Aberrant upregulation of the glycolytic enzyme PFKFB3 in CLN7 neuronal ceroid lipofuscinosis

CLN7 neuronal ceroid lipofuscinosis is an inherited lysosomal storage neurodegenerative disease highly prevalent in children. CLN7/MFSD8 gene encodes a lysosomal membrane glycoprotein, but the biochemical processes affected by CLN7-loss of function are unexplored thus preventing development of potential treatments. Here, we found, in the Cln7∆ex2 mouse model of CLN7 disease, that failure in autophagy causes accumulation of structurally and bioenergetically impaired neuronal mitochondria. In vivo genetic approach reveals elevated mitochondrial reactive oxygen species (mROS) in Cln7∆ex2 neurons that mediates glycolytic enzyme PFKFB3 activation and contributes to CLN7 pathogenesis. Mechanistically, mROS sustains a signaling cascade leading to protein stabilization of PFKFB3, normally unstable in healthy neurons. Administration of the highly selective PFKFB3 inhibitor AZ67 in Cln7∆ex2 mouse brain in vivo and in CLN7 patients-derived cells rectifies key disease hallmarks. Thus, aberrant upregulation of the glycolytic enzyme PFKFB3 in neurons may contribute to CLN7 pathogenesis and targeting PFKFB3 could alleviate this and other lysosomal storage diseases.


Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response. No statistical methods were used to determine sample size prior to experiments. Sample sizes were decided by the previous experience of the group in in vitro molecular experiments and in vivo experiments and/or on previously published similiar experiments. Exact information on the sample numbers being analyzed can be found in Figure legends and in Supplementary Information. The majority of biochemical assays were repeated at least three times in order to derive statistical information such as error bars, p values and significance.
No data were excluded from analyses.
In vitro: experiments were done from 3 to 5 times with independent biological samples and the necessary technical replicates for each technique (tipically, 4-6 replicas), in order to reproduce the results found. In vivo: the sample size of the behavioral studies was higher than in the in vitro experiments given the variability in the parameters measured in order to confirm a reliable result. This information has been added in the Statistical section of the manuscript. All attempts at replication were successfull.
For all mouse experiments, animals were chosen based on genotypes. Aged-matched wild-type and mutant littermates were compared to minimize variance in age, genetic background and environment. Then ageneral method of randomization to assign experimental groups was not performed because all experiments were conducted with appropiate positive and negative controls, therefore it was not applicable. For in vitro studies, randomization is not applicable as cells with different treatments or genetic knockdown cannot be randomized. However, for imaging experiments, cells were chosen at random within each condition.
No misidentified cell lines were used in this study.
No wild animals were used in the study No field collected samples were used in the study.

April 2020
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided. Mice cortical neurons from primary cultures or freshly isolated from brain were used. These cells were treated with the corresponding probes (Mitosox, DilC1),following manufacturer instructions, during a determined time. After incubation with the corresponding probe, the cells were centrifuged and the pellets were resuspended in PBS for further analysis.
At least 100,000 events were acquired in triplicate and by condition.
The threshold of the analyzer was adjusted in the corresponding channel of the flow cytometer to exclude most subcellular residues or cellular aggregates in the SSC/FSC plot. The median intensity values were obtained for each sample, and the FMO (unstained cells) substracted.
single imaging session per animal minimum of 2 spectra per animal per session. Imaging sessions were repeated after 1 week if spectra did not fit well by the LC Model software.