The Role of Neutrophil Heterogeneity and Molecular Mechanisms in Driving ALS

ABSTRACT
There is overwhelming evidence that the immune system plays a significant role in amyotrophic lateral
sclerosis (ALS) pathogenesis. However, information about the role of specific immune populations and the
underlying mechanisms is currently lacking. Neutrophils are innate immune cells that function as first-
responders to damage, including damage to neuronal tissue. These cells ultimately determine whether
damaged neurons undergo repair or whether damage is exacerbated. Additional damage can occur directly via
cytotoxicity or indirectly through the recruitment of other immune cell types, the release of pro-inflammatory
cytokines, and the suppression of repair mechanisms. Thus, neutrophils may play a central role in the
response to motor neuron damage in ALS. To support this, our previously published studies show that
neutrophils are increased in the blood of ALS patients, that these cells accumulate in the central nervous
system (CNS) of individuals with ALS, and that increased levels in the peripheral blood are associated with
shortened survival. In addition, preliminary data show that peripheral neutrophils are dysregulated in ALS with
increased expression of trafficking and activation markers. Finally, our predictive models of disease show that
neutrophil levels are the single best predictor of future ALS progression in patients. Therefore, I hypothesize
that neutrophils contribute to ALS pathology by accelerating motor neuron death and by suppressing pro-repair
immune responses in the CNS. To test this, I propose two Specific Aims which will assess the role of
neutrophils in ALS. In the first Aim, I will identify subpopulations of neutrophils or neutrophil-based mechanisms
that are associated with ALS progression and survival. To do this, I will use flow cytometry, single cell RNA-
Seq (scRNA-Seq), and analysis of neutrophil extracellular trap (NET) remnants to immunophenotype
neutrophils in the peripheral blood of control and ALS participants. Dysregulated pathways will first be
identified and then associated with ALS progression and survival using our existing biostatistical pipeline. In
the second Aim, I will identify the mechanisms by which neutrophils become activated and more pro-
inflammatory in ALS. To do so, I will culture primary and iPSC-derived neutrophils with and without ALS-
associated proteins such as TDP-43 aggregates. Activation and pro-inflammatory activity will be determined
via flow cytometry and assays for degranulation, reactive oxygen species release, and NET formation. Overall,
completion of these studies will elucidate mechanisms driving neutrophil-mediated damage in ALS and identify
potential therapeutic targets for future studies.

Grant Number: 5F31NS139629-02
NIH Institute/Center: NIH
Principal Investigator: Lillia Baird