Giovanni Palermo

Sonia Mazzucchi

Alessandra Della Vecchia

Gabriele Siciliano

Ubaldo Bonuccelli

Carole Azuar

Roberto Ceravolo

Simone Lista

Harald Hampel

Filippo Baldacci
email: filippo.baldacci@unipi.it

Different Clinical Contexts of Use of Blood Neurofilament Light Chain Protein in the Spectrum of Neurodegenerative Diseases

Keywords: Alzheimer's disease, Amyotrophic lateral sclerosis, Biomarkers, Creutzfeldt-Jakob disease, NFL, Parkinsonian syndromes 38

One of the most pressing challenges in the clinical research of neurodegenerative diseases (NDDs) is the validation and standardization of pathophysiological biomarkers for different contexts of use (CoUs), such as early detection, diagnosis, prognosis, and prediction of treatment response. Neurofilament light chain (NFL) concentration is a particularly promising candidate, an indicator of axonal degeneration, which can be analyzed in peripheral blood with advanced ultrasensitive methods. Serum/plasma

concentration is closely correlated with cerebrospinal fluid NFL and directly reflects neurodegeneration within the central nervous system. Here, we provide an update on the feasible CoU of blood NFL in NDDs and translate recent findings to potentially valuable clinical practice applications. As NFL is not a disease-specific biomarker, however, blood NFL is an easily accessible biomarker with promising different clinical applications for several NDDs: [START_REF] Glass | 1111 Mechanisms underlying inflammation in neurodegeneration[END_REF] early detection and diagnosis (i.e., amyotrophic lateral sclerosis, Creutzfeldt-Jakob disease, atypical parkinsonisms, sporadic late-onset ataxias), [START_REF] Heemels | Neurodegenerative diseases[END_REF] prognosis (Huntington's disease and Parkinson's disease), and (3) prediction of time to symptom onset (presymptomatic mutation carriers in genetic Alzheimer's disease and spinocerebellar ataxia type 3).

101 Keywords separated by ' -'

Alzheimer's disease -Amyotrophic lateral sclerosis -Biomarkers -Creutzfeldt-Jakob disease -NFL -Parkinsonian syndromes 102 Foot note information Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Introduction

Q2

Neurodegenerative diseases (NDDs) are currently considered as a continuum of disorders with common pathophysiological 30 mechanisms, including misfolded protein deposition, neuro-31 nal synaptic disruption, axonal degeneration, neuroinflamma-32 tion, and oxidative stress [START_REF] Glass | 1111 Mechanisms underlying inflammation in neurodegeneration[END_REF][START_REF] Heemels | Neurodegenerative diseases[END_REF][START_REF] Chi | Neuronal cell death mecha-1116 nisms in major neurodegenerative diseases[END_REF]. Therefore, the greatest current 33 challenge in the field of NDDs is to provide biomarkers for the 34 pathological mechanisms underlying each clinical picture [START_REF] Hampel | Alzheimer's disease 1119 biomarker-guided diagnostic workflow using the added value of 1120 six combined cerebrospinal fluid candidates: Aβ 1-42 , total-tau, 1121 phosphorylated-tau, NFL, neurogranin, and YKL-40[END_REF],

35
in order to improve the diagnostic and prognostic stratification 36 of the patients and to allow early diagnosis and disease mon- important steps in NDD pathogenesis, occurring long before neuronal cell death and often preceding detectable deposition of misfolded proteins [START_REF] Kanaan | Axonal degeneration in Alzheimer's disease: when 1135 signaling abnormalities meet the axonal transport system[END_REF]. During these processes, NFL is released into the extracellular space and, consequently, into body fluids, such as the cerebrospinal fluid (CSF) and blood.

Broadly speaking, mounting data reported increased CSF NFL levels in NDDs [START_REF] Khalil | Neurofilaments as 1124 biomarkers in neurological disorders[END_REF][START_REF] Gaetani | Neurofilament light chain as a biomarker in 1128 neurological disorders[END_REF][START_REF] Petzold | A sys-1147 tematic review and meta-analysis of CSF neurofilament protein 1148 levels as biomarkers in dementia[END_REF]. In the mid-1990s, through the first enzyme-linked immunosorbent assay (ELISA) developed for NFL, Rosengren and colleagues [START_REF] Rosengren | Patients with amyotrophic lateral sclerosis and other neu-1152 rodegenerative diseases have increased levels of neurofilament 1153 protein in CSF[END_REF] demonstrated that CSF NFL concentration was increased in amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and vascular cognitive impairment (VCI). Although with different magnitudes, further studies revealed that CSF NFL increased also in other several NDDs, such as frontotemporal dementia (FTD) [START_REF] Waldo | Cerebrospinal 1155 fluid neurofilament light chain protein levels in subtypes of 1156 frontotemporal dementia[END_REF][START_REF] Meeter | Neurofilament 1158 light chain: a biomarker for genetic frontotemporal dementia[END_REF], Parkinson's disease (PD) and atypical parkinsonisms (APs) [START_REF] Hall | Accuracy of a 1161 panel of 5 cerebrospinal fluid biomarkers in the differential diag-1162 nosis of patients with dementia and/or parkinsonian disorders[END_REF][START_REF] Magdalinou | A panel of 1165 nine cerebrospinal fluid biomarkers may identify patients with 1166 atypical parkinsonian syndromes[END_REF], Huntington's disease (HD) [START_REF] Vinther-Jensen | Selected CSF biomarkers indicate no evidence of early neuroinflammation in Huntington disease[END_REF], mild cognitive impairment (MCI) [START_REF] Lista | Diagnostic accuracy of CSF neurofilament light chain protein in the biomarker-guided classification system for Alzheimer's disease[END_REF][START_REF] Zetterberg | Association of cerebrospinal fluid neurofilament light concentration with Alzheimer disease progression[END_REF], and Creutzfeldt-Jakob disease (CJD) [START_REF] Van Eijk | CSF neurofilament proteins levels are elevated in sporadic Creutzfeldt-Jakob disease[END_REF][START_REF] Kanata | Cerebrospinal fluid neurofilament light in suspected sporadic Creutzfeldt-Jakob disease[END_REF], as well as in non-primary neurodegenerative disorders, such as multiple sclerosis (MS) [START_REF] Lycke | Neurofilament protein in cerebrospinal fluid: a potential marker of activity in multiple sclerosis[END_REF],

neuroinfectious conditions [START_REF] Yilmaz | Neurofilament light chain protein as a marker of neuronal injury: review of its use in HIV-1 infection and reference values for HIV-negative controls[END_REF], traumatic brain injury [START_REF] Shahim | Neurochemical aftermath of repetitive mild traumatic brain injury[END_REF],

acute stroke [START_REF] Pujol-Calderón | Neurofilament changes in serum and cerebrospinal fluid after acute ischemic stroke[END_REF], and cerebrovascular diseases [START_REF] Pawlitzki | CSF Neurofilament light chain level predicts axonal damage in cerebral vasculitis[END_REF].

Moreover, in NDDs, CSF NFL levels showed to correlate with poorer cognition, short survival times, brain atrophy, and disease severity and progression [START_REF] Skillbäck | CSF neurofilament light differs in neurodegenerative diseases and predicts severity and survival[END_REF][START_REF] Bacioglu | Neurofilament light chain in blood and CSF as marker of disease progression in mouse models and in neurodegenerative diseases[END_REF][START_REF] Bridel | Diagnostic value of cerebrospinal fluid neurofilament light protein in neurology: a systematic review and meta-analysis[END_REF], supporting the notion that it could be useful not only as a diagnostic biomarker but also as a prognostic and progression biomarker [START_REF] Mielke | Plasma and CSF neurofilament light: relation to longitudinal neuroimaging and cognitive measures[END_REF]. As a result, it has been proposed as a dynamic biomarker for axonal degeneration [START_REF] Khalil | Neurofilaments as 1124 biomarkers in neurological disorders[END_REF][START_REF] Gaetani | Neurofilament light chain as a biomarker in 1128 neurological disorders[END_REF][START_REF] Petzold | A sys-1147 tematic review and meta-analysis of CSF neurofilament protein 1148 levels as biomarkers in dementia[END_REF] with the potential capacity to monitor treatment effectiveness [START_REF] Zetterberg | Neurofilament light: a dynamic cross-disease 1140 fluid biomarker for neurodegeneration[END_REF][START_REF] Molinuevo | Current state of Alzheimer's fluid biomarkers[END_REF].

In the last few years, the interest in NFL research shifted toward blood. An ideal biomarker should be easily measurable, accurate, quantitative, reproducible, and employable to exactly categorize the population in line with a certain disease [START_REF] Hampel | Development of biomarkers to chart all Alzheimer's disease stages: the royal road to cutting the therapeutic Gordian Knot[END_REF][START_REF] Baldacci | Biomarker-guided classification scheme of neurodegenerative diseases[END_REF]. To this end, blood-based biomarkers would have significant advantages in time efficiency and cost efficiency compared to CSF and neuroimaging [START_REF] Hampel | Biomarkers for Alzheimer's disease: academic, industry and regulatory perspectives[END_REF][START_REF] Blennow | Cerebrospinal fluid and plasma biomarkers in Alzheimer disease[END_REF]. Moreover, they would offer potential applications at the population level as screening tools in primary care, as well as for longitudinal evaluations with repeated sampling during follow-up. It is not surprising that brain pathophysiological processes are reflected into the periphery. However, CSF proteins partially enters the blood flow, are subsequently diluted in a greater volume compared with CSF, and go through biochemical interactions with a large amount of plasma proteins. They are also cleared by blood cells and metabolized by other tissues.

Finally, these processes overall hamper their measurement in plasma or serum using traditional techniques. Nevertheless, in the past few years, the development of analytical tools for ultrasensitive quantification-the immunomagnetic reduction (IMR) and the single molecule array (Simoa) techniques-by allowing an efficient measurement of NFL in blood [38],

charted a tight correlation between CSF and blood NFL in different NDDs [39]. Therefore, blood NFL was suggested 105 as a proxy of any neurodegenerative process, paving the 106 way to its use in clinical practice as a reliable risk biomarker 107 for neurodegeneration [40,41]. Nonetheless, its potential ap-108 plication in real life remains unclear [42].

109

Biomarker is defined as "a characteristic that is objectively F dementia cases with an enormous health and economic impact [START_REF] Winblad | Defeating Alzheimer's disease and other dementias: a priority for European science and society[END_REF]. The scientific and clinical research is nowadays shifting from dementia to the prodromal or even preclinical phases of AD to find effective therapeutic interventions that can delay or halt neurodegenerative progression [START_REF] Fogel | Factors associated with clinical trials that fail and opportunities for improving the likelihood of success: a review[END_REF][START_REF] Dubois | The emergence of a new conceptual framework for Alzheimer's disease[END_REF].

Biomarkers hold promise for improving early diagnosis in AD and establishing a tailored approach. The use of specific surrogate biomarkers (neuroimaging, blood [plasma/serum],

and CSF) of AD pathology has been included in revised diagnostic criteria to distinguish AD from other forms of dementia since its early disease stages. However, postmortem studies demonstrate a high degree of neuropathologic heterogeneity in patients who received a clinical diagnosis of AD [START_REF] Beach | Accuracy of the clinical diagnosis of Alzheimer disease at National Institute on Aging Alzheimer Disease Centers, 2005-2010[END_REF]. The pathogenesis of AD involves interacting pathophysiological cascades in which the deposition of amyloid plaques (Aβ) and the formation of neurofibrillary tangles (NFTs) composed of hyperphosphorylated tau protein would represent only the core events. The recently established "A/T/N" scheme proposes three binary biomarker categories which reflect AD pathophysiology, where "A" refers to Aβ pathology, "T" to tau pathology, and N to neurodegeneration [START_REF] Jack Cr | A/T/N: an unbiased descriptive classification scheme for Alzheimer disease biomarkers[END_REF][START_REF] Jack Cr | NIA-AA research framework: toward a biological definition of Alzheimer's disease[END_REF]. However, emerging evidence stresses the existence of additional molecular pathophysiological pathways, such as synaptic dysfunction and degeneration, innate immune response and neuroinflammation, vascular and cell membrane dysregulation, brain metabolic dysfunction, and axonal disruption [START_REF] Hampel | Blood-based biomarkers for Alzheimer disease: mapping the road to the clinic[END_REF]. The latter is prominent in AD, and it is more closely related to cognitive decline than Aβ pathology [START_REF] Ashton | Increased plasma neurofilament light chain concentration correlates with severity[END_REF], thus leading to propose CSF NFL as a non-specific biomarker to detect early AD pathophysiological alterations [77]. In addition, an increased release of NFL molecules is a consequence of aging that contributes to an axonal degeneration due to subclinical cerebrovascular changes and neuronal atrophy [78]. In this regard, a recent gression along core biomarkers of AD pathology [START_REF] Zetterberg | Association of cerebrospinal fluid neurofilament light concentration with Alzheimer disease progression[END_REF]. -Patients with plasma NFL (London cohort) in the highest tertile had a HR = 3.78 (95% CI 1. 68-8.50).

AUTHOR'S PROOF U N C O R R E C T E D P R O O F t2:1

AUTHOR'S PROOF

U N C O R R E C T E D P R O O F t2:10
-Patients with serum NFL (Oxford cohort) in the middle tertile had a HR = 2.68 (95% CI 0.87-8.27).

-Patients with plasma NFL (London cohort) in the middle tertile had a HR = 1.91 (95% CI 0.86-4.23).

-Patients with combined blood NFL in the highest tertile had a HR = 3.82 (95% ).

-Patients with combined blood NFL in the middle tertile had a HR = 2.08 (95% CI -PD patients with baseline NFL levels > 21.84 pg/mL were at higher risk of motor symptom progression.

AUTHOR'S PROOF U N C O R R E C T E D P R O O F t2:23 Table 2
-PD patients with baseline NFL levels > 18.34 pg/mL were at higher risk of cognitive decline -At enrolment, participants with HTT CAG expansion mutations were classified as having premanifest or manifest HD based on the UHDRS-TMS.

Mol Neurobiol AUTHOR'S PROOF U N C O R R E C T E D P R O O F t2:29 Table 2
-18 (17%) subjects with premanifest disease at baseline were newly diagnosed as having manifest HD during the 3 years of follow-up t2:36 CJD t2:37

U N C O R R E C T E D P R O O F t2:38 Table 2

NFL as Risk/Screening Biomarker

Blood NFL levels seem to predict the progression to AD dementia in patients with subjective memory complaints [77].

An association between regional hypometabolism in the right hippocampus and higher plasma NFL levels was reported in cognitively normal participants from the ADNI database [87].

Hu and colleagues [90] Although not yet in humans, transgenic mice models treated 306 with a β-secretase (BACE) inhibitor showed beneficial effects 307 on AD-relevant downstream markers, including reduced plas-308 ma NFL concentrations [START_REF] Bacioglu | Neurofilament light chain in blood and CSF as marker of disease progression in mouse models and in neurodegenerative diseases[END_REF].

NFL as Diagnostic Biomarker

Serum NFL levels distinguished FTD patients from controls with good/optimal diagnostic accuracy (

NFL as Diagnostic Biomarker

Mounting evidence reports significantly higher blood NFL levels in ALS patients when compared to controls ( Actually, the first phenotype is characterized by a long-lasting and chronic disease course while the foster by an acute and highly aggressive onset. Additionally, subjects with infantileonset SMA report a significantly better response to nusinersen therapy when compared to individuals with adult-onset one.

In this regard, the finding of normal blood levels of NFL in SMA could be used in a diagnostic panel of biochemical markers to help differentiate patients presenting with motor neuron deficits, separating SMA from ALS. Indeed, a substantial proportion of patients with SMA initially receive a diagnosis of ALS [148].

Degenerative Parkinsonisms

PD is the most common degenerative parkinsonism, with evidence of progressive loss of dopaminergic neurons in the pars compacta of the substantia nigra. Diagnostic criteria have been recently revised to improve diagnostic accuracy imaging biomarkers as supportive features [START_REF] Postuma | MDS clinical diagnostic criteria for Parkinson's disease: MDS-PD clinical diagnostic criteria[END_REF]. However, early diagnosis and progression prediction remain challenging for physicians.

Of note, the differential diagnosis between PD and AP can be difficult, mainly at the early clinical stage. Similarly, evolution of diagnostic criteria for AP improved accuracy, but misdiagnosis rates are still high [149][150][151][152]. The APs that most commonly mimic PD are progressive supranuclear palsy (PSP)

and multiple system atrophy (MSA), whereas among APs, the lowest diagnostic accuracy regards the corticobasal degeneration (CBD).

Currently, biochemical biomarkers for PD and AP are an unmet need, but many CSF/serum molecules are under evaluation. CSF NFL concentration overlaps in patients with PD, PD with dementia (PDD), and DLB and are comparable with

AUTHOR'S PROOF U N C O R R E C T E D P R O O F 611
those in HC [START_REF] Bridel | Diagnostic value of cerebrospinal fluid neurofilament light protein in neurology: a systematic review and meta-analysis[END_REF]. In contrast, it has been demonstrated that 612 CSF NFL levels are markedly increased in AP patients.

613

Accordingly, it might discriminate between PD and AP with 614 a high degree of diagnostic accuracy [START_REF] Hall | Accuracy of a 1161 panel of 5 cerebrospinal fluid biomarkers in the differential diag-1162 nosis of patients with dementia and/or parkinsonian disorders[END_REF][START_REF] Magdalinou | A panel of 1165 nine cerebrospinal fluid biomarkers may identify patients with 1166 atypical parkinsonian syndromes[END_REF]153,154]. This and proved a significant correlation between plasma NFL concentration and cognitive dysfunction at MMSE [161,162]. In another study, PDD patients reported higher plasma NFL level compared with PD subjects without dementia [84], supporting an association between plasma NFL and cognitive function in PD patients [84]. Furthermore, higher baseline plasma NFL concentrations in PD patients were found to be longitudinally associated with a higher risk of progression for both motor and cognitive symptoms, suggesting that serum NFL may be a biomarker of clinical progression in PD ( In studies focusing exclusively on PSP patients, higher serum NFL levels were related to more severe motor, functional, and cognitive disability as well as shorter survival but not with age at symptom onset or disease duration (Table 2)

[163]. Notably, NFL levels in the higher tertile were

AUTHOR'S PROOF U N C O R R E C T E D P R O O F in

110

  measured and evaluated as an indicator of normal biologic and 111 pathogenic processes, or pharmacologic responses to a thera-112 peutic intervention" [43]. From a clinical perspective, a bio-113 marker can be also classified in further categories with some 114 practical and conceptual overlaps: (1) antecedent biomarkers 115 identifying a risk of disease development (risk biomarkers),

  differentiating PD vs HC, AUROC = 0.64 (95% CI 0.55-0.73), at the cutoff value of 15.6 pg/mL -The inclusion of serum NFL in a panel of CSF biomarkers (phosphorylated α-synuclein/total α-synuclein ratio and oligomeric α-synuclein/total α-synuclein ratio) yielded a sensitivity of 91% and a specificity of 81% (AUC 0NFL in differentiating PD vs HC, AUROC = 0.83 (95% CI 0.77-0.89) -At year 2, plasma NFL in discriminating TD subtype vs PIGD subtype, AUROC = 0.65 (95% CI 0.53-0.77) -Participants were included in the Early Parkinson's Disease Longitudinal Singapore Study which is an ongoing prospective cohort study analyzing the progression of early PD over a follow-up period of 5 years. -PD patients were classified into motor subtypes of "tremor dominant" (TD), "postural instability and gait disorders" (PIGD), or "indeterminate" based on MDS-UPDRS part II and III components t2:26 Marques, in differentiating AP vs PD, AUROC = 0.91 (95% CI 0.83-0.98), at the cutoff value of 14.8 ng/L -AP vs HC, AUROC = 0.88 (95% CI 0.80-0.96), at the cutoff value of 13.6 ng/L -Patients were recruited when the clinical diagnosis was still uncertain; definite clinical diagnosis was established after 3 years of follow-up and updated again after a maximum of 12 years of follow-up data t2:27 Li, in differentiating manifest SCA-3 vs HC, AUROC = 0.98 (95% CI 0.96-1.00), at the cutoff value of 20 pg/mL -Preclinical SCA-3 vs HC, AUROC = 0.83 (95% CI 0.72-0.95), at the cutoff value of 10 pg/mL -Participants in cohort B were classified into 3 subgroups according to the SARA scores: in differentiating MSA vs PD, AUROC = 0.80, at the cutoff value of 24.06 pg/mL -PD vs HC, AUROC = 0.75, at the cutoff value of 12.34 pg/mL -Higher baseline NFL levels were associated with a higher risk of motor symptom progression (adjusted HR 1.03, 95% CI 1.01-1.07) and cognition progression (adjusted HR 1.03, 95% CI 1.01-1.05)

60 -

 60 in differentiating PSP vs HC, AUROC = 0.87 (95% CI 0.83-0.92), at the cutoff value of 38.3 pg/mL -Higher NFL levels were associated with reduced survival (adjusted HR 1.5, 95% 1in differentiating c-MSA vs SAOA, AUROC = 0.74 (95% CI 0.59-0.89) -SCA vs HC, AUROC = 0.91 (95% CI 0.81-1in differentiating PD vs AP, AUROC = 0.91 (95% CI 0.87-0.95) (Lund cohort) -PD vs AP, AUROC = 0.85 (95% CI 0.72-0.98) (London cohort) -PD vs AP, AUROC = 0.81 (95% CI 0.73-0.90) (early-stage disease cohort) -The study included 2 independent prospective cohorts of PD and AP patients and HC: the Lund (n = 278) and London (n = 117) cohorts.-The third cohort consisted of PD and AP patients with a disease duration < 3 years (early-stage disease cohort, n = 109) baseline plasma NFL levels in premanifest HD were associated with clinical disease onset during the 3-year follow-up period, adjusted HR 3.03, 95% CI 1.07-8.Participants were enrolled in the TRACK-HD* study.

  in differentiating sCJD vs HC, AUROC = 0.99 (95% CI 0.98-1.0) -gCJD vs HC, AUROC = 1.0 (CI 1.0-1.0) -AD vs HC, AUROC = 0.99 (95% CI 0.97-1.0) -Other neurological disorders vs HC, AUROC = 0.96 (CI 0.90-1.0)

309 FTD310

 309 The term FTD indicates a heterogeneous spectrum of NDDs 311 inexorably conveying to a dementia syndrome characterized by 312 predominant behavioral-behavioral FTD (bvFTD) [94]-or 313 language-primary progressive aphasia (PPA)[START_REF] Gorno-Tempini | Classification of primary progressive aphasia and its variants[END_REF]is the third most common neurodegenerative dementia after AD and DLB and is typically diagnosed in middle age[95]. bvFTD is the most prevalent phenotype (55-60% of cases) whereas PPA (40-45% of cases) can be further classified as non-fluent/agrammatic variant (nfvFTD) and semantic variant (svFTD) [96]. Notably, an additional clinical PPA variant, named logopenic variant, can show AD pathological features in more than half of subjects [97]. Finally, a clinical overlap between FTD and ALS is described and about 10-15% of cases with ALS report a dementia syndrome in the FTD spectrum (ALS-FTD) [98]. The underlying pathologies in FTD, essentially abnormal accumulations of either tau or TAR DNA-binding protein 43 (TDP-43) proteins, may be recognized only on postmortem examination. These misfolded protein aggregates lead to atrophy (structural MRI) and hypometabolism ( 18F FDG-PET) of frontal and/or anterior temporal lobes depending on phenotype [41]. Unfortunately, with the exception of causative mutations in genetic forms (about 10% of cases: hexanucleotide repeat expansions near the chromosome 9 open reading frame gene (C9orf72), progranulin (GRN), and microtubule-associated protein tau (MAPT) [99]), CSF poly(GP) detection in C9ORF72 expansion carriers, and decreased CSF/blood progranulin levels in GRN mutation carriers, specific pathophysiological biomarkers for FTD are completely lacking. However, the diagnosis of FTD remains very challenging, especially for behavioral variant as its diagnosis is mainly based on clinical assessment and because its symptoms show a significant overlap with primary psychiatric disorders [100].In this context, blood NFL is a promising candidate biomarker for FTD, especially for disease differential diagnosis, monitoring, and prognosis[101, 102]. Recent evidence proved that blood and CSF levels of NFL are tightly related and significantly higher in FTD subjects than in HC [103], without gender differences[104]. Furthermore, although NFL levels generally increase with aging, this association seems not present for FTD patients[START_REF] Meeter | Neurofilament 1158 light chain: a biomarker for genetic frontotemporal dementia[END_REF][105][106][107]. Blood NFL levels tightly correlate with CSF values in almost all studies measuring this biomarker in both fluids, thus suggesting that the peripheral concentrations of NFL substantially reflect the pathophysiological modification within CNS leading to NFL increase in CSF.

615

  is in line with the remarkable axonal degeneration of large 616 myelinated axons occurring in AP as well as with the rapid 617 neuronal loss in such conditions [155]. Furthermore, CSF 618 NFL concentration correlates with measures of disease sever-619 ity and other clinical variables, demonstrating its capability to 620 reflect neurodegenerative mechanisms. However, to over-621 come the well-known limits related to CSF examination, 622 blood-derived NFL would be a more favorable biomarker. 623 In this regard, the strong correlation between blood and CSF 624 NFL levels in parkinsonian syndromes holds potential for an 625 application in clinical practice [156]. 626 PD 627 NFL as Diagnostic Biomarker 628 Similar to CSF results, NFL concentration in serum/plasma is 629 considered useful for a differential diagnosis between PD and 630 AP [157]. This has been tested for the first time in three inde-631 pendent prospective cohorts of PD, PSP, MSA, and CBD pa-632 tients, compared with HC. Blood NFL levels in AP were sig-633 nificantly elevated compared with those in PD, showing a di-634 agnostic accuracy ranging from good (in the early cohort with 635 disease duration < 3 years) to excellent (in the Lund cohort) 636 (

  and PD clinical features in three indepen-653 dent PD cohorts have been provided in the prospective and 654 longitudinal study of Hansson and colleagues [156]. In gen-655 eral, higher blood NFL levels were observed in more ad-656 vanced PD and, in the Lund PD cohort, a higher blood NFL 657 concentration was associated with disease duration and more severe motor symptoms (measured as Hoehn and Yahr (H&Y) stage, Unified Parkinson's Disease Rating Scale (UPDRS) III motor score, Timed Up and Go Test, and Tandem Gait Test). Conversely, no clinical correlations were described in the London cohort and the early-stage disease cohort [156]. However, further studies confirmed the positive relation between plasma NFL levels and motor symptom severity (measured as H&Y stage and UPDRS part III score)

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  Overview on the possible context of use of blood NFL as a biomarker in NDDs

	t1:2		Diagnostic value			Prognostic	Monitoring
						value	treatment
	t1:3		Preclinical	Prodromal	Full-blown
			phase	phase	picture
	t1:4	AD	±	+	+	+	±
	t1:5	P D	±	±	+	+	±
	t1:6	Atypical parkinsonisms	±	±	+	+	±
		(4R tauopathies)
	t1:7	D L B	±	±	±	±	±
	t1:8	FTD	-	±	+	±	±
	t1:9	A L S	±	±	+	±	±
	t1:10	CJD	±	+	+	-	±
	t1:11
			larger study in the Dominantly Inherited Alzheimer Network	205
			(DIAN) [82]. Other studies reported higher plasma/serum	206
			NFL levels in AD and MCI patients compared with controls	207
			[76, 83, 84], but with conflicting results about the differences	208
			between MCI and cognitively normal individuals. A recent	209
			meta-analysis by Wang and colleagues [85] confirmed these	210
			findings, supporting a possible contribute of plasma NFL in	211

193 Peripheral serum or plasma NFL strongly correlated with 194 CSF NFL concentration, suggesting that it reflects the same 195 pathological process [80]. In general, we found that the levels 196 of NFL are higher in serum than in plasma, but the majority of 197 studies used plasma to quantify NFL. 198 NFL as Diagnostic Biomarker 199 Current evidence revealed that plasma NFL allows to discrim-200 inate AD patients from HC subjects with a good/excellent 201 diagnostic accuracy [77, 81] (Table

2

). In addition, plasma 202 NFL levels showed to be higher in the AD dementia group 203 than in the MCI group and in Aβ-positive MCI patients than 204 HC [77]. More recently, this finding has been replicated in a 212 the AD diagnostic workup. Moreover, plasma NFL levels 213 could also reflect NFT pathology (as determined by 214 NFL immunostaining) and neurodegeneration at post-215 mortem evaluation

[START_REF] Ashton | Increased plasma neurofilament light chain concentration correlates with severity[END_REF]

.

t1:1 Table

1

Table 2

 2 The utility of NFL in the diagnostic and progression evaluation of neurodegenerative diseases

	Notes		-Mutation carriers were subdivided into 3	groups: presymptomatic mutation carriers	(individuals who scored 0 on the CDR scale	across all visits), converters (CDR = 0 at	baseline and CDR > 0 at subsequent visits),	and symptomatic mutation carriers (CDR > 0	across all visits)						-MCI-AD were AD patients at the	stage of MCI.	-ADD were AD patients at the stage	of early dementia.	-AD included MCI-AD + AD patients				-Patients were recruited from the ADNI cohort.	-MCI patients were divided into stable MCI	(sMCI, with no progression to dementia	during ≥ 2-year follow-up) and progressive	MCI (pMCI, with conversion to dementia)	-Multicenter cohort study on families with	genetic FTD in Europe and Canada	-At least 2 serum samples were taken with a	time interval of 6 months or more.	-9 presymptomatic carriers became	symptomatic during follow-up (converters).	-Presymptomatic carriers or non-carrier	mutations were healthy relatives of first	degree at risk of FTD				-The PPD group included patients with a	severe late-onset psychiatric disorder	(psychotic, mood disorders, or both).
	Diagnostic/prognostic value of blood NFL		-Rate of change of serum NFL in differentiating	non-mutation carriers vs presymptomatic	mutation carriers, AUROC = 0.7	-Non-mutation carriers vs symptomatic	non-mutation carriers, AUROC = 0.89	-Baseline serum NFL value in differentiating	non-mutation carriers vs presymptomatic	mutation carriers, AUROC = 0.49 (at the cutoff	value of 37.4 pg/mL)	-Non-mutation carriers vs symptomatic	non-mutation carriers, AUROC = 0.85 (at the	cutoff value of 27.9 pg/mL)	-Plasma NFL in differentiating patients vs HC,	AUROC = 0.85 (95% CI 0.772-0.934),	unadjusted for other variables	-AD vs HC, AUROC = 0.92 (95% CI	0.869-0.970), adjusted for age	-At the cutoff maximizing Youden index,	25.7 pg/mL, the unconditional	accuracy was 0.82	Plasma NFL in differentiating AD vs CNC,	AUROC = 0.87 (correcting for age, sex,	educational level, and APOE ε4 genotype). When	only correcting for age, sex, and educational	level, the AUROC was reduced to 0.79	-Serum NFL in differentiating symptomatic	mutation carriers vs presymptomatic mutation	carriers, AUROC = 0.93 (95% CI 0.90-0.97),	at the cutoff concentration	of 17 pg/mL	-Symptomatic mutation carriers vs non-mutation	carriers, AUROC = 0.95 (95% CI 0.92-0.98), at	the cutoff concentration of 17 pg/mL	-Baseline serum NFL converters vs	presymptomatic mutation carriers,	AUROC = 0.93 (95% CI 0.89-0.98), at the cutoff	level of 15.0 pg/mL	-Serum NFL in differentiating FTD vs PPD,	AUROC = 0.85 (95% CI 0.776-0.923), at the	cutoff level of 19.9 pg/mL
	Patients		AD presymptomatic	mutation carriers, n = 243											MCI-AD = 25	ADD = 33	AD = 58						AD = 180	MCI = 197	pMCI = 109	sMCI = 65		Presymptomatic carriers of	a mutation in GRN,	C9orf72, or MAPT = 149	FTD (symptomatic carriers	of a mutation in GRN,	C9orf72, or MAPT) = 59							FTD = 91 (bvFTD = 66;	nfvPPA = 16; svPPA = 4;	FTD-MND = 5)	PPD = 34
	Controls		HC (AD mutation	non-carriers) n = 162											HC = 41								HC = 193					Non-carriers of a mutation	in GRN, C9orf72, or	MAPT = 127
	t2:2 First author (type of study)	t2:3 AD	t2:4 Preische, 2019	(longitudinal study)											t2:5 Lewczuk, 2018	(cross-sectional study)							t2:6 Mattsson, 2017	(longitudinal study)			t2:7 FTD	t2:8 van der Ende, 2019	(longitudinal study)											t2:9 Katisko, 2019	(longitudinal study)

Table 2

 2

		Notes	-In the FTLD group, 26 patients had a definite	diagnosis due to the C9orf72 repeat	expansion	-20 bvFTD consisting of 9 possible bvFTD, 5	probable bvFTD, and 6 genetic bvFTD (4	due to C9orf72 mutations, 2 MAPT	mutations)	-Serum NFL levels did not differ between	psychiatric and control patients			-Genetic screening of 67 patients with bvFTD	revealed 7 carriers for C9orf72 repeat	expansion.	-Analysis of MAPT and GRN in 18 patients	revealed 1 carrier of the MAPT mutation and	1 carrier of the GRN mutation													-Patients with PPA met only clinical diagnostic	criteria without knowledge of fluid	biomarker concentration				-Participants were recruited as part of GENFI	or ascertained before participation in GENFI.	-4 subjects became symptomatic during	follow-up (converters)
		Diagnostic/prognostic value of blood NFL	-bvFTD vs PPD, AUROC = 0.82 (95% CI	0.732-0.908), at the cutoff level of 19.9 pg/mL		-Serum NFL in differentiating bvFTD vs HC,	AUROC = 0.94 (95% CI 0.87-0.99)	-bvFTD vs depression, AUROC = 0.89 (95% CI	0.8-0.98), at a cutoff level above 35.7 pg/mL	-bvFTD vs bipolar disorder, AUROC = 0.94 (95%	CI 0.81-1.01), at a cutoff level > 26.5 pg/mL	-bvFTD vs schizophrenia, AUROC = 0.9 (95% CI	0.77-1.03), at a cutoff level > 17.7 pg/mL	-Serum NFL in differentiating bvFTD vs AD,	AUROC = 0.67	-bvFTD vs MCI, AUROC = 0.9 (95% CI	0.84-0.97)	-bvFTD vs HC, AUROC = 0.85 (95% CI	0.72-0.97)	-bvFTD vs AD subgroups selected based on CSF	Aβ42 levels, AUROC = 0.79, at a cutoff value of	33 pg/mL	-bvFTD vs AD subgroups selected based on both	CSF Aβ42 and tau/p-tau levels, AUROC = 0.77,	at a cutoff value of 34.3 pg/mL							-Serum NFL in differentiating AUROC	nfvPPA/svPPA vs HC, AUROC = 0.84 (95% CI	0.69-0.99), at a cutoff value of 25 pg/mL	-nfvPPA/svPPA vs lvPPA, AUROC = 0.76 (95%	CI 0.64-0.88), at a cutoff level of 31 pg/mL	-Serum NFL in differentiating FTD vs HC,	AUROC = 0.81 (95% CI 0.72-0.91) at an	optimal cutoff value > 36 pg/mL	-ALS vs HC, AUROC = 0.99 (95% CI 0.98-1.00)	-Serum NFL in differentiating FTD vs HC,	AUROC = 0.97 (95% CI 0.93-1.00)	-FTD vs presymptomatic carriers, AUROC = 0.93	(95% CI 0.87-0.98), at the cutoff level of	18.0 pg/mL
		Patients				bvFTD = 20	Schizophrenia = 11	Depression = 28	Bipolar disorder = 11					bvFTD = 74	AD = 26 (excluding	patients with normal	Aβ42)	AD = 11 (without the	typical AD CSF	biomarker pattern)	MCI = 17	Subgroups were defined on	the basis of both CSF	Aβ42 and tau levels:	-30 bvFTD (excluding	patients with decreased	Aβ42)	-31 bvFTD (excluding	patients with decreased	Aβ42 and increased	tau or p-tau)	PPA = 99 (nfvPPA = 40,	svPPA = 38, lvPPA = 21)				FTD = 41	ALS = 25	FTD (caused by a	pathogenic mutation in	GRN, MAPT, or	C9orf72) = 101
		Controls				HC = 27								HC = 15																		HC = 35					HC = 46	HC = 71
	(continued)	First author (type of study)				t2:10 Al Shweiki, 2019	(cross-sectional study)							t2:11 Steinacker, 2018	(longitudinal study)																	t2:12 Steinacker, 2017	(longitudinal study)				t2:13 Wilke, 2016	(cross-sectional study)	t2:14 Meeter, 2016	(longitudinal study)

-Patients with serum NFL (Oxford cohort) in the highest tertile had a HR = 6.05 (95%

).

  UHDRS Unified Huntington's Disease Rating Scale-Total Motor Score *TRACK-HD is a multinational prospective observational study of HD that examines clinical and biological findings of disease progression in individuals with premanifest HD and early-stage HD

	AUTHOR'S PROOF
	-The present study included 132 autopsy NFL as Staging and Prognostic Biomarker cases with rapidly progressive neurological syndromes. -Cases with a wide range of neuropathological alterations, including cerebrovascular disease, inflammation (meningoencephalitis), primary age-related tauopathy, non-AD tauopathies or Lewy body pathology -39 CJD diagnoses were neuropathologically verified. -Patients with other diseases included AD Studies in AD and MCI-AD patients found a correlation between plasma NFL concentration and cognitive impair-ment, MRI hippocampal volume loss and brain atrophy, and cerebral 18F FDG-PET hypometabolism [76, 81-87]. Moreover, higher plasma NFL levels predicted faster cogni-(n = 12), MCI (n = 4), FTD (n = 3), tive deterioration and a higher rate of brain atrophy and hypometabolism in MCI patients over time [77]. Baseline plasma and CSF NFL levels were similarly associated with short-term declines in imaging measures of neurodegenera-and NPH (n = 1) tion and with global cognitive worsening, but not with
	change in amyloid ligand retention on PET [35], differently
	from CSF t-tau concentration that critically depends on ce-
	sCJD = 33 Serum NFL in differentiating O gCJD = 9 C N U R GSS mutation carrier = 1 Demented controls R (DCo) = 20 E C T E D sCJD + gCJD vs HC + DCo, AUROC = 0.95 P R rebral Aβ burden [88]. Instead, increased plasma NFL was related to baseline and longitudinal glucose hypometabolism, which is an unspecific neurodegeneration marker, in AD-O O F related regions of MCI Aβ+ individuals [87].
	HC = 40
	t2:41 Steinacker, 2016	(cross-sectional study)

Aβ42 amyloid β-peptide 1-42; AD Alzheimer's disease; ADNI Alzheimer's Disease Neuroimaging Initiative; ALS amyotrophic lateral sclerosis; AP atypical parkinsonism; APOE apolipoprotein E; AUROC area under the receiver operating curve; bvFTD behavioral variant of frontotemporal dementia; c-MSA cerebellar variant MSA; CBD corticobasal degeneration; CDR cognitive dementia rating; CI confidential interval; CIDP chronic inflammatory demyelinating polyneuropathy; CJD Creutzfeldt-Jakob disease; CSF cerebrospinal fluid; C9ORF72 chromosome 9 open reading frame 72; DLB dementia with Lewy body; FTD frontotemporal dementia; FTD-MND frontotemporal dementia with ALS; gCJD genetic Creutzfeldt-Jakob disease; GBS Guillain-Barre syndrome; GENFI genetic FTD initiative; GSS Gerstmann-Straussler-Scheinker disease; GRN progranulin; HC healthy controls; HD Huntington's disease; HR hazard ratio; HSP hereditary spastic paraplegia; HTT huntingtin; lvPPA logopenic variant PPA; MAPT microtubule-associated protein tau; MCI mild cognitive impairment; MCI-AD MCI converters to AD; MDS-UPDRS Movement Disorder Society-Unified Parkinson's Disease Rating Scale; MND motor neuron disease; MSA multiple system atrophy; NFL neurofilament light chain; nfvPPA non-fluent variant PPA; NINDS-SPSP National Institute of Neurological Disorders and Stroke and the Society for PSP; NPH normal-pressure hydrocephalus; PD Parkinson's disease; PLS primary lateral sclerosis; PPA primary progressive aphasia; PMA primary muscular atrophy; PPD primary psychiatric disorders; PRNP prion protein; PSP progressive supranuclear palsy; p-tau phospho-tau; sCJD sporadic Creutzfeldt-Jakob disease; SAOA sporadic adult-onset ataxia; SARA Scale for the Assessment and Rating of Ataxia; SCA spinocerebellar ataxia; SMA spinal muscular atrophy; svPPA semantic variant PPA; In a longitudinal analysis of NFL plasma levels in a large cohort of subjects enrolled in the Alzheimer's Disease Neuroimaging Initiative (ADNI), Mattsson and colleagues [82] found increasing rates of NFL changes from preclinical AD stage to frank AD dementia through prodromal phase, suggesting NFL as a dynamic biomarker sensitive to AD disease progression. Of note, longitudinal NFL variations correlated with several baseline AD-related features (CSF biomarkers, imaging measures, and cognition) in the whole population, though with significant differences regarding clinical stage. Actually, the strictest associations were reported in MCI participants. Longitudinal NFL level was generally increased in patients who were classified as N+ (using temporal brain atrophy as N-indicator) and in those who were only T+. Therefore, NFL might reflect a neurodegenerative process that occurred independently from Aβ pathology. Noteworthy, the NFL rate of change, rather than NFL absolute concentration, was subject to a significant increase in mutation carriers compared with non-carriers. Moreover, the NFL rate of change strongly correlated with longitudinal precuneus cortical thinning in both symptomatic and presymptomatic mutation carriers [89].

Table 2

 2

	) [105,

individuals in comparison with psychiatric patients affected by depression, schizophrenia, and bipolar disorders

[106]

, in which bvFTD misdiagnosis is common

[100]

. The diagnostic accuracy to differentiate bvFTD from psychiatric disorders was 364 above 80% (Table

2

), independently from the specific psychi-365 atric condition (mood or psychotic disorders) [106, 107]. 366 Only one study compared both FTD and AD subjects, 381 tween increased NFL concentration and TDP-43 pathology. 382 Further confirmation of this hypothesis is supported by ob-383 serving that FTD forms expected to be TDP-43 positive 384 (C9ORF72 and GRN mutation carriers, or svPPA phenotype) 385 reported higher concentration of this biomarker than FTD 386 subtypes expected to be tau positive (MAPT mutation carriers

387

and nfvPPA)

[START_REF] Meeter | Neurofilament 1158 light chain: a biomarker for genetic frontotemporal dementia[END_REF] 99, 104]

. 388 NFL as Staging and Prognostic Biomarker 389 In different studies, serum NFL increases overtime in FTD 390 subjects, independently from the phenotypes, with the excep-391 tion of lvPPA [105, 109]. A longitudinal Mini-Mental State 392 Examination Q3 (MMSE) decline was correlated with baseline 393 serum NFL levels [107]. Disease duration was not associated 394 with NFL concentration although one study reported a poor 395 survival in FTD subjects in the higher tertile of serum NFL 396 levels (Table

2

). 397 Additionally, peripheral blood NFL concentrations appeared 398 to reflect specific regional brain atrophy related to clinical phe-399 notypes (PPAs or bvFTD). In bvFTD patients, serum NFL was 400 associated with a low cognitive score and a reduction in whole-403 Serum NFL was correlated with baseline cognitive impair-410 NFL as Risk/Screening Biomarker 411 Noteworthy, serum NFL was higher in genetic FTD with a full-blown clinical picture but not in presymptomatic carriers 426 5 years from symptom onset. To date, there is no definitive 427 diagnostic test for ALS, and confirmation of diagnosis is 428 based on clinical findings, electromyography results, and ex-429 clusion of mimics [52]. Despite efforts to increase the sensi-430 tivity of diagnostic criteria, often the diagnosis is made only 431 after the onset of symptoms for both sporadic and familial 432 ALS [111, 112]. An early diagnosis would be paramount, 433 since it was observed that the benefit of riluzole is related to 434 its early administration. 435 A large body of research exists on neurochemical ALS 436 biomarkers [113, 114], among which phosphorylated neuro-437 filament heavy chain (pNFH) and NFL have been postulated 438 as the most interesting candidates [115, 116]. This is not sur-439 prising, given the axonal impairment that characterizes the 440 disease already at the early stage, with a large release of NF 441 in CSF [117]. Additionally, previous studies indicate cyto-442 skeletal proteins as one of the key factors contributing to neu-443 rodegeneration in ALS [118-123]. Other evidences provide 444 additional support that NFL aggregation is an early event in 445 motor neuron disease [124], and that NFL is involved in the 446 aggregation and neurotoxicity of other proteins in motor neu-447 rons [125]. At the same time, elevated NFL levels in ALS may 448 be explained by the higher content of axonal proteins in motor 449 neurons compared to other neuronal populations [126]. 450 Nonetheless, several data highlighted that NFL is able to dis-458 CNS, a high correlation between CSF and blood NFL concen-459 trations has been reported [132]. Moreover, at odds with other 460 neurodegenerative diseases such AD, NFL concentrations do 461 not correlate with age in ALS individuals [133-135].

Table 2

 2

	)

Accordingly, in a MRI-based study, elevated CSF and serum NFL concentrations were significantly associated with lower diffusion tensor imaging (DTI) fractional anisotropy and increased radial diffusivity in the corticospinal tract of ALS patients, as well as with clinically UMN score burden [133]. Furthermore, serum NFL levels at recruitment or at the time of diagnosis predicted survival independently from other clinical variables and were negatively associated with disease duration (Table 2) [136, 138, 142]. Thouvenot and colleagues 529 NFL as Risk/Screening Biomarker 530 In contrast with AD, CSF and blood NFL levels are reported 531 normal in presymptomatic ALS mutation carriers (C9orf72, 532 SOD1, FUS/TLS, or TARDBP), but they increase suddenly 533 with symptom onset in symptomatic mutation carriers as dem-534 onstrated by Weydt and colleagues [143]. Furthermore, using 535 the parental age of disease onset as a proxy for assumed age of 536 clinical onset, any trend toward an increase of NFL concen-537 tration was observed in asymptomatic mutation carriers. 538 Despite recent longitudinal data on a large cohort of presymp-539 tomatic, SOD1 mutation carriers provided evidence that an 540 increase in CSF and blood NFL levels occurs at least 1 year 541 before of any clinical manifestations of the disease [144]. 542 NFL as Predictive Biomarker 543 It would be interesting to determine whether riluzole reduces 544 blood NFL levels over time given its neuroprotective effects 545 and, although minimally, its capacity to slow disease progres-557 treated with nusinersen, with a greatest benefit found in chil-558 dren who received treatment earliest during the course of dis-559 ease [145]. Nusinersen, an antisense oligonucleotide delivered

Table 2 )

 2 [156]. Conversely, blood NFL levels were not able

637 to accurately separate PD from HC. A subsequent study in 638 subjects with an uncertain diagnosis at the time of inclusion 639 confirmed similar results (Table 2) [158]. Nevertheless, recent 640 studies support the promise of plasma NFL as a diagnostic 641 biomarker also in PD, demonstrating relatively higher NFL 642 levels in cases vs controls [159] and a good diagnostic accuracy 643 in differentiating PD patients from HC (Table 2) [160]. 644 Moreover, higher serum NFL levels were found even at early 645 stages of the disease and in participants at risk of disease pro-646 gression (prodromal PD and symptomatic and asymptomatic

Table 2 )

 2 [160][161][162].

	PSP
	NFL as Diagnostic Biomarker
	Two other studies confirmed the diagnostic value of blood
	NFL in PSP patients showing good capability to discriminate
	between PSP and HC (Table 2) [163, 164]. In contrast, blood
	NFL is not suitable to separate PSP from other forms of AP
	[156] and similar levels are reported in patients with MSA and
	PSP [158].
	NFL as Staging and Prognostic Biomarker
	Greater baseline NFL levels in serum/plasma seem to correlate
	with disease severity and clinical progression in PSP patients,
	though with conflicting results. Such heterogeneity may re-
	flect differences in study design, since PSP patients have been
	evaluated as a separate group in some studies but not in others
	where PSP, MSA, and CBD patients have been combined as a
	whole group. Specifically, blood NFL levels positively corre-
	lated with motor symptom severity, evaluated as H&Y stage
	and with UPDRS III motor score, but not with disease dura-
	tion or other clinical assessments, in the AP group (including
	also MSA and CBD patients) [156]. Similarly, serum NFL
	concentration at baseline correlated with motor performances,
	measured with the International Cooperative Ataxia Rating
	Scale score and Tandem Gait Test in another cohort of AP
	patients including PSP [158].

  large cohorts of SCA-3 subjects and demonstrated higher Because of the high negative predictive value of elevated 999 blood NFL concentrations in excluding PD, this candidate 1000 biomarker can represent a valid screening tool for clinicians 1001 in the early differential diagnosis between PD and AP in cases 1002 with confounding clinical presentations. In contrast, blood 1003 NFL measurements do not suffice to differentiate PD patients 1004 from controls and cannot be used to separate PSP, MSA, and 1005 CBD from each other. Nevertheless, blood NFL may be a 1006 prognostic tool in clinical practice in both PD and AP patients. 1007 Currently, to the best of our knowledge, there are no studies 1008 investigating blood NFL in patients with DLB, and few stud-1009 ies are available for PDD patients.

	AUTHOR'S PROOF AUTHOR'S PROOF AUTHOR'S PROOF
		Mol Neurobiol
	897 998	could have a role in this context. Previous reports on CSF colleagues [188]. However, despite a trend toward increasing	stable in a subgroup of 14 FA patients who received a 2-year CJD contribute to support ALS prognosis. Other confounding var-	849
	898	NFL indicate elevated concentrations in HD subjects [19, concentrations of both tau and NFL over the last 12 months	follow-up evaluation [169]. iables, such as the discordant disease progression in the dif-
	899	173, 174]. This is not surprising because mtHTT interacts with before death, at variance with tau, no association was found	Conversely, serum NFL concentration increased with dis-CJD is the most common human prion disease. Approximately ferent clinical subtypes of ALS, should be elucidated in fur-	850
	900	other proteins altering their function and finally leading to between serum NFL concentration and speed of decline on the	ease severity in a large cohort of SCA-3 patients, including 85% of cases are sCJD, but in a minority of cases, CJD can be ther studies. Actually, blood NFL could help better stratify the	851
	901	abnormal protein aggregation and impaired axonal transport Medical Research Council (MRC) Prion Disease Rating Scale	manifest and preclinical individuals, and correlated with both genetically determined (gCJD) [181]. The disease is a rapidly multifaceted clinical presentation of ALS phenotypes.	852
	902	[175]. Furthermore, the level of misfolded mtHTT protein [188]. Also, a recent study strengthens the tight association of	clinical scales (according to SARA and International progressive and fatal neurodegenerative condition, whose differ-However, further studies are needed to confirm its prognostic	853
	903	correlates with NFL concentration in CSF, thus suggesting a plasma tau levels with the rate of disease progression and	Cooperative Ataxia Rating Scale (ICARS) scores) and reduc-ent phenotypes depend, at least in part, by polymorphisms on value. In contrast with genetic forms of AD, blood NFL con-	854
	904	contemporary releasing of both proteins from damaged neu-survival time in sCJD [190].	tion of cerebellar and brainstem volume [167]. Preclinical the gene encoding prion protein (PrP) [182]. Diagnosis is fre-centrations are not increased in presymptomatic ALS muta-	855
		rons [176].	SCA-3 group was divided in early and late preclinical sub-quently tardive and relies on clinical World Health Organization tion carriers appearing to tightly link to the symptomatic phase	856
	905	NFL as Risk/Screening Biomarker	groups using the median predicted number of years to onset of (WHO) criteria supported by detection of the 14-3-3 protein of the disease.	857
			manifest disease. Serum NFL concentrations resulted higher and, more recently, t-tau in the CSF [66, 183, 184]. Also, CSF Another NDD where blood NFL increases result impres-	858
	906	NFL as Staging and Prognostic Biomarker gCJD forms are linked to mutations in the prion protein gene	in manifest than preclinical SCA-3 subjects and in late pre-NFL recently demonstrated to be a reliable biomarker in the sively is CJD. Because of early diagnosis remains challeng-	859
	907	(PRNP) inherited with an autosomal dominant pattern and	clinical SCA-3 subjects compared with early preclinical SCA-CJD diagnostic workup. Although few studies explored its role ing, NFL might be a reliable screening blood-based biomarker	860
	908	In the first retrospective study investigating NFL concentra-variable penetrance [191]. The relationship between genotype	3 individuals. However, no differences were observed be-as a biomarker in CJD patients so far, CSF NFL levels are with a potentially high negative predictive value for CJD sub-	861
	909 910 911 912 913 914 915	tions in the blood of premanifest HD (preHD) and early-stage HD patients enrolled in the TRACK-HD cohort, Byrne and colleagues [177] showed higher baseline NFL levels in 201 HTT mutation carriers, including 58 with early premanifest and 46 subjects with late premanifest disease, than in controls. Moreover, NFL concentration reflected baseline motor and cognitive deficits in HD patients and differed significantly and phenotype remains a matter of debate, and index cases do not always have a family history [192]. Under this scenario, the discovery of biomarkers serving as surrogates of outcome in clinical trials may be crucial because PRNP mutation car-riers might benefit from a presymptomatic intervention. PrP-lowering therapeutics are now in preclinical development, and to this end, CSF total PrP has demonstrated to be a strong	tween early preclinical subjects and HC [167]. Despite CAG repeat count is a well-known prognostic factor for SCA-3 and FA, a correlation between serum NFL and CAG repeat lengths has been inconsistently reported [168, 169]. NFL as Risk/Screening Biomarker P R O O F significantly increased in CJD (including those with more slow-ly progressive and atypical disease course) compared with AD, FTD, other NDDs (dementia), and controls, indicating a massive synaptic degeneration and neuroaxonal damage in CJD [22, 23, 185, 186]. Additionally, it is noteworthy that NFL concentra-tions in CSF appear highly variable among different sCJD sub-types, with higher NFL levels in those with more rapidly P R O O F jects, ruling out more common and less aggressive neurode-generative dementia, such as AD. Definitely, blood NFL could be of usefulness as a first-step examination to promptly detect CJD during its prodromal phase and to start future disease-modifying treatments. On the other hand, preliminary findings do not support a potential use of NFL as a predictor of longitudinal disease progression in CJD, and its specificity P R O O F	862 863 864 865 866 867 868
	733 734 735 736 737 738 739 740 741 742 934 933 932 931 930 929 916 917 922 923 924 925 926 927 928	levels in both preclinical and manifest SCA-3 individuals compared with HC [167]. Serum NFL levels discriminated manifest SCA-3 from HC with excellent accuracy, and the diagnostic performance remained good in distinguishing pre-quent occipital gray matter atrophy and widespread white mat-axonal degeneration and vascular changes in elderly. matched controls [168]. in regions known to be affected in HD and predicted subse-blood NFL concentrations correlate with aging due to a subtle sion in the ATXN3/MJD1 gene, compared with aged-tions of NFL in plasma were associated with lower volume frequently exhibiting vascular comorbidities. In general, mon autosomal recessive ataxia caused by CAG repeat expan-robust marker of brain atrophy. Notably, higher concentra-especially in subjects with NDDs, being generally old and affected by Friedreich's ataxia (FA), which is the most com-reduction, highlighting the value of NFL as a dynamic and thus representing a potential relevant methodological bias, plasma NFL concentrations resulted higher also in patients longitudinal MRI cortical thinning and white matter volume sociated with renal and hepatic dysfunctions remain unknown, clinical SCA-3 subjects from HC (Table 2) [167]. Recently, U N C O R R E C T E D huntingtin (mHTT) and extensive degeneration of neurons Moreover, serum NFL values have been elevated since the ical AD before the conversion phase. Moreover, NFL cap-(HTT) gene, leading to the expression of neurotoxic mutant than previously reported investigations (Table 2) [189]. in individuals with MCI at high risk and identify preclin-polyglutamine encoding CAG repeats in the huntingtin ation between prion and other disease cases resulted lower In AD, blood NFL may predict progression to dementia The etiological basis is the deleterious expansion of However, in this study, the diagnostic value in the differenti-psychiatric disorders. movements, psychiatric symptoms, and cognitive decline. AD cases) showing a rapidly progressive neurological picture. an underlying neurodegenerative disease in individuals with erative disease with the typical manifestations of involuntary of 132 pathologically classified patients (sCJD, gCJD, and NFL is assumed to be a promising screening tool to rule out In their study on 133 SCA-3 patients, Li and colleagues [167] demonstrated higher serum NFL concentrations in 26 preclin-ical ATXN3 mutation carriers (patients with SARA score < 3) compared with controls. Moreover, a correlation between mo-tor symptoms, neuroimaging markers, and serum NFL was found in all ATXN3 mutation carriers, suggesting that NFL may serve to track neurodegeneration and disease progression already in pre and prodromal SCA-3 phases. HD Among the most common neurodegenerative diseases, HD is unique, since the major part (≈ 99%) of individuals presenting a HD phenotype have a mutation in the same gene [170]. Indeed, HD is an autosomal dominant inherited neurodegen-with increasing disease stage. Positive associations were found between plasma NFL concentration, age, and CAG triplet repeat counts, with higher CAG lengths being associat-ed with earlier and steeper increases in plasma NFL [177]. Therefore, NFL is the first biofluid marker showing a direct relationship with a causative gene expansion [178]. Of note, baseline plasma NFL predicted rates of brain at-rophy, cognitive decline, and worsening of functional ability and motor performance in mtHTT carriers [177]. Interestingly, it was closely associated with the rate of whole-brain atrophy than with the rate of striatal one, suggest-ing that plasma NFL reflects more the rate of global neuronal degeneration than that of a specific brain area [177]. Additionally, in the same TRACK-HD cohort, Johnson and colleagues [179] showed voxel-wise region-specific associa-tions between plasma NFL levels and both cross-sectional and progressing disease [187]. Importantly, plasma NFL correlates with CSF NFL concentration and recent studies suggest that blood NFL can accurately reflect the massive neurodegeneration in CJD patients. NFL as Diagnostic Biomarker Diagnostic accuracy of serum NFL for discrimination be-tween CJD and controls was excellent (Table 2) [185]. These findings have been independently replicated in other two studies. Serum NFL distinguished patients from controls with 100% sensitivity and 100% specificity in 45 sCJD pa-tients enrolled in the National Prion Monitoring Cohort [188]. Noteworthy, Kovacs and colleagues [189] reported high sen-sitivity and specificity of plasma NFL concentration in dis-criminating CJD subjects from non-CJD controls in a cohort U N C O R R E C T E D candidate fluid biomarker showing stable low levels in an ongoing natural history study including presymptomatic mu-vestigated across published studies. Other variables possibly affecting its modification in peripheral blood, including sys-temic comorbidities and concomitant drug therapies, were not taken into account. Liver and renal clearance as well as blood cell counts and plasma protein composition could affect bio-marker concentrations [194]; however, these factors were not investigated. Indeed, blood NFL concentration alterations as-should be further substantiated in comparison with other high-ly aggressive forms of NDD. All reported studies showed a good or even excellent diag-nostic performance of blood NFL in distinguishing patients with neurodegenerative disorders from HC. The potential con-tribution of this biomarker candidate to discriminate between different dementia disorders remains ambiguous given the lack of pathognomonic specificity. However, mounting data suggest that blood NFL could be a useful diagnostic tool in the diagnostic workup of FTD, to distinguish FTD (especially FTD with a TDP-43 pathology) from AD patients and to iden-tify PPA with a likely underlying AD pathology, including lvPPA. Moreover, it can represent a biomarker tracking the disease progression and potentially identifying the transition the genetic forms of the disease. Most importantly, serum U phase from the presymptomatic to the symptomatic stage of N C O R R E C T E D	888 887 886 885 884 883 869 870 871 872 873 874 875 876 877 878 879 880 881 882
	935	ter reduction over the 3-year follow-up, independently of age However, such an association was clearly absent in CJD,	primarily occurring in the striatum and cortex [171]. The dis-early phases of the disease, suggesting a possible role as a tures early neurodegenerative changes in presymptomatic	889
	743 936	NFL as Staging and Prognostic Biomarker and CAG length repeats [179]. NFL increased significantly ALS, and AP. This suggests that the probable contribution	ease usually starts in midlife, with age of onset inversely cor-screening biomarker [188]. Conversely, serum NFL concen-familiar AD mutation carriers in which blood NFL con-	890
	937	from baseline both in individuals with premanifest HD and of aging on NFL concentrations and neurodegeneration be-	relating to CAG repeat number [171]. Although the cause is tration overlapped between ALS and CJD patients in a recent centrations correlate with the predicted time to symptom	891
	744 938	Serum NFL in c-MSA patients does not seem to correlate with in those with manifest HD [179]. Remarkably, in a subsequent comes trivial in highly aggressive forms of NDDs [195].	known, disease-modifying treatments are not yet available. In prospective study, even though the size of CJD group was onset. Further studies are crucial to calculate the negative	892
	745 939	clinical disease severity (as assessed by the Scale for the study, Byrne and colleagues [180], combining CSF/plasma Blood NFL concentrations are reported to be massively	HD, a reliable genetic test confirms a clinical diagnosis in very small [139]. predictive value of blood NFL as a screening tool in large	893
	746 940	Assessment and Rating of Ataxia (SARA)) or disease progres-NFL and CSF mutant huntingtin protein (mHTTp) as biofluid elevated in ALS patients, even in early disease stages, indicat-	symptomatic people or predicts disease onset in asymptomatic and selected cohorts of individuals at risk of neurodegen-
		biomarkers, demonstrated that NFL levels were more accurate	mutation carriers [172]. As a consequence, a novel biomarker
		than mHTTp to discriminate between premanifest and mani-	should be directed to track disease progression and predict a
		fest HDs and correlated with severity of symptoms better than	treatment response to targeted therapies. Although not conclu-
	750 944	duration [169]. Moreover, serum NFL concentration remains mHTTp in manifest HD. progression in ALS, suggesting that peripheral NFL could	sive, the results of the available studies display that blood NFL

747

sion

[165]

. Similarly, a recent study in 99 patients with genet-748 ically confirmed FA did not find a correlation with disease 749 severity (as defined by SARA score), age at onset, or disease 894 NFL as Staging and Prognostic Biomarker 895 Longitudinal changes in serum tau and NFL levels were in-896 vestigated in the aforementioned study of Thompson and 918 tation carriers and normal controls [193]. 919 Conclusions 920 The impact of physiological variables on blood NFL concen-921 tration, such as sex and age, has not been systematically in-941 ing its value as an efficacious, yet unspecific, biomarker in the 942 differential diagnosis of ALS from ALS mimics. Moreover, 943 blood NFL concentrations can reflect disease severity and/or eration and AD, such as individuals with subjective memory complaints and/or decline, late-onset psychiatric disorders, cerebrovascular disease, and diabetes, as well as in aging and elderly individuals in general.

JrnlID 12035_ArtID 2035_Proof# 1 -06/08/2020

Ladogana A, Kovacs GG (2018) Genetic Creutzfeldt-Jakob dis-

Kim MO, Takada LT, Wong K, Forner SA, Geschwind MD

Vallabh SM, Minikel EV, Schreiber SL, Lander ES (2020)

Q1. Please check if the affiliations are captured and presented correctly. Q2. Please check if the section headings are assigned to appropriate levels. Q3. Please check the provided expanded form for the abbreviation "MMSE" if correct. Q4. Please check the word "diseases" inserted after the word "neurodegenerative," which can be found in the sentence starting "Finally, serum NFL levels were reported relatively lower," for completeness if correct. Q5. References [START_REF] Vinther-Jensen | Selected CSF biomarkers indicate no evidence of early neuroinflammation in Huntington disease[END_REF]174], [START_REF] Van Eijk | CSF neurofilament proteins levels are elevated in sporadic Creutzfeldt-Jakob disease[END_REF]186], [200,203] based on original manuscript we received were identical. Hence, the latter was deleted and reference list and citations were adjusted. Please check if appropriate. Q6. Please provide complete bibliographic details of this reference.