**5. Future**

408 Neuroscience – Dealing with Frontiers

same individuals. The expression of each individual transcript can be regarded as an independent variable and tested against all the SNPs on the GWAs array. This experiment can detect 'cis' effects of SNPs influencing the expression of their own gene but also 'trans'

Alternatively an indirect approach can be used where the expression analysis is carried out in the brain tissue of neuropathologically confirmed cases and controls while the GWAs is in an independent AD cohort from (ideally) the same population. Such an indirect study has been carried out in AD and their major findings were three SNPs that associated significantly with IDE (insulin degrading enzyme) expression levels. This is a very interesting finding as IDE actually degrades A (Kurochkin and Goto, 1994) and a metaanalysis, although not the latest GWAs, suggests that it is associated with sporadic AD (Bertram *et al.,* 2007). Presumably such studies will soon be reported with the partial or

As will be seen in the next section of this chapter NGS will influence nearly all aspects of research on disease pathogenesis. However it may have the greatest impact in trancriptomic analysis (Sutherland *et al.*, 2011a). In comparison to microarrays, RNA-Seq with its linear dynamic range and sensitivity for expression changes in low-abundant transcripts, provides a much more accurate (digital) signal. All transcripts can be confidently assumed to be present regardless of their level of expression. Second, microarrays rely on known genomic sequences for their probe design whereas RNA-Seq, with no such limitation, can detect novel transcripts (Cloonan and Grimmond, 2008). Third, microarrays generally quantify only the total transcripts for each gene but RNA-Seq, with its single base resolution, can detect the exact location of transcription boundaries allowing all transcriptional outputs to be quantified. This includes variants due to alternative promoter usage, splicing patterns and 3' UTR lengths that are unique to the human brain. A recent 'proof of concept' RNA-Seq study compared commercial RNA samples from AD patients with pooled control samples (Twine *et al.,* 2011). One of their major findings was the dysregulation of *APOE* transcription in the temporal lobe of an AD patient. SNPs in and around the APOE gene seemingly associate with AD independently of the ε4 effect, although there is a 7 kb linkage disequilibrium block that covers the entire APOE gene locus (Belbin *et al.,* 2007). It is anticipated that more RNA-Seq studies of postmortem brain tissue will appear in the

The application of proteomics to AD has involved analyses of CSF, plasma and postmortem brain tissue. The driver for this research is to find biomarkers or a proteomic signature to improve AD diagnosis and potentially allow preclinical diagnosis (Zellner *et al.,* 2009).

It has been known for sometime that there are protein-based alterations in the CSF profile of AD patients. Given the role of excess A1-42 in the disease it is perhaps surprisingly to see that this specific peptide is reduced in patient (clinical) analytes (Blennow and Hampel, 2003). This has been explained by the pathogenic retention of A1-42 in the parenchyma

effects of SNPs that are spatially disparate from the gene of interest.

complete use of NGS.

literature in the near future.

**4.7 Proteomics** 

**4.6 RNA-Seq** 

As we contemplate the future of research in AD, it is worthwhile reiterating that there are currently no treatments that slow the progression of the disease. At their first clinical presentation AD sufferers will already have significant neuronal loss. Ideally, neuroprotective agents are required but these would still be relatively ineffective if only implemented at the onset of clinical signs. A successful treatment regime is dependent on the co-discovery of a therapeutic target(s) and a preclinical biomarker(s). These, in turn, are both predicated on gaining a greater understanding of early pathogenic events.

#### **5.1 Advances in understanding pathogenesis**

Without doubt the major technological advance in biology is NGS. It seems only a matter of time before the much-anticipated '\$1000 genome' is standard practice in both research and clinical practice (Pareek *et al.,* 2011). Furthermore NGS is about to become third generation sequencing where nucleic acids are sequenced without the need for preparatory PCR amplification of templates, a procedure that potentially introduces biases. These 'third-gen' technologies run at nanoscale proportions and rely on either detecting the exonuclease cleavage of a specific nucleotide from the template or their incorporation into a newly synthesised DNA strand. It is predicted that these new platforms will achieve read lengths of around 1000 base pairs making the subsequent alignment to a reference genome a more rapid and accurate process.

Alzheimer's Disease: Approaches to Pathogenesis in the Genomic Age 411

for use in a research context at this stage (Sperling *et al.,* 2011). When this detectable change in A metabolism actually begins is unknown but decreases in CSF A1-42 are seen as early as the 6th decade in apoE 4 carriers and their age-related decrease is more severe (Peskind *et al.,* 2006). Similarly PiB imaging of nondemented aged persons in Australia suggested that 33% of healthy controls had high PiB binding (Rowe *et al.,* 2010). Interesting a correlation

The increases in CSF phosphorylated and total tau are generally considered a later disease marker of the disease representative of neuronal degeneration, and likely to coincide with brain atrophy, hypometabolism or hypoperfusion on imaging (Weiner *et al.,* 2010). However at least one study of MCI suggests that by this stage there are already decreases in CSF A1- 42 and increases in tau (Herukka *et al.,* 2005). Rowe and colleagues suggested that A deposition (and increased CSF tau) are probably inevitable with ageing and other factors are

Although not the subject of this chapter it would seem remiss not to touch on the excitement currently generated by stem cell technology in medical research. The possibility of regenerative medicine in neurodegenerative disease has been stimulated by the demonstration of neurogenesis in the human adult brain (Eriksson *et al.,* 1998), the induction of stem cells from differentiated adult cells (Takahashi *et al.,* 2007) and the conversion of these cells to mature phenotypes such as dopaminergic (Soldner *et al.,* 2009) and motor neurons (Dimos *et al.,* 2008). Regenerative medicine or cell transplantation therapies may prove efficacious in many diseases although the chances in AD, with its extensive neuronal loss, do appear more remote. A therapy promoting endogenous neurogenesis looks more promising and its stimulation through physical activity or environmental enrichment seen in animal models may yet underlie the 'cognitive reserve' hypothesis. Furthermore the potential for modelling AD using patient-derived cell lines is an exciting field with applications in understanding basic pathogenesis through to preclinical drug screening and

The mechanism of action for the majority of drugs in current Phase II or III clinical trials is limiting the production or increasing the clearance of A (Gravitz, 2011). These include antibodies to A, inhibitors of A aggregation and inhibitors of -secretase. In comparison inhibitors of -secretase have proved more difficult to develop because of difficulties crossing the blood-brain-barrier and their poor affinity for the catalytic site, but two reports suggest that these roadblocks may have been solved (Atwal *et al.,* 2011; Yu *et al.,* 2011).

Two of the four drugs in Phase III trials, bapineuzumab and solanezumab are humanised monoclonal antibodies (derived from murine antibodies). These passive immunisation strategies were developed when initial studies testing an aggregated A vaccine were abandoned due to 6% of the participants developing sterile meningoencephalitis (Orgogozo *et al.,* 2003). An A vaccine had previously removed and prevented plaques in mutant APP transgenic mice, (Schenk *et al.,* 1999) and reversed their learning deficits (Janus *et al.,* 2000; Dodart *et al.,* 2002). Passive immunisation seemed to have a similar effect in mice but the

between PiB binding and cognition was only seen in *APOE* 4 carriers.

involved in conversion to dementia (Rowe *et al.,* 2010).

clinical toxicity studies (Sutherland and Sidhu, 2011).

**5.3 Treatments in development** 

In terms of AD research, NGS will spell the end of the current design of GWAs. All study subjects will soon be sequenced directly allowing both the 'rare' and 'common variant' hypotheses to be simultaneously tested. We are already seeing an intermediate combination of SNP global analyses with more focused sequencing efforts (Lupton *et al.,* 2011). For example, an investigation of hypertriglyceridemic patients by GWAs followed by the resequencing of candidate genes uncovered a significant burden of rare variants (Johansen *et al.,* 2010).

It is interesting to postulate whether the knowledge of all genetic variation between affected and unaffected individuals (in a cohort) will deliver us the complete understanding of AD pathogenesis. There is no doubt that we will uncover hitherto unknown genetic clues that could increase our focus towards one defective pathway. However the single mutation in the autosomal-dominant inherited Huntington's disease was discovered in 1993 (The Huntington's Disease Collaborative Research Group, 1993) and much remains to be known about the functional ramifications of that defect.

In terms of transcriptomics, RNA-Seq templates usually involve polyA pre-selection so that only the mRNA fraction is sequenced however total RNA approaches are available where ribosomal RNA, which accounts for as much as 90% of the transcriptome, is selectively depleted. The move to total RNA approaches seems prudent given the increasing importance of non-coding and non-polyadenylated RNA species in biology (Mattick *et al.,* 2010) and the human brain in particular (Mattick, 2011). Non-coding RNA is transcribed from what was previously called 'junk' DNA and there is an amazingly diverse and continually expanding list of different RNA species being discovered. Perhaps more daunting is that single RNA molecules can also exist as structural variants with different functional roles (Wan *et al.,* 2011). Notwithstanding the latter, the RNA-Seq platform finally gives us the opportunity to comprehensively investigate the transcriptome of the human brain (Sutherland *et al.*, 2011a).

#### **5.2 Diagnosis**

When the first comprehensive criteria for clinical diagnosis were formulated in 1984, the only imaging modality in routine use in neurology was computer-assisted tomography. Neuroimaging has come a long way since then with volumetric magnetic resonance imaging (MRI), functional MRI including diffusion tensor imaging, single-photon emission computed tomography (SPECT) and PET with the amyloid-binding ligand, 11C-Pittsburgh Compound B (PiB-PET) and brain glucose metabolism (FDG-PET)) all been applied to AD patients. Neuroimaging in AD has recently been the subject of a large international public/private partnership, based in the USA (Weiner *et al.,* 2010). Established in 2004, the Alzheimer's disease neuroimaging initiative (ADNI) seeks to validate MRI and PET images, in combination with CSF/blood biomarkers, as predictors and outcomes for use in clinical trials of AD treatments. They had a particular interest in establishing the criteria for preclinical AD and so recruited elderly controls and MCI patients, as well as AD sufferers, and followed them prospectively. ADNI consider, as per the amyloid cascade hypothesis, that the earliest detectable changes in at risk individuals are related to A. Moreover there is a detectable initial increase in CSF A1-42 (preclinical) and amyloid deposition using PiB-PET. As discussed above these changes have been included in preclinical criteria but only for use in a research context at this stage (Sperling *et al.,* 2011). When this detectable change in A metabolism actually begins is unknown but decreases in CSF A1-42 are seen as early as the 6th decade in apoE 4 carriers and their age-related decrease is more severe (Peskind *et al.,* 2006). Similarly PiB imaging of nondemented aged persons in Australia suggested that 33% of healthy controls had high PiB binding (Rowe *et al.,* 2010). Interesting a correlation between PiB binding and cognition was only seen in *APOE* 4 carriers.

The increases in CSF phosphorylated and total tau are generally considered a later disease marker of the disease representative of neuronal degeneration, and likely to coincide with brain atrophy, hypometabolism or hypoperfusion on imaging (Weiner *et al.,* 2010). However at least one study of MCI suggests that by this stage there are already decreases in CSF A1- 42 and increases in tau (Herukka *et al.,* 2005). Rowe and colleagues suggested that A deposition (and increased CSF tau) are probably inevitable with ageing and other factors are involved in conversion to dementia (Rowe *et al.,* 2010).

#### **5.3 Treatments in development**

410 Neuroscience – Dealing with Frontiers

In terms of AD research, NGS will spell the end of the current design of GWAs. All study subjects will soon be sequenced directly allowing both the 'rare' and 'common variant' hypotheses to be simultaneously tested. We are already seeing an intermediate combination of SNP global analyses with more focused sequencing efforts (Lupton *et al.,* 2011). For example, an investigation of hypertriglyceridemic patients by GWAs followed by the resequencing of candidate genes uncovered a significant burden of rare variants (Johansen

It is interesting to postulate whether the knowledge of all genetic variation between affected and unaffected individuals (in a cohort) will deliver us the complete understanding of AD pathogenesis. There is no doubt that we will uncover hitherto unknown genetic clues that could increase our focus towards one defective pathway. However the single mutation in the autosomal-dominant inherited Huntington's disease was discovered in 1993 (The Huntington's Disease Collaborative Research Group, 1993) and much remains to be known

In terms of transcriptomics, RNA-Seq templates usually involve polyA pre-selection so that only the mRNA fraction is sequenced however total RNA approaches are available where ribosomal RNA, which accounts for as much as 90% of the transcriptome, is selectively depleted. The move to total RNA approaches seems prudent given the increasing importance of non-coding and non-polyadenylated RNA species in biology (Mattick *et al.,* 2010) and the human brain in particular (Mattick, 2011). Non-coding RNA is transcribed from what was previously called 'junk' DNA and there is an amazingly diverse and continually expanding list of different RNA species being discovered. Perhaps more daunting is that single RNA molecules can also exist as structural variants with different functional roles (Wan *et al.,* 2011). Notwithstanding the latter, the RNA-Seq platform finally gives us the opportunity to comprehensively investigate the transcriptome of the human

When the first comprehensive criteria for clinical diagnosis were formulated in 1984, the only imaging modality in routine use in neurology was computer-assisted tomography. Neuroimaging has come a long way since then with volumetric magnetic resonance imaging (MRI), functional MRI including diffusion tensor imaging, single-photon emission computed tomography (SPECT) and PET with the amyloid-binding ligand, 11C-Pittsburgh Compound B (PiB-PET) and brain glucose metabolism (FDG-PET)) all been applied to AD patients. Neuroimaging in AD has recently been the subject of a large international public/private partnership, based in the USA (Weiner *et al.,* 2010). Established in 2004, the Alzheimer's disease neuroimaging initiative (ADNI) seeks to validate MRI and PET images, in combination with CSF/blood biomarkers, as predictors and outcomes for use in clinical trials of AD treatments. They had a particular interest in establishing the criteria for preclinical AD and so recruited elderly controls and MCI patients, as well as AD sufferers, and followed them prospectively. ADNI consider, as per the amyloid cascade hypothesis, that the earliest detectable changes in at risk individuals are related to A. Moreover there is a detectable initial increase in CSF A1-42 (preclinical) and amyloid deposition using PiB-PET. As discussed above these changes have been included in preclinical criteria but only

*et al.,* 2010).

about the functional ramifications of that defect.

brain (Sutherland *et al.*, 2011a).

**5.2 Diagnosis** 

Although not the subject of this chapter it would seem remiss not to touch on the excitement currently generated by stem cell technology in medical research. The possibility of regenerative medicine in neurodegenerative disease has been stimulated by the demonstration of neurogenesis in the human adult brain (Eriksson *et al.,* 1998), the induction of stem cells from differentiated adult cells (Takahashi *et al.,* 2007) and the conversion of these cells to mature phenotypes such as dopaminergic (Soldner *et al.,* 2009) and motor neurons (Dimos *et al.,* 2008). Regenerative medicine or cell transplantation therapies may prove efficacious in many diseases although the chances in AD, with its extensive neuronal loss, do appear more remote. A therapy promoting endogenous neurogenesis looks more promising and its stimulation through physical activity or environmental enrichment seen in animal models may yet underlie the 'cognitive reserve' hypothesis. Furthermore the potential for modelling AD using patient-derived cell lines is an exciting field with applications in understanding basic pathogenesis through to preclinical drug screening and clinical toxicity studies (Sutherland and Sidhu, 2011).

The mechanism of action for the majority of drugs in current Phase II or III clinical trials is limiting the production or increasing the clearance of A (Gravitz, 2011). These include antibodies to A, inhibitors of A aggregation and inhibitors of -secretase. In comparison inhibitors of -secretase have proved more difficult to develop because of difficulties crossing the blood-brain-barrier and their poor affinity for the catalytic site, but two reports suggest that these roadblocks may have been solved (Atwal *et al.,* 2011; Yu *et al.,* 2011).

Two of the four drugs in Phase III trials, bapineuzumab and solanezumab are humanised monoclonal antibodies (derived from murine antibodies). These passive immunisation strategies were developed when initial studies testing an aggregated A vaccine were abandoned due to 6% of the participants developing sterile meningoencephalitis (Orgogozo *et al.,* 2003). An A vaccine had previously removed and prevented plaques in mutant APP transgenic mice, (Schenk *et al.,* 1999) and reversed their learning deficits (Janus *et al.,* 2000; Dodart *et al.,* 2002). Passive immunisation seemed to have a similar effect in mice but the

Alzheimer's Disease: Approaches to Pathogenesis in the Genomic Age 413

Given the passage above the reader may become overly pessimistic about the possibilities for a cure of Alzheimer's in the immediate future. In its general use the term 'cure' implies that there is a readily identifiable agent that causes AD and that this agent can be attenuated

All AD research to date suggests that there is no clear single aetiology for the majority of sufferers. The major pathogenic hypothesis is that in susceptible individuals, on a background of ageing, there is an increased production or decreased clearance (Mawuenyega *et al.,* 2010) of a normal 'degradative' product called A whose build up in

An alternative or adjunctive interpretation is that A has a physiological role in the brain, perhaps synaptic pruning or inhibiting excitatory activity and that A metabolism itself becomes dysregulated in response to an unknown aetiological factor. The individuals who develop AD may have a greater propensity to deposit A or are less resistance to its downstream effects. In the latter scenario, monogenic forms of AD could still explained by a greater propensity to oligomerisation and deposition. The caveat is that prophylactic A immunotherapy may prove to have more serious side effects than if A is just a degradative

The amyloid cascade hypothesis is a very good one that no doubt encapsulates important aspects of the pathogenesis of sporadic AD. However technological advances such as GWAs suggests that A-independent mechanisms are (also) important in AD while RNA-Seq is only now offering a methodology where the transcriptome of the human brain can actually be comprehensively examined. It therefore seems too premature to place all our therapeutic

Access Economics (2009). Keeping Dementia Front of Mind: Incidence and Prevalence 2009 –

Albert MS, DeKosky ST, Dickson D, Dubois B, Feldman HH, Fox NC, Gamst A, Holtzman

Alladi S, Xuereb J, Bak T, Nestor P, Knibb J, Patterson K and Hodges JR (2007). Focal cortical

Altshuler DM, Gibbs RA, Peltonen L, Dermitzakis E, Schaffner SF, Yu F, Bonnen PE, de

presentations of Alzheimer's disease. *Brain* 130(Pt 10): 2636-2645.

DM, Jagust WJ, Petersen RC, Snyder PJ, Carrillo MC, Thies B and Phelps CH (2011). The diagnosis of mild cognitive impairment due to Alzheimer's disease: recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease. *Alzheimers Dement*

Bakker PI, Deloukas P, Gabriel SB, Gwilliam R, Hunt S, Inouye M, Jia X, Palotie A, Parkin M, Whittaker P, Chang K, Hawes A, Lewis LR, Ren Y, Wheeler D, Muzny DM, Barnes C, Darvishi K, Hurles M, Korn JM, Kristiansson K, Lee C, McCarrol SA, Nemesh J, Keinan A, Montgomery SB, Pollack S, Price AL, Soranzo N, Gonzaga-Jauregui C, Anttila V, Brodeur W, Daly MJ, Leslie S, McVean G, Moutsianas L, Nguyen H, Zhang Q, Ghori MJ, McGinnis R, McLaren W, Takeuchi F, Grossman SR, Shlyakhter I, Hostetter EB, Sabeti PC, Adebamowo CA, Foster MW, Gordon

2050. Report for Alzheimer's Australia. Canberra.

or even removed without any serious deleterious effects to the sufferer.

**6. Conclusion** 

product.

the brain becomes neurotoxic.

eggs in the A cascade basket.

7(3): 270-279.

**7. References** 

earliest reports from human trials suggest that, despite reducing amyloid load, these strategies have no effect on cognitive function (Rinne *et al.,* 2010). Furthermore trials determining the efficacy and safety of the -secretase inhibitor, semagacestat, had to be terminated due to actual cognitive decline (Cummings, 2010).

These results have been seen by some as serious threats to the validity of the amyloid cascade hypothesis in sporadic AD, although proponents have countered these criticisms by arguing that A therapy is failing because it is instituted too late in the disease process (Gandy, 2011; Golde *et al.*, 2011). They maintain that A therapy needs to be instituted as a prophylaxis and this, of course, means the preclinical identification of future AD patients and the treatment, or at least testing, of individuals without symptoms (Golde *et al.,* 2011).

According to Golde, Schneider and Koo, and based on the findings from imaging and CSF biomarker studies described above, individuals destined to have AD show detectable A deposition at least 10 years earlier. Furthermore amyloid plaques visualized by radioligand imaging and low CSF A1-42 can be monitored throughout potential trials of A therapies. It is not clear how an original cohort might be selected but aged *APOE* 4 carriers were suggested for screening (Golde *et al.,* 2011). This hypothesized approach raises some very interesting ethical questions, not to mention changes required to regulatory criteria that are currently based on clinical symptoms.

#### **5.4 Preventive health**

Epidemiological studies were instrumental in establishing the connection between serum lipid levels, particularly high cholesterol, and coronary heart disease. Proponents of prophylactic A immunotherapy might argue that their proposal is similar to the current use of statins in reducing blood cholesterol. However epidemiological studies have been relatively unsuccessful in finding factors that modify the risk for AD, meaning that any proposed preventative strategies remain speculative at best.

The statins story is an interesting one because individuals in the initial trials for heart disease appeared to have a reduced incidence of AD (Jick *et al.,* 2000; Wolozin *et al.,* 2000). This led to considerable research into cardiovascular risk factors in AD including hypertension, elevated serum cholesterol, smoking middle age obesity and type II diabetes. Interesting only type II diabetes survives meta-analyses (Sutherland *et al.*, 2011b).

Remaining physically and mentally active does seem to be relatively effective but it is likely that a cognitive reserve allows individuals to starve off the effects of AD pathology rather than decreasing the pathology itself. Of course this would still be an effective method to reduce the societal burden of AD.

Despite all the research, the current situation in AD could still be summarised by the following paragraph from a recent commentary in the Journal supplement, Nature Outlook:

*"A big part of the problem is that researchers don't know enough about the biology of Alzheimer's disease to identify the right targets. The disease is the result of a long chain of events, but some of the links in that chain are still a mystery — nobody is certain which link to cut to stop disease progression" (Gravitz, 2011).* 
