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Biomedical subjects

G M Pasinetti

Publications and source records attributed to G M Pasinetti.

At least 19 recordsLinked to original sources
PubMed2002

Randomized pilot study of nimesulide treatment in Alzheimer's disease.

BACKGROUND: Nonsteroidal anti-inflammatory drugs (NSAID) may be useful in the treatment of AD. Clinical and laboratory experience with nimesulide, an NSAID with preferential cyclooxygenase-2 inhibition, suggests that it may be a good candidate for AD therapy. METHODS: This pilot study investigated the clinical feasibility of nimesulide treatment in AD. Forty persons with probable AD, most of whom were taking cholinesterase inhibitors, were enrolled in a randomized, controlled, parallel-group trial designed to assess tolerability and short-term cognitive/behavioral effects of nimesulide. In the initial 12-week double-blind phase, participants were treated with nimesulide 100 mg by mouth twice daily or matching placebo; during the second 12-week phase all participants received active drug. Participants who tolerated the drug well and perceived benefit were invited to continue open-label nimesulide treatment. RESULTS: Short-term therapy with nimesulide, compared with placebo, had no significant effect on total assessment scores of measures of cognition, clinical status, activities of daily living, affect, and behavior. Long-term therapy was well tolerated for periods exceeding 2 years. CONCLUSION: These findings support the feasibility of nimesulide therapy in AD; assessment of efficacy will require a larger, long-term treatment study.

Aged↗
PubMed2001

Use of cDNA microarray in the search for molecular markers involved in the onset of Alzheimer's disease dementia.

Alzheimer's disease (AD) is the most common form of dementia, affecting as many as 4 million older persons and results from abnormal changes in the brain that most likely begin long before cognitive impairment and other clinical symptoms become apparent. Thus, efforts aimed at identifying methods of early detection and diagnosis for improving AD care might be the most appropriate strategy to initiate promising new treatments and/or prevention. We used cDNA microarray technology to investigate the sequence of changes in gene expression in brain that may take place during the transition from normal cognitive functioning through the early stages of impairment to frank AD. We examined the expression of approximately 7,000 genes in the brains of cases at the early stage of AD dementia using reference sample cases characterized by normal cognitive status. Genes that are differentially regulated in early AD cases were identified and were categorized into gene clusters based on similarities in biological functions. This analysis revealed that selected biological processes, including protein and amino acid metabolism, cytoskeleton integrity, and fatty acid metabolism, are involved in early phases of AD dementia. Most notable is the observation that selected genes involved in neurotransmitter release are differentially regulated in the brains of cases at high risk for dementia. This evidence supports the feasibility and usefulness of cDNA microarray techniques to study sequential changes of distinctive gene-expression patterns in the brain as a function of the progression of AD dementia. The study suggests new means to dissect and classify stages of AD dementia, or neuropathology, at the molecular level.

Alzheimer Disease↗
PubMed2001

Gene expression profiling of the tau mutant (P301L) transgenic mouse brain.

To provide a global analysis of the influence of Tau neuropathology at molecular level, we used cDNA arrays representing 8832 genes to determine the mRNA expression profile in transgenic mice expressing the most common frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) Tau mutation (P301L) (Nat. Genet. (2000) 402). Genes whose expression is associated with development of neurofibrillary tangles and neuron loss in P301L mice with motor and behavioral deficits were identified. The data suggest that a major mechanism underlying P301LTau neurodegeneration primarily involved altered expression of genes contributing to inhibition of apoptosis and intracellular transport. We propose that the expression of mutated P301L may lead to select altered expression of genes which may cause neurodegeneration in FTDP-17.

Animals↗
PubMed2001

Altered expression of a-type but not b-type synapsin isoform in the brain of patients at high risk for Alzheimer's disease assessed by DNA microarray technique.

Using a cDNA microarray representing 6794 distinct human genes, we identified candidate genes whose expression is altered in cerebral cortex of cases of early Alzheimer's disease (AD); among these was the synaptic vesicle protein synapsin II, which plays an important role in neurotransmitter release. While other candidate genes are presently under investigation in our lab, in this study we discuss the regulation of synapsin gene expression during the transition from normal cognitive function to early AD. We found a selective decrease in the expression of the synapsin splice variants I-III of the a-type isoform in the entorhinal (EC, BM36) but not visual cortex (VC, BM17) of cases characterized by the earliest clinically detectable stage of AD. In contrast, we found no changes in synapsin splice variant II of the b-type isoform. Alteration of synapsin expression at the earliest clinical stage of AD may suggest novel strategies for improved treatment.

Aged↗
PubMed2001

Neuronal cyclooxygenase 2 expression in the hippocampal formation as a function of the clinical progression of Alzheimer disease.

BACKGROUND: Prior studies have shown that cyclooxygenase 2 (COX-2), an enzyme involved in inflammatory mechanisms and neuronal activities, is up-regulated in the brain with Alzheimer disease (AD) and may represent a therapeutic target for anti-inflammatory treatments. OBJECTIVE: To explore COX-2 expression in the brain as a function of clinical progression of early AD. DESIGN AND MAIN OUTCOME MEASURES: Using semiquantitative immunocytochemistry, we analyzed COX-2 protein content in the hippocampal formation in 54 postmortem brain specimens from patients with normal or impaired cognitive status. SETTING AND PATIENTS: Postmortem study of nursing home residents. RESULTS: The immunointensity of COX-2 signal in the CA3 and CA2 but not CA1 subdivisions of the pyramidal layers of the hippocampal formation of the AD brain increased as the disease progressed from questionable to mild clinical dementia as assessed by Clinical Dementia Rating. COX-2 signal was increased in all 3 regions examined among cases characterized by severe dementia. CONCLUSION: Neuronal COX-2 content in subsets of hippocampal pyramidal neurons may be an indicator of progression of dementia in early AD.

Aged↗
PubMed2001

Complement anaphylatoxin C5a neuroprotects through mitogen-activated protein kinase-dependent inhibition of caspase 3.

We previously reported that pretreatment of murine cortico-hippocampal neuronal cultures with the complement-derived anaphylatoxin C5a, protects against glutamate neurotoxicity. In this study we explored the potential mechanisms involved in C5a-mediated neuroprotection. We found that C5a neuroprotects in vitro through inhibition of apoptotic death because pretreatment with human recombinant (hr)C5a prevented nuclear DNA fragmentation coincidental to inhibition of the pro-apoptotic caspase 3 activity mediated by glutamate treatment. Also, hrC5a-mediated responses appeared to be receptor-mediated because pretreatment of cultures with the specific C5a receptor antagonist C177, prevented hrC5a-mediated neuroprotection. Based on this evidence, we further explored possible signaling pathways involved in hrC5a inhibition of caspase 3 activation and apoptotic neuronal death. We found that treatment of cultures with the mitogen-activated protein kinase (MAPK) pathway inhibitor PD98059 prevented hrC5a-mediated inhibition of caspase 3 and apoptotic neuron death. MAPK pathways, whose activation by hrC5a is inhibited by PD98059 and C177, include the extracellular signal-regulated kinase (ERK)2 and, to a lesser extent, ERK1. The study suggests that C5a may protect against glutamate-induced apoptosis in neurons through MAPK-mediated regulation of caspase cascades.

Animals↗
PubMed2001

Non-steroidal anti-inflammatory drugs protect against chondrocyte apoptotic death.

Recent evidence suggests that the degradation of cartilage in osteoarthritis is characterized by chondrocyte apoptosis, but little is known about the molecular mechanisms involved or potential protective measures. In the present study, we used an immortalized chondrocyte cell line to explore the mechanisms of apoptotic chondrocyte cell death. We found that staurosporine-mediated chondrocyte death depended on the concentration and time of incubation, and coincided with increased Bax:Bcl-X mRNA expression, cytochrome C release, and activation of caspase-3. Pre-treatment of the cultures with nimesulide, a preferential cyclooxygenase (COX)-2 inhibitor, or with ibuprofen, a non-selective COX-1/COX-2 inhibitor, protected the chondrocytes against the staurosporine-mediated nuclear damage and cell death in a concentration-dependent manner (10(-12) to 10(-6) M). Cell protection coincided with inhibition of the staurosporine-mediated induction of caspase-3 activation. Notably, the selective COX-2 inhibitor NS-398 (10(-6) M, 24 hr pre-treatment) did not protect the cells against staurosporine-mediated apoptotic death. The data suggest that nimesulide and ibuprofen, in addition to their anti-inflammatory and analgesic benefits, may also have a protective effect in osteoarthritis through the inhibition of apoptosis in chondrocytes.

Animals↗
PubMed2001

From cDNA microarrays to high-throughput proteomics. Implications in the search for preventive initiatives to slow the clinical progression of Alzheimer's disease dementia.

Alzheimer's disease (AD) is the most common form of dementia, affecting as many as four million elderly people. lt results from abnormal changes in the brain that most likely begin long before cognitive impairment and other clinical symptoms become apparent. Little is known about the changes preceding or accompanying initiation of the disease. Using cDNA microarray, we previously reported candidate gene products whose expression is altered in the cerebral cortex of cases at risk for AD dementia. However, it is possible that the cDNA microarray evidence might have underestimated post-transcriptional modifications, and as a result, provided only a partial view of the biological problem of interest. Based on this hypothesis, we initiated a series of parallel high-throughput proteomic studies. We found that, consistent with the cDNA microarray evidence, the expression of proteins involved in synaptic activities was also altered in the brains of early AD cases. These studies support the feasibility and usefulness of high-throughput cDNA and protein microarray techniques to examine the sequential changes of distinctive gene expression patterns in the brain as a function of the progression of AD dementia. Our preliminary results also support the utility of high-throughput proteomic methodologies as a means to identify novel AD biomarkers from cerebral spinal fluid and/or from serum.

Alzheimer Disease↗
PubMed2000

The role of complement anaphylatoxin C5a in neurodegeneration: implications in Alzheimer's disease.

There is evidence that the complement system, a major component of inflammatory responses, may play an important role in neurodegenerative conditions such as Alzheimer's disease (AD). Work from our lab demonstrated that mice genetically deficient in the complement component C5 are more susceptible to hippocampal excitotoxic lesions (Pasinetti et al., 1996) and that the C5-derived ana;hylatoxin C5a may protect against excitotoxicity in vitro and in vivo (Osaka et al., 1999). Potential mechanisms identified in C5a-mediated neuroprotection include activation of mitogen activated protein (MAP)-kinase (Osaka et al., 1998; Osaka et al., 1999). This novel neuroprotective role of C5a complicates current theories that complement proteins augment beta-amyloid (Abeta) toxicity in AD. In view of the fact that the complement system represents a target for therapeutic interventions in AD, further characterization of the complex role of complement proteins is essential. Towards this aim, we have characterized a transgenic C5a receptor (C5aR) knockout (KO) mouse. Recent studies in the lab using C5aR-KO mice show that disruption of C5aR alters calcium calmodulin kinase (CaM-KII) signal transduction in brain cells. We are presently using C5aR-KO mice to study the role of C5a in caspase mediated apoptotic neuronal death. In this review we will attempt to delineate possible neuroprotective roles for C5a in mechanisms of neurotoxicity pertaining to AD.

Alzheimer Disease↗
PubMed2000

Cytokine gene expression as a function of the clinical progression of Alzheimer disease dementia.

BACKGROUND: Inflammatory cytokines have been linked to Alzheimer disease (AD) neurodegeneration, but little is known about the temporal control of their expression in relationship to clinical measurements of AD dementia progression. DESIGN AND MAIN OUTCOME MEASURES: We measured inflammatory cytokine messenger RNA (mRNA) expression in postmortem brain specimens of elderly subjects at different clinical stages of dementia and neuropathological dysfunction. SETTING AND PATIENTS: Postmortem study of nursing home patients. RESULTS: In brains of cognitively normal control subjects, higher interleukin 6 (IL-6) and transforming growth factor beta1 (TGF-beta1) mRNA expression was observed in the entorhinal cortex and superior temporal gyrus compared with the occipital cortex. Compared with age-matched controls, subjects with severe/terminal dementia, but not subjects at earlier disease stages, had higher IL-6 and TGF-beta1 mRNA expression in the entorhinal cortex (P<.01) and superior temporal gyrus (P<.01). When stratified by the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) neuropathological criteria, IL-6 mRNA expression in both the entorhinal cortex (P<.05) and superior temporal gyrus (P<.01) correlated with the level of neurofibrillary tangles but not neuritic plaques. However, in the entorhinal cortex, TGF-beta1 mRNA did not correlate with the level of either neurofibrillary tangles or neuritic plaques. Interestingly, in the superior temporal gyrus, TGF-beta1 mRNA expression negatively correlated with neurofibrillary tangles (P<.01) and showed no relationship to the pathological features of neuritic plaques. CONCLUSIONS: The data are consistent with the hypothesis that cytokine expression may differentially contribute to the vulnerability of independent cortical regions during the clinical progression of AD and suggest that an inflammatory cytokine response to the pathological effects of AD does not occur until the late stages of the disease. These findings have implications for the design of anti-inflammatory treatment strategies. Arch Neurol. 2000;57:1153-1160

Aged↗
PubMed1999

Regional distribution of cyclooxygenase-2 in the hippocampal formation in Alzheimer's disease.

Cyclooxygenase-2 (COX-2), a key enzyme in prostanoid biosynthesis, may represent an important therapeutic target in Alzheimer's disease (AD). In the present study, we explored the regulation of COX-2 in the hippocampal formation in sporadic AD. Using semiquantitative immunocytochemical techniques, we found that in AD cases (vs. age-matched controls) neurons of the CA1-CA4 subdivisions of the hippocampal pyramidal layer showed elevation of COX-2 signal; COX-2 levels correlated with amyloid plaque density. In contrast, the level of COX-2 immunostaining in the dentate gyrus granule neurons was not elevated in AD. No expression of COX-2 in cells with glial morphology was found in any case examined. In parallel, in vitro studies found that neurons derived from transgenic mice with neuronal overexpression of COX-2 are more susceptible to beta-amyloid (Abeta) toxicity, with potentiation of redox impairment. The results indicate elevated expression of neuronal COX-2 in subregions of the hippocampal formation in AD and that such elevation may potentiate Abeta-mediated oxidative stress.

Adult↗
PubMed1999

Complement-derived anaphylatoxin C5a protects against glutamate-mediated neurotoxicity.

Previous work from this laboratory indicates a role for the complement component C5 in neuroprotection against excitotoxicity. In the present study, we tested the hypothesis that the C5-derived anaphylatoxin C5a protects against kainic acid (KA)-induced neurodegeneration and investigated the mechanism of C5a neuronal activity in vitro. Brain intraventricular infusion of KA into adult mice caused neuronal morphological features of apoptosis in the pyramidal layer of the hippocampal formation as indicated by counts of neurons with pyknotic/condensed nuclei associated with cytoplasmic eosinophilia. Co-intraventricular infusion of human recombinant C5a with KA resulted in a marked reduction of morphological features of apoptotic neuronal death. In vitro studies confirmed C5a neuroprotection: treatment of primary murine corticohippocampal neurons with human or mouse recombinant C5a reduced glutamate neurotoxicity, as measured by trypan blue exclusion assay. This protection concurred with inhibition of glutamate-mediated induction of the caspase-3-related cysteine protease and coincided with marked reduction of neurons with morphological features of apoptosis, as found in vivo. Our studies indicate that C5a may inhibit glutamate-mediated neuronal death through partial inhibition of caspase-3 activity. These findings suggest a novel noninflammatory role for C5a in modulating neuronal responses to excitotoxins.

Alzheimer Disease↗
PubMed1999

Glial gene expression during aging in rat striatum and in long-term responses to 6-OHDA lesions.

The male rat striatum was examined for age-related changes in mRNAs expressed in astrocytes and microglia in two rat genotypes that differ by 35% in mean and maximum life spans: F344 and the longer-lived F1 (BN x F344) hybrid. The findings extend the established age-related increases in GFAP (glial fibrillary acidic protein) to other glial mRNAs: two lipoprotein mRNAs that are predominantly expressed in striatal astrocytes, apoE (apolipoprotein E) and apoJ (apolipoprotein J, clusterin, CLI, or SGP-2), and two mRNAs expressed in striatal microglia, TGF-beta1 and complement C1qB. By Northern blot hybridization, both genotypes showed progressive increases of GFAP mRNA to > 2.5-fold by the lifespan. Although the rat strains differed 35% in life span, the slope of the GFAP mRNA regression on age did not differ. Relative to GFAP, the increases of apoE, apoJ, C1q, and TGF-beta1 mRNAs were smaller, < or = 1.5-fold. Because prior studies showed that acute damage to striatal afferents induced astrocyte gene expression increases resembling those that also occur during aging, we examined long-term effects of damage to substantia nigra neurons on striatal astrocyte changes during aging. Young F344 rats were given 6-OHDA lesions that cause striatal dopamine deficits and induce GFAP. When examined 15 months later at age 18 months, there was no effect during prior lesions on the age-related elevation of GFAP mRNA. We conclude that aging changes in striatal GFAP mRNAs do not interact with loss of dopaminergic output to the striatum from 6-OHDA lesions and may be independent of the relatively modest dopaminergic losses during normal aging.

Aging↗
PubMed1999

Glycoprotein 330/megalin (LRP-2) has low prevalence as mRNA and protein in brain microvessels and choroid plexus.

Prior studies indicated that glycoprotein 330 (gp330)/megalin mediates transcytosis of apolipoprotein J (apoJ) with Alzheimer's amyloide-peptide (Abeta) across the vascular membranes of the central nervous system (CNS). Here we show the presence of gp330/megalin mRNA and gp330-like immunoepitopes in brain capillaries and choroid plexus and their absence from brain parenchyma. By polymerase chain reaction (PCR) we estimated 1.2 x 10(5) molecules (1 pg) of gp330/megalin mRNA/microg total brain capillary RNA, which is 3% of that in kidney RNA. However, gp330 mRNA was not detected by in situ hybridization in vascular CNS tissue, presumably because of low transcript prevalence. The ratio of gp330 protein:RNA was 17-fold higher in choroid plexus vs brain capillaries, which implies tissue specific regulation of the protein and mRNA prevalence. We conclude that gp330/megalin mRNA and protein are expressed in brain capillaries and choroid plexus in small amounts that are consistent with the observed activities of this endocytosing receptor in the regulation of apoJ and Abeta uptake by the CNS.

Animals↗
PubMed1999

Protein kinase C anchoring deficit in postmortem brains of Alzheimer's disease patients.

Protein kinase C (PKC) has been implicated in the pathophysiology of Alzheimer's disease (AD). The levels of particular isoforms and the activation of PKC are reduced in postmortem brain cortex of AD subjects. Receptors for activated C kinase (RACK) are a family of proteins involved in anchoring activated PKCs to relevant subcellular compartments. Recent evidence has indicated that the impaired activation (translocation) of PKC in the aging brain is associated with a deficit in RACK1, the most well-characterized member of this family. The present study was conducted to determine whether alterations in RACK1 occurred in cortical areas where an impaired translocation of PKC has been demonstrated in AD. Here we report the presence of RACK1 immunoreactivity in human brain frontal cortex for the first time and demonstrate a decrease in RACK1 content in cytosol and membrane extracts in AD when compared with non-AD controls. By comparison, the levels of the RACK1-related PKCbetaII were not modified in the same membrane extracts. These observations add a new perspective in understanding the disease-associated defective PKC signal transduction and indicate that a decrease in an anchoring protein for PKC is an additional determinant of this deficit.

Aged↗
PubMed1999

Potentiation of excitotoxicity in transgenic mice overexpressing neuronal cyclooxygenase-2.

In this study we describe the generation of a transgenic mouse model with neuronal overexpression of the human cyclooxygenase-2, h(COX)-2, to explore its role in excitotoxicity. We report that overexpression of neuronal hCOX-2 potentiates the intensity and lethality of kainic acid excitotoxicity in coincidence with potentiation of expression of the immediate early genes c-fos and zif-268. In vitro studies extended the in vivo findings and revealed that glutamate excitotoxicity is potentiated in primary cortico-hippocampal neurons derived from hCOX-2 transgenic mice, possibly through potentiation of mitochondrial impairment. This study is the first to demonstrate a cause-effect relationship between neuronal COX-2 expression and excitotoxicity. This model system will allow the systematic examination of the role of COX-2 in mechanisms of neurodegeneration that involve excitatory amino acid pathways.

Animals↗
PubMed1999

Expression of C5a receptor in mouse brain: role in signal transduction and neurodegeneration.

In this study we explored the potential role of the complement derived anaphylatoxin C5a and the expression of its receptor in mouse brain. Using in situ hybridization, we found that C5a receptor messenger RNA is expressed in mouse brain. In response to intraventricular kainic acid injection, there was marked increase in the C5a receptor messenger RNA expression, particularly in hippocampal formation and cerebral cortex. C5a ligand-binding autoradiography confirmed the functional expression and elevation of the C5a receptor post-lesioning. The expression of C5a receptor messenger RNA in brain was confirmed by northern blot hybridization of total RNA from neuronal and glial cells in vitro. Based on these findings we explored the role of C5a in mechanisms of signal transduction in brain cells. Treatment of primary cultures of mouse astrocytes with human recombinant C5a resulted in the activation of mitogen-activated extracellular signal-regulated protein kinase. This response appeared to be mediated by the C5a receptor since astrocyte cultures derived from C5a receptor knockout mice were not responsive to the treatment. Understanding the regulation of C5a receptor in brain and mechanisms by which pro-inflammatory C5a modulates specific signal transduction pathways in brain cells is crucial to studies of inflammatory mechanisms in neurodegeneration.

Animals↗