Building the Future Therapies for Down Syndrome: The Third International Conference of the T21 Research Society.

Research focused on Down syndrome has increased in the last several years to advance understanding of the consequences of trisomy 21 (T21) on molecular and cellular processes and, ultimately, on individuals with Down syndrome. The Trisomy 21 Research Society (T21RS) is the premier scientific organization for researchers and clinicians studying Down syndrome. The Third International Conference of T21RS, held June 6-9, 2019, in Barcelona, Spain, brought together 429 scientists, families, and industry representatives to share the latest discoveries on underlying cellular and molecular mechanisms of T21, define cognitive and behavioral challenges and better understand comorbidities associated with Down syndrome, including Alzheimer's disease and leukemia. Presentation of cutting-edge results in neuroscience, neurology, model systems, psychology, cancer, biomarkers and molecular and phar-ma-cological therapeutic approaches demonstrate the compelling interest and continuing advancement in all aspects of understanding and ameliorating conditions associated with T21.

Suppressing Aneuploidy-Associated Phenotypes Improves the Fitness of Trisomy 21 Cells.

An abnormal number of chromosomes, or aneuploidy, accounts for most spontaneous abortions, causes developmental defects, and is associated with aging and cancer. The molecular mechanisms by which aneuploidy disrupts cellular function remain largely unknown. Here, we show that aneuploidy disrupts the morphology of the nucleus. Mutations that increase the levels of long-chain bases suppress nuclear abnormalities of aneuploid yeast independent of karyotype identity. Quantitative lipidomics indicates that long-chain bases are integral components of the nuclear membrane in yeast. Cells isolated from patients with Down syndrome also show that abnormal nuclear morphologies and increases in long-chain bases not only suppress these abnormalities but also improve their fitness. We obtained similar results with cells isolated from patients with Patau or Edward syndrome, indicating that increases in long-chain bases improve the fitness of aneuploid cells in yeast and humans. Targeting lipid biosynthesis pathways represents an important strategy to suppress nuclear abnormalities in aneuploidy-associated diseases.

Nrf2 stabilization prevents critical oxidative damage in Down syndrome cells.

Mounting evidence implicates chronic oxidative stress as a critical driver of the aging process. Down syndrome (DS) is characterized by a complex phenotype, including early senescence. DS cells display increased levels of reactive oxygen species (ROS) and mitochondrial structural and metabolic dysfunction, which are counterbalanced by sustained Nrf2-mediated transcription of cellular antioxidant response elements (ARE). Here, we show that caspase 3/PKCδdependent activation of the Nrf2 pathway in DS and Dp16 (a mouse model of DS) cells is necessary to protect against chronic oxidative damage and to preserve cellular functionality. Mitochondria-targeted catalase (mCAT) significantly reduced oxidative stress, restored mitochondrial structure and function, normalized replicative and wound healing capacity, and rendered the Nrf2-mediated antioxidant response dispensable. These results highlight the critical role of Nrf2/ARE in the maintenance of DS cell homeostasis and validate mitochondrial-specific interventions as a key aspect of antioxidant and antiaging therapies.

Mitochondrial network complexity emerges from fission/fusion dynamics.

Mitochondrial networks exhibit a variety of complex behaviors, including coordinated cell-wide oscillations of energy states as well as a phase transition (depolarization) in response to oxidative stress. Since functional and structural properties are often interwinded, here we characterized the structure of mitochondrial networks in mouse embryonic fibroblasts using network tools and percolation theory. Subsequently we perturbed the system either by promoting the fusion of mitochondrial segments or by inducing mitochondrial fission. Quantitative analysis of mitochondrial clusters revealed that structural parameters of healthy mitochondria laid in between the extremes of highly fragmented and completely fusioned networks. We confirmed our results by contrasting our empirical findings with the predictions of a recently described computational model of mitochondrial network emergence based on fission-fusion kinetics. Altogether these results offer not only an objective methodology to parametrize the complexity of this organelle but also support the idea that mitochondrial networks behave as critical systems and undergo structural phase transitions.

Prion protein inhibits fast axonal transport through a mechanism involving casein kinase 2.

Prion diseases include a number of progressive neuropathies involving conformational changes in cellular prion protein (PrPc) that may be fatal sporadic, familial or infectious. Pathological evidence indicated that neurons affected in prion diseases follow a dying-back pattern of degeneration. However, specific cellular processes affected by PrPc that explain such a pattern have not yet been identified. Results from cell biological and pharmacological experiments in isolated squid axoplasm and primary cultured neurons reveal inhibition of fast axonal transport (FAT) as a novel toxic effect elicited by PrPc. Pharmacological, biochemical and cell biological experiments further indicate this toxic effect involves casein kinase 2 (CK2) activation, providing a molecular basis for the toxic effect of PrPc on FAT. CK2 was found to phosphorylate and inhibit light chain subunits of the major motor protein conventional kinesin. Collectively, these findings suggest CK2 as a novel therapeutic target to prevent the gradual loss of neuronal connectivity that characterizes prion diseases.

Metabolic and Growth Rate Alterations in Lymphoblastic Cell Lines Discriminate Between Down Syndrome and Alzheimer's Disease.

BACKGROUND: Deficits in mitochondrial function and oxidative stress play pivotal roles in Down syndrome (DS) and Alzheimer's disease (AD) and these alterations in mitochondria occur systemically in both conditions. OBJECTIVE: We hypothesized that peripheral cells of elder subjects with DS exhibit disease-specific and dementia-specific metabolic features. To test this, we performed a comprehensive analysis of energy metabolism in lymphoblastic-cell-lines (LCLs) derived from subjects belonging to four groups: DS-with-dementia (DSAD), DS-without-dementia (DS), sporadic AD, and age-matched controls. METHODS: LCLs were studied under regular or minimal feeding regimes with galactose or glucose as primary carbohydrate sources. We assessed metabolism under glycolysis or oxidative phosphorylation by quantifying cell viability, oxidative stress, ATP levels, mitochondrial membrane potential (MMP), mitochondrial calcium uptake, and autophagy. RESULTS: DS and DSAD LCLs showed slower growth rates under minimal feeding. DS LCLs mainly dependent on mitochondrial respiration exhibited significantly slower growth and higher levels of oxidative stress compared to other groups. While ATP levels (under mitochondrial inhibitors) and mitochondrial calcium uptake were significantly reduced in DSAD and AD cells, MMP was decreased in DS, DSAD, and AD LCLs. Finally, DS LCLs showed markedly reduced levels of the autophagy marker LC3-II, underscoring the close association between metabolic dysfunction and impaired autophagy in DS. CONCLUSION: There are significant mitochondrial functional changes in LCLs derived from DS, DSAD, and AD patients. Several parameters analyzed were consistently different between DS, DSAD, and AD lines suggesting that metabolic indicators between LCL groups may be utilized as biomarkers of disease progression and/or treatment outcomes.

Adaptive downregulation of mitochondrial function in down syndrome.

Mitochondrial dysfunction and oxidative stress are common features of Down syndrome (DS). However, the underlying mechanisms are not known. We investigated the relationship between abnormal energy metabolism and oxidative stress with transcriptional and functional changes in DS cells. Impaired mitochondrial activity correlated with altered mitochondrial morphology. Increasing fusion capacity prevented morphological but not functional alterations in DS mitochondria. Sustained stimulation restored mitochondrial functional parameters but increased reactive oxygen species production and cell damage, suggesting that reduced DS mitochondrial activity is an adaptive response for avoiding injury and preserving basic cellular functions. Network analysis of genes overexpressed in DS cells demonstrated functional integration in pathways involved in energy metabolism and oxidative stress. Thus, although preventing extensive oxidative damage, mitochondrial downregulation may contribute to increased susceptibility of individuals with DS to clinical conditions in which altered energy metabolism may play a role, such as Alzheimer's disease, diabetes, and some types of autistic spectrum disorders.

A role for thrombospondin-1 deficits in astrocyte-mediated spine and synaptic pathology in Down's syndrome.

BACKGROUND: Down's syndrome (DS) is the most common genetic cause of mental retardation. Reduced number and aberrant architecture of dendritic spines are common features of DS neuropathology. However, the mechanisms involved in DS spine alterations are not known. In addition to a relevant role in synapse formation and maintenance, astrocytes can regulate spine dynamics by releasing soluble factors or by physical contact with neurons. We have previously shown impaired mitochondrial function in DS astrocytes leading to metabolic alterations in protein processing and secretion. In this study, we investigated whether deficits in astrocyte function contribute to DS spine pathology. METHODOLOGY/PRINCIPAL FINDINGS: Using a human astrocyte/rat hippocampal neuron coculture, we found that DS astrocytes are directly involved in the development of spine malformations and reduced synaptic density. We also show that thrombospondin 1 (TSP-1), an astrocyte-secreted protein, possesses a potent modulatory effect on spine number and morphology, and that both DS brains and DS astrocytes exhibit marked deficits in TSP-1 protein expression. Depletion of TSP-1 from normal astrocytes resulted in dramatic changes in spine morphology, while restoration of TSP-1 levels prevented DS astrocyte-mediated spine and synaptic alterations. Astrocyte cultures derived from TSP-1 KO mice exhibited similar deficits to support spine formation and structure than DS astrocytes. CONCLUSIONS/SIGNIFICANCE: These results indicate that human astrocytes promote spine and synapse formation, identify astrocyte dysfunction as a significant factor of spine and synaptic pathology in the DS brain, and provide a mechanistic rationale for the exploration of TSP-1-based therapies to treat spine and synaptic pathology in DS and other neurological conditions.

Cryopreservation of cortical tissue blocks for the generation of highly enriched neuronal cultures.

In this study, we outline a standardized protocol for the successful cryopreservation and thawing of cortical brain tissue blocks to generate highly enriched neuronal cultures. For this protocol the freezing medium used is 10% dimethyl sulfoxide (DMSO) diluted in Hank's Buffered Salt Solution (HBSS). Blocks of cortical tissue are transferred to cryovials containing the freezing medium and slowly frozen at -1°C/min in a rate-controlled freezing container. Post-thaw processing and dissociation of frozen tissue blocks consistently produced neuronal-enriched cultures which exhibited rapid neuritic growth during the first 5 days in culture and significant expansion of the neuronal network within 10 days. Immunocytochemical staining with the astrocytic marker glial fibrillary acidic protein (GFAP) and the neuronal marker beta-tubulin class III, revealed high numbers of neurons and astrocytes in the cultures. Generation of neural precursor cell cultures after tissue block dissociation resulted in rapidly expanding neurospheres, which produced large numbers of neurons and astrocytes under differentiating conditions. This simple cryopreservation protocol allows for the rapid, efficient, and inexpensive preservation of cortical brain tissue blocks, which grants increased flexibility for later generation of neuronal, astrocyte, and neuronal precursor cell cultures.

NAP and ADNF-9 protect normal and Down's syndrome cortical neurons from oxidative damage and apoptosis.

NAP (Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln, single letter code: NAPVSIPQ) and ADNF-9 (activity-dependent neurotrophic factor-9; Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala; single letter code: SALLRSIPA) are peptides derived from naturally occurring glial proteins that have shown neuroprotection in rodent model systems. Here, the neuroprotective activity of ADNF-9 and NAP was tested in two human models of neuronal degeneration in culture mediated by oxidative stress: normal human cortical neurons treated with H2O2 and Down's syndrome (DS) cortical neurons. Incubation of normal cortical neurons with 50 microM H2O2 for 1 hour resulted in morphological and structural changes consistent with neuronal degeneration and loss of viability of more than 60% of the neurons present in the culture. Addition of ADNF-9 or NAP at femtomolar concentrations resulted in significant increases in survival of normal neurons treated with H2O2. Femtomolar concentrations of ADNF-9 or NAP exhibited a similar neuroprotective efficacy, comparable to the antioxidant N-tert-butyl-2-sulpho-phenylnitrone at 100 microM (s-PBN). Treatment of DS cortical neurons with ADNF-9 or NAP resulted in a significant increase in neuronal survival as well as reduction of degenerative morphological changes. The results suggest that ADNF-9 and NAP possess potent neuroprotective properties against oxidative damage in human neurons that may be useful to preserve neuronal function and prevent neuronal death associated with chronic neurodegenerative disorders.

Phosphorylation of actin-depolymerizing factor/cofilin by LIM-kinase mediates amyloid beta-induced degeneration: a potential mechanism of neuronal dystrophy in Alzheimer's disease.

Deposition of fibrillar amyloid beta (fAbeta) plays a critical role in Alzheimer's disease (AD). We have shown recently that fAbeta-induced dystrophy requires the activation of focal adhesion proteins and the formation of aberrant focal adhesion structures, suggesting the activation of a mechanism of maladaptative plasticity in AD. Focal adhesions are actin-based structures that provide a structural link between the extracellular matrix and the cytoskeleton. To gain additional insight in the molecular mechanism of neuronal degeneration in AD, here we explored the involvement of LIM kinase 1 (LIMK1), actin-depolymerizing factor (ADF), and cofilin in Abeta-induced dystrophy. ADF/cofilin are actin-binding proteins that play a central role in actin filament dynamics, and LIMK1 is the kinase that phosphorylates and thereby inhibits ADF/cofilin. Our data indicate that treatment of hippocampal neurons with fAbeta increases the level of Ser3-phosphorylated ADF/cofilin and Thr508-phosphorylated LIMK1 (P-LIMK1), accompanied by a dramatic remodeling of actin filaments, neuritic dystrophy, and neuronal cell death. A synthetic peptide, S3 peptide, which acts as a specific competitor for ADF/cofilin phosphorylation by LIMK1, inhibited fAbeta-induced ADF/cofilin phosphorylation, preventing actin filament remodeling and neuronal degeneration, indicating the involvement of LIMK1 in Abeta-induced neuronal degeneration in vitro. Immunofluorescence analysis of AD brain showed a significant increase in the number of P-LIMK1-positive neurons in areas affected with AD pathology. P-LIMK1-positive neurons also showed early signs of AD pathology, such as intracellular Abeta and pretangle phosphorylated tau. Thus, LIMK1 activation may play a key role in AD pathology.

ets-2 promotes the activation of a mitochondrial death pathway in Down's syndrome neurons.

Down's syndrome (DS) is characterized by mental retardation and development of Alzheimer's disease (AD). Oxidative stress and mitochondrial dysfunction are both related to neurodegeneration in DS. Several genes in chromosome 21 have been linked to neuronal death, including the transcription factor ets-2. Cortical cultures derived from normal and DS fetal brains were used to study the role of ets-2 in DS neuronal degeneration. ets-2 was expressed in normal human cortical neurons (HCNs) and was markedly upregulated by oxidative stress. When overexpressed in normal HCNs, ets-2 induced a stereotyped sequence of apoptotic changes leading to neuronal death. DS HCNs exhibit intracellular oxidative stress and increased apoptosis after the first week in culture (Busciglio and Yankner, 1995). ets-2 levels were increased in DS HCNs, and, between 7 and 14 d in vitro, DS HCNs showed increased bax, cytoplasmic translocation of cytochrome c and apoptosis inducing factor, and active caspases 3 and 7, consistent with activation of an apoptotic mitochondrial death pathway. Degeneration of DS neurons was reduced by dominant-negative ets-2, suggesting that increased ets-2 expression promotes DS neuronal apoptosis. In the human brain, ets-2 expression was found in neurons and astrocytes. Strong ets-2 immunoreactivity was observed in DS/AD and sporadic AD brains associated with degenerative markers such as bax, intracellular Abeta, and hyperphosphorylated tau. Thus, in DS/AD and sporadic AD brains, converging pathological mechanisms leading to chronic oxidative stress and ets-2 upregulation in susceptible neurons may result in increased vulnerability by promoting the activation of a mitochondrial-dependent proapoptotic pathway of cell death.