Fig 2: NDV replication level was decreased after knocking down SLC1A3. (a) A549 cells were transfected with siRNA (si-A3 and si-NC) followed by NDV (Herts/33, MOI= 0.1/1) infection, and cells were harvested to determine the effect of SLC1A3 siRNA using qRT-PCR. (b, c) After treatment with siRNA, NDV-infected A549 cell samples and supernatant were harvested for TCID50 measurement of NDV titers, and the cells were subjected to western blot analysis of the SLC1A3 and NP expression. (d, e) A549 cells (Control and shSLC1A3) were infected with NDV (Herts/33, MOI=0.1/1), and cell samples and supernatant were harvested for western blot and TCID50 detection. (f, g) Cell proliferation activity of A549 cells (Control and shSLC1A3) (F) and cells infected with NDV Herts/33 (MOI=0.1) (g) were measured using the Enhanced Cell Counting Kit-8. (H) the growth curve of NDV in A549 cells (Control and shSLC1A3). Cells were infected with NDV Herts/33 (MOI=0.1). Supernatants were harvested at the indicated times and were subjected to the TCID50 assay. (I) After treatment with TFB-TBOA, a glutamate transporter inhibitor, cells infected with NDV (Herts/33, MOI=1) were harvested at 12 h for western blot detection.

Fig 3: Human astrocytes express MHC-II in brain tissue from PD patients and healthy controls. The aSYN pathology was confirmed in the PD cases by immunostaining of aSYN and phosphorylated aSYN (p-aSYN) (a). The PD brains express higher levels of MHC-II compared to control brains (b). MHC-II levels correlate with the expression of p-aSYN in PD brains (c). There is a correlation between the MHC-II levels in the mesencephalon and striatum in the PD brains, but not in the control brains (d, e). Astrocytes labeled with GLAST (f) or GFAP (g) are expressing high levels of MHC-II, as well as Iba1-labeled microglia (h). Quantifications of the immunostainings revealed that 31% of the MHC-II area overlapped with the astrocyte marker GLAST (i), 15% overlapped with the reactive astrocyte marker GFAP (j), and 32 % overlapped with the microglia marker Iba1 in the PD brain (k). A similar overlap between MHC-II and GLAST, GFAP, and Iba1 (35%, 10%, and 33%, respectively) was revealed from the analysis of the control brains (j–k). Scale bars: a = 20 µm; f = 2 µm; g, h = 5 µm

Fig 4: Protein changes in ONHAs corroborate bioenergetics data. (A) Glucose transporter-1 protein levels in Stretched ONH astrocytes are significantly higher than Control (*p = 0.0225, n = 7 Control, n = 8 Stretch). Retinal lysate from a 2 month-old mouse was used as a positive control for each protein analyzed, while negative control was the signal obtained when no primary antibody was included in the capillary. (B) Lactate dehydrogenase-A, the astrocyte-specific isoform of the enzyme that catalyzes the interconversion of pyruvate and lactate, has equivalent protein levels in Control and Stretch ONH astrocytes. (C) Glucose-6-phosphate dehydrogenase, the enzyme that shunts glucose into the pentose phosphate pathway, is no different in Control and Stretch ONH astrocytes. (D) Glutamine synthetase, the enzyme that synthesizes glutamine from glutamate, is no different in Control and Stretch ONH astrocytes. (E) The monomeric form of glutamate-aspartate transporter (GLAST) has significantly higher protein levels in the Stretch as compared to the Control ONH astrocytes (p = 0.020; n = 4 Control, n = 5 Stretch). (F) GLAST dimer protein levels are no different in Control and Stretch ONH astrocytes.

Fig 5: NDV infection induced significant SLC1A3 upregulation. (a-c) A549 cells were harvested at 6, 12, 18 and 24 h.p.i. after NDV infection (Herts/33 or LaSota or SH15), and non-infected cells were used as the control. The mRNA levels of different amino acid transporters (SLC1A2, SLC1A3, SLC25A25, SLC7A14, SLC7A7, SLC6A19, SLC38A2) were determined using qRT-PCR. ß-actin was used as an internal reference gene. (d) A549 cells were harvested at 12 and 24 h.p.i. after NDV infection (Herts/33, MOI=1). The mRNA levels of the SLC1 family were detected after NDV infection in A549 cells. (e) A549 cells were harvested after NDV infection (Herts/33, MOI=0.1, 0.5, 1, 5) and were used to detect the mRNA levels of SLC1A3. (f) Amino acid transporters and their substrates. (g-h) the expression levels of SLC1A3 were detected in A549/DF-1 cells post-NDV infection.