National University of Singapore 2014/2015 Semester 1 GSN6501 A Selective Activity-Dependent Requirement for Dynamin-1 in Synaptic Vesicle Endocytosis

2. INTRODUCTION TO PAPER
READING OF THE TITLE AND ABSTRACT

3. TITLE OF THE PAPER
READING OF THE TITLE
TITLE
Novel/unique characteristic
Biological process in question
Protein of
Interest
Synaptic Vesicle Endocytosis
Dynamin-1
Activity-dependent

4. MAIN HYPOTHESIS
HYPOTHESIS OF THE PAPER
AUTHORS’ HYPOTHESIS
Dynamin-1 is involved in
mediating neck constriction
and fission of synaptic
vesicles at the presynaptic
terminus

5. ENTIRE ABSTRACT
ABSTRACT
READING OF THE ABSTRACT

6. FIRST SENTENCE
ABSTRACT
READING OF THE ABSTRACT
Dynamin-1
Background to understanding paper
Limited to
neurons GTPase
Fission reaction

7. BACKGROUND
BACKGROUND TO THE PAPER
SYNAPTIC VESICLE CYCLE
Dynamin-1
• mediates
neck
constriction
Ackermann, Gregory and Brodin. (2012). Key Events in Synaptic Vesicle Endocytosis, Molecular Regulation of Endocytosis

8. 4
GENERAL APPROACH
APPROACHES ADOPTED BY THE AUTHORS
Evaluation of synaptic vesicle
endocytosis/recycling
1
Generation of dynamin-1 KO
mice
2
Evaluate synaptic functionality of
KO neurons
3
Characterization of synaptic
vesicle morphology
Confirm for KO
Are synapses
functional?
What are the
morphological
changes?
What is the impact on
endocytosis recycling?

9. Generation of
dynamin-1 KO mice
Evaluation of
synaptic
transmission in KO
ABSTRACT
READING OF THE ABSTRACT
SECOND SENTENCE
Animal model adopted and indication of observed phenotype

10. METHODOLOGIES
APPROACHES EMPLOYED IN THE PAPER
1 Generation of dynamin-1 KO mice
(a) Phenotypic changes / defects
Developmental changes
(b) Confirmation of dynamin-1 knockout
Tissue localization
Isoform abundance
Effect on expression of
endocytic/synaptic proteins
Reduced milk ingestion
Poor motor coordination
Died within 2 weeks
Nervous system
Dynamin-1 predominant
isoform in nervous system
No effect on endogenous
expression levels

11. GENERATION OF KO MICE
DYNAMIN-1 KO MICE
Dynamin-1 is not required for embryonic development or
neurotransmission for perinatal life
milk
ingestion
poor motor
coordination KO pups unable to gain weight
Dynamin-1 KO pups died within 2 weeks

12. GENERATION OF KO MICE
Dynamin-1 is predominant isoform in nervous system
dynamin-1
In cortical neuron cultures
total
dynamin
Across tissues
No change in expression of other
synaptic/endocytic proteins
dynamin-3
DYNAMIN-1 KO MICE

13. METHODOLOGIES
APPROACHES EMPLOYED IN THE PAPER
2 Evaluation of synaptic transmission in dynamin-1 KO mice
(a) Evaluation of synaptic functionality
(i) WITHOUT presynaptic stimulus (spontaneous release)
mEPSC and mIPSC Increased mEPSC and mIPSC
(ii) WITH presynaptic stimulus
EPSC and IPSC
Recovery at high stimuli
Decreased IPSC peak
amplitudes
Decreased recovery of IPSC
peaks

14. SYNAPTIC TRANSMISSION
EVALUATION OF SYNAPTIC FUNCTIONALITY
Dynamin-1 KO neurons able to form functional synapses but
(1) with increased mEPSC and mIPSC
mean
mEPSC
amplitude
mean
mIPSC
amplitude
Indicative of increased vesicle size in KO neurons

15. SYNAPTIC TRANSMISSION
EVALUATION OF SYNAPTIC FUNCTIONALITY
Dynamin-1 KO neurons able to form functional synapses but
(2) with lower IPSC peaks
Peak
IPSC
Overall reduction in IPSC peak amplitudes
Peak
IPSC
EPSC IPSC IPSC

16. SYNAPTIC TRANSMISSION
EVALUATION OF SYNAPTIC FUNCTIONALITY
Dynamin-1 KO neurons able to form functional synapses but
(3) with impaired recovery
During stimulus Post-stimulus
Lower peak IPSC
Faster IPSC
depression time
Recovery of peak
IPSC
Impairment of IPSC recovery

17. SYNAPTIC TRANSMISSION
ACTIVITY-DEPENDENCE OF DYNAMIN-1
Dynamin-1 is not essential for synaptic transmission but is
required for efficient and sustained evoked release
Single presynaptic stimulus 1000 presynaptic stimuli
Peak IPSC Recovery of peak IPSC
Dynamin-1 KO neurons unable to
keep up with sustained stimuli
BUT Dynamin-1 KO neurons still
able to have synaptic transmission
under low stimulus

18. Characterization of
synaptic vesicle
morphology
ABSTRACT
READING OF THE ABSTRACT
THIRD SENTENCE
Synaptic vesicle morphology in dynamin-1 KO model

19. METHODOLOGIES
APPROACHES EMPLOYED IN THE PAPER
3 Characterization of synaptic vesicle morphology
Vesicle diameter
Presence of clathrin-coated
profiles
Continuous with membrane?
Larger than WT
Yes
Yes
(a) Vesicle morphology
(b) Formation of clathrin coated profiles
Dynamin-1 independent
processes
Involves Dynamin-3
(c) Formation of synaptic vesicles

20. CHARACTERIZATION OF
VESICLES
VESICLE MORPHOLOGY
Dynamin-1 KO neurons have less synaptic vesicles and
heterogeneous vesicle sizes with increased vesicle diameter
WT neurons Dynamin-1 KO neurons
synaptic vesicles (~20%)
heterogeneous vesicle sizes
homogeneous
vesicle sizes
vesicle diameter
in KO synapses

21. CHARACTERIZATION OF
VESICLES
VESICLE MORPHOLOGY
Dynamin-1 KO neurons have clathrin-coated profiles that are
interconnected and continuous with plasma membrane
clathrin-coated profiles
clathrin-coated
profiles
Dynamin-1 KO neurons
puncta distribution of
clathrin and AP-2

22. CHARACTERIZATION OF
VESICLES
VESICLE MORPHOLOGY
Dynamin-1 KO neurons have clathrin-coated profiles that are
interconnected and continuous with plasma membrane
Clathrin-coated
profiles
Addition of
extracellular
Interconnected clathrin buds
HRP

23. CHARACTERIZATION OF
VESICLES
VESICLE MORPHOLOGY
Dynamin-1 KO neurons have clathrin-coated profiles that are
interconnected and continuous with plasma membrane
Dynamin-1 KO neurons
Clathrin-coated
profiles able to take up
extracellular HRP
BUT NOT
normal vesicles
Clathrin-coated profiles are
continuous with plasma membrane
Defect in neck constriction and fission of vesicles

24. CHARACTERIZATION OF
Dynamin-1 independent processes involve dynamin-3 for
formation of synaptic vesicles
VESICLES
VESICLE MORPHOLOGY
AP
Blockade
of AP
Clathrin and
dynamin-3
co-localize in
KO neurons
Dynamin-1
independent
processes
involve
dynamin-3

25. CHARACTERIZATION OF
VESICLES
ACTIVITY-DEPENDENCE OF DYNAMIN-1
AP
Role of dynamin-1 in synaptic vesicle endocytosis
Blockade
of AP
is activity-dependent
No
co-localization
of dynamin-3
with clathrin
AP is the trigger for dynamin-1 activity
In absence of
AP, no more
clathrin-coated
profiles
Dynamin-3
compensating
for dynamin-1
in vesicle
formation

26. ABSTRACT
READING OF THE ABSTRACT
FOURTH SENTENCE
Synaptic vesicle endocytosis
Evaluation of synaptic vesicle
endocytosis/recycling

27. 4 Evaluation of synaptic vesicle endocytosis/recycling
(a) Synaptic vesicle recycling efficiency
Recovery of synaptic vesicles post-stimulation
Maximum frequency of stimulus
Delayed uptake of
HRP in KO neurons
10 Hz in KO neurons
(b) Activity-dependence
METHODOLOGIES
APPROACHES EMPLOYED IN THE PAPER
without bafilomycin
with bafilomycin
Decreased vesicle
endocytosis in KO neurons
(c) SynaptopHluorin assays

28. METHODOLOGIES
APPROACHES EMPLOYED IN THE PAPER
4 Evaluation of synaptic vesicle endocytosis/recycling
(d) Rescue experiments using dynamin isoforms
Rescue of KO neurons with
dynamin isoforms
Dynamin-1 and 3 able to
restore WT levels

29. SYNAPTIC ENDOCYTOSIS
RECYCLING OF SYNAPTIC VESICLE
Synaptic vesicle endocytosis is impaired in dynamin-1 KO
neurons during stimulation
WT neurons
Dynamin-1 KO
neurons
clathrin-coated
profiles
synaptic
vesicles
Stimulation with high K+ buffer (90mM KCl)
in presence of HRP
synaptic
vesicles

30. SYNAPTIC ENDOCYTOSIS
RECYCLING OF SYNAPTIC VESICLE
Synaptic vesicle endocytosis is impaired in dynamin-1 KO
neurons during stimulation
Recovery in presence of extracellular HRP
WT neurons
Dynamin-1 KO
neurons
(after 10 mins)
recovery
of synaptic
vesicles
recovery
of synaptic
vesicles
Impaired recovery of synaptic vesicles in dynamin-1 KO neurons

31. SYNAPTIC ENDOCYTOSIS
RECYCLING OF SYNAPTIC VESICLE
Synaptic vesicle endocytosis is impaired in dynamin-1 KO
neurons during stimulation
Recovery in
presence of
extracellular
HRP
HRP synaptic vesicles
recovery/recycling of
synaptic vesicles
Impaired recovery of
synaptic vesicles in
dynamin-1 KO neurons

32. SYNAPTIC ENDOCYTOSIS
RECYCLING OF SYNAPTIC VESICLE
Synapto-pH-luorin assay
Vesicle release Recycling
fluorescence
in low pH
vesicle
fluorescence
when
exocytosed
fluorescence
in low pH
vesicle
Δfluorescence

33. ACTIVITY-DEPENDENCE
FREQUENCY-DEPENDENT ENDOCYTIC BLOCKADE
Synaptic vesicle endocytosis is at its maximum at 30 Hz for
WT neurons and at 10 Hz for dynamin-1 KO neurons
Δfluorescence is at its
maximum at 30 Hz of
stimulation
Δfluorescence is at its
maximum at 10 Hz of
Dynamin-1 KO neurons are less able to copes taimt uhliagthio lnevels of
stimulation

34. ACTIVITY-DEPENDENCE
FREQUENCY-DEPENDENT ENDOCYTIC BLOCKADE
Synaptic vesicle endocytosis is at its maximum at 20 Hz for
WT neurons and at 10 Hz for dynamin-1 KO neurons
Endo/Exo is at its maximum at
20 Hz of stimulation for WT
neurons
Endo/Exo is at its maximum at
10 Hz of stimulation for KO
neurons

35. SYNAPTIC ENDOCYTOSIS
RECYCLING OF SYNAPTIC VESICLE
Dynamin-1 KO neurons have impaired synaptic vesicle
endocytosis compared to WT neurons
fluorescence fluorescence
Endocytosis of vesicle
membrane
Endocytosis of vesicle
membrane

36. SYNAPTIC ENDOCYTOSIS
Synapto-pH-luorin assay with Bafilomycin H+-ATPase inhibitor
Vesicle release Recycling
fluorescence
in low pH
vesicle
fluorescence
when
exocytosed
fluorescence
in low pH
vesicle
Bafilomycin
blocks
reacidification
Δfluorescence
RECYCLING OF SYNAPTIC VESICLE

37. SYNAPTIC ENDOCYTOSIS
RECYCLING OF SYNAPTIC VESICLE
Dynamin-1 KO neurons have impaired synaptic vesicle
endocytosis compared to WT neurons
Δfluorescence Δfluorescence
Endocytosis of vesicle
membrane
Endocytosis of vesicle
membrane
Δfluorescence
Δfluorescence

38. SYNAPTIC RECYCLING
RECYCLING OF SYNAPTIC VESICLE
Changes in fluorescence are not due to impaired acidification
or increased rates of exocytosis
ΔFluorescence same as pre-stimulus
level upon onset of AP Similar rates of exocytosis in
both WT and KO neurons
Acid-quench
Recovery of
vesicle
membrane for
KO
Acidification is normal

39. SYNAPTIC RECYCLING
RECYCLING OF SYNAPTIC VESICLE
Dynamin-1 and 3 are crucial for rescue of synaptic vesicle
endocytosis in dynamin-1 KO neurons
All dynamin isoforms are able to
participate in synaptic endocytosis
Dynamin-1 able to rescue
endocytosis in KO neurons to WT
levels
Dynamin-3 similarly able to rescue
endocytosis to near WT levels
Dynamin-1 and dynamin-3 have greater functional similarities that
enable dynamin-3 to participate in dynamin-1 independent processes

40. ABSTRACT
READING OF THE ABSTRACT
FIFTH SENTENCE
Key Finding / Conclusion
Dynamin-1 is essential for high
levels of neuronal activity to
mediate synaptic vesicle
endocytosis
Dynamin-1 independent
mechanisms can support low
levels of neuronal activity

41. CONCLUSION
KEY CONCLUSIONS MADE IN THE PAPER
Dynamin-1 activity is dependent on degree of stimulation
Basal Level
Previous view Updated view
Dynamin-1
High
Stimulation
Low
Stimulation
Dynamin-1
Dynamin-1
independent
processes

42. DISCUSSION
POINTS OF DISCUSSION MENTIONED
MOLECULAR REGULATION
Ca2+-dependent dephosphorylation by calcineurin
• Dephosphorylation of dynamin enhances recruitment to
endocytic sites
QUANTAL RELEASE OF NEUROTRANSMITTERS
Regulation of vesicle size
• Involvement of dynamin could be a key factor to
control/regulate vesicle size
CLATHRIN-COATED PROFILES
• Membrane tubulation offers strong support for clathrin-mediated
endocytosis

43. EVALUATION
EVALUATION OF THE PAPER
ON METHODOLOGIES
• Good methodologies
• Rigorous (Use of multiple assays to confirm)
• Bypasses dominant-negative effects associated with hetero-oligomerization
by mutant dynamin proteins
ON FINDINGS
• Highlights novel role of dynamin-3 in dynamin-1 independent
processes
• Findings offer strong support for clathrin-mediated
endocytosis
• Novel activity-dependent requirement for dynamin-1
involvement in synaptic vesicle endocytosis

44. EVALUATION
EVALUATION OF THE PAPER
LIMITATIONS
Use of primary cortical neuronal cultures
• Use of primary cortical neuronal cultures could lead to
variations in physiological and electrophysiological properties
compared to neurons in vivo
• Use of brain slice patch clamp to minimize changes to
microenvironment

45. EVALUATION
EVALUATION OF THE PAPER
If not dynamin-1, what is required for synaptic vesicle
endocytosis during embryonic development?
Dynamin-1 is not required for embryonic development or
neurotransmission for perinatal life
• What are the factors regulating
neurotransmission, specifically
synaptic vesicle endocytosis,
during embryonic development?

46. EVALUATION
EVALUATION OF THE PAPER
Role of Dynamin-3
What regulates the role of
dynamin-3 in dynamin-1
independent processes?
• Why are dynamin-3 rescue
experiments able to restore
endo/exo ratios to near WT
level but this is not seen in KO
animal models?
• What are the endogenous regulatory signals or factors tha
t
prevent dynamin-3 from acting in the same capacity as
dynamin-1?

47. EVALUATION
EVALUATION OF THE PAPER
CLATHRIN-INDEPENDENT PATHWAYS
BULK ENDOCYTOSIS
• Vesicles are taken up in the form of bulk endosomes
• Can occur simultaneously with clathrin-mediated endocytosis
• Not to be confused with ‘kiss-and-run’ model

49. ACKNOWLEDGEMENTS
RESOURCES USED
Images
• Frauke Ackermann, Joshua A. Gregory and Lennart Brodin
(2012). Key Events in Synaptic Vesicle Endocytosis, Molecu
lar Regulation of Endocytosis, Dr. Brian Ceresa (Ed.), ISBN:
978-953-51-0662-3, InTech, DOI: 10.5772/45785. Available
from: http://www.intechopen.com/books/molecular-regulatio
n-of-endocytosis/key-events-in-synaptic-vesicle-endocytosis