https://doi.org/10.5061/dryad.g1jwstr1h

Description of the data and file structure

Raw data used for the quantification and statistical analysis are presented in the figures and supplementary figures.

Files and variables

Content of Datasets CSV.zip file:

Each Fig##.zip file contains the datasets in csv format for the respective figure panels. Data were normalized to their respective control; units are shown in parentheses, and the statistical test used is shown for each dataset.

Fig1.zip

These datasets show the analysis of cytosolic viscosity measured by FRAP in giant presynaptic terminals upon mitochondria inhibition by FCCP treatment. Each column represents the measurement of fluorescence intensity and the cytosolic GFP mobile fraction in individual presynaptic terminals in the control untreated condition (CTRL) or treated with mitochondria inhibitor FCCP for 20 (FCCP20), 40 (FCCP40), and 60 (FCCP60) minutes, collected from 4 to 8 independent experiments.

Fig 1B: Fluorescence recovery intensity profiles (%), Mean ± 95% CI

Fig 1C: Mobile fraction (%), Median with min/max whiskers, Kruskal-Wallis with Dunn’s correction

Fig2.zip

These datasets show the analysis of the effect of mitochondria activity and ATP on the cytosolic viscosity and the condensation/decondensation of cytosolic aggregates in presynaptic terminals in control condition (CTRL), treated with FCCP (FCCP40), and supplemented with increasing concentration of ATP from 3 to 4 independent experiments. The data also reports the measurement of intracellular level of ATP in control condition (CTRL) or after treatment with FCCP for 40 minutes (FCCP40), measured in cellulo with the fluorescence sensor PercHR or in vitro by bioluminescence assay collected from 3 independent experiments.

Fig 2B: Size of condensates (mm), Mean ± sem, unpaired Student’s t-test

Fig 2C: Fluorescence recovery intensity profiles (%), Mean ± 95% CI

Fig 2D: Size of condensates (mm), Mean ± sem, one-way ANOVA with Tukey’s correction

Fig 2F: Fluorescence intensity (%), Mean ± sem, unpaired Student’s t-test

Fig 2G: Luminescence (%), Mean ± sem, unpaired Student’s t-test

Fig3.zip

These datasets show the analysis of purified proteins condensation/decondensation in vitro in the absence or presence of increasing concentration of ATP. Size and number of condensates for each protein were measured from 4 independent experiments.

Fig 3B: Size of condensates (mm), Mean ± sem, Kruskal-Wallis with Dunn’s correction

Fig 3C left and right: Number and size of condensates (%), Mean ± sem

Fig 3E left and right: Number of condensates and pre-fibrils (%), Mean ± sem, unpaired Student’s t-test

Fig4.zip

These datasets show the analysis of the formation of synapsin-1 protein condensate in the absence or presence of the crowding agent PEG and their sensitivity to ATP in vitro. Size and number of condensates for each protein were measured from 4 independent experiments.

Fig 4B: Frequency distribution of condensate size (%), Mean ± sem, unpaired Student’s t-test

Fig 4C: Size of condensates (mm), Mean ± sem, unpaired Student’s t-test

Fig 4D left and right: Number of condensates (%), Mean ± sem, unpaired Student’s t-test

Fig 4E left and right: Size of condensates (%), Mean ± sem, unpaired Student’s t-test

Fig5.zip

These datasets show the analysis of axoplasmic viscosity measured by FRAP in axons of iPSC-derived glutamatergic neurons in healthy control condition (Healthy), after mitochondria inhibition by FCCP treatment (FCCP), and in diseased neurons from Parkinson’s patients (PD PARK2). Each column represents the measurement of fluorescence intensity in individual axons from the different conditions mentioned above and collected from 3 independent experiments. The data also reports the measurement of intracellular level of ATP in control neurons (Healthy), after treatment with FCCP for 40 minutes (FCCP), and in diseased neurons (PD PARK2) measured in vitro by bioluminescence assay collected from 4 independent experiments.

Fig 5B: Fluorescence recovery intensity profiles (%), Mean ± 95% CI

Fig 5C: Mobile fraction (%), Median with min/max whiskers, Kruskal-Wallis with Dunn’s correction

Fig 5D: Luminescence (%), Mean ± sem, one-way ANOVA with Tukey’s correction

Fig 5E: Correlation between mobile fraction and luminescence, linear regression ± 95% CI 

Fig6.zip

These datasets show the analysis of axoplasmic viscosity measured by FRAP in axons of iPSC-derived motor neurons in healthy control condition (Healthy), after mitochondria inhibition by FCCP treatment (FCCP), and in diseased neurons from an ALS patient (ALS N390D). Each column represents the measurement of fluorescence intensity in individual axons from the different conditions mentioned above and collected from 3 independent experiments. The data also reports the measurement of intracellular level of ATP in control neurons (Healthy), after treatment with FCCP for 40 minutes (FCCP), and in diseased neurons (ALS N390D) measured in vitro by bioluminescence assay collected from 3 independent experiments.

Fig 6B: Fluorescence recovery intensity profiles (%), Mean ± 95% CI

Fig 6C: Mobile fraction (%), Median with min/max whiskers, Kruskal-Wallis with Dunn’s correction

Fig 6D: Luminescence (%), Mean ± sem, one-way ANOVA with Tukey’s correction

Fig 6E: Correlation between mobile fraction and luminescence, linear regression ± 95% CI

Fig7.zip

These datasets show the analysis of axoplasmic viscosity measured by FRAP in axons of iPSC-derived glutamatergic neurons from healthy individuals (Healthy) or Parkinson’s patients (PD PARK2) in the absence or presence of NAD precursor and mitochondria booster NMN. Each column represents the measurement of fluorescence intensity in individual axons from the different conditions mentioned above and collected from 3 independent experiments. The data also reports the measurement of intracellular level of ATP in control neurons (Healthy or PD PARK2) and after treatment with NMN, measured in vitro by bioluminescence assay collected from 3 independent experiments.

Fig 7B: Fluorescence recovery intensity profiles (%), Mean ± 95% CI

Fig 7C: Mobile fraction (%), Median with min/max whiskers, Kruskal-Wallis with Dunn’s correction

Fig 7D: Luminescence (%), Mean ± sem, one-way ANOVA with Tukey’s correction

Fig 7E: Correlation between mobile fraction and luminescence, linear regression ± 95% CI

Fig8.zip

These datasets show the analysis of axoplasmic viscosity measured by FRAP in axons of iPSC-derived motor neurons from a healthy individual (Healthy) or ALS patient (ALS N390D) in the absence or presence of NAD precursor and mitochondria booster NMN. Each column represents the measurement of fluorescence intensity in individual axons from the different conditions mentioned above and collected from 3 independent experiments. The data also reports the measurement of intracellular level of ATP in control neurons (Healthy or ALS N390D) and after treatment with NMN, measured in vitro by bioluminescence assay collected from 3 independent experiments.

Fig 8B: Fluorescence recovery intensity profiles (%), Mean ± 95% CI

Fig 8C: Mobile fraction (%), Median with min/max whiskers, Kruskal-Wallis with Dunn’s correction

Fig 8D: Luminescence (%), Mean ± sem, one-way ANOVA with Tukey’s correction

Fig 8E: Correlation between mobile fraction and luminescence, linear regression ± 95% CI

Fig9.zip

These datasets show the analysis of the pathological aggregation of TDP-43 protein in axons of iPSC-derived motor neurons from a healthy individual (Healthy) or an ALS patient (ALS N390D) in the absence or presence of NAD precursor and mitochondria booster NMN. Size and number of axonal TDP-43 aggregates were collected from 3 independent experiments.

Fig 9B: Frequency distribution of aggregate size (%), Mean ± sem

Fig 9C: Number of aggregates (#/mm^3), Median with min/max whiskers, Kruskal-Wallis with Dunn’s correction

Fig 9D: Correlation between number of aggregates and luminescence, linear regression ± 95% CI

Fig 9E: Correlation between number of aggregates and mobile fraction, linear regression ± 95% CI

Fig10.zip

These datasets show the analysis of axonal mitochondria number, size, and activity in axons of iPSC-derived motor neurons from healthy individuals (Healthy) or ALS patients (ALS N390D) collected from 5 independent experiments.

Fig 10B: Number of mitochondria (#/mm^3), Mean ± sem, unpaired Student’s t-test

Fig 10C: Volume of mitochondria (mm^3), Mean ± sem, unpaired Student’s t-test

Fig 10D: Fluorescence intensity (a.u.), Mean ± sem, unpaired Student’s t-test

FigS2.zip

These datasets show the analysis of cytosolic viscosity measured by FRAP in giant presynaptic terminal areas with (+ Mito) or without mitochondria (- Mito). Fluorescence intensity and the cytosolic GFP mobile fraction in individual presynaptic terminals were collected from 3 independent experiments.

Fig S2D: Mobile fraction (%), Median with min/max whiskers, Kruskal-Wallis with Dunn’s correction

FigS3.zip

These datasets show the analysis of neuronal cell death in control untreated condition (CTRL) or after treatment with mitochondria inhibitor FCCP for 40 minutes, collected from 3 independent experiments.

Fig S3B: Cell viability (%), Mean ± sem, unpaired Student’s t-test

FigS4.zip

These datasets show the analysis of the effect of mitochondria activity the cytosolic viscosity in presynaptic terminals in control condition (CTRL), treated with mitochondria inhibitor rotenone (ROT40), glycolysis inhibitor 2-Deoxy-glucose (2DG), or using red fluorescent protein (RFP) instead of green fluorescent protein collected from 3 independent experiments. The data also report the same measurement in the control condition (CTRL) or after treatment with another mitochondria blocker, oligomycin-A (OLIGO) or FCCP (FCCP) collected from 3 independent experiments. Each column represents the measurement of fluorescence intensity and the cytosolic GFP mobile fraction in individual presynaptic terminals or in individual axons in the different conditions mentioned above.

Fig S4A: Fluorescence recovery intensity profiles (%), Mean ± 95% CI

Fig S4B: Mobile fraction (%), Median with min/max whiskers, Kruskal-Wallis with Dunn’s correction

Fig S4C: Fluorescence recovery intensity profiles (%), Mean ± 95% CI

Fig S4D: Mobile fraction (%), Median with min/max whiskers, Kruskal-Wallis with Dunn’s correction

FigS5.zip

These datasets show the analysis of the synaptic vesicle pool viscosity and active zone viscosity measured by FRAP in giant presynaptic terminals upon mitochondria inhibition by FCCP treatment. Each column represents the measurement of fluorescence intensity and the mobile fraction of synaptic vesicles (VFP) or active zone (CFP-RIM1) components in individual presynaptic terminals in the control untreated condition (CTRL) or treated with mitochondria inhibitor FCCP for 40 (FCCP40), collected from 3 independent experiments.

Fig S5C: Mobile fraction (%), Median with min/max whiskers, Mann-Whitney

Fig S5F: Mobile fraction (%), Median with min/max whiskers, Mann-Whitney

FigS6.zip

These datasets show the analysis of the diffusion coefficient of synaptic vesicles in different regions of giant presynaptic terminals. Each column represents individual terminal measurements in the control untreated condition (CTRL) and after FCCP treatment (FCCP40) collected from 3 independent experiments.

Fig S6 letf and right: Diffusion coefficient (mm^2/s), Mean ± sem, unpaired Student’s t-test

FigS7.zip

These datasets show the analysis of intracellular pH measured with fluorescent sensor pHrodo in control untreated (CTRL) and after addition of ATP (ATP) neurons collected from 3 independent experiments.

Fig S7B: Fluorescence intensity (%), Mean ± sem, unpaired Student’s t-test

FigS10.zip

These datasets show the analysis of purified proteins condensation/decondensation in vitro in the absence or presence of increasing concentration of ATP. Size and number of condensates for each protein were measured from 4 independent experiments.

Fig S10B: Size of condensates (mm), Mean ± sem, Kruskal-Wallis with Dunn’s correction

Fig S10C left and right: Number and size of condensates (%), Mean ± sem

FigS13.zip

These datasets show the analysis of the condensation state of synapsin-1 protein in vitro measured by FRAP, and the size and number of condensates of synapsin-1 protein alone (SCNA) or mixed with synuclein wildtype (SNCA) or mutant (SNCA A53T) protein measured from 3 to 4 independent experiments.

Fig S13C: Fluorescence recovery intensity profiles (%), Mean ± 95% CI

Fig S13F left and right: Number and size of condensates (%), Mean ±

FigS14.zip

These datasets show the analysis of the formation of synuclein protein condensate in the absence or presence of the crowding agent PEG and their sensitivity to ATP in vitro. Size and number of condensates for each protein were measured from 4 independent experiments.

Fig S14B: Frequency distribution of condensate size (%), Mean ± sem, unpaired Student’s t-test

Fig S14C: Size of condensates (mm), Mean ± sem, unpaired Student’s t-test

Fig S14D left and right: Number of condensates (%), Mean ± sem, unpaired Student’s t-test

Fig S14E left and right: Size of condensates (%), Mean ± sem, unpaired Student’s t-test

FigS15.zip

These datasets show the analysis of the formation of TDP-43 protein condensate in the absence or presence of the crowding agent PEG and their sensitivity to ATP in vitro. Size and number of condensates for each protein were measured from 4 independent experiments.

Fig S15B: Frequency distribution of condensate size (%), Mean ± sem, unpaired Student’s t-test

Fig S15C: Size of condensates (mm), Mean ± sem, unpaired Student’s t-test

Fig S15D left and right: Number of condensates (%), Mean ± sem, unpaired Student’s t-test

Fig S15E left and right: Size of condensates (%), Mean ± sem, unpaired Student’s t-test

FigS17.zip

These datasets show the analysis of the cytoplasmic viscosity measured by microrheology in vitro. The mean square displacement and diffusion coefficient of nanobeads loaded onto purified cytosol fraction from untreated control (CTRL) or FCCP-treated (FCCP) neurons were collected from 3 independent experiments.

Fig S17B: MSD plot (mm^2/s), linear regression ± 95% CI

Fig S17C: Diffusion coefficient (mm^2/s), Mean ± sem, one-way ANOVA with Tukey’s correction

Fig S17D: Correlation between diffusion coefficient and mobile fraction, linear regression ± 95% CI

FigS18.zip

These datasets show the analysis of axoplasmic viscosity measured by FRAP in axons of iPSC-derived glutamatergic neurons in healthy control condition (Healthy), and in diseased neurons from Parkinson patient (PD SNCA 3x), as well as in iPSC-derived motor neurons in healthy control condition (Healthy) or diseased neurons from ALS patients with different mutations (ALS N390D, ALS M337V and ALS c9orf72). Each column represents the measurement of fluorescence intensity in individual axons from the different conditions mentioned above and collected from 3 to 4 independent experiments. The data also reports the measurement of the intracellular level of ATP in in vitro by bioluminescence assay collected from the same samples.

Fig S18A: Fluorescence recovery intensity profiles (%), Mean ± 95% CI

Fig S18B: Mobile fraction (%), Median with min/max whiskers, Mann-Whitney

Fig S18C: Fluorescence recovery intensity profiles (%), Mean ± 95% CI

Fig S18D: Mobile fraction (%), Median with min/max whiskers, Kruskal-Wallis with Dunn’s correction

Fig S18E: Luminescence (%), Mean ± sem, unpaired Student’s t-test

Fig S18F: Luminescence (%), Mean ± sem, one-way ANOVA with Tukey’s correction

FigS19.zip

These datasets show the analysis of axoplasmic viscosity measured by FRAP in axons of iPSC-derived glutamatergic neurons from healthy individual (Healthy), Parkinson patient (PD SNCA 3x), and ALS patients with different mutations (ALS N390D, ALS M337V and ALS c9orf72) in absence or presence of NAD precursor and mitochondria booster NMN. Each column represents the measurement of fluorescence intensity in individual axons from the different conditions mentioned above and collected from 3 to 4 independent experiments. The data also reports the measurement of intracellular level of ATP in control neurons (Healthy or PD or ALS ) and after treatment with NMN, measured in vitro by bioluminescence assay collected from the same samples.

Fig S19A: Fluorescence recovery intensity profiles (%), Mean ± 95% CI

Fig S19B: Mobile fraction (%), Median with min/max whiskers, Mann-Whitney

Fig S19C: Fluorescence recovery intensity profiles (%), Mean ± 95% CI

Fig S19D: Mobile fraction (%), Median with min/max whiskers, Kruskal-Wallis with Dunn’s correction

Fig S19E: Luminescence (%), Mean ± sem, unpaired Student’s t-test

Fig S19F: Luminescence (%), Mean ± sem, one-way ANOVA with Tukey’s correction

FigS20.zip

These datasets show the analysis of intracellular levels of ATP and NADH in iPSC-derived motor neurons from healthy individual (Healthy) and ALS patient (ALS N390D) in untreated control condition (CTRL), after treatment with NMN (NMN) and after co-treatment with NMN and 2DG (NMN + 2DG) measured in vitro by bioluminescence assay and collected from 6 independent experiments.

Fig S20A: Luminescence (%), Mean ± sem, one-way ANOVA with Tukey’s correction

Fig S20B: Luminescence (%), Mean ± sem, one-way ANOVA with Tukey’s correction