Hippocampal single-cell RNA Atlas of chronic methamphetamine abuse-induced cognitive decline in mice

Reviewer #1 (Public review):

Summary:

This manuscript focuses on single-cell RNA sequencing (scRNA-seq) analysis following chronic methamphetamine (METH) treatment in mice. The authors propose two hypotheses:

(1) METH induces neuroinflammation involving T and NKT cells, and (2) METH alters neuronal stem cell differentiation.

Strengths:

The authors provide a substantial dataset with numerous replicates, offering valuable resources to the research community.

Weaknesses:

Concerns persist regarding the interpretation of data and the validation of experiments. First, the presence of T cells, NKT cells, and neutrophils in both the control and METH-treated hippocampi suggests that blood contamination rather than immune cell infiltration is the cause. Since the authors claim that METH disrupts the blood-brain barrier, increasing the infiltration of these immune cells, identifying the source of these immune cells is critical.

Secondly, the pseudotime analysis, which suggests altered neural stem cell (NSC) differentiation, is not conclusively supported by the current data and requires further validation.

Overall, the authors provided comprehensive in vivo data on the impact of methamphetamine on the hippocampus; however, further in vivo and in vitro experimental validation of the key findings is needed.

Reviewer #2 (Public review):

Summary:

Chronic methamphetamine (METH) abuse leads to significant structural and functional deficits in the cortical and hippocampal regions in humans. However, the specific mechanisms underlying chronic METH-induced neurotoxicity in the hippocampus and its contribution to cognitive deficits remain poorly understood. The authors aim to address this knowledge gap using a single-cell transcriptomic atlas of the hippocampus under chronic METH exposure in mice. They present analyses of differential gene expression, cell-cell communication, pseudotemporal trajectories, and transcription factor regulation to characterize the cellular-level impact of METH abuse. However, the overall quality of the manuscript is currently very poor due to a lack of basic quality control, overly descriptive content, and unclear conclusions.

Strengths:

The major strength of this study is that it may represent the first report on the impact of METH on the hippocampus in mice. However, the authors should clarify whether similar studies have been previously conducted, as this point remains uncertain.

Weaknesses:

Despite this potential novelty, the study has numerous weaknesses. Notably, single-cell RNA sequencing was unable to capture an adequate number of neuronal populations. Neurons accounted for only approximately 0.6% of the total nuclei, representing a significant underrepresentation compared to their actual physiological proportion. Given that the behavioral effects of METH are likely mediated by neuronal dysfunction, readers would reasonably expect to see transcriptional changes in neurons. The authors should explain why they were unable to capture a sufficient number of neurons and justify how this incomplete dataset can still provide meaningful scientific insights for researchers studying METH-induced hippocampal damage and behavioral alterations.

Another significant weakness of this study is the lack of a cohesive hypothesis or overarching conclusion regarding how METH impacts neural populations. The authors provide a largely descriptive account of transcriptional alterations across various cell types, but the manuscript lacks clear, biologically meaningful conclusions. This descriptive approach makes it difficult for readers to identify the key findings or take-home messages. To improve clarity and impact, the authors should focus on developing and presenting a few plausible hypotheses or mechanistic scenarios regarding METH-induced neurotoxicity, grounded in their scRNA-seq data. Including schematic figures to illustrate these hypotheses would also help readers better understand and interpret the study.

The final major weakness of this study is its poor readability. It appears that the authors did not adequately proofread the manuscript, as there are numerous typographical errors (e.g., line 333: trisulting; line 756: essencial), unsupported scientific claims lacking citations (e.g., lines 485, 503, 749-753), and grammatically incorrect sentences (e.g., lines 470-472, 540-543, 749-753). In addition, many paragraphs are unorganized and overly descriptive, which further hinders clarity. Some figures are also problematic - too small in size and overcrowded with text in fonts that are difficult to read. It is recommended that the authors carry out quality control. There are too many typographical and grammatical errors to list individually; the authors should carefully review and revise the entire manuscript to address all of these issues.

Overall, this study could have offered some incremental new insights into neurotoxicity following chronic METH exposure, despite the poor capture of neuronal populations. However, the current manuscript feels more like a data dump than a thoughtfully constructed scientific narrative. I encourage the authors to extract and highlight meaningful biological insights from their dataset and clearly articulate these in the conclusion, ideally supported by an additional schematic figure. Furthermore, I strongly urge the authors to substantially improve the basic quality of the manuscript through careful proofreading and by seeking feedback from colleagues or other readers.

Reviewer #3 (Public review):

Summary:

This study aimed to elucidate the intricate mechanisms underlying cognitive decline induced by chronic METH abuse, focusing on the hippocampus at a single-cell resolution. The authors established a robust mouse model of chronic METH exposure. They observed significant impairments in working memory, spatial cognition, learning, and cognitive memory through Y-maze and novel object recognition tests. To gain deeper insights into the cellular and molecular changes, they utilized single-cell RNA sequencing to profile hippocampal cells. They performed extensive bioinformatics analyses, including cell clustering, differential gene expression, cellular communication, pseudotemporal trajectory, and transcription factor regulation.

Strengths:

(1) The authors performed a comprehensive suite of bioinformatics analyses, including differential gene expression, cellular cross-talk, pseudotime trajectory, and SCENIC analysis, which enable a multifaceted exploration of METH-induced changes at both the cellular and molecular levels.

(2) The study demonstrates an awareness of the potential influence of circadian rhythms, dedicating a specific section in the discussion to the disruption of circadian rhythms, which has rarely been mentioned in previous studies on METH. They highlight the frequent occurrence of circadian regulation in their analysis across several cell types.

(3) The pseudotime analysis provides valuable insights into hindered neurogenesis, showing a shift in NSC differentiation toward astrocytes rather than neuroblasts in METH-treated mice. The detailed analysis of BBB components (endothelial cells, mural cells, SMCs) and their heterogeneous responses to METH is also a significant contribution.

Weaknesses:

(1) While the bioinformatics analyses are extensive, the study is primarily descriptive at the molecular level. The absence of experimental validation, such as targeted mRNA/protein quantification and gene knockdown/overexpression to confirm the causal relationship between these identified genes and METH-induced cognitive deficits, is a notable limitation.

(2) While the discussion extensively covers the functional implications of specific molecular pathways and cell types, it would greatly benefit from a comparison of these findings with existing RNA sequencing data from other METH models in hippocampal tissue.

(3) The conclusion that "prolonged METH use may progressively impair cognitive function" may not be uniformly supported by the behavioral data: Figures 1C and F (discrimination and preference indexes) exhibited that the 4-week test further declined in the METH group compared to the 2-week. In contrast, Figure 1E and H present a contradictory pattern.