Suizhen Lin

Reduced levels of amyloid β-peptide antibody in Alzheimer disease

Objective: To investigate whether it was possible to detect the presence and different levels of naturally occurring anti-β-amyloid (Aβ) antibodies in the CSF of patients with AD and age-matched controls by employing a sensitive ELISA. Background: Immunization with preaggregated amyloid β-peptide (Aβ1–42) and administration of antibodies against Aβ into amyloid precursor protein APPV717F– transgenic mice (an animal model of AD) have recently been reported to dramatically reduce amyloid plaque deposition, neuritic dystrophy, and astrogliosis, most likely by enhancing Aβ clearance from brain. Methods: A sensitive ELISA was performed to detect levels of naturally occurring anti-Aβ antibodies in the CSF of patients with AD and age-matched controls. Additionally, an immunoprecipitation assay was performed to confirm that naturally occurring anti-Aβ antibodies also exist in the human blood. Result: Naturally occurring antibodies directed against Aβ were found in the CSF and plasma of patients with AD and healthy control subjects. Moreover, CSF anti-Aβ antibody titers are significantly lower in patients with AD compared with healthy control subjects. Conclusion: Naturally occurring antibodies directed against Aβ exist in human CSF and plasma. The CSF anti-Aβ antibody titers may be helpful in better understanding the effects of future immunologic therapies for AD.

Human APOE Isoform-Dependent Effects on Brain β-Amyloid Levels in PDAPP Transgenic Mice

To investigate the role of human apolipoprotein E (apoE) on Aβ deposition in vivo, we crossed PDAPP mice lacking mouse Apoe to targeted replacement mice expressing human apoE (PDAPP/TRE2, PDAPP/TRE3, or PDAPP/TRE4). We then measured the levels of apoE protein and Aβ peptides in plasma, CSF, and brain homogenates in these mice at different ages. We also quantified the amount of brain Aβ and amyloid burden in 18-month-old mice. In young PDAPP/TRE4 mice that were analyzed at an age before brain Aβ deposition, we observed a significant decrease in the levels of apoE in CSF and brain when compared with age-matched mice expressing either human E2 or E3. The brain levels of Aβ42 in PDAPP/TRE4 mice were substantially elevated even at this very early time point. In older PDAPP/TRE4 mice, the levels of insoluble apoE protein increased in parallel to the dramatic rise in brain Aβ burden, and the majority of apoE was associated with Aβ. In TRE4 only mice, we also observed a significant decrease in the level of apoE in brain homogenates. Since the relative level of apoE mRNA was equivalent in PDAPP/TRE and TRE only mice, it appears that post-translational mechanisms influence the levels of apoE protein in brain (E4 < E3 ≪ E2), resulting in early and dramatic apoE isoform-dependent effects on brain Aβ levels (E4 ≫ E3 > E2) that increase with age. Therapeutic strategies aimed at increasing the soluble levels of apoE protein, regardless of isoform, may effectively prevent and (or) treat Alzheimers disease.

Human antibodies against amyloid β peptide: A potential treatment for Alzheimer's disease

Naturally occurring antibodies directed against β‐amyloid (Aβ) were detected in intravenous immunoglobulin preparations. After intravenous immunoglobulin treatment in patients with different neurological diseases, total Aβ and Aβ1‐42 in the cerebrospinal fluid was reduced significantly compared with baseline values. In the serum, total Aβ levels increased after intravenous immunoglobulin treatment, whereas no significant change was observed in Aβ1‐42 levels. Antibodies against Aβ were found to be increased in the serum and cerebrospinal fluid after intravenous immunoglobulin treatment. This study provides evidence that intravenous immunoglobulin or purified Aβ antibodies may modify Aβ and Aβ1‐42 levels, suggesting potential utility as a therapy for Alzheimer disease.

N-methyl-D-aspartate induces a rapid, reversible, and calcium-dependent intracellular acidosis in cultured fetal rat hippocampal neurons

The ability of NMDA to alter intracellular pH (pHi) was studied in fetal rat hippocampal neurons and glia using the pH-sensitive fluorescent indicator 2′,7′-bis-(2-carboxyethyl)-5-(and-6)- carboxyfluorescein (BCECF). Brief exposure (60 sec) of hippocampal neurons to NMDA (2.5–250 microM) results in a rapid, and in most cells reversible, reduction in pHi, with full recovery to baseline pHi values taking several minutes following removal of NMDA. In contrast, little or no change in pHi was observed in glial cells exposed to these same concentrations of NMDA. The NMDA-induced acidification of neurons was concentration and time dependent, with an EC50 of 39 microM and Emax (delta pH) of -0.53. More prolonged exposure to NMDA (> or = 10 min) resulted in a more prolonged reduction in pHi values over the ensuing 20 min observation period. The intracellular acidification resulting from NMDA exposure of hippocampal neurons was blocked by the NMDA receptor antagonist 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid (CPP). Moreover, removal of extracellular Ca2+ eliminated both the selective NMDA-induced elevation in [Ca2+]i and the reduction in pHi, indicating that Ca2+ influx may be required for the decrease in pHi induced by NMDA receptor activation. Finally, the NMDA- induced reduction in pHi was not significantly attenuated when extracellular [H+] was decreased by increasing extracellular pH to 8.0. The latter suggests that an intracellular source of H+ is responsible for the NMDA-induced reduction in neuronal pHi. The reduction in neuronal pHi induced by NMDA receptor activation may mediate some of the physiological and (or) pathophysiological actions of glutamate.

Minocycline blocks 6-hydroxydopamine-induced neurotoxicity and free radical production in rat cerebellar granule neurons.

Neurotoxicity induced by 6-hydroxydopamine (6-OHDA) is believed to be due, in part, to the production of reactive oxygen species (ROS). Anti-oxidants by inhibiting free radical generation, protect neurons against 6-OHDA-induced neurotoxicity. In this study, we investigated whether or not minocycline, a neuroprotective compound, could directly protect neurons against 6-OHDA-induced neurotoxicity and inhibit 6-OHDA-induced free radical production in cultured rat cerebellar granule neurons (CGN). We now report that exposure of CGN to 6-OHDA (100 microM) resulted in a significant increase in free radical production with death of 86% of CGN. Pretreatment with minocycline (10 microM) for 2 h prevented 6-OHDA-induced free radical generation and neurotoxicity. Furthermore, minocycline also attenuated H(2)O(2)-induced neurotoxicity. Our results suggest that minocycline blocks 6-OHDA-induced neuronal death possibly by inhibiting 6-OHDA-induced free radical generation in CGN. Both the antioxidative and neuroprotective effects of minocycline may be beneficial in the therapy of Parkinsons disease and other neurodegenerative diseases.

Macrophage-Mediated Degradation of β-Amyloid via an Apolipoprotein E Isoform-Dependent Mechanism

Recent studies suggest that bone marrow-derived macrophages can effectively reduce β-amyloid (Aβ) deposition in brain. To further elucidate the mechanisms by which macrophages degrade Aβ, we cultured murine macrophages on top of Aβ plaque-bearing brain sections from transgenic mice expressing PDAPP [human amyloid precursor protein (APP) with the APP717V>F mutation driven by the platelet-derived growth factor promoter]. Using this ex vivo assay, we found that macrophages from wild-type mice very efficiently degrade both soluble and insoluble Aβ in a time-dependent manner and markedly eliminate thioflavine-S positive amyloid deposits. Because macrophages express and secrete apolipoprotein E (apoE), we compared the efficiency of Aβ degradation by macrophages prepared from apoE-deficient mice or mice expressing human apoE2, apoE3, or apoE4. Macrophages expressing apoE2 were more efficient at degrading Aβ than apoE3-expressing, apoE4-expressing, or apoE-deficient macrophages. Moreover, macrophage-induced degradation of Aβ was effectively blocked by an anti-apoE antibody and receptor-associated protein, an antagonist of the low-density lipoprotein (LDL) receptor family, suggesting involvement of LDL receptors. Measurement of matrix metalloproteinase-9 (MMP-9) activity in the media from human apoE-expressing macrophages cocultured with Aβ-containing brain sections revealed greater levels of MMP-9 activity in apoE2-expressing than in either apoE3- or apoE4-expressing macrophages. Differences in MMP-9 activity appear to contribute to the isoform-specific differences in Aβ degradation by macrophages. These apoE isoform-dependent effects of macrophages on Aβ degradation suggest a novel “peripheral” mechanism for Aβ clearance from brain that may also, in part, explain the isoform-dependent effects of apoE in determining the genetic risk for Alzheimers disease.

Activation of G proteins bidirectionally affects apoptosis of cultured cerebellar granule neurons

Abstract: Cultured cerebellar granule neurons maintained in depolarizing concentrations of K+ (25 mM) and then switched to physiological concentrations of K+ (5 mM) undergo apoptosis. We now report that activation of specific G proteins robustly and bidirectionally affects apoptosis of cultured rat cerebellar granule neurons. Stimulation of Gs with cholera toxin completely blocks apoptosis induced by nondepolarizing concentrations of K+, whereas stimulation of Go/Gi with the wasp venom peptide mastoparan induces apoptosis of cerebellar granule neurons even in high (depolarizing) concentrations of K+. Moreover, pretreatment of cerebellar granule neurons with cholera toxin attenuates neuronal death induced by mastoparan. By contrast, pertussis toxin, cell‐permeable analogues of cyclic AMP, and activators of protein kinase A do not affect apoptosis of cultured cerebellar granule neurons. These data suggest that G proteins may function as key switches for controlling the programmed death of mammalian neurons, especially in the developing CNS.

Characterization of the Excitoprotective Actions of N‐Methyl‐d‐Aspartate in Cultured Cerebellar Granule Neurons

Abstract: Exposure of cultured cerebellar granule neurons to subtoxic concentrations of N‐methyl‐d‐aspartate (NMDA) has been shown previously to result in a neuroprotective state, as measured by subsequent exposure to toxic concentrations of glutamate. In the present study, we have further characterized the excitoprotective actions of NMDA in these neurons. NMDA‐induced excitoprotection was concentration dependent (EC50∼30 µM) and time dependent, with maximal protection observed following 16 h of preexposure to NMDA. NMDA‐induced excitoprotection did not require continuous exposure to NMDA, as a 4‐h preincubation was sufficient to induce full excitoprotection when measured 8 h later. Maximal protection was manifest as a “right shift” in the concentration‐response relationship for glutamate toxicity of approximately three orders of magnitude (EC50∼30 µM in untreated neurons compared with ≥50 mM in NMDA‐treated neurons). After removal of NMDA, complete reversal of the excitoprotective state was observed by 48 h (t1/2≈24 h). The ability of NMDA to induce excitoprotection was observed in neurons maintained for up to 14 days in vitro (DIV) [postnatal day (PND) 22], but was absent at 21 and 32 DIV (PND 29–40), despite little to no difference in the toxicity of glutamate at any DIV examined. Preexposure of cerebellar granule neurons to a maximally excitoprotective concentration of NMDA (50 µM) failed to alter the density of NMDA receptors measured by the specific binding of [3H]MK‐801. Moreover, the immediate elevation in intracellular free calcium concentration ([Ca2+]i) induced by glutamate exposure and measured by microfluorimetry and the Ca2+‐sensitive indicator fura‐2 was similar in NMDA‐pretreated and untreated neurons. As reported previously, NMDA‐induced excitoprotection in cerebellar granule neurons was, however, reversed by coincubation with the protein synthesis inhibitor cycloheximide. Taken together, these data suggest that NMDA receptor‐mediated excitoprotection in cerebellar granule neurons is mediated via both a transcriptionally directed and a developmentally regulated postreceptor mechanism(s).

Mastoparan-induced apoptosis of cultured cerebellar granule neurons is initiated by calcium release from intracellular stores

We have recently reported that mastoparan, a peptide toxin isolated from wasp venom, induces apoptosis in cultured cerebellar granule neurons that can be blocked by cholera toxin, an activator of Gs. Measurements of intracellular free calcium concentration ([Ca2+]i) reveal that mastoparan induces a dramatic elevation of [Ca2+]i that is frequently followed by enhanced leakage of fura-2 out of the neurons, suggesting that this rise in [Ca2+]i may be due to a more generalized change in membrane permeability. However, the mastoparan-induced initial elevation of [Ca2+]i is maintained in the absence of extracellular Ca2+, suggesting that the rise of [Ca2+]i is from intracellular stores. This conclusion is supported by the observation that depletion of [Ca2+]i stores by pretreatment with either caffeine or thapsigargin attenuates both the rise in [Ca2+]i and cell death induced by mastoparan. Phospholipase C (PLC) inhibitors, neomycin and U73122 block mastoparan-induced increases of [Ca2+]i and protect against neuronal death. Pretreatment with cholera toxin, but not pertussis toxin, reduced the mastoparan-induced rise in [Ca2+]i. Taken together, our data suggest that mastoparan initiates cell death in cerebellar granule neurons by inducing Ca2+ release from intracellular stores, probably via activation of PLC and IP3. A secondary or parallel process results in disruption of plasma membrane integrity and may be ultimately responsible for the death of these neurons by mastoparan.

Inorganic Pi Increases Neuronal Survival in the Acute Early Phase Following Excitotoxic/Oxidative Insults

Abstract: Inorganic phosphate (Pi) plays a vital role in intracellular energy metabolism. Its many effects include stimulation of glucose use, enhancement of high‐energy phosphate concentrations, and modulation of cytosolic free [Ca2+]. Cultured fetal rat cortical neurons constitutively import Pi, and cytosolic levels positively correlate with [ATP], [NADPH], and energy charge. In the present study, we demonstrate that the concentration of intracellular Pi is an important determinant of acute neuronal survival after an excitotoxic or oxidative insult to cultured fetal rat cortical neurons. Extracellular Pi dose‐dependently enhanced survival of cortical neurons after exposure to NMDA at early (≤6 h) time points after termination of the insult. Pi similarly increased neuronal survival after exposure to kainic acid or H2O2. Pi‐exposed neurons had higher basal intracellular [Pi], [ATP], and [GSH], and slightly lower cytosolic free [Ca2+], compared with Pi‐deprived neurons. Pi‐exposed neurons maintained increased [ATP] after exposure to NMDA and displayed reduced formation of reactive oxygen species after exposure to kainic acid or H2O2, compared with Pi‐deprived neurons. These findings demonstrate that changes in extracellular and intracellular Pi can affect neuronal survival after excitotoxic or oxidative insults.