Dataset for the project "Dissecting molecular mechanisms triggered by progressive loss of progranulin and C9orf72 in frontotemporal dementia: novel therapeutic targets? (RF-2016-02361492)

Dataset for the project “Dissecting molecular mechanisms triggered by progressive loss of progranulin and C9orf72 in frontotemporal dementia: novel therapeutic targets?” Italian Ministry of Health, Italy, Ricerca Finalizzata (Grant RF-2016-02361492)

Specific Aim 1

To further collect and generate human cellular disease models (skin fibroblast and LCL) of progressive progranulin and C9orf72 loss.

Specific Aim 2

To test the hypothesis that EVs secreted from microglia, in which GRN or C9orf72 are downregulated, contain and deliver excessive C1q and C3 complements factors to the synapses, thereby causing aberrant synaptic pruning. Results of this aim will clarify whether microglia-derived EVs have a pathogenic role in the disease, thus indicating a novel therapeutic target to limit synaptic loss and disease progression.

Specific Aim 3

To identify sensitive biomarkers for patient stratification and treatment monitoring by a) characterization of exosomal and lysosomal pathways in human cellular disease models with progressive loss of progranulin and C9orf72; b) studying lysosomal and immune proteins in EVs circulating in body fluids of subjects with progressive loss of progranulin and C9orf72; c) studying the impact of variants in lysosomal genes on age of disease onset in subjects carrying GRN and C9orf72 mutations.

The present dataset contains the following folders:

• RawData_Aim2 [Unpublished Data]:
  • Materials and Methods_RawData_Aim2
  • RawData_Aim2:  a dataset (excel file) containing the following sheets:
    • Fig.1: Number of EVs from microglia (analysis with Tunable Resistive Pulse Sensing-TRPS technique, using a qNano instrument)
    • Fig.2: Genetic expression levels of homeostatic and inflammatory genes in microglia
    • Fig.3: WB analysis to confirm GRN silencing in microglia
    • Fig.4A: Quantification of immunofluorescence analysis for Lamp-1, CD68, and Cathepsin D
    • Fig.4B: WB analysis for Lamp-1, CD68, TMEM119 and Cathepsin D in microglia
    • Fig.5A: WB analysis for C3, C1q, and TMEM119 in microglia
    • Fig.5B: Quantification of immunofluorescence analysis for C1q and Cd11b in microglia
    • Fig.5C: WB analysis for C3, C1q, Alix, Flotillin, Annexin-A2, and TOM20 in EVs from microglia
    • Fig.6A-B: WB analysis for C3, C1q, Lamp1, CD68, Cathepsin D, Alix, and Annexin-A2 in EVs
    • Fig.8: densitometric analysis of presynaptic terminals (Basson) and postsynaptic terminals (Shank2) in neuron-microglia cocultures (no treatment)
    • Fig.9: densitometric analysis of presynaptic terminals (Basson) and postsynaptic terminals (Shank2) in neuron-microglia cocultures (treatments GW4869)
    • Fig.10: densitometric analysis of presynaptic terminals (Basson) and postsynaptic terminals (Shank2) in neuron-microglia cocultures (treatments with EVs)
    • Fig.11: Phagocytosis assay
  • Fig.4A: a folder containing original immunofluorescence pictures for Lamp-1, CD68, and Cathepsin D in microglia
  • Fig.5B: a folder containing original immunofluorescence pictures for C1q and Cd11b in microglia
  • Fig.11: a folder containing original pictures from phagocytosis assay

• RawData_Aim3a_Fibroblast [Unpublished Data]:
  • Materials and Methods_Aim3a_Fibroblasts
  • RawData_Aim3a_Fibroblasts_NTA_Complement_LC3_CathD: a folder containing:
    • RawData_Aim3a_Fibroblasts_NTA_Complement_LC3_CathD:  a dataset (excel file) containing the following variables referred to EVs from fibroblasts:
      • Sample (Col. A) and its Category (Col. B)
      • Raw NTA File name (Col. C)
      • Raw Values_C1q EVs from Bioplex assay (Col. D-F)
      • Raw Values_C3 EVs from Bioplex assay (Col. G-I)
      • Raw Values_C4 EVs from Bioplex assay (Col. J-L)
      • Raw Values_LC3 EVs from ELISA assay (Col. M-P)
      • Raw Values_Cathepsin D EVs from ELISA assay (Col. Q-T)
    • RawData_Aim3a_Fibroblasts_NTA_Files: a folder containing Raw NTA file for each sample (from these files we extracted EV concentration and size)
  • RawData_Aim3a_Fibroblasts_EVMarkers_PGRN_C9orf72_pTDP43: a folder containing:
    • WB_Aim3a_Fibroblasts_EVMarkers: a folder containing original WB pictures for EV biomarkers TSG101, Alix, CD63, Flotillin-1
    • ELISA_C9orf72_Fibroblasts: a dataset containing raw C9orf72 data from ELISA assay on fibroblasts lysates and EVs lysates
    • ELISA_pTDP43_Fibroblasts: a dataset containing raw pTDP43 data from ELISA assay on fibroblasts lysates and EVs lysates
    • WB_Analyses_Fibroblasts_PGRN: a dataset containing densitometric analysis from WB on fibroblasts lysates and EVs lysates for PGRN and Actin/TSG101 (for cells/EVs normalization respectively)

• 3.a RawData_Aim3a_LCL [Unpublished Data]:
  • Materials and Methods_Aim3a _LCL
  • RawData_Aim3a_LCL_NTA_Complement_LC3_CathD: a folder containing:
    • RawData_Aim3a_LCL_NTA_Complement_LC3_CathD: a dataset (excel file) containing the following variables referred to EVs from LCL:
      • Sample (Col. A)
      • Category (Col. B)
      • Raw NTA File name (Col. C)
      • Raw Values_C1q EVs from Bioplex assay (Col. D-F)
      • Raw Values_C3 EVs from Bioplex assay (Col. G-I)
      • Raw Values_C4 EVs from Bioplex assay (Col. J-L)
      • Raw Values_LC3 EVs from ELISA assay (Col. M-P)
      • Raw Values_Cathepsin D EVs from ELISA assay (Col. Q-T)
    • RawData_Aim3a_LCL_NTA_Files: a folder containing Raw NTA file for each sample (from these files we extracted EV concentration and size)
  • WB_Analyses_Aim3a_LCL_PGRN_C9orf72_TDP43_pTDP43_Lamp1: a dataset containing densitometric analysis from WB on LCL lysates and EVs lysates for PGRN, C9orf72, p-TDP43, TDP43, Lamp-1, Actin/TSG101 (for cells/EVs normalization respectively)

• 3.b RawData_Aim3b_Plasma_1 [Published]
  Results (NTA data) from aim 3.b are published in Bellini&Saraceno et al. Cells 2022 (doi:10.3390/cells11030488) and the raw data supporting the results are published in the Mendeley Data Repository at doi:10.17632/kds9sb4z6t.2
  • Materials and Methods_Aim3b_Plasma
  • RawData_Aim3b_Plasma: a folder containing:
    • RawData_Aim3b_Plasma: a dataset (excel file) containing the following variables:
      • Sample (Col. A)
      • Category (Col. B)
      • Age (Col. C)
      • Corresponding Raw NTA File name (Col. D)
    • RawData_Aim3b_NTA_Files: a folder containing Raw NTA file for each sample (from these files we extracted EV concentration and size)

• 3.b RawData_Aim3b_Plasma_2 [in press]
  Results (NTA and Cathepsin D data) from aim 3.b have been included in an article submitted to IJMS (Manuscript ID: ijms-1899683; in press) and the raw data supporting the results are published in the Zenodo Data Repository at doi: 10.5281/zenodo.6958338.
  • Materials and Methods_Aim3b_Plasma
  • RawData_Aim3b_Plasma: a folder containing:
    • RawData_Aim3b_Plasma: a dataset (excel file) containing the following variables:
      • Sample (Col. A)
      • Category (Col. B)
      • Age (Col. C)
      • Onset (Col. D)
      • Corresponding Raw NTA File name (Col. E)
      • Raw Values_Cathepsin D plasma EVs from ELISA assay (Col. F-J)
      • Raw Values_Cathepsin D plasma from ELISA assay (Col. K-O)
    • RawData_Aim3b_NTA_Files: a folder containing Raw NTA file for each sample (from these files we extracted EV concentration and size)

• 3.c RawData_Aim3c_NGS [Submitted]
  Results from aim 3.c have been included in an article submitted to IJMS (Manuscript ID: ijms-1922450; Under review) and the raw data supporting the results are openly available in the Zenodo Data Repository at doi: 10.5281/zenodo.7040532.
  • Materials and Methods_Aim3c_NGS
  • RawData­_ Aim3c_NGS: a folder containing:
    • Correspondence_RawData_NGS: a dataset (excel file) containing the following variables:
      • Sample (Col. A)
      • Group (Col. B)
      • Corresponding g.vcf File name (Col. C)
  • Gene_Selection: a file with the selection of the 98 genes related to the lysosomal pathway included in the NGS panel