This study compared the relative levels of ryanodine receptor (RyR) isoforms, inositol 1,4,5-trisphosphate receptor (IP 3 R) isoforms, and calcineurin, plus their association with FKBP12 in brain, skeletal and cardiac tissue. FKBP12 demonstrated a very tight, high affinity association with skeletal muscle microsomes, which was displaced by FK506. In contrast, FKBP12 was not tightly associated with brain or cardiac microsomes and did not require FK506 for removal from these organelles. Furthermore, of the proteins solubilised from skeletal muscle, cardiac muscle and brain microsomes, only skeletal muscle RyR1 bound to an FKBP12^glutathione-S-transferase fusion protein, in a high affinity FK506 displaceable manner. These results suggest that RyR1 has distinctive FKBP12 binding properties when compared to RyR2, RyR3, all IP 3 R isoforms and calcineurin. ß

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The clinical symptoms of all forms of Alzheimer's disease (AD) result from a slowly progressive neurodegeneration that is associated with the excessive deposition of ϐ-amyloid (Aϐ) in plaques and in the cerebrovasculature, and the formation of intraneuronal neurofibrillary tangles, which are composed primarily of abnormally hyperphosphorylated tau protein. The sequence of cellular events that cause this pathology and neurodegeneration is unknown. It is, however, most probably linked to neuronal signal transduction systems that become misregulated in the brains of certain individuals, causing excessive Aϐ to be formed and/or deposited, tau to become aggregated and hyperphosphorylated and neurons to degenerate. We hypothesize that a progressive alteration in the ability of neurons to regulate intracellular calcium, particularly at the level of the endoplasmic reticulum, is a crucial signal transduction event that is linked strongly to the initiation and development of AD pathology. In this chapter we will discuss the key findings that lend support to this hypothesis.

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Ryanodine receptors (RyRs) regulate calcium release from the endoplamic reticulum (ER) in brain and are dysfunctional and progrcssivley impaired as A D advances. Alterations in RyRs are apparent at the very earliest stages of AD pathology, with an initial up-regulation of RyR observed selective to areas of the hippocampal formation which first succumb to Stage 1-11 (Braak and Braak's protocol) neurofibrillary pathology. Following on from this, a progressive decline in RyR binding levels is observed which correlates with the development stages of neurofibrillary pathology in the hippocampal formation. This eventually culminates in a severe 74-94% loss of RyR binding in hippocampus with late isocortical (Stage V-VI) neurofibrillary pathology when compared to early Stage 1-11 cases. Although the staged development of beta-amyloid pathology in the hippocampus is not as defined as that of neurofibrillary pathology, loss of RyR binding sites also Correlates with the development of beta-amyloid pathologies in the entorhinal cortex and CA4. These results associated with the observation that RyR binding levels are unaffected in brain areas which d o not display AD pathology, suggest that alterations in RyR may be mechanistically involved in the pathogenesis of AD. This may in part be due to loss of the RyR2 isoform detected at end-stages of AD. RyRs have also been found to be dysfuctional in AD, showing a significantly decreased ability to be inhibited in response to ruthenium red and magnesium. The FKBPlZ protein and full-length presenilin 2 are significantly down-regulated in A D cases with dysfunctional RyRs, suggesting that the the impairment in RyRs observed in A D brain may involve loss of appropriate control of RyR by these accessory proteins F18 Modelling Alzheimer's Disease in multiple transgenic mice: is it possible? -Transgenic mice with neuronal overexpression of human Amyloid Precursor Protein (APP) develop an AD related phenotype (Moechars et al., 1996 , EMBO J, 151265-1274 J Biol Chem. 1999.2746483-6492). Early symptoms (3-9 months) include: disturbed behavior, neophobia, aggression, hypersensitivity to kainic acid, hypo-sensitivity to NMDA, defective cognition and memory, decreased L P . Differences between transgenic strains with different APP isoforms are quantitative (intensity, severity, age of onset) and dircetly related to APP expression levels. Late in life (12-15 months) amyloid plaques develop in the brain of high-expressing APP/london mice and correlate with high AB40/42 levels. Formation of amyloid plaques is thereby dissociated in time from and not involved in the early phenotype. The amyloid plaques are ATEpositive, indicating presence of hyperphosphorylated protein tau but no tangle pathology is obvious. In double transgenic mice, i.e. APP/london x mutant Presenilin-1. amyloid plaques develop at age 6 months due to increased production of AB42 peptide. Single PSI tg mice have essentially no phenotypic abnormalities while PSI-deficiency is embryonically lethal and reduces amyloid peptide pro...

The immunophilin FKBP12 (FK506 Binding Rotcin, M.W. 12kDa) is expressed to high levels in brain where it plays a role in neuroprotdon and neuroregeneration. This protein also interfaces with and regulates the function of the ryanodine and inositol 1,4,5-triphosphate receptors and colocalises with calcinewin, all of which arc dysfunctional in Alzheimer's disease (AD). To examine the functional integrity of FKBPl2 in AD, the present study examined FKBP12 levels and [3H]FK506 binding in pathologically a f f d and unaffected areas of matched AD and control post mortem brain. Western immunoblotting analysis of soluble fractions detsdad a single immuwnaCtive band (14kDa) d g r a t i n g with pure human recombinant FKBP12. A significant and selective (P