Intranasally delivered mesenchymal stromal cells decrease glial inflammation early in prion disease

Abstract

Mesenchymal stromal cells (MSCs) are an intriguing avenue for the treatment of neurological disorders due to their ability to migrate to sites of neuroinflammation and respond to paracrine signaling in those sites by secreting cytokines, growth factors, and other neuromodulators. We potentiated this ability by stimulating MSCs with inflammatory molecules, improving their migratory and secretory properties. We investigated the use of intranasally delivered adipose-derived MSCs (AdMSCs) in combating prion disease in a mouse model. Prion disease is a rare, lethal neurodegenerative disease that results from the misfolding and aggregation of the prion protein. Early signs of this disease include neuroinflammation, activation of microglia, and development of reactive astrocytes. Later stages of disease include development of vacuoles, neuronal loss, abundant aggregated prions, and astrogliosis. We demonstrate the ability of AdMSCs to upregulate anti-inflammatory genes and growth factors when stimulated with tumor necrosis factor alpha (TNFα) or prion-infected brain homogenates. We stimulated AdMSCs with TNFα and performed biweekly intranasal deliveries of AdMSCs on mice that had been intracranially inoculated with mouse-adapted prions. At early stages in disease, animals treated with AdMSCs showed decreased vacuolization throughout the brain. Expression of genes associated with Nuclear Factor-kappa B (NF-κB) and Nod-Like Receptor family pyrin domain containing 3 (NLRP3) inflammasome signaling were decreased in the hippocampus. AdMSC treatment promoted a quiescent state in hippocampal microglia by inducing changes in both number and morphology. Animals that received AdMSCs showed a decrease in both overall and reactive astrocyte number, and morphological changes indicative of homeostatic astrocytes. Although this treatment did not prolong survival or rescue neurons, it demonstrates the benefits of MSCs in combatting neuroinflammation and astrogliosis.

Targeting Neuroinflammation by Pharmacologic Downregulation of Inflammatory Pathways Is Neuroprotective in Protein Misfolding Disorders

“…Following this time, the food pellets left in the tubes were weighed, and the percentage burrowed was calculated. Prion-diseased mice are known to stop burrowing as the disease progresses, ,, but at 20, 21, and 22wpi, prion-diseased mice given therapeutic SB_NI_112 (i.n. and i.p.)…”

“…treatments displayed significantly lower scores at 22 wpi ( p < 0.0001) and 23 wpi ( p = 0.0197). Untreated prion clinical signs included tail rigidity, hyperactivity, ataxia, extensor reflex (clasping), tremors, righting reflex, kyphosis, and poor grooming. ,, Each sign was rated on a scale from 0 to 5. All clinical sign scores were combined to give a total score.…”

“…Briefly, mice were placed in a large cage with a PVC tube (2.33″ × 5.75″) full of food pellets, as described. ,, The percentage of burrowing activity is calculated from the difference in the weight of pellets in the tube before and after 2 h. To test nesting, we placed three fresh napkins in each cage. After 24 h, cages were examined for nesting activity.…”

“…Based on the extensive evidence supporting the central involvement of NLRP3 inflammasome formation following NF-κB activation in the progression of neurodegenerative diseases, it is important to develop potential therapeutics to inhibit NF-κB and NLRP3. Therapeutic intervention to target and inhibit NLRP3 inflammasome formation arises as a potential three-hit treatment against neurodegenerative diseases by (1) reducing inflammatory signaling among microglia and astrocytes, − (2) preventing propagation of misfolded proteins, − and (3) upregulating microglia autophagy to accelerate removal of misfolded proteins. − No studies to date have explored combined NF-κB and NLRP3 inhibition as a therapeutic intervention for neurodegenerative diseases.…”

“…During neurodegenerative disease-induced brain stress, there is a significant crosstalk between microglia, astrocytes, and neurons via critical inflammatory cell signaling events involving initiation of the nuclear factor κB (NF-κB) pathway and subsequent NOD (nucleotide-binding oligomerization domain), LRR (leucine-rich repeat), and pyrin-containing 3 (NLRP3) inflammasome formation. − NF-κB, when translocated into the nucleus, promotes transcription of several inflammatory cytokines and chemokines, including NLRP3, pro-interleukin-1β (pro-IL-1β), and pro-interleukin-18 (pro-IL-18), triggering a cascade of microglial and astrocytic inflammation . These reactive astrocytes are directly toxic to neurons and endothelial cells during prion disease. , Many recent studies, including human patient, in vitro, and in vivo animal models, have pointed to the assembly of the NLRP3 inflammasome in microglia and astrocytes as a significant contributor to inflammatory-related amplification of misfolded proteins in neurodegenerative diseases. − Formation of the NLRP3 inflammasome is a critical step for the activation of pro-inflammatory cytokines and perpetuation of the immune response through microglial pyroptosis. , Pro-caspase-1 is activated by proximity-induced autocatalytic activation upon recruitment to the NLRP3 inflammasome. Active caspase-1 cleaves pro-IL-1β and pro-IL-18 into their mature and biologically active forms, cytokines IL-1β and IL-18. , In mouse models of APP/PS1 mutation and a tauopathy, NLRP3 inflammasome assembly has been identified as a significant contributor to inflammatory-related amplification of Aβ plaques and neurofibrillary tangles. , Similarly, the NLRP3 inflammasome promotes α-Syn production in mouse models of PD. , Activation of NF-kB in prion disease augments neuronal death by increasing the expression of pro-apoptotic genes and triggering the release of cytochrome C, resulting in mitochondrial dysfunction and endoplasmic reticulum stress .…”