Peripheral neuropathy in the Twitcher mouse involves the activation of axonal caspase 3

Infantile Krabbe disease results in the accumulation of lipid-raft-associated galactosylsphingosine (psychosine), demyelination, neurodegeneration and premature death. Recently, axonopathy has been depicted as a contributing factor in the progression of neurodegeneration in the Twitcher mouse, a bona fide mouse model of Krabbe disease. Analysis of the temporal-expression profile of MBP (myelin basic protein) isoforms showed unexpected increases of the 14, 17 and 18.5 kDa isoforms in the sciatic nerve of 1-week-old Twitcher mice, suggesting an abnormal regulation of the myelination process during early postnatal life in this mutant. Our studies showed an elevated activation of the pro-apoptotic protease caspase 3 in sciatic nerves of 15- and 30-day-old Twitcher mice, in parallel with increasing demyelination. Interestingly, while active caspase 3 was clearly contained in peripheral axons at all ages, we found no evidence of caspase accumulation in the soma of corresponding mutant spinal cord motor neurons. Furthermore, active caspase 3 was found not only in unmyelinated axons, but also in myelinated axons of the mutant sciatic nerve. These results suggest that axonal caspase activation occurs before demyelination and following a dying-back pattern. Finally, we showed that psychosine was sufficient to activate caspase 3 in motor neuronal cells in vitro in the absence of myelinating glia. Taken together, these findings indicate that degenerating mechanisms actively and specifically mediate axonal dysfunction in Krabbe disease and support the idea that psychosine is a pathogenic sphingolipid sufficient to cause axonal defects independently of demyelination

The Sphingolipid psychosine inhibits fast axonal transport in krabbe disease by activation of GSK3 and deregulation of molecular motors

Loss of function of galactosylceramidase lysosomal activity causes demyelination and vulnerability of various neuronal populations in Krabbe disease. Psychosine, a lipid-raft-associated sphingolipid that accumulates in this disease, is thought to trigger these abnormalities. Myelin-free in vitro analyses showed that psychosine inhibited fast axonal transport through the activation of axonal PP1 and GSK3β in the axon. Abnormal levels of activated GSK3β and abnormally phosphorylated kinesin light chains were found in nerve samples from a mouse model of Krabbe disease. Administration of GSK3β inhibitors significantly ameliorated transport defects in vitro and in vivo in peripheral axons of the mutant mouse. This study identifies psychosine as a pathogenic sphingolipid able to block fast axonal transport and is the first to provide a molecular mechanism underlying dying-back degeneration in this genetic leukodystrophy

Pro-inflammatory activation following demyelination is required for myelin clearance and oligodendrogenesis

Remyelination requires innate immune system function, but how exactly microglia and macrophages clear myelin debris after injury and tailor a specific regenerative response is unclear. Here, we asked whether pro-inflammatory microglial/macrophage activation is required for this process. We established a novel toxin-based spinal cord model of de- and remyelination in zebrafish and showed that pro-inflammatory NF-κB-dependent activation in phagocytes occurs rapidly after myelin injury. We found that the pro-inflammatory response depends on myeloid differentiation primary response 88 (MyD88). MyD88-deficient mice and zebrafish were not only impaired in the degradation of myelin debris, but also in initiating the generation of new oligodendrocytes for myelin repair. We identified reduced generation of TNF-α in lesions of MyD88-deficient animals, a pro-inflammatory molecule that was able to induce the generation of new premyelinating oligodendrocytes. Our study shows that pro-inflammatory phagocytic signaling is required for myelin debris degradation, for inflammation resolution, and for initiating the generation of new oligodendrocytes

Enhancing protective microglial activities with a dual function TREM2 antibody to the stalk region

Triggering receptor expressed on myeloid cells 2 (TREM2) is essential for the transition of homeostatic microglia to a disease‐associated microglial state. To enhance TREM2 activity, we sought to selectively increase the full‐length protein on the cell surface via reducing its proteolytic shedding by A Disintegrin And Metalloproteinase (i.e., α‐secretase) 10/17. We screened a panel of monoclonal antibodies against TREM2, with the aim to selectively compete for α‐secretase‐mediated shedding. Monoclonal antibody 4D9, which has a stalk region epitope close to the cleavage site, demonstrated dual mechanisms of action by stabilizing TREM2 on the cell surface and reducing its shedding, and concomitantly activating phospho‐SYK signaling. 4D9 stimulated survival of macrophages and increased microglial uptake of myelin debris and amyloid β‐peptide in vitro. In vivo target engagement was demonstrated in cerebrospinal fluid, where nearly all oluble TREM2 was 4D9‐bound. Moreover, in a mouse model for Alzheimer's disease‐related pathology, 4D9 reduced amyloidogenesis, enhanced microglial TREM2 expression, and reduced a homeostatic marker, suggesting a protective function by driving microglia toward a disease‐associated state

Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity

The causative agent of coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For many viruses, tissue tropism is determined by the availability of virus receptors and entry cofactors on the surface of host cells. In this study, we found that neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, an effect blocked by a monoclonal blocking antibody against NRP1. A SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells facing the nasal cavity. Our data provide insight into SARS-CoV-2 cell infectivity and define a potential target for antiviral intervention.Peer reviewe

Mechanisms of Axonopathy in Krabbe Disease

In this work we describe the molecular mechanisms of axonal damage in Krabbe disease (KD). KD is a fatal genetic lysosomal storage disorder (LSD) caused by the loss of the lysosomal enzyme galactosyl-ceramidase (GALC). The deficiency causes the accumulation of galactosyl-sphingolipids. Psychosine, one of GALC substrates, is a lipid-raft associated neurotoxin believed to cause the death of myelinating cells in the CNS of affected patients. KD patients also suffer of neurodegeneration, due to axonal and neuronal deficiencies. Over the years, most studies have focused on the loss of myelin induced by psychosine, and have considered neurodegeneration as a consequence of demyelination. Recently, our laboratory showed that bone marrow transplantation (BMT) improved myelin preservation in Twitcher mice, the natural murine model for this disease, but it was insufficient to prevent neurodegeneration. There are various potential interpretations for this result. Neuronal loss and axonal degeneration in KD may start before oligodendroglia is affected, creating a progressive compounding factor contributing to the severity of the disease. Neurodegeneration may even be independent from demyelination. We hypothesized that GALC deficiency causes a cell-autonomous defect in the mutant neurons, leading to neurodegeneration. By studying the Twitcher mouse, we show that the Twitcher axons are affected by a decrease in axonal caliber and by morphological abnormalities from the first postnatal days. Importantly, we show that these defects are at least partially caused by changes in two critical components of neuronal function: the neurofilament (NF) cytoskeleton and fast axonal transport (FAT). In particular, we provide evidence that psychosine alters the activities of critical phosphotransferases, interfering with the phosphorylation state of NFs and kinesin, one of the molecular motors responsible for FAT. Finally, we identify protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A) and glycogen synthase kinase 3 β (GSK3β) as the phosphotransferases involved in psychosine toxicity. By injecting the newborn Twitcher mouse with an inhibitor of GSK3β, we rescue the functionality of the mutant nerve to a significant level, further demonstrating the relevance of altered phosphorylation in the development of axonopathy in the Twitcher mouse. In summary, these studies have shown that a sphingolipid can regulate NFs and FAT through specific phosphotransferases, and that this effect can cause axonal damage in KD. Pharmacological inhibition of these enzymes may provide new therapeutic alternatives to protect NFs and FAT in KD, therefore rescuing the mutant axons. Importantly, our studies also define a new mechanism by which psychosine, a substrate of a LSD, can cause axonopathy, raising the possibility that alterations in NFs and FAT might occur in other LSDs

Reparative inflammation in multiple sclerosis

Multiple sclerosis is a chronic inflammatory disease, in which repeated episodes of acute auto-inflammatory attacks trigger demyelinating injury in the central nervous system. Whereas our understanding of the disease-causing immune activation is constantly increasing, much less is known about the injury-induced reparative innate immune responses. Here, we discuss the essential function of microglia and monocyte-derived macrophages in orchestrating debris clearance and regeneration. Dampening reparative inflammation can result in insufficient clearance and, thus, to persisting damage, with the consequences of prolonged inflammation. Thus, an understanding of the entire spectrum of inflammatory responses is essential for the prevention of injury-inducing and the stimulation of repair promoting functions of the immune system

TREM2-dependent lipid droplet biogenesis in phagocytes is required for remyelination

Upon demyelinating injury, microglia orchestrate a regenerative response that promotes myelin repair, thereby restoring rapid signal propagation and protecting axons from further damage. Whereas the essential phagocytic function of microglia for remyelination is well known, the underlying metabolic pathways required for myelin debris clearance are poorly understood. Here, we show that cholesterol esterification in male mouse microglia/macrophages is a necessary adaptive response to myelin debris uptake and required for the generation of lipid droplets upon demyelinating injury. When lipid droplet biogenesis is defective, innate immune cells do not resolve, and the regenerative response fails. We found that triggering receptor expressed on myeloid cells 2 (TREM2)-deficient mice are unable to adapt to excess cholesterol exposure, form fewer lipid droplets, and build up endoplasmic reticulum (ER) stress. Alleviating ER stress in TREM2-deficient mice restores lipid droplet biogenesis and resolves the innate immune response. Thus, we conclude that TREM2-dependent formation of lipid droplets constitute a protective response required for remyelination to occur

Loss of NPC1 enhances phagocytic uptake and impairs lipid trafficking in microglia.

Niemann-Pick type C disease is a rare neurodegenerative disorder mainly caused by mutations in NPC1, resulting in abnormal late endosomal/lysosomal lipid storage. Although microgliosis is a prominent pathological feature, direct consequences of NPC1 loss on microglial function remain not fully characterized. We discovered pathological proteomic signatures and phenotypes in NPC1-deficient murine models and demonstrate a cell autonomous function of NPC1 in microglia. Loss of NPC1 triggers enhanced phagocytic uptake and impaired myelin turnover in microglia that precede neuronal death. Npc1-/- microglia feature a striking accumulation of multivesicular bodies and impaired trafficking of lipids to lysosomes while lysosomal degradation function remains preserved. Molecular and functional defects were also detected in blood-derived macrophages of NPC patients that provide a potential tool for monitoring disease. Our study underscores an essential cell autonomous role for NPC1 in immune cells and implies microglial therapeutic potential