Mapping Glial Autophagy Dynamics in an Amyotrophic Lateral Sclerosis Mouse Model Reveals Microglia and Astrocyte Autophagy Dysfunction

Abstract

Amyotrophic lateral sclerosis (ALS) is defined by motor neuron death. However, recent research has identified non-cell-autonomous mechanisms, with significant involvement of glia in disease progression. We link previous observations of intracellular protein aggregates in glia to the autophagy pathway, the primary mediator of intracellular degradation of large protein aggregates. While dysfunctional autophagy is reported in ALS motor neurons, pre-clinical and clinical outcomes of autophagy modulators have been inconsistent, indicating the need for a nuanced understanding of autophagy dynamics across CNS cell types and ALS-affected regions. We hypothesized that glial autophagy is defective in ALS, with glial-type-specific dysfunction. To investigate in vivo autophagy dynamics, we intercrossed SOD1^G93A mice with transgenic RFP-EGFP-LC3 autophagy reporter mice, enabling the quantification of autophagy degradation, termed flux. Investigation of autophagy dynamics in SOD1 oligodendrocytes, microglia, and astrocytes at key disease stages uncovered useful insights. While oligodendrocytes seemed to mount effective compensatory autophagic responses to combat mutant SOD1, significantly increased autophagy flux was observed in symptomatic spinal microglia and astrocytes in comparison to controls. Symptomatic SOD1 astrocytes displayed greater autophagy dysfunction compared to microglia, with subcellular analysis revealing cell compartment-specific, transient autophagy defects that returned to control levels by end stage. Interestingly, spinal glia showed more pronounced and earlier autophagy dysfunction compared to motor cortex glia, where autophagy dysfunction emerged later in disease end stage, aligning with greater spinal cord pathology reported in this model. Our results suggest that cell-type- and time-specific targeting might be essential when developing autophagy therapeutics for ALS, with prioritization of astrocytic autophagy modulation.

Keywords: ALS; SOD1; SOD1G93A; astrocytes; autophagy; microglia; oligodendrocytes.

FIGURE 1

SOD1 cortical oligodendrocytes show control comparable autophagy flux with subcellular differences at the pre‐symptomatic stage. Representative micrographs of APC labeled oligodendrocytes located next to CTIP2 labeled cortical motor neurons (asterisks) in layer V of the M1 motor cortex, depicting yellow autophagosomes (arrowheads) and red autolysosomes (arrows) at (a) pre‐symptomatic age, P60 and (b) symptomatic age, P120. Quantification of total number of autophagosomes, autolysosomes, and autophagic flux (ratio of red autolysosomes to yellow autophagosomes), per cell, in control and SOD1 mice at (c) pre‐symptomatic and (d) symptomatic stages. Data represent Mean ± SEM, n = 5 mice per group. *p < 0.05 using an unpaired t‐test. Subcellular compartment‐specific quantification of autophagosomes (yellow graphs), autolysosomes (red graphs), and autophagic flux (gray graphs) in soma and processes of oligodendrocytes at (e) pre‐symptomatic and (f) symptomatic stages. Data represent Mean ± SEM, n = 5 mice per group. *p < 0.05 using an unpaired t‐test.

FIGURE 2

Cell soma is the prominent site of autophagy dysfunction in SOD1 cortical microglia with autophagy flux defects appearing at end stage. Representative micrographs of IBA1 labeled microglia located around CTIP2 labeled motor neurons (asterisks) in layer V of the M1 motor cortex, depicting yellow autophagosomes (arrowheads) and red autolysosomes (arrows) at (a) pre‐symptomatic age, P60, (c) symptomatic age, P120 and (e) end stage, P150. Quantification of total number of autophagosomes, autolysosomes and autophagic flux (ratio of red autolysosomes to yellow autophagosomes), per cell, in control and SOD1 mice at (b) pre‐symptomatic, (d) symptomatic, and (f) end stages. Data represent Mean ± SEM, n = 5 mice per group. Subcellular compartment‐specific quantification of autophagosomes (yellow graphs), autolysosomes (red graphs), and autophagic flux (gray graphs) in soma and processes of microglia at (g) pre‐symptomatic, (h) symptomatic and (i) end stages. Data represent Mean ± SEM, n = 5 mice per group. *p < 0.05 using an unpaired t‐test.

FIGURE 3

SOD1 cortical astrocytes show no autophagy impairments until end stage. Representative micrographs of GFAP labeled astrocytes located around CTIP2 labeled motor neurons (asterisks) in the M1 motor cortex, depicting yellow autophagosomes (arrowheads) and red autolysosomes (arrows) at (a) the pre‐symptomatic age, P60, (c) symptomatic age, P120 and (e) end stage, P150. Quantification of total number of autophagosomes, autolysosomes and autophagic flux (ratio of autolysosomes to autophagosomes), per cell, in control and SOD1 mice at (b) pre‐symptomatic, (d) symptomatic, and (f) end stages. Data represent Mean ± SEM, n = 5 mice per group. Subcellular compartment‐specific quantification of autophagosomes (yellow graphs), autolysosomes (red graphs) and autophagic flux (gray graphs) in soma and processes of astrocytes at (g) pre‐symptomatic, (h) symptomatic and (i) end stages. Data represent Mean ± SEM, n = 5 mice per group. *p < 0.05 using an unpaired t‐test.

FIGURE 4

SOD1 spinal oligodendrocytes show control comparable autophagy flux. Representative micrographs of Olig2 labeled oligodendrocytes located in the ventral horn of lumbar spinal cord, depicting yellow autophagosomes (arrowheads) and red autolysosomes (arrows) at (a) the pre‐symptomatic age, P60 and (b) symptomatic age, P120. Quantification of total number of autophagosomes, autolysosomes, and autophagic flux (ratio of autolysosomes to autophagosomes), per cell, in control and SOD1 mice at (c) pre‐symptomatic and (d) symptomatic stages. Data represent Mean ± SEM, n = 5 mice per group. Subcellular compartment‐specific quantification of autophagosomes (yellow graphs), autolysosomes (red graphs) and autophagic flux (gray graphs) in soma and processes of oligodendrocytes at (e) pre‐symptomatic and (f) symptomatic stages. Data represent Mean ± SEM, n = 5 mice per group. *p < 0.05 using an unpaired t‐test.

FIGURE 5

SOD1 spinal microglia show increased autophagy flux at the symptomatic stage. Representative micrographs of IBA1 labeled microglia in the ventral horn of the lumbar spinal cord, depicting yellow autophagosomes (arrowheads) and red autolysosomes (arrows) at (a) the pre‐symptomatic age, P60, (c) symptomatic age, P120 and (e) end stage, P150. Quantification of total number of autophagosomes, autolysosomes and autophagic flux (ratio of autolysosomes to autophagosomes), per cell, in control and SOD1 mice at (b) pre‐symptomatic, (d) symptomatic, and (f) end stages. Data represent Mean ± SEM, n = 5 mice per group, *p < 0.05 using an unpaired t‐test. Subcellular compartment‐specific quantification of autophagosomes (yellow graphs), autolysosomes (red graphs) and autophagic flux (gray graphs) in soma and processes of microglia at (g) pre‐symptomatic, (h) symptomatic, and (i) end stages. Data represent Mean ± SEM, n = 5 mice per group. *p < 0.05 using an unpaired t‐test.

FIGURE 6

Symptomatic SOD1 spinal microglia contain misfolded SOD1 inclusions with control comparable levels of LAMP1, p62, and ubiquitin staining. (a) Representative micrographs of IBA1‐labeled microglia and C4F6 misfolded SOD1 staining in control and SOD1 mice. (b) LAMP1, (c) p62, and (d) ubiquitin staining intensities in IBA1‐labeled spinal microglia of control and SOD1 mice. Data represent Mean ± SEM, n = 5 mice per group.

FIGURE 7

SOD1 spinal astrocytes show increased autophagy flux at the symptomatic stage. Representative micrographs of GFAP labeled astrocytes in the ventral horn of the lumbar spinal cord, depicting yellow autophagosomes (arrowheads) and red autolysosomes (arrows) at (a) the pre‐symptomatic age, P60, (c) symptomatic age, P120, and (e) end stage, P150. Quantification of total number of autophagosomes, autolysosomes, and autophagic flux (ratio of autolysosomes to autophagosomes), per cell, in control and SOD1 mice at (b) pre‐symptomatic, (d) symptomatic, and (f) end stages. Data represent Mean ± SEM, n = 5 mice per group, **p < 0.01 using an unpaired t‐test. Subcellular compartment‐specific quantification of autophagosomes (yellow graphs), autolysosomes (red graphs), and autophagic flux (gray graphs) in soma and processes of astrocytes at (g) pre‐symptomatic, (h) symptomatic, and (i) end stages. Data represent Mean ± SEM, n = 5 mice per group. *p < 0.05, ***p < 0.001 using an unpaired t‐test.

FIGURE 8

Symptomatic SOD1 spinal astrocytes contain misfolded SOD1 inclusions with reduced p62 and increased ubiquitin staining compared to controls. (a) Representative micrographs of GFAP‐labeled astrocytes and C4F6 misfolded SOD1 staining in control and SOD1 mice. (b) LAMP1, (c) p62, and (d) ubiquitin staining intensities in GFAP‐labeled spinal astrocytes of control and SOD1 mice. Data represent Mean ± SEM, n = 4–5 mice per group. *p < 0.05 using an unpaired t‐test.

FIGURE 9

Healthy microglia show an age‐dependent increase in autophagosome size from P60 to P150. (a) Representative micrographs of yellow autophagosomes within IBA1 labeled microglia in the motor cortex of healthy control mice. Microglial autophagosome area (b) and autophagosome staining intensity (c) in control and SOD1 mice from P60 to P150. Data represent Mean ± SEM, n = 5 mice per group. *p < 0.05, **p < 0.01, ****p < 0.0001 using one‐way ANOVA with Tukey's multiple comparisons test. (d) Representative micrographs of yellow autophagosomes within IBA1 labeled microglia in the ventral horn of lumbar spinal cord in healthy control mice. Microglial autophagosome area (e) and autophagosome staining intensity (f) in control and SOD1 mice from P60 to P150. Data represent Mean ± SEM, n = 3–5 mice per group. *p < 0.05, **p ≤ 0.01, ***p < 0.001 using one‐way ANOVA with Tukey's multiple comparisons test. P; post‐natal day.

FIGURE 10

SOD1 spinal cords show key autophagy protein dysregulation from symptom onset. (a) Immunoblot analysis of autophagy‐related proteins in control and SOD1 from P30, P60 pre‐symptomatic stages to P90 symptom onset and P120 symptomatic stage. Quantification of (b) p62, (c) LC3‐II, (d) Hsc70, (e) LAMP2A, and (f) cathepsin D protein levels. Data represent mean ± SEM, n = 3 mice per group, *p < 0.05 and **p < 0.01 using two‐way ANOVA with Šídák's multiple comparisons test.