CLN7/MFSD8 may be an important factor for SARS-CoV-2 cell entry.

The SARS-CoV-2 virus has triggered a worldwide pandemic. According to the BioGrid database, CLN7 (MFSD8) is thought to interact with several viral proteins. The aim of this work was to investigate a possible involvement of CLN7 in the infection process. Experiments on a CLN7-deficient HEK293T cell line exhibited a 90% reduced viral load compared to wild-type cells. This observation may be linked to the finding that CLN7 ko cells have a significantly reduced GM1 content in their cell membrane. GM1 is found highly enriched in lipid rafts, which are thought to play an important role in SARS-CoV-2 infection. In contrast, overexpression of CLN7 led to an increase in viral load. This study provides evidence that CLN7 is involved in SARS-CoV-2 infection. This makes it a potential pharmacological target for drug development against COVID-19. Furthermore, it provides insights into the physiological function of CLN7 where still only little is known about.

Neuroinflammation in Gaucher disease, neuronal ceroid lipofuscinosis, and commonalities with Parkinson's disease.

Lysosomal storage diseases (LSDs) are rare genetic disorders caused by a disruption in cellular clearance, resulting in pathological storage of undegraded lysosomal substrates. Recent clinical and genetic studies have uncovered links between multiple LSDs and common neurodegenerative diseases such as Parkinson's disease (PD). Here, we review recent literature describing the role of glia cells and neuroinflammation in PD and LSDs, including Gaucher disease (GD) and neuronal ceroid lipofuscinosis (NCL), and highlight converging inflammation pathways that lead to neuron loss. Recent data indicates that lysosomal dysfunction and accumulation of storage materials can initiate the activation of glial cells, through interaction with cell surface or cytosolic pattern recognition receptors that detect pathogenic aggregates of cellular debris. Activated glia cells could act to protect neurons through the elimination of toxic protein or lipid aggregates early in the disease process. However prolonged glial activation that occurs over several decades in chronic-age related neurodegeneration could induce the inappropriate elimination of synapses, leading to neuron loss. These studies provide mechanistic insight into the relationship between lysosomal dysfunction and glial activation, and offer novel therapeutic pathways for the treatment of PD and LSDs focused on reducing neuroinflammation and mitigating cell loss.

Rescue of α-synuclein aggregation in Parkinson's patient neurons by synergistic enhancement of ER proteostasis and protein trafficking.

Neurodegenerative disorders are characterized by a collapse in proteostasis, as shown by the accumulation of insoluble protein aggregates in the brain. Proteostasis involves a balance of protein synthesis, folding, trafficking, and degradation, but how aggregates perturb these pathways is unknown. Using Parkinson's disease (PD) patient midbrain cultures, we find that aggregated α-synuclein induces endoplasmic reticulum (ER) fragmentation and compromises ER protein folding capacity, leading to misfolding and aggregation of immature lysosomal ß-glucocerebrosidase. Despite this, PD neurons fail to initiate the unfolded protein response, indicating perturbations in sensing or transducing protein misfolding signals in the ER. Small molecule enhancement of ER proteostasis machinery promotes ß-glucocerebrosidase solubility, while simultaneous enhancement of trafficking improves ER morphology, lysosomal function, and reduces α-synuclein. Our studies suggest that aggregated α-synuclein perturbs the ability of neurons to respond to misfolded proteins in the ER, and that synergistic enhancement of multiple proteostasis branches may provide therapeutic benefit in PD.

Inhibition of HDAC6 activity protects dopaminergic neurons from alpha-synuclein toxicity.

The neuropathological hallmarks of Parkinson's disease include preferential vulnerability of dopaminergic neurons of the substantia nigra pars compacta, and accumulation of intraneuronal protein inclusions known as Lewy bodies. These inclusions contain, among other proteins, aggregated alpha-synuclein and histone deacetylase 6 (HDAC6). In our study we found that selective inhibition of HDAC6 activity by Tubastatin A has protective effects in a rat model of Parkinson's disease. We provide evidence that this protection may be due to the activation of chaperone-mediated autophagy through the up-regulation of key members of this pathway. Moreover, Tubastatin A significantly inhibited the expression of a toxic form of alpha-synuclein that is phosphorylated at serine position 129. Tubastatin A treatment also permitted to partially modulate neuroinflammation. Taken together, our study highlights the neuroprotective effects of Tubastatin A in a rat model of Parkinson's disease and provides mechanistic insight in Tubastatin A-mediated protection against alpha-synuclein toxicity and substantia nigra degeneration. These findings are of potential therapeutic value in Parkinson's disease and other synucleinopathies.

The striatal kinase DCLK3 produces neuroprotection against mutant huntingtin.

The neurobiological functions of a number of kinases expressed in the brain are unknown. Here, we report new findings on DCLK3 (doublecortin like kinase 3), which is preferentially expressed in neurons in the striatum and dentate gyrus. Its function has never been investigated. DCLK3 expression is markedly reduced in Huntington's disease. Recent data obtained in studies related to cancer suggest DCLK3 could have an anti-apoptotic effect. Thus, we hypothesized that early loss of DCLK3 in Huntington's disease may render striatal neurons more susceptible to mutant huntingtin (mHtt). We discovered that DCLK3 silencing in the striatum of mice exacerbated the toxicity of an N-terminal fragment of mHtt. Conversely, overexpression of DCLK3 reduced neurodegeneration produced by mHtt. DCLK3 also produced beneficial effects on motor symptoms in a knock-in mouse model of Huntington's disease. Using different mutants of DCLK3, we found that the kinase activity of the protein plays a key role in neuroprotection. To investigate the potential mechanisms underlying DCLK3 effects, we studied the transcriptional changes produced by the kinase domain in human striatal neurons in culture. Results show that DCLK3 regulates in a kinase-dependent manner the expression of many genes involved in transcription regulation and nucleosome/chromatin remodelling. Consistent with this, histological evaluation showed DCLK3 is present in the nucleus of striatal neurons and, protein-protein interaction experiments suggested that the kinase domain interacts with zinc finger proteins, including the transcriptional activator adaptor TADA3, a core component of the Spt-ada-Gcn5 acetyltransferase (SAGA) complex which links histone acetylation to the transcription machinery. Our novel findings suggest that the presence of DCLK3 in striatal neurons may play a key role in transcription regulation and chromatin remodelling in these brain cells, and show that reduced expression of the kinase in Huntington's disease could render the striatum highly vulnerable to neurodegeneration.

Correction: The mechanism of sirtuin 2-mediated exacerbation of alpha-synuclein toxicity in models of Parkinson disease.

[This corrects the article DOI: 10.1371/journal.pbio.2000374.].

The mechanism of sirtuin 2-mediated exacerbation of alpha-synuclein toxicity in models of Parkinson disease.

Sirtuin genes have been associated with aging and are known to affect multiple cellular pathways. Sirtuin 2 was previously shown to modulate proteotoxicity associated with age-associated neurodegenerative disorders such as Alzheimer and Parkinson disease (PD). However, the precise molecular mechanisms involved remain unclear. Here, we provide mechanistic insight into the interplay between sirtuin 2 and α-synuclein, the major component of the pathognomonic protein inclusions in PD and other synucleinopathies. We found that α-synuclein is acetylated on lysines 6 and 10 and that these residues are deacetylated by sirtuin 2. Genetic manipulation of sirtuin 2 levels in vitro and in vivo modulates the levels of α-synuclein acetylation, its aggregation, and autophagy. Strikingly, mutants blocking acetylation exacerbate α-synuclein toxicity in vivo, in the substantia nigra of rats. Our study identifies α-synuclein acetylation as a key regulatory mechanism governing α-synuclein aggregation and toxicity, demonstrating the potential therapeutic value of sirtuin 2 inhibition in synucleinopathies.

Contribution of Neuroepigenetics to Huntington's Disease.

Unbalanced epigenetic regulation is thought to contribute to the progression of several neurodegenerative diseases, including Huntington's disease (HD), a genetic disorder considered as a paradigm of epigenetic dysregulation. In this review, we attempt to address open questions regarding the role of epigenetic changes in HD, in the light of recent advances in neuroepigenetics. We particularly discuss studies using genome-wide scale approaches that provide insights into the relationship between epigenetic regulations, gene expression and neuronal activity in normal and diseased neurons, including HD neurons. We propose that cell-type specific techniques and 3D-based methods will advance knowledge of epigenome in the context of brain region vulnerability in neurodegenerative diseases. A better understanding of the mechanisms underlying epigenetic changes and of their consequences in neurodegenerative diseases is required to design therapeutic strategies more effective than current strategies based on histone deacetylase (HDAC) inhibitors. Researches in HD may play a driving role in this process.

Identification of a conserved gene signature associated with an exacerbated inflammatory environment in the hippocampus of aging rats.

There have been a few descriptive studies in aged rodents about transcriptome changes in the hippocampus, most of them in males. Here, we assessed the age changes in spatial memory performance and hippocampal morphology in female rats and compared those changes with changes in the hippocampal transcriptome. Old rats displayed significant deficits in spatial memory. In both age groups, hole exploration frequency showed a clear peak at hole 0 (escape hole), but the amplitude of the peak was significantly higher in the young than in the old animals. In the hippocampus, there was a dramatic reduction in neurogenesis, whereas reactive microglial infiltrates revealed an inflammatory hippocampal state in the senile rats. Hippocampal RNA-sequencing showed that 210 genes are differentially expressed in the senile rats, most of them being downregulated. Our RNA-Seq data showed that various genes involved in the immune response, including TYROBP, CD11b, C3, CD18, CD4, and CD74, are overexpressed in the hippocampus of aged female rats. Enrichment analysis showed that the pathways overrepresented in the senile rats matched those of an exacerbated inflammatory environment, reinforcing our morphologic findings. After correlating our results with public data of human and mouse hippocampal gene expression, we found an 11-gene signature of overexpressed genes related to inflammatory processes that was conserved across species. We conclude that age-related hippocampal deficits in female rats share commonalities between human and rodents. Interestingly, the 11-gene signature that we identified may represent a cluster of immune and regulatory genes that are deregulated in the hippocampus and possibly other brain regions during aging as well as in some neurodegenerative diseases and low-grade brain tumors. Our study further supports neuroinflammation as a promising target to treat cognitive dysfunction in old individuals and some brain tumors. © 2017 Wiley Periodicals, Inc.

In vivo imaging of brain glutamate defects in a knock-in mouse model of Huntington's disease.

Huntington's disease (HD) is an inherited neurodegenerative disease characterized by motor, cognitive and psychiatric symptoms. Atrophy of the striatum has been proposed for several years as a biomarker to assess disease progression in HD gene carriers. However, it does not provide any information about the biological mechanisms linked to HD pathogenesis. Changes in brain metabolites have been also consistently seen in HD patients and animal models using Magnetic Resonance Spectroscopy (MRS), but metabolite measurements are generally limited to a single voxel. In this study, we used Chemical Exchange Saturation Transfer imaging of glutamate (gluCEST) in order to map glutamate distribution in the brain of a knock-in mouse model (Ki140CAG) with a precise anatomical resolution. We demonstrated that both heterozygous and homozygous mice with pathological CAG repeat expansion in gene encoding huntingtin exhibited an atrophy of the striatum and a significant alteration of their metabolic profile in the striatum as compared to wild type littermate controls. The striatal decrease was then confirmed by gluCEST imaging. Surprisingly, CEST imaging also revealed that the corpus callosum was the most affected structure in both genotype groups, suggesting that this structure could be highly vulnerable in HD. We evaluated for the first time gluCEST imaging as a potential biomarker of HD and demonstrated its potential for characterizing metabolic defects in neurodegenerative diseases in specific regions.

Striatal long noncoding RNA Abhd11os is neuroprotective against an N-terminal fragment of mutant huntingtin in vivo.

A large number of gene products that are enriched in the striatum have ill-defined functions, although they may have key roles in age-dependent neurodegenerative diseases affecting the striatum, especially Huntington disease (HD). In the present study, we focused on Abhd11os, (called ABHD11-AS1 in human) which is a putative long noncoding RNA (lncRNA) whose expression is enriched in the mouse striatum. We confirm that despite the presence of 2 small open reading frames (ORFs) in its sequence, Abhd11os is not translated into a detectable peptide in living cells. We demonstrate that Abhd11os levels are markedly reduced in different mouse models of HD. We performed in vivo experiments in mice using lentiviral vectors encoding either Abhd11os or a small hairpin RNA targeting Abhd11os. Results show that Abhd11os overexpression produces neuroprotection against an N-terminal fragment of mutant huntingtin, whereas Abhd11os knockdown is protoxic. These novel results indicate that the loss lncRNA Abhd11os likely contribute to striatal vulnerability in HD. Our study emphasizes that lncRNA may play crucial roles in neurodegenerative diseases.

Mutant huntingtin alters Tau phosphorylation and subcellular distribution.

Tau abnormalities play a central role in several neurodegenerative diseases, collectively known as tauopathies. In the present study, we examined whether mutant huntingtin (mHtt), which causes Huntington's disease (HD), modifies Tau phosphorylation and subcellular localization using cell and mouse HD models. Initially, we used novel bimolecular fluorescence complementation assays in live cells to evaluate Tau interactions with either wild type (25QHtt) or mutant huntingtin (103QHtt). While 25QHtt and Tau interacted at the level of the microtubule network, 103QHtt and Tau interacted and formed 'ring-like' inclusions localized in the vicinity of the microtubular organizing center (MTOC). Fluorescence recovery after photobleaching experiments also indicated that, whereas homomeric 103QHtt/103QHtt pairs rapidly re-entered into inclusions, heteromeric 103QHtt/Tau pairs remained excluded from the 'ring-like' inclusions. Interestingly, in vitro Tau relocalization was associated to Tau hyperphosphorylation. Consistent with this observation, we found strong Tau hyperphosphorylation in brain samples from two different mouse models of HD, R6/2 and 140CAG knock-in. This was associated with a significant reduction in the levels of Tau phosphatases (PP1, PP2A and PP2B), with no apparent involvement of major Tau kinases. Thus, the present study strongly suggests that expression of mHtt leads to Tau hyperphosphorylation, relocalization and sequestration through direct protein-protein interactions in inclusion-like compartments in the vicinity of the MTOC. Likewise, our data also suggest that Tau alterations may also contribute to HD pathogenesis.

Loss of the thyroid hormone-binding protein Crym renders striatal neurons more vulnerable to mutant huntingtin in Huntington's disease.

The mechanisms underlying preferential atrophy of the striatum in Huntington's disease (HD) are unknown. One hypothesis is that a set of gene products preferentially expressed in the striatum could determine the particular vulnerability of this brain region to mutant huntingtin (mHtt). Here, we studied the striatal protein µ-crystallin (Crym). Crym is the NADPH-dependent p38 cytosolic T3-binding protein (p38CTBP), a key regulator of thyroid hormone (TH) T3 (3,5,3'-triiodo-l-thyronine) transportation. It has been also recently identified as the enzyme that reduces the sulfur-containing cyclic ketimines, which are potential neurotransmitters. Here, we confirm the preferential expression of the Crym protein in the rodent and macaque striatum. Crym expression was found to be higher in the macaque caudate than in the putamen. Expression of Crym was reduced in the BACHD and Knock-in 140CAG mouse models of HD before onset of striatal atrophy. We show that overexpression of Crym in striatal medium-size spiny neurons using a lentiviral-based strategy in mice is neuroprotective against the neurotoxicity of an N-terminal fragment of mHtt in vivo. Thus, reduction of Crym expression in HD could render striatal neurons more susceptible to mHtt suggesting that Crym may be a key determinant of the vulnerability of the striatum. In addition our work points to Crym as a potential molecular link between striatal degeneration and the THs deregulation reported in HD patients.

Biological features of herpes simplex virus type 1 latency in mice according to experimental conditions and type of neurones.

PURPOSE: To compare the biological patterns of viral transcripts during herpes simplex virus type 1 (HSV1) latent infection according to experimental conditions. METHODS: Two types of murine models of HSV1 infection were used: the corneal scarification model, often used in studies of HSV1 latency, and the oro-ocular murine model. Two strains of HSV1 were used for the inoculation: SC16, a wild type strain considered as highly neuroinvasive, and KOS, previously described as poorly neurovirulent. The amounts of viral genomes, and those of four types of viral transcripts (immediate-early, early and late, together with latency-associated transcripts [LATs]), were measured by quantitative PCR and RT-PCR in the main sites of HSV1 latent infection at 6 days, 1 and 3 months post inoculation, and the number of LAT-expressing neurons was assessed by in-situ hybridization on histological sections of trigeminal ganglia (TG). RESULTS: Using the SC16 strain of HSV1 in the oro-ocular model, immediate-early transcripts were still present at 1 month post inoculation (early stage of latent infection), but were not detected at 3 months (late stage of latent infection). In both cases, early and late viral genes transcripts were not detected, demonstrating the latent nature of the infection with this combination of experimental conditions. In contrast, such progress in the viral gene expression was not observed in the corneal scarification model, particularly when the KOS strain of HSV1 was used. CONCLUSIONS: These results highlight that the behavior of the virus in the nervous system depends on the method inoculation, and the viral strain. All these parameters are likely to influence the establishment of latent infection.

Possible involvement of self-defense mechanisms in the preferential vulnerability of the striatum in Huntington's disease.

HD is caused by a mutation in the huntingtin gene that consists in a CAG repeat expansion translated into an abnormal poly-glutamine (polyQ) tract in the huntingtin (Htt) protein. The most striking neuropathological finding in HD is the atrophy of the striatum. The regional expression of mutant Htt (mHtt) is ubiquitous in the brain and cannot explain by itself the preferential vulnerability of the striatum in HD. mHtt has been shown to produce an early defect in transcription, through direct alteration of the function of key regulators of transcription and in addition, more indirectly, as a result of compensatory responses to cellular stress. In this review, we focus on gene products that are preferentially expressed in the striatum and have down- or up-regulated expression in HD and, as such, may play a crucial role in the susceptibility of the striatum to mHtt. Many of these striatal gene products are for a vast majority down-regulated and more rarely increased in HD. Recent research shows that some of these striatal markers have a pro-survival/neuroprotective role in neurons (e.g., MSK1, A2A, and CB1 receptors) whereas others enhance the susceptibility of striatal neurons to mHtt (e.g., Rhes, RGS2, D2 receptors). The down-regulation of these latter proteins may be considered as a potential self-defense mechanism to slow degeneration. For a majority of the striatal gene products that have been identified so far, their function in the striatum is unknown and their modifying effects on mHtt toxicity remain to be experimentally addressed. Focusing on these striatal markers may contribute to a better understanding of HD pathogenesis, and possibly the identification of novel therapeutic targets.

High throughput screening for inhibitors of REST in neural derivatives of human embryonic stem cells reveals a chemical compound that promotes expression of neuronal genes.

Decreased expression of neuronal genes such as brain-derived neurotrophic factor (BDNF) is associated with several neurological disorders. One molecular mechanism associated with Huntington disease (HD) is a discrete increase in the nuclear activity of the transcriptional repressor REST/NRSF binding to repressor element-1 (RE1) sequences. High-throughput screening of a library of 6,984 compounds with luciferase-assay measuring REST activity in neural derivatives of human embryonic stem cells led to identify two benzoimidazole-5-carboxamide derivatives that inhibited REST silencing in a RE1-dependent manner. The most potent compound, X5050, targeted REST degradation, but neither REST expression, RNA splicing nor binding to RE1 sequence. Differential transcriptomic analysis revealed the upregulation of neuronal genes targeted by REST in wild-type neural cells treated with X5050. This activity was confirmed in neural cells produced from human induced pluripotent stem cells derived from a HD patient. Acute intraventricular delivery of X5050 increased the expressions of BDNF and several other REST-regulated genes in the prefrontal cortex of mice with quinolinate-induced striatal lesions. This study demonstrates that the use of pluripotent stem cell derivatives can represent a crucial step toward the identification of pharmacological compounds with therapeutic potential in neurological affections involving decreased expression of neuronal genes associated to increased REST activity, such as Huntington disease.

Genetic and functional abnormalities of the melatonin biosynthesis pathway in patients with bipolar disorder.

Patients affected by bipolar disorder (BD) frequently report abnormalities in sleep/wake cycles. In addition, they showed abnormal oscillating melatonin secretion, a key regulator of circadian rhythms and sleep patterns. The acetylserotonin O-methyltransferase (ASMT) is a key enzyme of the melatonin biosynthesis and has recently been associated with psychiatric disorders such as autism spectrum disorders and depression. In this paper, we analysed rare and common variants of ASMT in patients with BD and unaffected control subjects and performed functional analysis of these variants by assaying the ASMT activity in their B-lymphoblastoid cell lines. We sequenced the coding and the regulatory regions of the gene in a discovery sample of 345 patients with BD and 220 controls. We performed an association study on this discovery sample using common variants located in the promoter region and showed that rs4446909 was significantly associated with BD (P= 0.01) and associated with a lower mRNA level (P< 10(-4)) and a lower enzymatic activity (P< 0.05) of ASMT. A replication study and a meta-analysis using 480 independent patients with BD and 672 controls confirmed the significant association between rs4446909 and BD (P= 0.002). These results correlate with the general lower ASMT enzymatic activity observed in patients with BD (P= 0.001) compared with controls. Finally, several deleterious ASMT mutations identified in patients were associated with low ASMT activity (P= 0.01). In this study, we determined how rare and common variations in ASMT might play a role in BD vulnerability and suggest a general role of melatonin as susceptibility factor for BD.