Enhancing Neuronal Cell Uptake of Therapeutic Nucleic Acids with Tetrahedral DNA Nanostructures.

The successful translation of therapeutic nucleic acids (NAs) for the treatment of neurological disorders depends on their safe and efficient delivery to neural cells, in particular neurons. DNA nanostructures can be a promising NAs delivery vehicle. Nonetheless, the potential of DNA nanostructures for neuronal cell delivery of therapeutic NAs is unexplored. Here, tetrahedral DNA nanostructures (TDN) as siRNA delivery scaffolds to neuronal cells, exploring the influence of functionalization with two different reported neuronal targeting ligands: C4-3 RNA aptamer and Tet1 peptide are investigated. Nanostructures are characterized in vitro, as well as in silico using molecular dynamic simulations to better understand the overall TDN structural stability. Enhancement of neuronal cell uptake of TDN functionalized with the C4-3 Aptamer (TDN-Apt), not only in neuronal cell lines but also in primary neuronal cell cultures is demonstrated. Additionally, TDN and TDN-Apt nanostructures carrying siRNA are shown to promote silencing in a process aided by chloroquine-induced endosomal disruption. This work presents a thorough workflow for the structural and functional characterization of the proposed TDN as a nano-scaffold for neuronal delivery of therapeutic NAs and for targeting ligands evaluation, contributing to the future development of new neuronal drug delivery systems based on DNA nanostructures.

Mitochondrial superoxide generation induces a parkinsonian phenotype in zebrafish and huntingtin aggregation in human cells.

Superoxide generation by mitochondria respiratory complexes is a major source of reactive oxygen species (ROS) which are capable of initiating redox signaling and oxidative damage. Current understanding of the role of mitochondrial ROS in health and disease has been limited by the lack of experimental strategies to selectively induce mitochondrial superoxide production. The recently-developed mitochondria-targeted redox cycler MitoParaquat (MitoPQ) overcomes this limitation, and has proven effective in vitro and in Drosophila. Here we present an in vivo study of MitoPQ in the vertebrate zebrafish model in the context of Parkinson's disease (PD), and in a human cell model of Huntington's disease (HD). We show that MitoPQ is 100-fold more potent than non-targeted paraquat in both cells and in zebrafish in vivo. Treatment with MitoPQ induced a parkinsonian phenotype in zebrafish larvae, with decreased sensorimotor reflexes, spontaneous movement and brain tyrosine hydroxylase (TH) levels, without detectable effects on heart rate or atrioventricular coordination. Motor phenotypes and TH levels were partly rescued with antioxidant or monoaminergic potentiation strategies. In a HD cell model, MitoPQ promoted mutant huntingtin aggregation without increasing cell death, contrasting with the complex I inhibitor rotenone that increased death in cells expressing either wild-type or mutant huntingtin. These results show that MitoPQ is a valuable tool for cellular and in vivo studies of the role of mitochondrial superoxide generation in redox biology, and as a trigger or co-stressor to model metabolic and neurodegenerative disease phenotypes.

Targeting the proteostasis network in Huntington's disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a polyglutamine expansion mutation in the huntingtin protein. Expansions above 40 polyglutamine repeats are invariably fatal, following a symptomatic period characterised by choreiform movements, behavioural abnormalities, and cognitive decline. While mutant huntingtin (mHtt) is widely expressed from early life, most patients with HD present in mid-adulthood, highlighting the role of ageing in disease pathogenesis. mHtt undergoes proteolytic cleavage, misfolding, accumulation, and aggregation into inclusion bodies. The emerging model of HD pathogenesis proposes that the chronic production of misfolded mHtt overwhelms the chaperone machinery, diverting other misfolded clients to the proteasome and the autophagy pathways, ultimately leading to a global collapse of the proteostasis network. Multiple converging hypotheses also implicate ageing and its impact in the dysfunction of organelles as additional contributing factors to the collapse of proteostasis in HD. In particular, mitochondrial function is required to sustain the activity of ATP-dependent chaperones and proteolytic machinery. Recent studies elucidating mitochondria-endoplasmic reticulum interactions and uncovering a dedicated proteostasis machinery in mitochondria, suggest that mitochondria play a more active role in the maintenance of cellular proteostasis than previously thought. The enhancement of cytosolic proteostasis pathways shows promise for HD treatment, protecting cells from the detrimental effects of mHtt accumulation. In this review, we consider how mHtt and its post translational modifications interfere with protein quality control pathways, and how the pharmacological and genetic modulation of components of the proteostasis network impact disease phenotypes in cellular and in vivo HD models.

Modulation of Molecular Chaperones in Huntington's Disease and Other Polyglutamine Disorders.

Polyglutamine expansion mutations in specific proteins underlie the pathogenesis of a group of progressive neurodegenerative disorders, including Huntington's disease, spinal and bulbar muscular atrophy, dentatorubral-pallidoluysian atrophy, and several spinocerebellar ataxias. The different mutant proteins share ubiquitous expression and abnormal proteostasis, with misfolding and aggregation, but nevertheless evoke distinct patterns of neurodegeneration. This highlights the relevance of the full protein context where the polyglutamine expansion occurs and suggests different interactions with the cellular proteostasis machinery. Molecular chaperones are key elements of the proteostasis machinery and therapeutic targets for neurodegeneration. Here, we provide a focused review on Hsp90, Hsp70, and their co-chaperones, and how their genetic or pharmacological modulation affects the proteostasis and disease phenotypes in cellular and animal models of polyglutamine disorders. The emerging picture is that, in principle, Hsp70 modulation may be more amenable for long-term treatment by promoting a more selective clearance of mutant proteins than Hsp90 modulation, which may further decrease the necessary wild-type counterparts. It seems, nevertheless, unlikely that a single Hsp70 modulator will benefit all polyglutamine diseases. Indeed, available data, together with insights from effects on tau and alpha-synuclein in models of Alzheimer's and Parkinson's diseases, indicates that Hsp70 modulators may lead to different effects on the proteostasis of different mutant and wild-type client proteins. Future studies should include the further development of isoform selective inhibitors, namely to avoid off-target effects on Hsp in the mitochondria, and their characterization in distinct polyglutamine disease models to account for client protein-specific differences.

Pharmacological modulation of HDAC1 and HDAC6 in vivo in a zebrafish model: Therapeutic implications for Parkinson's disease.

Histone deacetylases (HDACs) are key epigenetic enzymes and emerging drug targets in cancer and neurodegeneration. Pan-HDAC inhibitors provided neuroprotection in Parkinson's Disease (PD) models, however, the HDAC isoforms with highest neuroprotective potential remain unknown. Zebrafish larvae (powerful pharmacological testing tools bridging cellular and in vivo studies) have thus far been used in PD modelling with limited phenotypic characterization. Here we characterize the behavioural and metabolic phenotypes of a zebrafish PD model induced with MPP(+), assess the feasibility of targeting zebrafish HDAC1 and HDAC6 isoforms, and test the in vivo effects of their selective inhibitors MS-275 and tubastatin A, respectively. MPP(+) induced a concentration-dependent decrease in metabolic activity and sensorimotor reflexes, and induced locomotor impairments rescuable by the dopaminergic agonist apomorphine. Zebrafish HDAC1 and HDAC6 isoforms show high sequence identity with mammalian homologues at the deacetylase active sites, and pharmacological inhibition increased acetylation of their respective histone and tubulin targets. MS-275 and tubastatin rescued the MPP(+)-induced decrease in diencephalic tyrosine hydroxylase immunofluorescence and in whole-larvae metabolic activity, without modifying mitochondrial complex activity or biogenesis. MS-275 or tubastatin alone modulated spontaneous locomotion. When combined with MPP(+), however, neither MS-275 nor tubastatin rescued locomotor impairments, although tubastatin did ameliorate the head-reflex impairment. This study demonstrates the feasibility of pharmacologically targeting the zebrafish HDAC1 and HDAC6 isoforms, and indicates that their inhibition can rescue cellular metabolism in a PD model. Absence of improvement in locomotion, however, suggests that monotherapy with either HDAC1 or HDAC6 inhibitors is unlikely to provide strong benefits in PD. This study highlights parameters dependent on the integrity of zebrafish neuronal circuits as a valuable complement to cell-based studies. Also, the demonstrated feasibility of pharmacologically targeting HDAC1 and HDAC6 in this organism paves the way for future studies investigating HDAC inhibitors in other diseases modelled in zebrafish.