Plasma neurofilament light, glial fibrillary acidic protein and lysosphingolipid biomarkers for pharmacodynamics and disease monitoring of GM2 and GM1 gangliosidoses patients

GM2 and GM1 gangliosidoses are genetic, neurodegenerative lysosomal sphingolipid storage disorders. The earlier the age of onset, the more severe the clinical presentation and progression, with infantile, juvenile and late-onset presentations broadly delineated into separate phenotypic subtypes. Gene and substrate reduction therapies, both of which act directly on sphingolipidosis are entering clinical trials for treatment of these disorders. Simple to use biomarkers for disease monitoring are urgently required to support and expedite these clinical trials. Here, lysosphingolipid and protein biomarkers of sphingolipidosis and neuropathology respectively, were assessed in plasma samples from 33 GM2 gangliosidosis patients, 13 GM1 gangliosidosis patients, and compared to 66 controls. LysoGM2 and lysoGM1 were detectable in 31/33 GM2 gangliosidosis and 12/13 GM1 gangliosidosis patient samples respectively, but not in any controls. Levels of the axonal damage marker Neurofilament light (NF-L) were highly elevated in both GM2 and GM1 gangliosidosis patient plasma samples, with no overlap with controls. Levels of the astrocytosis biomarker Glial fibrillary acidic protein (GFAP) were also elevated in samples from both patient populations, albeit with some overlap with controls. In GM2 gangliosidosis patient plasma NF-L, Tau, GFAP and lysoGM2 were all most highly elevated in infantile onset patients, indicating a relationship to severity and phenotype. Plasma NF-L and liver lysoGM2 were also elevated in a GM2 gangliosidosis mouse model, and were lowered by treatment with a drug that slowed disease progression. These results indicate that lysosphingolipids and NF-L/GFAP have potential to monitor pharmacodynamics and pathogenic processes respectively in GM2 and GM1 gangliosidoses patients

Differentiation of mouse bone marrow derived stem cells toward microglia-like cells

<p>Abstract</p> <p>Background</p> <p>Microglia, the macrophages of the brain, have been implicated in the causes of neurodegenerative diseases and display a loss of function during aging. Throughout life, microglia are replenished by limited proliferation of resident microglial cells. Replenishment by bone marrow-derived progenitor cells is still under debate. In this context, we investigated the differentiation of mouse microglia from bone marrow (BM) stem cells. Furthermore, we looked at the effects of FMS-like tyrosine kinase 3 ligand (Flt3L), astrocyte-conditioned medium (ACM) and GM-CSF on the differentiation to microglia-like cells.</p> <p>Methods</p> <p>We assessed <it>in vitro-</it>derived microglia differentiation by marker expression (CD11b/CD45, F4/80), but also for the first time for functional performance (phagocytosis, oxidative burst) and <it>in situ </it>migration into living brain tissue. Integration, survival and migration were assessed in organotypic brain slices.</p> <p>Results</p> <p>The cells differentiated from mouse BM show function, markers and morphology of primary microglia and migrate into living brain tissue. Flt3L displays a negative effect on differentiation while GM-CSF enhances differentiation.</p> <p>Conclusion</p> <p>We conclude that <it>in vitro-</it>derived microglia are the phenotypic and functional equivalents to primary microglia and could be used in cell therapy.</p

Chronic Cyclodextrin Treatment of Murine Niemann-Pick C Disease Ameliorates Neuronal Cholesterol and Glycosphingolipid Storage and Disease Progression

BACKGROUND:Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder caused most commonly by a defect in the NPC1 protein and characterized by widespread intracellular accumulation of unesterified cholesterol and glycosphingolipids (GSLs). While current treatment therapies are limited, a few drugs tested in Npc1(-/-) mice have shown partial benefit. During a combination treatment trial using two such compounds, N-butyldeoxynojirimycin (NB-DNJ) and allopregnanolone, we noted increased lifespan for Npc1(-/-) mice receiving only 2-hydroxypropyl-beta-cyclodextrin (CD), the vehicle for allopregnanolone. This finding suggested that administration of CD alone, but with greater frequency, might provide additional benefit. METHODOLOGY/PRINCIPAL FINDINGS:Administration of CD to Npc1(-/-) mice beginning at either P7 or P21 and continuing every other day delayed clinical onset, reduced intraneuronal cholesterol and GSL storage as well as free sphingosine accumulation, reduced markers of neurodegeneration, and led to longer survival than any previous treatment regime. We reasoned that other lysosomal diseases characterized by cholesterol and GSL accumulation, including NPC disease due to NPC2 deficiency, GM1 gangliosidosis and mucopolysaccharidosis (MPS) type IIIA, might likewise benefit from CD treatment. Treated Npc2(-/-) mice showed benefits similar to NPC1 disease, however, mice with GM1 gangliosidosis or MPS IIIA failed to show reduction in storage. CONCLUSIONS/SIGNIFICANCE:Treatment with CD delayed clinical disease onset, reduced intraneuronal storage and secondary markers of neurodegeneration, and significantly increased lifespan of both Npc1(-/-) and Npc2(-/-) mice. In contrast, CD failed to ameliorate cholesterol or glycosphingolipid storage in GM1 gangliosidosis and MPS IIIA disease. Understanding the mechanism(s) by which CD leads to reduced neuronal storage may provide important new opportunities for treatment of NPC and related neurodegenerative diseases characterized by cholesterol dyshomeostasis

X-linked Angelman-like syndrome caused by Slc9a6 knockout in mice exhibits evidence of endosomal–lysosomal dysfunction

Mutations in solute carrier family 9 isoform 6 on chromosome Xq26.3 encoding sodium–hydrogen exchanger 6, a protein mainly expressed in early and recycling endosomes are known to cause a complex and slowly progressive degenerative human neurological disease. Three resulting phenotypes have so far been reported: an X-linked Angelman syndrome-like condition, Christianson syndrome and corticobasal degeneration with tau deposition, with each characterized by severe intellectual disability, epilepsy, autistic behaviour and ataxia. Hypothesizing that a sodium–hydrogen exchanger 6 deficiency would most likely disrupt the endosomal–lysosomal system of neurons, we examined Slc9a6 knockout mice with tissue staining and related techniques commonly used to study lysosomal storage disorders. As a result, we found that sodium–hydrogen exchanger 6 depletion leads to abnormal accumulation of GM2 ganglioside and unesterified cholesterol within late endosomes and lysosomes of neurons in selective brain regions, most notably the basolateral nuclei of the amygdala, the CA3 and CA4 regions and dentate gyrus of the hippocampus and some areas of cerebral cortex. In these select neuronal populations, histochemical staining for β-hexosaminidase activity, a lysosomal enzyme involved in the degradation of GM2 ganglioside, was undetectable. Neuroaxonal dystrophy similar to that observed in lysosomal disease was observed in the cerebellum and was accompanied by a marked and progressive loss of Purkinje cells, particularly in those lacking the expression of Zebrin II. On behavioural testing, Slc9a6 knockout mice displayed a discrete clinical phenotype attributable to motor hyperactivity and cerebellar dysfunction. Importantly, these findings show that sodium–hydrogen exchanger 6 loss of function in the Slc9a6-targeted mouse model leads to compromise of endosomal–lysosomal function similar to lysosomal disease and to conspicuous neuronal abnormalities in specific brain regions, which in concert could provide a unified explanation for the cellular and clinical phenotypes in humans with SLC9A6 mutations

Absence of Colony Stimulation Factor-1 Receptor Results in Loss of Microglia, Disrupted Brain Development and Olfactory Deficits

The brain contains numerous mononuclear phagocytes called microglia. These cells express the transmembrane tyrosine kinase receptor for the macrophage growth factor colony stimulating factor-1 (CSF-1R). Using a CSF-1R-GFP reporter mouse strain combined with lineage defining antibody staining we show in the postnatal mouse brain that CSF-1R is expressed only in microglia and not neurons, astrocytes or glial cells. To study CSF-1R function we used mice homozygous for a null mutation in the Csflr gene. In these mice microglia are >99% depleted at embryonic day 16 and day 1 post-partum brain. At three weeks of age this microglial depletion continues in most regions of the brain although some contain clusters of rounded microglia. Despite the loss of microglia, embryonic brain development appears normal but during the post-natal period the brain architecture becomes perturbed with enlarged ventricles and regionally compressed parenchyma, phenotypes most prominent in the olfactory bulb and cortex. In the cortex there is increased neuronal density, elevated numbers of astrocytes but reduced numbers of oligodendrocytes. Csf1r nulls rarely survive to adulthood and therefore to study the role of CSF-1R in olfaction we used the viable null mutants in the Csf1 (Csf1op) gene that encodes one of the two known CSF-1R ligands. Food-finding experiments indicate that olfactory capacity is significantly impaired in the absence of CSF-1. CSF-1R is therefore required for the development of microglia, for a fully functional olfactory system and the maintenance of normal brain structure

Disruption of Transient Serotonin Accumulation by Non-Serotonin-Producing Neurons Impairs Cortical Map Development

Polymorphisms that alter serotonin transporter SERT expression and functionality increase the risks for autism and psychiatric traits. Here, we investigate how SERT controls serotonin signaling in developing CNS in mice. SERT is transiently expressed in specific sets of glutamatergic neurons and uptakes extrasynaptic serotonin during perinatal CNS development. We show that SERT expression in glutamatergic thalamocortical axons (TCAs) dictates sensory map architecture. Knockout of SERT in TCAs causes lasting alterations in TCA patterning, spatial organizations of cortical neurons, and dendritic arborization in sensory cortex. Pharmacological reduction of serotonin synthesis during the first postnatal week rescues sensory maps in SERTGluΔ mice. Furthermore, knockdown of SERT expression in serotonin-producing neurons does not impair barrel maps. We propose that spatiotemporal SERT expression in non-serotonin-producing neurons represents a determinant in early life genetic programming of cortical circuits. Perturbing this SERT function could be involved in the origin of sensory and cognitive deficits associated with neurodevelopmental disorders