Modelling neurodegenerative disease using in vitro assays

Neurodegenerative drug development is held back due to the lack of appropriate models that help accelerate the pace at which novel therapeutic candidates move through the drug development pipeline. As such, in vitro assays prove to be both cost and time-effective, as they provide both target-specific insights and offer relatively higher throughput capabilities compared to in vivo models. However, in vitro models should not only be seen as a means to advance your therapeutic candidate to the in vivo stage of preclinical testing, but can also be viewed as a complementary tool to in vivo efficacy studies.  

Neurodegenerative disease research can extensively benefit from relatively high-throughput, biologically accurate in vitro assays that would allow for fast compound screening and decision-making. With this goal in mind, InnoSer offers an extensive neurodegenerative disease platform consisting of both in vitro and in vivo neurology contract research services.  

In this blog post, we present a selected number of in vitro assays from our portfolio that can be used to screen neurodegenerative disease-modifying test compounds. If you do not find your exact in vitro assay of choice on our website, InnoSer’s neurology study experts are always open to work with you on a customised study designs. 

Alzheimer’s and Parkinson’s disease in vitro assays  

The disease pathophysiology in two most prevalent neurodegenerative diseases, namely Alzheimer’s disease (AD) and Parkinson’s disease (PD), follows distinct underlying disease mechanisms. However, the primary disease mechanism in both AD and PD is characterised by the formation and presence of fibril aggregates.  

The histopathological hallmarks of AD are extracellular plaques composed of amyloid beta (Aβ) fibrils and intracellular inclusions of the protein Tau (neurofibrillary tangles). Similarly, treating neuronal and/or microglial cells in vitro with fibrillar Aβ (Figure 1) Tau (internal data) leads to fibril-induced cell toxicity. Furthermore, treatment of microglial cells with fibrillar Aβ induces an increase in phagocytic capacity that is significantly increased upon Aducanumab co-treatment (Figure 2), in line with previous research showing that Aducanumab increased microglial cells’ phagocytic capacity by stimulating the binding to Aβ-aggregates (1). 

PD is characterized by specific histopathological hallmarks observed in post-mortem brain tissue, including the presence of intracellular inclusions called Lewy bodies, consisting of the protein alpha-synuclein (α-syn), and cell death in the substantia nigra (SN). α-syn inclusions are the main histopathological hallmark correlating with clinical symptoms of not only PD, but also other synucleinopathies such as dementia with Lewy bodies (DLB), and multiple systems atrophy (MSA). Accordingly, treating neuronal and/or microglial cells in vitro with preformed α-syn fibrils leads to fibril-induced cell toxicity (Figure 3). Similarly, treatment of microglial cells with αSyn fibrils induces an increase in phagocytic capacity (Figure 4). Several lines of evidence suggest that glutamate is also implicated in the development of PD due to excitotoxicity, which we observe in our in vitro neurotoxicity assays as well (internal data).  

FIGURE 1. Amyloid-β-induced neurotoxicity in vitro assay can be used to test novel Alzheimer’s disease compounds’ efficacy in rescuing cell viability. 24-hour treatment with Aβ–fibrils induces toxicity in neuronal (SH-SY5Y) and microglial cells (HMC3). Data represent mean± SEM of n=4 per condition (*P=0.05, **P=0.005, ***P=0.001, ****P<0.0001 vs 0 µM). Cell viability was assessed using an MTT assay according to the manufacturer’s instructions.  

FIGURE 2.  Amyloid-β-induced phagocytosis assay can be used to test novel Alzheimer’s disease compounds’ efficacy in stimulating the clearance of Aβ fibrils. Aβ fibril treatment induces significant increase in HMC3 cells’ phagocytic capacity of pHrodo-labelled fibrils in comparison to no no fibril treatment. Simultaneous treatment of 0.1 µg/ml Aducanumab and 5.0 µM fibrils induces a significant increase in phagocytic capacity in comparison to 5.0 µM fibril treatment alone. (*P=0.05, **P=0.005, ***P=0.001, ****P<0.0001 vs 0 µM). Data represent mean± SEM of n=3 per condition. Phagocytic capacity of microglial cells was assessed using the IncuCyte live analysis cell system. The phagocytic capacity of the pHrodo-labelled fibrils was quantified as the Relative phagocytosis Integrated Intensity (RCUx µm2 /Image), defined as a normalized value relative to the initial fluorescent intensity at a specific time point (t) subtracted by the initial fluorescent intensity at time point zero (t=0). 

FIGURE 3. ⍺–synuclein-induced neurotoxicity in vitro assay can be used to test novel Parkinson’s disease compounds’ efficacy in rescuing cell viability. 24-hour treatment with ⍺-synuclein fibrils induces cell death in SH-SY5Y cells and HMC3 cells (*P=0.05, **P=0.005, ***P=0.001, ****P<0.0001 vs 0 µM). Data represent mean ± SEM of n=4 per condition. Cell viability was assessed using an MTT assay according to the manufacturer’s instructions. 

FIGURE 4. ⍺–synuclein-induced neurotoxicity in vitro assay can be used to test novel Parkinson’s disease compounds’ efficacy in stimulating the clearance of ⍺–synuclein fibrils. 2.5 µM α-Syn fibril treatment induces significant increase in HMC3 cells’ phagocytic capacity of pHrodo-labelled fibrils in comparison to no fibril treatment. (*P=0.05, **P=0.005, ***P=0.001, ****P<0.0001 vs 0 µM). Data represent mean ± SEM of n=3 per condition. Phagocytic capacity of microglial cells was assessed using the IncuCyte live analysis cell system. The phagocytic capacity of the pHrodo–labelled fibrils was quantified as the Relative phagocytosis Integrated Intensity (RCUx µm2 /Image), defined as a normalized value relative to the initial fluorescent intensity at a specific time point (t) subtracted by the initial fluorescent intensity at time point zero (t=0). 

To conclude, in vitro neurodegenerative disease models focusing on fibril-induced cell toxicity and phagocytosis can serve as a highly efficient method of testing multiple disease-modifying test compounds before screening in vivo. Besides focusing on fibril-induced toxicity and phagocytosis, InnoSer offers a larger variety of in vitro screening assays using human, rodent cell lines and primary cells with readouts focusing on fibril aggregation, neuroinflammation and neurotoxicity.