ANTAGONISM OF P38 MAPK ALPHA (P38α) REVERSES APP-INDUCED ENDOSOMAL ABNORMALITIES AND IMPROVES LYSOSOMAL FUNCTION IN DOWN SYNDROME FIBROBLASTS

Abstract

Endosomal abnormalities and lysosomal dysfunction are early events in Alzheimer's pathogenesis that lead to synaptic dysfunction (Pimplikar, 2010; Canter, 2016). In vitro, animal and human AD data indicate a key step in APP-induced endolysosomal dysfunction is activation of the endosomal regulatory protein Rab5 (Ginsberg 2010, Kim, 2016; Xu, 2016). As the stress-activated kinase p38α activates Rab5 (Cavalli, 2001; Macé, 2005; Pelksmans, 2005), p38α inhibition may impact APP-induced endosomal dysfunction. P38α otherwise is implicated in Aβ and inflammation-induced synaptic dysfunction (Watterson, 2013; Prieto, 2015; Colié, 2017), and in dysregulated autophagy resulting in amyloid-beta generation (Alam & Scheper, 2016). Accordingly, we evaluated the effects of a selective p38α-antagonist (neflamapimod, aka VX-745) in Down Syndrome (DS) human fibroblasts that express 3 copies of APP and recapitulate APP-induced endosomal dysfunction (Jiang, 2010). Neflamapimod is an investigational drug that in phase 2a clinical trials in early AD (CTAD, 2016) demonstrated evidence of improving episodic memory, a measure of hippocampal synaptic function, and reduction in brain amyloid plaque levels by PET scan. 2N or DS-fibroblasts were incubated 24–48 hours with 0–50 nM neflamapimod and then either (1) fixed and incubated with anti-EEA1 antibody, and then fluorescently imaged to assess the size and number of early-endosomes; or (2) incubated with Bodipy-pepstatinA for 30 minutes prior to fluorescence imaging to assess lysosomal function. Numbers and sizes of EAA1+ early-endosomes were increased in DS-fibroblasts and were lowered to values approaching those seen in 2N-fiboblasts at 1, 10 or 50 nM neflamapimod. Neflamapimod also demonstrated dose-dependent statistically significant improvement in lysosomal function in DS-fibroblasts. The IC50 for these pharmacological effects is >5-fold lower than the published IC50 of ∼50nM of neflamapimod for inhibition of cytokine production (Duffy, 2011), and correlates well with dose/concentration-response seen for episodic memory in the human clinical studies. The findings provide the first evidence that p38α antagonism could be a means to therapeutically target Alzheimer-related endosomal dysfunction. Further, the correlation between in vitro potency of neflamapomid on reversing endolysosomal dysfunction with clinical dose-response suggests that this pharmacological effect, rather than anti-inflammatory activity, may underlie the clinical activity of neflamapimod on episodic memory.