Mechanisms underlying motor neuron subtype-selective endoplasmic reticulum (ER) stress and associated axonal pathology in amyotrophic lateral sclerosis (ALS) remain unclear. Here we show that the molecular environment of the ER between motor neuron subtypes is distinct, with characteristic signatures. We identify cochaperone SIL1, mutated in Marinesco-Sjögren syndrome (MSS), as being robustly expressed in disease-resistant slow motor neurons but not in ER stress-prone fast-fatigable motor neurons. In a mouse model of MSS, we demonstrate impaired ER homeostasis in motor neurons in response to loss of SIL1 function. Loss of a single functional Sil1 allele in an ALS mouse model (SOD1-G93A) enhanced ER stress and exacerbated ALS pathology. In SOD1-G93A mice, SIL1 levels were progressively and selectively reduced in vulnerable fast-fatigable motor neurons. Mechanistically, reduction in SIL1 levels was associated with lowered excitability of fast-fatigable motor neurons, further influencing expression of specific ER chaperones. Adeno-associated virus-mediated delivery of SIL1 to familial ALS motor neurons restored ER homeostasis, delayed muscle denervation and prolonged survival.

Amyotrophic lateral sclerosis (ALS) is an adult onset progressive motor neuron disease with no cure. Transgenic mice overexpressing familial ALS associated human mutant SOD1 are a commonly used model for examining disease mechanisms. Presently, it is well accepted that alterations in motor neuron excitability and spinal circuits are pathological hallmarks of ALS, but the underlying molecular mechanisms remain unresolved. Here, we sought to understand whether the expression of mutant SOD1 protein could contribute to altering processes governing motor neuron excitability. We used the conformation specific antibody B8H10 which recognizes a misfolded state of SOD1 (misfSOD1) to longitudinally identify its interactome during early disease stage in SOD1G93A mice. This strategy identified a direct isozyme-specific association of misfSOD1 with Na(+)/K(+)ATPase-α3 leading to the premature impairment of its ATPase activity. Pharmacological inhibition of Na(+)/K(+)ATPase-α3 altered glutamate receptor 2 expression, modified cholinergic inputs and accelerated disease pathology. After mapping the site of direct association of misfSOD1 with Na(+)/K(+)ATPase-α3 onto a 10 amino acid stretch that is unique to Na(+)/K(+)ATPase-α3 but not found in the closely related Na(+)/K(+)ATPase-α1 isozyme, we generated a misfSOD1 binding deficient, but fully functional Na(+)/K(+)ATPase-α3 pump. Adeno associated virus (AAV)-mediated expression of this chimeric Na(+)/K(+)ATPase-α3 restored Na(+)/K(+)ATPase-α3 activity in the spinal cord, delayed pathological alterations and prolonged survival of SOD1G93A mice. Additionally, altered Na(+)/K(+)ATPase-α3 expression was observed in the spinal cord of individuals with sporadic and familial ALS. A fraction of sporadic ALS cases also presented B8H10 positive misfSOD1 immunoreactivity, suggesting that similar mechanism might contribute to the pathology.

In situ analysis of biomarkers is essential for clinical diagnosis and research purposes. The increasing need to understand the molecular signature of pathologies has led to the blooming of ultrasensitive and multiplexable techniques that combine in situ hybridization (ISH) and immunohistochemistry or immunocytochemistry (IHC or ICC). Most protocols are tailored to formalin-fixed paraffin embedded (FFPE) tissue sections. However, methods to perform such assays on non-adherent cell samples, such as patient blood-derived PBMCs, rare tumor samples, effusions or other body fluids, dissociated or sorted cells, are limited. Typically, a laboratory would need to invest a significant amount of time and resources to establish one such assay. Here, we describe a method that combines ultrasensitive RNAscope-ISH with ICC on cytospin cell preparations. This method allows automated, sensitive, multiplex ISH-ICC on small numbers of non-adherent cells. We provide guidelines for both chromogenic and fluorescent ISH/ICC combinations that can be performed either in fully automated or in manual settings. By using a CD8+ T cells in vitro stimulation paradigm, we demonstrate that this protocol is sensitive enough to detect subtle differences in gene expression and compares well to commonly used methods such as RT-qPCR and flow cytometry with the added benefit of visualization at the cellular level.