1. Analysis of Cellular and
Molecular Factors Influencing
Axonal Degeneration in
Neonatal Mice
Alexis Doggett
2. Background
Purpose:
Investigate cellular and molecular factors influencing axonal
degeneration, rather than NMJ degeneration, in response to
peripheral nerve injury in mice
WHY?
Neurodegenerative conditions affect millions of people world wide, and
motor neuron degeneration as a widely observed symptom in a subset
of these diseases, the cause of, which is still not fully understood.
Any research done to investigate influences in these disease
processes is critically important for finding cures.
Part 1:
Ex-vivo sciatic nerves from wild type mice over a period of 72 hours
from ages P9-P22
Part II:
Examining protein expression from three proteins implicated in
neurodegenerative processes, in brachial plexus nerves from
wild type mice ages P12- P24
ROCK2: neuronal survival and stability; actin regulator
Prp: cellular adhesion, synaptic metabolism, and signal transmission
SMN: mRNA splicing and axonal transport
3. Aims and Hypotheses
1. Identify changes in axonal
degeneration by quantifying levels of
WD in neonatal and adult mice.
2. Identify differences in protein
expression from ROCK2, Prp and SMN
across the range in which age-
dependent vulnerability shifts.
3. Discover whether there is a difference
in rate of axonal degeneration between
2B/+ & 2B/- (SMN deficient) mouse
models.
4. Methods
Axonal Imagining
◦ Ex-Vivo model with Fluorescent Imaging
◦ Statistical Analysis
The Axonal Integrity Ratio (AIR) was calculated by dividing the
sum total of fragmented lengths by the total length of the axon.
Protein Expression Quantification
◦ Western-Blot
◦ LiCOR Scanning
5. What to look for: Part I
What is Wallerian Degeneration
(WD)?
◦ Degeneration that appears along the axon
as fragmentation or broken bits
P14 Sciatic Axons at 21 Hours P14 Sciatic Axons at 72 Hours
6. AIM1: Identify changes in axonal degeneration by
quantifying levels of WD in neonatal and adult mice.
P9 P14 P18 P22
P9
P14
P18
P22
0.0
0.2
0.4
0.6
0.8
Axonal Integrity at 72hrs ExVivo
Age of YFP Wild Type Mice (Postnatal)
AIR(AxonIntegrityRatio)
*
ns
Comparison of AIR between
neonatal and adult mice: (A)
Fluorescent Microscopic images taken
of P9, P14, P18, and P22 Scale Bar:
100μm (B) Average AIR
quantifications from each time point
(P9-P22) compared against each
other, at 72 hours ex-vivo. Statistically
significant difference in degeneration
at 72-hours, between P9 and P22
axons (bars above data sets represent
mean + SEM).
7. What to look for: Part II
Changes in Protein Expression
◦ Sample intensity is greater (darker) with
larger protein expression
P12A
10μg 15μg 20μg 30μg
SMN Expression
P12B
8. AIM 2: Identify differences in protein expression from
ROCK2, Prp and SMN.
P12
P15
P18
P21
P24
0.0
0.5
1.0
1.5
SMN Expression In Brachial Plexus Nerves of Wild Type Mice
Age of Wild Type Mice (Postnatal)
ProteinExpression
P12
P15
P18
P21
P24
Figure 3.2.2. SMN Expression. This figure represents the mean with + SEM of SMN
expression across all five sample ages. Data from this graph was quantified using an
N=2, where P15-24 are normalized to the primary loading of P12. Due to the small N
number the data yielded no statistical significance for any sample sets. A general trend
can be seen where the SMN expression does decrease as age of mice increases.
SMN
α-Tub
P12 P15 P18 P21 P24
9. AIM 3: Discover whether there is a difference in rate
of axonal degeneration between 2B/+ & 2B/- (SMN
deficient) mouse models.
P18 2B+ at 0 Hrs P18 2B- at 0 Hrs
P18 2B+ at 72 Hrs P18 2B- at 72 Hrs
(A) Fluorescent Microscopy images taken under a green fluorescent channel of P18 2B/+ and 2B/-
Sciatic nerves following dissection and at 72-Hours ex-vivo. Images were taken at 20x
magnification. Scale bar: 100μm. (B) Graph displays differences in AIR quantification between 2B/-
(SMN deficient) and 2B/+ (Control) mouse models. Data was quantified using N=20 axons from 4
nerves of each phenotype. No significant difference noted in degeneration using a Mann-Whitney
analysis, p-value = 0.1292 (bars above data sets represent mean + SEM).
2B
-
2B
+
0.0
0.2
0.4
0.6
0.8
Axonal Integrity of 2B- vs 2B+ mice at 72 hours
Mouse Phenotype
AIR(AxonIntegrityRatio)
ns
A
B
10. Conclusion
This project utilized an ex-vivo model to observe
degeneration in neonatal and adult wild type
sciatic nerves
◦ Proved that age-dependent vulnerability does exist in
peripheral nerve axons, similar to that seen within the
NMJ.
This project also explored potential molecular
mechanisms influencing axonal degeneration
◦ Showed that ROCK2, Prp, and SMN protein
expression all change as age increases.
◦ SMN displayed a trend of decreasing as age of mice
increased
This project investigated whether levels of SMN
expression effect rates of degeneration
◦ Found that SMN deficient models do not present with
a statistically significant change in axonal
degeneration compared to mice with normal SMN
expression.
11. Relevance and Future
Studies
Better understanding of mechanisms that effect
neurodegeneration.
Investigation into proteins and their exact influence on
motor neurons can lead to potential treatments for
patients suffering from disease.
Future Studies:
◦ Other disease models and axonal protection
◦ Other protein expression changes over time (i.e.
ApoE, Htt, P-Tau)
