Journal: JCI Insight

Article Title: Neurofilament accumulation disrupts autophagy in giant axonal neuropathy

doi: 10.1172/jci.insight.177999

Figure Lengend Snippet: ( A ) Representative fluorescence microscopy images of DRG neurons from WT and Gan -null mice stained for neurofilament light (NFL) after 2, 4, and 7 days in vitro (DIV). Arrowheads denote NFL aggregation in the soma of Gan -null DRG neurons. Note that NFL aggregates are already present after 2 DIV. ( B ) The GAN phenotype can be recapitulated by lentiviral delivery of shRNA targeting the Gan gene. Scale bars: 30 μm ( A and B ). All insets are shown at ×3 magnification. After 12 DIV, axonal fragmentation, a sign of neurodegeneration, occurs in Gan -silenced DRG neurons, as denoted by arrowheads; large aggregate shown in zoom. ( C ) Quantification of axonal fragmentation was accomplished using Fiji’s measurement tool, reported here as the means of 3 independent experiments ± SEM. *** P < 0.001 by 2-tailed, unpaired Student’s t test.

Article Snippet: The following primary antibodies were used: neurofilament NFL chicken polyclonal (CPCA-NF-L, Encor), LC3A/B monoclonal (12741, Cell Signaling Technology), LAMP-1 rabbit polyclonal (ab24170, Abcam), GAPDH rabbit monoclonal (2118, Cell Signaling Technology), TFEB rabbit polyclonal (SAB2108453, Labome), TFEB rabbit polyclonal (A303-773A, Bethyl Laboratories), 14-3-3 rabbit polyclonal (51-0700, Cell Signaling Technology), cathepsin B mouse monoclonal (ab58802, Abcam), cathepsin D mouse monoclonal (ab75852, Abcam), and mucolipin-1 rabbit polyclonal (PA1-46474, Invitrogen).

Techniques: Fluorescence, Microscopy, Staining, In Vitro, shRNA

Journal: JCI Insight

Article Title: Neurofilament accumulation disrupts autophagy in giant axonal neuropathy

doi: 10.1172/jci.insight.177999

Figure Lengend Snippet: ( A ) Volcano plot showing the distribution of measured proteins extracted from DRG cultures in which Gan was silenced using shRNA (shGan). ( B ) Plot showing top-ranked altered pathways in GAN where pathway activation (change in z score) is shown (orange represents upregulation and blue represents downregulation). ( C and D ) Plots showing altered proteins for the phagosome formation and phagosome maturation pathways. * P < 0.05, ** P < 0.001, *** P < 0.0001; adjusted P values from 2-tailed, unpaired Student’s t test. NFL, neurofilament light; NFM, neurofilament medium; PRFN, peripherin.

Article Snippet: The following primary antibodies were used: neurofilament NFL chicken polyclonal (CPCA-NF-L, Encor), LC3A/B monoclonal (12741, Cell Signaling Technology), LAMP-1 rabbit polyclonal (ab24170, Abcam), GAPDH rabbit monoclonal (2118, Cell Signaling Technology), TFEB rabbit polyclonal (SAB2108453, Labome), TFEB rabbit polyclonal (A303-773A, Bethyl Laboratories), 14-3-3 rabbit polyclonal (51-0700, Cell Signaling Technology), cathepsin B mouse monoclonal (ab58802, Abcam), cathepsin D mouse monoclonal (ab75852, Abcam), and mucolipin-1 rabbit polyclonal (PA1-46474, Invitrogen).

Techniques: shRNA, Activation Assay

Journal: JCI Insight

Article Title: Neurofilament accumulation disrupts autophagy in giant axonal neuropathy

doi: 10.1172/jci.insight.177999

Figure Lengend Snippet: ( A ) Representative fluorescence images of DRG neurons from WT or Gan -null mice costained for the cytoskeleton marker neurofilament light (NFL) and the autophagosome marker LC3. Autophagosomes are excluded from neurofilament aggregates, and LC3 puncta are found at the periphery of the aggregates (white arrowhead). ( B ) WT or Gan -null mouse DRG neurons costained for the cytoskeletal marker NFL and the lysosomal marker LAMP-1. Two phenotypes are observed in Gan -null DRG neurons with neurofilament aggregates: lysosomes are either excluded (circular dotted line) or colocalized with neurofilament aggregates (arrowhead). Scale bars: 30 μm. Insets are shown at ×3 magnification. Representative images from 3 independent experiments.

Article Snippet: The following primary antibodies were used: neurofilament NFL chicken polyclonal (CPCA-NF-L, Encor), LC3A/B monoclonal (12741, Cell Signaling Technology), LAMP-1 rabbit polyclonal (ab24170, Abcam), GAPDH rabbit monoclonal (2118, Cell Signaling Technology), TFEB rabbit polyclonal (SAB2108453, Labome), TFEB rabbit polyclonal (A303-773A, Bethyl Laboratories), 14-3-3 rabbit polyclonal (51-0700, Cell Signaling Technology), cathepsin B mouse monoclonal (ab58802, Abcam), cathepsin D mouse monoclonal (ab75852, Abcam), and mucolipin-1 rabbit polyclonal (PA1-46474, Invitrogen).

Techniques: Fluorescence, Marker

Journal: JCI Insight

Article Title: Neurofilament accumulation disrupts autophagy in giant axonal neuropathy

doi: 10.1172/jci.insight.177999

Figure Lengend Snippet: Representative fluorescence images of DRG neurons silenced for gigaxonin (shGan) and control neurons (shScr) costained for NFL and ( A ) mucolipin-1 and vacuolar ATPase or ( B ) cathepsin B and cathepsin D. While cathepsin D is excluded from neurofilament aggregates, the 3 other lysosomal proteins colocalize with aggregates in shGan cells from mouse DRG. Scale bars: 30 μm. Insets are shown at ×3 magnification. Arrowheads highlight lysosomal proteins clumped in NFL aggregates in the shGan condition.

Article Snippet: The following primary antibodies were used: neurofilament NFL chicken polyclonal (CPCA-NF-L, Encor), LC3A/B monoclonal (12741, Cell Signaling Technology), LAMP-1 rabbit polyclonal (ab24170, Abcam), GAPDH rabbit monoclonal (2118, Cell Signaling Technology), TFEB rabbit polyclonal (SAB2108453, Labome), TFEB rabbit polyclonal (A303-773A, Bethyl Laboratories), 14-3-3 rabbit polyclonal (51-0700, Cell Signaling Technology), cathepsin B mouse monoclonal (ab58802, Abcam), cathepsin D mouse monoclonal (ab75852, Abcam), and mucolipin-1 rabbit polyclonal (PA1-46474, Invitrogen).

Techniques: Fluorescence, Control

Journal: JCI Insight

Article Title: Neurofilament accumulation disrupts autophagy in giant axonal neuropathy

doi: 10.1172/jci.insight.177999

Figure Lengend Snippet: ( A ) Representative live-imaging fluorescence images of control and shGan cells from mouse DRG transduced with NFL-GFP tagged lentivirus and treated with red LysoTracker to visualize lysosomes. Expression of the NFL-GFP construct in shGan cells from mouse DRG neurons allowed neurofilament aggregate visualization in living cells. Scale bar: 30 μm. Insets are shown at ×3 magnification. ( B and C ) shGan induces an increase in the number of lysosomes and the surface area covered by these organelles. Note that as observed after NFL and LAMP-1 costaining, LysoTracker dye is also mainly excluded from neurofilament aggregates (circular dotted line). Quantitative data are presented as mean ± SEM. *** P < 0.001 by 2-tailed, unpaired Student’s t test. Representative images from 3 independent experiments.

Article Snippet: The following primary antibodies were used: neurofilament NFL chicken polyclonal (CPCA-NF-L, Encor), LC3A/B monoclonal (12741, Cell Signaling Technology), LAMP-1 rabbit polyclonal (ab24170, Abcam), GAPDH rabbit monoclonal (2118, Cell Signaling Technology), TFEB rabbit polyclonal (SAB2108453, Labome), TFEB rabbit polyclonal (A303-773A, Bethyl Laboratories), 14-3-3 rabbit polyclonal (51-0700, Cell Signaling Technology), cathepsin B mouse monoclonal (ab58802, Abcam), cathepsin D mouse monoclonal (ab75852, Abcam), and mucolipin-1 rabbit polyclonal (PA1-46474, Invitrogen).

Techniques: Imaging, Fluorescence, Control, Transduction, Expressing, Construct

Journal: JCI Insight

Article Title: Neurofilament accumulation disrupts autophagy in giant axonal neuropathy

doi: 10.1172/jci.insight.177999

Figure Lengend Snippet: ( A ) Representative fluorescence images of control and shGan cells from DRG neurons costained for NFL and TFEB showing colocalization of transcription factor EB (TFEB) to neurofilaments (arrowhead). Scale bar: 30 μm. ( B ) Higher-magnification pictures showing decreased TFEB localization in the nuclear compartment in shGan cells. Accompanying plots show that TFEB has decreased intensity, and that nuclear size is similar between control and shGan cells. Quantitative data are presented as mean ± SEM. *** P < 0.001 by 2-tailed, unpaired Student’s t test. ( C ) Immunofluorescence images of control and shGan cells from mouse DRG costained for NFL and 14-3-3. Neurofilament aggregates recruit 14-3-3 (arrowhead). Scale bars: 30 μm. Insets are shown at ×3 magnification. Representative images from 3 independent experiments. ( D ) qRT-PCR analysis of Gan -null DRG neurons reveals downregulation of several TFEB targets: TFEB itself, mucolipin-1, LAMP-1, Beclin-1, and cathepsins B and D. Data represent fold change in Gan -null DRG neurons of the respective gene compared with WT and normalized to the expression level of GAPDH in 3 independent experiments plotted as mean ± SEM. * P < 0.05, ** P < 0.01 by 2-tailed, unpaired Student’s t test.

Article Snippet: The following primary antibodies were used: neurofilament NFL chicken polyclonal (CPCA-NF-L, Encor), LC3A/B monoclonal (12741, Cell Signaling Technology), LAMP-1 rabbit polyclonal (ab24170, Abcam), GAPDH rabbit monoclonal (2118, Cell Signaling Technology), TFEB rabbit polyclonal (SAB2108453, Labome), TFEB rabbit polyclonal (A303-773A, Bethyl Laboratories), 14-3-3 rabbit polyclonal (51-0700, Cell Signaling Technology), cathepsin B mouse monoclonal (ab58802, Abcam), cathepsin D mouse monoclonal (ab75852, Abcam), and mucolipin-1 rabbit polyclonal (PA1-46474, Invitrogen).

Techniques: Fluorescence, Control, Immunofluorescence, Quantitative RT-PCR, Expressing