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We have previously reported that levels of phosphatidylethanolamine (PE) in the erythrocyte membrane and of ceruloplasmin, a copper-transport antioxidant protein, in the serum are lower in children with autism than in control subjects. In the present study, we report that (a) copper oxidizes and reduces the levels of membrane PE and (b) copper-mediated oxidation of PE is higher in lymphoblasts from autistic subjects than from control subjects. The effect of copper was examined on the oxidation of liposomes composed of brain lipids from mice and also on the lymphoblasts from autism and control subjects. Among the various metal cations (copper, iron, calcium, cadmium and zinc), only copper was found to oxidize and decrease the levels of PE. The metal cations did not affect the levels of other phospholipids. The action of copper on PE oxidation was time-dependent and concentration-dependent. No difference was observed between copper-mediated oxidation of diacyl-PE and alkenyl-PE (plasmalogen), suggesting that plasmalogenic and non-plasmalogenic PE are equally oxidized by copper. Together, these studies suggest that ceruloplasmin and copper may contribute to oxidative stress and to reduced levels of membrane PE in autism.

Currently, there is no biochemical test to assist in the behavioral diagnosis of autism. We observed that levels of phosphatidylethanolamine (PE) were decreased while phosphatidylserine (PS) were increased in the erythrocyte membranes of children with autism as compared to their non-autistic developmentally normal siblings. A new method using Trinitrobenezene sulfonic acid (TNBS) for the quantification of PE and PS (amino-glycerophospholipids, i.e., AGP) in the plasma of children was developed and standardized. Wavelength scans of TNBS-PE and TNBS-PS complexes gave two peaks at 320 nm and 410 nm. When varying concentrations of PS and PE were used, a linear regression line was observed at 410 nm with TNBS. Using this assay, the levels of AGP were found to be significantly increased in the plasma of children with autism as compared to their non-autistic normal siblings. It is proposed that plasma AGP levels may function as a potential diagnostic marker for autism.

Bear serum alpha(2) macroglobulin (alpha(2)M) was purified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and partially characterized by tryptic digestion of alpha(2)M and analysis of the peptides by peptide mass fingerprinting. The molecular weight of bear serum alpha(2)M was 181 kDa, same as for human serum alpha(2)M, on SDS-PAGE. However, the MALDI mass spectrum of the tryptic digested bear serum alpha(2)M showed that it is different from human alpha(2)M or other data bank proteins. Liquid chromatography (LC)/mass spectrometry (MS)/MS of the proteolytic products of bear serum alpha(2)M showed eight peptides that had similarities to human alpha(2)M suggesting that the protein of interest was indeed alpha(2)M of bear. The polyclonal antibody against bear serum alpha(2)M recognized only one protein from the western blot of bear serum proteins. It also recognized human alpha(2)M. The levels of serum alpha(2)M were significantly increased during hibernating state as compared to active state of bears indicating its protective role from the consequences of the metabolic depression during hibernation.

Although the pathogenic mechanisms that underlie autism are not well understood, there is evidence showing that metabotropic and ionotropic glutamate receptors are hyper-stimulated and the GABAergic system is hypo-stimulated in autism. Memantine is an uncompetitive antagonist of NMDA receptors and is widely prescribed for treatment of Alzheimer's disease treatment. Recently, it has been shown to improve language function, social behavior, and self-stimulatory behaviors of some autistic subjects. However the mechanism by which memantine exerts its effect remains to be elucidated. In this study, we used cultured cerebellar granule cells (CGCs) from Fmr1 knockout (KO) mice, a mouse model for fragile X syndrome (FXS) and syndromic autism, to examine the effects of memantine on dendritic spine development and synapse formation. Our results show that the maturation of dendritic spines is delayed in Fmr1-KO CGCs. We also detected reduced excitatory synapse formation in Fmr1-KO CGCs. Memantine treatment of Fmr1-KO CGCs promoted cell adhesion properties. Memantine also stimulated the development of mushroom-shaped mature dendritic spines and restored dendritic spine to normal levels in Fmr1-KO CGCs. Furthermore, we demonstrated that memantine treatment promoted synapse formation and restored the excitatory synapses to a normal range in Fmr1-KO CGCs. These findings suggest that memantine may exert its therapeutic capacity through a stimulatory effect on dendritic spine maturation and excitatory synapse formation, as well as promoting adhesion of CGCs.

We have recently reported that fibrillar amyloid beta-protein (Abeta) inhibits the proteolytic activity of trypsin and high molecular weight bovine brain protease. We report here that trypsin binds to fibrillar Abeta (fAbeta) and the resulting complex of trypsin/fAbeta is sodium dodecyl sulfate (SDS)-stable. Electron microscopic analysis confirmed the binding of trypsin on the fibrils of both Abeta 1-40 and Abeta 1-42. SDS-polyacrylamide gel electrophoresis (PAGE) of fAbeta sample incubated in the presence of trypsin showed that major amount of trypsin was associated with fAbeta that did not enter the gel. The presence of trypsin in this protein complex was confirmed by Western blotting after its elution from the gel. Kinetic studies showed that the binding of trypsin to fibrillar Abeta was dependent on the degree of Abeta fibrillization and on the concentration of fAbeta. However, the trypsin binding to Abeta oligomers did not affect the fibril growth. The maximum binding (B(max)) of trypsin to fAbeta 1-40 and fAbeta 1-42 was 36 pmol and 40 pmol, and dissociation constant (K(d)) was 18.31 microM and 20 microM respectively. Similar to fAbeta, trypsin could also bind to fibrillar amylin. This binding was dependent on the concentration of fibrillar amylin. Under similar conditions, bovine serum albumin did not bind to fibrillar Abeta. These results suggest that fAbeta and fibrillar amylin have strong affinities for trypsin, and chelation of proteases by abnormal aggregated proteins may be a general mechanism for inflicting pathological conditions in various diseases.

The effect of soluble amyloid beta-protein (sAbeta) and fibrillar amyloid beta-protein (fAbeta) on the casein-digesting activity of high molecular weight bovine brain protease (HMW protease) and trypsin was studied. While sAbeta stimulated the casein-digesting activity of HMW protease in a concentration-dependent manner, it did not affect trypsin activity. Structure-activity relationship was studied by testing different soluble and fibrillar Abeta peptides. Various Abeta peptides affected casein-digesting activity of HMW protease differently: sAbeta 1-40 > sAbeta 22-35 = sAbeta 1-11 = sAbeta1-16 > sAbeta 1-28 = sAbeta 31-35, while sAbeta 12-28 and sAbeta 25-35 had no effect. On the other hand, among the fibrillar beta peptides, only fAbeta 1-40 significantly inhibited the casein-digesting activity of HMW protease. Tricine gel electrophoresis showed that sAbeta was digested by trypsin while it remained un-cleaved in the presence of HMW protease. However, fAbeta, a major component of amyloid plaques in Alzheimer's disease, inhibited the casein-digesting activity of both HMW protease and trypsin. fAbetawas found to be resistant to proteolysis by HMW protease and trypsin. The trypsin resistance starts in the early stage of fibrillization of Abeta, i.e., aggregated Abeta. Taken together, these results suggest that fibrillization of Abeta may affect the clearance of Abeta by inhibiting the brain proteases, thereby increasing the concentration of circulating Abeta, that may further increase the Abeta fibrillization.

During hibernation, fat is known to be the preferred source of energy. A detailed analysis of different phospholipids, as well as free and esterified cholesterol, was conducted to investigate lipid abnormalities during hibernation. The levels of total phospholipids and total cholesterol in the serum of black bears were found to increase significantly in hibernation as compared with the active state. Both free and esterified cholesterol were increased in the hibernating state in comparison with the active state (P < 0.05). The percentage increase during hibernation was more in free cholesterol (57%) than in esterified cholesterol (27%). Analysis of subclasses of serum phospholipids showed that choline containing phospholipids, i.e., sphingomyelin (SPG) (14%) and phosphatidylcholine (PC) (76%), are the major phospholipids in the serum of bear. The minor phospholipids included 8% of phosphatidylserine (PS) + phosphatidylinositol (PI), while phosphatidylethanolamine (PE) was only 2% of the total phospholipids. A comparison of phospholipid subclasses showed that PC, PS + PI and SPG were significantly increased, while PE was significantly decreased (P < 0.05) in the hibernating state as compared with the active state in black bears. These results suggest that the catabolism of phospholipids and cholesterol is decreased during hibernation in black bears, leading to their increased levels in the hibernating state as compared with the active state. In summary, our results indicate that serum cholesterol and phospholipid fractions (except PE) are increased during hibernation in bears. It is proposed that the increase of these lipids may be due to the altered metabolism of lipoproteins that are responsible for the clearance of the lipids.

Polyclonal antiserum against a high molecular weight glycosylated protease, purified from calf brain cytosol, was raised in rabbit and purified by immunoaffinity. The antibody specifically immunoreacted with the M r = 165,000 and 155,000 polypeptides of the protease. Immunocytochemical localization data revealed that the protease is localized in the pyramidal neurons, granular and glial cells of the hippocampus. Microscopic analysis of the pyramidal neurons indicates that the protease is present in the cytoplasm and extends to the dendrite and axon. The nuclei of these remain unstained.

The effect of the three forms (CANP1, CANP2 & CANP3) of calf brain calcium activated neutral protease (CANP) on the hydrolysis of purified neurofilament triplet proteins was investigated. It was observed that: 1) each of the purified neurofilament proteins, was hydrolyzed slowly by CANP2 whereas the hydrolysis of 150 KDa and 68 kDa proteins by CANP1 & CANP3 was rapid; 2) when assembled neurofilaments were used as a substrate, again differences in the rate and extent of degradation of the triplet proteins by the three proteases were observed. For example, little cleavage of the 68kDa protein by CANP2 and CANP3 was noted whereas 210kDa and 150kDa proteins remained largely intact. CANP1 degraded the 150kDa and 68kDa proteins more rapidly than 210kDa protein, where only a slight effect was noted. These data provide further proof of the existance of three different forms of CANP in the brain, and indications of the resistance of 210kDa protein to proteolysis which may be compatible with its proposed special role in crossbridge formation.

A third form (CANP3) of calcium-activated neutral proteinase (CANP) has been purified, 3900-fold, to near homogeneity from calf brain cortex. The purification procedure is based on the one recently developed for the purification of CANP1 and CANP2. The molecular weight of CANP3, as judged on SDS-polyacrylamide gel electrophoresis was Mr 78,000. A protein with an apparent Mr 17,000 co-purified with the proteinase. At neutral pH (7.2), it was maximally active at 260 microM CaCl2. In the presence of CaCl2, CANP1 and CANP3 were autolyzed very rapidly, whereas the autolysis of CANP2 was slow and gradual. The autolyzed CANP1 and CANP3 responded differently to CaCl2; CANP1 lost activity completely, whereas CANP3 was fully active at 0.5 microM CaCl2. Despite the opposite behavior of these proteinases in the presence of Ca2+, no significant differences in the peptide maps of the three proteinases were observed. Neurofilaments, neurotubules and myelin basic protein (MBP) were degraded by each of the proteinases. Monoclonal antibodies raised against CANP2 reacted almost equally with CANP1 and CANP3. As with CANP1 and CANP2, leupeptin and sulfhydryl-modifying compounds, NEM and iodoacetic acid, inhibited the activity of CANP3.

A Ca 2+ activated protease(s) capable of hydrolyzing several polypeptides at neutral pH including cytoskeletal proteins, actin, myosin, tubulin and neurofilament triplet was identified in calf brain cortex. The enzyme activity precipitates at 75 mM KCl, pH 6.5 – 7.0 and is inhibited by the sulfhydryl inhibitors, N-ethylmaleimide and para-chloromercuribenzoate and the protease inhibitors, antipain, pepstatin and leupeptin, leupeptin being the most effective.