CERTL reduces C16 ceramide, amyloid-β levels, and inflammation in a model of Alzheimer’s disease

Background: Dysregulation of ceramide and sphingomyelin levels have been suggested to contribute to the pathogenesis of Alzheimer’s disease (AD). Ceramide transfer proteins (CERTs) are ceramide carriers which are crucial for ceramide and sphingomyelin balance in cells. Extracellular forms of CERTs co-localize with amyloid-β (Aβ) plaques in AD brains. To date, the significance of these observations for the pathophysiology of AD remains uncertain. Methods: A plasmid expressing CERTL, the long isoform of CERTs, was used to study the interaction of CERTL with amyloid precursor protein (APP) by co-immunoprecipitation and immunofluorescence in HEK cells. The recombinant CERTL protein was employed to study interaction of CERTL with amyloid-β (Aβ), Aβ aggregation process in presence of CERTL, and the resulting changes in Aβ toxicity in neuroblastoma cells. CERTL was overexpressed in neurons by adeno-associated virus (AAV) in a mouse model of familial AD (5xFAD). Ten weeks after transduction, animals were challenged with behavior tests for memory, anxiety, and locomotion. At week 12, brains were investigated for sphingolipid levels by mass spectrometry, plaques, and neuroinflammation by immunohistochemistry, gene expression, and/or immunoassay. Results: Here, we report that CERTL binds to APP, modifies Aβ aggregation, and reduces Aβ neurotoxicity in vitro. Furthermore, we show that intracortical injection of AAV, mediating the expression of CERTL, decreases levels of ceramide d18:1/16:0 and increases sphingomyelin levels in the brain of male 5xFAD mice. CERTL in vivo over-expression has a mild effect on animal locomotion, decreases Aβ formation, and modulates microglia by decreasing their pro-inflammatory phenotype. Conclusion: Our results demonstrate a crucial role of CERTL in r

Expression of a Mutant SEMA3A Protein with Diminished Signalling Capacity Does Not Alter ALS-Related Motor Decline, or Confer Changes in NMJ Plasticity after BotoxA-Induced Paralysis of Male Gastrocnemic Muscle

<div><p>Terminal Schwann cells (TSCs) are specialized cells that envelop the motor nerve terminal, and play a role in the maintenance and regeneration of neuromuscular junctions (NMJs). The chemorepulsive protein semaphorin 3A (SEMA3A) is selectively up-regulated in TSCs on fast-fatigable muscle fibers following experimental denervation of the muscle (BotoxA-induced paralysis or crush injury to the sciatic nerve) or in the motor neuron disease amyotrophic lateral sclerosis (ALS). Re-expression of SEMA3A in this subset of TSCs is thought to play a role in the selective plasticity of nerve terminals as observed in ALS and following BotoxA-induced paralysis. Using a mouse model expressing a mutant SEMA3A with diminished signaling capacity, we studied the influence of SEMA3A signaling at the NMJ with two denervation paradigms; a motor neuron disease model (the G93A-hSOD1 ALS mouse line) and an injury model (BotoxA-induced paralysis). ALS mice that either expressed 1 or 2 mutant SEMA3A alleles demonstrated no difference in ALS-induced decline in motor behavior. We also investigated the effects of BotoxA-induced paralysis on the sprouting capacity of NMJs in the K108N-SEMA3A mutant mouse, and observed no change in the differential neuronal plasticity found at NMJs on fast-fatigable or slow muscle fibers due to the presence of the SEMA3A mutant protein. Our data may be explained by the residual repulsive activity of the mutant SEMA3A, or it may imply that SEMA3A alone is not a key component of the molecular signature affecting NMJ plasticity in ALS or BotoxA-induced paralysis. Interestingly, we did observe a sex difference in motor neuron sprouting behavior after BotoxA-induced paralysis in WT mice which we speculate may be an important factor in the sex dimorphic differences seen in ALS.</p></div

Sex differences observed in motor neuron sprouting behavior and risk for ALS.

<p>Sex differences observed in motor neuron sprouting behavior and risk for ALS.</p

G93A-hSOD1 ALS mice were bred with the K108N-SEMA3A line to create ALS mice homozygote or heterozygote for the mutant SEMA3A gene.

<p>G93A-hSOD1 ALS mice were bred with the K108N-SEMA3A line to create ALS mice homozygote or heterozygote for the mutant SEMA3A gene.</p

Overview of mice used in the BotoxA-induced paralysis paradigm.

<p>Overview of mice used in the BotoxA-induced paralysis paradigm.</p

ALS mice harboring the K108N-SEMA3A gene variant continue to display a similar decline in motor function compared to “normal” ALS mice.

<p>G93A-hSOD1 mice were crossbred with K108N-SEMA3A mice to produce homozygote and heterozygote ALS mice with respect to the K108N-SEMA3A gene. The expression of K108N-SEMA3A in ALS mice did not alter ALS-induced weight loss (panel A; compare red and blue curves with green curve). From 6 weeks of age, mice were subjected to weekly Rotarod (B) and Paw Grip Endurance (PaGE; C) behavioral testing. Rotarod performance of both ALS x N/N (homozygote; red curve; panel B) and ALS x N/- (heterozygote; blue curve; panel B) shows a similar decline over the course of the 14 weeks tested, starting at approximately 11 weeks of age. The progression of decline in performance is similar to that of ALS mice harboring the WT SEMA3A gene (green curve; panel B). Similarly, for PaGE performance, ALS x N/N (red curve; panel C) and ALS x N/- (blue curve; panel C) mice show a similar decline in motor performance. However, the initial decline in performance is delayed in ALS x N/- mice compared to ALS x N/N mice (* p>0.05; week 9). Overall, neither the ALS x N/N or ALS x N/- mice show a difference in performance compared to ALS mice harboring the WT SEMA3A gene (green curve; panel C).</p

Representative immune stainings of neuronal sprouts observed at mouse gastrocnemic muscle neuromuscular junctions 14 days after BotoxA-induced paralysis.

<p>Neuromuscular junctions (NMJs) were visualized with bungarotoxin (BTX; red) and antibody staining against neurofilament and SV2 (NF-2H3/SV2; green). Thin, neuronal sprouts (arrow) are clearly distinguishable from the original, thicker motor neuron synapsing onto the endplate (arrowhead; panels A, B and C). A normal NMJ is depicted inside the dashed oval which shows a complete overlap of the BTX and NF-2H3/SV2 positive regions (panel A). A neuronal sprout (green protrusion from the NMJ on the left of panel A, and in panel B) contains the beginnings of an ectopic endplate (faint red region; arrow). A neuronal sprout which has yet to synapse on an ectopic endplate is depicted in panel C (arrow). Scale bar is 10μm.</p

Repulsive Guidance Molecule a (RGMa) Induces Neuropathological and Behavioral Changes That Closely Resemble Parkinson's Disease

Repulsive guidance molecule member a (RGMa) is a membrane-associated or released guidance molecule that is involved in axon guidance, cell patterning, and cell survival. In our previous work, we showed that RGMa is significantly upregulated in the substantia nigra of patients with Parkinson's disease. Here we demonstrate the expression of RGMa in midbrain human dopaminergic (DA) neurons. To investigate whether RGMa might model aspects of the neuropathology of Parkinson's disease in mouse, we targeted RGMa to adult midbrain dopaminergic neurons using adeno-associated viral vectors. Overexpression of RGMa resulted in a progressive movement disorder, including motor coordination and imbalance, which is typical for a loss of DA release in the striatum. In line with this, RGMa induced selective degeneration of dopaminergic neurons in the substantia nigra (SN) and affected the integrity of the nigrostriatal system. The degeneration of dopaminergic neurons was accompanied by a strong microglia and astrocyte activation. The behavioral, molecular, and anatomical changes induced by RGMa in mice are remarkably similar to the clinical and neuropathological hallmarks of Parkinson's disease. Our data indicate that dysregulation of RGMa plays an important role in the pathology of Parkinson's disease, and antibody-mediated functional interference with RGMa may be a disease modifying treatment option.SIGNIFICANCE STATEMENTParkinson's disease (PD) is a neurodegenerative disease characterized by severe motor dysfunction due to progressive degeneration of mesencephalic dopaminergic (DA) neurons in the substantia nigra. To date, there is no regenerative treatment available. We previously showed that repulsive guidance molecule member a (RGMa) is upregulated in the substantia nigra of PD patients. Adeno-associated virus-mediated targeting of RGMa to mouse DA neurons showed that overexpression of this repulsive axon guidance and cell patterning cue models the behavioral and neuropathological characteristics of PD in a remarkable way. These findings have implications for therapy development as interfering with the function of this specific axon guidance cue may be beneficial to the survival of DA neurons

Repulsive guidance molecule a (RGMa) induces neuropathological and behavioral changes that closely resemble parkinson’s disease

Repulsive guidance molecule member a (RGMa) is a membrane-associated or released guidance molecule that is involved in axon guidance, cell patterning, and cell survival. In our previous work, we showed that RGMa is significantly upregulated in the substantia nigra of patients with Parkinson’s disease. Here we demonstrate the expression of RGMa in midbrain human dopaminergic (DA) neurons. To investigate whether RGMa might model aspects of the neuropathology of Parkinson’s disease in mouse, we targeted RGMa to adult midbrain dopaminergic neurons using adeno-associated viral vectors. Overexpression of RGMa resulted in a progressive movement disorder, including motor coordination and imbalance, which is typical for a loss of DA release in the striatum. In line with this, RGMa induced selective degeneration of dopaminergic neurons in the substantia nigra (SN) and affected the integrity of the nigrostriatal system. The degeneration of dopaminergic neurons was accompanied by a strong microglia and astrocyte activation. The behavioral, molecular, and anatomical changes induced by RGMa in mice are remarkably similar to the clinical and neuropathological hallmarks of Parkinson’s disease. Our data indicate that dysregulation of RGMa plays an important role in the pathology of Parkinson’s disease, and antibody-mediated functional interference with RGMa may be a disease modifying treatment option