Solid-organ transplantation in older adults: current status and future research.

An increasing number of patients older than 65 years are referred for and have access to organ transplantation, and an increasing number of older adults are donating organs. Although short-term outcomes are similar in older versus younger transplant recipients, older donor or recipient age is associated with inferior long-term outcomes. However, age is often a proxy for other factors that might predict poor outcomes more strongly and better identify patients at risk for adverse events. Approaches to transplantation in older adults vary across programs, but despite recent gains in access and the increased use of marginal organs, older patients remain less likely than other groups to receive a transplant, and those who do are highly selected. Moreover, few studies have addressed geriatric issues in transplant patient selection or management, or the implications on health span and disability when patients age to late life with a transplanted organ. This paper summarizes a recent trans-disciplinary workshop held by ASP, in collaboration with NHLBI, NIA, NIAID, NIDDK and AGS, to address issues related to kidney, liver, lung, or heart transplantation in older adults and to propose a research agenda in these areas.

Multiple caspases are involved in beta-amyloid-induced neuronal apoptosis.

beta-amyloid peptide (Abeta) has been implicated in the pathogenesis of Alzheimer disease and has been reported to induce apoptotic death in cell culture. Cysteine proteases, a family of enzymes known as caspases, mediate cell death in many models of apoptosis. Multiple caspases have been implicated in Abeta toxicity; these reports are conflicting. We show that treatment of cerebellar granule cells (CGC) with Abeta25-35 causes apoptosis associated with increased activity of caspases-2, -3 and -6. Selective inhibition of each of these three caspases provides significant protection against Abeta-mediated apoptosis. In contrast, no change in caspase-1 activity was seen after Abeta25-35 application, nor was inhibition of caspase-1 neuroprotective. Similar to CGC, cortical neuronal cultures treated with Abeta25-35 demonstrate increased caspase-3 activity but not caspase-1 activity. Furthermore, significant neuroprotection is elicited by selective inhibition of caspase-3 in cortical neurons administered Abeta25-35, whereas selective caspase-1 inhibition has no effect. Taken together, these findings indicate that multiple executioner caspases may be involved in neuronal apoptosis induced by Abeta.

Caspase pathways, neuronal apoptosis, and CNS injury.

Caspases are a family of mammalian proteases related to the ced-3 gene of Caenorhabditis elegans. They mediate many of the morphological and biochemical features of apoptosis, including structural dismantling of cell bodies and nuclei, fragmentation of genomic DNA, destruction of regulatory proteins, and propagation of other pro-apoptotic molecules. Based on their substrate specificities and DNA sequence homologies, the 14 currently identified caspases may be divided into three groups: apoptotic initiators, apoptotic executioners, and inflammatory mediators. Caspases are activated through two principal pathways, known as the "extrinsic pathway," which is initiated by cell surface death receptor ligation, and the intrinsic pathway, which arises from mitochondria. Endogenous inhibitors, such as the inhibitors of apoptosis (IAP) family, modulate caspase activity at various points within these pathways. Upon activation, caspases appear to play an important role in sequelae of traumatic brain injury, spinal cord injury, and cerebral ischemia. In addition, they may also play a role in mediating cell death in chronic neurodegenerative conditions such as Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis. This article reviews the current literature on the role of caspases in acute and chronic CNS injury, and provides evidence for the potential therapeutic use of caspase inhibitors in the setting of these conditions.

Ribozyme-mediated inhibition of caspase-3 protects cerebellar granule cells from apoptosis induced by serum-potassium deprivation.

Apoptosis is an important mechanism of physiological and pathological cell death. It is regulated by several gene products, including caspases and the bcl-2-like proteins, whose roles have been demonstrated in numerous systems. One of these is a model of cerebellar granule cells (CGCs) in which apoptosis is induced by acute removal of serum and depolarizing concentrations of potassium. Previous work by several authors showed that benzyloxycarbonyl-DEVD-fluoromethylketone, a somewhat selective caspase inhibitor, significantly protected CGCs from apoptosis; however, because this molecule targets multiple caspases, it is not known whether a single caspase is primarily responsible for effecting cell death in this model. We attempted to answer this question by cotransfecting CGCs with green fluorescent protein reporter and a hammerhead ribozyme directed against caspase-3 mRNA. Maximal protection by this ribozyme was observed after 24 hr of deprivation, at which time apoptosis was 18 +/- 0.7% compared with 32 +/- 2% in control cells. Significant protection was also observed with human inhibitor of apoptosis (IAP)-like protein-X-linked IAP, a specific inhibitor of caspase-3, -7, and -9, and with p35, a general caspase inhibitor. Overexpression of bcl-2 produced almost complete protection from apoptosis after 24 hr of serum-K(+) deprivation (5 +/- 2 vs 44 +/- 2% in control cells). These results confirm that caspases play an important role in CGC apoptosis and indicate that caspase-3 itself is a significant mediator of this process.

Beta-amyloid-induced apoptosis of cerebellar granule cells and cortical neurons: exacerbation by selective inhibition of group I metabotropic glutamate receptors.

Administration of beta-amyloid fragment 25-35 (Abeta25-35) to cultured rat cerebellar granule cells (CGC) or cortical neurons caused cell death that was characterized by morphological and nuclear changes consistent with apoptosis. Inhibition of NMDA receptors produced a mild exacerbation of Abeta25-35 toxicity in cortical neurons; a similar effect was induced by AMPA/kainate receptor inhibition in CGC. Selective activation of group I metabotropic glutamate receptors (mGluR) by dihyroxyphenylglycine (DHPG) had no effect on Abeta25-35-induced apoptosis in either cell type, and was unaffected by blockade of ionotropic glutamate receptors. In contrast, selective inhibition of group I mGluR by (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA) exacerbated Abeta toxicity in cortical neurons, whereas this treatment was without effect on CGC. However, AIDA significantly increased Abeta-induced apoptosis in CGC in the presence of either NMDA or AMPA/kainate receptor inhibition; blockade of both ionotropic glutamate receptor classes further increased the exacerbation of apoptosis following treatment with AIDA. These findings suggest that Abeta25-35-induced neuronal injury leads to activation of group I mGluR, which attenuates the resulting apoptosis.

The role of CED-3-related cysteine proteases in apoptosis of cerebellar granule cells.

The CED-3-related cysteine proteases (CRCPs) have been implicated as mediators of apoptosis, primarily in hematogenous cell systems, but their role in neuronal apoptosis remains unclear. The present study examined the role of two CRCP families-CPP32- and interleukin-1beta converting enzyme (ICE)-like cysteine proteases-in apoptosis of cerebellar granule cells (CGCs) caused by withdrawal of serum and/or potassium (K+). Serum deprivation potentiated apoptosis caused by K+ withdrawal, reducing cell viability by approximately one half of control values after 12 hr as measured by calcein fluorescence. Cell death after serum/K+ deprivation was significantly attenuated by the CPP32-like inhibitor z-DEVD-fmk; however, the ICE-like inhibitor z-YVAD-fmk had only slightly protective effects at the highest concentration used. Both inhibitors reduced CPP32-like activity directly in an in vitro fluorometric assay system, although z-DEVD-fmk showed much greater potency. K+ and serum/K+ deprivation each were accompanied by increased CPP32-like activity; however, ICE-like activity was absent after 12 hr of serum and/or K+ deprivation. CPP32 mRNA levels were unchanged after K+ deprivation but increased after serum and combined serum/K+ withdrawal as measured by reverse transcription-PCR (RT-PCR), with peak values at 4 hr reaching 210 +/- 37% and 269 +/- 42% of control levels, respectively. In contrast, ICE mRNA was undetectable by RT-PCR. These results are consistent with the hypothesis that CPP32-like proteases play an important role in apoptosis of CGCs caused by deprivation of K+ or serum/K+.

A new approach for the electrophoretic detection of apoptosis.

Apoptotic cell death is often characterized by internucleosomal cleavage of genomic DNA, which exhibits a distinctive ladder upon electrophoresis. However, techniques used for the isolation and detection of DNA to demonstrate laddering may not be sufficiently sensitive, particularly when cleaved DNA is present at modest levels. We propose a new approach for isolating total cellular DNA using a silica-based resin that improves the resolution of DNA laddering. In addition, we introduce a rapid DNA labeling method that can increase the sensitivity of detecting DNA laddering. Each of these methods can be used for DNA from cell cultures or tissues.

Search for viral nucleic acid sequences in brain tissues of patients with schizophrenia using nested polymerase chain reaction.

BACKGROUND: We used polymerase chain reaction to search for nucleic acid sequences of several viruses in DNA and RNA extracted from brain tissues of schizophrenic and control subjects. METHODS: We extracted DNA and RNA templates from frozen brain specimens of 31 patients with schizophrenia and 23 nonschizophrenic control patients with other diseases. The extracts were subjected to polymerase chain reaction with oligonucleotide primers for 12 different viruses (cytomegalovirus, Epstein-Barr virus, herpes simplex virus type 1, human herpesvirus type 6, varicellazoster virus, measles virus, mumps virus, rubella virus, the picornavirus group, influenza A virus, human T-cell lymphotropic virus type I, and St Louis encephalitis virus), several of which have been suspected of involvement in schizophrenia. Nested primers were used to increase the sensitivity of the method. RESULTS: No amplified nucleic acid sequences encoded by the selected viral genomes were detected in extracts of any brain specimens from either schizophrenic or control patients. CONCLUSIONS: These data agree with previous studies that failed to find sequences of a number of viruses in the cerebrospinal fluid or selected areas of the brains of schizophrenic patients. Additional efforts should be undertaken to identify other known and unknown pathogens in schizophrenia, sampling more areas of the brain from subjects with a variety of clinical types of schizophrenia.

Analysis by polymerase chain reaction of alpha 1 and alpha 6 GABAA receptor subunit mRNAs in individual cerebellar neurons after whole-cell recordings.

With the use of the single-cell polymerase chain reaction (PCR), the GABAA receptor subunit mRNA content was analyzed in granule and Purkinje neurons from rat cerebellar slices. We used an experimental protocol to assess simultaneously the presence of two subunits in each cell while electrophysiological recordings were performed with the whole-cell patch-clamp technique. Based on a computer alignment of the nucleotide sequence corresponding to alpha 1 and alpha 6 GABAA receptor subunits, homologous regions were identified that allowed coamplification of both mRNAs using a single primer combination. The presence of selective restriction sites within the targeted templates allowed us to identify which receptor subunit mRNAs were coamplified by performing restriction enzyme-mediated cleavage of the amplification products. In all Purkinje neurons assayed, alpha 1 subunit mRNA but not alpha 6 mRNA was detected. In contrast, among individual granule neurons we found a heterogeneous distribution of the mRNA for the alpha 1 and alpha 6 GABAA receptor subunits. A comparison of the results of the PCR amplification and the analysis of GABA-mediated inhibitory synaptic currents does not allow us to identify kinetic characteristics of synaptic currents that clearly correlate with the presence or the absence of alpha 6 subunit mRNA.