Adenosine Deaminase-Mediated Purine Dysfunction Leads to DNA Repair Inhibition and Senescence in Sporadic Amyotrophic Lateral Sclerosis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors investigated downstream functional consequences of ADA loss in SALS iAstrocytes, and assess whether the in vitro findings could be recapitulated in patient tissue, biofluids and in vivo. They found that along with loss of ADA, key enzymes involved in the purine metabolism pathway are dysregulated at both the protein and RNA level. The purine metabolism dysfunction is intrinsically linked to ADA level, which can influence DNA repair mechanisms and senescence. These alterations were recapitulated in SALS CSF and post-mortem tissue, with aging and sex effecting purine metabo-lite levels downstream of ADA and positively correlating with disease progression. The authors also found that that ADA-mediated purine metabolism correlates with age of death and disease progression in SALS cases. They suggest a link between ADA mediated dysfunction and TDP43 pathology.

This is a comprehensive study with numerous results, but it relies heavily on the experimental design and findings of previous studies. Due to the large number of figures and results, it is somewhat difficult to follow the main objective throughout the manuscript.

Some suggestions: 

The abstract does not provide a clear conclusion or highlight the main findings of the study.

The Introduction should clearly state the aim and/or hypothesis of the study, while the Results should be focused on addressing and substantiating this specific objective.

There is no clear final conclusion or take-home message that the reader can draw from the study.

I am curious why the authors present the blot bands as white bands on a black background rather than the opposite, which would be much clearer and easier to interpret.

 

 

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study of Hall and colleagues represent a step ahead in their efforts to link alterations of adenosine deaminase with the onset and progression of ALS. The study is rather extensive by approaching several mechanistic processes and its extent is probably the reason for the perception that there are several open issues that may require attention, as detailed below:

1-lines 52-54 ‘ADA mediated purine dysfunction represents an important pathway in ALS, as it influences energy metabolism, antioxidant synthesis, DNA methylation, and the DNA damage response’: Raising the intracellular levels of adenosine is also expected to trigger an increase in the extracellular levels of adenosine (through equilibrative nucleoside transporters) and increased activation of adenosine receptors. Accordingly, antagonists of adenosine receptors have been shown to afford benefits in animal models of ALS (e.g. Exp Neurol, 2015, 267:115-22, doi: 10.1016/j.expneurol.2015.03.004; Neuropharmacology, 2020, 171:108106, doi: 10.1016/j.neuropharm.2020.108106; Neurobiol Dis, 2024, 199:106603, doi: 10.1016/j.nbd.2024.106603; JMIR Res Protoc, 2025, 14:e76105, doi: 10.2196/76105).

2-Lower CD73 levels seems to be associated with longevity (Immun Ageing, 2017, 14:11, doi: 10.1186/s12979-017-0094-3) and CD73 inhibition is neuroprotective (e.g. Neurobiol Dis, 2019, 132:104570, doi: 10.1016/j.nbd.2019.104570; Br J Pharmacol, 2019, 176(18):3666-3680, doi: 10.1111/bph.14771). This should be commented.

3-Some side effects of pentostatin are centrally mediated, namely alterations of mood, memory and motor performance. This seems to match the proposed role of decreased ADA levels in the progression of ALS.

4-In cancer cells, inosine is capable of activating mechanistic target of rapamycin complex 1 (mTORC1), thereby promoting survival and growth during nutrient stress (Cell Death Dis, 2023, 14:492, doi: 10.1038/s41419-023-06017-2).

5-Probably the most relevant purine metabolite in the control of brain function (both neuronal and glial) is adenosine. Detailing the data on the levels of adenosine in the CSF would be most informative.

6-White matter tracks in the motor cortex are mostly constituted by oligodendrocytes. This makes the choice of investigating alterations in iAstrocytes somewhat odd. An explanation or clarification seems required.

7-line 282 ‘6 hours treatment’: Does this cause a loss of viability of the treated cells or a metabolic dysfunction that could be the direct cause of the adaptive changes of ADA expression and protein levels? In other words, is there a negative control of other purinergic enzymes that is preserved with this treatment?

8-I cannot see the scale bars in the immunocytochemistry pictures. Please make it well-visible.

9-A key process linking DNA damage and senescence is the activity of PARPs and exhaustion of NAD, but neither of these processes were evaluated.

10-Most of the 'purinergic dysfunction' was due to alterations of purine metabolites produced within cells. In contrast CD73 activity is solely related to extracellular metabolism of adenine nucleotides and formation of extracellular adenosine coupled to the activation of adenosine A2A receptors (e.g. J Neurosci, 2013, 33:11390-9, doi: 10.1523/JNEUROSCI.5817-12.2013). One would expect to see a quantification of intracellular 5'-nucleotidase, an enzyme responsible for the formation of intracellular adenosine and that is molecularly different from CD73.

 

11-Previous studies have systematically shown that CD73 levels and activity are up-regulated at early stages of different animal models of brain diseases in particular in nerve terminals (e.g. Br J Pharmacol, 2019, 176:3666-80, doi: 10.1111/bph.14771; Neurobiol Dis, 2019, 132:104570, doi: 10.1016/j.nbd.2019.104570; Neurobiol Dis, 2021, 157:105441, doi: 10.1016/j.nbd.2021.105441; ACS Chem Neurosci, 2023, 14:1299-309, doi: 10.1021/acschemneuro.2c00810). Here, there is a decrease of CD73 in astrocytes, although astrytes exposed to amyloid peptides do upregulate CD73 (Mol Neurobiol, 2021, 58:6232-6248, doi: 10.1007/s12035-021-02538-z). This difference needs to be commented in the Discussion.

12-Alterations of the energy charge are coupled to alterations of metabolic flow in pathways of primary metabolism through alteration of the intracellular levels of AMP. Apart from a direct regulation of key regulatory enzymes, AMP also signals through the activation of AMPK, which activity is also paramount to define senescent phenotypes. Neither the levels of AMP nor the activity of AMPK are considered in this study.

13-lines 444-6: There are elegant studies on alterations of the purinome upon aging that were not considered.

14-The discussion should at least entertain the possibility that alterations in the purinome may be linked to ALS through the alteration of adenosine A2A receptor function.

15-Expression has a defined meaning in biology, designating the process of gene readout and mRNA formation. Thus, proteins are not expressed but have densities, levels or amounts upon their translation. Correct throughout the manuscript

Author Response

See attached document

Author Response File: Author Response.pdf

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

I congratulate the authors for addressing and mostly resolving all the questions raised by this Reviewer.