Richard F. Cowburn

Distribution of active glycogen synthase kinase 3β (GSK-3β) in brains staged for Alzheimer disease neurofibrillary changes

Accumulation of paired helical filaments (PHFs) in neurofibrillary tangles, neuropil threads, and dystrophic neurites is one of the major ncuropathological hallmarks of Alzheimer disease (AD). The principal protein subunit of PHFs is the abnormally hyperphosphorylated tau. Glycogen synthase kinase 3B (GSK-3B) is one of the candidate kinases involved in PHF-tau formation. To play a role in PHF-tau formation, it would be expected that GSK-3B is active in tangle bearing neurons. In the present study, we investigated the regional and intracellular distributions of active and inactive forms of GSK-3B in brains staged for neurofibrillary changes. We found that neurons with tangle-like inclusions positive for active, but not inactive, GSK-3β appear initially in the Pre-α layer of the entorhinal cortex and extend to other brain regions, coincident with the sequence of the development of neurofibrillary changes. Active, but not inactive, GSK-3β was found to initially accumulate in the cytoplasm of pretangle neurons. These data provide direct in situ evidence that is consistent with the involvement of GSK-3β in PHF-tau formation.

A mutation in Alzheimer's disease destroying a splice acceptor site in the presenilin-1 gene

A series of mutations has been reported in the presenilin-1 (PS-1) gene which cause early onset Alzheimers disease (AD). The mutations reported to date have encoded missense mutations which alter residues conserved between PS-1 and the presenilin-2 (PS-2) gene. We have recently determined the intron/exon structure of the PS-1 gene and this information has been used to identify a mutation in the splice acceptor site for exon 9 in a family with early onset AD. Amplification of cDNA from lymphoblasts of affected individuals revealed that the effect of the mutation was to cause splicing out of exon 9, however it does not change the open reading frame of the mRNA. The importance of this observation is discussed.

Accumulation of cyclin-dependent kinase 5 (cdk5) in neurons with early stages of Alzheimer's disease neurofibrillary degeneration

Cyclin-dependent kinase 5 (cdk5) is one of the candidate kinases involved in the abnormal hyperphosphorylation of tau. To have a direct effect on tau hyperphosphorylation, cdk5 protein levels and enzyme activity should be upregulated in especially those neurons that develop neurofibrillary tangles (NFTs). We studied the distribution of cdk5 immunoreactivity in neurons with or without early- and late-stage NFTs in hippocampal, entorhinal, transentorhinal, temporal and frontal cortices, and cerebellum of Alzheimers disease (AD) and control brain. The immunocytochemical localisation of cdk5 was compared with that obtained using antibodies to PHF-tau (tau in paired helical filaments of NFTs, mAb AT8) and ubiquitin as markers of early and late stage NFTs, respectively. Immunoreactivities of cdk5 and PHF-tau were found in neuronal perikarya and processes of hippocampal, entorhinal, transentorhinal, temporal and frontal, and cerebellar cortices. An apparent increase of cdk5 immunoreactivity was seen in pretangle neurons and in neurons bearing early stage NFTs. These findings suggest that this kinase might be involved in the formation of NFTs at a relatively early stage in the neocortex.

Up-Regulation of Phosphorylated/Activated p70 S6 Kinase and Its Relationship to Neurofibrillary Pathology in Alzheimer’s Disease

The ribosomal S6 protein kinase p70 S6 kinase is known for its role in modulating cell-cycle progression, cell size, and cell survival. In response to mitogen stimulation, p70 S6 kinase activation up-regulates ribosomal biosynthesis and enhances the translational capacity of the cell. In Alzheimers disease (AD), there is a marked increase in total tau protein in the form of abnormally hyperphosphorylated tau (PHF-tau) in neurons with neurofibrillary tangles (NFTs). In the present study, we investigated whether p70 S6 kinase activation is associated with PHF-tau accumulation in AD. By immunohistochemistry, we found that the levels of phosphorylated p70 S6 kinase (at Thr389 or at Thr421/Ser424) were increased in accordance with the progressive sequence of neurofibrillary changes according to Braaks criteria. Confocal microscopy showed that in AD brain, phosphorylated p70 S6 kinase appeared especially in neurons that are known to later develop NFTs. This pattern of neurons showed dot-like structures of phosphorylated p70 S6 kinase and hyperphosphorylated tau, which partially correlated with rab5 (endosome marker), lamp-1 (lysosome marker), and ubiquitin (ubiquitin-proteasomal system marker). By indirect enzyme-linked immunosorbent assay, phosphorylated p70 S6 kinase (Thr389 or Thr421/Ser424), total tau, and PHF-tau were found to be significantly increased in AD brain as compared to control cases. The levels of total p70 S6 kinase and p70 S6 kinase phosphorylated at Thr421/Ser424 showed significant correlations with the levels of both total tau and PHF-tau. Regression analyses revealed a significant dependence of total tau or PHF-tau on p70 S6 kinase phosphorylated at Thr421/Ser424 rather than at Thr389. The levels of ribosomal protein S6 as well as the levels of markers for the proteolytic system were also significantly increased in AD as compared to control brain. Using a SH-SY5Y neuroblastoma cell model, we found that 100 micro mol/L zinc sulfate could induce p70 S6 kinase phosphorylation and activation, in particular at Thr421/Ser424. This up-regulation of the activated kinase resulted in an increased expression and phosphorylation of tau. Pretreatment of cells with rapamycin (an inhibitor of FRAP/mTOR which is the immediate upstream kinase of the p70 S6 kinase) attenuated the effects induced by zinc. In primary cultured neurons of rat cortical cortex, zinc sulfate treatment could repeat p70 S6 kinase phosphorylation and activation at Thr421/Ser424, followed by increased expression and phosphorylation of tau. Taken together, these data suggest that activated p70 S6 kinase could mediate an up-regulation of tau translation. The partial co-localization of phosphorylated p70 S6 kinase with rab5, lamp-1 and ubiquitin, or PHF-tau with ubiquitin suggests that the activated proteolytic system might not be sufficient to degrade the over-produced and over-phosphorylated tau protein. A p70 S6 kinase modulated up-regulation of tau translation might contribute to PHF-tau accumulation in neurons with neurofibrillary changes.

Up-regulation of mitogen-activated protein kinases ERK1/2 and MEK1/2 is associated with the progression of neurofibrillary degeneration in Alzheimer’s disease

The abnormal hyperphosphorylation of tau in Alzheimers disease (AD) has been proposed to involve the extracellular-signal-regulated protein kinase (ERK) of the mitogen-activated protein (MAP) kinase family. ERK is phosphorylated and thereby activated by MAP kinase kinase (MEK). In the present study, we determined the intracellular and regional distribution of the active forms of both MEK1/2 and ERK1/2, i.e. p-MEK1/2 and p-ERK1/2 in the entorhinal, hippocampal, and temporal cortices of 49 brains staged for neurofibrillary changes according to Braak and Braaks protocol. We found that p-MEK1/2 and p-ERK1/2 were present in the initial stages of neurofibrillary degeneration in the projecting neurons in the transentorhinal region, and extended into other brain regions co-incident with the progressive sequence of neurofibrillary changes up to and including Braak stage VI. It appeared that the accumulation of p-MEK1/2 and p-ERK1/2 was initiated in the cytoplasm of pretangle neurons in varying size granules, which grew into large aggregates co-existing with the progressive development of neurofibrillary tangles. Accumulation of p-MEK1/2 and p-ERK1/2 was found in cases with stages I-III neurofibrillary degeneration, which were devoid of amyloid deposition. These data provide direct in situ evidence consistent with the possible involvement of MAP kinase pathway in the hyperphosphorylation of tau and the presence of this lesion before deposition of beta-amyloid in AD.

Mitochondrial Alterations in PINK1 Deficient Cells Are Influenced by Calcineurin-Dependent Dephosphorylation of Dynamin-Related Protein 1

PTEN-induced novel kinase 1 (PINK1) mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1) exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium-dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential.

Localization of active forms of C-jun kinase (JNK) and p38 kinase in Alzheimer's disease brains at different stages of neurofibrillary degeneration.

The principal protein component of paired helical filaments (PHFs) in Alzheimer disease is abnormally hyperphosphorylated tau (PHF-tau). The stress activated protein kinases JNK and p38 have been shown to phosphorylate tau at some sites only seen in PHF-tau. If JNK and p38 are involved in the abnormal hyperphosphorylation of tau, they should be activated in neurons undergoing neurofibrillary degeneration. In the present study, we determined the intracellular and regional distribution of the active forms of JNK and p38 kinase in entorhinal, hippocampal, and temporal cortices of brains staged for neurofibrillary changes according to Braak and Braak. Neurons with tangle-like inclusions positive for active forms of JNK and p38 kinase were found to appear first in the Pre-alpha layer of the entorhinal cortex, and then extend into other brain regions co-incident with the progressive sequence of neurofibrillary changes. The intraneuronal accumulation of active forms of JNK and p38 kinase apeared to precede the deposition of amyloid in the extracellular space. These data indicate that increased activation of the stress related kinases JNK and p38 occurs very early in the disease and might be involved in the intraneuronal protein phosphorylation/dephosphorylation imbalance that leads to neurofibrillary degeneration in Alzheimer disease.

Role of protein kinase B in Alzheimer's neurofibrillary pathology

Protein kinase B (PKB) is an important intermediate in the phosphatidylinositol-3 kinase signaling cascade that acts to phosphorylate glycogen synthase kinase-3 (GSK-3) at its serine 9 residue, thereby inactivating it. Activated GSK-3 has been previously shown to be preferentially associated with neurofibrillary tangles (NFTs) in Alzheimers disease (AD) brain. In the present study, we performed immunohistochemistry with an antibody to the active form of PKB in brains with different stages of neurofibrillary degeneration. We found that the amount of activated PKB (p-Thr308) increased in correlation to the progressive sequence of AT8 immunoreactivity and neurofibrillary changes assessed according to Braaks criteria. By confocal microscopy, activated PKB (p-Thr308) was found to appear in particular in neurons that are known to later develop NFTs in AD. Western blotting showed that activated PKB was increased by more than 50% in the 16,000-g supernatants of AD brains as compared with normal aged and Huntingtons disease controls. This increase in PKB levels corresponded with a several-fold increase in the levels of total tau and abnormally hyperphosphorylated tau at the Tau-1 site. These studies suggest the involvement of PKB/GSK-3 signaling in Alzheimer neurofibrillary degeneration.

Region-specific loss of glutamate innervation in Alzheimer's disease

Synaptosomal D-aspartate has been used as a marker for glutamate neurons in control and in postmortem Alzheimers disease brains. This technique shows a marked (60%) decrease of the glutamate uptake site in cortical and hippocampal regions. There were no significant changes in subcortical regions. We interpret these results as indicating loss of, or damage to, cortical glutamatergic innervation. These losses probably represent the biochemical correlate of pyramidal neuron damage in Alzheimers disease.

Okadaic-Acid-Induced Inhibition of Protein Phosphatase 2A Produces Activation of Mitogen-Activated Protein Kinases ERK1/2, MEK1/2, and p70 S6, Similar to That in Alzheimer’s Disease

In Alzheimers disease (AD) brain the activity of protein phosphatase (PP)-2A is compromised and that of the extracellular signal-regulated protein kinase (ERK1/2) of the mitogen-activated protein kinase (MAPK) family, which can phosphorylate tau, is up-regulated. We investigated whether a decrease in PP-2A activity could underlie the activation of these kinases and the abnormal hyperphosphorylation of tau. Rat brain slices, 400-microm-thick, kept under metabolically active conditions in oxygenated (95% O(2), 5% CO(2)) artificial CSF were treated with 1.0 micromol/L okadaic acid (OA) for 1 hour at 33 degrees C. Under this condition, PP-2A activity was decreased to approximately 35% of the vehicle-treated control slices, and activities of PP-1 and PP-2B were not affected. In the OA-treated slices, we observed a dramatic increase in the phosphorylation/activation of ERK1/2, MEK1/2, and p70 S6 kinase both immunohistochemically and by Western blots using phosphorylation-dependent antibodies against these kinases. Treatment of 6-microm sections of the OA-treated slices with purified PP-2A reversed the phosphorylation/activation of these kinases. Hyperphosphorylation of tau at several abnormal hyperphosphorylation sites was also observed, as seen in AD brain. These results suggest 1) that PP-2A down-regulates ERK1/2, MEK1/2, and p70 S6 kinase activities through dephosphorylation at the serine/threonine residues of these kinases, and 2) that in AD brain the decrease in PP-2A activity could have caused the activation of ERK1/2, MEK1/2, and p70 S6 kinase, and the abnormal hyperphosphorylation of tau both via an increase in its phosphorylation and a decrease in its dephosphorylation.