Scrapie time mouse adapted ME7 scrapie strain to

Scrapie
can be experimentally induced from one species to another species. Inter-specie
transmission when successful result in prolonged incubation periods, a phenomenon
referred to as species barrier (Dickinson AG, 1976; Kimberlin RH, 1979;
Beringue V et al., 2008). This phenomenon also known as transmissibility
barrier has been discovered to be abrogated with the adaptation of the strain
to the new host after 2-3 passages (Bruce ME et al., 1991).  Serial passages of sheep scrapie showed
distinct diseases phenotypes and perpetuation of the same biological properties
upon serial passage in experimental animals of same genetic background (Bruce
ME et al., 1992). Strain typing parameters includes incubation period, vacuolar
lesion profiles, PrPSc immunohistochemistry
  and molecular PrPSc profile (Siso S et al., 2012). In
this study the transmissibility of long time mouse adapted ME7 scrapie strain
to TgSShp PrP mice at second passage
was examined.  We observed there was
reduction in the incubation time from 227.0 ±
6.35 days at first passage to 185.3 ± 5.41 days at second passage. Consistent
with our result is the report that cross-species transmission leads to an
inefficient disease process resulting in long incubation time at first passage
but becomes shorten after initial prion adaptation in the new host.  This is probably because there is increased
efficient propagation of the donor PrPSc and the experimentally
induced scrapie at second passage became more adapted to the environment of the
recipient host (Marsh RF et al., 1991; Taylor DM et al., 1986; Shi et al.,
2015).

Glycosylation profile is one of the biochemical features used to
discriminate between scrapie strains. Results from our study indicate a change
in glycoform patterns. PrPSc from ME7 infected TgSShp PrP Mice brains were predominantly
di-glycosylated in contrast to the mono-glycosylated band pattern observed C57BL/6
–ME7 infected mice brains. Similar to our results is the findings reported by
Shi et al, (2012). Same glycosylation pattern was observed at the third passage
(data not shown) suggesting that glycosylation profile formed during
interspecies transmission is maintained over serial passaging in the same host
(Beck KE et al., 2013). This may also support the observation that the brain
microenvironment of a new host obviously affects the PrPSc
properties during inter-species transmission. Also our results showed that PrPSc
from ME7 infected C57BL/6 mice were more resistant to proteinase k digestion
even at high concentration of 500µg/ml as
against that of TgSShp PrP Mice in which at same concentration relatively
weaker signal of PrPSc were detected. The slightly higher
resistance ability of mouse adapted ME7 scrapie strain might be attributed to
stability resulting from repeated passaging.

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To
determine the neuropathological changes induced by this interspecies
transmission, hematoxylin staining was performed on formalin-fixed tissues of TgSShp PrP- ME7 infected and control mice.
Spongiform degeneration, a porous sponge like lesion in brain of diseased
animals is one of the pathological hallmarks of prion diseases and has been
found to be more pronounced in the hippocampal region of brains infected with
ME7 scrapie strain. Dentate gyrus, CA3 and CA1 hippocampal region were examined
for spongiform degeneration.  In this
study, sponge like lesions were more pronounced in the CA1 region of terminally
sick TgSShp PrP- ME7 mice examined. 
Numerous oval shaped vacuoles were observed in the fields of view most
especially in the CA1 hippocampal region of the infected brain. Another
criterion considered for strain peculiarity was the vacuolar lesion profile. As
reported by Fraser and Dickson (1968) the pattern of vacuolar lesion in the
brain is characteristic for a given strain in a given host using a
non-transgenic mice transmission experimental model. Validating this in a prion
transgenic mice model, the nine grey areas examined for vacuolar lesion showed
distinct vulnerabilities towards the ME7 scrapie agent. From our result, severe
vacuolation was observed in the hippocampal, brain stem, substantia nigra
regions of the infected brains. Other grey areas such as hypothalamus, thalamus
and septal area also showed marked vacuolation but less conspicuous vacuolation
in the cerebellar cortex. In agreement with our findings was a similar results
reported by C. Cunningham et al., (2005) where they observed that ME7 strain
showed marked vacuolation of the hippocampal, septal and thalamic neuropil.

Reports from experimental studies done has showed
that the deposition pattern of PrPSc  across
brains of infected mice is agent strain dependent (Beck KE et al., 2010; Brown
DA et al., 2003; Bruce ME et al., 1994). In this study, we detected PrPSc accumulation
with formation of plaques.  PrPSc aggregation
accompanied with plaques was more prominent in the CA1 hippocampal region. This is consistent with the report that, in the
hippocampus, ME7 predominantly targeted the polymorph layer of the dentate
gyrus and stratum lucidum, the CA3 field and the pyramidal layer with granular
labeling and small aggregates (Beck et al., 2012). In a similar study carried
out by Siso S. et al., (2012), they reported that amyloid plaques are abundant
and characteristics of murine 87V but do inconsistently occur in ME7 and 22A
infections, vascular plaques were occasionally present in ME7 infections of
VRQ/VRQ Cheviot and ARQ/ARQ Suffolk sheep. Also, C. Cunningham and colleagues,
(2005) reported that ME7 infected brains showed a more PrPSc
punctuate labeling pattern with some small plaques deposition both in the
hippocampus and thalamic brain regions. Results
from studies using mouse-adapted prion strains has demonstrated that
neuroinvasive strains formed diffuse, non fibrillar PrP aggregates in the
Central nervous system and mice rapidly progress to terminal disease, while
weakly neuroinvasive strains form dense, congophilic, fibrillar plaques and
mice progressed to terminal disease more slowly. These findings suggest that
the non-fibrillar PrP aggregates  are
more toxic which is in agreement with studies indicating that smaller
aggregates of the mammalian prion protein, which could be more readily
generated by strains that form fragile particles, are markedly more infectious
than larger aggregates (Laura Solforosi et al., 2013; Silveira JR et al., 2005).
 A strong counter stain is a
well-known marker of sick cells about to die by an apoptotic pathway (Liu et
al., 2015; Sharma et al., 2016). Our results showed decreased in population of
neuronal cells and a significant higher accumulation of hematoxylin-counter stain.
Also, there was decrease in the
expression level of neuronal marker used to measure extent of neuronal loss
between infected and control mice in this study. Hippocampal neuronal cell
death has been observed to be a late stage phenomenon in ME7 scrapie infections.
Hippocampal CA1 pyramidal neuronal loss was reported to be significant in ME7
and 79A infected mice (C. Cunningham et al., 2003, 2005; Jeffrey M et al.,
2000; Hilton KJ et al., 2013). Most likely, the severe aggregation of PrPSc in CA1 in this study could have resulted
from the death of CA1 neurons.

The increase in population of reactive astrocytes
was well pronounced in brain tissues of the infected mice. GFAP upregulation
was severe in the dentate gyrus of the brain. In the brains from the age
matched control mice, GFAP positive astrocytes are detectable in the dentate
gyrus, CA3 and CA1 brain region but not as severe as the degree of detection in
the terminally diseased mice. This result agrees with similar findings reported
by Hilton K J et al., (2013). They demonstrated that the hippocampus and
dentate gyrus are sites of GFAP upregulation in ME7- and 79A- animals.

Synaptic loss has been found to be a key feature
of many neurodegenerative diseases and it occurs before neuronal death in many
diseases state (Hilton K J et al., 2013). 
Biochemical studies of the Hippocampus of ME7 infected animals revealed
that proteins of the pre-synaptic terminal compartment declines first
especially those of the synaptic vesicles (Gray BC et al., 2009).  Experimental studies of ME7 infected animals
using electron microscopy has clearly shown loss of synaptic vesicles’
integrity to be the first apparent morphological feature of synaptic
dysfunction in stratum radiatum (Siskova Z et al.,2009) Different studies have
shown the degeneration of asymmetric terminals to be a detectable and early
pathological hallmark in ME7 infection. It precedes any observable degeneration
of cell bodies of CA3 or CA1 pyramidal neurons (Siskova Z et al., 2009; Gray BC
et al., 2009). Various studies has revealed PrPC to be highly
concentrated in presynaptic terminals where it has been found to co-localize
with Synaptophysin and also widespread in brain structures associated with
synaptic plasticity (Fournier JG et al., 2000; Haeberle AM et al., 2000;  Herms J et al., 1999;  Sales N et al., 1998). From our western blot results there was no significant difference in
the expression level of pre-synaptic protein marker: Synaptophysin in ME7
infected brains when compared with the age matched control but in contrast, we
observed marked difference in the expression level of post synaptic protein
marker PSD 95 in ME7 infected brains relative to the age matched control
brains. This result is in contrast with a study findings reported by C.
Cunningham et al., (2005) in which they observed a clear loss of Synaptophysin
in the hippocampus of mice infected with scrapie strain 22L, 79A and ME7 but
the loss was most severe in the ME7 infected animals. One reason for this
difference could be because whole brain homogenates were used for our western
blot but specific brain regions were examined during the Immunohistochemical
staining for the study results reported by C. Cunningham et al.

Results from a study conducted by Peretz D and
colleagues (2002) reported that a change in conformation accompanied the
emergence of new prion strain during interspecies transmission while the
conformational stability of PrPSc was preserved when a strain bred true in a new
host. Strain specific differences in PrPSc conformation may
reasonably evoke differences in neuronal targeting, trafficking and
processing/degradation of protein (Bruce ME et al., 1994; DeArmond SJ et al.,
1993; Bessen RA et al., 1994; Gonzalez L et al., 2003).Our results showed a
change in conformation stability of ME7 infected C57BL/6 mice after
interspecies transmission to TgSShp PRP mice. The resulting PrPSc
specie from the cross specie transmission was relatively more stable and
resistant to denaturation by Guanidine Hydrochloride. Although, conformational
stability has been reported to be directly proportional to the length of
incubation in mice (Legname G et al., 2006) results from this study in agreement
with similar findings by Laura Pirisinu and others (2010, 2013) reported
contrawise.

Differences in pathological features, that is,
pattern of histological lesions and PrPSc accumulation in scrapie infected mice has been
reported to be related to the interaction between the host (genotype, age, sex
and breed) and the agent strain (passage history, dose, route of inoculation,
donor genotype and organ used for preparing inoculum) (Fraser H, 1976; Jeffrey
M et al., 2002; Beringue V et al., 2012). Also, it has been suggested that some
features of disease phenotype might be strain-encoded and propagated in the
recipient host (Siso S. et al., 2012).

In conclusion, Interspecies transmission of long term mouse
adapted ME7 scrapie strain was successful in transgenic mice expressing ovine
prion protein but with accompanying biophysical and neuropathological changes.
Differences in survival time, 
biophysical (Glycoform change, conformational stability) and
neuropathological features (plaque formation) reported in this study could be
due to inherent strain or host characteristics or it could have resulted from
some kind of strain mutation leading to a change in the properties in the new
host. Further studies are needed to confirm if the observed changes are
maintained in subsequent passages within the same hScrapie
can be experimentally induced from one species to another species. Inter-specie
transmission when successful result in prolonged incubation periods, a phenomenon
referred to as species barrier (Dickinson AG, 1976; Kimberlin RH, 1979;
Beringue V et al., 2008). This phenomenon also known as transmissibility
barrier has been discovered to be abrogated with the adaptation of the strain
to the new host after 2-3 passages (Bruce ME et al., 1991).  Serial passages of sheep scrapie showed
distinct diseases phenotypes and perpetuation of the same biological properties
upon serial passage in experimental animals of same genetic background (Bruce
ME et al., 1992). Strain typing parameters includes incubation period, vacuolar
lesion profiles, PrPSc immunohistochemistry
  and molecular PrPSc profile (Siso S et al., 2012). In
this study the transmissibility of long time mouse adapted ME7 scrapie strain
to TgSShp PrP mice at second passage
was examined.  We observed there was
reduction in the incubation time from 227.0 ±
6.35 days at first passage to 185.3 ± 5.41 days at second passage. Consistent
with our result is the report that cross-species transmission leads to an
inefficient disease process resulting in long incubation time at first passage
but becomes shorten after initial prion adaptation in the new host.  This is probably because there is increased
efficient propagation of the donor PrPSc and the experimentally
induced scrapie at second passage became more adapted to the environment of the
recipient host (Marsh RF et al., 1991; Taylor DM et al., 1986; Shi et al.,
2015).

Glycosylation profile is one of the biochemical features used to
discriminate between scrapie strains. Results from our study indicate a change
in glycoform patterns. PrPSc from ME7 infected TgSShp PrP Mice brains were predominantly
di-glycosylated in contrast to the mono-glycosylated band pattern observed C57BL/6
–ME7 infected mice brains. Similar to our results is the findings reported by
Shi et al, (2012). Same glycosylation pattern was observed at the third passage
(data not shown) suggesting that glycosylation profile formed during
interspecies transmission is maintained over serial passaging in the same host
(Beck KE et al., 2013). This may also support the observation that the brain
microenvironment of a new host obviously affects the PrPSc
properties during inter-species transmission. Also our results showed that PrPSc
from ME7 infected C57BL/6 mice were more resistant to proteinase k digestion
even at high concentration of 500µg/ml as
against that of TgSShp PrP Mice in which at same concentration relatively
weaker signal of PrPSc were detected. The slightly higher
resistance ability of mouse adapted ME7 scrapie strain might be attributed to
stability resulting from repeated passaging.

To
determine the neuropathological changes induced by this interspecies
transmission, hematoxylin staining was performed on formalin-fixed tissues of TgSShp PrP- ME7 infected and control mice.
Spongiform degeneration, a porous sponge like lesion in brain of diseased
animals is one of the pathological hallmarks of prion diseases and has been
found to be more pronounced in the hippocampal region of brains infected with
ME7 scrapie strain. Dentate gyrus, CA3 and CA1 hippocampal region were examined
for spongiform degeneration.  In this
study, sponge like lesions were more pronounced in the CA1 region of terminally
sick TgSShp PrP- ME7 mice examined. 
Numerous oval shaped vacuoles were observed in the fields of view most
especially in the CA1 hippocampal region of the infected brain. Another
criterion considered for strain peculiarity was the vacuolar lesion profile. As
reported by Fraser and Dickson (1968) the pattern of vacuolar lesion in the
brain is characteristic for a given strain in a given host using a
non-transgenic mice transmission experimental model. Validating this in a prion
transgenic mice model, the nine grey areas examined for vacuolar lesion showed
distinct vulnerabilities towards the ME7 scrapie agent. From our result, severe
vacuolation was observed in the hippocampal, brain stem, substantia nigra
regions of the infected brains. Other grey areas such as hypothalamus, thalamus
and septal area also showed marked vacuolation but less conspicuous vacuolation
in the cerebellar cortex. In agreement with our findings was a similar results
reported by C. Cunningham et al., (2005) where they observed that ME7 strain
showed marked vacuolation of the hippocampal, septal and thalamic neuropil.

Reports from experimental studies done has showed
that the deposition pattern of PrPSc  across
brains of infected mice is agent strain dependent (Beck KE et al., 2010; Brown
DA et al., 2003; Bruce ME et al., 1994). In this study, we detected PrPSc accumulation
with formation of plaques.  PrPSc aggregation
accompanied with plaques was more prominent in the CA1 hippocampal region. This is consistent with the report that, in the
hippocampus, ME7 predominantly targeted the polymorph layer of the dentate
gyrus and stratum lucidum, the CA3 field and the pyramidal layer with granular
labeling and small aggregates (Beck et al., 2012). In a similar study carried
out by Siso S. et al., (2012), they reported that amyloid plaques are abundant
and characteristics of murine 87V but do inconsistently occur in ME7 and 22A
infections, vascular plaques were occasionally present in ME7 infections of
VRQ/VRQ Cheviot and ARQ/ARQ Suffolk sheep. Also, C. Cunningham and colleagues,
(2005) reported that ME7 infected brains showed a more PrPSc
punctuate labeling pattern with some small plaques deposition both in the
hippocampus and thalamic brain regions. Results
from studies using mouse-adapted prion strains has demonstrated that
neuroinvasive strains formed diffuse, non fibrillar PrP aggregates in the
Central nervous system and mice rapidly progress to terminal disease, while
weakly neuroinvasive strains form dense, congophilic, fibrillar plaques and
mice progressed to terminal disease more slowly. These findings suggest that
the non-fibrillar PrP aggregates  are
more toxic which is in agreement with studies indicating that smaller
aggregates of the mammalian prion protein, which could be more readily
generated by strains that form fragile particles, are markedly more infectious
than larger aggregates (Laura Solforosi et al., 2013; Silveira JR et al., 2005).
 A strong counter stain is a
well-known marker of sick cells about to die by an apoptotic pathway (Liu et
al., 2015; Sharma et al., 2016). Our results showed decreased in population of
neuronal cells and a significant higher accumulation of hematoxylin-counter stain.
Also, there was decrease in the
expression level of neuronal marker used to measure extent of neuronal loss
between infected and control mice in this study. Hippocampal neuronal cell
death has been observed to be a late stage phenomenon in ME7 scrapie infections.
Hippocampal CA1 pyramidal neuronal loss was reported to be significant in ME7
and 79A infected mice (C. Cunningham et al., 2003, 2005; Jeffrey M et al.,
2000; Hilton KJ et al., 2013). Most likely, the severe aggregation of PrPSc in CA1 in this study could have resulted
from the death of CA1 neurons.

The increase in population of reactive astrocytes
was well pronounced in brain tissues of the infected mice. GFAP upregulation
was severe in the dentate gyrus of the brain. In the brains from the age
matched control mice, GFAP positive astrocytes are detectable in the dentate
gyrus, CA3 and CA1 brain region but not as severe as the degree of detection in
the terminally diseased mice. This result agrees with similar findings reported
by Hilton K J et al., (2013). They demonstrated that the hippocampus and
dentate gyrus are sites of GFAP upregulation in ME7- and 79A- animals.

Synaptic loss has been found to be a key feature
of many neurodegenerative diseases and it occurs before neuronal death in many
diseases state (Hilton K J et al., 2013). 
Biochemical studies of the Hippocampus of ME7 infected animals revealed
that proteins of the pre-synaptic terminal compartment declines first
especially those of the synaptic vesicles (Gray BC et al., 2009).  Experimental studies of ME7 infected animals
using electron microscopy has clearly shown loss of synaptic vesicles’
integrity to be the first apparent morphological feature of synaptic
dysfunction in stratum radiatum (Siskova Z et al.,2009) Different studies have
shown the degeneration of asymmetric terminals to be a detectable and early
pathological hallmark in ME7 infection. It precedes any observable degeneration
of cell bodies of CA3 or CA1 pyramidal neurons (Siskova Z et al., 2009; Gray BC
et al., 2009). Various studies has revealed PrPC to be highly
concentrated in presynaptic terminals where it has been found to co-localize
with Synaptophysin and also widespread in brain structures associated with
synaptic plasticity (Fournier JG et al., 2000; Haeberle AM et al., 2000;  Herms J et al., 1999;  Sales N et al., 1998). From our western blot results there was no significant difference in
the expression level of pre-synaptic protein marker: Synaptophysin in ME7
infected brains when compared with the age matched control but in contrast, we
observed marked difference in the expression level of post synaptic protein
marker PSD 95 in ME7 infected brains relative to the age matched control
brains. This result is in contrast with a study findings reported by C.
Cunningham et al., (2005) in which they observed a clear loss of Synaptophysin
in the hippocampus of mice infected with scrapie strain 22L, 79A and ME7 but
the loss was most severe in the ME7 infected animals. One reason for this
difference could be because whole brain homogenates were used for our western
blot but specific brain regions were examined during the Immunohistochemical
staining for the study results reported by C. Cunningham et al.

Results from a study conducted by Peretz D and
colleagues (2002) reported that a change in conformation accompanied the
emergence of new prion strain during interspecies transmission while the
conformational stability of PrPSc was preserved when a strain bred true in a new
host. Strain specific differences in PrPSc conformation may
reasonably evoke differences in neuronal targeting, trafficking and
processing/degradation of protein (Bruce ME et al., 1994; DeArmond SJ et al.,
1993; Bessen RA et al., 1994; Gonzalez L et al., 2003).Our results showed a
change in conformation stability of ME7 infected C57BL/6 mice after
interspecies transmission to TgSShp PRP mice. The resulting PrPSc
specie from the cross specie transmission was relatively more stable and
resistant to denaturation by Guanidine Hydrochloride. Although, conformational
stability has been reported to be directly proportional to the length of
incubation in mice (Legname G et al., 2006) results from this study in agreement
with similar findings by Laura Pirisinu and others (2010, 2013) reported
contrawise.

Differences in pathological features, that is,
pattern of histological lesions and PrPSc accumulation in scrapie infected mice has been
reported to be related to the interaction between the host (genotype, age, sex
and breed) and the agent strain (passage history, dose, route of inoculation,
donor genotype and organ used for preparing inoculum) (Fraser H, 1976; Jeffrey
M et al., 2002; Beringue V et al., 2012). Also, it has been suggested that some
features of disease phenotype might be strain-encoded and propagated in the
recipient host (Siso S. et al., 2012).

In conclusion, Interspecies transmission of long term mouse
adapted ME7 scrapie strain was successful in transgenic mice expressing ovine
prion protein but with accompanying biophysical and neuropathological changes.
Differences in survival time, 
biophysical (Glycoform change, conformational stability) and
neuropathological features (plaque formation) reported in this study could be
due to inherent strain or host characteristics or it could have resulted from
some kind of strain mutation leading to a change in the properties in the new
host. Further studies are needed to confirm if the observed changes are
maintained in subsequent passages within the same host.ost.