Jan-Olof Karlsson

Patients with amyotrophic lateral sclerosis and other neurodegenerative diseases have increased levels of neurofilament protein in CSF.

Abstract: In the present study we describe an ELISA to quantify the light subunit of the neurofilament triplet protein (NFL) in CSF. The method was validated by measuring CSF NFL concentrations in healthy individuals and in two well‐characterized groups of patients with amyotrophic lateral sclerosis (ALS) and Alzheimers disease (AD). The levels were increased in ALS (1,743 ± 1,661 ng/L; mean ± SD) and AD (346 ± 176 ng/L) compared with controls (138 ± 31 ng/L; p < 0.0001 for both). Within the ALS group, patients with lower motor neuron signs only had lower NFL levels (360 ± 237 ng/L) than those with signs of upper motor neuron disease (2,435 ± 1,633 ng/L) (p < 0.05). In a second study patients with miscellaneous neurodegenerative diseases were investigated (vascular dementia, olivopontocerebellar atrophy, normal pressure hydrocephalus, cerebral infarctions, and multiple sclerosis), and the CSF NFL level was found to be increased (665 ± 385 ng/L; p < 0.0001). NFL is a main structural protein of axons, and we suggest that CSF NFL can be used to monitor neurodegeneration in general, but particularly in ALS with involvement of the pyramidal tract.

Transforming growth factor-beta and epidermal growth factor synergistically stimulate epithelial to mesenchymal transition (EMT) through a MEK-dependent mechanism in primary cultured pig thyrocytes

Enhancement of tumor cell growth and invasiveness by transforming growth factor-β (TGF-β) requires constitutive activation of the ras/MAPK pathway. Here we have investigated how MEK activation by epidermal growth factor (EGF) influences the response of fully differentiated and growth-arrested pig thyroid epithelial cells in primary culture to TGF-β1. The epithelial tightness was maintained after single stimulation with EGF or TGF-β1 (both 10 ng/ml) for 48 hours. In contrast, co-stimulation abolished the transepithelial resistance and increased the paracellular flux of [3H]inulin within 24 hours. Reduced levels of the tight junction proteins claudin-1 and occludin accompanied the loss of barrier function. N-cadherin, expressed only in few cells of untreated or single-stimulated cultures, was at the same time increased 30-fold and co-localised with E-cadherin at adherens junctions in all cells. After 48 hours of co-stimulation, both E- and N-cadherin were downregulated and the cells attained a fibroblast-like morphology and formed multilayers. TGF-β1 only partially inhibited EGF-induced Erk phosphorylation. The MEK inhibitor U0126 prevented residual Erk phosphorylation and abrogated the synergistic responses to TGF-β1 and EGF. The observations indicate that concomitant growth factor-induced MEK activation is necessary for TGF-β1 to convert normal thyroid epithelial cells to a mesenchymal phenotype.

Impairment of mitochondrial respiration after cerebral hypoxia-ischemia in immature rats: relationship to activation of caspase-3 and neuronal injury.

Mitochondrial damage may play a key role in the development of necrotic and apoptotic hypoxic-ischemic (HI) brain damage. It has previously been shown that mitochondrial respiration is depressed in the cerebral cortex after HI in neonatal animals. The aim of the present study was to further characterize the time course of the mitochondrial impairment during reperfusion and the correlation between the respiratory control ratio and brain injury and activation of caspase-3. Rat pups were subjected to unilateral carotid artery ligation and exposed to hypoxia (7.7% oxygen). Mitochondrial respiration was measured 0-72 h after HI in a mitochondrial fraction isolated from cerebral cortex. Microtubule associated protein-2 (MAP2) and caspase-3 were analyzed with immunoblotting in cerebral cortex homogenates. In addition, the time course of caspase-3 activation was measured as DEVD cleavage. The mitochondrial respiratory control ratio in cerebral cortex decreased immediately after HI followed by a partial recovery at 3-8 h. Thereafter, a secondary drop occurred with a minimum reached at 24 h of reperfusion. The secondary loss of respiratory function was accompanied by depletion of MAP2, cleavage of caspase-3 and an increased caspase-3 -like activity at 3-24 h after the insult. In conclusion, the primary phase of mitochondrial dysfunction was paralleled by a moderate decrease of MAP2 and a limited activation of caspase-3. The secondary mitochondrial impairment was associated with neuronal injury and pronounced activation of caspase-3.

N-acetylcysteine reduces lipopolysaccharide-sensitized hypoxic-ischemic brain injury.

Maternal inflammation/infection alone or in combination with birth asphyxia increases the risk for perinatal brain injury. Free radicals are implicated as major mediators of inflammation and hypoxia‐ischemia (HI)–induced perinatal brain injury. This study evaluated the neuroprotective efficacy of a scavenging agent, N‐acetylcysteine (NAC), in a clinically relevant model.

Rapid intracellular transport of fucose-containing glycoproteins in retinal ganglion cells.

[3H]Fucose was incorporated into glycoproteins in the rabbit retinal ganglion cells and subsequently transported at a rapid rate along the optic pathway to the nerve terminals of the lateral geniculate body and the superior colliculus. Radioautographic results indicated a preferential labelling of the terminal part of the axon. Cell fractionation showed that the major part of the transported fucose‐containing glycoproteins were associated with membranes. Sodium dodecy 1 sulphate electrophoresis of rapidly transported glycoproteins showed that most of the polypeptides had a mol. wt. of more than 40,000.

Transport of labelled proteins in the optic nerve and tract of the rabbit

Abstract The transport of labelled material in the optic nerve and tract of the rabbit after an intraocular injection of [ 3 H]leucine has been studied. It was demonstrated that the precursor was incorporated into proteins, which were transported down along the axons of the retinal ganglion cells. Two migrating protein components are described. One with and average transport rate of 1.5–2 mm per day and a second very rapidly moving component with a transport rate of at least 110–150 m per day.

Cyclosporin A prevents calpain activation despite increased intracellular calcium concentrations, as well as translocation of apoptosis-inducing factor, cytochrome c and caspase-3 activation in neurons exposed to transient hypoglycemia.

Blockade of mitochondrial permeability transition protects against hypoglycemic brain damage. To study the mechanisms downstream from mitochondria that may cause neuronal death, we investigated the effects of cyclosporin A on subcellular localization of apoptosis‐inducing factor and cytochrome c, activation of the cysteine proteases calpain and caspase‐3, as well as its effect on brain extracellular calcium concentrations. Redistribution of cytochrome c occurred at 30 min of iso‐electricity, whereas translocation of apoptosis‐inducing factor to nuclei occurred at 30 min of recovery following 30 min of iso‐electricity. Active caspase‐3 and calpain‐induced fodrin breakdown products were barely detectable in the dentate gyrus and CA1 region of the hippocampus of rat brain exposed to 30 or 60 min of insulin‐induced hypoglycemia. However, 30 min or 3 h after recovery of blood glucose levels, fodrin breakdown products and active caspase‐3 markedly increased, concomitant with a twofold increase in caspase‐3‐like enzymatic activity. When rats were treated with neuroprotective doses of cyclosporin A, but not with FK 506, the redistribution of apoptosis‐inducing factor and cytochrome c was reduced and fodrin breakdown products and active caspase‐3 immuno‐reactivity was diminished whereas the extracellular calcium concentration was unaffected. We conclude that hypoglycemia leads to mitochondrial permeability transition which, upon recovery of energy metabolism, mediates the activation of caspase‐3 and calpains, promoting cell death.

Fodrin degradation and subcellular distribution of calpains after neonatal rat cerebral hypoxic-ischemia

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (unilateral occlusion of the common carotid artery + 7.70% O2 for 100 min). Ipsi-and contralateral parietal cerebral cortex was assayed with Western blotting for fodrin breakdown product (FBDP). Calpain immunoreactivity was assayed in the cytosolic fraction (CF) and the membrane and microsomal fraction (MMF). Calpain immunoreactivity decreased bilaterally in the CF during the insult (62-68% of controls) and remained significantly lower during early recovery, whereas the MMF showed no significant changes. This relative redistribution of calpains coincided with the appearance of FBDP in the left, ipsilateral hemisphere, displaying a significantly higher level of FBDP from immediately after the insult until at least 1 day of recovery (204-292% of controls). No significant changes in FBDP could be detected in the right, contralateral hemisphere, indicating that although redistribution of calpains occurred, hypoxia per se did not suffice to initiate fodrin degradation in this model of neonatal hypoxic-ischemia.

Degradation of fodrin and MAP 2 after neonatal cerebral hypoxic-ischemia

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (unilateral occlusion of the common carotid artery + 7.70% O2 for 100 min) and allowed to recover for 3 h, 24 h, 2 days or 14 days. Consecutive tissue sections were stained with antibodies against alpha-fodrin, the 150 kDa breakdown product of alpha-fodrin (FBDP, marker of calpain proteolysis) or microtubule associated protein 2 (MAP 2, marker of dendrosomatic neuronal injury). Cortical tissue pieces were subjected to Western blotting using the antibody against the FBDP. Areas with brain injury displayed a distinct loss of MAP 2 which clearly delineated the infarct. FBDP accumulated in injured and borderline regions ipsilaterally and a less conspicuous, transient increase in FBDP also occurred in the contralateral hemisphere, especially in the white matter. A reciprocal staining pattern could be seen in the cerebral cortex, i.e. loss of MAP 2 and accumulation of FBDP, most pronounced 14 days after the insult. Fodrin and MAP 2 are known calpain substrates, and degradation of these proteins preceded neuronal degeneration, indicating that these proteases may be involved in the early events triggering the cascades leading to neuronal death.

Upregulation of calpain activity in neonatal rat brain after hypoxic-ischemia

Neonatal rats were subjected to transient cerebral hypoxic-ischemia (unilateral occlusion of the common carotid artery plus 7.7% O2 for 2 h) and allowed to recover for 0 min, 30 min or 20 h. The calpain and calpastatin activities were assayed in subcellular fractions of the ipsilateral, hypoxic-ischemic and the contralateral, hypoxic hemisphere. An upregulation of calpain activity occurred in the hypoxic hemisphere, both in the major, cytosolic fraction and in the hypotonic, membrane associated fraction (110% and 133% of controls, respectively). The hypoxic-ischemic hemisphere displayed a decrease in calpain activity in the cytosolic fraction but an increase in the hypotonic fraction (90% and 111% of controls, respectively). The changes in calpastatin activity were less pronounced. This indicates that an upregulation of calpain activity occurs in parallel with development of hypoxic-ischemic damage. However, this upregulation is not necessarily coupled to development of injury as lesions are not seen in the hypoxic hemisphere.