ARF role as tumor suppressor has been challenged in the last years by several findings of different groups ultimately showing that its functions can be strictly context dependent. We previously showed that ARF loss in HeLa cells induces spreading defects, evident as rounded morphology of depleted cells, accompanied by a decrease of phosphorylated Focal Adhesion Kinase (FAK) protein levels and anoikis. These data, together with previous finding that a PKC dependent signalling pathway can lead to ARF stabilization, led us to the hypothesis that ARF functions in cell proliferation might be regulated by phosphorylation. In line with this, we show here that upon spreading ARF is induced through PKC activation. A constitutive-phosphorylated ARF mutant on the conserved threonine 8 (T8D) is able to mediate both cell spreading and FAK activation. Finally, ARF-T8D expression confers growth advantage to cells thus leading to the intriguing hypothesis that ARF phosphorylation could be a mechanism through which pro-proliferative or anti proliferative signals could be transduced inside the cells in both physiological and pathological conditions. Further studies indicated that, apart from p53, ARF functionally interacts with a number of factors, thus mediating cellular response also through p53-independent activities3. ARF has unexpectedly been found over expressed or stabilized in several types of cancers6, to be involved in autophagy9 and, recently, to have a role in protecting human melanocytes from free radicals arising upon mitochondrial dysfunction10. In addition to this, it also appears to play a role during development13. These observations led to the conclusion that, somehow, ARF role within the cell can be highly pleomorphic or context-dependent, ranging from halting uncontrolled cell proliferation in some cases to favour cancer growth in others. We recently demonstrated that ARF plays an unexpected role in the cytoplasm in the organization of the cytoskeleton. During cell adhesion, ARF accumulates at sites of polymerized actin such as focal adhesions, where it co-localizes with and induces activation of the Focal Adhesion Kinase (FAK). Interestingly, this mechanism appears to be conserved in mouse. By aiding cytoskeleton assembly during spreading, ARF protects cells from anoikis blocking DAPK (Death Associated Protein Kinase) dependent apoptosis14.The p14ARF protein, encoded by the INK4a/ARF locus, was initially described as a tumor suppressor that, in response to different oncogenic stimuli, by protecting p53 from proteasome mediated degradation, initiates a cell pathway leading to cell cycle block and/or apoptosis15. The involvement of phosphorylation in controlling ARF activities has been suggested by different experimental approaches19. In vitro kinase assay shows that three PKC consensus sites identified in silico within ARF sequence are specifically phosphorylated by PKC. In addition, we show that the protein is phosphorylated in vivo15. Mimicking the un-phosphorylatable status of the protein on Threonine 8 (T8A mutant), confers instability to the protein while not affecting its ability to restrain cell proliferation. Conversely, the T8D ARF mutant, that corresponds to the constitutive phosphorylation status of the protein, accumulates in the cytoplasm and is less efficient than the wt in halting cell proliferation. These data led to the hypothesis that ARF function might be regulated by phosphorylation on this conserved residue. PKC plays important role in a number of cell functions20. Among these, it has been shown that it is involved in the regulation of cell morphology21 through the phosphorylation of a high number of proteins involved in cell migration and in the generation of focal adhesion23.We previously demonstrated that ARF is regulated through the activation of PKC pathway in both cancer and transformed cell linesOn the basis of this evidence, we sought to investigate if ARF role in cell spreading and its functional relation with FAK could be regulated by PKC activity. Here we show that during cytoskeleton remodelling induced by cell spreading, ARF protein levels increase in the cytoplasm through a PKC dependent mechanism. Mimicking the phosphorylation status of the protein is sufficient to drive its localization in the cytoplasm and to rescue spreading defect as well as FAK phosphorylation of ARF silencing in HeLa cells, thus resulting in an increased proliferative ability. Taken together these data indicate that PKC activation can prime ARF involvement in cell spreading leading to increased FAK activation and cell proliferation.15), we constructed double (T8-S52) and triple (T8-S52-S127) mutants in which each single potential PKC site was replaced either with an alanine (“A” serie), that cannot be phosphorylated, or with an aspartic acid (“D”), that mimics the phosphorylation status of the protein. ARF protein displays various degree of accumulation in nucleoli and/or scattered throughout the nucleoplasm25. We then tested if the inserted mutations could affect ARF subcellular localization evaluating subcellular localization of tagged WT and mutant ARF proteins transfected in U2OS cells by IF with anti Hys antibody. For each mutant, we counted the number of transfected cells displaying nuclear . To this aim, we monitored levels of pPKC during cell spreading by western blot using the anti pPKC pan antibody that recognizes all the PKC isoforms phosphorylated at a carboxyl-terminal residue homologous to serine 660 of PKC β II (activated pPKC). Cytoplasmic and nuclear protein extracts were collected from untreated (nt) and detached cells, as well as from replated cells five hours after seeding, when spreading process is almost completed. As shown in Fig. To better analyse if the observed PKC activation is required to induce ARF increase, we knocked down PKC expression by RNA interference and analysed ARF cytoplasmic levels by western blot in HeLa cells. As control, cells were also treated with ARF specific siRNA. Control (siLuc and siSCR), PKCalpha and ARF depleted cells were collected 72hrs after transfection or subjected to trypsinization and divided in two aliquots. One was subjected to direct lysis Fig.  and S3. 14. Interestingly, upon PKC downregulation by siRNA or by bisindolylmaleimide I treatment, cells showed spreading defects similarly to ARF depletion , FAK is activated through auto-phosphorylation of tyrosine 397 (Y397). This activation is followed by increased Src binding to FAK resulting in its massive phosphorylation onto several tyrosine residues within FAK sequence27. We thus preliminary checked if and which ARF mutant is also able to rescue FAK activation by western blot of crude extracts with anti pFAK antibodies. Results showed that in T8A expressing cells devoid of ARF expression, lower levels of both total and pFAK on tyrosine 397 are achieved, while no difference between siSCR and siARF could be detected in T8D expressing cells , thus suggesting that the effect of ARF phosphorylation on FAK activation can be disengaged from the effect on cell proliferation in these cells.The evidence that ARF functions can be thus modulated by phosphorylation events taking place on the conserved Threonine 8 leaves us to the concept that ARF role in cell survival may more realistically depends on the cellular and/or tissue type and context, thus accounting for the controversy surrounding the topic. It is interesting to underline how also the cyclin-dependent kinase inhibitor p21, in addition to the well-known growth inhibitor function, displays cancer promoting features in some cell context, as recently reported37. Given the context dependency of ARF role within the cell, we focused on ARF function in cell spreading in a cell context in which ARF functions as tumor suppressor are blocked, such as HeLa cells, thanks to the inactivation of the p53 pathway. Our data show that during cytoskeleton remodelling induced by detachment, both the activated form of PKC and ARF protein levels increase in the cytoplasm. We previously showed that ARF ability to favour cell spreading is accompanied by the transduction of growth signals arising from the integrin/FAK functional interaction14. We now added data showing that T8A can only in part rescue FAK activation, although this is not enough to allow cell spreading. This can be explained by decreased stability of the T8A mutant respect to T8D protein38. Our data depict a scenario in which during cell spreading or uncontrolled PKC activation, ARF stabilization and thus FAK activation could allow the pro-proliferative signal transduction within the cells. The effect of ARF and its mutants on cell proliferation suggested us that the T8D behaves as a constitutive active mutant. As the WT has no effect on cell proliferation this could mean that in HeLa cells something can block this ARF function. Interestingly, in H1299 cells we did not observed difference in cell growth profiles of WT and mutant proteins transfected cells. We previously demonstrated that ARF-FAK pro-proliferative axis is interrupted due undetectable levels of the Death Associated Protein Kinase (DAPK) in this cell context. DAPK is a Serine/threonine kinase playing important roles in tumor suppression and apoptosis. We previously showed that ARF expression prevents DAPK mediated anoikis. It has been shown that FAK activation can be counteracted by DAP kinase expression40 by disrupting signal transduction between integrin and FAK upon ECM interaction during spreading. This leads to the interesting hypothesis that ARF and/or T8D could be able to protect FAK by DAPK negative effect while T8A being less efficient. Interestingly, when we analysed transfected cells 24 hrs after transfection, we observed that while both controls and ARF expressing cells displayed a certain percentage of dying cells, T8D expressing cells did not (data not shown). This suggested us that T8D expression could protect cells from cell death and thus results in the observed increased cell number.By means of pharmacological studies, protein kinase C has been implicated as a key molecule involved in cell spreading and migration, in part through interaction with beta1-integrin42. The involvement of PKC in tumor progression has thus gained recognition as potential therapeutic targets for the treatment of various malignancies. A deeper understanding of the PKC dependent ARF functions, both in physiological or pathological contexts, may provide useful information about the environmental cues that determine ARF functions as tumor suppressor or tumor promoter.Our data led us to conceive a model in which, during cell spreading and PKC activation, p14ARF protein levels increase in the cytoplasm. It has been widely reported that PKC mediated phosphorylation regulates composition and turn-over of focal adhesions where assembly and disassembly of actin fibers take place upon different environmental inputs. In particular, cellular motility and invasivity, two of worst signals of cancer progression, are sustained by this highly dynamic phenomenon15 and were routinely tested for mycoplasma contamination by PCR based method and kept in culture for no more than 6 weeks after resuscitation.HeLa cells were purchased from SIGMA. U2OS and H1299 were purchased from the American Type Culture Collection (ATTC) and authenticated by STR DNA Profiling Analysis. Cells were grown as described15. The cells were transfected with Lipofectamine 2000 reagent (Invitrogen) or with electroporation  as described in43.ARF mutants used in this study were generated as describedet al., 2009. PKC alfa and luciferase siRNAs are available by Qiagen . For rescue experiment, the cells were transfected with an ARF siRNA, that anneals in the 5′UTR region of ARF endogenous transcript, as previously described in Vivo et al., 2017 and Kobayashi. All siRNAs were transfected using RNAiMAX reagent (Invitrogen). Rescue was performed as described in Vivo et al., 2017.For RNA interference experiments, ARF siRNA , that anneals in exon 1ß of p14ARF transcript, and scrambled siRNA (negative control) sequences has been reported in Vivo in Vivo et al. 2017.Treatments in this study were performed as follows: treatment with bisindolylmaleimide I : 24 hrs after plating, HeLa cells were treated either with DMSO or bisindolylmaleimide I at 5 μM and 2,5 μM final concentrations for 2 hours then were detached by trypsin to synchronize the adhesion/spreading process. An aliquot of cells was harvested and total extracts prepared for subsequent analysis as described. Another aliquot of cells was replaced in presence of bisindolylmaleimide I at 2,5 μM as final concentration and, after 5 hrs and 24 hrs post plating, the cells were harvested and total extracts prepared as described. Live phase-contrast images were acquired using a Nikon Eclipse microscope  with 20x objective. 5 fields were randomly selected in the plates for each experimental point and images acquired with the Image Pro Plus software (Media Cybernetics). Cell spreading was quantified as described 6 HeLa cells were transiently transfected by electroporation as described14. For H1299, 4 × 106 cells were transiently transfected by electroporation with the indicated plasmid at 500 ng. After 72 hrs from transfection (48 hrs for H1299), the cells were counted using ScepterTM automatic cell counter (Millipore) following the manufacturer’s protocol. The data obtained were analysed using Scepter™ Software Pro (from Millipore).4 × 106 cells per sample). Subcellular fractionation was done as previously described and cytoplasmic extracts were incubated with anti-FAK C-20 antibody as described in14 and in45.Immunoprecipitation assays were performed with cytoplasmic extracts . Proteins were visualized by enhanced chemiluminescence (western blotting substrate Thermo Scientific). Images were acquired by ChemiDoc Imaging System equipped with a CCD camera (Bio-rad) through the Quantity One software. Representative experiments are shown for each blot. Full-length western blots are represented in supplementary information , normalized respect to loading control and reported as fold enrichment respect to control sample. List of antibodies used in this study: anti-ARF C-18, anti-FAK C-20, anti-actin I-19, anti-ß-Tubulin H-235, anti-GAPDH 6C5, anti-Lamin A/C H-110, anti-Histone H1 N-16 , anti-pFAK (Tyr397) , anti-pTyr 05-321 , anti- PKC alpha, beta and gamma M110 , anti- 6XHIS monoclonal antibody (Clontech), anti-AKT, anti-phospho PKC pan ßII Ser660; .Western blot (WB) analysis was performed as previously described  with 20x objective. 5 fields were randomly selected in the plates for each experimental point and images acquired with LAS X life scence (Leica). To quantify the percentage of rounded cells, for each transfection point, we counted rounded (and adherent) cells and pooled data from three to five experiments as described in14.HeLa cells were detached by trypsinization and replated at a density of 1 × 1046.Real time experiment was performed as describedData presented in this work derive from experiments performed at least in triplicate , except when differently stated. The sample size of each experimental point is reported in the relative figure legend, as well as the specific statistical analysis performed. In all the experiments in which single cells were analysed 5 to 10 fields were randomly selected in the coverslip for each experimental point. t-test and ANOVA were performed using GraphPad Prism 5.0 software.All data generated or analysed during this study are included in this published article (and its Supplementary Information files).Supplementary information


Glial cell line-derived neurotrophic factor (GDNF) is a potent survival and regeneration-promoting factor for dopaminergic neurons in cell and animal models of Parkinson disease (PD). GDNF is currently tested in clinical trials on PD patients with so far inconclusive results. The receptor tyrosine kinase Ret is the canonical GDNF receptor, but several alternative GDNF receptors have been proposed, raising the question of which signaling receptor mediates here the beneficial GDNF effects. To address this question we overexpressed GDNF in the striatum of mice deficient for Ret in dopaminergic neurons and subsequently challenged these mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Strikingly, in this established PD mouse model, the absence of Ret completely abolished GDNF's neuroprotective and regenerative effect on the midbrain dopaminergic system. This establishes Ret signaling as absolutely required for GDNF's effects to prevent and compensate dopaminergic system degeneration and suggests Ret activation as the primary target of GDNF therapy in PD. In addition, the in vivo function of the alternative GDNF receptors in the dopaminergic system under physiological and pathophysiological conditions, like PD, and their dependence on GDNF has not yet been addressed in detail. This raised the important question which GDNF receptor might be required to mediate GDNF's reported neuroprotective and regenerative effect in the dopaminergic system in PD animal models and potentially in PD patients.29GDNF signaling is highly complex as this neurotrophic factor can bind to a variety of receptors, thus being able to induce pleiotropic effects. GDNF efficiently binds to the GPI-linked GDNF family receptor 30 In adult mice endogenous GDNF levels are rather low.31 Therefore, we could not rule out in that study the possibility, that higher levels of GDNF—as also used in the clinical GDNF trials in PD patients—might have neuroprotective and regenerating effects even in the absence of the Ret receptor. Here we addressed now this question by viral overexpression of GDNF in MPTP-treated mice lacking expression of Ret again specifically in dopaminergic neurons.30 We found that in the absence of Ret in dopaminergic neurons even a substantial overexpression of GDNF in the striatum does not have a neuroprotective and regenerative effect. Thus, despite the expression of alternative GDNF receptors on midbrain dopaminergic neurons, the presence of the canonical GDNF receptor Ret seems to be mandatory for mediating GDNF's beneficial survival and axonal resprouting effect in these neurons.Previously, we showed in dopaminergic neuron-specific Ret knockout mice that Ret receptor loss does not result in a higher vulnerability of midbrain dopaminergic neurons against MPTP but to less resprouting of left over dopaminergic neuron axons in the striatum after MPTP intoxication.lx/lx)23 by crossing mice carrying the floxed allele of Ret (Retlx/lx)32 with dopamine transporter (DAT) promoter-driven Cre mice (DAT-Cre).33 We overexpressed GDNF in these mice by stereotactic injection of a recombinant adeno-associated virus serotype 5 (AAV5) encoding the mouse GDNF cDNA under the control of the glial fibrillary acidic protein (GFAP) promoter (AAV5-GDNF) unilaterally in the striatum of the left mouse brain hemisphere  in some mice confirming astrocyte-specific transgene expression, we also unilaterally sham-injected some mice  as a control .30 The n of GDNF .30lx/lx and DAT-Retlx/lx) MPTP reduced the number of dopaminergic neurons by 46%  compared with saline (NaCl)-treated mice (lx/lx) and MPTP induced <20% loss of dopaminergic neurons (lx/lx). Thus, in the absence of Ret, even substantial amounts of GDNF did not protect dopaminergic neurons from MPTP toxicity. The same cell numbers were also observed in mice analyzed 3 months after MPTP treatment confirming the long-term neuroprotective effect of GDNF/Ret on dopaminergic neurons and also the general lack of dopaminergic cell regeneration after MPTP treatment independent of GDNF overexpression or not -positive dopaminergic neurons of the SNpc in the mouse brains 2 weeks after MPTP treatment. In all three genotypes  and homovanillic acid (HVA) in the striatum two weeks after MPTP treatment . Consist/lx mice . This sh30 resprouting of the residual dopaminergic fibers after partial denervation by MPTP strictly depends on the presence of the Ret receptor and is already induced by endogenous levels of GDNF , exogenous GDNF must be provided for the clinical trials by specific routes. GDNF protein can be intrastriatal or intranasal infused, modified or packaged GDNF that can pass the BBB might be supplied systemically, GDNF encoding DNA constructs might be provided by viral vectors, or GDNF expressing cells might be transplanted into the striatum.41 Previously we compared astrocytic (human GFAP promoter) and neuronal (human synapsin 1 promoter) expression levels of GDNF with the AAV5 serotype vector and found similar GDNF expression levels in the injected side but less spreading to the contralateral side using the GFAP promoter.34 It has already been previously shown that AAV serotype 5 allows wide spreading of the virus in the striatum and efficient targeting of astrocytes.42 The GFAP promoter led to high- and long-term GDNF expression in astrocytes without adverse effects as also shown previously in adeno- and lentivirus vectors.44 This is in contrast to high expression of GDNF in striatal and nigral neurons which can lead to negative side effects, such as downregulation of TH protein levels.48 If in clinical trials the amount of bioactive GDNF in the striatum is limiting, the here used AAV5-GDNF serotype 5 vector with the GFAP promoter seems to be an efficient and safe alternative to provide GDNF.34 Further experiments are needed to clarify if the mild striatal gliosis found in our mice is also observed in humans and if this results in negative side effects.Interestingly, endogenous GDNF has been shown to be expressed in the adult striatum only in fast spiking, parvalbumin-positive GABAergic interneurons—representing 0.7% of all striatal neurons—and to a lower extent in some somatostatinergic and cholinergic interneurons, but not in medium spiny neurons.51 It is also well established that the Ret receptor it essential for maintaining aged SNpc dopaminergic neurons and seems to crosstalk for this maintenance function with proteins such as the chaperon DJ-1, PINK-1 (PTEN-induced putative kinase 1) and parkin, an E3 ubiquitin-protein ligase, for which mutated forms were found to lead to PD.53 Ret and DJ-1 (deglycase) trigger dopaminergic cell survival in mice by activating together the Ras/MAPK pathway.24 Ret crosstalks with PINK-1 and parkin to ensure mitochondrial morphological and physiological integrity in dopaminergic neurons.53 Ret and parkin are not only important for survival of aged dopaminergic cell body in the SNpc but also for the maintenance of the striatal dopaminergic innervation.53 Therefore, we suspect that these signaling events downstream of the Ret receptor play also an important role in mediating GDNF's neuroprotective and regenerative function in the dopaminergic system.Previous work has already shown that GDNF's neuroprotective function requires not only Ras-MAPK activation but also PI3K-Akt signaling.28 supports the idea, that alternative GDNF receptors might even function without the need to bind GDNF as important signaling recognition or cell adhesion molecules in dopaminergic neurons. Further studies are needed to clarify the physiological function of alternative GDNF receptors and GDNF itself in mice and man.Although we have shown here the requirement for Ret for mediating GDNF's neuroprotective and regenerative effect in midbrain dopaminergic neurons, this does not rule out an additional function for alternative GDNF receptors in this context and also for mediating survival during development and maintenance of these neurons. The recent finding, that GDNF might be dispensable for the development and maintenance of the dopaminergic system in miceα1 agonists have already been developed.55 Such substances would not only simplify the application procedure but would presumably also decrease the chance of unwanted side effects that might result from interaction with alternative GDNF receptors. If the Ret protein on dopaminergic neurons is limiting, Ret might also be provided by a viral vector approach targeting directly dopaminergic neurons.10 Such scenario has been created by high viral overexpression of α-synuclein in rats leading to downregulation of the orphan nuclear receptor Nurr-1 and its downstream target Ret.56α-Synuclein is aggregating in Lewy bodies and neurites frequently observed in PD patients and gene amplifications and mutations of α-synuclein are found in some familiar forms of PD.57 However, so far no58 or only mild56 reduction of Ret mRNA and protein levels have been found in PD patients, so that more investigations are needed to clarify this issue.Small molecules that specifically activate Ret and can cross the BBB would be ideal and seem to be an achievable goal as small non-peptidyl quinol based GFR12 and emphasize the central role of Ret in mediating GDNF's survival and regeneration signal in SNpc dopaminergic neurons under neurodegenerative conditions that can cause PD.Taken together, our data support the ongoing clinical phase I trials on PD patients testing AAV-encoded GDNF and phase II trials using recombinant GDNF protein53 Further experiments are needed to support this hypothesis.It is likely that the beneficial effect of GDNF/Ret signaling might be further enhanced and more broadly applicable for the heterogeneity of idiopathic PD patients if combined with other neuroprotective and regenerative signals such as for example parkin overexpression.34Recombinant AAV5 viruses expressing GDNF and EGFP were produced as described.30 All experimental animal procedures were conducted according to approved experimental animal licenses issued by the responsible animal welfare authority (Niedersächsisches Landesamt für Verbraucherschutz und Lebensmittelsicherheit) and controlled by the local animal welfare committee of the University Medical Center Göttingen.Transgenic mice were kept on a C57Bl/6J background and were previously described.9 vector genomes (vg) of AAV5- GDNF or AAV5-EGFP in 2–3-month-old male mice and MPTP treatment was done as previously described.34 Animals were either sacrificed by cervical dislocation, the striata dissected, frozen and stored at −80 °C or animals were sacrificed using CO2 and after transcardial perfusion  the whole brain was fixed and cryoprotected (24 h in 4% PFA and 48 h in 30% sucrose at 4 °C).Unilateral stereotactical injection of 3 × 1034GDNF was quantified by IHC staining and ELISA as described.30 Glia cell and dopaminergic fiber density quantification in the striatum was performed as described.30Stereological counting of dopaminergic neurons was performed by the optical fractionation method using StereoInvestigator as described.Primary antibodies were used in the following concentrations: anti-TH , anti-DAT , anti-GFAP , and anti-Iba-1 .30Catecholamine levels were determined by ion-pair HPLC with electrochemical detection essentially as described.post hoc test (GraphPad Prism 4.0). Significance levels were set at *P<0.05, **P<0.01 and ***P<0.001.Data are expressed as means±S.D. To compare different group means for independent samples, the statistical analysis was performed by ANOVA, followed by Tukey's


In this Part II review, as a complement to the Part I published in thissupplement, the authors cover the imaging techniques that evaluates theAlzheimer's disease according to the different metabolic and molecular profiles.In this section MR spectroscopy, FDG-PET and amyloid PET are deeplydiscussed. These tests are most accurate in only theadvanced stages of the disease. Advanced neuroimaging modalities pose a challengefor traditional AD diagnosis and monitoring.More than 5.0 million Americans are currently afflicted by AD. AD affects 5 millionpeople aged more than 65 years and 200,000 individual aged less than 65 years whohas younger-onset of AD.Besides neuronal loss, the other hallmark histological changes in AD are theaccumulation of abnormal amyloid-β (Aβ) proteins forming the plaques(AP) and neurofibrillary tangles (NFTs).3An ideal neuroimaging marker should be able to accurately detect earlyneurodegenerative pathology, reflect pathological stages across the entire severityspectrum, predict when an individual with early pathology will become demented, andmonitor the effect of a therapeutic intervention on the neurodegenerativepathology.In this part of the review, the roles and limitations of the biomarkers used in PETand 1H (hydrogen) MR spectroscopy for management of AD are discussed.1 The most consistent finding of MRSmeasurements reported for AD is decreased NAA in many brain regions, which mayindicate neuronal loss or mitochondria dysfunction. Subjects with AD has shownreduced NAA in the hippocampus,3 posterior cingulated,5 temporal lobe,7 mesial temporal lobe,8 occipital lobe,10 parietallobe,12 andfrontal lobe.13 Decrease in NAA ofwhite matter (WM) is observed to be smaller than grey matter (GM) but some authorsreported no WM change in NAA.8Other concordant result is increase in mI concentration at several brain locations,which links to gliosis or membrane abnormalities (Recent date suggest a role of 1H MR spectroscopy (1H MRS) in clinical evaluation ofAlzheimer's disease (AD). 1H MRS can detect different metabolic substrates such asN-Acetylaspartate (NAA), creatine and phosphocreatine (Cr) and choline (Cho).Additional metabolites that can be measured with more complex technique aremyoinositol (ml), glutamate and glutamine complex (Glx) and lactate (Lac).malities .14 anterior and posteriorcingulated,15 parietal lobe,11 occipital10 and white matter.11 The resonance peak of mI consistsof multiple peaks or so called multiplet structures that yield a complex and closelyspaced group of resonance lines at clinical field strengths. This broad spectrumpattern is not measured accurately using only single peak of model, which mayaccount for variability in earlier reports. Even recently, despite improvements inautomated processing software, clinical group have reported difficulties inobtaining consistent analysis of the mI peak.1The areas involving with increased mI include mesial temporal lobe,16 found higher myoinositol/creatinine ratio in the posteriorcingulate in AD compared to controls (p<0.001), in AD compared to MCI (p=0.048)and in MCI versus controls (p=0.006). The ratio of NAA /mI was also inferior in ADpatients compared to controls (p<0.001), in AD compared to MCI (p=0.002), and inMCI compared to controls (p=0.008). NAA/mI at posterior cingulate provided thehighest sensitivity for distinguish AD and control of 82% at the fixed specificityof 80%. Other studies by Wang et al.17 found different values in NAA/Cr, mI/Cr and mI/NAA ratios athippocampus among AD, MCI and normal subjects. However at posterior cingulate, therewere different results only in mI/NAA while comparing AD with controls, and AD withMCI. Moreover, they also noted good correlation between mI/NAA and level ofcognitive impairment in subjects with AD and MCI.Some investigators used ratios between MRS-visible metabolites for distinguish ADfrom normal subjects. Kantarci et al.18 For example, study of Mackey etal.18 found elevatedCho/Cr ratio at posterior cingulate and precuneous in AD versus controls. It issuggested that the increase of Cho peak is due to membrane phosphotidylcholinecatabolism with the purpose to offer free choline for the insufficient acetylcholineproduction commonly seen in AD. Cho/Cr decreases with the use of cholinergic agonistdrugs in AD which may imply that down regulation of choline acetyltransferaseactivity may be responsible for the rising of Cho19 However, other report no changes20 ordecreases.7 This discrepancy may be results of differences inprotocol MRS or anatomical variation from voxel selection.Conflicting reports about changes of Cho in AD patient has been noted. Some studiesreport increased Cho.16NAA/mI or mI/NAA ratios seem to be the most useful parameters due to some reasons.They are independent of Cr values, decreasing variability resulting from age andother factors without having to calculate absolute concentrations. They are alsoshown to be a dependable diagnostic measure for AD versus controls with highaccuracy.21 determined the peakvalues of NAA in subcortical ischemic vascular dementia (SIVD) compared with ADgroup. SIVD had reduced peak of NAA by 13% in frontal cortex and by 20% in the leftparietal cortex as compared with AD subjects. Kattapong et al.22 showed lower ratios of NAA/Cr andNAA/Cho in vascular dementia than in AD (p<0.02). Study by Waldman23 found higher mI/Cr ratio in ADpatients than vascular dementia patients. In contrast with results ofKattapong,22 they reportedsimilar findings of NAA/Cr or NAA/Cho between clinical groups. They mentioned thatit may reflect the small sample of control subjects and possibly the method ofmeasuring peak heights from spectra, which are scaled to the amplitude of NAA. Ernstet al.20 found reduction of NAAand Glx and increasing of mI at frontal lobe in frontotemporal dementia patientswhile there was no statistically significant frontal abnormality in AD subjects.Some patients in frontotemporal dementia group also showed Lac peak. They reportedthe overall accuracy for discrimination among group of 84%. Coulthard etal.24 reported reductionof NAA/Cr in frontotemporal regions, but not in parietal lobes in frontotemporaldementia. In contrast, study of Garrard et al.25 who used MRS to measure metabolites in the posteriorcingulate in patients with subtypes of frontotemporal dementia; semantic dementiaand progressive nonfluent aphasia subtypes in comparison with AD patients, reportedindistinguishable findings between frontotemporal dementia and AD due to overlappedfindings of decreased NAA/Cr and increased mI/Cr.Many studies compared the MRS findings in different types of dementia. Schuff etal.26 examined 53patients with aMCI and followed them up for average 3 years, They found by measuringthe occipital NAA/Cr ration that MRS could be highly accurate in identifying thetrue converters. The striking finding was a 100% negative predictive value and anoverall accuracy of 88.7%. Interestingly, they found no significant results by doinganalysis in the hippocampal and parietal regions. They explained that theseinconsistent results with the early involvement of hippocampal and parietal area inAD may be caused by partial volume effects which the large size of the voxelprobable included the non-targeted tissue in the analysis or no difference inneuropathological alterations at hippocampus and parietal between converters andnon-converters. Longitudinal study by Fayed et al.27 recruited 110 subjects with aMCI with a follow upperiod of 29 months. They reported that MRS measuring the NAA/Cr in the posteriorcingulate had sensitivity higher than 80% for predicting who is going to convert toprobable AD. However, the distinction of different types of MCY was not possibleusing MRS.MRS has been studied as a tool to predict which patient with MCI would convert to AD.Modrego et al.28 used MRS ingenetic mutated carriers who have a very high risk of developing AD. Theinvestigators demonstrated that NAA/Cr and NAA/mI ratios of carriers weresignificant lower relative to controls groups. Mean reductions in NAA/Cr and NAA/mIwere 10% and 25%, respectively. The reduction of NAA/mI in carriers was related toproximity of expected age at onset.Godbolt et al.29 Theauthors found association among decrease in NAA/Cr and increase in mI/Cr, and higherBraak stage, higher neuritic plaque score and more typical histological findings ofAD. The NAA/mI proved to be the strongest predictor of the pathologic likelihood ofAD. The best correlation noted was that between NAA/mI ratio and Braak stage.Correlation between antemortem MRS results and postmortem neuropathology has beenstudied by Kantarci et al.30 studied the correlation between medial temporallobe and verbal memory, parietotemporal lobe and language and visuoconstructionalskills, and frontal lobe and executive functions in patients with AD, and foundstrong correlation between regional MRS changes and the associated-cognitivedeficits mentioned above.The concordance between MRS and neuropsychological tests are dependent on the type ofcognitive deficit the patient presents. Chantal et al.31 measuredthe level of NAA, Cho, NAA/Cr, Cho/Cr, and mI/Cr in non-treated AD patients andfollowed them after four months of Cholinesterase inhibitor treatment; nameddonepezil. 1H MRS was acquired at right hippocampus. After treatment it could not befound any cognitive improvement. Decreased level of all the metabolites measured wasobserved. They concluded that the reduced levels of NAA indicated continued declinein neuronal loss. The decrease in mI level after treatment might indicate asubsequent reduction in reactive gliosis. However, limitations due to small numberof subjects and limited time of follow-up should be considered.The ability of MRS in monitoring effectiveness of therapies in drug trials hasstudied. Bartha et al.Limitation. Although recent data suggest that MRS may have a role inclinical diagnosis and prognosis of AD, some limitations have to be discussed. It isimportant to mention that metabolites ratios provide robust in vivo markers ofbiochemistry but it has to be interpreted with caution because the ratios areintrinsically ambiguous and prone to misinterpretation.32 Technical problems to adjust the TE MRS mightcontribute to the decrease of the test-retest reproducibility of metabolitemeasurements. Medial temporal region is one of the most interested site for ADpatients. The anterior and mesial portion of the temporal lobe is situated nearby tothe tissue-air interface close to the petrous bone. Due to the differences betweenbrain tissue and air magnetic susceptibility, setting a homogenous magnetic fieldand water suppression within the 1H MRS voxel is complex. MRS can be performed by 2techniques; single-voxel spectroscopy (SVS) or alternatively, multiple-voxeltechnique or known as chemical shift imaging (CSI). One of the limitations of SVS isthe size of the voxel. Usually it is bigger than the majority of mesial temporalstructures, promoting then an effect of partial volume averaging of the adjacenttissue. That also impairs the regional specificity of SVS. 1H MRS at higher Teslamachines would potentially give comparable SNR using smaller voxels. The duration ofspectroscopic study is sometimes too long, and that can be a major limitation forless-cooperative AD patients.33Pitfall of MRS could be minimized by applying standard protocols.It has been shown very high diagnostic value of 18F-[2]-fluorodeoxyglucose positronemission tomography (FDG PET) in establishing presence of absence of AD and otherneurodegenerative disease with autopsy confirmation. PET is sensitive to change overtime, thus, it has value in monitoring disease worsening and therapeuticinterventions. FDG PET provides glucose metabolic activity and patients withneurodegenerative dementia show reduced regional cerebral metabolism.34 or MCI due to AD classified as new AD criteria,35 may not have the characteristicsof more severe AD. However, PET scans performed with FDG show a significant decreasein metabolism in the posterior association cortex, precuneus, and posteriorcingulated.34 Thesecritical early-diagnostic features may be easily overlooked, as the aforementionedregions generally have a higher glucose metabolic rate than surrounding tissue;impairment would lead them to merely "blend in" to the surrounding regions ratherthan stand out in a qualitative assessment of an FDG PET scan.36 Additionally, patients diagnosedwith MCI with AD-like patterns in FDG PET produced scans have been found toeventually develop AD.34 Thesefindings demonstrate that FDG PET can potentially be used to predict conversion fromMCI to later-stage AD.Prodromal AD, a pre-dementia state of mild memory loss while still retaining theability to perform a daily routine37 proposed a imaging classifier that correlates regionalmetabolic changes over time, termed regional 18F-FDG time correlation coefficient(rFTC). They have performed a baseline scan and repeated it within an average timeof 4.3 ± 1 year. They used linear mixed-effects models to determine differentdecline rates of rFTC between controls and individuals at risk for AD, then foundthe association between each subjects' rFTC and cognitive test results. ConstantrFTC of controls subjects were found over time whereas in MCI, the values droppedmuch faster than seen in controls by an additional annual change of -0.02. Thedecline in rFTC of MCI subjects was also associated with change of cognition. Theinvestigators concluded that this classifier method could be used to monitorcognitive deterioration and disease progression.Recent study carried out by Shokouhi et al.38It was found that while the FAQ might not catch small changes in a patient'scognitive decline and that FDG measures were strongly associated with a change inFAQ results, illustrating FDG PET's potential to supplement more subjective,clinical forms of diagnosis.Characteristic findings in regions mentioned above highlight the importance ofintegrating FDG PET more in clinical settings because of its power as an earlydiagnostic tool. Landau et al. compared the performance of FDG PET with theFunctional Activities Questionnaire (FAQ), which is often used to monitor functionalabilities in a clinical setting.39Hallmarks of progressed AD shown by FDG PET include evidence of hypometabolism inposterior regions of the brain, more particularly the temporoparietal region andposterior cingulate .39Impairment of the frontal cortex may also be included, but this is associated withlater-stage AD and may not occur initially .36 PET imaging also demonstrates that certain areas of thebrain that have been spared impairment in AD, especially the basal ganglia,thalamus, cerebellum, and cortex.40 Mosconi et al. suggests that AD-related processes may affectthe entorhinal cortex and other regions of the brain, which may facilitatefunctional impairment.41Herholz et al. concluded that hemispheric asymmetry might be present, which could beresponsible for language and visual impairment.40 Therefore, in addition to showing key characteristics ofAD-caused neurological damage, FDG PET has the ability to map the progressivecognitive decline of AD. FDG PET reveals that as AD progresses, parietotemporalhypometabolism becomes increasingly bilateral in addition to the frontal cortexbecoming more hypometabolic.34Comorbid conditions can affect the specificity of these predictions. Depressedpatients and individuals with abnormal thyroid function have a higher false positiverate for being expected to experience progressing cognitive decline.The initial degree of hypometabolism determined by PET has been shown to correlatewith the magnitude of future decline.In addition to FDG PET's ability to develop image-based diagnostic criteria for AD,it also has the ability to distinguish AD from similar neurodegenerative conditions.AD and other types of dementia have a characteristic pattern of FDG PET imagingwhich can be used to differentiate diagnosis in early stage when the specific typeremain unclear. FTD, which is often misdiagnosed as AD in its early stages, ischaracterized by behavioral and language disturbance. Therefore, it could bedifficult to distinguish from early AD symptoms in a clinical setting. However, thatdistinction is easier with FDG PET since reduced regional glucose uptake is seen infrontal and anterior portion of temporal lobes in FTD while that metabolic deficitis seen more in the posterior areas of the brain in AD .39 Other similar conditions, Dementiawith Lewy Bodies (DLB), FDG PET shows reduced metabolism in parieto-occipital areaslike the primary visual cortex and occipital association areas with normal glucoseuptake at association temporal and posterior cingulate cortex, whereas occipitalcortex is preserved in AD from a negative PET scan.Alzheimer's Disease Neuroimaging Initiative (ADNI) and post-mortem studiesdemonstrated evidence for the power of FDG PET as a biomarker for AD. From reviewsliterature,45 by assessment the brain in ADpatients and age-matched controls. They found that by multiplying segmented brainvolumes from MRI by mean cerebral metabolic rates for glucose, significantdifferences between two groups can be demonstrated. This approach requirescalculating tissue volume by utilizing modern computer based algorithms and partialvolume corrected measurement of metabolic activities at each site of interest. Bythe same concept, other study by Alavi et al.46 investigated 20 patients with probable AD and 17 similarage controls who underwent FDG PET and MRI. They found that atrophy-weighted totalbrain metabolism  showed a very significant difference between two groups . Absolute wholebrain metabolism  also showed significant difference which were 37.24 ± 9.65 inAD and 45.09 ± 8.52 in controls, p <0.014). These measurements correlatedwith mini-mental status exam (MMSE) score. Recent studies carries out by Musiekfound that whole brain metabolic volumetrix product (MVP) were significantly lowerin AD and accurately distinguished AD patients from controls.47Global disease assessment enhances the accuracy of measurement of FDG PET imaging.The principle of the global metabolic activity is based on multiplying partialvolume corrected average SUV to the volume of the organ of interest obtained fromanatomical modalities (CT/MRI), the result of multiplying can be named as metabolicvolumetric product (MVP). It was first introduced by Alavi et al.Limitation. Some of the limitations include the creation of artifactsand noise during FDG PET image construction, the disadvantages and potential sourcesof error in both qualitative and quantitative analysis techniques, and disadvantagesin semi-quantitative methods. FDG PET imaging can also be affected by pre-existingpatient conditions or errors made in protocol during the scanning process.41 especially in atrophic brain of elderly subjects or ADpatients. PVE can also be caused by an incorrect superposition of voxel parametersonto brain tissue causing voxels to contain different tissue types, or tissuefraction. Additionally, patient motion or the movement of either the circulatory orrespiratory systems can generate PVE.48 Because analysis PET images are dependent on measurements ofmetabolic activity, and because differential patterns of glucose uptake serve asimportant characteristics for neurological conditions, it is important that PVE becorrected in order to prevent misdiagnosis or images that show no evidence ofabnormality for cases where abnormalities are truly present. Currently, there exista variety of methods for partial volume correction (PVC), which seeks to curb theproblems caused by PVE.Another notable limitation, which has been studied extensively, is partial volumeerror (PVE). Incorrect measurements of tissue activity are due to the limitations ofscanners to process structures smaller than 2-3 times the full-width-at-half-maximumspatial resolution of the scanner,48Methods to reduce PVE can include techniques which utilize anatomical information tocorrect individual voxels, specific regions of interest (multiple or single), orwhole images. Other techniques include post-construction methods, using projectiondata to obtain region of interest (ROI) mean values, or methods that allow for agradient of activity levels within each region to correct for the assumption thatactivity within each region is uniform. Techniques to address tissue fraction havealso been developed, including methods where edge voxels are treated as multipletissue types.49Mosconi et al. notes the relative lack of studies used to examine individual cases ofMCI, which may be preventing a more detailed understanding of MCI features on anindividual level, as well as the dearth of studies which compare MCI to disordersother than AD.The first amyloid-β (Aβ) PET exam in human was introduced in anindividual with probable AD using the 11C-labeled radiopharmaceutical PittsburghCompound B (PiB). Amyloid imaging was repeatedly claimed that it is very sensitivetechnique for the in vivo identification of amyloid plaques into the brain tissue,non invasively, therefore allowing an early confirmation of AD. The normal patternof amyloid imaging is the white matter deposition of PIB compound, with no corticaluptake .54 In AD group, the highest tracerbinding is observed at prefrontal cortex, precuneus and posterior cingulate,followed by lateral parietal cortex, temporal cortex and striatum -2-naphthylethylidene)malononitrile (DDNP), as a PET tracer to track the deposit sites of neurofibrillarytangles (NFTs) and Aβ senile plaques in the living AD patients. 18F FDDNPhave been postulated to recognize amyloid plaque as well as NFTs in living human.Moreover, it is the only imaging agent which visualizes AD pathology in hippocampalregion in vivo. 18F FDDNP accumulates significantly in several cortical areas ofpatients with AD.56 Small etal.57 reportedsignificantly lower values of FDDNP-PET binding in the whole brain in control groupcompared to the MCI group as well as lower values in MCI group compared to AD.The other cortical regions including the hippocampal and amygdala did not show anyremarkable PiB uptake compared to controls. Subcortical WM, pons and cerebellumwhich are unaffected by amyloid deposition showed low PiB binding. At the mean timethat PiB was developed, Shoghi-Jadid et al.58 used 18F Florbetapirto predict the presence of Aβ in the brain at autopsy. A good correlation wasobtained between the visual interpretation of 18F Florbetapir PET imaging and theautopsy findings that confirmed the deposition of Aβ in the brain tissue,according to the standard pathological criteria to define AD. A very high rate ofagreement (96%) was seen between amyloid PET imaging and histological confirmationof Aβ. Another study in correlation of 18F Florbetapir and postmortemhistopathology was performed by Choi et al.59 There was very good correlation of Aβ plaquesidentified by specific pathological staining techniques, including silver stainingand special immunohistochemical assays, and Florbetapir PET imaging pattern.Fleisher et al.60 brought 18FFlorbetapir PET to clinical cohort of 210 subjects including probable AD, mild MCIand older healthy controls. The data were pooled from four phase I and II clinicaltrials that used 18F Florbetapir PET imaging under similar protocols. The authorsreported that mean (SD) cortical-to-whole-cerebellar SUVRs were significantlydistinct among the 3 groups and in the expected direction: 1.39 (0.24) for theprobable AD group, 1.17 (0.27) for the MCI group, and 1.05 (0.16) for the controlsgroup (P=2.9x10−14). There also found significant difference of percentage meetinglevels of amyloid associated with AD by SUVR criteria  and percentage meeting SUVr criteria for the presence of any identifiableAβ (SUVrs greater than 1.08) among three groups. There was also a strong anddirect correlation of florbetapir cortical retention with aging and the presence ofAPOE ε4 allele (p=0.048).Recently three new, longer-lived 18F tracers including 18F florbetapir, 18Fflorbetaben and 18F flutemetamol have been brought to research and clinical use. In2012, the Food and Drug Administration (FDA) approved the clinical use of Aβprobe AmyvidTM (18F Florbetapir) for evaluation of patients suspected AD. Clark etal.61 Phase IIstudy62 proposedsensitivity of 80% and specificity of 91% for discriminating individuals withprobable AD form age-matched controls. Phase III studies in 238 patients from 17centers have reached completed.63The investigators claimed 100% sensitivity and 92% specificity of 18F florbetabenPET at subject level analysis but 77% sensitivity and 94% specificity for regionaldetected Aβ as compare with postmortem diagnosis. Ong et al.64 found high Aβ burden in 53%of MCI subjects when using SUVr 1.45 as a threshold. There is a good directcorrelation between 18F florbetaben and PiB global SUBr values with almost samediagnostic power to differentiate AD from healthy subjects.6518F florbetaben has also been shown to bind with Aβ in brain and selectivelylabeled Aβ plaques and cerebral amyloid angiopathy (CAA) in ADtissue.66 Duara etal.67 suggested anadditive information from 18F flutemetamol PET and sMRI in classifying amnestic MCIsubjects. The overall correct classification rate for amnestic MCI from 18Fflutemetamol PET using SUVr 1.4 and medial temporal atrophy derived from sMRI was86%. Longitudinal study68 in ADand amnestic MCI with 2-year follow-up reported 18F flutemetamol PET SUVr showedclear group clustering while hippocampal volume showed extensive overlap betweengroup. A longitudinal study showed that more that 89% of the converters came fromthe positive flutemetamol group. Pooled results of phase III studies in 18Fflutemetamol have not been announced yet.18F flutemetamol PET with visual assessment has been reported 93.1% sensitivity and93.3% specificity against standard of truth among AD, MCI and healthy controlssubjects.69 reviewed recentpublications in clinical dementia setting and reported 96% of AD patients wereamyloid positive. On the other hand, amyloid-negative scans in patients with thediagnosis of probable AD would represent imprecise clinical diagnosis or thatpatients bear very small amount of tissue amyloid plaques that PET could not detect,and by following them up it will be detected years ahead.Johnson et al.70 reviewed the technicalaspects and described several potential problems, such as partial volume effectsresulting in underestimated SUV data, high ratio of nonspecific to specific WMuptake and discordance between the concentration of Aβ in the brain withhistopathological and immunohistochemical studies and question about the specificityof these tracers. Investigators in amyloid imaging field have answered someMoghbel's questions,71 however,some issues still need to be clarified. Kepe et al.72 proposed the lack of in vivo binding validation ofthese probes and the consequent deficiency in the understanding of their tissuebinding and specificity. It is uncertain how amyloid agents interact with many formof Aβ. Lockhart et al.73demonstrated that PiB clearly delineated classical plaque as well as diffuse plaqueand CCA. It was also found to label NFTs with lower intensity than Aβpathology. Cairns et al.74reported case diagnosed mild AD whose PiB PET showed unremarkable but positivebiofluid markers. However, autopsy performed 2.5 years after scan showed lesionsthat met neurofibrillary stage III and Braak and Braak stage C. There was noevidence of any other neurodegenerative or clinically meaningful vascular disease.Aβ deposition is also an important pathology in Downs's syndrome. Inaddition, Aβ has been reported as an additional pathology in Parkinson'sdisease, dementia with Lewy Bodies, Pick's disease, corticobasal degeneration,amyotrophic lateral sclerosis and progressive supranuclear palsy.75 Ly et al.76 found nearly most ischemic strokepatients in their study has a high PiB uptake within the peri-infarct regioncompared to the contralateral side, particularly in the WM around the infarctregion. The cause of the focal PiB retention was uncertain and requires furtherinvestigation. There are also evidence that suggests even cognitively-normalpatients may have high levels of 11C-PIB, ligand used to detect Aβ,suggesting that a large degree of Aβ buildup may not always translate intothe development of AD symptoms. Healthy elderly controls can also show high PIBretention.78 Some PIB positive elderly healthycontrols have demonstrated normal cognition.79 Moreover, it is common to see numerous degenerative changesincluding NFTs and Aβ plaque in a large number of cognitively normalindividuals.80Although a number of new PET probes are currently under investigation in academia andunder development by pharma companies, there are some concerns with respect to theclinical value of Aβ imaging and questions have been recently raised. Moghbelet al.72 Many studiesfound non-negligible WM uptake in both AD and controls.81 Recent study by Barthel62 reported the highest 18Fflorbetaben SUVr in cerebral WM as compared to other cortical and subcorticalregions. Nonspecific WM uptake can produce spillover and partial volume effect intoneighboring GM which should be concerned in atrophic AD brain. The extensive WMuptake can make unreliable imaging interpretation. Moreover, this phenomenon provideadditional evidence that PiB and stilbene derivatives are nonspecific to Aβtarget as some studies showed these probes can bind to myelin with highaffinity.84 Villemagne et al.71 have addressed that this WM uptakeis similar between AD and normal controls and that partial volume effect is not anexclusive limitation to amyloid PET imaging but affects equally all PET imageprocedures. They claimed even by knowing that this limitation had not proven to be amajor obstacle to the quantitative analysis of Aβ deposits in cortical GM,visual assessment was of higher priority than absolute quantification andlocalization for many clinical purposes. However, the authors accepted that a lot ofimprovements must be accomplished regarding the development of more sensitive andspecific probes, with lesser WM concentration, and that will allow the incorporationof more suitable imaging tools to quantify and better classify patients withcognitive impairment.Additionally, the rapid peripheral and central metabolism of these probes and thebrain transport of metabolites are severe limitations at the very heart of thetracer design and development. These limitations cause extensive and nonspecificuptake of amyloid agents in WM which affects both AD patients andcontrols.In present days, it well recognized that Aβ deposition starts in thepreclinical AD, increases up to the time when the AD diagnosis is confirmedclinically, and then remains under a plateau as disease progresses. Cerebralamyloidosis itself is not sufficient to promote cognitive deficits in AD which ismore related with FDG PET and sMRI as the biomarker of neurodegeneration. Anti-Aβ therapies have been repeatedly reported to be ineffective. Thus, there isno validated clinical value of amyloid imaging in monitoring diseaseprogression.85 It is suitable for individuals with stable orprogressive unexplained MCI, satisfying core clinical presentation either anatypical clinical course or an etiologically mixed presentation and progressivedementia, and atypically early age of onset. Patients with one of these appropriatecriteria should have the following characteristics:Considering the limitations discussed above, the amyloid imaging demands carefuldiscussions in the proper clinical utility. Recently, the Society of NuclearMedicine and Molecular Imaging (SNMMI) and the Alzheimer's Association (AA) havedeveloped the appropriate use criteria for amyloid PET.1) a cognitive deficit confirmed by an objective neuropsychologicaltest;2) A diagnosis of possible AD, but when the diagnosis is uncertain aftera comprehensive evaluation by a dementia expert; and3) when the recognition of the pathological status of Aβ isexpected to increase diagnostic certainty and change management. Theinappropriate situations include patient that fulfill the diagnosticcriteria for probable AD under typical age of onset, to determine thelevel of cognitive impairment, based solely on a positive family historyof dementia or APOE ε4 presentation, unconfirmed clinicalexamination of cognitive impairment, suspected autosomal mutationcarriers, asymptomatic individuals and nonmedical use such as legal,insurance coverage, or employment screening.86 Then in July 2013, the Centers forMedicare and Medicaid Services (CMS) released a draft decision memo indicating thatMedicare would pay for contrast-enhanced PET scans aimed at visualizing beta-amyloidprotein plaques in patients brains only in the contest of rigorous clinical trials,under the agency's "coverage with evidence development" (CED) policy. The decisionmainly focuses on the role of positive scan, while the guideline of SNMMI and AAconsiders both on positive and negative findings which negative finding would ruleout an AD. CMS reported that use of the scans to exclude AD in narrowly defined andclinically difficult differential diagnoses is promising. Nevertheless, CMSacknowledged that more evidences need to be discovered, including when the scanwould replace or complement other biomarker for particular patientsubpopulations.However, there is a lot of skepticism regarding the value of amyloid imaging tosignificantly change outcomes and management of patients with prodromal and even AD.The main issue is that Aβ PET findings are not specific to AD and about 30%of older people have Aβ and do not have AD and will not have AD.Limitation. Amyloid imaging tracers do not meet the fundamentaladvantage of PET that is different from other imaging modalities as the ability ofquantitative functional assessment of specific tissue in human. Appropriate amyloidPET probe should provide the signal only from Aβ retention and its peripheralmetabolites should be minimize or pass blood-brain-barrier that can be predicted forquantification. For recent evidence, the in vivo specificity of the amyloid agentshas not been fully established and the sources of non-specific uptake have not beenidentified. Moreover, the technical limitation in PET system has not beencorrected.87 uses 3 parameters including (A)immunohistochemistry-derived Aβ plaque score described by Thal etal.,88 (B) NFTs stage fromimmunohistochemistry for tau or phosphor-tau, and (C) neuritic plaque score fromThioflavin S or modified Bielschowsky as recommended by Consortium to Establish aRegistry for Alzheimer's disease (CERAD) protocol to obtain "ABC" score andtransform into one of four levels of AD neuropathologic change: Not, Low,Intermediate or High. For Aβ plaque score, other method that identifiesprogressive accumulation of Aβ deposition in medial temporal lobe only isrecommended as it is highly correlated with Thal phases.88 Present status of amyloid imaging may provideinformation of neuritic plaque that fulfills only criteria (C), however, it cannotyield appropriate signal in the medial temporal lobe and insensitive to taudeposition. Thus, amyloid imaging shows no enough strong evidence that it issuitable for AD diagnosis which is the most indication that described inliteratures.Even though all limitations are not considered, the diagnostic value of amyloidimaging is still questionable. Current criteria for the neuropathological diagnosisof AD by National Institutes of Aging-Alzheimer's Association


Nuclear protein in testis carcinoma is a rare and very aggressive undifferentiated cancer which characteristically arises in the midline of the head, neck, and mediastinum.We describe the case of a 46-year-old white woman admitted for superior vena cava syndrome revealing a mediastinal tumor. Pathological examination of specimens obtained by mediastinoscopy revealed an undifferentiated tumor with solid growth and positive immunoreactivity for p40 and negative immunoreactivity for cytokeratin markers. Immunohistochemical staining was positive for nuclear protein in testis, allowing the diagnosis of nuclear protein in testis midline carcinoma of the mediastinum.We present a rare case of mediastinal nuclear protein in testis carcinoma with diagnosis based on nuclear protein in testis protein positivity and atypical immunohistochemical features including p40 positivity and anti-cytokeratin negativity. Physicians must remain aware of the possibility of nuclear protein in testis carcinoma especially in young patients with thoracic symptoms and suspicion of neoplasm. NUT gene on chromosome 15q14 with members of the BRD gene family (BRD4 and BRD3) resulting in a BRD–NUT fusion product, which decreases histone acetylation and therefore suppresses squamous cell differentiation [Nuclear protein in testis (NUT) carcinoma is a rare and very aggressive undifferentiated cancer, which characteristically arises in the midline of the head, neck, and mediastinum  and showntiation . We repoA 46-year-old white woman with no medical history presented to our hospital with complaints of dyspnea, chest pain, dysphagia, cyanosis of the trunk and head, and distended superficial veins over her neck and chest of 3 weeks’ duration; these were all features of superior vena cava obstruction. She had no history of tobacco smoking, alcohol consumption, or illicit substance use. A physical examination showed decreased breathing sounds and dull percussion on the lower lobe of her right lung as well as diffuse wheezing of her right hemithorax. Laboratory tests did not reveal anemia, hydroelectrolytic, or coagulation disorders. A chest X-ray revealed widening of anterosuperior mediastinum Fig. . A contrAfter multidisciplinary cancer team meeting discussions, our patient underwent a course of radiation therapy to her chest (20 Gy in five fractions) and received three cycles of carboplatin and paclitaxel. Her symptoms of vena cava syndrome improved and a chest CT scan showed that the size of the mediastinal mass had decreased by 32%: a partial response according to Response Evaluation Criteria in Solid Tumors (RECIST) 1.1 criteria . After tNUT carcinoma is a rare carcinoma with only 39 cases of intrathoracic tumors published to date . ChronicHistological findings typically show two types of tumor cell populations: (1) poorly differentiated carcinoma, and (2) well-differentiated squamous cell islands with focal keratinization , 6. PosiIn our case, immunohistochemistry revealed positivity for p40 and negativity for CK. To the best of our knowledge, our patient is the third reported case of intrathoracic NUT carcinoma with positivity for p40 .in situ hybridization (FISH) analysis allows the diagnosis of NUT carcinoma with 100% specificity [BRD4–NUT and BRD3–NUT, and more rarely NSD3–NUT, is not mandatory for the diagnosis, but is recommended for its possible association with unique prognostic features [BRD4 fusion gene rearrangements are more differentiated and therefore possibly less aggressive [Nuclear positivity of more than 50% for anti-NUT antibody with fluorescence cificity . Charactfeatures . NUT carBRD4–NUT fusion NUT carcinoma [At present, there is no consensus concerning the optimal treatment strategy. A combination of multidrug chemotherapy with gemcitabine, docetaxel, and cisplatin and locoregional radiation therapy achieved complete response in a case of NUT midline carcinoma . Adminisarcinoma .We present a rare case of mediastinal NUT carcinoma revealed by superior vena cava obstruction with a definitive diagnosis based on NUT protein positivity and atypical immunohistochemical features including p40 positivity and anti-CK negativity.


Diets high in advanced glycation end products (AGEs) are thought to be detrimental to cardiovascular health. However, there remains uncertainty about the beneficial effect of a low AGE diet on cardiovascular risk factors and inflammatory markers in overweight individuals. We thus performed a randomised, double blind, crossover trial to determine whether consumption of low AGE diets reduce inflammation and cardiovascular risks in overweight and obese otherwise healthy adults. All participants (n = 20) consumed low and high AGE diets alternately for two weeks and separated by a four week washout period. Low AGE diets did not change systolic (p = 0.2) and diastolic blood pressure (p = 0.3), mean arterial pressure (p = 0.8) and pulse pressure (p = 0.2) compared to high AGE diets. Change in total cholesterol (p = 0.3), low-density lipoprotein (p = 0.7), high-density lipoprotein (p = 0.2), and triglycerides (p = 0.4) also did not differ and there was no difference in inflammatory markers: interleukin-6 (p = 0.6), monocyte chemoattractant protein-1 (p = 0.9), tumour necrosis factor α (p = 0.2), C-reactive protein (p = 0.6) and nuclear factor kappa beta (p = 0.2). These findings indicate that consumption of low AGE diets for two weeks did not improve the inflammatory and cardiovascular profiles of overweight and obese adults. Consumption of foods high in sugar and/or foods exposed to high temperature cooking methods such as deep-frying, broiling, roasting, baking and grilling can increase the total daily AGE intake by 25% compared to the average adult daily intake3. High AGE intake from food accelerates the production of endogenous AGEs and increases the level of circulating AGEs in the blood stream5. This intake of high AGE contributes to the progression of type 2 diabetes (T2DM) and cardiovascular diseases (CVD) although the exact mechanisms are not clearly understood11. AGE formation can be reduced by shortening the cooking time and lowering temperature to reduce food browning, or by addition of acidic (low pH) ingredients and by high humidity or food moisture content12. Given the ease of such changes, reduction of AGE intake may be a promising intervention to lower cardiovascular risk15.Advanced glycation end products (AGEs) are formed endogenously from non-enzymatic reactions of amino acids with sugars17 but did not change plasma lipid levels18 or blood pressure19. Whereas, other studies showed a reduction in plasma lipid levels after low AGE diets21 but not of markers of inflammation22. In patients with T2DM, low AGE diets improved inflammatory markers9, but not blood pressure9, and lipid levels9. Cai and colleagues however reported a reduction in oxidised low-density lipoprotein after 6 weeks of consumption of low AGE diets (5 fold lower in AGE content) in patients with T2DM23. In all these studies, test and control diets were either not matched for energy and macronutrient content or it was not stated whether similar macronutrient content was achieved between the diets, which could potentially influence the results24. We have therefore investigated the impact of low and high AGE diets carefully matched for both energy and macronutrient profile. These matched diets were given for two weeks each to healthy obese adults in a randomised cross-over design trial to determine effects on cardiometabolic parameters. We have previously reported the main outcomes of the trial which showed that dietary AGEs improved insulin sensitivity but not insulin secretion in overweight and obese non-diabetic individuals24. In this study, we conducted analyses of secondary outcomes of the trial and investigated if dietary AGEs improve blood pressure, plasma lipid profiles and inflammatory markers.In humans, the impact of low AGE diets on inflammation and cardiovascular risk factors is not clearly understood. In some studies involving healthy obese individuals, low AGE diets reduced inflammatory markers24, the mean age, body mass index, waist-to-hip ratio and % body fat of the participants were 34 (10) years, 31.3 (3.8) kg/m2, 0.9 (0.1) and 31.1 (6.7), respectively. There was no difference in levels of circulating AGEs and metabolic profiles such as insulin sensitivity and secretion between group 1 and 2 before each test diet24. Anthropometric and blood pressure measurements, plasma lipid levels and inflammatory markers were also not different between the two interventions at the beginning of each dietary period , C-reactive protein (CRP), monocyte chemoattractant protein 1 (MCP-1) and interleukin 6 (IL-6) levels and nuclear factor kappa beta (NFκβ) activity in peripheral blood mononuclear cells (PBMCs) did not change after consumption of either low or high AGE diets. Between groups comparison also showed that the changes in these markers were not significantly different between low and high AGE diets Table .Table 2EAnthropometric measures such as weight and body mass index were significantly reduced only in the low AGE group compared to high AGE group. Percentage body fat and waist-to-hip ratio did not change after consumption of either diet. Between groups comparison have shown that none of these anthropometric measures were different between the diets Table . SystoliThe total plasma cholesterol levels decreased in both groups, however, the change between the two diets was not significant. Both plasma low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol levels decreased with low AGE diet but not changed with high AGE diet whereas the overall difference between the diets was not significant Table . Plasma The correlation analyses indicated that the change in diastolic blood pressure and MCP-1 is positively correlated with % body fat  and waist-to-hip ratio , respectively. Change in systolic blood pressure is also positively correlated with NFκβ  whereas plasma lipid levels negatively correlate with CRP  and IL-6 . After adjusting either for change in weight, body mass index, % body fat or waist-to-hip ratio, neither systolic blood pressure nor lipid parameters correlated with inflammatory markers. Other inflammation parameters did not show any correlation with cardiovascular parameters . Circulating and urinary AGE levels also did not correlate with any cardiovascular parameters .19, prediabetes21, obese people with metabolic syndrome26, and patients with T2DM9.The present study aimed to measure the effect of consuming low AGE diets for 2 weeks on markers of inflammation and cardiovascular risk factors in healthy obese and overweight individuals. No significant difference was found in changes in blood pressure, plasma lipid levels  or inflammatory markers  between low and high AGE diets. Our findings on blood pressure measurements were consistent with other studies conducted in healthy overweight individuals18, obese individuals with metabolic syndrome26, and patients with T2DM9 have also reported that consumption of low AGE diets did not change plasma lipid levels compared to high AGE diets. In contrast, two other studies that involved young healthy obese volunteers20 and prediabetic individuals21 noted a reduction in plasma lipid levels after a low AGE diet. In the first study, the difference between the 2 diets could have been due to the higher intake of dietary fat and carbohydrate on the high AGE diets20. The patients in the second trial in the low AGE arm of the study had relatively lower intima-media thickness compared to those who were in the high AGE diet21. This may exaggerated the effect of high AGE diets on lipid profile. In addition, consumption of low AGE diets combined with aerobic exercise program for 12 weeks showed a decrease in plasma lipid levels in overweight and obese men19. This effect however could have been due to the synergistic effect of physical activity with low AGE diets as it has been widely known for its beneficial effect on metabolic changes27. Therefore, these studies21 were not able to differentiate between the true effect of low and high AGE diet as they were confounded by other differences in diet, exercise or disease state of the patients.Regarding plasma lipid profile, we did not find a significant difference between low and high AGE diets. Some studies conducted in healthy individuals22. Other studies conducted in healthy individuals showed an improvement in inflammatory markers after intake of low AGE diets17. However, in these studies, the diets were not matched for macronutrient content, which might have influenced the findings. Studies in obese people with metabolic syndrome26, prediabetes21 and patients with T2DM9 showed an improvement in inflammatory markers after intake of low AGE diets compared to high AGE diets. Therefore, it could be suggested that intake of low AGE diets was beneficial for those who had higher baseline values in inflammatory markers such as patients with metabolic syndrome or T2DM.In this study, intake of low AGE diets for 2 weeks did not change inflammatory markers. This finding was consistent with some studies conducted in healthy overweight and obese individualsCompared to the previous studies, our study has several strengths that include standardised diets in energy and macronutrient content were provided, study personnel including the investigators were unaware of the dietary allocation, and baseline characteristics such as anthropometric, circulating AGEs and metabolic parameters were not different at baseline during each dietary period. We have also used a crossover design to control for possible confounding factors in addition to the rigorous randomisation process. The small sample size and relatively short duration could be considered as main limitations of this study as one can argue that the change in physiologic processes after intake of low AGE diets may require longer duration.In conclusion, we have shown that consumption of low AGE diets for 2 weeks did not have beneficial effects in reducing markers of inflammation and cardiovascular risk factors in healthy overweight and obese adults. Long-term well designed trials with larger sample sizes are needed to confirm our findings.24, according to a protocol as previously published elsewhere28. Participants did not have diabetes as indicated by a 75 g oral glucose tolerance test (OGTT) (WHO 1999 criteria). All were non-smokers and had no clinical and laboratory signs of infection and none took supplements/medications during the study period. The participants were recruited from the general community through advertisements between January 2006 and December 2010. Ethical approval was obtained from the Alfred Hospital Ethics Committee, Melbourne, Australia and complied with the Declaration of Helsinki. All participants provided written informed consent prior to participation.Twenty overweight and obese but otherwise healthy and normoglycemic adults, aged 18–50 years completed the study24.This study employed a two-period randomised cross-over double-blind design. All participants underwent both diets; one low in AGE content and one high in AGE content  and diets were matched in energy and macronutrient content. Participants commenced the study after a two-week run-in on their habitual diet but with limitation of their alcohol, fast food and coffee intake. Test diets were consumed for two weeks separated by a four-week washout period . The primary outcomes of this study that focused on insulin resistance/sensitivity have been publishedRandomisation occurred for 7 blocks of 4 participants stratified by gender and diet order. Participants were masked to the allocation of diet type and to how the diet might affect glucose metabolism. Clinical and laboratory investigators were also masked to diet allocation.All participants underwent medical and laboratory screening including a 75 g oral glucose tolerance test (OGTT). Prior to metabolic testing, participants were asked to abstain from strenuous exercise and caffeine for 3 days.4 and use of differently processed foods. All food for the two test diets was provided weekly to the participants as ready-to-eat items or as packed food portions, to assist with dietary compliance. For food that required cooking, detailed instructions for storage and heating  were provided. Participants were asked to eat to appetite throughout both dietary periods to maintain constant body weight. They were required to keep a detailed dietary record indicating cooking method and number of portions eaten for each food item supplied, unconsumed foods or additional foods eaten. Later foods incorporated in these diets were also chemically analysed for their AGE content. The dietician made telephone contact twice a week to provide support and resolve difficulties as well as to ensure dietary compliance. To analyse the dietary AGE content, food items were obtained from local supermarkets and prepared according to the instructions provided to the participants in the study.Prior to starting the ‘run-in’ to the first diet, participants kept a 3-day diet record of their habitual diet  based on household measures. Nutrient content was analysed with SERVE , based on Australian food composition tables. With the use of Australian food composition data from SERVE as well as data from the United States on the N (ε)-carboxymethyllysine  content of common foods, a menu of carefully matched alternative food choices (of low AGE vs high AGE content) individualised to suit the preferences and habitual diet of each participant was designed. These alternative food choices were matched for macronutrient content and total energy but greatly differed in calculated AGE content, and were provided for each meal of the day, including snacks and beverages. Food choices on the high AGE diet had higher dietary AGE content, while the isoenergetic low AGE diet, matched for macronutrient content, had reduced AGE content through altered cooking techniques29. Plasma lipid levels such as total cholesterol, LDL, HDL and triglycerides were measured using a standard commercial enzymatic assay  and Systems Lipid and Multi Calibrators (Beckman Coulter Diagnostics).Systolic and diastolic blood pressure were measured using an automated oscillometric measurement system (Omron) after a 30 minute rest. Pulse pressure was calculated by subtracting the diastolic blood pressure from the systolic blood pressure. Mean arterial pressure was computed as diastolic blood pressure + pulse pressure/330.Markers of inflammation  were analysed using a commercial automated chemiluminescent enzyme immunoassay and immulite analyser (Diagnostic Products Corporation). Whereas, highly sensitive near infrared particle immunoassay rate methodology was used to measure plasma CRP levels. NFκβ activity in PBMCs was detected and quantified using TransAM NF- κβ DNA-binding activity assay . Measurement of urinary and serum AGEs were quantified with the use of ultraperformance liquid chromatography–tandem mass spectrometry as previously describedChange from baseline values was calculated for each parameter in both dietary periods. Paired t-tests were used to compare the participants’ characteristics at the beginning of each dietary period and to determine the change in parameters after each study diet. Repeated measures ANOVA was employed to determine the change difference between the two diets after adjusting for carryover, sequence and period effects. The observed carryover effect in this study was very minimal (29.8%) and thus the data in both study periods were combined. Results are presented using means and standard deviations unless and otherwise stated. Appropriate data transformation was undertaken in the event of skewed data to approximate a normal distribution. Statistical analyses were performed using Stata 14 software. P-value of ≤ 0.05 is used to describe statistical significant difference.The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.


Class C G protein-coupled receptors (GPCRs) regulate important physiological functions and allosteric modulators binding to the transmembrane domain constitute an attractive and, due to a lack of structural insight, a virtually unexplored potential for therapeutics and the food industry. Combining pharmacological site-directed mutagenesis data with the recent class C GPCR experimental structures will provide a foundation for rational design of new therapeutics.We uncover one common site for both positive and negative modulators with different amino acid layouts that can be utilized to obtain selectivity. Additionally, we show a large potential for structure-based modulator design, especially for four orphan receptors with high similarity to the crystal structures.http://gpcrdb.org/mutations/ and can be analyzed online or downloaded in excel format.All collated mutagenesis data is available in the GPCRdb mutation browser at Bioinformatics online. This alThe binding sites of class C GPCR AMs have been extensively studied using site-directed mutagenesis and receptor structure models , but theB2 , GPRC6, mGlu1, mGlu2, mGlu4, mGlu5 and TAS1R3 receptors. Residue positions, numbered according to the GPCRdb generic scheme  and mGlu1 (PDB code 4OR2) crystal structures were downloaded from the Protein Data Bank (www.rcsb.org) and the PyMOL Molecular Graphics System, version 1.8, Schrödinger, LLC was used to prepare The mGluB receptors. This confirms that the established receptor families can indeed serve as a basis to map allosteric data, and as target profiles for ligand selectivity and inference.Class C GPCRs are classified into families by endogenous ligand , which bAn overall TMD allosteric site in the class C GPCRs can be delineated from the mGlu structures and the collated mutagenesis data. In total, 28 residue positions line the TMD pocket and together represent the overall potential AM contacts . NotablyThe second extracellular loop of GPCRs typically forms a disulphide bridge to the top of TM3, placing the loop as a lid on the TMD pocket ; indicat5 receptor structures contain a unique site extension located between TM2, 3 and 7 . Together, this indicates that these subtype specific Gly residues are associated with distinct receptor structures, such as a helical kink or rotation implicating additional class C GPCRs with a Gly at 4x42 (GPR179) or 7x43 (CaS and GPRC6 receptors)  . It woulThe remaining mutated positions in 1 and mGlu5 structures could be valid templates also for the design of PAMs. However, to avoid bias toward NAM activity, it is advisable to first optimize the structural templates around a high-affinity PAM, which has previously been successful in GPCR virtual screening (5 (The available structural templates, which are all NAM-complexes, map as well to the PAM as the NAM mutagenesis data . This isening (5  receptorAn advantage of allosteric ligands is their ability to achieve higher receptor target selectivity. As shown in Modelling of class C GPCR-AM complexes has become more accessible with the possibility to utilize the mGlu structures in combination with our collation of literature mutations, which are available for visualization and download in GPCRdb. We previously demonstrated that such data was sufficient to build conclusive models explaining mGlu NAM selectivity . This isIn conclusion, the accumulated mutagenesis data for AMs on class C GPCRs almost exclusively maps to one common inter-helical allosteric site for both PAMs and NAMs, which corresponds very well with the allosteric site in the mGlu crystal structures. The combined mGlu-NAM structure complexes, mutagenesis data, sequence comparisons and known NAMs/PAMs lays a stronger foundation for rational design of novel modulators through the construction of high quality homology models. Specifically, structures, mutagenesis data and sequence comparisons have been combined in GPCRdb to create an online tool to design new mutagenesis experiments (Supplementary DataClick here for additional data file.