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Reconstitution of TDP-43 filaments that exhibit sequence and morphological features similar to those found in the brain helps to uncover a new mechanism for the formation and propagation of pathology in amyotrophic lateral sclerosis and other neurodegenerative diseases. Despite the strong evidence linking the transactive response DNA-binding protein 43 (TDP-43) aggregation to the pathogenesis of frontotemporal lobar degeneration with TDP-43, amyotrophic lateral sclerosis and several neurodegenerative diseases, our knowledge of the sequence and structural determinants of its aggregation and neurotoxicity remains incomplete. Herein, we present a new method for producing recombinant full-length TDP-43 filaments that exhibit sequence and morphological features similar to those of brain-derived TDP-43 filaments. We show that TDP-43 filaments contain a beta-sheet-rich helical amyloid core that is fully buried by the flanking structured domains of the protein. We demonstrate that the proteolytic cleavage of TDP-43 filaments and exposure of this amyloid core are necessary for propagating TDP-43 pathology and enhancing the seeding of brain-derived TDP-43 aggregates. Only TDP-43 filaments with exposed amyloid core efficiently seeded the aggregation of endogenous TDP-43 in cells. These findings suggest that inhibiting the enzymes mediating cleavage of TDP-43 aggregates represents a viable disease-modifying strategy to slow the progression of amyotrophic lateral sclerosis and other TDP-43 proteinopathies.

We introduce a new automated machine learning analysis pipeline to precisely classify cellular structures captured through single molecule localization microscopy, which we call ECLiPSE (Enhanced Classification of Localized Pointclouds by Shape Extraction). ECLiPSE leverages 67 comprehensive shape descriptors encompassing geometric, boundary, skeleton and other properties, the majority of which are directly extracted from the localizations to accurately characterize individual structures. We validate ECLiPSE through unsupervised and supervised classification on a dataset featuring five distinct cellular structures, achieving exceptionally high classification accuracies nearing 100%. Moreover, we demonstrate the versatility of our approach by applying it to two novel biological applications: quantifying the clearance of tau protein aggregates, a critical marker for neurodegenerative diseases, and differentiating between two distinct morphological features (morphotypes) of TAR DNA-binding protein 43 proteinopathy, potentially associated to different TDP-43 strains, each exhibiting unique seeding and spreading properties. We anticipate that this versatile approach will significantly enhance the way we study cellular structures across various biological contexts, elucidating their roles in disease development and progression.

TAR-DNA binding protein-43 (TDP-43) proteinopathy is seen in multiple brain diseases. A standardized terminology was recommended recently for common age-related TDP-43 proteinopathy: limbic-predominant, age-related TDP-43 encephalopathy (LATE) and the underlying neuropathological changes, LATE-NC. LATE-NC may be co-morbid with Alzheimer’s disease neuropathological changes (ADNC). However, there currently are ill-defined diagnostic classification issues among LATE-NC, ADNC, and frontotemporal lobar degeneration with TDP-43 (FTLD-TDP). A practical challenge is that different autopsy cohorts are composed of disparate groups of research volunteers: hospital- and clinic-based cohorts are enriched for FTLD-TDP cases, whereas community-based cohorts have more LATE-NC cases. Neuropathological methods also differ across laboratories. Here, we combined both cases and neuropathologists’ diagnoses from two research centres—University of Pennsylvania and University of Kentucky. The study was designed to compare neuropathological findings between FTLD-TDP and pathologically severe LATE-NC. First, cases were selected from the University of Pennsylvania with pathological diagnoses of either FTLD-TDP (n = 33) or severe LATE-NC (mostly stage 3) with co-morbid ADNC (n = 30). Sections from these University of Pennsylvania cases were cut from amygdala, anterior cingulate, superior/mid-temporal, and middle frontal gyrus. These sections were stained for phospho-TDP-43 immunohistochemically and evaluated independently by two University of Kentucky neuropathologists blinded to case data. A simple set of criteria hypothesized to differentiate FTLD-TDP from LATE-NC was generated based on density of TDP-43 immunoreactive neuronal cytoplasmic inclusions in the neocortical regions. Criteria-based sensitivity and specificity of differentiating severe LATE-NC from FTLD-TDP cases with blind evaluation was ∼90%. Another proposed neuropathological feature related to TDP-43 proteinopathy in aged individuals is ‘Alpha’ versus ‘Beta’ in amygdala. Alpha and Beta status was diagnosed by neuropathologists from both universities (n = 5 raters). There was poor inter-rater reliability of Alpha/Beta classification (mean κ = 0.31). We next tested a separate cohort of cases from University of Kentucky with either FTLD-TDP (n = 8) or with relatively ‘pure’ severe LATE-NC (lacking intermediate or severe ADNC; n = 14). The simple criteria were applied by neuropathologists blinded to the prior diagnoses at University of Pennsylvania. Again, the criteria for differentiating LATE-NC from FTLD-TDP was effective, with sensitivity and specificity ∼90%. If more representative cases from each cohort (including less severe TDP-43 proteinopathy) had been included, the overall accuracy for identifying LATE-NC was estimated at >98% for both cohorts. Also across both cohorts, cases with FTLD-TDP died younger than those with LATE-NC (P

Limbic-predominant age-related TDP-43 encephalopathy (LATE) is characterized by the accumulation of TAR-DNA-binding protein 43 (TDP-43) aggregates in older adults. LATE coexists with Lewy body disease (LBD) as well as other neuropathological changes including Alzheimer’s disease (AD). We aimed to identify the pathological, clinical, and genetic characteristics of LATE in LBD (LATE-LBD) by comparing it with LATE in AD (LATE-AD), LATE with mixed pathology of LBD and AD (LATE-LBD + AD), and LATE alone (Pure LATE). We analyzed four cohorts of autopsy-confirmed LBD (n = 313), AD (n = 282), LBD + AD (n = 355), and aging (n = 111). We assessed the association of LATE with patient profiles including LBD subtype and AD neuropathologic change (ADNC). We studied the morphological and distributional differences between LATE-LBD and LATE-AD. By frequency analysis, we staged LATE-LBD and examined the association with cognitive impairment and genetic risk factors. Demographic analysis showed LATE associated with age in all four cohorts and the frequency of LATE was the highest in LBD + AD followed by AD, LBD, and Aging. LBD subtype and ADNC associated with LATE in LBD or AD but not in LBD + AD. Pathological analysis revealed that the hippocampal distribution of LATE was different between LATE-LBD and LATE-AD: neuronal cytoplasmic inclusions were more frequent in cornu ammonis 3 (CA3) in LATE-LBD compared to LATE-AD and abundant fine neurites composed of C-terminal truncated TDP-43 were found mainly in CA2 to subiculum in LATE-LBD, which were not as numerous in LATE-AD. Some of these fine neurites colocalized with phosphorylated α-synuclein. LATE-LBD staging showed LATE neuropathological changes spread in the dentate gyrus and brainstem earlier than in LATE-AD. The presence and prevalence of LATE in LBD associated with cognitive impairment independent of either LBD subtype or ADNC; LATE-LBD stage also associated with the genetic risk variants of TMEM106B rs1990622 and GRN rs5848. These data highlight clinicopathological and genetic features of LATE-LBD.

The neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with TAR DNA-binding protein-43 (TDP-43) inclusions (FTLD-TDP) share the neuropathological hallmark of aggregates of TDP-43. However, factors governing the severity and regional distribution of TDP-43 pathology, which may account for the divergent clinical presentations of ALS and FTLD-TDP, are not well-understood. Here, we investigated the influence of genotypes at TMEM106B, a locus associated with risk for FTLD-TDP, and hexanucleotide repeat expansions in C9orf72, a known genetic cause for both ALS and FTLD-TDP, on global TDP-43 pathology and regional distribution of TDP-43 pathology in 899 postmortem cases from a spectrum of neurodegenerative diseases. We found that, among the 110 ALS cases, minor (C)- allele homozygotes at the TMEM106B locus sentinel SNP rs1990622 had more TDP-43 pathology globally, as well as in select brain regions. C9orf72 expansions similarly associated with greater TDP-43 pathology in ALS. However, adjusting for C9orf72 expansion status did not affect the relationship between TMEM106B genotype and TDP-43 pathology. In order to elucidate the direction of causality for this association, we directly manipulated TMEM106B levels in an inducible cell system that expresses mislocalized TDP-43 protein. We found that partial knockdown of TMEM106B, to levels similar to what would be expected in rs1990622 C allele carriers, led to development of more TDP-43 cytoplasmic aggregates, which were more insoluble, in this system. Taken together, our results support a causal role for TMEM106B in modifying the development of TDP-43 proteinopathy.

Aim The heterogeneity in the distribution and morphological features of TAR DNA-binding protein-43 (TDP-43) pathology in the brains of frontotemporal lobar degeneration (FTLD-TDP) patients and their different clinical manifestations suggest that distinct pathological TDP-43 strains could play a role in this heterogeneity between different FTLD-TDP subtypes (A-E). Our aim was to evaluate the existence of distinct TDP-43 strains in the brains of different FTLD-TDP subtypes and characterise their specific seeding properties in vitro and in vivo. Methods and results We used an inducible stable cell line expressing a mutant cytoplasmic TDP-43 (iGFP-NLSm) to evaluate the seeding properties of distinct pathological TDP-43 strains. Brain-derived TDP-43 protein extracts from FTLD-TDP types A (n = 6) and B (n = 3) cases induced the formation of round/spherical phosphorylated TDP-43 aggregates that morphologically differed from the linear and wavy wisps and bigger heterogeneous filamentous (skein-like) aggregates induced by type E (n = 3) cases. These morphological differences correlated with distinct biochemical banding patterns of sarkosyl-insoluble TDP-43 protein recovered from the transduced cells. Moreover, brain-derived TDP-43 extracts from type E cases showed higher susceptibility to PK digestion of full-length TDP-43 and the most abundant C-terminal fragments that characterise type E extracts. Finally, we showed that intracerebral injections of different TDP-43 strains induced a distinctive morphological and subcellular distribution of TDP-43 pathology and different spreading patterns in the brains of CamKIIa-hTDP-43NLSm Tg mice. Conclusions We show the existence of distinct TDP-43 strains in the brain of different FTLD-TDP subtypes with distinctive seeding and spreading properties in the brains of experimental animal models.

Two ways to get tangled? Neurodegeneration in Alzheimer's disease dementia is associated with neurofibrillary tangles composed of aggregated tau protein. Darwich et al. describe an additional form of autosomal-dominant dementia with neurofibrillary tangles linked to a hypomorph mutation in valosin-containing protein (VCP). VCP was found to disaggregate pathologic tau, and the hypomorph mutation increased tau accumulation in cells and mice. These findings highlight the role of protein turnover in maintaining neuronal health and suggest that VCP may provide a therapeutic target for Alzheimer's disease. Science , this issue p. eaay8826

Frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP) can typically be categorized into one of four distinct histopathologic patterns of TDP-43 pathology, types A to D. The strength of this histopathologic classification lies in the association between FTLD-TDP subtypes and various clinical and genetic features of disease. Seven cases of FTLD-TDP were identified here which were difficult to classify based on existing pathologic criteria. Distinct features common to these cases included TDP-43 aggregates over a wide neuroanatomic distribution comprised of granulofilamentous neuronal inclusions, abundant grains, and oligodendroglial inclusions. TDP-43 aggregates were phosphorylated and associated with loss of normal nuclear TDP-43 protein (nuclear clearance) but were negative for ubiquitin. Biochemical analysis confirmed the presence of insoluble and phosphorylated TDP-43 and also revealed a distinct pattern of TDP-43 C-terminal fragments relative to other FTLD-TDP subtypes. Finally, these cases were uniformly associated with a very rapid clinical course culminating in death within ~3 years of disease onset. We suggest that these cases may represent a unique clinicopathologic subtype of FTLD-TDP which we provisionally call “type E.” The immature appearance of TDP-43 aggregates, widespread distribution, uniform biochemical profile and rapid clinical course highlights the clinical and pathologic variability within FTLD-TDP, and raises the possibility that type E neuropathology is the sequelae of a particularly virulent strain of TDP-43 proteinopathy.

Supplemental digital content is available in the text., Frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) are 2 neurodegenerative disorders that share clinical, genetic, and neuropathologic features. The presence of abnormal expansions of GGGGCC repeats (G4C2 repeats) in a noncoding region of the Chromosome 9 open reading frame 72 (C9orf72) gene is the major genetic cause of both FTLD and ALS. Transcribed G4C2 repeats can form nuclear RNA foci and recruit RNA-binding proteins, thereby inhibiting their normal function. Moreover, through a repeat-associated non-ATG translation mechanism, G4C2 repeats translation leads to dipeptide-repeat protein aggregation in the cytoplasm of neurons. Here, we identify Drosha protein as a new component of these dipeptide-repeat aggregates. In C9orf72 mutation cases of FTLD-TDP (c9FTLD-TDP) and ALS (c9ALS), but not in FTLD or ALS cases without C9orf72 mutation, Drosha is mislocalized to form neuronal cytoplasmic inclusions in the hippocampus, frontal cortex, and cerebellum. Further characterization of Drosha-positive neuronal cytoplasmic inclusions in the hippocampus, frontal cortex, and cerebellum revealed colocalization with p62 and ubiquilin-2, 2 pathognomonic signatures of c9FTLD-TDP and c9ALS cases; however, Drosha inclusions rarely colocalized with TDP-43 pathology. We conclude that Drosha may play a unique pathogenic role in the onset or progression of FTLD-TDP/ALS in patients with the C9orf72 mutation.

Regulation of chromatin structure is critical for brain development and function. However, the involvement of chromatin dynamics in neurodegeneration is less well understood. Here we find, launching from Drosophila models of amyotrophic lateral sclerosis and frontotemporal dementia, that TDP-43 impairs the induction of multiple key stress genes required to protect from disease by reducing the recruitment of the chromatin remodeler Chd1 to chromatin. Chd1 depletion robustly enhances TDP-43-mediated neurodegeneration and promotes the formation of stress granules. Conversely, upregulation of Chd1 restores nucleosomal dynamics, promotes normal induction of protective stress genes, and rescues stress sensitivity of TDP-43-expressing animals. TDP-43-mediated impairments are conserved in mammalian cells, and, importantly, the human ortholog CHD2 physically interacts with TDP-43 and is strikingly reduced in level in temporal cortex of human patient tissue. These findings indicate that TDP-43-mediated neurodegeneration causes impaired chromatin dynamics that prevents appropriate expression of protective genes through compromised function of the chromatin remodeler Chd1/CHD2. Enhancing chromatin dynamics may be a treatment approach to amyotrophic lateral scleorosis (ALS)/frontotemporal dementia (FTD).

Adenosine A2A receptor (A2AR) is a G-protein coupled receptor that stimulates adenylyl cyclase activity. In the brain, A2ARs are found highly enriched in striatal GABAergic medium spiny neurons, related to the control of voluntary movement. Pharmacological modulation of A2ARs is particularly useful in Parkinson's disease (PD) due to their property of antagonizing dopamine D2 receptor activity. Increases in A2AR levels have been described in PD patients showing an important loss of dopaminergic denervation markers, but no data have been reported about A2AR levels in incidental PD brains. In the present report, we show that increased A2ARs protein levels were also detected in the putamen of incidental PD cases (Braak PD stages 1-2) with respect to age-matched controls. By contrast, A2ARs mRNA levels remained unchanged, suggesting that posttranslational mechanisms could be involved in the regulation of A2ARs. It has been described how miR-34b/c downregulation is an early event in PD cases. We found that miR-34b levels are also significantly reduced in the putamen of incidental PD cases and along disease progression. Given that 3'UTR of A2AR contains a predicted target site for miR-34b, the potential role of this miRNA in protein A2AR levels was assessed. In vitro studies revealed that endogenous A2AR protein levels increased when miR-34b function was blocked using a specific anti-miR-34b. Moreover, using a luciferase reporter assay with point mutations in a miR-34b predicted binding site within the 3'UTR region of A2AR mRNA abolished the effect of the miRNA using a miR-34b mimic. In addition, we showed a reduced percentage of DNA methylation in the 5'UTR region of ADORA2A in advanced PD cases. Overall, these findings reveal that increased A2AR protein levels occur in asymptomatic PD patients and provide new insights into the molecular mechanisms underlying A2AR expression levels along the progression of this neurodegenerative disease.

The original version of this Article contained an error in the author affiliations. The affiliation of Alice Chen-Plotkin with the Department of Neurology, Perelman School of Medicine, Philadelphia, PA, 19104 USA was inadvertently omitted. This has now been corrected in both the PDF and HTML versions of the Article.

Huntington disease (HD) is a neurodegenerative disorder that predominantly affects neurons of the forebrain. We have applied the Illumina massively parallel sequencing to deeply analyze the small RNA populations of two different forebrain areas, the frontal cortex (FC) and the striatum (ST) of healthy individuals and individuals with HD. More than 80% of the small-RNAs were annotated as microRNAs (miRNAs) in all samples. Deep sequencing revealed length and sequence heterogeneity (IsomiRs) for the vast majority of miRNAs. Around 80-90% of the miRNAs presented modifications in the 3'-terminus mainly in the form of trimming and/or as nucleotide addition variants, while the 5'-terminus of the miRNAs was specially protected from changes. Expression profiling showed strong miRNA and isomiR expression deregulation in HD, most being common to both FC and ST. The analysis of the upstream regulatory regions in co-regulated miRNAs suggests a role for RE1-Silencing Transcription Factor (REST) and P53 in miRNAs downregulation in HD. The putative targets of deregulated miRNAs and seed-region IsomiRs strongly suggest that their altered expression contributes to the aberrant gene expression in HD. Our results show that miRNA variability is a ubiquitous phenomenon in the adult human brain, which may influence gene expression in physiological and pathological conditions.

Oxidative stress (OS) underlies neuronal dysfunction in many neurodegenerative disorders. Regulator of Calcineurin 1 (RCAN1 or DSCR1) is a dose-sensitive gene whose overexpression has been linked to Down syndrome (DS) and Alzheimer's disease (AD) neuropathology and to the response of cells to stress stimuli. Here, we show that RCAN1 mRNA and protein expression are sensitive to OS in primary neurons, and we evaluate the involvement of RCAN1 dosage in neuronal death induced by OS. We find that Rcan1(-/-) neurons display an increased resistance to damage by H(2)O(2), which can be reverted by RCAN1 overexpression or by exogenous inhibitors of calcineurin. Although increased intracellular Ca(2+) concentration is an important factor in OS-mediated cell death, our results show that Ca(2+) loading after exposure to H(2)O(2) was similar in Rcan1(+/+) and Rcan1(-/-) neurons. Our data further suggest that CaN and NFAT signaling protect against OS in both Rcan1(+/+) and Rcan1(-/-) neurons. To explain the observed differential vulnerability, we therefore propose a mechanism downstream of H(2)O(2)-mediated Ca(2+) entry, involving calcineurin-NFAT signaling. These findings highlight the importance of RCAN1 gene dosage in the modulation of cell survival and death pathways and suggest that changes in the amount of RCAN1 could represent an important mechanism for regulating susceptibility to neurodegeneration, especially in DS and AD.

People with Down syndrome (DS) exhibit abnormal brain structure. Alterations affecting neurotransmission and signalling pathways that govern brain function are also evident. A large number of genes are simultaneously expressed at abnormal levels in DS; therefore, it is a challenge to determine which gene(s) contribute to specific abnormalities, and then identify the key molecular pathways involved. We generated RCAN1-TG mice to study the consequences of RCAN1 over-expression and investigate the contribution of RCAN1 to the brain phenotype of DS. RCAN1-TG mice exhibit structural brain abnormalities in those areas affected in DS. The volume and number of neurons within the hippocampus is reduced and this correlates with a defect in adult neurogenesis. The density of dendritic spines on RCAN1-TG hippocampal pyramidal neurons is also reduced. Deficits in hippocampal-dependent learning and short- and long-term memory are accompanied by a failure to maintain long-term potentiation (LTP) in hippocampal slices. In response to LTP induction, we observed diminished calcium transients and decreased phosphorylation of CaMKII and ERK1/2- proteins that are essential for the maintenance of LTP and formation of memory. Our data strongly suggest that RCAN1 plays an important role in normal brain development and function and its up-regulation likely contributes to the neural deficits associated with DS. © The Author 2012. Published by Oxford University Press. All rights reserved., This work was supported by the Fondation Jérôme Lejeune; philanthropic grants from the Judith Jane Mason and Harold Stannett Williams Memorial Foundation managed by ANZ Trustees; the APEX Foundation for Research into Intellectual Disability; the CASS Foundation; and the L.E.W. Carty Charitable Fund.

12 páginas, 5 figuras.-- Miñones-Moyano, Elena et al., MicroRNAs (miRNAs) are post-transcriptional gene expression regulators, playing key roles in neuronal development, plasticity and disease. Parkinson's disease (PD) is the second most common neurodegenerative disorder, characterized by the presence of protein inclusions or Lewy bodies and a progressive loss of dopaminergic neurons in the midbrain. Here, we have evaluated miRNA expression deregulation in PD brain samples. MiRNA expression profiling revealed decreased expression of miR-34b and miR-34c in brain areas with variable neuropathological affectation at clinical (motor) stages (Braak stages 4 and 5) of the disease, including the amygdala, frontal cortex, substantia nigra and cerebellum. Furthermore, misregulation of miR-34b/c was detected in pre-motor stages (stages 1–3) of the disease, and thus in cases that did not receive any PD-related treatment during life. Depletion of miR-34b or miR-34c in differentiated SH-SY5Y dopaminergic neuronal cells resulted in a moderate reduction in cell viability that was accompanied by altered mitochondrial function and dynamics, oxidative stress and reduction in total cellular adenosin triphosphate content. MiR-34b/c downregulation was coupled to a decrease in the expression of DJ1 and Parkin, two proteins associated to familial forms of PD that also have a role in idiopathic cases. Accordingly, DJ1 and Parkin expression was reduced in PD brain samples displaying strong miR-34b/c downregulation. We propose that early deregulation of miR-34b/c in PD triggers downstream transcriptome alterations underlying mitochondrial dysfunction and oxidative stress, which ultimately compromise cell viability. A better understanding of the cellular pathways controlling and/or controlled by miR-34b/c should allow identification of targets for development of therapeutic approaches., This work was supported by the Spanish Ministry of Health ‘Fondo de Investigaciones Sanitarias’ (PI081367); the Sixth Framework Programme of the European Commission through the SIROCCO integrated project LSHG-CT-2006-037900; the Spanish Ministry of Science and Innovation (SAF2008-00357); the Generalitat of Catalonia (Department of Health and Department of Economy and knowledge); the Spanish Ministry of Health: ‘Programa Miguel Servet’ (E.M.). Instituto de Salud Carlos III: ‘Contrato Postdoctoral Sara Borrell’ (S.P.) and BEFI contract (E.M.-M.).

Previous studies have demonstrated the presence of hemoglobin α-chain and β-chain in neurons of the rodent and human brain thus indicating that hemoglobin is a normal component of nerve cells and that hemoglobin may play a role in intraneuronal oxygen homeostasis. Progressing with these studies, hemoglobin expression has been examined in selected cell population in the brains of Alzheimer's disease (AD), argyrophilic grain disease (AGD), Parkinson's disease (PD) and Dementia with Lewy bodies (DLB). Double labeling immunofluorescence and confocal microscopy revealed reduced hemoglobin α-chain and β-chain in practically all neurons with small amounts of granular or punctuate hyperphosphorylated tau deposits and in neurons with tangles in the hippocampus and frontal cortex in AD and in the hippocampus in AGD; in ballooned neurons containing αB-crystallin in the amygdala in AD and AGD; and in about 80% of neurons with punctuate α-synuclein deposits and in neurons with Lewy bodies in the substantia nigra pars compacta and in vulnerable neurons of the medulla oblongata in PD and DLB; and in neurons with Lewy bodies in the frontal cortex in DLB. Hemoglobin immunoreactivity was also observed in the core of neuritic plaques and in diffuse plaques, but not in dystrophic neurites. Loss of hemoglobin was specific as neuroglobin was present equally in neurons with and without abnormal protein inclusions, and erythropoietin receptor was expressed equally in neurons without and in neurons with abnormal protein aggregates in AD, AGD, PD, and DLB.

RCANs, also called Down Syndrome Critical Region-1 (DSCR1)-like proteins, Modulatory Calcineurin Interacting Proteins (MCIPs) or calcipressins, are regulators of calcineurin, a Ca(2+)-dependent protein phosphatase involved in several neuronal functions. Despite the potential importance of the RCAN proteins in brain physiology, very little is known about their relative abundance and distribution patterns in the central nervous system. In this study we report the expression and distribution of RCAN mRNA transcripts and proteins in the mouse brain. RT-PCR and Western blot analysis showed that all Rcan mRNAs (Rcan1-1, Rcan1-2, Rcan2-1, Rcan2-3 and Rcan3) and their corresponding protein products (RCAN1-L, RCAN1-S, RCAN2-L, RCAN2-S and RCAN3) are present in every adult mouse brain region examined. All protein isoforms are also expressed in these same brain regions at early postnatal stages. Within regions, RCAN1-L, RCAN1-S, RCAN2-L and RCAN3 are differentially expressed depending on the region and developmental stage, whereas RCAN2-S is distributed homogeneously. Detailed immunohistochemical analysis revealed significant differences in the cellular and subcellular distributions of RCAN proteins. In the adult, RCAN1 was mainly expressed in the neuropil throughout the brain. Although at lower levels, RCAN3 was also detected throughout the neuropil. In contrast, RCAN2 was highly expressed in scattered neurons, in both the nucleus and the cytoplasm. Interestingly, RCAN2 is the only member of the RCAN family that was detected in glial cells. Finally, the expression patterns of RCANs at early postnatal stages differed from those of the adult, in different brain areas, in both their distributions and relative abundance, suggesting that the expression of these proteins could be regulated during neuronal differentiation. The nonoverlapping expression patterns of the RCAN proteins shown here highlight the existence of different physiological scenarios and therefore suggest different RCAN functional activities in the brain, depending on the cellular context and developmental stage.

Huntington's disease (HD) is an autosomal dominantly inherited disorder caused by the expansion of CAG repeats in the Huntingtin (HTT) gene. The abnormally extended polyglutamine in the HTT protein encoded by the CAG repeats has toxic effects. Here, we provide evidence to support that the mutant HTT CAG repeats interfere with cell viability at the RNA level. In human neuronal cells, expanded HTT exon-1 mRNA with CAG repeat lengths above the threshold for complete penetrance (40 or greater) induced cell death and increased levels of small CAG-repeated RNAs (sCAGs), of ≈21 nucleotides in a Dicer-dependent manner. The severity of the toxic effect of HTT mRNA and sCAG generation correlated with CAG expansion length. Small RNAs obtained from cells expressing mutant HTT and from HD human brains significantly decreased neuronal viability, in an Ago2-dependent mechanism. In both cases, the use of anti-miRs specific for sCAGs efficiently blocked the toxic effect, supporting a key role of sCAGs in HTT-mediated toxicity. Luciferase-reporter assays showed that expanded HTT silences the expression of CTG-containing genes that are down-regulated in HD. These results suggest a possible link between HD and sCAG expression with an aberrant activation of the siRNA/miRNA gene silencing machinery, which may trigger a detrimental response. The identification of the specific cellular processes affected by sCAGs may provide insights into the pathogenic mechanisms underlying HD, offering opportunities to develop new therapeutic approaches.

Genes located on chromosome 21, over-expressed in Down syndrome (DS) and Alzheimer's disease (AD) and which regulate vesicle trafficking, are strong candidates for involvement in AD neuropathology. Regulator of calcineurin activity 1 (RCAN1) is one such gene. We have generated mutant mice in which RCAN1 is either over-expressed (RCAN1(ox)) or ablated (Rcan1-/-) and examined whether exocytosis from chromaffin cells, a classic cellular model of neuronal exocytosis, is altered using carbon fibre amperometry. We find that Rcan1 regulates the number of vesicles undergoing exocytosis and the speed at which the vesicle fusion pore opens and closes. Cells from both Rcan1-/- and RCAN1(ox) mice display reduced levels of exocytosis. Changes in single-vesicle fusion kinetics are also evident resulting in the less catecholamine released per vesicle with increasing Rcan1 expression. Acute calcineurin inhibition did not replicate the effect of RCAN1 overexpression. These changes are not due to alterations in Ca2+ entry or the readily releasable vesicle pool size. Thus, we illustrate a novel regulator of vesicle exocytosis, Rcan1, which influences both exocytotic rate and vesicle fusion kinetics. If Rcan1 functions similarly in neurons then overexpression of this protein, as occurs in DS and AD brains, will reduce both the number of synaptic vesicles undergoing exocytosis and the amount of neurotransmitter released per fusion event. This has direct implications for the pathogenesis of these diseases as sufficient levels of neurotransmission are required for synaptic maintenance and the prevention of neurodegeneration and vesicle trafficking defects are the earliest hallmark of AD neuropathology.