Deletion of Kif5c Does Not Alter Prion Disease Tempo or Spread in Mouse Brain

Abstract

In prion diseases, the spread of infectious prions (PrPSc) is thought to occur within nerves and across synapses of the central nervous system (CNS). However, the mechanisms by which PrPSc moves within axons and across nerve synapses remain undetermined. Molecular motors, including kinesins and dyneins, transport many types of intracellular cargo. Kinesin-1C (KIF5C) has been shown to transport vesicles carrying the normal prion protein (PrPC) within axons, but whether KIF5C is involved in PrPSc axonal transport is unknown. The current study tested whether stereotactic inoculation in the striatum of KIF5C knock-out mice (Kif5c^-/-) with 0.5 µL volumes of mouse-adapted scrapie strains 22 L or ME7 would result in an altered rate of prion spreading and/or disease timing. Groups of mice injected with each strain were euthanized at either pre-clinical time points or following the development of prion disease. Immunohistochemistry for PrP was performed on brain sections and PrPSc distribution and tempo of spread were compared between mouse strains. In these experiments, no differences in PrPSc spread, distribution or survival times were observed between C57BL/6 and Kif5c^-/- mice.

Keywords: KIF5C; PrP; PrPC; PrPSc; kinesin; prion; scrapie.

Figure 1

PrPSc staining of 22 L scrapie 7 days post-inoculation. IHC using anti-PrP antibody D13 was performed on coronal sections containing the needle track (nt) from B6 mice (left) and Kif5c^−/− mice (middle) and PrP null mice (right). (A,D) Overviews of the cerebral cortex (cx), corpus callosum (cc) and dorsal aspect of the striatum (st). The nt is shown with a dashed line, red blood cells (rbc) can be seen in A from hemorrhage post inoculation. (B,C,E,F) Higher magnification of the cx, cc and st showing PrPSc closely associated with the nt (blue arrowheads). Similar staining was observed in B6 and Kif5c^−/− mice. (G) No cellular prion protein (PrPC) staining is present with D13 IHC on PrP null mouse brain tissue, 7 days post-inoculation. The scale bar in A is 100 µm and applies to panels A,D and G, the bar in C is 50 µm and applies to panels B, C, E and F.

Figure 2

Schematic of the sagittal brain depicting inoculation and coronal section locations. The approximate location of the nt in the striatum (dotted line) and the levels of the three coronal sections (solid lines) analyzed for PrP deposition are shown. The midbrain sections included the substantia nigra and the rostral pons sections included the locus coeruleus.

Figure 3

PrPSc staining of mouse brains 25 days post 22 L scrapie inoculation. IHC using anti-PrP antibody D13 was performed on coronal sections from four areas. A representative B6 mouse is shown on the left side, and a Kif5c^−/− mouse on the right. (A,B) Coronal sections including the striatum. The nt is depicted with a blue box. The insets show higher magnification of the cerebral cortex with normal PrPC staining. (C,D) Coronal sections through the thalamus. The black oval depicts the approximate borders of the thalamus and the high magnification insets show focal PrPSc staining (blue arrowheads). (E,F) Midbrain sections including the substantia nigra (black ovals). Insets show higher magnification and PrPSc present in the substantia nigra. (G,H) Rostral pons sections and higher magnification insets showing subtle PrPSc. The scale bar in A is 1 mm and applies to all the large panels. The scale bar in the inset within panel A is 50 µm and applies to all the insets.

Figure 4

PrPSc staining of mouse brains 40 days post 22 L scrapie inoculation. IHC using anti-PrP antibody D13 was performed on coronal sections from four areas. A representative B6 mouse is shown on the left side, and a Kif5c^−/− mouse on the right. (A,B) Coronal sections including the striatum. The nt is depicted with a blue box. The insets show higher magnification of the cerebral cortex with subtle PrPSc staining (blue arrowheads). (C,D) Coronal sections through the thalamus. The black oval depicts the approximate borders of the thalamus and the high magnification insets show focal PrPSc staining. (E,F) Midbrain sections including the substantia nigra (black ovals). Insets show higher magnification and PrPSc present in the substantia nigra. (G,H) Rostral pons sections and higher magnification insets showing PrPSc. The scale bar in B is 1 mm and applies to all the large panels. The scale bar in the inset within panel B is 50 µm and applies to all the insets.

Figure 5

PrPSc staining of mouse brains 40 days post ME7 scrapie inoculation. IHC using anti-PrP antibody D13 was performed on coronal sections from three areas. A representative B6 mouse is shown on the left side, and a Kif5c^−/− mouse on the right. (A,B) Coronal sections through the thalamus. The black oval depicts the approximate borders of the thalamus and the high magnification insets show very subtle, focal PrPSc staining (blue arrowheads). (C,D) Midbrain sections including the substantia nigra (black ovals). Insets show higher magnification, no PrPSc was observed in the mice shown. (E,F) Rostral pons sections and higher magnification insets showing perineuronal PrPSc. The scale bar in B is 1 mm and applies to all the large panels. The scale bar in the inset within panel B is 50 µm and applies to all the insets.

Figure 6

Survival curve of B6 and Kif5c^−/− mice following 22 L scrapie infection. Twelve mice of each strain were inoculated stereotactically in the striatum with 0.5 µL of 10% 22 L scrapie brain homogenate. Mice were euthanized when advanced signs of terminal disease were present. Black squares indicate Kif5c^−/− mice, black circles indicate B6 mice. The mean ± standard deviation (SD) for each strain are shown adjacent to the legend. Statistical analysis of the survival curve using the Log-rank (Mantel–Cox) test indicated no difference (p = 0.3152).

Figure 7

PrPSc deposition and neuropathology in brain collected from terminally sick 22 L-infected B6 and Kif5c^−/− mice at 129 dpi. A representative B6 mouse is shown on the left side, and a Kif5c^−/− mouse on the right. (A,B) IHC using anti-PrP antibody D13 (brown) performed on sagittal brain sections. PrPSc is widely distributed in both strains of mice. The black rectangle indicates the approximate area of thalamus shown at higher magnification in panels C–J. (C,D) High magnification D13 IHC. (E,H) Anti-Iba1 IHC showing activated microglia in thalamus. (F,I) Anti-GFAP IHC showing astrocytosis. (G,J) Hematoxylin and eosin (H&E) staining demonstrating spongiform lesions. No differences were observed in the distribution or amount of PrPSc, gliosis or spongiform degeneration between the two mouse strains. The scale bar in A is 1 mm and applies to panels A and B. The scale bar in J is 50 µm and applies to panels C–J.