Melvin L. Billingsley

Biopsy-derived adult human brain tau is phosphorylated at many of the same sites as Alzheimer's disease paired helical filament tau

Tau from Alzheimers disease (AD) paired helical filaments (PHF-tau) is phosphorylated at sites not found in autopsy-derived adult tau from normal human brains, and this suggested that PHF-tau is abnormally phosphorylated. To explore this hypothesis, we examined human adult tau from brain biopsies and demonstrated that biopsy-derived tau is phosphorylated at most sites thought to be abnormally phosphorylated in PHF-tau. These sites also were phosphorylated in autopsy-derived human fetal tau and rapidly processed rat tau. The hypophosphorylation of autopsy-derived adult human tau is due to rapid dephosphorylation postmortem, and protein phosphatases 2A (PP2A) and 2B (PP2B) in human brain biopsies dephosphorylate tau in a site-specific manner. The down-regulation of phosphatases (i.e., PP2A and PP2B) in the AD brain could lead to the generation of maximally phosphorylated PHF-tau that does not bind microtubules and aggregates as PHFs in neurofibrillary tangles and dystrophic neurites.

Phosphorylation of Syk Activation Loop Tyrosines Is Essential for Syk Function AN IN VIVO STUDY USING A SPECIFIC ANTI-Syk ACTIVATION LOOP PHOSPHOTYROSINE ANTIBODY

Syk is an important protein-tyrosine kinase in immunoreceptor signaling. FcεRI aggregation in mast cells induces tyrosine phosphorylation and increased enzymatic activity of Syk. The two adjacent tyrosines in the Syk activation loop are thought to be important for the propagation of FcεRI signaling. To evaluate the phosphorylation of these tyrosines in vivo and further understand the relationship of Syk tyrosine phosphorylation with its function, an antibody was developed specific for phosphorylated tyrosines in the activation loop of Syk. FcεRI aggregation on mast cells induced the phosphorylation of both tyrosine residues of the activation loop. The kinase activity of Syk played the major role in phosphorylating its activation loop tyrosines both in vivoand in vitro. In FcεRI-stimulated mast cells, the total Syk tyrosine phosphorylation paralleled the phosphorylation of its activation loop tyrosines and downstream propagation of signals for histamine release. In contrast, the cell surface binding of anti-ganglioside monoclonal antibody AA4 induced only strong general tyrosine phosphorylation of Syk and minimal histamine release and weak phosphorylation of activation loop tyrosines. These results demonstrate that phosphorylation of the activation loop tyrosines is important for mediating receptor signaling and is a better marker of Syk function than is total Syk tyrosine phosphorylation.

Mechanisms of Injury in the Central Nervous System

Neurotoxicants with similar structural features or common mechanisms of chemical action frequently produce widely divergent neuropathologic outcomes. Methylmercury (MeHg) produces marked cerebellar dysmorphogenesis during critical periods of development. The pathologic picture is characterized by complete architectural disruption of neuronal elements within the cerebellum. MeHg binds strongly to protein and soluble sulphydryl groups. Binding to microtubular -SH groups results in catastrophic depolymerization of immature tyrosinated microtubules. However, more mature acetylated microtubules are resistant to MeHg-induced depolymerization. In contrast to MeHg, the structurally similar organotin trimethyltin (TMT) elicits specific apoptotic destruction of pyramidal neurons in the CA3 region of the hippocampus and in other limbic structures. Expression of the phylogenetically conserved protein stannin is required for development of TMT-induced lesions. Inhibition of expression using antisense oligonucleotides against stannin protects neurons from the effects of TMT, suggesting that this protein is required for expression of neurotoxicity. However, expression of stannin alone is insufficient for induction of apoptotic pathways in neuronal populations. The aromatic nitrocompound 1,3-dinitrobenzene (DNB) has 2 independent nitro groups that can redox cycle in the presence of molecular oxygen. Despite its ability to deplete neural glutathione stores, DNB produces edematous gliovascular lesions in the brain stem of rats. Glial cells are susceptible despite high concentrations of reduced glutathione compared with neuronal somata in the central nervous system (CNS). The severity of lesions produced by DNB is modulated by the activity of neurons in the affected pathways. The inherent discrepancy between susceptibility of neuronal and glial cell populations is likely mediated by differential control of the mitochondrial permeability transition in astrocytes and neurons. Lessons learned in the mechanistic investigation of neurotoxicants suggest caution in the evaluation and interpretation of structure-activity relationships, eg, TMT, MeHg, and DNB all induce oxidative stress, whereas TMT and triethyltin produce neuronal damage and myelin edema, respectively. The precise CNS molecular targets of cell-specific lipophilic neurotoxicants remain to be determined.

Induction and variants of neuronal nitric oxide synthase type I during synaptogenesis.

In the adult central nervous system, nitric oxide (NO) is formed from L‐arginine by the so‐called constitutive or type I NO synthase (NOS‐I155). However, expression of NOS‐I155 immunore‐activity and activity was low or not detectable in developing mouse and rat brain. NOS‐I155 was sharply induced coincident with the onset of synapto‐genesis in specific brain regions. This was followed by a second phase in which total NOS‐I155 expression decreased both in specific cell populations and in the total synaptosomal subcellular fraction. Furthermore, two putative variants of NOS‐I were transiently observed: an NOS‐I‐immunoreactive protein with increased electrophoretic mobility (NOS‐I144) and a transient hypersensitivity of NOS‐I155 to the competitive substrate inhibitor Nω‐nitro‐L‐arginine. It is concluded that NOS‐I expression is not constitutive but locally induced. In the central nervous system, this regionally specific, biphasic pattern of postnatal NOS‐I induction is consistent with a role for NO in synap‐togenesis and synaptic plasticity.—Ogilvie, P., Schilling, K., Billingeley, M. L., Schmidt, H. H. H. W. Induction and variants of neuronal nitric oxide synthase type I during synaptogenesis. FASEBJ. 9, 799‐806 (1995)

τ Phosphorylation in Human, Primate, and Rat Brain: Evidence that a Pool of τ Is Highly Phosphorylated In Vivo and Is Rapidly Dephosphorylated In Vitro

Abstract: The extent of τ phosphorylation is thought to regulate the binding of τ to microtubules: Highly phosphorylated τ does not bind to tubules, whereas dephosphorylated τ can bind to microtubules. It is interesting that the extent of τ phosphorylation in vivo has not been accurately determined. τ was rapidly isolated from human temporal neocortex and hippocampus, rhesus monkey temporal neocortex, and rat temporal neocortex and hippocampus under conditions that minimized dephosphorylation. In brain slices, we observed that τ isolated under such conditions largely existed in several phosphorylated states, including a pool that was highly phosphorylated; this was determined using epitope‐specific monoclonal and polyclonal antibodies. This highly phosphorylated τ was dephosphorylated during a 120‐min time course in vitro, presumably as a result of neuronal phosphatase activity. The slow‐mobility forms of τ were shifted to faster‐mobility forms following in vitro incubation with alkaline phosphatase. Laser densitometry was used to estimate the percent of τ in slow‐mobility, highly phosphorylated forms. Approximately 25% of immunoreactive τ was present as slow‐mobility (66‐ and 68‐kDa) forms of τ. The percentage of immunoreactive τ in faster‐mobility pools (42–54 kDa) increased in proportion to the decrease in content of 66–68‐kDa τ as a function of neuronal phosphatases or alkaline phosphatase treatment. These data suggest that the turnover of phosphorylated sites on τ is rapid and depends on neuronal phosphatases. Furthermore, τ is highly phosphorylated in normal‐appearing human, primate, and rodent brain. The presence of a highly phosphorylated pool of τ in adult brain may modify the present hypotheses on how paired helical filaments of Alzheimers disease are formed.

Expression of the calmodulin-dependent protein phosphatase, calcineurin, in rat brain : developmental patterns and the role of nigrostriatal innervation

The distribution of neurons expressing the calmodulin-dependent protein phosphatase, calcineurin (CN) was characterized in developing and adult rat brain using a combination of immunocytochemical, immunoblot and in situ hybridization approaches. Immunoblot analysis revealed a strong increase postnatally in CN protein expression. Four differently-charged isoforms of CN were observed in adult brain with apparent regional differences in isoform expression. Immunocytochemistry showed highest levels of CN in hippocampus, striatum, substantia nigra, amygdala and septal nuclei with immunoreactivity first appearing in striatum and septal nuclei, followed by hippocampus, neocortex and limbic structures. In situ hybridization demonstrated that mRNA for the catalytic subunit of CN was seen as early as postnatal day (PND) 1 in striatum, cortex and hippocampus. Since immunoreactivity was not detectable until day 4, this suggests that mRNA expression may precede that of protein by several days in these regions. Lesioning of developing and adult nigrostriatal dopamine neurons either with 6-hydroxydopamine or by surgical hemitransection had little effect on expression of CN, suggesting that CN expression is not influenced transsynaptically by dopamine. Collectively, these findings demonstrate that CN protein and mRNA expression are subject to regional and temporal control during brain development suggesting that specific synaptic connections may influence CN gene expression. However, in striatum, dopaminergic innervation does not appear to affect CN levels.

Calcium-dependent interaction of calcineurin with Bcl-2 in neuronal tissue.

Calcineurin, a calmodulin-dependent protein phosphatase, regulates transcription and possibly apoptosis. Previous studies demonstrated that in baby hamster kidney-21 cells after co-transfection calcineurin interacts with Bcl-2, thereby altering transcription and apoptosis. Using co-immunoprecipitation and subcellular fractionation techniques, we observed that calcineurin occurred as a complex with Bcl-2 in various regions of rat and mouse brain. The calcineurin-Bcl-2 complex was identified in mitochondrial, nuclear, microsomal and cytosol fractions. In vitro induction of hypoxia and aglycia or N-methyl-D-aspartate treatment markedly altered both extent of complex formation and its subcellular localization. These observations suggest that Bcl-2 either sequesters calcineurin, that calcineurin dephosphorylates Bcl-2, or that Bcl-2 shuttles calcineurin to specific substrates. Calcineurin also co-immunoprecipitated with the inositol-tris-phosphate receptor. This interaction increased after in vitro hypoxia/aglycia. In Bcl-2 (-/-) mice, interactions between calcineurin- and inositol-tris-phosphate receptor occurred less frequently than in wild-type mice under both control and hypoxic conditions. Experiments involving cell-free systems, as well as brain slices treated with thapsigargin or with N-methyl-D-aspartate suggested that calcium and calmodulin activation of calcineurin leads to interactions between calcineurin and Bcl-2. These data indicate that during times of cellular stress and damage, Bcl-2 targets activated calcineurin to specific compartments and substrates.

Alterations in hippocampal expression of SNAP-25, GAP-43, stannin and glial fibrillary acidic protein following mechanical and trimethyltin-induced injury in the rat

A set of well-defined antisera against neuronal and glial proteins were used to characterize patterns of protein expression in rat hippocampus following transection of the fimbira-fornix and perforant pathways or after administration of the selective neurotoxicant trimethyltin (8 mg/kg, i.p.). SNAP-25 (synaptosomal protein, mol. wt 25,000) is a neuron-specific, developmentally regulated presynaptic protein, stannin is a protein enriched in cells sensitive to trimethyltin, and GAP-43 (growth-associated protein, mol. wt 43,000) is associated with axonal growth and regeneration. Glial fibrillary acidic protein is an astrocyte-specific intermediate filament protein and a marker for reactive gliosis. SNAP-25 immunoreactivity was altered following both neurotoxicant and mechanical injury. Three days after fimbria-fornix/perforant path lesions, there was a loss of SNAP-25 immunoreactivity in hippocampal efferent pathways and in the lesioned entorhinal cortex. By day 12, there was evidence of reinnervation of hippocampal subfields by SNAP-25-immunopositive commissural afferent fibers. On day 3, immunoblots showed the appearance of SNAP-25a, a developmental isoform produced by alternative splicing of nine amino acids in exon 5, in lesioned tissues. This isoform declined by day 12 and was not found in contralateral control hippocampus or non-lesioned brain regions. Stannin immunoreactivity was unchanged, while GAP-43 was prominent on day 12 post-lesion. Glial fibrillary acidic protein immunoreactivity indicated gliosis near the site of pathway transection. In contrast, trimethyltin induced a marked loss of stannin immunoreactivity in hippocampal neurons seven days after injection. Trimethyltin increased glial fibrillary acidic protein staining in the hippocampus and other damaged regions. SNAP-25 immunoreactivity was markedly increased in mossy fibers and other hippocampal fields seven days following trimethyltin. Immunoblot analysis showed that only the adult SNAP-25b isoform was expressed after trimethyltin intoxication. These data suggest that SNAP-25 is a useful marker for presynaptic damage. Furthermore, reexpression of developmental isoforms of SNAP-25a may precede functional reinnervation when the postsynaptic target remains intact.

Distribution and expression of SNAP-25 immunoreactivity in rat brain, rat PC-12 cells and human SMS-KCNR neuroblastoma cells

Immunocytochemical, immunoblotting and in situ hybridization studies were used to map the distribution of SNAP-25 protein and mRNA in the rodent nervous system. These experiments demonstrated that subsets of neurons expressed SNAP-25, and that several patterns of expression emerged: SNAP-25 expression in caudate nucleus was initially concentrated in axons, which subsequently was localized in presynaptic regions of these axons. Other regions, typified by neocortex, showed developmental increases and persistent adult neuronal immunoreactivity for SNAP-25. Finally, olfactory bulb contained neurons which initially expressed SNAP-25, but lost expression during maturation. Additional studies in cultured human and rat cell lines derived from neural crest suggested that SNAP-25 is expressed in such lines, but not in glial or fibroblast lines. Differentiation of rat PC-12 cells with nerve growth factor failed to alter steady-state levels of SNAP-25 protein; similar responses were seen in human SMS-KCNR neuroblastoma cells differentiated using retinoic acid. The presence of SNAP-25 in presynaptic regions of numerous neuronal subsets and in neural crest cell lines suggests that this protein subserves an important function in neuronal tissues.

Alterations in tau phosphorylation in rat and human neocortical brain slices following hypoxia and glucose deprivation.

Tau is a microtubule-associated protein which is regulated by phosphorylation. Highly phosphorylated tau does not bind microtubules and is the main component of the paired helical filaments seen in Alzheimers and related neurodegenerative diseases. Recent reports suggested that patterns of tau phosphorylation changed following ischemia and/or reperfusion in vivo. We used an in vitro model employing rat and human neocortical slices to investigate changes in tau phosphorylation which accompany oxygen and glucose deprivation. Western blotting with polyclonal and phosphorylation-sensitive Tau-1 monoclonal antisera was used to monitor changes in tau which accompanied conditions of oxygen and glucose deprivation and reestablishment of these nutrients. In vitro hypoglycemia/hypoxia caused tau to undergo significant dephosphorylation in both rat and human neocortical slices after 30 and 60 min of deprivation. This dephosphorylation was confirmed using immunoprecipitation experiments after radiolabeling tau and other proteins with 32Pi. Okadaic acid, a phosphatase inhibitor, was able to prevent tau dephosphorylation in both control and ischemic slices. Lubeluzole, a benzothiazole derivative with in vivo neuroprotective activity, did not significantly alter patterns of tau phosphorylation. Restoration of oxygen and glucose following varied periods of in vitro hypoxia/hypoglycemia (15-60 min) led to an apparent recovery in phosphorylated tau. These data suggest that tau undergoes a rapid, but reversible dephosphorylation following brief periods of in vitro hypoxia/hypoglycemia in brain slices and that changes in tau phosphorylation help determine the extent of recovery following oxygen and glucose deprivation.