Increase of cdk5 is related to neurofibrillary pathology in progressive supranuclear palsy.

BACKGROUND: Progressive supranuclear palsy (PSP) is characterized by a pure neurofibrillary tau pathology involving mainly basal ganglia and brainstem nuclei. In addition to a haplotype of the tau gene potentially favoring tau aggregation, lipoperoxidation has been shown to be associated with PSP tau pathology. OBJECTIVE: To analyze cdk5/p35 complex, a kinase that regulates neurite outgrowth, as a potential cellular mechanism underlying tau phosphorylation in brain tissues from PSP and control cases and comparatively in cerebral cortex from subjects with AD. METHODS: Cdk5/p35 protein levels and distribution were evaluated by immunoblotting and immunocytochemistry in brain regions from seven PSP, six AD, and seven control cases, with similar postmortem intervals. RESULTS: Total cdk5 protein levels were significantly increased by more than threefold in PSP tissue and were augmented in PSP neurons, codistributed with tau immunoreactivity. P35, the regulatory subunit of cdk5, was degraded by postmortem proteolysis to the same extent in PSP, AD, and control tissues. CONCLUSIONS: The proteolysis in vivo of p35, the regulatory subunit of the kinase, is not ascertainable because it is masked by its postmortem degradation. The study, however, indicates that in PSP, the alteration of cdk5 is different from that described in AD and suggests that the absence of amyloid beta protein deposition may account for the different pathways responsible for the same kinase activation.

Changes of retinal neurons and glial fibrillary acid protein immunoreactive astrocytes in spontaneously hypertensive rats.

OBJECTIVE: The influence of arterial hypertension on retinal neurons and glial fibrillary acid protein (GFAP) immunoreactive astrocytes was investigated in spontaneously hypertensive rats (SHRs). METHODS: The retinas of 4- and 6-month-old SHRs and age-matched Wistar-Kyoto rats (WKY) were investigated. A group of SHRs, treated from 4 to 6 months with the hypotensive drug hydralazine, was also examined. Microanatomical and immunohistochemical techniques associated with image analysis and the terminal deoxyribonucleotidyl transferase (TdT)-mediated biotin-16-dUTP nick-end labelling (TUNEL) technique for apoptosis or necrosis were used, as well as astrocyte molecular biology (Western blot) techniques. RESULTS: In 4-month-old SHR and WKY rats, retinal morphology and the number of retinal neurons and of GFAP-immunoreactive astrocytes were similar, with the exception of the occurrence of 1% of TUNEL-positive ganglionic neurons in SHRs. In 6-month-old SHRs a decrease of retinal volume and of the number of ganglionic neurons and photoreceptors was observed, compared with age-matched normotensive WKY rats or younger SHR and WKY rats. Two per cent of ganglionic neurons and 5% of photoreceptors were also TUNEL positive. In 6-month-old SHRs, hypertrophic perivascular GFAP-immunoreactive astrocytes were found, whereas their number was unchanged compared to younger cohorts or WKY rats. An increased expression of GFAP was also noticeable in SHRs by Western blot analysis. Hypotensive treatment with hydralazine partly countered retinal changes occurring in SHRs. CONCLUSIONS: The occurrence of neuronal and astroglial changes when a stable hypertension was developed, and their sensitivity to antihypertensive treatment, suggest that they may represent a hypertension-related phenomenon.

Microanatomical changes of intracerebral arteries in spontaneously hypertensive rats: a model of cerebrovascular disease of the elderly.

Changes occurring in intracerebral arteries of 24-week-old spontaneously hypertensive rats (SHR) compared with age-matched normotensive Wistar-Kyoto (WKY) rats were assessed using microanatomical techniques associated with image analysis. Morphometric parameters investigated included arterial diameter, lumen area, wall area, and wall-to-lumen ratio. Intracerebral arteries (lumen diameter>46 microm) and arterioles (lumen diameter 46-10 microm) of frontal cortex, striatum, and hippocampus were examined. In frontal cortex of SHR arterial wall hypertrophy and luminal narrowing were observed. In striatum, an increase of wall area not accompanied by luminal narrowing predominates resulting in arterial hypertrophy without vasoconstriction. In hippocampal arteries of SHR, luminal narrowing, without changes of wall area was found indicating the occurrence of remodeling. In brain areas investigated, hypertensive changes affected primarily arterioles. The demonstration of a sensitivity of intracerebral arteries to hypertension suggests that changes of these vessels may represent a cause of brain structural alterations occurring in hypertension. The specificity of alterations occurring in intracerebral arteries of brain areas investigated may account for the different localization of cerebral lesions in cerebrovascular disease. The possibility that microanatomical changes developed in intracerebral arteries of SHR may represent a model of cerebrovascular disease of the elderly is discussed.

Androgen responsiveness and intrarenal localization of transcripts coding for the enzymes of polyamine metabolism in the mouse.

Polyamines, spermidine (SPD), and spermine (SPM) are intracellular polycations required for cell growth and differentiation. Their biosynthetic precursor, the diamine putrescine (PUT), is produced by regulatory ornithine decarboxylase (ODC). Spermidine/spermine N1-acetyltransferase (SSAT) is the ODC counterpart in the degradation pathway which retroconverts SPM and SPD into PUT. Castration of male mice for 7 days resulted in a 40% decrease of the renal levels of both SSAT and ODC transcripts. Administration of 5-alpha-dihydrotestosterone (DHT) to castrated mice for the last 3 days before sacrifice caused the levels of ODC and SSAT mRNAs to increase by 250% and 180%, respectively. Thus activation of the retroconversion pathway of polyamine metabolism appears to contribute towards the increase in PUT production known to be caused by androgens in the mouse kidney. In situ hybridization histochemistry experiments showed that the SSAT transcript is expressed only by the epithelial cells of the straight and convoluted distal tubules of the nephron, while the expression of the ODC transcript is confined to the epithelium of the convoluted and straight portion of the proximal tubules. The separation of the biosynthetic from the degradation pathway along the nephron suggests that PUT is mostly produced in the distal tubule, where it may play a physiological role, independent of androgen action, in protecting tubular cells from the very low osmolarity to which they are exposed in this nephron segment.

Glial reaction to volkensin-induced selective degeneration of central neurons.

Volkensin, a highly toxic protein retrogradely transported through axons, was used to target primary neuronal death in brainstem precerebellar relays after injection in the cerebellar cortex of rats. The reaction of astrocytes and microglia was studied with immunohistochemistry in the inferior olivary and pontine nuclei from 6 h to 14 days. Neurodegenerative features were evident since the first hours, especially in the pontine nuclei, and neuronal loss reached a plateau at 7 days in the inferior olive and at 10 days in the pons. Astrocytic activation, revealed by glial fibrillary acidic protein immunoreactivity, was concomitant with early signs of neuronal death and gradually increased. Microglia activation, revealed by OX-42 immunoreactivity, was evident at 2 days and became rapidly intense in precerebellar relays. At 1 week, marked ED-1 immunoreactivity also revealed phagocytic features of microglia, which persisted during the second week. In addition, major histocompatibility complex antigens (MHC) class I and II were induced in cells exhibiting microglial features. In the inferior olive, MHC I immunoreactivity was evident since 4 days and persisted at 14 days, whereas MHC II induction was intense at 7 days and subsided at 2 weeks. In the pontine nuclei high expression of both MHC antigens persisted instead at 14 days, probably reflecting the progression of neuronal death. Thus, targeted lethal injury of central neurons elicited prompt activation of both astrocytes and microglia; the marked microglia activation resulted in phagocytic features and immunophenotypic changes, with a temporal regulation that paralleled the evolution of neurodegenerative phenomena.

Occupancy by oral administration of nicardipine of L-type calcium channels in rat brain.

The occupancy of L-type Ca2+ channels by treatment with an oral dose of the dihydropyridine-type Ca2+ antagonist nicardipine (sustained-release formulation) was evaluated in membrane preparations of rat frontal cortex and hippocampus using a radioligand binding assay technique, with [3H]-nicardipine as a ligand. Three hours after nicardipine administration, specific binding was decreased by about 15-20%, both in the frontal cortex and hippocampus. This indicates that oral nicardipine occupied approximately 15-20% of L-type Ca2+ channels. A progressive occupancy of Ca2+ channels was observed between six and 12 h after nicardipine administration. Twelve hours after drug administration, approximately 65-70% of Ca2+ channels were occupied. These findings indicate that oral treatment with 3 mg/kg of nicardipine (sustained-release formulation) occupies L-type Ca2+ channels in rat brain by more than 40% from the 6th to the 24th h after drug administration. This suggests that an oral dose of nicardipine (sustained-release formulation) in duces a significant occupancy of L-type Ca2+ channels in rat frontal cortex and hippocampus for about one day. The possible clinico-therapeutic relevance of this observation is discussed.

The hippocampus in spontaneously hypertensive rats: a quantitative microanatomical study.

The influence of hypertension on the morphology of hippocampus was assessed in spontaneously hypertensive rats of two, four and six months and in age-matched normotensive Wistar-Kyoto rats. Values of systolic pressure were slightly increased in two-month-old spontaneously hypertensive rats in comparison with age-matched Wistar-Kyoto rats and augmented progressively with age in spontaneously hypertensive rats. No microanatomical changes were observed in the hippocampus of spontaneously hypertensive rats of two months in comparison with age-matched Wistar-Kyoto rats, whereas a decrease of white matter volume was observed in the CA(1) subfield and in the dentate gyrus of four-month-old spontaneously hypertensive rats. In the hippocampus of six-month-old spontaneously hypertensive rats a reduction of grey matter volume both in the CA(1) subfield and in the dentate gyrus, a loss of neurons affecting to a greater extent the CA(1) subfield and an increase of glial fibrillary acid protein-immunoreactive astrocytes was found. The occurrence of apoptosis and/or necrosis identified using the terminal deoxyribonucleotidyl transferase-mediated biotin-16-dUTP nick end labelling technique was also observed in the CA(1) subfield and to a lesser extent in the dentate gyrus. The only change noticeable in the CA(3) subfield of six-month-old spontaneously hypertensive rats was a slight increase in the number of glial fibrillary acid protein-immunoreactive astrocytes. These findings indicate the occurrence of neuronal loss and of astrocyte changes in the hippocampus of spontaneously hypertensive rats of six months, being the CA(1) subfield the area most affected. The relevance of these neurodegenerative changes in hypertension and the possible occurrence of apoptosis and/or necrosis as expression of hypertensive brain damage is discussed.

Oxidative stress induces increase in intracellular amyloid beta-protein production and selective activation of betaI and betaII PKCs in NT2 cells.

Amyloid beta-protein (Abeta) aggregation produces an oxidative stress in neuronal cells that, in turn, may induce an amyloidogenic shift of neuronal metabolism. To investigate this hypothesis, we analyzed intra- and extracellular Abeta content in NT2 differentiated cells incubated with 4-hydroxy-2,3-nonenal (HNE), a major product of lipid peroxidation. In parallel, we evaluated protein kinase C (PKC) isoenzymes activity, a signaling system suspected to modulate amyloid precursor protein (APP) processing. Low HNE concentrations (0.1-1 microM) induced a 2-6 fold increase of intracellular Abeta production that was concomitant with selective activation of betaI and betaII PKC isoforms, without affecting either cell viability or APP full-length expression. Selective activation of the same PKC isoforms was observed following NT2 differentiation. Our findings suggest that PKC beta isoenzymes are part of cellular mechanisms that regulate production of the intracellular Abeta pool. Moreover, they indicate that lipid peroxidation fosters intracellular Abeta accumulation, creating a vicious neurodegenerative loop.

Neuronal loss up-regulates clusterin mRNA in living neurons and glial cells in the rat brain.

The aim of this study was to examine the time course and cellular localization of clusterin mRNA after neurodegeneration. Selective neuronal death was achieved in the rat inferior olivary complex after volkensin injection in the contralateral cerebellar cortex. Quantitative analysis of the in situ hybridization signal demonstrated over-expression of clusterin mRNA in living neurons at 6 days and outside the neuronal cell bodies at 10 days post-injection. We conclude that, in our experimental model, clusterin over-expression occurs as an early and transient neuronal and as a delayed glial response to selective neuronal death, supporting the view that clusterin may be involved in cytoprotection and tissue remodeling.

Determination of xanthine oxidase in human serum by a competitive enzyme-linked immunosorbent assay (ELISA).

Xanthine oxidase was purified from human milk and used to immunise rabbits. A competitive immunoenzymatic assay with purified enzyme and rabbit antiserum was optimised to measure xanthine oxidase in human serum, the lowest detectable amount being 0.03 pmol of enzymatic protein. Thus, the test (i) is sensitive enough to determine xanthine oxidase in human serum, being more sensitive than the spectrophotometric method, (ii) it is more convenient for clinical laboratories than other sensitive tests and (iii) it has the advantage over the enzyme activity-based assays of also detecting inactive enzyme molecules. A competitive enzyme-linked immunosorbent assay (ELISA) was used to measure the serum xanthine oxidase level in healthy donors and in patients with liver diseases, and it was found that any concentration below 1 mg/L is in the normal range.

Immunocytochemical discrimination between early and late S phase: a new approach to the study of gingival epithelium proliferation in rats.

The renewal of the free gingival margin epithelium in rats was studied evaluating 5-bromodeoxyuridine (BrdU) incorporation in proliferating cells by means of an immunocytochemical method. We found a close correspondence between light and electron microscopy patterns of BrdU incorporation at a nuclear level. BrdU was localized in the inner interchromatin regions in cells starting DNA synthesis, while it was localized in the peripheral heterochromatin domains in cells terminating the S phase. This possibility of discriminating cells in early S phase from cells in late S is able to provide far more information as to the time at which a labeled cell starts proliferation than that obtainable with 3H-thymidine autoradiography. This, in turn, permits detection of cells that start proliferation in a wide period of time by means of a single BrdU administration. Rats treated at 7 a.m. demonstrated higher proliferation than rats treated at 7 p.m., supporting the existence of circadian variations in the epithelial renewal. Proliferative events take place by consecutive activation of at least three replication waves, producing clusters of labeled cells which could be observed in rats sacrificed at 10 a.m. In rats treated once with BrdU at 7 a.m., the clusters were localized in both the basal and suprabasal layer of the epithelium; in rats further injected with BrdU at the same time, the clusters increased in size, progressively extending throughout the epithelium. In this way, the renewal of the free gingival margin epithelium does not proceed randomly, but by consecutive activation of discrete units or clusters of basal cells, which then extend to the upper layers. This can be followed at a morphological level as a progression of labeled cells, which move from the basal layer to the epithelium surface in approximately 82-85 hours.

Changes in the activity of cdk2 and cdk5 accompany differentiation of rat primary oligodendrocytes.

Oligodendrocytes, the myelinating cells of the central nervous system, are terminally differentiated cells that originate through asynchronous waves of proliferation and differentiation of precursors present at birth. Withdrawal from cell cycle and onset of differentiation are tightly linked and depend on an intrinsic program modulated by the action of growth factors. p27 plays a central and obligatory role in the initiation of oligodendrocyte differentiation and cessation of proliferation. In this paper, we have characterized the role of modulation of cdk2 and cdk5 kinase activity during the process of oligodendrocyte precursor differentiation. As rat primary oligodendrocytes differentiate in culture there is a fall in cdk2 activity and a rise in cdk5 activity as well as an increase in the cdk inhibitor, p27 protein. The decline in cdk2 activity is not accompanied by a drop in cdk2 protein level, suggesting that it results from inhibition of cdk2 activation rather than decreased protein expression. Taken together, these data suggest that oligodendrocytes may withdraw from the cell cycle at G1-S transition through inactivation of cdk2 activity, possibly initiated by increasing amount of p27, and that cdk5 may have a role until now unrecognized in the differentiation of oligodendrocytes.

Vascular and neuronal hypertensive brain damage: protective effect of treatment with nicardipine.

AIM: The brain is sensitive to hypertension, which causes a variety of vascular and neuronal cerebral changes. The present study was designed to assess the effect of long-term treatment with the Ca2+ channel blocker nicardipine on intracerebral (intraparenchymal) arteries in spontaneously hypertensive rats (SHR) by using microanatomical techniques associated with image analysis. The effects of hypertension and treatment with nicardipine on nerve cells and glial fibrillary acid protein (GFAP)-immunoreactive glial cells were also evaluated. EFFECTS OF NICARDIPINE ON BLOOD PRESSURE: In SHR a significant increase in systolic blood pressure in comparison with age-matched normotensive Wistar-Kyoto (WKY) rats was noticeable. Treatment with nicardipine significantly reduced systolic pressure in the SHR. The media: lumen ratio and the thickness of the tunica media were increased in medium (diameter between 150 and 50 microns and small (diameter < 50 microns intracerebral arteries. This phenomenon was accompanied by luminal narrowing. Treatment with nicardipine significantly reduced the thickness of the tunica media, the media: lumen ratio and increased the luminal area, primarily at the level of small pial arteries and of intracerebral arteries. EFFECTS OF NICARDIPINE IN THE BRAIN: In control SHR, the number of neurones in the frontal and occipital cortex was reduced in comparison with normotensive WKY rats. GFAP-immunoreactive astrocytes were increased in number (hyperplasia) and in size (hypertrophy), both in the frontal cortex and in the occipital cortex of control SHR. In the CA1, field of the hippocampus, the number of neurones and their size were decreased in SHR in comparison with normotensive WKY rats. Hyperplasia of GFAP-immunoreactive astrocytes of white matter and hypertrophy of those of grey matter was also noticeable. No important changes were found in other portions of the hippocampus. Treatment with nicardipine increased the number of neurones in the frontal cortex and in the occipital cortex of SHR and countered hyperplasia and hypertrophy of GFAP-immunoreactive astrocytes. Moreover, it increased the number of neurones in the CA1 field of the hippocampus and decreased the number and the size of astrocytes of the white matter and grey matter, respectively. CONCLUSIONS: These findings show that treatment of SHR with nicardipine significantly reduced systolic blood pressure and induced moderate vasodilation of both extracerebral and intracerebral arteries regulating cerebrovascular resistance. The compound also countered some microanatomical changes occurring in the hypertensive brain. The frontal and occipital (visual) cortex and the CA1 field of the hippocampus were the cerebral areas more sensitive to treatment with nicardipine. This suggests that nicardipine induces moderate cerebrovascular dilation and exerts neuroprotective effects on SHR neurones. The possible relevance of the neuroprotective actions of nicardipine in the hypertensive brain deserves to be evaluated in future studies.

Different localization of spermidine/spermine N1-acetyltransferase and ornithine decarboxylase transcripts in the rat kidney.

In situ hybridization histochemistry of transverse sections from male rat kidney showed that the mRNA of the regulatory enzyme of polyamine degradation, spermidine/spermine N1-acetyltransferase, has a spotty distribution in the cortex, is low and diffused in the outer stripe and high and diffused in the inner stripe of the outer medulla. At the cellular level, this mRNA is solely expressed by the epithelium of the distal straight and convoluted nephron tubules. Since biosynthetic ornithine decarboxylase mRNA is solely found in the proximal straight tubules, it is proposed that polyamine biosynthesis and degradation occur at separate sites along the nephron.

Suicide retrograde transport of volkensin in cerebellar afferents: direct evidence, neuronal lesions and comparison with ricin.

Volkensin and ricin, either free or conjugated with colloidal gold, were injected into the cerebellar cortex of rats. The inferior olive and pontine nuclei were examined to verify the retrograde axonal transport of these two toxins, and the consequent neuronal damage. No evidence was obtained of a retrograde axonal transport of ricin in these pathways. Injection of gold-conjugated volkensin in the cerebellar cortex resulted in retrogradely labelled neurones in the inferior olive after 3 h, and in the pontine nuclei after 6 h. Degenerative changes were very severe in the retrogradely labelled neurones 48 h after the gold-conjugated volkensin injection. In the Nissl-stained material, neuronal degeneration started to be evident in the inferior olive 12 h, and in pontine nuclei 6 h, after volkensin injection. The neuronal degeneration in both the inferior olive and pons increased up to 4 days after the injection. These findings provide direct evidence of the retrograde axonal transport of volkensin in the central nervous system, and the time course of the consequent degenerative changes in the afferents to the cerebellar cortex.

Nicardipine and treatment of cerebrovascular diseases with particular reference to hypertension-related disorders.

Nicardipine is a second generation dihydropyridine-type Ca2+ antagonist with high vascular selectivity and strong cerebral and coronary vasodilatory activity. The compound is used in the treatment of hypertension, primarily in the elderly. In this review the main evidence of the cerebrovascular activity of nicardipine in preclinical studies using in vitro and in vivo models is detailed. A particular physico-chemical property of nicardipine is the almost complete protonation in acid environment. This allows its accumulation in ischemic brain regions and makes it a candidate for the treatment of cerebrovascular disorders characterised by impaired brain perfusion. The main clinical data on the use of nicardipine in cerebral ischemia and related disorders, subarachnoid haemorrhage and stroke, are also reviewed. These studies included 5940 patients affected by chronic cerebrovascular insufficiency (cerebral ischemia, cerebral atherosclerosis mainly associated with hypertension, transient ischemic attacks, sequelae of cerebral infarction, thrombosis or embolia, hypertensive encephalopathy), 1540 patients affected by sequelae of subarachnoid haemorrhage and 206 patients affected by stroke. Both preclinical studies and clinical trials have shown that nicardipine is a safe Ca2+ antagonist with powerful cerebrovascular activity. This suggests its possible use in cerebrovascular disorders in which blockade of Ca2+ channels of the L-type and/or selective cerebral vasodilatation is desirable. Further studies are necessary to establish if modulation of neuronal Ca2+ channels of the L-type by nicardipine may have a neuroprotective effect independent by the cerebrovascular activity of the compound.

Molecular diagnosis of transthyretin Met30 mutation in an Italian family with familial amyloidotic polyneuropathy.

We report the molecular analysis of the transthyretin gene in a large Italian pedigree with familial amyloidotic polyneuropathy and demonstrate the presence of a Met30 mutation. The usefulness of the genetic analysis in the identification of presymptomatic persons and the diagnosis of individuals with partial symptoms is discussed.

Increased levels of clusterin mRNA in the ventral prostate of the aging rat are associated to increases in cuboidal (atrophic) cell population and not to changes in apoptotic activity.

In the ventral prostate of the intact rat, clusterin mRNA is expressed in a small population of cuboidal epithelial cells undergoing apoptosis, but not in the columnar cells comprising the vast majority of the glandular epithelium. Upon castration, clusterin mRNA expression and apoptotic activity are turned off in the cuboidal cells and turned on in the columnar ones. We show here that the progressive enhancements in the abundance of clusterin mRNA, occurring in the rat ventral prostate upon aging, are based on increases of the cuboidal cells at the expense of the columnar ones. DNA fragmentation, a typical sign of apoptosis, assayed both by agarose gel electrophoresis and in situ staining, was undetectable in 3-month-old rats but was evident among the cuboidal cells of 24-month-old animals and columnar cells of 1-day castrates. The DNA content of the ventral prostate did not change significatively between young and old rats, indicating that no increase in the rate of cell death occurs within the age interval examined. It is concluded that the enhancement in cuboidal cell population, the consequent augmented accumulation of clusterin mRNA, and the increased frequency of DNA fragmentation that we have detected in the aging rat ventral prostate are not directly related to the rate of apoptosis.

Gene relaxation and aging: changes in the abundance of rat ventral prostate SGP-2 (clusterin) and ornithine decarboxylase mRNAs.

Sulfated glycoprotein 2 (SGP-2) mRNA progressively increased in the ventral prostate of the aging rat, reaching, at 24 months, 4-fold higher than at 3 months. Ornithine decarboxylase (ODC) mRNA peaked at 6 months (4-fold increase), and at 12 and 24 months was maintained at higher levels than at 3 months. ODC enzymatic activity was enhanced at 6 months to a much smaller extent than its own mRNA, the values at 12 and 24 months dropping to below those at 3 months. Putrescine (Put), spermidine (Spd) and spermine (Sp) concentrations also peaked at 6 months (100% increase for Put, 50% for Sp and Spd). At 24 months, Put and Spd were diminished, and Sp was unchanged with respect to the 3-month values. Under the same conditions, glyceraldehyde-3-phosphate dehydrogenase mRNA did not undergo significant alterations.

Immunological properties of ribosome-inactivating proteins and a saporin immunotoxin.

Antibodies have been raised in rabbits against plant ribosome-inactivating proteins (RIPs) and used to demonstrate cross-reactivity between RIPs from plants belonging to the same family, but little or no cross-reactivity between RIPs from taxonomically unrelated plants. When an immunotoxin consisting of saporin conjugated to bovine IgG was injected into rabbits, the animals formed antibodies against both saporin and bovine IgG.