Biology subjects

Pereira de Almeida, L.

Publications and source records attributed to Pereira de Almeida, L..

5 recordsLinked to original sources

bioRxiv2023

Nuclear aging in polyglutamine-induced neurodegeneration

Machado-Joseph disease (MJD) is an autosomal dominantly-inherited neurodegenerative disorder characterized by an over-repetition of the CAG trinucleotide of the ATXN3 gene, conferring a toxic gain-of-function to the resulting ataxin-3 protein. Despite the significant advances produced over the last years, the molecular mechanisms involved in MJD are still unclear and no treatment able to modify the disease progression is available. Aging is the major risk factor for neurodegenerative disorders, being associated with the occurrence and progression of several diseases, such as Alzheimers, Huntingtons, among others. The nuclear membrane proteins - lamins - and lamin-processing related proteins, such as ZMPSTE24, have been shown to be altered, not only during normal aging, but also in neurodegenerative disorders, such as Alzheimers disease. Taking this into account, we aimed at investigating the role of aging in MJD by evaluating the presence of age-related markers in human and animal MJD models. Decreased levels of lamins B and C, together with decreased ZMPSTE24 levels were identified in the different MJD models. Accordingly, abnormalities in nuclear circularity, a hallmark of aging, were also observed in a N2a MJD cellular model, supporting an age-related phenotype. Furthermore, overexpressing progerin, the abnormal lamin A, generated in Hutchinson Guilford Progeria Syndrome patients that present premature and accelerated aging, in a relevant brain area of a lentiviral MJD mouse model, induced an aggravation of MJD-associated neuropathology. Our results suggest that aging is a key player in the context of MJD pathogenesis, unveiling new pathways for the development of future therapies for the disease.

neuroscience↗

bioRxiv2023

Isolation of Biologically Active Extracellular Vesicles-Associated AAVs for Gene Delivery to the Brain by Size Exclusion Chromatography

Extracellular vesicles-associated adeno-associated viral vectors (EV-AAVs) emerged as a new opportunity for non-invasive gene therapy targeting the central nervous system (CNS). However, in previous reports, only AAV serotypes with known ability to cross the blood-brain barrier (BBB) have been used for EV-AAV production and testing through non-invasive strategies. In this work, we aimed at optimizing a size exclusion chromatography (SEC) protocol for the production and isolation of natural and biologically active brain-targeting EV-AAVs, that could be applied to any AAV serotype and further used for non-invasive gene delivery to the CNS. We performed a comparison between SEC and differential ultracentrifugation (UC) isolation protocols in terms of yield, contaminants, and transgene expression efficiency. We found that SEC allows a higher recovery of EV-AAVs, free of cell contaminating proteins and with less solo AAVs than UC. Remarkably, SEC-purified EV-AAVs also showed to be more potent at transgene expression than solo AAVs in neuronal cell lines. EV-AAVs exhibited the ability to cross the BBB in neonatal mice upon intravenous administration. In conclusion, SEC-purified brain-targeting EV-AAVs show to be a promising gene delivery vector for therapy of brain disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/542901v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@7016b8org.highwire.dtl.DTLVardef@e3a5f3org.highwire.dtl.DTLVardef@1a43286org.highwire.dtl.DTLVardef@f110c5_HPS_FORMAT_FIGEXP M_FIG C_FIG During the production of AAV vectors, a small percentage of AAVs is secreted in association with extracellular vesicles, named "EV-AAVs". EV-AAVs can be efficiently isolated by size exclusion chromatography (SEC). When intravenously injected in mice, brain targeting EV-AAVs can cross the blood brain barrier (BBB) and transduce neuronal cells.

molecular biology↗

bioRxiv2023

Ghrelin delays premature aging in Hutchinson-Gilford progeria syndrome

Hutchinson-Gilford progeria syndrome (HGPS) is a rare and fatal genetic condition arising from a single nucleotide alteration in the LMNA gene, which leads to the production of a defective lamin A protein known as progerin. The buildup of progerin hastens the onset of premature and expedited aging. Patients with HGPS exhibit short stature, low body weight, lipodystrophy, metabolic dysfunction, and skin and musculoskeletal abnormalities and, in most cases, die of cardiovascular disease by their early teenage years. Currently, no effective cure or treatment for the disease highlights the importance of discovering new therapeutic strategies. Herein, we present evidence that the hormone ghrelin, besides promoting autophagy and progerin clearance, rescued several cellular hallmarks of premature aging of human HGPS fibroblasts. Using an HGPS mouse model, LmnaG609G/G609G mice, we also show that ghrelin administration rescued the short-lived mice molecular and histopathological progeroid features, prevented progressive weight loss at later stages, reverted the lipodystrophic phenotype, and extended lifespan. Thus, we disclose that modulation of ghrelin signaling may give rise to new treatment targets and translational approaches that may improve outcomes and the health quality of HGPS patients and natural aging pathologies.

physiology↗

bioRxiv2023

Blood and cerebellar abundance of ATXN3 splice variants in spinocerebellar ataxia type 3/Machado-Joseph disease

BackgroundSpinocerebellar ataxia type 3 (SCA3)/Machado-Joseph disease (MJD) is an autosomal dominant polyglutamine disease. SCA3/MJD causative gene, ATXN3, is known to undergo alternative splicing (AS) and 54 transcripts are currently annotated. Differences in the toxicity of ataxin-3 protein isoforms, harbouring on its C-terminus two or three ubiquitin interacting motifs (UIMs), were previously uncovered, raising the hypothesis that specific ATXN3 splice variants play key roles in promoting the selective toxicity displayed in SCA3/MJD. MethodsUsing RNA-seq datasets we identified and determined the abundance of annotated ATXN3 transcripts in blood (n=60) and cerebellum (n=12) of SCA3/MJD subjects and controls. ResultsGlobally, the number and the abundance of individual ATXN3 transcripts were higher in the cerebellum than in the blood. While the most abundant transcript in the cerebellum was a protein with a coding sequence not defined of unknown function (ATXN3-208), the transcript with the highest abundance in blood was the reference transcript (ATXN3-251) which translates into an ataxin-3 isoform harboring three UIMs. Noteworthy, the abundance of ATXN3-251 and ATXN3-214, two out of the four transcripts that encode full-length ataxin-3 protein isoforms but differ in the C-terminus were strongly related with tissue expression specificity: ATXN3-251 (3UIM) was expressed in blood 50-fold more than in cerebellum, whereas ATXN3-214 (2UIM) was expressed in the cerebellum 20-fold more than in blood. ConclusionsThese findings provide new insights into the elucidation of ATXN3 AS in different tissues, contributing for a better understanding of SCA3/MJD pathogenesis and providing information for the development of future effective ATXN3 mRNA-lowering therapies.

genetics↗

bioRxiv2023

Extracellular communication between brain cells through functional transfer of Cre mRNA

1In the central nervous system (CNS), the crosstalk between neural cells is mediated by extracellular mechanisms, including brain-derived extracellular vesicles (bdEVs). To study endogenous communication across the brain and periphery, we explored Cre-mediated DNA recombination to permanently record the functional uptake of bdEVs cargo overtime. To elucidate functional cargo transfer within the brain at physiological levels, we promoted the continuous secretion of physiological levels of neural bdEVs containing Cre mRNA from a localized region in the brain by in situ lentiviral transduction of the striatum of Flox-tdTomato Ai9 mice reporter of Cre activity. Our approach efficiently detected in vivo transfer of functional events mediated by physiological levels of endogenous bdEVs throughout the brain. Remarkably, a spatial gradient of persistent tdTomato expression was observed along the whole brain exhibiting an increment of more than 10-fold over 4 months. Moreover, bdEVs containing Cre mRNA were detected in the bloodstream and extracted from brain tissue to further confirm their functional delivery of Cre mRNA in a novel and highly sensitive Nanoluc reporter system. Overall, we report a sensitive method to track bdEVs transfer at physiological levels which will shed light on the role of bdEVs in neural communication within the brain and beyond.

cell biology↗