PrPSc spreading patterns in the brain of sheep linked to different prion types

Abstract

Scrapie in sheep and goats has been known for more than 250 years and belongs nowadays to the so-called prion diseases that also include e.g. bovine spongiform encephalopathy in cattle (BSE) and Creutzfeldt-Jakob disease in humans. According to the prion hypothesis, the pathological isoform (PrPSc) of the cellular prion protein (PrPc) comprises the essential, if not exclusive, component of the transmissible agent. Currently, two types of scrapie disease are known--classical and atypical/Nor98 scrapie. In the present study we examine 24 cases of classical and 25 cases of atypical/Nor98 scrapie with the sensitive PET blot method and validate the results with conventional immunohistochemistry. The sequential detection of PrPSc aggregates in the CNS of classical scrapie sheep implies that after neuroinvasion a spread from spinal cord and obex to the cerebellum, diencephalon and frontal cortex via the rostral brainstem takes place. We categorize the spread of PrPSc into four stages: the CNS entry stage, the brainstem stage, the cruciate sulcus stage and finally the basal ganglia stage. Such a sequential development of PrPSc was not detectable upon analysis of the present atypical/Nor98 scrapie cases. PrPSc distribution in one case of atypical/Nor98 scrapie in a presumably early disease phase suggests that the spread of PrPSc aggregates starts in the di- or telencephalon. In addition to the spontaneous generation of PrPSc, an uptake of the infectious agent into the brain, that bypasses the brainstem and starts its accumulation in the thalamus, needs to be taken into consideration for atypical/Nor98 scrapie.

Figure 1

Characteristic PrP^Sc deposition patterns in classical and atypical/Nor98 scrapie: The same lymph follicle in the tonsil of a sheep with classical scrapie is shown stained either with the PET blot method (a) or conventional immunohistochemistry (b) (both mAb P4, bars = 250 μm). In the cervical spinal cord segment of a sheep with atypical/Nor98 scrapie (c) stained with the PET blot method (mAb P4, bar = 800 μm) synaptic PrP^Sc aggregates are present in the substantia gelatinosa (vertical arrow) and granular PrP^Sc in the corticospinal tract (horizontal arrows). In the cerebellar cortex of a classical scrapie sheep stained with the PET blot (d) complex PrP^Sc deposits are visible. Glia-associated PrP^Sc deposits take a stellate form in the molecular layer (mAb P4, bar = 150 μm; M = molecular layer, P = layer of Purkinje cells, G = granular cell layer). In the majority of the atypical/Nor98 scrapie sheep the cerebellar cortices show a more intense staining of the molecular layer than the granular layer (e) (PET blot, mAb P4; bar 600 μm). Intraneuronal, perineuronal and glia-associated PrP^Sc aggregates (f) in the reticular formation of a classical scrapie sheep (PET blot, mAb P4; bar = 50 μm). Tissue sections derived from sheep with the genotypes VRQ/ARH (a,b), AHQ/AHQ (c,e) and ARQ/ARQ (d,f).

Figure 2

Classification of the PrPSc spread during disease development in classical scrapie.The examined classical scrapie cases classified into four stages of PrP spread according to certain affected neuroanatomical sites (PET blots, mAb P4). In the CNS entry stage (a - d) only discrete PrP^Sc deposits are visible in the obex region, while in the brainstem stage (e - h) PrP^Sc aggregates are clearly visible in the brainstem and start to appear in more rostral structures. Once PrP^Sc deposits can be found in the deep cortical layers of the frontal cortex (i), the cruciate sulcus stage (i - l) is reached. In the basal ganglia stage, intense deposits in basal ganglia and thalamic nuclei can be found (m - p). Brain sections shown for the first, third and fourth stage derived from sheep with the genotype ARQ/ARQ while the sheep whose brain sections are depicted in the brainstem stage carried the genotype ARH/VRQ (bar = 5 mm).

Figure 3

Progression of classical scrapie in the brain shown for certain affected neuroanatomical sites. The colour code agrees with the one in Figure 5.

Figure 4

Accumulation of PrP^Sc in different brain regions during disease progression: The four stages of the examined classical scrapie cases are depicted in four overlying graphs that illustrate how PrP^Sc aggregates (PET blot method, mAb P4) are increasingly accumulated in the brains from caudal (left) to rostral (right). Evaluation of PrP^Sc intensity was performed on a scale from 0.5 - 4 (see material and methods) and shown for the following brain areas: 1 dorsal motor nucleus of the vagus nerve (DMNV), 2 inferior olive, 3 dorsal tegmental nucleus, 4 cerebellar molecular layer, 5 cerebellar granular layer, 6 cerebral peduncle, 7 central grey (mesencephalon), 8 caudate nucleus, 9 ventral pallidum, 10 rostral commissure, 11 cruciate sulcus, 12 frontal white matter.

Figure 5

Form and appearance of PrP^Sc deposits in classical and atypical/Nor98 scrapie sheep presented for representative CNS regions. As a sequential development of PrP^Sc distribution could not be observed upon analysis of the present atypical/Nor98 scrapie cases, no coding colours were used for the results of this scrapie type in contrast to classical scrapie sheep. PrP^Sc deposits are detectable in the respective location in classical scrapie sheep belonging to the stages of spread indicated in the bottom of the figure using the same colour code as in Figure 3.

Figure 6

PrP^Sc distribution in the brain of atypical/Nor98 scrapie: Brain sections of an atypical/Nor98 scrapie case stained with the PET blot (mAb P4) have a different PrP^Sc distribution than the ones of classical scrapie cases as shown in Figure 2 (bar = 5 mm). Brain section derived from a sheep with the genotype ARQ/AHQ.

Figure 7

Differences in the neuroanatomical distribution of PrP^Sc deposit in atypical/Nor98 and classical sheep scrapie: In atypical/Nor98 scrapie, white matter structures like the external capsule or rostral commissure contain substantially more PrP^Sc than the subcortical nuclei or basal ganglia respectively (a and c), whereas this is the reverse in classical scrapie (b and d). The external capsule (a and b) and the rostral commissure (c and d) are marked with arrows (mAb P4, bars = 1 mm). Tissue derived from sheep with the genotypes ARQ/AHQ (a), ARQ/ARQ (b and d) and AHQ/AFRQ (c).