The finding that a GGGGCC (G 4 C 2 ) hexanucleotide repeat expansion in the chromosome 9 ORF 72 (C9ORF72) gene is a common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) links ALS/FTD to a large group of unstable microsatellite diseases. Previously, we showed that microsatellite expansion mutations can be bidirectionally transcribed and that these mutations express unexpected proteins by a unique mechanism, repeat-associated non-ATG (RAN) translation. In this study, we show that C9ORF72 antisense transcripts are elevated in the brains of C9ORF72 expansion-positive [C9(+)] patients, and antisense GGCCCC (G 2 C 4 ) repeat-expansion RNAs accumulate in nuclear foci in brain. Additionally, sense and antisense foci accumulate in blood and are potential biomarkers of the disease. Furthermore, we show that RAN translation occurs from both sense and antisense expansion transcripts, resulting in the expression of six RAN proteins (antisense: Pro-Arg, Pro-Ala, Gly-Pro; and sense: Gly-Ala, Gly-Arg, Gly-Pro). These proteins accumulate in cytoplasmic aggregates in affected brain regions, including the frontal and motor cortex, hippocampus, and spinal cord neurons, with some brain regions showing dramatic RAN protein accumulation and clustering. The finding that unique antisense G 2 C 4 RNA foci and three unique antisense RAN proteins accumulate in patient tissues indicates that bidirectional transcription of expanded alleles is a fundamental pathologic feature of C9ORF72 ALS/FTD. Additionally, these findings suggest the need to test therapeutic strategies that target both sense and antisense RNAs and RAN proteins in C9ORF72 ALS/FTD, and to more broadly consider the role of antisense expression and RAN translation across microsatellite expansion diseases.cytoplasmic inclusions | clustered aggregates | noncoding RNA T he chromosome 9p21-linked form of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the most common cause of familial FTD and ALS identified to date, is caused by an expanded GGGGCC (G 4 C 2 ) hexanucleotide repeat in intron 1 of chromosome 9 ORF 72 (C9ORF72) (1, 2). The C9ORF72 mutation is found in 40% of familial and 7% of sporadic ALS cases and 21% of familial and 5% of sporadic FTD patients (3). The discovery of the C9ORF72 expansion has generated substantial excitement because it connects ALS and FTD to a large group of disorders caused by microsatellite expansion mutations (4).Traditionally, microsatellite expansion mutations located in predicted coding and noncoding regions were thought to cause disease by protein gain-or loss-of-function or RNA gain-of-function mechanisms (4). Protein loss-of-function has been proposed to underlie C9ORF72-driven ALS/FTD because the expansion mutation leads to decreased levels of variant 1 transcripts and potential decreases in C9ORF72 protein expression (1, 5). Additionally, because the C9ORF72 G 4 C 2 expansion mutation is located in an intron, several studies have pursued the hypothesis that C9-linked ALS/FTD results f...

Parkin is an E3 ubiquitin ligase involved in the ubiquitination of proteins that are important in the survival of dopamine neurons in Parkinson's disease (PD). We show that parkin is S-nitrosylated in vitro, as well as in vivo in a mouse model of PD and in brains of patients with PD and diffuse Lewy body disease. Moreover, S-nitrosylation inhibits parkin's ubiquitin E3 ligase activity and its protective function. The inhibition of parkin's ubiquitin E3 ligase activity by S-nitrosylation could contribute to the degenerative process in these disorders by impairing the ubiquitination of parkin substrates.

Cytoplasmic aggregation of TDP-43, accompanied by its nuclear clearance, is a key common pathological hallmark of amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). However, a limited understanding of this RNA-binding protein (RBP) impedes the clarification of pathogenic mechanisms underlying TDP-43 proteinopathy. In contrast to RBPs that regulate splicing of conserved exons, we found that TDP-43 repressed the splicing of nonconserved cryptic exons, maintaining intron integrity. When TDP-43 was depleted from mouse embryonic stem cells, these cryptic exons were spliced into messenger RNAs, often disrupting their translation and promoting nonsense-mediated decay. Moreover, enforced repression of cryptic exons prevented cell death in TDP-43–deficient cells. Furthermore, repression of cryptic exons was impaired in ALS-FTD cases, suggesting that this splicing defect could potentially underlie TDP-43 proteinopathy.

␣-synucleinopathy ͉ mass spectrometry ͉ proteolysis ͉ Lewy body P arkinson's disease (PD) is a common progressive neurodegenerative disease characterized by the loss of dopaminergic neurons of substantia nigra and the presence of the fibrillar cytoplasmic aggregates of ␣-synuclein (␣-Syn) in multiple brain regions (1, 2). Mutations in the ␣-Syn gene (3-7) and the abnormal aggregation of ␣-Syn are implicated in the pathogenesis of PD, and other related diseases are classified as ␣-synucleinopathies (1, 8-10). ␣-Syn is a highly conserved protein of 140 amino acids that is predominantly expressed in neurons, particularly in presynaptic terminals (11), and may have a role in synaptic plasticity and modulation of dopaminergic neurotransmission (11).Although the bulk of previous studies focused on the aggregation and the biology of the full-length ␣-Syn (␣-SynFL) (12, 13), the conspicuous presence of lower molecular mass ␣-Syn species in ␣-Syn aggregates (14, 15), and the enhanced in vitro fibril assembly of recombinant C-terminally truncated ␣-Syn (16, 17) suggests that the low-molecular mass ␣-Syn species may be of pathogenic significance. However, because postpathogenic and͞or postmortem processes could potentially generate a variety of ␣-Syn species, the significance of low-molecular mass ␣-Syn species to the development of ␣-synucleinopathy is uncertain.Herein, we demonstrate that C-terminally truncated lowmolecular mass ␣-Syn species (␣-Syn⌬C) with aggregationpromoting properties are normally generated in vivo. The expression of familial PD (FPD)-linked mutant human (Hu) ␣-Syn is associated with the higher cellular accumulation of ␣-Syn⌬C. Moreover, human cases with ␣-Syn lesions show preferential accumulation of ␣-Syn⌬C in aggregates and higher relative levels of soluble ␣-Syn⌬C. Our findings show that ␣-Syn⌬Cs are not an artifact of postpathologic processes and are likely to participate in the disease-linked aggregation of ␣-Syn. Materials and MethodsAdditional details are provided in Supporting Materials and Methods, which is published as supporting information on the PNAS web site.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset, autosomal dominant familial Parkinson's disease (PD) and also contribute to idiopathic PD. LRRK2 mutations represent the most common cause of PD with clinical and neurochemical features that are largely indistinguishable from idiopathic disease. Currently, transgenic mice expressing wild-type or disease-causing mutants of LRRK2 have failed to produce overt neurodegeneration, although abnormalities in nigrostriatal dopaminergic neurotransmission have been observed. Here, we describe the development and characterization of transgenic mice expressing human LRRK2 bearing the familial PD mutations, R1441C and G2019S. Our study demonstrates that expression of G2019S mutant LRRK2 induces the degeneration of nigrostriatal pathway dopaminergic neurons in an age-dependent manner. In addition, we observe autophagic and mitochondrial abnormalities in the brains of aged G2019S LRRK2 mice and markedly reduced neurite complexity of cultured dopaminergic neurons. These new LRRK2 transgenic mice will provide important tools for understanding the mechanism(s) through which familial mutations precipitate neuronal degeneration and PD.

The pathologic accumulation and aggregation of α-synuclein (α-syn) underlies Parkinson’s disease (PD). The molecular mechanisms by which pathologic α-syn causes neurodegeneration in PD is not known. Here we found that pathologic α-syn activates poly (ADP-ribose) (PAR) polymerase-1 (PARP-1) and PAR generation accelerates the formation of pathologic α-syn resulting in cell death via parthanatos. PARP inhibitors or genetic deletion of PARP-1 prevented pathologic α-syn toxicity. In a feed-forward loop, PAR converted pathologic α-syn to a more toxic strain. PAR levels were increased in the cerebral spinal fluid and brains of PD patients suggesting that PARP activation plays a role in PD pathogenesis. Thus, strategies aimed at inhibiting PARP-1 activation could hold promise as a disease modifying therapy to prevent the loss of dopamine (DA) neurons in PD.

SUMMARY Huntington disease (HD) is caused by a CAG·CTG expansion in the huntingtin (HTT) gene. While most research has focused on the HTT polyGln-expansion protein, we demonstrate that four additional, novel, homopolymeric expansion proteins (polyAla, polySer, polyLeu, and polyCys) accumulate in HD human brains. These sense and antisense repeat-associated non-ATG (RAN) translation proteins accumulate most abundantly in brain regions with neuronal loss, microglial activation and apoptosis, including caudate/putamen, white matter, and, in juvenile-onset cases, also the cerebellum. RAN protein accumulation and aggregation are length dependent, and individual RAN proteins are toxic to neural cells independent of RNA effects. These data suggest RAN proteins contribute to HD and that therapeutic strategies targeting both sense and antisense genes may be required for efficacy in HD patients. This is the first demonstration that RAN proteins are expressed across an expansion located in an open reading frame and suggests RAN translation may also contribute to other poly-glutamine diseases.

BackgroundThe metabolic basis of Alzheimer disease (AD) is poorly understood, and the relationships between systemic abnormalities in metabolism and AD pathogenesis are unclear. Understanding how global perturbations in metabolism are related to severity of AD neuropathology and the eventual expression of AD symptoms in at-risk individuals is critical to developing effective disease-modifying treatments. In this study, we undertook parallel metabolomics analyses in both the brain and blood to identify systemic correlates of neuropathology and their associations with prodromal and preclinical measures of AD progression.Methods and findingsQuantitative and targeted metabolomics (Biocrates AbsoluteIDQ [identification and quantification] p180) assays were performed on brain tissue samples from the autopsy cohort of the Baltimore Longitudinal Study of Aging (BLSA) (N = 44, mean age = 81.33, % female = 36.36) from AD (N = 15), control (CN; N = 14), and “asymptomatic Alzheimer’s disease” (ASYMAD, i.e., individuals with significant AD pathology but no cognitive impairment during life; N = 15) participants. Using machine-learning methods, we identified a panel of 26 metabolites from two main classes—sphingolipids and glycerophospholipids—that discriminated AD and CN samples with accuracy, sensitivity, and specificity of 83.33%, 86.67%, and 80%, respectively. We then assayed these 26 metabolites in serum samples from two well-characterized longitudinal cohorts representing prodromal (Alzheimer’s Disease Neuroimaging Initiative [ADNI], N = 767, mean age = 75.19, % female = 42.63) and preclinical (BLSA) (N = 207, mean age = 78.68, % female = 42.63) AD, in which we tested their associations with magnetic resonance imaging (MRI) measures of AD-related brain atrophy, cerebrospinal fluid (CSF) biomarkers of AD pathology, risk of conversion to incident AD, and trajectories of cognitive performance. We developed an integrated blood and brain endophenotype score that summarized the relative importance of each metabolite to severity of AD pathology and disease progression (Endophenotype Association Score in Early Alzheimer’s Disease [EASE-AD]). Finally, we mapped the main metabolite classes emerging from our analyses to key biological pathways implicated in AD pathogenesis. We found that distinct sphingolipid species including sphingomyelin (SM) with acyl residue sums C16:0, C18:1, and C16:1 (SM C16:0, SM C18:1, SM C16:1) and hydroxysphingomyelin with acyl residue sum C14:1 (SM (OH) C14:1) were consistently associated with severity of AD pathology at autopsy and AD progression across prodromal and preclinical stages. Higher log-transformed blood concentrations of all four sphingolipids in cognitively normal individuals were significantly associated with increased risk of future conversion to incident AD: SM C16:0 (hazard ratio [HR] = 4.430, 95% confidence interval [CI] = 1.703–11.520, p = 0.002), SM C16:1 (HR = 3.455, 95% CI = 1.516–7.873, p = 0.003), SM (OH) C14:1 (HR = 3.539, 95% CI = 1.373–9.122, p = 0.009), and SM C18:1 (HR = 2....