Peripheral immune challenges elicit differential up-regulation of hippocampal cytokine and chemokine mRNA expression in a mouse model of the 15q13.3 microdeletion syndrome.

The human heterozygous 15q13.3 microdeletion is associated with neuropathological disorders, most prominently with epilepsy and intellectual disability. The 1.5 Mb deletion encompasses six genes (FAN1 [MTMR15], MTMR10, TRPM1, KLF13, OTUD7A, and CHRNA7); all but one (TRPM1) are expressed in the brain. The 15q13.3 microdeletion causes highly variable neurological symptoms, and confounding factors may contribute to a more severe phenotype. CHRNA7 and KLF13 are involved in immune system regulation and altered immune responses may contribute to neurological deficits. We used the Df[h15q13]/+ transgenic mouse model with a heterozygous deletion of the orthologous region (Het) to test the hypothesis that the microdeletion increases innate immune responses compared to wild type (WT). Male and female mice were acutely challenged with the bacteriomimetic lipopolysaccharide (LPS, 0.1 mg/kg, i.p.) or the viral mimetic polyinosinic:polycytidylic acid (Poly(I:C), 5 mg/kg). Hippocampal mRNA expression of pro-inflammatory cytokines and chemokines were determined three hours after injection using quantitative PCR analysis. In controls, expression was not affected by sex or genotype. LPS and Poly(I:C) resulted in significantly increased hippocampal expression of cytokines, chemokines, and interferon-γ (IFNγ), with more robust increases for TNF-α, IL-6, IL-1ß, CXCL1, and CCL2 by LPS, higher induction of IFNγ by Poly(I:C), and similar increases of CCL4 and CCL5 by both agents. Generally, Hets exhibited stronger responses than WT mice, and significant effects of genotype or genotype × treatment interactions were detected for CXCL1 and CCL5, and IL-6, IL-1ß, and CCL4, respectively, after LPS. Sex differences were detected for some targets. LPS but not Poly(I:C), reduced overnight burrowing independent of sex or genotype, suggesting that LPS induced sickness behavior. Thus, mice carrying the microdeletion have an increased innate immune response following a LPS challenge, but further studies will have to determine the extent and mechanisms of altered immune activation and subsequent contributions to 15q13.3 microdeletion associated deficits.

Central and peripheral immune responses to low-dose lipopolysaccharide in a mouse model of the 15q13.3 microdeletion.

Carriers of the human 15q13.3 microdeletion (MD) present with a variable spectrum of neuropathological phenotypes that range from asymptomatic to severe clinical outcomes, suggesting an interplay of genetic and non-genetic factors. The most common 2 MB 15q13.3 MD encompasses six genes (MTMR10, FAN1, TRPM1, KLF13, OTUD7A, and CHRNA7), which are expressed in neuronal and non-neuronal tissues. The nicotinic acetylcholine receptor (nAChR) α7, encoded by CHRNA7, is a key player in the cholinergic anti-inflammatory pathway, and the transcription factor KLF13 is also involved in immune responses. Using a mouse model with a heterozygous deletion of the orthologous region of the human 15q13.3 (Df[h15q13]/+), the present study examined peripheral and central innate immune responses to an acute intraperitoneal (i.p.) injection of the bacteriomimetic, lipopolysaccharide (LPS) (100 µg/kg) in adult heterozygous (Het) and wildtype (WT) mice. Serum levels of inflammatory markers were measured 2 h post injection using a Multiplex assay. In control saline injected animals, all measured cytokines were at or below detection limits, whereas LPS significantly increased serum levels of interleukin 1beta (IL-1ß), tumor necrosis factor alpha (TNF-α), IL-6 and IL-10, but not interferon-γ. There was no effect of genotype but a sexual dimorphic response for TNF-α, with females exhibiting greater LPS-induced TNF-α serum levels than males. In situ hybridization revealed similar increases in LPS-induced c-fos mRNA expression in the dorsal vagal complex in all groups. The hippocampal expression of the pro-inflammatory cytokines was evaluated by real-time quantitative PCR. LPS-treatment resulted in significantly increased mRNA expression for IL-1ß, IL-6, and TNF-α compared to saline controls, with no effect of genotype, but a significant sex-effect was detected for IL-1ß. The present study provided no evidence for interactive effects between the heterozygous 15q13.3 MD and a low-dose LPS immune challenge in innate peripheral or central immune responses, although, sex-differential effects in males and females were detected.

Molecular, physiological and behavioral characterization of the heterozygous Df[h15q13]/+ mouse model associated with the human 15q13.3 microdeletion syndrome.

The human 15q13.3 microdeletion syndrome (DS) is caused by a heterozygous microdeletion (MD) affecting six genes: FAN1; MTMR10; TRPM1; KLF13; OTUD7A; and CHRNA7. Carriers are at risk for intellectual disability, epilepsy, autism spectrum disorder, and schizophrenia. Here we used the Df[h15q13]/+ mouse model with an orthologous deletion to further characterize molecular, neurophysiological, and behavioral parameters that are relevant to the 15q13.3 DS. First, we verified the expression and distribution of the α7 nicotinic acetylcholine receptor (nAChR), a gene product of the CHRNA7, in cortical and subcortical areas. Results revealed similar mRNA distribution pattern in wildtype (WT) and heterozygous (Het) mice, with about half the number of α7 nAChR binding sites in mutants. Hippocampal recordings showed similar input/output responses of field excitatory post-synaptic potentials and theta-burst induced long-term potentiation in WT and Het mice. Het males exhibited impaired spatial learning acquisition in the Barnes Maze. Indicative of increased seizure susceptibility, Het mice developed secondary seizures after 6-Hz corneal stimulation, and had significantly increased sensitivity to the chemoconvulsant pentylenetetrazol resulting in increased spiking in hippocampal EEG recordings. Basal mRNA expression of brain derived neurotrophic factor and activity regulated immediate early genes (c-fos, Arc, Erg-1 and Npas4) during adolescence, a critical period of brain maturation, was unaffected by genotype. Thus, the MD did not show gross neuroanatomical, molecular, and neurophysiological abnormalities despite deficits in spatial learning and increased susceptibility to seizures. Altogether, our results verify the phenotypic profile of the heterozygous Df[h15q13]/+ mouse model and underscore its translational relevance for human 15q13.3 DS.

Role of the Cell Asymmetry Apparatus and Ribosome-Associated Chaperones in the Destabilization of a Saccharomyces cerevisiae Prion by Heat Shock.

Self-perpetuating transmissible protein aggregates, termed prions, are implicated in mammalian diseases and control phenotypically detectable traits in Saccharomyces cerevisiae Yeast stress-inducible chaperone proteins, including Hsp104 and Hsp70-Ssa that counteract cytotoxic protein aggregation, also control prion propagation. Stress-damaged proteins that are not disaggregated by chaperones are cleared from daughter cells via mother-specific asymmetric segregation in cell divisions following heat shock. Short-term mild heat stress destabilizes [PSI+ ], a prion isoform of the yeast translation termination factor Sup35 This destabilization is linked to the induction of the Hsp104 chaperone. Here, we show that the region of Hsp104 known to be required for curing by artificially overproduced Hsp104 is also required for heat-shock-mediated [PSI+ ] destabilization. Moreover, deletion of the SIR2 gene, coding for a deacetylase crucial for asymmetric segregation of heat-damaged proteins, also counteracts heat-shock-mediated destabilization of [PSI+ ], and Sup35 aggregates are colocalized with aggregates of heat-damaged proteins marked by Hsp104-GFP. These results support the role of asymmetric segregation in prion destabilization. Finally, we show that depletion of the heat-shock noninducible ribosome-associated chaperone Hsp70-Ssb decreases heat-shock-mediated destabilization of [PSI+ ], while disruption of a cochaperone complex mediating the binding of Hsp70-Ssb to the ribosome increases prion loss. Our data indicate that Hsp70-Ssb relocates from the ribosome to the cytosol during heat stress. Cytosolic Hsp70-Ssb has been shown to antagonize the function of Hsp70-Ssa in prion propagation, which explains the Hsp70-Ssb effect on prion destabilization by heat shock. This result uncovers the stress-related role of a stress noninducible chaperone.

The redox behavior of the heme in cystathionine beta-synthase is sensitive to pH.

Human cystathionine beta-synthase (CBS) is a unique pyridoxal-5'-phosphate-dependent enzyme in which heme is also present as a cofactor. Because the function of heme in this enzyme has yet to be elucidated, the study presented herein investigated possible relationships between the chemistry of the heme and the strong pH dependence of CBS activity. This study revealed, via study of a truncation variant, that the catalytic core of the enzyme governs the pH dependence of the activity. The heme moiety was found to play no discernible role in regulating CBS enzyme activity by sensing changes in pH, because the coordination sphere of the heme is not altered by changes in pH over a range of pH 6-9. Instead, pH was found to control the equilibrium amount of ferric and ferrous heme present after reaction of CBS with one-electron reducing agents. A variety of spectroscopic techniques, including resonance Raman, magnetic circular dichroism, and electron paramagnetic resonance, demonstrated that at pH 9 Fe(II) CBS is dominant while at pH 6 Fe(III) CBS is favored. At low pH, Fe(II) CBS forms transiently but reoxidizes by an apparent proton-gated electron-transfer mechanism. Regulation of CBS activity by the iron redox state has been proposed as the role of the heme moiety in this enzyme. Given that the redox behavior of the CBS heme appears to be controlled by pH, interplay of pH and oxidation state effects must occur if CBS activity is redox regulated.