In this study, compared with those in control astrocytes, the proportions of iNs generated from NP-mediated astrocytes in the D4-OE and D7-OE groups were 36% and 39.3%, respectively. Dynamic gene expression during the conversion process was described, and it was found that the expression of certain neuronal markers (Map2, Cux1) gradually increased during the conversion. Through pseudotemporal heatmaps, we observed that during astrocyte-to-iN conversion, a set of genes were simultaneously activated in D4-OE cells, participated in glial cell differentiation and neurotransmitter uptake. On the other hand, in D7-OE, another set of genes was simultaneously activated, involving pathways such as forebrain development, telencephalon development, hindbrain development and centrosome duplication. By constructing a weighted coexpression network, we determined the major molecular features of iNs during D4-OE and D7-OE. On D4-OE, genes related to glial cell differentiation, regulation of cell growth, and regulation of neurogenesis were associated with iNs. Moreover, in D7-OE, genes related to the positive regulation of nervous system development, regulation of neurogenesis, positive regulation of neurogenesis, axon guidance, and neuron projection guidance were associated with iNs. These results revealed the molecular characteristics and regulatory mechanisms underlying the astrocyte-to-iN conversion.

Nng2 is essential for the glutamatergic neurotransmitter phenotype in the embryonic neocortex and effectively converts astrocytes into glutamatergic neurons, underscoring its role in excitatory neurogenesis. Additionally, Ngn2 induces both excitatory and inhibitory neurons in the spinal cord [22]. Through the CRISPRa system, the activation of Ngn2 and Isl1 can convert astrocytes into functional motor neurons [23]. Furthermore, when Ngn2 combines with Nurr1, it can induce the formation of dopaminergic neurons [24]. As a key regulator of neurogenesis, Pax6 serves as an intrinsic determinant of the neurogenic potential of glial cells [13]. Pax6 transduction in proliferative glial cells induces the expression of early neuronal markers [25].

This study employed the CytoTRACE method, a novel approach to delineate the developmental trajectory and pinpoint the initiation points of astrocytes as they undergo reprogramming into iNs. Our findings in the D4-OE and D7-OE groups revealed that astrocytes with elevated CytoTRACE scores are less differentiated, supporting the premise that these cells are poised at the onset of their cellular developmental pathway. Genes associated with cell differentiation processes, such as H3f3b, Cdkn1a, and Prdx5, were identified. H3f3b, the gene encoding histone H3.3, is crucial for establishing the identity of mitotic neurons after key developmental stages [26]. CDKN1A/p21, a cyclin-dependent kinase inhibitor, plays a pivotal role in the suppression of ferroptosis through its induction by p53 [27]. This is particularly relevant in the context of neuronal reprogramming, where the generation of reactive oxygen species (ROS) has been reported to impede the reprogramming process [28].

Additionally, PRDX5, an antioxidant protein, is associated with aberrant ROS [29]. Given that oxidative stress-induced ferroptosis can impose limitations on neuronal reprogramming [30], the potential of PRDX5 as a key mediator in safeguarding cells against ROS, particularly those originating from mitochondrial sources, is highlighted [31]. These insights collectively enhance our understanding of the molecular dynamics during the reprogramming of astrocytes into iNs and offer avenues for developing targeted strategies to optimize this process.

The reprogramming of astrocytes into iNs is a complex and intricate process that involves a delicate interplay of transcription factors and cellular markers, which are pivotal in determining cell fate. Following the overexpression of NP in astrocytes, we observed a rapid downregulation of astrocytic genes in both the D4-OE and D7-OE groups. The rapid transcriptomic changes observed after overexpressing neural transcription factors are consistent with previous reports on NeuroD1, Ascl1, or Ngn2 [10, 32,33,34]. The observed downregulation of astrocytic markers such as S100a10, Aqp4, and Gfap, coupled with the upregulation of neuronal markers such as Sox2, Map2, and Crym, underscores the phenotypic metamorphosis that astrocytes undergo as they are reprogrammed into iNs. As revealed by our Monocle 2 analysis, the temporal dynamics of gene expression provided deeper insights into the molecular timeline of the reprogramming process from astrocytes to iNs. There was early upregulation of Ascl1, Cux1, Map2, and Tubb3 in the D4-OE group, with increased expression of Map2 and Cux1 in the D7-OE group during the transformation process. Previous studies have shown that the direct reprogramming of fibroblasts into neurons involves two stages: an initiation stage in which Ascl1 induces neuronal fate, and a maturation stage in which reprogrammed fibroblasts permanently acquire a neuronal identity [12]. This may also hold true for astrocytes, as Ascl1 expression increases and then decreases during the process of astrocyte-to-iN differentiation. In the initial stage, Ascl1 expression increases, and Ascl1 functions as a core driver of reprogramming [10], with its overexpression capable of inducing rapid neuronal differentiation [35]. Map2, which is predominantly expressed in neurons, is integral to axonal and dendritic growth, synaptic plasticity, and overall neuronal development [36]. Cux1 promotes the integration of layer II–III neurons in the cortical network in a highly specific manner [37]. The orchestrated modulation of astrocytic and neuronal markers during the reprogramming of astrocytes into iNs highlights the complexity of cell fate determination. Understanding the molecular choreography that drives this process is vital for harnessing the therapeutic potential of cellular reprogramming in neurological applications.

The pseudotemporal heatmaps allowed us to dissect the temporal dynamics of this transition, revealing significant insights into the genes and pathways involved. In the D4-OE stage, the activation of genes such as Prdx1 and Slc1a3 is noteworthy. Antioxidant proteins play a crucial role in maintaining body homeostasis. PRDX1 possesses an antioxidant function and is capable of clearing ROS within the body [38]. It interacts with various kinases and enzymes and can prevent cell apoptosis caused by oxidative stress [39]. Its upregulation during the astrocyte-to-iN transition suggests a potential protective mechanism against oxidative stress, which is known to be a critical factor in neurogenesis [40]. Glutamate levels are regulated by SLC1A3, which reduces excitotoxicity and helps to prevent damage to the nervous system caused by excitotoxicity [41]. The functional enrichment analysis revealed that these activated genes are predominantly involved in pathways associated with glial cell differentiation, neuron apoptotic process, response to oxidative stress and neurotransmitter uptake. Moreover, direct neuronal reprogramming to obtain neurons is a process that can induce oxidative stress [42], which necessitates an appropriate response to oxidative stress to maintain cellular homeostasis and ensure the successful differentiation of reprogrammed cells.

In the subsequent D7-OE stage, genes such as Fth1, Tmsb4x, Hpca, Cadm3, and Gm42418 were upregulated. TMSB4X plays a crucial role in the transdifferentiation of human fibroblasts into myogenic cells, enhancing the efficiency of the process [43]. The expression level of HPCA increases during neuronal differentiation, and overexpression of HPCA enhances neuronal differentiation [44]. CADM3 mediates direct contact and interaction between axons and glial cells [47].

The hdWGCNA was used to identify gene modules and hub genes that play a central role in the reprogramming process. GO enrichment analysis of the hub genes in the D7-OE group revealed functions that are essential for the development and maturation of the nervous system, including positive regulation of nervous system development, regulation of neurogenesis, axon guidance, and neuron projection guidance. Neurons undergo processes such as neuronal migration, axonal elongation, axon pruning, dendritic morphogenesis, synaptic maturation, and plasticity to form neural circuits [45]. Moreover, KEGG pathway enrichment analysis revealed pathways that are implicated in neurodegenerative diseases, such as ferroptosis, and other pathways related to the JAK–STAT and PI3K–Akt signaling pathways. The JAK–STAT3 pathway is not only recognized as the core signaling pathway for maintaining pluripotency but also has recently been shown to be essential for the complete reprogramming of mouse somatic cells [46]. The dedifferentiation of astrocytes into an undifferentiated state is induced through the activation of the PI3K/Akt/p21 signaling pathway, thereby promoting their transdifferentiation into neurons [48].

There are limitations to this study. Our research is based on in vitro experiments, and the results need to be validated in vivo to ensure their reliability and reproducibility. The specific mechanisms and processes of transformation still require further investigation. There may be unknown factors and obstacles during the cellular conversion process, which could affect the efficiency of conversion and the stability of cell characteristics. Our study revealed the potential of the NP to reprogram astrocytes into iNs, explored the molecular features and regulatory mechanisms of astrocyte-to-iN conversion.