Charlotte Delay

In vivo regulation of amyloid precursor protein neuronal splicing by microRNAs.

J. Neurochem. (2011) 116, 240–247.

Phosphorylation, lipid raft interaction and traffic of α-synuclein in a yeast model for Parkinson

Parkinsons disease is a neurodegenerative disorder characterized by the formation of Lewy bodies containing aggregated alpha-synuclein. We used a yeast model to screen for deletion mutants with mislocalization and enhanced inclusion formation of alpha-synuclein. Many of the mutants were affected in functions related to vesicular traffic but especially mutants in endocytosis and vacuolar degradation combined inclusion formation with enhanced alpha-synuclein-mediated toxicity. The screening also allowed for identification of casein kinases responsible for alpha-synuclein phosphorylation at the plasma membrane as well as transacetylases that modulate the alpha-synuclein membrane interaction. In addition, alpha-synuclein was found to associate with lipid rafts, a phenomenon dependent on the ergosterol content. Together, our data suggest that toxicity of alpha-synuclein in yeast is at least in part associated with endocytosis of the protein, vesicular recycling back to the plasma membrane and vacuolar fusion defects, each contributing to the obstruction of different vesicular trafficking routes.

MicroRNAs and Alzheimer's Disease Mouse Models: Current Insights and Future Research Avenues

Evidence from clinical trials as well as from studies performed in animal models suggest that both amyloid and tau pathologies function in concert with other factors to cause the severe neurodegeneration and dementia in Alzheimers disease (AD) patients. Accumulating data in the literature suggest that microRNAs (miRNAs) could be such factors. These conserved, small nonprotein-coding RNAs are essential for neuronal function and survival and have been implicated in the regulation of key genes involved in genetic and sporadic AD. The study of miRNA changes in AD mouse models provides an appealing approach to address the cause-consequence relationship between miRNA dysfunction and AD pathology in humans. Mouse models also provide attractive tools to validate miRNA targets in vivo and provide unique platforms to study the role of specific miRNA-dependent gene pathways in disease. Finally, mouse models may be exploited for miRNA diagnostics in the fight against AD.

Synphilin-1 Enhances α-Synuclein Aggregation in Yeast and Contributes to Cellular Stress and Cell Death in a Sir2-Dependent Manner

Background Parkinsons disease is characterized by the presence of cytoplasmic inclusions, known as Lewy bodies, containing both aggregated α-synuclein and its interaction partner, synphilin-1. While synphilin-1 is known to accelerate inclusion formation by α-synuclein in mammalian cells, its effect on cytotoxicity remains elusive. Methodology/Principal Findings We expressed wild-type synphilin-1 or its R621C mutant either alone or in combination with α-synuclein in the yeast Saccharomyces cerevisiae and monitored the intracellular localization and inclusion formation of the proteins as well as the repercussions on growth, oxidative stress and cell death. We found that wild-type and mutant synphilin-1 formed inclusions and accelerated inclusion formation by α-synuclein in yeast cells, the latter being correlated to enhanced phosphorylation of serine-129. Synphilin-1 inclusions co-localized with lipid droplets and endomembranes. Consistently, we found that wild-type and mutant synphilin-1 interacts with detergent-resistant membrane domains, known as lipid rafts. The expression of synphilin-1 did not incite a marked growth defect in exponential cultures, which is likely due to the formation of aggresomes and the retrograde transport of inclusions from the daughter cells back to the mother cells. However, when the cultures approached stationary phase and during subsequent ageing of the yeast cells, both wild-type and mutant synphilin-1 reduced survival and triggered apoptotic and necrotic cell death, albeit to a different extent. Most interestingly, synphilin-1 did not trigger cytotoxicity in ageing cells lacking the sirtuin Sir2. This indicates that the expression of synphilin-1 in wild-type cells causes the deregulation of Sir2-dependent processes, such as the maintenance of the autophagic flux in response to nutrient starvation. Conclusions/Significance Our findings demonstrate that wild-type and mutant synphilin-1 are lipid raft interacting proteins that form inclusions and accelerate inclusion formation of α-synuclein when expressed in yeast. Synphilin-1 thereby induces cytotoxicity, an effect most pronounced for the wild-type protein and mediated via Sir2-dependent processes.

Increased LINGO1 in the cerebellum of essential tremor patients.

Essential tremor (ET) is the most prevalent adult‐onset movement disorder. Despite its health burden, no clear pathognomonic sign has been identified to date because of the rarity of clinicopathological studies. Moreover, treatment options are still scarce and have not significantly changed in the last 30 years, underscoring the urgent need to develop new treatment avenues. In the recent years, leucine‐rich repeat (LRR) and immunoglobulin (Ig) domain‐containing Nogo receptor‐interacting proteins 1 and 2 (LINGO1 and LINGO2, respectively) have been increasingly regarded as possible ET modulators due to emerging genetic association studies linking LINGO with ET. We have investigated LINGO protein and messenger RNA (mRNA) expression in the cerebellum of patients with ET, patients with Parkinsons disease (PD), and a control group using Western immunoblotting and in situ hybridization. Protein levels of LINGO1, but not LINGO2, were significantly increased in the cerebellar cortex of ET patients compared with controls, particularly in individuals with longer disease duration. Compared with controls, LINGO1 protein levels were increased in the cerebellar white matter of PD and ET patients but, for the latter, only when disease duration exceeded 20 years. However, no alteration in LINGO1 mRNA was observed between groups in either the cerebellar cortex or the white matter. We observed alterations in LINGO expression in diseased brain that seemed to progress along with the disease, being initiated in the cerebellar cortex before reaching the white matter. Because LINGO up‐regulation has been identified as a potential pathological response to ongoing neurodegenerative processes, the present data suggest that LINGO1 is a potential drug target for ET.

Gene Network and Pathway Analysis of Mice with Conditional Ablation of Dicer in Post-Mitotic Neurons

Background The small non-protein-coding microRNAs (miRNAs) have emerged as critical regulators of neuronal differentiation, identity and survival. To date, however, little is known about the genes and molecular networks regulated by neuronal miRNAs in vivo, particularly in the adult mammalian brain. Methodology/Principal Findings We analyzed whole genome microarrays from mice lacking Dicer, the enzyme responsible for miRNA production, specifically in postnatal forebrain neurons. A total of 755 mRNA transcripts were significantly (P<0.05, FDR<0.25) misregulated in the conditional Dicer knockout mice. Ten genes, including Tnrc6c, Dnmt3a, and Limk1, were validated by real time quantitative RT-PCR. Upregulated transcripts were enriched in nonneuronal genes, which is consistent with previous studies in vitro. Microarray data mining showed that upregulated genes were enriched in biological processes related to gene expression regulation, while downregulated genes were associated with neuronal functions. Molecular pathways associated with neurological disorders, cellular organization and cellular maintenance were altered in the Dicer mutant mice. Numerous miRNA target sites were enriched in the 3′untranslated region (3′UTR) of upregulated genes, the most significant corresponding to the miR-124 seed sequence. Interestingly, our results suggest that, in addition to miR-124, a large fraction of the neuronal miRNome participates, by order of abundance, in coordinated gene expression regulation and neuronal maintenance. Conclusions/Significance Taken together, these results provide new clues into the role of specific miRNA pathways in the regulation of brain identity and maintenance in adult mice.

Association of Neuropathological Markers in the Parietal Cortex With Antemortem Cognitive Function in Persons With Mild Cognitive Impairment and Alzheimer Disease

The associations between cognitive function and neuropathological markers in patients with mild cognitive impairment (MCI) and Alzheimer disease (AD) remain only partly defined. We investigated relationships between antemortem global cognitive scores and &bgr;-amyloid (A&bgr;), tau, TDP-43, synaptic proteins and other key AD neuropathological markers assessed by biochemical approaches in postmortem anterior parietal cortex samples from 36 subjects (12 MCI, 12 AD and 12 not cognitively impaired) from the Religious Orders Study. Overall, the strongest negative correlation coefficients associated with global cognitive scores were obtained for insoluble phosphorylated tau (r2 = −0.484), insoluble A&bgr;42 (r2 = −0.389) and neurofibrillary tangle counts (r2 = −0.494) (all p < 0.001). Robust inverse associations with cognition scores were also established for TDP-43-positive cytoplasmic inclusions (r2 = −0.476), total insoluble tau (r2 = −0.385) and A&bgr; plaque counts (r2 = −0.426). Sarkosyl (SK)- or formic acid (FA)-extracted tau showed similar interrelations. On the other hand, synaptophysin (r2 = +0.335), pS403/404 TDP-43 (r2 = +0.265) and septin-3 (r2 = +0.257) proteins positively correlated with cognitive scores. This study suggests that tau and A&bgr;42 in their insoluble aggregated forms, synaptic proteins and TDP-43 are the markers in the parietal cortex that are most strongly associated with cognitive function. This further substantiates the relevance of investigating these markers to understand the pathogenesis of AD and develop therapeutic tools.

Accumulation of amyloid-β in the cerebellar cortex of essential tremor patients

The accumulation of insoluble amyloid-beta (Aβ) peptides is associated with neurodegenerative disorders, such as Alzheimers disease (AD). As essential tremor (ET) could involve neurodegenerative processes in the cerebellum, we quantified soluble and insoluble Aβ in cerebellar cortices from patients diagnosed with ET (n=9), compared to Controls (n=16) or individuals with Parkinsons disease (n=10). Although ante-mortem cognitive performance was not documented, all individuals included had the diagnosis of AD ruled out by a neuropathologist. ELISA-determined concentrations of insoluble Aβ42 in ET patients displayed a bimodal distribution, with a median 246-fold higher than in Controls (P<0.01, Kruskal-Wallis). Higher Aβ42 concentrations were measured in the parietal cortex of the same ET patients, compared to Controls (107-fold median increase, P<0.01, Kruskal-Wallis), but similar phosphorylated tau levels were detected. The rise in cerebellar insoluble Aβ42 concentrations is not associated to APP expression and processing or the ApoE4 status. However, Aβ42 levels in ET individuals were correlated with cerebellar insoluble phosphorylated tau (r(2)=0.71, P=0.005), unphosphorylated neurofilament heavy chain (NF-H; r(2)=0.50, P=0.030) and Lingo-1 (r(2)=0.73, P=0.007), indicative of a generalized neurodegenerative process involving the cerebellum. Our results suggest prevalent accumulations of insoluble Aβ42 in the cerebellum of ET, but not in age-matched PD. Whether this anomaly plays a role in ET symptoms warrants further investigations.

MiRNA regulation of APP, BACE1 and Nicastrin and the effect of 3'UTR polymorphisms

Background: Familial Alzheimer’s disease associated with PSEN1 mutations has a wide spectrum of clinical presentations. Cognitive deficits are the dominant features; however, other signs and symptoms can be seen including some associated with movement disorders. Methods: Affected individuals from a family were studied clinically (2), genetically (3), and neuropathologically (1). Results: The proband, a female, reportedly began to have seizures, headaches and psychiatric problems at age 14. She attempted suicide and was considered to have conversion disorder at age 15. Her seizure disorder was treated with phenytoin and carbamazepine at age 19. Also, she was diagnosed by a psychiatrist as having adolescent adjustment reaction and reactive depression. Her family noted episodes of confusion and memory difficulty at age 27. Her memory worsened, speech became dysarthric and spasticity developed in the lower extremities at age 29. A diagnosis of spinocerebellar degeneration was considered at that time. Her condition progressed until she died at age 36. The proband’s mother was reported to have had ataxia and spasticity at age 18, dementia by 30 and died at 55. The proband’s sister developed a gait disorder at age 21, dementia at 25 and died at 47 after having been in a vegetative state for 9 years. The proband’s brother was known to have a gait disorder at age 28, but was lost to follow-up. Genetic analysis of the proband and two siblings identified a single nucleotide substitution (C for T) in exon 6 in the PSEN1 gene leading to a proline for leucine substitution at codon 166 (L166P). Autopsy of the proband showed corticospinal tract degeneration with atrophy of the cerebral hemispheres, pons, cerebellum, and rostral spinal cord. Histopathology revealed advanced stage neurofibrillary tangle and neuritic plaque pathology accompanied by prominent amyloid-b (Ab) deposition as well as “cotton wool” plaques and severe amyloid angiopathy. Conclusions: This mutation in PSEN1 results in one of the earliest onsets of symptoms and most “malignant” clinical courses reported to date. This report underscores the importance of close clinical, genetic, and neuropathologic studies to diagnose atypical presentations of a neurodegenerative disease.