Astrocytes, the largest and most numerous glial cells in the central nervous system (CNS), play a variety of important roles in regulating homeostasis, increasing synaptic plasticity and providing neuroprotection, thus helping to maintain normal brain function. At the same time, astrocytes can participate in the inflammatory response and play a key role in the progression of neurodegenerative diseases. Reactive astrocytes are strongly induced by numerous pathological conditions in the CNS. Astrocyte reactivity is initially characterized by hypertrophy of soma and processes, triggered by different molecules. Recent studies have demonstrated that neuroinflammation and ischemia can elicit two different types of reactive astrocytes, termed A1s and A2s. However, in the case of astrocyte reactivity in different neurodegenerative diseases, the recently published research issues remain a high level of conflict and controversy. So far, we still know very little about whether and how the function or reactivity of astrocytes changes in the progression of different neurodegenerative diseases. In this review, we aimed to briefly discuss recent studies highlighting the complex contribution of astrocytes in the process of various neurodegenerative diseases, which may provide us with new prospects for the development of an excellent therapeutic target for neurodegenerative diseases.

The clustered regularly interspaced short palindromic repeat (CRISPR) gene editing technique, based on the non-homologous end-joining (NHEJ) repair pathway, has been used to generate gene knock-outs with variable sizes of small insertion/deletions with high efficiency. More precise genome editing, either the insertion or deletion of a desired fragment, can be done by combining the homology-directed-repair (HDR) pathway with CRISPR cleavage. However, HDR-mediated gene knock-in experiments are typically inefficient, and there have been no reports of successful gene knock-in with DNA fragments larger than 4 kb. Here, we describe the targeted insertion of large DNA fragments (7.4 and 5.8 kb) into the genomes of mouse embryonic stem (ES) cells and zygotes, respectively, using the CRISPR/HDR technique without NHEJ inhibitors. Our data show that CRISPR/HDR without NHEJ inhibitors can result in highly efficient gene knock-in, equivalent to CRISPR/HDR with NHEJ inhibitors. Although NHEJ is the dominant repair pathway associated with CRISPR-mediated double-strand breaks (DSBs), and biallelic gene knock-ins are common, NHEJ and biallelic gene knock-ins were not detected. Our results demonstrate that efficient targeted insertion of large DNA fragments without NHEJ inhibitors is possible, a result that should stimulate interest in understanding the mechanisms of high efficiency CRISPR targeting in general.

Restriction enzymes have two major limitations for cloning: they cannot cleave at any desired location in a DNA sequence and may not cleave uniquely within a DNA sequence. In contrast, the clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme 9 (Cas9), when coupled with single guide RNAs (sgRNA), has been used in vivo to cleave the genomes of many species at a single site, enabling generation of mutated cell lines and animals. The Cas9/sgRNA complex recognizes a 17-20 base target site, which can be of any sequence as long as it is located 5' of the protospacer adjacent motif (PAM; sequence 5'-NRG, where R = G or A). Thus, it can be programmed to cleave almost anywhere with a stringency higher than that of one cleavage in a sequence of human genome size. Here, the Cas9 enzyme and a specific sgRNA were used to linearize a 22 kb plasmid in vitro. A DNA fragment was then inserted into the linearized vector seamlessly through Gibson assembly. Our technique can be used to directly, and seamlessly, clone fragments into vectors of any size as well as to modify existing constructs where no other methods are available.

Reported is a highly chemoselective intermolecular annulation of indole-based biaryls with bromoalkyl alkynes by using palladium/norbornene (Pd/NBE) cooperative catalysis. This reaction is realized through a sequence of Catellani-type C-H alkylation, alkyne insertion, and indole dearomatization, by forming two C(sp )-C(sp ) and one C(sp )-C(sp ) bonds in a single chemical operation, thus providing a diverse range of pentacyclic molecules, containing a spiroindolenine fragment, in good yields with excellent functional-group tolerance. Preliminary mechanistic studies reveal that C-H bond cleavage is likely involved in the rate-determining step, and the indole dearomatization might take place through an olefin coordination/insertion and β-hydride elimination Heck-type pathway.