Iba1+ cells envelop neurons in SSLOW-infected brains

a. Immunostaining for microglia (IBA1, red) and neurons (NeuN, green) showing neuronal envelopment by myeloid cells in Cx3cr1/EGFP mice infected by SSLOW via intraperitoneal route at the terminal stage of the disease. b. Immunostaining for microglia (IBA1, red) and neurons (NeuN, green) of non-infected, age-matched Cx3cr1/EGFP mice. Confocal microscopy imaging followed by 3D reconstruction was used for both a and b.

Reactive myeloid cells are highly mobile

Brain slices were prepared acutely using non-infected Cx3cr1/EGFP (normal) mice, or Cx3cr1/EGFP mice infected with SSLOW via ip route and examined at sub-clinical, early clinical or advanced stages of the disease. Analysis of mean speed (a) and total distance (b) traveled by individual EGFP+ cells over 3-hour period. The midline of the box-and-whisker plot denotes the median, the + represents the mean, and the ends of the box plot denote the 25th and 75th percentiles. c. Examples of tracks recorded from EGFP+ cells. Colored lines represent tracks of individual cells recorded within 3-hour period. d. Principal component analysis of mobility parameters. n=70-90 cells per group, *p<0.05, **p<0.01, ****p<0.0001, ns - non-significant by non-parametric Kruskal-Wallis test with Dunn’s multiple comparison.

High- and low-mobility behavioral patterns of myeloid cells in prion-infected brains

Acute brain slices were prepared using normal, non-infected Cx3cr1/EGFP mice, or SSLOW-infected Cx3cr1/EGFP mice and examined at the sub-clinical, early clinical or advanced stages of the disease. a Rose plot of individual cell trajectories. In brain slices from SSLOW-infected animals, EGFP+ cells showed two behavioral patterns – with high and low mobility. b Change in the percentage of high-mobility EGFP+ cells with the disease progression. N=3 animals per group. The data presented as Means ± SD, **p<0.01, ****p<0.0001, ns - non-significant by Tukeys’ multiple comparisons test. c, d, e Analysis of mean speed (c), total distance travelled over 3-hour period (d), and mean directional change rate (e) for individual high- and low-mobility EGFP+ cells in slices from SSLOW-infected mice at three disease stages, and normal mice. The midline of the box-and-whisker plot denotes the median, the + represents the mean, and the ends of the box plot denote the 25th and 75th percentiles. n=25-30 cells per group, *p<0.05, **p<0.01, ***p<0.01, ****p<0.0001, ns - non-significant by non-parametric Kruskal-Wallis test with Dunn’s multiple comparison.

Behavioral patterns of high-mobility reactive myeloid cells

Time-lapse imaging of acute brain slices of SSLOW-infected Cx3cr1/EGFP mice captured at the early clinical (a), advanced (b, d), and subclinical (c) stages of disease. a (Video S3): An EGFP+ cell (indicated by an arrow) extends a process toward neurons #1 and #2, migrates along this process, and subsequently envelops both neurons while simultaneously extending processes toward neurons #3, #4, and #5. b (Video S10): EGFP+ cell #1 migrates toward and surveys a neuron (circled), then departs. Subsequently, a second EGFP+ cell (#2) migrates to and interacts with the same neuron. White and blue curves trace the respective migration paths of the two cells. c Upper panels: An EGFP+ cell approaches, envelops, and then retracts from a neuron. Middle panels: A migrating EGFP+ cell (indicated by an arrow) surveys five distinct neurons (circled), interacting simultaneously with neurons #1 and #2, followed by #3 and #4. Lower panels: An EGFP+ cell maintains prolonged contact with a neuron. d (Video S5): An EGFP+ cell (arrow) migrates across the field, initially extending processes (arrowhead) toward a neuron. The cell body then translocates along these processes, briefly contacts one neuron, and continues movement toward another. All videos were recorded over a 3- hour period with 5-minute intervals. Nuclei were visualized using Hoechst staining. Scale bars = 20 μm.

Behavioral patterns of low-mobility reactive myeloid cells

Time-lapse imaging of acute brain slices of SSLOW-infected Cx3cr1/EGFP mice, captured at early clinical (a, b) and advanced (c) stages of disease. a (Video S8): An EGFP+ cell exhibits prolonged interactions simultaneously with two neurons. b (Video S3): An EGFP+ cell shows sustained envelopment of neuron #1, then initiates simultaneous envelopment of neuron #2 while maintaining contact with neuron #1 and possibly neuron #3. c An EGFP+ cell moves toward neurons #1 and #2, partially envelops neuron #1, and fully envelops neuron #2. All videos were recorded over a 3-hour period with 5-minute intervals. Nuclei were visualized using Hoechst staining. Scale bars = 20 μm. d, e 3D reconstructions from confocal microscopy images of fixed brain slices immunostained for myeloid cells (IBA1, red) and neurons (NeuN, green) in WT mice infected with SSLOW via the intraperitoneal route, analyzed at early clinical and advanced disease stages. Scale bar = 25 μm.

Analysis of cell morphology

Acute brain slices were prepared from normal, non-infected Cx3cr1/EGFP mice and from SSLOW-infected Cx3cr1/EGFP mice at the at sub-clinical, early clinical or advanced stages of the disease. Analysis of cell radius (a), cell area (b), cell perimeter (c) and shape index (d) of high- and low-mobility EGFP+ cells in slices from SSLOW-infected mice at three disease stages, and normal mice. The midline of the box-and-whisker plot denotes the median, the + represents the mean, and the ends of the box plot denote the 25th and 75th percentiles. n=30-90 cells per group, *p<0.05, **p<0.01, ***p<0.01, ****p<0.0001, ns - non-significant by non-parametric Kruskal-Wallis test with Dunn’s multiple comparison.

Sustained Ca2⁺ bursts in high-mobility myeloid cells

Acute brain slices were prepared from non-infected Cx3cr1/EGFP mice and from SSLOW-infected Cx3cr1/EGFP mice at the advanced stage of disease. a Quantification of signal intensity of Ca2⁺ puncta within high- or low-mobility EGFP⁺ cells. Data from normal, non-infected mice are shown for reference. Sustained Ca2⁺ bursts were detected using Calbryte-590 AM and averaged per cell over a 3-hour period. N = 24-28 cells per group. *p < 0.05, **p < 0.01, ns = not significant by non-parametric Kruskal-Wallis test with Dunn’s multiple comparisons. b Changes in Ca2⁺ signal intensity in individual EGFP⁺ cells over the 3-hour imaging session. Images were acquired at 5-minute intervals. c Time-lapse imaging of acute brain slice from a SSLOW-infected Cx3cr1/EGFP mouse recorded over 3 hours. Upper panels (Video S19): EGFP⁺ cell (#1) envelops a neuronal soma and exhibits low Ca2⁺ activity. Lower panels: highly mobile EGFP⁺ cell (#2) displays sustained somatic Ca2⁺ bursts. Scale bars = 50 μm.

Inhibition of the P2Y6 receptor reduces motility of reactive myeloid cells

Acute brain slices were prepared from non-infected Cx3cr1/EGFP mice and from SSLOW-infected Cx3cr1/EGFP mice at the early clinical stage of disease. a, b Quantification of mean speed (a) and total distance traveled (b) by individual EGFP⁺ cells over a 3-hour period in brain slices from SSLOW-infected mice treated with either MRS-2578 (2 μM) or vehicle control. Data from normal, non-infected mice are shown for reference. n=100- 250 cells per group. **p < 0.001, ***p < 0.0001 by Tukey’s multiple comparisons test. c, Time-lapse imaging of MRS-2578-treated acute brain slice from a SSLOW-infected Cx3cr1/EGFP mouse recorded over 3 hours. The EGFP⁺ cell exhibits bidirectional movement between two neuronal somas (labeled #1 and #2), sequentially enveloping each soma. Scale bars = 20 μm.

In vitro analysis of CD11b⁺ cell mobility

CD11b⁺ cells were acutely isolated from Cx3cr1/EGFP mice at the clinical stage of SSLOW infection or from non-infected Cx3cr1/EGFP mice. a,b,c Quantification of cell mobility parameters, including mean speed (a), total track distance (b), and mean directional change rate (c) of CD11b⁺/EGFP⁺ cells over a 3-hour period, in the presence or absence of N2a cells. In box-and-whisker plots, the midline indicates the median, the “x” denotes the mean, and the box limits represent the 25th and 75th percentiles. n = 178-354 cells per group. Statistical significance: p < 0.05; p< 0.01; *p < 0.001; **p < 0.0001; ns, not significant by non-parametric Kruskal-Wallis test with Dunn’s multiple comparison. (d) Representative cell tracks of CD11b⁺/EGFP⁺ cells over 3 hours. Colored lines indicate trajectories of individual cells.