Chaperone-like activity of immunophilin FKBP12 from bovine brain, a cytoplasmic receptor of immunosuppressor FK506

The chaperone-like activity of brain immunophilin—a cytoplasmic receptor of immunosuppressor FK506 (FK506-binding protein, FKBP12) was evaluated in a test-system in vitro based on suppression of insulin aggregation as a result of A-and B-chain dissociation induced by disulfide bond reduction by dithiotreitol. Using dynamic light scattering technique we have demonstrated the concentration dependent suppression of light scattering intensity and the decrease of the hydrodynamic radius of insulin B-chain amorphous aggregates upon addition of FKBP12 to the incubation medium, a significant effect being revealed even in the substoichiometric concentration range. The results provide evidence that FKBP12 possesses chaperone-like activity preventing insulin aggregation.     

Postischemic hyperthermia induces Alzheimer-like pathology in the rat brain

This study addresses the effects of induced hyperthermia on post-ischemic rat brain evaluated histologically and/or immunohistochemically after 7-day, 2-month or 6-month survival. Hyperthermia (38.5 degrees - 40 degrees C) maintained (by heating the cage environment to 34-35 degrees C) for two consecutive periods of 5 and 9 h timed, respectively, from 4- and 21-h recirculation following 10-min global ischemia (two-vessel occlusion + hypotension) induced chronic neuronal death that became apparent in the rat forebrain from 7-day to 2-month survival. Associated immunohistochemical findings after 2 or 6 months of recovery included: (1) complement activation (membrane attack complex formation); (2) generalized overexpression of ubiquitin in surviving forebrain neurons; (3) persistent activation of macrophages; (4) presence of gemistocytic astrocytes in the hippocampus; (5) maturation of amyloid plaques (identified by immunohistochemistry using anti-human beta-A4 primary antibody) in cerebral cortex; and (6) intracellular deposits identified by anti-human hyperphosphorylated tau protein antibodies. This novel non-transgenic, self-sustained model of neurodegeneration triggered by the association of two prevalent insults to the aging human brain (ischemia and hyperthermia) presents morphological features similar to those of Alzheimer's disease. This finding raises the possibility that febrile complications of acute brain injuries may similarly impair human cognitive function in the long run.     

Decrease of the immunophilin FKBP52 accumulation in human brains of Alzheimer's disease and FTDP-17

Human neurodegenerative diseases characterized by abnormal intraneuronal inclusions of the tau protein, or "tauopathies", include Alzheimer's disease (AD), Pick's disease, progressive supranuclear palsy, corticobasal degeneration as well as fronto-temporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Several abnormalities of tau may contribute to the pathological processes, yet the mechanisms involved in tau cellular toxicity remain unclear. Previously, we demonstrated an interaction between various isoforms of tau and the immunophilin FKBP52 (FK506-Binding Protein), suggesting a direct involvement of FKBP52 in tau function. Here we analyze the expression of FKBP52 in human brains of patients with different tauopathies, including AD. Immunohistofluorescence studies carried out on cerebral cortex in different tauopathies reveal that FKBP52 is not sequestered by filamentous tau inclusions while FKBP52 is colocalized with tau in the control case brains. We found that FKBP52 expression level is abnormally low in frontal cortex of AD and FTDP-17 brains, as compared to controls, despite no alteration in the FKBP52 mRNA expression level. The possible involvement of FKBP52 in pathological tau expression/function is discussed.     

The Hsp90-binding immunophilin FKBP52 enhances neurodifferentiation and neuroregeneration in murine models

The term immunophilin involves a family of proteins whose domain shows peptidylprolyl-(cis/trans)-isomerase (PPIase)enzymatic activity,i.e., the reversible cis/trans interconversion of Xaa-Pro bonds (Annett et al.,2020).The PPlase domain of these proteins usually binds to immunosuppressive drugs,such as the macrolide FK506 (referred to as the FKBP subfamily) or the cyclic undecapeptide cyclosporine A (called CyP subfamily).     

Postischemic production of eicosanoids in gerbil brain

The postischemic production of PGE2, PGD2, 6-keto-PGF1 alpha, and TXB2 in brain tissue was studied in Mongolian gerbils using tissue extraction as well as a new ex vivo method. This new method permits the study of prostaglandin synthesis in slices from discrete areas of the brain (cortex, hippocampus, striatum, hypothalamus). Gerbils were sacrificed at 0, 5, and 30 minutes, and 4 and 24 hours after a 15-minute occlusion of both carotid arteries. Apart from 6-keto-PGF1 alpha, tissue prostaglandins determined by the extraction method were significantly increased 3 and 30 minutes after reperfusion. The most pronounced changes found by the ex vivo method were increased productions of PGD2 immediately after reperfusion (285% in cortex, 215% in hypothalamus) and PGE2 (350% in hippocampus, 320% in striatum) 4 hours after reperfusion. At 24 hours after reperfusion PGE2 and PGD2 synthesis were significantly decreased by 23-70% of the values obtained from sham-operated controls. Thromboxane increased slightly in all areas after recirculation and subsequently decreased to values below the control level in striatum. The results obtained by ex vivo incubation of tissue slices demonstrate that ischemia and subsequent recirculation cause site-, time-, and PG-specific changes of tissue eicosanoid production.     

Early decrease of the immunophilin FKBP 52 in the spinal cord of a transgenic model for amyotrophic lateral sclerosis

Intrathecal administration of 5-HT(2) receptor agonists produces an anti-allodynic effect in a rat model of neuropathic pain. Several non-serotonergic neurotransmitters have been implicated these anti-nociceptive effects. In the present study, intrathecal pre-treatment with the muscarinic receptor antagonist atropine (10 and 30 microg) and pirenzepine (10 microg) reversed the anti-allodynic effect of the 5-HT(2) receptor agonist alpha-methyl-5-hydroxytryptamine, unlike various other antagonists. Thus, muscarinic receptors may be involved in the anti-allodynic action of intrathecally injected 5-HT(2) receptor agonist.     

Postischemic glucose utilization in rat hippocampal layers

The influence on hippocampal glucose utilization was determined in male Wistar rats 7 days after a 10-min forebrain ischemia. Ischemia was induced by clamping of the carotid arteries and lowering blood pressure to 40 mm Hg. Despite severe neuronal damage as assessed by histological techniques, local cerebral glucose utilization (LCGU) was significantly increased in the pyramidal and radiatum layer of the CA1 sector, while in layers of the CA2, CA3 and CA4 sector and dentate gyrus. LCGU was reduced compared to non-ischemic controls. The increases in LCGU are suggested to reflect long-lasting hyperexcitation in the selectively vulnerable CA1 sector, implicating a correlation between cellular hypermetabolism and neuronal damage.     

Glycogen changes in X-Irradiated rat brain

Summary X-irradiation of the head of rats resulted in the accumulation of histochemically demonstrable PAS-positive granules in the brain, which were identified as glucogen. The glycogen granules were confined predominantly to the neuroglial cells; they did not appear in neurons. The amount of glycogen granules was approximately proportional to the dose administered. Although such changes are known to occur under conditions of ionizing particle radiation, this is the first demonstration that they appear following X-irradiation.Quantitative chemical analysis of the brains by the glucose oxydase method demonstrated an increase of glycogen of approximately 40%, 24 hours after exposure to 3.000 r.The biochemical change responsible for the glycogen accumulation may consist in an impairment of the enzymes mediating the incorporation and release of glucose from glycogen.

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Zusammenfassung Röntgenbestrahlung des Rattenkopfes hatte im Gehirn die Anhäufung von histochemisch nachweisbaren PAS-positiven Granula zur Folge, die sich als Glykogen erwiesen.Die Glykogen-Granula fanden sich hauptsächlich in Gliazellen; sie traten nicht in Nervenzellen auf. Die Anzahl der Glykogen-Granula war ungefähr proportional der verabreichten Strahlendosis. Obgleich das Vorkommen derartiger Veränderungen unter den Bedingungen von ionisierenden Teilchenbestrahlungen bekannt ist, stellt dies den ersten Nachweis nach Röntgenbestrahlung dar.Quantitative chemische Analysen des Gehirnes mittels der Glucose-Oxydase-Methode zeigten — 24 Std nach Applikation von 3.000 r — einen Anstieg des Glykogengehaltes um ca. 40%.

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Postischemic changes of calcium and endogenous antagonist in the rat hippocampus studied by proton-induced X-ray emission analysis

Proton-induced X-ray emission analysis (PIXE) was applied to determine ischemia-induced changes in the content of calcium, iron, nickel and zinc in the rat hippocampus. After 30 min or 2 h reperfusion following 20 min of ischemia, the nickel content decreased and the zinc content increased. The calcium content was increased 50% after 30 min but was normal after 2 h reperfusion.     

Mounting evidence of FKBP12 implication in neurodegeneration

Intrinsically disordered proteins, such as tau or α-synuclein, have long been associated with a dysfunctional role in neurodegenerative diseases. In Alzheimer's and Parkinson's' diseases, these proteins, sharing a common chemical-physical pattern with alternating hydrophobic and hydrophilic domains rich in prolines, abnormally aggregate in tangles in the brain leading to progressive loss of neurons. In this review, we present an overview linking the studies on the implication of the peptidyl-prolyl isomerase domain of immunophilins, and notably FKBP12, to a variety of neurodegenerative diseases, focusing on the molecular origin of such a role. The involvement of FKBP12 dysregulation in the aberrant aggregation of disordered proteins pinpoints this protein as a possible therapeutic target and, at the same time, as a predictive biomarker for early diagnosis in neurodegeneration, calling for the development of reliable, fast and cost-effective detection methods in body fluids for community-based screening campaigns.     

Postischemic administration of idazoxan, an alpha-2 adrenergic receptor antagonist, decreases neuronal damage in the rat brain

The effect of an alpha-2 receptor antagonist, idazoxan, on ischemic neuronal damage in the hippocampus and neocortex was studied in rats following 10 min of forebrain ischemia. Idazoxan was given 0.1 mg/kg i.v. immediately after recirculation, followed by 48 h of continuous infusion at a rate of 10 micrograms/kg/min. A histopathological examination of the CA1 region of the dorsal hippocampus and neocortex from each hemisphere was made on paraffin-embedded sections following 7 days of survival. In ischemic animals receiving an infusion of saline, 71% of the neurons in the hippocampal CA1 region were degenerated. In contrast, in the idazoxan-treated animals only 31% of the neurons were irreversibly damaged (p less than 0.01). We conclude that postischemic administration of the alpha-2 antagonist idazoxan protects neurons against damage following cerebral ischemia. Rapid postischemic administration of alpha-2 adrenergic receptor antagonists could be an effective treatment after stroke and cardiac arrest.     

FKBP12 mRNA expression is upregulated by intrinsic CNS neurons regenerating axons into peripheral nerve grafts in the brain

We have examined the expression of the immunophilin FKBP12 in adult rat intrinsic CNS neurons stimulated to regenerate axons by the implantation of segments of autologous tibial nerve into the thalamus or cerebellum. After survival times of 3 days to 6 weeks, the brains were fresh-frozen. In some animals the regenerating neurons were retrogradely labelled with cholera toxin subunit B 1 day before they were killed. Sections through the thalamus or cerebellum were used for in situ hybridization with digoxygenin-labelled riboprobes for FKBP12 or immunohistochemistry to detect cholera toxin subunit B-labelled neurons. FKBP12 was constitutively expressed by many neurons, and was very strongly expressed in the hippocampus and by Purkinje cells. Regenerating neurons were found in the thalamic reticular nucleus and deep cerebellar nuclei of animals that received living grafts. Neurons in these nuclei upregulated FKBP12 mRNA; such neurons were most numerous at 3 days post grafting but were most strongly labelled at 2 weeks post grafting. Regenerating neurons identified by retrograde labelling were found to have upregulated FKBP12 mRNA. No upregulation was seen in neurons in animals that received freeze-killed grafts, which do not support axonal regeneration. We conclude that FKBP12 is a regeneration-associated gene in intrinsic CNS neurons.     

Postischemic accumulation of lipid peroxidation products in the rat brain: immunohistochemical detection of 4-hydroxy-2-nonenal modified proteins

We report an immunohistochemical study on the distribution and alterations of 4-hydroxy-2-nonenal (HNE)-modified proteins, an indicator of lipid peroxidation, in the rat brain after 3 h of middle cerebral artery (MCA) occlusion followed by reperfusion. HNE immunoreactivity was not observed in intact neurons, but it appeared in some shrunken neurons within the infarcted zone at 3 h after reperfusion. The number of HNE-positive neurons increased with the spread of the infarcted area. The pyramidal neurons in the third layer of the frontoparietal cortex were HNE-positive and the intensity of their HNE immunoreactivity was highest at 24 h after reperfusion. At 48 h, HNE-positive neurons were observed in the medial part of the striatum, the lateral side of the frontoparietal cortex, and at the boundary between the infarcted and noninfarcted zones. In addition, strong HNE immunoreactivity was seen in microglia (identified by OX-42 immunostaining). This method seems to be useful to follow the progress of lipid peroxidation at the cellular level after ischemic injury.     

Age dependent changes in the methylation of rat brain phospholipids

Phospholipid methyltransferase I and II enzymes methylate phosphatidylethanolamine three times to form phosphatidylcholine. The activity of these two enzymes was determined in synaptosome-enriched fractions from rats 1, 3, 7, 15 and 21 months old. The activity of phospholipid methyltransferase I was significantly greater in 7-, 15- and 21-month-old rats than in 1- and 3-month-old rats. In contrast, the activity of phospholipid methyltransferase II did not change with age. These changes in methyltransferase activity with increasing age may be related to changes in beta-receptor function with increasing age.     

Presynaptic excitability changes following traumatic brain injury in the rat

Pathological processes affecting presynaptic terminals may contribute to morbidity following traumatic brain injury (TBI). Posttraumatic widespread neuronal depolarization and elevated extracellular potassium and glutamate are predicted to alter the transduction of action potentials in terminals into reliable synaptic transmission and postsynaptic excitation. Evoked responses to orthodromic single- and paired-pulse stimulation were examined in the CA1 dendritic region of hippocampal slices removed from adult rats following fluid percussion TBI. The mean duration of the extracellularly recorded presynaptic volley (PV) increased from 1.08 msec in controls to 1.54 msec in slices prepared at 1 hr postinjury. There was a time-dependent recovery of this injury effect, and PV durations at 2 and 7 days postinjury were not different from controls. In slices removed at 1 hr postinjury, the initial slopes of field excitatory postsynaptic potentials (fEPSPs) were reduced to 36% of control values, and input/output plots revealed posttraumatic deficits in the transfer of excitation from pre- to postsynaptic elements. Manipulating potassium currents with 1.0 mM tetraethylammonium or elevating potassium ion concentration to 7.5 mM altered evoked responses but did not replicate the injury effects to PV duration. Paired-pulse facilitation of fEPSP slopes was significantly elevated at all postinjury survivals: 1 hr, 2 days, and 7 days. These results suggest two pathological processes with differing time courses: 1) a transient impairment of presynaptic terminal functioning affecting PV durations and the transduction of afferent activity in the terminals to reliable synaptic excitation and 2) a more protracted deficit to the plasticity mechanisms underlying paired-pulse facilitation.     

Age-related changes of the nitric oxide system in the rat brain

This work examines the age-related changes of the NO pathway in the central nervous system (CNS), analyzing nitric oxide synthase (NOS) isoform expression, the level of nitrotyrosine-modified proteins, and the NOS activity in the cerebral cortex, decorticated brain (basal ganglia, thalamus, hypothalamus, tegtum and tegmentum) and cerebellum of young, adult and aged rats. Our data demonstrate that the different NOS isoforms are not uniformly expressed across the CNS. In this sense, the nNOS and eNOS isoenzymes are expressed mainly in the cerebellum and decorticated brain, respectively, while the iNOS isoenzyme shows the highest level in cerebellum. Concerning age, in the cerebral cortex nNOS significantly increased its expression only in adult animals; meanwhile, in the cerebellum the eNOS expression decreased whereas iNOS increased in adult and aged rats. No age-related changes in any isoform were found in decorticated brain. NOS activity, determined by nitrate plus nitrite quantification, registered the highest levels in the cerebellum, where the significant increase detected with aging was probably related to iNOS activity. The number of nitrotyrosine-modified immunoreactive bands differed among regions; thus, the highest number was detected in the decorticated brain while the cerebellum showed the least number of bands. Finally, bulk protein nitration increased in cerebral cortex only in adult animal. No changes were found in the decorticated brain, and the decrease detected in the cerebellum of aged animals was not significant. According to these results, the NO pathway is differently modified with age in the three CNS regions analyzed.     

Age-related changes in bindings of second messengers in the rat brain

Age-related alterations in bindings of major second messengers in the brain were studied in 3-week- and 6-, 12-, 18- and 24-month-old Fisher 344 rats using receptor autoradiography. [³H]Phorbol 12,13-dibutyrate (PDBu) and [3H]forskolin were used to label protein kinase C (PKC) and adenylate cyclase, respectively. In immature rats (3-week-old), [³H]PDBu binding showed a significant decrease only in the cerebellum as compared to adult rats (6-month-old), whereas [³H]forskolin binding exhibited a significant reduction in the neocortex, nucleus accumbens, thalamus and substantia nigra. In aged rats, [³H]PDBu binding showed no significant change in all brain areas. In contrast, [³H]forskolin binding showed a conspicuous reduction in various brain areas in 18-month-old rats as compared to adult animals. The age-related reduction was especially observed in the cerebral cortex, hippocampal CA3 pyramidal cell layer, dentate gyrus, thalamus and molecular layer of cerebellum of 24-month-old rats. The results indicate that adenylate cyclase system in the rat brain is more susceptible to aging processes than phosphoinositide cycle system. Furthermore, our data demonstrate that the change in the adenylate cyclase system is more pronounced than that in the phosphoinositide cycle system in immature rat brain. These findings suggest that the adenylate cyclase system is primarily affected in aging processes and this may lead to age-related neurological deficits.