Molecular biomarkers of neurodegeneration

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Molecular biomarkers of neurodegeneration Expert Review of Molecular Diagnostics Downloaded from informahealthcare.com by Washington University Library on 01/10/15 For personal use only.

Expert Rev. Mol. Diagn. 13(8), 845–861 (2013)

Kina Ho¨glund and Hugh Salter* AstraZeneca Translational Science Centre, Personalised Healthcare & Biomarkers, AstraZeneca R&D Innovative Medicines, Tomtebodava¨gen 23a 17165 Solna, Sweden and Department of Clinical Neuroscience, Science for Life Laboratory, Karolinska Institutet, Sweden *Author for correspondence: Tel.: +46 855 323 406 Fax: +46 852 481 425 [email protected]

Neuronal dysfunction and degeneration are central events of a number of major diseases with significant unmet need. Neuronal dysfunction may not necessarily be the result of cell death, but may also be due to synaptic damage leading to impaired neuronal cell signaling or long-term potentiation. Once degeneration occurs, it is unclear whether axonal or synaptic loss comes first or whether this precedes neuronal cell death. In this review we summarize the pathophysiology of four major neurodegenerative diseases; Alzheimer’s disease, Parkinson’s disease, multiple sclerosis and amyotrophic lateral sclerosis (Lou Gehrig’s disease) For each of these diseases, we describe how biochemical biomarkers are currently understood in relation to the pathophysiology and in terms of neuronal biology, and we discuss the clinical and diagnostic utility of these potential tools, which are at present limited. We discuss how markers may be used to drive drug development and clinical practice. KEYWORDS: Alzheimer’s disease • amyotrophic lateral sclerosis • axon • biomarkers • cerebrospinal fluid • clinical trials • multiple sclerosis • neurodegeneration • Parkinson’s disease • plasma • synapse.

Biomarkers are objective measures of biological states, used as indicators of normal or pathogenic processes, and used to measure or predict response to a therapeutic intervention. By this definition, there is nothing novel about the concept of a biomarker per se; blood pressure and sugar load of urine (taste test) have been clinically useful biomarkers since a long time. The use of molecularly defined biomarkers is a recent concept, directed by knowledge of the pathophysiology of a disease and which is specifically targeted toward informing and improving the probability of success in developing new drug treatments. In this sense, a biomarker is a measure that selects a patient for the treatment, where the biomarker identifies a homogenous group of patients diagnostically or prognostically with respect to the disease mechanism and proposed intervention, or which monitors the effect of a treatment. The molecular species that may be a biomarker, can reflect any of the informational and functional states in the cell (gene, protein etc.) or a downstream systemic consequence (image or other functional or physiological measure), and can be measured directly or indirectly. Obviously, every measurable domain will not reflect biology equally, so biomarkers that cross or integrate biological domains, for www.expert-reviews.com

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example, the combination of a PET image with a genotype [1] can be more informative than confining measures to a single domain. In recent years, there has been a tendency to drive biomarker discovery with ‘-omics’ approaches that systematically address a particular biological domain. While this generates huge amounts of data, there is a need for a program to understand disease per se as well; integrating and applying technology to the natural history of a disease in order to understand the pathophysiology over time. This is particularly true for neurodegenerative disease, for reasons that we discuss below. Such analyses are huge endeavors, and no single organization, or perhaps even country, can reasonably succeed alone. Therefore for the mutual benefit, national and transnational public/private consortia are emerging to drive underlying molecular understanding of the progression and measurement of disease. Relevant examples to neurodegeneration include the Alzheimer’s Disease Neuroimaging Initiative (ADNI) and the Parkinson’s Progression Markers Initiative (PPMI) [2,3]. Biomarkers in drug development & clinical practice

The use of biomarkers in pharmaceutical development can be broadly categorized. Firstly,

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biomarkers can be used to support the clinical development of pharmaceuticals directly. Thus, biomarkers which are surrogates of an ongoing disease process, but which have either a greater accuracy (specificity/selectivity) or a more rapid readout for identifying a drug response than a clinical endpoint, can contribute to shortening of timelines to accurate decisions. Such a biomarker can also be used to claim disease modification, it reflects that the biomarker support effects on disease pathology – this aspect is further discussed in a recent US FDA guidance on the development of treatments for early Alzheimer’s disease (AD) [201]. Biomarkers (variously called PK/PD, proof of mechanism or proof of pharmacology markers) are also widely used to establish, early in clinical development, a quantitative relationship between dose and proximal effect at the drug target in the desired compartment. Such biomarkers enable accurate decision making, and indeed are an occasionally neglected prerequisite, along with an adequate margin to toxicity, for drug development to succeed. Schema to categorize these types of biomarkers are widely used, in an attempt to create clarity of thinking [4], however such schema reflects the imperatives of drug development more than the clinical utility. The second major use of biomarkers is that those are intended for clinical decision-making with existing or new medicinal products, that is, those are intended (or have the potential to be) clinically useful diagnostic tools paired to new treatments (companion diagnostics), and arguably two major subtypes of biomarkers can be envisaged. From this perspective, biomarkers can be further subdivided into: those that are diagnostic of a current status, and those that are prognostic of a future outcome. Both types have utility for selecting the right patient for the right medicine. The intention of both types of biomarkers is to identify a particular sub-group of patients that would benefit from a particular type of treatment. Such markers can be identified in parallel with conventional clinical trials, which can themselves be designed to test particular hypotheses. While in an ideal world, this investigative paradigm reduces the complexity of clinical trials, and enables shorter and more precise trials, at least in the near future the development of diagnostics may in fact result in more complex clinical trials. This can be envisaged to be the case where a particular marker needs to be tested, but there is insufficient confidence in the underlying biology. Since such a test may require multiple arms of a trial to be powered separately and fully for testing an outcome versus a primary readout (e.g., apoE4 status in Alzheimer’s patients), and for which recruitment may need to be increased. Recent regulatory guidance in this area has been presented [202]. There are thus different, although fundamentally related and overlapping, drivers for how biomarkers should be applied to clinical practice and drug development in order to produce new treatments which can identify more effectively novel-safe and effective treatments that have a high probability of success in addressing unmet medical need. Within neurodegeneration, an ideal biomarker should reflect the neuropathology of the disease and should be validated 846

against neuropathologically confirmed cases. In addition, such a marker should have high sensitivity and specificity for the prediction of neurological disability. Furthermore, a biomarker should be ideally reliable, reproducible, non-invasive, genetically invariant, simple to use and in expensive, though naturally there are no such examples present. A challenge in drug discovery dealing with central targets is the reassurance of central target engagement. Pre-clinically, it is quite straightforward to establish the link between peripheral body and brain, as brain tissue can be sampled directly. Therefore, to be able to translate to humans alternative readout needs to be used. For molecular biomarkers, the cerebrospinal fluid (CSF) is considered to be the most appropriate body fluid, since it is sampleable via clinically established lumbar puncture. The CSF fills the ventricles, surrounds the external surface of the brain [5] and is directly connected to the extracellular (interstitial) fluid. Since the extracellular fluid surrounds the neurons and glia pathological changes in the brain are believed to be reflected in the CSF, as measured by proteins, lipids or neuropeptides. Although CSF sampling is becoming more accepted, it is still considered semiinvasive, and can have poor patient acceptance and so implementing CSF sampling in large clinical trials is challenging. Ultimately, brain specific proteins (or catalytic peptides derived from them) in plasma could provide a non-invasive, cheap and straightforward alternative. However, proteins found in the CSF and predominantly produced in the CNS are not influenced by blood concentrations or blood–brain barrier (BBB) permeability; hence it is very rare that CSF levels of these brain specific proteins are related to blood levels. The search for pathologically related biomarkers in the plasma is therefore challenging. Neurodegeneration

Neurodegeneration, the loss of functional neurons with consequent clinical deficit, is the hallmark of a number of major diseases with significant unmet medical need, creating huge personal impact on the lives of patients and their caregivers, as well as a social economic burden that, given an aging population, is predicted to become unsustainable in the future. It is perhaps no exaggeration to suggest that the ability to develop new treatments for neurodegenerative diseases is a key to the future of society. Neurodegenerative diseases can be pharmacologically addressed in essentially two different approaches. Symptomatic treatments aim to mitigate impaired function of the already damaged neuron, for example, by modulating the activity of NMDA receptors (memantine as a treatment for Alzheimer’s), or by increasing neurotransmitter levels in an attempt to compensate for ongoing damage (L-DOPA treatment for Parkinson’s disease [PD]; ACE therapy for AD). In contrast, disease modification approaches aim to prevent further progression or even reverse the course of a disease. Such approaches are at present few and far between, for example, blocking further disease progression by inhibiting the passage of activated immune cells over the BBB (natalizumab blockade of endothelial a-4 integrin for treatment of multiple sclerosis). A notable recent Expert Rev. Mol. Diagn. 13(8), (2013)

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consensus is that pharmacological intervention to prevent disease progression in neurodegenerative disease likely needs to be applied very early in disease progression, certainly in prodromal/very early symptom state but conceivably even in asymptomatic middle age. Tools to recognize who will be benefited are therefore required. Beyond pharmacological approaches, remodeling or rebuilding the damaged CNS can be imagined, for example, by using targeted stem cell approaches. There are no effective diseasemodification treatments for most neurodegenerative diseases. The fundamental reasons for this are the relatively poor understanding of underlying biology of the diseases, compounded with the difficulty of creating safe and effective centrally acting medicines per se, given the BBB. Moreover, directly addressing synaptic biology is an unexplored option [4]. In this review, we summarize how biochemical biomarkers may enable drug development and clinical application in neurodegeneration. We summarize the pathophysiology of four major neurodegenerative diseases; AD, PD, multiple sclerosis (MS) and amyotrophic lateral sclerosis (Lou Gehrig’s disease; ALS). For each of these diseases, we describe how molecular biomarkers are currently understood in relation to that pathophysiology, and in terms of neuronal biology, and we speculate on our understanding of how these markers may be used and integrated in the future to drive drug development and clinical practice. There are several common themes including the relative paucity of true diagnostics, the paucity of prognostic knowledge of pathophysiology in treatment paradigms, the need to understand prodromal disease states biologically and epidemiologically, the central role of inflammation and the need for broad collaboration in the life science community, both public and private, to succeed. Neurodegenerative disease

Neurodegenerative diseases such as PD, ALS, MS and AD cause major suffering. They share a progressive course of the disease that most often leads to a common pathological pathway of neurodegeneration. The cause of the non-familiar forms of these neurodegenerative diseases is still not known. It is believed that the interplay between genetic susceptibility and environmental factors are important drivers. Several mechanisms of neurodegeneration, like protein aggregation, impaired mitochondrial function and oxidative stress, are shared characteristics. They all result in neuronal neurodegeneration including ultimately both axonal and synaptic degeneration. Multiple sclerosis

MS is a neurological, demyelinating disease of the CNS. It is most commonly diagnosed during young adulthood and is more common among women than men with a ratio of 2:1. MS is also the most common non-traumatic cause of disability in younger adults [6]. The clinical course of the disease is divided into three main categories: relapsing-remitting MS (RRMS); secondary progressive MS (SPMS); and primary www.expert-reviews.com

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progressive MS (PPMS). RRMS is the most common form at onset (80–90%) of the disease and the majority of these patients later develop SPMS [7]. Patients show a broad range of symptoms which included motor disturbances, sensory disturbances, pain, balance disturbances as well as cognitive impairment and fatigue [6]. At present there are no laboratory biomarkers available for a specific diagnosis of MS, and the current clinical diagnosis is a combination of clinical neurological examination and supporting laboratory tests including structural imaging. The current clinical diagnostic criteria for MS, the McDonald criteria, were published in 2001 [8] and last revised in 2010 [9]. MS is usually considered to be an autoimmune inflammatory demyelinating disease of the CNS. The disease is associated with focal lesions in the white matter, plaques which are characterized by a demyelinated area with glial scars and axonal loss, accompanying the demyelinating process. The demyelination process is a result of a number of processes including immune-mediated mechanisms such as cytokines and T cells as well as antibody mediated damage to myelin. The destruction of the myelin sheets leads to reduced conduction velocity of the axonal action potential [10]. Previously demyelination was believed to be the major cause of neurological impairment in MS, but this has been challenged and primary neurodegeneration has been suggested to be the initial event in lesion formation [11]. Axonal loss has also been observed to be occurring early in disease as well as ongoing in older lesions, and it is presumed to be secondary to demyelination [12]. MS is traditionally considered to be a white matter disease but even so, lesions in the gray matter, primarily cortical lesions, are present. Gray matter lesions have been associated with less macrophage and T-cell infiltration and as such less inflammation [13]. A number of genetic loci are strongly linked to MS, but have not yet led to easy identification of biomarkers. For further reading we refer to the following review [14]. Amyotrophic lateral sclerosis

ALS is a severe, rapidly progressing neurodegenerative disease. Diagnosis of ALS is made using the El Escorial criteria and subsequent modifications [15]. ALS is also known as Lou Gehrig’s disease and motor neuron disease (MND), characterized by rapid loss of the upper and lower motor neurons in the motor cortex and spinal cord, leading to the loss of voluntary muscle control. ALS is one of several muscular dystrophies, but has a strong central component. Because of the rapid progression of ALS, diagnostic biomarkers would have utility in enabling rapid application of therapeutic options. Pathophysiological markers reflecting disease progression linked to underlying mechanism would also enable assessment of novel therapeutics, for a disease for which options are currently limited and of modest effect [16], including enabling selection of patients to allow for differences in underlying pathology. Understanding of the underlying pathology has been strongly informed by familial genetics, although most cases (90%) are sporadic. The most established and most frequently observed 847

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mutations in ALS are in the SOD1 gene, encoding superoxide dismutase. Mutations in this gene account for approximately 20% of familial cases, and are generally inherited in an autosomal dominant fashion, though many other loci have been implicated, without a clear rationale to identify a single underlying trigger or mechanism. Nevertheless the bulk of familial cases are caused by four genes, including SOD1 [16,17]. Other genes identified as causing familial forms of ALS include: TARDBP (encoding TAR DNA-binding protein, TDP-43) [18] and C9orf72 [19,20]; in the latter a hexanucleotide repeat is associated with both FTD (frontotemporal dementia) and ALS, suggesting a common route to neurodegeneration. Moreover, mutations in TRPM7 have been proposed to relate Parkinsonlike cognitive deficits and an ALS-like syndrome in isolated kindred’s, implying some relationship in underlying mechanism [21]. It has also been suggested [22] that failure in ubiquitin-mediated repair is a common mechanism in ALS and other forms of neurodegeneration. The accumulation of toxic levels of extracellular glutamate has also been proposed as a unifying feature. Recently, duplications in SMN1 have been shown (counter intuitively) to be associated with ALS susceptibility [23], again indicating that the underlying mechanism remains poorly understood. Thus, biomarker research to understand disease progression and the clinical relevance of underlying mechanisms to prognosis, diagnosis and treatment options remains a field where much research is needed. For further reading we refer to following review [24]. Parkinson’s disease

PD represent a large group of neurodegenerative diseases such as multiple system atrophy (MSA), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD) and PD itself. All Parkinsonian disorders are characterized by Parkinsonism where the fundamental signs include resting tremor, bradykinesia, rigity and postural instability. PD is one of the most common disorder, with a prevalence of approximately 1% in people aged 65 years and older. The onset of PD usually occurs in patients over the age of 50 years and its incidence slowly increases with age. The etiology of PD is largely unknown, with the exception of toxin-induced causes, for example, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and some rare familial forms of PD. Mutations in the PARK8 gene encoding leucine-rich repeat kinase 2 (LRRK2) are the most prevalent cause of autosomal dominant PD but LRRK2 is also a risk factor for common PD in two large GWAS studies [25]. The reported incidence of PD varies from 4.5–19 per 100,000 population/year and the prevalence is 100–200/100,000 population [26]. Most studies indicate that PD is two-times more common in men than in women. The differential diagnosis of PD is based on clinical features and the golden standard still remains neuropathological confirmation. High sensitivity and specificity can be obtained but only at specialized centers and after several years of follow-up. There are two widely accepted and used sets of clinical diagnostic criteria, the United Kingdom 848

Parkinson’s Disease Society Brain Bank (UKPDS) clinical diagnostic criteria [27] and the criteria commissioned by the Advisory Council of the National Institute of Neurological Disorders and Stroke (NINDS) National Institute of Health (NIH) [28]. The primary motor characteristics of the disease are mainly due to the progressive degeneration of dopaminergic neurons in the substantia nigra (SN) and projections to the caudate and putamen, with subsequent decline in dopamine (DA) content. Non-dopaminergic degeneration has also been reported on multiple sites in the PD-affected brain and is linked to primarily non-motor symptoms such as dementia, sleep disturbances, mood swings and severe anxiety [29,30]. The pathological hallmark is Lewy bodies (LBDs) with the major constituent being aggregated a-synuclein [31,32]. LBDs have been found to be comprised of tubulin, neurofilaments (Nfs) [33] and ubiquitin [34]. Studies on autopsy material from patients with PD demonstrate that LBDs are formed prior to appearance of motor symptoms [35]. The LBDs first emerge in the brain stem and olfactory bulb followed by the gradual spreading throughout the brain. Olfactory loss occurs in up to 90% of PD patients and is very early sign of disease preceding clinical (motor) PD symptoms by 2–7 years [36,37]. Therefore, combination of olfactory testing with other biomarkers might be used for selection of early PD patients in the future. It has also been demonstrated that olfactory testing can be useful in differential diagnosis and where sleep disturbance is also a potential early marker, linked to a-synuclein pathology. For further reading on PD we suggest the following review [38]. Alzheimer’s disease

AD is the most common form of dementia with a prevalence of 1–2% at the age of 65 years, doubling every 5 years to >35% at the age of 85 years. The majority (>95%) of all AD cases are sporadic and where the etiology is largely unknown, though apoE4 genotype status clearly increases incidence risk and age of onset (but not disease severity per se). The familiar form of AD is caused by mutations in three genes encoding proteins linked to the processing of the amyloid precursor protein (APP). The processing of APP generates the b-amyloid (Ab) peptide which is the main constituent of the extracellular senile plaques found in the brain and an early pathological hallmark of AD. A second pathological hallmark of AD is neurofibrillary tangles located intracellular and where the major building block is the hyperphosphorylated tau protein. Although more than a century has passed since AD was first described, definitive diagnosis of AD can still only be made post mortem by neuropathological confirmation of individuals who have been examined during their lifetime and found to have fulfilled the criteria for probable dementia of the Alzheimer’s type. The diagnosis of AD is based on the clinical examination using the criteria of the National Institute of Neurological Expert Rev. Mol. Diagn. 13(8), (2013)

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Biomarkers of neurodegeneration

and Communicative Disorders and Stroke (NINCDS) and the Alzheimer’s Disease and Related Disorders Association (ADRDA) Work Group [39]. These criteria have been found to be reliable and valid in general. However, any measure increasing the prognostic sensitivity and specificity would be highly valuable, improving early detection and intervention. This would especially apply to very early cognitive impairment or even asymptomatic status. In AD, there are three widely accepted biomarkers, total tau, phosphorylated tau Ab1-42 used to support clinical diagnosis and they provide fairly good specificity and sensitivity. These CSF markers are firmly established to the point that the newly revised criteria for diagnosing AD now partly rely on them [40]. Recent evidence suggests that these CSF biomarkers predict a poor long-term clinical prognosis from an early disease stage, however, and critically, they do not per se predict the individual patient’s response. For further reading on AD please refer to Ballard and colleagues [41]. Biochemical biomarkers of neurodegeneration

Neuronal dysfunction and degeneration are central events in AD, PD, MS and ALS. Neuronal dysfunction may not necessarily be the result of cell death, but may also be due to synaptic loss, impaired neuronal cell signaling or impairment of longterm potentiation. Once degeneration occurs, there are many uncertainties around whether axonal or synaptic loss comes first or whether any of these two proceeds or follows neuronal cell death. There are also uncertainties around the molecular mechanism of cell death in these diseases, mainly because neuronal cell death is a slow process, affecting a very small proportion of neurons at any time (reviewed in [42]). Most data from in vitro and in vivo studies support apoptosis to be a key mechanism while more recent data suggests that there are other forms of cell death that may participate, such as necrosis, excitotoxicity and autophagic cell death. Potential markers of axonal and synaptic degeneration and cell death are the cytoskeletal proteins. They play an important role in maintaining structural polarity of neurons, important for their normal physiological function, as well as for stability, axonal transport and plasticity. Following damage to the axon these proteins are released into the intracellular space in the brain and from there diffuse over the brain-CSF barrier. The microtubules (MTs) and actin are two important components of the cytoskeleton. Their interaction plays an important role in the development and maintenance of the nervous system. This is exemplified by the accumulation of neurofibrillary tangles in AD, consisting of paired helical filaments of the MT-stabilizing protein tau [43] and the presence of tubulin, MT-associated proteins (MAP) and Nfs in LBDs, a pathophysiological hallmark of PD [44]. Additionally, increasing amount of evidence, links these cytoskeletal components to impairment of neurotransmission leading to neuronal degeneration [45,46]. In general, clinical manifestation and diagnosis can be seen as a reflection of interrelated underlying degenerative pathology. www.expert-reviews.com

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Many studies aggregate to highlight the central importance of synapse loss in neurodegeneration. Some studies in AD suggest that synapse loss precedes neuron loss. Biochemical markers reflecting synaptic loss are most likely proteins found in the pre-or post-synapse and involved in maintaining synaptic function. In the next section, we review and summarize biochemical biomarkers in CSF reflecting neurodegeneration in AD, PD, ALS and MS. Neuronal structure

Neurons are highly specialized cells and their main function is receiving and transmitting information, forming the basis of the CNS (FIGURE 1). The two key structures involved in neurotransmission are the dendritic tree, covered with spines which are receiving information and transmitting it to the soma of the cell, and the axon, an elongated fiber stretching from the cell soma to the terminal endings (presynaptic terminal). At the synapse, neurotransmitter release communicates the neuronal signal to the next neuron. The neuron constantly rebuilds and reorganizes its synapses and thus demands a highly flexible structure. The cytoskeleton contributing to this flexibility depends on three types of protein filaments: actin filaments; microtubules; and intermediate filaments. In principal they have three distinct functions where the actin filaments are essential for many movements of the cell, especially those of its surface. The MTs are thought to be the primary organizers of the cytoskeleton. Finally the intermediate filaments provide mechanical strength to the cell. In brain, the intermediate filaments are mainly composed of Nfs together with a-internexin and peripherin. Many structural proteins are therefore highly abundant in the brain and also play a key role in synaptic function. As reviewed below, the exploration of most of these proteins as biomarkers of neurodegeneration, especially in the context of treatment decisions in existing or emerging treatments, is currently limited. Axonal markers Neurofilaments

Neurofilaments (Nfs) are the major axonal cytoskeleton protein and found exclusively in neurons [47]. Besides Nfs, aa-internexin, nestin and peripherin belong to a class of intermediate filaments expressed in the CNS [43], however their expression is limited to neuronal development. Finally, vimentin and Glial fibrillary acidic protein (GFAP) also form intermediate filament type 3. Vimentin is not exclusively found in brain while GFAP is expressed in astrocytes and Schwann cells. The neurofilament protein is a heteropolymer composed of four subunits: Nf light (NfL); medium (NfM); heavy (NfH) and a-internexin [48]. The NfL is the most abundant form (ratio: 4:2:1, NfL:NfM: NfH) containing only four sites for phosphorylation. NfH contains >100 and might therefore be extensively phosphorylated. The phosphorylation degree of the protein has been shown to correlate to increased susceptibility to proteases [49]. In accordance with this, NfL levels in CSF tends to be unstable which reduces its reliability as a biomarker. Earlier studies have shown 849

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P

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P P P P P

P P

P P

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P

Aβ plaques

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P Neurofilament =

α-synuclein =

Tubulin (mt) =

Neuromodulin =

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Figure 1. Distribution of biomarkers within the neuron. Shows the distribution in the soma, synapse and axon of candidate biomarkers of neurodegeneration within a normal neuron but also what is believed to happen in AD when tau relocates to the soma and Ab deposits into extracellular plaques. In AD, increased levels of a-synuclein have been found in CSF, maybe reflecting neurodegeneration. While in PD, the reduced levels of a-synuclein mirrors the buildup of Lewy bodies. AD: Alzheimer’s disease; CSF: Cerebrospinal fluid; PD: Parkinson’s disease.

that Ffs might work as a diagnostic marker for AD where both NfL and NfH have been studied. However, there is no study where simultaneous measurement of both phosphorylated and non-phosphorylated forms of Ffs is performed and where the ratio is used as a marker. There are results indicating that this might further increase its attribute as a therapeutic/clinical marker. In ALS, a limited number of studies have been published on the CSF levels of Nfs comparing patients versus healthy controls [50–57]. All studies demonstrated increased levels of NfH and/or NfL in patients with ALS. In the study by Brettschneider et al., it was also demonstrated that higher levels of NfH were linked to a more rapid progressive course of the disease [50]. In agreement with a more recent article by Tortelli et al. who also report a positive correlation with increased NfH levels in CSF and disease progression rate, hence increased CSF Nf levels are believed to reflect the burden of neurodegeneration. The significant relation between CSF NfL levels and disease progression suggests that NfL may be a useful marker of disease activity and progression in ALS. Levels of phosphorylated NfH are also found to be increased in ALS, both in plasma, serum and CSF [55–57]. In agreement with Tortelli, Boylan and colleagues also demonstrate that pNfH levels provide a prognostic value, not only in CSF, but also in the serum and plasma [56]. The CSF levels in patients with MS have been quite extensively studied (reviewed in [58,59]). They consistently reveal increased levels of NfL [60–66] and NfH [54,60,63,67–72] compared to controls. Nf levels have been found 850

to correlate with degree of disability and elevated levels of NfH may indicate a poor prognosis. There are also studies evaluating CSF levels of NfL in AD reporting increases in the levels as compared to controls [51,54,73–77]. Only a few studies have evaluated levels of NfH which also have been found to be increased compared to controls [54,74,76,77]. However, CSF levels of Nf are also increased in other types of dementia and therefore have low clinical utility as a diagnostic biomarker. Still, Nf proteins may be of value for investigating disease progression (reviewed in [78], where the ratio between phosphorylated forms and non-phosphorylated forms should be evaluated. In PD, there is a rather consistent result indicating no difference between disease and controls [79–82]. However, data so far indicate that CSF levels of NfL proteins enables discrimination between other PD and PD, especially MSA and PSP [79,80,82,83], where levels in PSP and MSA are significantly elevated compared to PD and controls. There are also a number of studies evaluating the phosphorylated form of NfH [77,81,82] which also increases discrimination between PD and MSA or PSP, but reveals no difference between PD and control. Tubulin

The second major component of the axonal cytoskeleton is the microtubule, structurally consisting of polymers formed by the interaction between aand b tubulin subunits. They exist in six isoforms where class II is the major neuronal isotype, expressed mainly in the brain while class III is a neuron specific marker Expert Rev. Mol. Diagn. 13(8), (2013)

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found in brain and dorsal root ganglia. The polymerization, directed by polarity is the basis for the very dynamic property of microtubules and which make them critical for specific biological functions. Microtubules are important for maintaining cell structure and provide platforms for intracellular transport but they have also been found to be important for vesicle transport. There are a very limited number of studies examining the CSF levels of microtubules in neurodegeneration. Levels of tubulin have been found to be increased in CSF from patients with progressive MS compared to controls [66]. These results are in agreement with other studies evaluating the levels of auto-antibodies toward tubulin [84–86]. In one study, increased levels of auto-antibodies toward b-tubulin II were found compared to controls, while levels of auto-antibodies toward b-tubulin III were linked to the scoring of patients and suggested as a prognostic biomarker. Terryberry and colleagues [86] present a large and comprehensive study where the presence of auto-antibodies is examined in several neurological diseases including AD, PD, ALS and RRMS and CRMS. The study reveals that 60–70% of individuals with AD, PD, ALS or RRMS present auto antibodies in CSF, compared to 0–5% in control groups. Together, these data suggest that tubulin levels in CSF might be useful biomarker of neurodegeneration. Acetylation of tubulin has been quite extensively studied in neurodegeneration. Acetylation has been found to promote axonal transport. To our knowledge there are no studies on the CSF levels of acetylated tubulin, but there are several studies indicating a central role of tubulin and posttranslational modifications thereof in neurodegeneration. For example, neuroprotective effects of genetic silencing of Sirt2 or Hdac6, two major a-tubulin deacetylase, have been reported in a model of PD (reviewed in [87,88], respectively). There are additional evidence linking tubulin to PD. Parkin is a gene, responsible for a familiar and recessive form of PD, binds to tubulin and stabilizes microtubule in vitro. Actin

Actin is the monomeric subunit of microfilaments and thin filaments and it exists in three isoforms: a, b and g. The a actions are found in muscle tissues while b and g actins coexist in most cell types. In brain the Mfs are found in presynaptic terminals and dendritic spines (post synaptic) (FIGURE 1) and are most probably a key player in forming and organizing these structures. The regulation of actin dynamics in the growth cone is necessary for axon outgrowth. A limited number of studies have evaluated changes in CSF of actin in neurodegenerative disorders. To our knowledge, there is only one study where CSF levels have been investigated in AD revealing increased CSF levels in patients homozygous for the APOE4 allele. These high levels of actin may be due to the extensive damage in the brain cells of these patients in relation to the e4 allele [89]. In a study by Semra and colleagues [66] increased levels of actin was demonstrated in CSF from patients with progressive MS compared www.expert-reviews.com

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to patients with relapsed remitting MS, inflammatory disease or healthy individuals. Further supporting a role of actin in neurodegeneration is a paper by Fulga et al. [90] which provides evidence that tau induces changes in neuronal actin filaments which in turn contribute to AD like neurodegeneration in vivo. This is further supported by the pathological structures named Hirano bodies and intra-neuronal structures containing actin filaments. Hirano bodies have been termed a ‘non-specific manifestation of neuronal degeneration’ since they have been noted in autopsy examinations of brains from patients with a variety of conditions like AD, PD, ALS and other tauopathies. Tau

Tau is a microtubule associated protein and it functions as a microtubule stabilizing protein. Six different isoforms of tau, created by alternative splicing are found in the CNS. Commercially available immunoassays measures all six isoforms of tau in totality, but not full length tau. The binding to microtubule is regulated by phosphorylation of tau and when tau becomes hyperphosphorylated and released from the microtubule, it causes destabilization of the cytoskeleton, impaired axonal transport and subsequently axonal degeneration. It has also been demonstrated that when tau, preferentially found in axons, is phosphorylated, it is redistributed to the synapse. The consequence of this redistribution is not yet fully understood. When comparing CSF levels of tau between patients with ALS and controls, two studies have been published demonstrating increased levels in ALS [50,91], while three studies found them to be normal [92,93]. In the study by Brettschneider et al., data indicates that t-tau appears to be inferior to Nf, monitoring neuroaxonal damage in ALS. Evaluation of tau CSF levels in MS has been performed in a respectable number of studies with contradictory results shown in (reviewed in [59,58]. Several studies reveal increased CSF tau in patients with MS compared to controls [72,94–97], while other studies reported no difference in MS compared to controls [60,98–102]. A few number of studies have compared tau CSF levels between clinically isolated syndrome (CIS) and controls [72,77], and these studies consistently demonstrate increase in CSF levels in patients compared to controls. These data indicate that the intensity of neuronal damage is most prominent in early phase of MS as reflected by the increase in CSF tau levels. This is further supported by magnetic resonance spectroscopy studies of patients with MS [103]. Further studies on CIS and early MS are needed to reveal the utility of CSF tau levels as indicator of progression. A quite extensive number of studies have explored levels of total tau in CSF from patients with PD and the majority of publications indicate no difference when compared to controls (reviewed in [104]. This has also been repeatedly shown in more recent papers [105,106]. Some studies indicate that tau levels may to be able to differentiate between different Parkinsonian disorders [82,83], although contradictory data exist as well [105]. However, in dementia with LBDs, where tau pathology is present, tau CSF levels are 851

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increased compared to controls [105,107]. This has also been seen in PD with dementia patients [107]. A more recent article could not find any correlations with memory impairment and CSF tau levels [108]. Another approach was given by Vranova and colleagues who identified increased CSF tau in non-tremor PD, a sub phenotype of PD. The non-tremor phenotype is linked to a more rapid clinical progression and pronounced neuropathological changes thereby further indicating that CSF tau levels are a marker of neurodegeneration in subtypes of PD or in other tauopathies [109]. A vast number of studies have evaluated the CSF levels of tau in AD. In 2005 >1300 controls and 2500 AD patients have been included in >40 studies, using the most commonly applied ELISA for measurement of T-tau in CSF: INNOTEST hTAU-Ag (Innogenetics, Ghent, Belgium) [110]. Consistently, studies demonstrate two–threefold increases of tau in CSF compared to controls (reviewed in [59,110], and tau analyses provide 40–86% sensitivity, depending on study design. The measurement of total tau and phospho-tau in CSF, at present, is one of the few established biomarkers that could be expected to realistically monitor overt neurodegeneration during interventional trials for early AD, though no regulatory consensus as to whether a new medicine could be approved for the disease modification on this basis, exists. Regulatory guidance also indicates that tau load can also be used to select AD patients for intent to treat, alongside amyloid load. It is important to note that measures of tau in CSF likely reflect a relatively advanced disease state, being possibly subsequent to propagated pathology. Hence, there is an opportunity for more sensitive measures of intact tau pathology, for which PET-ligands are starting to emerge. The role of tau genetics in relation to biomarker performance is also poorly understood, though some recent work [111], suggests that genetic variants in the machinery that modulates tau phosphorylation may be important. Amyloid precursor protein

Immunohistochemical studies very often use the APP as a marker for axonal degeneration. Evaluation of the levels of APP or the soluble fragments sAPPa or sAPPb in CSF is limited in diseases other than AD. In a recent study CSF levels of sAPPa and sAPPb were found to be decreased in patients with MS compared to controls [112]. In a study by Henriksson et al., levels of APP were shown to be decreased in PD patients [113]. The decrease of sAPP in CSF is a bit surprising, if the hypothesis is that it should reflect axonal degeneration. In AD, where amyloid pathology is wide spread, CSF levels of APP (total) and sAPPa/sAPPb seem unchanged although levels in the prodromal state (mild cognitive impairment) may be increased [114]. There is little evidence that APP load, as represented by Ab42 and Ab40 in the CSF, is reflective of overt neurodegeneration. Rather, it clearly inversely correlates with load of amyloid plaque burden. PET imaging is an orthogonally directional measure and closely correlates to CSF measures, suggesting that CSF can accurately reflect brain biochemistry. As a biomarker, APP products in CSF have direct clinical 852

utility in evaluating whether an individual is in the disease progression of AD. However, amyloid load per se is not predictive of neurodegeneration, but rather may be employed as an early inclusion measure [203]. Synaptic markers

Several synaptic proteins have been shown to be decreased in neurodegenerative disorders [115], reflected by immunohistochemical techniques in brain tissue. The proteins that have been studied are most often involved in the machinery of neurotransmitter release. a-synuclein

a-synuclein is a 140 amino acid protein and has been localized to the presynaptic site. a-?synuclein has been found to be implicated in the control of synaptic membrane processes and biogenesis [116,117], thus suggested to play a role in synaptic activity. Furthermore, it has been found to be implicated in the control of neurotransmitter release [45] and has been hypothesized to contribute to synaptic plasticity. In recent years, several studies have reported on CSF a -synuclein levels in patients with PD compared with controls and other synucleinopathies. Most studies demonstrate decreased levels of a-synuclein in PD [105,118–123], but without the sensitivity and specificity to give its clinical relevance. Studies from reviews [91,93,95,104,105,107,119,120,122,124] and some results have been conflicting those from reviews by [125,126]. Applying CSF levels of a-synuclein for differentiating between different Parkinsonian disorders also shows inconsistent results [125,127]. In a study by Hall et al., and Tateno et al., a-synuclein levels in CSF have shown to be increased in CSF from patients with AD compared to controls [121,128]. This could be a sign of increased neurodegeneration and similar findings have been seen in Creutzfeldt–Jakob disease (CJD) where neurodegeneration is ¨ hrfelt et al. reported unchanged widespread [120]. In contrast, O levels of a -synuclein in CSF from patients with AD [124]. To our knowledge, there is no data on a-synuclein in CSF from patients with ALS. However pathological studies indicate that LBD like inclusions are found in ALS and they have been found to be positive for a-synuclein [129]. We have only identified one study on CSF levels of a-synuclein in MS [130]. They reported on increased levels, which is the same as for AD. Since both diseases lack LBD pathology, this increase most likely reflects synaptic degeneration. This is further supported by a study demonstrated increased levels of a-synuclein after traumatic brain injury [131]. N-acetylaspartic acid

N-acetylaspartic acid (NAA) is the amino acid synthesized and almost exclusively localized in neurons [132], and is one of the most abundant molecules in the CNS. NAA is produced by neuronal mitochondria and catabolized by oligodendrocytes. In a cross sectional study, Teunissen et al., hypothesized that dynamic changes of CSF NAA levels during different stages of axonal degeneration in MS and reported a decrease in CSF Expert Rev. Mol. Diagn. 13(8), (2013)

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NAA in the more advanced stages of the disease [60]. Previous study by the same group also demonstrated increased NAA levels in CSF in the SPMS group compared to RRMS [133]. A third study in MS revealed increased NAA levels in MS compared to controls. As speculated by Teunissen et al., MRS techniques revealed regional changes while CSF reflects changes from a whole brain perspective. Comparing CIS patients with controls revealed no difference [134]. Tortorella et al. reported increased serum and CSF levels of NAA in MS compared to neuromyelitis optica suggesting a differential diagnostic value [135]. Serum levels of NAA have also been investigated in ALS where levels were found to be increased [136]. In this study they also found a correlation between NAA serum levels and disease progression. As seen for MS patients, brain NAA is reduced in patients with PD with dementia and the authors suggest that reduced NAA/Cr of the posterior cingulate could be used as a marker for dementia in patients with PD [137]. To our knowledge there are no studies on NAA CSF in any other neurodegenerative disease because MRS is a non-invasive technique, it attracts as a clinical modality, however, it remains to be demonstrated whether early state disease changes are useful to predict treatment outcomes which can be detected robustly by MRS with sufficient sensitivity. The more recent findings of NAA as a potential marker in serum are therefore of great importance. Other biomarkers of synaptic degeneration

There are a very limited number of studies on other potential biomarkers for neuronal or synaptic damage. One of the first studies on synaptic proteins in CSF was performed in the late 90’s [138], based on the earlier findings from post mortem studies of brain tissue. Neuron specific enolase (NSE) is a glycolytic enzyme which exists in three isoforms: (a, b and g) g-enolase is found in neurons, glial cells and neuroendocrine cells. Early studies indicate that changes in CSF levels of NSE are linked to neurodegeneration [139]. A limited number of studies have evaluated NSE levels in CSF in MS and reported no alterations [61,140,141]. Contradictory data has been reported on CSF levels of NSE in AD. Studies reported increased [142], no difference [143] as well as decreased [144], levels in CSF from patients compared to controls. Only one study has been found reporting on NSE CSF levels in patients with PD where no difference was found between PD and controls [83].Growthassociated protein 43, also known as neuromodulin, is a marker associated with growth cones, synaptic plasticity and synaptic regeneration. An early study using immunohistochemistry demonstrated decreased levels in cortex and hippocampus [145], in patients with AD. Its presence in CSF was first revealed in 1999 [146], followed by a couple of papers with quantitative measurements indicating increases in AD [147] and PD [148]. Associations between tau and neuromodulin were also found. Neurogranin is the postsynaptic analogue of the neuromodulin [149] and is involved in activity-dependent synaptic plasticity. Studies in brain tissue from patients with AD demonstrate reduced levels of neurogranin compared to www.expert-reviews.com

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controls [117,150]. In a paper on the CSF levels of neurogranin in AD and MCI [151], levels of neurogranin were increased in patients with AD compared to patients with MCI and healthy controls. Detectable levels of synaptotagmin in CSF were for the first time demonstrated by Davidsson et al. [138] who at the same time, using a semi quantitative approach, also reported on decreased levels in CSF from patients with AD compared to controls. In this publication they also demonstrated that full length synaptotagmin is present in CSF. Expert commentary

A limited number of clinical studies have been performed to explore the potential of axonal and/or synaptic proteins and their use as biomarkers in neurodegeneration, with few exceptions (TABLE 1). The proteins that have been more thoroughly studied, like tau, a-synuclein and Ffs, do reveal some interesting differences. However, there is sometimes inconsistency which leads to limited clinical value at present. After reviewing the literature we find it quite obvious that the evaluation of synaptic proteins as markers in neurodegenerative disease is a field which requires more attention. Synaptic proteins may serve as early markers for neurodegenerative process, which has been demonstrated to be an early event in these diseases [152,153]. There are also studies revealing links to the cognitive state [154,155], which suggests that synaptic proteins may serve as biomarkers for differentiation between subgroups, that is, personalized healthcare. They may also serve as prognostic markers, helping us to identify individuals who are more likely to develop cognitive symptoms or individuals who are likely to progress at a higher rate. Much learning has been and can be taken, from the previous biomarker work within AD (Ab) and MS (Nfs). One important approach, when exploring the potential value of synaptic proteins in neurodegenerative disease, is quantitative studies of well characterized brain tissue. Results from these types of studies will help in generating biomarker candidates for further evaluation in CSF. The potentially low concentrations in CSF require the availability of both immunoassays as well as mass spectrometry based technologies to secure the development of sensitive assays. Pathological studies on brain tissue suggest that different regions are affected. This may partly explain the lack of consistency between different markers of axonal degeneration between different diseases. For example, increases of axonal proteins in CSF are hypothesized to reflect neuronal degeneration such as increased levels of tau in AD. However, this hypothesis does not take into account the lack of increased levels of CSF tau in other tauopathies where the extent of neurodegeneration is similar or greater than in AD. Further studies are needed on subtypes of disease to improve our understanding of pathological process and the clinical utility of these markers. A challenge here is, of course, the limitation of performing accurate clinical diagnosis, hence longitudinal studies are very important and where clinical diagnosis is confirmed post mortem. 853

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Table 1. Changes in CSF protein levels reflecting neurodegeneration.

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Axonal degeneration

Synaptic degeneration

Biomarker

AD

NfL

"

Tau

""

Actin

PD

MS

ALS

§

"

"



No change

" Early in disease (CIS)

Conflicting data

"

"



"

"‡

Tubulin

ND

ND

"

ND

NAA

ND

ND

" Late in disease

ND

a-synuclein

"

#

"

ND

NSE

Conflicting data

No change



# Early (CIS)

The table summarizes the state of knowledge of biochemical biomarkers of neurodegeneration with respect to the different diseases summarized within the text. † However increases in PD with dementia. ‡ Auto antibodies. § Helps discriminating between PD and other a-synucleinopathies. AD: Alzheimer’s disease; ALS: Amytrophic lateral sclerosis; CIS: Clinically isolated syndrome; CSF: Cerebrospinal fluid; MS: Multiple sclerosis; NAA: N-acetylaspartic acid; ND: No data to our knowledge; NSE: Neuron specific enolas; PD: Parkinson’s disease.

The limited clinical value demonstrated for a biomarker, although repeatedly shown to differ in disease state and control, may also be due to lack of knowledge about: presence of isoforms, truncated versions or post-translationally modified species in CSF; and what the (immuno)assay which is used, is actually measuring. The conflicting data on CSF levels of asynuclein is one example. Firstly, it is not fully understood if fragments of a-synuclein are present in CSF. There are several studies indicating that a-synuclein is a substrate for protease activity, some of which affects the aggregation state of the protein and secondly, different assays are used in the different studies, and it is not always completely understood that what form of a-synuclein, the assay, is measuring. Hence, more in depth protein characterization of a-synuclein (or protein of interest) is needed, combining immunoprecipitation, mass spectrometry and western blotting. The levels of a protein in CSF are affected by the presence of pathology in brain. The low levels of Ab42 in CSF from patients with AD are one example, which have been shown to correlate to amyloid pathology in the brain measured by PET. This means that the same protein may serve as a biochemical marker for different pathogenic or neurodegenerative processes. One example is a-synuclein that is the major protein component of LB patients displaying synopathies, such as PD. These patients display reduced levels in CSF. As for Ab42 and its link to amyloid pathology in the brain, the reduction in CSF is hypothesized to be due to the aggregation and accumulation of a-synuclein in LBD in the brain. However, a-synuclein is also a synaptic protein hence, a potential marker for synaptic degeneration. This might be the reason for the increase of a-synuclein in CSF in patients with AD. Further research is needed to elucidate the relative roles of the different proteins during the progression and treatment of different diseases. It is also important to recognize that only a very small fraction of the protein complement of the neuron has yet been studied and still many more potential markers remain to be explored. As (in particular) proteomics approaches become more developed, including the ability to monitor post-translational modification, 854

it seems likely that more potential markers will emerge (for recent reviews on proteomics in neurodegeneration) [156–158]. In essence, clinical studies alongside developing technologies have the potential to make this type of markers much more useful. Moreover, integration of biochemical observations with functional measures of brain activity such as EEG and other electrophysiological modalities as well as imaging modalities such as PET will certainly have a role in developing a whole organism perspective of how disease can be monitored. In particular, the detailed relationship between functional measures of synaptic plasticity (such as LTP) and synaptic and axonal damage as measured by released protein in the CSF, remain to be elucidated, but is a strong avenue for future research. Synaptic and axonal markers reflect neurodegeneration within the brain. While it has been established that CSF measures can (though not necessarily so) accurately reflect the biology of the brain tissue, this is a difficult area to study. When considering the difficulties of using biochemical measures to understand central biology, despite their obvious face validity, several aspects need to be considered. Firstly, the absolute levels of protein released by the damaged neuron, especially in early (or even asymptomatic) disease, can be presumed to be relatively low and hence CSF detection methods require extreme sensitivity. Even measures considered clinically relevant and relatively easy (such as Ab42 peptide in CSF) are in the pg/ml range, corresponding in molar proportionality to ‘measuring a sugar cube dissolved in a swimming pool’. Methods that can accurately detect sub picogram amounts of analyte would therefore enable the field. Secondly, dynamic breakdown of released proteins occurs and can be expected to be specific to each analyte. Hence, knowledge of the dynamics of turnover is also needed. It has been proposed that systemic measures (i.e., using the peripheral circulation as a summed surrogate for the brain state) may be a way forward; this in turn implies either that a detectable signal reaches the periphery despite the BBB, or that a peripheral reaction is triggered as a specific and sensitive downstream consequence of neuronal degeneration. Thirdly, the standardization of clinical diagnosis, pre-analytical procedures (sampling, handling and Expert Rev. Mol. Diagn. 13(8), (2013)

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Hypothesis driven Discovery storage) and the technical validation of the Preclinical model -omics driven biomarker assays have highest importance. Many of the consortia established today Independent datasets Replication are well aware of this and scientists work collaboratively to establish these types of Back-translation standardized procedures. However, it is Robust assay Technical validation also important to improve the validation work of the assays and the reporting (pubApplication lication) thereof so that the scientific community is aware of the limitations of the assays, which is an important aspect when Research use in GCP-assayfor interpreting clinical outcomes. The first of clinical practice clinical trials these aspects has been explored using soQualification called neo-epitopes, which are peripherally identifiable, compartment (CSF/brain)IVD development Development of for diagnostic use specific catabolic fragments and which by non-invasive equivalents virtue of size can become theoretically Figure 2. Idealized biomarker development and validation pathway. Shows the equilibrated between CSF and plasma. generic process for development of biomarkers for decision making and ultimately for Peripheral systemic reaction to neuroinuse as clinical or companion diagnostics. flammation is an obvious avenue. However, the immediate (and indeed sustained) peripheral consequences of neurodegeneration are less Five-year view obvious. Hence, significant methodological development will be With a 5 year perspective, several factors will significantly needed to make measures of neurodegeneration peripherally alter the landscape, and make the availability of markers of observable. This is, however, important because in the end, clini- synaptic biology, an important component of the drug discal practice requires cheap and convenient markers that ideally covery landscape. From the perspective of disease understanding, longitudinal studies (such as the ADNI initiatives) will can be used as early as in primary care. In FIGURE 2, we visualize the generic process whereby bio- have reached further maturity, and the ability to monitor markers that originally represent ‘standalone’ observations of synaptic biology will add value to the interpretation of this biology become accepted both by the scientific community and type of study, and particularly in the ability to relate early by the regulatory authorities as drug development tools and as cognitive changes to synaptic damage in neurodegeneration. clinical diagnostics. The process can be broadly divided into This will enable the use of biochemical biomarkers to monithree phases. Initially, discoveries are made in initial cohorts tor treatments in the long term. Moreover, in the 5 year per(or in animal studies and then translated into observations in spective, it is likely that several interventional trials in early man). Secondly, the clinical relevance of observations is estab- (and in dominantly inherited) AD will have completed [204]. lished in independent cohorts, and the technical quality of Understanding the extent and nature of synaptic biology in assays is raised to acceptable standards, such as GCP. Thirdly, these cohorts will have value, as this will offer the opportuthe complex process of providing robust qualification of the nity to understand, again, the relationship between cognition marker for a particular context of use via the regulatory author- and pathophysiology as new treatments seek to enter the ities takes place. The process is complex and is a commercially clinic with demonstrated effect on disease modification. driven interplay between the biopharma industry, the diagnos- Finally, it is plausible that in the 5-year perspective, there tics industry, academia and regulatory authorities. Within the will be increased understanding of how peripheral measures field covered by this review, at present, there are no qualified reflect CSF biology, which in turn opens up the possibility biomarkers accepted by the FDA, and with the exception of for the availability of primary or point-of-care diagnostics amyloid-b assays (and in particular amyloid b-42 assays, reflective of synaptic biology in the long term, for treatment though possibly also tau and synuclein) this is unlikely to initiation or continuation decisions. change over the next few years, though it can be expected that b-amyloid assays – already widely used within clinical Financial & competing interests disclosure practice – will reach IVD status within a few years from now. The authors have no relevant affiliations or financial involvement with This will represent a significant transition for the field, recog- any organization or entity with a financial interest in or financial connizing the importance of CSF biochemistry in neurodegenera- flict with the subject matter or materials discussed in the manuscript. tion as a clinical tool and potentially as a companion This includes employment, consultancies, honoraria, stock ownership or diagnostic. Nevertheless, it is a sobering thought that >20 years options, expert testimony, grants or patents received or pending, or have passed since reduction of CSF amyloid was clearly royalties. No writing assistance was utilized in the production of this manuscript. accepted as a diagnostic hallmark of AD.

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Key issues • Synaptic damage is common to the major neurodegenerative diseases. • Biochemical biomarkers of synaptic damage exist, and can be measured in cerebrospinal fluid. • The exact relationship between underlying disease and biomarker levels is relatively unknown. • Key biomarkers include (structural proteins [ex actin, tubulin, tau and neurofilaments]). • Proteins involved in transmitter release (ex Rab3a, synaptotagmin, synaptophysin, synapsin, SNARE’s).

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• Pre- and post-synaptic proteins (ex GAP43, synaptodpodin, drebrin). • Because synaptic status functionally aligns to cognitive symptoms, synaptic biomarkers have the possibility to select patients for emerging therapies, both prognostically and diagnostically. • Synaptic status also has the possibility to be used a surrogate markers for monitoring treatment effects. • Integrated biomarker programs monitoring multiple modalities, including synaptic markers, are expected to increase the quality of future biomarker programs. • Peripheral correlates of cerebrospinal fluid markers is a relatively unexplored field, but one with the possibility to open up diagnostics into clinical practice.

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