Mouse erythrocyte tropomodulin in the brain reported by lacZ knocked-in downstream from the E1 promoter

Erythrocyte tropomodulin (E-Tmod, Tmod1) is a tropomyosin-binding protein that caps the slow-growing end of actin filaments. In erythrocytes, it may favor the formation of short actin protofilaments needed for elastic cell deformation. Previously we created a knockout mouse model in which lacZ was knocked-in downstream of the E1 promoter to report the expression of full length E-Tmod. Here we utilize E-Tmod^+/lacZ mice to study E-Tmod expression patterns in the CNS. X-gal staining and in situ hybridization of adults revealed its restricted expression in the olfactory bulb, hippocampus, cerebral cortex, basal ganglia, nuclei of brain stem and cerebellum. In neonates, signals in the cortex and caudate putamen increased from days 15 to 40. Immunohistochemistry also revealed that signals for β-galactosidase coincided with that of NeuN, a post-mitotic nuclear marker for neurons, but not that for GFAP+ astrocytes or APC+ oligodendrocytes, suggesting E-Tmod/lacZ-positive cells in the CNS were neurons. Large neurons, e.g., mitral cells in olfactory bulb and mossy cells in hilus of the dentate gyrus are among those that expressed very high levels of E-Tmod in the CNS.     

Mouse erythrocyte tropomodulin in the brain reported by lacZ knocked-in downstream from the E1 promoter

Erythrocyte tropomodulin (E-Tmod, Tmod1) is a tropomyosin-binding protein that caps the slow-growing end of actin filaments. In erythrocytes, it may favor the formation of short actin protofilaments needed for elastic cell deformation. Previously we created a knockout mouse model in which lacZ was knocked-in downstream of the E1 promoter to report the expression of full length E-Tmod. Here we utilize E-Tmod(+/lacZ) mice to study E-Tmod expression patterns in the CNS. X-gal staining and in situ hybridization of adults revealed its restricted expression in the olfactory bulb, hippocampus, cerebral cortex, basal ganglia, nuclei of brain stem and cerebellum. In neonates, signals in the cortex and caudate putamen increased from days 15 to 40. Immunohistochemistry also revealed that signals for beta-galactosidase coincided with that of NeuN, a post-mitotic nuclear marker for neurons, but not that for GFAP+ astrocytes or APC+ oligodendrocytes, suggesting E-Tmod/lacZ-positive cells in the CNS were neurons. Large neurons, e.g., mitral cells in olfactory bulb and mossy cells in hilus of the dentate gyrus are among those that expressed very high levels of E-Tmod in the CNS.     

Similar phenotypes of Girdin germ-line and conditional knockout mice indicate a crucial role for Girdin in the nestin lineage

Girdin is an Akt substrate and actin-binding protein. Mice with germ-line deletions of Girdin (a non-conditional knockout, (ncKO)) exhibit complete postnatal lethality accompanied by growth retardation and neuronal cell migration defects, which results in hypoplasia of the olfactory bulb and granule cell dispersion in the dentate gyrus. However, the physiological and molecular abnormalities in Girdin ncKO mice are not fully understood. In this study, we first defined the distribution of Girdin in neonates (P1) and adults (6 months or older) using β-galactosidase activity in tissues from ncKO mice. The results indicate that Girdin is expressed throughout the nervous system (brain, spinal cord, enteric and autonomic nervous systems). In addition, β-galactosidase activity was detected in non-neural tissues, particularly in tissues with high tensile force, such as tendons, heart valves, and skeletal muscle. In order to identify the cellular population where the Girdin ncKO phenotype originates, newly generated Girdin flox mice were crossed with nestin promoter-driven Cre transgenic mice to obtain Girdin conditional knockout (cKO) mice. The phenotype of Girdin cKO mice was almost identical to ncKO mice, including postnatal lethality, growth retardation and decreased neuronal migration. Our findings indicate that loss of Girdin in the nestin cell lineage underlies the phenotype of Girdin ncKO mice.     

Increased Nitric Oxide Production and GFAP Expression in the Brains of Influenza A/NWS Virus Infected Mice

The cause of influenza to the brain was investigated using the A/NWS/33 influenza virus infected BALB/c mouse model. NOS-2 mRNA levels in the infected mouse brain was greater than in control mice in all brain regions examined, particularly in the olfactory bulb and hippocampus by 1 day p.i. On the contrary, no differences in NOS-1 or NOS-3 mRNA levels were found between infected and control mice. There was also a marked increase in the levels of metabolites of nitric oxide in the olfactory bulb and hippocampus. Immunohistochemistry showed positive staining for anti-NOS-2 primarily in the hippocampus of infected mice. Further, anti-NOS-2 and GFAP staining was mostly found around capillary blood vessels of the hippocampus starting early in the course of the disease. These results indicate that the NWS enhances the activation of astrocytes and NOS-2 expression which in turn enhances NO production and the expansion of capillary blood vessels.     

Phospho-eIF2α level is important for determining abilities of BACE1 reduction to rescue cholinergic neurodegeneration and memory defects in 5XFAD mice

β-Site APP-cleaving enzyme 1 (BACE1) initiates amyloid-β (Aβ) generation and thus represents a prime therapeutic target in treating Alzheimer's disease (AD). Notably, increasing evidence indicates that BACE1 levels become elevated in AD brains as disease progresses; however, it remains unclear how the BACE1 upregulation may affect efficacies of therapeutic interventions including BACE1-inhibiting approaches. Here, we crossed heterozygous BACE1 knockout mice with AD transgenic mice (5XFAD model) and compared the abilities of partial BACE1 reduction to rescue AD-like phenotypes at earlier (6-month-old) and advanced (15-18-month-old) stages of disease, which expressed normal (～100%) and elevated (～200%) levels of BACE1, respectively. BACE1(+/-) deletion rescued memory deficits as tested by the spontaneous alternation Y-maze task in 5XFAD mice at the earlier stage and prevented their septohippocampal cholinergic deficits associated with significant neuronal loss. Importantly, BACE1(+/-) deletion was no longer able to rescue memory deficits or cholinergic neurodegeneration in 5XFAD mice at the advanced stage. Moreover, BACE1(+/-) deletion significantly reduced levels of Aβ42 and the β-secretase-cleaved C-terminal fragment (C99) in 6-month-old 5XFAD mouse brains, while these neurotoxic β-cleavage products dramatically elevated with age and were not affected by BACE1(+/-) deletion in 15-18-month-old 5XFAD brains. Interestingly, although BACE1(+/-) deletion lowered BACE1 expression by ～50% in 5XFAD mice irrespective of age in concordance with the reduction in gene copy number, BACE1 equivalent to wild-type controls remained in BACE1(+/-)·5XFAD mice at the advanced age. In accord, phosphorylation of the translation initiation factor eIF2α, an important mediator of BACE1 elevation, was dramatically increased (～9-fold) in 15-18-month-old 5XFAD mice and remained highly upregulated (～6-fold) in age-matched BACE1(+/-)·5XFAD mice. Together, our results indicate that partial reduction of BACE1 is not sufficient to block the phospho-eIF2α-dependent BACE1 elevation during the progression of AD, thus limiting its abilities to reduce cerebral Aβ/C99 levels and rescue memory deficits and cholinergic neurodegeneration.     

Spatiotemporal Alterations in Primary Odorant Representations in Olfactory Marker Protein Knockout Mice

Olfactory marker protein (OMP) is highly and selectively expressed in primary olfactory sensory neurons (OSNs) across species, but its physiological function remains unclear. Previous studies in the olfactory epithelium suggest that it accelerates the neural response to odorants and may modulate the odorant-selectivity of OSNs. Here we used a line of gene-targeted mice that express the fluorescent exocytosis indicator synaptopHluorin in place of OMP to compare spatiotemporal patterns of odorant-evoked neurotransmitter release from OSNs in adult mice that were heterozygous for OMP or OMP-null. We found that these patterns, which constitute the primary neural representation of each odorant, developed more slowly during the odorant presentation in OMP knockout mice but eventually reached the same magnitude as in heterozygous mice. In the olfactory bulb, each glomerulus receives synaptic input from a subpopulation of OSNs that all express the same odor receptor and thus typically respond to a specific subset of odorants. We observed that in OMP knockout mice, OSNs innervating a given glomerulus typically responded to a broader range of odorants than in OMP heterozygous mice and thus each odorant evoked synaptic input to a larger number of glomeruli. In an olfactory habituation task, OMP knockout mice behaved differently than wild-type mice, exhibiting a delay in their onset to investigate an odor stimulus during its first presentation and less habituation to that stimulus over repeated presentations. These results suggest that the actions of OMP in olfactory transduction carry through to the primary sensory representations of olfactory stimuli in adult mice in vivo.     

Hyperpolarization-activated and cyclic nucleotide-gated channels are differentially expressed in juxtaglomerular cells in the olfactory bulb of mice

In the olfactory bulb, input from olfactory receptor neurons is processed by neuronal networks before it is relayed to higher brain regions. In many neurons, hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels generate and control oscillations of the membrane potential. Oscillations also appear crucial for information processing in the olfactory bulb. Four channel isoforms exist (HCN1–HCN4) that can form homo- or heteromers. Here, we describe the expression pattern of HCN isoforms in the olfactory bulb of mice by using a novel and comprehensive set of antibodies against all four isoforms. HCN isoforms are abundantly expressed in the olfactory bulb. HCN channels can be detected in most cell populations identified by commonly used marker antibodies. The combination of staining with marker and HCN antibodies has revealed at least 17 different staining patterns in juxtaglomerular cells. Furthermore, HCN isoforms give rise to an unexpected wealth of co-expression patterns but are rarely expressed in the same combination and at the same level in two given cell populations. Therefore, heteromeric HCN channels may exist in several cell populations in vivo. Our results suggest that HCN channels play an important role in olfactory information processing. The staining patterns are consistent with the possibility that both homomeric and heteromeric HCN channels are involved in oscillations of the membrane potential of juxtaglomerular cells.     

Filipin recognizes both GM1 and cholesterol in GM1 gangliosidosis mouse brain

Filipin is an antibiotic polyene widely used as a histochemical marker for cholesterol. We previously reported cholesterol/filipin-positive staining in brain of β-galactosidase (β-gal) knockout ((-/-)) mice (GM1 gangliosidosis). The content and distribution of cholesterol and gangliosides was analyzed in plasma membrane (PM) and microsomal (MS) fractions from whole-brain tissue of 15 week-old control (β-gal(+/-)) and GM1 gangliosidosis (β-gal(-/-)) mice. Total ganglioside content (μg sialic acid/mg protein) was 3-fold and 7-fold greater in the PM and MS fractions, respectively, in βgal(-/-) mice than in βgal(+/-) mice. GM1 content was 30-fold and 50-fold greater in the PM and MS fractions, respectively. In contrast, unesterified cholesterol content (μg/mg protein) was similar in the PM and the MS fractions of the βgal(-/-) and βgal(+/-) mice. Filipin is known to bind to various sterol derivatives and phospholipids on thin-layer chromatograms. Biochemical evidence is presented showing that filipin also binds to GM1 with an affinity similar to that for cholesterol, with a corresponding fluorescent reaction. Our data suggest that the GM1 storage seen in the β-gal(-/-) mouse contributes to the filipin ultraviolet fluorescence observed in GM1 gangliosidosis brain. The data indicate that in addition to cholesterol, filipin can also be useful for detecting GM1.     

Pattern of compensatory expression of voltage-dependent Ca2+ channel alpha1 and beta subunits in brain of N-type Ca2+ channel alpha1B subunit gene-deficient mice with a CBA/JN genetic background.

The Ca(2+) channel alpha(1B) subunit is a pore-forming component capable of generating N-type Ca(2+) channel activity. Although the N-type Ca(2+) channel plays a role in a variety of neuronal functions, alpha(1B)-deficient mice with a CBA/JN genetic background show no apparent behavioral or anatomical-histological abnormality, presumably owing to compensation by other Ca(2+) channels. In this study, we examined the mRNA expression of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits in the olfactory bulb, cerebral cortex, hippocampus and cerebellum of alpha(1B)-deficient mice. We found that the mRNA expression levels of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits were the same in the olfactory bulbs of wild, heterozygous and homozygous alpha(1B)-deficient mice. In the cerebral cortex, alpha(1A) mRNA in homozygous alpha(1B)-deficient mice was expressed at a higher level than in wild or heterozygous mice, but no difference in the expression levels of the alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2), beta(3) and beta(4) subunits was found among wild, heterozygous and homozygous mice. In hippocampus and cerebellum, beta(4) mRNA in homozygous alpha(1B)-deficient mice was expressed at a higher level than in wild or heterozygous mice, but no difference in the expression levels of the alpha(1A), alpha(1C), alpha(1D), alpha(1E), beta(1), beta(2) and beta(3) subunits was found among wild, heterozygous and homozygous mice. These results suggest that the compensatory mechanisms differ in different brain regions of alpha(1B)-deficient mice with a CBA/JN genetic background.     

Patterns of heterogeneous expression of pannexin 1 and pannexin 2 transcripts in the olfactory epithelium and olfactory bulb

Pannexins form membrane channels that release biological signals to communicate with neighboring cells. Here, we report expression patterns of pannexin 1 (Panx1) and pannexin 2 (Panx2) in the olfactory epithelium and olfactory bulb of adult mice. In situ hybridization revealed that mRNAs for Panx1 and Panx2 were both expressed in the olfactory epithelium and olfactory bulb. Expression of Panx1 and Panx2 was mainly found in cell bodies below the sustentacular cell layer in the olfactory epithelium, indicating that Panx1 and Panx2 are expressed in mature and immature olfactory neurons, and basal cells. Expression of Panx2 was observed in sustentacular cells in a few locations of the olfactory epithelium. In the olfactory bulb, Panx1 and Panx2 were expressed in spatial patterns. Many mitral cells, tufted cells, periglomerular cells and granule cells were Panx1 and Panx2 positive. Mitral cells located at the dorsal and lateral portions of the olfactory bulb showed weak Panx1 expression compared with those in the medial side. However, the opposite was true for the distribution of Panx2 positive mitral cells. There were more Panx2 mRNA positive mitral cells and granule cells compared to those expressing Panx1. Our findings on pannexin expression in the olfactory system of adult mice raise the novel possibility that pannexins play a role in information processing in the olfactory system. Demonstration of expression patterns of pannexins in the olfactory system provides an anatomical basis for future functional studies.