The multiple sclerosis degradome: enzymatic cascades in development and progression of central nervous system inflammatory disease.

An array of studies implicate different classes of protease and their endogenous inhibitors in multiple sclerosis (MS) pathogenesis based on expression patterns in MS lesions, sera, and/or cerebrospinal fluid (CSF). Growing evidence exists regarding their mechanistic roles in inflammatory and neurodegenerative aspects of this disease. Proteolytic events participate in demyelination, axon injury, apoptosis, and development of the inflammatory response including immune cell activation and extravasation, cytokine and chemokine activation/inactivation, complement activation, and epitope spreading. The potential significance of proteolytic activity to MS therefore relates not only to their potential use as important biomarkers of disease activity, but additionally as prospective therapeutic targets. Experimental data indicate that understanding the net physiological consequence of altered protease levels in MS development and progression necessitates understanding protease activity in the context of substrates, endogenous inhibitors, and proteolytic cascade interactions, which together make up the MS degradome. This review will focus on evidence regarding the potential physiologic role of those protease families already identified as markers of disease activity in MS; that is, the metallo-, serine, and cysteine proteases.

Dynamic role of kallikrein 6 in traumatic spinal cord injury.

Kallikrein 6 (K6) is a member of the kallikrein gene family that comprises 15 structurally and functionally related serine proteases. In prior studies we showed that, while this trypsin-like enzyme is preferentially expressed in neurons and oligodendroglia of the adult central nervous system (CNS), it is up-regulated at sites of injury due to expression by infiltrating immune and resident CNS cells. Given this background we hypothesized that K6 is a key contributor to the pathophysiology of traumatic spinal cord injury (SCI), influencing neural repair and regeneration. Examination of K6 expression following contusion injury to the adult rat cord, and in cases of human traumatic SCI, indicated significant elevations at acute and chronic time points, not only at the injury site but also in cord segments above and below. Elevations in K6 were particularly prominent in macrophages, microglia and reactive astrocytes. To determine potential effects of elevated K6 on the regeneration environment, the ability of neurons to adhere to and extend processes on substrata which had been exposed to recombinant K6 was examined. Limited (1 h) or excess (24 h) K6-mediated proteolytic digestion of a growth-facilitatory substrate, laminin, significantly decreased neurite outgrowth. By contrast, similar hydrolysis of a growth-inhibitory substrate, aggrecan, significantly increased neurite extension and cell adherence. These data support the hypothesis that K6 enzymatic cascades mediate events secondary to spinal cord trauma, including dynamic modification of the capacity for axon outgrowth.

Potential scope of action of tissue kallikreins in CNS immune-mediated disease.

The objective of this study was to define the potential scope of action of tissue kallikreins in T cell-mediated disease of the CNS. We demonstrate quantitatively the differential expression of all 15 human tissue kallikreins within brain, spinal cord and immune compartments. In human Jurkat T cells we demonstrate differential regulation of select kallikreins by CD3 receptor, Concanavilin A (Con A), interleukin 2 (IL2), and lipopolysaccharide (LPS)-mediated activation and by exposure to steroid hormones, dexamethasone, norgestrel, androstan and estradiol. The patterns of co-expression and co-regulation described point to novel effector roles for select tissue kallikreins in neurological disorders involving T cells, such as multiple sclerosis.

Distinct promoters regulate tissue-specific and differential expression of kallikrein 6 in CNS demyelinating disease.

Kallikrein 6 is a serine protease expressed abundantly in normal adult human and rodent CNS, and therein is regulated by injury. In the case of CNS demyelinating disease, K6 expression in CNS occurs additionally in perivascular and parenchymal inflammatory cells suggesting a role in pathogenesis. Herein we describe two unique transcripts that occur within the human and mouse K6 genes that differ in their 5'-untranslated regions. These transcripts have identical translation initiation sites in exon 3, are expressed in a tissue-specific fashion and are differentially regulated in response to CNS injury. While the human and mouse 5'-transcripts differ in sequence they are identical in genomic organization and tissue-specific expression. The most 5'-transcript, designated transcript 1, includes exon 1-7, and was detectable in all CNS regions, but not in any non-CNS tissues examined (spleen, thymus, liver, kidney, pancreas, submandibular gland and peripheral nerve). In contrast, transcript 2 lacks exon 1, but contains a unique sequence at the 5'-end of exon 2, designated exon 2A. Transcript 2 was expressed both in CNS and in each peripheral tissue. In a murine model of human CNS demyelinating inflammatory disease induced by Theiler's picornovirus, mouse K6 transcript 1 was up-regulated in brain and spinal cord at acute and more chronic phases of CNS inflammation and demyelination, while overall transcript 2 expression was not significantly altered. However, in isolated splenocyte cultures, transcript 2 was up-regulated two-fold by cellular activation. Tissue-specific expression patterns and differential regulation in CNS disease indicates that each K6 5'-transcript is probably regulated by unique promoter elements and may serve as a molecular target to treat inflammatory demyelinating disease.

Hit-Hit and hit-Run: viruses in the playing field of multiple sclerosis.

Viruses have been major players in the search for the cause of multiple sclerosis (MS). In support of the viral theory is the predominance of CD8+ T cells and class-I major histocompatibility complex in lesions, the powerful therapeutic effects of beta interferons, the ease of inducing demyelination in experimental models following virus challenge, and the documented examples of several human demyelinating diseases conclusively demonstrated to be of viral origin. We propose two hypotheses of how viruses may cause MS. In the "Hit-Hit" hypothesis, the virus persists or may be reactivated in the central nervous system (CNS). Injury is the result of direct viral damage and by an attempt of the immune response to clear the infectious agent. In the "Hit-Run" hypothesis, virus infects the periphery but never enters the CNS. The virus sets up an abnormal immunologic milieu for subsequent autoimmunity. In both scenarios, knowing the inciting virus would be expected to eliminate disease if the population were vaccinated to prevent infection. In the treatment of patients with fully established disease, the Hit-Hit hypothesis would require that antiviral agents enter the CNS and stop replication. In the case of the Hit-Run hypothesis, treatment of patients with established disease with antiviral agents would be futile.

Activity of a newly identified serine protease in CNS demyelination.

We have identified a novel serine protease, myelencephalon-specific protease (MSP), which is preferentially expressed in the adult CNS, and therein, is abundant in both neurones and oligodendroglia. To determine the potential activity of MSP in CNS demyelination, we examined its expression in multiple sclerosis lesions and in two animal models of multiple sclerosis: Theiler's murine encephalomyelitis virus (TMEV) and myelin oligodendrocyte glycoprotein (MOG)-induced experimental allergic encephalomyelitis (EAE) in marmosets. High levels of MSP were present within infiltrating mononuclear cells, including macrophages and T cells, which characteristically fill sites of demyelination, both in multiple sclerosis lesions and in animal models of this disease. The functional consequence of excess MSP on oligodendroglia was determined in vitro by evaluating the effects of recombinant MSP (r-MSP) on oligodendrocyte survival and process number. Application of excess r-MSP resulted in a dramatic loss of processes from differentiated oligodendrocytes, and a parallel decrease in process outgrowth from immature cells. Transfection of oligodendrocyte progenitors with an MSP-green fluorescent protein construct produced similar changes in oligodendrocyte process number. Importantly, r-MSP did not affect oligodendrocyte survival or differentiation towards the sulphatide-positive lineage. We further demonstrate that myelin basic protein, and to a lesser extent myelin oligodendrocyte glycoprotein, can serve as MSP substrates. These studies support the hypothesis that excess MSP, as is present in inflammatory CNS lesions, promotes demyelination.

MSP, a trypsin-like serine protease, is abundantly expressed in the human nervous system.

The goal of the present investigation was to determine the regional and cellular specific expression patterns of the newly identified serine protease, myelencephalon-specific protease (MSP), in the adult human brain (Scarisbrick et al. [1997b] J. Neurosci. 17:8156-8168). To assess the potential scope of MSP activity, Northern blot techniques were used to determine the relative abundance of MSP mRNA in 16 different adult human brain regions, and in the brain and peripheral tissues of the midgestation human fetus. The regional and temporal specific expression patterns of MSP mRNA were directly compared with those of tissue plasminogen activator (tPA), a serine protease strongly implicated in the development, ongoing plasticity, and response of the nervous system to injury and disease. mRNA encoding each protease was distributed widely throughout the normal adult human central nervous system (CNS), but the expression of each was only partially overlapping. Additionally, compared with tPA, MSP exhibited a more restricted distribution and delayed developmental onset. By immunohistochemical localization, MSP was present at moderate to high levels in neurons and oligodendroglia of the adult human brain, at a level closely resembling the relative abundance indicated by Northern blot. MSP was most abundantly expressed in the spinal cord, hippocampus, substantia nigra, and basal ganglia. The robust expression of MSP in clinically significant regions of the adult human CNS indicates that further study of this protease in terms of both normal brain physiology and neurodegenerative disorders is warranted.

Neurotrophin-4/5 promotes proliferation of oligodendrocyte-type-2 astrocytes (O-2A).

In this study, the ability of neurotrophin-5 (NT-4/5) to promote the proliferation of oligodendrocyte precursor (O-2A) cells has been examined. This has been accomplished by the addition of exogenous NT-4/5 to purified cultures of O-2A cells maintained in an undifferentiated state by the addition of the mitogens platelet derived growth factor (PDGF-AA) and basic fibroblast growth factor (bFGF). Counts of cells incorporating bromodeoxyuridine (BrdU) showed that the addition of NT-4/5 for 24 h increased O-2A cell proliferation by 1.8-fold above that seen in PDGF-AA and bFGF alone. These data demonstrate a previously unidentified role for the neurotrophin NT-4/5 in oligodendrocyte biology.

Preferential expression of myelencephalon-specific protease by oligodendrocytes of the adult rat spinal cord white matter.

Myelencephalon-specific protease (MSP) is a novel serine protease that is expressed predominantly in the nervous system. In the adult rat spinal cord, MSP mRNA expression was dramatically upregulated, in both the white and gray matter, after systemic exposure to the glutamate receptor agonist, kainic acid (KA) (Scarisbrick et al. J Neurosci 17: 8156-8168, 1997b). To determine the cell-specific expression patterns of MSP, we generated MSP-specific monoclonal antibodies. These have been used in immunohistochemical and in situ hybridization colocalization studies, to demonstrate that MSP mRNA and protein are produced predominantly by CNP-immunoreactive oligodendroglia, but not by GFAP-immunoreactive astrocytes, in the white matter of the normal adult cord. In vitro, the soma of oligodendrocytes were also densely MSP immunoreactive, as were their growth tips, while astrocytes were associated with lower levels. These findings suggest that the enzymatic activity of MSP is likely to be important in the biology of oligodendrocytes and/or in the maintenance of the nerve fiber tracts of the adult spinal cord.

Differential expression of brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 in the adult rat spinal cord: regulation by the glutamate receptor agonist kainic acid.

Previous in vitro studies indicate that select members of the neurotrophin gene family, namely brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4/5 (NT-4/5), contribute to survival and differentiation of spinal cord motoneurons. To investigate the potential roles of these factors in the adult spinal cord, we examined their cellular localization and regulation after systemic exposure to an excitotoxic stimulus, kainic acid (KA). Of the neurotrophins examined, NT-4/5 mRNA was most robustly expressed in the lumbosacral spinal cord of the normal adult rat, including expression by neurons throughout the gray matter, and in a subpopulation of white and gray matter glia. Both BDNF and NT-3 mRNAs were also densely expressed by alpha motoneurons of lamina IX, but were detected at lower levels elsewhere in the gray matter. NT-3 mRNA was additionally expressed by spinal cord glia, but was less widespread compared to NT-4/5. In response to systemic administration of KA, NT-4/5 and BDNF mRNAs were dramatically upregulated in a spatially and temporally restricted fashion, whereas levels of NT-3 mRNA were unchanged. These results provide strong in vivo evidence to support the idea that BDNF, NT-3, and in particular, NT-4/5, play a role in the normal function of the adult spinal cord. Furthermore, our results indicate that the actions of BDNF and NT-4/5 participate in the response of the cord to excitotoxic stimuli, and that those of NT-4/5 and NT-3 include both neurons and glia.

Nervous system-specific expression of a novel serine protease: regulation in the adult rat spinal cord by excitotoxic injury.

A full-length cDNA clone of a previously unidentified serine protease, myelencephalon-specific protease (MSP), has been isolated by using a PCR cloning strategy and has been shown to be expressed in a nervous system and spinal cord-specific pattern. Sequence analysis demonstrated that MSP is most similar in sequence to neuropsin, trypsin, and tissue kallikrein and is predicted to have trypsin-like substrate specificity. MSP mRNA was found to be approximately 10-fold greater in the CNS of the rat and human, as compared with most peripheral tissues, and within the CNS was found to be highest by a factor of four in the medulla oblongata and spinal cord. Levels of mRNA encoding tissue plasminogen activator (tPA) also were elevated in the spinal cord but were more widespread in peripheral tissues as compared with MSP. In the adult rat lumbosacral spinal cord, in situ localization of MSP mRNA demonstrated 2-fold higher levels in the white, as compared with the gray, matter. MSP mRNA expression was shown to increase 3-fold in the white matter and 1.5-fold in the gray laminae at 72 hr after intraperitoneal injection of the AMPA/kainate glutamate receptor-specific agonist, kainic acid (KA). MSP mRNA remained elevated in the ventral gray matter, including expression associated with the motor neurons of lamina IX, at 7 d after the initial excitotoxic insult. Together, these observations indicate that MSP is in a position to play a fundamental role in normal homeostasis and in the response of the spinal cord to injury.

Coexpression of mRNAs for NGF, BDNF, and NT-3 in the cardiovascular system of the pre- and postnatal rat.

The expression of NGF, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) mRNAs was examined in whole rat embryos and in the heart and great vessels of postnatal and adult rats, using in situ hybridization of cRNA probes. The patterns of expression were correlated with innervation patterns as revealed by immunostaining for neural cell adhesion molecule (NCAM) and with the HNK-1 antibody, which demonstrates derivatives of the neural crest. The patterns of neurotrophin mRNA localization were different from those of mRNAs for the low-molecular-weight NGF receptor. Hybridization indicating the presence of mRNAs for all three neurotrophins is particularly prominent within the tunica media of the aorta, pulmonary, and other major elastic arteries of the thorax and abdomen and is first observed on embryonic day 13 (E13) when innervation is being established and rises to maximum by E15. In the fetus, there is little or no detectable expression in the CNS or PNS. NT-3 expression in the vessels is relatively constant and high from embryonic to adult stages, while levels of BDNF increase and those of NGF decrease over the same time course. During the fetal period, hybridization in the heart is absent. In the postnatal period, additional label becomes detectable in the coronary arteries but not in the walls of the atria or ventricles, other than at the base of the aorta and pulmonary trunk.(ABSTRACT TRUNCATED AT 250 WORDS)

NCAM immunoreactivity during major developmental events in the rat maxillary nerve-whisker system.

The distribution of immunoreactivity for neural cell adhesion molecule (NCAM) has been characterized during the formation of the trigeminal ganglion and during the process of axon outgrowth and target differentiation in the maxillary nerve-whisker system, in rat fetuses of known gestational age. Proliferating cells within the trigeminal placode are NCAM immunoreactive when first observed on embryonic day (E) 10. NCAM immunoreactivity is lost from placode-derived cells as they migrate to the trigeminal ganglion. It re-appears on ganglion cell somata and on centrally and peripherally projecting axons at the time of neurite outgrowth. NCAM-immunoreactive centrally projecting axons reach the developing brain stem two days before peripheral axons encounter the presumptive whisker pad. NCAM immunoreactivity on axons and somata is down regulated after P0, following target contact and whisker follicle differentiation. The presumptive dermis of the whisker pad at E13 appears as a sheet-like condensation of intensely NCAM immunostained cells. Discrete infraorbital row nerves can be identified on E13. These form in the subdermal region which contains only low levels of NCAM immunoreactivity. Condensations of NCAM immunostained mesenchyme replace the dermal sheet on E14 and each condensation is associated with a plexus of infraorbital nerve fibers. The epithelium overlying each condensation grows downward on E15. Focal epithelial regions become NCAM immunoreactive by E18. NCAM immunostaining within epithelial components of the whisker follicle is temporally correlated with contact by NCAM-immunoreactive infraorbital nerve fibers. The site restricted expression of NCAM immunoreactivity during trigeminal embryogenesis is consistent with the idea that NCAM plays an integral role in critical aspects of pattern formation in the maxillary nerve-whisker system, particularly in the organization of placode and non-placode derived trigeminal neuroblasts, axon outgrowth and in the differentiation of the vibrissae follicles.

Effects of self- and cross-reinnervation on the numbers of motoneurons regenerating to forearm muscles in young and adult rats.

The present study was carried out to compare the ability of motoneurons to regenerate to functionally appropriate and inappropriate muscles, following axotomy at different stages of postnatal development. Five-, 10-, 21-day-old and adult rats of both sexes were used. In one group, the right median and radial nerves were cut and reunited. In a second group, the cut nerves were cross reunited and, in a third group the nerves were merely exposed. Following survival periods of up to one year, the extent of motoneuron regeneration through the repaired nerves was determined by injecting the retrogradely transported tracers horseradish peroxidase (HRP) and Fast Blue into the flexor and extensor muscles of the right forearm. The results were expressed in terms of the difference between the number of labelled motoneurons on the experimental side of the spinal cord and the number on the control side, the latter having been labelled by injection of HRP and Fast Blue into the muscles of the left forearm. Comparisons were then made between the groups with respect to the age at which axotomy occurred, and the target of regeneration. The results showed that when axotomy was performed in 5- and 10-day-old rats, significantly fewer motoneurons were labelled, irrespective of whether or not the target was functionally appropriate, than when axotomy was performed in adulthood. The difference was most likely due to a lower survival rate of motoneurons following axotomy in neonates. No difference was found, however, between the numbers of labelled median and radial nerve motoneurons following self- versus cross-reinnervation in any age group. This suggests that, in both adult and neonatal rats, motoneurons which survive axotomy are able to regenerate equally well to functionally appropriate or inappropriate muscles.

The arrangement of forearm motoneurons in young and adult rats and the possibility of naturally occurring motoneuron death.

The normal number and arrangement of motoneurons contributing fibres, via the median and radial nerves, to the forearm muscles of the rat were determined at different stages of postnatal development. Horseradish peroxidase was applied directly to the proximal cut ends of the nerves in 5, 10, 21 days old and adult rats. At each stage of development, the motoneuronal pool for each nerve was confined to the ipsilateral, dorsolateral area of Lamina IX. In general, median nerve motoneurons were found dorsal and medial to those of the radial nerve and were located between the rostral parts of the C6-T1 segments of the spinal cord. The radial nerve motoneuronal pool extended between the caudal end of the C4 and rostral tip of the T1 segment. There was considerable overlap between the two populations, especially along the dorsoventral axis. Counts of labelled motoneurons revealed that significantly fewer were present in adult compared to 5 days old rats (P less than 0.05 for each nerve). Approximately 50% of the motoneurons were lost from each nerve over this period. Although results from other studies suggest that part of the apparent loss may have been due to deficiencies in the HRP tracing technique, the possibility of motoneuronal death cannot be entirely excluded in this study.