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Amyotrophic lateral sclerosis (ALS) is characterized by progressive dysfunction and degeneration of motor neurons in the central nervous system (CNS). In the absence of effective drug treatments for ALS, stem cell treatment has emerged as a candidate therapy for this disease. To date, however, there is no consensus protocol that stipulates stem cell types, transplantation timing, or frequency. Using an ALS mouse model carrying a high copy number of a mutant human superoxide dismutase-1 (SOD1)(G93A) transgene, we investigated the effect of neural induction on the innate therapeutic potential of mesenchymal stem cells (MSCs) in relation to preclinical transplantation parameters. In our study, the expression of monocyte chemoattractant protein-1 (MCP-1) was elevated in the ALS mouse spinal cord. Neural induction of MSCs with neurogenin 1 (Ngn1) upregulated the expression level of the MCP-1 receptor, CCR2, and enhanced the migration activity toward MCP-1 in vitro. Ngn1-expressing MSCs (MSCs-Ngn1) showed a corresponding increase in tropism to the CNS after systemic transplantation in ALS mice. Notably, MSCs-Ngn1 delayed disease onset if transplanted during preonset ages,whereas unprocessed MSCs failed to do so. If transplanted near the onset ages, a single treatment with MSCs-Ngn1 was sufficient to enhance motor functions during the symptomatic period (15–17 weeks), whereas unprocessed MSCs required repeated transplantation to achieve similar levels of motor function improvement. Our data indicate that systemically transplanted MSCs-Ngn1 can migrate to the CNS and exert beneficial effects on host neural cells for an extended period of time through paracrine functions, suggesting a potential benefit of neural induction of transplanted MSCs in long-term treatment of ALS.

Transmission from photoreceptors to ON bipolar cells in mammalian retina is mediated by a sign-inverting cascade. Upon binding glutamate, the metabotropic glutamate receptor mGluR6 activates the heterotrimeric G-protein Gαoβ3γ13, and this leads to closure of the TRPM1 channel (melastatin). TRPM1 is thought to be constitutively open, but the mechanism that leads to its closure is unclear. We investigated this question in mouse rod bipolar cells by dialyzing reagents that modify the activity of either Gαo or Gβγ and then observing their effects on the basal holding current. After opening the TRPM1 channels with light, a constitutively active mutant of Gαo closed the channel, but wild-type Gαo did not. After closing the channels by dark adaptation, phosducin or inactive Gαo (both sequester Gβγ) opened the channel while the active mutant of Gαo did not. Co-immunoprecipitation showed that TRPM1 interacts with Gβ3 and with the active and inactive forms of Gαo. Furthermore, bioluminescent energy transfer assays indicated that while Gαo interacts with both the N- and the C- termini of TRPM1, Gβγ interacts only with the N-terminus. Our physiological and biochemical results suggest that both Gαo and Gβγ bind TRPM1 channels and cooperate to close them.

The Cre/LoxP system is a well-established approach to spatially and temporally control genetic inactivation. The calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIα) promoter limits expression to specific regions of the forebrain and thus has been utilized for the brain-specific inactivation of the genes. Here, we show that CaMKIIα-Cre can be utilized for simultaneous inactivation of genes in the adult brain and in male germ cells. Double transgenic Rosa26+/stop-lacZ::CaMKIIα-Cre+/Cre mice generated by crossing CaMKIIα-Cre+/Cre mice with floxed ROSA26 lacZ reporter (Rosa26+/stop-lacZ) mice exhibited lacZ expression in the brain and testis. When these mice were mated to wild-type females, about 27% of the offspring were whole body blue by X-gal staining without inheriting the Cre transgene. These results indicate that recombination can occur in the germ cells of male Rosa26+/stop-lacZ::CaMKIIα-Cre+/Cre mice. Similarly, when double transgenic Gnao+/f::CaMKIIα-Cre+/Cre mice carrying a floxed Go-alpha gene (Gnaof/f) were backcrossed to wild-type females, approximately 22% of the offspring carried the disrupted allele (GnaoΔ) without inheriting the Cre transgene. The GnaoΔ/Δ mice closely resembled conventional Go-alpha knockout mice (Gnao−/−) with respect to impairment of their behavior. Thus, we conclude that CaMKIIα-Cre mice afford recombination for both tissue- and time-controlled inactivation of floxed target genes in the brain and for their permanent disruption. This work also emphasizes that extra caution should be exercised in utilizing CaMKIIα-Cre mice as breeding pairs.

In mammals, initial detection of olfactory stimuli is mediated by sensory neurons in the main olfactory epithelium (MOE) and the vomeronasal organ (VNO). The heterotrimeric GTP-binding protein G o is widely expressed in the MOE and VNO of mice. Early studies indicated that G o expression in VNO sensory neurons is critical for directing social and sexual behaviors in female mice [Oboti L, et al. (2014) BMC Biol 12:31]. However, the physiological functions of G o in the MOE have remained poorly defined. Here, we examined the role of G o in the MOE using mice lacking the α subunit of G o . Development of the olfactory bulb (OB) was perturbed in mutant mice as a result of reduced neurogenesis and increased cell death. The balance between cell types of OB interneurons was altered in mutant mice, with an increase in the number of tyrosine hydroxylase-positive interneurons at the expense of calbindin-positive interneurons. Sexual behavior toward female mice and preference for female urine odors by olfactory sensory neurons in the MOE were abolished in mutant male mice. Our data suggest that G o signaling is essential for the structural and functional integrity of the MOE and for specification of OB interneurons, which in turn are required for the transmission of pheromone signals and the initiation of mating behavior with the opposite sex.heterotrimeric G o protein | olfactory mucosa | olfactory bulb interneuron | tyrosine monooxygenase | sexual behavior P heromones evoke diverse social and sexual behaviors in animals of the same species. In mammals, initial detection of olfactory stimuli is mediated by sensory neurons in the main olfactory epithelium (MOE) and vomeronasal organ (VNO). Early studies indicated that the VNO is largely responsible for the detection of pheromones and the consequent direction of social and sexual behaviors (1). VNO sensory neurons (VSNs) express distinct types of vomeronasal receptors: type 1 (V1Rs) in the apical region and type 2 (V2Rs) in the basal region. These receptors couple, respectively, to G i and G o members of the heterotrimeric GTPbinding protein family. It has been suggested that, following ligand binding to V1Rs and V2Rs, the Gβγ subunits released from G i and G o activate phospholipase C and increase the intracellular concentration of Ca 2+ . Ca 2+ ions induce opening of small Ca 2+ -activated K + (SK3) channels, subsequently activating Ca 2+ -activated Cl − channels (CACCs). In the VNO, Gβγ can also directly induce the opening of G protein-activated inward rectifying K + (GIRK) channels independently of transient receptor potential cation channel C2 (TRPC2) (2). Because of the VNO's high luminal K + concentration (2), opening of SK3 and GIRK channels leads to depolarization of VSNs and transmission of pheromone signals. Targeted deletion of the TRPC2 gene abrogates gender identification and intermale aggression without affecting male-to-female mating behavior in male mice (3, 4). Targeted deletion of the GIRK1 gene partially attenuates mating behavior of the male toward fem...

24Western-style diets cause disruptions in myelinating cells and astrocytes within the CNS. We 25 identified increased CD38 expression in the mouse spinal cord following chronic high fat 26 consumption or focal demyelinating injury. CD38-catalytically inactive mice are significantly 27 protected from high fat-induced NAD + depletion, oligodendrocyte loss, oxidative damage, and 28 astrogliosis. 78c, a CD38 inhibitor, increased NAD + and attenuated neuroinflammatory changes 29 in astrocytes induced by saturated fat. Conditioned media from saturated fat-treated astrocytes 30 impaired oligodendrocyte differentiation pointing to indirect mechanisms of 31 oligodendrogliopathy. Combined saturated fat and lysolecithin demyelination in cerebellar slices 32 resulted in additional deficits in myelin proteins that were mitigated by concomitant 78c 33 treatment. Importantly, oral 78c increased counts of oligodendrocytes and remyelinated axons 34 after focal demyelination. Our findings suggest that high fat diet impairs oligodendrocyte 35 survival and differentiation through astrocyte-linked mechanisms mediated by the NAD + ase 36 CD38, and highlight the use of CD38 inhibitors as potential therapeutic candidates to improve 37 myelin regeneration. 38 39 Keywords 40 CD38, Glia, high fat diet, NAD + , remyelination 41 42 43Results 96 Astrocytes increase CD38 in the spinal cord following chronic HFD or demyelination 97Our recent studies revealed spinal cord changes involving ER stress, mitochondrial 98 dysfunction, and oxidative stress in the spinal cords of mice chronically consuming a HFD and 99