Incorporating upper motor neuron health in ALS drug discovery

Abstract

Amyotrophic lateral sclerosis (ALS) is a complex disease, that affects the motor neuron circuitry. After consecutive failures in clinical trials for the past 20 years, edaravone was recently approved as the second drug for ALS. This generated excitement in the field revealed the need to improve preclinical assays for continued success. Here, we focus on the importance and relevance of upper motor neuron (UMN) pathology in ALS, and discuss how incorporation of UMN survival in preclinical assays will improve inclusion criteria for clinical trials and expedite the drug discovery effort in ALS and related motor neuron diseases.

Figure 1

UCHL1-eGFP mice allow visualization and cellular assessment of corticospinal motor neurons in vivo [33] and in vitro. (a) eGFP expression is under the control of the UCHL1 promoter in the UCHL1-eGFP mice, which results in stable and long-lasting eGFP expression at the UCHL1 promoter. (b) eGFP expression is restricted to corticospinal motor neurons (CSMNs) in the motor cortex. They are located in layer V of the motor cortex and have large pyramidal soma with long apical dendrites. (c) Representative images of dissociated cortical cultures isolated from the motor cortex of UCHL1-eGFP mice. CSMNs retain their eGFP expression in vitro. Therefore, among all other cortical cells and neurons, CSMNs can be identified based on their fluorescence (blue = Hoescht). (d) CSMNs retain neuronal identity in culture. They maintain their pyramidal shape, extend a long axon and, most importantly, continue to express molecular markers, such as CTIP2. Abbreviations: Amp, ampicillin; R6Ky, origin of replication; PA, polyadenylated tail; eGFP, enhanced green fluorescent protein; Chlor(+), chloramphenicol; UTR, untranslated region.

Figure 2

The experimental scheme summarizing generation of reporter lines of diseased corticospinal motor neurons (CSMNs). (a) Mouse models of amyotrophic lateral sclerosis (ALS) that display CSMN vulnerability and progressive degeneration are crossed with the UCHL1-eGFP mice to generate CSMN reporter lines of ALS mouse models. This simple approach is used to generate hSOD1^G93A-UeGFP, TDP43-UeGFP, Alsin^KO-UeGFP mice, and the same approach can be used to generate other reporter lines of diseased CSMNs after other mouse models are generated and the timing and extent of their CSMN loss is well reported.

Figure 3

Reporter lines of diseased corticospinal motor neurons (CSMNs) offer great advantages for compound selection and verification in vitro and in vivo. (a) Dissociated cortical cells isolated from the motor cortex of reporter lines of diseased CSMNs include many different types of neurons and non-neuronal cells. However, CSMNs are distinguished among them by their enhanced green fluorescent protein (eGFP) expression. Upon compound administration, overall survival of CSMNs that become diseased owing to different underlying factors, the mode of their improved health and their interaction with non-neuronal cells such as astrocytes and microglia can be studied with precision. Their response to treatment can be assessed at a cellular level and compounds that display efficacy for a specific underlying cause or causes can be identified. (b) Compounds of interest can be administered to reporter lines of diseased CSMNs by oral delivery, intraperitoneal (IP) injection or even via viral gene delivery. It is possible that some compounds will display better outcome measures on different cellular pathologies. Their impact on overall motor function, improved CSMN and SMN survival together with improved health and integrity of neuromuscular junctions can be assessed to make a better judgement on the efficacy of compounds tested.