Rajiv Joseph

Platelet Glycine, Glutamate and Aspartate in Primary Headache

Platelet levels of glutamic and aspartic acid and glycine were measured in patients with migraine with aura, migraine without aura, tension headache and cluster headache. High levels of these amino acids were found in patients with migraine with aura compared to normal subjects and other headache groups. During headache, glutamate levels further increased in migraine with aura patients. These findings may have relevance to the neurological symptoms of migraine with aura.

Amyloid β-protein fragment 25–35 causes activation of cytoplasmic calcium in neurons

The cellular mechanism by which beta-amyloid has its effect on neurons is unknown. Based on observations that endogenous neurotoxins, such as glutamate and platelet activating factor (PAF), cause activation of cytoplasmic calcium, we tested if this was true with beta-amyloid. Using nerve growth factor-treated PC12 cells, we noted that the active beta-amyloid fragment, containing residues 25 to 35, caused a specific and dose-dependent increase in intracellular calcium due to an influx of extracellular calcium.

Neuronatin mRNA : alternatively spliced forms of a novel brain-specific mammalian developmental gene

Neurogenesis begins with the closure of the neural tube around mid gestation and continues in the rat for about two weeks postnatally. Therefore, we investigated the role of neuronatin, a novel cDNA that we cloned from neonatal rat brain (Joseph et al., Biochem. Biophys. Res. Commun., 201 (1994) 1227-1234), in brain development. Further studies described in the present manuscript, lead to the identification of two alternatively spliced forms of neuronatin mRNA, alpha and beta, with the same open reading frame. Neuronatin-alpha encoded a novel protein of 81 aa, and the beta-form encoded 54 aa. Both forms were identical, except that the alpha-form had an additional 81 bp sequence inserted into the middle of the coding region. On Northern analyses, neuronatin mRNA was relatively selective for the brain. It first appeared at E11-14, a time when the neural tube has closed and neuroepithelial proliferation initiated, became pronounced at E16-19 with a surge in neurogenesis, and declined postnatally to adult levels with the completion of neurogenesis. In order to determine whether there were other forms of neuronatin mRNA, and to study the expression of the alpha and beta forms separately during development, reverse transcriptase-polymerase chain reaction was carried out using primers flanking the coding region of the alpha and beta forms. The RT-PCR results clearly indicated that there were only two forms of neuronatin. The beta-form first appeared at E11-14, whereas the alpha-form was present even earlier at E7-10. Together, these findings indicate that the two forms of neuronatin mRNA are regulated differently during brain development.(ABSTRACT TRUNCATED AT 250 WORDS)

Neuronal death, cytoplasmic calcium and internucleosomal DNA fragmentation: evidence for DNA fragments being released from cells

Neuronal death, secondary to endogenous agents such as glutamate, may involve changes in cytoplasmic calcium. Besides its well recognized role as a second messenger mediating cellular response, calcium is necessary for the activation of endonuclease(s), resulting in DNA fragmentation and cell death. Therefore, we investigated the relationship between changes in cytoplasmic calcium, DNA fragmentation and neuronal death, using PC12 and NCB-20 cell lines. The calcium ionophore, A23187, caused a dose-dependent increase in cytoplasmic calcium, loss of cell viability, increased lactate dehydrogenase (LDH)-release, and DNA fragmentation. DNA fragments, typical of internucleosomal digestion of genomic DNA, characteristic of endonuclease(s) activation, were consistently detected in the incubating medium. Release of DNA fragments into the medium was seen with A23187 in concentrations as low as 10 nM, and within an hour of treatment. Furthermore, calcium added to preparations of PC12 nuclei also produced DNA fragmentation, although, less pronounced than when intact cells were treated with A23187. The findings indicate that A23187-induced neuronal death involves the activation of endonuclease(s). The role of cytoplasmic calcium in this process is supported by evidence that A23187 selectively mobilizes cytoplasmic calcium, and that calcium can directly activate endonuclease(s) in nuclear preparations.

Whole blood platelet function in acute ischemic stroke. Importance of dense body secretion and effects of antithrombotic agents.

We studied platelet function in whole blood, a situation that better reflects the in vivo state, from 85 patients with acute ischemic stroke and from 19 healthy controls. Patients receiving no antithrombotic drugs demonstrated increased platelet dense body secretion without an associated increase in platelet aggregation, thus raising the possibility that dense body secretion may be of separate importance in cerebral infarction. Our results also suggest that dense body secretion may occur independently of aggregation. Heparin and heparin plus warfarin were ineffective in reducing the high level of dense body secretion seen in acute cerebral infarction, whereas treatment with aspirin plus dipyridamole inhibited both dense body secretion and platelet aggregation. It seems worthwhile to investigate the usefulness of antiplatelet drugs in the treatment of acute ischemic stroke wherein clinical outcome is correlated with the extent of suppression of platelet dense body secretion.

Migraine and the lupus anticoagulant. Case reports and review of the literature.

Lupus anticoagulants (LA) are antiphospholipid serum immunoglobulins generally associated with autoimmune conditions, especially systemic lupus erythematosus (SLE). They have recently been linked to thrombotic events, including stroke. A possible association of migraine with LA is now forwarded with the presentation of two cases and a literature review. Our two patients, both in their forties, had migrainous phenomena without SLE or thrombotic events. Eight other cases were found in the literature, suggesting more than a chance association. Relevance to migraine pathophysiology is discussed and may come from the ability of the LA to alter prostaglandins and platelet activity and to interact with neuronal phospholipids. Further, larger studies are needed to support this association.

Personality and Memory in Childhood Migraine

A series of neuropsychological tests were administered to a group of healthy children and another group suffering from common migraine. The tests demonstrated that children with common migraine do not have definitely abnormal personality traits even though inhibition of aggressivity and greater anxiety levels following certain environmental stimuli were seen. We also observed a decreased short- and long-term memory function in children with common migraine.

Serotonergic Hypofunction in Migraine: A Synthesis of Evidence Based on Platelet Dense Body Dysfunction

We studied the platelet dense body as a model for serotonergic vesicular function. Headache-free migraine sufferers had increased numbers of dense bodies, decreased dense body secretion, a defective link between cytosolic ionized calcium and platelet activation, an abnormal sensitivity to activation by platelet-activating factor and decreased serotonin metabolism in the presence of an unactivated platelet. These findings are interpreted as evidence for low platelet serotonin turnover. Also an abnormality in coupling of secretion to cytosolic ionized calcium caused by a membranal defect results in reduced platelet secretory function. A similar abnormality is postulated for serotonergic vesicles in central neurons. Central serotonergic hypofunction in migraine sufferers may reduce this normally present inhibitory influence on the intrinsic noradrenergic system, the activation of which may initiate the neuronal mechanisms of migraine.

Platelet activity and stroke severity.

Although platelets constitute the major component of a thrombus, its role in determining the clinical severity of thrombotic stroke is unknown. Therefore, we investigated the relationship between platelet ionized calcium ([Ca2+i]), a measure of platelet activity and presumably proneness to thrombosis, and clinical stroke severity in 45 consecutively studied acute ischemic stroke patients. Even though there was no correlation between the clinical neurological scores and the levels of baseline and activated platelet [Ca2+i], stroke was less severe in patients who had been taking aspirin at the time of stroke onset. These results raise several important questions: (a) is the extent of platelet activation a reflection of thrombus volume, (b) does the clinical severity of neurological deficit reflect the causative thrombus volume, and (c) whether the beneficial effect of aspirin in stroke prophylaxis is through its inhibition of platelets alone.

Platelet secretory products may contribute to neuronal injury.

Background: We do not fully understand the mechanisms for neuronal damage following cerebral arterial occlusion by a thrombus that consists mainly of platelets. The view that certain endogenous substances, such as glutamate, may also contribute to neuronal injury is now reasonably well established. Blood platelets are known to contain and secrete a number of substances that have been associated with neuronal dysfunction. Therefore, we hypothesize that a high concentration (approximately several thousand-fold higher than in plasma, in our estimation) of locally released platelet secretory products derived from the causative thrombus may contribute to neuronal injury and promote reactive gliosis. Summary of Comment: We have recently been able to report some direct support for this concept. When organotypic spinal cord cultures were exposed to platelet and platelet products, a significant reduction in the number and the size of the surviving neurons occurred in comparison with those in controls. We further observed that serotonin, a major platelet product, has neurotoxic properties. There may be other platelet components with similar effect. Conclusions: The hypothesis of platelet-mediated neurotoxicity gains some support from these recent in vitro findings. The concept could provide a new area of research in stroke, both at the clinical and basic levels.