Leukotriene A4 hydrolase inhibition improves age-related cognitive decline via modulation of synaptic function.

Leukotrienes, a class of inflammatory bioactive lipids, are well studied in the periphery, but less is known of their importance in the brain. We identified that the enzyme leukotriene A4 hydrolase (LTA4H) is expressed in healthy mouse neurons, and inhibition of LTA4H in aged mice improves hippocampal dependent memory. Single-cell nuclear RNA sequencing of hippocampal neurons after inhibition reveals major changes to genes important for synaptic organization, structure, and activity. We propose that LTA4H inhibition may act to improve cognition by directly inhibiting the enzymatic activity in neurons, leading to improved synaptic function. In addition, LTA4H plasma levels are increased in both aging and Alzheimer's disease and correlated with cognitive impairment. These results identify a role for LTA4H in the brain, and we propose that LTA4H inhibition may be a promising therapeutic strategy to treat cognitive decline in aging related diseases.

Noncanonical Activity of Tissue Inhibitor of Metalloproteinases 2 (TIMP2) Improves Cognition and Synapse Density in Aging.

Peripheral administration of tissue inhibitor of metalloproteinases 2 (TIMP2), a protein inhibitor of matrix metalloproteinases (MMPs), has previously been shown to have beneficial effects on cognition and neurons in aged mice. Here, to better understand the potential of recombinant TIMP2 proteins, an IgG4Fc fusion protein (TIMP2-hIgG4) was developed to extend the plasma half-life of TIMP2. Following one month of administration of TIMP2 or TIMP2-hIgG4 via intraperitoneal injections, 23-month-old male C57BL/6J mice showed improved hippocampal-dependent memory in a Y-maze, increased hippocampal cfos gene expression, and increased excitatory synapse density in the CA1 and dentate gyrus (DG) of the hippocampus. Thus, fusion to hIgG4 extended the half-life of TIMP2 while retaining the beneficial cognitive and neuronal effects. Moreover, it retained its ability to cross the blood-brain barrier. To deepen the mechanistic understanding of the beneficial function of TIMP2 on neuronal activity and cognition, a TIMP2 construct lacking MMP inhibitory activity, Ala-TIMP2, was generated, which provides steric hindrance that prevents inhibition of MMPs by the TIMP2 protein while still allowing MMP binding. A comprehensive assessment of the MMP inhibitory and binding capacity of these engineered proteins is outlined. Surprisingly, MMP inhibition by TIMP2 was not essential for its beneficial effects on cognition and neuronal function. These findings both confirm previously published research, expand on the potential mechanism for the beneficial effects of TIMP2, and provide important details for a therapeutic path forward for TIMP2 recombinant proteins in aging-related cognitive decline.

CCR3 plays a role in murine age-related cognitive changes and T-cell infiltration into the brain.

Targeting immune-mediated, age-related, biology has the potential to be a transformative therapeutic strategy. However, the redundant nature of the multiple cytokines that change with aging requires identification of a master downstream regulator to successfully exert therapeutic efficacy. Here, we discovered CCR3 as a prime candidate, and inhibition of CCR3 has pro-cognitive benefits in mice, but these benefits are not driven by an obvious direct action on central nervous system (CNS)-resident cells. Instead, CCR3-expressing T cells in the periphery that are modulated in aging inhibit infiltration of these T cells across the blood-brain barrier and reduce neuroinflammation. The axis of CCR3-expressing T cells influencing crosstalk from periphery to brain provides a therapeutically tractable link. These findings indicate the broad therapeutic potential of CCR3 inhibition in a spectrum of neuroinflammatory diseases of aging.

Molecular and histological correlates of cognitive decline across age in male C57BL/6J mice.

INTRODUCTION: Increasing age is the number one risk factor for developing cognitive decline and neurodegenerative disease. Aged humans and mice exhibit numerous molecular changes that contribute to a decline in cognitive function and increased risk of developing age-associated diseases. Here, we characterize multiple age-associated changes in male C57BL/6J mice to understand the translational utility of mouse aging. METHODS: Male C57BL/6J mice from various ages between 2 and 24 months of age were used to assess behavioral, as well as, histological and molecular changes across three modalities: neuronal, microgliosis/neuroinflammation, and the neurovascular unit (NVU). Additionally, a cohort of 4- and 22-month-old mice was used to assess blood-brain barrier (BBB) breakdown. Mice in this cohort were treated with a high, acute dose of lipopolysaccharide (LPS, 10 mg/kg) or saline control 6 h prior to sacrifice followed by tail vein injection of 0.4 kDa sodium fluorescein (100 mg/kg) 2 h later. RESULTS: Aged mice showed a decline in cognitive and motor abilities alongside decreased neurogenesis, proliferation, and synapse density. Further, neuroinflammation and circulating proinflammatory cytokines were increased in aged mice. Additionally, we found changes at the BBB, including increased T cell infiltration in multiple brain regions and an exacerbation in BBB leakiness following chemical insult with age. There were also a number of readouts that were unchanged with age and have limited utility as markers of aging in male C57BL/6J mice. CONCLUSIONS: Here we propose that these changes may be used as molecular and histological readouts that correspond to aging-related behavioral decline. These comprehensive findings, in the context of the published literature, are an important resource toward deepening our understanding of normal aging and provide an important tool for studying aging in mice.

Safety and Tolerability of GRF6019 Infusions in Severe Alzheimer's Disease: A Phase II Double-Blind Placebo-Controlled Trial.

BACKGROUND: The plasma fraction GRF6019 shows multiple benefits on brain aging in mice, including enhanced cognition, neurogenesis, and synaptic density, as well as reduced neuroinflammation. OBJECTIVE: To evaluate the safety, tolerability, and preliminary efficacy of GRF6019 in patients with severe Alzheimer's disease (AD). METHODS: A phase II, double-blind, placebo-controlled study in patients with severe AD (Mini-Mental State Examination score 0-10). Patients were randomized 2 : 1 to GRF6019 (N = 18) or placebo (N = 8) and received daily 250 mL intravenous infusions over 5 days. The primary endpoints were the rates of adverse events (AEs) and the tolerability of GRF6019 as assessed by the number of patients completing the study. Change from baseline in cognitive and functional assessments was also evaluated. RESULTS: All patients completed 100%of study visits and infusions. The rate of AEs was similar in the GRF6019 (8/18 patients [44.4%]) and placebo (3/8 patients [37.5%]) groups, and there were no deaths or serious AEs. The most common AEs considered related to treatment were mild, transient changes in blood pressure in the GRF6019 group (hypotension: 2 patients [11.1%]; hypertension: 1 patient [5.6%]); there were no related AEs in the placebo group. The trial was not powered to detect statistically significant differences between treatment groups. At the end of the study, patients in both treatment groups remained stable or improved on all cognitive and functional endpoints. CONCLUSION: GRF6019 demonstrated excellent safety, feasibility, and tolerability. Future trials designed to characterize the potential functional benefits of GRF6019 and related plasma fractions in severe AD are warranted.

Safety and tolerability of GRF6019 in mild-to-moderate Alzheimer's disease dementia.

INTRODUCTION: This phase 2 trial evaluated the safety, tolerability, and feasibility of repeated infusions of the plasma fraction GRF6019 in mild-to-moderate Alzheimer's disease. METHODS: In this randomized, double-blind, dose-comparison trial, 47 patients were randomized 1:1 to receive daily infusions of 100 mL (n = 24) or 250 mL (n = 23) of GRF6019 for 5 consecutive days over two dosing periods separated by a treatment-free interval of 3 months. RESULTS: The mean (standard deviation [SD]) age of the enrolled patients was 74.3 (6.9), and 62% were women. Most adverse events (55%) were mild, with no clinically significant differences in safety or tolerability between the two dose levels. The mean (SD) baseline Mini-Mental State Examination score was 20.6 (3.7) in the 100 mL group and 19.6 (3.7) in the 250 mL group; at 24 weeks, the within-patient mean change from baseline was -1.0 points (95% confidence interval [CI], -3.1 to 1.1) in the 100 mL group and +1.5 points (95% CI, -0.4 to 3.3) in the 250 mL group. The within-patient mean change from baseline on the Alzheimer's Disease Assessment Scale-Cognitive subscale was -0.4 points (95% CI, -2.9 to 2.2) in the 100 mL group, while in the 250 mL group it was -0.9 points (95% CI, -3.0 to 1.2). The within-patient mean change from baseline on the Alzheimer's Disease Cooperative Study-Activities of Daily Living was -0.7 points in the 100 mL group (95% CI, -4.3 to 3.0) and -1.3 points (95% CI, -3.4 to 0.7) in the 250 mL group. The mean change from baseline on the Category Fluency Test, Clinical Dementia Rating Scale-Sum of Boxes, Alzheimer's Disease Cooperative Study-Clinical Global Impression of Change, and Neuropsychiatric Inventory Questionnaire was similar for both treatment groups and did not show any worsening. DISCUSSION: GRF6019 was safe and well tolerated, and patients experienced no cognitive decline and minimal functional decline. These results support further development of GRF6019.

Aging: therapeutics for a healthy future.

Increased healthcare and pharmaceutical understanding has led to the eradication of many childhood, infectious and preventable diseases; however, we are now experiencing the impact of aging disorders as the lifespan increases. These disorders have already become a major burden on society and threaten to become a defining challenge of our generation. Indications such as Alzheimer's disease gain headlines and have focused the thinking of many towards dementia and cognitive decline in aging. Indications related to neurological function and related behaviors are thus an extremely important starting point in the consideration of therapeutics.However, the reality is that pathological aging covers a spectrum of significant neurological and peripheral indications. Development of therapeutics to treat aging and age-related disorders is therefore a huge need, but represents a largely unexplored path. Fundamental scientific questions need to be considered as we embark towards a goal of improving health in old age, including how we 1) define aging as a therapeutic target, 2) model aging preclinically and 3) effectively translate from preclinical models to man. Furthermore, the challenges associated with identifying novel therapeutics in a financial, regulatory and clinical sense need to be contemplated carefully to ensure we address the unmet need in our increasingly elderly population. The complexity of the challenge requires different perspectives, cross-functional partnerships and diverse concepts. We seek to raise issues to guide the field, considering the current state of thinking to aid in identifying roadblocks and important challenges early. The need for therapeutics that address aging and age-related disorders is acute, but the promise of effective treatments provides huge opportunities that, as a community, we all seek to enable effectively as soon as possible.

Plasma-Based Strategies for Therapeutic Modulation of Brain Aging.

Age is the primary risk factor for the vast majority of disorders, including neurodegenerative diseases impacting brain function. Whether the consequences of aging at the biological level can be reversed, or age-related changes prevented, to change the trajectory of such disorders is thus of extreme interest and value. Studies using young plasma, the acellular component of blood, have demonstrated that aging is malleable, with the ability to restore functions in old animals. Fascinatingly, this functional improvement is even observed in the brain, despite the blood-brain barrier, indicating that peripheral sources can effectively impact central sites leading to clinically relevant changes such as enhancement of cognitive function. A plasma-based approach is also attractive as aging is inherently complex, with an array of mechanisms dysregulated in diverse cells and organs throughout the body leading to disturbed function. Plasma, containing a natural mixture of components, has the ability to act multimodally, modulating diverse mechanisms that can converge to change the trajectory of age-related diseases. Here we review the evidence that plasma modulates aging processes in the brain and consider the therapeutic applications that derive from these observations. Plasma and plasma-derived therapeutics are an attractive translation of this concept, requiring critical consideration of benefits, risks, and ethics. Ultimately, knowledge derived from this science will drive a comprehensive molecular understanding to deliver optimized therapeutics. The potential of highly differentiated, multimodal therapeutics for treatment of age-related brain disorders provides an exciting new clinical approach to address the complex etiology of aging.

Safety, Tolerability, and Feasibility of Young Plasma Infusion in the Plasma for Alzheimer Symptom Amelioration Study: A Randomized Clinical Trial.

Importance: Young mouse plasma restores memory in aged mice, but, to our knowledge, the effects are unknown in patients with Alzheimer disease (AD). Objective: To assess the safety, tolerability, and feasibility of infusions of young fresh frozen plasma (yFFP) from donors age 18 to 30 years in patients with AD. Design, Setting, and Participants: The Plasma for Alzheimer Symptom Amelioration (PLASMA) study randomized 9 patients under a double-blind crossover protocol to receive 4 once-weekly infusions of either 1 unit (approximately 250 mL) of yFFP from male donors or 250 mL of saline, followed by a 6-week washout and crossover to 4 once-weekly infusions of an alternate treatment. Patients and informants were masked to treatment and subjective measurements. After an open-label amendment, 9 patients received 4 weekly yFFP infusions only and their subjective measurements were unmasked. Patients were enrolled solely at Stanford University, a tertiary academic medical center, from September 2014 to December 2016, when enrollment reached its target. Eighteen consecutive patients with probable mild to moderate AD dementia, a Mini-Mental State Examination (score of 12 to 24 inclusive), and an age of 50 to 90 years were enrolled. Thirty-one patients were screened and 13 were excluded: 11 failed the inclusion criteria and 2 declined to participate. Interventions: One unit of yFFP from male donors/placebo infused once weekly for 4 weeks. Main Outcome and Measures: The primary outcomes were the safety, tolerability, and feasibility of 4 weekly yFFP infusions. Safety end point analyses included all patients who received the study drug/placebo. Results: There was no difference in the age (mean [SD], 74.17 [7.96] years), sex (12 women [67%]), or baseline Mini-Mental State Examination score (mean [SD], 19.39 [3.24]) between the crossover (n = 9) and open-label groups (n = 9). There were no related serious adverse events. One patient discontinued participation because of urticaria and another because of an unrelated stroke. There was no statistically significant difference between the plasma (17 [94.4%]) and placebo (9 [100.0%]) cohorts for other adverse events, which were mild to moderate in severity. The most common adverse events in the plasma group included hypertension (3 [16.7%]), dizziness (2 [11.1%]), sinus bradycardia (3 [16.7%]), headache (3 [16.7%]), and sinus tachycardia (3 [16.7%]). The mean visit adherence (n = 18) was 86% (interquartile range, 87%-100%) and adherence, accounting for a reduction in the total visit requirement due to early patient discontinuation, was 96% (interquartile range, 89%-100%). Conclusions and Relevance: The yFFP treatment was safe, well tolerated, and feasible. The study's limitations were the small sample size, short duration, and change in study design. The results warrant further exploration in larger, double-blinded placebo-controlled clinical trials. Trial Registration: ClinicalTrials.gov Identifier: NCT02256306.

A Guide to Single-Cell Transcriptomics in Adult Rodent Brain: The Medium Spiny Neuron Transcriptome Revisited.

Recent advances in single-cell technologies are paving the way to a comprehensive understanding of the cellular complexity in the brain. Protocols for single-cell transcriptomics combine a variety of sophisticated methods for the purpose of isolating the heavily interconnected and heterogeneous neuronal cell types in a relatively intact and healthy state. The emphasis of single-cell transcriptome studies has thus far been on comparing library generation and sequencing techniques that enable measurement of the minute amounts of starting material from a single cell. However, in order for data to be comparable, standardized cell isolation techniques are essential. Here, we analyzed and simplified methods for the different steps critically involved in single-cell isolation from brain. These include enzymatic digestion, tissue trituration, improved methods for efficient fluorescence-activated cell sorting in samples containing high degree of debris from the neuropil, and finally, highly region-specific cellular labeling compatible with use of stereotaxic coordinates. The methods are exemplified using medium spiny neurons (MSN) from dorsomedial striatum, a cell type that is clinically relevant for disorders of the basal ganglia, including psychiatric and neurodegenerative diseases. We present single-cell RNA sequencing (scRNA-Seq) data from D1 and D2 dopamine receptor expressing MSN subtypes. We illustrate the need for single-cell resolution by comparing to available population-based gene expression data of striatal MSN subtypes. Our findings contribute toward standardizing important steps of single-cell isolation from adult brain tissue to increase comparability of data. Furthermore, our data redefine the transcriptome of MSNs at unprecedented resolution by confirming established marker genes, resolving inconsistencies from previous gene expression studies, and identifying novel subtype-specific marker genes in this important cell type.

Protein Phosphatase 2A and Its Methylation Modulating Enzymes LCMT-1 and PME-1 Are Dysregulated in Tauopathies of Progressive Supranuclear Palsy and Alzheimer Disease.

Hyperphosphorylated tau aggregates are characteristic of tauopathies including progressive supranuclear palsy (PSP) and Alzheimer disease (AD), but factors contributing to pathologic tau phosphorylation are not well understood. Here, we studied the regulation of the major tau phosphatase, the heterotrimeric AB55αC protein phosphatase 2 A (PP2A), in PSP and AD. The assembly and activity of this PP2A isoform are regulated by reversible carboxyl methylation of its catalytic C subunit, while the B subunit confers substrate specificity. We sought to address whether the decreases in PP2A methylation and its methylating enzyme, leucine carboxyl methyltransferase (LCMT-1), which are reported in AD, relate to tau pathology or to concomitant amyloid pathology by comparing them in the relatively pure tauopathy PSP. Immunohistochemical analysis of frontal cortices showed that methyl-PP2A is reduced while demethyl-PP2A is increased, with no changes in total PP2A or B55α subunit, resulting in a reduction in the methyl/demethyl PP2A ratio of 63% in PSP and 75% in AD compared to controls. Similarly, Western blot analyses showed a decrease of methyl-PP2A and an increase of demethyl-PP2A with a concomitant reduction in the methyl/demethyl PP2A ratio in both PSP (74%) and AD (76%) brains. This was associated with a decrease in LCMT-1 and an increase in the demethylating enzyme, protein phosphatase methylesterase (PME-1), in both diseases. These findings suggest that PP2A dysregulation in tauopathies may contribute to the accumulation of hyperphosphorylated tau and to neurodegeneration.

Dysregulation of protein phosphatase 2A in parkinson disease and dementia with lewy bodies.

OBJECTIVE: Protein phosphatase 2A (PP2A) is a heterotrimeric holoenzyme composed of a catalytic C subunit, a structural A subunit, and one of several regulatory B subunits that confer substrate specificity. The assembly and activity of PP2A are regulated by reversible methylation of the C subunit. α-Synuclein, which aggregates in Parkinson disease (PD) and dementia with Lewy bodies (DLB), is phosphorylated at Ser129, and PP2A containing a B55α subunit is a major phospho-Ser129 phosphatase. The objective of this study was to investigate PP2A in α-synucleinopathies. METHODS: We compared the state of PP2A methylation, as well as the expression of its methylating enzyme, leucine carboxyl methyltransferase (LCMT-1), and demethylating enzyme, protein phosphatase methylesterase (PME-1), in postmortem brains from PD and DLB cases as well as age-matched Controls. Immunohistochemical studies and quantitative image analysis were employed. RESULTS: LCMT-1 was significantly reduced in the substantia nigra (SN) and frontal cortex in both PD and DLB. PME-1, on the other hand, was elevated in the PD SN. In concert with these changes, the ratio of methylated PP2A to demethylated PP2A was markedly decreased in PD and DLB brains in both SN and frontal cortex. No changes in total PP2A or total B55α subunit were detected. INTERPRETATION: These findings support the hypothesis that PP2A dysregulation in α-synucleinopathies may contribute to the accumulation of hyperphosphorylated α-synuclein and to the disease process, raising the possibility that pharmacological means to enhance PP2A phosphatase activity may be a useful disease-modifying therapeutic approach.

Dual κ-agonist/µ-antagonist opioid receptor modulation reduces levodopa-induced dyskinesia and corrects dysregulated striatal changes in the nonhuman primate model of Parkinson disease.

OBJECTIVE: Effective medical management of levodopa-induced dyskinesia (LID) remains an unmet need for patients with Parkinson disease (PD). Changes in opioid transmission in the basal ganglia associated with LID suggest a therapeutic opportunity. Here we determined the impact of modulating both mu and kappa opioid receptor signaling using the mixed agonist/antagonist analgesic nalbuphine in reducing LID and its molecular markers in the nonhuman primate model. METHODS: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated macaques with advanced parkinsonism and reproducible LID received a range of nalbuphine doses or saline subcutaneously as: (1) monotherapy, (2) acute coadministration with levodopa, and (3) chronic coadministration for 1 month. Animals were assessed by blinded examiners for motor disability and LID severity using standardized rating scales. Plasma levodopa levels were determined with and without nalbuphine, and postmortem brain samples were subjected to Western blot analyses. RESULTS: Nalbuphine reduced LID in a dose-dependent manner by 48% (p < 0.001) without compromising the anti-PD effect of levodopa or changing plasma levodopa levels. There was no tolerance to the anti-LID effect of nalbuphine given chronically. Nalbuphine coadministered with levodopa was well tolerated and did not cause sedation. Nalbuphine monotherapy had no effect on motor disability. Striatal tissue analyses showed that nalbuphine cotherapy blocks several molecular correlates of LID, including overexpression of ΔFosB, prodynorphin, dynorphin A, cyclin-dependent kinase 5, and increased phosphorylation of DARPP-32 at threonine-34. INTERPRETATION: Nalbuphine reverses the molecular milieu in the striatum associated with LID and is a safe and effective anti-LID agent in the primate model of PD. These findings support repurposing this analgesic for the treatment of LID.

A pharmacological screening approach for discovery of neuroprotective compounds in ischemic stroke.

With the availability and ease of small molecule production and design continuing to improve, robust, high-throughput methods for screening are increasingly necessary to find pharmacologically relevant compounds amongst the masses of potential candidates. Here, we demonstrate that a primary oxygen glucose deprivation assay in primary cortical neurons followed by secondary assays (i.e. post-treatment protocol in organotypic hippocampal slice cultures and cortical neurons) can be used as a robust screen to identify neuroprotective compounds with potential therapeutic efficacy. In our screen about 50% of the compounds in a library of pharmacologically active compounds displayed some degree of neuroprotective activity if tested in a pre-treatment toxicity assay but just a few of these compounds, including Carbenoxolone, remained active when tested in a post-treatment protocol. When further examined, Carbenoxolone also led to a significant reduction in infarction size and neuronal damage in the ischemic penumbra when administered six hours post middle cerebral artery occlusion in rats. Pharmacological testing of Carbenoxolone-related compounds, acting by inhibition of 11-ß-hydroxysteroid dehydrogenase-1 (11ß-HSD1), gave rise to similarly potent in vivo neuroprotection. This indicates that the increase of intracellular glucocorticoid levels mediated by 11ß-HSD1 may be involved in the mechanism that exacerbates ischemic neuronal cell death, and inhibiting this enzyme could have potential therapeutic value for neuroprotective therapies in ischemic stroke and other neurodegenerative disorders associated with neuronal injury.

Neuroprotective and anti-inflammatory properties of a coffee component in the MPTP model of Parkinson's disease.

Consumption of coffee is associated with reduced risk of Parkinson's disease (PD), an effect that has largely been attributed to caffeine. However, coffee contains numerous components that may also be neuroprotective. One of these compounds is eicosanoyl-5-hydroxytryptamide (EHT), which ameliorates the phenotype of α-synuclein transgenic mice associated with decreased protein aggregation and phosphorylation, improved neuronal integrity and reduced neuroinflammation. Here, we sought to investigate if EHT has an effect in the MPTP model of PD. Mice fed a diet containing EHT for four weeks exhibited dose-dependent preservation of nigral dopaminergic neurons following MPTP challenge compared to animals given control feed. Reductions in striatal dopamine and tyrosine hydroxylase content were also less pronounced with EHT treatment. The neuroinflammatory response to MPTP was markedly attenuated, and indices of oxidative stress and JNK activation were significantly prevented with EHT. In cultured primary microglia and astrocytes, EHT had a direct anti-inflammatory effect demonstrated by repression of lipopolysaccharide-induced NFκB activation, iNOS induction, and nitric oxide production. EHT also exhibited a robust anti-oxidant activity in vitro. Additionally, in SH-SY5Y cells, MPP(+)-induced demethylation of phosphoprotein phosphatase 2A (PP2A), the master regulator of the cellular phosphoregulatory network, and cytotoxicity were ameliorated by EHT. These findings indicate that the neuroprotective effect of EHT against MPTP is through several mechanisms including its anti-inflammatory and antioxidant activities as well as its ability to modulate the methylation and hence activity of PP2A. Our data, therefore, reveal a strong beneficial effect of a novel component of coffee in multiple endpoints relevant to PD.

An ultra-specific avian antibody to phosphorylated tau protein reveals a unique mechanism for phosphoepitope recognition.

Highly specific antibodies to phosphoepitopes are valuable tools to study phosphorylation in disease states, but their discovery is largely empirical, and the molecular mechanisms mediating phosphospecific binding are poorly understood. Here, we report the generation and characterization of extremely specific recombinant chicken antibodies to three phosphoepitopes on the Alzheimer disease-associated protein tau. Each antibody shows full specificity for a single phosphopeptide. The chimeric IgG pT231/pS235_1 exhibits a K(D) of 0.35 nm in 1:1 binding to its cognate phosphopeptide. This IgG is murine ortholog-cross-reactive, specifically recognizing the pathological form of tau in brain samples from Alzheimer patients and a mouse model of tauopathy. To better understand the underlying binding mechanisms allowing such remarkable specificity, we determined the structure of pT231/pS235_1 Fab in complex with its cognate phosphopeptide at 1.9 Å resolution. The Fab fragment exhibits novel complementarity determining region (CDR) structures with a "bowl-like" conformation in CDR-H2 that tightly and specifically interacts with the phospho-Thr-231 phosphate group, as well as a long, disulfide-constrained CDR-H3 that mediates peptide recognition. This binding mechanism differs distinctly from either peptide- or hapten-specific antibodies described to date. Surface plasmon resonance analyses showed that pT231/pS235_1 binds a truly compound epitope, as neither phosphorylated Ser-235 nor free peptide shows any measurable binding affinity.

α-Synuclein phosphorylation as a therapeutic target in Parkinson's disease.

Phosphorylation is a key post-translational modification necessary for normal cellular signaling and, therefore, lies at the heart of cellular function. In neurodegenerative disorders, abnormal hyperphosphorylation of pathogenic proteins is a common phenomenon that contributes in important ways to the disease process. A prototypical protein that is hyperphosphorylated in the brain is α-synuclein (α-syn) - found in Lewy bodies and Lewy neurites - the pathological hallmarks of Parkinson's disease (PD) and other α-synucleinopathies. The genetic linkage of α-syn to PD as well as its pathological association in both genetic and sporadic cases have made it the primary protein of interest. In understanding how α-syn dysfunction occurs, increasing focus is being placed on its abnormal aggregation and the contribution of phosphorylation to this process. Studies of both the kinases and phosphatases that regulate α-syn phosphorylation are beginning to reveal the roles of this post-translational modification in disease pathogenesis. Modulation of α-syn phosphorylation may ultimately prove to be a viable strategy for disease-modifying therapeutic interventions. In this review, we explore mechanisms related to α-syn phosphorylation, its biophysical and functional consequences, and its role in neurodegeneration.

Protein phosphatases and Alzheimer's disease.

Alzheimer's Disease (AD) is characterized by progressive loss of cognitive function, linked to marked neuronal loss. Pathological hallmarks of the disease are the accumulation of the amyloid-ß (Aß) peptide in the form of amyloid plaques and the intracellular formation of neurofibrillary tangles (NFTs). Accumulating evidence supports a key role for protein phosphorylation in both the normal and pathological actions of Aß as well as the formation of NFTs. NFTs contain hyperphosphorylated forms of the microtubule-binding protein tau, and phosphorylation of tau by several different kinases leads to its aggregation. The protein kinases involved in the generation and/or actions of tau or Aß are viable drug targets to prevent or alleviate AD pathology. However, it has also been recognized that the protein phosphatases that reverse the actions of these protein kinases are equally important. Here, we review recent advances in our understanding of serine/threonine and tyrosine protein phosphatases in the pathology of AD.

Targeting phosphatases as the next generation of disease modifying therapeutics for Parkinson's disease.

Phosphorylation is a key post-translational modification for cellular signaling, and abnormalities in this process are observed in several neurodegenerative disorders. Among these disorders, Parkinson's disease (PD) is particularly intriguing as there are both genetic causes of disease that involve phosphorylation, and pathological hallmarks of disease composed of a hyperphosphorylated protein. Two of the major genes linked to PD are themselves kinases - leucine rich repeat kinase 2 (LRRK2) and phosphatase and tensin induced homolog kinase 1 (PINK1). Mutations in LRRK2 lead to its increased kinase activity and dominantly inherited PD, while mutations in PINK1 lead to loss of function and recessive PD. A third genetic linkage to disease is α-synuclein, a protein that is heavily phosphorylated in Lewy bodies and Lewy neurites, the pathological hallmarks of PD. The phosphorylation of α-synuclein at various residues influences its aggregation, either positively or negatively, thereby impacting its central role in disease pathogenesis. Given these associations of phosphorylation with PD, modulation of this modification is an attractive therapeutic strategy. The kinases that act in these disease relevant pathways have been the primary target for such approaches. But, the development of kinase inhibitors has been complicated by the necessary specificity to retain safety, the redundancy of kinases leading to lack of efficacy, and the difficulties in overcoming the blood-brain barrier. The field of modulating phosphatases has the potential to overcome some of these issues and provide the next generation of therapeutic targets for PD. In this review, we address the phosphorylation pathways involved in PD, the kinases and issues related to their inhibition, and the evolving field of the phosphatases relevant in PD and how they may be targeted pharmacologically.