Wendy Noble

Long-term clinical efficacy of grass-pollen immunotherapy.

BACKGROUND Pollen immunotherapy is effective in selected patients with IgE-mediated seasonal allergic rhinitis, although it is questionable whether there is long-term benefit after the discontinuation of treatment. METHODS We conducted a randomized, double-blind, placebo-controlled trial of the discontinuation of immunotherapy for grass-pollen allergy in patients in whom three to four years of this treatment had previously been shown to be effective. During the three years of this trial, primary outcome measures were scores for seasonal symptoms and the use of rescue medication. Objective measures included the immediate conjunctival response and the immediate and late skin responses to allergen challenge. Cutaneous-biopsy specimens obtained 24 hours after intradermal allergen challenge were examined for T-cell infiltration and the presence of cytokine-producing T helper cells (TH2 cells) (as evidenced by the presence of interleukin-4 messenger RNA). A matched group of patients with hay fever who had not received immunotherapy was followed as a control for the natural course of the disease. RESULTS Scores for seasonal symptoms and the use of rescue antiallergic medication, which included short courses of prednisolone, remained low after the discontinuation of immunotherapy, and there was no significant difference between patients who continued immunotherapy and those who discontinued it. Symptom scores in both treatment groups (median areas under the curve in 1995, 921 for continuation of immunotherapy and 504 for discontinuation of immunotherapy; P=0.60) were markedly lower than those in the group that had not received immunotherapy (median value in 1995, 2863). Although there was a tendency for immediate sensitivity to allergen to return late after discontinuation, there was a sustained reduction in the late skin response and associated CD3+ T-cell infiltration and interleukin-4 messenger RNA expression. CONCLUSIONS Immunotherapy for grass-pollen allergy for three to four years induces prolonged clinical remission accompanied by a persistent alteration in immunologic reactivity.

Tau phosphorylation: the therapeutic challenge for neurodegenerative disease

The microtubule-associated protein tau is integral to the pathogenesis of Alzheimers disease (AD), as well as several related disorders, termed tauopathies, in which tau is deposited in affected brain regions. In the tauopathies, pathological tau is in an elevated state of phosphorylation and is aberrantly cleaved. It also exhibits abnormal conformations and becomes aggregated, resulting in neurofibrillary tau pathology. Recent evidence suggests that relatively early disease-associated changes in soluble tau proteins, including phosphorylation, are involved in the induction of neuronal death. Here, we summarize recent developments that suggest new therapeutic strategies to prevent or reduce the progression of pathology in the tauopathies. A list of tau phosphorylation sites identified in the tauopathies and in controls accompanies this review.

Cdk5 Is a Key Factor in Tau Aggregation and Tangle Formation In Vivo

Tau aggregation is a common feature of neurodegenerative diseases such as Alzheimers disease, and hyperphosphorylation of tau has been implicated as a fundamental pathogenic mechanism in this process. To examine the impact of cdk5 in tau aggregation and tangle formation, we crossed transgenic mice overexpressing the cdk5 activator p25, with transgenic mice overexpressing mutant (P301L) human tau. Tau was hyperphosphorylated at several sites in the double transgenics, and there was a highly significant accumulation of aggregated tau in brainstem and cortex. This was accompanied by increased numbers of silver-stained neurofibrillary tangles (NFTs). Insoluble tau was also associated with active GSK. Thus, cdk5 can initiate a major impact on tau pathology progression that probably involves several kinases. Kinase inhibitors may thus be beneficial therapeutically.

Physiological release of endogenous tau is stimulated by neuronal activity

Propagation of tau pathology is linked with progressive neurodegeneration, but the mechanism underlying trans‐synaptic spread of tau is unknown. We show that stimulation of neuronal activity, or AMPA receptor activation, induces tau release from healthy, mature cortical neurons. Notably, phosphorylation of extracellular tau appears reduced in comparison with intracellular tau. We also find that AMPA‐induced release of tau is calcium‐dependent. Blocking pre‐synaptic vesicle release by tetanus toxin and inhibiting neuronal activity with tetrodotoxin both significantly impair AMPA‐mediated tau release. Tau secretion is therefore a regulatable process, dysregulation of which could lead to the spread of tau pathology in disease.

Astrocytes are important mediators of Aβ-induced neurotoxicity and tau phosphorylation in primary culture.

Alzheimers disease (AD) is pathologically characterised by the age-dependent deposition of β-amyloid (Aβ) in senile plaques, intraneuronal accumulation of tau as neurofibrillary tangles, synaptic dysfunction and neuronal death. Neuroinflammation, typified by the accumulation of activated microglia and reactive astrocytes, is believed to modulate the development and/or progression of AD. We have used primary rat neuronal, astrocytic and mixed cortical cultures to investigate the contribution of astrocyte-mediated inflammatory responses during Aβ-induced neuronal loss. We report that the presence of small numbers of astrocytes exacerbate Aβ-induced neuronal death, caspase-3 activation and the production of caspase-3-cleaved tau. Furthermore, we show that astrocytes are essential for the Aβ-induced tau phosphorylation observed in primary neurons. The release of soluble inflammatory factor(s) from astrocytes accompanies these events, and inhibition of astrocyte activation with the anti-inflammatory agent, minocycline, reduces astrocytic inflammatory responses and the associated neuronal loss. Aβ-induced increases in caspase-3 activation and the production of caspase-3-truncated tau species in neurons were reduced when the astrocytic response was attenuated with minocycline. Taken together, these results show that astrocytes are important mediators of the neurotoxic events downstream of elevated Aβ in models of AD, and suggest that mechanisms underlying pro-inflammatory cytokine release might be an important target for therapy.

Tyrosine 394 Is Phosphorylated in Alzheimer's Paired Helical Filament Tau and in Fetal Tau with c-Abl as the Candidate Tyrosine Kinase

Tau is a major microtubule-associated protein of axons and is also the principal component of the paired helical filaments (PHFs) that comprise the neurofibrillary tangles found in Alzheimers disease and other tauopathies. Besides phosphorylation of tau on serine and threonine residues in both normal tau and tau from neurofibrillary tangles, Tyr-18 was reported to be a site of phosphorylation by the Src-family kinase Fyn. We examined whether tyrosine residues other than Tyr-18 are phosphorylated in tau and whether other tyrosine kinases might phosphorylate tau. Using mass spectrometry, we positively identified phosphorylated Tyr-394 in PHF-tau from an Alzheimer brain and in human fetal brain tau. When wild-type human tau was transfected into fibroblasts or neuroblastoma cells, treatment with pervanadate caused tau to become phosphorylated on tyrosine by endogenous kinases. By replacing each of the five tyrosines in tau with phenylalanine, we identified Tyr-394 as the major site of tyrosine phosphorylation in tau. Tyrosine phosphorylation of tau was inhibited by PP2 (4-amino-5-(4-chlorophenyl-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), which is known to inhibit Src-family kinases and c-Abl. Cotransfection of tau and kinases showed that Tyr-18 was the major site for Fyn phosphorylation, but Tyr-394 was the main residue for Abl. In vitro, Abl phosphorylated tau directly. Abl could be coprecipitated with tau and was present in pretangle neurons in brain sections from Alzheimer cases. These results show that phosphorylation of tau on Tyr-394 is a physiological event that is potentially part of a signal relay and suggest that Abl could have a pathogenic role 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 motors implicated in the axonal transport of tau and alpha-synuclein.

Tau and α-synuclein are both proteins implicated in the pathology of neurodegenerative disease. Here we have investigated the mechanisms of axonal transport of tau and α-synuclein, because failure of axonal transport has been implicated in the development of several neurodegenerative disorders. We found that the transport of both of these proteins depend on an intact microtubule- but not actin-cytoskeleton, and that tau and α-synuclein both move at overall slow rates of transport. We used time-lapse video microscopy to obtain images of live neurons that had been transfected with plasmids expressing proteins tagged with enhanced green fluorescent protein. We found that particulate structures containing tau or α-synuclein travel rapidly when moving along axons but spend the majority of the time paused, and these structures have similar characteristics to those previously observed for neurofilaments. The motile particles containing tau or α-synuclein colocalise with the fast-transporting molecular motor kinesin-1 in neurons. Co-immunoprecipitation experiments demonstrate that tau and α-synuclein are each associated with complexes containing kinesin-1, whereas only α-synuclein appears to interact with dynein-containing complexes. In vitro glutathione S-transferase-binding assays using rat brain homogenate or recombinant protein as bait reveals a direct interaction of kinesin-1 light chains 1 and 2 with tau, but not with α-synuclein. Our findings suggest that the axonal transport of tau occurs via a mechanism utilising fast transport motors, including the kinesin family of proteins, and that α-synuclein transport in neurons may involve both kinesin and dynein motor proteins.

The importance of tau phosphorylation for neurodegenerative diseases

Fibrillar deposits of highly phosphorylated tau are a key pathological feature of several neurodegenerative tauopathies including Alzheimer’s disease (AD) and some frontotemporal dementias. Increasing evidence suggests that the presence of these end-stage neurofibrillary lesions do not cause neuronal loss, but rather that alterations to soluble tau proteins induce neurodegeneration. In particular, aberrant tau phosphorylation is acknowledged to be a key disease process, influencing tau structure, distribution, and function in neurons. Although typically described as a cytosolic protein that associates with microtubules and regulates axonal transport, several additional functions of tau have recently been demonstrated, including roles in DNA stabilization, and synaptic function. Most recently, studies examining the trans-synaptic spread of tau pathology in disease models have suggested a potential role for extracellular tau in cell signaling pathways intrinsic to neurodegeneration. Here we review the evidence showing that tau phosphorylation plays a key role in neurodegenerative tauopathies. We also comment on the tractability of altering phosphorylation-dependent tau functions for therapeutic intervention in AD and related disorders.

Co-localization of cholesterol, apolipoprotein E and fibrillar Aβ in amyloid plaques

Recent evidence strongly suggests a role for cholesterol and apolipoprotein E in the etiology of Alzheimers disease. We have demonstrated the co-localization of cholesterol and apolipoprotein E with beta-amyloid immunoreactivity and thioflavin S immunofluorescence in AD type plaques of a transgenic mouse model. Cholesterol and apolipoprotein E co-localized to the core of thioflavin S-positive (fibrillar) plaques, but not thioflavin S-negative (diffuse) plaques from an early age. By 18 months of age, there was extensive coverage of fibrillar plaques immunopositive for apolipoprotein E and cholesterol oxidase. These findings support evidence that cholesterol and apolipoprotein E are involved in fibrillar plaque formation or maintenance, and suggest that cholesterol may impact amyloid formation extracellularly, as well as through an intracellular effect.