Molecular dissection of pericyte-to-neuron reprogramming reveals cellular identity safeguarding mechanisms

Neurodegenerative diseases, strokes, and injuries affect millions of people worldwide and current treatment options are insufficient. Since death of neurons in the brain is a common feature of all these disorders, a potential therapy could replace the lost neurons by newly generated ones to restore brain function. Natural adult neurogenesis in humans has been proven inadequate to deal with a major loss of brain cells. Therefore, for many years, transplantation of fetal tissue or stem cell-derived neural progenitors have been the focus of investigations regarding new treatments. More recently, new methods and insights have rendered brain-resident cells a promising means of an alternative therapeutic approach. While cellular identity was believed to be irreversible once differentiated for a long time, this view has changed gradually over the last decades. Among other cells, it has been shown for human brain pericytes that retroviral expression of the transcription factors (TFs) Ascl1 and Sox2 (AS) is sufficient to generate functional induced neurons (iNs) by direct reprogramming, and that this process is accompanied by a neural stem cell (NSC)-like state. While it is clear now that even a terminal cellular identity can be changed, the exact mechanisms remain elusive. Therefore, in this study we aimed at (i) identifying barriers and molecular mechanisms involved in cellular identity conversion from somatic cells into induced neurons, (ii) improving the efficiency of pericyte-to-neuron reprogramming, and (iii) directing the reprogramming process towards the desired cell types. By single cell RNA sequencing, we generated a high-resolution dataset of cells during pericyte-to-iN conversion. Using RNA velocity analysis, we were able to predict the progression of cells towards the neuronal fate and could identify blocker and facilitator genes that obstruct or enable cells to pass past a designated decision point. Among the facilitator genes, we identified several chromatin remodelers and cytoskeleton genes, and revealed a temporal heterogeneity regarding their expression pattern. Interestingly, we show that the blocker genes are part of a cellular identity safeguarding mechanism triggered by AS reprogramming. We demonstrate that the metabolic transition from glycolysis to oxidative phosphorylation is an essential barrier cells must overcome to transit from a pericyte towards a neuronal identity. Our findings suggest that any failure to meet metabolic requirements results in cells being either unable to change their identity or adopting a confused fate. To impact on the NSC-like state, we used either modulation of NOTCH signaling or TGF-β signaling by inhibition of the γ-secretase or dual SMAD inhibition, respectively, via small molecules. Strikingly, both treatments counteracted pericyte identity safeguarding mechanisms and significantly lowered reprogramming barriers. Consequently, our results show a strong increase in the number of generated iNs. Interestingly, we demonstrate that TGF-β signaling inhibition is more potent in lowering these metabolic barriers than NOTCH signaling inhibition, re-routing cells onto an entirely different route towards neurons. Additionally, TGF-β signaling inhibition almost completely suppresses the generation of undesired off-target cells without a clear identity, likely due to antioxidant regulon activity, which supports the metabolic transition. Remarkably, we illustrate that despite different treatments, iNs are transcriptionally similar and that both neuronal subtypes can be mapped to developing human brain regions. Finally, we used a different approach and reprogrammed pericytes into TUBB3+ cells using Neurog2/Sox2 (NS). We show that NS generated cells have a distinct transcriptomic identity from AS generated ones: While they are more likely to lose their original identity, the NS-generated iNs exhibit more progenitor-like properties, pointing at the different reprogramming capacities of proneural TFs. Altogether, this thesis emphasizes not only that cellular identity even in terminally differentiated cells can still be altered without returning to a pluripotent state. It further illustrates several previously unknown mechanisms during direct pericyte-to-iN reprogramming and opens new ways to improve its efficiency. Every new insight into cross-lineage cellular identity conversion paves the way for future neuronal replacement therapies

TLR5 decoy receptor as a novel anti-amyloid therapeutic for Alzheimer\u27s disease.

There is considerable interest in harnessing innate immunity to treat Alzheimer\u27s disease (AD). Here, we explore whether a decoy receptor strategy using the ectodomain of select TLRs has therapeutic potential in AD. AAV-mediated expression of human TLR5 ectodomain (sTLR5) alone or fused to human IgG4 Fc (sTLR5Fc) results in robust attenuation of amyloid β (Aβ) accumulation in a mouse model of Alzheimer-type Aβ pathology. sTLR5Fc binds to oligomeric and fibrillar Aβ with high affinity, forms complexes with Aβ, and blocks Aβ toxicity. Oligomeric and fibrillar Aβ modulates flagellin-mediated activation of human TLR5 but does not, by itself, activate TLR5 signaling. Genetic analysis shows that rare protein coding variants in human TLR5 may be associated with a reduced risk of AD. Further, transcriptome analysis shows altered TLR gene expression in human AD. Collectively, our data suggest that TLR5 decoy receptor-based biologics represent a novel and safe Aβ-selective class of biotherapy in AD

Results of Anatomic Lateral Ankle Ligament Reconstruction with Tendon Allograft

Chronic ankle instability can be addressed surgically through direct lateral ligament repair, non-anatomic reconstruction, or anatomic reconstruction. The goal of this study was to assess the radiographic, functional, and clinical results of patients undergoing an anatomic lateral ankle ligament reconstruction using an anterior tibial tendon allograft. Eleven patients (12 feet; mean age, 48.9 ± 11.4 years) undergoing lateral ankle ligament reconstruction were followed at a mean of 3.5 ± 1.7 years after surgery (range, 1.2 to 5.0 years). Indications for surgery were previous failed repair (i.e., Broström; one case), hyperlaxity (seven cases), and high-demand patients (four cases). Subjective outcomes including the Foot and Ankle Outcome Score (FAOS), SF-36, and activity level were assessed. Mortise and lateral ankle stress radiographs were performed. The FAOS daily activity and sports activity subscores were 93.4 (range, 77.9 to 100) and 78.6 (range, 30 to 100), respectively. The SF-36v2 physical health and mental health components were 50.4 (range, 30.6 to 65.7) and 45.0 (range, 24.8 to 68.0), respectively. Four patients (five feet) reported no restriction; six patients reported mild restrictions, and one patient reported moderate activity restrictions. Tibiotalar tilt improved significantly from 20.2° to 4.6° after surgery (p < 0.01). The radiographic anterior displacement of the talus from the tibia was 6.5 mm postoperatively. The technique described restores mechanical stability in patients with chronic lateral ankle instability and may be considered in a select group of patients