Enhanced anxiety, depressive-like behaviour and impaired recognition memory in mice with reduced expression of the vesicular glutamate transporter 1 (VGLUT1).

Three isoforms of a vesicular glutamate transporter (VGLUT1-3) have been identified. Of these, VGLUT1 is the major isoform in the cerebral cortex and hippocampus where it is selectively located on synaptic vesicles of excitatory glutamatergic terminals. Variations in VGLUT1 expression levels have a major impact on the efficacy of glutamate synaptic transmission. Given evidence linking alterations in glutamate neurotransmission to various neuropsychiatric disorders, we investigated the possible influence of a down-regulation of VGLUT1 transporter on anxiety, depressive-like behaviour and learning. The behavioural phenotype of VGLUT1-heterozygous mice (C57BL/6) was compared to wild-type (WT) littermates. Moreover, VGLUT1-3 expression, hippocampal excitatory terminal ultrastructure and neurochemical phenotype were analysed. VGLUT1-heterozygous mice displayed normal spontaneous locomotor activity, increased anxiety in the light-dark exploration test and depressive-like behaviour in the forced swimming test: no differences were shown in the elevated plus-maze model of anxiety. In the novel object recognition test, VGLUT1(+/-) mice showed normal short-term but impaired long-term memory. Spatial memory in the Morris water maze was unaffected. Western blot analysis confirmed that VGLUT1 heterozygotes expressed half the amount of transporter compared to WT. In addition, a reduction in the reserve pool of synaptic vesicles of hippocampal excitatory terminals and a 35-45% reduction in GABA in the frontal cortex and the hippocampus were observed in the mutant mice. These observations suggest that a VGLUT1-mediated presynaptic alteration of the glutamatergic synapses, in specific brain regions, leads to a behavioural phenotype resembling certain aspects of psychiatric and cognitive disorders.

An essential role for vesicular glutamate transporter 1 (VGLUT1) in postnatal development and control of quantal size.

Quantal neurotransmitter release at excitatory synapses depends on glutamate import into synaptic vesicles by vesicular glutamate transporters (VGLUTs). Of the three known transporters, VGLUT1 and VGLUT2 are expressed prominently in the adult brain, but during the first two weeks of postnatal development, VGLUT2 expression predominates. Targeted deletion of VGLUT1 in mice causes lethality in the third postnatal week. Glutamatergic neurotransmission is drastically reduced in neurons from VGLUT1-deficient mice, with a specific reduction in quantal size. The remaining activity correlates with the expression of VGLUT2. This reduction in glutamatergic neurotransmission can be rescued and enhanced with overexpression of VGLUT1. These results show that the expression level of VGLUTs determines the amount of glutamate that is loaded into vesicles and released and thereby regulates the efficacy of neurotransmission.

Performance and evaluation of small construction safety training simulations.

BACKGROUND: Back- and fall-related injuries occur frequently in construction and are costly in terms of workers' compensation claims and lost productivity. Interventions are needed that address the susceptibility to these injuries. AIMS: The purpose of this study was to develop and test a safety training intervention for small construction companies (

Discovery of a novel murine keratin 6 (K6) isoform explains the absence of hair and nail defects in mice deficient for K6a and K6b.

The murine genome is known to have two keratin 6 (K6) genes, mouse K6 (MK6)a and MK6b. These genes display a complex expression pattern with constitutive expression in the epithelia of oral mucosa, hair follicles, and nail beds. We generated mice deficient for both genes through embryonic stem cell technology. The majority of MK6a/b-/- mice die of starvation within the first two weeks of life. This is due to a localized disintegration of the dorsal tongue epithelium, which results in the build up of a plaque of cell debris that severely impairs feeding. However, approximately 25% of MK6a/b-/- mice survive to adulthood. Remarkably, the surviving MK6a/b-/- mice have normal hair and nails. To our surprise, we discovered MK6 staining both in the hair follicle and the nail bed of MK6a/b-/- mice, indicating the presence of a third MK6 gene. We cloned this previously unknown murine keratin gene and found it to be highly homologous to human K6hf, which is expressed in hair follicles. We therefore termed this gene MK6 hair follicle (MK6hf). The presence of MK6hf in the MK6a/b-/- follicles and nails offers an explanation for the absence of hair and nail defects in MK6a/b-/- animals.

Delayed wound healing in keratin 6a knockout mice.

Keratin 6 (K6) expression in the epidermis has two components: constitutive expression in the innermost layer of the outer root sheath (ORS) of hair follicles and inducible expression in the interfollicular epidermis in response to stressful stimuli such as wounding. Mice express two K6 isoforms, MK6a and MK6b. To gain insight into the functional significance of these isoforms, we generated MK6a-deficient mice through mouse embryonic stem cell technology. Upon wounding, MK6a was induced in the outer ORS and the interfollicular epidermis including the basal cell layer of MK6a(+/+) mice, whereas MK6b induction in MK6a(-/-) mice was restricted to the suprabasal layers of the epidermis. After superficial wounding of the epidermis by tape stripping, MK6a(-/-) mice showed a delay in reepithelialization from the hair follicle. However, the healing of full-thickness skin wounds was not impaired in MK6a(-/-) animals. Migration and proliferation of MK6a(-/-) keratinocytes were not impaired in vitro. Furthermore, the migrating and the proliferating keratinocytes of full-thickness wounds in MK6a(-/-) animals expressed neither MK6a nor MK6b. These data indicate that MK6a does not play a major role in keratinocyte proliferation or migration but point to a role in the activation of follicular keratinocytes after wounding. This study represents the first report of a keratin null mutation that results in a wound healing defect.

Expression of MK6a dominant-negative and C-terminal mutant transgenes in mice has distinct phenotypic consequences in the epidermis and hair follicle.

Mouse keratin 6a (MK6a) is constitutively expressed in a single cell layer of the outer root sheath (ORS) of hair follicles, but its synthesis can be induced in interfollicular epidermis including the basal cell layer in response to perturbing stimuli. A basally inducible human K6 (HK6) isoform has not been described, and it is not clear which of the known HK6 isoforms is expressed in the ORS. In this study we show that expression of a dominant-negative MK6a construct (Delta2B-P) in the interfollicular epidermis caused severe blistering and neonatal lethality, suggesting that mutations in a yet to be identified basally expressed HK6 isoform might result in a severe blistering phenotype. Surviving Delta2B-P animals showed transgene expression only in isolated epidermal cells and not in all cells of the ORS, but nevertheless developed severe alopecia. Expression of two different C-terminal mutant transgenes also caused alopecia while a third C-terminal mutant had no phenotypic conse- quences. Electron microscopy revealed that Delta2B-P expression resulted in the collapse of keratin filaments, while destruction of hair follicles in the two phenotypic C-terminal mutant lines occurred in the absence of filament abnormalities. The latter finding indicates that the innermost ORS cells are uniquely sensitive to expression of even slightly altered K6 proteins, suggesting that mutations affecting an HK6 isoform expressed in this cell layer could result in alopecia in humans as well.

The mouse keratin 6 isoforms are differentially expressed in the hair follicle, footpad, tongue and activated epidermis.

Keratin 6 (K6) is expressed constitutively in a variety of internal stratified epithelia as well as in palmoplantar epidermis and in specialized cells of the hair follicle. K6 expression can also be induced by hyperproliferative conditions as in wound healing or by conditions that perturb normal keratinocyte function. The functional significance of the expression of K6 on keratinocyte biology under these disparate conditions is not known. Here we report on the characterization of two isoforms of mouse K6 that are encoded by separate genes. The two genes (denoted K6a and K6b) are linked, have the same orientation and are actively transcribed. Sequence analysis revealed, that although they encode almost identical products, they have distinctly different regulatory regions, suggesting that the two K6 genes would be differentially expressed. In an attempt to define the expression characteristics of the K6 isoforms, we produced transgenic mice with each gene after modifying the C-terminal sequences to enable detection of the transgenic proteins with specific antibodies. The constitutive expression of the K6a transgene paralleled that of the endogenous genes in all K6 expressing tissues, except in the tongue. The K6b transgene was also expressed in these tissues but, in contrast to K6a, was only expressed in suprabasal cells. Both K6 transgenes were also induced in the interfollicular epidermis in response to phorbol esters, with K6a induced in all layers of the treated epidermis, while K6b was expressed only in suprabasal cells. These studies suggest that the K6 isoforms have overlapping yet distinct expression profiles.

Biochemical surface modification of Ti-6Al-4V for the delivery of protein to the cell-biomaterial interface.

Biochemical surface modification involves delivery of biomolecules to the tissue-implant interface to induce desired cell and tissue responses. We have previously had success in immobilizing and retaining bioactive molecules on Co-Cr-Mo but not on Ti-6Al-4V. The purpose of this study was to modify the gamma-aminopropyltriethoxysilane (APS) scheme to enable successful attachment of protein to the surface of Ti-6Al-4V. Ti-6Al-4V samples were silanized with organic (acetone) solutions of APS and dried at increasing temperatures. Concentrations resulting in 2-4 NH2 per nm2 of nominal surface area were incubated in physiological saline for up to 96 hr to assess retention of amino groups. A model protein, trypsin, was coupled to silanized Ti-6Al-4V via glutaraldehyde. The samples were then incubated in saline, and the activity of residual immobilized enzyme was quantified. After drying at 45, 80, or 115 degrees C, the NH2 groups were lost from the surface by 24 hr of incubation in saline. On samples dried at 150 degrees C, with 4% APS, the number of NH2 groups increased after 8 hr and remained relatively constant through 96 hr. Likewise, at 150 degrees C with 2% APS the surface density of NH2 groups reached a maximum at 24 hr and remained relatively constant up to 96 hr. When incubated for 96 hr, Ti-6Al-4V with 4% APS and dried at 150 degrees C retained approximately 31% of the activity initially immobilized, whereas protein on 45 degrees C or adsorbed samples was lost by 24-48 hr.