Journal of Alzheimer's Disease - Volume 46, issue 2

Authors: Tomasini, Maria Cristina | Borelli, Andrea Celeste | Beggiato, Sarah | Ferraro, Luca | Cassano, Tommaso | Tanganelli, Sergio | Antonelli, Tiziana

Article Type: Research Article

Abstract: Background: Considering the heterogeneity of pathological changes occurring in Alzheimer’s disease (AD), a therapeutic approach aimed both to neuroprotection and to neuroinflammation reduction may prove effective. Palmitoylethanolamide (PEA) has attracted attention for its anti-inflammatory/neuroprotective properties observed in AD animal models. Objective and Methods: We evaluated the protective role of PEA against amyloid-β42 (Aβ42 ) toxicity on cell viability and glutamatergic transmission in primary cultures of cerebral cortex neurons and astrocytes from the triple-transgenic murine model of AD (3xTg-AD) and their wild-type littermates (non-Tg) mice. Results: Aβ42 (0.5 μM; 24 h) affects the cell …viability in cultured cortical neurons and astrocytes from non-Tg mice, but not in those from 3xTg-AD mice. These effects were counteracted by the pretreatment with PEA (0.1 μM). Basal glutamate levels in cultured neurons and astrocytes from 3xTg-AD mice were lower than those observed in cultured cells from non-Tg mice. Aβ42 -exposure reduced and increased glutamate levels in non-Tg mouse cortical neurons and astrocytes, respectively. These effects were counteracted by the pretreatment with PEA. By itself, PEA did not affect cell viability and glutamate levels in cultured cortical neurons and astrocytes from non-Tg or 3xTg-AD mice. Conclusion: The exposure to Aβ42 induced toxic effects on cultured cortical neurons and astrocytes from non-Tg mice, but not in those from 3xTg-AD mice. Furthermore, PEA exerts differential effects against Aβ42 -induced toxicity in primary cultures of cortical neurons and astrocytes from non-Tg and 3xTg-AD mice. In particular, PEA displays protective properties in non-Tg but not in 3xTg-AD mouse neuronal cultured cells overexpressing Aβ. Show more

Keywords: Alzheimer’s disease, cell viability, GFAP immunoreactivity, glutamate, MAP2 immunoreactivity

DOI: 10.3233/JAD-143039

Citation: Journal of Alzheimer's Disease, vol. 46, no. 2, pp. 407-421, 2015

Authors: Forlenza, Orestes Vicente | Miranda, Aline Silva | Guimar, Izabela | Talib, Leda Leme | Diniz, Breno Satler | Gattaz, Wagner Farid | Teixeira, Antonio Lucio

Article Type: Research Article

Abstract: Background: There is evidence of decreased neurotrophic support in Alzheimer’s disease (AD), including its prodromal stages, but it is not clear whether this abnormality represents a marker of this process. Objective: To determine serum concentrations of a panel of neurotrophic factors (BDNF, NGF, and GDNF) in a cross-section of elderly patients with mild cognitive impairment (MCI) and AD compared to cognitively healthy controls, and to evaluate whether abnormal levels of these factors at baseline predict the transition from MCI to dementia. Methods: A total of 134 older adults were enrolled in this study. Twenty-six patients …with mild to moderate AD, 62 with MCI, and 46 cognitively healthy older adults (controls) were subjected to a clinical evaluation including several cognitive tests. Peripheral blood was drawn and serum levels of BDNF, NGF, and GDNF were measured by enzyme-linked immunosorbent assay. APOE genotyping was performed by PCR assays. Results: Serum concentrations of BDNF, NGF, and GDNF were significantly reduced in cognitively impaired subjects (i.e., MCI and AD) as compared to controls, although only the former two remained statistically different after controlling for age, gender, and cognitive performance (p  = 0.05 and p  = 0.01, respectively). Lower BDNF and NGF levels were also observed in the sub-sample of MCI patients who progressed to dementia upon follow-up (p  = 0.02 and p  = 0.002, respectively). Conclusion: Abnormalities in neurotrophic systems are observed at early stages of AD and may represent a marker of cognitive deterioration. Show more

Keywords: Alzheimer’s disease, BDNF, GDNF, mild cognitive impairment, NGF, physiopathology

DOI: 10.3233/JAD-150172

Citation: Journal of Alzheimer's Disease, vol. 46, no. 2, pp. 423-429, 2015

Authors: Savage, Mary J. | Holder, Daniel J. | Wu, Guoxin | Kaplow, June | Siuciak, Judith A. | Potter, William Z. | the Foundation for the National Institutes of Health (FNIH) Biomarkers Consortium CSF Proteomics Project Team for the Alzheimer’s Disease Neuroimaging Initiative

Article Type: Research Article

Abstract: β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) plays an important role in the development of Alzheimer’s disease (AD), freeing the amyloid-β (Aβ) N-terminus from the amyloid-β protein precursor (AβPP), the first step in Aβ formation. Increased BACE1 activity in AD brain or cerebrospinal fluid (CSF) has been reported. Other studies, however, found either no change or a decrease with AD diagnosis in either BACE1 activity or sAβPPβ, the N-terminal secreted product of BACE1 (sBACE1) activity on AβPP. Here, sBACE1 enzymatic activity and secreted AβPPβ (sAβPPβ) were measured in Alzheimer’s Disease Neuroimaging Initiative-1 (ADNI-1) baseline CSF samples and no statistically significant …changes were found in either measure comparing healthy control, mild cognitively impaired, or AD individual samples. While CSF sBACE1 activity and sAβPPβ demonstrated a moderate yet significant degree of correlation with each other, there was no correlation of either analyte to CSF Aβ peptide ending at residue 42. Surprisingly, a stronger correlation was demonstrated between CSF sBACE1 activity and tau, which was comparable to that between CSF Aβ42 and tau. Unlike for these latter two analytes, receiver-operator characteristic curves demonstrate that neither CSF sBACE1 activity nor sAβPPβ concentrations can be used to differentiate between healthy elderly and AD individuals. Show more

Keywords: ADNI, Alzheimer’s disease, amyloid-β, amyloid-β protein precursor, BACE1, cerebrospinal fluid, ELISA, mild cognitive impairment, sAβPPβ, sBACE1, secretase

DOI: 10.3233/JAD-142778

Citation: Journal of Alzheimer's Disease, vol. 46, no. 2, pp. 431-440, 2015

Authors: Gago, Miguel F. | Fernandes, Vítor | Ferreira, Jaime | Yelshyna, Darya | Silva, Hélder David | Rodrigues, Maria Lurdes | Rocha, Luís | Bicho, Estela | Sousa, Nuno

Article Type: Research Article

Abstract: Background: Postural stability requires the integration of multisensory input information and translation into appropriate motor responses. Surprisingly, few previous studies have addressed the role of auditory input on postural stability in healthy subjects, and none has investigated this in Alzheimer’s disease (AD). Objective: To assess the influence of the visual and auditory systems on postural stability in patients with AD and healthy subjects. Methods: Twenty-four patients with AD and healthy age-matched subjects were examined by kinematic postural analysis (inertia measurement units placed at the center of mass of the body) under four different conditions: …stance with eyes open and eyes closed, with and without suppression of background noise (using ear defenders). The effects of visual and auditory influences were analyzed independently and in conjunction. Results: In both groups, visual suppression had a negative impact on postural stability, while suppression of background noise, non-specifically and without spatial cues, significantly benefited postural stability. We also observed that in both groups, the positive effect of background noise suppression was insufficient to compensate for the negative effect of visual suppression, to which the patients were significantly more vulnerable. Conclusions: Audition, albeit less significant than vision, also plays a role in the multi-sensorial dynamic control of postural stability by the central nervous system. In everyday life, audition is likely to be a relevant factor in postural stability. This is especially relevant in AD in which, even when the peripheral sensory system is intact, the central processing is impaired and sensory dependence is re-weighted. Show more

Keywords: Alzheimer’s disease, auditory system, postural stability, visual system

DOI: 10.3233/JAD-150131

Citation: Journal of Alzheimer's Disease, vol. 46, no. 2, pp. 441-449, 2015

Authors: Thyrian, Jochen René | Eichler, Tilly | Hertel, Johannes | Wucherer, Diana | Dreier, Adina | Michalowsky, Bernhard | Killimann, Ingo | Teipel, Stefan | Hoffmann, Wolfgang

Article Type: Research Article

Abstract: Background: There is limited knowledge about the range and effects of neuropsychiatric symptoms shown by persons with dementia (PWD) living in the community and their related caregiver burden. Objective: To examine neuropsychiatric symptoms in PWD in primary care with regard to frequency, severity, and burden to caregiver; to compare PWD with and without symptoms with regard to sociodemographics, care-related, and disease-related variables; and to identify variables associated with symptoms. Methods: A general physician-based epidemiological cohort of 248 people screened positive for dementia over the age of 70 (living at home) and their caregivers, was assessed …using the Neuropsychiatric Interview (NPI), sociodemographics, and disease-related variables. Results: In preliminary analyses, neuropsychiatric symptoms were frequent in PWD. Prevalence numbers ofdysphoria/depression, apathy, and agitation/aggression were each more than 30% . The severity of neuropsychiatric symptoms in people screened positive for dementia in primary care is moderate with a mean NPI score of m = 11.91 (SD = 16.0). Overall, caregiver distress is low, indicated by a total distress score of m = 5.94 (SD = 7.2, range 0–39). Common or frequent symptoms are not necessarily the most distressing symptoms. Conclusions: Neuropsychiatric symptoms are common in people screened positive for dementia in primary care. While frequency, severity, and perceived distress might be low in the total sample, we identified the dimensions delusions, aggression, anxiety, disinhibition, and depression to be perceived “severely” to “extremely” distressing in more than 30% of the caregivers affected. The association between activities of daily living and symptoms needs further attention. Show more

Keywords: Behavioral symptoms, caregiver burden, Neuropsychiatric Interview, population-based, primary care

DOI: 10.3233/JAD-143114

Citation: Journal of Alzheimer's Disease, vol. 46, no. 2, pp. 451-459, 2015

Authors: Terni, Beatrice | Ferrer, Isidro

Article Type: Research Article

Abstract: Previous studies have shown that metalloproteinases (MMPs) participate in the clearance of amyloid-β (Aβ) in Alzheimer’s disease (AD); MMP2 and MMP3 cleave soluble Aβ, and both MMP9 and MT1-MMP are able to degrade soluble and fibrillar forms of Aβ. The present study shows increased expression levels of active MMP2 in the entorhinal cortex at early stages of AD-related pathology (Braak and Braak stages I/II-0 and III/IV-A) as revealed by western blotting and gelatin zymography. Confocal microscopy discloses co-localization of MMP2 and phospho-tau in neurofibrillary tangles and dystrophic neurites. MMP2 has the capacity to cleave recombinant tau in vitro in …a dose-dependent manner, consistent with a physiological function of MMP2 in normal tau proteolysis. However, MMP2 does not cleave hyperphosphorylated and dephosphorylated tau from enriched paired helical filament fractions. These observations raise the possibility that accumulation of MMP2 in neurofibrillary tangles and concomitant loss of proteolytic capacity on tau protein is a response geared to eliminating production of toxic truncated tau species in AD brains. Show more

Keywords: Alzheimer’s disease, metalloproteinases, MMP2, neurofibrillary tangles, tau

DOI: 10.3233/JAD-142460

Citation: Journal of Alzheimer's Disease, vol. 46, no. 2, pp. 461-469, 2015

Authors: Balducci, Claudia | Paladini, Alessandra | Micotti, Edoardo | Tolomeo, Daniele | La Vitola, Pietro | Grigoli, Emanuele | Richardson, Jill C. | Forloni, Gianluigi

Article Type: Research Article

Abstract: Alzheimer’s disease (AD) is the most common form of dementia characterized by synaptic dysfunction, memory loss, neuroinflammation, and neuronal cell death. Amyloid-β (Aβ), recognized as the main culprit of AD, aggregates and accumulates in the extracellular compartment as neuritic plaques, after deregulation of its production or clearance. Apolipoprotein E (ApoE) plays a major role in Aβ clearance and its expression is transcriptionally regulated by the liver X receptor and retinoid X receptors (RXRs) system. Bexarotene (BEXA), an RXR agonist that increases ApoE expression and microglia phagocytosis has been proposed as a promising therapy for AD, resolving both the amyloid pathology …and memory loss. Despite the first compelling report, however, multiple failures have been documented, raising concern about whether BEXA could in fact become a novel disease-modifying strategy for AD. To help clarify this, we investigated the effect of BEXA in vivo at multiple levels in TASTPM transgenic mice. Seven-day oral administration of BEXA to these mice did not achieve any significant memory improvement, plaque reduction, or enhancement of microglial cell activation. No differences were found when specifically investigating the microglial phagocytic state in vivo . In addition, a brain structural analysis with magnetic resonance did not detect any BEXA-mediated change in the volume reduction of the main affected brain areas in our mice. These results suggest that BEXA has no beneficial effects on the multi-factorial pathologic phenotype of AD mice. Show more

Keywords: Alzheimer’s disease, amyloid-β, apolipoprotein E, bexarotene, magnetic resonance imaging, memory, TASTPM mice, therapy

DOI: 10.3233/JAD-150029

Citation: Journal of Alzheimer's Disease, vol. 46, no. 2, pp. 471-482, 2015

Authors: Denvir, James | Neitch, Shirley | Fan, Jun | Niles, Richard M. | Boskovic, Goran | Schreurs, Bernard G. | Primerano, Donald A. | Alkon, Daniel L.

Article Type: Research Article

Abstract: Background: Early onset dementias have variable clinical presentations and are often difficult to diagnose. We established a family pedigree that demonstrated consistent recurrence of very early onset dementia in successive generations. Objective and Method: In order to refine the diagnosis in this family, we sequenced the exomes of two affected family members and relied on discrete filtering to identify disease genes and the corresponding causal variants. Results: Among the 720 nonsynonymous single nucleotide polymorphisms (SNPs) shared by two affected members, we found a C to T transition that gives rise to a Thr147Ile missense substitution …in the presenilin 1 (PS1) protein. The presence of this same mutation in a French early-onset Alzheimer’s disease family, other affected members of the family, and the predicted high pathogenicity of the substitution strongly suggest that it is the causal variant. In addition to exceptionally young age of onset, we also observed significant limb spasticity and early loss of speech, concurrent with progression of dementia in affected family members. These findings extend the clinical presentation associated with the Thr147Ile variant. Lastly, one member with the Thr147Ile variant was treated with the PKC epsilon activator, bryostatin, in a compassionate use trial after successful FDA review. Initial improvements with this treatment were unexpectedly clear, including return of some speech, increased attentional focus, ability to swallow, and some apparent decrease in limb spasticity. Conclusions: Our findings confirm the role of the PS1 Thr147Ile substitution in Alzheimer’s disease and expand the clinical phenotype to include expressive aphasia and very early onset of dementia. Show more

Keywords: Bryostatin, early-onset Alzheimer’s disease, expressive aphasia, PSEN1, whole exome sequencing

DOI: 10.3233/JAD-150051

Citation: Journal of Alzheimer's Disease, vol. 46, no. 2, pp. 483-490, 2015

Authors: Wang, Chong | Tan, Lan | Wang, Hui-Fu | Yu, Wan-Jiang | Liu, Ying | Jiang, Teng | Tan, Meng-Shan | Hao, Xiao-Ke | Zhang, Dao-Qiang | Yu, Jin-Tai | Disease Neuroimaging Initiative Alzheimer’s

Article Type: Research Article

Abstract: The phospholipase D3 (PLD3 ) gene has shown association with Alzheimer’s disease (AD). However, the role of PLD3 common variants in amyloid-β (Aβ ) pathology remains unclear. We examined the association of thirteen common single nucleotide polymorphisms (SNPs) with cerebrospinal fluid (CSF) Aβ 1 - 42 levels and florbetapir retention on florbetapir 18 F amyloid positron emission tomography (AV45-PET) in a large population. We found that one SNP (rs11667768) was significantly associated with CSF Aβ 1 - 42 levels in the normal cognition group. We did not observe an association of any SNP with florbetapir retention. Our study …predicted the potential role of PLD3 variants in Aβ pathology. Show more

Keywords: Alzheimer’s disease, amyloid-β , association, PLD3

DOI: 10.3233/JAD-150110

Citation: Journal of Alzheimer's Disease, vol. 46, no. 2, pp. 491-495, 2015

Authors: Altamura, Claudia | Ventriglia, Mariacarla | Martini, Maria Giulia | Montesano, Domenico | Errante, Yuri | Piscitelli, Fabiana | Scrascia, Federica | Quattrocchi, Carlo | Palazzo, Paola | Seccia, Serenella | Vernieri, Fabrizio | Di Marzo, Vincenzo

Article Type: Research Article

Abstract: Background: Growing evidence suggests that the endocannabinoid system is involved in the pathogenesis of Alzheimer’s disease (AD) and atherosclerosis. Objective: The purpose of this study was to investigate the activation of the endocannabinoid system in AD in vivo and the possible intermediate role of atherosclerosis. Methods: We enrolled 41 patients with probable AD, and 30 age- and gender-matched controls. All subjects underwent: ultrasound examination of cerebral and neck vessels (including intima-media thickness and plaque stenosis evaluation); blood sampling to measure levels of endocannabinoid [anandamide (AEA), 2-arachidonoylglycerol (2-AG)] and endogenous AEA analogues [N-palmitoyl-ethanolamide (PEA); N-oleoyl-ethanolamide]; …neuropsychological evaluation and brain MRI (atrophy, white matter hyperintensity volume). Results: 2-AG levels were higher in AD patients compared to controls (Mann-Whitney test p  = 0.021). In the AD group, 2-AG correlated to white matter hyperintensity volume (r  = 0.415, p  = 0.015) and was higher in patients with chronic heart ischemic disease (p  = 0.023). In AD patients, 2-AG was also positively related to memory (r  = 0.334, p  = 0.05) and attention (r  = 0.423, p  = 0.018) performances. Constructional praxia test scores were lower in patients with higher levels of PEA (r  =−0.389, p  = 0.019). Conclusion: AD patients present high plasma 2-AG levels, also in relation to heart ischemic disease and cerebral leukoaraiosis. This may be a protective mechanism hindering neurodegeneration, but it may also play an ambivalent role on cerebrovascular circulation. The increase in 2-AG and PEA levels observed with ongoing pathological processes may differently modulate cognitive performances. Show more

Keywords: Alzheimer’s disease, atherosclerosis, endocannabinoids, leukoaraiosis

DOI: 10.3233/JAD-142349

Citation: Journal of Alzheimer's Disease, vol. 46, no. 2, pp. 497-506, 2015

Authors: Zhan, Xinhua | Cox, Christopher | Ander, Bradley P. | Liu, Dazhi | Stamova, Boryana | Jin, Lee-Way | Jickling, Glen C. | Sharp, Frank R.

Article Type: Research Article

Abstract: Background: Ischemia, white matter injury, and Alzheimer’s disease (AD) pathologies often co-exist in aging brain. How one condition predisposes to, interacts with, or perhaps causes the others remains unclear. Objectives: To better understand the link between ischemia, white matter injury, and AD, adult rats were administered lipopolysaccharide (LPS) to serve as an inflammatory stimulus, and 24 h later subjected to 20-min focal cerebral ischemia (IS) followed by 30-min hypoxia (H). Methods: Myelin and axonal damage, as well as amyloid-β (Aβ) and amyloid-β protein precursor (AβPP) deposition were examined by Western blot and immunocytochemistry following LPS/IS/H. Findings …were compared to the 5XFAD mouse AD brain. Results: Myelin/axonal injury was observed bilaterally in cortex following LPS/IS/H, along with an increase in IL-1, granzyme B, and LPS. AβPP deposition was present in ischemic striatum in regions of myelin loss. Aβ1-42 and AβPP were deposited in small foci in ischemic cortex that co-localized with myelin aggregates. In the 5XFAD mouse AD model, cortical amyloid plaques also co-localized with myelin aggregates. Conclusions: LPS/IS/H produce myelin injury and plaque-like aggregates of myelin. AβPP and Aβ co-localize with these myelin aggregates. Show more

Keywords: Alzheimer’s disease, amyloid plaques, amyloid-β, amyloid-β protein precursor, hypoxia, lipopolysaccharide, myelin, myelin basic protein

DOI: 10.3233/JAD-143072

Citation: Journal of Alzheimer's Disease, vol. 46, no. 2, pp. 507-523, 2015

Authors: Starks, Erika J. | Patrick O’Grady, J. | Hoscheidt, Siobhan M. | Racine, Annie M. | Carlsson, Cynthia M. | Zetterberg, Henrik | Blennow, Kaj | Okonkwo, Ozioma C. | Puglielli, Luigi | Asthana, Sanjay | Dowling, N. Maritza | Gleason, Carey E. | Anderson, Rozalyn M. | Davenport-Sis, Nancy J. | DeRungs, LeAnn M. | Sager, Mark A. | Johnson, Sterling C. | Bendlin, Barbara B.

Article Type: Research Article

Abstract: Background: Insulin resistance (IR) is linked with the occurrence of pathological features observed in Alzheimer’s disease (AD), including neurofibrillary tangles and amyloid plaques. However, the extent to which IR is associated with AD pathology in the cognitively asymptomatic stages of preclinical AD remains unclear. Objective: To determine the extent to which IR is linked with amyloid and tau pathology in late-middle-age. Method: Cerebrospinal fluid (CSF) samples collected from 113 participants enrolled in the Wisconsin Registry for Alzheimer’s Prevention study (mean age = 60.6 years), were assayed for AD-related markers of interest: Aβ 42 , P-Tau181 , …and T-Tau. IR was determined using the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR). Linear regression was used to test the effect of IR, and APOE ɛ 4, on tau and amyloid pathology. We hypothesized that greater IR would be associated with higher CSF P-Tau181 and T-Tau, and lower CSF Aβ 42 . Results: No significant main effects of HOMA-IR on P-Tau181 , T-Tau, or Aβ 42 were observed; however, significant interactions were observed between HOMA-IR and APOE ɛ 4 on CSF markers related to tau. Among APOE ɛ 4 carriers, higher HOMA-IR was associated with higher P-Tau181 and T-Tau. Among APOE ɛ 4 non-carriers, HOMA-IR was negatively associated with P-Tau181 and T-Tau. We found no effects of IR on Aβ 42 levels in CSF. Conclusion: IR among asymptomatic APOE ɛ 4 carriers was associated with higher P-Tau181 and T-Tau in late-middle age. The results suggest that IR may contribute to tau-related neurodegeneration in preclinical AD. The findings may have implications for developing prevention strategies aimed at modifying IR in mid-life. Show more

Keywords: Alzheimer’s disease, apolipoprotein ɛ4, cerebrospinal fluid, glucose, insulin, insulin resistance, tau protein

DOI: 10.3233/JAD-150072

Citation: Journal of Alzheimer's Disease, vol. 46, no. 2, pp. 525-533, 2015

Authors: Armbrecht, Harvey J. | Siddiqui, Akbar M. | Green, Michael | Farr, Susan A. | Kumar, Vijaya B. | Banks, William A. | Patrick, Ping | Shah, Gul N. | Morley, John E.

Article Type: Research Article

Abstract: The senescence-accelerated mouse (SAMP8) strain exhibits an age-related decrease in memory accompanied by an increase in hippocampal amyloid-β protein precursor (AβPP) and amyloid-β peptide (Aβ). We have shown that administration of an antisense oligonucleotide against the Aβ region of AβPP (AβPP antisense) reverses the memory deficits. The purpose of this study was to determine the effect of peripheral (IV) administration of AβPP antisense on hippocampal gene expression. The AβPP antisense reversed the memory deficits and altered expression of 944 hippocampal genes. Pathway analysis showed significant gene expression changes in nine pathways. These include the MAPK signaling pathway (p  = 0.0078) and …the phosphatidylinositol signaling pathway (p  = 0.043), which we have previously shown to be altered in SAMP8 mice. The changes in these pathways contributed to significant changes in the neurotropin (p  = 0.0083) and insulin signaling (p  = 0.015) pathways, which are known to be important in learning and memory. Changes in these pathways were accompanied by phosphorylation changes in the downstream target proteins p70S6K, GSK3β, ERK, and CREB. These changes in hippocampal gene expression and protein phosphorylation may suggest specific new targets for antisense therapy aimed at improving memory. Show more

Keywords: Antisense oligonucleotides, gene expression, MAPK signaling, memory loss, phosphatidylinositol signaling, SAMP8 mouse

DOI: 10.3233/JAD-142760

Citation: Journal of Alzheimer's Disease, vol. 46, no. 2, pp. 535-548, 2015

Article Type: Meeting Report

DOI: 10.3233/JAD-150271

Citation: Journal of Alzheimer's Disease, vol. 46, no. 2, pp. 549-552, 2015