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MicroRNA-19a-PTEN axis is involved in the developmental decline of axon
regenerative capacity in retinal ganglion cells
Heather K. Mak, PhD, Jasmine SY. Yung, PhD, Robert N. Weinreb, MD, Shuk Han
Ng, MSc, Xu Cao, PhD, Tracy YC. Ho, PhD, Tsz Kin Ng, PhD, Wai Kit Chu, DPhil,
Wing-Ho Yung, DPhil, Kwong Wai Choy, PhD, Chi-Chiu Wang, M.B.B.S. PhD, Tin-
Lap Lee, PhD, Christopher Kai-shun Leung, MD, MB ChB
PII: S2162-2531(20)30157-8
DOI: https://doi.org/10.1016/j.omtn.2020.05.031
Reference: OMTN 901
To appear in: Molecular Therapy: Nucleic Acid
Received Date: 27 February 2020
Accepted Date: 28 May 2020
Please cite this article as: Mak HK, PhD Yung JS, PhD Weinreb RN, MD Ng SH, MSc Cao X, PhD Ho
TY, PhD Ng TK, PhD Chu WK, DPhil Yung W-H, DPhil Choy KW, PhD Wang C-C, PhD M, Lee T-L, PhD
Leung CK-s, MicroRNA-19a-PTEN axis is involved in the developmental decline of axon regenerative
capacity in retinal ganglion cells, Molecular Therapy: Nucleic Acid (2020), doi: https://doi.org/10.1016/
j.omtn.2020.05.031.
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1
MicroRNA-19a-PTEN axis is involved in the developmental decline of axon
1
regenerative capacity in retinal ganglion cells
2
3
4
Heather K Mak
1
, PhD, Jasmine SY Yung
1
, PhD, Robert N Weinreb
2
, MD, Shuk Han Ng
1
, MSc, Xu
5
Cao
1
, PhD, Tracy YC Ho
1
, PhD, Tsz Kin Ng
1
, PhD, Wai Kit Chu
1
, DPhil, Wing-Ho Yung
3,4
, DPhil,
6
Kwong Wai Choy
5
, PhD, Chi-Chiu Wang
5
, PhD, MBBS, Tin-Lap Lee
3
, PhD, Christopher Kai-shun
7
Leung
1
, MD, MB ChB
8
9
Affiliation:
10
1. Department of Ophthalmology and Visual Sciences, The Chinese University of Hong Kong,
11
Hong Kong, PRC.
12
2. Hamilton Glaucoma Center, Shiley Eye Institute, and Department of Ophthalmology,
13
University of California, San Diego, La Jolla, CA, USA.
14
3. School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong,
15
Hong Kong, PRC.
16
4. Gerald Choa Neuroscience Centre, The Chinese University of Hong Kong, Hong Kong, PRC.
17
5. Department of Obstetrics and Gynecology, Prince of Wales Hospital, The Chinese University
18
of Hong Kong, Hong Kong, PRC.
19
20
Short title:
21
miR-19a enhances axon regeneration in aged RGCs
22
23
Keywords:
24
Retinal ganglion cells; microRNA-19; Phosphatase and tensin homolog; axon regenerative
25
capacity; adeno-associated virus; optic nerve crush.
26
27
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29
30
31
Correspondence should be addressed to:
32
Christopher Kai-shun Leung
33
E-mail: cksleung@cuhk.edu.hk
34
35
Department of Ophthalmology and Visual Sciences
36
The Chinese University of Hong Kong
37
Hong Kong, PRC.
38
Tel: +852 2762 3181
39
Fax: +852 2715 9490
40
41
2
ABSTRACT
42
Irreversible blindness from glaucoma and optic neuropathies is attributed to retinal ganglion
43
cells (RGCs) losing the ability to regenerate axons. While several transcription factors and
44
proteins have demonstrated enhancement of axon regeneration after optic nerve injury,
45
mechanisms contributing to the age-related decline in axon regenerative capacity remain
46
elusive. Here, we show that microRNAs are differentially expressed during RGC development,
47
and identify microRNA-19a (miR-19a) as a heterochronic marker; developmental decline of
48
miR-19a relieves suppression of PTEN, a key regulator of axon regeneration, and serves as a
49
temporal indicator of decreasing axon regenerative capacity. Intravitreal injection of miR-19a
50
promotes axon regeneration after optic nerve crush in adult mice, and increases axon extension
51
in RGCs isolated from aged human donors. This study uncovers a previously unrecognized
52
involvement of the miR-19a-PTEN axis in RGC axon regeneration, and demonstrates therapeutic
53
potential of microRNA-mediated restoration of axon regenerative capacity via intravitreal
54
injection in patients with optic neuropathies.
55
56
57
58
59
60
61
62
63
3
INTRODUCTION
64
Developmental decline in axon regenerative capacity is well-recognized in retinal ganglion cells
65
(RGCs),
1,2
which leads to permanent loss in visual function in all forms of optic neuropathies
66
including glaucoma – a leading cause of irreversible blindness. Failure to regenerate axons in
67
RGCs has been attributed to external growth inhibiting factors in the central nervous system
68
(CNS)
3-8
and intrinsic molecular mechanisms that regulate cell growth.
9-13
Although a growing
69
number of transcription factors and proteins have been demonstrated to enhance axon
70
regenerative potential in injured RGCs,
14-20
the signaling pathways that govern axon extension
71
during development remain poorly understood.
72
MicroRNAs (miRNAs) are short non-coding RNA molecules that function primarily as
73
posttranscriptional regulators.
21,22
Although many studies have demonstrated the involvement
74
of miRNAs in the development and differentiation of cortical neurons in mice and C. elegans,
23-
75
33
it is largely unclear whether miRNAs are involved in the regulation of developmental decline
76
in axon regenerative capacity in the RGCs. We set off to identify miRNAs that are differentially
77
expressed in the RGCs during development and investigate whether manipulating their levels
78
can restore a molecular environment conducive for axon regeneration in aged or injured RGCs.
79
Using microarray analysis, we show that the levels of microRNA-19a (miR-19a) are substantively
80
downregulated in the RGCs during development and that its downregulation is inversely
81
associated with the expression of PTEN, a key suppressor of optic nerve regeneration.
14
PTEN is
82
a predicted target of miR-19a;
34
miR-19a has been implicated in the regulation of PTEN
83
expression in a variety of pathological and physiological conditions, such as the PTEN
84
hamartoma tumor syndromes,
35
regulation of glycogen synthesis in hepatocytes,
36
and
85
4
regulation of axon outgrowth in embryonic cortical neurons.
32
However, our understanding of
86
the involvement of the miR-19a-PTEN axis in the developmental decline of axon regenerative
87
capacity remains incomplete. Specifically, it is unclear whether miR-19a can resuscitate the loss
88
of axon regenerative capacity in adult RGCs. Here, we demonstrate the inverse relationship
89
between miR-19a and PTEN to be developmentally regulated, which coincides with the age-
90
related decline of axon regenerative capacity in RGCs; increasing the levels of miR-19a in RGCs
91
suppresses PTEN expression and significantly improves axon regeneration in vivo after optic
92
nerve crush in mice, as well as in RGCs isolated from aged human donors. Our results reveal a
93
previously unrecognized involvement of the miR-19a-PTEN axis as a heterochronic marker for
94
the developmental regulation of axon regenerative capacity.
95
96
RESULTS
97
Developmental decline in axon regenerative capacity coincides with a decreased expression
98
of microRNA-17-92 in retinal ganglion cells
99
To examine developmental decline of axon regenerative capacity in RGCs, we isolated RGCs
100
from Sprague Dawley (SD) rats using CD90.1 magnetic microbeads (Miltenyi Biotech) (Figure
101
S1A) and showed that neurites extended from post-natal day 6 (P6) and post-natal day 30 (P30)
102
RGCs were 74.4±2.7% (mean ± s.e.m.) and 88.4±0.7% shorter, respectively, than those from
103
embryonic day 21 (E21) RGCs (p<0.001) on day 14 in vitro (Figure S1B). We hypothesized that
104
miRNAs, as key regulators of post-transcriptional gene expression during development, axon
105
extension, and degeneration in cortical neurons,
31,33,37-42
would contribute to the
106
developmental decline of axon regenerative capacity in RGCs. Using microarray (Agilent) to
107
5
screen for differential expression of miRNAs in the RGCs during development, we found that 76
108
miRNAs had more than 4-fold difference in the expression levels between E21 and P30 RGCs,
109
among which 32 (42%) were up- and 44 (58%) were down-regulated (Figure 1A). The top three
110
miRNAs with the greatest fold change – miR-17, miR-20b, and miR-19a – were downregulated
111
from E21 to P30 RGCs (Table S1). Two of these miRNAs (miR-17 and miR-19a) belong to a highly
112
conserved single polycistronic cluster, the miR-17-92 cluster (Figure 1B).
43
The other members
113
of the miR-17-92 cluster (miR-18a, miR-19b, miR-20a and miR-92a) were also downregulated
114
from E21 to P30 (p≤0.025) (Figure 1C). TaqMan qRT-PCR confirmed that the expression levels of
115
miR-17, miR-18a, miR-19a, miR-19b, miR-20a and miR-92a in the RGCs decreased substantively
116
from E21 to P30 (p≤0.007) and from E21 to P6 (p≤0.014) (Figure 1C). Together, the
117
developmental decline of axon regenerative capacity in the RGCs parallels the substantial
118
decreases in the expression levels of the miR-17-92 family members.
119
We searched potential downstream targets of the miR-17-92 family members using
120
TargetScan that may connect to axon projection or regeneration and clustered a total of 6759
121
predicted targets according to their associated biological processes using the Gene Ontology
122
Consortium (Version 11).
44
There were 314 downstream targets with a significant association
123
(2.12-fold enrichment) with neuron projection development (Table S2). Within the 314 targets,
124
7 were found to be significantly related to regulation of axon regeneration (17.13-fold
125
enrichment): EPHA4, IGF1R, MAP2K1, NDEL1, PTEN, RGMA, and SCARF1.
14,45-50
The TargetScan
126
context++ score percentile rank
51,52
was then referenced to evaluate the preferential binding of
127
each of the miR-17-92 miRNA members to the relative efficacy of mRNA repression, in which
128
PTEN showed the highest score (76%) compared with the other targets (range: 15%-32%). PTEN
129
6
is a negative regulator of mTOR (mechanistic target of rapamycin), a key regulator of cell
130
growth.
53-58
Unlike other miR-17-92 family members having only 1 binding site on PTEN 3'UTR,
131
miR-19a and miR-19b have 3 binding sites on PTEN 3'UTR in human and 2 in mouse. As genetic
132
deletion of Pten and Socs3, a suppressor of cytokine signaling inhibiting STAT3 activation, has
133
been shown to synergistically promote axon regeneration after optic nerve crush,
17
we also
134
searched for potential binding of the miR-17-92 family members on SOCS3 3’UTR and found
135
that only miR-19a and miR-19b contain predicted binding sites on the 3’UTR sequences of PTEN
136
and SOCS3. miR-19a and miR-19b differ from each other only by one nucleotide and the
137
predicted locations of complementary binding onto PTEN and SOCS3 3’UTR are the same in
138
rodents and humans. With the expression level of miR-19a in RGCs showing a greater reduction
139
during development compared with miR-19b (Figure 1C and Table S1), we hypothesized miR-
140
19a to be an important upstream regulator controlling the expression of PTEN and SOCS3
141
during development that contributes to the decline in axon regenerative capacity in mature
142
RGCs.
143
144
Endogenous expression of microRNA-19a is inversely associated with PTEN in retinal ganglion
145
cells
146
To validate direct binding of miR-19a onto the 3’UTR binding sites of PTEN and SOCS3 in the
147
RGCs, we performed dual luciferase reporter assays in purified RGCs transfected with dual
148
luciferase reporter plasmids containing PTEN or SOCS3 3’UTR sequences and oligos of miR-19a
149
mimics or scramble (control) sequences (Figure S2A and S2B). PTEN luciferase activity
150
decreased by 55.9±6.4% (p<0.001) whereas SOCS3 luciferase activity showed no significant
151
7
difference (p=0.976) in RGCs transfected with miR-19a oligos versus those transfected with
152
scramble oligos (Figure 2A). Suppression of PTEN by miR-19a was relieved when the miR-19a
153
binding site was mutated (Figure 2A). Among the members of the miR-17-92 cluster, miR-19a
154
attained the greatest PTEN suppression in the RGCs (Figure S2C). We then examined if
155
overexpression of miR-19a in RGCs via transduction with enhanced green fluorescent protein-
156
tagged adeno-associated virus (AAV-eGFP) (Figure S3A) would decrease the protein expression
157
levels of PTEN and SOCS3. Immunoblots of AAV-miR-19a-eGFP-transduced RGCs revealed a
158
significant reduction in PTEN (25.9±0.04%, p=0.001), but not SOCS3 (p=0.869) protein
159
expression, compared with RGCs transduced with AAV-eGFP (Figure 2B). Single-cell
160
immunocytofluorescence staining further supported that overexpression of miR-19a decreased
161
the level of PTEN expression by 10.8±2.3% (p=0.002) (Figure 2C), whereas inhibition of miR-19a
162
in RGCs, via AAV transduction of a Tough Decoy miRNA inhibitor
59
(AAV-miR-19TuD-eGFP)
163
containing two miR-19a binding sites per transcribed RNA secondary structure (Figures S3A and
164
S3B), resulted in a 14.7±1.4% increase in PTEN expression (p<0.001) (Figure 2D), compared with
165
controls. The endogenous expression of miRNA-19a and PTEN exhibited a reciprocal
166
relationship in the retina during development (Figure 2E). In agreement with the TaqMan qRT-
167
PCR assay of miR-19a levels in purified RGCs, in situ hybridization (ISH) showed that miR-19a
168
expression, which was largely localized to the ganglion cell layer, decreased from E21 to P6 and
169
to P30 retinas. PTEN expression detected by immunohistochemistry, by contrast, increased
170
from E21 to P6 and to P30 retinas. ISH in purified RGCs revealed that miR-19a was strongly
171
expressed in the cytoplasm, axons, and growth cones of E21 RGCs, but was only weakly
172
expressed in the cytoplasm, and undetectable in the growth cones, in P6 and P30 RGCs (Figure
173
8
2F). PTEN, by contrast, was minimally expressed in E21 RGCs, weakly expressed in the
174
cytoplasm of P6 RGCs, and moderately expressed in the cytoplasm, axons and growth cones in
175
P30 RGCs (Figure 2G). P30 RGCs showed 1.6-fold and 1.4-fold increases in endogenous PTEN
176
expression compared with E21 and P6 RGCs, respectively (p<0.001) (Figure 2H). Collectively,
177
our data support that the developmental downregulation of miR-19a is connected to an
178
upregulation of PTEN expression in RGCs.
179
180
MicroRNA-19a augments axon regeneration in mature rodent retinal ganglion cells
181
We reasoned that increasing the level of miR-19a in mature RGCs would partially restore their
182
capacity to regenerate axons after optic nerve injury. To study axon regeneration, we measured
183
(1) the length of axon extension of RGCs in a microfluidic chamber, (2) the rate of axon
184
extension in isolated RGCs using time-lapse imaging, and (3) the number of regenerating RGC
185
axons after optic nerve crush in C57BL/6J mice. The microfluidic chamber is composed of two
186
compartments separated by microgrooves (Figure S4A). This creates a difference in hydrostatic
187
pressure and permits only axons to extend into the opposing compartment, isolating axons
188
from cell bodies. Immunofluorescence staining confirmed the segregation of TAU-positive
189
axons from MAP2-positive somata/dendrites in their respective compartments (Figures S4B
190
and S4C). At day-in-vitro 14, axon lengths of P6 RGCs transduced with AAV-miR-19a-eGFP were
191
2.9±0.5-fold longer compared with those transduced with AAV-eGFP (p=0.034) (Figure 3A).
192
Inhibiting miR-19a with AAV-miR-19a-TuD significantly decreased axon extension by 59.6±1.4%
193
compared with the control vector (p<0.001) (Figures S4D and S4E). To study the rate of axon
194
extension, P6 RGCs at day-in-vitro 14 were trypsinized and replated onto glass bottom dishes
195
9
for time-lapse imaging of individual RGCs. RGCs transduced with AAV-miR-19a-eGFP extended
196
axons at a faster rate (17.4±2.1 μm/hr) compared with those transduced with AAV-eGFP
197
(7.0±2.1 μm/hr) (p<0.001) (Figures 3B and 3C) (Movies S1 and S2). The mean length of axon
198
extension was also greater in AAV-miR-19a-eGFP-transduced RGCs (108.4±18.1 μm) than the
199
AAV-eGFP-transduced RGCs (47.8±11.5 μm) (p=0.019). There were no significant differences in
200
soma diameter, soma circularity, and baseline axon lengths (p≥0.273) between the two groups.
201
To investigate the effects of overexpression of miR-19a on RGC axon regeneration in
202
vivo, AAV-miR-19a-eGFP (n=8 mice) or AAV-eGFP (n=8 mice) was injected into the vitreous of a
203
randomly selected eye in 16 C57BL/6J mice (Figure 3D). Optic nerve crush was performed at 3-4
204
weeks after intravitreal injection (in vivo imaging using confocal scanning laser ophthalmoscopy
205
showed fluorescent intensity of AAV-transduced RGCs peaked at 2-3 weeks) (Figure S5A).
206
Cholera toxin subunit B (CTB) was then injected into the vitreous 3-4 weeks after optic nerve
207
crush and the number of CTB positive axons was measured at every 0.5mm from the crush site.
208
Immunofluorescence staining of GAP43 confirmed that CTB-positive axons were regenerating
209
axons (Figure S5B). AAV-miR-19a-eGFP-injected eyes had a greater number of regenerating
210
axons compared with AAV-eGFP-injected eyes (multivariable linear mixed modeling adjusting
211
for multiple comparisons at different distances from the crush site, p=0.004) (Figures 3E and
212
3F). Overexpression of miR-19a also improved RGC survival following optic nerve crush
213
compared with controls (p=0.006) (Figures S5C and S5D).
214
215
MicroRNA-19a promotes axon regeneration in human adult retinal ganglion cells
216
10
Similar to rodent RGCs, we observed a significantly higher level of miR-19a expression in human
217
fetal RGCs compared with aged RGCs purified from human donor eyes (Figure 4A). We next
218
examined whether overexpression of miR-19a was able to augment the axon regenerative
219
potential of aged RGCs isolated from adult human donor retinas. RGC axon length and total
220
neurite length (i.e. length of axons and dendrites) were measured and analyzed after two
221
weeks of AAV-mediated transduction of miR-19a-eGFP or eGFP. The frequency distributions of
222
axon length and total neurite length were right shifted for RGCs transduced with miR-19a-eGFP
223
(n=60 RGCs) compared with those transduced with the control vector (n=61 RGCs) (n=121 total
224
RGCs isolated from two human donors aged 69 years and 75 years) (Figure 4B). Axon length
225
and total neurite length of the miR-19a-eGFP transduced RGCs were 1.3-fold (p=0.041) and 1.7-
226
fold (p<0.001) longer, respectively, compared with those of the controls.
227
228
DISCUSSION
229
While a number of transcription factors and proteins have been implicated in the regulation of
230
axon regeneration, why mature RGCs lose their intrinsic ability to extend axons remains largely
231
unexplained. By isolating RGCs from the retina at different ages during development, our study
232
uncovers miR-19a to be a heterochronic marker that drastically decreases in expression during
233
the maturation of RGCs, which relieves the suppression of PTEN and contributes to the
234
developmental decline of axon regenerative capacity (Figure 4C).
235
MicroRNA-19a has been demonstrated to regulate neural progenitor cell migration
236
during neuronal development in the hippocampus and enhance axon growth in embryonic
237
cortical neurons.
32,42
Its role in the retina, however, has not been previously investigated. It is
238
11
worth noting that downregulation of miR-19a in the RGCs during development was not only
239
observed in rodents, but also in RGCs isolated from fetal and aged humor donors (Figure 4A).
240
Increasing the levels of miR-19a in the RGCs not only promoted axon regeneration in young
241
adult rodents (optic nerve crush was performed at the age of ~2 months), but also enhanced
242
axon regeneration in RGCs isolated from human donors at 69-75 years of age (Figure 4B). In
243
other words, the involvement of the miR-19a-PTEN axis in the regulation of axon extension or
244
regeneration is conserved in rodent and human RGCs. Enhancement of axon regeneration
245
following overexpression of miR-19a is likely to be translatable in clinical practice.
246
A caution in considering intravitreal injection of miR-19a for axon regeneration is its
247
oncogenic potential. While co-expressing other members of the miR-17-92 cluster may further
248
extend axon outgrowth, overexpression of the miR-17-92 cluster has been implicated in human
249
retinoblastoma although the oncogenic function in retinoblastoma is not mediated by the miR-
250
19a-PTEN axis.
60
With a selective tropism of AAV2/2 to the inner retina,
61
intravitreal injection
251
of AAV-miR-19a-eGFP attained a high transduction efficiency in RGCs, and we did not observe
252
tumor formation or other abnormal ocular findings in mice intravitreally injected with AAV-miR-
253
19a-eGFP for up to one year.
254
As U6-driven miRNAs are known to induce non-specific transcriptome changes,
62-64
the
255
lack of a scramble control to rule out potential off-targeting effects is a limitation to this study.
256
Further, as there was a total of 76 miRNAs that were differentially expressed in RGCs during
257
development (Figure 1A), modifying the level of miR-19a alone may not be sufficient to recover
258
visual function following optic nerve crush. Modulation of other intrinsic signaling pathways
259
that mediate axon growth capacity, refinement of the extrinsic growth-prohibiting
260
12
environment, and provision of growth cone guidance cues for synapse formation at the right
261
location would be required to fully restore the functional integrity of the optic nerve.
65-67
262
Although PTEN protein levels significantly increased in P30 RGCs by 60% and 40% from
263
E21 RGCs and from P6 RGCs, respectively (p<0.001) (Figure 2H), the developmental decline in
264
axon regenerative capacity would not be solely accountable by an increase in PTEN expression.
265
Growing evidence suggests that PTEN is not only post-transcriptionally but also post-
266
translationally regulated for regulation of axon growth and regeneration. Post-translational
267
modifications, such as phosphorylation, acetylation, oxidation, S-nitrosylation, ubiquitination,
268
or sumoylation, may influence PTEN phosphatase activity, binding to the membrane,
269
subcellular localization, or protein interactions in the regulation of axon growth and
270
regeneration.
68
Antagonizing the PDZ-motif interactions of PTEN using cell-permeable peptides,
271
for example, has been shown to increase neuronal survival, optic nerve regeneration, and visual
272
acuity after optic nerve injury in adult rats.
69
273
To summarize, our study demonstrates that the mir19a-PTEN-axis is involved in the
274
developmental decline of axon regenerative capacity in RGCs. That overexpression of miR-19a
275
augments axon regeneration in mature rodent RGCs and aged human RGCs underscores the
276
therapeutic potential of local administration of miRNAs via intravitreal injection to rejuvenate
277
RGCs for axon regeneration in the treatment of optic neuropathies.
278
279
MATERIAL AND METHODS
280
Experimental design
281
13
Sample sizes were determined for each study, based on the principle of using the minimum
282
number of animals to provide adequate statistical power, being mindful of the
283
recommendations of IACUC (animal studies). A minimum of 3 mice were used for each
284
treatment group, according to standard scientific conventions. More mice were used to
285
augment statistical power (final n=5-8 mice per treatment group). Sample size on human
286
biospecimen data was limited by availability. Sample sizes of all other experiments were based
287
on effect sizes and sample-to-sample variability during pilot experiments. Please see below and
288
figure legends for more details.
289
290
Data inclusion and exclusion
291
We excluded any data that failed to adhere to criteria that were established prior to data
292
collection. Microfluidic chambers excluded batches with microfluidic chambers that did not
293
fully adhere to culture plates, leading to inconsistent cell density and total cell number. Time
294
lapse imaging excluded RGCs that were connected to other cells, RGCs without eGFP signal, or
295
RGCs without an extended axon and visible growth cone prior to time-lapse imaging. Our in vivo
296
mice model excluded one mouse from the optic nerve count analyses due to an incomplete
297
optic nerve crush (i.e. residual axons remained in optic nerve sections), and another mouse due
298
to retinal hemorrhaging after optic nerve crush, leading to eyeball atrophy before time of
299
sample retrieval. No other data were excluded from analyses.
300
301
Randomization and blinding
302
14
C57BL/6J mice were allocated randomly to receive an intravitreal injection of AAV-miR-19a-
303
eGFP or AAV-eGFP in one eye. All mice in each treatment group were age and sex matched. All
304
manual tracings of single cell neurite lengths and microfluidic chamber axon lengths, and the
305
counting of regenerated optic nerve axons were analyzed by blinded investigators.
306
307
Postmortem human retinal tissues
308
Human adult retinal tissues were collected from the Hospital Authority Eye Bank, Hong Kong
309
Eye Hospital with written informed consent collected from a closest family member. Human
310
fetal retinal tissues from aborted fetuses were collected from the Department of Obstetrics and
311
Gynecology, Prince of Wales Hospital, Hong Kong, with written informed consent obtained from
312
the mother. All study protocols for human adult and fetal retinal tissues were approved by the
313
Joint CUHK-NTEC Clinical Research Ethics Committee and the Cluster Research Ethics
314
Committee/Institutional Review Board (REC/IRB). Purified RGCs that were cultured for human
315
adult RGC axon growth experiments in this study were collected from retina donors at 69 years
316
(female), 73 years (male), and 75 years (male). There were no known ocular or brain disorders
317
for each donor. Purified human fetal RGCs that were cultured for in situ hybridization staining in
318
this study were collected from a fetus at gestation week 14.6.
319
320
Experimental animals
321
All experimental procedures were approved by The Chinese University of Hong Kong Animal
322
Experimentation Ethics Committee and Hong Kong Department of Health, which adhere to The
323
International Guiding Principles for Biomedical Research Involving Animals and The Hong Kong
324
15
Code of Practice for Care and Use of Animals for Experimental Purposes. Sprague-Dawley (SD)
325
rats and C57BL/6J mice were fed standard diet ad libitum and housed in a 12-hr light/12-hr dark
326
light cycle.
327
328
Purification of retinal ganglion cells
329
Retinal ganglion cells were purified using a magnetic cell sorting technique. Briefly, retinas from
330
Sprague Dawley (SD) rats or human donor eyeballs were dissected and dissociated using Neural
331
Tissue Dissociation Kit Postnatal Neurons (Miltenyi Biotec, #130-094-802). All retinal tissues
332
were transferred into Neurobasal-A media (GIBCO) supplemented with 5% bovine serum
333
albumin (BSA) (Sigma) and placed on ice immediately upon collection. The dissociated retinas
334
were passed through a 40 μm filter (Fisher Scientific) to obtain a single-cell suspension before
335
RGC purification. For rat retinal tissue, primary antibody rat and mouse CD90.1 Microbeads
336
(Miltenyi Biotec #130-094-523) and biotinylated Depletion Antibody (Miltenyi Biotec #130-096-
337
209) were used for purification of RGCs and depletion of non-RGC neuronal cells, respectively,
338
according to manufacturer’s instructions. For human retinal tissue, primary antibodies human
339
CD90 Microbeads (Miltenyi Biotec #130-096-253) and biotinylated Human CX3CR1 (clone 2A9-
340
1, Miltenyi Biotec #130-096-446) were used for purification of RGCs and depletion of non-RGC
341
neuronal cells, respectively. Retinal ganglion cells were eluted from MS Columns (Miltenyi
342
Biotec, #130-042-201) using Neurobasal-A media (GIBCO) supplemented with 1X B27
343
(Invitrogen), 1X penicillin/streptomycin (GIBCO), 6mM sodium bicarbonate (Sigma), 1% BSA
344
(Sigma), 1X GlutaMAX (GIBCO), 10ng/ml brain-derived neurotrophic factor (BDNF) (Peprotech),
345
10ng/ml ciliary neurotrophic factor (CNTF) (Peprotech), 10ng/ml insulin-like growth factor 1
346
16
(IGF-1) (Peprotech), and 0.5µM forskolin (Sigma). Cells were plated at a density of 20-30
347
cells/mm
2
on culture plates (Corning) pre-coated with 0.2 mg/ml poly-D-lysine (PDL) (Millipore)
348
and 20 μg/ml laminin (Invitrogen). Approximately 30, 20, and 40 retinas from embryonic day 21
349
(E21), post-natal day 6 (P6), and post-natal day 30 (P30) SD rats, respectively, are required for
350
an average total yield of 8-10 x 10
5
RGCs.
351
352
Rodent RGC neurite length analysis
353
Images of cultured E21, P6, and P30 RGCs were captured using an inverted Leica DM IRB
354
microscope using the Spot software with a 20X objective (N Plan L 20X/0.4, Leica) on day 3, 7,
355
and 14 for comparisons of total neurite length. Images were assembled into montages and
356
adjusted for brightness and contrast before manual tracing. Total neurite length was measured
357
using a custom program written in MATLAB (Data file S1, cellbody_maxlength_3color.m).
358
359
Quantification of endogenous microRNA in retinal ganglion cells using microarray and
360
TaqMan qRT-PCR
361
For RNA extraction of purified E21, P6, and P30 RGCs, RNeasy Mini Kit (Qiagen) was used
362
according to manufacturer’s instructions. The quantity (ng/μl) and quality (A
260
/A
280
) of the
363
extracted RNA was measured using a spectrophotometer (Nanodrop ND-1000; Thermo
364
Scientific). Three biological replicates were analyzed for E21 (40 retinas per replicate) and P6
365
(20 retinas per replicate) RGCs. Two biological replicates were analyzed for P30 RGCs (40
366
retinas per replicate). After RNA extraction, total RNA from E21, P6, and P30 RGCs (100 ng per
367
biological replicate) was used for microarray analysis using miRNA Complete Labeling and
368
17
Hybridization Kit (5190-0456, Agilent) and SurePrint G3 Rat v16.0 miRNA Array Kit (Agilent),
369
according to manufacturer’s instructions. Rat microRNA Microarray v2.0 slides contained
370
probes for 677 rat miRNAs (Sanger miRBase database v16.0). Values from scanned images were
371
extracted using Agilent Feature Extraction Software (v9.5.3.1) with the default protocols and
372
settings for background subtraction and signal intensity processing. Non-uniform outliers were
373
excluded. Data was further analyzed using GeneSpring GX Software (v11.5). The data set was
374
normalized using a 75
th
percentile shift normalization method, which normalizes miRNA
375
expression values of each independent sample to the 75
th
percentile of the expression intensity
376
across all probes. P-values were calculated using a moderated t-test with a Benjamini-Hochberg
377
correction.
70-73
For TaqMan qRT-PCR, complementary DNA (cDNA) was synthesized from total
378
RNA using TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems), according to
379
manufacturer’s instructions. TaqMan® primer sequences were: miR-17-1-5p 5’-
380
CAAAGUGCUUACAGUGCAGGUAG-3’, miR-18a-5p 5’-UAAGGUGCAUCUAGUGCAGAUAG-3’, miR-
381
19a-3p 5’-UGUGCAAAUCUAUGCAAAACUGA-3’, miR-19b-3p 5’-
382
UGUGCAAAUCCAUGCAAAACUGA-3’, miR-20a-5p 5’-UAAAGUGCUUAUAGUGCAGGUAG-3’, miR-
383
92a-3p 5’-UAUUGCACUUGUCCCGGCCU-GU-3’, and U6 snRNA (endogenous control) 5’-
384
GTGCTCGCTTCGGCAGCACATATACTAAAATTGGAACGATACAGAGAAGATTAGCATGGCCCCTGCGCA
385
AGGATGACACGCAAATTCGTGAAGCGTTCCATATTTT-3’. Newly synthesized cDNA was qPCR-
386
amplified using TaqMan® MicroRNA Assay Kit (Applied Biosystems) and Roche PCR machine
387
(LC480 II), according to manufacturer’s instructions. Three biological replicates were assayed
388
per miRNA per PCR reaction (triplicate measurements). Results were averaged across all
389
18
replicate PCR reactions. The relative gene expression was calculated using the 2
−ΔΔCt
method
74
390
with normalization to U6 snRNA and relativity to E21.
391
392
Validation of miR-19a binding onto PTEN 3’UTR and quantification of PTEN downregulation
393
For dual luciferase assay, PTEN 3’UTR was amplified from human cDNA (forward primer, 5’-
394
TAATGAGCTCAATGCTCAGAAAGGAAATAA-3’; reverse primer, 5’-TAATTCTAGATGCCACAGCAAA-
395
GAATGGTG-3’; 2.2kb length), and contained two miR-19a binding sites; SOCS3 3’UTR was
396
amplified from human cDNA (forward primer, 5’-TAATGAGCTCACTCACTGGAGGGCAC-3’;
397
reverse primer, 5’TAATTCTAGATTTTTCATTAAAAAAT-3’; 0.7kb length), and contained one miR-
398
19a binding site (upstream SacI and XbaI restriction sites in forward and reverse primers,
399
respectively, are in boldface). Full length human PTEN and SOCS3 3’UTR contain three and one
400
miR-19a binding sites, respectively. PTEN 3’UTR with miR-19a binding sites mutated (mPTEN)
401
replaced both miR-19a binding sites with AAAAAAA. All three 3’UTR sequences were cloned
402
into pmirGLO plasmid (Promega) that contained the Firefly luciferase (luc2) gene as the primary
403
reporter monitoring mRNA regulation whereas the Renilla luciferase (hRluc-neo) gene acted as
404
a control reporter for endogenous normalization control (Figures S2A and S2B). DNA plasmids
405
were purified using QIAfilter Plasmid Midi Kit (Qiagen). Purified P6 RGCs were cultured at 20-30
406
cells/mm
2
in 24-well plates (Corning) for 3 days before co-transfection of 0.8 μg pmirGLO
407
reporter plasmid containing PTEN, mPTEN, or SOCS3 3’UTRs with 100 nM of miR-19a-3p mimic
408
oligo (mirVana®, 4464066) or scramble miRNA oligo 5’-TAACACGTCTATACGCCCA-3’ (Exiqon,
409
199006-011) using NeuroMag transfection reagent (1:3; OZ Biosciences). Experiments were
410
performed in 4 replicates with data averaged across 3 culture replicates per oligo per gene per
411
19
experimental replicate. Firefly and Renilla luciferase expression was assayed at 36 hours post-
412
transfection using the Dual-Luciferase® Reporter Assay System (Promega), according to
413
manufacturer’s instructions. Luciferase expression was measured using GloMax® 20/20
414
luminometer (Promega). Relative Firefly-to-Renilla luciferase ratio was calculated to normalize
415
3’UTR expression to endogenous gene expression for each sample. Relative luciferase
416
expression with miRNA targets was relative to luciferase expression with scramble oligos.
417
Empty vector pmirGLO was used as a positive control for transfection efficiency and signal
418
detection. For immunoblot, purified P6 RGCs were transduced by AAV-miR-19a-eGFP or AAV-
419
eGFP at a multiplicity of infection of 1x10
5
and cultured at 40 cells/mm
2
. On day 14, RGCs were
420
lysed with RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% Triton-X 100, 0.5% sodium
421
deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 1 mM sodium orthovanadate, 1 mM NaF,
422
and protease inhibitors (Complete
TM
, Roche)) for total protein extraction. Concentration of
423
protein was quantified using a total protein assay (Bio-Rad, Hercules, CA). Forty micrograms of
424
protein were used per sample with an equal volume of 2X Laemmli buffer containing 4% SDS,
425
10% 2-mercaptoethanol, 20% glycerol, 0.004% bromophenol blue, and 0.125M Tris-HCl at pH
426
6.8. For gel electrophoresis and blotting, equal amounts of protein were loaded into a 12.5%
427
acrylamide resolving gel (0.3% bis-acrylamide, 0.375 M Tris-HCl, 0.1% SDS, 0.05% ammonium
428
persulfate, and 0.02% TEMED) and 4% acrylamide stacking gel (0.15% bis-acrylamide, 0.125 M
429
Tris-HCl, 0.1% SDS, 0.05% ammonium persulfate, and 0.02% TEMED). After electrophoresis, the
430
gel was transferred to a nitrocellulose membrane (Amersham Pharmacia; Cleveland, OH) for
431
blotting. Membranes were blocked in 5% non-fat milk powder (Santa Cruz Biotechnology) in
432
TBST buffer at room temperature for 2 hours before primary antibody incubation at 4˚C
433
20
overnight with rabbit monoclonal PTEN (1:1000; Cell Signaling #9559) or mouse monoclonal
434
SOCS3 (1:1000; Abcam #ab14939) diluted in 1% non-fat milk. After incubation, membranes
435
were rinsed and incubated with horse-radish peroxidase (HRP)-conjugated secondary antibody
436
(1:1000; Jackson Immuno. Res, West Grove, PA) at room temperature for 1 hour. GAPDH
437
(1:10,000; Thermo Fisher #AM4300) or beta-Actin (1:10,000; Sigma #A3854) was used as an
438
internal loading control. Chemiluminescent signals from bands were detected using Enhanced
439
Chemiluminescence (ECL) (Amersham Pharmacia) and imaged using CCD camera ChemiDoc
440
(Biorad). ImageJ was used to quantify band intensity. All antibodies used in this study can be
441
found in Table S3.
442
443
Immunofluorescence staining
444
Cultured RGCs were fixed in 4% paraformaldehyde (PFA) for 20 minutes at room temperature,
445
and retinal whole mounts and optic nerves were fixed in 4% paraformaldehyde overnight at 4°C
446
before a 5% normal goat serum (The Jackson Laboratory) and 1% BSA (Sigma) solution in PBS
447
was used for blocking. Primary antibodies included rabbit monoclonal BRN3A (1:50; Abcam
448
#ab81213), rabbit polyclonal GAP43 (1:200; Abcam, ab12274), mouse monoclonal MAP2
449
(1:200; Upstate, 05-346), rabbit monoclonal PTEN (1:100; Cell Signaling #9559), rabbit
450
monoclonal TAU (1:200; Abcam, ab32057), and mouse monoclonal TUJ1 (1:200; Millipore
451
#MAB5564). Primary antibodies were diluted using 2% NGS and 1% BSA in PBS (0.03% Triton-X
452
100 was supplemented for BRN3A) and incubated overnight at 4°C. Secondary antibodies were
453
diluted 1:500 (Alexa Fluor® 488 or Alexa Fluor® 555 conjugates; Molecular Probes,
454
ThermoFisher) with Hoechst (1:1000; Sigma #H6024) using 2% NGS and 1% BSA in PBS at room
455
21
temperature (2 hrs). Coverslips or slides were mounted using GB-Mount (GBI Labs). Images
456
were captured using a Nikon A1MP confocal microscope using the NIS Elements software with a
457
40X objective (NIR Apo 40X/0.8W, Nikon), or Nikon ECLIPSE Ti microscope using the NIS
458
Elements software with a 20X objective (S Plan Fluor 20X, Nikon).
459
460
In situ hybridization of miR-19a in human and rodent purified RGCs and rodent retinas
461
MicroRNA in situ hybridization (ISH) optimization kit (Exiqon) and TSA plus fluorescein
462
evaluation kit (Perkin Elmer) were used for chromogenic and fluorescent detection of miR-19a
463
in the retinas and cultured RGCs, respectively, according to manufacturer’s instructions. Retinas
464
were fixed in 4% PFA at 4°C overnight, cryoprotected in serial dilutions of sucrose (5%, 15%, and
465
30%), and snap frozen in Tissue-Tek® optimal cutting temperature compound (OCT compound)
466
(Fisher Scientific). Only cryosections (8 μm) containing both pupil and optic nerve structures
467
were examined. Before probe hybridization, cryosections were heated at 40°C for 20 minutes
468
before OCT compound removal by PBS. Purified RGCs cultured on 13mm coverslips
469
(Sondheim/Rhön, Germany) for 7 days were fixed using 4% PFA at room temperature for 20
470
minutes before fluorescent ISH detection. Double digoxigenin (DIG)-labeled miRCURY locked
471
nucleic acid (LNA)
TM
microRNA detection probes (Exiqon) included miR-19a-3p 5’-
472
TCAGTTTTGCATAGATTTGCAC-3’ (40nM; Exiqon #611510-360), scramble miRNA negative
473
control 5’-GTGTAACACGTCTATACGCCCA-3’ (40nM; Exiqon #699004-360), and U6 snRNA
474
positive control 5’-CACGAATTTGCGTGTCATCCTT-3’ (4nM; Exiqon #699002-360). All procedures
475
before the addition of primary antibody is the same for chromogenic and fluorescent ISH.
476
Primary antibodies used for chromogenic and fluorescent ISH were anti-DIG-AP (1:800; Roche)
477
22
and anti-DIG-POD (1:800; Roche), respectively. Samples were mounted with GB-Mount (GBI
478
Labs) after counterstaining with Hoechst (1:1000; Sigma #H6024). For fluorescent samples,
479
images were captured using a Nikon A1MP confocal microscope using the NIS Elements
480
software with a 40X objective (NIR Apo 40X/0.8W, Nikon). For chromogenic samples, images
481
were captured using an upright Carl Zeiss Axioplan 2 microscope using the Spot software with a
482
20X objective (Plan-ApoChromat 20X/0.75, Carl Zeiss).
483
484
Adeno-associated virus (AAV) design and packaging
485
AAV-miR-19a-eGFP was packaged by Virovek (Hayward, CA). Mature miR-19a-3p sequence 5’-
486
TCAGTTTTGCATAGATTTGCACA-3’ was cloned into a pFastBac (pFB) AAV2/2 shuttle vector with
487
a hybrid construct consisting the cytomegalovirus (CMV) enhancer fused to the chicken β-actin
488
promoter (CAG) promoter and an enhanced green fluorescent protein (eGFP) fluorescent
489
marker. An empty vector control without a miRNA insert was cloned using the same backbone
490
with either eGFP (green) or mCherry (red) as a fluorescent marker (Figure S3A). Validation with
491
TaqMan qRT-PCR demonstrated a 2.5±0.3-fold increase of miR-19a expression levels in RGCs
492
transduced with AAV-miR-19a-eGFP compared with those transduced with an AAV-eGFP empty
493
vector control (Figure S3C). AAV-miR-19aTuD-eGFP was packaged by University of Iowa, Viral
494
Vector Core (Iowa City, IA). The miR-19aTuD sequence 5’-
495
GGATCCGACGGCGCTAGGATCATCAACCAGTTTTcccaATAGATTTGCACACAAGTATTCTGGTCACAGA
496
ATACAACCAGTTTTcccaATAGATTTGCACACAAGATGATCCTAGCGCCGTCTTTTTTGAATTC-3’
497
(binding site of miR-19a is italicized, seed sequence is underlined, and small caps indicate
498
location of induced bulge to increase miRNA binding affinity by imperfect complementarity)
499
23
was designed based on previously described methods of AAV-mediated expression of miRNA
500
TuD constructs
59
and cloned into a pFB AAV2/2 shuttle vector with a CMV promoter and an
501
eGFP fluorescent marker (Figure S3A and S3B). An empty vector control was cloned using the
502
same backbone with an eGFP fluorescent marker. Validation with TaqMan qRT-PCR
503
demonstrated a 74±9.9% reduction in miR-19a expression levels in RGCs transduced with AAV-
504
miR-19aTuD-eGFP compared with those transduced with AAV-eGFP empty vector control
505
(Figure S3C). All RGCs in vitro were transduced at a multiplicity of infection of 1x10
5
viral
506
genomes/cell.
507
508
Retinal ganglion cell culture in microfluidic chamber
509
Axon length was measured by plating purified P6 RGCs into a microfluidic chamber device
510
(Xona Microfluidics, RD450) (Figure S4A) for the separation of RGC soma and dendrites from
511
axons (Figure S4B and Figure S4C). The device is composed of two compartments joined by
512
microgrooves that extend 450 μm from one compartment to the other. With RGC somas and
513
dendrites localized in one compartment, only axons extended into the adjacent compartment
514
through microgrooves. Purified P6 RGCs were transduced by AAV-miR-19a-eGFP or AAV-eGFP
515
at a multiplicity of infection of 1x10
5
and plated at a density of 3-4 x 10
5
cells/chamber. Images
516
were captured and automatically stitched using a Nikon ECLIPSE Ti microscope and NIS
517
Elements software on day 14 using a 20X objective (S Plan Fluor 20X, Nikon). All axons extended
518
into the axon compartment were manually traced before measurement of total axon length
519
(i.e. sum of all manually traced axon extensions) using a custom program written in MATLAB
520
(Data file S2, N_skeleton.m).
521
24
522
Time-lapse imaging of retinal ganglion cells
523
The rate of axon extension of purified RGCs (cultured at 20 cells/mm
2
) from P6 SD rats
524
transduced by AAV-miR-19a-eGFP or AAV-eGFP (multiplicity of infection of 1x10
5
) was
525
measured with time-lapse imaging. RGCs cultured for 14 days were trypsinized and replated
526
onto glass bottom dishes (P35G-1.0-14, MatTek) pre-coated with 0.2 mg/ml poly-D-lysine (PDL)
527
(Millipore) and 20 μg/ml laminin (Invitrogen) in a 37˚C heated chamber (Chamlide UM, Live Cell
528
Instrument, Korea) with 5% CO
2
. Only RGCs with eGFP expression and an extended neurite with
529
a visible growth cone were selected for time-lapse imaging. Images were automatically
530
captured every 20 minutes for 8 hours by a Nikon A1MP confocal microscope using the NIS
531
Elements software with a 40X objective (NIR Apo 40X/0.8W, Nikon). The longest neurite length
532
and soma area were manually traced and measured at each time point using the NIS Elements
533
Analysis software (NIS AR, Nikon). Circularity is defined as a measure of shape that is derived
534
from the area a and perimeter p, such that circularity = 4 π a / p
2
. Circularity equals 1 for circles;
535
whereas all other shapes have a circularity less than 1 (NIS AR, Nikon).
536
537
Intravitreal injection of AAV and optic nerve crush
538
Sixteen one-month-old C57BL/6J mice (males) were anaesthetized with 100 mg/kg ketamine
539
and 9 mg/kg xylazine. Intravitreal injection of 1μl (2.13 x 10
13
vg/ml) of AAV-miR-19a-eGFP (n=8
540
mice) or AAV-eGFP (n=8 mice) was performed in one eye of each animal. The optic nerve of the
541
injected eye was crushed 3-4 weeks after the intravitreal injection. After anaesthetizing the
542
animals (as described above), inferior limbal conjunctival peritomy was performed. The
543
25
conjunctiva was gently peeled back to allow access to the retrobulbar region. The optic nerve
544
was exposed through a small window after a gentle blunt dissection dissociating the
545
surrounding blood sinuses and connective tissue. The optic nerve was crushed using a pair of
546
Dumant No. 5 self-closing tweezers (Ted Pella Inc., Redding, CA) for 2 seconds at a site
547
approximately 1 mm posterior to the globe. Antibiotic ointment was applied to the surgical site
548
after injection. During the postoperative period, mice were monitored for normal eating and
549
drinking behavior.
550
551
Quantification of regenerated axons in the optic nerve
552
After 3-4 weeks of optic nerve crush, mice were anesthetized with 100 mg/kg ketamine and 9
553
mg/kg xylazine. The eye with optic nerve crush received intravitreal injection of 1 μl of cholera
554
toxin β subunit (CTB) Alexa Fluor® 555 Conjugate (2 μg/μl; Invitrogen). Four days after
555
intravitreal injection of CTB, mice were perfused using 4% PFA. The eyeballs were removed and
556
immersed in 4% PFA at 4°C overnight for TUJ1 staining (see Immunofluorescence staining for
557
quantification of RGC survival below). The optic nerves were dissected, immersed in 4% PFA at
558
4°C overnight, cryoprotected in serial dilutions of sucrose (5%, 15%, and 30%), and snap frozen
559
in Tissue-Tek® OCT Compound (Fisher Scientific). The cryosections were cut at 8μm thickness t
560
using a cryostat (CryoStar NX50, Thermo Scientific). Three to five sections of each optic nerve
561
were used for analysis by two blinded observers. The number of CTB-labeled axons were
562
counted at distances of 0.2mm, 0.5mm, 1mm, 1.5mm, 2mm, 2.5mm, 3mm, 3.5mm, and 4mm
563
from the optic nerve crush site. The cross-sectional width of the optic nerve was measured at
564
every recorded distance from the optic nerve crush site. The radius r (µm) of the optic nerve at
565
26
every recorded distance was defined as the measured width w (µm) divided by 2. The total
566
number of regenerated axons a at every recorded distance d (mm) from the optic nerve crush
567
site was estimated by averaging all sections with a radius r (µm), width w (µm), and thickness t
568
(µm): a
d
= πr
2
x [average number of counted regenerated axons per section per recorded
569
distance / (w x t)].
570
571
Immunofluorescence staining for quantification of RGC survival
572
Retinas were dissected for TUJ1 immunofluorescence staining to quantify the number of
573
surviving RGCs after optic nerve crush. Retinas were blocked in 5% NGS and 1% BSA for 1 hour
574
at room temperature, incubated with monoclonal TUJ1 antibody (1:500; Covance #PRB-425P) in
575
2% NGS and 1% BSA overnight at 4°C. Anti-rabbit secondary antibody (Alexa Fluor
®
350, Thermo
576
Fisher) was used in 2% NGS and 1% BSA at 1:500 for 2 hours at room temperature. Retinas
577
were flat-mounted with the RGC layer facing upwards and mounted onto a glass slide using GB-
578
Mount (GBI Labs). Images were captured with a Nikon A1MP confocal microscope using the NIS
579
Elements software with a 40X objective (NIR Apo 40X/0.8W, Nikon). A total of 24 fields were
580
imaged per retina (6 fields per retinal quadrant). The total number of TUJ1-positive cells per
581
field was measured across all images per eye.
582
583
Human RGC axon and neurite length analysis
584
Purified human adult RGCs were plated at 40 cells/mm
2
and transduced with either AAV-eGFP
585
or AAV-miR-19a-eGFP at an MOI of 1 x 10
5
. Two weeks post-transduction, RGCs were fixed and
586
imaged using a Nikon ECLIPSE Ti microscope using the NIS Elements software with a 20X
587
27
objective (S Plan Fluor 20X, Nikon). Only RGCs with eGFP expression were included for analysis.
588
All RGC cell bodies and neurites were manually traced before axon length and total neurite
589
length analysis using written MATLAB program (Data file S1, cellbody_maxlength_3color.m).
590
The longest neurite of the RGC was identified as the axon.
591
592
Statistical analysis
593
Statistical analyses were performed using Stata 13.0 (StataCorp; College Station, TX). A two-
594
tailed moderated t-test with a Benjamini-Hochberg correction for false discovery rate was used
595
to compare the means of miRNA microarray expression values between E21 and P6, and E21
596
and P30.
70-73
An unpaired two-tailed Student’s t-test was used for comparisons between two
597
groups of equal sample size: (1) miR-17-92 expression levels measured by TaqMan qRT-PCR
598
between E21 and P6/P30 RGCs, (2) RGC neurite lengths in vitro between E21 and P6/P30 RGCs,
599
(3) dual luciferase PTEN and SOCS3 3’UTR expression levels between AAV-eGFP- and AAV-miR-
600
19a-eGFP-transduced RGCs, (4) miR-19a fold change measured by TaqMan qRT-PCR between
601
AAV-eGFP- and AAV-miR-19a-eGFP-transduced RGCs and between AAV-eGFP- and AAV-miR-
602
19aTuD-eGFP-transduced RGCs, (5) PTEN and SOCS3 immunoblots between AAV-eGFP- and
603
AAV-miR-19a-eGFP-transduced RGCs, (6) immunocytofluorescent staining of PTEN between
604
AAV-eGFP- and AAV-miR-19a-eGFP-transduced RGCs and between AAV-eGFP- and AAV-miR-
605
19aTuD-eGFP-transduced RGCs, (7) immunocytofluorescent staining of PTEN between E21 and
606
P6, E21 and P30, and P6 and P30 RGCs, (8) microfluidic chamber axon lengths between AAV-
607
eGFP- and AAV-miR-19a-eGFP-transduced RGCs and between AAV-eGFP- and AAV-miR-19aTuD-
608
eGFP-transduced RGCs, (9) RGC soma diameter, soma circularity, duration, baseline length, and
609
28
length of axon extension between AAV-eGFP- and AAV-miR-19a-eGFP-transduced RGCs, (10)
610
TUJ1-positive RGCs after ON crush between AAV-eGFP- and AAV-miR-19a-eGFP-transduced
611
retinas, and (11) human RGC axon lengths between AAV-eGFP- and AAV-miR-19a-eGFP-
612
transduced RGCs. Multivariable linear mixed modeling was used for analysis of (1) time-lapse
613
imaging comparing the rates of axon extension between AAV-eGFP- and AAV-miR-19a-eGFP-
614
transduced RGCs and (2) optic nerve count of regenerated axonal fibers, controlling for
615
repeated measurements. For time-lapse imaging for measurement of axon extension, the
616
model was fitted with axon length as the dependent variable; time, types of AAV transduction
617
(AAV-eGFP or AAV-miR-19a-eGFP), the interaction between time and types of AAV transduction
618
(to determine if the types of AAV transduction would affect the rate of axon extension),
619
baseline axon length, and the interaction between baseline axon length and time as fixed
620
effects variables, including random intercepts (axon lengths measured at multiple time points
621
nested within cell) and random coefficients for time for individual RGCs. For optic nerve counts,
622
the model was fitted with CTB-labelled axon length as the dependent variable; distance from
623
optic nerve crush site and types of AAV transduction (AAV-eGFP or AAV-miR-19a-eGFP) as fixed
624
effect variables, with random intercepts for individual mice (number of CTB-labeled axons
625
nested within mice). In all analyses, P < 0.05 was considered to be statistically significant.
626
627
Data and software availability
628
All microarray data that support the findings of this study have been deposited in the National
629
Center for Biotechnology Information Gene Expression Omnibus (GEO) and are accessible
630
through the GEO Series accession number (GEO ID: GSE102458). All other relevant data are
631
29
available from the corresponding author upon request. All MATLAB software coding that
632
support the findings of this study are provided in Supplemental Information.
633
634
Author contributions
635
H.M. performed the purification and culture of retinal ganglion cells, image and data analyses,
636
data interpretation, and wrote the manuscript. J.Y. assisted with cell culture, immunoblotting,
637
and luciferase plasmid designs. C.X. assisted with cell culture and neurite imaging and tracing.
638
S.N. assisted with optic nerve crush and performed sample retrieval and optic nerve sectioning
639
and imaging. K.C. and C.W. provided fetal retinal tissue samples and performed the microarray.
640
H.M. performed all other experiments. T.N. and T.H. assisted with purification of retinal
641
ganglion cells and data analyses. W.C., T.L., T.N., R.W., and W.Y. contributed to the design of
642
the study and proofing of the manuscript. C.L. conceived and designed the study, performed
643
intravitreal injections and optic nerve crush, assisted with data analysis and interpretation,
644
supervised the work, wrote the manuscript, and gave final approval of the manuscript.
645
646
Acknowledgements
647
We would like to thank Ms. Catherine Wong and her team (Hospital Authority Eye Bank, Hong
648
Kong Eye Hospital) for providing human adult retinal tissues and for their continued support
649
and collaboration, and Prof. Calvin Chi Pui Pang for his contribution in providing suggestions to
650
improve the manuscript. This work was supported by funding from the Hong Kong General
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Research Fund (14109814).
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653
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Conflict of interest
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The authors declare no competing financial interests.
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FIGURE TITLES AND LEGENDS
851
Figure 1. MicroRNAs are differentially expressed in retinal ganglion cells during development.
852
(A) A representative heat map of microRNA expression profiles constructed from a microarray
853
analysis of retinal ganglion cells (RGCs) purified from embryonic day 21 (E21, n=3 biological
854
replicates), post-natal day 6 (P6, n=3 biological replicates), and post-natal day 30 (P30, n=2
855
biological replicates) Sprague Dawley (SD) rats, showing 76 endogenously expressed microRNAs
856
with significant differential expression (≥4-fold changes in expression levels) during
857
development. Developmental ages (as biological replicates) are indicated in columns, and
858
differentially expressed microRNAs are indicated in rows. All 6 members of the miR-17-92
859
cluster (red asterisks) were found to have significant downregulation from E21 to P30 (right
860
panel). Blue (-8.5) and red (+8.5) in the color coding scale represent relative low and high
861
normalized microRNA expressions, respectively. A two-tailed moderated t-test with a Benjamini-
862
Hochberg correction for false discovery rate was used to compare the means of miRNA
863
microarray expression values between E21 and P6, and E21 and P30. List of the 76 microRNAs
864
can be found in “Table S1” and all other microarray data that support the findings of this study
865
have been deposited in the National Center for Biotechnology Information Gene Expression
866
40
Omnibus (GEO) and are accessible through the GEO Series accession number (GEO ID:
867
GSE102458). (B) Illustration of the polycistronic miR-17-92 cluster region on human
868
chromosome 13 (chromosome 15 for rats). Precursor transcripts derived from the miR-17-92
869
gene cluster contains six tandem stem-loop hairpin structures that yield six mature miRNAs:
870
miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a. The miRNAs can be categorized into
871
four miRNA families according to their conserved seed sequences (underlined). (C)
872
Downregulation of the 6 miRNAs from E21 to P30, and from E21 to P6 was confirmed by
873
TaqMan qRT-PCR (E21 vs. P6, miR-17-5p, p=0.0053, t=5.5129, df=4; miR-18a, p=0.0029,
874
t=6.4974, df=4; miR-19a, p=0.0136, t=4.2087, df=4; miR-19b, p=0.0126, t=4.3057, df=4; miR-
875
20a, p=0.005, t=5.5871, df=4; miR-92a, p=0.014, t=4.1772, df=4; E21 vs. P30, miR-17-5p,
876
p=0.0004, t=10.9137, df=4; miR-18a, p=0.0005, t=10.5594, df=4; miR-19a, p=0.0072, t=5.0504,
877
df=4; miR-19b, p=0.0046, t=5.7335, df=4; miR-20a, p=0.0011, t=8.3661, df=4; miR-92a,
878
p=0.0032, t=6.3286, df=4; n=3 biological replicates for each age group). Unpaired two-tailed
879
Student’s t-test was used for TaqMan comparisons. All values are in mean ± s.e.m.
880
881
Figure 2. Developmental decline of miR-19a is associated with increased PTEN expression in
882
retinal ganglion cells.
883
(A) Dual luciferase assays of PTEN, mPTEN, and SOCS3 3’UTR after transfection with miR-19a
884
mimic or scramble oligos in P6 RGCs. Only PTEN 3’UTR, but not mPTEN nor SOCS3 3’UTR was
885
significantly suppressed by miR-19a (PTEN, p=0.0001, t=8.8019, df=6; mPTEN, p=0.9873, t=-
886
0.0169, df=4; SOCS3, p=0.9763, t=-0.0310, df=6; n=3-4 experimental replicates averaged across
887
3 culture replicates per oligo per gene). Firefly luciferase was normalized to Renilla activity. (B)
888
41
Western blots of PTEN and SOCS3 in P6 RGCs after transduction with AAV-miR-19a-eGFP or
889
AAV-eGFP (left). miR-19a significantly reduced PTEN (PTEN, p=0.0012, t=4.4722, df=10; n=6
890
experimental replicates), but not SOCS3 (SOCS3, p=0.8691, t=-0.1684, df=12; n=7 experimental
891
replicates) (right). Values normalized to GAPDH or β-actin. (C-D) Immunofluorescence images of
892
PTEN (red) in P6 RGCs with AAV-miR-19a-eGFP (green) (C) or AAV-miR-19aTuD-eGFP (green) (D).
893
Scale bar:10µm. Single-cell PTEN intensity was significantly lower in AAV-miR-19a-eGFP-
894
transduced RGCs (p=0.0018, t=3.1704, df=202; n=100 RGCs), and higher in AAV-miR-19aTuD-
895
eGFP-transduced RGCs (p<0.0001, t=-8.5435, df=404; n=183 RGCs), compared with control
896
(n=327 RGCs). (E) In situ hybridization (ISH) and immunohistofluorescence images of
897
endogenous miR-19a (upper panel) and PTEN (lower panel), respectively, in E21, P6, and P30
898
retinas. Boxed areas are magnified on the right. Scale bar: 25µm. GCL=ganglion cell layer;
899
INL=inner nuclear layer; ONL=outer nuclear layer. (F) ISH images of endogenous miR-19a (green)
900
in E21, P6, and P30 RGCs. Boxed areas are magnified above. Scale bars:10µm. Axon growth
901
cones are indicated by arrowheads. (G) Immunofluorescence images of endogenous PTEN (red)
902
in E21, P6, and P30 RGCs. Boxed areas are magnified above. Scale bar:10µm. Axon growth cones
903
are indicated by arrowheads. (H) Single-cell PTEN intensity significantly increased from E21
904
(n=78 RGCs) to P6 (E21 vs. P6, p=0.0465, t=-2.0057, df=165; n=89 RGCs) and to P30 (E21 vs. P30,
905
p<0.0001, t=-7.7080, df=164; P6 vs. P30, p<0.0001, t=-6.3310, df=175; n=88 RGCs). All values
906
are in mean ± s.e.m. Unpaired two-tailed Student’s t-test was used for all comparisons; *p<0.05;
907
**p<0.01; ***p<0.001; N.S.=not significant.
908
909
Figure 3. miR-19a augments axon regeneration in mature rodent retinal ganglion cells.
910
42
(A) Serial fluorescence images of cropped image areas of the axonal compartment of
911
microfluidic chambers with P6 RGCs transduced with AAV-miR-19a-eGFP or AAV-eGFP at 7, 14,
912
and 21 days in vitro. Total axon lengths in the axonal compartment were significantly longer in
913
RGCs transduced with AAV-miR-19a-eGFP (day 7, p=0.2636, t=-1.2996, df=4; day 14, p=0.0343,
914
t=-3.156, df=4; day 21, p=0.0024, t=-6.8128, df=4; n=3 experimental replicates) than with AAV-
915
eGFP (n=3 experimental replicates). Scale bar:100μm. Unpaired two-tailed Student’s t-test was
916
used for all comparisons. (B) Time-lapse microscopy images (left) showing axon extension of P6
917
RGCs transduced with AAV-miR-19a-eGFP (top) or AAV-eGFP (bottom). Scale bar:25μm. (C) The
918
rate of axon extension of AAV-miR-19a-eGFP-transduced RGCs (n=9 RGCs from 7 experimental
919
replicates; 17.4 μm/hr, 95% CI: 13.2-21.5 μm/hr) was significantly faster than that of AAV-eGFP-
920
transduced RGCs (n=7 RGCs from 5 experimental replicates; 7.0 μm/hr, 95% CI: 2.8-11.2 μm/hr).
921
Multivariable linear mixed model was used for comparison after adjusting for comparisons at
922
multiple time points after axotomy, p<0.0001. (D) Timeline for evaluation of axon regeneration
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in C57BL/6 mice after optic nerve crush. (E) Fluorescence images of optic nerve sections from
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eyes intravitreally injected with AAV-miR-19a-eGFP (top) or AAV-eGFP (bottom). Regenerating
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axons were labeled with cholera toxin B (black). The crush site is indicated by an asterisk. Scale
926
bar:100μm. (F) The numbers of regenerating axons were significantly greater in AAV-miR-19a-
927
eGFP-transduced eyes (n=8 mice) than AAV-eGFP-transduced eyes (n=8 mice). Multivariable
928
linear mixed modeling was used for comparison after adjusting for comparisons at multiple
929
distances from the crush site, p=0.004. All values are in mean ± s.e.m. *p<0.05; **p<0.01;
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***p<0.001; N.S.=not significant.
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43
Figure 4. miR-19a promotes axon regeneration in human adult retinal ganglion cells.
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(A) In situ hybridization images of endogenous miR-19a expression (green) in purified fetal (15-
934
week-old) and adult (73-year-old) human RGCs at 7 days in vitro. Images on the right are
935
magnifications of boxed areas. miR-19a was most prominently detected in the cytoplasm and
936
decreased in expression from fetal to adult human RGCs. Scale bar:10μm. (B) Fluorescence
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images of purified human adult RGCs transduced with AAV-eGFP or AAV-miR-19a-eGFP (left).
938
Scale bar:25μm. Single-cell analysis showed that AAV-miR-19a-eGFP-transduced human adult
939
RGCs (n=60 RGCs; 2 experimental replicates) had longer axon lengths and total neurite lengths
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compared with AAV-eGFP-transduced human adult RGCs (n=61 RGCs; 2 experimental replicates)
941
at 14 days in vitro (top) (axon length, p=0.0407, t=-2.069, df=119; total neurite length, p=0.0002,
942
t=-3.7888, df=119; total n=121 RGCs purified from two donors aged 69 years and 75 years).
943
Unpaired two-tailed Student’s t-test was used for all comparisons; *p<0.05; ***p<0.001. All
944
values are in mean ± s.e.m. The proportions of RGCs by axon length and total neurite length
945
between AAV-eGFP-transduced and AAV-miR-19a-eGFP-transduced human RGCs are
946
represented by histograms (bottom). The Gaussian distribution of AAV-eGFP and AAV-miR-19a-
947
eGFP is indicated by an overlapping curve. (C) A schematic illustrating the reciprocal relationship
948
of endogenous expression of miR-19a and PTEN in RGCs during development and their
949
association with the decline in axon regenerative capacity.
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eTOC synopsis
This study demonstrates a previously unrecognized involvement of the miR-19a-PTEN axis in
axon regenerative capacity of retinal ganglion cells (RGCs) during development. Mak and
colleagues show that miR-19a promotes axon regeneration after optic nerve crush in adult mice,
and increases axon extension in RGCs isolated from aged human donors.
