Mid1 deletion leads to cognitive dysfunction in Opitz syndrome by regulates neural rhythms through the inhibition of p-Creb by PP2Ac

Reviewer #1 (Public review):

Summary:

The authors demonstrated that a mouse model of Opitz syndrome induced by Mid1 gene knockout exhibited a significant decrease in α rhythm in HPC and abnormal synchronization of γ rhythm in the prefrontal cortex and hippocampus, showing decreased synaptic plasticity and learning and memory dysfunction. All these effects were attributed to the inhibition of p Creb by PP2Ac.

Strengths:

The authors used Mid1 gene knockout mice as a mouse model of Opitz syndrome. They carried out RNA seq analysis and found cAMP signaling pathway, calcium signaling pathway, and 100 other pathways have changed significantly.

Weaknesses:

(1) A Mid1 supplementation experiment in Mid1 knockout mice was lacking in this study.

(2) Enzymes that regulate Creb phosphorylation include not only phosphatases such as PP2A, but also kinases such as CaMKII, PKA, and ERK1/2. These protein kinases should be detected, especially CaMKII, their bioinformatics data show calcium signaling pathways have significantly changed.

Reviewer #2 (Public review):

Summary:

The manuscript investigates the role of the Mid1 gene in hippocampal (HPC) development and its contribution to Opitz G/BBB syndrome (OS), which is characterized by neurological deficits and structural abnormalities. The authors use a knockout mouse model (Mid1-/y) to elucidate the underlying molecular mechanisms that contribute to learning and memory impairments. They demonstrate that Mid1 gene deletion leads to reduced synaptic plasticity, abnormal neural rhythms, and decreased cognitive functions, providing a mechanistic explanation for the neurological deficits seen in OS patients. This study addresses an important gap in understanding the neural mechanisms underlying Opitz G/BBB syndrome and provides substantial evidence that the Mid1 gene plays a critical role in hippocampal function and cognition.

Strengths:

Understanding the role of Mid1 in HPC development could have broader implications for neurodevelopmental disorders beyond OS, particularly in conditions associated with synaptic dysfunction or memory impairments. The study's focus on the impact of Mid1 on the cAMP signaling pathway, BDNF expression, and synaptic plasticity offers novel mechanisms relevant to both neurodevelopment and neurodegeneration. Moreover, the combination of RNA-seq, electrophysiological measurements, and histological staining provides a multidimensional approach to understanding how Mid1 influences neuronal function and structure.

Weaknesses:

(1) The introduction is insufficient, and the number of references is too low. With only nine references, there isn't enough context to adequately explain the background and previous evidence.

(2) The specificity of behavioral deficits is lacking. The authors indicate learning and memory dysfunction, yet the Y-maze and Morris water maze primarily assess spatial memory. Additional behavioral tests, such as the novel object recognition test for recognition memory or fear conditioning for associative learning, should be included to provide a more comprehensive assessment.

(3) The manuscript mentions decreased synaptic plasticity but lacks thorough investigation; a more detailed analysis of long-term potentiation (LTP) or depression (LTD) would strengthen the claims. Additionally, while spine morphology is analyzed, incorporating electrophysiological measurements of synaptic strength would better correlate structural changes with functional outcomes.

(4) The authors performed H&E staining to count the number of hippocampal pyramidal neurons; however, H&E lacks specificity for identifying pyramidal neurons. Neuronal-specific IHC staining would be more appropriate for this quantification. Additionally, the manuscript does not mention the counting method used, which should be clarified.

(5) Information on the knockout mice used in the study is missing from the Methods section. Additionally, the sex of the mice should be specified, as exploring potential sex-specific differences in the impact of Mid1 deletion could significantly enhance the study's findings.

Reviewer #3 (Public review):

Summary:

The authors tried to characterize the neuronal deficiency in Mid1 knockout mice. They performed behavioral, neuroelectrophysiological, and pathological experiments to show that Mid1 knockout mice have cognitive function, impaired synaptic plasticity, and changes in gene expression.

Strengths:

The evidence provides insight into the mechanisms of cognitive impairments in Opitz syndrome. Overall, the manuscript is well-organized.

Weaknesses:

(1) The major weakness is that the proposed molecular mechanism is not fully supported by the current data. The data presented here only show that changes in gene expression levels, cognitive impairments, and electrophysiological impairments are correlated with each other, but do not support causality.

(2) The main conclusion is that "The main reason is that the deletion of Mid1 gene will increase the accumulation of Pp2ac protein, inhibit the activity of p-Creb, affect the downstream cAMP pathway, lead to the decrease of synaptic density and plasticity, and ultimately affect the learning and memory ability". This should be toned down, since causality is not supported here.

(3) The description of the results should be improved. Only one figure is presented in the manuscript. Some key information in the supplementary figures should be moved to the main figures. This is very strange since four display items are allowed even for a short report.

Author response:

First of all, I'd like to express my heartfelt thanks to you for your meticulous and professional review comments. Your feedback is very important to our work. It not only helps us identify the shortcomings in the paper, but also provides valuable guidance for improving the quality of the paper.

We carefully read every suggestion you made and were deeply inspired. Please rest assured that we will carefully consider and revise each opinion to ensure that our research work is more rigorous and clear. We promise to revise the manuscript accordingly to meet the standards of the journal and enhance the credibility and influence of the research.

The main modifications include the experiment of A Mid1 supplementation experiment in Mid1 knockout micesupplementing Mid1 in Mid1 knockout mice; Detection of kinases such as CaMKII, PKA and ERK1/2; Supplementary references; Supplement the behavioral experiment of new object recognition; Electrophysiological measurement experiment of supplementing LTP; Supplementary neuron-specific immunohistochemical staining experiment; Supplementing the information of knockout mice used in the study; Modify the language expression of the article and the problem of too few pictures.

Thank you again for your valuable time and professional advice. We look forward to submitting the revised manuscript to you for further review.