Familial autoimmune myastenia gravis: four patients involving three generations.

BACKGROUND: Familial autoimmune myasthenia gravis (MG) is rare, although a genetic role for the development of autoimmune MG is suggested by concordance in monozygotic twins and the increased frequency of other autoimmune diseases in family members of myasthenics. METHODS: A patient with a family history of MG was evaluated in hospital. Relatives were interviewed and medical records examined for details regarding the diagnosis of MG in three other family members. RESULTS: The index case first experienced symptoms of MG at age 75 years. She developed generalized MG and required corticosteroids and immunosuppressive therapy to control her disease. Her father developed predominantly bulbar symptoms of MG at age 75 years. He died of complications experienced following a gastrostomy placed for continued difficulty swallowing. His brother developed similar symptoms of MG in his early 60s and died shortly after thymectomy. A 46-year-old nephew of the index case is also beginning to exhibit signs of generalized MG. Acetylcholine receptor antibodies were strongly positive in the index case and her nephew. (The assay was not available for her father and uncle). CONCLUSIONS: Four individuals in three successive generations had diagnoses of autoimmune MG. Study of familial cases such as these may clarify the contribution of genetic factors to the development of this disease.

Colocalization of TrkB and brain-derived neurotrophic factor proteins in green-red-sensitive cone outer segments.

PURPOSE: To examine the distribution of neurotrophins (NTs) and their catalytic receptors in adult rat photoreceptors. METHODS: Immunocytochemistry and Western blot analyses were performed using primary antibodies raised against NTs (nerve growth factor [NGF], brain-derived neurotrophic factor [BDNF], NT-3, and NT-4/5) and NT receptors (TrkA, TrkB, TrkC, and p75NTR). Double-labeling of retinal sections with opsin-specific antibodies was performed to identify each photoreceptor type. Competitive experiments using excess recombinant NT or Trk receptors confirmed the binding specificity of each antibody. RESULTS: TrkB and BDNF immunoreactivity was colocalized in cone outer segments. TrkB and BDNF were detected in all green-red-sensitive cones, but not in blue-UV cones or rods, and other NTs and NT receptors were not detected in any of the photoreceptor types. CONCLUSIONS: The findings suggest a specific role for BDNF through its signaling receptor TrkB in the function and maintenance of green-red cones, the predominant cone type in the rat retina.

Prolonged administration of NT-4/5 fails to rescue most axotomized retinal ganglion cells in adult rats.

The survival of axotomized RGCs was increased by intravitreal NT-4/5 given by repeated injections or osmotic minipumps, but the effects were less complete than predicted. Compared to a single injection of the neurotrophin on day 0, second injections on days 3 or 7 only sustained an additional 10-20% of the RGCs on day 10. Minipumps augmented RGC survival up to 4-fold (50%) at 2 weeks but most RGCs were lost by 1 month. Thus, specific neurotrophins can rescue many RGCs soon after injury but long-term neuronal survival may require a better understanding of changes in neurotrophin receptors and interactions with other molecules.

Prolonged delivery of brain-derived neurotrophic factor by adenovirus-infected Müller cells temporarily rescues injured retinal ganglion cells.

In this study, we demonstrate that: (i) injection of an adenovirus (Ad) vector containing the brain-derived neurotrophic factor (BDNF) gene (Ad.BDNF) into the vitreous chamber of adult rats results in selective transgene expression by Müller cells; (ii) in vitro, Müller cells infected with Ad.BDNF secrete BDNF that enhances neuronal survival; (iii) in vivo, Ad-mediated expression of functional BDNF by Müller cells, temporarily extends the survival of axotomized retinal ganglion cells (RGCs); 16 days after axotomy, injured retinas treated with Ad.BDNF showed a 4.5-fold increase in surviving RGCs compared with control retinas; (iv) the transient expression of the BDNF transgene, which lasted approximately 10 days, can be prolonged with immunosuppression for at least 30 days, and such Ad-mediated BDNF remains biologically active, (v) persistent expression of BDNF by infected Müller cells does not further enhance the survival of injured RGCs, indicating that the effect of this neurotrophin on RGC survival is limited by changes induced by the lesion within 10-16 days after optic nerve transection rather than the availability of BDNF. Thus, Ad-transduced Müller cells are a novel pathway for sustained delivery of BDNF to acutely-injured RGCs. Because these cells span the entire thickness of the retina, Ad-mediated gene delivery to Müller cells may also be useful to influence photoreceptors and other retinal neurons.

Regenerated retinal ganglion cell axons form normal numbers of boutons but fail to expand their arbors in the superior colliculus.

Regenerated retinal ganglion cell (RGC) axons can re-form functional synapses with target neurons in the superior colliculus (SC). Because preterminal axon branching determines the size, shape and density of innervation fields, we investigated the branching patterns and bouton formation of individual RGC axons that had regrown along peripheral nerve (PN) grafts to the SC. Within the superficial layers of the SC, the regenerated axons formed terminal arbors with average numbers of terminal boutons that were similar to the controls. However, axonal branches were shorter than normal so that the mean area of the regenerated arbors was nearly one-tenth that of control arbors and the resulting fields of innervation contained greater than normal numbers of synapses concentrated in small areas of the target. Our results have delineated a critical defect in the reconstitution of retino-collicular circuitry in adult mammals: the failure of terminal RGC branches to expand appropriately. Because recent studies have documented that brain-derived neurotrophic factor (BDNF) can specifically lengthen RGC axonal branches not only during development in the SC but also within the adult retina after axotomy, the present quantitative studies should facilitate experimental attempts to correct this deficit of the regenerative response.

A distinct pattern of trophic factor expression in myelin-deficient nerves of Trembler mice: implications for trophic support by Schwann cells.

Distal to a peripheral nerve transection, myelin degradation and Schwann cell (SC) proliferation are accompanied by a marked upregulation of brain-derived neurotrophic factor (BDNF) and a decrease of ciliary neurotrophic factor (CNTF) in non-neuronal cells. To investigate the role of SC differentiation in trophic factor regulation, we studied BDNF and CNTF expression in sciatic nerves from Trembler-J (Tr-J) mice. In these animals, a mutation in the pmp-22 gene causes a failure of myelination and continuous SC proliferation, but axonal continuity is preserved. In spite of the severe abnormalities in Tr-J nerves, BDNF levels remained as low as in the intact controls. Thus, the primary SC disorder in Tr-J produces a different pattern of BDNF expression from that caused by axonal breakdown due to nerve transection. Furthermore, the upregulation of BDNF mRNA triggered by transection was 70-fold in control nerves, but only 30-fold in Tr-J sciatic nerves. Because these results raised the possibility that axonal loss may influence neurotrophin expression only in SCs that have differentiated toward a myelinating phenotype, we measured BDNF mRNA after axotomy in the cervical sympathetic trunk (CST), a predominantly unmyelinated autonomic nerve. In contrast to the sciatic nerves, the BDNF mRNA level barely increased in the injured CST, supporting the idea that not all SCs are equal sources of trophic molecules. In Tr-J sciatic nerves, CNTF mRNA levels were fourfold lower than normal, implying that the downregulation of this cytokine is a sensitive indicator of a spectrum of SC perturbations that affect myelinating cells.

Brain-derived neurotrophic factor and neurotrophin-4/5 stimulate growth of axonal branches from regenerating retinal ganglion cells.

To investigate the influences of growth factors on axonal regeneration in the mammalian CNS, we used intracellular tracers to quantitate the effects of brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-4/5, or NT-3 on individual retinal ganglion cell (RGC) axons in the retinas of adult rats after optic nerve transection. A single injection of BDNF or the prolonged administration of NT-4/5 by mini-pump increased axon branch median lengths by eightfold but had no effect on the number of branches formed by the RGC axons. NT-3 did not significantly influence axonal regrowth. These specific in vivo effects of BDNF and NT-4/5 on axonal regeneration from injured RGCs may be used to promote growth and expand the abnormally small terminal arbors observed when RGCs regrow into their CNS targets.

Effects of neurotrophins on the survival and regrowth of injured retinal neurons.

The focus of this short review is the role of certain neurotrophins and their receptors on the survival and regrowth of retinal ganglion cells (RGCs) whose axons are damaged in the optic nerve. Initial experiments in our laboratory documented patterns of RGC death after axotomy. Subsequent studies were designed to investigate the distribution of high-affinity neurotrophin receptors in neurons and glial cells of the retina and optic nerve. This information was used both in vitro and in vivo to study the effects of specific trophic molecules on the survival and regrowth of injured RGCs. During the course of experiments involving neurotrophin administration, an endogenous source of trophic support--independent of the exogenous administration of growth factors--was found within the eye. Several experiments were subsequently undertaken to define further this survival effect and determine its nature and source within the eye. Finally, anatomical techniques that help visualize fine axonal processes within the retina have provided insights into the specific effects of neurotrophins on the growth and branching of injured CNS axons.

Neurotrophin-4/5 (NT-4/5) increases adult rat retinal ganglion cell survival and neurite outgrowth in vitro.

Retinal ganglion cell (RGC) survival and neurite outgrowth were investigated in retinal explants from adult rats. Neurotrophin-4/5 (NT-4/5) caused dose-dependent increases in neurite outgrowth with one-half maximal effects at approximately 0.5 ng/ml and maximal effects at 5 ng/ml. In explants treated for 7 days, the actions of NT-4/5 were similar to those of brain-derived neurotrophic factor (BDNF); with either neurotrophin, nearly twice as many RGCs survived and there was a two- to threefold increase in the number of neurites formed by RGCs. Combinations of saturating concentrations of NT-4/5 and BDNF did not enhance these in vitro effects, implying that both neurotrophins share a common signaling pathway. In contrast, nerve growth factor (NGF), neurotrophin-3 (NT-3), or ciliary neurotrophic factor (CNTF) appeared to exert minimal influences on RGC survival or neurite outgrowth.

Axotomy results in delayed death and apoptosis of retinal ganglion cells in adult rats.

Using quantitative anatomical techniques, we show that after intraorbital optic nerve transection in adult rats, virtually all retinal ganglion cells (RGCs) survive for 5 d and then die abruptly in large numbers, reducing the RGC population to approximately 50% of normal by day 7 and to less than 10% on day 14. During this period of rapid cell loss, some RGCs show cytochemical alterations indicative of apoptosis ("programmed cell death"), a change not previously categorized after axotomy in adult mammals. With intracranial lesions 8-9 mm from the eye, the onset of cell death is delayed until day 8 and is greater with cut than crush. The demonstration that axotomy results in apoptosis, the long interval between axonal injury and RGC death, and the different time of onset of the massive RGC loss with optic nerve lesions near or far from the eye suggest that axonal interruption triggers a cascade of molecular events whose outcome may be critically dependent on the availability of neuronal trophic support from endogenous or exogenous sources. The role of such molecules in RGC survival and the reversible nature of these injury-induced changes is underscored by the temporary rescue of most RGCs by a single intravitreal injection of brain-derived neurotrophic factor during the first 5 d after intraorbital optic nerve injury (Mansour-Robaey et al., 1994). The delayed pattern of RGC loss observed in the present experiments likely explains such a critical period for effective neurotrophin administration.

Effects of ocular injury and administration of brain-derived neurotrophic factor on survival and regrowth of axotomized retinal ganglion cells.

Optic nerve transection in adult rats results in the death of approximately 50% of the axotomized retinal ganglion cells (RGCs) by 1 week and nearly 90% by 2 weeks after injury. The capacity of brain-derived neurotrophic factor (BDNF) to prevent this early, severe loss of RGCs was investigated in vivo by intravitreal injections of BDNF [5 micrograms in 5 microliters of bovine serum albumin/phosphate-buffered saline (BSA/PBS)] or vehicle (5 microliters of BSA/PBS). Using quantitative anatomical techniques, we show that (i) all RGCs survived 1 week after a single injection of BDNF at the time of axotomy. (ii) RGC densities decreased in the BDNF-treated retinas by 2 weeks but remained significantly greater than in the untreated controls. (iii) An enhanced RGC survival was obtained with single injections of BDNF from 6 days before to 5 days after axotomy. (iv) Repeated injections resulted in greater numbers of surviving RGCs, an effect that declined to undetectable levels by 6 weeks. (v) There were indications for an endogenous local source of trophic support whose expression was triggered by ocular injury, particularly to the anterior part of the eye. (vi) With multiple BDNF injections, there was profuse axonal sprouting around the optic disc. This remarkable intraretinal growth was not, however, reflected in increased RGC innervation of the peripheral nerve grafts, which are known to facilitate regeneration when used as optic nerve substitutes.

Long-term growth and remodeling of regenerated retino-collicular connections in adult hamsters.

The capacity of regenerating axons for long-term growth and synaptic plasticity was investigated in the visual system of adult hamsters. Four to six and 8-10 months after the eye and the superior colliculus (SC) were linked by a peripheral nerve (PN) graft, the retinal ganglion cell (RGC) axons that had regrown into the SC were examined ultrastructurally. Together with the data from hamsters with similar PN grafts for 2 months (Carter et al., 1989), this study spans most of the life of these animals. The overall findings indicate that (1) the RGC axons extended twice as far into the SC and the number of RGC terminals increased 30-fold between 2 and 4-6 months. These parameters did not change thereafter. The highest density of regenerated RGC terminals observed in the SC was 11.5% of controls. (2) The new RGC terminals acquired most of their normal ultrastructural characteristics by 2 months. (3) The mean size of the terminals was larger than in controls but decreased gradually, and there was a small increase in the size of the regenerated synapses. (4) At all times, the RGC terminals remained confined to the layers of the SC that normally receive retinal inputs, and their synapses were formed in normal proportions with the dendritic shafts and spines of SC neurons. Thus, there is a protracted long-term growth and remodeling of the RGC axons that have regenerated into the SC of these adult mammals.(ABSTRACT TRUNCATED AT 250 WORDS)

Different forms of the neurotrophin receptor trkB mRNA predominate in rat retina and optic nerve.

The expression of TrkB mRNAs was investigated in rat retina and optic nerve. A 11.5 kb transcript that encodes full-length TRKB was found to predominate in Northern blots of retinal RNA. By in situ hybridization, this trkB expression was concentrated in the ganglion cell and inner nuclear layers. Furthermore, an antibody to the full-length TRKB immunostained retinal ganglion cells and their axons. In contrast, Northern blots of optic nerve RNA showed a prominent 9.5 kb band that encoded a form of the TRKB receptor lacking the tyrosine kinase domain. This species was also detected in both the sciatic nerve and cultured astrocytes and C6 glioma cells. These results suggest that neurons express the full-length TRKB containing the tyrosine kinase domain, while non-neuronal cells express the truncated form of the receptor. These two classes of TRKB may mediate different neurotrophic actions in the retina and optic nerve.

Rapid and protracted phases of retinal ganglion cell loss follow axotomy in the optic nerve of adult rats.

To investigate the short- and long-term effects of axotomy on the survival of central nervous system (CNS) neurons in adult rats, retinal ganglion cells (RGCs) were labelled retrogradely with the persistent marker diI and their axons interrupted in the optic nerve (ON) by intracranial crush 8 or 10 mm from the eye or intraorbital cut 0.5 or 3 mm from the eye. Labelled RGCs were counted in flat-mounted retinas at intervals from 2 weeks to 20 months after axotomy. Two major patterns of RGC loss were observed: (1) an initial abrupt loss that was confined to the first 2 weeks after injury and was more severe when the ON was cut close to the eye; (2) a slower, persistent decline in RGC densities with one-half survival times that ranged from approximately 1 month after intraorbital ON cut to 6 months after intracranial ON crush. A small population of RGCs (approximately 5%) survived for as long as 20 months after intraorbital axotomy. The initial loss of axotomized RGCs presumably results from time-limited perturbations related to the position of the ON injury. A persistent lack of terminal connectivity between RGCs and their targets in the brain may contribute to the subsequent, more protracted RGC loss, but the differences between intraorbital cut and intracranial crush suggest that additional mechanisms are involved. It is unclear whether the various injury-related processes set in motion in both the ON and the retina exert random effects on all RGCs or act preferentially on subpopulations of these neurons.

Synapse formation and preferential distribution in the granule cell layer by regenerating retinal ganglion cell axons guided to the cerebellum of adult hamsters.

To investigate constraints and preferences for synaptogenesis in the injured mammalian CNS, regenerating retinal ganglion cell (RGC) axons of adult hamsters were guided through a peripheral nerve (PN) graft to a target they do not usually innervate: the cerebellum (Cb). When identified by the presence of HRP anterogradely transported from the retina 2-9 months later, such RGC axons were found to have extended into the cerebellar cortex for up to 650 microns. Most of this growth was in the granule cell layer (GCL) and only a few axons entered the molecular layer. The preference for the GCL could not be explained by the position of the PN graft in the Cb, a selective denervation of the GCL, local damage to other neurons, or the distribution of reactive gliosis in the vicinity of the graft. Furthermore, by EM, more than 95% of the labeled retinocerebellar terminals and synapses were in the GCL. Retinocerebellar terminals were larger and contained more synapses than the regenerated RGC terminals previously studied in the superior colliculus. These results indicate that regenerating axons of CNS neurons can form persistent synapses with novel targets. The preferential synaptogenesis in the GCL suggests that such unusual connections are not formed randomly in the CNS of these adult mammals.

Calcium binding protein (calbindin D28k) immunoreactivity in the hamster superior colliculus: ultrastructure and lack of co-localization with GABA.

The expression of specific calcium binding proteins is being used increasingly as a potential neuroanatomical marker for neurons with similar functions. In this study, the distribution of calbindin D28k in the superior colliculus (SC) of adult hamsters was examined by light and electron microscopy. Calbindin immunoreactivity was prominent in specific regions and laminae of the SC throughout its rostrocaudal extent, and was found to label horizontal, vertical and stellate cell types. In addition, calbindin label highlighted "bridges" of neuronal processes in the intermediate layers. The most frequent calbindin-immunoreactive profiles seen in the electron microscope were dendrites, some of which were post-synaptic to apparent retinal ganglion cell axon terminals. Labelled axons and axon terminals were less frequently encountered. There was considerable overlap between the size distribution of calbindin D28k-immunoreactive neurons and that of GABA-immunoreactive or Nissl stained neurons in the SC. However, using a double fluorescent labelling technique, and examination of the tissue with confocal laser microscopy, no neurons were observed in the hamster SC that showed immunoreactivity for both calbindin and GABA. In this regard, the SC is similar to the mammalian lateral geniculate nucleus and the pretectum, but differs from the neocortex, where calbindin and GABA are colocalized. The demonstration in the SC, as well as other parts of the nervous system, of sub-populations of neurons that contain distinct calcium-binding proteins suggests that these neurons have different functional properties. Correlative studies may clarify the relevance of these cytoplasmic components as cell markers, as well as their different patterns of association with neurotransmitters and peptides.

Regenerated synapses persist in the superior colliculus after the regrowth of retinal ganglion cell axons.

Synapse formation by retinal ganglion cell axons was sought in the superior colliculus of four adult rats 16-18 months after the optic nerve was transected and replaced by a peripheral nerve graft that guided regenerating RGC axons from the eye to the superior colliculus. The terminals of retinal ganglion cell axons were labelled by intravitreal injections of tritiated amino acids and studied by light and electron microscopic autoradiography. We found that (i) retinal ganglion cell axons had extended from the tips of the peripheral nerve grafts into the superior colliculus for approximately 350 microns; (ii) within the superior colliculus, some regenerated retinal ganglion cell axons became ensheathed by CNS myelin; (iii) retinal ganglion cell terminals formed asymmetric synapses with dendrites of neurons in the superficial layers of the superior colliculus, mainly the stratum griseum superficialis. Regenerated (n = 418) and normal retinal ganglion cell terminals (n = 1775) in the superior colliculus were compared in terms of their size (area, perimeter, and maximum diameter), contacts per terminal, contacts per 10 microns terminal perimeter, and post-synaptic structure contacted (dendritic spine, shaft, or soma). No statistically significant differences in the ultrastructural characteristics of the pre-synaptic profiles were apparent between the two groups. The post-synaptic structures contacted by axon terminals were similar in regenerated and control animals, although there were quantitative differences in the distributions of these contacts among dendritic spines and shafts. These results suggest that the regeneration of retinal ganglion cell axons in adult rats can lead to the formation of ultrastructurally normal synapses in the appropriate layers of the superior colliculus. The re-formed connections appear to persist for the life-span of these animals.

Retinal ganglion cell terminals in the hamster superior colliculus: an ultrastructural study.

The distribution, cross-sectional area, and presynaptic and postsynaptic characteristics of retinal ganglion cell axon terminals in the superior colliculus of normal adult female Syrian hamsters were investigated by quantitative ultrastructural techniques. After an intravitreal injection of horseradish peroxidase, most labelled axon terminals were found in the stratum griseum superficiale and stratum opticum of the contralateral superior colliculus. However, a small proportion (approximately 2%) of retinal ganglion cell axon terminals were located in deeper layers of the superior colliculus between the stratum opticum and the periaqueductal grey matter. Terminals were smaller in the upper two-thirds of the stratum griseum superficiale than in the lower one-third of this layer, the stratum opticum, and the stratum griseum intermedium. Presynaptic characteristics such as the length and number of contacts and the postsynaptic neuronal domains (somata, dendritic spines, or shafts) contacted by retinal ganglion cell axons in the superior colliculus were similar in all layers.