Amyotrophic lateral sclerosis (ALS) is characterized by predominant vulnerability and central degeneration of both corticospinal/corticobulbar motor neurons (CSMN; “upper motor neurons”) in cerebral cortex, and spinal/bulbar motor neurons (SMN; “lower motor neurons”) in spinal cord and brainstem. Increasing evidence indicates broader cerebral cortex pathology in cognitive, sensory, and association systems in select cases. It remains unclear whether widely accepted transgenic ALS models, in particular hSOD1^G93Amice, undergo degeneration of CSMN and molecularly/developmentally closely related populations of nonmotor projection neurons [e.g., other subcerebral projection neurons (SCPN)], and whether potential CSMN/SCPN degeneration is specific and early. This relative lack of knowledge regarding upper motor neuron pathology in these ALS model mice has hindered both molecular-pathophysiologic understanding of ALS and their use toward potential CSMN therapeutic approaches. Here, using a combination of anatomic, cellular, transgenic labeling, and newly available neuronal subtype-specific molecular analyses, we identify that CSMN and related nonmotor SCPN specifically and progressively degenerate in hSOD1^G93Amice. Degeneration starts quite early and presymptomatically, by postnatal day 30. Other neocortical layers, cortical interneurons, and other projection neuron populations, even within layer V, are not similarly affected. Nonneuronal pathology in neocortex (activated astroglia and microglia) is consistent with findings in human ALS cortex and in affected mouse and human spinal cord. These results indicate previously unknown neuron type-specific vulnerability of CSMN/sensory and association SCPN, and identify that characteristic dual CSMN and SMN degeneration is conserved in hSOD1^G93Amice. These results provide a foundation for detailed investigation of CSMN/SCPN vulnerability and toward potential CSMN therapeutics in ALS.

Apical dendrites of Betz cells are important sites for the integration of cortical input, however their health has not been fully assessed in ALS patients. We investigated the primary motor cortices isolated from post-mortem normal control subjects, patients with familial ALS (fALS), sporadic ALS (sALS), ALS with frontotemporal dementia (FTD-ALS), and Alzheimer’s disease (AD), and found profound apical dendrite degeneration of Betz cells in both fALS and sALS, as well as FTD-ALS patients. In contrast, Betz cells of AD patients and normal controls retain cellular integrity in the motor cortex, and CA1 pyramidal neurons show abnormalities predominantly within their soma, rather than the apical dendrite. In line with extensive vacuolation and cytoarchitectural disintegration, the numbers of synapses were also significantly reduced only in ALS patients. Our findings indicate apical dendrite degeneration as a novel cellular pathology that distinguishes ALS and further support the importance of cortical dysfunction for disease pathology.

Understanding mechanisms that lead to selective motor neuron degeneration requires visualization and cellular identification of vulnerable neurons. Here we report generation and characterization ofUCHL1-eGFPandhSOD1^G93A-UeGFPmice, novel reporter lines for cortical and spinal motor neurons. Corticospinal motor neurons (CSMN) and a subset of spinal motor neurons (SMN) are genetically labeled inUCHL1-eGFPmice, which express eGFP under theUCHL1promoter. eGFP expression is stable and continues through P800in vivo. Retrograde labeling, molecular marker expression, electrophysiological analysis, and cortical circuit mapping confirmed CSMN identity of eGFP⁺neurons in the motor cortex. Anatomy, molecular marker expression, and electrophysiological analysis revealed that the eGFP expression is restricted to a subset of small-size SMN that are slow-twitch α and γ motor neurons. Crossbreeding ofUCHL1-eGFPandhSOD1^G93Alines generatedhSOD1^G93A-UeGFPmice, which displayed the disease phenotype observed in ahSOD1^G93Amouse model of ALS. eGFP⁺SMN showed resistance to degeneration inhSOD1^G93A-UeGFPmice, and their slow-twitch α and γ motor neuron identity was confirmed. In contrast, eGFP⁺neurons in the motor cortex ofhSOD1^G93A-UeGFPmice recapitulated previously reported progressive CSMN loss and apical dendrite degeneration. Our findings using these two novel reporter lines revealed accumulation of autophagosomes along the apical dendrites of vulnerable CSMN at P60, early symptomatic stage, suggesting autophagy as a potential intrinsic mechanism for CSMN apical dendrite degeneration.

Corticospinal motor neurons (CSMN) receive, integrate, and relay cerebral cortex's input toward spinal targets to initiate and modulate voluntary movement. CSMN degeneration is central for numerous motor neuron disorders and neurodegenerative diseases. Previously, 5 patients with mutations in the ubiquitin carboxy-terminal hydrolase-L1 (UCHL1) gene were reported to have neurodegeneration and motor neuron dysfunction with upper motor neuron involvement. To investigate the role of UCHL1 on CSMN health and stability, we used both in vivo and in vitro approaches, and took advantage of the Uchl1^nm3419 (UCHL1^−/−) mice, which lack all UCHL1 function. We report a unique role of UCHL1 in maintaining CSMN viability and cellular integrity. CSMN show early, selective, progressive, and profound cell loss in the absence of UCHL1. CSMN degeneration, evident even at pre-symptomatic stages by disintegration of the apical dendrite and spine loss, is mediated via increased ER stress. These findings bring a novel understanding to the basis of CSMN vulnerability, and suggest UCHL1^−/− mice as a tool to study CSMN pathology.