Robert B. Nelson

Anesthesia leads to tau hyperphosphorylation through inhibition of phosphatase activity by hypothermia.

Postoperative cognitive dysfunction, confusion, and delirium are common after general anesthesia in the elderly, with symptoms persisting for months or years in some patients. Even middle-aged patients are likely to have postoperative cognitive dysfunction for months after surgery, and Alzheimers disease (AD) patients appear to be particularly at risk of deterioration after anesthesia. Several investigators have thus examined whether general anesthesia is associated with AD, with some studies suggesting that exposure to anesthetics may increase the risk of AD. However, little is known on the biochemical consequences of anesthesia on pathogenic pathways in vivo. Here, we investigated the effect of anesthesia on tau phosphorylation and amyloid precursor protein (APP) metabolism in mouse brain. We found that, regardless of the anesthetic used, anesthesia induced rapid and massive hyperphosphorylation of tau, rapid and prolonged hypothermia, inhibition of Ser/Thr PP2A (protein phosphatase 2A), but no changes in APP metabolism or Aβ (β-amyloid peptide) accumulation. Reestablishing normothermia during anesthesia completely rescued tau phosphorylation to normal levels. Our results indicate that changes in tau phosphorylation were not a result of anesthesia per se, but a consequence of anesthesia-induced hypothermia, which led to inhibition of phosphatase activity and subsequent hyperphosphorylation of tau. These findings call for careful monitoring of core temperature during anesthesia in laboratory animals to avoid artifactual elevation of protein phosphorylation. Furthermore, a thorough examination of the effect of anesthesia-induced hypothermia on the risk and progression of AD is warranted.

A selective increase in phosphorylation of protein F1, a protein kinase C substrate, directly related to three day growth of long term synaptic enhancement

Increased in vitro phosphorylation of the 47 kdalton, 4.5 pI protein F1 was observed in dorsal hippocampal tissue from animals exhibiting long term enhancement (LTE) three days after high frequency stimulation of the perforant pathway, as compared to tissue from low frequency stimulated controls or from unoperated animals. The increase in protein F1 phosphorylation was related to LTE rather than simple activation of perforant path-dentate gyrus synapses. This is the first report of a change in brain protein phosphorylation accompanying synaptic enhancement lasting days. The extent of growth of LTE over the three days following stimulation was directly related (r = +0.66, P less than 0.05) to protein F1 phosphorylation. Among the phosphoproteins studied this relationship between LTE and phosphorylation was selective for protein F1. This suggests that protein F1 may regulate growth of synaptic plasticity for at least a three day period. The mechanism for the LTE-related increase in protein F1 phosphorylation has not been established. However, recent evidence from this laboratory indicates: that protein F1 is phosphorylated by the calcium/phospholipid-dependent protein kinase C; and that kinase C is activated 1 h after LTE. Therefore, the increase in protein F1 phosphorylation following LTE may result from long term activation of protein C kinase.

Direct relation of long-term synaptic potentiation to phosphorylation of membrane protein F1, a substrate for membrane protein kinase C

One hour after long-term potentiation (LTP) in the intact hippocampus, a selective increase in protein F1 in vitro phosphorylation was observed in homogenate prepared from dorsal hippocampus. Protein F1 phosphorylation was directly related to the magnitude and persistence of potentiation. No other phosphoprotein studied exhibited a relationship with synaptic enhancement. Low-frequency, non-potentiating stimulation did not increase protein F1 phosphorylation, and phosphorylation of F1 was not elevated when high-frequency stimulation did not produce potentiation. We also confirmed our earlier demonstration of a similar pattern of results 5 min after LTP. In related work we have previously observed: that protein F1 is a substrate for protein kinase C (PKC); that membrane PKC activity was increased by translocation from the cytosol following LTP; and that membrane PKC activity was directly related to the persistence of enhancement. We therefore predicted in the present study that protein F1 phosphorylation in a dorsal hippocampal membrane fraction would be related to LTP. Hippocampal membrane protein F1 was found to be directly related to both the magnitude and persistence of response enhancement. Thus the molecular events leading to prolonged potentiation may involve increased PKC/protein F1 association. Persistence of potentiation may be related to synaptic growth processes involving the growth-associated function of protein F1.

Gradients of protein kinase C substrate phosphorylation in primate visual system peak in visual memory storage areas

Two protein kinase C (PKC) substrates of 50 and 81 kDa display topographical gradients in 32P-incorporation along the occipitotemporal visual processing pathway in rhesus monkey cerebral cortex. The 50 kDa protein appears to be homologous to protein F1 from rat (47 kDa) on the basis of isoelectric point, two-dimensional phosphopeptide maps, and kinase specificity, while the 81 kDa protein is probably the same as a previously described PKC substrate. The phosphorylation of protein F1 and 81 kDa was significantly higher in temporal regions of the occipitotemporal pathway, which have been implicated in the storage of visual representations, than in occipital regions, which appear to be less important for visual memory functions. These results suggest that the PKC phosphorylation system, which has been related previously to changes in neural plasticity, plays a progressively greater role in later stages of visual processing, and that this role may involve the storage of visual information in inferotemporal cortical areas.

Selective decline in protein F1 phosphorylation in hippocampus of senescent rats.

Certain forms of neuronal plasticity have been found to be expressed through alterations in brain protein phosphorylation, and its regulation by protein kinase activity. Of interest in this regard is the possibility that the decline in neuronal plasticity and cognitive function that occurs in advanced age may result in part from altered phosphorylation of specific proteins. As a first attempt to identify age-related changes in phosphoproteins, we assayed in vitro phosphorylation of proteins in hippocampus, cerebellum, entorhinal cortex, and frontal cortex from Fischer-344 rats of 5 months, 11 months, and 25 months of age. Compared to the middle-aged animals, the aged rats showed a selective 46% decline in phosphorylation of the 47 kDa protein (F1) in hippocampus, with no change in the phosphorylation of other proteins measured in this structure. Aged animals also showed decreased phosphorylation relative to young animals. No age-related change was observed in any protein band for the other brain areas examined. Since protein F1 is phosphorylated by protein kinase C (PKC), the cytosolic and membrane distribution of this enzyme was compared across age groups. The activity of PKC in hippocampus did not change across age. The explanation of this age-related decline in protein F1 phosphorylation is likely to be a decline in the substrate protein itself. The results are discussed in terms of protein F1s possible role in age-related decline of hippocampal synaptic plasticity.

Phosphoproteins localized to presynaptic terminal linked to persistence of long-term potentiation (LTP): quantitative analysis of two-dimensional gels

Previous findings suggest: (1) that altering protein kinase C (PKC) activity alters the persistence of long-term potentiation (LTP) in the intact hippocampal formation; and (2) that PKC activity is directly correlated with persistence of LTP in vivo as measured by the in vitro phosphorylation of two major PKC substrates in adult hippocampus, protein F1 and 80k. Using quantitative analysis of two-dimensional gels, we report here two additional phosphoproteins of 72 and 55 kDa which were directly correlated to persistence of LTP induced in the intact dorsal hippocampal formation. The phosphorylation of both proteins in response to addition of different kinase stimulators was distinct from that of protein F1 and 80k. Moreover, neither protein was a substrate for exogenous PKC. The physicochemical properties of these phosphoproteins suggest they are identical to the previously described synaptic vesicle proteins IIIa and IIIb, and as such are immunologically indistinguishable. Because proteins IIIa and IIIb are known to be phosphorylated by a Ca2+/calmodulin (CaM)-stimulated kinase, and protein F1 is known to be a plasma membrane-associated protein (P-57) which releases bound CaM in response to phosphorylation by PKC, the present findings suggest a potential mechanism in which PKC-mediated changes in plasma membrane proteins produce CaM kinase-mediated changes in synaptic vesicle proteins through a phosphorylation cascade. These membrane/vesicle alterations are postulated to underlie the increased synaptic efficacy which marks persistent LTP.

Contrasting patterns of protein phosphorylation in human normal and Alzheimer brain : focus on protein kinase C and protein F1/GAP-43

We introduce a new procedure to study kinase substrates in postmortem human brain. By adding purified exogenous protein kinase C (PKC) and the phospholipid phosphatidylserine to brain homogenates in vitro we are able to analyze PKC substrates. A human 53-kDa phosphoprotein is described that appears to be homologous to rat and monkey protein F1 (GAP-43). This identity is based on molecular weight, isoelectric point, phosphorylation by exogenous protein kinase C, enhancement of its phosphorylation by three activators (phospholipids, calcium and phorbol esters), phosphopeptide maps, and cross-reactivity with an antibody raised against rat protein F1. Protein F1 is a PKC substrate associated with synaptic plasticity and nerve growth. Its phosphorylation in rat brain has been correlated with long-term potentiation, an electrophysiological model of memory. In the present study of normal brain, human protein F1 shows an occipitotemporal in vitro phosphorylation gradient. This is consistent with previous observations in nonhuman primates. This gradient is less pronounced in Alzheimers disease (AD). Changes in the in vitro phosphorylation pattern of three other non-PKC substrates in Alzheimers disease, including one with characteristics similar to microtubule-associated protein tau, are also reported. These results suggest that protein phosphorylation can be studied in postmortem human brain and that PKC-mediated phosphorylation of protein F1, already linked to synaptic plasticity and memory, may be altered in AD.

The protein F1/protein kinase C module and neurite growth: potential pathway for facilitating brain transplantation.

Publisher Summary This chapter describes that regulation of protein kinase C (PKC) substrates such as protein F1, may also prove to be effective in regulating neurite growth, reviews some evidence that protein F1 is identical to pp 46, GAP-43 and B-50, and considers the implications of these new findings for transplantation. PKC is a multifunctional enzyme with a long list of putative physiological substrates. It is important to investigate the relationship between successful brain transplants and PKC and its substrates. If PKC and protein F1 in the transplanted cells play a critical role in facilitating axonal outgrowth in the transplant, then manipulation of gene expression of cells prior to transplantation could prove to be of particular value. Thus, transfection of a given neuron with the cloned gene on a recombinant plasmid offers the possibility of a wider range of cell types from which to select for transplantation.