Retrograde transport of transmissible mink encephalopathy within descending motor tracts

Abstract

The spread of the abnormal conformation of the prion protein, PrP(Sc), within the spinal cord is central to the pathogenesis of transmissible prion diseases, but the mechanism of transport has not been determined. For this report, the route of transport of the HY strain of transmissible mink encephalopathy (TME), a prion disease of mink, in the central nervous system following unilateral inoculation into the sciatic nerves of Syrian hamsters was investigated. PrP(Sc) was detected at 3 weeks postinfection in the lumbar spinal cord and ascended to the brain at a rate of approximately 3.3 mm per day. At 6 weeks postinfection, PrP(Sc) was detected in the lateral vestibular nucleus and the interposed nucleus of the cerebellum ipsilateral to the site of sciatic nerve inoculation and in the red nucleus contralateral to HY TME inoculation. At 9 weeks postinfection, PrP(Sc) was detected in the contralateral hind limb motor cortex and reticular thalamic nucleus. These patterns of PrP(Sc) brain deposition at various times postinfection were consistent with that of HY TME spread from the sciatic nerve to the lumbar spinal cord followed by transsynaptic spread and retrograde transport to the brain and brain stem along descending spinal tracts (i.e., lateral vestibulospinal, rubrospinal, and corticospinal). The absence of PrP(Sc) from the spleen suggested that the lymphoreticular system does not play a role in neuroinvasion following sciatic nerve infection. The rapid disease onset following sciatic nerve infection demonstrated that HY TME can spread by retrograde transport along specific descending motor pathways of the spinal cord and, as a result, can initially target brain regions that control vestibular and motor functions. The early clinical symptoms of HY TME infection such as head tremor and ataxia were consistent with neuronal damage to these brain areas.

FIG. 1.

Incubation period following sciatic nerve inoculation of HY TME. In trial 1 (A), hamsters inoculated in the sciatic nerve with HY TME segregated into short and long incubation groups based on the onset of clinical signs. The short incubation group (open circle; n = 4) had an incubation period of 73 ± 7 days, while the long incubation group (filled circle; n = 3) had an incubation period of 143 ± 12 days. The incubation period for the latter group was not statistically different (P > 0.01) from that for the intramuscularly inoculated hamsters (142 ± 14 days; diamond, n = 5). Mock-infected hamsters (open square; n = 5) were clinically normal for the duration of the experiment. In trial 2 (B), with use of a modified sciatic nerve inoculation procedure as described in the text, hamsters developed clinical disease at 68 ± 2 days postinfection (filled triangle; n = 16). Intracerebral and intraperitoneal inoculation with HY TME resulted in incubation periods of 59 ± 2 days (filled diamond; n = 5) and 101 ± 2 days (filled pentagon; n = 6), respectively. Mock-infected hamsters were clinically normal for the duration of the experiment (filled square; n = 9).

FIG. 2.

Temporal distribution of PrP^Sc in the spinal cord following sciatic nerve inoculation of HY TME. Western blot analysis (A) and quantification (B) of PrP^Sc (0.5-mg tissue equivalent) in spinal cord between 3 and 10 weeks postinfection (p.i.). The relative amount of PrP^Sc in each spinal cord segment (indicated as vertebral level) was expressed as a percentage of the PrP^Sc signal from a HY TME-infected brain (0.5-mg brain equivalent) at terminal disease. PrP^Sc signal was measured using a Storm PhosphorImager and ImageQuant software. Illustrated are Western blots from individual hamsters (A) and the relative PrP^Sc signal intensities (PrP^Sc) from an average of three animals (B) at each week postinfection. Standard errors are indicated. Spinal cord vertebral segments refer to lumbar (L), thoracic (T), and cervical (C). Uninfected (U) brain homogenate controls are indicated.

FIG. 3.

Western blot analysis of spinal cord segments enriched for PrP^Sc after sciatic nerve infection with HY TME. Thoracic (T) and cervical (C) spinal cord homogenates were enriched for PrP^Sc by detergent extraction and PK digestion as described in Materials and Methods and analyzed by Western blotting for PrP^Sc levels at the indicated intervals postinfection (Wk p.i.). The amounts in milligrams (mg) of tissue equivalents that were analyzed are indicated for each lane. Brain (B) homogenates from HY TME-infected hamsters with clinical symptoms were prepared as described for Fig. 2.

FIG. 4.

Immunodetection of PrP^Sc in spleen and sciatic nerve after infection with HY TME. (A) Western blot analysis of spleen from uninfected (U) and HY TME-infected hamsters following intracerebral inoculation. Spleens were prepared for analysis as described for spinal cord homogenates in Fig. 3. (B) Western blot analysis of spleen at 5 and 6 weeks postinfection from hamsters inoculated in the sciatic nerve with HY TME. A control HY TME-infected brain (lane B) was prepared as described for Fig. 2. Western blot analysis (C) and PhosphorImager and ImageQuant quantification (D) of PrP^Sc in the sciatic nerve at 10 weeks postinfection. Hamsters were inoculated in the sciatic nerve with HY TME and the ipsilateral (Ip), contralateral (Co), and uninfected (U) sciatic nerves were removed and prepared for Western blot analysis as described for spinal cord homogenates in Fig. 3. The amounts in milligrams (mg) of tissue equivalents that were analyzed are indicated for each lane.

FIG. 5.

PrP^Sc deposition in the midbrain following HY TME inoculation of the sciatic nerve. (A) PrP^Sc immunohistochemistry indicates the location of PrP^Sc (red deposits) at 10 weeks postinfection in both red nuclei (RN). The red nucleus located contralateral (asterisk) to sciatic nerve inoculation was viewed at higher magnification using differential interference contrast microscopy (B) to illustrate PrP^Sc deposition in the ventral and ventrolateral (asterisk) areas of the red nucleus. (C) Hematoxylin-eosin staining of an adjacent brain section illustrates spongiform lesions in the same area of the red nucleus. Abbreviations: Contra, contralateral; Ipsi, ipsilateral; PAG, periaqueductal gray matter; IP, interpeduncular nucleus; SNr, substantia nigra par reticulata. Bar, 100 micrometers.

FIG. 6.

PrP^Sc deposition in pons, cerebellum, and cerebrum following HY TME inoculation of the sciatic nerve. Cresyl violet (A) and PrP^Sc immunostaining (red deposits) (D) of the ipsilateral (Ipsi) cerebellum and pons in adjacent brain sections at 8 weeks postinfection, demonstrating the distribution of PrP^Sc in the interposed nucleus of the cerebellum (Int) and lateral vestibular nucleus (LVe), is shown. Cresyl violet (B) and PrP^Sc immunostaining (E) of the contralateral (Contra) hind limb motor cortex in adjacent brain sections at 9 weeks postinfection, demonstrating the PrP^Sc distribution, is shown. (F) Higher magnification from a different brain section illustrates PrP^Sc immunostaining in the hind limb motor cortex with respect to the third (III), fifth (V), and sixth (VI) cortical cell layers as viewed by differential interference contrast microscopy. An adjacent cresyl violet-stained brain section is illustrated (C). Abbreviations: 4V, fourth ventricle; HL-AgL, hind limb and lateral agranular cortex; AgM, medial agranular cortex. Bar, 100 micrometers.