A simple high throughput assay to evaluate water consumption in the fruit fly.

Water intake is essential for survival and thus under strong regulation. Here, we describe a simple high throughput system to monitor water intake over time in Drosophila. The design of the assay involves dehydrating fly food and then adding water back separately so flies either eat or drink. Water consumption is then evaluated by weighing the water vessel and comparing this back to an evaporation control. Our system is high throughput, does not require animals to be artificially dehydrated, and is simple both in design and implementation. Initial characterisation of homeostatic water consumption shows high reproducibility between biological replicates in a variety of experimental conditions. Water consumption was dependent on ambient temperature and humidity and was equal between sexes when corrected for mass. By combining this system with the Drosophila genetics tools, we could confirm a role for ppk28 and DopR1 in promoting water consumption, and through functional investigation of RNAseq data from dehydrated animals, we found DopR1 expression in the mushroom body was sufficient to drive consumption and enhance water taste sensitivity. Together, we provide a simple high throughput water consumption assay that can be used to dissect the cellular and molecular machinery regulating water homeostasis in Drosophila.

Sucralose Promotes Food Intake through NPY and a Neuronal Fasting Response.

Non-nutritive sweeteners like sucralose are consumed by billions of people. While animal and human studies have demonstrated a link between synthetic sweetener consumption and metabolic dysregulation, the mechanisms responsible remain unknown. Here we use a diet supplemented with sucralose to investigate the long-term effects of sweet/energy imbalance. In flies, chronic sweet/energy imbalance promoted hyperactivity, insomnia, glucose intolerance, enhanced sweet taste perception, and a sustained increase in food and calories consumed, effects that are reversed upon sucralose removal. Mechanistically, this response was mapped to the ancient insulin, catecholamine, and NPF/NPY systems and the energy sensor AMPK, which together comprise a novel neuronal starvation response pathway. Interestingly, chronic sweet/energy imbalance promoted increased food intake in mammals as well, and this also occurs through an NPY-dependent mechanism. Together, our data show that chronic consumption of a sweet/energy imbalanced diet triggers a conserved neuronal fasting response and increases the motivation to eat.

PI4KIIα phosphorylation by GSK3 directs vesicular trafficking to lysosomes.

Glycogen synthase kinase 3 (GSK3) is essential for normal development and function of the central nervous system. It is especially important for regulating neurotransmission, although the downstream substrates mediating this function are not yet clear. In the present paper, we report the lipid kinase phosphatidylinositol 4-kinase II α (PI4KIIα) is a novel substrate of GSK3 that regulates trafficking and cell-surface expression of neurotransmitter receptors in neurons. GSK3 phosphorylates two distinct sites in the N-terminus of PI4KIIα (Ser5 and Ser47), promoting binding to the adaptor protein 3 (AP-3) complex for trafficking to the lysosome to be degraded. Blocking phosphorylation reduces trafficking to the lysosome, stabilizing PI4KIIα and its cargo proteins for redistribution throughout the cell. Importantly, a reduction in PI4KIIα expression or phosphorylation increases α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor expression at the surface of hippocampal neurons. These studies implicate signalling between GSK3 and PI4KIIα as a novel regulator of vesicular trafficking and neurotransmission in the brain.

Parkinson's disease-linked human PARK9/ATP13A2 maintains zinc homeostasis and promotes α-Synuclein externalization via exosomes.

α-Synuclein plays a central causative role in Parkinson's disease (PD). Increased expression of the P-type ATPase ion pump PARK9/ATP13A2 suppresses α-Synuclein toxicity in primary neurons. Our data indicate that ATP13A2 encodes a zinc pump; neurospheres from a compound heterozygous ATP13A2(-/-) patient and ATP13A2 knockdown cells are sensitive to zinc, whereas ATP13A2 over-expression in primary neurons confers zinc resistance. Reduced ATP13A2 expression significantly decreased vesicular zinc levels, indicating ATP13A2 facilitates transport of zinc into membrane-bound compartments or vesicles. Endogenous ATP13A2 localized to multi-vesicular bodies (MVBs), a late endosomal compartment located at the convergence point of the endosomal and autophagic pathways. Dysfunction in MVBs can cause a range of detrimental effects including lysosomal dysfunction and impaired delivery of endocytosed proteins/autophagy cargo to the lysosome, both of which have been observed in cells with reduced ATP13A2 function. MVBs also serve as the source of intra-luminal nanovesicles released extracellularly as exosomes that can contain a range of cargoes including α-Synuclein. Elevated ATP13A2 expression reduced intracellular α-Synuclein levels and increased α-Synuclein externalization in exosomes >3-fold whereas ATP13A2 knockdown decreased α-Synuclein externalization. An increased export of exosome-associated α-Synuclein may explain why surviving neurons of the substantia nigra pars compacta in sporadic PD patients were observed to over-express ATP13A2. We propose ATP13A2's modulation of zinc levels in MVBs can regulate the biogenesis of exosomes capable of containing α-Synuclein. Our data indicate that ATP13A2 is the first PD-associated gene involved in exosome biogenesis and indicates a potential neuroprotective role of exosomes in PD.

Glycogen synthase kinase 3 substrates in mood disorders and schizophrenia.

The dominant genetic and environmental causes of mood disorders and schizophrenia have not been forthcoming, so alternative approaches are required to elucidate the mechanisms underlying these diseases and to develop improved treatments for use in the clinic. Pharmacological evidence implicates glycogen synthase kinase 3 (GSK3) as a key target of current therapeutics, and this is well supported by genetic studies in animal models. Several upstream regulators of GSK3 are also genetically associated with mood disorders and schizophrenia, further suggesting convergence on GSK3 signalling. Whereas pathways upstream of GSK3 are being elucidated, relatively little progress has been made in identifying targets downstream of GSK3 that mediate its functional effects. This is important, because these substrates themselves could become next-generation therapeutic targets that are more potent and specific than current therapeutics targeting GSK3. Here, a few likely candidates and their connection to mood disorders and schizophrenia are discussed.

GSK3 as a Sensor Determining Cell Fate in the Brain.

Glycogen synthase kinase 3 (GSK3) is an unusual serine/threonine kinase that controls many neuronal functions, including neurite outgrowth, synapse formation, neurotransmission, and neurogenesis. It mediates these functions by phosphorylating a wide range of substrates involved in gene transcription, metabolism, apoptosis, cytoskeletal dynamics, signal transduction, lipid membrane dynamics, and trafficking, amongst others. This complicated list of diverse substrates generally follow a more simple pattern: substrates negatively regulated by GSK3-mediated phosphorylation favor a proliferative/survival state, while substrates positively regulated by GSK3 favor a more differentiated/functional state. Accordingly, GSK3 activity is higher in differentiated cells than undifferentiated cells and physiological (Wnt, growth factors) and pharmacological inhibitors of GSK3 promote the proliferative capacity of embryonic stem cells. In the brain, the level of GSK3 activity influences neural progenitor cell proliferation/differentiation in neuroplasticity and repair, as well as efficient neurotransmission in differentiated adult neurons. While defects in GSK3 activity are unlikely to be the primary cause of neurodegenerative diseases, therapeutic regulation of its activity to promote a proliferative/survival versus differentiated/mature functional environment in the brain could be a powerful strategy for treatment of neurodegenerative and other mental disorders.

CRMP2 hyperphosphorylation is characteristic of Alzheimer's disease and not a feature common to other neurodegenerative diseases.

Collapsin response mediator protein 2 (CRMP2) is an abundant brain-enriched protein that regulates neurite outgrowth. It is phosphorylated by Cdk5 and GSK3, and these modifications are abnormally high in the brains of Alzheimer's disease (AD) patients. Increased phosphorylation of CRMP2 is also apparent in mouse models of AD that express mutated AßPP and PSEN1, but not AßPP or tau alone, where it is detectable before the appearance of amyloid plaques and neurofibrillary tangles, suggesting it is an early event in AD pathogenesis. Here, we have extended these observations by showing that CRMP2 is not hyperphosphorylated in mice overexpressing mutated PSEN1 alone, or in cultured neurons treated with soluble, oligomeric Aß42 peptide. Similarly, CRMP2 phosphorylation was not increased in a mouse model of severe neurodegeneration (PMSC-1 knockout) or in cultured neurons subjected to neurotoxic concentrations of NMDA or staurosporine. Most interestingly, CRMP2 phosphorylation was not increased in frontal cortex from patients with frontotemporal lobar degeneration associated with mutations in MAPT or with Pick bodies. Together, these observations are consistent with the hypothesis that abnormal phosphorylation of CRMP2 is specific to AD and occurs downstream of excessive processing of AßPP, but that neither excessive Aß42 peptide nor neurotoxicity alone are sufficient to promote hyperphosphorylation.

Bioinformatic prediction and confirmation of beta-adducin as a novel substrate of glycogen synthase kinase 3.

It is important to identify the true substrates of protein kinases because this illuminates the primary function of any kinase. Here, we used bioinformatics and biochemical validation to identify novel brain substrates of the Ser/Thr kinase glycogen synthase kinase 3 (GSK3). Briefly, sequence databases were searched for proteins containing a conserved GSK3 phosphorylation consensus sequence ((S/T)PXX(S/T)P or (S/T)PXXX(S/T)P), as well as other criteria of interest (e.g. brain proteins). Importantly, candidates were highlighted if they had previously been reported to be phosphorylated at these sites by large-scale phosphoproteomic studies. These criteria identified the brain-enriched cytoskeleton-associated protein ß-adducin as a likely substrate of GSK3. To confirm this experimentally, it was cloned and subjected to a combination of cell culture and in vitro kinase assays that demonstrated direct phosphorylation by GSK3 in vitro and in cells. Phosphosites were mapped to three separate regions near the C terminus and confirmed using phosphospecific antibodies. Prior priming phosphorylation by Cdk5 enhanced phosphorylation by GSK3. Expression of wild type, but not non-phosphorylatable (GSK3 insensitive), ß-adducin increased axon and dendrite elongation in primary cortical neurons. Therefore, phosphorylation of ß-adducin by GSK3 promotes efficient neurite outgrowth in neurons.

Identification of a proline-rich inositol polyphosphate 5-phosphatase (PIPP)•collapsin response mediator protein 2 (CRMP2) complex that regulates neurite elongation.

Neuron polarization is essential for the formation of one axon and multiple dendrites, establishing the neuronal circuitry. Phosphoinositide 3-kinase (PI3K) signaling promotes axon selection and elongation. Here we report in hippocampal neurons siRNA knockdown of the proline-rich inositol polyphosphate 5-phosphatase (PIPP), which degrades PI3K-generated PtdIns(3,4,5)P(3), results in multiple hyperelongated axons consistent with a polarization defect. We identify collapsin response mediator protein 2 (CRMP2), which regulates axon selection by promoting WAVE1 delivery via Kinesin-1 motors to the axon growth cone, as a PIPP-interacting protein by Y2H screening, direct binding studies, and coimmunoprecipitation of an endogenous PIPP, CRMP2, and Kinesin-1 complex from brain lysates. The C-terminal growth cone-targeting domain of PIPP facilitates its interaction with CRMP2. PIPP growth cone localization is CRMP2-dependent. PIPP knockdown in PC12 cells promotes neurite elongation, WAVE1 and Kinesin-1 growth cone localization, whereas knockdown of CRMP2 exhibits the opposite phenotype, with shorter neurites and decreased WAVE1/Kinesin-1 at the growth cone. In contrast, CRMP2 overexpression promotes neurite elongation, a phenotype rescued by full-length PIPP, or expression of the CRMP2-binding PIPP domain. Therefore this study identifies PIPP and CRMP2 exert opposing roles in promoting axon selection and neurite elongation and the complex between these proteins serves to regulate the localization of effectors that promote neurite extension.

Scapinin-induced inhibition of axon elongation is attenuated by phosphorylation and translocation to the cytoplasm.

Scapinin is an actin- and PP1-binding protein that is exclusively expressed in the brain; however, its function in neurons has not been investigated. Here we show that expression of scapinin in primary rat cortical neurons inhibits axon elongation without affecting axon branching, dendritic outgrowth, or polarity. This inhibitory effect was dependent on its ability to bind actin because a mutant form that does not bind actin had no effect on axon elongation. Immunofluorescence analysis showed that scapinin is predominantly located in the distal axon shaft, cell body, and nucleus of neurons and displays a reciprocal staining pattern to phalloidin, consistent with previous reports that it binds actin monomers to inhibit polymerization. We show that scapinin is phosphorylated at a highly conserved site in the central region of the protein (Ser-277) by Cdk5 in vitro. Expression of a scapinin phospho-mimetic mutant (S277D) restored normal axon elongation without affecting actin binding. Instead, phosphorylated scapinin was sequestered in the cytoplasm of neurons and away from the axon. Because its expression is highest in relatively plastic regions of the adult brain (cortex, hippocampus), scapinin is a new regulator of neurite outgrowth and neuroplasticity in the brain.

Dynamin I phosphorylation by GSK3 controls activity-dependent bulk endocytosis of synaptic vesicles.

Glycogen synthase kinase 3 (GSK3) is a critical enzyme in neuronal physiology; however, it is not yet known whether it has any specific role in presynaptic function. We found that GSK3 phosphorylates a residue on the large GTPase dynamin I (Ser-774) both in vitro and in primary rat neuronal cultures. This was dependent on prior phosphorylation of Ser-778 by cyclin-dependent kinase 5. Using both acute inhibition with pharmacological antagonists and silencing of expression with short hairpin RNA, we found that GSK3 was specifically required for activity-dependent bulk endocytosis (ADBE) but not clathrin-mediated endocytosis. Moreover we found that the specific phosphorylation of Ser-774 on dynamin I by GSK3 was both necessary and sufficient for ADBE. These results demonstrate a presynaptic role for GSK3 and they indicate that a protein kinase signaling cascade prepares synaptic vesicles for retrieval during elevated neuronal activity.

PCTK proteins: the forgotten brain kinases?

PCTAIRE kinases (PCTKs) are highly conserved serine/threonine kinases that are closely related to cyclin-dependent kinases. They are enriched in post-mitotic neurons of adult brains, suggesting they might perform important neuron-specific functions independent of the cell cycle. So far, the biological functions of PCTKs in the brain have been largely neglected and remain to be discovered. This review summarises preliminary investigations into the expression and characterisation of PCTK kinase activity, providing a basis for further investigations. In particular, it identifies three key areas of priority for further research: (1) do PCTKs require activating phosphorylation by an upstream kinase or binding to an essential co-factor for maximal kinase activity; (2) what are the physiological substrates of PCTKs, and (3) what is the functional effect of PCTK kinase activity in primary neurons?

Measuring GSK3 expression and activity in cells.

Glycogen synthase kinase (GSK)-3 is a key signalling intermediate in the action ofWnts. This protein kinase is ubiquitously expressed and has high inherent activity but is inhibited by activation of Wnt signalling or activation of growth factor receptor tyrosine kinases (e.g. insulin, nerve growth factor [NGF], platelet-derived growth factor [PDGF], etc.). The degree of inhibition of GSK3 in cells treated with such reagents is dependent on the cell type and the stimulus used. Therefore, the ability to accurately measure GSK3 activity in cells is an important aspect of GSK3 research. The activity of GSK3 is reduced by posttranslational modification (phosphorylation) and this can be measured by immunoblot with specific reagents (indirect), or by immunoprecipitation and assay (direct), as long as the modification is protected during these procedures. However, inhibition by phosphorylation is specific to cellular activation by growth factors and nutrients. Wnt inhibition of GSK3 does not involve phosphorylation of these residues on GSK3 and therefore it cannot be measured using this modification. Currently, the simplest way to assess Wnt inhibition of GSK3 is to monitor phosphorylation of specific GSK3 substrates in cells (e.g. beta-catenin). Alternatively, Wnt inhibition of GSK3 can be measured by partial purification of cellular GSK3 by ion exchange chromatography and assay of fractions or possibly by immunoprecipitation and assay. In this chapter, we demonstrate the use of the different approaches to measure GSK3 activity in SH-SY5Y cells, describe the best antibodies currently available, and discuss the potential drawbacks of each method.

Relative resistance of Cdk5-phosphorylated CRMP2 to dephosphorylation.

Collapsin response mediator protein 2 (CRMP2) binds to microtubules and regulates axon outgrowth in neurons. This action is regulated by sequential phosphorylation by the kinases cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3 (GSK3) at sites that are hyperphosphorylated in Alzheimer disease. The increased phosphorylation in Alzheimer disease could be due to increases in Cdk5 and/or GSK3 activity or, alternatively, through decreased activity of a CRMP phosphatase. Here we establish that dephosphorylation of CRMP2 at the residues targeted by GSK3 (Ser-518/Thr-514/Thr-509) is carried out by a protein phosphatase 1 family member in vitro, in neuroblastoma cells, and primary cortical neurons. Inhibition of GSK3 activity using insulin-like growth factor-1 or the highly selective inhibitor CT99021 causes rapid dephosphorylation of CRMP2 at these sites. In contrast, pharmacological inhibition of Cdk5 using purvalanol results in only a gradual and incomplete dephosphorylation of CRMP2 at the site targeted by Cdk5 (Ser-522), suggesting a distinct phosphatase targets this residue. A direct comparison of dephosphorylation at the Cdk5 versus GSK3 sites in vitro shows that the Cdk5 site is comparatively resistant to phosphatase treatment. The presence of the peptidyl-prolyl isomerase enzyme, Pin1, does not affect dephosphorylation of Ser-522 in vitro, in cells, or in Pin1 transgenic mice. Instead, the relatively high resistance of this site to phosphatase treatment is at least in part due to the presence of basic residues located nearby. Similar sequences in Tau are also highly resistant to phosphatase treatment. We propose that relative resistance to phosphatases might be a common feature of Cdk5 substrates and could contribute to the hyperphosphorylation of CRMP2 and Tau observed in Alzheimer disease.

Novel procedure to investigate the effect of phosphorylation on protein complex formation in vitro and in cells.

The identification of phosphorylation state-dependent interacting proteins provides clues as to the function of the phosphorylation. Techniques such as yeast two hybrid and co-immunoprecipitation do not employ a single species of fully phosphorylated proteins. This is a particular problem for substrates of glycogen synthase kinase-3 (GSK3), where multiple Ser/Thr residues can be targeted, almost always subsequent to a priming phosphorylation by an alternative kinase. We previously identified the brain enriched collapsin response mediator proteins (CRMP2 and CRMP4) as physiological substrates of GSK3. Cdk5 phosphorylates CRMP2 at Ser522, priming for subsequent phosphorylation at three residues by GSK3 in vitro and in vivo. It is clear that phosphorylation of CRMP2 influences axonal growth; however, the molecular processes underlying this action are not fully established. In addition, the role of phosphorylation in other actions of CRMPs has not been elucidated. We developed a novel procedure to isolate CRMP2 and CRMP4 fully phosphorylated at four sites, namely, Ser522 (by CDK5), Ser518, Thr514, and Thr509 (by GSK3). These phosphoproteins were then used to identify binding partners in rat brain lysates in direct comparison with the non-phosphorylated isoforms. We validated the approach by confirming that a previously reported interaction with tubulin-beta is regulated by phosphorylation. We also show that CRMPs (CRMP1, CRMP2, and CRMP4) form heteromers and found that these complexes may also be regulated by phosphorylation. We identified DYRK and Pin1 as novel CRMP4 binding proteins with DYRK interacting preferentially with dephospho-CRMP4 and Pin1 with phospho-CRMP4. Finally, we used this approach to identify the mitochondrial protein ANT as a novel CRMP2 and CRMP4 binding protein. We believe that this approach could be applied generally to the study of phosphorylation-dependent interactions.

Collapsin response mediator protein-2 hyperphosphorylation is an early event in Alzheimer's disease progression.

Collapsin response mediator protein 2 (CRMP2) is an abundant brain-enriched protein that can regulate microtubule assembly in neurons. This function of CRMP2 is regulated by phosphorylation by glycogen synthase kinase 3 (GSK3) and cyclin-dependent kinase 5 (Cdk5). Here, using novel phosphospecific antibodies, we demonstrate that phosphorylation of CRMP2 at Ser522 (Cdk5-mediated) is increased in Alzheimer's disease (AD) brain, while CRMP2 expression and phosphorylation of the closely related isoform CRMP4 are not altered. In addition, CRMP2 phosphorylation at the Cdk5 and GSK3 sites is increased in cortex and hippocampus of the triple transgenic mouse [presenilin-1 (PS1)(M146V)KI; Thy1.2-amyloid precursor protein (APP)(swe); Thy1.2tau(P301L)] that develops AD-like plaques and tangles, as well as the double (PS1(M146V)KI; Thy1.2-APP(swe)) transgenic mouse. The hyperphosphorylation is similar in magnitude to that in human AD and is evident by 2 months of age, ahead of plaque or tangle formation. Meanwhile, there is no change in CRMP2 phosphorylation in two other transgenic mouse lines that display elevated amyloid beta peptide levels (Tg2576 and APP/amyloid beta-binding alcohol dehydrogenase). Similarly, CRMP2 phosphorylation is normal in hippocampus and cortex of Tau(P301L) mice that develop tangles but not plaques. These observations implicate hyperphosphorylation of CRMP2 as an early event in the development of AD and suggest that it can be induced by a severe APP over-expression and/or processing defect.

Molecular connexions between dementia and diabetes.

Recent evidence suggests that the molecular defects associated with the development of diabetes also contribute to an increased risk of all types of dementia, including Alzheimer's disease, vascular dementia and Pick's disease. Indeed, the presence of type II diabetes mellitus results in a two to three fold higher risk of developing dementia [Fontbonne et al., 2001. Changes in cognitive abilities over a 4-year period are unfavourably affected in elderly diabetic subjects: results of the Epidemiology of Vascular Aging Study. Diabetes Care 24, 366-370; Gregg et al., 2000. Is diabetes associated with cognitive impairment and cognitive decline among older women? Study of Osteoporotic Fractures Research Group. Archives of Internal Medicine 160, 174-180; Peila et al., 2002. Type 2 diabetes, APOE gene, and the risk for dementia and related pathologies: The Honolulu-Asia Aging Study. Diabetes 51, 1256-1262]. There are currently 250 million people worldwide (>2 million in the UK) diagnosed with diabetes, and this number is predicted to double within the next 20 years, therefore the associated risk translates into a potential explosion in the appearance of dementia in the population. This review primarily focuses on the proposed molecular links between insulin action, Diabetes and Alzheimer's disease, while discussing the potential for therapeutic intervention to alleviate these disorders. In particular, we will review the regulation of glycogen synthase kinase-3 (GSK-3) and its neuronal substrates.

Distinct priming kinases contribute to differential regulation of collapsin response mediator proteins by glycogen synthase kinase-3 in vivo.

Collapsin response mediator proteins (CRMPs) are a family of neuron-enriched proteins that regulate neurite outgrowth and growth cone dynamics. Here, we show that Cdk5 phosphorylates CRMP1, CRMP2, and CRMP4, priming for subsequent phosphorylation by GSK3 in vitro. In contrast, DYRK2 phosphorylates and primes CRMP4 only. The Cdk5 and DYRK2 inhibitor purvalanol decreases the phosphorylation of CRMP proteins in neurons, whereas CRMP1 and CRMP2, but not CRMP4, phosphorylation is decreased in Cdk5(-/-) cortices. Stimulation of neuroblastoma cells with IGF1 or TPA decreases GSK3 activity concomitantly with CRMP2 and CRMP4 phosphorylation. Conversely, increased GSK3 activity is not sufficient to increase CRMP phosphorylation. However, the growth cone collapse-inducing protein Sema3A increases Cdk5 activity and promotes phosphorylation of CRMP2 (but not CRMP4). Therefore, inhibition of GSK3 alters phosphorylation of all CRMP isoforms; however, individual isoforms can be differentially regulated by their respective priming kinase. This is the first GSK3 substrate found to be regulated in this manner and may explain the hyperphosphorylation of CRMP2 observed in Alzheimer's disease.

GSK-3 phosphorylation of the Alzheimer epitope within collapsin response mediator proteins regulates axon elongation in primary neurons.

Elevated glycogen synthase kinase-3 (GSK-3) activity is associated with Alzheimer disease. We have found that collapsin response mediator proteins (CRMP) 2 and 4 are physiological substrates of GSK-3. The amino acids targeted by GSK-3 comprise a hyperphosphorylated epitope first identified in plaques isolated from Alzheimer brain. Expression of wild type CRMP2 in primary hippocampal neurons or SH-SY5Y neuroblastoma cells promotes axon elongation. However, a GSK-3-insensitive CRMP2 mutant has dramatically reduced ability to promote axon elongation, a similar effect to pharmacological inhibition of GSK-3. Hence, we propose that phosphorylation of CRMP proteins by GSK-3 regulates axon elongation. This work provides a direct connection between hyperphosphorylation of these residues and elevated GSK-3 activity, both of which are observed in Alzheimer brain.