Necroptosis inhibition as a therapy for Niemann-Pick disease, type C1: Inhibition of RIP kinases and combination therapy with 2-hydroxypropyl-β-cyclodextrin

Abstract

Niemann-Pick disease, type C1 (NPC1) is an inborn error of metabolism that results in endolysosomal accumulation of unesterified cholesterol. Clinically, NPC1 manifests as cholestatic liver disease in the newborn or as a progressive neurogenerative condition characterized by cerebellar ataxia and cognitive decline. Currently there are no FDA approved therapies for NPC1. Thus, understanding the pathological processes that contribute to neurodegeneration will be important in both developing and testing potential therapeutic interventions. Neuroinflammation and necroptosis contribute to the NPC1 pathological cascade. Receptor Interacting Protein Kinase 1 and 3 (RIPK1 and RIPK3), are protein kinases that play a central role in mediating neuronal necroptosis. Our prior work suggested that pharmacological inhibition of RIPK1 had a significant but modest beneficial effect; however, the inhibitors used in that study had suboptimal pharmacokinetic properties. In this work we evaluated both pharmacological and genetic inhibition of RIPK1 kinase activity. Lifespan in both Npc1−/−mice treated with GSK’547, a RIPK1 inhibitor with better pharmacokinetic properties, and Npc1−/− :Ripk1kd/kd double mutant mice was significantly increased. In both cases the increase in lifespan was modest, suggesting that the therapeutic potential of RIPK1 inhibition, as a monotherapy, is limited. We thus investigated the potential of combining RIPK1 inhibition with 2-hydroxypropyl-β-cyclodextrin (HPβCD) therapy HPβCD has been shown to slow neurological disease progression in NPC1 mice, cats and patients. HPpβCD appeared to have an additive positive effect on the pathology and survival of Npc1−/−:Ripk1kd/kd mice. RIPK1 and RIPK3 are both critical components of the necrosome, thus we were surprised to observe no increase survival in Npc1−/−;Ripk3−/− mice compared to Npc1−/− mice. These data suggest that although necroptosis is occurring in NPC1, the observed effects of RIPK1 inhibition may be related to its RIPK3-independent role in neuroinflammation and cytokine production.

Single Cell Transcriptome Analysis of Niemann–Pick Disease, Type C1 Cerebella

“…In stark contrast to the general lack of genotype related difference in neuronal transcriptomes, at three weeks of age, Npc1 −/− microglia already show reduced expression of linage (Tmem119) and increased expression of activation (Cd68, Igf1 and Gapdh) markers. These observations are consistent with our prior data showing corresponding changes at the protein level [27]. This observation of microglial activation preceding significant changes in neuronal transcriptome changes is consistent with the recent observation by Kavetsky et al [102] that showed increased interactions and engulfment of dendrites by microglia in the molecular layer prior to Purkinje neuron degeneration.…”

“…No significant gene expression differences were observed comparing Npc1 control and null monocytes. However, consistent with our prior work characterizing transcript levels in FACS isolated microglia [27], we observed major gene expression differences between Npc1 +/+ and Npc1 −/− microglia.…”

“…The Npc1 −/− microglia transcriptomes from seven-week-old mice can be classified as disease-associated microglia initially described in Alzheimer disease [29]. Our current data support the concept that NPC1 microglia have both neuroprotective and neurotoxic activity [27]. This dual role will confound efforts to treat NPC1 by general approaches to reduce microglia activity.…”

“…This expression pattern is consistent with a subset of the microglia in Npc1 −/− being classified as disease associate microglia (DAM). DAM were initially described in an Alzheimer disease mouse model and are thought to be neuroprotective [29], thus supporting the concept that subpopulations of microglia in NPC1 have neurotoxic or neuroprotective activity [27]. Decreased expression of Csf1r in Npc1 −/− microglia likely explains a prior unpublished observation that treatment of Npc1 −/− mice with PLX3397, a CSF1R inhibitor used to deplete microglia, did not have a demonstratable effect on either cerebellar histopathology or survival of Npc1 −/− mice ( Figure S2A,B).…”

Necroptosis: a crucial pathogenic mediator of human disease

“…In a genetic model of Niemann-Pick disease (Npc1 -/mice), RIPK1-specific inhibitor GSK′547 or transgenic overexpression of kinase-deficient RIPK1, but not Ripk3 deficiency, improved lifespan (126). These studies affirmed a RIPK3-independent role for RIPK1-dependent necroinflammation in disease pathogenesis (126).…”