Neurodegeneration​ Overview

Neurodegenerative diseases, such as Alzheimer’s (AD) or Parkinson’s disease (PD), are chronic and debilitating disorders that progressively cause degeneration and/or death of neuronal cells. These conditions often occur in elderly populations and symptoms can include memory loss, issues with movement, behavioral changes and difficulties with everyday tasks. 

Many neurodegenerative disorders can be characterized by the abnormal formation and aggregation of proteins in the brain, however determining the complex pathological mechanisms involved in both the onset of the disease and its progression is challenging. Improved in-vitro models that recapitulate the chronic nature of the disease, in combination with advanced cell models such as primary cells or iPSCs, may increase our insight into disease pathology, the mechanisms at play and contribute to drug discovery.


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Key Advantages

Investigate peptide- and drug-induced effects

Study disease-relevant neurotoxicity models using non-perturbing reagents to gain insight into morphological changes

Figure 1. Chronic in-vitro model of aggregated tau peptide-induced effects on neurite length. Primary rat cortical neurons (rCortical, Sartorius) were seeded in PDL coated 96-well plates at 20,000 cells/well and placed in the Incucyte® for the duration of the studies. 8 days post-seeding, cells were treated with solubilized or aggregated Tau (Heparin 4:1 ratio, 37°C with sporadic rotation for 5d). Automatic segmentation and quantification of neurite outgrowth was performed. Aggregated Tau yielded a concentration-dependent decrease of neurite length (80 ± 5 vs. 166 ± 3 %, 2-3 replicates) and Non-aggregated only yielded substantial inhibition of neurite formation at 150 µg/mL.

Gain dynamic insights

Multiplex kinetic readouts of neurite length and cell health for deeper understanding

Figure 2. Oxidopamine (6-OHDA) treatment increases cell death and decreases neurite outgrowth in a kinetic model of Parkinson’s disease. Co-cultures of rat primary striatum and astrocytes were seeded in PDL coated 96-well plates (20,000 and 15,000 cells/well, respectively) and infected with Incucyte® Neurolight-Orange (Sartorius). 10 days post-infection cultures were treated with 6-OHDA (2 – 500 µM) in media containing the apoptotic marker Incucyte® Annexin V Green (0.5%; Sartorius). Fluorescent images were captured in real-time using the Incucyte® live-cell analysis system. Representative images shown comparing 6-OHDA treatment (19, 56, and 167 µM) to vehicle at 21 days post-treatment. Time-courses and drug-response curves show a concentration-dependent decrease in neurite length (orange) with the corresponding increase of Annexin V signal (green), showing the drugs detrimental effect. Data is presented as Mean ± SEM (3 replicates).

Figure 3. Brain region specificity in a model of Parkinson's disease: 6-OHDA selectively affects substantia nigra and striatal neurite outgrowth compared to cortical regions. Co-cultures of rat primary striatum, nigra or cortical neurons and astrocytes were seeded in PDL-coated 96-well plates (20,000 neuronal and 15,000 astrocytic cells/well, respectively) and infected with Incucyte® Neurolight-Orange (Sartorius). 10 days post-infection cultures were treated with 6-OHDA (56 – 500 µM). Fluorescent images were captured in real-time using the Incucyte® live-cell analysis system. Bar graphs and drug-response curves show the selective profile effect of the drug to the neurite length (orange) of the different brain regions. Vehicle data demonstrates differential development of neurite formation across the brain regions. Data is presented as Mean ± SEM (3 replicates).

Assess activity in chronic disease models

Obtain insights into neuronal activity in relevant neurodegenerative disease models to assess health of neuronal networks

Figure 4. Functional assessments revealed Tau induced loss of neuronal activity whilst retaining connectivity. Rat cortical neurons and rat astrocytes (Neuroprime®, Sartorius) were seeded as a co-culture (25,000 and 15,000 cells/well, respectively) in PDL coated 96-well plates. Neurons were infected with the genetically-encoded calcium indicator Incucyte® Neuroburst-Orange (Sartorius) to monitor spontaneous neuronal activity over time through measuring calcium fluctuations. Images were taken every 24 hours in the Incucyte® system (3-minute scans at 3 frames per second). Once mature, functional networks had formed (14 days), cells were treated with either the AD-related peptide tau (aggregated, 300 μg/mL) or the protein phosphatase inhibitor OKA (50 nM). Images show the active range (maximum/minimum fluorescence) over a complete scan. Calcium traces represent calcium fluctuation of all active objects within the field of view. Bar graphs provide the quantification of the number of active objects (1/image) and the correlation (connectivity). This data shows that, compared to vehicle, tau treatment decreased the number of active nodes (1407 ± 94 objects/image vs. 920 ± 43 objects/image) and their mean intensity (13.7 ± 1.7 OCU vs. 7.2 ± 3.6 OCU), while not affecting correlation (0.95 ± 0.01 vs. 0.97 ± 0.01, 2 replicates). In contrast, OKA decreased the number of active nodes (180 ± 24 objects/image), mean intensity (3.8 ± 0.3 OCU) as well as correlation (0.27 ± 0.02, 6 replicates) compared to vehicle. Data presented as Mean ± SEM.

Utilize relevant cell-based models

Use your in-vitro neurodegeneration cell models of choice - compatible with iPSC-derived or primary cells, in mono- or co-culture.

Figure 5. Patient-derived AD iPSC models in 2D and 3D reveal distinct neurite development and spheroid formation. Healthy and AD (PSEN1 mutation) derived iPSC neurons (Axol Bioscience) were seeded in ReadySet + SureBond coated 96-well plates at 25,000 cells/well (2D) or ULA 96-well plates at 50,000 cells/well and centrifuged (250 g; 10 mins). Spheroids were allowed to form for 3 days (3D), and neuronal differentiation was induced as per supplier’s protocol (supplements A+B). 2D studies: neurite development was automatically quantified using the Incucyte® Software Analysis Module (Sartorius) for up to 15 days. Time-course showed that the patient derived AD iPSC line yielded a lower neurite length than the healthy control (48 ± 3 mm/mm2 vs. 80 ± 3 mm/mm2 respectively, 3 replicates). 3D spheroid growth: the difference in size was quantified in a non-perturbing manner using brightfield analysis for up to 15 days. Matrigel® (2.25 mg/mL) was added to selected wells at 6 days, and spheroid morphology and process development from the spheroid body was observed for a further 9 days. Time-course showed a distinct spheroid size for both cell lines. Pictures also show the differential process development capability for healthy and Alzheimer’s lines.

Ordering Information

Product

Qty.

Cat. No.

Incucyte® NeuroTrack Software Module

Each

9600-0010

Incucyte® Spheroid Analysis Software Module

Each

9600-0019

Incucyte® NeuroLight Orange Lentivirus

Two vials (0.45 mL/vial)

4808

Incucyte® NeuroLight Red Lentivirus

Two vials (0.45 mL/vial)

4807

Incucyte® NeuroPrime Orange Kit

Each

4760

Incucyte® NeuroPrime Red Kit

Each

4585

Incucyte® Annexin V NIR Reagent

1 vial

4768

Incucyte® rCortical Neurons

1 vial

4753

Incucyte® rAstrocytes

1 vial

4586


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Resources and Products

Application Notes

White Paper: Improving in-Vitro Models for Alzheimer’s Disease

White Paper: Improving In-Vitro Models for Alzheimer’s Disease

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White Paper: Unraveling the Complexities of Neurological Disease...

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Application Note: Long-Term Live Cell Visualization and Quantification of Neuronal Activity

Long-Term Live Cell Visualization and Quantification of Neuronal...

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Protocols

Incucyte® Annexin V NIR and Incucyte® NeuroLight Orange Multiplex...

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Incucyte® Single Spheroid Assay Protocol

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Incucyte® Neuronal Activity Assay

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Label-Free Neurite Analysis Protocol

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Fluorescence Neurite Analysis Protocol

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SFN 2019 - Improved Alzheimer's Disease Models Using Neuronal and...

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SFN 2017 | Long-Term Live Cell Visualization and Quantification...

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SFN 2014 - Kinetic, Label-Free Assays for Neurite Outgrowth in...

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