation factor 1A (eEF1A), the mRNA of which contains a 5' terminal oligopyrimidine tract. This effect is blocked by rapamycin, indicating that the increase in EF1A expression is mediated by the mammalian target of rapamycin (mTOR) pathway. We find that mRNA for eEF1A is present in pyramidal cell dendrites and that the LTP-associated increase in eEF1A expression was intact in dendrites that had been severed from their cell bodies before stimulation. eEF1A levels increased within 5 min after stimulation in a translation-dependent manner, and this effect remained stable for 3 h. These results suggest a mechanism whereby synaptic stimulation, by signaling through the mTOR pathway, produces an increase in dendritic translational capacity that contributes to LTP maintenance.

Epsilon-sarcoglycan immunoreactivity and mRNA expression in mouse brain.

Myoclonus dystonia (M-D) is a hereditary movement disorder caused by a maternally imprinted gene that is often associated with psychiatric symptoms. Most cases of M-D are believed to result from mutations of the epsilon-sarcoglycan protein. The neuroanatomical distribution of epsilon-sarcoglycan-like immunoreactivity in mouse was investigated by using an antiserum against the epsilon-sarcoglycan protein. The expression of epsilon-sarcoglycan mRNA was studied by a sensitive fluorescence in situ hybridization (FISH) method. Immunohistochemistry and FISH revealed a wide distribution of epsilon-sarcoglycan protein and mRNA throughout the mouse brain. High expression levels of epsilon-sarcoglycan mRNA and immunoreactivity were found in the mitral cell layer of the olfactory bulb, the Purkinje cell layer in cerebellum, and the monoaminergic neurons in the mouse midbrain. Immunohistochemistry revealed a similar distribution of epsilon-sarcoglycan protein. Double-labeling FISH showed colocalization of tyrosine hydroxylase and epsilon-sarcoglycan mRNAs within all the midbrain dopaminergic (DAergic) cell groups. By combining FISH with fluorescence immunohistochemistry, coexpression of epsilon-sarcoglycan mRNA and tryptophan hydroxylase immunoreactivity was found in the serotonergic (5-HTergic) neurons within the dorsal raphe nucleus. The distribution of epsilon-sarcoglycan in the mouse brain suggests that the symptom complex of M-D may be related to the effects of decreased epsilon-sarcoglycan activity on the development or function of monoaminergic neurons.

Novel compounds in the treatment of lung cancer: current and developing therapeutic agents.

Lung cancer is the leading cause of cancer-related death in the United States. Though incremental advances have been made in the treatment of this devastating disease during the past decade, new therapies are urgently needed. Traditional cytotoxic agents have been combined with other modalities with improved survival for early-stage patients. Newer cytotoxic agents targeting the same or different mechanisms have been developed at different stages. Optimization of various chemotherapy regimens in different settings is one of the aims of current clinical trials. Some predictive biomarkers (eg, excision repair cross-complementing 1, ERCC1) and histotypes (eg, adenocarcinoma) are found to be associated with resistance/response to some cytotoxic drugs. Another notable advance is the addition of targeted therapy to lung cancer treatment. Targeted agents such as erlotinib and bevacizumab have demonstrated clinical benefits and gained Food and Drug Administration approval for lung cancer. More agents targeting various signaling pathways critical to lung cancer are at different stages of development. Along with the effort of new targeted drug discovery, biomarkers such as epidermal growth factor receptor and anaplastic lymphoma kinase mutations have proven useful for patient selection, and more predictive biomarkers have been actively evaluated in non-small cell lung cancer. The paradigm of lung cancer treatment has shifted towards biomarker-based personalized medicine.