A protein quality control pathway regulated by linear ubiquitination.

Neurodegenerative diseases are characterized by the accumulation of misfolded proteins in the brain. Insights into protein quality control mechanisms to prevent neuronal dysfunction and cell death are crucial in developing causal therapies. Here, we report that various disease-associated protein aggregates are modified by the linear ubiquitin chain assembly complex (LUBAC). HOIP, the catalytic component of LUBAC, is recruited to misfolded Huntingtin in a p97/VCP-dependent manner, resulting in the assembly of linear polyubiquitin. As a consequence, the interactive surface of misfolded Huntingtin species is shielded from unwanted interactions, for example with the low complexity sequence domain-containing transcription factor Sp1, and proteasomal degradation of misfolded Huntingtin is facilitated. Notably, all three core LUBAC components are transcriptionally regulated by Sp1, linking defective LUBAC expression to Huntington's disease. In support of a protective activity of linear ubiquitination, silencing of OTULIN, a deubiquitinase with unique specificity for linear polyubiquitin, decreases proteotoxicity, whereas silencing of HOIP has the opposite effect. These findings identify linear ubiquitination as a protein quality control mechanism and hence a novel target for disease-modifying strategies in proteinopathies.

Decreased IL-8 levels in CSF and serum of AD patients and negative correlation of MMSE and IL-1ß.

BACKGROUND: It is widely accepted that neuroinflammatory processes play an important role in the pathogenesis of Alzheimer's disease (AD) and high levels of cytokines and chemokines are detected around Aß plaques. METHODS: As neuroinflammation is involved in the development and progression of AD, we measured the pro-inflammatory cytokines interleukin 1ß (IL-1ß), IL-8 and tumor necrosis factor α (TNF-α) in serum and cerebrospinal fluid (CSF) samples from 45 AD patients and 53 age-matched control subjects using a highly sensitive multiplex electrochemiluminescence assay. To address the association with disease progression we correlated cognitive status with cytokine levels. RESULTS: CSF as well as serum IL-8 levels were found to be significantly lower in AD patients than in controls (p = 0.02). A statistically significant inverse correlation was observed between the CSF level of IL-1ß and the MMSE score (rs = -0.03, p = 0.02). We therefore stratified the AD patients by their MMSE scores into three equal groups and found that in the AD group with the most severe cognitive impairment CSF-IL-1ß was significantly increased compared to age-matched controls (p < 0.05), whereas in the other investigated groups the increase was not statistically significant. CONCLUSION: Our results confirm data suggesting that cytokine alterations are involved in AD pathogenesis and may be helpful as a biomarker for monitoring disease progression.

The role of TREM2 R47H as a risk factor for Alzheimer's disease, frontotemporal lobar degeneration, amyotrophic lateral sclerosis, and Parkinson's disease.

A rare variant in TREM2 (p.R47H, rs75932628) was recently reported to increase the risk of Alzheimer's disease (AD) and, subsequently, other neurodegenerative diseases, i.e. frontotemporal lobar degeneration (FTLD), amyotrophic lateral sclerosis (ALS), and Parkinson's disease (PD). Here we comprehensively assessed TREM2 rs75932628 for association with these diseases in a total of 19,940 previously untyped subjects of European descent. These data were combined with those from 28 published data sets by meta-analysis. Furthermore, we tested whether rs75932628 shows association with amyloid beta (Aß42) and total-tau protein levels in the cerebrospinal fluid (CSF) of 828 individuals with AD or mild cognitive impairment. Our data show that rs75932628 is highly significantly associated with the risk of AD across 24,086 AD cases and 148,993 controls of European descent (odds ratio or OR = 2.71, P = 4.67 × 10(-25)). No consistent evidence for association was found between this marker and the risk of FTLD (OR = 2.24, P = .0113 across 2673 cases/9283 controls), PD (OR = 1.36, P = .0767 across 8311 cases/79,938 controls) and ALS (OR = 1.41, P = .198 across 5544 cases/7072 controls). Furthermore, carriers of the rs75932628 risk allele showed significantly increased levels of CSF-total-tau (P = .0110) but not Aß42 suggesting that TREM2's role in AD may involve tau dysfunction.

Engulfment adaptor phosphotyrosine-binding-domain-containing 1 (GULP1) is a nucleocytoplasmic shuttling protein and is transactivationally active together with low-density lipoprotein receptor-related protein 1 (LRP1).

APP (amyloid precursor protein) and LRP1 (low-density lipoprotein receptor-related protein 1) have been implicated in the pathogenesis of AD (Alzheimer's disease). They are functionally linked by Fe65, a PTB (phosphotyrosine-binding)-domain-containing adaptor protein that binds to intracellular NPxY-motifs of APP and LRP1, thereby influencing expression levels, cellular trafficking and processing. Additionally, Fe65 has been reported to mediate nuclear signalling in combination with intracellular domains of APP and LRP1. We have previously identified another adaptor protein, GULP1 (engulfment adaptor PTB-domain-containing 1). In the present study we characterize and compare nuclear trafficking and transactivation of GULP1 and Fe65 together with APP and LRP1 and report differential nuclear trafficking of adaptors when APP or LRP1 are co-expressed. The observed effects were additionally supported by a reporter-plasmid-based transactivation assay. The results from the present study indicate that Fe65 might have signalling properties together with APP and LRP1, whereas GULP1 only mediates LRP1 transactivation.

Globular and protofibrillar aß aggregates impair neurotransmission by different mechanisms.

In Alzheimer's disease, substantial evidence indicates the causative role of soluble amyloid ß (Aß) aggregates. Although a variety of Aß assemblies have been described, the debate about their individual relevance is still ongoing. One critical issue hampering this debate is the use of different methods for the characterization of endogenous and synthetic peptide and their intrinsic limitations for distinguishing Aß aggregates. Here, we used different protocols for the establishment of prefibrillar Aß assemblies with varying morphologies and sizes and compared them in a head-to-head fashion. Aggregation was characterized via the monomeric peptide over time until spheroidal, protofibrillar, or fibrillar Aß aggregates were predominant. It could be shown that a change in the ionic environment induced a structural rearrangement, which consequently confounds the delineation of a measured neurotoxicity toward a distinct Aß assembly. Here, neuronal binding and hippocampal neurotransmission were found to be suitable to account for the synaptotoxicity to different Aß assemblies, based on the stability of the applied Aß aggregates in these settings. In contrast to monomeric or fibrillar Aß, different prefibrillar Aß aggregates targeted neurons and impaired hippocampal neurotransmission with nanomolar potency, albeit by different modalities. Spheroidal Aß aggregates inhibited NMDAR-dependent long-term potentiation, as opposed to protofibrillar Aß aggregates, which inhibited AMPAR-dominated basal neurotransmission. In addition, a provoked structural conversion of spheroidal to protofibrillar Aß assemblies resulted in a time-dependent suppression of basal neurotransmission, indicative of a mechanistic switch in synaptic impairment. Thus, we emphasize the importance of addressing the metastability of prefacto characterized Aß aggregates in assigning a biological effect.

Vertigoheel promotes rodent cognitive performance in multiple memory tests.

Introduction: Cognitive impairment associated with old age or various brain disorders may be very disabling for affected individuals, placing their carers and public health services under considerable stress. The standard-of-care drugs produce only transient improvement of cognitive impairment in older people, so the search for novel, safe and effective therapeutics that would help to reverse or delay cognitive impairment is warranted. Repurposing pharmacological therapies with well-established safety record for additional indications is a promising recent trend in drug development. Vertigoheel (VH-04), a multicomponent drug made of Ambra grisea, Anamirta cocculus L., Conium maculatum, and Petroleum rectificatum, has been successfully used for several decades in the treatment of vertigo. Here, we investigated effects of VH-04 on cognitive performance in standard behavioral tests assessing different types of memory and explored cellular and molecular underpinnings of VH-04's biological activity. Methods: In the majority of behavioral experiments, namely in the spontaneous and rewarded alternation tests, passive avoidance test, contextual/cued fear conditioning, and social transmission of food preference, we examined the ability of single and repeated intraperitoneal administrations of VH-04 to improve cognitive parameters of mice and rats disrupted by the application of the muscarinic antagonist scopolamine. In addition, we also assessed how VH-04 affected novel object recognition and influenced performance of aged animals in Morris water maze. Furthermore, we also studied the effects of VH-04 on primary hippocampal neurons in vitro and mRNA expression of synaptophysin in the hippocampus. Results: Administration of VH-04 positively influenced visual recognition memory in the novel object recognition test and alleviated the impairments in spatial working memory and olfactory memory caused by the muscarinic antagonist scopolamine in the spontaneous alternation and social transmission of food preference tests. In addition, VH-04 improved retention of the spatial orientation memory of old rats in the Morris water maze. In contrast, VH-04 did not have significant effects on scopolamine-induced impairments in tests of fear-aggravated memory or rewarded alternation. Experiments in vitro showed that VH-04 stimulated neurite growth and possibly reversed the age-dependent decrease in hippocampal synaptophysin mRNA expression, which implies that VH-04 may preserve synaptic integrity in the aging brain. Discussion: Our findings allow a cautious conclusion that in addition to its ability to alleviate manifestations of vertigo, VH-04 may be also used as a cognitive enhancer.

Identification of novel substrates for Cdk5 and new targets for Cdk5 inhibitors using high-density protein microarrays.

Cyclin-dependent kinase (Cdk) 5 is a serine/threonine kinase that plays an important role during CNS development and its dysregulation is causally involved in the process of neuronal degeneration. To date more than 20 Cdk5 substrates have been identified and the number of Cdk5 substrates is still increasing. The different cellular functions of Cdk5 and its substrates are not completely known at present. High-throughput protein microarray technology is a powerful tool to identify a large number of potential kinase substrates in parallel under the same experimental conditions. Using Protoarray protein microarrays we identified protein phosphatase 1, regulatory subunit 14A (PPP1R14A) as a novel substrate of Cdk5/p25. Phosphorylation was confirmed in two secondary assays. Our findings may contribute to the elucidation of the physiological function of Cdk5 in synaptic signalling. Functional Kinome Arrays were validated in a second set of experiments to characterize the selectivity of the Cdk5 inhibitor indolinone A. This lead to the identification of two additional kinases that are targeted by this compound and may provide a deeper understanding of its neuroprotective mode of action. However, several false negative results possibly due to a denatured or inactive conformation of the arrayed proteins, sound a note of caution when using protein array techniques.

Functional protein kinase arrays reveal inhibition of p-21-activated kinase 4 by alpha-synuclein oligomers.

There is increasing evidence that aggregation of alpha-synuclein contributes to the functional and structural deterioration in the CNS of Parkinson's disease patients and transgenic animal models. alpha-Synuclein binds to various cellular proteins and aggregated alpha-synuclein species may affect their physiological function. In the present study, we used protein arrays spotted with 178 active human kinases for a large-scale analysis of the effects of recombinant alpha-synuclein on kinase activities. Incubation with globular alpha-synuclein oligomers significantly inhibited autophosphorylation of p21-activated kinase (PAK4) compared to treatment with monomeric alpha-synuclein or beta-synuclein. A concentration-dependent inhibition was also observed in a solution-based kinase assay. To show in vivo relevance, we analyzed brainstem protein extracts from alpha-synuclein (A30P) transgenic mice where accumulation of alpha-synuclein oligomers has been demonstrated. By immunoblotting using a phospho-specific antibody, we detected a significant decline in phosphorylation of LIM kinase 1, a physiological substrate for PAK4. Suppression of PAK activity by amyloid-beta oligomers has been reported in Alzheimer's disease. Thus, PAKs may represent a target for various neurotoxic protein oligomers, and signaling deficits may contribute to the behavioral defects in chronic neurodegenerative diseases.

Reduced cGMP levels in CSF of AD patients correlate with severity of dementia and current depression.

BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative disorder, primarily affecting memory. That disorder is thought to be a consequence of neuronal network disturbances and synapse loss. Decline in cognitive function is associated with a high burden of neuropsychiatric symptoms (NPSs) such as depression. The cyclic nucleotides cyclic adenosine-3',5'-monophosphate (cAMP) and cyclic guanosine-3',5'-monophosphate (cGMP) are essential second messengers that play a crucial role in memory processing as well as synaptic plasticity and are potential therapeutic targets. Biomarkers that are able to monitor potential treatment effects and that reflect the underlying pathology are of crucial interest. METHODS: In this study, we measured cGMP and cAMP in cerebrospinal fluid (CSF) in a cohort of 133 subjects including 68 AD patients and 65 control subjects. To address the association with disease progression we correlated cognitive status with cyclic nucleotide levels. Because a high burden of NPSs is associated with decrease in cognitive function, we performed an exhaustive evaluation of AD-relevant marker combinations in a depressive subgroup. RESULTS: We show that cGMP, but not cAMP, levels in the CSF of AD patients are significantly reduced compared with the control group. Reduced cGMP levels in AD patients correlate with memory impairment based on Mini-Mental State Examination score (r = 0.17, p = 0.048) and tau as a marker of neurodegeneration (r = -0.28, p = 0.001). Moreover, we were able to show that AD patients suffering from current depression show reduced cGMP levels (p = 0.07) and exhibit a higher degree of cognitive impairment than non-depressed AD patients. CONCLUSION: These results provide further evidence for an involvement of cGMP in AD pathogenesis and accompanying co-morbidities, and may contribute to elucidating synaptic plasticity alterations during disease progression.

Novel Blood-Based Biomarkers of Cognition, Stress, and Physical or Cognitive Training in Older Adults at Risk of Dementia: Preliminary Evidence for a Role of BDNF, Irisin, and the Kynurenine Pathway.

 Psychosocial stress and physical, cognitive, and social activity predict the risk of cognitive decline and dementia. The aim of this study was to elucidate brain-derived neurotrophic factor (BDNF), irisin, and the kynurenine pathway (KP) as potential underlying biological correlates. We evaluated associations of irisin and the KP with BDNF in serum and with cognition, stress, and activities. Furthermore, changes in serum concentrations of BDNF, irisin, and KP metabolites were investigated after physical or cognitive training. Forty-seven older adults at risk of dementia were assigned to 10 weeks of physical training, cognitive training, or a wait-list control condition. Previous physical, cognitive, and social activities and stressful life events were recorded; global cognition, episodic memory, and executive functions were assessed. Serum levels of L-kynurenine, kynurenic acid, 3-hydroxykynurenine (3-HK), and quinolinic acid (QUIN) were determined by validated assays based on liquid chromatography coupled to tandem mass spectrometry. BDNF and irisin serum levels were determined with enzyme-linked immunosorbent assays. BDNF and irisin correlated positively with global cognition and episodic memory, while the neurotoxic metabolite QUIN correlated negatively with executive functions. Stressful life events were associated with reduced BDNF and increased 3-HK. 3-HK decreased after cognitive training, while BDNF tended to increase after physical training. This suggests that psychosocial stress as well as cognitive and physical training may impact BDNF serum levels and the KP. Irisin and QUIN may constitute novel serum biomarkers of cognitive impairment, in addition to BDNF. Larger scale trials are needed to replicate and extend these novel findings.

Increased levels of antigen-bound ß-amyloid autoantibodies in serum and cerebrospinal fluid of Alzheimer's disease patients.

Recent studies have suggested a protective role of physiological ß-amyloid autoantibodies (Aß-autoantibodies) in Alzheimer's disease (AD). However, the determination of both free and dissociated Aß-autoantibodies in serum hitherto has yielded inconsistent results regarding their function and possible biomarker value. Here we report the application of a new sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of antigen-bound Aß-autoantibodies (intact Aß-IgG immune complexes) in serum and cerebrospinal fluid (CSF) of a total number of 112 AD patients and age- and gender-matched control subjects. Both serum and CSF levels of Aß-IgG immune complexes were found to be significantly higher in AD patients compared to control subjects. Moreover, the levels of Aß-IgG complexes were negatively correlated with the cognitive status across the groups, increasing with declining cognitive test performance of the subjects. Our results suggest a contribution of IgG-type autoantibodies to Aß clearance in vivo and an increased immune response in AD, which may be associated with deficient Aß-IgG removal. These findings may contribute to elucidating the role of Aß-autoantibodies in AD pathophysiology and their potential application in AD diagnosis.

The role of clusterin, complement receptor 1, and phosphatidylinositol binding clathrin assembly protein in Alzheimer disease risk and cerebrospinal fluid biomarker levels.

CONTEXT: Two recent and simultaneously published genome-wide association studies independently implicated clusterin (CLU), complement receptor 1 (CR1), and phosphatidylinositol binding clathrin assembly protein (PICALM) as putative novel Alzheimer disease (AD) risk loci. Despite their strong statistical support, all 3 signals emerged from heterogeneous case-control populations and lack replication in different settings. OBJECTIVE: To determine whether genetic variants in CLU, CR1, and PICALM confer risk for AD in independent data sets (n = 4254) and to test the impact of these markers on cerebrospinal fluid (CSF)-Aß42 and total-tau protein levels (n = 425). DESIGN: Genetic association study using family-based and case-control designs. SETTING: Ambulatory or hospitalized care. PARTICIPANTS: Family samples originate from mostly multiplex pedigrees recruited at different centers in the United States (1245 families, 2654 individuals with AD, and 1175 unaffected relatives). Unrelated case-control subjects originate from 1 clinical center in Germany (214 individuals with AD and 211 controls). All subjects were of European descent. MAIN OUTCOME MEASURES: The association between 5 genetic variants in CLU, CR1, and PICALM and risk for AD, and the correlation between these 5 genetic variants and CSF-Aß42 and tau levels. RESULTS: All 3 investigated loci showed significant associations between risk for AD (1-tailed P values ranging from <.001 to .02) and consistent effect sizes and direction. For each locus, the overall evidence of association was substantially strengthened on meta-analysis of all available data (2-tailed P values ranging from 1.1 × 10(-16) to 4.1 × 10⁻7). Of all markers tested, only rs541458 in PICALM was shown to have an effect on CSF protein levels, suggesting that the AD risk allele is associated with decreased CSF Aß42 levels (2-tailed P = .002). CONCLUSIONS: This study provides compelling independent evidence that genetic variants in CLU, CR1, and PICALM are genetically associated with risk for AD. Furthermore, the CSF biomarker analyses provide a first insight into the potentially predominant pathogenetic mechanism(s) underlying the association between AD risk and PICALM.

MicroRNA and proteome expression profiling in early-symptomatic α-synuclein(A30P)-transgenic mice.

The α-synuclein has been implicated in the pathophysiology of Parkinson's disease (PD), because mutations in the alpha-synuclein gene cause autosomal-dominant hereditary PD and fibrillary aggregates of alpha-synuclein are the major component of Lewy bodies. Since presynaptic accumulation of α-synuclein aggregates may trigger synaptic dysfunction and degeneration, we have analyzed alterations in synaptosomal proteins in early symptomatic α-synuclein(A30P)-transgenic mice by two-dimensional differential gel electrophoresis. Moreover, we carried out microRNA expression profiling using microfluidic chips, as microRNA have recently been shown to regulate synaptic plasticity in rodents and to modulate polyglutamine-induced protein aggregation and neurodegeneration in flies. Differentially expressed proteins in α-synuclein(A30P)-transgenic mice point to alterations in mitochondrial function, actin dynamics, iron transport, and vesicle exocytosis, thus partially resembling findings in PD patients. Oxygen consumption of isolated brain mitochondria, however, was not reduced in mutant mice. Levels of several microRNA (miR-10a, -10b, -212, -132, -495) were significantly altered. One of them (miR-132) has been reported to be highly inducible by growth factors and to be a key regulator of neurite outgrowth. Moreover, miR-132-recognition sequences were detected in the mRNA transcripts of two differentially expressed proteins. MicroRNA may thus represent novel biomarkers for neuronal malfunction and potential therapeutic targets for human neurodegenerative diseases.

Enhanced expression and shedding of the transmembrane chemokine CXCL16 by reactive astrocytes and glioma cells.

The transmembrane chemokine CXCL16 is expressed by dendritic and vascular cells and mediates chemotaxis and adhesion of activated T cells via the chemokine receptor CXCR6/Bonzo. Here we describe the expression and shedding of this chemokine by glioma cells in situ and in vitro. By quantitative RT-PCR and immunohistochemistry, we show that CXCL16 is highly expressed in human gliomas, while expression in normal brain is low and mainly restricted to brain vascular endothelial cells. In cultivated human glioma cells as well as in activated mouse astroglial cells, CXCL16 mRNA and protein is constitutively expressed and further up-regulated by tumour necrosis factor alpha (TNFalpha) and interferon-gamma (IFNgamma). CXCL16 is continuously released from glial cells by proteolytic cleavage which is rapidly enhanced by stimulation with phorbol-12-myristate-13-acetate (PMA). As shown by inhibitor studies, two distinct members of the disintegrin-like metalloproteinase family ADAM10 and 17 are involved in the constitutive and PMA-induced shedding of glial CXCL16. In addition to the chemokine, its receptor CXCR6 could be detected by quantitative RT-PCR in human glioma tissue, cultivated murine astrocytes and at a lower level in microglial cells. Functionally, recombinant soluble CXCL16 enhanced proliferation of CXCR6-positive murine astroglial and microglial cells. Thus, the transmembrane chemokine CXCL16 is expressed in the brain by malignant and inflamed astroglial cells, shed to a soluble form and targets not only activated T cells but also glial cells themselves.