Serum neuronal exosomes predict and differentiate Parkinson's disease from atypical parkinsonism.

OBJECTIVE: Parkinson's disease is characterised neuropathologically by α-synuclein aggregation. Currently, there is no blood test to predict the underlying pathology or distinguish Parkinson's from atypical parkinsonian syndromes. We assessed the clinical utility of serum neuronal exosomes as biomarkers across the spectrum of Parkinson's disease, multiple system atrophy and other proteinopathies. METHODS: We performed a cross-sectional study of 664 serum samples from the Oxford, Kiel and Brescia cohorts consisting of individuals with rapid eye movement sleep behavioural disorder, Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, frontotemporal dementia, progressive supranuclear palsy, corticobasal syndrome and controls. Longitudinal samples were analysed from Parkinson's and control individuals. We developed poly(carboxybetaine-methacrylate) coated beads to isolate L1 cell adhesion molecule (L1CAM)-positive extracellular vesicles with characteristics of exosomes and used mass spectrometry or multiplexed electrochemiluminescence to measure exosomal proteins. RESULTS: Mean neuron-derived exosomal α-synuclein was increased by twofold in prodromal and clinical Parkinson's disease when compared with multiple system atrophy, controls or other neurodegenerative diseases. With 314 subjects in the training group and 105 in the validation group, exosomal α-synuclein exhibited a consistent performance (AUC=0.86) in separating clinical Parkinson's disease from controls across populations. Exosomal clusterin was elevated in subjects with non-α-synuclein proteinopathies. Combined neuron-derived exosomal α-synuclein and clusterin measurement predicted Parkinson's disease from other proteinopathies with AUC=0.98 and from multiple system atrophy with AUC=0.94. Longitudinal sample analysis showed that exosomal α-synuclein remains stably elevated with Parkinson's disease progression. CONCLUSIONS: Increased α-synuclein egress in serum neuronal exosomes precedes the diagnosis of Parkinson's disease, persists with disease progression and in combination with clusterin predicts and differentiates Parkinson's disease from atypical parkinsonism.

Neurochemical and behavioral profiling of the selective GlyT1 inhibitors ALX5407 and LY2365109 indicate a preferential action in caudal vs. cortical brain areas.

Selective inhibitors of the glycine transporter 1 (GlyT1) have been implicated in central nervous system disorders related to hypoglutamatergic function such as schizophrenia. The selective GlyT1 inhibitors ALX5407 (NFPS) and LY2365109 {[2-(4-benzo[1,3]dioxol-5-yl-2-tert-butylphenoxy)ethyl]-methylamino}-acetic acid increased cerebrospinal fluid levels of glycine and potentiated NMDA-induced increases in dialysate levels of neurotransmitters in the prefrontal cortex (PFC) and the striatum. However, higher doses produced both stimulatory and inhibitory effects on motor performance and impaired respiration, suggesting significant involvement of cerebellar and brain stem areas. A dual probe microdialysis study showed that ALX5407 transiently elevated extracellular levels of glycine in the PFC with more sustained increases in the cerebellum. In support of these findings, immuno-staining with pan-GlyT1 and GlyT1a antibodies showed a higher abundance of immunoreactivity in the brain stem/cerebellum as compared to the frontal cortical/hippocampal brain areas in four different species studied, including the mouse, rat, monkey and human. In addition, the inhibitory effects of ALX5407 on cerebellar levels of cGMP in the mouse could be reversed by the glycine A receptor antagonist strychnine but not the glycine B receptor antagonist L-701324. We propose that the adverse events seen with higher doses of ALX5407 and LY2365109 are the result of high GlyT1 inhibitory activity in caudal areas of the brain with sustained elevations of extracellular glycine. High levels of glycine in these brain areas may result in activation of strychnine-sensitive glycine A receptors that are inhibitory on both motor activity and critical brain stem functions such as respiration.

Loss of Trem2 in microglia leads to widespread disruption of cell coexpression networks in mouse brain.

Rare heterozygous coding variants in the triggering receptor expressed in myeloid cells 2 (TREM2) gene, conferring increased risk of developing late-onset Alzheimer's disease, have been identified. We examined the transcriptional consequences of the loss of Trem2 in mouse brain to better understand its role in disease using differential expression and coexpression network analysis of Trem2 knockout and wild-type mice. We generated RNA-Seq data from cortex and hippocampus sampled at 4 and 8 months. Using brain cell-type markers and ontology enrichment, we found subnetworks with cell type and/or functional identity. We primarily discovered changes in an endothelial gene-enriched subnetwork at 4 months, including a shift toward a more central role for the amyloid precursor protein gene, coupled with widespread disruption of other cell-type subnetworks, including a subnetwork with neuronal identity. We reveal an unexpected potential role of Trem2 in the homeostasis of endothelial cells that goes beyond its known functions as a microglial receptor and signaling hub, suggesting an underlying link between immune response and vascular disease in dementia.

Genome-wide RNAseq study of the molecular mechanisms underlying microglia activation in response to pathological tau perturbation in the rTg4510 tau transgenic animal model.

BACKGROUND: Activation of microglia, the resident immune cells of the central nervous system, is a prominent pathological hallmark of Alzheimer's disease (AD). However, the gene expression changes underlying microglia activation in response to tau pathology remain elusive. Furthermore, it is not clear how murine gene expression changes relate to human gene expression networks. METHODS: Microglia cells were isolated from rTg4510 tau transgenic mice and gene expression was profiled using RNA sequencing. Four age groups of mice (2-, 4-, 6-, and 8-months) were analyzed to capture longitudinal gene expression changes that correspond to varying levels of pathology, from minimal tau accumulation to massive neuronal loss. Statistical and system biology approaches were used to analyze the genes and pathways that underlie microglia activation. Differentially expressed genes were compared to human brain co-expression networks. RESULTS: Statistical analysis of RNAseq data indicated that more than 4000 genes were differentially expressed in rTg4510 microglia compared to wild type microglia, with the majority of gene expression changes occurring between 2- and 4-months of age. These genes belong to four major clusters based on their temporal expression pattern. Genes involved in innate immunity were continuously up-regulated, whereas genes involved in the glutamatergic synapse were down-regulated. Up-regulated innate inflammatory pathways included NF-κB signaling, cytokine-cytokine receptor interaction, lysosome, oxidative phosphorylation, and phagosome. NF-κB and cytokine signaling were among the earliest pathways activated, likely driven by the RELA, STAT1 and STAT6 transcription factors. The expression of many AD associated genes such as APOE and TREM2 was also altered in rTg4510 microglia cells. Differentially expressed genes in rTg4510 microglia were enriched in human neurodegenerative disease associated pathways, including Alzheimer's, Parkinson's, and Huntington's diseases, and highly overlapped with the microglia and endothelial modules of human brain transcriptional co-expression networks. CONCLUSION: This study revealed temporal transcriptome alterations in microglia cells in response to pathological tau perturbation and provides insight into the molecular changes underlying microglia activation during tau mediated neurodegeneration.

Preclinical profile of a dopamine D1 potentiator suggests therapeutic utility in neurological and psychiatric disorders.

DETQ, an allosteric potentiator of the dopamine D1 receptor, was tested in therapeutic models that were known to respond to D1 agonists. Because of a species difference in affinity for DETQ, all rodent experiments used transgenic mice expressing the human D1 receptor (hD1 mice). When given alone, DETQ reversed the locomotor depression caused by a low dose of reserpine. DETQ also acted synergistically with L-DOPA to reverse the strong hypokinesia seen with a higher dose of reserpine. These results indicate potential as both monotherapy and adjunct treatment in Parkinson's disease. DETQ markedly increased release of both acetylcholine and histamine in the prefrontal cortex, and increased levels of histamine metabolites in the striatum. In the hippocampus, the combination of DETQ and the cholinesterase inhibitor rivastigmine increased ACh to a greater degree than either agent alone. DETQ also increased phosphorylation of the AMPA receptor (GluR1) and the transcription factor CREB in the striatum, consistent with enhanced synaptic plasticity. In the Y-maze, DETQ increased arm entries but (unlike a D1 agonist) did not reduce spontaneous alternation between arms at high doses. DETQ enhanced wakefulness in EEG studies in hD1 mice and decreased immobility in the forced-swim test, a model for antidepressant-like activity. In rhesus monkeys, DETQ increased spontaneous eye-blink rate, a measure that is known to be depressed in Parkinson's disease. Together, these results provide support for potential utility of D1 potentiators in the treatment of several neuropsychiatric disorders, including Parkinson's disease, Alzheimer's disease, cognitive impairment in schizophrenia, and major depressive disorder.

Association of expression of CD44v6 with systemic anaplastic large cell lymphoma: comparison with primary cutaneous anaplastic large cell lymphoma.

CD44 is a ubiquitous multifunctional cell surface adhesion molecule family. High expression of the standard form, CD44s (CD44), and its variant form, CD44v6, has been reported to be associated with tumor dissemination in non-Hodgkin lymphoma. To evaluate the potential role of CD44 and/or CD44v6 in different entities of anaplastic large cell lymphoma (ALCL), 30 cases of systemic ALCL (sALCL; 20 cases) and primary cutaneous ALCL (cALCL; 10 cases) were compared for expression of CD44 and CD44v6 by immunohistochemical staining. Expression of CD44v6 also was analyzed with respect to expression of anaplastic lymphoma kinase (ALK) in sALCL. No difference of CD44 expression was noted between sALCL and cALCL In contrast, expression of CD44v6 was found in 18 (90%) of sALCL cases and in 5 (50%) of cALCL cases. There was no correlation between expression of CD44v6 and expression of ALK in sALCL. These results indicate that expression of CD44v6 rather than CD44 correlates with sALCL. Furthermore, these results suggest that CD44v6 and ALK may be independent predictors of risk for the systemic phenotype of ALCL.

Assessment of t(2;5)(p23;q35) translocation and variants in pediatric ALK+ anaplastic large cell lymphoma.

To evaluate t(2;5) and its variants, we studied 21 pediatric cases of anaplastic lymphoma kinase (ALK)+ anaplastic large cell lymphoma (ALCL) by using immunohistochemical staining, fluorescence in situ hybridization, cytogenetics, and reverse transcriptase-polymerase chain reaction. Results showed 7 (33%) cases with t(2;5), 6 (29%) with variant gene rearrangements, 7 (33%) with uncharacterized rearrangements, and 1 with ALK protein expression but no ALK rearrangement. Among 6 variant gene rearrangements, 1 had TPM4-ALK/t(2;19)(p23;p13) and 2 had inv(2) with the breakpoint proximate to ATIC-ALK and an unknown partner gene separately. The genetic features of the remaining 3 cases were as follows: ins(8;2) with an unknown partner gene; conversion from ALK- at diagnosis to ALK+ at recurrence with unspecified gene rearrangement; complex karyotype without involvement of 2p23, suggesting a cryptic translocation. Concordance between different laboratory results varied from 47% to 81%. These data suggest that ALK variants are not uncommon and underscore the necessity of integrating immunohistochemical, cytogenetic, and molecular genetic approaches to detect, characterize, and confirm t(2;5) and its variant translocations.

Quantitative measurement of intact alpha-synuclein proteoforms from post-mortem control and Parkinson's disease brain tissue by intact protein mass spectrometry.

A robust top down proteomics method is presented for profiling alpha-synuclein species from autopsied human frontal cortex brain tissue from Parkinson's cases and controls. The method was used to test the hypothesis that pathology associated brain tissue will have a different profile of post-translationally modified alpha-synuclein than the control samples. Validation of the sample processing steps, mass spectrometry based measurements, and data processing steps were performed. The intact protein quantitation method features extraction and integration of m/z data from each charge state of a detected alpha-synuclein species and fitting of the data to a simple linear model which accounts for concentration and charge state variability. The quantitation method was validated with serial dilutions of intact protein standards. Using the method on the human brain samples, several previously unreported modifications in alpha-synuclein were identified. Low levels of phosphorylated alpha synuclein were detected in brain tissue fractions enriched for Lewy body pathology and were marginally significant between PD cases and controls (p = 0.03).

Pharmacological characterization of cGMP regulation by the biarylpropylsulfonamide class of positive, allosteric modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

The biarylpropylsulfonamide class of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) potentiators represented by N-2-(4-(4-cyanophenol)phenol)propyl-2-propanesulfonamide (LY404187) and (R)-4'-[1-fluoro-1-methyl-2-(propane-2-sulfonylamino)-ethyl]-biphenyl-4-carboxylic acid methylamide (LY503430) are positive, allosteric AMPA receptor activators, which enhance AMPA receptor-mediated neurotransmission by reducing desensitization of the ion channel. Although these compounds have efficacy in in vivo rodent models of cognition, depression, and Parkinson's disease, little is known about biochemical pathways activated by these agents. Given the well established regulation of the nitric oxide/cGMP pathway by excitatory neurotransmission, the current study characterized AMPA receptor potentiator-mediated cGMP response in mouse cerebellum. Acute treatment by both LY404187 and LY503430 [2.0, 5.0, or 10 mg/kg subcutaneously (s.c.)] elevated basal cerebellar cGMP levels in a dose-dependent manner. Pretreatment with the noncompetitive, allosteric AMPA receptor-selective antagonist 7H-1,3-dioxolo[4,5-h][2,3]benzodiazepine-7-carboxamide, 5-(4-aminophenyl)-8,9-dihydro-N,8-dimethyl-monohydrochloride-(9CI) (GYKI 53655) [3.0 mg/kg intraperitoneally (i.p.)], completely blocked the effect of LY404187, demonstrating that activation of AMPA receptors induces cGMP levels. Interestingly, pretreatment with the N-methyl-d-aspartate (NMDA) open channel blocker dizocilpine (0.3 and 1.0 mg/kg i.p.) also abolished the AMPA receptor potentiator-mediated cGMP accumulation, indicating that activation of AMPA receptors leads to NMDA receptor-mediated transmission involved in cGMP regulation. Pharmacological augmentation of the endogenous glutamate tone via the alkaloid harmaline (20-60 mg/kg i.p.) synergized with AMPA potentiator activity and provided further direct evidence of in vivo allosteric activation of AMPA receptors by LY404187. The synergism between harmaline and LY404187 was specific, since cGMP accumulation induced by foot-shock stress was not augmented by the AMPA receptor potentiator. Taken together, these data indicate that the cGMP system may play an important role in pharmacological efficacy of the biarylpropylsulfonamide class of AMPA receptor potentiators.

Nonsteroidal anti-inflammatory drugs can lower amyloidogenic Abeta42 by inhibiting Rho.

A subset of nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to preferentially reduce the secretion of the highly amyloidogenic, 42-residue amyloid-beta peptide Abeta42. We found that Rho and its effector, Rho-associated kinase, preferentially regulated the amount of Abeta42 produced in vitro and that only those NSAIDs effective as Rho inhibitors lowered Abeta42. Administration of Y-27632, a selective Rock inhibitor, also preferentially lowered brain levels of Abeta42 in a transgenic mouse model of Alzheimer's disease. Thus, the Rho-Rock pathway may regulate amyloid precursor protein processing, and a subset of NSAIDs can reduce Abeta42 through inhibition of Rho activity.