The histone deacetylase SIRT6 is a tumor suppressor that controls cancer metabolism.

Reprogramming of cellular metabolism is a key event during tumorigenesis. Despite being known for decades (Warburg effect), the molecular mechanisms regulating this switch remained unexplored. Here, we identify SIRT6 as a tumor suppressor that regulates aerobic glycolysis in cancer cells. Importantly, loss of SIRT6 leads to tumor formation without activation of known oncogenes, whereas transformed SIRT6-deficient cells display increased glycolysis and tumor growth, suggesting that SIRT6 plays a role in both establishment and maintenance of cancer. By using a conditional SIRT6 allele, we show that SIRT6 deletion in vivo increases the number, size, and aggressiveness of tumors. SIRT6 also functions as a regulator of ribosome metabolism by corepressing MYC transcriptional activity. Lastly, Sirt6 is selectively downregulated in several human cancers, and expression levels of SIRT6 predict prognosis and tumor-free survival rates, highlighting SIRT6 as a critical modulator of cancer metabolism. Our studies reveal SIRT6 to be a potent tumor suppressor acting to suppress cancer metabolism.

The histone deacetylase Sirt6 regulates glucose homeostasis via Hif1alpha.

SIRT6 is a member of a highly conserved family of NAD(+)-dependent deacetylases with various roles in metabolism, stress resistance, and life span. SIRT6-deficient mice develop normally but succumb to a lethal hypoglycemia early in life; however, the mechanism underlying this hypoglycemia remained unclear. Here, we demonstrate that SIRT6 functions as a histone H3K9 deacetylase to control the expression of multiple glycolytic genes. Specifically, SIRT6 appears to function as a corepressor of the transcription factor Hif1alpha, a critical regulator of nutrient stress responses. Consistent with this notion, SIRT6-deficient cells exhibit increased Hif1alpha activity and show increased glucose uptake with upregulation of glycolysis and diminished mitochondrial respiration. Our studies uncover a role for the chromatin factor SIRT6 as a master regulator of glucose homeostasis and may provide the basis for novel therapeutic approaches against metabolic diseases, such as diabetes and obesity.

Unraveling the hidden role of a uORF-encoded peptide as a kinase inhibitor of PKCs.

Approximately 40% of human messenger RNAs (mRNAs) contain upstream open reading frames (uORFs) in their 5' untranslated regions. Some of these uORF sequences, thought to attenuate scanning ribosomes or lead to mRNA degradation, were recently shown to be translated, although the function of the encoded peptides remains unknown. Here, we show a uORF-encoded peptide that exhibits kinase inhibitory functions. This uORF, upstream of the protein kinase C-eta (PKC-η) main ORF, encodes a peptide (uPEP2) containing the typical PKC pseudosubstrate motif present in all PKCs that autoinhibits their kinase activity. We show that uPEP2 directly binds to and selectively inhibits the catalytic activity of novel PKCs but not of classical or atypical PKCs. The endogenous deletion of uORF2 or its overexpression in MCF-7 cells revealed that the endogenously translated uPEP2 reduces the protein levels of PKC-η and other novel PKCs and restricts cell proliferation. Functionally, treatment of breast cancer cells with uPEP2 diminished cell survival and their migration and synergized with chemotherapy by interfering with the response to DNA damage. Furthermore, in a xenograft of MDA-MB-231 breast cancer tumor in mice models, uPEP2 suppressed tumor progression, invasion, and metastasis. Tumor histology showed reduced proliferation, enhanced cell death, and lower protein expression levels of novel PKCs along with diminished phosphorylation of PKC substrates. Hence, our study demonstrates that uORFs may encode biologically active peptides beyond their role as translation regulators of their downstream ORFs. Together, we point to a unique function of a uORF-encoded peptide as a kinase inhibitor, pertinent to cancer therapy.

SIRT6 recruits SNF2H to DNA break sites, preventing genomic instability through chromatin remodeling.

DNA damage is linked to multiple human diseases, such as cancer, neurodegeneration, and aging. Little is known about the role of chromatin accessibility in DNA repair. Here, we find that the deacetylase sirtuin 6 (SIRT6) is one of the earliest factors recruited to double-strand breaks (DSBs). SIRT6 recruits the chromatin remodeler SNF2H to DSBs and focally deacetylates histone H3K56. Lack of SIRT6 and SNF2H impairs chromatin remodeling, increasing sensitivity to genotoxic damage and recruitment of downstream factors such as 53BP1 and breast cancer 1 (BRCA1). Remarkably, SIRT6-deficient mice exhibit lower levels of chromatin-associated SNF2H in specific tissues, a phenotype accompanied by DNA damage. We demonstrate that SIRT6 is critical for recruitment of a chromatin remodeler as an early step in the DNA damage response, indicating that proper unfolding of chromatin plays a rate-limiting role. We present a unique crosstalk between a histone modifier and a chromatin remodeler, regulating a coordinated response to prevent DNA damage.

Impaired innate and conditioned social behavior in adult C57Bl6/J mice prenatally exposed to chlorpyrifos.

BACKGROUND: Signs of pervasive developmental disorder and social deficits were reported in toddlers and children whose mothers were exposed to organophosphate pesticides during pregnancy. Deficits in social preference were reported in adult male mice exposed to chlorpyrifos on gestational days 12-15. This study aimed (a) to test the hypothesis that adult female and male mice that were exposed prenatally to subtoxic doses of chlorpyrifos would be impaired in social behavior and (b) to determine if prenatal chlorpyrifos altered the expression of transcripts for oxytocin in the hypothalamus. Pregnant mice were treated by gavage with corn oil vehicle or 2.5 mg/kg or 5 mg/kg of CPF on gestational days 12-15. Social preference, social and non-social conditioned place preference tasks were tested in adults. Expression of oxytocin transcripts in hypothalamus was measured by qPCR. RESULTS: Chlorpyrifos (5 mg/kg on GD 12-15) reduced the innate preference for a conspecific in a dose and sex dependent manner. Adult males exposed prenatally to 5 mg/kg CPF showed a reduction in social preference. Socially conditioned place preference was impaired in offspring of dams treated with either dose of CPF. Non-social appetitive place conditioning was impaired in offspring of dams exposed to 2.5 mg/kg, but not to 5 mg/kg chlorpyrifos. Prenatal chlorpyrifos treatment did not alter the expression of the oxytocin mRNA in the hypothalamus, although expression was significantly lower in females. CONCLUSIONS: Prenatal chlorpyrifos induced innate and learned social deficits and non-specific conditioning deficits in adult mice in a sex-dependent manner. Males showed specific social deficits following the higher dose whereas both males and females showed a more generalized conditioning deficit following the intermediate dose.

A SIRT1-LSD1 corepressor complex regulates Notch target gene expression and development.

Epigenetic regulation of gene expression by histone-modifying corepressor complexes is central to normal animal development. The NAD(+)-dependent deacetylase and gene repressor SIRT1 removes histone H4K16 acetylation marks and facilitates heterochromatin formation. However, the mechanistic contribution of SIRT1 to epigenetic regulation at euchromatic loci and whether it acts in concert with other chromatin-modifying activities to control developmental gene expression programs remain unclear. We describe here a SIRT1 corepressor complex containing the histone H3K4 demethylase LSD1/KDM1A and several other LSD1-associated proteins. SIRT1 and LSD1 interact directly and play conserved and concerted roles in H4K16 deacetylation and H3K4 demethylation to repress genes regulated by the Notch signaling pathway. Mutations in Drosophila SIRT1 and LSD1 orthologs result in similar developmental phenotypes and genetically interact with the Notch pathway in Drosophila. These findings offer new insights into conserved mechanisms of epigenetic gene repression and regulation of development by SIRT1 in metazoans.

Epigenetic control of early neurodegenerative events in diabetic retinopathy by the histone deacetylase SIRT6.

Diabetic retinopathy (DR) is one of the common complications associated with diabetes mellitus and the leading cause of blindness worldwide. Recent research has demonstrated that DR is not only a microvascular disease but may be a result of neurodegenerative processes. Moreover, glucose-induced neuron and glial cell damage may occur shortly after the onset of diabetes which makes the disease hard to diagnose at early stages. SIRT6, a NAD-dependent sirtuin deacylase, modulates aging, energy metabolism, and neurodegeneration. In previous studies we showed that SIRT6 deficiency causes major retinal transmission defects, changes in the expression of glycolytic genes, and elevated levels of apoptosis. Given the importance of glucose availability for retinal function and the critical role of SIRT6 in modulating glycolysis, we aimed to analyze SIRT6 participation in the molecular machinery that regulates the development of experimental DR. Using non-obese diabetic mice, we determined by western blot that 2 weeks after the onset of the disease, high glucose concentrations induced retinal increase in a neovascularization promoting factor (vascular endothelial growth factor, VEGF), and the loss of a neuroprotective factor (brain-derived neurotrophic factor, BDNF) associated with reduced levels of SIRT6 and increased acetylation levels of its substrates (H3K9 and H3K56) suggesting a deregulation of key neural factors. Noteworthy, retinas from CNS conditionally deleted SIRT6 mice showed a resemblance to diabetic retinas exhibiting lower protein levels of BDNF factor and increased protein levels of VEGF. Moreover, cultured Müller glial cells subjected to high glucose concentrations exhibited decreased levels of SIRT6 and increased levels of H3K56 acetylation. In addition, the increment of VEGF levels induced by high glucose was reverted by the over-expression of SIRT6 in this cell type. Accordingly, siRNA experiments showed that, when SIRT6 was silenced, VEGF levels increased. Our findings suggest that epigenetically regulated neurodegenerative events may occur at an early diabetic stage prior to the characteristic proliferative and vascular changes observed at a later diabetic stage.

Short versus long double-stranded RNA activation of a post-transcriptional gene knockdown pathway.

RNA interference (RNAi) utilizes a conserved cellular autoimmune defense mechanism involving the internalization of dsRNA into cells and the activation of a set of RNAi related genes. Using RNAi, complete sex reversal is achievable in males of the prawn Macrobrachium rosenbergii by knocking down the transcript level of an insulin-like androgenic gland hormone (Mr-IAG) through injections of dsRNA of the entire Mr-IAG ORF sequence (dsMr-IAG - 518bp). Interestingly, in-vivo knockdown success and dsMr-IAG lengths seemed to correlate, with long dsRNA being the most effective and short dsRNA fragments showing no effect. However, little is known about the RNAi machinery in M. rosenbergii. We discovered the Mr-Dicer and Mr-Argonaute gene families, associated with the major knockdown pathways, in our M. rosenbergii transcriptomic library. In response to dsMr-IAG administration, only post-transcriptional pathway-related gene transcript levels were upregulated. In addition, a passive dsRNA channel (a SID1 gene ortholog) that allows external dsRNA to enter cells was found. Its function was validated by observing Mr-SID1 specific upregulation dependent on dsRNA lengths, while attempted loss-of-function experiments were lethal. Our results, which suggest differential systemic responses to dsRNA lengths, provide evidence that the above RNAi-based manipulation occurs via the post-transcriptional pathway. The temporal nature of the latter pathway supports the safety of using such RNAi-based biotechnologies in aquaculture and environmental applications. Unlike reports of RNAi driven by the administration of small dsRNA fragments in-vitro, the case presented here demonstrates length dependency in-vivo, suggesting further complexity in the context of the entire organism.

SIRT6 is a key regulator of mitochondrial function in the brain.

The SIRT6 deacetylase has been implicated in DNA repair, telomere maintenance, glucose and lipid metabolism and, importantly, it has critical roles in the brain ranging from its development to neurodegeneration. Here, we combined transcriptomics and metabolomics approaches to characterize the functions of SIRT6 in mouse brains. Our analysis reveals that SIRT6 is a central regulator of mitochondrial activity in the brain. SIRT6 deficiency in the brain leads to mitochondrial deficiency with a global downregulation of mitochondria-related genes and pronounced changes in metabolite content. We suggest that SIRT6 affects mitochondrial functions through its interaction with the transcription factor YY1 that, together, regulate mitochondrial gene expression. Moreover, SIRT6 target genes include SIRT3 and SIRT4, which are significantly downregulated in SIRT6-deficient brains. Our results demonstrate that the lack of SIRT6 leads to decreased mitochondrial gene expression and metabolomic changes of TCA cycle byproducts, including increased ROS production, reduced mitochondrial number, and impaired membrane potential that can be partially rescued by restoring SIRT3 and SIRT4 levels. Importantly, the changes we observed in SIRT6-deficient brains are also occurring in aging human brains and particularly in patients with Alzheimer's, Parkinson's, Huntington's, and Amyotrophic lateral sclerosis disease. Overall, our results suggest that the reduced levels of SIRT6 in the aging brain and neurodegeneration initiate mitochondrial dysfunction by altering gene expression, ROS production, and mitochondrial decay.

4-phenylbutyric acid-Identity crisis; can it act as a translation inhibitor?

Loss of proteostasis can occur due to mutations, the formation of aggregates, or general deficiency in the correct translation and folding of proteins. These phenomena are commonly observed in pathologies, but most significantly, loss of proteostasis characterizes aging. This loss leads to the chronic activation of stress responses and has a generally deleterious impact on the organism. While finding molecules that can alleviate these symptoms is an important step toward solutions for these conditions, some molecules might be mischaracterized on the way. 4-phenylbutyric acid (4PBA) is known for its role as a chemical chaperone that helps alleviate endoplasmic reticulum (ER) stress, yet a scan of the literature reveals that no biochemical or molecular experiments have shown any protein refolding capacity. Here, we show that 4PBA is a conserved weak inhibitor of mRNA translation, both in vitro and in cellular systems, and furthermore-it does not promote protein folding nor prevents aggregation. 4PBA possibly alleviates proteostatic or ER stress by inhibiting protein synthesis, allowing the cells to cope with misfolded proteins by reducing the protein load. Better understanding of 4PBA biochemical mechanisms will improve its usage in basic science and as a drug in different pathologies, also opening new venues for the treatment of different diseases.

Characterization of nuclear sirtuins: molecular mechanisms and physiological relevance.

Sirtuins are protein deacetylases/mono-ADP-ribosyltransferases found in organisms ranging from bacteria to humans. This group of enzymes relies on nicotinamide adenine dinucleotide (NAD(+)) as a cofactor linking their activity to the cellular metabolic status. Originally found in yeast, Sir2 was discovered as a silencing factor and has been shown to mediate the effects of calorie restriction on lifespan extension. In mammals seven homologs (SIRT1-7) exist which evolved to have specific biological outcomes depending on the particular cellular context, their interacting proteins, and the genomic loci to where they are actively targeted. Sirtuins biological roles are highlighted in the early lethal phenotypes observed in the deficient murine models. In this chapter, we summarize current concepts on non-metabolic functions for sirtuins, depicting this broad family from yeast to mammals.

SIRT6 Through the Brain Evolution, Development, and Aging.

During an organism's lifespan, two main phenomena are critical for the organism's survival. These are (1) a proper embryonic development, which permits the new organism to function with high fitness, grow and reproduce, and (2) the aging process, which will progressively undermine its competence and fitness for survival, leading to its death. Interestingly these processes present various similarities at the molecular level. Notably, as organisms became more complex, regulation of these processes became coordinated by the brain, and failure in brain activity is detrimental in both development and aging. One of the critical processes regulating brain health is the capacity to keep its genomic integrity and epigenetic regulation-deficiency in DNA repair results in neurodevelopmental and neurodegenerative diseases. As the brain becomes more complex, this effect becomes more evident. In this perspective, we will analyze how the brain evolved and became critical for human survival and the role Sirt6 plays in brain health. Sirt6 belongs to the Sirtuin family of histone deacetylases that control several cellular processes; among them, Sirt6 has been associated with the proper embryonic development and is associated with the aging process. In humans, Sirt6 has a pivotal role during brain aging, and its loss of function is correlated with the appearance of neurodegenerative diseases such as Alzheimer's disease. However, Sirt6 roles during brain development and aging, especially the last one, are not observed in all species. It appears that during the brain organ evolution, Sirt6 has gained more relevance as the brain becomes bigger and more complex, observing the most detrimental effect in the brains of Homo sapiens. In this perspective, we part from the evolution of the brain in metazoans, the biological similarities between brain development and aging, and the relevant functions of Sirt6 in these similar phenomena to conclude with the evidence suggesting a more relevant role of Sirt6 gained in the brain evolution.

FACS-based isolation of fixed mouse neuronal nuclei for ATAC-seq and Hi-C.

The organization of chromatin structure plays a crucial role in gene expression, DNA replication, and repair. Chromatin alterations influence gene expression, and modifications could be associated with genomic instability in the cells during aging or diseases. Here, we provide a modified protocol to isolate fixed neuronal nuclei from a single mouse cortex to investigate the spatial organization of chromatin structure on a genome-wide scale by ATAC-seq (the assay for transposase-accessible chromatin with high-throughput sequencing) and chromatin conformation by Hi-C (high-throughput chromosome conformation capture).

TP73-AS1 is induced by YY1 during TMZ treatment and highly expressed in the aging brain.

Aging is a factor associated with poor prognosis in glioblastoma (GBM). It is therefore important to understand the molecular features of aging contributing to GBM morbidity. TP73-AS1 is a long noncoding RNA (lncRNA) over expressed in GBM tumors shown to promote resistance to the chemotherapeutic temozolomide (TMZ), and tumor aggressiveness. How the expression of TP73-AS1 is regulated is not known, nor is it known if its expression is associated with aging. By analyzing transcriptional data obtained from natural and pathological aging brain, we found that the expression of TP73-AS1 is high in pathological and naturally aging brains. YY1 physically associates with the promoter of TP73-AS1 and we found that along with TP73-AS1, YY1 is induced by TMZ. We found that the TP73-AS1 promoter is activated by TMZ, and by YY1 over expression. Using CRISPRi to deplete YY1, we found that YY1 promotes up regulation of TP73-AS1 and the activation of its promoter during TMZ treatment. In addition, we identified two putative YY1 binding sites within the TP73-AS1 promoter, and used mutagenesis to find that they are essential for TMZ mediated promoter activation. Together, our data positions YY1 as an important TP73-AS1 regulator, demonstrating that TP73-AS1 is expressed in the natural and pathological aging brain, including during neurodegeneration and cancer. Our findings advance our understanding of TP73-AS1 expression, bringing forth a new link between TMZ resistance and aging, both of which contribute to GBM morbidity.

Aging and pathological aging signatures of the brain: through the focusing lens of SIRT6.

Brain-specific SIRT6-KO mice present increased DNA damage, learning impairments, and neurodegenerative phenotypes, placing SIRT6 as a key protein in preventing neurodegeneration. In the aging brain, SIRT6 levels/activity decline, which is accentuated in Alzheimer's patients. To understand SIRT6 roles in transcript pattern changes, we analyzed transcriptomes of young WT, old WT and young SIRT6-KO mice brains, and found changes in gene expression related to healthy and pathological aging. In addition, we traced these differences in human and mouse samples of Alzheimer's and Parkinson's diseases, healthy aging and calorie restriction (CR). Our results define four gene expression categories that change with age in a pathological or non-pathological manner, which are either reversed or not by CR. We found that each of these gene expression categories is associated with specific transcription factors, thus serving as potential candidates for their category-specific regulation. One of these candidates is YY1, which we found to act together with SIRT6 regulating specific processes. We thus argue that SIRT6 has a pivotal role in preventing age-related transcriptional changes in brains. Therefore, reduced SIRT6 activity may drive pathological age-related gene expression signatures in the brain.

A Mechanism for the Inhibition of Tau Neurotoxicity: Studies with Artificial Membranes, Isolated Mitochondria, and Intact Cells.

It is currently believed that molecular agents that specifically bind to and neutralize the toxic proteins/peptides, amyloid ß (Aß42), tau, and the tau-derived peptide PHF6, hold the key to attenuating the progression of Alzheimer's disease (AD). We thus tested our previously developed nonaggregating Aß42 double mutant (Aß42DM) as a multispecific binder for three AD-associated molecules, wild-type Aß42, the tauK174Q mutant, and a synthetic PHF6 peptide. Aß42DM acted as a functional inhibitor of these molecules in in vitro assays and in neuronal cell-based models of AD. The double mutant bound both cytotoxic tauK174Q and synthetic PHF6 and protected neuronal cells from the accumulation of tau in cell lysates and mitochondria. Aß42DM also reduced toxic intracellular levels of calcium and the overall cell toxicity induced by overexpressed tau, synthetic PHF6, Aß42, or a combination of PHF6and Aß42. Aß42DM inhibited PHF6-induced overall mitochondrial dysfunction: In particular, Aß42DM inhibited PHF6-induced damage to submitochondrial particles (SMPs) and suppressed PHF6-induced elevation of the ζ-potential of inverted SMPs (proxy for the inner mitochondrial membrane, IMM). PHF6 reduced the lipid fluidity of cardiolipin/DOPC vesicles (that mimic the IMM) but not DOPC (which mimics the outer mitochondrial membrane), and this effect was inhibited by Aß42DM. This inhibition may be explained by the conformational changes in PHF6 induced by Aß42DM in solution and in membrane mimetics. On this basis, the paper presents a mechanistic explanation for the inhibitory activity of Aß42DM against Aß42- and tau-induced membrane permeability and cell toxicity and provides confirmatory evidence for its protective function in neuronal cells.

Metabolic Imbalance Effect on Retinal Müller Glial Cells Reprogramming Capacity: Involvement of Histone Deacetylase SIRT6.

Retinal Müller glial cells (MGs) are among the first to demonstrate metabolic changes during retinal disease and are a potential source of regenerative cells. In response to a harmful stimulus, they can dedifferentiate acquiring neural stem cells properties, proliferate and migrate to the damaged retinal layer and differentiate into lost neurons. However, it is not yet known how this reprogramming process is regulated in mammals. Since glucose and oxygen are important regulatory elements that may help directing stem cell fate, we aimed to study the effect of glucose variations and oxidative stress in Müller cells reprogramming capacity and analyze the participation the histone deacetylase SIRT6, as an epigenetic modulator of this process. We found that the combination of high glucose and oxidative stress induced a decrease in the levels of the marker glutamine synthetase, and an increase in the migration capacity of the cells suggesting that these experimental conditions could induce some degree of dedifferentiation and favor the migration ability. High glucose induced an increase in the levels of the pluripotent factor SOX9 and a decrease in SIRT6 levels accompanied by the increase in the acetylation levels of H3K9. Inhibiting SIRT6 expression by siRNA rendered an increase in SOX9 levels. We also determined SOX9 levels in retinas from mice with a conditional deletion of SIRT6 in the CNS. To further understand the mechanisms that regulate MGs response under metabolic impaired conditions, we evaluated the gene expression profile and performed Gene Ontology enrichment analysis of Müller cells from a murine model of Diabetes. We found several differentially expressed genes and observed that the transcriptomic change involved the enrichment of genes associated with glucose metabolism, cell migration, development and pluripotency. We found that many functional categories affected in cells of diabetic animals were directly related to SIRT6 function. Transcription factors enrichment analysis allowed us to predict several factors, including SOX9, that may be involved in the modulation of the differential expression program observed in diabetic MGs. Our results underline the heterogeneity of Müller cells response and the challenge that the study of metabolic impairment in vivo represents.

SIRT6-CBP-dependent nuclear Tau accumulation and its role in protein synthesis.

Several neurodegenerative diseases present Tau accumulation as the main pathological marker. Tau post-translational modifications such as phosphorylation and acetylation are increased in neurodegeneration. Here, we show that Tau hyper-acetylation at residue 174 increases its own nuclear presence and is the result of DNA damage signaling or the lack of SIRT6, both causative of neurodegeneration. Tau-K174ac is deacetylated in the nucleus by SIRT6. However, lack of SIRT6 or chronic DNA damage results in nuclear Tau-K174ac accumulation. Once there, it induces global changes in gene expression, affecting protein translation, synthesis, and energy production. Concomitantly, Alzheimer's disease (AD) case subjects show increased nucleolin and a decrease in SIRT6 levels. AD case subjects present increased levels of nuclear Tau, particularly Tau-K174ac. Our results suggest that increased Tau-K174ac in AD case subjects is the result of DNA damage signaling and SIRT6 depletion. We propose that Tau-K174ac toxicity is due to its increased stability, nuclear accumulation, and nucleolar dysfunction.

Engineering DYRK1A overdosage yields Down syndrome-characteristic cortical splicing aberrations.

Down syndrome (DS) associates with impaired brain functions, but the underlying mechanism(s) are yet unclear. The "gene dosage" hypothesis predicts that in DS, overexpression of a single gene can impair multiple brain functions through a signal amplification effect due to impaired regulatory mechanism(s). Here, we report findings attributing to impairments in the splicing process such a regulatory role. We have used DS fetal brain samples in search for initial evidence and employed engineered mice with MMU16 partial trisomy (Ts65Dn) or direct excess of the splicing-associated nuclear kinase Dyrk1A, overdosed in DS for further analyses. We present specific albeit modest changes in the DS brain's splicing machinery with subsequently amplified effects in target transcripts; and we demonstrate that engineered excess of Dyrk1A can largely recapitulate these changes. Specifically, in both the fetal DS brains and the Dyrk1A overdose models, we found ample modestly modified splicing-associated transcripts which apparently induced secondary enhancement in exon inclusion of key synaptic transcripts. Thus, DS-reduced levels of the dominant-negative TRKBT1 transcript, but not other TRKB mRNA transcripts, were accompanied by corresponding decreases in BDNF. In addition, the DS brains and Dyrk1A overdosage models showed selective changes in the transcripts composition of neuroligin mRNAs as well as reductions in the "synaptic" acetylcholinesterase variant AChE-S mRNA and corresponding increases in the stress-inducible AChE-R mRNA variant, yielding key synaptic proteins with unusual features. In cotransfected cells, Dyrk1A overdosage caused parallel changes in the splicing pattern of an AChE mini-gene, suggesting that Dyrk1A overdosage is both essential and sufficient to induce the observed change in the composition of AChE mRNA variants. Furthermore, the Dyrk1A overdosage animal models showed pronounced changes in the structure of neuronal nuclear speckles, where splicing events take place and in SR proteins phosphorylation known to be required for the splicing process. Together, our findings demonstrate DS-like brain splicing machinery malfunctioning in Dyrk1A overexpressing mice. Since individual splicing choices may alter cell fate determination, axon guidance, and synaptogenesis, these findings suggest the retrieval of balanced splicing as a goal for DS therapeutic manipulations early in DS development.

Acetylcholinesterase variants in Alzheimer's disease: from neuroprotection to programmed cell death.

BACKGROUND: In Alzheimer's disease (AD), cholinergic neurons are particularly vulnerable for as yet unclear reasons. Here, we report that modified composition, localization and properties of alternative splice variants encoding the acetylcholine-hydrolyzing enzyme acetylcholinesterase (AChE) may be variably involved in disease progression or in systemic efforts to attenuate its progression. OBJECTIVE: The purpose of this study was to explore the implications for AD of the cellular and biochemical properties of the various AChE proteins, differing in their N and C termini. METHODS: We have used cell transfection with genetically engineered vectors as well as microinjection to overexpress specific AChE variants and explore the consequences to cellular well-being and survival. Additionally, we employed highly purified recombinant AChE-R and AChE-S to explore putative interactions with the AD beta-amyloid peptide. RESULTS: Our findings demonstrate distinct, and in certain cases inverse cell fate outcome under enforced expression of the human N- and C-terminally modified AChE variants, all of which have similar enzymatic activities. CONCLUSION: The N-terminal extension in conjunction with the primary helical C-terminal peptide of 'tailed' AChE-S facilitates, whereas the shorter, naturally unfolded C-terminus of the stress-induced AChE-R variant attenuates Alzheimer's pathology.