Specific Mutations in the Cholesterol-Binding Site of APP Alter Its Processing and Favor the Production of Shorter, Less Toxic Aß Peptides.

Excess brain cholesterol is strongly implicated in the pathogenesis of Alzheimer's disease (AD). Here we evaluated how the presence of a cholesterol-binding site (CBS) in the transmembrane and juxtamembrane regions of the amyloid precursor protein (APP) regulates its processing. We generated nine point mutations in the APP gene, changing the charge and/or hydrophobicity of the amino-acids which were previously shown as part of the CBS. Most mutations triggered a reduction of amyloid-ß peptides Aß40 and Aß42 secretion from transiently transfected HEK293T cells. Only the mutations at position 28 of Aß in the APP sequence resulted in a concomitant significant increase in the production of shorter Aß peptides. Mass spectrometry (MS) confirmed the predominance of Aßx-33 and Aßx-34 with the APPK28A mutant. The enzymatic activity of α-, ß-, and γ-secretases remained unchanged in cells expressing all mutants. Similarly, subcellular localization of the mutants in early endosomes did not differ from the APPWT protein. A transient increase of plasma membrane cholesterol enhanced the production of Aß40 and Aß42 by APPWT, an effect absent in APPK28A mutant. Finally, WT but not CBS mutant Aß derived peptides bound to cholesterol-rich exosomes. Collectively, the present data revealed a major role of juxtamembrane amino acids of the APP CBS in modulating the production of toxic Aß species. More generally, they underpin the role of cholesterol in the pathophysiology of AD.

The amyloid precursor protein is a conserved Wnt receptor.

The Amyloid Precursor Protein (APP) and its homologues are transmembrane proteins required for various aspects of neuronal development and activity, whose molecular function is unknown. Specifically, it is unclear whether APP acts as a receptor, and if so what its ligand(s) may be. We show that APP binds the Wnt ligands Wnt3a and Wnt5a and that this binding regulates APP protein levels. Wnt3a binding promotes full-length APP (flAPP) recycling and stability. In contrast, Wnt5a promotes APP targeting to lysosomal compartments and reduces flAPP levels. A conserved Cysteine-Rich Domain (CRD) in the extracellular portion of APP is required for Wnt binding, and deletion of the CRD abrogates the effects of Wnts on flAPP levels and trafficking. Finally, loss of APP results in increased axonal and reduced dendritic growth of mouse embryonic primary cortical neurons. This phenotype can be cell-autonomously rescued by full length, but not CRD-deleted, APP and regulated by Wnt ligands in a CRD-dependent manner.

Combination of acamprosate and baclofen (PXT864) as a potential new therapy for amyotrophic lateral sclerosis.

There is currently no therapy impacting the course of amyotrophic lateral sclerosis (ALS). The only approved treatments are riluzole and edaravone, but their efficacy is modest and short-lasting, highlighting the need for innovative therapies. We previously demonstrated the ability of PXT864, a combination of low doses of acamprosate and baclofen, to synergistically restore cellular and behavioral activity in Alzheimer's and Parkinson's disease models. The overlapping genetic, molecular, and cellular characteristics of these neurodegenerative diseases supported investigating the effectiveness of PXT864 in ALS. As neuromuscular junction (NMJ) alterations is a key feature of ALS, the effects of PXT864 in primary neuron-muscle cocultures injured by glutamate were studied. PXT864 significantly and synergistically preserved NMJ and motoneuron integrity following glutamate excitotoxicity. PXT864 added to riluzole significantly improved such protection. PXT864 activity was then assessed in primary cultures of motoneurons derived from SOD1G93A rat embryos. These motoneurons presented severe maturation defects that were significantly improved by PXT864. In this model, glutamate application induced an accumulation of TDP-43 protein in the cytoplasm, a hallmark that was completely prevented by PXT864. The anti-TDP-43 aggregation effect was also confirmed in a cell line expressing TDP-43 fused to GFP. These results demonstrate the value of PXT864 as a promising therapeutic strategy for the treatment of ALS.

Synergistic PXT3003 therapy uncouples neuromuscular function from dysmyelination in male Charcot-Marie-Tooth disease type 1A (CMT1A) rats.

Charcot-Marie-Tooth disease 1 A (CMT1A) is caused by an intrachromosomal duplication of the gene encoding for PMP22 leading to peripheral nerve dysmyelination, axonal loss, and progressive muscle weakness. No therapy is available. PXT3003 is a low-dose combination of baclofen, naltrexone, and sorbitol which has been shown to improve disease symptoms in Pmp22 transgenic rats, a bona fide model of CMT1A disease. However, the superiority of PXT3003 over its single components or dual combinations have not been tested. Here, we show that in a dorsal root ganglion (DRG) co-culture system derived from transgenic rats, PXT3003 induced myelination when compared to its single and dual components. Applying a clinically relevant ("translational") study design in adult male CMT1A rats for 3 months, PXT3003, but not its dual components, resulted in improved performance in behavioral motor and sensory endpoints when compared to placebo. Unexpectedly, we observed only a marginally increased number of myelinated axons in nerves from PXT3003-treated CMT1A rats. However, in electrophysiology, motor latencies correlated with increased grip strength indicating a possible effect of PXT3003 on neuromuscular junctions (NMJs) and muscle fiber pathology. Indeed, PXT3003-treated CMT1A rats displayed an increased perimeter of individual NMJs and a larger number of functional NMJs. Moreover, muscles of PXT3003 CMT1A rats displayed less neurogenic atrophy and a shift toward fast contracting muscle fibers. We suggest that ameliorated motor function in PXT3003-treated CMT1A rats result from restored NMJ function and muscle innervation, independent from myelination.

CYP46A1 protects against NMDA-mediated excitotoxicity in Huntington's disease: Analysis of lipid raft content.

Huntington's Disease (HD) is an autosomal dominant neurodegenerative disease caused by abnormal polyglutamine expansion in huntingtin (mHtt) protein leading to degeneration of striatal neurons. Excitotoxicity, consecutive to overstimulation of N-methyl d-aspartate receptors (NMDARs) has a pivotal role in many neurological disorders including HD. Mutant Htt causes enhanced NMDA sensitivity, alteration of NMDAR expression and localization in neurons. Excitotoxic events initiate neuronal death in numerous ways, including activation of apoptotic cascades. Among the NMDAR subunits involved in glutamatergic-mediated excitotoxicity, GluN2B has been extensively reported. In addition to excitotoxicity, alteration of cholesterol metabolism has been observed in HD, with a decrease of cholesterol precursor synthesis along with an increase of cholesterol accumulation, which is deleterious for neurons. Expression of Cholesterol Hydroxylase enzyme, CYP46A1, which converts cholesterol into 24 S-hydroxycholesterol is down-regulated in HD. We found that CYP46A1 overexpression is beneficial in HD neurons and mouse model, but the mechanisms involved still remain unclear. In this study we addressed the effect of CYP46A1 on NMDAR-mediated excitotoxicity in HD primary neurons and its role in modulating cholesterol and localization of GLUN2B in lipid rafts. We showed that CYP46A1 is protective against NMDAR-mediated excitotoxicity in two different HD neuronal cell models. Cholesterol as well as GluN2B level in lipid raft, are significantly increased by mHtt. Despite a clear effect of CYP46A1 in reducing cholesterol content in lipid raft extracts from wild type neurons, CYP46A1 overexpression in HD neurons could not normalize the increased cholesterol levels in lipid rafts. This study highlights the beneficial role of CYP46A1 against NMDAR-mediated excitotoxicity and gives further insights into the cellular mechanisms underlying CYP46A1-mediated neuroprotection.

CYP46A1, the rate-limiting enzyme for cholesterol degradation, is neuroprotective in Huntington's disease.

Huntington's disease is an autosomal dominant neurodegenerative disease caused by abnormal polyglutamine expansion in huntingtin (Exp-HTT) leading to degeneration of striatal neurons. Altered brain cholesterol homeostasis has been implicated in Huntington's disease, with increased accumulation of cholesterol in striatal neurons yet reduced levels of cholesterol metabolic precursors. To elucidate these two seemingly opposing dysregulations, we investigated the expression of cholesterol 24-hydroxylase (CYP46A1), the neuronal-specific and rate-limiting enzyme for cholesterol conversion to 24S-hydroxycholesterol (24S-OHC). CYP46A1 protein levels were decreased in the putamen, but not cerebral cortex samples, of post-mortem Huntington's disease patients when compared to controls. Cyp46A1 mRNA and CYP46A1 protein levels were also decreased in the striatum of the R6/2 Huntington's disease mouse model and in SThdhQ111 cell lines. In vivo, in a wild-type context, knocking down CYP46A1 expression in the striatum, via an adeno-associated virus-mediated delivery of selective shCYP46A1, reproduced the Huntington's disease phenotype, with spontaneous striatal neuron degeneration and motor deficits, as assessed by rotarod. In vitro, CYP46A1 restoration protected SThdhQ111 and Exp-HTT-expressing striatal neurons in culture from cell death. In the R6/2 Huntington's disease mouse model, adeno-associated virus-mediated delivery of CYP46A1 into the striatum decreased neuronal atrophy, decreased the number, intensity level and size of Exp-HTT aggregates and improved motor deficits, as assessed by rotarod and clasping behavioural tests. Adeno-associated virus-CYP46A1 infection in R6/2 mice also restored levels of cholesterol and lanosterol and increased levels of desmosterol. In vitro, lanosterol and desmosterol were found to protect striatal neurons expressing Exp-HTT from death. We conclude that restoring CYP46A1 activity in the striatum promises a new therapeutic approach in Huntington's disease.

Synaptic scaling up in medium spiny neurons of aged BACHD mice: A slow-progression model of Huntington's disease.

Huntington's disease (HD) is an autosomal dominant disease that develops in midlife (~ 40 years-old at onset) and then progresses slowly. It is still unclear how striatal medium spiny neurons (MSNs), the most vulnerable neurons in HD, maintain their function for decades despite the chronic expression of mutant huntingtin (mHTT). In this study, we used aged BACHD mice, a HD model expressing the full-length human mHTT gene, to investigate the molecular, morphological and functional properties of striatal MSNs. We report that the density of dendritic spines in MSNs is substantially lower in aged BACHD mice than in wild-type (WT) mice, in the absence of major dendritic changes and neuronal loss. This spine loss is accompanied by changes in transcription, resulting in a low expression of the striatum-specific G protein-coupled receptor 88 (Gpr88) as well as a reorganization of the composition of AMPAR subunits (high Gria1/Gria2 mRNA ratio). We also detected functional changes in BACHD MSNs. Notably, BACHD MSNs were hyperexcitable and the amplitude of AMPAR-mediated synaptic currents was higher than in WT MSNs. Altogether, these data show that both the intrinsic properties and the strength of the remaining synapses are modified in MSNs with low dendritic spine density in aged BACHD mice. These homeostatic mechanisms may compensate for the substantial loss of synaptic inputs and thus alleviate the deleterious effects of mHTT expression on the activity of MSNs and also possibly on the motor phenotype in aged BACHD.

Impaired brain energy metabolism in the BACHD mouse model of Huntington's disease: critical role of astrocyte-neuron interactions.

Huntington's disease (HD) is caused by cytosine-adenine-guanine (CAG) repeat expansions in the huntingtin (Htt) gene. Although early energy metabolic alterations in HD are likely to contribute to later neurodegenerative processes, the cellular and molecular mechanisms responsible for these metabolic alterations are not well characterized. Using the BACHD mice that express the full-length mutant huntingtin (mHtt) protein with 97 glutamine repeats, we first demonstrated localized in vivo changes in brain glucose use reminiscent of what is observed in premanifest HD carriers. Using biochemical, molecular, and functional analyses on different primary cell culture models from BACHD mice, we observed that mHtt does not directly affect metabolic activity in a cell autonomous manner. However, coculture of neurons with astrocytes from wild-type or BACHD mice identified mutant astrocytes as a source of adverse non-cell autonomous effects on neuron energy metabolism possibly by increasing oxidative stress. These results suggest that astrocyte-to-neuron signaling is involved in early energy metabolic alterations in HD.

Aging of the dopaminergic system and motor behavior in mice intoxicated with the parkinsonian toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication of mice is a standard model of Parkinson's disease (PD). However, it does not reproduce functionally PD. Given the occurrence of PD during aging, symptoms might only be detected in MPTP-intoxicated mice after aging. To address this, mice injected with MPTP at 2.5 months were followed up to a maximum age of 21 months. There was no loss of dopamine cells with aging in control mice; moreover, the initial post-MPTP intoxication decrease in dopamine cell was no longer significant at 21 months. With aging, striatal dopamine level remained constant, but concentrations of the dopamine metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) were markedly reduced in both groups. There was also a late impairment of fine motor skills. After MPTP intoxication, hyperactivity was immediately detected and it became greater than in control mice from 14 months of age; fine motor skills were also more impaired; both these symptoms were correlated with striatal dopamine, DOPAC and HVA concentrations. In bothgroups, neither motor symptoms nor dopamine changes worsened with age. These findings do not support the notion that PD develops with age in mice after MPTP intoxication and that the motor deficits seen are because of an aging process.