Differential brain region-specific expression of MeCP2 and BDNF in Rett Syndrome patients: a distinct grey-white matter variation.

INTRODUCTION AND OBJECTIVES: Rett Syndrome (RTT) is a neurodevelopmental disorder caused by Methyl CpG Binding Protein 2 (MECP2) gene mutations. Previous studies of MeCP2 in the human brain showed variable and inconsistent mosaic-pattern immunolabelling, which has been interpreted as a reflection of activation-state variability. We aimed to study post mortem MeCP2 and BDNF (MeCP2 target) degradation and brain region-specific detection in relation to RTT pathophysiology. METHODS: We investigated MeCP2 and BDNF stabilities in non-RTT human brains by immunohistochemical labelling and compared them in three brain regions of RTT and controls. RESULTS: In surgically excised samples of human hippocampus and cerebellum, MeCP2 was universally detected. There was no significantly obvious difference between males and females. However, post mortem delay in autopsy samples had substantial influence on MeCP2 detection. Immunohistochemistry studies in RTT patients showed lower MeCP2 detection in glial cells of the white matter. Glial fibrillary acidic protein (GFAP) expression was also reduced in RTT brain samples without obvious change in myelin basic protein (MBP). Neurons did not show any noticeable decrease in MeCP2 detection. BDNF immunohistochemical detection showed an astroglial/endothelial pattern without noticeable difference between RTT and controls. CONCLUSIONS: Our findings indicate that MeCP2 protein is widely expressed in mature human brain cells at all ages. However, our data points towards a possible white matter abnormality in RTT and highlights the importance of studying human RTT brain tissues in parallel with research on animal and cell models of RTT.

Different pathological processes for acute white matter lesions in multiple sclerosis.

INTRODUCTION: Multiple sclerosis (MS) has variable clinical presentations, prognoses, pathogeneses, and pathological patterns. We conducted a pathological review of acute MS-associated lesions that focused on the degree of axonal injury, myelin loss, and glial reaction to determine whether the observed demyelination was of the primary or secondary type. MATERIALS AND METHODS: After searching the records for a 15-year period at the London Health Sciences Centre Pathology Department, we identified 8 cases of surgical acute lesion biopsies in which clinical MS diagnoses were made before or after the biopsy. RESULTS: The white matter pathologies in these cases could be sorted into 3 morphological patterns. The first pattern, which represents typical demyelinated plaques, was observed in 4 cases and was characterised by nearly complete demyelination accompanied by variable degrees of axon preservation and axonal swelling. The second pattern was observed in 3 cases and was characterised by demyelinating lesions containing variable numbers of myelinated axons mixed with a few demyelinated axons and variable numbers of axonal swellings. The myelinated axons ranged from scattered fibres to bands of variable thickness, and the demyelination was a mixture of primary and secondary demyelination. The third pattern was observed in 1 case and was characterised by well-demarcated areas of reduced myelin staining and numerous apoptotic nuclei. Axonal staining revealed many fragmented axons with reduced myelin staining but no definitely demyelinated axons. CONCLUSIONS: This report shows that the predominant pathology underlying acute MS-related lesions is not limited to demyelination but can include axonal degeneration alone or in combination with primary demyelination which reflect different pathogenesis for these acute lesions.

Progressive supranuclear palsy in a family with TDP-43 pathology.

UNLABELLED: A member of a family with an autosomal dominant pattern of frontotemporal dementia (FTD) with a TDP-43 pathological substrate in other members and no mutations in FTD-associated genes developed behavioral variant FTD followed by Progressive Supranuclear Palsy. Autopsy revealed a pure tauopathy of PSP pattern. CONCLUSIONS: The findings raise the possibility of shared pathogenic pathways and a proximal genetic abnormality between PSP and FTLD-43.

Mefloquine in the treatment of progressive multifocal leukoencephalopathy.

Mefloquine, an antimalarial medication with efficacy against JC virus, was used to treat progressive multifocal leukoencephalopathy. A 54-year-old woman with sarcoidosis presented with a progressive cerebellar syndrome. MRI showed lesions affecting the right cerebellum that progressed over time to the brainstem. JC virus was found in the cerebrospinal fluid (CSF), and brain biopsy confirmed the diagnosis of progressive multifocal leukoencephalopathy. Mefloquine 1000 mg/week was initiated 6 months after symptom onset. Clinical progression stopped immediately, and JC virus became undetectable in the CSF. No clinical or imaging evidence of disease progression has occurred over 20 months of follow-up. This is the first report of successful treatment of progressive multifocal leukoencephalopathy with mefloquine.

Progressive supranuclear palsy: a review of co-existing neurodegeneration.

OBJECTIVES: The neuropathological findings of 32 progressive supranuclear palsy (PSP) cases over a period of 17 years were reviewed. RESULTS: Of the 26 cases with adequate clinical data, 20 patients either presented with cognitive dysfunction or developed a cognitive impairment subsequently during the course of the disease. Co-existing changes of argyrophilic grains and corticobasal degeneration (CBD) were found in 28% and 32% of the cases respectively. Alzheimer-related pathology was found in 69% of cases but only 18.75% of cases fulfilled the consortium to establish a registry for Alzheimer's disease (CERAD) criteria for either definite or probable Alzheimer's disease. Lewy bodies were noted in four cases (12.5%), all in the subcortical regions. Only seven cases of PSP showed no pathological evidence of other co-existing neurodegenerative diseases. The severity of the cerebrovascular pathology in this cohort was insufficient to explain any clinical symptomatology. CONCLUSIONS: As in previous studies, this study has demonstrated the frequent co-existence of pathological changes usually noted in other neurodegenerative diseases in PSP. Whether these co-existing pathological changes contribute to the cognitive impairment in PSP remains uncertain.

Human glaucoma and neural degeneration in intracranial optic nerve, lateral geniculate nucleus, and visual cortex.

The pathology of glaucoma has been extensively studied at the level of the retina and optic nerve head. Here the first clinicopathological case of human glaucoma is reported demonstrating degenerative changes in the brain involving the intracranial optic nerve, lateral geniculate nucleus, and visual cortex. Pathological evidence of neural degeneration in this patient is correlated with clinical, optic nerve head, visual field, and neuroradiology findings. Neuropathology in the glaucoma brain is compared to age matched controls. In the presence of advanced human glaucoma with 50% visual field loss, neural damage is evident in multiple vision stations within the brain.

Motor and sensory specificity of host nerve axons influence nerve allograft rejection.

Previous studies have shown both survival and loss of regenerated host axons within nerve allograft segments after withdrawal of Cyclosporin A (CsA) immunosuppression. We hypothesized that the nature of end-organ reinnervation may influence the response of the axon, with survival of axons for appropriate innervation vs degeneration for inappropriate innervation. The rat femoral nerve model was chosen, as it has approximately equal sensory (S) and motor (M) divisions. Four ACI rat peroneal nerve allografts were sutured in straight (right leg: MM and SS) or switched (left leg; MS and SM) orientation in each femoral nerve transection gap in each Lewis rat recipient. Rats received CsA for 8 weeks to allow end-organ reinnervation, after which immunosuppression was discontinued. Rats were killed at various times thereafter, and underwent histologic and morphometric analysis of the graft segment axons. The regenerated axon population in the allograft reflected the nerve of origin: significantly more but smaller fibers when the proximal nerve was sensory and fewer but larger fibers when the proximal nerve was motor. After CsA withdrawal, there was a marked decrease of host axons as part of an ensuing rejection episode. The overall proportional decrease of axons was similar across all nerve orientation groups and, therefore, did not appear to be influenced by the nerve of origin or by the end-organ. However, the sensory proximal groups (SS and SM) contained more mature, noninjured fibers, while the motor proximal groups (MM and MS) contained significantly more degeneration and newly regenerating axons. We conclude that the motor or sensory nerve origin of the host axon, rather than the end-organ, influences axon survival after immunosuppression cessation. It is hypothesized that sensory axons may be more resilient while motor axons are selectively vulnerable to this second injury.

Versican enhances locomotion of astrocytoma cells and reduces cell adhesion through its G1 domain.

Versican is a large extracellular proteoglycan and is expressed in a variety of tissues including the central nervous system. A malignant astrocytoma cell line U87 with high motility expressed a higher level of versican than another malignant astrocytoma cell line U343 with lower motility. We observed that the U87 cells were less adherent to tissue culture plates than the U343 cells. To investigate the role of versican in astrocytoma cell migration, we generated recombinant products of a mini-versican construct expressed in COS-7 cells. We found that the mini-versican products enhanced astrocytoma cell migration. Furthermore, enhanced migration was promoted by the G1 domain but not the G3 domain of versican. We introduced culture medium containing products of the mini-versican, the G1, and the G3 constructs separately into the astrocytoma cell lines U87 and U343. The mini-versican and the G1 construct, but not the G3 construct, were shown to reduce astrocytoma cell adhesion. The present data suggest that versican exerts its effect on astrocytoma cell migration and adhesion through the G1 domain.

Phenotypic variability of Gerstmann-Sträussler-Scheinker disease is associated with prion protein heterogeneity.

Gerstmann-Sträussler-Scheinker disease (GSS), a cerebello-pyramidal syndrome associated with dementia and caused by mutations in the prion protein gene (PRNP), is phenotypically heterogeneous. The molecular mechanisms responsible for such heterogeneity are unknown. Since we hypothesize that prion protein (PrP) heterogeneity may be associated with clinico-pathologic heterogeneity, the aim of this study was to analyze PrP in several GSS variants. Among the pathologic phenotypes of GSS, we recognize those without and with marked spongiform degeneration. In the latter (i.e. a subset of GSS P102L patients) we observed 3 major proteinase-K resistant PrP (PrPres) isoforms of ca. 21-30 kDa, similar to those seen in Creutzfeldt-Jakob disease. In contrast, the 21-30 kDa isoforms were not prominent in GSS variants without spongiform changes, including GSS A117V, GSS D202N, GSS Q212P, GSS Q217R, and 2 cases of GSS P102L. This suggests that spongiform changes in GSS are related to the presence of high levels of these distinct 21-30 kDa isoforms. Variable amounts of smaller, distinct PrPres isoforms of ca. 7-15 kDa were seen in all GSS variants. This suggests that GSS is characterized by the presence PrP isoforms that can be partially cleaved to low molecular weight PrPres peptides.

End-Organ reinnervation does not prevent axonal degeneration in nerve allografts following immunosuppression withdrawal.

Previous work indicated that appropriate end-organ reinnervation fails to influence axonal degeneration in nerve allografts following immunosuppression withdrawal. In the present study, we examined if differences existed in axonal degeneration when axons regenerated across nerve allografts are allowed or completely denied end-organ reinnervation. Two ACI rat nerve allografts (3 cm long) were sutured into gaps created in both peroneal nerves in Lewis rats. In the right leg, the distal end of the graft was connected to the distal host nerve stump to allow end-organ reinnervation. In the left leg, the distal end was turned back and double ligated (unconnected) to prevent end-organ reinnervation. Rats received Cyclosporin A daily for 12 weeks to allow for regeneration and were sacrificed at 16 (n = 5) or 18 (n = 5) weeks following engraftment to assess axonal degeneration following immunosuppression withdrawal. Five Lewis rats receiving autografts served as control and were sacrificed at 12 weeks. Morphometric analysis was performed. In the control group (autografts) the cross-sectional area of and the number of myelinated fibres in the unconnected grafts was double that of the connected grafts, suggesting a sprouting effect. There was a tenfold reduction in the mean number of fibres at weeks 16 and 18 in the allografts compared to controls, without any significant differences in the connected versus unconnected sides. End-organ reinnervation decreases sprouting of axons within the graft but does not protect axons from degeneration following immunosuppression withdrawal.

A simple, yet versatile, co-culture method for examining neuron-glia interactions.

There is increasing interest in examining neuron-glial interactions in the developing and the mature central nervous system. Here, we describe a system that allows examination of interactions mediated by diffusible molecules even when such interactions involve more than 2 cell populations. Our procedure involves culturing highly enriched populations of neurons and glial cells on separate coverslips. Such coverslips can be readily moved from one petri dish to another, thus enabling the investigator to subject the neurons and the glial cells to different experimental manipulations. The coverslips can then be placed in any desired combination within a petri dish. This gives one great flexibility in examining neuron-glial interactions. Using this approach, we unequivocally demonstrate that astrocytes protect neurons from glutamate excitotoxicity.

Peptidergic neurons of subcortical white matter in aging and Alzheimer's brain.

Most of the neurons in the subcortical white matter of the adult cerebrum are remnants of the transient subplate cortex which appears during early cortical development. The peptidergic neurons in the subcortical white matter, beneath the striate cortex were examined qualitatively and qualitatively with immunohistochemistry for substance P, cholecystokinin, somatostatin and neuropeptide Y in seven control patients and eight patients with Alzheimer's disease. The different peptidergic subcortical neurons still persisted in normal aging. In Alzheimer's disease, however, the substance P- and somatostatin-immunoreactive neurons were decreased in numbers and showed degenerative changes.

Chromogranin A, a soluble synaptic vesicle protein, is found in cortical neurons other than previously defined peptidergic neurons in the human neocortex.

Neuropeptides in the cerebral cortex have previously been identified in non-pyramidal neurons only. By comparing the location of chromogranin A (CgA), a soluble protein of large dense-core synaptic vesicles, with that of SMI-32, neuropeptide Y (NPY), parvalbumin (PV) and calbindin (CaBP) using double label immunohistochemistry, we demonstrate that CgA is present in pyramidal neurons as well as in several subtypes of non-pyramidal neurons.

Aluminum pretreatment impairs the ability of astrocytes to protect neurons from glutamate mediated toxicity.

A number of laboratories have shown that astrocytes protect neurons from glutamate excitotoxicity. The experiments described in this paper were designed to address the question whether prior exposure of astrocytes to aluminum (in the form of aluminum citrate) interfered with the ability of astrocytes to protect neurons from glutamate excitotoxicity. Our culture paradigm consisted of highly enriched cultures of neurons and astrocytes grown on separate coverslips; this design enables one to subject either the neurons or the astrocytes to specific treatments and recombine the cells into the same petri dish simply by moving coverslips from dish to dish. We have confirmed findings of other laboratories that astrocytes could protect neurons from glutamate-induced death when glutamate (100 microM) is added to the culture medium. We have also demonstrated that prior treatment of astrocytes with 100 microM aluminum citrate impairs this ability of astrocytes to promote neuronal survival. No differences, however, were observed in the ability of control and aluminum-treated astrocytes to take up glutamate. These findings suggest that aluminum may cause astrocytes to: (i) secrete a factor that makes neurons more susceptible to glutamate-induced toxicity; (ii) secrete a neuronotoxic factor in the presence of glutamate; or (iii) reduce secretion of a factor that protects neurons from glutamate excitotoxicity.

Increase in enkephalin-like immunoreactivity in hippocampi of adults with generalized epilepsy.

The changes of opioid peptide reactivity in seizure activity have been well studied in animals. Increased enkephalin and dynorphin immunoreactivity in the hippocampi of animals are interpreted as the result of seizure induced mossy fibre sprouting. We studied the hippocampi of six patients with a history of long-standing grand mal seizures and six age-matched control patients with no history of epilepsy or neurologic disease, using frozen sections which were immunostained with antibodies against Leu-enkephalin and Met-enkephalin. The staining intensity in the CA3, CA4 and internal molecular layer of the dentate fascia in each case was quantified using optical densitometry image analysis. The CA3 and CA4 of the epileptic hippocampi showed highly significant increase in Leu-enkephalin-like immunoreactivity compared to the controls (P < 0.005) while the inner molecular layer showed only significant increase (P < 0.05). Met-Enkephalin-like immunoreactivity was only significantly increased in CA4 of the epileptic hippocampi (P < 0.05).

Neurite promoting activity of insulin, insulin-like growth factor I and nerve growth factor on spinal motoneurons is astrocyte dependent.

Mouse motoneurons were isolated from dissociated E15 mouse spinal cord and grown on polyornithine-coated round coverslips in a growth medium (DMEM/F12) supplemented with progesterone, trans-ferrin, selenium, horse serum and muscle extract. Astrocytes from newborn mouse neopallium were grown on rectangular coverslips. The motoneuron neurite growth was determined at day 8 of culture by counting, using the light microscope, the intersections produced by neurites radiating from the perikaryon placed centrally in a graticule eyepiece of concentric circles. The mean intersections for cultures without addition of astrocytes, insulin, insulin-like growth factor I (IGF-I) or nerve growth factor (NGF) was 12.6 +/- 0.8. When astrocytes on a separate coverslip were introduced from day 1, there was a small increase in neurite growth (16.3 +/- 0.9). The neurite growth was further increased significantly with the addition of insulin (27.3 +/- 1.4), IGF-I (31.5 +/- 1.4) or NGF (21.8 +/- 1.1) to cultures with astrocytes. Insulin, IGF-I or NGF in the absence of astrocytes did not greatly increase the neurite growth. We conclude that insulin, IGF-I and NGF promote neurite growth through some interactions with astrocytes.

SMI-32 immunoreactivity in human striate cortex during postnatal development.

SMI-32, an antibody which recognizes the non-phosphorylated epitopes on the neurofilament proteins was used to study the morphological changes in the human striate cortex during postnatal development. Striate cortices from 12 autopsied patients with ages ranging from 1 day to 70 years were obtained. Using the avidin-biotin-peroxidase method, the first SMI-32 immunoreactive neurons were identified at sublayers Vb/VIa on the first postnatal day. At 5 months, the next group of neurons to develop immunoreactivity were in IVb. By 15 months, SMI-32 immunoreactive neurons were observed at III, IVa, IVb, V and VI. The changes in SMI-32 immunoreactivity (ir) were stabilized from 3 years and after. The SMI-32 ir in the striate cortex could be a useful morphological correlate for studying developmental diseases affecting the neocortex.

Delayed changes of chromogranin A immunoreactivity (CgA ir) in human striate cortex during postnatal development.

The changes in chromogranin A expression in the human striate cortex from birth till 67 years were studied by immunohistochemical method in 18 autopsied patients. The first chromogranin A immunoreactivity (CgA ir) was identified at birth in layer IV (especially IVc) mainly as fine nerve terminals. By 6 months, the first perikaryal reactivity was noted in the large pyramidal neurons of layer V. The smaller neurons in layers IV, V and VI showed a progressive increase in CgA ir from 15 months to about 17 years. At approximately 9 years, immunoreactivity began to be noted in supragranular neurons in layers II and III. The final laminar distribution of CgA ir seemed to be attained at about 25 years with relatively little change thereafter. The CgA ir in the striate cortex demonstrates a prolonged period of developmental changes, lasting from birth to about 25 years.

Motoneuron survival in vitro: effects of pyruvate, alpha-ketoglutarate, gangliosides and potassium.

When grown in a serum and muscle extract containing medium, about 20% of chick motoneurons survived for one week. The presence of pyruvate during the first hour of culture doubles the number of motoneurons that survive. The subsequent addition of 5 mM alpha-ketoglutarate, 10-20 mM potassium, or 100 microM GM1 ganglioside results in a further increase in motoneurons surviving. These effects of alpha-ketoglutarate, potassium and GM1 ganglioside are not additive. Using such compounds in the culture medium, one can have a three-fold increase in the survival of motoneurons.