Aging alters mRNA expression of amyloid transporter genes at the blood-brain barrier.

Decreased clearance of potentially toxic metabolites, due to aging changes, likely plays a significant role in the accumulation of amyloid-beta (Aß) peptides and other macromolecules in the brain of the elderly and in the patients with Alzheimer's disease (AD). Aging is the single most important risk factor for AD development. Aß transport receptor proteins expressed at the blood-brain barrier are significantly altered with age: the efflux transporters lipoprotein receptor-related protein 1 and P-glycoprotein are reduced, whereas the influx transporter receptor for advanced glycation end products is increased. These receptors play an important role in maintaining brain biochemical homeostasis. We now report that, in a rat model of aging, gene transcription is altered in aging, as measured by Aß receptor gene messenger RNA (mRNA) at 3, 6, 9, 12, 15, 20, 30, and 36 months. Gene mRNA expression from isolated cerebral microvessels was measured by quantitative polymerase chain reaction. Lipoprotein receptor-related protein 1 and P-glycoprotein mRNA were significantly reduced in aging, and receptor for advanced glycation end products was increased, in parallel with the changes seen in receptor protein expression. Transcriptional changes appear to play a role in aging alterations in blood-brain barrier receptor expression and Aß accumulation.

Amyloid-beta accumulation, neurogenesis, behavior, and the age of rats.

The goals of this research were to describe age-related changes in brain biochemistry and behavior, and the relationships between them. The chronological ages of greatest change are particularly important for targeting interventions. In this experiment, 36 Fischer 344/Brown-Norway rats (3, 12, 20, and 30 months old) were trained in lever boxes on temporal discrimination tasks. The greatest response rate decrease and response pattern change occurred between 12 and 20 months. The biochemical results showed that amyloid-beta peptides (Aß40 and Aß42) increased with age. The endothelial expression of the Aß influx transporter (RAGE) also increased, and the expression of Aß efflux transporter (LPR-1) decreased, with age. The greatest change in the biochemical measures also were between 12 and 20 months. Twenty additional rats were analyzed for stem cell proliferation, and neurogenesis decreased with age, particularly between about 12 and 20 months. These early changes in brain, biochemistry, and behavior provide opportunity for new therapies or prophylaxis.

Amyloid efflux transporter expression at the blood-brain barrier declines in normal aging.

Reduced clearance of amyloid ß peptides (Aß) across the blood-brain barrier contributes to amyloid accumulation in Alzheimer disease. Amyloid ß efflux transport is via the endothelial low-density lipoprotein receptor-related protein 1 (LRP-1) and P-glycoprotein (P-gp), whereas Aß influx transport is via the receptor for advanced glycation end products. Because age is the major risk factor for developing Alzheimer disease, we measured LRP-1 and P-gp expression and associated transporter expression with Aß accumulation in aging rats. Quantitative LRP-1 and P-gp microvessel expression was measured by immunohistochemistry (IHC); LRP-1 and P-gp expression were assessed in microvessel isolates by Western blotting. There was an age-dependent loss of capillary LRP-1 across all ages (3-36 months) by IHC (linear trend p = 0.0004) and between 3 and 20 months by Western blotting (linear trend p < 0.0001). There was a late (30-36 months) P-gp expression loss by IHC (p < 0.05) and Western blotting (p = 0.0112). Loss of LRP-1 correlated with Aß42 accumulation (p = 0.0121) and very nearly with Aß40 (p = 0.0599) across all ages. Expression of LRP-1 correlated negatively with the expression of receptor for advanced glycation end products (p < 0.0004). These data indicate that alterations in LRP-1 and P-gp expression seem to contribute progressively to Aß accumulation in aging.

Amyloid deposition and influx transporter expression at the blood-brain barrier increase in normal aging.

Aging is the most important single risk factor for developing Alzheimer disease. We measured amyloid-beta peptide (Abeta) levels in rat cerebral cortex and hippocampus during normal aging of Brown-Norway/Fischer rats. Amyloid-beta accumulation was associated with expression of the Abeta influx transporter, the receptor for advanced glycation end-products (RAGEs) at the blood-brain barrier. Rats at selected ages from 3 to 36 months were analyzed by 1) immunohistochemistry for amyloid deposition and quantitative microvessel surface area RAGE expression, 2) ELISA for cortical Abeta40 and Abeta42 concentrations, and 3) Western blotting of microvessel proteins for RAGE expression. Immunohistochemistry showed increasing accumulation of brain Abeta with aging. By ELISA analysis, both Abeta40 and Abeta42 concentrations in cortical homogenates rose sharply from 9 to 12 months. The Abeta42 continued to rise up to age 30 months, whereas Abeta40 stabilized after 12 months. The expression of RAGE initially decreased between 3 and 12 months but then increased between 12 and 34 months by immunohistochemistry. On immunoblotting, RAGE decreased up to 9 months and then progressively increased up to 36 months. These data indicate an association between amyloid and microvessel RAGE during aging. An increase in capillary RAGE expression seems to play a role in the later Abeta accumulation but not in the initial increase.

Shedding of tumor necrosis factor type 1 receptor after experimental spinal cord injury.

In a number of stress conditions, the biological effects of tumor necrosis factor-alpha (TNF-alpha), such as the induction of neuronal apoptosis, are presumably attenuated by the soluble fragments of TNF receptors (sTNFRs). Within 1 h after spinal cord injury, increased synthesis and/or secretion of TNF-alpha is detectable at the injury site. However, the shedding of ectodomains of TNFRs in the traumatized spinal cord has not yet been reported. In the present study, adult Sprague-Dawley rats were subjected to acute spinal cord injury (ASCI) by applying a 25-g Walsh-Tator aneurysm clip at the C8-T1 level. Sham-injured animals underwent laminectomy and facetectomy only. A PE10 catheter was placed in the subarachnoid space to collect the samples of cerebrospinal fluid (CSF) from near the injury site. These CSF samples were analyzed by ELISA for the presence of TNF-alpha and soluble TNFR1 and TNFR2 (sTNFR1 and sTNFR2, respectively). The spinal cord tissue was analyzed by immunohistochemistry for the expression of TNF-alpha, TNFR1, and TNFR2, and by the TUNEL technique for the occurrence of neuronal death. The levels of TNFR1 and sTNFR1 in the injured tissue were determined by Western blotting. Immunohistochemistry demonstrated the increased neuronal expression of TNF-alpha and its receptors at 6 h post-ASCI. No changes in the intensity of staining were observed in the sham-injured rats. In addition, at 6 h after the injury, a significant increase in the number of TUNEL-positive neurons was observed. Numerous neurons in traumatized tissue were also immunoreactive for activated caspase-3, suggesting that the TUNEL-positive neurons were undergoing an apoptotic death. At 1 h after ASCI, TNF-alpha levels in the CSF were significantly higher than those found in the sham-injured animals, indicating the release of this cytokine into the interstitial fluid. This was followed by a significant increase, compared to the sham-injured controls, in sTNFR1 levels in the CSF at 3 and 6 h after the insult. Unlike sTNFR1, the levels of sTNFR2 in the CSF were unchanged at any time point post-ASCI. The increased shedding of TNFR1 was confirmed by Western blotting. It is concluded that the shedding of TNFR1 receptor may represent an important post-traumatic physiological response aimed at reducing the proapoptotic effect of TNF-alpha.

Physiological and behavioral evidence for focal nociception induced by epidural glutamate infusion in rats.

STUDY DESIGN: Blinded animal study. OBJECTIVES: To determine if an increased concentration of epidural glutamate can cause a focal nociceptive response in the lower extremities that is consistent with sciatica. SUMMARY OF BACKGROUND DATA: It is believed that the origin of sciatic pain is related to more than physical pressure on the nerve roots. Recently, it was determined that disc material may be a significant source of free glutamate, resulting from the enzymatic degradation of matrix aggrecan proteins. We believe that this free glutamate acts as a neurotransmitter at glutamate receptors on the dorsal root ganglion (DRG) cell bodies, thereby initiating a nociceptive response. METHODS: Rats were subject to a 72-hour epidural glutamate infusion via a mini osmotic pump. Von Frey behavioral testing was performed 24 hours before, and 24 and 72 hours after the onset of the infusion. DRG and dorsal horn tissues were analyzed for changes in receptor expression, which have been previously shown to correlate with a nociceptive state. RESULTS: Von Frey behavioral tests showed focal hyperalgesia that was maximal at the 0.02 mmol/L glutamate concentration. Significant changes in DRG glutamate receptor expression were seen for alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid, kainite, and N-methyl-D aspartate receptors. Analysis of dorsal horn glutamate receptors also showed patterns in alpha-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid and kainate receptor expression that were consistent with a nociceptive state. CONCLUSIONS: Epidural glutamate elicits a focal nociceptive response. Free glutamate that has been liberated from the disc material may be an important factor in the development of sciatic pain.