PrP is a central player in toxicity mediated by soluble aggregates of neurodegeneration-causing proteins.

Neurodegenerative diseases are an enormous public health problem, affecting tens of millions of people worldwide. Nearly all of these diseases are characterized by oligomerization and fibrillization of neuronal proteins, and there is great interest in therapeutic targeting of these aggregates. Here, we show that soluble aggregates of α-synuclein and tau bind to plate-immobilized PrP in vitro and on mouse cortical neurons, and that this binding requires at least one of the same N-terminal sites at which soluble Aß aggregates bind. Moreover, soluble aggregates of tau, α-synuclein and Aß cause both functional (impairment of LTP) and structural (neuritic dystrophy) compromise and these deficits are absent when PrP is ablated, knocked-down, or when neurons are pre-treated with anti-PrP blocking antibodies. Using an all-human experimental paradigm involving: (1) isogenic iPSC-derived neurons expressing or lacking PRNP, and (2) aqueous extracts from brains of individuals who died with Alzheimer's disease, dementia with Lewy bodies, and Pick's disease, we demonstrate that Aß, α-synuclein and tau are toxic to neurons in a manner that requires PrPC. These results indicate that PrP is likely to play an important role in a variety of late-life neurodegenerative diseases and that therapeutic targeting of PrP, rather than individual disease proteins, may have more benefit for conditions which involve the aggregation of more than one protein.

Cellular Prion Protein Mediates the Disruption of Hippocampal Synaptic Plasticity by Soluble Tau In Vivo.

Intracellular neurofibrillary tangles (NFTs) composed of tau protein are a neuropathological hallmark of several neurodegenerative diseases, the most common of which is Alzheimer's disease (AD). For some time NFTs were considered the primary cause of synaptic dysfunction and neuronal death, however, more recent evidence suggests that soluble aggregates of tau are key drivers of disease. Here we investigated the effect of different tau species on synaptic plasticity in the male rat hippocampus in vivo Intracerebroventricular injection of soluble aggregates formed from either wild-type or P301S human recombinant tau potently inhibited hippocampal long-term potentiation (LTP) at CA3-to-CA1 synapses. In contrast, tau monomers and fibrils appeared inactive. Neither baseline synaptic transmission, paired-pulse facilitation nor burst response during high-frequency conditioning stimulation was affected by the soluble tau aggregates. Similarly, certain AD brain soluble extracts inhibited LTP in a tau-dependent manner that was abrogated by either immunodepletion with, or coinjection of, a mid-region anti-tau monoclonal antibody (mAb), Tau5. Importantly, this tau-mediated block of LTP was prevented by administration of mAbs selective for the prion protein (PrP). Specifically, mAbs to both the mid-region (6D11) and N-terminus (MI-0131) of PrP prevented inhibition of LTP by both recombinant and brain-derived tau. These findings indicate that PrP is a mediator of tau-induced synaptic dysfunction.SIGNIFICANCE STATEMENT Here we report that certain soluble forms of tau selectively disrupt synaptic plasticity in the live rat hippocampus. Further, we show that monoclonal antibodies to cellular prion protein abrogate the impairment of long-term potentiation caused both by recombinant and Alzheimer's disease brain-derived soluble tau. These findings support a critical role for cellular prion protein in the deleterious synaptic actions of extracellular soluble tau in tauopathies, including Alzheimer's disease. Thus, approaches targeting cellular prion protein, or downstream pathways, might provide an effective strategy for developing therapeutics.

Soluble tau aggregates inhibit synaptic long-term depression and amyloid ß-facilitated LTD in vivo.

Soluble synaptotoxic aggregates of the main pathological proteins of Alzheimer's disease, amyloid ß-protein (Aß) and tau, have rapid and potent inhibitory effects on long-term potentiation (LTP). Although the promotion of synaptic weakening mechanisms, including long-term depression (LTD), is posited to mediate LTP inhibition by Aß, little is known regarding the action of exogenous tau on LTD. The present study examined the ability of different assemblies of full-length human tau to affect LTD in the dorsal hippocampus of the anaesthetized rat. Unlike Aß, intracerebroventricular injection of soluble aggregates of tau (SτAs), but not monomers or fibrils, potently increased the threshold for LTD induction in a manner that required cellular prion protein. However, MTEP, an antagonist of the putative prion protein coreceptor metabotropic glutamate receptor 5, did not prevent the disruption of synaptic plasticity by SτAs. In contrast, systemic treatment with Ro 25-6981, a selective antagonist at GluN2B subunit-containing NMDA receptors, reduced SτA-mediated inhibition of LTD, but not LTP. Intriguingly, SτAs completely blocked Aß-facilitated LTD, whereas a subthreshold dose of SτAs facilitated Aß-mediated inhibition of LTP. Overall, these findings support the importance of cellular prion protein in mediating a range of, sometimes opposing, actions of soluble Aß and tau aggregates with different effector mechanisms on synaptic plasticity.

Preclinical development of a high affinity α-synuclein antibody, MEDI1341, that can enter the brain, sequester extracellular α-synuclein and attenuate α-synuclein spreading in vivo.

There are no approved drug therapies that can prevent or slow the progression of Parkinson's disease (PD). Accumulation and aggregation of α-synuclein protein is observed throughout the nervous system in PD. α-Synuclein is a core component of Lewy bodies and neurites that neuropathologically define PD, suggesting that α-synuclein may be a key causative agent in PD. Recent experimental data suggest that PD progression may arise due to spreading of pathological forms of extracellular α-synuclein throughout the brain via a cellular release, uptake and seeding mechanism. We have developed a high affinity α-synuclein antibody, MEDI1341, that can enter the brain, sequester extracellular α-synuclein and attenuate α-synuclein spreading in vivo. MEDI1341 binds both monomeric and aggregated forms of α-synuclein. In vitro, MEDI1341 blocks cell-to-cell transmission of pathologically relevant α-synuclein preformed fibrils (pffs). After intravenous injection into rats and cynomolgus monkeys, MEDI1341 rapidly enters the central nervous system and lowers free extracellular α-synuclein levels in the interstitial fluid (ISF) and cerebrospinal fluid (CSF) compartments. Using a novel lentiviral-based in vivo mouse model of α-synuclein spreading in the brain, we show that treatment with MEDI1341 significantly reduces α-synuclein accumulation and propagation along axons. In this same model, we demonstrate that an effector-null version of the antibody was equally as effective as one with effector function. MEDI1341 is now in Phase 1 human clinical trial testing as a novel treatment for α-synucleinopathies including PD with the aim to slow or halt disease progression.

Learnings about the complexity of extracellular tau aid development of a blood-based screen for Alzheimer's disease.

INTRODUCTION: The tau protein plays a central role in Alzheimer's disease (AD), and there is huge interest in measuring tau in blood and cerebrospinal fluid (CSF). METHODS: We developed a set of immunoassays to measure tau in specimens from humans diagnosed based on current best clinical and CSF biomarker criteria. RESULTS: In CSF, mid-region- and N-terminal-detected tau predominated and rose in disease. In plasma, an N-terminal assay (NT1) detected elevated levels of tau in AD and AD-mild cognitive impairment (MCI). Plasma NT1 measurements separated controls from AD-MCI (area under the curve [AUC] = 0.88) and AD (AUC = 0.96) in a discovery cohort and in a Validation Cohort (with AUCs = 0.79 and 0.75, respectively). DISCUSSION: The forms of tau in CSF and plasma are distinct, but in each specimen type, the levels of certain fragments are increased in AD. Measurement of plasma NT1 tau should be aggressively pursued as a potential blood-based screening test for AD/AD-MCI.

Pyrazolopyridazine alpha-2-delta-1 ligands for the treatment of neuropathic pain.

Optimization of the novel alpha-2-delta-1 ligand 4 provided compounds 37 and 38 which have improved DMPK profiles, good in vivo analgesic activity and in vitro selectivity over alpha-2-delta-2. An in-house P-gp prediction programme and the MetaSite software package were used to help solve the specific problems of high P-gp efflux and high in vivo clearance.

Anti-PrPC antibody rescues cognition and synapses in transgenic alzheimer mice.

Objective: Amyloid-beta oligomers (Aßo) trigger the development of Alzheimer's disease (AD) pathophysiology. Cellular prion protein (PrPC) initiates synaptic damage as a high affinity receptor for Aßo. Here, we evaluated the preclinical therapeutic efficacy of a fully human monoclonal antibody against PrPC. This AZ59 antibody selectively targets the Aßo binding site in the amino-terminal unstructured domain of PrPC to avoid any potential risk of direct toxicity. Methods: Potency of AZ59 was evaluated by binding to PrPC, blockade of Aßo interaction and interruption of Aßo signaling. AZ59 was administered to mice by weekly intraperitoneal dosing and brain antibody measured. APP/PS1 transgenic mice were treated with AZ59 and assessed by memory tests, by brain biochemistry and by histochemistry for Aß, gliosis and synaptic density. Results: AZ59 binds PrPC with 100 pmol/L affinity and blocks human brain Aßo binding to PrPC, as well as prevents synaptotoxic signaling. Weekly i.p. dosing of 20 mg/kg AZ59 in a murine form achieves trough brain antibody levels greater than 10 nmol/L. Aged symptomatic APP/PS1 transgenic mice treated with AZ59 for 5-7 weeks show a full rescue of behavioral and synaptic loss phenotypes. This recovery occurs without clearance of plaque pathology or elimination of gliosis. AZ59 treatment also normalizes synaptic signaling abnormalities in transgenic brain. These benefits are dose-dependent and persist for at least 1 month after the last dose. Interpretation: Preclinical data demonstrate that systemic AZ59 therapy rescues central synapses and memory function from transgenic Alzheimer's disease pathology, supporting a disease-modifying therapeutic potential.

Extracellular Monomeric and Aggregated Tau Efficiently Enter Human Neurons through Overlapping but Distinct Pathways.

In Alzheimer's disease, neurofibrillary tangle pathology appears to spread along neuronal connections, proposed to be mediated by the release and uptake of abnormal, disease-specific forms of microtubule-binding protein tau MAPT. It is currently unclear whether transfer of tau between neurons is a toxic gain-of-function process in dementia or reflects a constitutive biological process. We report two entry mechanisms for monomeric tau to human neurons: a rapid dynamin-dependent phase typical of endocytosis and a second, slower actin-dependent phase of macropinocytosis. Aggregated tau entry is independent of actin polymerization and largely dynamin dependent, consistent with endocytosis and distinct from macropinocytosis, the major route for aggregated tau entry reported for non-neuronal cells. Anti-tau antibodies abrogate monomeric tau entry into neurons, but less efficiently in the case of aggregated tau, where internalized tau carries antibody with it into neurons. These data suggest that tau entry to human neurons is a physiological process and not a disease-specific phenomenon.

Central inhibition of granulocyte-macrophage colony-stimulating factor is analgesic in experimental neuropathic pain.

With less than 50% of patients responding to the current standard of care and poor efficacy and selectivity of current treatments, neuropathic pain continues to be an area of considerable unmet medical need. Biological therapeutics such as monoclonal antibodies (mAbs) provide better intrinsic selectivity; however, delivery to the central nervous system (CNS) remains a challenge. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is well described in inflammation-induced pain, and early-phase clinical trials evaluating its antagonism have exemplified its importance as a peripheral pain target. Here, we investigate the role of this cytokine in a murine model of traumatic nerve injury and show that deletion of the GM-CSF receptor or treatment with an antagonizing mAb alleviates pain. We also demonstrate enhanced analgesic efficacy using an engineered construct that has greater capacity to penetrate the CNS. Despite observing GM-CSF receptor expression in microglia and astrocytes, the gliosis response in the dorsal horn was not altered in nerve injured knockout mice compared with wild-type littermate controls as evaluated by ionized calcium binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein, respectively. Functional analysis of glial cells revealed that pretreatment with GM-CSF potentiated lipopolysaccharide-induced release of proinflammatory cytokines. In summary, our data indicate that GM-CSF is a proinflammatory cytokine that contributes to nociceptive signalling through driving spinal glial cell secretion of proinflammatory mediators. In addition, we report a successful approach to accessing CNS pain targets, providing promise for central compartment delivery of analgesics.

TREM2 shedding by cleavage at the H157-S158 bond is accelerated for the Alzheimer's disease-associated H157Y variant.

We have characterised the proteolytic cleavage events responsible for the shedding of triggering receptor expressed on myeloid cells 2 (TREM2) from primary cultures of human macrophages, murine microglia and TREM2-expressing human embryonic kidney (HEK293) cells. In all cell types, a soluble 17 kDa N-terminal cleavage fragment was shed into the conditioned media in a constitutive process that is inhibited by G1254023X and metalloprotease inhibitors and siRNA targeting ADAM10. Inhibitors of serine proteases and matrix metalloproteinases 2/9, and ADAM17 siRNA did not block TREM2 shedding. Peptidomimetic protease inhibitors highlighted a possible cleavage site, and mass spectrometry confirmed that shedding occurred predominantly at the H157-S158 peptide bond for both wild-type and H157Y human TREM2 and for the wild-type murine orthologue. Crucially, we also show that the Alzheimer's disease-associated H157Y TREM2 variant was shed more rapidly than wild type from HEK293 cells, possibly by a novel, batimastat- and ADAM10-siRNA-independent, sheddase activity. These insights offer new therapeutic targets for modulating the innate immune response in Alzheimer's and other neurological diseases.

Further validation of LABORAS using various dopaminergic manipulations in mice including MPTP-induced nigro-striatal degeneration.

The automated behavioural apparatus, LABORAS (Laboratory Animal Behaviour Observation, Registration and Analysis System), has been further validated with respect to the ability of the system to detect behavioural impairments in mice, following various dopaminergic manipulations. Initially data were obtained from mice administered with amphetamine, haloperidol, SCH23390, apomorphine and L-DOPA, with the focus on locomotor and grooming activities. The data recorded by LABORAS on administration of these pharmacological tool compounds, is comparable with published findings using standard LMA systems and conventional observer methods. In addition the home cage behaviour of mice administered with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) using an acute dosing regimen was also investigated. In LABORAS, mice subjected to MPTP lesioning showed deficits in spontaneous motor activity at day 6-7 post-MPTP administration, over a 24 h test period, as compared to saline treated controls. The data captured and analysed using LABORAS, suggests that the automated system is able to detect both pharmacologically and lesion-induced changes in behaviour of mice, reliably and efficiently.

A novel behavioural registration system LABORAS and the social interaction paradigm detect long-term functional deficits following middle cerebral artery occlusion in the rat.

Following stroke, patients suffer a wide range of disabilities including motor impairment, anxiety and depression. However, to date, characterisation of rodent stroke models has concentrated mainly on the investigation of motor deficits. The aim of the present studies was therefore to investigate home cage behaviour (as assessed by a recently developed automatic behavioural classification system, LABORAS) and social behaviour (as a measure of anxiety) in rats following transient middle cerebral artery occlusion (tMCAO). Rats subjected to tMCAO (90 min) showed deficits in general home cage behaviours including locomotion, rearing, grooming and drinking for up to 7 weeks post occlusion, as compared with sham operated controls. In addition, a significant decrease in the total duration of social interaction was also observed in occluded rats compared with shams. The data shows that in addition to motor deficits, animals display changes in home cage behaviour and decreased social behaviour which, in contrast to motor function, are prolonged over time. Transient MCAO in rats may therefore provide a pre-clinical model to investigate agents offering symptomatic relief for ischaemia-induced motor deficits and anxiety over time following injury.

LABORAS: Initial pharmacological validation of a system allowing continuous monitoring of laboratory rodent behaviour.

A newly developed apparatus for automated behavioural analysis, Laboratory Animal Behaviour Observation, Registration and Analysis System (LABORAS), has been further validated with respect to the ability of the system to detect the pharmacodynamic effects of standard pharmacological tools. Data were obtained from rats administered with mCPP (reversal with SB242084), 8-OH-DPAT (reversal with WAY100635), amphetamine (reversal with haloperidol) and angiotensin, with the focus on locomotor activity, feeding and drinking behaviours. The data captured and analysed by LABORAS, suggests that the automated system is able to detect pharmacologically induced changes in behaviour, reliably and efficiently, with a significant reduction in the number of animals required, and reduced operator input.

High-performance liquid chromatography/tandem mass spectrometry assay for the determination of 1-methyl-4-phenyl pyridinium (MPP+) in brain tissue homogenates.

A high-throughput liquid chromatography/tandem mass spectrometry method has been developed for the quantitative assessment of 1-methyl-4-phenylpyridinium (MPP+) in brain tissue samples. This separation is based on reversed phase chromatography using formic acid and acetonitrile as the mobile phase. Using gradient separation conditions, MPP+ was resolved within 5 min and detected using tandem mass spectrometry in the positive ion electrospray mode. The limit of detection for MPP+ was found to be 1 fmol on column with a signal to noise ratio of 3:1. The assay has been used routinely in our laboratory for the measurement of MPP+ levels in brain tissue from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice, and can be used to distinguish neuroprotective efficacy and monoamine oxidase inhibition.

Lead discovery for microsomal prostaglandin E synthase using a combination of high-throughput fluorescent-based assays and RapidFire mass spectrometry.

Microsomal prostaglandin E synthase-1 (mPGES-1) represents an attractive target for the treatment of rheumatoid arthritis and pain, being upregulated in response to inflammatory stimuli. Biochemical assays for prostaglandin E synthase activity are complicated by the instability of the substrate (PGH(2)) and the challenge of detection of the product (PGE(2)). A coupled fluorescent assay is described for mPGES-1 where PGH(2) is generated in situ using the action of cyclooxygenase 2 (Cox-2) on arachidonic acid. PGE(2) is detected by coupling through 15-prostaglandin dehydrogenase (15-PGDH) and diaphorase. The overall coupled reaction was miniaturized to 1536-well plates and validated for high-throughput screening. For compound progression, a novel high-throughput mass spectrometry assay was developed using the RapidFire platform. The assay employs the same in situ substrate generation step as the fluorescent assay, after which both PGE(2) and a reduced form of the unreacted substrate were detected by mass spectrometry. Pharmacology and assay quality were comparable between both assays, but the mass spectrometry assay was shown to be less susceptible to interference and false positives. Exploiting the throughput of the fluorescent assay and the label-free, direct detection of the RapidFire has proved to be a powerful lead discovery strategy for this challenging target.

Discovery of 1-[4-(3-chlorophenylamino)-1-methyl-1H-pyrrolo[3,2-c]pyridin-7-yl]-1-morpholin-4-ylmethanone (GSK554418A), a brain penetrant 5-azaindole CB2 agonist for the treatment of chronic pain.

We report the synthesis and SAR of a series of novel azaindole CB(2) agonists. 6-Azaindole 18 showed activity in an acute pain model but was inactive in a chronic model. 18 is a Pgp substrate with low brain penetration. The template was redesigned, and the resulting 5-azaindole 36 was a potent CB(2) agonist with high CNS penetration. This compound was efficacious in the acute model and the chronic joint pain model.