Antibodies designed for neurodegenerative-disease research

Neurodegenerative diseases are a growing burden worldwide. Common conditions such as Parkinson’s and Alzheimer’s diseases, epileptic encephalopathy and amyotrophic lateral sclerosis (ALS) threaten the lives of millions of people, and current therapies are only minimally effective. Tremendous amounts of research time and money are being invested in this area, driven by an urgent need to understand these diseases at the molecular level.

Research in this field has already uncovered some biological insights. Most neurodegenerative diseases share common pathogenic mechanisms. Defects in axonal transport have been implicated in Parkinson’s and Alzheimer’s diseases, and there is evidence that vesicular transport across synapses — the junctions between neurons — is also impaired in many neurodegenerative conditions.

Antibodies are important reagents to further neuroscience research. Not only can they help researchers study proteins involved in the pathogenesis of these diseases, but they can also be used in biomarker tests for early detection. Thermo Fisher Scientific has developed antibodies against three critical targets: SNAP25, VAMP1 and OPTN. Both SNAP25 (synaptosomal-associated protein, molecular mass of 25 kDa) and VAMP1 (vesicle-associated membrane protein-1) are important members of the SNARE family of proteins, which are involved in vesicular transport of neurotransmitters across synapses¹. OPTN (optineurin) is a Golgi complex-associated protein that is involved in many intracellular processes including autophagy flux, which is disrupted in several neurodegenerative disorders². OPTN mutation has also been reported as a causative factor in glaucoma and ALS³.

Finding antibodies against these targets is only the first step. For them to be useful to the neuroscience community, the antibodies need to be well characterized, highly specific, and certain to work in relevant applications such as western blotting and cell and tissue immunofluorescence. As such, Thermo Fisher extensively tests its Invitrogen™ antibodies using a two-part validation approach: using target specificity models and relevant functional applications. Using tools such as CRISPR-Cas9- or RNAi-mediated gene knockdown, and relative expression of specific proteins in different cell types, Thermo Fisher can provide neuroscientists with high-quality antibodies to use in their experiments to obtain reliable, reproducible results.

Three tests for three antibodies

SNAP25 is known to be enriched in specific areas of neurons. As a control, Thermo Fisher scientists added Invitrogen SNAP25 monoclonal antibodies to a primary culture of mouse hippocampal neurons, and confirmed that they localized to the hippocampal membrane and presynaptic terminals as expected. In order to test their specificity, Thermo Fisher scientists used the PC12 cell line. These cells are of embryonic origin and easily divide until treated with nerve growth factor (NGF), whereupon they terminally differentiate into neuron-like cells. In undifferentiated PC12 cells, the SNAP25 antibody was distributed throughout the cytoplasm. When NGF was added and the cells differentiated, the antibodies were redistributed to the membrane of developing axons and nerve endings (Figure 1).

The same model system was used to test the VAMP1 oligoclonal antibody, where tissue from rat and mouse brains serves as the control. In a western blot experiment, the antibody detected a single band at 16 kDa, corresponding to the molecular mass of VAMP1. To validate the specificity, the antibody was again added to PC12 cells. In undifferentiated cells, no VAMP1 was detected, but in NGF-treated, differentiated cells, western blotting showed a band at 16 kDa. Tissue immunofluorescence of mouse brain sections confirmed that VAMP1 was localized in the hippocampal regions (data not shown).

HCT116 cell lysates were used to test the specificity of the OPTN monoclonal antibody. Western blot analysis showed a single band at the expected size of 66 kDa. The signal was absent in lysates of cells in which OPTN had been knocked out using CRISPR-Cas9, confirming the specificity of this antibody. OPTN was distinctively localized to the retinal ganglion cells (RGCs) in adult mouse eye tissue. Consistent with this, tissue immunofluorescence of mouse eye sections using the OPTN antibody showed a strong signal in the ganglion cell layer. Similarly, immunofluorescence in RGCs differentiated from induced pluripotent stem cells (iPSCs) picked up a strong signal that was absent in undifferentiated iPSCs (Figure 2).

By using the appropriate test for each antibody, based on the biology of its target protein and the intended application, Thermo Fisher is able to confirm specificity. Having reliable antibodies will give researchers confidence in their studies of the mechanisms of neurodegenerative disease, as well as in applying them to downstream diagnostic tests or treatments.

For a detailed explanation of these validation strategies, please go to thermofisher.com/antibodyvalidation.