Differential effects of amyloid-ß peptide aggregation status on in vivo retinal neurotoxicity.

The present study examined the relationship between amyloid beta (Aß)-peptide aggregation state and neurotoxicity in vivo using the rat retinal-vitreal model. Following single unilateral intravitreal injection of either soluble Aß1-42 or Aß1-42 preaggregated for different periods, retinal pathology was evaluated at 24 hours, 48 hours, and 1-month postinjection. Injection of either soluble Aß (sAß) or preaggregated Aß induced a rapid reduction in immunoreactivity (IR) for synaptophysin, suggesting that direct contact with neurons is not necessary to disrupt synapses. Acute neuronal ionic and metabolic dysfunction was demonstrated by widespread loss of IR to the calcium buffering protein parvalbumin (PV) and protein gene product 9.5, a component of the ubiquitin-proteosome system. Injection of sAß appeared to have a more rapid impact on PV than the preaggregated treatments, producing a marked reduction in PV cell diameters at 48 hours, an effect that was only observed for preaggregated Aß after 1-month survival. Extending the preaggregation period from 4 to 8 days to obtain highly fibrillar Aß species significantly increased the loss of choline acteyltransferase IR, but had no effect on PV-IR. These findings prompt the conclusion that Aß assembly state has a significant impact on in vivo neurotoxicity by triggering distinct molecular changes within the cell.

Characterization of mesenchymal stromal cells derived from full-term umbilical cord blood.

BACKGROUND: Multipotent mesenchymal stromal cells (MSC) are of interest for their potential to repair bone and cartilage, and also their immunosuppressive properties. Umbilical cord blood (UCB) is reported to contain MSC, and therefore may be a useful source of these cells for clinical applications. METHODS: We evaluated protocols for isolating MSC from UCB and characterized the surface phenotype, differentiation potential and immunoregulatory properties of the cells obtained. RESULTS: Ten of 25 UCB units processed yielded MSC-like colonies, with depletion of lineage+ cells providing a higher efficiency. Only two of the cultures could be expanded satisfactorily; the remainder failed to proliferate. One culture generated transformed lines that were grossly aneuploid, had up-regulated TERT transcripts and had lost CD90 expression and the capacity to differentiate. The two propagated UCB-MSC lines were similar to those from bone marrow but were not identical to each other, with differences seen in expression of surface markers and cytoskeletal proteins. Both underwent osteogenesis, but at different rates and to different degrees, while neither generated adipocytes. When added as a third party to a mixed lymphocyte culture, both suppressed proliferation. DISCUSSION: MSC-like cells can be isolated from UCB, but at low efficiencies, and they exhibit a variety of morphologies, growth rates and differentiation potentials and can transform in culture.

Amyloid beta peptide causes chronic glial cell activation and neuro-degeneration after intravitreal injection.

We have previously demonstrated that amyloid beta (Abeta) peptide is acutely toxic to retinal neurones in vivo and that this toxicity is mediated by an indirect mechanism. We have now extended these studies to look at the chronic effect of intravitreal injection of Abeta peptides on retinal ganglion cells (RGC), the projection neurones of the retina and the glial cell response. 5 months after injection of Abeta1-42 or Abeta42-1 there was no significant reduction in RGC densities but there was a significant reduction in the retinal surface area after both peptides. Phosphate-buffered saline (PBS) injection had no effect on retinal size or RGC density. There was a pronounced reduction in the number of large RGCs with a concomitant significant increase in medium and small RGCs. There was no change in cell sizes 5 months after injection with PBS. At 5 months after injection of both peptides, there was marked activation of Muller glial cells and microglia. There was also expression of the major histocompatibility complex (MHC) class II molecule on some of the microglial cells but we saw no evidence of T-cell infiltration into the injected retinas. In order to elucidate potential toxic mechanisms, we have looked at levels of glutamine synthetase and nitric oxide synthase. As early as 2 days after injection we noted that activation of Muller glia was associated with a decrease in glutamine synthetase immuno-reactivity but there was no detectable expression of inducible nitric oxide synthase in any retinal cells. These results suggest that chronic activation of glial cells induced by Abeta peptides may result in chronic atrophy of projection neurones in the rat retina.

Alteration of Bcl-2 expression in the nigrostriatal system after kainate injection with or without melatonin co-treatment.

In order to understand further the role of the anti-apoptotic Bcl-2 proto-oncogene protein in excitotoxin-induced brain injury and possible interaction between Bcl-2 and the antioxidant melatonin, the expression of Bcl-2 in various brain parts was studied after intrastriatal injection of kainate (KA, 2.5 nmol) with or without co-treatment of melatonin (10 mg/kg, intraperitoneally (i.p.)). Three days after unilateral injection of KA to the striatum in the rat, a dramatic direct cytotoxic effect was observed, as indicated an expression of Bcl-2 immunoreactivity in TUNEL- and OX-42-positive cells in the KA-injected striatum and traumatized cortical region. A less severe detrimental effect was also observed in the ipsilateral substantia nigra and peritraumatic cortex, as reflected by an upregulation of Bcl-2-immunostained neurons. Surprisingly, a reduction in Bcl-2-immunoreactive neurons that was accompanied by a less severe loss of tyrosine hydroxylase-immunoreactive neurons in the nigrostriatal pathway was observed after co-treatment with melatonin. Western blot analysis confirmed that Bcl-2 expression is elevated in striatum and cortex on the lesioned side, and that its expression was attenuated substantially after systemic administration of melatonin. The results showing an upregulation of Bcl-2 in nigral neurons and reactive microglia after KA lesion are consistent with the view that Bcl-2 is protective in function in the central nervous system.

Expression of beta-amyloid precursor and Bcl-2 proto-oncogene proteins in rat retinas after intravitreal injection of aminoadipic acid.

In order to investigate the role of glia in relation to factors that affect the expression of beta-amyloid precursor protein (betaAPP) and B cell lymphoma oncogene protein (Bcl-2) in the central nervous tissue, the patterns of expression of betaAPP and Bcl-2 in developing and mature rat retinas were studied immunocytochemically after intravitreal injection of alpha-aminoadipic acid (alpha-AAA), a glutamate analogue and gliotoxin that is known to cause injury of retinal Müller glial cells. In normal developing retinas, betaAPP and Bcl-2 were expressed primarily but transiently in a small number of neurons in the ganglion cell layer during the first postnatal week. Immunoreactivity of betaAPP and Bcl-2 appeared in the endfeet and proximal part of the radial processes of Müller glial cells from the second postnatal week onwards. In rats that received intravitreal injection of alpha-AAA at birth, there was a loss of immunoreactivity to vimentin, and a delayed expressed on betaAPP or Bcl-2 in Muller glial cells until 3-5 weeks post-injection. Immunoreactive neurons were also observed in the inner retina especially in the ganglion cell layer from 5 to 35 days after injection. A significant reduction in numerical density of cells with large somata in the ganglion cell layer was observed in the neonatally injected retinas at P56, which was accompanied by an increased immunostaining in radial processes of Müller glial cells. In contrast, no detectable changes in the expression of betaAPP and Bcl-2 were observed in retina that received alpha-AAA as adults. These results indicate that the gliotoxin alpha-AAA has long lasting effects on the expression of betaAPP and Bcl-2 in Müller glial cells as well as neurons in the developing but not mature retinas. The loss of vimentin and delayed expression of betaAPP and Bcl-2 in developing Müller glial cells suggests that the metabolic integrity of Müller cells was temporarily compromised, which may have adverse effects on developing neurons that are vulnerable or dependent on trophic support from the Müller glial cells.

NADPH-diaphorase activity in normally developing and intracranially transplanted retinas.

The activity and distribution of nicotinamide dinucleotide phosphate diaphorase (NADPH-d), an enzyme that is widely distributed in the central nervous system and involved in the production of the free radical nitric oxide, were investigated histochemically in the normal developing and intracranially transplanted retinas. In the normal rat retina, NADPH-d activity was first detected in cells in the ganglion cells layer (GCL) and blood vessels on the first postnatal day (P0). A small but distinct population of NADPH-d positive cells were observed along the inner border of the inner nuclear layer at P7. NADPH-d positive sublaminae began to appear in the inner plexiform layer during the second postnatal week, and several strongly reactive sublaminae resembling those observed in the adult were observed by the fourth postnatal week. The overall spatio- temporal sequence of development of NADPH-d positive cells in the transplanted retina was similar to that of the normal retina, except a lack of reactive in the inner plexiform layer in more mature transplants as compared with normal retinas of corresponding ages. These results indicate that the time course of development and distribution of NADPH-d cells in early postnatal retina requires signals mainly of intraretinal origin and is independent of influence from the surroundings. While this finding is supportive to the notion that neurons that are rich in NADPH-d are resistant to injury or perturbation, the observation of a lack of well organized NADPH-d reactive sublaminae in the inner plexiform layer in older transplants suggests a possible alteration in the synaptic circuitry in the inner retina with increasing postgrafting survival time.

Effects of bFGF and TGFbeta on the expression of amyloid precursor and B-cell lymphoma protooncogene proteins in the rat retina.

The pattern of immunoreactivity for amyloid precursor (APP) and B-cell lymphoma protooncogene (Bcl-2) proteins in the rat retina was studied after intravitreal injection of basic fibroblast growth factor (bFGF) or transforming growth factor-beta (TGFbeta). In normal control retinas, intense immunostaining of APP and Bcl-2 was observed primarily in the endfeet and proximal part of radial processes of Müller glial cells. A dose-dependent reduction in immunostaining of APP and Bcl-2 in Müller cells was observed after injection of bFGF and TGFbeta. These results provide the first evidence that APP and Bcl-2 can be down-regulated by cytokines in vivo.

Factors that affect the expression of beta-amyloid precursor protein immunoreactivity in the rat retina.

Expression of beta-amyloid precursor protein immunoreactivity in the rat retina was studied after intravitreal injection of substances known to influence neural function in different ways. The substances were the excitatory amino acid glutamate, the inflammatory agent lipopolysaccharide, the depolarizing agent potassium chloride, and the potassium channel blocker barium chloride. In comparison with controls, more beta-amyloid precursor protein immunoreactivity was observed in the radial process of Müller glial cells 24 hours after injection of glutamate or lipopolysaccharide. In contrast, administration of barium chloride greatly reduced immunostaining in Müller cells. Further, an increase in immunostaining was observed in the inner and outer plexiform layers in retinas treated with any of the 3 chemicals, and in blood vessels after injection of glutamate and lipopolysaccharide. These observations suggest that multiple but specific signaling pathways are involved in regulating expression of beta-amyloid precursor protein in distinct cell types and regions in the retina.

Immunoreactivity to synaptosomal-associated protein-25 in developing rat retinas: effects of a glutamate agonist and retinal transplantation to a host brain.

In the postnatal rat retina immunoreactivity to synaptosomal-associated protein-25 (SNAP-25) was expressed primarily in the photoreceptors, inner and outer plexiform layers, and optic nerve fibres in a highly specific and age related manner. Of special interest are observations of characteristic changes of intracellular immunostaining in photoreceptors and distinct immunoreactive sublaminae in the inner plexiform layer during early postnatal stages. The overall pattern of immunoreactivity in the photoreceptor and plexiform layers remained unchanged after neonatal intravitreal injection of the glutamate agonist 2-amino-4-phosphonobutyric acid or optic tract lesion, and after adult intraorbital optic nerve section. However, a significant reduction in immunostaining in the inner plexiform and optic nerve fibre layers was observed several weeks after transplantation of the retina to a neonatal host brain. These results provide new insights into the role of SNAP-25 in membrane remodelling in relation to specific cell types and functions, and into intraretinal regulatory mechanisms that seem to be independent of the levels of glutamate and the influence of retinal ganglion cells.

Cell-specific expression of beta-amyloid precursor protein isoform mRNAs and proteins in neurons and astrocytes.

The abnormal accumulation of beta-amyloid (A beta) in senile plaques appears to be a central pathological process in Alzheimer's disease. A beta is formed by proteolysis of beta-amyloid precursor protein (APP) with several isoforms generated by alternative splicing of exons 7, 8 and 15. A semi-quantitative reverse transcription (RT)-polymerase chain reaction (PCR) analysis showed that APP695 mRNA lacking exon 7 and 8 was most abundant in primary cultures of rat neurons, while APP770 and APP751 representing, respectively, the full length and exon 8 lacking isoforms predominated in cultured astroglial cells. Antisera AP-2 and AP-4 were produced by immunizing rabbits with keyhole limpet haemocyanin coupled with synthetic peptides representing KPI region APP301-316 and A beta region APP670-686 of APP770, respectively. These polyclonal antisera were purified against the corresponding peptide using affinity chromatography. Western blot analysis of homogenates of relatively enriched neuronal and astroglial cultures showed that these antibodies discretely stained bands of proteins in a cell-specific manner. Dot-blot analysis using AP-2, AP-4 and 22C11 antibodies indicated that, in comparison with neurons, cultured astrocytes contained 3-fold greater KPI-containing APP isoform proteins. The amount of total APP proteins, which include both KPI-containing and KPI-lacking APP isoforms, was approximately 90% higher in astrocytes than in neurons. Consistent with these in vitro findings in cultured astrocytes, in fimbria-fornix lesioned rat hippocampus, labelling with AP-2 antibody, which specifically reacts with KPI-containing APP proteins, was mainly observed in glial fibrillary acidic protein-positive reactive astrocytes in vivo. The results showed that APP isoforms are expressed in a cell type-specific manner in the brain and, since deposition of A beta is closely associated with the expression of KPI-containing APP isoforms, provide further evidence for the involvement of astrocytes in plaque biogenesis.

Nitric oxide synthase in developing retinas and after optic tract section.

We compared the pattern of nitric oxide synthase (NOS) immunoreactivity in retinas of rats during normal development and after unilateral transection of the optic tract at postnatal day 7. NOS was first detected in the second postnatal week in the inner nuclear and inner plexiform layers. There was no detectable difference in the overall pattern of immunoreactivity between normal retinas and retinas with severe loss of ganglion cells due to the lesion. We suggest that NOS may have a role in synaptic and vascular development in the inner retina, but is unlikely to play a major role in normal physiological retinal ganglion cell death or axotomy-induced cell death.

Expression of beta-amyloid precursor protein immunoreactivity in the retina of the rat during normal development and after neonatal optic tract lesion.

Immunoreactivity to beta-amyloid precursor protein (APP) was present in the inner plexiform, ganglion cell and optic fibre layers, as well as in blood vessels, at birth in normally developing rat retinas. In the inner plexiform layer immunoreactivity disappeared by postnatal day (P) 14. A small population of ganglion cells was immunoreactive at birth, but none were visible at P7. From P14 onwards, however, there was weak immunoreactivity in ganglion cells again, and strong staining in Müller glia. Retinas affected by neonatal optic tract lesions contained more immunoreactive ganglion cells at P4 than did controls, but by P14 there was a severe loss of ganglion cells. These observations are consistent with APP being involved in retinal differentiation, including maturation of glia and neurones, synaptogenesis and possibly neuronal survival.

Distribution of beta-amyloid precursor and B-cell lymphoma protooncogene proteins in the rat retina after optic nerve transection or vascular lesion.

The expression of beta-amyloid precursor protein (APP) and B-cell lymphoma protooncogene protein (Bcl-2) in retinal cells in the rat was studied using immunocytochemistry at different times after intraorbital optic nerve transection or vascular lesion. Three hours to one month after transection of the optic nerve, a significant increase in APP and Bcl-2 immunostaining was observed in retinal Müller glia but not in retinal neurons. In contrast, injury to blood vessels that supply the eye without cutting the optic nerve resulted in a complete loss of APP and Bcl-2 immunostaining in Müller cells and an increase in immunoreactivity in distinct populations of retinal neurons. The overall pattern of APP immunostaining in Müller cells and neurons was essentially the same as that of Bcl-2 under identical experimental conditions. These results suggest that the expression of APP and Bcl-2 in retinal cells is dependent on the nature and severity of injury, and that rapid and common mechanisms are involved in regulating the expression of these molecules.

Prenatal development of NADPH-diaphorase-reactive neurons in human frontal cortex.

Nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) histochemistry was used to study the morphology and development of neurons that metabolize nitric oxide (NO) in the frontal cortex of human fetuses aged from 13 weeks of gestation (13W) to term, to investigate whether the two distinct types of NO neuron described in the adult develop differently. Large, heavily stained, sparsely spiny, non-pyramidal neurons (Type I) develop by 15W mainly in the subplate (SP) of the cortical Anlage. They achieve an adult-like pattern by 32W, distributed thoughout the cortex and subcortical white matter, but with the highest concentration in the white matter. Small, lightly stained cells (Type II) develop later (32W) thoughout the cortex, but especially in layers II-IV, and increase in number to term. NADPH-d-positive dendrites and axons appear in the cortex and white matter by 15W. They include thick, radially oriented, dendritic processes from Type I neurons in SP and CP. Their arbors expand and mature between 17 and 28W. Fine horizontal axons are visible in layer I by 17W. Others develop in layers II-IV from 28W, and have reached a high degree of development by term. NADPH-d-positive axons in the cortex seem to have both intrinsic and extrinsic origins. Thus the two types of NADPH-d neurons found in adult primate, including human, cortex are reflected by different developmental forms prenatally. It is concluded that NO-metabolizing neurons in the human cortex may be involved in various aspects of development, including morphological and functional maturation, and that the late-developing Type II neurons may represent a cell line specific to primates, perhaps related to the development of their higher cortical activity and of potential importance in the pathophysiology of diseases of cognitive function.

NADPH-diaphorase-positive neurons in primate cerebral cortex colocalize with GABA and calcium-binding proteins.

Neurons in the monkey cerebral cortex containing nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) can be divided into two distinct types, both nonpyramidal. Type I neurons have a large soma (diameter 20-50 microm), a dense NADPH-d histochemical reaction, and are distributed throughout the cortex, but mainly in the subcortical white matter, and are mostly aspiny. Type II cells have a small soma ( Together with previous observations that almost all cortical NADPH-d cells in various subprimates are like type I cells, we suggest that type II cells may form a group of NADPH-d-rich neurons differentiated in higher mammalian cortex from a subpopulation of calbindin-containing GABAergic interneurons, and these nitric oxide-synthesizing cells may play a role in control of intracortical neuronal activity characteristic of higher cerebral functions in advanced mammals.

Spatial periodicity of NADPH-diaphorase and synaptophysin, but not SNAP-25, reactivity in the monkey cerebellar cortex.

We recently described a parasagittal patchy organisation of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d) activity in the granular layer of the rat cerebellum. We now report the pattern of NADPH-d distribution in the primate cerebellum and its relationship to two synaptic proteins, synaptophysin and synaptosomal associated protein 25 kDa (SNAP-25), using histochemistry and immunocytochemistry. NADPH-d reactivity was localised in the molecular and granular layers (ML, GL) and a subset of infraganglionic plexuses (IGPs), but not in the Purkinje cell layer and the white matter. In ML, the histochemical reactivity was dense and relatively homogeneous in the neuropil, and moderate in the stellate cells. A patchy organisation of NADPH-d in GL was detected in both horizontal and parasagittal sections. In the IGPs staining for NADPH-d revealed modular positive zones alternating with negative ones. The positive and negative IGP zones were usually congruent with the high and low NADPH-d reactivity in GL, respectively. Both synaptic proteins were strongly expressed in the neuropil in ML and GL, and their patterns were relatively homogeneous. However, synaptophysin was present in a subpopulation of IGPs organised in modules which corresponded to those expressing NADPH-d. Our results indicate that the NADPH-d modular system is more complicated in the primate cerebellum than in the rat. In addition, we have provided suggestive evidence of a co-expression of NADPH-d and synaptophysin in selected IGP modules in primate cerebellum, which suggests that nitric oxide may be involved in the activity of the Purkinje cells by affecting the basket cell synaptic input.

Increased expression of NADPH-diaphorase in visual centres after unilateral optic nerve transection in the rat.

The present study shows an increase NADPH-d histochemical staining in the optic layers of the superior colliculus, the ventral nucleus of the lateral geniculate body and the primary visual cortex in the rat after transection of the contralateral optic nerve. The alteration was observed 4-60 days after the lesion, with a maximum at 30 days. These results suggest that retinal activity has a transneuronal and time-dependent regulatory effect on nitric oxide synthesis in denervated or visually deprived visual centres, an increase that may play a role in enhancing local blood flow and could be neuroprotective in function.

Bcl-2 proto-oncogene protein immunoreactivity in normally developing and axotomised rat retinas.

This study demonstrates immunocytochemically that bcl-2 (B cell lymphoma) proto-oncogene protein is expressed by two populations of retinal cells, one in the inner retina representing developing neurons or glia at perinatal stages, and the other identified as Müller cells from postnatal day 10 onward. An increase in bcl-2 immunoreactivity was detected in the Müller cell processes after neonatal optic tract lesion or optic nerve transection in adult rats. These results suggest that bcl-2 protein may function on cellular differentiation, maturation and homeostasis in the inner retina, and its expression by Müller cells can be up-regulated by the degeneration of ganglion cells.