Sadaki Fujimoto

Phospholipase C isozymes in the human brain and their changes in Alzheimer's disease

Phosphoinositide-specific phospholipase C is a key enzyme in signal transduction. We have previously demonstrated that an isozyme of phospholipase C, phospholipase C-delta1, accumulates aberrantly in the brains of patients with Alzheimers disease. In the present study, we examined the property of phospholipase C isozymes in human brains using the methods of chromatofocusing and gel filtration chromatography, and investigated their changes in Alzheimers disease brains. The chromatofocusing profile of human brain phospholipase C activity on a Mono P HR column demonstrated that phospholipase C-gamma1, exhibiting an isoelectric point value of 5.2, and phospholipase C-delta1, exhibiting isoelectric point values of 5.2 and 4.6, are partly overlapped in their elution. In contrast, the elution profiles of control and Alzheimers disease brain phospholipase C on Superdex 200 pg column gel filtration chromatography indicated that phospholipase C-gamma1 and phospholipase C-delta1 can be separated with the elution position having a molecular weight of about 240,000 and 140,000, respectively, in the human brain. Using this gel filtration chromatography it was revealed that the phospholipase C-gamma1 activity was significantly decreased and the phospholipase C-delta1 activity was significantly increased in Alzheimers disease brains compared with controls. These results suggest that the phospholipase C isozymes are differentially involved in Alzheimers disease.

Alteration of Phospholipase C-δ Protein Level and Specific Activity in Alzheimer's Disease

Abstract: Phosphoinositide‐specific phospholipase C (PLC) is a key enzyme in signal transduction. We have previously demonstrated that an antibody to an isozyme of PLC, PLC‐δ, produced intense staining of neurofibrillary tangles in the brains of patients with Alzheimers disease. In the present study, we investigated the protein level and activity of this enzyme in control and Alzheimer brains. Western blot analysis using a specific antibody for PLC‐δ showed that the concentration of PLC‐δ protein was significantly higher in the cytosolic fraction of Alzheimers disease cortical tissue than in control brains. The activity of PLC‐δ, which hydrolyzes phosphatidylinositol, was also investigated, and we found that PLC‐δ activity was not significantly different in the Alzheimer and control cytosolic fractions. These results indicate that the specific activity of PLC‐δ is decreased in Alzheimer brains and suggest that inactivation of PLC‐δ might be related to the pathophysiology of this disease.

Differential subcellular localization of caspase family proteins in the adult rat brain

The distribution of the caspase family (caspase-2, -3, -6, -7, -8, -9, -10) was assessed using immunochemical detection of subcellular fractions of 8-week-old rat brain tissues. The present study demonstrated that the relative protein level of caspase-2, -3, -6, -8 and -10 was highest in the soluble cytosolic fraction, while that for caspase-9 was highest in the nucleus. We also found that caspase-3 and -6 were present at high levels and caspase-2, -8 and -9 at moderate levels in the nerve endings fraction as well as in the soluble cytosolic fraction. These results suggest that rat brain caspases are differentially expressed in the subcellular fractions of the rat brain, and that caspases not only contribute to the regulation of neuronal death, but also to synaptic plasticity.

Mitochondrial dysfunction is involved in P2X7 receptor-mediated neuronal cell death.

J. Neurochem. (2012) 122, 1118–1128.

Phosphatidylinositol-specific phospholipase C activity in the postmortem human brain: no alteration in Alzheimer's disease

The activity of phospholipase C (PLC) which hydrolyzes exogenous phosphatidylinositol (PI), was investigated in samples prepared from postmortem normal human brains and Alzheimer disease brains. The enzyme activity did not change significantly after rat brains were left for 24 h at room temperature. The PI-specific PLC activity in the Alzheimer cytosolic and particulate fractions was not significantly different from that in the control fractions. The PI-specific PLC activity as a function of the free Ca2+ concentration was also similar between control and Alzheimer brains. These results suggest that the PI-specific PLC activity is not altered in Alzheimers disease.

Transport mechanism for lovastatin acid in bovine kidney NBL-1 cells: kinetic evidences imply involvement of monocarboxylate transporter 4.

We previously indicated that lovastatin acid, a 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, was transported by a monocarboxylate transporter (MCT) in cultured rat mesangial cells. In this study, to identify the MCT isoform(s) responsible for the lovastatin acid uptake, the transport mechanism was investigated using bovine kidney NBL-1 cells, which have been reported to express only MCT4 at the protein level. On RT-PCR analysis, the message of mRNAs for MCT1 and MCT4 was detected in the NBL-1 cells used in this study, which was confirmed by kinetic analysis of [14C]L-lactic acid uptake, consisting of high- and low-affinity components corresponding to MCT1 and MCT4, respectively. The lovastatin acid uptake depended on an inwardly directed H+-gradient, and was inhibited by representative monocarboxylates, but not by inhibitors/substrates for organic anion transporting polypeptides and organic anion transporters. In addition, L-lactic acid competitively inhibited the uptake of lovastatin acid and lovastatin acid inhibited the low affinity component of [14C]L-lactic acid uptake dose dependently. The inhibition constant of L-lactic acid for lovastatin acid uptake was almost the same as the Michaelis constant for [14C]L-lactic acid uptake by the low-affinity component. These kinetic evidences imply that lovastatin acid was taken up into NBL-1 cells via MCT4.

Characterization of guanine and guanosine transport in primary cultured rat cortical astrocytes and neurons.

In this study, we examined the transport mechanisms for guanine and guanosine in rat neurons and astrocytes, and compared their characteristics. In the both types of cell, the uptake of [3H]guanine and [3H]guanosine was time‐, temperature‐, and concentration‐dependent, and Na+‐independent. Their uptake decreased on the addition of purine and pyrimidine nucleobases or nucleosides, and the inhibitory effect of the purine analogues was greater than that of the pyrimidine ones. In both cell types, equilibrative nucleoside transporter (ENT) 1 and ENT2 expression was confirmed at the mRNA level, and nitrobenzylmercaptopurine riboside, a representative inhibitor for ENT, decreased their uptake at concentrations of over 10 μM. Comparing uptake characteristics between the substrates, [3H]guanine uptake exhibited higher affinity and clearance than [3H]guanosine uptake in each type of cell. Although between neurons and astrocytes, there was no difference in the apparent uptake clearance for [3H]guanine and [3H]guanosine, which was calculated based upon the cellular protein content, the cellular uptake clearance was significantly greater in astrocytes than in neurons. These findings indicate that guanine and guanosine, of which the former is a preferable substrate, are taken up into both neurons and astrocytes via ENT2, and that the extracellular concentrations of guanine and guanosine are mainly regulated by astrocytes to maintain brain physiology.

Alteration of myo-inositol monophosphatase in Alzheimer's disease brains.

myo-Inositol monophosphatase (E.C.3.1.3.25) catalyzes the hydrolysis of myo-inositol 1-phosphate in the presence of Mg2+ at a physiologic pH to form free myo-inositol, maintaining a supply that represents the precursor for inositol phospholipid second messenger signaling systems. In the present study the activity and protein level of myo-inositol monophosphatase were investigated in samples from normal human and Alzheimers disease (AD) postmortem brains. The separation profile on Sephadex G-100 gel filtration chromatography revealed one major form of myo-inositol monophosphatase in crude extracts from both normal human and AD brains. In AD brains myo-inositol monophosphatase activity and its protein level were significantly higher than in control brains. The activity of myo-inositol monophosphatase per enzyme molecule was similar in control and AD brains. These results suggest that myo-inositol monophosphatase is upregulated in AD, probably reflecting compensatory mechanisms concerned with phospholipid metabolism.

Reduction of platelet phospholipase C activity in patients with Alzheimer disease.

Summary Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in signal transduction. We have previously demonstrated that a PLC isozyme is abnormally accumulated in the brain tissue in Alzheimers disease (AD). AD has been suggested to be a systemic disease in which the expression of abnormalities is most prominent in neuronal tissues. In a recent study, we have revealed the increase of the cytosolic protein kinase C (PKC) concentration in platelets of AD patients, suggesting the change of PLC, which is upstream to PKC in phosphoinositide metabolism. In this study, we examined phosphatidylinositol-specific PLC activity in platelets from patients with AD and age-matched controls by measuring the formation of radioactive inositol phosphate. The PLC activity was significantly lower in the AD platelets than in the controls. These findings suggest that aberrant phosphoinositide metabolism is present in nonneuronal tissues as well as the brain in AD.

The endogenous substrate of low molecular weight acid phosphatase in the brain is an epidermal growth factor receptor

We have recently reported that low molecular weight (LMW) acid phosphatase, which is supposed to possess phosphotyrosine protein phosphatase activity, showed a significant decrease of activity in Alzheimer brains compared to control brains [Ann. Neurol., 33 (1993) 616-621]. In the present study, we investigated the endogenous substrate of LMW acid phosphatase in the brain. LMW acid phosphatase was purified from bovine brain, and the enzyme was obtained with both a high specific activity and a good yield. The bovine brain enzyme was a monomer with a molecular mass of 17 kDa. We used a specific monoclonal anti-phosphotyrosine antibody to detect phosphotyrosine protein in rat brain extracts. The LMW acid phosphatase from bovine brain dephosphorylated a M(r) 170 kDa phosphotyrosine protein in rat brain extracts. This M(r) 170 kDa protein was considered to be the epidermal growth factor (EGF) receptor using a specific antibody. These results suggest that LMW acid phosphatase in the brain may regulate EGF receptor-dependent transmembrane signalling by dephosphorylating the phosphorylated receptor.