Calcineurin Immunoreactivity Research Articles

Calcineurin Immunoreactivity in Alzheimer's Disease

Aberrant phosphorylation of tau is linked to formation of the paired helical filaments (PHF) seen in Alzheimer's disease. Protein kinases such as mitogenactivated protein kinase, and calcium-regulated protein kinases may, in part, be responsible for addition of phosphate groups to serine residues of PHFtau; however, less is known concerning the phosphatases which regulate tau. In this report, we used several well-characterized antibodies to document calcineurin immunoreactivity in brain tissue from patients with Alzheimer's disease. We now report that levels of immunoreactive calcineurin are not significantly altered in neocortex and cerebellum of Alzheimer's patients relative to similar regions of age-matched controls. Immunocytochemical studies indicated that calcineurin immunoreactivity was present in dendrites and perikarya of many different neuronal populations in both control and Alzheimer brain. When specific antibodies against PHFtau were used in double-labeling experiments with anti-calcineurin antibodies, calcineurin immunoreactivity was seen in association with neurofibrillary tangles. However, calcineurin was not seen in all tangle bearing neurons. These data suggest that calcineurin levels per se are not significantly altered in Alzheimer's disease, but that calcineurin is distributed around some neurofibrillary tangles and may play a role in regulation of tau phosphorylation.

Immunocytochemical localization of calcineurin in the adult and developing primary visual cortex of cats.

An immunocytochemical method was used to localize calcineurin, a calcium-dependent calmodulin-stimulated protein phosphatase, in the primary visual cortex of developing and adult cats. In the adult calcineurin immunoreactivity exhibits a laminar distribution with dense labeling in the upper half of layers II/III and two lightly labeled bands in lower layer IV and in layer VI. Most of the immunoreactive neurons are pyramidal in shape and appear to form a subpopulation of cortical neurons, but non-pyramidal neurons were also labeled, especially during early stages of postnatal development. The distribution pattern of calcineurin immunoreactivity showed developmental changes until at least 3 months of age. The number of calcineurin-positive cells abruptly increased at 3 weeks, and heavily labeled neurons appeared in a well-delineated band in layer IV between 3 and 5 weeks of age. At 6 to 10 weeks, neurons in layers II/III also became strongly immunoreactive. At this developmental stage intensely stained cells were thus distributed throughout layers II to IV. Thereafter, there was a marked decrease in the number of immunoreactive cells in layer IV and beyond 12 weeks the distribution pattern of calcineurin immunoreactivity became similar to that of adult animals. These changes of calcineurin expression show some relation with the inside-out pattern of cortical maturation and with the time course and the laminar selectivity of use-dependent malleability. Therefore, we suggest that calcineurin may be involved in processes of neuronal differentiation and experience-dependent plasticity.

Striatonigral involvement following transient focal cerebral ischemia in the rats: an immunohistochemical study on a reversible ischemia model.

A topographical and cellular immunohistochemical analysis was performed on the striatonigral system of rats with unilateral, reversible middle cerebral artery (MCA) occlusion. Antibodies to calcineurin (CaN), parvalbumin (PV), choline acetyltransferase (ChAT) and glial fibrillary acidic protein (GFAP) were used in this study. Sixty days after the operation, the ipsilateral striatum showed a characteristic cell type-specific injury in the dorsolateral part of the nucleus (i.e., non-limbic striatum): a marked reduction in the number of medium-sized spinous neurons expressing CaN immunoreactivity and a selective sparing of PV- and ChAT-positive interneurons. There was also a marked depletion of striatonigral afferents visualized by CaN immunostaining in the lateral portion of the substantia nigra pars reticulata, which is considered to be implicated with motor function. In addition, it was noted that such striatonigral involvement was accompanied by marked gliosis showing strong GFAP immunolabeling. The present data suggest that rats with reversible MCA occlusion can be a useful animal model for studying cell type-specific ischemic injury and subdivisional involvement of the striatonigral pathway as a part of the cortico-subcortical loop subserving motor function.

Calcineurin, a calcium/calmodulin-regulated protein phosphatase, in mammalian neuroendocrine cells and neoplasms.

Calcineurin is a calcium/calmodulin-regulated protein phosphatase. By using enzyme-immunoassay and immunocytochemistry with an affinity-purified specific antibody to this protein, we have found that calcineurin is expressed in the central and peripheral neuroendocrine cells, also termed amine precursor uptake and decarboxylation cells. In addition, calcineurin immunoreactivity was found in the central neuroendocrine neoplasms such as pineocytoma, olfactory neuroblastoma and paraganglioma. The present findings indicate that the activity of phosphatase regulated by calcium and calmodulin is involved in neuroendocrine functions, and that the enzyme can be useful for the identification and characterization of neuroendocrine cell tumors.

Alteration in the immunoreactivity of the calcineurin subunits after ischemic hippocampal damage

Dephosphorylation processes of target proteins are critical to the reversible regulation of intracellular signal transduction systems. Further, brain damage such as ischemic insult induces marked changes in protein kinase activity. To study these changes more thoroughly, specific monoclonal antibodies of the A and B subunits of calcineurin (protein phosphatase 2B) were raised, and regional alterations in the immunoreactivity of calcineurin in the rat hippocampus were investigated after a transient forebrain ischemic insult causing selective and delayed hippocampal CA1 pyramidal cell damage. In normal rats it was found that both the calcineurin A and the B subunits showed high immunoreactivity in the dendritic fields of the hippocampal formation. The immunoreactivity of subunit A in the strata oriens, the radiatum of the CA1 subfield and in the stratum lucidum of the CA3 subfield was most intense, whereas the immunoreactivity in the other CA3 subfields and in the dentate gyrus was relatively low. In contrast, the dendritic fields of the hippocampal formation were equally immunoreactive to calcineurin subunit B, although the stratum lucidum of the CA3, where the mossy fibers from the dentate granule cells terminate, showed a very high immunoreactivity of the B subunit. After transient forebrain ischemia in the CA1 subfield, where selective pyramidal cell death occurred two days after this ischemia, a marked loss of immunoreactivity in both subunits was observed, along with morphological pyramidal cell damage. A recovery of the immunoreactivity of A and B subunits in the strata oriens and radiatum was later noted 30 days after ischemia. In the stratum lucidum of the CA3, the immunoreactivity of both the A and B subunits was transiently depressed from 6 to 24 h, followed by a marked immunoreactivity enhancement from four to 30 days after ischemia. Further, in the histologically intact dentate gyrus, both the immunoreactivity of the A and B subunits in the molecular layer were transiently enhanced from four to 14 days after ischemia, particularly in the supragranular layer. The results clearly indicate that the protein dephosphorylation systems were markedly altered in the whole hippocampal formation during the recirculation period following ischemia. Further, the transient depression in the calcineurin immunoreactivity seen in the mossy fiber terminals may reflect modulated synaptic activity of the dentate granule cells, which may play a pivotal role in the delayed and selective death of the CA1 pyramidal cells. Thus, calcineurin appears to be an excellent marker enzyme for the detection of neuronal activity and synaptic plasticity after brain damage, such as an ischemic insult.

Inhomogeneity of the putaminal lesion in striatonigral degeneration

An immunohistochemical topographic study was carried out in the putamen from three patients with striatonigral degeneration (SND) using antibody to calcineurin (CaN), a neurochemical marker for the striatal medium-size spinous neurons. In patients with SND, there was significant depletion of CaN immunoreactivity in the putamen with the caudal and lateral portion of the putamen being consistently and severely affected. In addition, the SND patients showed an inhomogeneous distribution pattern of residual CaN staining in the putamen, where remaining CaN immunoreactivity appeared as a characteristic patchwork of "islands" resembling the "striosomes" observed by the tyrosine hydroxylase or Met-enkephalin immunostaining in the putamen from normal individuals. This finding may account for the fact that there are subregional and compartmental differences in susceptibility of the medium-sized spinous neurons in the putamen with SND.

Calcineurin and synaptophysin in the human spinal cord of normal individuals and patients with familial dysautonomia

This report concerns the immunohistochemical demonstration of two neuronal Ca2(+)-binding proteins, calcineurin and synaptophysin, in the spinal cord of normal controls and from patients with familial dysautonomia. In controls, calcineurin immunoreactivity was highly concentrated in small nerve cells and fibers of the substantia gelatinosa. Synaptophysin immunoreactivity was normally distributed throughout the spinal cord gray matter, being highly concentrated in the substantia gelatinosa, the dorsal nucleus of Clarke and the anterior horn. In patients with familial dysautonomia, no apparent changes in calcineurin immunoreactivity were found in the substantia gelatinosa. By contrast, there was a significant depletion of synaptophysin-positive axon terminals in the substantia gelatinosa and in the dorsal nucleus of Clarke of patients with familial dysautonomia.

Subdivisional involvement of nigrostriatal loop in idiopathic parkinson's disease and striatonigral degeneration

A topographical immunocytochemical analysis was performed on the substantia nigra from patients with idiopathic Parkinson's disease and striatonigral degeneration. Antibodies to tyrosine hydroxylase, a marker for nigrostriatal dopaminergic neurons, and to calcineurin, a marker for striatonigral projection fibers, were used in this study. There was a marked depletion of dopaminergic neurons in the substantia nigra of parkinsonian patients compared with control subjects, the reduction being greater in the lateral portion than in the medial portion (p less than 0.001). Calcineurin immunoreactivity was densely distributed throughout the substantia nigra of patients with Parkinson's disease and control subjects. The numbers of dopaminergic neurons and of calcineurin-immunoreactive fibers were markedly reduced in the lateral portion of the substantia nigra in all patients with striatonigral degeneration. Our results suggest that many symptoms of these two diseases may be due to disruption of the functions of the putamen and the lateral portion of the substantia nigra, which have dense reciprocal connections as part of the dopamine-related nigrostriatal loop.

Calcineurin in the postnatal striatum of the rat: an immunohistochemical study

This report concerns the postnatal expression of calcineurin (CaN) in rat striatum. For this purpose an immunohistochemical procedure was used. In neonatal striatum CaN immunoreactivity was found in discrete patches composed of many immature cells. With development there was an increase in staining intensity and in the proportion of positively reacting cells. CaN expression and localization in the striatum of 22-25 day old rats were similar to those of adult animals.

Evidence for transsynaptic regulation of calmodulin-dependent cyclic nucleotide phosphodiesterase in cerebellar Purkinje cells

Calmodulin-dependent phosphodiesterase (CaM-PDE) is selectively expressed in specific neuronal populations in adult rat brain. In cerebellar cortex, it is expressed at high levels in Purkinje cells (soma and dendrites). Climbing fiber ablation by intraperitoneal injections of 3-acetylpyridine resulted in a selective depression of cerebellar CaM-PDE expression using Western immunoblot procedures; neither calcineurin (calmodulin-dependent protein phosphatase) nor other calmodulin binding proteins, detected by biotinylated calmodulin overlays, were affected. Immunocytochemical staining of cerebellum revealed a loss of detectable CaM-PDE immunoreactivity in Purkinje cells, with no appreciable change in calcineurin immunoreactivity. Cerebral cortex was examined as a control for a direct effect of 3-acetylpyridine on CaM-PDE expression, independent of climbing fiber deafferentation. There were no detectable changes in CaM-PDE or calcineurin immunoreactivity in cortical pyramidal cells, and no changes were detected, either in Western blot analyses for CaM-PDE or calcineurin or in biotinylated calmodulin overlays. These data suggest that CaM-PDE expression in Purkinje cells is regulated transsynaptically by climbing fiber inputs.

An immunocytochemical demonstration of calcineurin in human nerve cell tumors. A comparison with neuron-specific enolase and glial fibrillary acidic protein

Human central and peripheral nerve cell tumors were examined in detail using antibodies to calcineurin, glial fibrillary acidic protein (GFAP) and neuron-specific enolase (NSE). Forty-eight formalin-fixed and paraffin-embedded specimens of human neuronal tumors, including 27 medulloblastomas, were examined. Calcineurin-positive cells were found in all peripheral nerve cell tumors and the two gangliogliomas, whereas 20 of the 27 medulloblastomas and one of the two cerebral neuroblastomas did not contain calcineurin-positive cells. Differentiation of cells along the neuronal lines was positively correlated with calcineurin immunoreactivity. NSE-positive cells were found in all of the tumors with the exception of the one cerebral neuroblastoma. NSE immunoreactivity was not invariably consistent with calcineurin immunoreactivity and non-neuronal cells were often positive. Calcineurin-positive cells were all devoid of GFAP, but NSE-positive cells expressed GFAP in some tumors. GFAP-immunoreactive cells were found only in central nerve cell tumors, and not in peripheral tumors. In addition, GFAP-positive cells in some tumors such as retinoblastoma and medulloblastoma morphologically revealed not only neoplastic but also reactive astrocytic features.

The distribution of calcineurin in rat brain by light and electron microscopic immunohistochemistry and enzyme-immunoassay

Calcineurin is the calcium (divalent cations)-dependent calcodulin-stimulated phosphoprotein phosphatase which is capable of dephosphorylating various substrate proteins. The subcellular and regional distribution of calcineurin in the rat brain has been studied by light and electron microscopic immunohistochemistry using antiserum against calcineurin. Immunoreactivity was observed in many neurons but was not detected in glial cells, such as astrocytes, oligodendrocytes and ependymal cells by the PAP method. Light microscopy demonstrates strong immunoreactivity in neuronal somata and neurites. By electron microscopy, calcineurin immunoreactivity was found to be present in dendrites including postsynaptic densities, somata, spines, axons and terminals. Calcineurin immunoreactivity was present in neurons throughout the brain, but marked regional variation in strength of the immunoreactivity was observed. The caudatoputamen, hippocampal formation, and substantia nigra were strongly stained. Cerebral and cerebellar neocortex showed moderate immunoreactivity. In substantia nigra and globus pallidus, only neurites were stained, but neuronal somata not. The staining of the substantia nigra was thought to be due to that of the nerve terminals originating from the caudatoputamen, in view of the findings by cerebral hemitransection and electron microscopic immunohistochemistry. We developed an enzyme-immunoassay (EIA) for calcineurin. The sensitivity of the EIA was 1 ng (13 fmol) of calcineurin. We determined the level of calcineurin in various regions of the rat brain. The caudate nucleus, putamen and hippocampal formation showed a high concentration of calcineurin. The results are consistent with those obtained by immunohistochemistry.

Calcineurin in human brain and its relation to extrapyramidal system. Immunohistochemical study on postmortem human brains.

Calcineurin immunoreactivity has been successfully detected in formalin-fixed paraffin-embedded postmortem human brain tissue using the peroxidase-antiperoxidase method. We have examined two autopsy cases with Huntington's disease (HD), three cases with Parkinson's disease, and two senile patients as controls. In the controls, calcineurin immunoreactivity was present in neuronal cells only and highly concentrated in the caudate nucleus, putamen, globus pallidus (striato-pallidal pathway), substantia nigra (striato-nigral pathway) and hippocampal formation. These localizations were similar to those identified in rat brain. There was a marked depletion of neurons containing calcineurin in the caudate nucleus and putamen, and a marked reduction of calcineurin-immunoreactive nerve fibers in the globus pallidus and substantia nigra were found in the cases with HD, but not in those with Parkinson's disease. These findings suggest that calcineurin can be a useful and specific index of neuronal degeneration in the caudato-putamen resulting from extrapyramidal disease, and that the calcineurin-immunostaining method can be a valuable tool for clarifying the anatomy of the human extrapyramidal system.