Neurodegenerative Disease_Management

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October 2013 Vol.3 Issue 5

Neurodegenerative Disease_Management ASSOCIATE EDITORS Bruno Dubois & Anthony HV Schapira

Highlights in this issue Editorial J Stamford & P Willocks Trials and tribulations of clinical research: promoting Parkinson’s patient participation Review I H Blasco etaL Biological and neuroimaging biomarkers for amyotrophic lateral sclerosis: 2013 and beyond Review I 5K Khoo eta!. Could miRNA expression changes be a reliable clinical biomarker for Parkinson’s disease?

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Could m1RNA expression changes be a reliable clinical biomarker for Parkinson’s disease? a, Leslie A 4 2 Sok Kean Khoo*l Neuman Lars Forsgren , , David Petillo 5 6 & Patrik Brund in 1 V.,

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Current diagnosis of Parkinson’s disease (PD) is based on subjective clinical grounds, determined primarily by the presence of motor features.

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Approximately 50—80% of a patient’s substantia nigra dopaminergic neurons are already lost and neurodegeneration has reached an advanced stage by the time of diagnosis.

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Clinical biomarkers that are objective and quantifiable have yet to be established for early detection of PD.

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miRNAs have shown promise as diagnostic/prognostic clinical biomarkers in oncology, but remain relatively undeveloped for use in rieurodegenerative diseases.

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miRNAs as clinical biomarkers for PD may facilitate early detection and track PD progression, and could provide insights leading to the development of new neuroprotective strategies.

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Practicing standardization and harmonization of clinical cohorts, sample collection and biomarker assays in multiple clinical settings may remain the ultimate challenge.

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SUMMARY Parkinson’s disease (PD) is a complex neurological disorder involving progressive loss of dopaminergic and other neurons, as well as intraneuronal aggregation of ct-synuclein. These changes lead to motor, cognitive and multiple non-motor clinical issues. Currently, PD diagnosis is primarily based on its motor symptoms, which are delayed and subtle; neurodegeneration is believed to reach a relatively advanced stage by the time of clinical diagnosis. Developing reliable clinical biomarkers that are objective, measurable, specific and sensitive for early detection of PD remains a challenge. miRNAs are small, noncoding RNAs involved in development and gene regulation. miRNAs possess unique and ideal biomarker characteristics: highly abundant, stable and quantifiable. Here, we review a list of miRNA candidates that could potentially represent clinical biomarkers for PD.

Center for Neurodegenerative Science, Van Andel Institute, Grand Rapids, MI, USA 2 G eriomic Microarray Core, Van Andel Institute, Grand Rapids, Ml, USA Department of Cell & Molecular Biology, Grand Valley State University, Grand Rapids, Ml, USA 3 Neuroscience Program, Mercy Health Saint Mary’s, Grand Rapids, Ml, USA Department of Pharmacology & Clinical Neuroscience, Umet University, UmeB, Sweden Center for Cancer & Cell Biology, Van Andel Institute, Grand Rapids, Ml, USA ‘Author for correspondence:Tel.: +1 616 234 5536; Fax: +1 616234 5537; [email protected]

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ISSN 1758-2024

Neurodegen. Dis. Manage. (2013) 3(5), 455—465

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REVIEW Khoo, Neuman, Forsgren, Petillo & Brundin Parkinson’s disease (PD) is rhe second mosr com mon neurodegenerative disorder, after Alzheim er’s disease (AD), and the most common motor degenerative disease. The CDC rated PD as the 14th leading cause of death, with 22,032 deaths (7.1 out of 100,000 individuals) in 2010 [tofl. Globally, the incidence rate is 4.5—19 out of 100,000 per year, as estimated by WHO [102]. The annual economic burden of PD in the USA was approximately US$10.78 billion in 2009, with an average annual cost of $21,626 per patient [1]. With the number of PD patients pro jected to double by 2030 [2], the monetary costs attributable to PD are likely to increase substan tially. Regrettably, PD is currently incurable; its insidious onset makes early detection of PD a challenging task for treating physicians. In this review, we will highlight some specific miRNA biomarker candidates and identil5r a few obstacles to apply miRNA expression as a molecular bio marker for PD. Emerging biofluid-based miRNA biomarkers, especially circulating miRNAs, as trait and state biomarkers, will also be discussed.

sensitive and quantifiable clinical biomarkers for PD are strongly desired at its earliest stage (premotor/presympromatic). In the absence of such biomarkers, besides delay and/or inaccurate early diagnosis, it will be more difficult to evaluate the effects of neuroprorecrive therapies for PD. miRNA biomarkers: the silver lining? There are many potential biomarkers for PD, ranging from a variety of image monitoring (functional, ultrasound and scintigraphy) to phe notypic assessing (psychological, motor, neuro physiological, olfactory and vision) to molecular testing (blood-based, cerebrospinal fluid (CSF) and genetics) [6]. However, due to the complexity of PD, each category has its advantages and limi tations, and none has yet completely reflected the ideal features of a clinical biomarker: sensitive, specific, inexpensive, minimally/noninvasive, quantifiable and reproducible. miRNAs are small, conserved RNAs (18—22 nucleotides) that mediate posttranslational gene regulation and are involved in cell development, differentiation, prolifera tion and apoptosis [7]. miRNAs interact with target sequences on mRNAs and affect their stability/translation. Interestingly, a single miRNA alone can target anything between a few up to several hundred mRNAs, while the expression of a specific mRNA can be regulated by more than one miRNA [8,9]. Thus, miRNA expression changes may be used as a biomarker of interest when it correlates with specific mRNAs. More importantly, miRNAs are known to be highly abundant, quantifiable, tissue-specific and intrinsically stable with nonpostprocess modifi cation [10]; all are ideal features to fulfill clinical biomarker development.

Why are clinical biomarkers for PD urgently needed? PD is a complex and heterogeneous disease. Progressive loss of the midbrain dopaminergic neurons in the substantia nigra pars compacta produces its characteristic motor symptoms of resting tremor, rigidity, bradykinesia and postural instability. The presence of two of these motor features supports the clinical diagnosis for PD. Preclinical features, such as anosmia, REM sleep behavior disorder and constipation, often precede the insidious onset of motor features by years i. However, none of these premotor signs are suf ficiently sensitive or specific to allow a reliable diagnosis before the appearance of the cardinal biomarker signs of PD. By the time of clinical diagnosis, an miRNA expression & PD al in potenti inergic dopam estimated 5 0—80% of a patient’s NAs in PD has emerged neurons are lost [4] and neurodegeneration is at Interest in the role ofmiR gh their mechanism of an advanced stage. Moreover, essential tremor and in the past 5 years. Althou understood, miRNAs atypical parkinson syndromes such as multiple action has yet to be fully begun to provide valu system atrophy, progressive supra nuclear palsy and their expression have pathology of this disease. and corticobasal degeneration mimic PD features, able insights into the has been studied can making differentiation of PD from these disor miRNA expression that following various sources ders a challenge. Overall, standard clinical assess be obtained from the ment such as Unified Parkinson’s Disease Rat (Table 1]. ing Scale (UPDRS) or UK Parkinson’s Disease ary & sporadic Society Brain Bank Clinical Diagnostic Criteria • Source one: heredit genes PD is for PD sis is subjective; the accuracy of diagno of hereditary syndromes is not 100%, even by experienced movement dis The genetic study most critical steps that order specialists [5]. Therefore, objective, specific, known to be one of the —

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Blood plasma of nontreated PD patients (n

Multiple

=

4)

32)

31) vs healthy controls (n

=

25)

19) vs healthy controls (n 32) vs healthy controls (n

Blood plasma of treated PD patients (n

Unknown

=

=

=

8)

=

13)

Potential

Diagnostic

Diagnostic, progression Diagnostic, progression

Surrogate

Diagnostic Diagnostic Diagnostic

Diagnostic? Diagnostic?

Diagnostic

Diagnostic

Diagnostic

Diagnostic? Diagnostic?

Diagnostic?

Progression? Progression

Diagnostic?

Diagnostic

?: Its potential as a biomarker needs furiher study/conhrmatiori; MPTP: 1-methyl-4phenyI-1, 2,3, 6-tetrahydropyridine; PD: Parkinson’s disease; SNCA: cx-synuclein, SNP: Single nucleotide polymorphism.

miR-1 826, miR-450b-3p, miR-626, miR-505 miR-331 -5p

Peripheral blood mononuclear cells of treated PD patients (n

8) vs treated PD (n

USP3/6, NEDD4

=

Peripheral blood of nontreated PD patients (n

BDNF, IGF-1, CDC42, TPPP/p25, CHC, TPS3BP2, GRIA SLC6A3, FGF2O, IGF-1

TFEB

Ventrolateralsubventricularzoneofpax6mutantmice Midbrain dopaminergic neurons of tfeb mutant rats Peripheral blood of nontreated PD patients (n 8) vs healthy controls (n

LAMP-2A

miR-21, miR-224, mir-373 miR-26b, miR1O6a*, miR-301b miR-64/65 miR-lOa/b, miR-212, miR-132, miR-495 miR-7a miR-128 miR-1, miR22*, miR-29a miR16_2*, miR26a2*, miR-30a miR126*

Pax6

Pitx3

miR-133b

Brainstem of early-symptomatic snca mutant mice

FGF2O BAF53a, REST, CoREST, PTBP-1

miR-433 miR9/9*, miR-124

snca mutant worms

LRRK2

miR-205

Unknown

DP

E2F1, pdr-J

miR-1 84* let-7

mdl-1, ptc-1

PARK2, PARK7, SNCA, PINKI

miR-34b/c, miR-34b-3p

HEK293T, SH-SY5Y, NS2OY, substantia nigra, striatum, olfactory bulb of MPTP-induced mice; HEK293, midbrain, hippocampus, cortex, cortical neurons of snca mutant mice SH-SY5Y, amygdala, frontal cortex, substantia nigra, cerebellum of human PD brain; HEK2O3, SH-SY5Y; SH-SY5Y, snca mutant worms HEK293T, dopaminergic neurons of Irrk2 mutant flies HEK293T, dopaminergic neurons of Irrk2 mutant flies; snco mutant worms HEK293T, HeLa, M17, frontal cortex of human PD brains, dopaminergic neurons of Irrk2 mutant mice Brain and peripheral blood of PD patients, Neuro2A, different genotypic (SNP) fibroblasts Mouse, rat and human brains; neurons of adult mouse brain; human fibroblast-induced neurons Midbrain dopaminergic neuron differentiated from mouse embryonic stem cells, midbrain of human PD brains, aphakia mutant mice, 6-hydroxydopamine-induced mice SH-SY5Y, substantia nigra and amygdala of human PD brains SH-SY5Y, substantia nigra and amygdala of human PD brains

SNCA

miR-7, miR-153

Sources of miRNA expression

HSC7O

Putative target gene(s)

miRNA

Table 1. m1RNA expression and its potential biomarker utilities in Parkinson’s disease. Ref.

[78]

[77]

[74]

[73]

[731

[661 [681

[65]

[621

[58]

[581

[541

[46-48,53]

[39]

[33]

[30,62]

[30]

[21,22,38]

[19,20]

m

m

Ln

(D

o

-

>

3

o

REVIEW Khoo, Neuman, Forsgren, Petillo & Brundin leads to a better understanding of the patho role in synuclein production. Hence, evaluating physiology of a particular human disease. Iden their expression (e.g., via nasal biopsy; also see the be tification of a hereditary gene is usually followed ‘Conclusion & future perspective’ section) may stage. earlier an at PD detecting to with screening sporadic cases to determine the one approach Recently, decreased expression of miR-34b/c frequency of the genetic aberrations/risk factor in the population and eventually to study the was linked to brain samples of idiopathic PD common (or sometimes different) underlying patients at premotor and advanced stages :21]. mechanisms between hereditary and sporadic Early deregulation of miR-34b/c was suggested forms. In PD, identification of genes related to to trigger mitochondrial dysfunction and oxida familial PD and their functional studies have tive stress, leading to reduced neuronal viability. elucidated some molecular pathways, especially Downregulation of miR-34b/c is also correlated with regard to dopaminergic neurodegeneration. with decreased expression of parkin and DJ-1 (functional proteins of PARK2 and PARK7 that cause familial PD; see below) that can impair x-synucIei n The gene coupled with PD was first mapped in mitochondrial dynamics and induce oxidative bind to a 1996 using linkage analysis and was later shown damage. In addition, miR-34b can with SNCA of isoform transcript to be cr-synuclein (SNCA) [11]. The first point specific RNA expres SNCA and mutation in SNCA, A53T, was identified to a long 3’-UTR (aSynL), cause an autosomal dominant form of hereditary sion increased with the addition of miR-34b-3p PD [12]. Subsequently, additional missense muta precursor, while it decreased with miR-34b-3p tions [13,141 and genomic multiplications [15—18] inhibitor in HEK293 cells [221. The aSynL:total were identified in families with PD. On the other SNCA expression ratio has been reported to be hand, currently, there are approximately 30 publi significantly higher in the peripheral blood of PD cations on genetic variants of SNCA related to PD, patients versus healthy controls. However, miR thanks to the advancement of high-throughput 34b expression in blood that also corresponds genornic technologies in recent years, especially with this elevation needs to be confirmed to jus those allowing genome-wide association studies tify its potential as a blood-based biornarker for to be performed in PD patients with sporadic early detection of PD. occurrence. SNCA is currently the most wellstudied gene for PD; aggregation of the SNCA LRRK2 protein is known to play an important role in PD LRRK2 was the second gene identified to cause autosomal-dominant inherited PD [23-251. pathogenesis and progression. miR-7, which is enriched in the brain, has Its known mutations are concentrated in the been reported to downregulate SNCA expres GTPase, kinase and carboxy-terminal of Roc sion and its protein by binding to the 3’-untrans- domains [26]. LRRK2 mutations are reported lated region (3’-UTR; the usual binding site to impair dopaminergic neurotransmission and of targeting miRNAs) of SNCA [19,20]. In the neuronal differentiation [27,28], and inhibition of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine- LRRK2 is neuroprotective in the Lrrk2G2Ol9S induced neurotoxin model of PD, miR-7 expres mouse model [29]. The mechanistic link between LRRK2 sion is decreased and is likely to contribute to the upregulation of SNCA expression. miR-153 and miRNA was recently investigated in is another miRNA that binds to the 3’-UTR of coimmunoprecipitation studies, showing asso SNCA and downregulates its mRNA and protein ciation of LRRK2 with Drosophila melanogaster expression [20]. A combination of both miRNAs argonaute-1 or human argonaute-2, compo has an additive effect on repressing SNCA expres nents of the RNA-induced silencing complex sion post-transcriptionally. Quantitative real-time in miRNA biogenesis [30]. Overexpression of PCR has revealed higher miR-7 expression in Dicer 1, another RNA-induced silencing com the substantia nigra, striatum and olfactory bulb plex component, suppressed the neurodegen miR_184* and compared with the cerebral cortex and cerebel erative effects in Lrrk2 mutants. to bind to the identified lum and, miR-7 and miR-153 expression corre let-7 were subsequently and E2F1, DP factors lates with SNCA expression throughout devel 3’-UTR of transcription transla two key are E2FJ and DP opment. Since their expression patterns mirror respectively. overand LRRK2, pathogenic of SNCA expression in different tissues and during tional targets miR_184* in specifically or let-7, neuronal development, they may play a tuning expression of

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Could miRNA expression changes be a reliable clinical biomarker for Parkin son’s disease? the dopaminergic neurons, can partially rescue the neurodegeneration in Lrrk2 flies. let-7 was recently found to activate TLR7 and induce neurodegeneration, with increased expression levels of let-7 in the CSF of AD patients [311. A progressive increase of let-7 also contributed to the impairment of axon regeneration [32]. Hence, let-7 expression may be an indicator for neuronal cell death and degeneration, and can be evaluated as a potential progression biomarker for PD. miR-205 is a newly identified miRNA that suppresses LRRK2 protein expression via bind ing to the 3’-UTR of LRRK2 [33]. The expression of mir-205 is significantly lower in the frontal cortex of sporadic PD patients than controls and its expression is inversely correlated with LRRK2. It will be interesting to examine the correlation of miR-205 and LRRK2 expres sion in CSF or blood to justify its potential as a candidate biomarker for PD. PARK2 & PARK7 PARK2 is an autosomal recessive gene first detected in early-onset PD in 1998 [34]. Its pro tein, parkin, is a component of the E3 ubiquitin ligase complex. Parkin is believed to be involved in mitochondrial homeostasis by protecting mtDNA from oxidative stress and stimulating mtDNA repair in proliferating neuronat cells [35]; overexpression ofparkin can stimulate tran scription of mitochondrial genes. PARK7, which encodes the DJ-1 protein, is another autosomal recessive gene identified in early-onset PD fami lies 1361. DJ-1 was recently reported to act paral lel with PINK] (another early-onset PD-related autosomal recessive gene involved in mitochon drial autophagy) Iparkin to regulate mitochon drial function and autophagy [37]. Reduction of miR-34b/c in SH-SY5Y cells causes a significant decline of parkin and DJ-1 expression, but not of PINK1. Depletion of miR-34b/c is detected in the PD brain, with a significant decrease of parkin and DJ-1 expression as well [21]. Downregulation ofSNCA (where miR-34b can regulate its expression) suppresses rnitochondrial fusion, and this inhibition can be rescued by coexpres sion ofPINK1, parkin or DJ-1 138]. These studies strengthen the notion that miR-34b should be explored further in the biofluid samples of these patients to determine its potential as a biomarker. FGF2O Changes in FGF2O have been associated with risk of PD based on the evidence that miR-433

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binding to the FGF2O 3’-UTR may lead to increased expression of endogenous SNCA [39]. However, more recent studies showed no direct association of miR-433 with PD orSNC’A [40,41]. Nevertheless, miR-433 may indirectly suppress SNCA through FGF2O [42], but further studies are needed before miR-433 should be viewed as a candidate biomarker for PD. • Source two: brain tissues & neuronal cultured cells miRNAs are known to be tissue-specific, including in brain samples. In oncology, differ entially-expressed miRNAs are identified from tumor/normal cells and tissues as molecular biomarkers to discriminate diseased elements from healthy controls [43,44]. Although the cur rent method to study miRNA expression in a PD patient’s brain is from post-mortem brain tissues, miRNAs are known to be very stable (even in degraded RNAs) [45] and may still pro vide valuable information from these samples. Hence, differential changes of miRNA expres sion between PD and normal brains or neuronal cell cultures may reflect pathological changes in PD patients. This may facilitate downstream biomarker development, especially in CSF, which immerses the CNS and directly contacts these cells and tissues. Northern blot analyses revealed miR-9, -124a, -124b, -135, -153 and -219 as brain-specific miR NAs in humans, while miR-124 and -128 are most highly expressed in neurons in the embry onic stem cell line D3 [46,47]. Sequencing tech nology has confirmed that miR-9, -124, -128 and -128b are highly and specifically expressed in all brain regions [48]. miR-9/-9 and -124 are known to regulate multiple target genes to promote neuronal differentiation [49-51] and miR-1 24 expression is progressively upregulated during embryonic development of the murine neocortex [521. Although miR9/9* and -124 mediate neurogenesis in a synergistic fashion, neither the expression of tyrosine hydroxylase nor DOPA decarboxylase can be detected in miR9/9*124DAM induced midbrain dopa minergic cells [531. Hence, it remains unclear whether miR-9, 9* or -124 can be used as PD biomarkers. Besides decreased miR-34b/c expression detected in PD-affected brains (mentioned above in the ‘cL-synuclein’ section), miR-133b is also deficient in PD patient midbrains, while enriched in normal, healthy midbrains [54).

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REVIEW Khoo, Neuman, Forsgren, Petillo & Brundin miR-133b and the transcriptional factor Pitx3 forms a negative-feedback ioop to regulate dopaminergic neuron differentiation; Pitx3 induces transcription of miR-133b, which in turn inhibits Pitx3 expression. Although trans fer of miR-133b from multipotent mesenchymal stromal cells to neural cells may regulate neurite outgrowth 1551, gene variants of miR-133b and Pitx3 do not contribute to the risk of develop ing PD [56], and mir-133b conditional knock out mice also show unaffected dopaminergic neuron gene expression, including Pitx3 [57]. Thus, it is uncertain how miRl33b or its expres sion may contribute to PD, and merits further investigation. miR-21, -224 and 373*, which target mem brane receptor LAMP-2A, and mir-26b, lO6a* and -301b, which target hsc7O, are expressed at significantly higher levels in the substantia nigra of PD patients compared with healthy controls [58]. Both LAMP-2A and hsc7O are pos sibly involved in SNCA turnover; transfection of these miRNAs in SH-SY5Y cells results in decreased protein levels of lamp-2a and hsc7O, and significantly increased SNCA accumulation. Thus, these newly reported miRNAs constitute another set of biomarker candidates for PD. • Source three: animal models Since many genes are conserved across species, animal models are usually developed to study the contribution of genetic and environmental factors to a particular disease to better under stand its etiology. Expression profiling is fre quently performed using appropriate animal models with various cancers for biomarker dis coveries; these biomarkers usually have a direct relevance to their corresponding human cancers. In AD, potential stage-specific biomarkers have been identified in the brain of an AD mouse model [591. In PD, a number of mammalian (rodents and primates) and non-mammalian models (flies, worms, zebra fish and frogs) have been established for many years [60,61]. However, only a few studies have globally profiled miRNA expression in animal models of PD. Global miRNA profiling in Caenorhabdi tis elegans with overexpressed human A53T SNCA, mutated vesicular cat-i or mutated parkin showed underexpression of miR-64 and -65 in SNCA transgenic and parkin mutants [62]. Interestingly, let-7 was also significantly underexpressed in SNCA transgenic worms and mem bers of the let-7 family (miR-241 and miR-48)

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in parkin mutants. Hence, downregulation of let-7 with overexpression of SNCA is consistent in both human and worm models. Although the function of let-7 in neurons is unclear, its expression may still represent a biomarker for ct-synucleinopathy in PD. In the D. melanogaster model, although miR 34 has recently been reported to control aging and neurodegeneration [63], deep sequencing of fly heads revealed no evidence of miRNA dysregulation in overexpressed SNCA or early-pathological stage PD fly models [64]. miRNA expression profiling in the brainstem of early-symptomatic SNCA A3OP transgenic mice shows downregulation of miR-lOb, -lOa, -212, -132 and -495 in the absence of neuro nal cell loss [65], but their expression has yet to be investigated in humans. On the other hand, Pax6 is a transcription factor that was recently found to regulate survival of dopaminergic olfac tory bulb neurons [66]. In Pax6-GFP transgenic mice, downregulation of Pax6 protein expres sion can be induced by miR-7a inhibition, and miR-7a may be involved in the control of forebrain dopaminergic neuron generation [67]. In an adeno-associated virus rat model of PD, overexpression of miR-1 28 significantly downregulates TFEB, a potential mediator of SNGA toxicity [68]. miR-7a and -128 expression in rodent tissues reflects their expression findings in humans and, hence, supports their potential as reliable clinical biomarkers for PD. • Source four: biofluids Biomarker discoveries using biofluids have been rapidly emerging as these biosamples (e.g., CSF and blood) are readily accessible in most clin ics. Protein biomarkers such as SNCA and DJ-1 in CSF [69,70] have been leading the ‘wet’ bio marker field in PD. However, it is not unusual to find reports of conflicting results regarding these potential biomarkers, which might be attributed to differences in assay and sample handling, besides blood contamination [71]. Genomic biomarkers such as miRNAs have since been attracting interest due to the feasibil ity of screening biofluids using high-throughput genomic technologies. mIRNAs in the CSF

In healthy donors, 212 detectable miRNAs were shown in CSF [72]. Unfortunately, a study on the differential expression of miRNAs in the CSF of PD patients is yet to be reported.

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Could m1RNA expression changes be relia a ble clinical biomarker for Parkinson’s disease? mIRNAs in peripheral blood Blood is an ideal source for biomarker disco very as it captures the biological and chemical con tents released by cells which may relate directly or indirectly to a disease. Additionally, the stan dard blood collection procedure is simple, fast, low-cost and minimally invasive. The expres sion of miR-1, _22* and -29a are found to be significantly lower in the blood of de novo PD than healthy controls, while the expression of a2* and -30a are significa 6 miR-16-2, 2 ntly higher in treated versus nontreated PD [73]. miRNAs that change their expression on med i cation can be used as surrogate biomarke rs to measure the effectiveness of investigationa l drugs in clinical trials. In another study, glob al miRNA expression profiling in peripheral bloo d mononuclear cells of 19 PD patients (from early to advanced stage; Hoehn and Yahr stage one to five) showed miR126* to be significantly downregulated compared with 13 healthy con trols [74]. Although the miRNA expression can distinguish PD subjects from normal controls, the possibility for using them as biomarke rs, such as testing specificity and sensitivity, was not evaluated. Circulating miRNAs in blood plasma It is known that miRNAs detected in vario us cells and tissues can also be found as circulat ing miRNAs in blood serum and plasma [75]. miRNAs can be derived from tumor cells and transfer into biofluids to reflect the pathological changes in cells or tissues. Hence, circulatin g miRNAs in plasma or serum have aggressiv ely emerged in the oncology field to show promise as robust biomarkers and therapeutic targets. How ever, the question of how circulating miRNAs could potentially reflect pathogenetic changes in the brain is largely unknown. Since exosome s are known to be secreted from neuronal cells and involved in the removal of pathogenic pro teins [76], one can speculate that miRNAs may be transported by exosomes into the peripheral blood to reflect changes in the brain. This is a question worthy of investigation and needs to be proven. The first proof-of-concept study of PD bio marker discovery using plasma-based circulatin g miRNAs was performed in 2012 [77]. A pane l of circulating miRNA biomarkers (miR-182 6, -450b-3p, -626 and -505) was identified in 32 treated PD versus 32 healthy controls and showed 100% specificity and 91% sensitivity

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in its replication set. Unfortunately, the bio marker performance was lower in a small, new, independent validation cohort. The prospect of these miRNAs as PD-specific biomarkers may be strengthened by using a standardized, larger sample size of validation to increase the power of statistics (see ‘Challenges’ section). A more recent study showed miR-331-5p significantly overexpressed in the plasma of nontreated PD [78], but the result is yet to be valid ated in a new cohort of patients. One of the reasons to explain the reported differences in circulating miRNA biomarkers between these two studies is the dif ferent technologies used (microarray based vs real time [RTJ-PCR based) in the discovery phase. Moreover, the number of miRNAs examined was 866 in microarray compared with 384 in RT-PCR. In the RT-PCR validation, a differ ent endogenous control was used to demonstrate minimal interindividual variability. There is no ‘universal’ endogenous control that fits all experimental conditions (e.g., blood, cells or tis sues), and yet normalization to an endogenous control is one of the most important approaches to correcting for RT efficiency biases. Hence, establishing an optimal normalization control or set of controls to compare miRNA biornarker expression in blood plasma is highly desired.

Challenges The study of miRNA expression is an emerg ing field in PD research. Changes in miRNA expression may be related to the pathological changes in the cells or organisms to become potential powerful PD biomarkers as mentioned above. However, to develop and establish these biomarkers, at least two significant challenge s should be overcome: • Validation One of the biggest challenges in biomarke r research is validation. First, a majority of bio marker studies are pilot studies that involve small sample sizes without validation or follow up studies. Second, when validation is per formed, usually in small sample sizes as well, biomarker discovery or performance cannot be replicated. Thus, it is inevitable that large and prospective samples are needed from new, inde pendent cohorts to obtain statistically significa nt results to confirm the robustness and reproduc ibility of biomarker candidates. Due to the complexity/heterogeneity of PD, instead of tra ditional sample size calculation, comprehensiv e

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REVIEW Khoo, Neuman, Forsgren, Petillo & Brundin computer simulations may be a more practical tool to predict adequate sample sizes to sensi tively and specifically discriminate PD from healthy controls. I Standardization & harmonization Molecular assay performance can be affected by multiple preanalytical parameters from the initial phase of sample collection at the clinical sites, followed by transportation to the laborato ries, and then by handling and processing of the biospecimen before the final sample storage [79]. More variability follows in performing specific molecular protocols for biomarkers in individual laboratories (e.g., reagents, equipment and per sonnel), causing large data variability and sub sequently poor reproducibility of results. There fore, implementation of standardization from sample collection to data interpretation is highly recommended. Sample and assay variability can be minimized if standardized guidelines such as standard operating procedures and GLP are strictly adhered to. In addition, the concept of ‘assay harmonization’, which involves iterative testing procedures to identify variables related to assay performance, has been introduced to facilitate the comparability and integration of biomarker data across multiple laboratories [80]. Most recently, analytical harmonization of amy bid 42 and tau biomarkers in CSF for AD was adopted by the Alzheimer’s Association Global Biomarkers Standardization Consortium [81].

Conclusion & future perspective Presently, there are no objective and quantifiable molecular tools to detect or track PD (although neuroimaging can provide such an index to some extent) [82]. With the rapid progress made during the past 5 years, miRNAs as a stable and mea surable analyte remain attractive as molecular biomarkers for PD. As interesting as it may seem to examine the miRNA expression in brains of PD patients to see whether they reflect PD pathology/progression, obtaining brain samples from living PD patients for testing miRNAs as biomarkers is unlikely to be possible. However, a recent study has shown the feasibility of using olfactory neurons from a simple nasal biopsy to obtain a miRNA signature for schizophre nia patients [83]. This new diagnostic method may overcome the current limitation of utilizing postmortem brain samples by using olfactory neuroepithelium as surrogate samples of the brain for early diagnosis/confirmation of PD.

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Due to the complexity of PD, a combina tion of biomarkers (e.g., ‘wet’ biomarkers such as plasma-based miRNAs, brain imaging and olfactory testing) may eventually be required to attain clinically relevant biomarker performance. Therefore, recruitment for PD biomarker stud ies should include highly standardized clinical, biological and neuroimaging protocols, which can be modeled after the Parkinson’s Progression Markers Initiative (PPMI). The PPMI recently reached its milestone of recruiting 400 de nova PD and 200 control subjects, and launched enrollment in a prodromal PD cohort to exam ine biomarker progression prior to its motor onset. The availability of standardized cohorts and evolving technologies such as next-genera tion sequencing for detection may accelerate the next phase of PD clinical biomarker discoveries. In summary, miRNAs have great potential to become specific clinical biomarker candidates for early detection and progression of PD. A blood test for PD is undeniably a ‘holy grail’ for researchers, clinicians and patients alike, and miRNAs are likely to fulfill most criteria of blood-based biomarkers (e.g., quantifiable, stable, minimally invasive and cost effective). In addition, a PD-predictive biomarker, espe cially for its prod romal phase, should benefit the development of disease-modifying treatments or neuroprotective therapies to limit or slow its progression. Hence, if miRNA research is pri oritized in more funding agencies to support more miRNA-related biomarker studies (which has occured in the current translational oncol ogy field), it may be just a matter of time for miRNAs to become a game changer in the field of PD and other neurodegenerative research. Acknowledgements The authors would like to thank all participants who ton tributed samples for their plasma-based miRNA biornarker study.

Financial & competing interests disclosure This article was supported by the Van Andelinstitute. Some ofthe authors’ work mentioned in this review was partially supported by the MichaelJ Fox Foundati on for Parkinson s Research Rapid Response Innovation Award. The authors have no other relevant affiliations orfinancial involvement with any organization or entity with afinancial interest in or financial conflict with the subject matter or materials discussed in the man uscrzpt apartfrom those disclosed. No writing assistance was utilized in the production of this manuscript.

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Could miRNA expression changes be a reliable clinica bioma l rker for Parkinson’s disease? References

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