eBook: Advancing Neuroscience Research - 13

Figure 2. Immunohistochemical GFAP staining of the
substantia nigra pars compacta region in a mouse brain.
Optical density analysis of GFAP-positive cells reveals an
increased amount of astrogliosis in PFF α-syn treated mice
in comparison to monomer α-syn, revealing neuroinflammation
associated with α-syn accumulation. Reproduced
from Earls 2019.4
Figure 3. Transmission electron microscopy of human tau441/2N4R
pre-formed fibrils. Protein aggregates with distinct
fibrous structures are visible with accurate morphology.
some complications. This is most notable with the
lack of NFT formation due to the inhibition of expressed
human tau by endogenous mouse tau.
On the other hand, toxin-induced models simulate
the clinical symptoms in a reproducible format but
lack the molecular pathology needed to understand
disease progression. PFFs are a more direct
methodology, injecting pre-formed pathogenic
fibrils to form protein aggregates and induce neurodegeneration.
This method not only induces the
molecular pathology faster than transgenic mice,
but also reduces the scientific burden and length
of time for mouse maturation. Similarly, PFFs are
useable at the in vitro level, enabling a direct, more
reproducible method to identify and measure disease
pathology while providing scientists a model
to test new drug candidates.
PFFs are easily formed from protein monomers by
incubating at 37°C and shaking with or without
heparin. Each protocol depending on the protein
monomer of choice depends on the monomers'
ability and tendency to aggregate. For example,
tau-441 PFFs require a concentration of 2 mg·mL-1
with heparin, while shaking and incubating for 7
days. In contrast, alpha-synuclein PFFs are incubated
without heparin at an initial monomer concentration
of 5 mg·mL-1
. For other mutated monomers
or pathogenic fragments such as alpha-synuclein
A53T or tau K18 P301L, incubation time can be
much shorter (4-5 days) due to its increased tendency
to aggregate.
Verification of PFFs is also critical before implementation
as disease models. The presence of fibrillary
structures should be visually verified through microscopy
and chemically through ThT assays. ThT
assays help analyze the structure of high molecular
weight species and determine whether the typical
cross-beta structure has been formed. Similarly,
size and morphology of aggregates should be confirmed
through transmission electron microscopy
(TEM) or atomic force microscopy. In addition, the
length of the formed fibrils is important in pathogenicity.
Thus, before use, PFFs should be sonicated
into 50 nm or smaller seeds before experiments;
both of which can be visualized through TEM or
dynamic light scattering.
Current applications of
pre-formed fibrils
Tau PFF models for AD study and drug development
are well established in cellular systems including
neurons, microglia, and astrocytes. Studies
evaluating PFF addition to cultured cells have indicated
that the tau PFFs are internalized by cells
through endocytosis and serve as a seed to recruit
13
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