Adam R. Cole

Further evidence that the tyrosine phosphorylation of glycogen synthase kinase-3 (GSK3) in mammalian cells is an autophosphorylation event

Phosphorylation of the endogenous GSK3alpha (glycogen synthase kinase-3alpha) at Tyr279 and GSK3beta at Tyr216 was suppressed in HEK-293 or SH-SY5Y cells by incubation with pharmacological inhibitors of GSK3, but not by an Src-family inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4- d ]pyrimidine (PP2), or a general protein tyrosine kinase inhibitor (genistein). GSK3beta transfected into HEK-293 cells or Escherichia coli became phosphorylated at Tyr216, but catalytically inactive mutants did not. GSK3beta expressed in insect Sf 21 cells or E. coli was extensively phosphorylated at Tyr216, but the few molecules lacking phosphate at this position could autophosphorylate at Tyr216 in vitro after incubation with MgATP. The rate of autophosphorylation was unaffected by dilution and was suppressed by the GSK3 inhibitor kenpaullone. Wild-type GSK3beta was unable to catalyse the tyrosine phosphorylation of catalytically inactive GSK3beta lacking phosphate at Tyr216. Our results indicate that the tyrosine phosphorylation of GSK3 is an intramolecular autophosphorylation event in the cells that we have studied and that this modification enhances the stability of the enzyme.

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.

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 Alzheimers disease.

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)M146VKI; Thy1.2‐amyloid precursor protein (APP)swe; Thy1.2tauP301L] that develops AD‐like plaques and tangles, as well as the double (PS1M146VKI; Thy1.2‐APPswe) 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 β peptide levels (Tg2576 and APP/amyloid β‐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 Alzheimers disease, vascular dementia and Picks 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 Alzheimers 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.

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.

Increased CRMP2 phosphorylation is observed in Alzheimer's disease; does this tell us anything about disease development?

Collapsin response mediator protein-2 (CRMP2) was recently identified as a physiological substrate for GSK3 and Cdk5, two protein kinases suggested to exhibit greater activity in Alzheimers disease (AD). Indeed, phosphorylation of CRMP2, at the residues targeted by GSK3 and Cdk5, is relatively high in cortex isolated from human AD brain, as well as in the brains of animal models of AD, while phospho-CRMP2 is found in neurofibrillary tangles. In mouse models of AD, increased phosphorylation occurs prior to pathology. Although CRMP2 has no known enzymatic activity, a great deal of information is appearing on its importance in neuronal development and polarity, as well as in axon growth and guidance. In this mini-review, we examine what is known about CRMP2 function, how that is controlled by phosphorylation, what alterations in molecular mechanisms could lead to the abnormally high CRMP2 phosphorylation in AD, and whether this is likely to be specific to AD or occur in other forms of neurodegeneration. This will include discussion of the evidence for increased GSK3 or Cdk5 activity, for decreased phosphatase activity, or the upregulation of other CRMP2 protein kinases in AD. Importantly, we will compare the processes that may contribute to increased CRMP2 phosphorylation with those known to increase tau hyperphosphorylation in AD, and whether these are likely to be part of disease development or a useful early marker for AD.

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.

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 Alzheimers disease (AD) patients. Increased phosphorylation of CRMP2 is also apparent in mouse models of AD that express mutated AβPP and PSEN1, but not AβPP 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 Aβ42 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 AβPP, but that neither excessive Aβ42 peptide nor neurotoxicity alone are sufficient to promote hyperphosphorylation.

Glycogen synthase kinase-3.

Glycogen synthase kinase-3 (GSK3) is a ubiquitous and promiscuous kinase that has been studied extensively for over four decades. Initial reports beginning in the 1970s described its role in cellular metabolic pathways fundamental to glucose metabolism, but in more recent years the number of reports describing aberrant GSK3 activity in pathological conditions has risen dramatically. Interest in GSK3 in the field of Alzheimers disease was first sparked in the early 1990s by papers that described the ability of GSK3 to phosphorylate tau. Excessive tau phosphorylation is present in Alzheimers-disease-affected brain. These early papers provided new insight to the mechanisms that may contribute to tau pathology of Alzheimers, with GSK3 as a potential central figure. Since then, the research effort invested into GSK3 in Alzheimers disease has expanded, and mechanistic studies now demonstrate a functional relationship between not only GSK3 and tau, but also GSK3 and amyloid-β. Through weight of numbers, strong evidence now indicates that GSK3 is associated with the two key pathological features of Alzheimers-disease-affected brain: neurofibrillary tangles and amyloid plaques. The scope of this special issue is to provide an overview of the data that implicate GSK3 in Alzheimers disease. As an introduction, the special issue begins with a review of the regulation of GSK3 activity (M. Medina and F. Wandosell). This is followed by a report that provides caution by articulating the need to demonstrate the bona fide substrates of GSK3 (C. Sutherland). The role of GSK3 in the brain and neuronal function is then introduced by two reports. The first is on presynaptic function of GSK3 (K. J. Smillie and M. A. Cousin) and the second on GSK3 in brain development and neuronal plasticity (P. Salcedo-Tello et al.). After this the contribution of GSK3 to neurodegenerative diseases is described in four reviews. The first describes GSK3 in neurodegenerative diseases in general (P. Lei et al.) while the following three discuss more specific aspects of Alzheimers disease, including cell survival mechanisms (M. A. Mines et al.), inflammation (j. Koistinaho et al.), and tau phosphorylation (D. P. Hanger and W. Nobel). Finally, the special issue concludes with two reviews on therapeutic strategies for Alzheimers disease that focus on GSK3. The potential of targeting GSK3 for therapeutic benefit against oxidative stress is presented (K. Kanninen et al.), followed by an appraisal of GSK3 inhibitors in the next horizon (A. Martinez et al.). Since several GSK3 inhibitors are currently in clinical trials for treatment of neurological and other disorders, we feel this special issue is a timely “snapshot” of our current knowledge of GSK3 function in healthy and diseased brain, and highlights outstanding issues for future research on this important brain kinase. Peter Crouch Adam Cole Michael Cousin Ana Martinez Katja Kanninen