Laquinimod Treatment Improves Myelination Deficits at the Transcriptional and Ultrastructural Levels in the YAC128 Mouse Model of Huntington Disease.

Laquinimod, an immunomodulatory agent under clinical development for Huntington disease (HD), has recently been shown to confer behavioural improvements that are coupled with prevention of atrophy of the white matter (WM)-rich corpus callosum (CC) in the YAC128 HD mice. However, the nature of the WM improvements is not known yet. Here we investigated the effects of laquinimod on HD-related myelination deficits at the cellular, molecular and ultrastructural levels. We showed that laquinimod treatment improves motor learning and motor function deficits in YAC128 HD mice, and confirmed its antidepressant effect even at the lowest dose used. In addition, we demonstrated for the first time the beneficial effects of laquinimod on myelination in the posterior region of the CC where it reversed changes in myelin sheath thickness and rescued Mbp mRNA and protein deficits. Furthermore, the effect of laquinimod on myelin-related gene expression was not region-specific since the levels of the Mbp and Plp1 transcripts were also increased in the striatum. Also, we did not detect changes in immune cell densities or levels of inflammatory genes in 3-month-old YAC128 HD mice, and these were not altered with laquinimod treatment. Thus, the beneficial effects of laquinimod on HD-related myelination abnormalities in YAC128 HD mice do not appear to be dependent on its immunomodulatory activity. Altogether, our findings describe the beneficial effects of laquinimod treatment on HD-related myelination abnormalities and highlight its therapeutic potential for the treatment of WM pathology in HD patients.

Altered proliferation and networks in neural cells derived from idiopathic autistic individuals.

Autism spectrum disorders (ASD) are common, complex and heterogeneous neurodevelopmental disorders. Cellular and molecular mechanisms responsible for ASD pathogenesis have been proposed based on genetic studies, brain pathology and imaging, but a major impediment to testing ASD hypotheses is the lack of human cell models. Here, we reprogrammed fibroblasts to generate induced pluripotent stem cells, neural progenitor cells (NPCs) and neurons from ASD individuals with early brain overgrowth and non-ASD controls with normal brain size. ASD-derived NPCs display increased cell proliferation because of dysregulation of a ß-catenin/BRN2 transcriptional cascade. ASD-derived neurons display abnormal neurogenesis and reduced synaptogenesis leading to functional defects in neuronal networks. Interestingly, defects in neuronal networks could be rescued by insulin growth factor 1 (IGF-1), a drug that is currently in clinical trials for ASD. This work demonstrates that selection of ASD subjects based on endophenotypes unraveled biologically relevant pathway disruption and revealed a potential cellular mechanism for the therapeutic effect of IGF-1.

Dual epithelial and immune cell function of Dvl1 regulates gut microbiota composition and intestinal homeostasis.

Homeostasis of the gastrointestinal (GI) tract is controlled by complex interactions between epithelial and immune cells and the resident microbiota. Here, we studied the role of Wnt signaling in GI homeostasis using Disheveled 1 knockout (Dvl1-/-) mice, which display an increase in whole gut transit time. This phenotype is associated with a reduction and mislocalization of Paneth cells and an increase in CD8+ T cells in the lamina propria. Bone marrow chimera experiments demonstrated that GI dysfunction requires abnormalities in both epithelial and immune cells. Dvl1-/- mice exhibit a significantly distinct GI microbiota, and manipulation of the gut microbiota in mutant mice rescued the GI transit abnormality without correcting the Paneth and CD8+ T cell abnormalities. Moreover, manipulation of the gut microbiota in wild-type mice induced a GI transit abnormality akin to that seen in Dvl1-/- mice. Together, these data indicate that microbiota manipulation can overcome host dysfunction to correct GI transit abnormalities. Our findings illustrate a mechanism by which the epithelium and immune system coregulate gut microbiota composition to promote normal GI function.

Loss of Dishevelleds disrupts planar polarity in ependymal motile cilia and results in hydrocephalus.

Defects in ependymal (E) cells, which line the ventricle and generate cerebrospinal fluid flow through ciliary beating, can cause hydrocephalus. Dishevelled genes (Dvls) are essential for Wnt signaling, and Dvl2 has been shown to localize to the rootlet of motile cilia. Using the hGFAP-Cre;Dvl1(-/-);2(flox/flox);3(+/-) mouse, we show that compound genetic ablation of Dvls causes hydrocephalus. In hGFAP-Cre;Dvl1(-/-);2(flox/flox);3(+/-) mutants, E cells differentiated normally, but the intracellular and intercellular rotational alignments of ependymal motile cilia were disrupted. As a consequence, the fluid flow generated by the hGFAP-Cre;Dvl1(-/-);2(flox/flox);3(+/-) E cells was significantly slower than that observed in control mice. Dvls were also required for the proper positioning of motile cilia on the apical surface. Tamoxifen-induced conditional removal of Dvls in adult mice also resulted in defects in intracellular rotational alignment and positioning of ependymal motile cilia. These results suggest that Dvls are continuously required for E cell planar polarity and may prevent hydrocephalus.

Age-dependent brain gene expression and copy number anomalies in autism suggest distinct pathological processes at young versus mature ages.

Autism is a highly heritable neurodevelopmental disorder, yet the genetic underpinnings of the disorder are largely unknown. Aberrant brain overgrowth is a well-replicated observation in the autism literature; but association, linkage, and expression studies have not identified genetic factors that explain this trajectory. Few studies have had sufficient statistical power to investigate whole-genome gene expression and genotypic variation in the autistic brain, especially in regions that display the greatest growth abnormality. Previous functional genomic studies have identified possible alterations in transcript levels of genes related to neurodevelopment and immune function. Thus, there is a need for genetic studies involving key brain regions to replicate these findings and solidify the role of particular functional pathways in autism pathogenesis. We therefore sought to identify abnormal brain gene expression patterns via whole-genome analysis of mRNA levels and copy number variations (CNVs) in autistic and control postmortem brain samples. We focused on prefrontal cortex tissue where excess neuron numbers and cortical overgrowth are pronounced in the majority of autism cases. We found evidence for dysregulation in pathways governing cell number, cortical patterning, and differentiation in young autistic prefrontal cortex. In contrast, adult autistic prefrontal cortex showed dysregulation of signaling and repair pathways. Genes regulating cell cycle also exhibited autism-specific CNVs in DNA derived from prefrontal cortex, and these genes were significantly associated with autism in genome-wide association study datasets. Our results suggest that CNVs and age-dependent gene expression changes in autism may reflect distinct pathological processes in the developing versus the mature autistic prefrontal cortex. Our results raise the hypothesis that genetic dysregulation in the developing brain leads to abnormal regional patterning, excess prefrontal neurons, cortical overgrowth, and neural dysfunction in autism.

ApoE4-Driven Accumulation of Intraneuronal Oligomerized Aß42 following Activation of the Amyloid Cascade In Vivo Is Mediated by a Gain of Function.

Activating the amyloid cascade by inhibiting the Aß-degrading enzyme neprilysin in targeted replacement mice, which express either apoE4 or apoE3, results in the specific accumulation of oligomerized Aß42 in hippocampal CA1 neurons of the apoE4 mice. We presently investigated the extent to which the apoE4-driven accumulation of Aß42 and the resulting mitochondrial pathology are due to either gain or loss of function. This revealed that inhibition of neprilysin for one week triggers the accumulation of Aß42 in hippocampal CA1 neurons of the apoE4 mice but not of either the corresponding apoE3 mice or apoE-deficient mice. At 10 days, Aß42 also accumulated in the CA1 neurons of the apoE-deficient mice but not in those of the apoE3 mice. Mitochondrial pathology, which in the apoE4 mice is an early pathological consequence following inhibition of neprilyisn, also occurs in the apoE-deficient but not in the apoE3 mice and the magnitude of this effect correlates with the levels of accumulated Aß42 and oligomerized Aß42 in these mice. These findings suggest that the rate-limiting step in the pathological effects of apoE4 on CA1 neurons is the accumulation of intracellular oligomerized Aß42 which is mediated via a gain of function property of apoE4.

Following activation of the amyloid cascade, apolipoprotein E4 drives the in vivo oligomerization of amyloid-ß resulting in neurodegeneration.

According to the amyloid hypothesis, the accumulation of oligomerized amyloid-ß (Aß) is a primary event in the pathogenesis of Alzheimer's disease (AD). The trigger of the amyloid cascade and of Aß oligomerization in sporadic AD, the most prevalent form of the disease, remains elusive. Here, we examined the hypothesis that apolipoprotein E4 (ApoE4), the most prevalent genetic risk factor for AD, triggers the accumulation of intraneuronal oligomerized Aß following activation of the amyloid cascade. We investigated the intracellular organelles that are targeted by these processes and govern their pathological consequences. This revealed that activation of the amyloid cascade in vivo by inhibition of the Aß degrading enzyme neprilysin specifically results in accumulation of Aß and oligomerized Aß and of ApoE4 in the CA1 neurons of ApoE4 mice. This was accompanied by lysosomal and mitochondrial pathology and the co-localization of Aß, oligomerized Aß, and ApoE4 with enlarged lysosomes and of Aß and oligomerized Aß with mitochondria. The time course of the lysosomal effects paralleled that of the loss of CA1 neurons, whereas the mitochondrial effects reached an earlier plateau. These findings suggest that ApoE4 potentiates the pathological effects of Aß and the amyloid cascade by triggering the oligomerization of Aß, which in turn, impairs intraneuronal mitochondria and lysosomes and drives neurodegeneration.

Possible role of tau in mediating pathological effects of apoE4 in vivo prior to and following activation of the amyloid cascade.

Injection of the neprilysin inhibitor thiorphan into the brain induces the accumulation of Abeta in hippocampal CA1 neurons and septal neurons in apoE4 knock-in mice but not in mice that express the corresponding Alzheimer's disease benign isoform apoE3. We investigated the possible role of tau phosphorylation in mediating this synergistic pathological cross talk between apoE4 and the amyloid cascade. This revealed that in both apoE4 and apoE3 mice, activating the amyloid cascade by inhibiting neprilysin triggers the accumulation of AT100 phosphorylated tau in the perikarya of CA1 neurons. In contrast, in the septum this treatment elevated the level of phosphorylation of the tau AT100 epitope only in the apoE4 mice. This suggests that tau-related processes by themselves do not mediate the synergistic pathological effects of apoE4 and Abeta in CA1 neurons. However, tau and cytoskeletal-related mechanisms may mediate the synergistic pathological effects of apoE4 and Abeta in the septum. The basal levels of tau phosphorylation are also affected by the apoE genotype. This effect, which is associated with hyperphosphorylation of the tau AT8 epitope, is most prominent in hippocampal CA3 neurons. This suggests that the apoE4 mice are already stressed under nonstimulated conditions and that AT8 tau phosphorylation may contribute to their increased susceptibility to brain insults.

Pathological synergism between amyloid-beta and apolipoprotein E4--the most prevalent yet understudied genetic risk factor for Alzheimer's disease.

This review focuses on apolipoprotein E4 (apoE4), the most prevalent genetic risk factor of Alzheimer's disease, and on in vivo and in vitro model studies of the mechanisms underlying its pathological phenotype. The review will first center on in vivo studies with transgenic mice that express human apoE4 and other human apoE alleles, and on the extent to which this model mimics and reproduces the human apoE4 phenotypes. The second part of this review will address apoE4-related in vitro studies, with particular emphasis on the effects of the state of lipidation of apoE4 on its biochemical properties and on the extent to which the in vitro results can be generalized and applied to the in vivo situation. The third part of this review will focus on a novel pharmacological in vivo system that was recently developed in our laboratory, which is based on activation of the amyloid cascade in apoE transgenic mice by prolonged inhibition of the Abeta-degrading enzyme neprilysin and on what this system and its high spatio-temporal resolution has taught us about the mechanisms underlying the pathological effects of apoE4 in vivo.

ApoE4-dependent Abeta-mediated neurodegeneration is associated with inflammatory activation in the hippocampus but not the septum.

Apolipoprotein E4 (ApoE4), the most prevalent genetic risk factor for Alzheimer's disease, is histopathologically associated with increased deposition of amyloid-beta and brain inflammation and with impaired neuronal plasticity and repair. We have recently shown that the activation of the amyloid cascade by inhibition of the Abeta-degrading enzyme, neprilysin, stimulates the isoform-specific degeneration of hippocampal CA1 neurons and septal neurons in apoE4 transgenic mice and that this effect is accompanied by the accumulation of intracellular Abeta in the affected neurons. We presently examined the extent to which this apoE4-dependent Abeta-mediated neurodegeneration is associated with brain area specific inflammatory activation. This revealed that the activation of the amyloid cascade in apoE transgenic mice results in the activation of microgliosis and astrogliosis in the hippocampus of apoE4, but not in apoE3 transgenic mice. The effect was most pronounced in the hippocampal CA1 subfield and its initial kinetics followed that of the accumulation of Abeta in CA1 neurons. In contrast, the corresponding apoE4-dependent Abeta degeneration of septal neurons was not associated with the activation of either gliosis or astrogliosis in this brain area. These animal model findings, that the association between brain inflammation and neurodegeneration is brain area specific, suggest that neuropathological inflammatory interactions in AD may also be brain area specific and that consequently the efficacy of putative anti-inflammatory intervention may also be brain area selective.

Activation of the amyloid cascade in apolipoprotein E4 transgenic mice induces lysosomal activation and neurodegeneration resulting in marked cognitive deficits.

The allele E4 of apolipoprotein E (apoE4), the most prevalent genetic risk factor for Alzheimer's disease, is associated histopathologically with elevated levels of brain amyloid. This led to the suggestion that the pathological effects of apoE4 are mediated by cross-talk interactions with amyloid beta peptide (Abeta), which accentuate the pathological effects of the amyloid cascade. The mechanisms underlying the Abeta-mediated pathological effects of apoE4 are unknown. We have shown recently that inhibition of the Abeta-degrading enzyme neprilysin in brains of wild-type apoE3 and apoE4 mice results in rapid and similar elevations in their total brain Abeta levels. However, the nucleation and aggregation of Abeta in these mice were markedly affected by the apoE genotype and were specifically enhanced in the apoE4 mice. We presently used the neprilysin inhibition paradigm to analyze the neuropathological and cognitive effects that are induced by apoE4 after activation of the amyloid cascade. This revealed that apoE4 stimulates isoform specifically the degeneration of hippocampal CA1 neurons and of entorhinal and septal neurons, which is accompanied by the accumulation of intracellular Abeta and apoE and with lysosomal activation. Furthermore, these neuropathological effects are associated isoform specifically with the occurrence of pronounced cognitive deficits in the ApoE4 mice. These findings provide the first in vivo evidence regarding the cellular mechanisms underlying the pathological cross talk between apoE4 and Abeta, as well as a novel model system of neurodegeneration in vivo that is uniquely suitable for studying the early stages of the amyloid cascade and the effects thereon of apoE4.

Activation of the amyloid cascade by intracerebroventricular injection of the protease inhibitor phosphoramidon.

We presently investigated the pathological effects of prolonged inhibition of brain beta-amyloid (Abeta) degradation in vivo. The results obtained revealed that intracerebroventricular injection of the protease inhibitor phosphoramidon into wild-type mice for up to a month elevated the soluble and deposited brain Abeta levels and concomitantly induced the neurodegeneration of distinct hippocampal neurons as well as neuroinflammation. These findings reproduce pathological effects associated with the initial stages of the amyloid cascade and provide a novel model system for studying their underlying mechanisms.

Intraneuronal amyloid-beta plays a role in mediating the synergistic pathological effects of apoE4 and environmental stimulation.

The allele E4 of apolipoprotein E4 (apoE4), which is the most prevalent genetic risk factor of Alzheimer's disease (AD), inhibits synaptogenesis and neurogenesis and stimulates apoptosis in brains of apoE4 transgenic mice that have been exposed to an enriched environment. In the present study, we investigated the hypothesis that the brain activity-dependent impairments in neuronal plasticity, induced by apoE4, are mediated via the amyloid cascade. Importantly, we found that exposure of mice transgenic for either apoE4, or the Alzheimer's disease benign allele apoE3, to an enriched environment elevates similarly the hippocampal levels of amyloid-beta peptide (Abeta) and apoE of these mice, but that the degree of aggregation and spatial distribution of Abeta in these mice are markedly affected by the apoE genotype. Accordingly, environmental stimulation triggered the formation of extracellular plaque-like Abeta deposits and the accumulation of intra-neuronal oligomerized Abeta specifically in brains of apoE4 mice. Further experiments revealed that hippocampal dentate gyrus neurons are particularly susceptible to apoE4 and environmental stimulation and that these neurons are specifically enriched in both oligomerized Abeta and apoE. These findings show that the impairments in neuroplasticity which are induced by apoE4 following environmental stimulation are associated with the accumulation of intraneuronal Abeta and suggest that oligomerized Abeta mediates the synergistic pathological effects of apoE4 and environmental stimulation.