DOI: 10.5061/dryad.g1jwstr1h
title: Loss of intracellular ATP affects axoplasmic viscosity and pathological protein aggregation in mammalian neurons

description: Neurodegenerative diseases display synaptic deficits, mitochondrial
 defects, and protein aggregation. We show that intracellular ATP regulates
 axoplasmic viscosity and protein aggregation in mammalian neurons.
 Decreased intracellular ATP upon mitochondria inhibition leads to
 axo-terminal cytosol, synaptic vesicles, and active zone components
 condensation, modulating the functional organization of mouse
 glutamatergic synapses. Proteins involved in the pathogenesis of
 Parkinson’s disease (PD), Alzheimer's disease (AD), and Amyotrophic
 Lateral Sclerosis (ALS) condensed and underwent ATP-dependent LPS in
 vitro. Human iPSC-derived neurons from PD and ALS patients displayed a
 reduction in their axoplasmic fluidity and decreased intracellular ATP.
 Finally, nicotinamide mono-nucleotide (NMN) treatment successfully rescued
 intracellular ATP levels and axoplasmic viscosity in neurons from PD and
 ALS patients, and reduced TDP-43 aggregation in human motor neurons
 derived from an ALS patient. Thus, our data suggest that the hydrotropic
 activity of ATP contributes to the regulation of neuronal homeostasis in
 both physiological and pathological conditions.
description: # Loss of intracellular ATP affects axoplasmic viscosity and pathological
 protein aggregation in mammalian neurons
 [https://doi.org/10.5061/dryad.g1jwstr1h](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