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Background: A rare variant in the Triggering Receptor Expressed on Myeloid cells 2 (TREM2) gene has been reported to be a genetic risk factor for Alzheimer's disease by two independent groups (Odds ratio between 2.9-4.5). Given the key role of TREM2 in the

Microglia are resident immune cells of the brain that play important roles in mediating inflammatory responses in several neurological diseases via direct and indirect mechanisms. One indirect mechanism may involve extracellular vesicle (EV) release, so that the molecular cargo transported by microglia-derived EVs can have functional effects by facilitating intercellular communication. The molecular composition of microglia-derived EVs, and how microglial activation states impact EV composition and EV-mediated effects in neuroinflammation, remain poorly understood. We hypothesize that microglia-derived EVs have unique molecular profiles that are determined by microglial activation state. Using size-exclusion chromatography to purify EVs from BV2 microglia, combined with proteomic (label-free quantitative mass spectrometry or LFQ-MS) and transcriptomic (mRNA and noncoding RNA seq) methods, we obtained comprehensive molecular profiles of microglia-derived EVs. LFQ-MS identified several classic EV proteins (tetraspanins, ESCRT machinery, and heat shock proteins), in addition to over 200 proteins not previously reported in the literature. Unique mRNA and microRNA signatures of microglia-derived EVs were also identified. After treating BV2 microglia with lipopolysaccharide (LPS), interleukin-10, or transforming growth factor beta, to mimic pro-inflammatory, anti-inflammatory, or homeostatic states, respectively, LFQ-MS and RNA seq revealed novel state-specific proteomic and transcriptomic signatures of microglia-derived EVs. Particularly, LPS treatment had the most profound impact on proteomic and transcriptomic compositions of microglia-derived EVs. Furthermore, we found that EVs derived from LPS-activated microglia were able to induce pro-inflammatory transcriptomic changes in resting responder microglia, confirming the ability of microglia-derived EVs to relay functionally relevant inflammatory signals. These comprehensive microglia-EV molecular datasets represent important resources for the neuroscience and omics communities and provide novel insights into the role of microglia-derived EVs in neuroinflammation., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Different brain cell types play distinct roles in brain development and disease. Molecular characterization of cell-specific mechanisms using cell type-specific approaches at the protein (proteomic) level can provide biological and therapeutic insights. To overcome the barriers of conventional isolation-based methods for cell type-specific proteomics, in vivo proteomic labeling with proximity-dependent biotinylation of cytosolic proteins using biotin ligase TurboID, coupled with mass spectrometry (MS) of labeled proteins, emerged as a powerful strategy for cell type-specific proteomics in the native state of cells without the need for cellular isolation. To complement in vivo proximity labeling approaches, in vitro studies are needed to ensure that cellular proteomes using the TurboID approach are representative of the whole-cell proteome and capture cellular responses to stimuli without disruption of cellular processes. To address this, we generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing cytosolic TurboID to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under homeostatic conditions. TurboID labeled endolysosome, translation, vesicle, and signaling proteins in BV2 microglia and synaptic, neuron projection, and microtubule proteins in N2A neurons. TurboID expression and biotinylation minimally impacted homeostatic cellular proteomes of BV2 and N2A cells and did not affect lipopolysaccharide-mediated cytokine production or resting cellular respiration in BV2 cells. MS analysis of the microglial biotin-labeled proteins captured the impact of lipopolysaccharide treatment (>500 differentially abundant proteins) including increased canonical proinflammatory proteins (Il1a, Irg1, and Oasl1) and decreased anti-inflammatory proteins (Arg1 and Mgl2)., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Mitochondria influence cellular function through both cell-autonomous and non-cell autonomous mechanisms, such as production of paracrine and endocrine factors. Here, we demonstrate that mitochondrial regulation of the secretome is more extensive than previously appreciated, as both genetic and pharmacological disruption of the electron transport chain caused upregulation of the Alzheimer's disease risk factor apolipoprotein E (APOE) and other secretome components. Indirect disruption of the electron transport chain by gene editing of SLC25A mitochondrial membrane transporters as well as direct genetic and pharmacological disruption of either complexes I, III, or the copper-containing complex IV of the electron transport chain elicited upregulation of APOE transcript, protein, and secretion, up to 49-fold. These APOE phenotypes were robustly expressed in diverse cell types and iPSC-derived human astrocytes as part of an inflammatory gene expression program. Moreover, age- and genotype-dependent decline in brain levels of respiratory complex I preceded an increase in APOE in the 5xFAD mouse model. We propose that mitochondria act as novel upstream regulators of APOE-dependent cellular processes in health and disease., Competing Interests: MW, OO, AL, AG, SZ, CX, ZW, DD, SR, AI, EO, ED, NS, BR, AC, VS, MP, NS, SK, SC, AB, AW, TW, SR, AL, EW, VF No competing interests declared, (© 2023, Wynne et al.)

MECP2 loss-of-function mutations cause Rett syndrome, a neurodevelopmental disorder resulting from a disrupted brain transcriptome. How these transcriptional defects are decoded into a disease proteome remains unknown. We studied the proteome of Rett cerebrospinal fluid (CSF) to identify consensus Rett proteome and ontologies shared across three species. Rett CSF proteomes enriched proteins annotated to HDL lipoproteins, complement, mitochondria, citrate/pyruvate metabolism, synapse compartments, and the neurosecretory protein VGF. We used shared Rett ontologies to select analytes for orthogonal quantification and functional validation. VGF and ontologically selected CSF proteins had genotypic discriminatory capacity as determined by receiver operating characteristic analysis in Mecp2 -/y and Mecp2 -/+ . Differentially expressed CSF proteins distinguished Rett from a related neurodevelopmental disorder, CDKL5 deficiency disorder. We propose that Mecp2 mutant CSF proteomes and ontologies inform putative mechanisms and biomarkers of disease. We suggest that Rett syndrome results from synapse and metabolism dysfunction., Competing Interests: Mustafa Sahin reports grant support from Novartis, Biogen, Astellas, Aeovian, Bridgebio, and Aucta. He has served on Scientific Advisory Boards for Novartis, Roche, Regenxbio, SpringWorks Therapeutics, Jaguar Therapeutics and Alkermes. James J. Fink is an employee of Q-State Biosciences. VF is a member of the iScience editorial board., (© 2022 The Author(s).)

In Alzheimer's disease (AD), a multitude of genetic risk factors and early biomarkers are known. Nevertheless, the causal factors responsible for initiating cognitive decline in AD remain controversial. Toxic plaques and tangles correlate with progressive neuropathology, yet disruptions in circuit activity emerge before their deposition in AD models and patients. Parvalbumin (PV) interneurons are potential candidates for dysregulating cortical excitability as they display altered action potential (AP) firing before neighboring excitatory neurons in prodromal AD. Here, we report a novel mechanism responsible for PV hypoexcitability in young adult familial AD mice. We found that biophysical modulation of K v 3 channels, but not changes in their mRNA or protein expression, were responsible for dampened excitability in young 5xFAD mice. These K + conductances could efficiently regulate near-threshold AP firing, resulting in gamma-frequency-specific network hyperexcitability. Thus, biophysical ion channel alterations alone may reshape cortical network activity prior to changes in their expression levels. Our findings demonstrate an opportunity to design a novel class of targeted therapies to ameliorate cortical circuit hyperexcitability in early AD., Competing Interests: VO, AG, SR, ED, NS, SR, JD, MR No competing interests declared, (© 2022, Olah, Goettemoeller et al.)

Proteomic profiling of brain cell types using isolation-based strategies pose limitations in resolving cellular phenotypes representative of their native state. We describe a mouse line for cell type-specific expression of biotin ligase TurboID, for in vivo biotinylation of proteins. Using adenoviral and transgenic approaches to label neurons, we show robust protein biotinylation in neuronal soma and axons throughout the brain, allowing quantitation of over 2000 neuron-derived proteins spanning synaptic proteins, transporters, ion channels and disease-relevant druggable targets. Next, we contrast Camk2a-neuron and Aldh1l1-astrocyte proteomes and identify brain region-specific proteomic differences within both cell types, some of which might potentially underlie the selective vulnerability to neurological diseases. Leveraging the cellular specificity of proteomic labeling, we apply an antibody-based approach to uncover differences in neuron and astrocyte-derived signaling phospho-proteins and cytokines. This approach will facilitate the characterization of cell-type specific proteomes in a diverse number of tissues under both physiological and pathological states., (© 2022. The Author(s).)

Kv1.3 potassium channels, expressed by proinflammatory central nervous system mononuclear phagocytes (CNS-MPs), are promising therapeutic targets for modulating neuroinflammation in Alzheimer's disease (AD). The molecular characteristics of Kv1.3-high CNS-MPs and their cellular origin from microglia or CNS-infiltrating monocytes are unclear. While Kv1.3 blockade reduces amyloid beta (Aβ) burden in mouse models, the downstream immune effects on molecular profiles of CNS-MPs remain unknown. We show that functional Kv1.3 channels are selectively expressed by a subset of CD11b + CD45 + CNS-MPs acutely isolated from an Aβ mouse model (5xFAD) as well as fresh postmortem human AD brain. Transcriptomic profiling of purified CD11b + Kv1.3 + CNS-MPs, CD11b + CD45 int Kv1.3 neg microglia, and peripheral monocytes from 5xFAD mice revealed that Kv1.3-high CNS-MPs highly express canonical microglial markers ( Tmem119 , P2ry12 ) and are distinct from peripheral Ly6c high /Ly6c low monocytes. Unlike homeostatic microglia, Kv1.3-high CNS-MPs express relatively lower levels of homeostatic genes, higher levels of CD11c , and increased levels of glutamatergic transcripts, potentially representing phagocytic uptake of neuronal elements. Using irradiation bone marrow CD45.1/CD45.2 chimerism in 5xFAD mice, we show that Kv1.3 + CNS-MPs originate from microglia and not blood-derived monocytes. We show that Kv1.3 channels regulate membrane potential and early signaling events in microglia. Finally, in vivo blockade of Kv1.3 channels in 5xFAD mice by ShK-223 reduced Aβ burden, increased CD11c + CNS-MPs, and expression of phagocytic genes while suppressing proinflammatory genes ( IL1b ). Our results confirm the microglial origin and identify unique molecular features of Kv1.3-expressing CNS-MPs. In addition, we provide evidence for CNS immunomodulation by Kv1.3 blockers in AD mouse models resulting in a prophagocytic phenotype., Competing Interests: The authors declare no competing interest.

The repeated failures of amyloid-targeting therapies have challenged our narrow understanding of Alzheimer's disease (AD) pathogenesis and inspired wide-ranging investigations into the underlying mechanisms of disease. Increasing evidence indicates that AD develops from an intricate web of biochemical and cellular processes that extend far beyond amyloid and tau accumulation. This growing recognition surrounding the diversity of AD pathophysiology underscores the need for holistic systems-based approaches to explore AD pathogenesis. Here we describe how network-based proteomics has emerged as a powerful tool and how its application to the AD brain has provided an informative framework for the complex protein pathophysiology underlying the disease. Furthermore, we outline how the AD brain network proteome can be leveraged to advance additional scientific and translational efforts, including the discovery of novel protein biomarkers of disease.

Microglia and central nervous system (CNS)-infiltrating macrophages, collectively called CNS mononuclear phagocytes (CNS-MPs), play central roles in neurological diseases including neurodegeneration and stroke. CNS-MPs are involved in phagocytic clearance of pathological proteins, debris and neuronal synapses, each with distinct underlying molecular pathways. Characterizing these phagocytic properties can provide a functional readout that compliments molecular profiling of microglia using traditional flow cytometry, transcriptomics and proteomics approaches. Phagocytic profiling of microglia has relied on microscopic visualization and in vitro cultures of mouse neonatal microglia. The former approach suffers from limited sampling while the latter approach is inherently poorly reflective of the true in vivo state of adult CNS-MPs. This paper describes optimized protocols to phenotype phagocytic properties of acutely-isolated mouse CNS-MPs by flow cytometry. CNS-MPs are acutely isolated from adult mouse brain using mechanical dissociation followed by density gradient centrifugation, incubated with fluorescent microspheres or fluorescent Aβ fibrils, washed, and then labeled with panels of antibodies against surface markers (CD11b, CD45). Using this approach, it is possible to compare phagocytic properties of brain-resident microglia with CNS-infiltrating macrophages and then assess the effect of aging and disease pathology on these phagocytic phenotypes. This rapid method also holds potential to functionally phenotype acutely-isolated human CNS-MPs from post-mortem or surgical brain specimens. Additionally, specific mechanisms of phagocytosis by CNS-MP subsets can be investigated by inhibiting select phagocytic pathways.

Background: Proteomic characterization of microglia provides the most proximate assessment of functionally relevant molecular mechanisms of neuroinflammation. However, microglial proteomics studies have been limited by low cellular yield and contamination by non-microglial proteins using existing enrichment strategies., Methods: We coupled magnetic-activated cell sorting (MACS) and fluorescence activated cell sorting (FACS) of microglia with tandem mass tag-mass spectrometry (TMT-MS) to obtain a highly-pure microglial proteome and identified a core set of highly-abundant microglial proteins in adult mouse brain. We interrogated existing human proteomic data for Alzheimer's disease (AD) relevance of highly-abundant microglial proteins and performed immuno-histochemical and in-vitro validation studies., Results: Quantitative multiplexed proteomics by TMT-MS of CD11b + MACS-enriched (N = 5 mice) and FACS-isolated (N = 5 mice), from adult wild-type mice, identified 1791 proteins. A total of 203 proteins were highly abundant in both datasets, representing a core-set of highly abundant microglial proteins. In addition, we found 953 differentially enriched proteins comparing MACS and FACS-based approaches, indicating significant differences between both strategies. The FACS-isolated microglia proteome was enriched with cytosolic, endoplasmic reticulum, and ribosomal proteins involved in protein metabolism and immune system functions, as well as an abundance of canonical microglial proteins. Conversely, the MACS-enriched microglia proteome was enriched with mitochondrial and synaptic proteins and higher abundance of neuronal, oligodendrocytic and astrocytic proteins. From the 203 consensus microglial proteins with high abundance in both datasets, we confirmed microglial expression of moesin (Msn) in wild-type and 5xFAD mouse brains as well as in human AD brains. Msn expression is nearly exclusively found in microglia that surround Aβ plaques in 5xFAD brains. In in-vitro primary microglial studies, Msn silencing by siRNA decreased Aβ phagocytosis and increased lipopolysaccharide-induced production of the pro-inflammatory cytokine, tumor necrosis factor (TNF). In network analysis of human brain proteomic data, Msn was a hub protein of an inflammatory co-expression module positively associated with AD neuropathological features and cognitive dysfunction., Conclusions: Using FACS coupled with TMT-MS as the method of choice for microglial proteomics, we define a core set of highly-abundant adult microglial proteins. Among these, we validate Msn as highly-abundant in plaque-associated microglia with relevance to human AD.

Mutations in MATR3 have been associated with amyotrophic lateral sclerosis (ALS) as well as a form of distal myopathy termed vocal cord pharyngeal distal myopathy (VCPDM). To begin to understand how mutations in MATR3 may cause disease, here we provide initial characterization of transgenic (Tg) mice expressing human wild-type (WT) MATR3 (MATR3 WT ) and ALS-mutant F115C MATR3 (MATR3 F115C ) proteins under the control of the mouse prion promoter (MoPrP). For each construct, we established multiple independent lines of mice that stably transmitted the transgene. Unexpectedly, for all stably-transmitting lines examined, MATR3 transgenic mRNA expression was more robust in muscle, with minimal expression in spinal cord. The levels of transgenic mRNA in muscle did not differ between mice from our lead MATR3 F115C line and lead MATR3 WT line, but mice from the lead MATR3 F115C line had significantly higher levels of MATR3 protein in muscle over the lead MATR3 WT line. Mice from the three independent, established lines of MATR3 F115C mice developed weakness in both fore- and hind-limbs as early as < 1 months of age; whereas, MATR3 WT mice aged to > 20 months were not overtly distinguishable from non-transgenic (NT) littermates based on basic motor phenotype. Muscle of both MATR3 WT and MATR3 F115C mice showed vacuoles by 2 months of age which worsened by ~ 10 months, but vacuolation was noticeably more severe in MATR3 F115C mice. Overall, our results indicate that increasing the levels of MATR3 in muscle can cause pathologic changes associated with myopathy, with MATR3 F115C expression causing overt muscle atrophy and a profound motor phenotype. The findings suggest that analysis of muscle pathology in individuals harboring ALS-linked MATR3 mutations should be routinely considered.

Loss-of-function mutations in ATP13A2 are associated with three neurodegenerative diseases: a rare form of Parkinson's disease termed Kufor-Rakeb syndrome (KRS), a lysosomal storage disorder termed neuronal ceroid lipofuscinosis (NCL), and a form of hereditary spastic paraplegia (HSP). Furthermore, recent data suggests that heterozygous carriers of mutations in ATP13A2 may confer risk for the development of Parkinson's disease, similar to the association of mutations in glucocerebrosidase (GBA) with both Parkinson's disease and Gaucher's disease, a lysosomal storage disorder. Mutations in ATP13A2 are generally thought to be loss of function; however, the lack of human autopsy tissue has prevented the field from determining the pathological consequences of losing functional ATP13A2. We and others have previously neuropathologically characterized mice completely lacking murine Atp13a2, demonstrating the presence of lipofuscinosis within the brain - a key feature of NCL, one of the diseases to which ATP13A2 mutations have been linked. To determine if loss of one functional Atp13a2 allele can serve as a risk factor for disease, we have now assessed heterozygous Atp13a2 knockout mice for key features of NCL. In this report, we demonstrate that loss of one functional Atp13a2 allele leads to both microgliosis and astrocytosis in multiple brain regions compared to age-matched controls; however, levels of lipofuscin were only modestly elevated in the cortex of heterozygous Atp13a2 knockout mice over controls. This data suggests the possibility that partial loss of ATP13A2 causes inflammatory changes within the brain which appear to be independent of robust lipofuscinosis. This study suggests that heterozygous loss-of-function mutations in ATP13A2 are likely harmful and indicates that glial involvement in the disease process may be an early event that positions the CNS for subsequent disease development., (Copyright © 2018. Published by Elsevier Inc.)

The authors are retracting this article. The article describes mice expressing wild-type human MATR3. However, since publication the authors have become aware that all of the lines of mice described express human MATR3 containing the F115C mutation. Transgenic mice expressing wild-type and mutant Matrin were created simultaneously in their laboratory and, at a crucial stage of generating the DNA for embryo injection, as confirmed by an investigation by the University of Florida, the DNA preparations were accidentally mislabelled. All of the founders created were mosaic, requiring extensive breeding to isolate stable lines. Mice mislabelled as expressing wild-type MATR3 were the first to produce lines that stably transmitted the transgene and thus were the first to be characterized. However, as lines of mice that were mislabelled as expressing the mutant (F115C) MATR3 were ultimately established, the data began to suggest that an error had been made. Sequence analysis of amplified tail DNA from mice descended from the lines reported in the article have revealed that they express the F115C variant. The data described are therefore an accurate description of the pathology of mice that express the F115C variant of MATR3, but not of mice expressing wild-type MATR3. The authors are preparing a new manuscript reporting data from both mice expressing the F115C variant of MATR3 and mice expressing wild-type MATR3.

Serpins are a superfamily of proteins, most of which inhibit cognate serine proteases by forming inactive acyl-enzyme complexes. In the tobacco hornworm Manduca sexta, serpin-1, -3 through -7 negatively regulate a hemolymph serine protease system that activates precursors of the serine protease homologs (SPHs), phenoloxidases (POs), Spätzles, and other cytokines. Here we report the cloning and characterization of M. sexta serpin-9 and -13. Serpin-9, a 402-residue protein most similar to Drosophila Spn77Ba, has R 366 at the P1 position right before the cleavage site; Serpin-13, a 444-residue ortholog of Drosophila Spn28Dc, is longer than the other seven serpins and has R 410 as the P1 residue. Both serpins are mainly produced in fat body and secreted into plasma to function. While their mRNA and protein levels were not up-regulated upon immune challenge, they blocked protease activities and affected proPO activation in hemolymph. Serpin-9 inhibited human neutrophil elastase, cathepsin G, trypsin, and chymotrypsin to different extents; serpin-13 reduced trypsin activity to approximately 10% at a molar ratio of 4:1 (serpin: enzyme). Serpin-9 was cleaved at Arg 366 by the enzymes with different specificity, but serpin-13 had four P1 sites (Arg 410 for trypsin-like proteases, Gly 406 and Ala 409 for the elastase and Thr 404 for cathepsin G). Supplementation of induced cell-free hemolymph (IP, P for plasma) with recombinant serpin-9 did not noticeably affect proPO activation, but slightly reduced the PO activity increase after 0-50% ammonium sulfate fraction of the IP had been elicited by bacteria. In comparison, addition of recombinant serpin-13 significantly inhibited proPO activation in IP and the suppression was stronger in the fraction of IP. Serpin-9- and -13-containing protein complexes were isolated from IP using their antibodies. Hemolymph protease-1 precursor (proHP1), HP6 and HP8 were found to be associated with serpin-9, whereas proHP1, HP2 and HP6 were pulled downed with serpin-13. These results indicate that both serpins regulate immune proteases in hemolymph of M. sexta larvae., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The autosomal dominant spinocerebellar ataxias (SCAs) are a diverse group of neurological disorders anchored by the phenotypes of motor incoordination and cerebellar atrophy. Disease heterogeneity is appreciated through varying comorbidities: dysarthria, dysphagia, oculomotor and/or retinal abnormalities, motor neuron pathology, epilepsy, cognitive impairment, autonomic dysfunction, and psychiatric manifestations. Our study focuses on SCA13, which is caused by several allelic variants in the voltage-gated potassium channel KCNC3 (Kv3.3). We detail the clinical phenotype of four SCA13 kindreds that confirm causation of the KCNC3R423H allele. The heralding features demonstrate congenital onset with non-progressive, neurodevelopmental cerebellar hypoplasia and lifetime improvement in motor and cognitive function that implicate compensatory neural mechanisms. Targeted expression of human KCNC3R423H in Drosophila triggers aberrant wing veins, maldeveloped eyes, and fused ommatidia consistent with the neurodevelopmental presentation of patients. Furthermore, human KCNC3R423H expression in mammalian cells results in altered glycosylation and aberrant retention of the channel in anterograde and/or endosomal vesicles. Confirmation of the absence of plasma membrane targeting was based on the loss of current conductance in cells expressing the mutant channel. Mechanistically, genetic studies in Drosophila, along with cellular and biophysical studies in mammalian systems, demonstrate the dominant negative effect exerted by the mutant on the wild-type (WT) protein, which explains dominant inheritance. We demonstrate that ocular co-expression of KCNC3R423H with Drosophila epidermal growth factor receptor (dEgfr) results in striking rescue of the eye phenotype, whereas KCNC3R423H expression in mammalian cells results in aberrant intracellular retention of human epidermal growth factor receptor (EGFR). Together, these results indicate that the neurodevelopmental consequences of KCNC3R423H may be mediated through indirect effects on EGFR signaling in the developing cerebellum. Our results therefore confirm the KCNC3R423H allele as causative for SCA13, through a dominant negative effect on KCNC3WT and links with EGFR that account for dominant inheritance, congenital onset, and disease pathology.

Mutations in the MATR3 gene encoding the nucleotide binding protein Matrin 3 have recently been identified as causing a subset of familial amyotrophic lateral sclerosis (fALS) and more rarely causing distal myopathy. Translating the identification of MATR3 mutations into an understanding of disease pathogenesis and the creation of mouse models requires a complete understanding of normal Matrin 3 levels and distribution in vivo. Consequently, we examined the levels of murine Matrin 3 in body tissues and regions of the central nervous system (CNS). We observed a significant degree of variability in Matrin 3 protein levels among different tissues of adult animals, with the highest levels found in reproductive organs and the lowest in muscle. Within the adult CNS, Matrin 3 levels were lowest in spinal cord. Further, we found that Matrin 3 declines significantly in CNS through early development and young adulthood before stabilizing. As previously reported, antibodies to Matrin 3 primarily stain nuclei, but the intensity of staining was not uniform in all nuclei. The low levels of Matrin 3 in spinal cord and muscle could mean that that these tissues are particularly vulnerable to alterations in Matrin 3 function. Our study is the first to characterize endogenous Matrin 3 in rodents across the lifespan, providing the groundwork for deciphering disease mechanisms and developing mouse models of MATR3-linked ALS. J. Comp. Neurol. 524:2740-2752, 2016. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

Protein beverages have been in demand due to an increasing consumers' interest in healthy eating habit. However, there is an increased concern on the use of genetic modified (GM) ingredient in the food product. This study aimed to develop protein hydrolysate beverages using a non-GM soybean (R08-4004/high protein line) grown in Arkansas. Protein isolate was prepared from the soybean using alkaline method (pH 9.5). Due to its poor solubility in acidic condition, alcalase 2.4 L (food grade protease) hydrolyzed soy protein was used to develop a beverage containing 20 g protein per serving (500 mL). Three flavored beverages: Chai tea (C), tangerine (T), and mixed berries (MB) were prepared using bitter blocker, masking agent, and citric acid to minimize an unpleasant bitter taste developed in the soy hydrolysates. Protein solubility, pH, microbial growth, instrumental color parameters, and turbidity were measured to evaluate the shelf-life stability of the beverages at refrigerated storage (5 °C) for 42 days. Beverages T and MB received overall highest scores from the sensory panel. Citric acid alone or in combination with bitter blocker or masking agent lowered the bitterness. Pasteurization (90-95 °C for 5 min) was effective in preventing microbial growth. Although pH remained constant, decrease in protein solubility and color changes were observed over the storage time in all the three flavored beverages. Cloudiness in beverage C increased over the storage period while beverages T and MB were very stable. Overall, T and MB flavored beverages have the potential for commercial application., Competing Interests: The authors declare no conflict of interest.

Dementia with Lewy bodies (DLB) is the second leading cause of neurodegenerative dementia in the elderly and is clinically characterized by the presence of cognitive decline, parkinsonism, REM sleep behavior disorder, and visual hallucinations.(1,2) At autopsy, α-synuclein-positive Lewy-related pathology is observed throughout the brain. Concomitant Alzheimer disease-related pathology including amyloid plaques and, to a lesser degree, neurofibrillary tangles are often present.(2) The clinical characteristics of DLB share overlapping features with Alzheimer disease dementia (AD) and Parkinson disease (PD). A recent genetic association study examining known hits from PD and AD identified variants at both the α-synuclein (SNCA) and APOE loci as influencing the individual risk to DLB.(3) These findings would suggest that DLB may be a distinct disease with shared genetic risk factors with PD and AD.

A pentapeptide prepared from rice bran demonstrated growth inhibition on human lung, liver, breast, and colon cancer cell lines. The objectives of this study were to evaluate the human prostate cancer growth inhibition by the pentapeptide and its 6-mo storage stability by incorporating spray-dried orange juice, and determining sensory acceptability. The pentapeptide showed inhibition of human prostate cancer cells by 45% at 460 μg/mL concentration. When incorporated in spray-dried orange juice, and reconstituted with water and tested, there was an approximately 10% degradation of the peptide at 620 μg/mL concentration under refrigerated conditions over a 6 mo storage period, whereas at ambient temperature the degradation was 30%. Larger degradation was observed when 240 or 460 μg/mL pentapeptide was used. Overall, consumer panelists liked sensory aspect of the reconstituted pentapeptide incorporated orange juice beverage. Also consumer panelists liked the color and mouthfeel attributes, their hedonic impression of flavor attribute was slightly low due to unpalatable bitter note caused by the presence of the peptide. Incorporation of the pentapeptide in spray-dried orange juice has the potential to serve as a functional food ingredient that can offer health benefits to consumers. It is possible that the structural instability can be minimized by encapsulation., (© 2016 Institute of Food Technologists®)

Introduction: Dopamine and glutamate are crucial neurotransmitters in Parkinson disease (PD). While recent large meta-analyses reported that genetic variation of dopamine (DRD2, DRD3) and glutamine (NMDA, GRIN2B) neurotransmitter receptors was not associated with PD risk, they could conceivably influence PD phenotype. We studied the association of these receptor polymorphisms relating to PD age of onset., Methods: There were 664 PD patients and 718 controls, all Caucasian, with stored DNA at Mayo Clinic, Jacksonville, Florida. Genotyping was performed for DRD2 (Taq 1A, rs1800497), DRD3 (rs6280), and NMDA (GRIN2B, rs7301328) polymorphisms with ABI Taqman assays. Single nucleotide polymorphism associations with age of onset were evaluated using dominant, recessive, and additive genotypic models., Results: DRD3 variant carriers had an approximate 4.4-year decrease in mean age of onset when both copies of the minor allele were present (P = 0.0034) and an approximate 1.5-year decrease in mean age at onset for every additional minor allele (P = 0.023) (recessive and additive models, respectively). There was no association with age of onset for DRD2 or GRIN2B under any statistical model (all P ≥ 0.22)., Conclusions: The DRD3 (rs6280) polymorphism, but not DRD2 (Taq1A) or GRIN2B, influences younger PD age of onset in the US Caucasian population. Validation of these findings in larger studies with other ethnic groups is indicated., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Electrostatic spraying which has an even and retained surface coverage could be an effective novel technique to completely cover the surface of fresh produce to disrupt biofilm formation by pathogenic bacteria. Spinach leaves and cantaloupe rind were spot-inoculated with a bacterial culture and stored at 8°C for 72h to allow biofilm formation. Among various green fluorescent protein-labeled strains, ED 14 strain of E. coli O157:H7 and SD 10strain of Salmonella Typhimurium had the best attachment based on colony counts. The produce samples were electrostatically sprayed with malic (MA) and lactic (LA) acid solutions alone (1.0/2.0/3.0/4.0% w/v) or in combination (0.5+0.5/1.0+1.0/1.5+1.5/2.0+2.0% w/v) to test for a reduction in the attached bacteria. A combined treatment of LA 2.0% w/v+MA 2.0% w/v had the highest log reduction (CFU/disk) of 4.14 and 3.6 on the attached E. coli strain ED 14 (spinach) and Salmonella strain SD 10 (cantaloupe), respectively. Crystal violet assay demonstrated the disruptive effect of organic acids on biofilms formed by the pathogenic bacteria. Application of electrostatic spray with a combination of malic and lactic acids resulting in a log reduction (CFU/disk) of 3.6 or higher can improve the microbial safety of spinach and cantaloupe by preventing the pathogenic biofilm formation and bacterial growth., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: To assess the importance of MAPT variant p.A152T in the risk of synucleinopathies., Methods: In this case-control study, we screened a large global series of patients and controls, and assessed associations between p.A152T and disease risk. We included 3,229 patients with clinical Parkinson disease (PD), 442 with clinical dementia with Lewy bodies (DLB), 181 with multiple system atrophy (MSA), 832 with pathologically confirmed Lewy body disease (LBD), and 2,456 healthy controls., Results: The minor allele frequencies (MAF) in clinical PD cases (0.28%) and in controls (0.2%) were not found to be significantly different (odds ratio [OR] 1.37, 95% confidence interval [CI] 0.63-2.98, p = 0.42). However, a significant association was observed with clinical DLB (MAF 0.68%, OR 5.76, 95% CI 1.62-20.51, p = 0.007) and LBD (MAF 0.42%, OR 3.55, 95% CI 1.04-12.17, p = 0.04). Additionally, p.A152T was more common in patients with MSA compared to controls (MAF 0.55%, OR 4.68, 95% CI 0.85-25.72, p = 0.08) but this was not statistically significant and therefore should be interpreted with caution., Conclusions: Overall, our findings suggest that MAPT p.A152T is a rare low penetrance variant likely associated with DLB that may be influenced by coexisting LBD and AD pathology. Given the rare nature of the variant, further studies with greater sample size are warranted and will help to fully explain the role of p.A152T in the pathogenesis of the synucleinopathies., (© 2015 American Academy of Neurology.)

Functional properties of the soy protein need to improve to have better applications in food industry. Alkali extracted and acid precipitated soy protein isolate (SPI) was glycosylated using D-glucose (G) and Xanthan gum (X) via Maillard reaction to improve solubility. The effects of SPI to G and SPI to X ratios (SPI:G = 2:1, 1:1, and 1:2; SPI:X = 100:1 and 10:1) and incubation time (0, 6, 12, and 24 h) on the solubility and functional properties of glycosylated SPI were evaluated. The SPI:G ratio of 1:2 yielded a maximum degree of glycosylation of 71.1 %. The solubility of SPI after glycosylation significantly increased (P < 0.05) at pH 4.0-8.0 compared to SPI alone. Although the emulsion stability of glycosylated SPIs has not significantly increased (P > 0.05), the emulsifying activity improved significantly (P < 0.05). Glycosylation with SPI-X at a ratio of 10: 1 showed maximum emulsifying activity of 191.6 m(2)/g (SPI alone: 66.3 m(2)/g). Moreover, the SPI:X (ratio of 100:1) showed the maximum foaming activity (205 mL) compared to SPI alone (155 mL). The foaming stability of SPI (2.6 %) increased to 5.5 and 8.2 % when using xanthan gum at the ratio of 100:1 and 10:1, respectively. Glycosylated SPI with enhanced emulsifying and foaming properties has potential to improve the functional quality of the food products.

Exome-sequencing analyses have identified vacuolar protein sorting 35 homolog (VPS35) and DnaJ (Hsp40) homolog, subfamily C, member 13 (DNAJC13) harboring disease-causing variants for Parkinson disease (PD). Owing to the suggested clinical, pathological and genetic overlap between PD and essential tremor (ET) we assessed the presence of two VPS35 and DNAJC13 disease-causing variants in ET patients. TaqMan probes were used to genotype VPS35 c.1858G>A (p.(D620N)) (rs188286943) and DNAJC13 c.2564A>G (p.(N855S)) (rs387907571) in 571 ET patients of European descent, and microsatellite markers were used to define the disease haplotype in variant carriers. Genotyping of DNAJC13 identified two ET patients harboring the c.2564A>G (p.(N855S)) variant previously identified in PD patients. Both patients appear to share the disease haplotype previously reported. ET patients with the VPS35 c.1858G>A (p.(D620N)) variants were not observed. Although a genetic link between PD and ET has been suggested, DNAJC13 c.2564A>G (p.(N855S)) represents the first disease-causing variant identified in both, and suggests the regulation of clathrin dynamics and endosomal trafficking in the pathophysiology of a subset of ET patients.

Background: Loss of function COQ2 mutations results in primary CoQ10 deficiency. Recently, recessive mutations of the COQ2 gene have been identified in two unrelated Japanese families with multiple system atrophy (MSA). It has also been proposed that specific heterozygous variants in the COQ2 gene may confer susceptibility to sporadic MSA. To assess the frequency of COQ2 variants in patients with MSA, we sequenced the entire coding region and investigated all exonic copy number variants of the COQ2 gene in 97 pathologically-confirmed and 58 clinically-diagnosed MSA patients from the United States., Results: We did not find any homozygous or compound heterozygous pathogenic COQ2 mutations including deletion or multiplication within our series of MSA patients. In two patients, we identified two heterozygous COQ2 variants (p.S54W and c.403 + 10G > T) of unknown significance, which were not observed in 360 control subjects. We also identified one heterozygous carrier of a known loss of function p.S146N substitution in a severe MSA-C pathologically-confirmed patient., Conclusions: The COQ2 p.S146N substitution has been previously reported as a pathogenic mutation in primary CoQ10 deficiency (including infantile multisystem disorder) in a recessive manner. This variant is the third primary CoQ10 deficiency mutation observed in an MSA case (p.R387X and p.R197H). Therefore it is possible that in the heterozygous state it may increase susceptibility to MSA. Further studies, including reassessing family history in patients of primary CoQ10 deficiency for the possible occurrence of MSA, are now warranted to resolve the role of COQ2 variation in MSA.

We recently showed that mutation of the VPS35 gene can cause late-onset Parkinson's disease. In the present study we sequenced 702 affected subjects from the Mayo Clinic Parkinson's disease patient-control series for the VPS29 and VPS26A/B genes. We identified only 2 rare nonsynonymous variants in the VPS26A p.K93E and VPS29 p.N72H. The results show that mutations in the genes composing the retromer cargo recognition subunit are not a common cause of Parkinson's disease., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: To use the rs1229984 variant in the alcohol dehydrogenase 1B gene (ADH1B) as an instrument to investigate the causal role of alcohol in cardiovascular disease., Design: Mendelian randomisation meta-analysis of 56 epidemiological studies., Participants: 261 991 individuals of European descent, including 20 259 coronary heart disease cases and 10 164 stroke events. Data were available on ADH1B rs1229984 variant, alcohol phenotypes, and cardiovascular biomarkers., Main Outcome Measures: Odds ratio for coronary heart disease and stroke associated with the ADH1B variant in all individuals and by categories of alcohol consumption., Results: Carriers of the A-allele of ADH1B rs1229984 consumed 17.2% fewer units of alcohol per week (95% confidence interval 15.6% to 18.9%), had a lower prevalence of binge drinking (odds ratio 0.78 (95% CI 0.73 to 0.84)), and had higher abstention (odds ratio 1.27 (1.21 to 1.34)) than non-carriers. Rs1229984 A-allele carriers had lower systolic blood pressure (-0.88 (-1.19 to -0.56) mm Hg), interleukin-6 levels (-5.2% (-7.8 to -2.4%)), waist circumference (-0.3 (-0.6 to -0.1) cm), and body mass index (-0.17 (-0.24 to -0.10) kg/m(2)). Rs1229984 A-allele carriers had lower odds of coronary heart disease (odds ratio 0.90 (0.84 to 0.96)). The protective association of the ADH1B rs1229984 A-allele variant remained the same across all categories of alcohol consumption (P=0.83 for heterogeneity). Although no association of rs1229984 was identified with the combined subtypes of stroke, carriers of the A-allele had lower odds of ischaemic stroke (odds ratio 0.83 (0.72 to 0.95))., Conclusions: Individuals with a genetic variant associated with non-drinking and lower alcohol consumption had a more favourable cardiovascular profile and a reduced risk of coronary heart disease than those without the genetic variant. This suggests that reduction of alcohol consumption, even for light to moderate drinkers, is beneficial for cardiovascular health., (© Holmes et al 2014.)

A recent genome-wide association study of patients with essential tremor (ET) from Germany has nominated SLC1A2 rs3794087 as a novel risk factor for disease. This association was independently replicated in the Chinese population, albeit with an opposite direction of effect. To further define the role of SLC1A2 in ET, we genotyped rs3794087 in a North American series consisting of 1347 patients with ET and controls. Statistical analysis did not identify significant differences in genotype or allele frequencies between healthy controls and patients with ET (p > 0.36). These findings therefore do not support a role for SLC1A2 rs3794087 in susceptibility to ET in the North American population. Further studies in ethnically distinct populations of patients with ET are necessary to understand whether genetic variability in SLC1A2 affects disease risk for ET., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Polymorphisms within the apolipoprotein-E (APOE), Methylenetetrahydrofolate reductase (MTHFR) and Angiotensin I-converting enzyme (ACE) genes has been associated with cardiovascular and cerebrovascular disorders, Alzheimer's disease and other complex diseases in various populations. The aim of the study was to analyze the allelic and genotypic frequencies of APOE, MTHFR C677T and ACE I/D gene polymorphisms in the Zambian population., Results: The allele frequencies of APOE polymorphism in the Zambian populations were 13.8%, 59.5% and 26.7% for the ε2, ε3 and ε4 alleles respectively. MTHFR C677T and ACE I/D allele frequencies were 8.6% and 13.8% for the T and D minor alleles respectively. The ε2ε2 genotype and TT genotype were absent in the Zambian population. The genetic distances between Zambian and other African and non-African major populations revealed an independent variability of these polymorphisms., Conclusion: We found that the APOE ε3 allele and the I allele of the ACE were significantly high in our study population while there were low frequencies observed for the MTHFR 677 T and ACE D alleles. Our analysis of the APOE, MTHFR and ACE polymorphisms may provide valuable insight into the understanding of the disease risk in the Zambian population.

Saskatoon berries (Amelanchier alnifolia Nutt.) have significantly higher levels of anthocyanins (ACY) among berries with potential health benefits. The pomace is a by-product of juice extracted from berries and is a potential source of inexpensive polyphenols. The objectives of this study were to extract the maximum amount of total phenolics from saskatoon pomace, to determine the antioxidant activity, and to identify individual phenolic components. Pomace extracts showed high content of total phenolics, total ACY, and total flavonoids of 43.3, 2.8, and 10.3 g/kg of dried weight (DW) of pomace. A high oxygen radical absorbing capacity value of 119.4 μmol Trolox equivalents/g DW and free radical scavenging activity of pomace extract (200 ppm, 86.8%) were observed. Five major ACY, two flavonols, and three chlorogenic acids were identified and quantified in pomace extracts. This study shows that saskatoon berries pomace rich in antioxidant phenolics could be extracted by "green" solvents (water and ethanol) and used as suitable food product applications.

Mutations of the FUS gene were first reported to cause amyotrophic lateral sclerosis (ALS). Subsequent studies confirmed the role of mutations in ALS and also implicated them in frontotemporal dementia (FTD). Recently, through Next-Generation Exome sequencing approaches a mutation resulting in a substitution (p.Q290X) in the nuclear export domain of the FUS protein was nominated as a cause of autosomal dominant essential tremor (ET) in a large kindred. In addition, recent reports suggest a possible role for TDP-43 mutations in parkinsonism; TDP-43 is another RNA-binding protein implicated in ALS. Given these findings we investigated the role of FUS variants in Parkinson's disease (PD). We sequenced specific regions of the gene encoding three functional domains of the FUS protein in 702 patients with PD. Our sequencing study did not identify any novel non-synonymous variant that would appear to affect the subjects' susceptibility to Parkinson's disease. These findings and previous studies have shown that variants within the FUS gene are not a common cause of PD or ET, in comparison to their role in ALS., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Parkinson's disease (PD) is a multifactorial movement disorder characterized by progressive neurodegeneration. Genome-wide association studies (GWAS) have nominated over fifteen distinct loci associated with risk of PD, however the biological mechanisms by which these loci influence disease risk are mostly unknown. GWAS are only the first step in the identification of disease genes: the specific causal variants responsible for the risk within the associated loci and the interactions between them must be identified to fully comprehend their impact on the development of PD. In the present study, we first attempted to replicate the association signals of 17 PD GWAS loci in our series of 1381 patients with PD and 1328 controls. BST1, SNCA, HLA-DRA, CCDC62/HIP1R and MAPT all showed a significant association with PD under different models of inheritance and LRRK2 showed a suggestive association. We then examined the role of coding LRRK2 variants in the GWAS association signal for that gene. The previously identified LRRK2 risk mutant p.M1646T and protective haplotype p.N551K-R1398H-K1423K did not explain the association signal of LRRK2 in our series. Finally, we investigated the gene-gene interaction between PARK16 and LRRK2 that has previously been proposed. We observed no interaction between PARK16 and LRRK2 GWAS variants, but did observe a non-significant trend toward interaction between PARK16 and LRRK2 variants within the protective haplotype. Identification of causal variants and the interactions between them is the crucial next step in making biological sense of the massive amount of data generated by GWAS studies. Future studies combining larger sample sizes will undoubtedly shed light on the complex molecular interplay leading to the development of PD.

The aim of the present study was to investigate the association of APOE, MTHFR and ACE polymorphisms with stroke in the Zambian population. We analyzed 41 stroke patients and 116 control subjects all of Zambian origin for associations between the genotype of the APOE, MTHFR and ACE polymorphisms and stroke. The APOE ε2ε4 genotype showed increased risk for hemorrhagic stroke (P<0.05) and also a high risk for ischemic stroke (P=0.05). There was complete absence of the APOE ε2ε2 and the MTHFR TT genotypes in the Zambian population. The difference between cases and controls was not significant for the other genetic variants when analyzed for relationship between stroke, stroke subtype and genotype. We show that genetic variation at the APOE locus affects susceptibility to stroke. No detectable association were observed for the MTHFR and ACE genotypes and stroke in the Zambian population.

Background: Mutations within the NOTCH3 gene cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). CADASIL mutations appear to be restricted to the first twenty-four exons, resulting in the gain or loss of a cysteine amino acid. The role of other exonic NOTCH3 variation not involving cysteine residues and mutations in exons 25-33 in ischemic stroke remains unresolved., Methods: All 33 exons of NOTCH3 were sequenced in 269 Caucasian probands from the Siblings With Ischemic Stroke Study (SWISS), a 70-center North American affected sibling pair study and 95 healthy Caucasian control subjects. Variants identified by sequencing in the SWISS probands were then tested for association with ischemic stroke using US Caucasian controls collected at the Mayo Clinic (n=654), and further assessed in a Caucasian (n=802) and African American (n=298) patient-control series collected through the Ischemic Stroke Genetics Study (ISGS)., Results: Sequencing of the 269 SWISS probands identified one (0.4%) with small vessel type stroke carrying a known CADASIL mutation (p.R558C; Exon 11). Of the 19 common NOTCH3 variants identified, the only variant significantly associated with ischemic stroke after multiple testing adjustment was p.R1560P (rs78501403; Exon 25) in the combined SWISS and ISGS Caucasian series (Odds Ratio [OR] 0.50, P=0.0022) where presence of the minor allele was protective against ischemic stroke. Although only significant prior to adjustment for multiple testing, p.T101T (rs3815188; Exon 3) was associated with an increased risk of small-vessel stroke (OR: 1.56, P=0.008) and p.P380P (rs61749020; Exon 7) was associated with decreased risk of large-vessel stroke (OR: 0.35, P=0.047) in Caucasians. No significant associations were observed in the small African American series., Conclusion: Cysteine-affecting NOTCH3 mutations are rare in patients with typical ischemic stroke, however our observation that common NOTCH3 variants may be associated with risk of ischemic stroke warrants further study.

Essential Tremor is the most common form of movement disorder. Aggregation in families suggests a strong genetic component to disease. Linkage and association studies have identified several risk loci but the specific causal variants are still unknown. A recent study using whole exome sequencing identified a rare nonsense variant in the FUS gene (p.Q290X) that segregated with Essential Tremor in a large French Canadian family. In addition, two other rare FUS variants were identified (p.R216C and p.P431L) in Essential Tremor patients however co-segregation analysis with disease was not possible. In the present study, we sequenced all 15 exons of FUS in 152 familial probands with Essential Tremor and genotyped three reported FUS variants in 112 sporadic Essential Tremor patients and 716 control subjects recruited at Mayo Clinic Florida. Only known synonymous SNPs unlikely to be pathogenic were detected in our sequencing and not any of the recently identified mutations or novel ones. We conclude that the FUS mutations associated with risk of Essential Tremor are probably a rare occurrence., (Copyright © 2013 Elsevier Ltd. All rights reserved.)