Hypoxia-ischemia alters distribution of lysosomal proteins in rat cortex and hippocampus.

Neuronal excitotoxicity induced by glutamatergic receptor overstimulation contributes to brain damage. Recent studies have shown that lysosomal membrane permeabilization (LMP) is involved in ischemia-associated neuronal death. In this study we evaluated the effect of neonatal hypoxia-ischemia (HI), as a model of excitotoxicity, on the lysosomal integrity throughout the distribution of the lysosomal proteins cathepsin D and prosaposin. Rat pups (7 days old) of the Wistar Kyoto strain were submitted to HI and they were euthanized 4 days after treatment and the cerebral cortex (Cx) and hippocampus (HIP) were processed for immunohistochemistry or immunoblotting. Treatment induced an increase of gliosis and also a redistribution of both prosaposin and cathepsin D (as intermediate and mature forms), into the cytosol of the HIP and Cx. In addition, HI induced a decrease of LAMP-1 in the membranous fraction and the appearance of a reactive band to anti-LAMP-1 in the cytosolic fraction, suggesting a cleavage of this protein. From these results, we propose that the abnormal release of Cat D and PSAP to the cytosol is triggered as a result of LAMP-1 cleavage in HI animals, which leads to cell damage. This could be a common mechanism in pathological conditions that compromises neuronal survival and brain function.

Prosaposin: a protein with differential sorting and multiple functions.

In eukaryotes the delivery of newly synthesized proteins to their final destination is dependent on a series of functionally distinct compartments, including the endoplasmic reticulum and the Golgi apparatus, which plays a role in post-translational modification, sorting and distribution of proteins. Most cargo is sorted within, and exits from, the trans-Golgi network (TGN). Proteins delivered to lysosomes include hydrolytic enzymes and nonenzymic activator proteins. They are directed away from the cell surface by their binding to mannose-6-phosphate receptors (MPR). However, in I-cell disease, in which the MPR pathway is disrupted, the nonenzymic sphingolipid activator protein, prosaposin, continue to traffic to lysosomes. This observation led to discovery of a new lysosomal sorting receptor, sortilin. The targeting prosaposin to the lysosomes results from the interaction of its C-terminus with sortilin. Deletion of the C-terminus did not interfere with its secretion, but abolished its transport to the lysosomes. Mutational analysis revealed that the first half of the prosaposin C-terminus contains a motif required for its binding to sortilin and its transport to the lysosomes. Prosaposin can be also secreted to the extracellular space as oligomers. Extracellular prosaposin showed to exert a variety of responses in nervous tissues including the activation of G protein-coupled receptors and ERK phosphorylation. Lastly, prosaposin has been found to be expressed in other fluids of the body such as pancreatic juice, bile, cerebrospinal fluid, milk and seminal fluid, indicating that prosaposin is not only a house keeping lysosomal protein but an essential factor in the development and maintenance of the nervous systems and other systems of the body.

Testosterone influences the expression and distribution of the cation-dependent mannose-6-phosphate receptor in rat epididymis. Implications in the distribution of enzymes.

The mammalian epididymis plays a role in sperm maturation through its secretory activity. Among the proteins secreted by the epithelium, there are significant amounts of acid hydrolases. In most cell types, the normal distribution of lysosomal enzymes is mediated by mannose-6-phosphate receptors (MPRs). In this study, we analysed the expression and distribution of the cation-dependent MPR (CD-MPR) in epididymis from control, castrated or castrated rats with testosterone replacement. It was observed that expression of CD-MPR increased due to castration in all regions of the epididymis, which was reversed by injection of testosterone. We also measured the activity of α-mannosidase and observed that the castration tends to increase the retention of this enzyme in the tissue, which is reversed by the hormone replacement. In corpus, this resulted in a reduced secretion of the enzyme. Immunohistochemistry showed that CD-MPR has a supranuclear location (different from the cation-independent MPR), most likely in principal cells, and low reactivity in other cell types. The signal in castrated animals was more intense and tended to redistribute towards the apical cytoplasm. Thus, we concluded that expression and distribution of CD-MPR is affected by decrease of testosterone in rat epididymis, and this could change the distribution of lysosomal enzymes.

Developmental differences between cation-independent and cation-dependent mannose-6-phosphate receptors in rat brain at perinatal stages.

Mannose-6-phosphate receptors (MPRs) play a role in the selective transport of macromolecules bearing mannose-6-phosphate residue to lysosomes. To date, two types of MPRs have been described in most of cells and tissues: the cation-dependent (CD-MPR) and cation-independent mannose-6-phosphate receptor (CI-MPR). In order to elucidate their possible role in the central nervous system, the expression and binding properties of both MPRs were studied in rat brain along perinatal development. It was observed that the expression of CI-MPR decreases progressively from fetuses to adults, while the CD-MPR increases around the 10th day of birth, and maintains these values up to adulthood. Binding assays showed differences in the Bmax and KD values between the ages studied, and they did not correlate with the expression levels of both MPRs. Variations in lysosomal enzyme activities and expression of phosphomannosylated ligands during development correlated more with CD-MPR than with CI-MPR expression. These results suggest that both receptors play a different role in rat brain during perinatal development, being CD-MPR mostly involved in lysosome maturation.

Transgenic SOD1 G93A mice develop reduced GLT-1 in spinal cord without alterations in cerebrospinal fluid glutamate levels.

Glutamate-induced excitotoxicity is suggested to play a central role in the development of amyotrophic lateral sclerosis (ALS), although it is still unclear whether it represents a primary cause in the cascade leading to motor neurone death. We used western blotting, immunocytochemistry and in situ hybridization to examine the expression of GLT-1 in transgenic mice carrying a mutated (G93A) human copper-zinc superoxide dismutase (TgSOD1 G93A), which closely mimic the features of ALS. We observed a progressive decrease in the immunoreactivity of the glial glutamate transporter (GLT-1) in the ventral, but not in the dorsal, horn of lumbar spinal cord. This effect was specifically found in 14- and 18-week-old mice that had motor function impairment, motor neurone loss and reactive astrocytosis. No changes in GLT-1 were observed at 8 weeks of age, before the appearance of clinical symptoms. Decreases in GLT-1 were accompanied by increased glial fibrillary acidic protein (GFAP) levels and no change in the levels of GLAST, another glial glutamate transporter. The glutamate concentration in the cerebrospinal fluid (CSF) of TgSOD1 G93A mice was not modified at any of the time points examined, compared with age-matched controls. These findings indicate that the loss of GLT-1 protein in ALS mice selectively occurs in the areas affected by neurodegeneration and reactive astrocytosis and it is not associated with increases of glutamate levels in CSF. The lack of changes in GLT-1 at the presymptomatic stage suggests that glial glutamate transporter reduction is not a primary event leading to motor neurone loss.

Increased peripheral benzodiazepine binding sites and pentraxin 3 expression in the spinal cord during EAE: relation to inflammatory cytokines and modulation by dexamethasone and rolipram.

We have studied the mRNA expression of pentraxin 3 (PTX3) and the binding of the peripheral-type benzodiazepine receptor (PBR) ligand, [3H]-PK11195, in the spinal cord of Lewis rats where EAE was actively induced. PTX3 was induced during the active phase of EAE (day 10-14), it remained high up to 30 days and disappeared only 60 days later. Similarly, PK11195 binding peaked at day 14-17 during the recovery and it disappeared by day 60. On the other hand, the levels of TNF and IL-6 in the spinal cord were elevated at the peak and at the onset of clinical signs and returned to non-detectable by day 14-17. Dexamethasone abolished all these changes, while treatment with rolipram, delayed the appearance of the disease and then decreased its severity. However the peaks of TNF, IL-6, PBR and PTX3 levels in spinal cord were only delayed, but not reduced, by rolipram treatment. In conclusion, we show two types of inflammatory changes in EAE: acute, short term changes (TNF and IL-6), that correlate with the disease; and effects such as PTX3 expression and PK11195 binding that last longer after recovery from the disease.

Amphetamine-induced loss of human dopamine transporter activity: an internalization-dependent and cocaine-sensitive mechanism.

The dopamine transporter (DAT) is a target of amphetamine (AMPH) and cocaine. These psychostimulants attenuate DAT clearance efficiency, thereby increasing synaptic dopamine (DA) levels. Re-uptake rate is determined by the number of functional transporters at the cell surface as well as by their turnover rate. Here, we present evidence that DAT substrates, including AMPH and DA, cause internalization of human DAT, thereby reducing transport capacity. Acute treatment with AMPH reduced the maximal rate of [(3)H]DA uptake, decreased AMPH-induced currents, and significantly redistributed the immunofluorescence of an epitope-tagged DAT from the plasma membrane to the cytosol in human embryonic kidney 293 cells. Conversely, DAT inhibitors, such as cocaine, mazindol, and nomifensine, when administered with AMPH, blocked the reduction in [(3)H]DA uptake and the redistribution of DAT immunofluorescence to the cytosol. The reductions of [(3)H]DA uptake and AMPH-induced DAT internalization also were inhibited by coexpression of a dominant negative mutant of dynamin I (K44A), indicating that endocytosis modulates transport capacity, likely through a clathrin-mediated pathway. With this mechanism of regulation, acute application of AMPH would reduce DA uptake not only by direct competition for uptake, but also by reducing the available cell-surface DAT. Moreover, AMPH-induced internalization might diminish the amount of DAT available for DA efflux, thereby modulating the cytotoxic effects of elevated extracellular DA.

Biochemical and pharmacological evidence of a functional role of AMPA receptors in motor neuron dysfunction in mnd mice.

We studied ionotropic glutamate receptor subtypes and the effect of chronic treatment with NBQX [6-nitro-7-sulphamoyl-benzo(F)quinoxaline-2,3-dione], a selective (rs)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist, in the spinal cord of mnd mice. NBQX (8 mg/kg daily i.p. for 3 weeks starting from 24 weeks old) significantly improved the behavioural scores (hind leg extension reflex, cage rung grasping and gait) in mnd mice, measured after the last drug injection, and increased the number of mice with 'normal' gait (from 50% to 90%, P < 0.05). Receptor binding autoradiography of the competitive N-methyl-D-aspartate (NMDA) antagonist, [3H]CGP 39653, of [3H]AMPA and [3H]kainic acid in spinal cord sections, measured after 1 week of drug washout, were not significantly different in control and mnd mice, and were not modified by NBQX. GluR2/3 immunoreactivity, assessed using Western blotting, was significantly enhanced (by 59%, P < 0.01) in the spinal cord but not in the brain of 28-week-old mnd mice compared to age-matched control mice. NBQX treatment increased GluR2/3 immunoreactivity in the spinal cord of control mice and mnd mice by 327 +/- 74% (P < 0.01) and 212 +/- 52% (P < 0.01), respectively. The changes in GluR2/3 subunits may involve adaptive mechanisms of the receptor and play some role in the protective effect of NBQX. These findings suggest that selective antagonism of ionotropic non-NMDA receptors may be of value in the treatment of motor neuron disease.