Åsa Sandelius

Comparison of three analytical platforms for quantification of the neurofilament light chain in blood samples: ELISA, electrochemiluminescence immunoassay and Simoa

Abstract Background: Neuronal damage is the morphological substrate of persisting neurological disability. Neurofilaments (Nf) are specific cytoskeletal proteins of neurons and their quantification has shown encouraging results as a biomarker for axonal injury. Methods: We aimed at comparing a widely used conventional ELISA for Nf light chain (NfL) with an electrochemiluminescence-based method (ECL assay) and a newly developed single-molecule array (Simoa) method in clinically relevant cerebrospinal fluid (CSF) and serum samples. Results: Analytical sensitivity was 0.62 pg/mL for Simoa, 15.6 pg/mL for the ECL assay, and 78.0 pg/mL for the ELISA. Correlations between paired CSF and serum samples were strongest for Simoa (r=0.88, p<0.001) and the ECL assay (r=0.78, p<0.001) and weaker for ELISA measurements (r=0.38, p=0.030). CSF NfL measurements between the platforms were highly correlated (r=1.0, p<0.001). Serum NfL levels were highly related between ECL assay and Simoa (r=0.86, p<0.001), and this was less visible between ELISA-ECL assay (r=0.41, p=0.018) and ELISA-Simoa (r=0.43, p=0.013). Multiple sclerosis (MS) patients had significantly higher serum NfL levels than controls when measured with Simoa (p=0.001) but not with the other platforms. Conclusions: We found Simoa to be more sensitive than ELISA or the ECL assay. Our results support the feasibility of quantifying NfL in serum; the results correlate with the more-established CSF NfL test. The highly sensitive Simoa technology deserves further studies in larger patient cohorts to clarify whether serum NfL could be used in the future to measure disease severity and determine prognosis or response to treatment interventions in neurological diseases.

Neurofilament light protein in CSF and blood is associated with neurodegeneration and disease severity in Huntington’s disease R6/2 mice

There is an unmet need to reliably and non-invasively monitor disease progression in preclinical Huntington’s disease (HD) models. As a marker of axonal damage, neurofilament light chain (NfL) has been suggested a marker for neurodegeneration. NfL concentrations in blood and CSF were recently shown to have prognostic value for clinical HD progression and brain atrophy. We therefore hypothesized that CSF and blood NfL concentrations could be useful preclinical HD markers, reflecting underlying pathology. To test our hypothesis we utilized the R6/2 mouse model of HD and measured NfL concentrations in CSF and serum using the ultrasensitive Single molecule array (Simoa) platform. In addition, we assessed HD mouse disease characteristics. We found robust increases of NfL in CSF and serum in R6/2 mice compared to wild-type littermates. CSF and serum concentrations of NfL were significantly correlated, suggesting similar marker potential of serum NfL. CSF and serum concentrations of NfL correlated with disease severity, as assessed by striatal volume and body weight loss. We here provide evidence that CSF and blood NfL concentrations can be used as accessible and reliable pre-clinical HD markers. This will be of potential use for monitoring HD mouse model disease progression and evaluating preclinical disease-modifying treatment response.

Serum concentrations of the axonal injury marker neurofilament light protein are not influenced by blood-brain barrier permeability

A blood biomarker to monitor individual susceptibility to neuronal injury from cranial radiotherapy could potentially help to individualize radiation treatment and thereby reduce the incidence and severity of late effects. An important feature of such a blood biomarker is that its concentration is not confounded by varying degrees of release from the brain into the blood across the blood-brain barrier (BBB). In this study, we investigated serum neurofilament light protein (NFL) concentrations in 21-day old mice following a single dose of cranial irradiation (8Gy). Cranial irradiation resulted in acute cell injury measured as a 12.9-fold increase in caspase activity 6h after irradiation; activation of inflammation measured by levels of CCL2 and increased BBB permeability measured by 14C-sucrose concentration ratios in brain and cerebrospinal fluid (CSF). Serum levels of NFL peaked at 6h after both anesthesia and cranial irradiation, but no timely correlation of serum NFL concentration with BBB permeability was found. Further, three groups of patients with different degrees of BBB impairment (measured as the CSF/serum albumin ratio) were investigated. There was no correlation between serum NFL concentration and CSF/serum albumin ratio (r=0.139, p=0.3513), however a strong correlation was found for NFL concentration in serum and NFL concentration in CSF (r=0.6303, p<0.0001). In conclusion, serum NFL appears to be a reliable blood biomarker for neuronal injury, and its concentration is not confounded by BBB permeability.

Expression and secretion of synaptic proteins during stem cell differentiation to cortical neurons

ABSTRACT Synaptic function and neurotransmitter release are regulated by specific proteins. Cortical neuronal differentiation of human induced pluripotent stem cells (hiPSC) provides an experimental model to obtain more information about synaptic development and physiology in vitro. In this study, expression and secretion of the synaptic proteins, neurogranin (NRGN), growth‐associated protein‐43 (GAP‐43), synaptosomal‐associated protein‐25 (SNAP‐25) and synaptotagmin‐1 (SYT‐1) were analyzed during cortical neuronal differentiation. Protein levels were measured in cells, modeling fetal cortical development and in cell‐conditioned media which was used as a model of cerebrospinal fluid (CSF), respectively. Human iPSC‐derived cortical neurons were maintained over a period of at least 150 days, which encompasses the different stages of neuronal development. The differentiation was divided into the following stages: hiPSC, neuro‐progenitors, immature and mature cortical neurons. We show that NRGN was first expressed and secreted by neuro‐progenitors while the maximum was reached in mature cortical neurons. GAP‐43 was expressed and secreted first by neuro‐progenitors and its expression increased markedly in immature cortical neurons. SYT‐1 was expressed and secreted already by hiPSC but its expression and secretion peaked in mature neurons. SNAP‐25 was first detected in neuro‐progenitors and the expression and secretion increased gradually during neuronal stages reaching a maximum in mature neurons. The sensitive analytical techniques used to monitor the secretion of these synaptic proteins during cortical development make these data unique, since the secretion of these synaptic proteins has not been investigated before in such experimental models. The secretory profile of synaptic proteins, together with low release of intracellular content, implies that mature neurons actively secrete these synaptic proteins that previously have been associated with neurodegenerative disorders, including Alzheimers disease. These data support further studies of human neuronal and synaptic development in vitro, and would potentially shed light on the mechanisms underlying altered concentrations of the proteins in bio‐fluids in neurodegenerative diseases. HighlightsHuman iPSC‐derived cortical neurons express and secrete synaptic proteins.NRGN, GAP‐43 and SNAP‐25 are first expressed and secreted by neuro‐progenitors.SYT‐1 is expressed and secreted by stem cells.Synaptic proteins are secreted the highest when mature cortical neurons are formed.

Cerebrospinal fluid GAP-43 in early multiple sclerosis

Background/Objective Novel biomarkers identifying and predicting disease activity in multiple sclerosis (MS) would be valuable for primary diagnosis and as outcome measures for monitoring therapeutic effects in clinical trials. Axonal loss is present from the earliest stages of MS and correlates with disability measures. Growth-associated protein 43 (GAP-43) is a presynaptic protein with induced expression during axonal growth. We hypothesized this protein could serve as a biomarker of axonal regeneration capacity in MS. Methods We developed a novel GAP-43 enzyme-linked immunosorbent assay for quantification in cerebrospinal fluid (CSF) and measured GAP-43 levels in 71 patients with clinically isolated syndrome, 139 MS patients and 51 controls. Results GAP-43 concentrations were similar in patients and controls. Nevertheless, GAP-43 levels were higher in patients with >10 T2-magnetic resonance imaging (MRI) lesions (p = 0.005). CSF GAP-43 concentrations correlated with CSF mononuclear cell counts (p = 0.031) and were inversely correlated with patient age (p = 0.038) with a trend for higher CSF GAP-43 concentrations in patients with gadolinium-enhancing MRI lesions and positive CSF oligoclonal immunoglobulin G status. Conclusion Our results suggest that axonal regeneration capacity is relatively preserved in early MS. CSF GAP-43 concentration is positively associated with markers of inflammation, suggesting possible inflammatory-driven expression of this growth-associated protein in early MS.

Cerebrospinal fluid markers of extracellular matrix remodelling, synaptic plasticity and neuroinflammation before and after cranial radiotherapy

Advances in the treatment of brain tumours have increased the number of long‐term survivors, but at the cost of side effects following cranial radiotherapy ranging from neurocognitive deficits to outright tissue necrosis. At present, there are no tools reflecting the molecular mechanisms underlying such side effects, and thus no means to evaluate interventional effects after cranial radiotherapy. Therefore, fluid biomarkers are of great clinical interest.

Neurofilament light chain as disease biomarker in a rodent model of chemotherapy induced peripheral neuropathy

Abstract The objective of this study is to test the feasibility of using serum neurofilament light chain (NfL) as a disease biomarker in Chemotherapy Induced Peripheral Neuropathy (CIPN) since this easy accessible biological test may have a large impact on clinical management and safety of cancer patients. We performed this preclinical study using a well‐characterized rat model based on repeated administration of the cytostatic drug vincristine (VCR, 0.2 mg/kg intravenously via the tail vein once/week for 4 times). Serial NfL serum concentration was measured using the in‐house Simoa NfL assay and peripheral neuropathy onset was measured by sensory and motor nerve conduction studies. Serum NfL measure in untreated and VCR‐treated rats demonstrated a steady, and significant increase during the course of VCR administration, with a final 4‐fold increase with respect to controls (p < .001) when sign of axonopathy and loss of intraepidermal nerve fibers were clearly evident and verified by behavioral, neurophysiological and pathological examination. This simple monitoring approach based on serum NfL concentration measures may be easily translated to clinical practice and should be considered as a putative marker of CIPN severity in a typical oncology outpatient setting. Further studies are needed to validate its utility in cancer patients treated with different neurotoxic drugs.

Elevated CSF GAP-43 is Alzheimer's disease specific and associated with tau and amyloid pathology

The level of the presynaptic protein growth‐associated protein 43 (GAP‐43) in cerebrospinal fluid (CSF) has previously been shown to be increased in Alzheimers disease (AD) and thus may serve as an outcome measure in clinical trials and facilitate earlier disease detection.

PRESYNAPTIC DEGRADATION IN ALZHEIMER’S DISEASE MEASURED BY NOVEL GAP-43 ELISA IN CSF

Commission, Joint Research Centre, Geel, Belgium; Roche Diagnostics GmbH, Penzberg, Germany; ADx NeuroSciences, Gent, Belgium; Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, Gothenburg, Sweden; Clinical Neurochemistry Laboratory, Sahlgrenska University Hospital, M€olndal, Sweden; Institute of Neuroscience and Physiology, The Sahlgrenska Academy at University of Gothenburg, M€olndal, Sweden; University of Pennsylvania, Philadelphia, PA, USA. Contact e-mail: sebastien.boulo@ec. europa.eu