The loss of other HtrA family members reduces the severity of the Parkinsonian phenotype observed in HtrA2 knockout mice. Multiple HtrA gene knockout mice show a rescue of OPA1 processing, improved mitochondrial respiration, and reduced severity of Parkinsonian phenotypes compared to HtrA2 knockout mice. Deletion of an additional HtrA gene leads to increased mitochondrial activity, improved neuromuscular strength, and modulation of the Parkinsonian phenotype presented in HtrA2 knockout mice.
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is an open access international journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
For more information contact: Slideshare@marcusevans.com.
Roy Pettipher
CRTH2 Antagonists for the Treatment of Asthma and Allergic Rhinoconjunctivitis
Drug Discovery Summit Lisbon March 16-18 2015
IOSR Journal of Pharmacy and Biological Sciences(IOSR-JPBS) is an open access international journal that provides rapid publication (within a month) of articles in all areas of Pharmacy and Biological Science. The journal welcomes publications of high quality papers on theoretical developments and practical applications in Pharmacy and Biological Science. Original research papers, state-of-the-art reviews, and high quality technical notes are invited for publications.
For more information contact: Slideshare@marcusevans.com.
Roy Pettipher
CRTH2 Antagonists for the Treatment of Asthma and Allergic Rhinoconjunctivitis
Drug Discovery Summit Lisbon March 16-18 2015
Mervyn Singer discusses the use of biomarkers in critical care.
Multiple biomarkers - physiological, biochemical, biological - can prognosticate early in critical illness, even in the ED.
These biomarkers are numerous - lipids, progesterone, troponin, thyroid stimulating hormone, inflammatory cytokines, mitochondrial dysfunction… so on and so forth!
Prognostication can happen as early as the Emergency Department.
Studies from the States have found high levels of inflammatory cytokines can predict death, separately from clinical presentation. Therefore, we can predict when critically ill patients are destined to die. So, does this mean that we are just prolonging the life of those destined to die in critical care? Perhaps.
Mervyn discusses this being the possible reason for many failed ICU studies. Concurrently, the only progress in critical care in the past 20 years may be due only to less iatrogenic harm.
Furthermore, he explains his experiments with rats demonstrating the use of cardiovascular parameters, cytokines, troponins and even cholesterol being accurate prognostic biomarkers.
Then, Mervyn goes on to identify the use of steroids in sepsis. He talks about research that demonstrates a benefit to steroid use, but only in those patients predicted to die using the aforementioned biomarkers. This could be a key to selecting an appropriate patient group to allocate a specific treatment too.
Furthermore, we examine treating sepsis with beta blockers. Giving beta blockers to everyone has no effect at best and a harmful effect at worst. However, giving beta-blockers to those who were predicted to die conferred benefit!
In conclusion, we can predict outcome early in disease. This may allow better selection of patients for certain treatments! We thus need to adopt a completely different strategy for such patients predetermined to die. This also applies to trial design, especially where survival is the endpoint.
For more like this, head to our podcast page. #CodaPodcast
1. Introduction
The High temperature requirement protein A (HtrA)
family of proteins contains four family members: HtrA
Serine peptidase 1 (HtrA1), HtrA Serine peptidase 2
(HtrA2), HtrA Serine peptidase 3 (HtrA3) and Human
Serine peptidase 4 (HtrA4).
Mutations in HtrA1, HtrA2, HtrA3 and HtrA4 have
been associated with age-related macular
degeneration, Parkinson’s Disease, various
malignancies and preeclampsia respectively.
HtrA2 has an anti-apoptotic role when localized
to the the inner membrane space of mitochondria
The loss of HtrA2 is sufficient to induce Parkinsonian
Phenotypes in mice.
It has been suggested that HtrA3 has mitochondrial
localization.
HtrA1, HtrA3 do not exhibit gross phenotypes.
Q: Does the loss of HtrA family members modulate
the Parkinsonian phenotype displayed by Htra2
knock out mice?
Generating compound knockouts to
test modulation of the HtrA2 KO
phenotype
HtrA1 mRNA (arrow) is deleted in HtrA1/HtrA2/HtrA3
and HtrA1/HtrA2 KO mice (A). HtrA3 mRNA (red arrow)
is deleted in HtrA1/HtrA2/HtrA3 and HtrA2/HtrA3 KO
mice (A). HtrA1/HtrA2 (C), HtrA2/HtrA3 (D) and
HtrA1/HtrA2/HtrA3 (E) KO mice appear to display a
positive divergence in weight gain from HtrA2 KO mice
at P21, P25 and P20 respectively (C-E).
The HtrA Family and Its Relationship to Aberrant
Mitochondrial Morphology
Brian Thompson1, Hoh J1, Patterson VL1
1Dept of Environmental Health Sciences, Yale School of Medicine, New Haven, CT
Conclusions
Future Studies:
• Continue to investigate the nature of the HtrA
gene family complementation and its relationship
to mitochondrial morphology
• Investigate differences in the proteosomes
between the different genotypes
The loss of other HtrA family members reduces the
severity of the Parkinsonian phenotype observed in
the HtrA2 mouse model.
The Parkinsonian phenotype presented in HtrA2 KO
mice is modulated in multiple HtrA gene knockout mice:
A rescue of OPA1 processing
Improvement in respiration
Reduced severity of Parkinsonian phenotypes
Deletion of an additional HtrA gene
may rescue neuromuscular strength
The deletion of an additional HtrA
gene leads to increased
mitochondrial respiration
The loss of HtrA2 leads to a reduction in mitochondrial
respiration as compared to WT mice. Multiple gene
knockout mice present with an increase in Complex I
(NADH) function in the cerebellum as compared with
HtrA2 KO mice. Scale Bar 100 μm.
Disrupted OPA1 processing is
attenuated upon the deletion of an
additional HtrA gene
The loss of HtrA2 leads to an change in OPA-1
processing, the accumulation of S-OPA1 at the expense
of L-OPA1, this shift is not observed in WT mice. The
ratio of S-OPA1/L-OPA1 is reduced in multiple gene
knockout mice. (N=1)
Deletion of an additional HtrA gene
may rescue neuromuscular strength
continued
Deletion of an additional HtrA gene
leads to increased activity
The deletion of HtrA2 leads to a decrease in HtrA2 KO
mice activity as compared to HtrA2 WT mice. When
compared to HtrA2 KO mice, HtrA2/HtrA3 and
HtrA1/HtrA2/HtrA3 KO mice display a marked increase in
total activities scores.
Grip strength is improved upon the
deletion of an additional HtrA gene
The deletion of HtrA2 leads to a decrease in the latency to
fall by HtrA2 KO mice as compared to HtrA2 WT mice.
Multiple gene knockout mice display increased
neuromuscular strength as compared to HtrA2 KO mice
when subjected to a grip strength test (A-D). HtrA1/HtrA2
(B) and HtrA2/HtrA3 (C) display a significant increase in
their latency to fall at both P24 and P26.
HtrA1
Β-Actin
1/2/3 2/31/2
HtrA3
HtrA2 KO HtrA1/HtrA2 KO HtrA2/HtrA3 KO
HtrA1/HtrA2/HtrA3
KO
ComplexI(NADH)
OPA1
HtrA2
B
D
nWT nKO
P25 brain
PHB2
OPA1
L
S
nWT nKO
0
1
2
3
S-OPA1/L-OPA1ratio
**
C
WT KO
0
1
2
3
S-OPA1/L-OPA1ratio
***
WT KO
P9 brain
PHB2
L
S OPA1
Brain Heart Kidney Liver Muscle Retina
0
1
2
3
4
5
S-OPA1/L-OPA1ratio
HTRA2 WT
HTRA2 KO
***
VDAC
MFN2
HTRA2
WT KO
brain
WT KO WT KO WT KO
heart kidney liver
WT KO WT KO
muscle retina
OPA1
L
S
P25 HTRA2 KO
A
KOWT KOWT
P33 brain
β-actin
LC3β
P62
I
II
B
D
nWT nKO
P25 brain
PHB2
OPA1
L
S
nWT nKO
0
1
2
3
S-OPA1/L-OPA1ratio
**
C
WT KO
0
1
2
3
S-OPA1/L-OPA1ratio
***
WT KO
P9 brain
PHB2
L
S OPA1
Brain Heart Kidney Liver Muscle Retina
0
1
2
3
4
5
S-OPA1/L-OPA1ratio
HTRA2 WT
HTRA2 KO
***
VDAC
MFN2
HTRA2
WT KO
brain
WT KO WT KO WT KO
heart kidney liver
WT KO WT KO
muscle retina
OPA1
L
S
P25 HTRA2 KO
A
KOWT KOWT
P33 brain
β-actin
LC3β
P62
I
II
H2WT
H1/H2KO
H2KO
H2/H3KO
H1/H2/H3
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
0
2
4
6
8
10
Age (Days)
Weight(Grams)
Weight
H2 KO
H1-H2 KO
H2-H3 KO
H1-H2-H3 KO
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
0
2
4
6
8
10
Age (Days)
Weight(Grams)
HtrA2/HtrA3 KO vs. HtrA2 KO Weight
H2 KO
H2-H3 KO
****
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
0
2
4
6
8
10
Age (Days)
Weight(Grams)
HtrA1/HtrA2 KO vs. HtrA2 KO Weight
H2 KO
H1-H2 KO
****
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40
0
2
4
6
8
10
Age (Days)
Weight(Grams)
HtrA1/HtrA2/HtrA3 KO vs. HtrA2 KO Weight
H2 KO
H1-H2-H3 KO
****
B
D
C
E
H
2
K
O
H
1-H
2
K
O
H
2-H
3
K
O
H
1-H
2-H
3
K
O
0
50
100
Genotype
TotalActivityScore
Weanling Observation
****
****
4 6 8 10 12
0
2
4
6
8
Days
PullAttempts
Hindlimb Pulls
H2 KO
H1-H2 KO
H2-H3 KO
H1-H2-H3 KO
4 6 8 10 12
0
2
4
6
8
Days
PullAttempts
HtrA2/HtrA3 KO vs. HtrA2 KO Hindlimb Pulls
H2 KO
H2-H3 KO
****
*
4 6 8 10 12
0
2
4
6
8
Days
PullAttempts
HtrA1/HtrA2 KO vs. HtrA2 KO Hindlimb Pulls
H2 KO
H1-H2 KO
****
4 6 8 10 12
0
2
4
6
8
Days
PullAttempts
HtrA1/HtrA2/HtrA3 KO vs. HtrA2 KO Hindlimb Pulls
H2 KO
H1-H2-H3 KO
**
****
A B
C D
6 8 10 12
0
4
8
12
16
20
24
Age (Days)
LatencyToFall(sec)
Hind Limb Latency To Fall
H2 KO
H1-H2 KO
H2-H3 KO
H1-H2-H3 KO
6 8 10 12
0
4
8
12
16
20
24
Age (Days)
LatencyToFall(sec)
HtrA2/HtrA3 KO vs. HtrA2 KO Latency to Fall
H2 KO
H2-H3 KO
****
6 8 10 12
0
4
8
12
16
20
24
Age (Days)
LatencyToFall(sec)
HtrA1/HtrA2 KO vs. HtrA2 KO Latency to Fall
H2 KO
H1-H2 KO
**
6 8 10 12
0
4
8
12
16
20
24
Age (Days)
LatencyToFall(sec)
HtrA1/HtrA2/HtrA3 KO vs. HtrA2 KO Latency to Fall
H2 KO
H1-H2-H3 KO
***
A
C
B
D
22 24 26
0
10
20
30
40
Days
LatencyToFall(sec)
Grip Strength
H2 KO
H1-H2 KO
H2-H3 KO
H1-H2-H3 KO
22 24 26
0
10
20
30
40
Days
LatencyToFall(sec)
Grip Strength
H2 KO
H2-H3 KO
****
**
22 24 26
0
10
20
30
40
Days
LatencyToFall(sec)
Grip Strength
H2 KO
H1-H2 KO
****
****
22 24 26
0
10
20
30
40
Days
LatencyToFall(sec)
Grip Strength
H2 KO
H1-H2-H3 KO
A
C
B
D
The deletion of HtrA2 leads to a decrease in the
number of pull attempts made by HtrA2 KO mice as
compared to HtrA2 WT mice. Multiple gene knockout
mice attempt a greater number of pulls in the hindlimb
suspension test as compared to HtrA2 KO mice (A-D).
A significant improvement in pull attempts is observed
at P12, P4 and P7 in HtrA1/HtrA2 (B), HtrA2/HtrA3 (C)
and HtrA1/HtrA2/HtrA3 (D) KO mice respectively.
The deletion of HtrA2 leads to a decrease in the latency
to fall in HtrA2 KO mice as compared to HtrA2 WT
mice. Multiple gene knockout mice have an increased
latency to fall in the hindlimb suspension test as
compared to HtrA2 KO mice (A-D)). A significant
improvement in the latency to fall is observed at P7, P5
and P6 in HtrA1/HtrA2 (B), HtrA2/HtrA3 (C) and
HtrA1/HtrA2/HtrA3 (D) KO mice respectively.
4 6 8 10 12
0
2
4
6
8
Days
PullAttempts
Hindlimb Pulls
H2 KO
H1-H2 KO
H2-H3 KO
H1-H2-H3 KO
4 6 8 10 12
0
2
4
6
8
Days
PullAttempts
HtrA2/HtrA3 KO vs. HtrA2 KO Hindlimb Pulls
H2 KO
H2-H3 KO
****
*
4 6 8 10 12
0
2
4
6
8
Days
PullAttempts
HtrA1/HtrA2 KO vs. HtrA2 KO Hindlimb Pulls
H2 KO
H1-H2 KO
****
4 6 8 10 12
0
2
4
6
8
Days
PullAttempts
HtrA1/HtrA2/HtrA3 KO vs. HtrA2 KO Hindlimb Pulls
H2 KO
H1-H2-H3 KO
**
****
A B
C D
H1/H2/H3 KO H2/H3 KO H1/H2 KO H2 KO H2 WT
0.0
0.5
1.0
1.5
2.0
Genotype
S-OPA/L-OPA1Ratio
P25 Brain Lysate
2. Structure and size of
mitochondria are disrupted in
KO brain
Electron microscopy of the cerebellum shows distorted
mitochondrial structure in HtrA2 KO brain at P20 and
more severely at P32. Mitochondria are swollen and
internal membrane structure is disrupted. Scale bar: 1
μm.
Normal Normal-vesicular Vesicular Vesicular-swollen Swollen
Structure Size
Swollen mitochondria are only present in the absence
of HtrA2 and the incidence increases with age. Size is
increased in KO brain, consistent with mitochondrial
swelling. Scale bar: 500 nm.
P20 P32
WTKO
Brian Thompson:
Post Graduate Fellow
School of Public Health
Department of Environmental Health Sciences
Contact Information:
Brian.thompson@yale.edu
Advisors:
Dr. Josephine Hoh
Dr. Victoria Patterson