Deciphering Neuronal Deficit and Protein Profile Changes in Human Brain Organoids from Patients with Creatine Transporter Deficiency

Reviewer #1 (Public Review):

This well written and designed study by Broca-Brisson et al describes the generation of a new in vitro model for creatine transporter deficiency (CTD), making use of human brain organoid cultures derived from CTD patients. This new model will certainly prove itself very useful to better understand this genetic disease essentially affecting CNS. As CTD has no satisfactory treatment so far (despite more than 20 years of research), this new model will also be very useful to design and develop new treatments.

In particular, through the use of immunohistochemistry, real time PCR, and proteomics combined with integrative bioinformatic and statistical analysis, authors provide very interesting new informations on the brain pathways affected in CTD (e.g. neurogenesis with down-regulation of SOX2 and PAX6 but up-regulation of GSK3b; and proteins involved in autistic spectrum, epilepsies or intellectual disabilities).

While the CTD human brain organoids show a decrease in Cr (in absence of Cr in the culture medium) as compared to control organoids (4 times less), they are not devoid of Cr. Do these organoids express the two enzymes allowing Cr synthesis (AGAT and GAMT), and in which brain cell types? If yes, how to explain the decrease in Cr in the CTD organoids?

The rescue experiment, re-establishing a functional Cr transporter (CRT or SLC6A8) in the CTD human brain organoids, is very interesting, as this may help the design and development of new treatments for CTD. However, authors claim that the functional CRT expressed in the rescued CTD organoids was expressed in each cell. This may be a difficulty in the development of new CTD treatments, as CRT should be expressed in neurons and oligodendrocytes, but not in astrocytes. Authors may want to comment on this point.

Reviewer #2 (Public Review):

In their recent manuscript, Broca-Brisson et al. deliver a multidisciplinary approach to investigate creatine transporter deficiency (CTD) using human-derived brain organoids. The authors have provided a compelling CTD human brain organoid model using induced pluripotent stem cells (iPSCs) derived from individuals with CTD. This model shows distinct differences in creatine uptake between organoids originating from CTD patients and their healthy counterparts. Furthermore, the researchers effectively restored creatine uptake by reintroducing the wild-type CRT in the iPSCs.

The team used advanced molecular biology techniques and sophisticated mass spectrometry to identify changes in protein regulation within these CTD brain organoids. They propose an intriguing theory linking reduced creatine uptake to abnormalities in the GSK3β kinase pathway and mitochondrial function, which might underlie intellectual disability seen in CTD patients.
This study is well-structured and easy to follow, with clear and concise explanations of the experiments. The authors present an important idea: a dysfunction in just one protein transporter (CRT) can cause significant biochemical changes in the brain. Their findings are well-presented and backed by high-quality figures and comprehensive data analysis.

Author Response

Reviewer #1 (Public Review):

While the CTD human brain organoids show a decrease in Cr (in absence of Cr in the culture medium) as compared to control organoids (4 times less), they are not devoid of Cr. Do these organoids express the two enzymes allowing Cr synthesis (AGAT and GAMT), and in which brain cell types? If yes, how to explain the decrease in Cr in the CTD organoids?

There is a lack of functional CRT in the CTD human brain organoids. The basal level of creatine in CTD human brain organoid is significantly lower than in healthy human brain organoids. The intracerebral creatine synthesis is due to different expression of the AGAT and GAMT enzymes and relies on functional CRT for the transport of the GAA intermediate Litterature pointed out that both enzymes are rarely co-expressed (Braissant et al., 2001, PMID: 11165387) meaning that GAA intermediate needs to be transported by CRT to neurones for complete creatine synthesis. Even if we evidenced a slight mRNA expression of AGAT and GAMT enzymes, the creatine synthesis is not effective since the GAA intermediate could not be transporterd in cell expressing GAMT due to the non functional creatine transporter in the CTD human brain organoids.

The rescue experiment, re-establishing a functional Cr transporter (CRT or SLC6A8) in the CTD human brain organoids, is very interesting, as this may help the design and development of new treatments for CTD. However, authors claim that the functional CRT expressed in the rescued CTD organoids was expressed in each cell. This may be a difficulty in the development of new CTD treatments, as CRT should be expressed in neurons and oligodendrocytes, but not in astrocytes. Authors may want to comment on this point.

As shown in Figure S2C, the whole brain organoid in the resue experiment shows the expression of the GFP protein, thus also the co-expressed wild-type CRT. In these experiments we did not make a detailed cellular characterization of the rescued organoids, and this may be a task in our next experiments for an exact characterization of the cell-specific CRT expresion and function in the rescued brain organoids. According to this, we will correct in the revision version of manuscript the statement on page 6: “SLC6A8 expressing brain organoids showed GFP fluorescence in the whole area of the organoid (Fig S2C).”