Repressing PTBP1 is incapable to convert reactive astrocytes to dopaminergic neurons in a mouse model of Parkinson’s disease

Abstract

Lineage reprograming of resident glia cells to induced dopaminergic neurons (iDAns) holds attractive prospect for cell-replacement therapy of Parkinson’s disease (PD). Recently, whether repressing polypyrimidine tract binding protein 1 (PTBP1) could truly achieve efficient astrocyte-to-iDAn conversion in substantia nigra and striatum aroused widespread controversy. Although reporter positive iDAns were observed by two groups after delivering adeno-associated virus (AAV) expressing a reporter with shRNA or Crispr-CasRx to repress astroglial PTBP1, the possibility of AAV leaking into endogenous DAns could not be excluded without using a reliable lineage tracing method. By adopting stringent lineage tracing strategy, two other studies showed that neither knockdown nor genetic deletion of quiescent astroglial PTBP1 fails to obtain iDAns under physiological condition. However, the role of reactive astrocyte might be underestimated since upon brain injury, reactive astrocyte could acquire certain stem cell hallmarks which may facilitate the lineage conversion process. Therefore, whether reactive astrocytes could be genuinely converted to DAns after PTBP1 repression in a PD model needs further validation. In this study, we used Aldh1l1-CreERT2-mediated specific astrocyte-lineage tracing method to investigate whether reactive astrocytes could be converted to DAns in the 6-OHDA PD model. However, we found that no astrocyte-originated DAn was generated after effective knockdown of astroglial PTBP1 either in the substantia nigra or in the striatum, while AAV “leakage” to nearby neurons was observed. Our results further confirmed that repressing PTBP1 is unable to convert astrocytes to DAns no matter in physiological or PD-related pathological conditions.Graphic abstractHighlightsAAV-shPtbp1 rapidly and efficiently induces viral-reporter-labeled DAns in mouse brain under physiological conditionViral-reporter-positive DAns are not originated from PTBP1 repressed and lineage traced reactive astrocytes in a mouse PD model

Ptbp1 knockdown in mouse striatum did not induce astrocyte-to-neuron conversion using HA-tagged labeling system

“…Since AtN intermediate cells might emerge earlier than 1 month, we also tracked the HA signal at 1 week and 2 weeks post-injection, in the early stages of Ptbp1 suppression. Again, no significant differences in HA+ NeuN+ cell proportions could be detected between Ptbp1 knockdown and non-target control group (Figure S1G, and S1H), which aligned well with several recent reports of no AtN conversion in transgenic mouse models 4–7 . In addition, NeuN+ cell density was not significantly changed (Figure 1G).…”

“…Aldh1l1-CreER T2 ;LSL-YFP transgenic mouse lines exhibit high specificity in astrocyte, with relatively low (4.3%) neuron mislabeling 6,11 . Indeed, three independent groups observed no AtN conversion through Ptbp1 knockdown or knockout in vivo using Aldh1l1-CreER T2 labeling systems 4,6,7 . In contrast, in another common astrocyte labeling systems using GFAP promoter, AtN conversion have been able to occur .…”

“…Recent studies have reported that shRNA-, CasRx-, or ASO-mediated Ptbp1 suppression could reprogram resident astrocytes to neurons 1-3 . However, some groups have disputed the data interpretation of the reported AtN conversion events [4][5][6][7] . These controversies surrounding AtN conversion may due to differences in the astrocyte fate-mapping systems they applied from that in the original study, i.e., recombinant mouse strains with astrocyte specific reporter constructs versus AAV-based labeling systems.…”

Ectopic insert-dependent neuronal expression of GFAP promoter-driven AAV constructs in adult mouse retina

“…This could reflect the action of cis-regulatory elements that bind transcription factors that promote neuronal-specific expression, the activity of the vector-encoded transcription factors on the GFAP minipromoter, or some combination of the two. Similar conclusions have been drawn following re-examination of claims of astrocyte-to-neuron conversion in brain (Chen et al, 2021;Hoang et al, 2021;Wang et al, 2021a), and these findings raise serious concerns about the accuracy of previously reported claims of AAV-mediated Müller glia-to-neuron conversion (Yao et al, 2018;Zhou et al, 2020;Xiao et al, 2021).…”

“…First, while studies reporting Müller glia-to-neuron conversion using transgenic mice have used well-characterized inducible transgenic Cre lines to infer cell lineage relationships (Jorstad et al, 2017; Hoang et al, 2020), AAV-based studies have used GFAP mini promoter-driven Cre constructs for this purpose. Second, the GFAP minipromoter, which is used to drive glial-specific expression of constructs used for reprogramming, can show leaky neuronal expression in brain (Taschenberger et al, 2017; Griffin et al, 2019), most notably when overexpressing Neurod1 or disrupting Ptbp1 expression in brain astrocytes (Chen et al, 2021; Wang et al, 2021a), both of which have been claimed to induce astrocyte-to-neuron conversion (Puls et al, 2020; Qian et al, 2020; Zhou et al, 2020; Xiang et al, 2021). This raises the question of whether reports of AAV-based Müller glia-to-neuron conversion actually represent cases of ectopic expression of GFAP reporter constructs in endogenous neurons (Blackshaw and Sanes, 2021; Qian et al, 2021).…”