ABSTRACT
Virus-Induced Gene Silencing (VIGS) is a versatile tool in plant science, yet its application to non-model species like sunflower demands extensive optimization due to transformation challenges. In this study, we aimed to elucidate the factors that significantly affect the efficiency of Agrobacterium-VIGS in sunflowers. After testing a number of approaches, we concluded that the seed vacuum technique followed by 6 h of co-cultivation produced the most efficient VIGS results. Genotype-dependency analysis revealed varying infection percentages (62-91%) and silencing symptom spreading in different sunflower genotypes. Additionally, we explored the mobility of tobacco rattle virus (TRV) and phenotypic silencing manifestation (photo-bleaching) across different tissues and regions of VIGS-infected sunflower plants. We showed the presence of TRV is not necessarily limited to tissues with observable silencing events. Finally, time-lapse observation demonstrated a more active spreading of the photo-bleached spots in young tissues compared to mature ones. This study not only offers a robust VIGS protocol for sunflowers but also provides valuable insights into genotype-dependent responses and the dynamic nature of silencing events, shedding light on TRV mobility across different plant tissues.
ABSTRACT
BACKGROUND: Sleep-wake dysfunction is an early and common event in Alzheimer's disease (AD). The lateral hypothalamic area (LHA) regulates the sleep and wake cycle through wake-promoting orexinergic neurons (OrxN) and sleep-promoting melanin-concentrating hormone or MCHergic neurons (MCHN). These neurons share close anatomical proximity with functional reciprocity. This study investigated LHA OrxN and MCHN loss patterns in AD individuals. Understanding the degeneration pattern of these neurons will be instrumental in designing potential therapeutics to slow down the disease progression and remediate the sleep-wake dysfunction in AD. METHODS: Postmortem human brain tissue from donors with AD (across progressive stages) and controls were examined using unbiased stereology. Formalin-fixed, celloidin-embedded hypothalamic sections were stained with Orx-A/MCH, p-tau (CP13), and counterstained with gallocyanin. Orx or MCH-positive neurons with or without CP13 inclusions and gallocyanin-stained neurons were considered for stereology counting. Additionally, we extracted RNA from the LHA using conventional techniques. We used customized Neuropathology and Glia nCounter (Nanostring) panels to study gene expression. Wald statistical test was used to compare the groups, and the genes were considered differentially expressed when the p-value was <.05. RESULTS: We observed a progressive decline in OrxN alongside a relative preservation of MCHN. OrxN decreased by 58% (p=0.03) by Braak stages (BB) 1-2 and further declined to 81% (p=0.03) by BB 5-6. Conversely, MCHN demonstrated a non-statistical significant decline (27%, p=0.1088) by BB 6. We observed a progressive increase in differentially expressed genes (DEGs), starting with glial profile changes in BB2. While OrxN loss was observed, Orx-related genes showed upregulation in BB 3-4 compared to BB 0-1. GO and KEGG terms related to neuroinflammatory pathways were mainly enriched. CONCLUSIONS: To date, OrxN loss in the LHA represents the first neuronal population to die preceding the loss of LC neurons. Conversely, MCHN shows resilience to AD p-tau accumulation across Braak stages. The initial loss of OrxN correlates with specific neuroinflammation, glial profile changes, and an overexpression of HCRT, possibly due to hyperexcitation following compensation mechanisms. Interventions preventing OrxN loss and inhibiting p-tau accumulation in the LHA could prevent neuronal loss in AD and, perhaps, the progression of the disease.