Barney E. Dwyer

Increased Iron and Free Radical Generation in Preclinical Alzheimer Disease and Mild Cognitive Impairment

It is now established that oxidative stress is one of the earliest, if not the earliest, change that occurs in the pathogenesis of Alzheimers disease (AD). Consistent with this, mild cognitive impairment (MCI), the clinical precursor of AD, is also characterized by elevations in oxidative stress. Since such stress does not operate in vacuo, in this study we sought to determine whether redox-active iron, a potent source of free radicals, was elevated in MCI and preclinical AD as compared to cognitively-intact age-matched control patients. Increased iron was found at the highest levels both in the cortex and cerebellum from the pre-clinical AD/MCI cases. Interestingly, glial accumulations of redox-active iron in the cerebellum were also evident in preclinical AD patients and tended to increase as patients became progressively cognitively impaired. Our findings suggests that an imbalance in iron homeostasis is a precursor to the neurodegenerative processes leading to AD and that iron imbalance is not necessarily unique to affected regions. In fact, an understanding of iron deposition in other regions of the brain may provide insights into neuroprotective strategies. Iron deposition at the preclinical stage of AD may be useful as a diagnostic tool, using iron imaging methods, as well as a potential therapeutic target, through metal ion chelators.

Differential expression of heme oxygenase-1 in cultured cortical neurons and astrocytes determined by the aid of a new heme oxygenase antibody. Response to oxidative stress.

Heme oxygenase exists as two isoenzymes designated heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2). HO-2 is made constitutively in many cell types whereas HO-1 is a stress protein inducible by heat, heavy metals, ultraviolet irradiation, and oxidative stress. Recombinant rat HO-1 was expressed in bacteria and antiserum designated HO-1713 was raised against the purified protein. HO-1713 detected recombinant rat HO-1 and recombinant rat HO-2. In rat tissues it detected HO-1 and a second, unidentified band designated HO-L (heme oxygenase-like immunoreactivity) which was not HO-2. Cultured rat cortical neurons and forebrain astrocytes were exposed to hydrogen peroxide (0.14-0.7 micromolar for 30 or 60 min). Neurons which contained little detectable HO-1 and which were sensitive to hydrogen peroxide at the high end of the dose curve failed to induce HO-1 by Western blot analysis. In contrast, cultured rat forebrain astrocytes which contained HO-1 under normal culture conditions and which were resistant to injury by hydrogen peroxide, increased their content of immunoreactive HO-1 by 7-fold within 3 h after exposure. Our results support a protective role for HO-1 in oxidative injury and suggest that the relative inability of neurons to increase HO-1 after oxidative stress may contribute to their selective vulnerability vis-a-vis astrocytes. They also suggest that differential expression of heme oxygenase in studies utilizing CNS cultures may alter normal cell physiology and cell survival.

Synthesis of the major inducible heat shock protein in rat hippocampus after neonatal hypoxia-ischemia.

Rats aged 7 days were exposed to 3.5 h of cerebral hypoxia-ischemia produced by unilateral common carotid artery ligation combined with hypoxia (8% oxygen). The major inducible heat-shock protein, HSP-68, was synthesized in ipsilateral but not contralateral (control) hippocampus during early recovery (1 and 3 h). HSP-68 synthesis was not detected during longer recovery periods. The presence of HSP-68 was confirmed by Western blotting and immunostaining with a polyclonal antibody to HSP-68.

Comparison of the heat shock response in cultured cortical neurons and astrocytes

Cultured cortical neurons and astrocytes were compared for synthesis of the major inducible 68 kDa heat shock protein. By one- and two-dimensional electrophoresis the inducible 68 kDa protein appeared similar, but astrocytes produced greater amounts of the protein by 3 h than did neurons. Antibodies raised against HeLa cell inducible 72 and constitutive 73 kDa heat shock proteins were used to characterizes the inducible heat shock proteins in neurons and astrocytes. Unlike the gels, major differences were noted of the major inducible heat shock protein in astrocytes compared with neurons when analyzed by Western immunoblots. Heat shock protein 68 kDa mRNA induction in neurons was less than astrocytes suggesting an attenuated inducible 68 kDa heat shock protein response in neurons. The neuronal protein may be a different isoform of the 70 kDa family of heat shock proteins.

Neuroprotection against CA1 injury with metalloporphyrins.

The hippocampal slice was used to examine neuroprotection with metalloporphyrins, a class of drug which inhibits heme oxygenase and which has been found to be effective in the treatment of neonatal hyperbilirubi-nernia. Tin-protoporphyrin given during hypoxia significantly improved recovery of CA1 antidromic PS to a mean of 82 ± 2% of initial amplitude, while unmedicated slices regained only 6 ± 3% of initial amplitude. Tin- protoporphyrin also protected against fluid percussion injury with an EC50 of 10 μM when given after trauma. This protection extended to induction of long-term potentiation. Tin-mesoporphyrin and zinc-protopor-phyrin protected against trauma with EC50s of 4 and 32 μM. Treatment with Sn-PP also protected against exposure to hydrogen peroxide, but not NMDA, AMPA, glycine or nitric oxide. These findings indicate that metalloporphyrins protect against CA1 neuronal injury through direct neural effects.

Generalized seizures deplete brain energy reserves in normoxemic newborn monkeys

The cerebral metabolic response to bicuculline (BC)-induced status epilepticus (SE) was studied in two-week-old ketamine-anesthetized marmoset monkeys. During 30-min clonic seizures, mean blood pressure, plasma glucose and paO2 did not decrease and plasma lactate doubled. Brains were funnel-frozen and punch biopsies of frontoparietal cortex, temporal cortex and thalamus were analyzed for ATP, phosphocreatine (PCr), glucose and lactate. There were marked reductions of ATP (to 56-77% of controls), PCr (to 23-28% of controls) and glucose (to 1-4% of controls), and lactate increased 3- to 6-fold in seizure animals. NADH fluorescence increased during seizures in cerebral cortex, thalamus, amygdaloid nuclei, hippocampus, posterior striatum and hemispheric white matter. This suggests a reduced tissue redox state in these regions and is correlated with the high energy phosphate depletion and elevated lactate in cortex and thalamus. Our results demonstrate a significant depletion of energy reserves and glucose in cerebral cortex and thalamus during neonatal seizures in the absence of adverse systemic factors. These seizure-induced metabolic changes in brain could have adverse long-term effects on brain development and function.

Angiotensin II directly increases rabbit renal brush-border membrane sodium transport: presence of local signal transduction system.

SummaryIn the present study, we have examined the direct actions of angiotensin II (AII) in rabbit renal brush border membrane (BBM) where binding sites for AII exist. Addition of AII (10−11–10−7m) was found to stimulate22Na− uptake by the isolated BBM vesicles directly. AII did not affect the Na+-dependent BBM glucose uptake, and the effect of AII on BBM22Na+ uptake was inhibited by amiloride, suggesting the involvement of Na+/H+ exchange mechanism. BBM proton permeability as assessed by acridine orange quenching was not affected by AII, indicating the direct effect of AII on Na+/H+ antiport system.In search of the signal transduction mechanism, it was found that AII activated BBM phospholipase A2 (PLA) and that BBM contains a 42-kDa guanine nucleotide-binding regulatory protein (G-protein) that underwent pertussis toxin (PTX)-catalyzed ADP-ribosylation. Addition of GTP potentiated, while GDP-ßS or PTX abolished, the effects of AII on BBM PLA and22Na+ uptake, suggesting the involvement of G-protein in AIIs actions. On the other hand, inhibition of PLA by mepacrine prevented AIIs effect on BBM22Na+ uptake, and activation of PLA by mellitin or addition of arachidonic acid similarly enhanced BBM22Na+ uptake, suggesting the role of PLA activation in mediating AIIs effect on BBM22Na+ uptake.In summary, results of the present study show a direct stimulatory effect of AII on BBM Na+/H+ antiport system, and suggest the presence of a local signal transduction system involving G-protein mediated PLA activation.

Rat Brain Protein Synthesis Declines During Postdevelopmental Aging

: Using improved methods to measure brain protein synthesis in vivo (Dunlop et al., 1975) we have established that brain protein synthesis significantly declines in forebrain, cerebellum and brain stem when mature rats (3 months old) are compared to old rats (22.5 months old). The incorporation of (3H) L‐lysine into forebrain protein is reduced 11% in 10.5 month old rats compared to 3 month old rats. A further reduction of 9% occurred between 16.5 months and 22.5 months. Our data suggest that reduced levels of protein synthesis initiation may be responsible, at least in part, for this age‐related decline.

Transient induction of heme oxygenase after cortical stab wound injury

Heme oxygenase (HO) exists as two isoenzymes designated heme oxygenase-1 (HO-1) and heme oxygenase-2 (HO-2). HO-1 has been identified as a heat shock or stress protein and is inducible whereas HO-2 is largely refractory to induction. HO-2 is the predominant isoenzyme in normal brain and appears to have a predominantly neuronal distribution in cerebral cortex. Cortical stab wound injury resulted in HO-1 induction as determined by Western blot analysis. Immunohistochemical analysis suggested that induced HO-1 was largely restricted to reactive astrocytes and macrophage-like cells. Enhanced HO-1 immunoreactivity was observed in hypertrophied, GFAP+ reactive astrocytes near the wound margin as early as 12 h after injury. Very rarely were HO-1+ neurons observed and then only up to 6 h after stabbing. Maximal numbers of HO-1+ astrocytes were found 3 days after stabbing. Their numbers declined thereafter. By 5 days after stab injury few HO-1+ reactive astrocytes were observed although GFAP+ reactive astrocytes were still prominent near the wound margin. HO-1+ macrophage-like cells were initially observed between 1 and 3 days after injury and they persisted in the margin of the wound for at least 14 days. The proximity of HO-1+ cells to the wound margin suggests that factors associated with injury contribute to the regulation of HO-1 in injured cortex.

Hematoma removal, heme, and heme oxygenase following hemorrhagic stroke.

Abstract: The hemorrhagic strokes, intracerebral (ICH) and subarachnoid hemorrhage (SAH), often have poor outcomes. Indeed, the most common hemorrhagic stroke, ICH, has the highest mortality and morbidity rates of any stroke subtype. In this report, we discuss the evidence for the staging of red blood cell removal after ICH and the significance of control of this process. The protective effects of clinically relevant metalloporphyrin heme oxygenase inhibitors in experimental models of ICH and in superficial siderosis are also discussed. We also examine literature paradoxes related to both heme and heme oxygenase in various disorders of the central nervous system. Last, new data are presented that support the concept that heme, although primarily a pro‐oxidant, can also have antioxidant properties.