Morphometric analysis of nerve fibers in neural leprosy.

BACKGROUND: The present study assesses the contributions of axonal degeneration and demyelination in leprosy nerve damage. New clinical strategies can emerge from an in-depth understanding of the pathogenesis of neural leprosy (NL). METHODS: Morphometric analysis of myelinated nerve fibers was performed on 44 nerve biopsy samples collected from leprosy patients. Measures of density, diameter distribution, g-ratios, and the counting of axonal ovoids on the myelinated fibers were taken and compared to those in the control group. RESULTS: The proportion of small myelinated fibers increased in the leprosy group while large fiber frequency decreased. Indicative of axonal atrophy, the g-ratio was lower in the leprosy group. The frequency of axonal ovoids was identical to that found in the non-leprosy neuropathies. CONCLUSIONS: Axonal atrophy, Wallerian degeneration, and demyelination coexist in NL. Axonal degeneration predominates over demyelination in the chronic course of the disease; however, this may change during leprosy reactive episodes. This study regards demyelination and axon degeneration as concurrent mechanisms of damage to nerve fibers in leprosy. It also calls into question the view that demyelination is the primary and predominant mechanism in the complex pathogeny of NL.

A Macrophage Response to Mycobacterium leprae Phenolic Glycolipid Initiates Nerve Damage in Leprosy.

Mycobacterium leprae causes leprosy and is unique among mycobacterial diseases in producing peripheral neuropathy. This debilitating morbidity is attributed to axon demyelination resulting from direct interaction of the M. leprae-specific phenolic glycolipid 1 (PGL-1) with myelinating glia and their subsequent infection. Here, we use transparent zebrafish larvae to visualize the earliest events of M. leprae-induced nerve damage. We find that demyelination and axonal damage are not directly initiated by M. leprae but by infected macrophages that patrol axons; demyelination occurs in areas of intimate contact. PGL-1 confers this neurotoxic response on macrophages: macrophages infected with M. marinum-expressing PGL-1 also damage axons. PGL-1 induces nitric oxide synthase in infected macrophages, and the resultant increase in reactive nitrogen species damages axons by injuring their mitochondria and inducing demyelination. Our findings implicate the response of innate macrophages to M. leprae PGL-1 in initiating nerve damage in leprosy.

LRRK2 and RAB7L1 coordinately regulate axonal morphology and lysosome integrity in diverse cellular contexts.

Leucine-rich repeat kinase 2 (LRRK2) has been linked to several clinical disorders including Parkinson's disease (PD), Crohn's disease, and leprosy. Furthermore in rodents, LRRK2 deficiency or inhibition leads to lysosomal pathology in kidney and lung. Here we provide evidence that LRRK2 functions together with a second PD-associated gene, RAB7L1, within an evolutionarily conserved genetic module in diverse cellular contexts. In C. elegans neurons, orthologues of LRRK2 and RAB7L1 act coordinately in an ordered genetic pathway to regulate axonal elongation. Further genetic studies implicated the AP-3 complex, which is a known regulator of axonal morphology as well as of intracellular protein trafficking to the lysosome compartment, as a physiological downstream effector of LRRK2 and RAB7L1. Additional cell-based studies implicated LRRK2 in the AP-3 complex-related intracellular trafficking of lysosomal membrane proteins. In mice, deficiency of either RAB7L1 or LRRK2 leads to prominent age-associated lysosomal defects in kidney proximal tubule cells, in the absence of frank CNS pathology. We hypothesize that defects in this evolutionarily conserved genetic pathway underlie the diverse pathologies associated with LRRK2 in humans and in animal models.

M. leprae components induce nerve damage by complement activation: identification of lipoarabinomannan as the dominant complement activator.

Peripheral nerve damage is the hallmark of leprosy pathology but its etiology is unclear. We previously identified the membrane attack complex (MAC) of the complement system as a key determinant of post-traumatic nerve damage and demonstrated that its inhibition is neuroprotective. Here, we determined the contribution of the MAC to nerve damage caused by Mycobacterium leprae and its components in mouse. Furthermore, we studied the association between MAC and the key M. leprae component lipoarabinomannan (LAM) in nerve biopsies of leprosy patients. Intraneural injections of M. leprae sonicate induced MAC deposition and pathological changes in the mouse nerve, whereas MAC inhibition preserved myelin and axons. Complement activation occurred mainly via the lectin pathway and the principal activator was LAM. In leprosy nerves, the extent of LAM and MAC immunoreactivity was robust and significantly higher in multibacillary compared to paucibacillary donors (p = 0.01 and p = 0.001, respectively), with a highly significant association between LAM and MAC in the diseased samples (r = 0.9601, p = 0.0001). Further, MAC co-localized with LAM on axons, pointing to a role for this M. leprae antigen in complement activation and nerve damage in leprosy. Our findings demonstrate that MAC contributes to nerve damage in a model of M. leprae-induced nerve injury and its inhibition is neuroprotective. In addition, our data identified LAM as the key pathogen associated molecule that activates complement and causes nerve damage. Taken together our data imply an important role of complement in nerve damage in leprosy and may inform the development of novel therapeutics for patients.

Mechanisms of nerve injury in leprosy.

All patients with leprosy have some degree of nerve involvement. Perineural inflammation is the histopathologic hallmark of leprosy, and this localization may reflect a vascular route of entry of Mycobacterium leprae into nerves. Once inside nerves, M. leprae are ingested by Schwann cells, with a wide array of consequences. Axonal atrophy may occur early in this process; ultimately, affected nerves undergo segmental demyelination. Knowledge of the mechanisms of nerve injury in leprosy has been greatly limited by the minimal opportunities to study affected nerves in man. The nine-banded armadillo provides the only animal model of the pathogenesis of M. leprae infection. New tools available for this model enable the study and correlation of events occurring in epidermal nerve fibers, dermal nerves, and nerve trunks, including neurophysiologic parameters, bacterial load, and changes in gene transcription in both neural and inflammatory cells. The armadillo model is likely to enhance understanding of the mechanisms of nerve injury in leprosy and offers a means of testing proposed interventions.

Schwann cells: origins and role in axonal maintenance and regeneration.

The Schwann cell plays a vital role in maintaining the peripheral nervous system (PNS). Schwann cells are derived from neural crest cells, and come in two types either myelinating or non-myelinating Schwann cells. Both play a pivotal role in the maintenance and regeneration of axons of the neurons in the PNS. The regulation of Schwann cells is mediated a number of different neurotrophic factors which signal to transcription factors such as Krox-20, Oct-6 and Sox-10. Schwann cells are affected in a number of demyelinating disorders, such as Charcot-Marie-Tooth disease and Guillain-Barré Syndrome, infected by Mycobacterium leprae to cause leprosy and are responsible for the tumors seen in patients with neurofibromatosis type 1 and neurofibromatosis type 2. The Schwann cell is under investigation as a therapeutic agent for demyelinating diseases and spinal cord injuries. Further research on Schwann cells will help understand these diseases and perhaps lead to new treatments.

Alterations in neurofilament protein(s) in human leprous nerves: morphology, immunohistochemistry and Western immunoblot correlative study.

Using a specific antibody (SMI 31), the state of phosphorylation of high and medium molecular weight neurofilaments (NF-H and NF-M) was studied in 22 leprous and four nonleprous human peripheral nerves by means of immunohistochemistry, sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot (WB). The results thus obtained were compared with morphological changes in the respective nerves studied through light and electron microscopy. Many of the leprous nerves showing minimal pathology revealed lack of or weak staining with SMI 31, denoting dephosphorylation. Remyelinated fibres stained intensely with SMI 31 antibody. The WB analysis of Triton X-100 insoluble cytoskeletal preparation showed absence of regular SMI 31 reactive bands corresponding to 200 and 150 kDa molecular weight (NF-H and NF-M, respectively) in 10 nerves. Three of the 10 nerves revealed presence of NF protein bands in SDS-PAGE but not in WB. Presence of additional protein band (following NF-M) was seen in four nerves. Two nerves revealed NF-H band but not NF-M band and one nerve showed trace positivity. In the remaining five nerves presence of all the three NF bands was seen. Thus, 77.3% (17/22) of human leprous nerves studied showed abnormal phosphorylation of NF protein(s). The ultrastructural study showed abnormal compaction and arraying of NF at the periphery of the axons in the fibres with altered axon to myelin thickness ratio (atrophied fibres) as well as at the Schmidt-Lantermann (S-L) cleft region. Such NF changes were more pronounced in the severely atrophied axons suggesting a direct correlation. The observed well-spaced NF in the remyelinated fibres under ultrastructural study was in keeping with both intense SMI 31 staining and presence of NF triplet bands seen in WBs in four of leprous nerves that showed a large number of regenerating fibres suggesting reversal of changes with regeneration. Findings in the present study suggest that atrophy, that is, the reduction in axonal calibre and paranodal demyelination, seen in leprous nerves may result from dephosphorylation of NF-H and NF-M proteins.

Axonal spherical bodies in the peripheral nerves of leprosy patients.

Spherical bodies, roughly 10 micro m in diameter, which have not been reported before, were found in the peripheral nerve axons of specimens collected during post-mortem examination of leprosy patients. These bodies were found in the fascicles of all peripheral nerves of the extremities examined (median, radial, ulnar, peroneal and sciatic nerves). Their incidence was not related to the type of leprosy. The area immediately below the thickened perineurium, a feature associated with leprosy, often showed a large number of spherical bodies. When observed under a transmission electron microscope, the spherical lesions often showed a lamellar structure, although some of them were amorphous. No structure resembling organelles was seen within the bodies. Observation with the merge technique showed a clearly lamellar structure in most of the spherical bodies. These bodies and the surrounding myelin sheaths were partially polarized. The axonal spherical bodies observed in our study seem to represent lesions gradually formed due to glycoprotein denaturation over long periods of time and to be associated with leprosy-caused thickening of the perineurium of peripheral nerves.

Mycobacterium leprae-induced demyelination: a model for early nerve degeneration.

The molecular events that occur at the early phase of many demyelinating neurodegenerative diseases are unknown. A recent demonstration of rapid demyelination and axonal injury induced by Mycobacterium leprae provides a model for elucidating the molecular events of early nerve degeneration which might be common to neurodegenerative diseases of both infectious origin and unknown etiology. The identification of the M. leprae-targeted Schwann cell receptor, dystroglycan, and its associated molecules in myelination, demyelination and axonal functions suggests a role for these molecules in early nerve degeneration.

It's all in the assay: a new model for retinotectal topographic mapping.

Ephrin-As have been implicated as topographic mapping labels in the retinotectal system, but the underlying molecular mechanisms for their activities in this context remain somewhat mysterious. Hansen et al. (this issue of Neuron) developed an assay that reveals new mechanisms for ephrins in topographic mapping and suggest a model whereby retinal axons grow and terminate in the tectum via a balance of growth promotion and repulsion, with the balance point depending on retinal position and concentration of ephrin-As.

Alterations in neurofilament protein(s) in human leprous nerves: morphology, immunohistochemistry and Western immunoblot correlative study

Using a specific antibody (SMI 31), the state of phosphorylation of high and medium molecular weight neurofilaments (NF-H and NF-M) was studied in 22 leprous and four nonleprous human peripheral nerves by means of immunohistochemistry, sodium dodecyl sulfate-poly acrylamide gel electrophoresis (SDS-PAGE) and Western immunoblot (WB). The results thus obtained were compared with morphological changes in the respective nerves studied through light and electron microscopy. Many of the leprous nerves showing minimal pathology revealed lack of or weak staining with SMI 31, denoting dephosphorylation. Remyelinated fibres stained intensely with SMI 31 antibody. The WB analysis of Triton X-100 insoluble cytoskeletal preparation showed absence of regular SMI 31 reactive bands corresponding to 200 and 150 kDa molecular weight (NF-H and NF-M, respectively) in 10 nerves. Three of the 10 nerves revealed presence of NF protein bands in SDS-PAGE but not in WB. Presence of additional protein band (following NF-M) was seen in four nerves. Two nerves revealed NF-H band but not NF-M band and one nerve showed trace positivity. In the remaining five nerves presence of all the three NF bands was seen. Thus, 77.3% (17/22) of human leprous nerves studied showed abnormal phosphorylation of NF protein(s). The ultrastructural study showed abnormal compaction and arraying of NF at the periphery of the axons in the fibres with altered axon to myelin thickness ratio (atrophied fibres) as well as at the Schmidt-Lantermann (S-L) cleft region. Such NF changes were more pronounced in the severely atrophied axons suggesting a direct correlation. The observed well-spaced NF in the remyelinated fibres under ultrastructural study was in keeping with both intense SMI 31 staining and presence of NF triplet bands seen in WBs in four of leprous nerves that showed a large number of regenerating fibres suggesting reversal of changes with regeneration. Findings in the present study suggest that atrophy, that is, the reduction in axonal calibre and paranodal demyelination, seen in leprous nerves may result from dephosphorylation of NF-H and NF-M proteins.

Mycobacterium leprae-induced demyelination: a model for early nerve degeneration

The molecular events that occur at the early phase of many demyelinating neurodegenerative diseases are unknown. A recent demonstration of rapid demyelination and axonal injury induced by Mycobacterium leprae provides a model for elucidating the molecular events of early nerve degeneration which might be common to neurodegenerative diseases of both infectious origin and unknown etiology. The identification of the M. leprae-targeted Schwann cell receptor, dystroglycan, and its associated molecules in myelination, demyelination and axonal functions suggests a role for these molecules in early nerve degeneration.

The expression of NGFr and PGP 9.5 in leprosy reactional cutaneous lesions: an assessment of the nerve fiber status using immunostaining.

The effects of reactional episodes on the cutaneous nerve fibers of leprosy patients was assessed in six patients (three with reversal reactions and three with erythema nodosum leprosum). Cryosections of cutaneous biopsy of reactional lesions taken during the episode and of another sample during the remission period were immunostained with anti-NGFr and anti-PGP 9.5 (indirect immunofluorescence). We found no significant statistical difference in the number of NGFr- and PGP 9.5-positive fibers between the reactional and post-reactional groups. A significant difference was detected between the number of NGFr and PGP 9.5-stained fibers inside of the reactional group of biopsy cryosections but this difference was ascribed to the distinct aspects of the nerve fibers displayed whether stained with anti-NGFr or with anti-PGP 9.5; NGFr-positive branches looked larger and so interpreted as containing more fibers. In addition, a substantial number NGFr-positive fibers were PGP 9.5-negative. No differences in the number of stained fibers among the distinct cutaneous regions examined (epidermis + upper dermis, mid and deep dermis) was detected. In conclusion, the number of PGP- and NGFr-positive fibers were not significantly different in the reactional and post-reactional biopsies in the present study. NGFr-staining of the nerve fibers is different from their PGP-imunoreactivity and the evaluation of the nerve fiber status on an innervated target organ should be carried out choosing markers for both components of nerve fibers (Schwann cells and axons).

The expression of NGFr and PGP 9.5 in leprosy reactional cutaneous lesions: an assessment of the nerve fiber status using immunostaining

The effects of reactional episodes on the cutaneous nerve fibers of leprosy patients was assessed in six patients (three with reversal reactions and three with erythema nodosum leprosum). Cryosections of cutaneous biopsy of reactional lesions taken during the episode and of another sample during the remission period were immunostained with anti-NGFr and anti-PGP 9.5 (indirect immunofluorescence). We found no significant statistical difference in the number of NGFr- and PGP 9.5-positive fibers between the reactional and post-reactional groups. A significant difference was detected between the number of NGFr and PGP 9.5-stained fibers inside of the reactional group of biopsy cryosections but this difference was ascribed to the distinct aspects of the nerve fibers displayed whether stained with anti-NGFr or with anti-PGP 9.5; NGFr-positive branches looked larger and so interpreted as containing more fibers. In addition, a substantial number NGFr-positive fibers were PGP 9.5-negative. No differences in the number of stained fibers among the distinct cutaneous regions examined (epidermis + upper dermis, mid and deep dermis) was detected. In conclusion, the number of PGP- and NGFr-positive fibers were not significantly different in the reactional and post-reactional biopsies in the present study. NGFr-staining of the nerve fibers is different from their PGP-imunoreactivity and the evaluation of the nerve fiber status on an innervated target organ should be carried out choosing markers for both components of nerve fibers (Schwann cells and axons)

Contact-dependent demyelination by Mycobacterium leprae in the absence of immune cells.

Demyelination results in severe disability in many neurodegenerative diseases and nervous system infections, and it is typically mediated by inflammatory responses. Mycobacterium leprae, the causative organism of leprosy, induced rapid demyelination by a contact-dependent mechanism in the absence of immune cells in an in vitro nerve tissue culture model and in Rag1-knockout (Rag1-/-) mice, which lack mature B and T lymphocytes. Myelinated Schwann cells were resistant to M. leprae invasion but undergo demyelination upon bacterial attachment, whereas nonmyelinated Schwann cells harbor intracellular M. leprae in large numbers. During M. leprae-induced demyelination, Schwann cells proliferate significantly both in vitro and in vivo and generate a more nonmyelinated phenotype, thereby securing the intracellular niche for M. leprae.

Role of the cell wall phenolic glycolipid-1 in the peripheral nerve predilection of Mycobacterium leprae.

The cell wall of pathogenic mycobacteria is abundant with complex glycolipids whose roles in disease pathogenesis are mostly unknown. Here, we provide evidence for the involvement of the specific trisaccharide unit of the phenolic glycolipid-1 (PGL-1) of Mycobacterium leprae in determining the bacterial predilection to the peripheral nerve. PGL-1 binds specifically to the native laminin-2 in the basal lamina of Schwann cell-axon units. This binding is mediated by the alpha(2LG1, alpha2LG4, and alpha2LG5 modules present in the naturally cleaved fragments of the peripheral nerve laminin alpha2 chain, and is inhibited by the synthetic terminal trisaccharide of PGL-1. PGL-1 is involved in the M. leprae invasion of Schwann cells through the basal lamina in a laminin-2-dependent pathway. The results indicate a novel role of a bacterial glycolipid in determining the nerve predilection of a human pathogen.

Do nerve growth factor-related mechanisms contribute to loss of cutaneous nociception in leprosy?

While sensory loss in leprosy skin is the consequence of invasion by M. leprae of Schwann cells related to unmyelinated fibres, early loss of cutaneous pain sensation, even in the presence of nerve fibres and inflammation, is a hallmark of leprosy, and requires explanation. In normal skin, nerve growth factor (NGF) is produced by basal keratinocytes, and acts via its high affinity receptor (trk A) on nociceptor nerve fibres to increase their sensitivity, particularly in inflammation. We have therefore studied NGF- and trk A-like immunoreactivity in affected skin and mirror-site clinically-unaffected skin from patients with leprosy, and compared these with non-leprosy, control skin, following quantitative sensory testing at each site. Sensory tests were within normal limits in clinically-unaffected leprosy skin, but markedly abnormal in affected skin. Sub-epidermal PGP 9.5- and trk A- positive nerve fibres were reduced only in affected leprosy skin, with fewer fibres contacting keratinocytes. However, NGF-immunoreactivity in basal keratinocytes, and intra-epidermal PGP 9.5-positive nerve fibres, were reduced in both sites compared to non-leprosy controls, as were nerve fibres positive for the sensory neurone specific sodium channel SNS/PN3, which is regulated by NGF, and may mediate inflammation-induced hypersensitivity. Keratinocyte trk A expression (which mediates an autocrine role for NGF) was increased in clinically affected and unaffected skin, suggesting a compensatory mechanism secondary to reduced NGF secretion at both sites. We conclude that decreased NGF- and SNS/PN3-immunoreactivity, and loss of intra-epidermal innervation, may be found without sensory loss on quantitative testing in clinically-unaffected skin in leprosy; this appears to be a sub-clinical change, and may explain the lack of cutaneous pain with inflammation. Sensory loss occurred with reduced sub-epidermal nerve fibres in affected skin, but these still showed trk A-staining, suggesting NGF treatment may restore pain sensation.