**6. Timing and other influencing factors of biomarker use**

human diseases such as diabetes, obesity, atherosclerosis, and AD (Wenk et al., 2005; Watson., 2006; Steinberg,. 2005; Sato et al., 2010). In support of the hypothesis that lipid dysfunction plays an important role in AD pathogenesis, previous studies with post-mortem brain tissue samples have demonstrated altered lipidomes at the different stage of AD pathogenesis. For example, multiple classes of sphingolipids are altered not only at the late stage of the disease but also at the earliest clinically recognizable stage of AD. All major classes of phospholipids are ubiquitously decreased at the late stage of AD. Among these, the levels of plasmalogen (a major component in nerve tissue membranes counting for up to 85% of ethanolamine glycer‐ ophospholipid, or ∼30% of total phospholipids of these membranes) are gradually reduced as progress of AD severity (Han et al., 2011). Sato et al (2011) established a lipidomics method for comprehensive phospholipids evaluation that identified 31 phospholipids as AD biomarker candidates in human plasma using LC/MS (Satoet al., 2010). Moreover, additional studies have suggested that AD associates with other lipid metabolism pathways and lipid carrier proteins such as apoE (Bertram et al., 2008; Corder et al., 1993; Farrer et al., 1997; Strittmatter et al., 1993). A very recent study by Sato et al (2011) were able to find a biomarker desmosterol that changes in AD compared with plasma from healthy elderly controls. They have shown that desmosterol plasma level and the desmosterol/cholesterol ratio in the same patients was significantly decreased. This study is the first report that plasma desmosterol levels are decreased in AD and MCI. And future studies are needed to confirm whether desmosterol could become an attractive plasma AD biomarker that could perhaps also be utilized for diagnosis and as well as for monitoring noninvasively the effect of future AD drugs on disease progression.

MicroRNAs (miRNAs) are a class of small, endogenous, noncoding RNA molecules that serve as posttranscriptional regulators of gene expression (Lee etal.,1993;Giannakakis etal.,2007). miRNAs are acquiring important and determinant roles in the regulation of brain gene transcription in health and disease: the fact that approximately 80% of the human brain genome is transcribed into RNA, but only about 2% of the genome is transcribed into protein, under‐ scores the potential of various levels of RNA signaling and epigenetic mechanisms to contrib‐ ute to physiological gene control (Makeyevetal., 2008). In the last few years, miRNAs have been emerging as important regulators of various aspects of neuronal development and dysfunction (Gao.,2007; Lukiw.,2007). The role of miRNAs in neurodegenerative diseases has been investigated using miRNA microarray profiling in brain tissue samples derived from patients and controls. Using miRNA expression profiling in cortex samples from a wellcharacterized clinicopathological series of elderly controls, MCI subjects and AD patients, Wang et al (2008) identified miR-107 to be specifically decreased early in the course of AD. Computational analyses predicted BACE1 mRNA as a target of miR-107 and correlative mRNA expression studies confirmed its role in regulating BACE1 expression. An independent miRNA profiling study by Hebert et al (2008) confirmed the importance of BACE1 regulation by miRNAs. The presence of a modulation of miRNA in regions of brain targeted by AD neuropathology was further demonstrated (Lukiw et al., 2008; Lukiw., 2009), thus suggesting

**5. MicroRNAs as biomarkers for AD**

206 Understanding Alzheimer's Disease

Disease modifying drugs are likely to be most effective in the earlier stages of AD, before neurodegeneration is too severe and widespread, so trials for this type of drug will need to include AD cases in the earlier stages of the disease. Validated biomarkers that could enable accurate identification of AD pathology at an early stage would be of great use (Hampel et al., 2011). Alternatively, baseline biomarker measurements can be used for enrichment and stratification in proof-of concept studies, as well as for supporting go/no-go decision making of phase III trials. Biomarkers should be used in all stages of drug development including phase I, phase II and phase III. They can be used to enhance inclusion and exclusion criteria, for stratification. Biomarkers can also be used as outcome markers to detect treatment effects. Particularly, if biomarkers are intended to be used as surrogate endpoints in pivotal studies, they must have been qualified to be a substitute for a clinical standard of truth and as such reasonably predict a clinical meaningful outcome. Finally, biomarkers can be used to identify adverse effects. Nevertheless there are several pitfalls to be faced in the interpretation of biomarker data in AD drug development, such as the fact that biomarkers may be nonspecific to AD, it may not be feasible to measure them in the appropriate system (i.e. the central nervous system) and the risk of over-interpreting biomarker data in phase II trials if statistical significance levels are not adjusted for multiple comparisons (Aisen, 2009). Failure to consider these issues could contribute to false conclusions and costly errors (Hampel et al., 2011; Hampel et al., 2004)

## **7. Conclusion and future directions**

Several promising drug candidates with disease-modifying effect, such as A*β* immunother‐ apy, secretase modulators, and tau aggregation inhibitors, have now reached the stage of being tested in clinical trials. The promise of disease-modifying therapy has created a need for biomarkers to enable the clinical identification of the disease at an early stage. Early diagnosis will be of great importance since disease-modifying drugs are likely to be most effective in the earlier stages of the disease, before neurodegeneration is too severe and widespread. A large number of studies have demonstrated that tests based on

CSF t-tau protein, p-tau and CSF beta-amyloid1–42 have reasonable specificity and sensitivity when differentiating AD from normal aging. A smaller number of studies show similar accuracy when distinguishing AD from major depression. These tests may also be useful in detecting MCI patients who go on to develop AD.

years, growing studies have been focused to establish a direct link between tissue specific oxidation and systemic oxidative damage (Blennow et al., 2010; Korolainen et al 2010; Ahmed et al., 2005; Aksenov et al., 2001). Correlations between total levels of oxidation markers in the brain and in the periphery have been shown. Although some of the reported results in AD are controversial, most of them support the presence of peripheral oxidative damage and of a characteristic panel of systemic oxidation that correlates with the occurrence of the disease. Studies investigating oxidative stress outside of the CNS, particularly in blood, while prove the occurrence of oxidative reactions, are not fully elucidating the complex cascade of events. Thus, one hypothesis is that oxidative stress first develops in the periphery as a result of different causes, and then it will contribute to perturb neuronal homeostasis, either by increasing the production of ROS or by depleting antioxidant defense, which will eventually lead to oxidative damage of the brain and neurodegeneration. The development of new plasma biomarkers could facilitate early detection, risk assessment and therapeutic monitoring in AD. On the other hand, it is also possible to imagine that oxidative stress starts in the CNS where several different metabolic end-products are formed and released into the blood stream. In this context, an important issue is to perform further studies in order to investigate the timing of appearance of oxidative damage signatures at systemic level during the onset of AD early

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Recently, important steps have been accomplished but there is still a lot of work to be directed towards the discovery, testing and validation of a panel of novel and old assays that could serve all the requirements for ideal biomarkers. However, the emerging trend which results from the collection of multiple data from different source is the wide variability among different studies that led to contrasting results. Thus, there is an urgent need to standardize protocols for replicate experiments on large population, which may allow to better under‐ standing the effect of systemic oxidative damage in the pathogenesis and progression of AD. Indeed, this is also evident by the lack of redox proteomics and microRNA studies applied to biological fluids. This approach has the power to search for specific microRNA and protein oxidative modification thus allowing the identification of altered miroRNA and protein in complex matrices such as body fluids, which may discriminate AD vs healthy condition.

There are several different reasons to support the development of more sensitive method to detect a biochemical marker in AD: to increase diagnostic accuracy; to identify MCI subjects who will progress to clinical AD; to monitor pharmacological and biological effects of drugs. There is an urgent need to add further peripheral markers of oxidative stress as useful diagnostic biomarker. There is clearly a growing interest among clinicians and basic scientists to tap on each other's expertise in the area of ageing neurobiology research. Such collaborations between geriatricians, neuroimaging specialists, neuropsychiatrists as well as molecular and cellular neurobiologists are being fostered. Further research is necessary to improve especially the early/differential biochemical diagnosis of AD. Some considerations need to be taken into account when designing future studies. These should include high numbers of relevant AD of different origin, a combination of biomarkers and other risk factors, long-term follow- up of patients and if possible neuropathological verification of the diagnosis. Standardization of methods seems critical to reducing inconsistency and increasing reliability. It is necessary to

stages and the progression to late stages.

Unfortunately, the value of these biomarkers to clinicians is limited, because they are not specific enough to accurately separate AD from other common forms of dementia, such as VaD and LBD. Sometimes the combination of both CSF t-tau protein and CSF Aβ1–42 markers does not markedly improve on their individual sensitivity. CSF p-tau, based on different phos‐ phorylation epitopes of tau protein, has now been examined in a number of independent studies. Initial results are extremely promising, showing that different p-tau protein epitopes may substantially contribute to improved diagnostic accuracy of AD in comparison with healthy aged controls, elderly depressed patients and those with other types of dementia. Compared with CSF t-tau protein and CSF Ab1–42 markers, CSF p-tau is more specific and less influenced by age or degree of cognitive decline (Hampel et al., 2004). This has an important implication for the value of CSF p-tau to clinicians. If the marker becomes abnormal very early in the course of disease relatively independent from the degree of cognitive decline than the marker may be ideal as a diagnostic test. If, however, the marker is closely linked to current or future cognitive decline, then it may be better suited as a prognostic tool. Studies of all possible biomarkers to date in AD, suggest p-tau comes the closest to the ideal diagnostic marker. However, different epitopes of p-tau may have different strengths and weaknesses. CSF p-tau231 may be most useful in distinguishing AD from frontotemporal dementia (FTD). CSF p-tau181 may improve separation between AD and LBD. In addition, CSF p-tau231 may be the most useful prognostic marker candidates that predicts cognitive decline to AD in MCI subjects.Further studies are needed to decide whether detection of multiple phosphoepitopes may allow a distinct representation of AD related pathology at different stages of the disease (Augustinack et al., 2002).

NFTs contain aberrantly hyperphosphorylated Tau as paired helical filaments. Although NFs have been shown immunohistologically to be part of NFTs, there has been debate that the identity of NF proteins in NFTs is due to the cross-reactivity of phosphorylated NF antibodies with phospho-Tau. Our laboratory recently reported (Rudrabhatla et al., 2010, 2011) the direct evidence of NFs in NFTs. Moreover, neuronal death and degeneration may release fragments of these proteins into body fluids at sufficient levels to be easily detected by specific antibodies at early, preclinical stages of AD. A battery of antibodies to NF-specific phosphoepitopes and Tau in NFTs may offer a unique approach to the design of effective early biomarkers.

The rapidly developing fields of large-scale and massive-scale genomics, proteomics, and metabolomics are now joining functional neuroimaging, structural neuroimaging, and neuropsychometric contenders in the race to establish useful biomarkers of AD and other dementing illnesses. Redox proteomics studies have provided insights into the role of oxidative stress in AD pathology. Posttranslational modifications of brain proteins, induced by oxidative damage, lead to impairment and dysfunction of several cellular functions thus providing clues about important molecular basis of neurodegeneration associated to AD. In addition, these studies have identified specific therapeutic targets in this disorder. In recent years, growing studies have been focused to establish a direct link between tissue specific oxidation and systemic oxidative damage (Blennow et al., 2010; Korolainen et al 2010; Ahmed et al., 2005; Aksenov et al., 2001). Correlations between total levels of oxidation markers in the brain and in the periphery have been shown. Although some of the reported results in AD are controversial, most of them support the presence of peripheral oxidative damage and of a characteristic panel of systemic oxidation that correlates with the occurrence of the disease. Studies investigating oxidative stress outside of the CNS, particularly in blood, while prove the occurrence of oxidative reactions, are not fully elucidating the complex cascade of events. Thus, one hypothesis is that oxidative stress first develops in the periphery as a result of different causes, and then it will contribute to perturb neuronal homeostasis, either by increasing the production of ROS or by depleting antioxidant defense, which will eventually lead to oxidative damage of the brain and neurodegeneration. The development of new plasma biomarkers could facilitate early detection, risk assessment and therapeutic monitoring in AD. On the other hand, it is also possible to imagine that oxidative stress starts in the CNS where several different metabolic end-products are formed and released into the blood stream. In this context, an important issue is to perform further studies in order to investigate the timing of appearance of oxidative damage signatures at systemic level during the onset of AD early stages and the progression to late stages.

CSF t-tau protein, p-tau and CSF beta-amyloid1–42 have reasonable specificity and sensitivity when differentiating AD from normal aging. A smaller number of studies show similar accuracy when distinguishing AD from major depression. These tests may also be

Unfortunately, the value of these biomarkers to clinicians is limited, because they are not specific enough to accurately separate AD from other common forms of dementia, such as VaD and LBD. Sometimes the combination of both CSF t-tau protein and CSF Aβ1–42 markers does not markedly improve on their individual sensitivity. CSF p-tau, based on different phos‐ phorylation epitopes of tau protein, has now been examined in a number of independent studies. Initial results are extremely promising, showing that different p-tau protein epitopes may substantially contribute to improved diagnostic accuracy of AD in comparison with healthy aged controls, elderly depressed patients and those with other types of dementia. Compared with CSF t-tau protein and CSF Ab1–42 markers, CSF p-tau is more specific and less influenced by age or degree of cognitive decline (Hampel et al., 2004). This has an important implication for the value of CSF p-tau to clinicians. If the marker becomes abnormal very early in the course of disease relatively independent from the degree of cognitive decline than the marker may be ideal as a diagnostic test. If, however, the marker is closely linked to current or future cognitive decline, then it may be better suited as a prognostic tool. Studies of all possible biomarkers to date in AD, suggest p-tau comes the closest to the ideal diagnostic marker. However, different epitopes of p-tau may have different strengths and weaknesses. CSF p-tau231 may be most useful in distinguishing AD from frontotemporal dementia (FTD). CSF p-tau181 may improve separation between AD and LBD. In addition, CSF p-tau231 may be the most useful prognostic marker candidates that predicts cognitive decline to AD in MCI subjects.Further studies are needed to decide whether detection of multiple phosphoepitopes may allow a distinct representation of AD related pathology at different stages of the disease

NFTs contain aberrantly hyperphosphorylated Tau as paired helical filaments. Although NFs have been shown immunohistologically to be part of NFTs, there has been debate that the identity of NF proteins in NFTs is due to the cross-reactivity of phosphorylated NF antibodies with phospho-Tau. Our laboratory recently reported (Rudrabhatla et al., 2010, 2011) the direct evidence of NFs in NFTs. Moreover, neuronal death and degeneration may release fragments of these proteins into body fluids at sufficient levels to be easily detected by specific antibodies at early, preclinical stages of AD. A battery of antibodies to NF-specific phosphoepitopes and

Tau in NFTs may offer a unique approach to the design of effective early biomarkers.

The rapidly developing fields of large-scale and massive-scale genomics, proteomics, and metabolomics are now joining functional neuroimaging, structural neuroimaging, and neuropsychometric contenders in the race to establish useful biomarkers of AD and other dementing illnesses. Redox proteomics studies have provided insights into the role of oxidative stress in AD pathology. Posttranslational modifications of brain proteins, induced by oxidative damage, lead to impairment and dysfunction of several cellular functions thus providing clues about important molecular basis of neurodegeneration associated to AD. In addition, these studies have identified specific therapeutic targets in this disorder. In recent

useful in detecting MCI patients who go on to develop AD.

(Augustinack et al., 2002).

208 Understanding Alzheimer's Disease

Recently, important steps have been accomplished but there is still a lot of work to be directed towards the discovery, testing and validation of a panel of novel and old assays that could serve all the requirements for ideal biomarkers. However, the emerging trend which results from the collection of multiple data from different source is the wide variability among different studies that led to contrasting results. Thus, there is an urgent need to standardize protocols for replicate experiments on large population, which may allow to better under‐ standing the effect of systemic oxidative damage in the pathogenesis and progression of AD. Indeed, this is also evident by the lack of redox proteomics and microRNA studies applied to biological fluids. This approach has the power to search for specific microRNA and protein oxidative modification thus allowing the identification of altered miroRNA and protein in complex matrices such as body fluids, which may discriminate AD vs healthy condition.

There are several different reasons to support the development of more sensitive method to detect a biochemical marker in AD: to increase diagnostic accuracy; to identify MCI subjects who will progress to clinical AD; to monitor pharmacological and biological effects of drugs. There is an urgent need to add further peripheral markers of oxidative stress as useful diagnostic biomarker. There is clearly a growing interest among clinicians and basic scientists to tap on each other's expertise in the area of ageing neurobiology research. Such collaborations between geriatricians, neuroimaging specialists, neuropsychiatrists as well as molecular and cellular neurobiologists are being fostered. Further research is necessary to improve especially the early/differential biochemical diagnosis of AD. Some considerations need to be taken into account when designing future studies. These should include high numbers of relevant AD of different origin, a combination of biomarkers and other risk factors, long-term follow- up of patients and if possible neuropathological verification of the diagnosis. Standardization of methods seems critical to reducing inconsistency and increasing reliability. It is necessary to implement common protocols for sample preparation, experimental design and generation of proteomics data. Thus, global initiatives of standardization are of critical importance and large multicenter studies are needed to further define the added diagnostic value when multiple biomarker modalities are combined.

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The essential goal in biomarker discovery studies is the identification of preclinical marker, which facilitates disease diagnosis at earlystages, is hoped that markers of prognosis will enable clinicians to monitor whether new candidate treatments of AD are working, effectively and inexpensively and assesses the response to treatments by the time that disease-modifying treatments become available in clinical practice.
