Expression of cyclin-dependent kinase 5 and its activator p35 in models of induced apoptotic death in neurons of the substantia nigra in vivo.

Cyclin-dependent kinase 5 is predominantly expressed in postmitotic neurons and plays a role in neurite elongation during development. It has also been postulated to play a role in apoptosis in a variety of cells, including neurons, but little is known about the generality and functional significance of cdk5 expression in neuronal apoptosis in living brain. We have therefore examined its expression and that of its known activators, p35, p39 and p67, in models of induced apoptosis in neurons of the substantia nigra. We find that cdk5 is expressed in apoptotic profiles following intrastriatal injection of 6-hydroxydopamine and axotomy. It is expressed exclusively in profiles which are in late morphologic stages of apoptosis. In these late stages, derivation of the profiles from neurons, and localization of expression to the nucleus, can be demonstrated by co-labeling with a neuron-specific nuclear marker, NeuN. In another model of induced apoptotic death in nigra, produced by developmental striatal lesion, kinase activity increases in parallel with cell death. While mRNAs for all three cdk5 activators are expressed in nigra during development, only p35 protein is expressed in apoptotic profiles. We conclude that cdk5/p35 expression is a general feature of apoptotic neuron death in substantia nigra neurons in vivo.

Upregulation of cytosolic branched chain aminotransferase in substantia nigra following developmental striatal target injury.

We have previously shown that apoptotic cell death can be induced in substantia nigra (SN) by developmental striatal target lesion. In this model, only a portion of nigral neurons dies, so it provides a paradigm to examine not only the molecular basis of cell death, but also the cellular responses of adjacent neurons which survive. Using a differential display approach, we have found that cytosolic branched chain aminotransferase (BCATc) mRNA is upregulated in SN in this model. This upregulation is associated with an increased number of BCATc-positive neuronal profiles, demonstrated by immunostaining. BCATc-positive neurons show normal morphology and rarely contain apoptotic chromatin. We conclude that BCATc is upregulated in neurons, which are likely to survive, and plays a role in either maintenance of viability or restoration of normal function.

Alpha-synuclein up-regulation in substantia nigra dopaminergic neurons following administration of the parkinsonian toxin MPTP.

Mutations in alpha-synuclein cause a form of familial Parkinson's disease (PD), and wild-type alpha-synuclein is a major component of the intraneuronal inclusions called Lewy bodies, a pathological hallmark of PD. These observations suggest a pathogenic role for alpha-synuclein in PD. Thus far, however, little is known about the importance of alpha-synuclein in the nigral dopaminergic pathway in either normal or pathological situations. Herein, we studied this question by assessing the expression of synuclein-1, the rodent homologue of human alpha-synuclein, in both normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. In normal mice, detectable levels of synuclein mRNA and protein were seen in all brain regions studied and especially in ventral midbrain. In the latter, there was a dense synuclein-positive nerve fiber network, which predominated over the substantia nigra, and only few scattered synuclein-positive neurons. After a regimen of MPTP that kills dopaminergic neurons by apoptosis, synuclein mRNA and protein levels were increased significantly in midbrain extracts; the time course of these changes paralleled that of MPTP-induced dopaminergic neurodegeneration. In these MPTP-injected mice, there was also a dramatic increase in the number of synuclein-immunoreactive neurons exclusively in the substantia nigra pars compacta; all synuclein-positive neurons were tyrosine hydroxylase-positive, but none coexpressed apoptotic features. These data indicate that synuclein is highly expressed in the nigrostriatal pathway of normal mice and that it is up-regulated following MPTP-induced injury. In light of the synuclein alterations, it can be suggested that, by targeting this protein, one may modulate MPTP neurotoxicity and, consequently, open new therapeutic avenues for PD.

Increased expression of rat synuclein in the substantia nigra pars compacta identified by mRNA differential display in a model of developmental target injury.

Human alpha-synuclein was identified on the basis of proteolytic fragments derived from senile plaques of Alzheimer's disease, and it is the locus of mutations in some familial forms of Parkinson's disease. Its normal function and whether it may play a direct role in neural degeneration remain unknown. To explore cellular responses to neural degeneration in the dopamine neurons of the substantia nigra, we have developed a rodent model of apoptotic death induced by developmental injury to their target, the striatum. We find by mRNA differential display that synuclein is up-regulated in this model, and thus it provides an opportunity to examine directly whether synuclein plays a role in the death of these neurons or, alternatively, in compensatory responses. Up-regulation of mRNA is associated with an increase in the number of neuronal profiles immunostained for synuclein protein. At a cellular level, synuclein is almost exclusively expressed in normal neurons, rather than apoptotic profiles. Synuclein is up-regulated throughout normal postnatal development of substantia nigra neurons, but it is not further up-regulated during periods of natural cell death. We conclude that up-regulation of synuclein in the target injury model is unlikely to mediate apoptotic death and propose that it may be due to a compensatory response in neurons destined to survive.

Alpha-synuclein expression in substantia nigra and cortex in Parkinson's disease.

Mutations in the human alpha-synuclein gene have been identified in several families of European descent with early-onset Parkinson's disease (PD). We sequenced the complete alpha-synuclein cDNA from substantia nigra and cortex from nine patients with PD and eight control subjects. No mutations were found. We then analyzed alpha-synuclein mRNA levels using a ribonuclease protection assay. Two major protected bands of alpha-synuclein mRNA, possibly representing two splice variants of the gene, were observed. Alpha-synuclein mRNA was significantly diminished in the substantia nigra of patients with PD compared with control subjects but not in the cortex. Our findings suggest that decreased synuclein mRNA may be an early alteration in the SN in PD, and imply that decreased levels of the protein may play a role in the pathogenesis of sporadic cases of the disease.

Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17.

Thirteen families have been described with an autosomal dominantly inherited dementia named frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), historically termed Pick's disease. Most FTDP-17 cases show neuronal and/or glial inclusions that stain positively with antibodies raised against the microtubule-associated protein Tau, although the Tau pathology varies considerably in both its quantity (or severity) and characteristics. Previous studies have mapped the FTDP-17 locus to a 2-centimorgan region on chromosome 17q21.11; the tau gene also lies within this region. We have now sequenced tau in FTDP-17 families and identified three missense mutations (G272V, P301L and R406W) and three mutations in the 5' splice site of exon 10. The splice-site mutations all destabilize a potential stem-loop structure which is probably involved in regulating the alternative splicing of exon10. This causes more frequent usage of the 5' splice site and an increased proportion of tau transcripts that include exon 10. The increase in exon 10+ messenger RNA will increase the proportion of Tau containing four microtubule-binding repeats, which is consistent with the neuropathology described in several families with FTDP-17.

Stroke associated with cardiac surgery. Determinants, timing, and stroke subtypes.

OBJECTIVE: To determine the nature of strokes complicating cardiac surgery. DESIGN: A medical record review was undertaken of all patients who underwent cardiac surgery (either coronary bypass, valvular surgery, or both) between January 1990 and July 1995. Univariate and multivariate analyses were done using odds ratios (ORs) and logistic regression. RESULTS: A total of 2211 patients underwent cardiac surgery. A total of 44 patients (2%) had postoperative strokes. They were compared with 104 surgical controls. Univariate analysis revealed that the patients with stroke were significantly older and had significantly higher rates of transient ischemic attack, congestive heart failure, and peripheral vascular disease by history. A multivariate logistic regression revealed the significant variables associated with stroke were congestive heart failure (OR, 6.8) and transient ischemic attack (OR, 1.2). Analyses of surgical variables revealed that bypass pump time of more than 120 minutes (OR, 1.40) was the only significant predictor. The majority of strokes (61%) had occurred by postoperative day 2, but 39% occurred between postoperative days 3 to 9. Hemispheric syndromes occurred in 70%, brain stem and cerebellar syndromes in 14%, and lacunar syndromes in 16%. Computed tomographic scans revealed that 29 patients had new infarcts, of which 20 (69%) were territorial, 5 (17%) were small deep, and 4 (14%) were border zone. Hemorrhagic infarction was found in 2 cases (5%). One patient (2.5%) had a cardiorespiratory arrest while undergoing computed tomography. Discharge disposition was good in 88% of patients with stroke (12% with poor outcomes) vs 97% of controls with good outcomes (3% with poor outcomes) (P = .04). CONCLUSIONS: Strokes may be delayed following cardiac surgery perhaps because of ongoing risk of embolism or a hypercoagulable state. A surprisingly high proportion of patients have lacunar syndromes or small-deep infarcts shown on computed tomography. Early computed tomographic imaging may be useful to exclude hemorrhage if anticoagulation is considered, but is not without risk in these potentially unstable patients. Greater understanding of risk factors for stroke, timing, and subtypes may ultimately allow identification of patients at particularly high risk for perioperative stroke.

Chromosome 12-linked autosomal dominant scapuloperoneal muscular dystrophy.

Scapuloperoneal syndromes are characterized by their distribution of muscle weakness and wasting. The reported pattern of inheritance has been variable. Both neurogenic and myopathic forms of autosomally dominantly inherited scapuloperoneal syndrome have been described. It has been suggested that these are variants of other neuromuscular diseases. We examined 44 members from a family with 14 members affected with a scapuloperoneal syndrome. Physiological and histological analysis implied that this condition is predominantly myopathic. Linkage analysis was done to confirm the genetic etiology of the disease in this family and to evaluate the possibility that it is a allelic variant of other neuromuscular diseases. Genetic analysis demonstrated linkage of the disease to chromosome 12, which makes it genetically distinct from other loci known to cause neuromuscular disease. Muscle fibers with hyaline desmin-containing cytoplasmic inclusions in combination with focal myopathic changes may be a disease-specific morphological marker of the disease.

A high-resolution genetic linkage map of the pericentromeric region of the human X chromosome.

We generated a high-resolution genetic linkage map of the pericentromeric region of the human X chromosome from approximately Xp11.4 to Xq22. This map contains 41 loci defined by 50 marker systems genotyped in the CEPH families. For this study we have generated 3 new markers (DXS1689, DXS1690, and DXS159) and 2 new primer sequences for previously described markers (PGK1P1 and AR). Using two different mapping algorithms based on genotype data alone, we developed two well-supported framework maps containing 15 and 18 markers with average interval sizes of 2.7 and 1.7 cM. The 18 marker map is [DXS426, PFC]-2.0-DXS255-1.2-DXS991-1.6-AR-1.3-DXS153-2.0-DX S106-1.5-DXS132-0.6- DXS1690-0.8-DXS453-1.4-DXS559-1.5-[PGK1, DXS56]-0.6-DXS1002-1.8-DXYS1X-2.0-DXS3-7.5-DX S458-2.8-DXS454, where the distance between adjacent loci is in centimorgans. As a third approach, we used physical mapping data to define bins for markers; this approach permitted us to place 26 markers on our framework map. Finally, we constructed a map based on the physical order of 35 markers from the fifth international workshop on human X chromosome mapping. A comparison of the physical and genetic maps indicates a relationship of 2 cM per megabase in this region, with two regions of reduced recombination. The first is around the centromere (DXZ1), and the second is in the region around PGK1 (DXS441 to DXS995). Our maps should aid in the fine-mapping of the many disease loci that localize to this region of the X chromosome.

Production of the STA2-encoded glucoamylase in Saccharomyces cerevisiae is subject to feed-back control.

Three modes of production of the extracellular glucoamylase (GA) in Saccharomyces cerevisiae have been identified; repressed, basal and induced. The repressed mode is found with cells grown in rich media containing non-limiting concentrations of monosaccharides or disaccharides, including GA-hydrolysable maltose, as a sole carbon source. Both the basal and the induced modes (spanned by some seven-fold differences in the rate of GA production) can be displayed by either glucose-limited or glycerol-plus ethanol-consuming cultures; the induced mode is switched over to the basal one due to a feed-back inhibition by extracellularly accumulated GA. It is proposed that the feed-back control involved in GA production can be attenuated by starch which can thus 'induce' higher rates of GA production compared to the basal mode.