BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major components of plaque, beta-amyloid peptides (Abetas), are produced from amyloid precursor protein (APP) by the activity of beta- and gamma-secretases. beta-secretase activity cleaves APP to define the N-terminus of the Abeta1-x peptides and, therefore, has been a long- sought therapeutic target for treatment of AD. The gene encoding a beta-secretase for beta-site APP cleaving enzyme (BACE) was identified recently. However, it was not known whether BACE was the primary beta-secretase in mammalian brain nor whether inhibition of beta-secretase might have effects in mammals that would preclude its utility as a therapeutic target. In the work described herein, we generated two lines of BACE knockout mice and characterized them for pathology, beta-secretase activity and Abeta production. These mice appeared to develop normally and showed no consistent phenotypic differences from their wild-type littermates, including overall normal tissue morphology and brain histochemistry, normal blood and urine chemistries, normal blood-cell composition, and no overt behavioral and neuromuscular effects. Brain and primary cortical cultures from BACE knockout mice showed no detectable beta-secretase activity, and primary cortical cultures from BACE knockout mice produced much less Abeta from APP. The findings that BACE is the primary beta-secretase activity in brain and that loss of beta-secretase activity produces no profound phenotypic defects with a concomitant reduction in beta-amyloid peptide clearly indicate that BACE is an excellent therapeutic target for treatment of AD.

Use of gamma-glutamyl transpeptidase activity as a marker of hair cycle and anagen induction in mouse hair follicles.

gamma-glutamyl transpeptidase activity was monitored in cycling mice by histologic localization and biochemical assay. Our objective for this study is to establish the relationship between gamma-glutamyl transpeptidase activity and hair growth and to determine whether its activity can be correlated to induced hair growth. In cycling mouse skin, gamma-glutamyl transpeptidase activity is pronounced during anagen and greatly diminished during telogen. In the skin, the enzyme is present exclusively in the outer and inner root sheaths of hair follicles. gamma-glutamyl transpeptidase is limited to the follicle below the level of the sebaceous gland and is completely absent in the follicle above the sebaceous gland level. During anagen, the outer root sheath in the hypodermis is intensely positive for gamma-glutamyl transpeptidase activity whereas the hair matrix cells and dermal papillar are negative. The inner root sheath above the bulb shows distinctive membrane staining for gamma-glutamyl transpeptidase. gamma-glutamyl transpeptidase activity can be seen to vary only in cycling follicles. Inducing anagen by plucking hair shafts results in an increase in gamma-glutamyl transpeptidase activity directly correlated to hair regrowth. In a similar manner, mice were plucked and treated with a daily dose of 2% minoxidil. A slight difference in cycle lengths was seen in animals treated with minoxidil when compared to vehicle control. Minoxidil treatment may cause an early initiation of anagen, but both the minoxidil-treated skin and the vehicle-treated skin entered telogen at the same time. Together, these studies indicate that gamma-glutamyl transpeptidase is a specific marker of anagen in growing hair.

A method to detect areas high in sulfhydryl groups in mouse epithelium.

We have recently modified a non-fluorescent, non-radioactive histochemical method to detect sulfhydryl (S-H) groups in tissues. This method was originally intended to detect chloramphenicol acetyltransferase (CAT) in transgenic mice. Temporal developmental differences in the keratinization of mouse digits can be seen in the staining pattern of the skin about the toes of neonatal mice. The basal cells of the epidermis exposed to the air show intense staining while the epidermis that is still attached to an adjacent toe shows no staining. The degree of S-H presence can be determined by the tissues' resistance to blocking of the S-H groups by iodoacetic acid. Areas that contain very high numbers of S-H groups still show staining following blocking by iodoacetic acid. We have found that this method shows clear differences in the S-H distribution of various epithelium, including skin, hair, nails, and tongue epithelium.

Enhanced in vitro hair growth at the air-liquid interface: minoxidil preserves the root sheath in cultured whisker follicles.

Inasmuch as hair follicles are difficult to maintain in culture, the study of hair biology using cultured hair follicles has met with only limited success. In our attempts to solve the problem of follicle degeneration, we cultured follicles at the air-surface interface on a modified collagen matrix (Gelfoam). In follicles cultured at the air-surface or submerged, we examined follicular morphology, hair shaft growth, sulfotransferase levels, cysteine incorporation, an expression of a tissue inhibitor of metalloproteinase (TIMP), and ultra-high sulfur keratin (UHSK). Follicles cultured at the air-liquid interface produced a 2.7-fold increase in hair growth and maintained an anagen-like morphology. Substrates such as nylon mesh seeded with fibroblasts, Full Thickness Skin, or 5-microns polycarbonate filter also supported hair growth, whereas Gelfilm, GF-A glass filter, filter paper, or 1-micron polycarbonate filter did not. The UHSK expression was significantly higher in the air-liquid interface cultures compared to the submerged culture. Several potassium channel openers, including minoxidil, a minoxidil analog, and the pinacidil analog (P-1075), all stimulated significant cysteine incorporation in follicles. Minoxidil and its analog specifically preserved the follicular root sheath, in contrast to P-1075 which did not, indicating a difference in the two drug types. The preservation of the root sheath was measured by increased TIMP expression and sulfotransferase activity and indicates that the root sheath is a target tissue for minoxidil. Our results show that follicles cultured at the air-liquid interface maintain a better morphology and produced greater hair growth than follicles cultured on tissue culture plastic.

Interaction of minoxidil with pigment in cells of the hair follicle: an example of binding without apparent biological effects.

To identify minoxidil target cells in hair follicles we followed the uptake of radiolabeled drug in mouse vibrissae follicles both in vitro and in vivo. Autoradiography showed that both 3H-minoxidil and 3H-minoxidil sulfate accumulated in the differentiating epithelial matrix cells superior to the dermal papilla, a distribution similar to that of pigment. Minoxidil localized in melanocytes, melanocyte processes, and areas of greater melanin concentrations within the epithelial cells. Although uptake of minoxidil was significantly less in unpigmented follicles, the drug stimulated proliferation and differentiation of both pigmented and unpigmented follicles. Labeled minoxidil bound to Sepia melanin and was displaced with unlabeled minoxidil and other electron donor drugs. This interaction with melanin acts as a targeting mechanism of minoxidil to pigmented hair follicles but has no apparent functional significance in hair growth. This work illustrates how measurement of drugs in hair may be biased by pigmentation.

High-sulfur protein gene expression in a transgenic mouse.

We analyzed the effect of minoxidil on hair follicles isolated from transgenic mice. These transgenic animals synthesize the reporter enzyme CAT in their hair follicles only during the active phases of hair growth. The recombinant gene used to generate these mice contained the bacterial enzyme CAT under the control of the promoter from the gene of UHS protein. Studies using in situ hybridization showed that UHS proteins are expressed specifically in the matrix cells of the hair follicle during the terminal stages of hair differentiation. Hence the expression of the UHS proteins is a clear sign of active hair growth. With other in situ hybridization studies we demonstrated that CAT mRNA is expressed in differentiating matrix cells of the hair shaft in a location similar to that in which mRNA encodes UHS proteins. Thus we can use the levels of CAT activity as a measure of hair growth. We have confirmed that expression of the transgene is found in hair that is high in anagen and low in catagen follicles. The usefulness of our model was further demonstrated by showing that minoxidil, a drug that stimulates hair growth, increased the expression of CAT in cultured hair follicles. Thus we have demonstrated that expression of this reporter gene is sensitive, hair specific, and also useful for monitoring effects in cultured hair follicles. Hence these transgenic mice provide a model system for studying the biology of hair growth.

Hair-specific keratins: characterization and expression of a mouse type I keratin gene.

A genomic clone for a member of the mouse type I hair keratin protein family has been isolated and analyzed in order to study the regulation of this keratin during the hair growth cycle. The coding sequence is divided into seven exons. The gene structure is typical of keratins in particular and intermediate filaments in general in that the intron-exon borders are not located at the domain borders of the protein. Comparison with a sheep wool keratin gene shows that the splice sites in the two hair keratin genes are found at identical locations relative to the amino acid sequence of the proteins. Similarly, comparison of the promoter areas of these genes shows several areas of nucleotide sequence conservation, including the area around the TATA box and an SV40 core enhancer sequence. In addition, a high degree of sequence identity exists in the fourth intron. In situ hybridization shows that transcripts of this gene are first found in the relatively undifferentiated proximal cortex area in the keratogenous zone of mouse vibrissae.

Localization of TIMP in cycling mouse hair.

TIMP (tissue inhibitor of metalloproteinase) is a glycoprotein inhibitor of metalloproteinases that we hypothesize to be involved in the tissue remodeling that occurs during each hair growth cycle. We examined this hypothesis by studying the expression of TIMP at selected times during a single hair cycle using TIMP-lacZ transgenic mice to localize TIMP gene activity in the hair follicle. TIMP gene induction was visualized by staining mouse back skin for beta-galactosidase (beta-gal) activity. Paraffin sections were analyzed for the localization of TIMP expression. TIMP gene activation appears in hair follicles only during the mid-anagen (the growing stage of the hair cycle) primarily in Henle's layer of the inner root sheath. Some expression of TIMP is also seen in a few connective tissue cells, in the sebaceous gland and in cells at the proximity of the dermal papilla cells in catagen (regressing) and telogen (resting) follicles. These results are consistent with a role for TIMP in cyclic remodeling of connective tissue in hair follicles.

Immunohistochemical and autoradiographic findings suggest that minoxidil is not localized in specific cells of vibrissa, pelage, or scalp follicles.

Immunohistochemistry with a minoxidil antibody suggested that minoxidil-immunoreactivity is associated with the root sheaths, laterally orientated differentiating matrix cells, and dividing epithelial cells of cultured vibrissa follicles of pigmented and albino neonatal mice. The dermal papilla and connective tissue sheath were devoid of minoxidil-immunoreactivity. To verify that minoxodil-immunoreactivity in the follicles was specific, immunostaining was conducted with dissected whisker pads, formalin-fixed "dead" follicles, and sections of spleen, liver and kidney (non-haired organs) cultured with minoxidil. Microscopic examination revealed minoxidil-immunoreactivity in all of these tissues. Follicles and whisker pads cultured with minoxidil, then washed for one h in media were devoid of minoxidil-immunoreactivity. These data suggest that minoxidil-immunoreactivity in cultured vibrissa follicles is probably non-specific. Sections of skin from C3H and CF1 mice which were topically dosed with minoxidil (in vivo) showed no minoxidil-immunoreactivity. Autoradiography demonstrated that tritiated minoxidil was bound in vivo and in vitro only to melanin granules in pigmented follicles of rodent and human tissue. This is probably non-specific binding since melanin is known to accumulate several chemically and pharmacologically unrelated drugs. It is reasonable to conclude that, under the conditions of these experiments, minoxidil is not specifically localized in any cells of whisker, pelage or, scalp follicles.

Hair-specific expression of chloramphenicol acetyltransferase in transgenic mice under the control of an ultra-high-sulfur keratin promoter.

We have generated a transgenic mouse line by microinjection of a chimeric DNA fragment (KER-CAT) containing a hair-specific, murine ultra-high-sulfur keratin promoter (KER) fused to the coding region of the bacterial chloramphenicol acetyltransferase (CAT) gene. A 671-base pair (bp) stretch of the 5' promoter region was used to direct the expression of the CAT gene in this construct. Of the tissues tested for CAT activity in these transgenic animals only skin with growing hair, isolated hair follicles, and microdissected vibrissae showed substantial levels of activity. These are the same tissues where the endogenous ultra-high-sulfur keratin gene is expressed as shown by in situ hybridization. Furthermore, analysis of the CAT activity during the developmental stages of the hair growth cycle shows that the chimeric gene is expressed during the anagen phase of the hair growth cycle; this is the expected time during development for its expression. From these results we conclude that 671 bp of the promoter sequence from the ultra-high-sulfur keratin gene is sufficient to direct the correct development-specific and tissue-specific expression of the reporter gene construct in transgenic mice. The appropriate expression of the KER-CAT construct in transgenic mice is an important step in understanding the regulation of this gene during hair organogenesis.

Minoxidil stimulates mouse vibrissae follicles in organ culture.

Minoxidil, a potent vasodilator, stimulates the growth of terminal hair from vellus or miniaturized follicles in balding scalp. To study minoxidil's action on isolated follicles we developed and validated an organ culture system using mouse whisker follicles. Control follicles cultured without minoxidil showed macroscopic changes including kinking of the hair shafts and bending of the follicles. Necrosis was evident in the differentiating epithelial elements forming the cuticle, cortex, and inner root sheath. These abnormalities were eliminated or greatly reduced in minoxidil-treated follicles. The morphology of these follicles was consistent with the production of new hair during culture. Direct measurement demonstrated that minoxidil-treated follicles grew significantly longer than control follicles during the 3-d culture. Minoxidil increased the incorporation of radiolabeled cysteine and glycine in follicles compared with control treatment. Doses of minoxidil up to 1 mM caused increased cysteine incorporation, while higher doses were inhibitory. Experiments with labeled thymidine indicated that minoxidil induced proliferation of hair epithelial cells near the base of the follicle. Autoradiography also showed that cysteine accumulated in the keratogenous zone above the dermal papilla. These studies demonstrate that organ cultured follicles are suitable for determining minoxidil's mechanism of action and may be useful for studying other aspects of hair biology. The results also show that minoxidil's effect on hair follicles is direct. This suggests that minoxidil's action in vivo includes more than just increasing blood flow to hair follicles.

Variation in basement membrane topography in human thick skin.

Samples of human plantar and palmar skin were excised and incubated in 20 mM EDTA after which the epidermis was gently separated from the dermis with the plane of separation occurring in the lamina lucida. Scanning electron microscopic examination of the dermal component revealed the classically described series of regularly spaced grooves and papillae that characterize the epidermal-dermal junction in thick skin. Primary dermal grooves exhibited evenly spaced tunnels that were originally occupied by sweat gland ducts. The basement membrane (basal lamina) in the primary grooves was relatively smooth but did exhibit a flattened, reticulated pattern at high magnifications. The basement membrane of secondary dermal grooves and papillae was in the form of numerous, elevated microridges off of which septae arose at roughly right angles. The surface appearance of the basement membrane in these areas was that of a honeycomb owing to the numerous compartments and recesses formed by the ridges and septae. Degradation of the basement membrane by trypsin demonstrated that the foundation for the highly folded and compartmentalized basement membrane was composed of dermal collagen fibrils, 60-70 nm in diameter, that were arranged in a series of variably sized, interconnected collagen bundles or walls. Epidermal basal cells extended cytoplasmic (foot) processes into two or more compartments, formed by the ridges and septae, which considerably amplified the basement membrane surface available for epidermal attachment. Scanning electron microscopic studies of the epidermal-dermal junction confirm the variable surface character of this interface previously reported by others using sectioned material.(ABSTRACT TRUNCATED AT 250 WORDS)