Expression of beta-galactosidase in neurons of dorsal root ganglia which are latently infected with herpes simplex virus type 1.

Explanation into culture of dorsal root ganglia (DRG) latently infected with herpes simplex virus type 1 (HSV-1) causes reactivation of the virus. Previous studies have suggested that either latency-associated transcripts (LATs) were removed as an early consequence of reactivation or, alternatively, there was a population of latently infected cells which did not contain LATs. We have now attempted to detect this population of neurons by inserting a reporter gene (Escherichia coli lacZ gene), under the control of promoters other than LAT, into the HSV-1 strain 17 mutant in 1814, which was used in the earlier studies. One of these promoters, the human cytomegalovirus enhancer, resulted in weak expression of beta-galactosidase in DRG neurons for at least 5 months. The pattern of staining was predominantly homogeneous in neurons at 3 or 5 days post-infection or at 3 days post-explanation, and was predominantly speckled in latently infected neurons (1 to 5 months post-infection). About 30% of the beta-galactosidase-positive neurons did not contain LATs by in situ hybridization. However, the detergents used to enable penetration of the substrate for beta-galactosidase had also reduced the levels of the LATs; in neurons which originally had only small numbers of LATs this may have reduced levels to below those detectable by the methods used. There was, therefore, no unequivocal evidence for a population of latently HSV-1-infected cells which did not express LATs.

Neurons containing latency-associated transcripts are numerous and widespread in dorsal root ganglia following footpad inoculation of mice with herpes simplex virus type 1 mutant in1814.

The herpes simplex virus type 1 (HSV-1) mutant in1814 lacks the ability to trans-activate immediate early gene transcription and enter lytic replication but it can establish and reactivate from latency. We therefore investigated the number of neurons that expressed latency-associated transcripts (LATs) in animals latently infected with in1814, the rescued revertant (1814R), or wild-type (wt) HSV-1. The percentage of LAT+ neurons increased with increasing doses of each of the viruses. After inoculation of equal amounts of infectious virus many more LAT+ neurons were observed in animals infected with in1814 than with 1814R or wt HSV-1. Whereas the LAT+ neurons in animals infected with 1814R or wt HSV-1 were largely confined to lumbar dorsal root ganglia (DRG) L4/L5/L6 (those which innervate the lower leg), in animals infected with in1814 they were also present in DRG not directly involved with such innervation (thoracic 12 and 13, L1, L2 and L3). We concluded that the large number of LAT+ neurons observed with in1814 was related to the high particle numbers in the inoculum and that spread of virus was related to limited replication as well as to the low neurovirulence of in1814. This spread was not unique to in1814 but when it occurred with more virulent viruses such as 1814R or wt HSV-1, it resulted in the death of the host.

Reactivation in vivo and in vitro of herpes simplex virus from mouse dorsal root ganglia which contain different levels of latency-associated transcripts.

In the dorsal root ganglia (DRG) of mice latently infected with the herpes simplex virus type 1 mutant in1814, there are more neurons that contain latency-associated transcripts (LATs) than in DRG of mice infected with a dose of equal infectivity of either a revertant or a wild-type virus. We investigated whether higher levels of LAT+ neurons resulted in more extensive reactivation either in vivo following neurectomy of the sciatic nerve or in vitro after explantation into culture. Neurectomy appeared to induce expression of immediate early 1 mRNA (IE1mRNA) in neurons of mice latently infected with each of three viruses. However IE1mRNA was detected in no more than 0.25% of the neurons of DRG from animals 2 to 4 days after neurectomy, irrespective of the percentage of LAT+ neurons present. Of the 22 neurons shown to express IE1mRNA, none expressed LATs also. However the lack of expression of viral antigen and the absence of a reduced potential for reactivation on explanation suggested that neurectomy had not induced full reactivation involving lytic replication leading to the death of the latently infected neurons. When DRG were explanted into culture, the distribution of the frequency of reactivation was similar to the distribution of DRG that contained LAT+ neurons. The presence of a high proportion of LAT+ neurons was not directly associated with earlier detection of reactivation but such experiments cannot be regarded as quantitative. We therefore concluded that neurectomy did not result in a reduced reactivation potential as described by others and that the frequency of expression of IE1mRNA following neurectomy did not correlate with the number of LAT+ neurons present.

P0 gene expression in cultured Schwann cells.

This study examines the expression of the major myelin protein gene P0 in cultured Schwann cells, grown on their own or in association with neurons. Many freshly dissociated Schwann cells from actively myelinating nerves express Po mRNA in high abundance. If neurons are not present, signal intensity falls markedly with time so that by 7 days in culture only a basal expression is evident which is negligible compared to the level in vivo. Dorsal root ganglia from embryo day 16 (E16) rats contain no significant levels of Po mRNA but when grown in full myelinating medium (containing serum and embryo extract) increasing expression is seen from 4 to 5 days onward even though myelination does not occur until after the second week. In this intervening period the intensity of P0 mRNA expression is lower than that found in the actively myelinating cell. Neurons from sympathetic ganglia are also capable of inducing P0 mRNA expression. Schwann cells in dorsal root ganglia explants grown in serum-free defined medium do not assemble a basal lamina and will not wrap or myelinate axons. Nevertheless P0 mRNA, but not protein, is expressed in levels similar to those found in full myelinating medium prior to myelination. Such Schwann cells also exhibit galactocerebroside and the sulphatide recognised by the 04 antibody. It appears that in defined medium or in myelinating medium prior to myelination axonal signals can induce P0 mRNA expression to a certain degree. However, full up-regulation is usually associated with the rapid membrane expansion accompanying myelination. Whether this augmented up-regulation is due to further axonal signalling or events in the Schwann cell is unknown, but the results suggest that P0 expression can be regulated at several stages of synthesis.

Regeneration of cat posterior temporalis muscle in culture.

Cat posterior temporalis muscle has a rapid speed of contraction associated with a unique superfast myosin isoform. Superfast myosin expression appears to be an intrinsic property of the muscle fibres and satellite cells, though in culture they failed to express superfast myosin. We have, therefore, cultured this muscle in a system which had previously been shown to encourage the expression of an adult phenotype. The presence of nerve cells resulted in effective regeneration of cat posterior temporalis muscle and even the formation of functional neuromuscular junctions. However, superfast myosin was not found even in mature, contracting, innervated cultures. Thyroid hormone, a known regulator of myosin isoform expression, also failed to elicit superfast myosin expression. Different culture conditions may allow a different outcome, but under circumstances in which mouse muscle expresses an adult phenotype, cat posterior temporalis muscle fails to do so.

Localization of dystrophin in cultures of human muscle.

Dystrophin is a protein present in normal human muscle but absent in patients with Duchenne muscular dystrophy (DMD). Using a specific antibody, we have investigated the expression of dystrophin in human muscle which had regenerated in culture in the presence of nerve cells. Dystrophin was present and was correctly localized in the cultures of normal muscle but was absent from cultures of muscle from patients with DMD. Its control and function can now be studied.

Parvalbumin in mouse muscle in vivo and in vitro.

Parvalbumin is a cytosolic calcium-binding protein found in adult fast-twitch mammalian muscle. Using an antibody to paravalbumin, we have shown that its distribution in adult mouse muscles is associated with certain fibre types. It is absent from slow-twitch type 1 fibres, is absent or at low levels in fast-twitch type 2A fibres, but is present at moderate or high levels in fast-twitch type 2B fibres. When adult mouse muscle is cultured with embryonic mouse spinal cord, the regenerated fibres become innervated, express the adult fast isoform of myosin heavy chain and appear histochemically as fast-twitch fibres. We therefore investigated whether these apparently mature fibres also contained parvalbumin. Parvalbumin was not found in any fibres of twenty mature cultures, suggesting that neurotrophic activity in the absence of specific adult nerve activity patterns was insufficient to cause the expression of parvalbumin in the cultures.

Morphological differentiation of human muscle cocultured with mouse spinal cord.

Human muscle fibres have been cocultured with sections of embryonic mouse spinal cord for periods of up to 2 months. The muscle fibres regenerated to form a bundle of myotubes, a proportion of which developed cross-striations and contractions. This proportion was variable between biopsies, and morphological differentiation was not as successful as when mouse muscle and mouse nerve were cultured together. Regeneration and morphological differentiation were unaffected by storing samples in liquid nitrogen, and were not improved by the presence of original synaptic areas in the explanted bundle or by alterations in the growth media. These involved changing the levels of serum and embryo extract, using different sources of serum, and the incorporation of additives in the medium. A comparison of the growth characteristics of samples of muscle from 30 patients (including some control samples) indicated that although muscle from younger patients (less than 14 years) regenerated more quickly, the myotubes did not have better differentiation. It also indicated that the growth characteristics of regenerated myotubes from diseased and normal muscle were indistinguishable within the 4-8 weeks observation period. Muscle from patients with Duchenne muscular dystrophy regenerated and differentiated less well than would be expected from age-matched controls, but this was not thought to reflect an intrinsic abnormality in the regenerative capacity of the muscle.

Atypical persisting fibres in explants of human muscle cocultured with embryonic nerve cells.

When samples of human muscle are cocultured with embryonic mouse spinal cord, the muscle fibres usually regenerate to form a bundle of new myotubes which become innervated and develop cross-striations and contractions. However, we have noticed that in some cultures of 6 biopsies of human muscle, there were fibres which "persisted" in the cultures for long periods of time without being replaced by regenerated myotubes. At both the light and electron microscopic levels, they appeared to be mature muscle with well-organized myofibrils, intact plasma membranes and closely-apposed basal laminae. At least some of the fibres contained adult myosin heavy chains. They were only found in freshly-cultured samples, and appeared to be associated with younger patients, but were not associated with any particular muscle condition. The nature of these persisting fibres is discussed, and we emphasize the need for sequential observation of cultures to ensure that fibres which have regenerated in culture are distinguished from those which have persisted from the original biopsy.

5'-Nucleotidase in skin fibroblasts from patients with Duchenne muscular dystrophy.

The 5'-nucleotidase of plasma membranes of cultured skin fibroblasts from patients with Duchenne muscular dystrophy had a reduced affinity for its substrate, 5'-AMP. The Arrhenius plot of the temperature dependence of this enzyme activity was normal. There was no difference between patients and controls in the specific 5'-nucleotidase activity in the whole cell homogenates.

Neonatal and adult myosin heavy chain isoforms in a nerve-muscle culture system.

When adult mouse muscle fibers are co-cultured with embryonic mouse spinal cord, the muscle regenerates to form myotubes that develop cross-striations and contractions. We have investigated the myosin heavy chain (MHC) isoforms present in these cultures using polyclonal antibodies to the neonatal, adult fast, and slow MHC isoforms of rat (all of which were shown to react specifically with the analogous mouse isoforms) in an immunocytochemical assay. The adult fast MHC was absent in newly formed myotubes but was found at later times, although it was absent when the myotubes myotubes were cultured without spinal cord tissue. When nerve-induced muscle contractions were blocked by the continuous presence of alpha-bungarotoxin, there was no decrease in the proportion of fibers that contained adult fast MHC. Neonatal and slow MHC were found at all times in culture, even in the absence of the spinal cord, and so their expression was not thought to be nerve-dependent. Thus, in this culture system, the expression of adult fast MHC required the presence of the spinal cord, but was probably not dependent upon nerve-induced contractile activity in the muscle fibers.