CPPS 325 Signalling #21 FYI Luman Ying et al UPR J Neurosci 2015 (1) Flashcards
(155 cards)
1
Q
What role does the transcription factor Luman/CREB3 play in sensory axon regeneration?
A
Luman serves as a unique retrograde injury signal regulating the intrinsic elongating form of sensory axon regeneration.
Luman is activated in injured axons and regulates the unfolded protein response (UPR) and cholesterol biosynthesis.
2
Q
What is the unfolded protein response (UPR)?
A
The UPR is a cellular response to stress in the endoplasmic reticulum (ER) aimed at increasing protein folding capacity and maintaining lipid homeostasis.
Activation of UPR can have both beneficial and detrimental effects in different contexts.
3
Q
What happens to UPR-associated mRNA and protein expression following sciatic nerve crush injury?
A
There is a robust increase in UPR-associated mRNA and protein expression in both neuronal cell bodies and the injured axons.
This increase indicates an activation of the UPR in response to injury.
4
Q
What effect does knockdown of Luman expression have on axonal outgrowth?
A
Knockdown of Luman expression in injury-conditioned neurons attenuates axonal outgrowth to 48% of control injured neurons.
This reduction is linked to decreased UPR and cholesterol biosynthesis-associated gene expression.
5
Q
Which transcripts involved in cholesterol regulation were downregulated by Luman siRNA treatment?
A
Four transcripts involved in cholesterol regulation were downregulated by more than 2-fold by Luman siRNA treatment.
These transcripts are critical for cholesterol biosynthesis.
6
Q
What can rescue the Luman siRNA-attenuated outgrowth?
A
Outgrowth can be significantly rescued by either cholesterol supplementation or by the UPR inducer tunicamycin.
Tunicamycin elevates UPR gene expression to levels equivalent to those observed with crush injury.
7
Q
What are the three ER-resident proteins that act as UPR transducers?
A
The three ER-resident proteins are:
* Protein kinase RNA-like ER kinase (PERK)
* Inositol-requiring protein-1 (IRE1)
* Activating transcription factor-6 (ATF6)
These proteins play key roles in mediating the UPR.
8
Q
True or False: The UPR has only negative implications in neurodegenerative diseases.
A
False
The UPR can also have beneficial effects, despite being implicated negatively in many neurodegenerative diseases.
9
Q
What is the significance of Luman in the context of ER stress and axonal regeneration?
A
Luman is implicated in linking ER stress to axon repair and regulates the UPR and cholesterol biosynthesis beneficial for axonal regeneration.
This suggests that Luman could be a therapeutic target for enhancing nerve repair.
10
Q
Fill in the blank: Peripheral nerve injury induces complex pathophysiologic changes in ______.
A
sensory neurons
These changes include increased synthesis of axonal and cell body proteins and lipids.
11
Q
What is the role of activated PERK in the UPR?
A
Activated PERK phosphorylates eukaryotic initiation factor 2 alpha (eIF2α), suppressing protein translation, and regulates the apoptotic regulator CHOP.
This action is part of the cellular response to restore ER homeostasis.
12
Q
What type of injury was performed in the study?
A
Aseptic unilateral mid-thigh sciatic nerve crush injuries were performed
The procedure involved using a smooth-jaw hemostat under anesthesia.
13
Q
What anesthesia was used during the nerve injury procedure?
A
2% isoflurane anesthesia was used.
14
Q
What substance was used to kill animals post-surgery?
A
Euthanyl Forte (sodium pentobarbital) was used.
15
Q
What were the control animals in the study?
A
Naive animals served as controls.
16
Q
What was the purpose of adding cytosine β-D-arabinofuranoside in the culture?
A
To inhibit non-neuronal cell proliferation.
17
Q
What did the siRNA treatment target in the DRG cultures?
A
Luman-specific siRNA was used.
18
Q
What method was used to assess Luman knockdown efficiency?
A
Western blot and immunohistochemistry were used.
19
Q
What was the concentration of cholesterol used in the study?
A
Cholesterol was used at 2 µg/ml.
20
Q
What was the final concentration of tunicamycin used?
A
Tunicamycin was used at final concentrations of 0.4, 2, and 10 ng/ml.
21
Q
What method was used to calculate total axon length/neuron?
A
Total axon length/neuron was calculated using NeurphologyJ.
22
Q
What type of membrane was used in the axon isolation procedure?
A
A polyethylene tetraphthalate membrane with 3 µm pores was used.
23
Q
What was the purpose of the in vitro axotomy model?
A
To study axon growth and response to injury.
24
Q
How were the explants maintained during the culture?
A
The volume of medium was kept low for the first 24 hours.
25
What was done to the explants before imaging?
Explants were fixed for 1 hour in 4% PFA.
26
What was the method used for neuronal counts?
All β-III tubulin-immunopositive neurons were counted within 38–40 fields.
27
What kit was used for cholesterol mass determination?
Amplex Red Cholesterol assay kit was used.
28
What was the fixation time for dissociated DRG neurons?
Dissociated DRG neurons were fixed in 4% PFA for 20 minutes.
29
What were the blocking agents used in immunofluorescence cytochemistry?
2% goat serum, 2% horse serum, and 1% BSA were used.
30
What software was used for immunofluorescence signal analysis?
Northern Eclipse version 7.0 software was used.
31
What was the purpose of creating a mask with β-III-tubulin signal?
To quantify alterations in immunofluorescence signal within axons.
32
What was the microscopy technique used for two-photon imaging?
A custom-modified Olympus BX51WIF upright research microscope was used.
33
What laser source was used for two-photon imaging?
A Mai Tai XF mode-locked Ti:Sapphire laser source was used.
34
What type of microscope was used in the research?
BX51WIF upright research microscope
## Footnote
Interfaced with an Ultima-X-Y laser-scanning module
35
What laser source was coupled to the microscope?
Mai Tai XF mode-locked Ti:Sapphire laser source
## Footnote
Provided by Spectra Physics
36
What type of objective lenses were used for imaging?
High numerical aperture Olympus objective lenses
## Footnote
Examples include LUMPLFL 40× W/IR-2 40×
37
How were the epifluorescence images detected?
With two top-mounted low-dark current high-sensitivity external PMT detectors
## Footnote
Provided by Hamamatsu
38
What was the spatial resolution of the Z-stack images?
0.29 µm/pixel
39
What buffer was used for homogenizing L4–L6 DRGs?
RIPA buffer containing protease inhibitor mixture
40
What method was used to determine protein concentration?
Bradford assay
41
What type of gels were used for electrophoresis?
12% SDS polyacrylamide gels
42
What membranes were proteins transferred onto?
PVDF membranes
## Footnote
Provided by Bio-Rad
43
What was the incubation condition for the primary antibodies?
Overnight at 4°C
44
Which protein was used as a loading control?
Goat anti-Lamin B or mouse anti-GAPDH
45
What type of arrays were used for UPR qRT-PCR?
UPR qRT-PCR arrays containing primers for 84 gene transcripts
46
What was used to extract total RNA?
RNeasy Kit from QIAGEN
47
What was the purpose of the qRT-PCR analysis?
To compare mRNA purified from dissociated DRG neurons transfected with Luman-specific siRNA or nontargeting control siRNA
48
What statistical method was used to assess differences between means?
One-way ANOVA with post hoc Tukey’s analysis
49
What significant p-value threshold was considered in the study?
p < 0.05
50
What was the main finding regarding sciatic nerve crush injury?
It triggers the UPR in DRGs and axons
51
Which UPR-related genes showed increased expression after injury?
GRP78, CHOP, xbp1, and the splice product of xbp1
52
What proteins were confirmed to increase in response to nerve injury?
PERK and IRE1
53
What type of imaging confirmed increased GRP78 and CHOP expression?
Immunofluorescence imaging
54
What does CHOP stand for?
C/EBP homologous protein
55
What does GRP78 stand for?
Glucose-regulated protein 78
56
What was used to verify the purity of axonal RNA extracts?
Absence of cell body-restricted γ-actin mRNA and presence of β-actin mRNA
57
What did the qRT-PCR analysis on pure axonal mRNAs reveal?
Increased levels of GRP78, CHOP, and spliced xbp1 in injury-conditioned neurons
58
Fill in the blank: The Odyssey Infrared Imaging System was used for ______.
visualizing proteins
59
What does qRT-PCR analysis reveal in injury-conditioned neurons?
Increased levels of GRP78, CHOP, and spliced xbp1
## Footnote
Indicates that nerve injury triggers the UPR in both axotomized DRGs and axons.
60
What proteins were analyzed in tissue sections of injured nerve?
GRP78 and CHOP
## Footnote
These proteins were analyzed using β III-tubulin immunofluorescence to define axonal regions.
61
What significant changes were observed in injured axons?
Significant injury-associated increases in GRP78 and CHOP
## Footnote
CHOP was also highly expressed in extra-axonal cells.
62
What was posited about the role of axonal injury in protein transport?
Axonal injury may induce retrograde transport of UPR proteins to the cell body
## Footnote
This is similar to the transport of axonal ER-resident transcription factor Luman.
63
What experimental method was used to further investigate the role of Luman?
Luman-selective siRNA treatment
## Footnote
This was to assess the impact on Luman expression at mRNA, protein, and functional levels.
64
What were the survival rates of neurons treated with Luman siRNA?
103 ± 5.51% (intact) and 102 ± 9.58% (injury-conditioned)
## Footnote
These percentages indicate survival relative to control cultures.
65
What effect did Luman siRNA have on Luman protein levels?
Knocked down expression of both full-length and cleaved/activated forms
## Footnote
This was confirmed by Western blot analysis.
66
What was observed about Luman localization in the nucleus post-axotomy?
Significantly lower levels of Luman in the nuclei of Luman siRNA-treated cultures
## Footnote
Compared to the axotomy control neurons.
67
What was the outcome of the qRT-PCR arrays assessing UPR-associated gene expression?
Many UPR-related genes were downregulated in Luman knockdown neurons
## Footnote
Included Insig1, Srebf1, Ubxn4, Insig2, Canx, and Mbtp2.
68
What specific UPR markers showed significant downregulation?
GRP78, CHOP, xbp1, and spliced xbp1
## Footnote
Confirmed by qRT-PCR analysis.
69
What indicates the purity of the 'axon only' extract in the experiments?
The absence of γ-actin
## Footnote
Confirms that only axons extend through the pores to grow on the underside of the membrane.
70
What was the purpose of using β III-tubulin in the analysis?
To detect neuron/axon markers in the cultures
## Footnote
This helps confirm the presence of axons and absence of glial cells.
71
What does the presence of intense CHOP or GRP78 signal indicate?
Retrograde transport of CHOP and GRP78 from the initial crush site toward the cell body
## Footnote
This supports the idea of injury-induced UPR response.
72
What is Luman associated with in the context of UPR?
Luman is a UPR-associated transcription factor
## Footnote
Activated in sensory neurons by axotomy.
73
Fill in the blank: The UPR is ______ in injured axons.
elevated
## Footnote
This indicates a response to cellular stress due to injury.
74
True or False: CHOP was only found in injured axons and not in extra-axonal cells.
False
## Footnote
CHOP was also highly expressed in extra-axonal cells.
75
What marker is colocalized with the axonal marker β-III tubulin?
UPR-associated proteins
## Footnote
This indicates the presence of UPR markers in axons following injury.
76
What was the significance level for the expression of markers in 1 d injured axons compared to intact control?
p < 0.01
## Footnote
This indicates a statistically significant difference in expression levels.
77
How many animals were analyzed in the study?
N = 3 animals
## Footnote
37–44 fields were analyzed per condition.
78
Which proteins showed significantly elevated expression in 1 d injured axons?
UPR-associated proteins
## Footnote
CHOP also showed increased extra axonal expression.
79
What are some of the genes downregulated by more than 2-fold?
* Insig1
* Insig2
* Srebf1
* Mbtps2
## Footnote
These genes are involved in the regulation of cholesterol synthesis.
80
What effect does supplemental cholesterol have on Luman knockdown neurons?
Rescues axon outgrowth
## Footnote
This occurs in vitro, indicating cholesterol's role in axon growth.
81
What was the percentage reduction in free and total cholesterol levels in Luman knockdown neurons?
34 ± 7% and 33 ± 5%
## Footnote
This reduction was observed in injury-conditioned sensory neurons.
82
What was the impact of Luman knockdown on SREBP1 expression?
Reduced at transcript and protein levels
## Footnote
This indicates a link between Luman and cholesterol biosynthesis.
83
What was the result of Luman knockdown on axon length?
Axons were 48 ± 6% shorter than injury-conditioned controls
## Footnote
This suggests impaired axon growth due to cholesterol deficiency.
84
What was the effect of cholesterol supplementation in Luman knockdown neurons?
Significantly rescued axon outgrowth to 74 ± 9% of control
## Footnote
This indicates the importance of cholesterol for axon regeneration.
85
What UPR inducer was used to investigate its effects on axon outgrowth?
Tunicamycin
## Footnote
Tunicamycin activates the UPR through pathways not involving Luman.
86
Which UPR markers showed decreased mRNA expression due to Luman knockdown?
* Canx
* Ubxn4
* GRP78
* CHOP
* xbp1
* spliced xbp1
## Footnote
These markers are crucial for the UPR response.
87
What was the effect of 2 ng/ml tunicamycin on UPR gene expression?
Increased expression by 1.4 ± 0.5-fold to 3.1 ± 0.4-fold
## Footnote
This brought expression levels close to those in control injured neurons.
88
What was the percentage of rescued axon outgrowth in Luman knockdown neurons treated with tunicamycin?
69 ± 9% of control injury-conditioned neurons
## Footnote
This indicates tunicamycin's role in promoting axon growth.
89
What was the combined effect of cholesterol and tunicamycin on axon growth?
Axons grew to 83 ± 10% of control injury-conditioned neurons
## Footnote
This suggests an interplay between cholesterol and UPR pathways.
90
What transcription factor is associated with cholesterol biosynthesis that was analyzed?
SREBP1
## Footnote
SREBP1 is crucial for driving cholesterol biosynthesis.
91
What type of transport was suggested by the accumulation of GRP78 proximal to ligation?
Anterograde transport
## Footnote
This occurs in response to prior injury.
92
What was used to analyze Luman expression levels in neurons?
qRT-PCR and Western blot analysis
## Footnote
These methods confirmed effective knockdown of Luman expression.
93
What is the observed effect of Luman siRNA treatment on nuclear Luman levels in axotomized neurons?
Visibly reduced nuclear Luman levels in the siRNA-treated injury conditioned neurons.
## Footnote
Scale bar, 25 µm.
94
What does the quantification of Luman immunofluorescence signal in importin-β-positive nuclei indicate?
A significant reduction in levels of Luman immunofluorescence signal in the nucleus (N = 97–98 importin-β-positive neuronal nuclei assessed/condition).
## Footnote
p < 0.001.
95
What was the effect of tunicamycin on axon outgrowth in Luman siRNA-treated neurons?
The rescue of axon outgrowth with 2 ng/ml tunicamycin was associated with increased SREBP1 expression in both the cell bodies and axons of these injury-conditioned neurons.
## Footnote
Qualitative and quantitative analysis revealed these changes.
96
How does cholesterol supplementation affect SREBP1 expression in Luman siRNA-treated, injury-conditioned neurons?
SREBP1 expression was reduced even further relative to Luman siRNA-treated, injury-conditioned control neurons.
## Footnote
This suggests a negative feedback loop in the endogenous cholesterol biosynthetic pathway.
97
What was the observed effect of cholesterol supplementation on CHOP expression in Luman siRNA-treated neurons?
CHOP expression was elevated in both the cell bodies and axon compartments of Luman siRNA + cholesterol-supplemented neurons relative to Luman siRNA injury-conditioned control neurons.
## Footnote
Quantification of the immunofluorescence signal over the neuronal cell body revealed these changes are significant.
98
What is the primary function of the UPR in neurons during ER stress?
To reestablish proteostasis while meeting the demand for increased protein production and/or proper protein folding under times of ER stress.
99
What role does Luman play in the UPR and sensory neuron regeneration?
Luman regulates the UPR and is involved in the intrinsic ability of injured sensory neurons to undergo robust axon regenerative growth.
100
What is the impact of the UPR on neurodegenerative disorders?
The deleterious impact of the UPR on neuronal pathological states is generally the focus of ER stress/UPR research.
101
How does axonal application of BDNF affect UPR components?
Axonal application of BDNF causes xbp1 splicing.
102
What is the effect of lysophosphatidic acid treatment on UPR components in axons?
It increases eIF2 phosphorylation and calreticulin translation.
103
What was the result of knocking down Luman expression in terms of UPR activation?
Knockdown of Luman expression decreases the injury-induced UPR.
104
What is the effect of Luman overexpression on cells under ER stress?
Luman overexpression protects cells against ER stress-induced apoptosis.
105
What does the dependency of sensory axon outgrowth on cholesterol indicate?
Knockdown of Luman decreased total and free cholesterol levels and axon outgrowth that could be partially rescued by supplemented cholesterol.
106
What does the assessment of UPR-associated gene mRNA levels in neurons treated with tunicamycin indicate?
It indicates the effectiveness of tunicamycin in inducing UPR-associated gene expression.
107
What is the significance of the relationship between cholesterol levels and axon outgrowth in the study?
Increased cholesterol levels were linked to enhanced axon growth in injury-conditioned neurons.
108
What is the effect of Luman knockdown on total axon outgrowth?
Luman knockdown decreased total axon outgrowth that could be partially rescued by tunicamycin.
109
What does UPR stand for?
Unfolded Protein Response.
110
What role does tunicamycin play in relation to UPR?
Tunicamycin is a UPR inducer.
111
True or False: Luman knockdown increases UPR in injury-conditioned neurons.
False.
112
Which protein is linked to axon growth and was mentioned in the study?
GRP78.
113
What is the relationship between UPR and axon growth according to the study?
UPR regulation is linked to the intrinsic regenerative axonal growth capacity of sensory neurons.
114
Fill in the blank: Luman knockdown was associated with lower levels of ______ in the injury-conditioned neuronal nuclei.
Luman.
115
What cellular event triggers the synthesis of axonal transcripts held in a translationally repressed state?
Axotomy.
116
What was observed regarding the expression of SREBP1 in Luman siRNA treated neurons?
SREBP1 expression significantly decreased in response to Luman siRNA treatment.
117
How does cholesterol supplementation affect SREBP1 expression in Luman siRNA treated neurons?
Cholesterol supplementation further reduced SREBP1 expression to nearly undetectable levels.
118
What is the consequence of exposing injury-conditioned neurons to high doses of tunicamycin?
It leaves the neurons in a growth incapacitated and compromised state.
119
What does the activation of SREBPs require?
Proteolysis by S1P and S2P.
120
True or False: Exogenous cholesterol has no impact on SREBP1 regulation.
False.
121
What is the primary function of SREBPs mentioned in the study?
Regulating sterol biosynthesis.
122
What was the dose of tunicamycin that benefited regenerative axonal growth?
2 ng/ml.
123
What happens to axon growth capacity in naive neurons when treated with tunicamycin?
There was no discernible impact on axon growth capacity.
124
What type of proteins are involved in transporting cargo proteins from the site of injury to the cell body?
Importin-β proteins.
125
Fill in the blank: The UPR is believed to have a ______ relationship with the intrinsic ability of sensory neurons to regenerate axons.
causal.
126
What is one potential compensatory mechanism for defective cholesterol biosynthesis in mature neurons?
Astrocytic cholesterol transfer.
127
What is the significance of the study by Ying et al. in relation to UPR and axon growth?
It demonstrates the connection between UPR and the ability of sensory neurons to regenerate axons.
128
True or False: The study suggests that Luman regulates UPR components involved in axon growth.
True.
129
What were treated injury-conditioned neurons observed with?
RNA + cholesterol
## Footnote
This suggests a potential link between lipid metabolism and neuronal response to injury.
130
What is inhibited in lipid repletion models according to Ye and Debose-Boyd (2011)?
Proteolytic cleavage/activation of SREBPs
## Footnote
SREBPs are key regulators of lipid homeostasis.
131
What may be an attempt by neurons to mitigate an excessive UPR response?
Cholesterol supplementation
## Footnote
A neuroprotective strategy during high UPR.
132
What elevated levels were observed in Luman knock-down neurons in response to cholesterol supplementation?
CHOP
## Footnote
CHOP is associated with the UPR and is indicative of stress responses in cells.
133
What does the lack of growth increase in control siRNA-transfected neurons suggest?
Additional factors beyond cholesterol limit regenerative growth
## Footnote
This indicates that cholesterol alone is not sufficient for axon regeneration.
134
What can endogenous axotomized DRG neurons synthesize enough of under the examined conditions?
Cholesterol
## Footnote
This suggests a self-sufficient mechanism for supporting regenerative axon growth.
135
What is transported back to the cell in an importin-β-dependent manner after axonal injury?
Activated Luman
## Footnote
Luman acts as a retrograde injury signal essential for neuronal regeneration.
136
What do the accumulation of axonal GRP78 and CHOP distal to a nerve injury site support?
That they may serve as retrograde injury signals
## Footnote
This highlights the role of UPR proteins in signaling during injury response.
137
What phase of sensory axon regeneration is indicated by the cellular events examined in the study?
Inductive phase
## Footnote
This phase is dependent on early rises in BDNF expression following injury.
138
What are the injury-associated increases in Luman, UPR, and SREBP1 expression in vivo characterized as?
Transient
## Footnote
Marked reductions in expression are observed by 4 days after axotomy.
139
What do these findings demonstrate regarding the UPR in axotomized DRG neurons?
An extensive Luman-regulated UPR is activated
## Footnote
This includes cholesterol regulation and is crucial for neuronal regeneration.
140
What do UPR induction and cholesterol supplementation contribute to?
Intrinsic regenerative axon growth capacity of sensory neurons
## Footnote
These findings provide novel therapeutic targets for improved nerve regeneration.
141
What protein does Luman/CREB3 induce transcription of through an ER stress response element?
Herp
## Footnote
Herp is an endoplasmic reticulum stress response protein.
142
Which family does Luman belong to?
CREB/ATF family
## Footnote
Luman is a new member of this family.
143
What is the main mechanism by which LRP1 regulates Schwann cell survival after injury?
The unfolded protein response
## Footnote
This response is crucial for cellular stress management.
144
What does ER stress protect against according to Mendes et al. (2009)?
Retinal degeneration
## Footnote
This indicates a protective role of ER stress in retinal health.
145
What are peripheral nerve axons capable of containing according to Merianda and Twiss (2013)?
Machinery for co-translational secretion of axonally-generated proteins
## Footnote
This suggests that axons can produce and secrete proteins directly.
146
Fill in the blank: The unfolded protein response is relevant to _______ and sleep disorders.
sleep
## Footnote
Naidoo (2009) discusses the relationship between cellular stress and sleep.
147
What new therapeutic target is suggested in diabetic peripheral neuropathy?
ER stress
## Footnote
O’Brien et al. (2014) emphasize the potential of targeting ER stress in treatment.
148
True or False: Restoring endoplasmic reticulum homeostasis improves functional recovery after spinal cord injury.
True
## Footnote
This conclusion is drawn from the research by Ohri et al. (2013).
149
What does membrane lipid saturation activate?
Endoplasmic reticulum unfolded protein response transducers
## Footnote
Volmer et al. (2013) describe this activation mechanism.
150
What does lysophosphatidic acid differentially regulate in axons?
Axonal mRNA translation
## Footnote
Vuppalanchi et al. (2012) studied this regulation through 5' UTR elements.
151
What is the role of Luman in relation to herpes simplex virus VP16?
Cellular counterpart processed by regulated intramembrane proteolysis
## Footnote
Raggo et al. (2002) investigate this relationship.
152
What does ER stress induce cleavage of?
Membrane-bound ATF6
## Footnote
Ye et al. (2000) highlight the role of proteases in this process.
153
What does activated Luman/CREB3 serve as according to Ying et al. (2014)?
A novel axonally synthesized retrograde regeneration signal
## Footnote
This indicates Luman's role in signaling for regeneration following nerve injury.
154
What is a functional role of intra-axonal protein synthesis during axonal regeneration?
Supports regeneration from adult sensory neurons
## Footnote
Zheng et al. (2001) provide insights into this functional role.
155
What cytokines are discussed by Zigmond (2012) in relation to nerve regeneration?
Cytokines that promote nerve regeneration
## Footnote
This highlights the role of specific cytokines in facilitating recovery.
