Kathryn M. Albers

Sympathetic Innervation of Lymphoreticular Organs Is Rate Limiting for Prion Neuroinvasion

Transmissible spongiform encephalopathies are commonly propagated by extracerebral inoculation of the infectious agent. Indirect evidence suggests that entry into the central nervous system occurs via the peripheral nervous system. Here we have investigated the role of the sympathetic nervous system in prion neuroinvasion. Following intraperitoneal prion inoculation, chemical or immunological sympathectomy delayed or prevented scrapie. Prion titers in spinal cords were drastically reduced at early time points after inoculation. Instead, keratin 14-NGF transgenic mice, whose lymphoid organs are hyperinnervated by sympathetic nerves, showed reduction in scrapie incubation time and, unexpectedly, much higher titers of prion infectivity in spleens. We conclude that sympathetic innervation of lymphoid organs is rate limiting for prion neuroinvasion and that splenic sympathetic nerves may act as extracerebral prion reservoirs.

NEUROTROPHINS, NOCICEPTORS, AND PAIN

It is now well established that neurotrophins play a crucial role in the development of the nervous system. However, there is increasing evidence that the function of neurotrophins persists throughout adulthood. The broad scope of neurotrophin action is well documented in the case of nerve growth factor (NGF) and its effect on nociceptors and nociception. Here, we review the evidence for these multiple roles for NGF. Two manipulations influencing NGF levels are discussed in detail. The first involves the use of transgenic mice that overexpress or underexpress neurotrophins. A second strategy involves administration of NGF or its antibody in vivo to increase or decrease its level. During prenatal development, NGF is required for survival of nociceptors. In the early postnatal period, NGF is required for expression of the appropriate nociceptor phenotype. In adults, NGF acts as an important intermediate in inflammatory pain, contributing to both peripheral and central sensitization. The sensitization of peripheral nociceptors can be very rapid and can involve non‐neural cells such as mast cells, neutrophils, fibroblasts, and macrophages. Recent evidence indicates that other neurotrophins also play key supporting roles in the development of nociceptors (e.g., NT‐3) and in inflammatory pain (e.g., BDNF, NT‐4/5). Furthermore, molecules from other superfamilies (e.g., GDNF) also are required to assure survival of certain classes of nociceptors. The diverse effects of neurotrophins on nociceptive processing emphasize their broad importance in the development and function of the nervous system. Microsc Res Tech 45:252–261, 1999.u2003

Altered expression of nerve growth factor in the skin of transgenic mice leads to changes in response to mechanical stimuli

It has recently become clear that the neurotrophic factor, nerve growth factor, interacts specifically with nociceptive sensory neurons during development and maturity. Indeed, it may serve as a critical link between inflammation and the hyperalgesia that ensues in adult animals. Nerve growth factor is normally expressed in limiting amounts in target tissues of sensory and postganglionic sympathetic neurons. In the present study we have altered the basal level of nerve growth factor expression in the skin by producing transgenic mice that express a fusion gene construct containing either a sense or antisense nerve growth factor complementary DNA linked to the K14 keratin promoter. The K14-nerve growth factor transgene (sense or antisense) is abundantly expressed in skin from approximately embryonic day 15 and is then constitutively expressed throughout the life of the animal. In light of the fact that systemic administration of nerve growth factor to neonatal or adult rats leads to hyperalgesia, we have asked whether mice expressing the sense K14-nerve growth factor transgene exhibit similar sensory abnormalities and whether mice expressing the antisense nerve growth factor complementary DNA were hypoalgesic. Here we show that mice over-expressing nerve growth factor in skin display a profound hyperalgesia to noxious mechanical stimulation. Additionally, K14-nerve growth factor antisense mice displayed a profound hypoalgesia to the same stimuli.

A role for p75 neurotrophin receptor in the control of apoptosis-driven hair follicle regression

To examine the mechanisms that underlie the neurotrophin‐induced, apoptosis‐driven hair follicle involution (catagen), the expression and function of p75 neurotrophin receptor (p75NTR), which is implicated in apoptosis control, were studied during spontaneous catagen development in murine skin. By RT‐PCR, high steady‐state p75NTR mRNA skin levels were found during the anagen– catagen transition of the hair follicle. By immunohistochemistry, p75NTR alone was strongly expressed in TUNEL+/Bcl2— keratinocytes of the regressing outer root sheath, but both p75NTR and TrkB and/or TrkC were expressed by the nonregressing TUNEL‐/Bcl2+ secondary hair germ keratinocytes. To determine whether p75NTR is functionally involved in catagen control, spontaneous catagen development was compared in vivo between p75NTR knockout (— /—) and wild‐type mice. There was significant catagen retardation in p75NTR knockout mice as compared to wild‐type controls (P<0.05). Instead, transgenic mice‐overexpressing NGF (promoter: K14) showed substantial acceleration of catagen (P<0.001). Although NGF, brain‐derived neurotrophic factor (BDNF), and neurotrophin 3 (NT‐3) accelerated catagen in the organ‐cultured skin of C57BL/6 mice, these neurotrophins failed to promote catagen development in the organ‐cultured p75NTR null skin. These findings suggest that p75NTR signaling is involved in the control of kerotinocyte apoptosis during catagen and that pharmacological manipulation of p75NTR signaling may prove useful for the treatment of hair disorders that display premature entry into catagen.—Botchkarev, V. A., Botchkareva, N. V., Albers, K. M., Chen, L.‐H., Welker, P., Paus, R. A role for p75 neurotrophin receptor in the control of apoptosisdriven hair follicle regression. FASEB J. 14, 1931–1942 (2000)

A New Role for Neurotrophin-3 : Involvement in the Regulation of Hair Follicle Regression (Catagen)

Nervous system and hair follicle epithelium share a common ectodermal origin, and some neurotrophins (NTs) can modulate keratinocyte proliferation and apoptosis. Therefore, it is reasonable to ask whether NTs are also involved in hair growth control. Here, we show that the expression of NT-3 and its high-affinity receptor, tyrosine kinase C, in the skin of C57BL/6 mice is strikingly hair cycle-dependent, with maximal transcript and protein expression seen during spontaneous hair follicle regression (catagen). During catagen, NT-3 and tyrosine kinase C are co-expressed by terminal deoxynucleotidyl transferase-mediated in situ nick end labeling-positive keratinocytes in the club hair and secondary germ. NT-3-overexpressing transgenic mice show precocious catagen development during the postnatal initiation of hair follicle cycling, whereas heterozygous NT-3 knockout (+/-) mice display a significant catagen retardation. Finally, NT-3 stimulates catagen development in organ culture of normal C57BL/6 mouse skin. These observations suggest that the hair follicle is both a source and target of NT-3 and that NT-3/tyrosine kinase C signaling is functionally important in the control of hair follicle regression. Therefore, tyrosine kinase C agonists and antagonists deserve systematic exploration for the management of hair growth disorders that are related to premature (alopecia/effluvium) or retarded catagen (hirsutism/hypertrichosis).

OVEREXPRESSION OF NERVE GROWTH FACTOR IN SKIN CAUSES PREFERENTIAL INCREASES AMONG INNERVATION TO SPECIFIC SENSORY TARGETS

The impact of increased levels of skin‐derived nerve growth factor (NGF) neurotrophin on sensory and sympathetic innervation to the mouse mystacial pad and postero‐orbital vibrissae was determined. Consistent with an approximate doubling of neuron number in trigeminal and superior cervical ganglia, many components of the sensory and sympathetic innervation were substantially enhanced. Although the increased number of neurons raised the possibility that all types of innervation were increased, immunohistochemical analysis indicated that enhanced NGF production had a differential effect upon sensory innervation, primarily increasing unmyelinated innervation. This increased innervation occurred in specific locations known to be innervated by small, unmyelinated fibers, suggesting that NGF modulated sensory innervation density, but not targeting. In contrast, sympathetic innervation was not only increased but also was distributed to some aberrant locations. In the intervibrissal fur of the mystacial pad, both the number of sensory axons and branches appeared increased, whereas in vibrissal follicle sinus complexes, only branching increased. In some areas, sensory ending density was lower than expected based upon the size of the source nerve bundles suggesting that many axons and branches were surviving but failing to form functional endings. Furthermore, the immunochemical profile of innervation was altered in some sensory populations as demonstrated by the coexistence of RT‐97 neurofilament labeling in calcitonin gene‐related peptide (CGRP) positive axons, by the loss of substance P colocalization in some CGRP axons, and by an absence of neuropeptide Y labeling in tyrosine hydroxylase positive sympathetic axons. Collectively, these results indicate that the NGF mediated increase in neuron number may be selective for particular sets of innervation and that increases among some populations may result from phenotypic switching. J. Comp. Neurol. 387:489–506, 1997.

Overexpression of nerve growth factor in skin increases sensory neuron size and modulates Trk receptor expression.

Sensory neuron development and differentiation is dependent on a family of growth factors known as neurotrophins. Neurotrophins modulate neuron development via trk tyrosine kinase receptor proteins trkA, trkB and trkC. To determine how elevated levels of a target‐derived neurotrophin might affect neuronal differentiation, we analysed trk expression in the trigeminal ganglion of transgenic mice that overexpressed nerve growth factor (NGF) in the skin. increased levels of NGF caused a five‐fold increase in neurons expressing trkA mRNA and a two‐fold increase in neurons expressing trkC. In control mice, cell size distributions of neuronal subpopulations expressing each trk mRNA showed the three subpopulations distributed over a narrow, overlapping range. In contrast, cell size distribution in NGF‐transgenic mice was significantly divergent due in large part to hypertrophy of trkA neurons and, to a lesser extent, trkC neurons. In addition, we examined neurons that bound the isolectin B4 from Bandeiraea simplicifolia (BS‐IB4) because most of these neurons do not express any trk receptor in the adult. There was a significant increase in the size of BS‐IB4–positive neurons in transgenic mice; however, there was no increase in their number. These studies indicate that an increased level of target‐derived NGF affects the development of sensory neurons that in the adult express trkA or trkC, as well as neurons that do not express trk receptors.

Sodium channel expression in NGF-overexpressing transgenic mice

Dorsal root ganglion (DRG) neurons depend on nerve growth factor (NGF) for survival during development, and for the maintenance of phenotypic expression of neuropeptides in the adult. NGF also plays a role in the regulation of expression of functional sodium channels in both PC12 cells and DRG neurons. Transgenic mice that overexpress NGF under the keratin promoter (hyper‐NGF mice) show increased levels of NGF in the skin from embryonic day 11 through adulthood, hypertrophy of the peripheral nervous system and mechanical hyperalgesia. We show here that mRNA levels for specific sodium channel isotypes are greater in small (<30 μm diameter) DRG neurons from hyper‐NGF mice compared to wild‐type mice. Hybridization signals for sodium channel subunits αII and β2 displayed the most substantial enhancement in hyper‐NGF mice, compared to wild‐type mice DRG, and mRNA levels for αI, NaG, Na6, SNS/PN3, NaN, and β1 were also greater in hyper‐NGF DRG. In contrast, the levels of αII and PN1 mRNAs were similar in neurons from hyper‐NGF and wild‐type DRG. Whole‐cell patch‐clamp studies showed no significant differences in the peak sodium current densities in hyper‐NGF vs. wild‐type DRG neurons. These data demonstrate that DRG neurons in wild‐type mice have a heterogeneous pattern of sodium channel expression, which is similar to that previously described in rat, and suggest that transcripts of some, but not all, sodium channel mRNAs can be modulated by long‐term overexpression of NGF. J. Neurosci. Res. 57:39–47, 1999.

Age-dependent phenotypic switching of mast cells in NGF-transgenic mice

Effects of overexpression of nerve growth factor (NGF) on mast cell phenotype and numbers were investigated in nasal and oral mucosae and skin of 3- and 6-week-old transgenic mice in which NGF expression in epithelial basal cells was driven by the keratin-14 promoter. Mast cell phenotypes were identified by Alcian blue/safranin and berberine sulfate histochemistry. In the 3-week-old transgenic mice, NGF overexpression had no effect on phenotype except in tongue, where mast cells exhibited mixed or connective tissue phenotypes compared with the mucosal phenotype in the non-transgenic. In 6-week-old transgenic animals, NGF overexpression resulted in the mucosal phenotype in tissues which contained connective tissue or mixed mast cells in non-transgenics. Mast cell hyperplasia occurred at both ages. NGF effects on mast cell phenotype were age-dependent and involve complex microenvironmental interactions.

Cellular localization of pan-trk immunoreactivity and trkC mRNA in the enteric nervous system

The members of the trk family of tyrosine receptor kinases, trkA, trkB, and trkC, are the functional receptors for neurotrophins, a family of related neurotrophic factors. In this study, we investigated 1) the distribution of neurotrophin receptors in the developing and adult rat digestive tract with a pan‐trk antibody that recognizes all known trks and 2) the cellular localization of trk‐encoding mRNAs in the adult gut with single‐stranded RNA probes specific for trkA, trkB, and trkC. In the developing myenteric plexus, trk immunoreactivity was present at embryonic day (ED) 14. Cells and fibers immunoreactive for trk could be visualized in the myenteric plexus at ED 16. At this age, dense staining was found in thick bundles of fibers in proximity to the myenteric plexus in the longitudinal muscle and in association with blood vessels in the mesentery. At ED 18, trk immunoreactivity was also seen in thin processes running from the myenteric plexus into the circular muscle, and in fibers and cells in intrapancreatic ganglia. By ED 20, immunoreactive staining was quite dense in both the myenteric and submucosal plexuses. At birth, virtually all enteric ganglia displayed strong trk immunoreactivity; the intensity of the staining at this age made it difficult to discern individual cells. During postnatal development, there was a decrease in cell body staining and an increase in the density of trk‐containing fibers that became widely distributed to the gut wall and pancreas. The adult pattern of trk immunoreactivity was established between postnatal days 5 and 10. In adults, trk immunoreactivity was found in numerous enteric and intrapancreatic ganglion cells and in dense networks of fibers innervating all the layers of the gut, the pancreas, and vasculature. The trkC mRNA was expressed in adult enteric ganglion cells of both the myenteric and submucous plexus. By contrast, the trkA and trkB mRNAs could not be detected in enteric ganglia. All three trk mRNAs were expressed in dorsal root ganglia, which were used as positive controls. The density and wide distribution of trk immunoreactivity together with its persistence in adulthood support the concept that neurotrophins play a broad role in the digestive system from development through adult life, perhaps being involved in differentiation, phenotypic expression, and tissue maintenance. The presence of trkC mRNA in enteric neurons along with recent evidence that neurotrophin‐3 plays a role in the development of the enteric nervous system suggest that trkC and neurotrophin‐3 are a major neurotrophin system in the gastrointestinal tract.