Let-7i inhibition enhances progesterone-inducedfunctional recovery in a mouse model of ischemiaTrinh Nguyena,b, Chang Sua,b, and Meharvan Singha,b,1

aDepartment of Pharmacology and Neuroscience, University of North Texas Health Science Center, Fort Worth, TX 76107; and bInstitute for Healthy Aging,University of North Texas Health Science Center, Fort Worth, TX 76107

Edited by Bruce McEwen, The Rockefeller University, New York, NY, and approved August 17, 2018 (received for review March 13, 2018)

Progesterone (P4) is a potent neuroprotectant and a promisingtherapeutic for stroke treatment. However, the underlying mecha-nism(s) remain unclear. Our laboratory recently reported that brain-derived neurotrophic factor (BDNF) is a critical mediator of P4’sprotective actions and that P4-induced BDNF release from corticalastrocytes is mediated by a membrane-associated progesterone re-ceptor, Pgrmc1. Here, we report that the microRNA (miRNA) let-7i is anegative regulator of Pgrmc1 and BDNF in glia and that let-7i disruptsP4-induced BDNF release and P4’s beneficial effects on cell viabilityand markers of synaptogenesis. Using an in vivo model of ischemia,we demonstrate that inhibiting let-7i enhances P4-induced neuropro-tection and facilitates functional recovery following stroke. The dis-covery of such factors that regulate the cytoprotective effects ofP4 may lead to the development of biomarkers to differentiate/pre-dict those likely to respond favorably to P4 versus those that do not.

progesterone | BDNF | Pgrmc1 | ischemic stroke | let-7i

Stroke has been reported as one of the leading causes of deathand a major cause of disability in the United States (1),

costing approximately $34 billion annually (according to theCenters for Disease Control). A number of studies have shownthat the ovarian hormone progesterone (P4) is neuroprotective ina variety of experimental models of stroke (2–4). However, theunderlying mechanisms for P4’s protective effects remain unclear.One known mediator of P4’s protective function is brain-

derived neurotrophic factor (BDNF) (5). A deficit in BDNFhas been linked to a more severe stroke pathophysiology (6, 7).This neurotrophin also has an established role in promotingneuronal differentiation, survival, synaptic plasticity (8–10), andsynaptogenesis (11–13). Synaptogenesis occurring in the pen-umbra is known to strongly contribute to enhanced functionalrecovery from stroke (14–17). Based on these observations, it isplausible that the P4/BDNF signaling-mediated enhanced syn-aptogenesis and neuroprotection may contribute to P4’s pro-tective effects during poststroke brain repair.We recently reported that P4 elicits the release of BDNF from

primary astrocytes via a putative membrane progesterone re-ceptor consisting of progesterone receptor membrane compo-nent 1 (Pgrmc1) (18). Our results also suggest that conditionedmedium derived from P4-treated astrocytes, when applied toprimary cortical neurons, increases the expression of synapticmarkers in these neural cells and enhances their survival againstoxidative stress. These findings support the model wherebyP4 elicits its (neuro)protective effects through a mechanism thatinvolves Pgrmc1-dependent BDNF release from glia.While it is clear that Pgrmc1 plays an important role in P4’s

protective effects on the brain, knowledge regarding the regu-lation of Pgrmc1 in the brain and the consequence of such regu-lation is limited. Studies suggest that microRNA (miRNA) mightbe involved (19, 20). MicroRNAs are a class of small noncodingRNAs with mature transcripts consisting of 18 to 25 nucleotides(21). Indeed, there exists support for the role of miRNA in stroke(21–23) where manipulation of miRNA in experimental models ofstroke resulted in neuroprotection (19, 22–24).

In the present study, we aimed to investigate the role of miRNAlet-7i in regulating the protective function of progesterone in is-chemia. Using an in vitro two-cell model system, which consisted ofastrocytes and neurons, we found that let-7i negatively regulatesexpression of both Pgrmc1 and BDNF in glia, leading to suppres-sion of P4-induced BDNF release from glia and attenuation of P4’sbeneficial effects on cell viability and markers of synaptogenesis. Inour in vivo model of ischemia [middle cerebral artery occlusion(MCAo), followed by reperfusion], combined treatment of P4 andthe let-7i inhibitor/antagomir led to reduced ischemic injury andcomplete recovery of motor function. These findings support thetherapeutic value of let-7i inhibition as an approach to enhance P4’sprotective efficacy in ischemic brain.

ResultsBDNF and Pgrmc1 Are Negatively Regulated by Let-7i in Primary CorticalAstrocytes. An in silico analysis using three prediction softwareprograms (miRDB, TargetScan, and microRNA.org) revealed pu-tative let-7 binding sites in the 3′UTR of pgrmc1 and bdnf that wereconserved in rat, mouse, and human sequences. Based on this ob-servation, coupled with a prior report citing the regulation ofPgrmc1 by let-7i in ovarian cancer cells, we evaluated the effect oflet-7i on both Pgrmc1 and BDNF. We evaluated the effect of let-7fas a control, recognizing that the Sohrabji laboratory had pre-viously shown an inverse relationship between let-7f and BDNF(19). Our studies showed that overexpression of let-7i, by trans-fecting a let-7i mimic, led to decreased mRNA levels of bothpgrmc1 (Fig. 1A) and BDNF (Fig. 1B). Interestingly, over-expression of let-7f had no effect. Additionally, inhibition of let-7i(anti–let-7i) and let-7f (anti–let-7f), using miRNA inhibitor, did notalter basal mRNA levels of pgrmc1 and bdnf (Fig. 1 A and B).

Significance

Pgrmc1 plays an important role in mediating progesterone’s pro-tective effects in that it is a critical mediator of progesterone-induced BDNF release. Here, we identified the microRNA let-7i,which increased in stroke, as a negative regulator of Pgrmc1 andBDNF expression. Conversely, inhibition of let-7i enhanced pro-gesterone’s protective effects against stroke. In addition to en-hancing progesterone’s neuroprotective effects, the fact that let-7ialso diminishes the expression of BDNF suggests that inhibition oflet-7i may also be useful to any intervention that targets the en-hancement of BDNF signaling and, as such, may be relevant to thetreatment of a variety of brain disorders where BDNF is dimin-ished, to include depression, traumatic brain injury, and Alzheimer’sdisease.

Author contributions: T.N., C.S., and M.S. designed research; T.N., C.S., and M.S. per-formed research; T.N., C.S., and M.S. analyzed data; and T.N. and M.S. wrote the paper.

Conflict of interest statement: The authors of this manuscript are coinventors on a patentapplication (application no. PCT/US18/46456).

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence should be addressed. Email: Meharvan.Singh@unthsc.edu.

Published online September 20, 2018.

E9668–E9677 | PNAS | vol. 115 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1803384115

Dow

nloa

ded

by g

uest

on

Dec

embe

r 1,

202

1

Western blot analysis (Fig. 1C) showed a consistent finding in thatreduction in Pgrmc1 protein level was only observed in the grouptransfected with let-7i mimic. Quantitative assessments of BDNF,using an ELISA (Fig. 1D), showed that BDNF levels were down-regulated in cultures transfected with let-7i mimic. These data sug-gested that let-7i, and not let-7f, negatively regulates BDNF/Pgrmc1 system in cortical astrocytes.

Progesterone-Induced BDNF Release Is Inhibited in Cultures OverexpressingLet-7i. Since our laboratory has shown that Pgrmc1 plays a central rolein mediating the effect of P4 on the release of BDNF from corticalastrocytes (18) and let-7i negatively regulates expression of thiscomponent (Fig. 1 A and C), we tested whether overexpressionof let-7i inhibits P4-elicited BDNF release from these cells. In situassessment of BDNF release (Fig. 2) showed that P4 (10 nM, 24 h)elicited a significant release of BDNF into the culture media com-pared with the vehicle control (DMSO), an effect that was notblocked by overexpressing the let-7i antagomir. In contrast, let-7ioverexpression led to the inhibition of P4’s effect on BDNF re-lease. These findings support our hypothesis that overexpression oflet-7i, through the negative regulation of the Pgrmc1/BDNF axis,abolishes P4-induced BDNF release from primary cortical astrocytes.

The Let-7i Antagomir Inhibits Oxygen–Glucose Deprivation-Induced Increasein Let-7i Expression. Oxygen–glucose deprivation (OGD), used in theprimary cortical astrocytes as an in vitro model of ischemia, revealedan increase in let-7i expression. Importantly, the data also verified theeffectiveness of the let-7i antagomir to attenuate the OGD-inducedincrease in let-7i expression (Fig. 3A). Moreover, as a complement todata presented in Fig. 2, data in Fig. 3B demonstrate that OGD(which increases let-7i expression) compromised the ability ofprogesterone (P4)-induced BDNF release from primary corticalastrocytes, similar to what was noted when let-7i was specificallyoverexpressed.

Let-7i Represses Progesterone’s Neuroprotection and Its Enhancementon Synaptogenesis. To investigate the role of let-7i in P4’s neuro-protective effects, we manipulated miRNA expression in primary

cortical astrocytes and then treated them with either vehicle(DMSO) or P4, following which astrocyte-derived conditionedmedium (ACM) was collected. The conditioned medium was thenapplied to primary cortical neurons [days in vitro (DIV)14] thathad been exposed to oxygen–glucose deprivation (OGD). Theneurons were then assessed for cell viability to ascertain if con-ditioned media from P4-treated astrocytes elicited greater neu-roprotection relative to neurons treated with conditioned mediafrom DMSO-treated astrocytes (Fig. 4). We found that condi-tioned media collected from P4-treated astrocytes conferredsimilar neuroprotection as seen in the positive control group[consisting of direct administration of BDNF (50 ng/mL) to theneuronal cultures]. However, conditioned media collected fromP4-treated astrocytes that overexpressed let-7i failed to promotethe protection of neurons from OGD.Next, we determined if conditioned media from the different

experimental groups represented in Fig. 5 resulted in changes inexpression of synaptophysin (SYP), a presynaptic marker closelylinked to synaptogenesis (4). We observed that conditionedmedia derived from P4-treated astrocytes (P4-ACM) resulted ina robust increase in SYP (green) immunofluorescence (Fig. 5A),relative to neurons treated with conditioned media from DMSO-treated, and mock-transfected astrocytes. Quantitative analysisrevealed that P4-ACM significantly increased both SYP proteinlevel (Fig. 5C) and the number of SYP puncta (Fig. 5B). Thesame observations were seen in the positive control group[consisting of direct application of BDNF (50 ng/mL) to theprimary neuronal cultures]. Application of conditioned mediacollected from P4-treated astrocytes that overexpressed let-7i(group label: let-7i + P4), however, failed to elicit the increasein synaptophysin expression.

Combined Treatment of Progesterone and Let-7i Inhibition AlleviatesIschemia-Induced Suppression of Pgrmc1 and BDNF Expressions in thePenumbra of the Ischemic Brain. Since we determined that let-7i is anegative regulator of BDNF and Pgrmc1 (Fig. 1) and thatOGD, as an in vitro model of ischemia, induced expression of

pgrmc1 mRNAA

bdnf mRNAB

C

D

Rel

ativ

e m

RN

A le

vel

(% o

f con

trol

)

Control

let-7i

mim

ic

Anti-let-

7i

let-7f

mim

ic

Anti-let-

7f 0

50

100

150

***

n.s n.sn.s###

Rel

ativ

e m

RN

A le

vel

(% o

f con

trol

)

Control

let-7i

mim

ic

Anti-let-

7i

let-7f

mim

ic

Anti-let-

7f0

50

100

150

***

n.s n.sn.s

###

Pgrm

c1/G

apdh

ratio

(% o

f con

trol

)

Control

let-7i

mimic

Anti-let-

7i

let-7f

mim

ic

Anti-let-

7f0

50

100

150

***

n.s n.s n.s

###

Control

let-7i

mim

ic

Anti-let-

7i

let-7f

mimic

Anti-let-

7f0

50

100

150

**

n.s

n.sn.s

###

Fig. 1. Let-7i negatively regulates the expression ofPgrmc1 and BDNF in primary cortical astrocytes. Theeffect of let-7 mimics and antagomirs on pgrmc1 (A)and bdnf (B) mRNA (n = 4). (C) Representative im-munoblot for Pgrmc1 protein and associated quan-titation depicting the signal (densitometric) intensity,expressed as the ratio of Pgrmc1 to GAPDH (n = 4). (D)Total cellular BDNF measured by ELISA (n = 5). n.s,not significant; ***P < 0.001 compared with control;###P < 0.001 compared with let-7i mimic. Data arepresented as the mean ± SEM.

Nguyen et al. PNAS | vol. 115 | no. 41 | E9669

NEU

ROSC

IENCE

Dow

nloa

ded

by g

uest

on

Dec

embe

r 1,

202

1

let-7i (Fig. 3A), we next determined the expression of let-7i inthe middle cerebral artery occlusion model of ischemic stroke,focusing on changes in the penumbra. Assessments of let-7i ex-pression were conducted at different time points – 2, 7, and 14 dfollowing stroke. Representative images of immunoblots probedfor Pgrmc1, along with pro-BDNF and mature BDNF, are shownin Fig. 6A. We found that, compared with sham (nonstrokedcontrols), ischemic injury resulted in an up-regulation of let-7iexpression (Fig. 6E) starting at day 7 and remained elevated upto 14 d following stroke. P4 treatment alone [P4 + a controlsequence for let-7i (scrambled)] did not attenuate the stroke-induced increase in let-7i. As expected, ischemia-induced in-crease in let-7i expression was repressed in the group receivingcombined treatment of P4 and let-7i inhibition (P4 + –let-7i) (Fig.6E). Importantly, along with up-regulating let-7i level, ischemiaalso resulted in a reduction of Pgrmc1 protein level observed atday 7 and day 14 (Fig. 6B). P4 treatment alone did not restorePgrmc1 level at either of the two time points. Combinedtreatment (P4 + anti–let-7i), however, reversed ischemia-induced suppression of Pgrmc1 protein levels. Furthermore,expression of mature BDNF was reduced as a consequence ofstroke at the 14-d poststroke evaluation period (Fig. 6D) while

pro-BDNF levels (Fig. 6C) remained unchanged across all timepoints and all treatments. Compared with sham, the treatmentof P4 alone was able to maintain the same level of matureBDNF, even at 14 d poststroke. Remarkably, combined treat-ment (P4 + anti–let-7i) led to a robust increase in expression ofmature BDNF observed at day 7 and day 14.

Combined Treatment of Progesterone and Let-7i Inhibition ReducesIschemic Injury and Enhances Functional Recovery. To examine theeffect of P4 with or without the let-7i antagomir on the extent ofischemic injury, we utilized 2,3,5-triphenyltetrazolium chloride(TTC) staining to visualize the size of the ischemic lesion.Representative images of TTC stained are shown in Fig. 7A.Quantification of relative infarct size (Fig. 7B) revealed that thecombined treatment (P4 + anti–let-7i) significantly reduced is-chemic injury whereas P4 treatment alone did not.Motor function (grip strength) was also evaluated using the wire

suspension test. Results (Fig. 8) showed that, compared with the ve-hicle group (DMSO + scrambled), treatment of P4 led to a partialrecovery of motor function, observed on day 7 and day 14. In-terestingly, the combined treatment of P4 and the let-7i antagomirresulted in a rapid, but partial, motor function recovery as early as 3 dposttreatment. By day 7, combined treatment led to complete func-tional recovery, and the improvement was still evident at day 14.Results from Figs. 7 and 8 support our hypothesis that let-7i inhibitionenhances P4’s neuroprotective effects, and enhances functionalrecovery.

Inhibition of Let-7i Enhances Progesterone’s Effect on a SynaptogenicMarker. Synaptic plasticity in the ischemic penumbra region haslong been known to influence the functional recovery afterstroke (14, 16, 25). Therefore, to determine whether synapto-genesis occurring in the penumbra could be a factor contributingto functional recovery observed in Fig. 8, we extended our invitro findings to evaluate the expression of synaptophysin (SYP),a synaptogenic marker, in the penumbra of stroked mice. To doso, we performed immunofluorescence to visualize SYP expres-sion (red) (Fig. 9A) and quantified the relative number of SYPpuncta, which is an indication of potential synapses (Fig. 9B). Inparallel, Western blot analysis was performed to evaluate totalSYP protein levels. Representative immunoblots probed for SYPare shown in Fig. 9C, and its relative quantification of proteinlevel is depicted in Fig. 9D. Results revealed that ischemiaresulted in a sustained down-regulation of synaptophysin puncta(Fig. 9B) in the penumbra at day 2, 7, and 14 poststroke. Inaddition, ischemic injury led to decreased SYP protein level atday 2 and 14. There was a transient increase in SYP expression atday 7, which could be due to a compensatory response to theischemic injury. P4 treatment alone led to a delayed, but sus-tained, restoration in SYP total protein expression, observed atday 7 and day 14. With regard to the number of SYP puncta, the

0

10

20

30

40

50

Mock Anti-let-7ilet-7imimic

n.s ##

Fig. 2. Let-7i overexpression abolished progesterone (P4)-induced BDNF releasefrom primary cortical astrocytes. Quantitation of BDNF release measured byBDNF in situ ELISA (n = 4). n.s, not significant; ***P < 0.001; ##P < 0.01 comparedwith corresponding DMSO groups. Data are presented as the mean ± SEM.

BA Fig. 3. Oxygen–glucose deprivation (OGD) resultsin an increase in let-7i expression and suppressesprogesterone (P4)-induced BDNF release from pri-mary cortical astrocytes. Primary cortical astrocyteswere exposed to 1 h of OGD. Immediately afterreinstatement of normal oxygen and glucose con-centrations, these cells were either mock trans-fected (control) or transfected with the let-7iantagomir. Twelve hours later, expression of let-7iwas evaluated (A) (n = 4). n.s, not significant;****P < 0.0001 compared with normoxic control.(B) Quantitation of BDNF release measured byBDNF in situ ELISA (n = 4). n.s, not significantcompared with DMSO group. Data are presented asmean ± SEM.

E9670 | www.pnas.org/cgi/doi/10.1073/pnas.1803384115 Nguyen et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 1,

202

1

positive effect of P4 was only evident at day 14 posttreatment.Interestingly, at day 7 and 14, combined treatment (P4 + anti–let-7i)resulted in significantly higher expression of SYP, compared withsham controls and P4 treatment alone. This combined treatmentalso led to a complete restoration of synaptophysin puncta at day 7,an effect that was further enhanced at day 14. Taken together, thesefindings indicate that P4 induces synaptogenesis in the penumbra ofischemic brain and that let-7i inhibition further enhances this ben-eficial function of P4.

DiscussionAn increasing number of publications infer the brain-protectiveeffects of P4, including studies showing that P4 is neuro-protective in a variety of experimental models of stroke (2–4).However, the underlying mechanism(s) for P4’s protective ef-fects remain unclear. In point of fact, our ability to optimize theeffectiveness of P4 requires a better understanding of the factorsthat influence the expression of key mediators (e.g., receptors) ofP4’s protective effects. In addition, most of the literature asso-ciated with P4’s protective effects has focused on a direct effectof P4 on neurons. The notion that glia may be an equally im-portant target underlying P4’s protective effects on the brain hasonly been studied minimally. Indeed, astrocytes have been con-sidered an important component of postischemic recovery asthese cells are critical for regeneration and remodeling of neuralcircuits following stroke (26).One mechanism that underlies the protective function of P4 is

its ability to initiate intercellular cross-talk between astrocytesand neurons, where BDNF is a key mediator (27). For example,we have reported that P4 elicits a significant release of matureBDNF from astrocytes through a Pgrmc1-dependent ERK5 sig-naling mechanism. From this same study, BDNF from the con-ditioned media of P4-treated astrocytes was identified specificallyas the protective factor that not only promoted neuronal viabilitybut also increased the expression of markers of synaptogenesis(27). In this study, we identified an upstream regulator of this

Pgrmc1/BDNF axis in glia and characterized its influence in amouse model of ischemic stroke (MCAo). Our results support therole of let-7i as a negative regulator of Pgrmc1 and BDNF inprimary cortical astrocytes. Interestingly, despite sharing a similarseeding sequence, let-7f did not have the same effect, suggestingthat regulation of Pgrmc1/BDNF in astrocytes is specific to let-7i.Not only did increased expression of let-7i significantly reduce P4-induced BDNF release from cultured astrocytes, it preventedP4 from protecting neurons against oxygen–glucose deprivation(OGD) and from increasing the expression of synaptophysin, asurrogate marker for synaptogenesis. We also found that OGD,serving as an in vitro model of ischemia, resulted in an up-regulation of let-7i. Consistent with our working model thatlet-7i, by inhibiting the expression of Pgrmc1, inhibits P4-inducedBDNF release from astrocytes (as shown in Fig. 2), OGD alsoinhibited the P4-induced BDNF release, an effect correlated withthe effect of OGD on let-7i expression. Collectively, these findingssuggest that let-7i and a condition that leads to the elevation of thismiRNA, such as ischemia, repress P4-induced BDNF release fromglia, thus leading to attenuation of the beneficial effects of P4 onneuronal survival and markers of synaptogenesis. It is noteworthythat inhibition of let-7i alone (using the let-7i antagomir/inhibitor)did not confer any protection against OGD. Rather, let-7i in-hibition enhanced the protective effects of P4.The results from our in vivo studies, using the MCAo model of

stroke in mice, corroborated the results from our in vitro ex-periments. For example, like OGD, ischemic injury induced inthe mouse led to an up-regulation of let-7i in the penumbra,which was correlated with decreased Pgrmc1 expression and areduction in the level of mature BDNF which was seen at day14 following stroke. Given that BDNF has such a vital role onbrain function (8–10), the observed reduction in Pgrmc1 could notonly compromise the protective efficacy of hormones like P4 but,more generally, could underlie the long term sequelae that lead tofunctional impairment over time in stroke victims. It is worthnoting, however, that the reduction in BDNF levels was observedonly at day 14. This could be due to a compensatory effort of thebrain to maintain BDNF at earlier time points (day 2 and 7).Treatment of P4 was able to restore the level of mature

BDNF. Despite the reduction in Pgrmc1 levels in ischemic brain,P4’s mild restorative effect on BDNF expression might be dueto its action via classical progesterone receptor (PR), as sup-ported by our previous work (28). Importantly, combined treat-ment (P4 and let-7i inhibition) restored Pgrmc1 expression andresulted in a marked increase in mature BDNF level, despitepro-BDNF levels not changing appreciably. This interest-ing finding, where the let-7i antagomir, when coapplied withprogesterone (P4), enhanced mature BDNF expression withoutaffecting pro-BDNF, is somewhat consistent with our priorpublished studies where progesterone increased the ratio ofmature to pro-BDNF released from glia (27). However, anotherpossible consequence of elevated let-7i is to influence theexpression of proneurotrophin convertases, such that increasedlet-7i may inhibit the convertases responsible for processing pro-BDNF to mature BDNF. Indeed, our data show that the reducedexpression of mature BDNF (at day 14) in the stroke-only group(Fig. 6D, open bar) is a time point associated with a significantelevation of let-7i. Conversely, it is possible that inhibiting let-7i,which would alleviate inhibition of the convertases, could re-sult in greater processing of the pro-BDNF molecule to ma-ture BDNF. The striking increase in mature BDNF expressionobserved with the combined treatment could also be due tothe effect of the intervention on the stability of matureBDNF (versus pro-BDNF). Nevertheless, taken together withour in vitro results, the observations from the in vivo studiessupport the hypothesis that inhibition of let-7i alleviates ischemia-induced suppression of Pgrmc1 expression, thereby allowing

Fig. 4. Let-7i prevents progesterone (P4)-induced neuroprotection againstoxygen–glucose deprivation (OGD). Conditioned media derived from hormoneor control-treated astrocytes were applied to primary cortical neurons (DIV 14)after 1-h exposure to OGD. BDNF (50 ng/mL) was directly added to neuronsafter OGD to serve as positive control. Neuronal viability was measured byCellTiter-Glo viability assay (n = 5). n.s, not significant; ***P < 0.001 and **P <0.01 compared with normoxia. Data are presented as the mean ± SEM.

Nguyen et al. PNAS | vol. 115 | no. 41 | E9671

NEU

ROSC

IENCE

Dow

nloa

ded

by g

uest

on

Dec

embe

r 1,

202

1

P4 to increase expression (and release) of mature BDNF viaPgrmc1 signaling.In agreement with other published studies (2, 29), our results

showed that treatment of P4 alone led to a modest and delayedimprovement in functional recovery, along with an increasein synaptogenesis depicted by the increase in expression andnumber of synaptophysin puncta. However, treatment withP4 alone failed to reduce the size of the ischemic lesion. In-terestingly, the combined treatment of the let-7i antagomir andP4 not only significantly reduced the size of the ischemic lesion butalso led to a quicker and more pronounced functional recovery, aswell as a more pronounced increase in markers of synaptogenesisin the penumbra region. These findings are consistent with otherstudies proposing the link between synaptogenesis in the penum-bra and functional recovery following stroke (14, 30).While progesterone has been shown to exert protective effects

in animal models of ischemic stroke (3, 31), this effect is notalways consistent (32, 33). Moreover, there is yet to be a clinical

evaluation of the effectiveness of P4 intervention for ischemicstroke. We recognize, however, that prior efforts to translaterobust preclinical data that support the neuroprotective effectsof steroid hormones, like P4, have been somewhat disappointing.For example, a recent phase III clinical trial (ProTECT III)assessing the efficacy of P4 treatment for acute traumatic braininjury (TBI) showed rather disappointing results, with no fa-vorable effects noted (34) despite significant support for thetherapeutic efficacy of P4 from preclinical studies. Similarly, theSYNAPSE trial (35), which evaluated the efficacy of P4 againstsevere TBI, also failed to demonstrate improvement in func-tional outcomes. The apparent discrepancy between the pre-clinical studies and the clinical trials may be attributed tonumerous variables, including the complexity and variability ofthe injuries noted in human subjects within the trials. In addition,we suggest that the biological heterogeneity of the subject cohortwithin multicenter trials, despite laudable efforts to define thestudy cohort with cited inclusionary and exclusionary criteria,

DAPISYP Merge

BDNF

Mock + DMSO

Mock + P4

Let-7i + DMSO

Let-7i + P4

Anti-let-7i+ DMSO

Anti-let-7i+ P4

A B

C

Ave

rage

num

ber o

f SYP

pun

cta

per c

ell

Mock +

DMSO

Mock +

P4

Let-7i

+ DMSO

Let-7i

+ P4

Anti-let-

7i + D

MSO

Anti-let-

7i + P

4BDNF

Fig. 5. Let-7i inhibits progesterone (P4)-induced synaptophysin (SYP) expression in primary cortical neurons. (A) Representative confocal images of primarycortical neurons (DIV 14) immunostained with synaptophysin (SYP, green) and DAPI (blue). (Magnification: 60×.) (Scale bars: 30 μm.) (B) Quantification ofaverage number of SYP puncta per neuron (n = 3). n.s, not significant; ***P < 0.001 compared with mock transfected + DMSO group. (C) Representativeimmunoblots probed for SYP and quantification graph of relative SYP protein ratio to Gapdh (n = 4). n.s, not significant; ****P < 0.0001 and ***P <0.001 compared with mock transfected + DMSO group. Data are presented as the mean ± SEM.

E9672 | www.pnas.org/cgi/doi/10.1073/pnas.1803384115 Nguyen et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 1,

202

1

may also play a role. Such heterogeneity may have its basis in thebiology of the individual, through variable expression of factorsthat modify the effects of P4. Our data offer potential insightinto how differences in the expression of microRNA let-7i couldalter the efficacy of progesterone. Since some studies havereported an elevated expression of let-7i in traumatic brain injury(TBI) models (36, 37), we suggest that intersubject differences inlet-7i expression may predict who may be responsive to P4. Morespecifically, our data would suggest that higher expression of let-7ileads to impaired Pgrmc1/BDNF signaling, thus dampening theprotective function of P4.Collectively, our findings reported herein identify let-7i as a

negative upstream regulator of both Pgrmc1 and BDNF in glia,leading to suppression of P4-induced BDNF release from gliaand attenuation of P4’s beneficial effects on neuronal viabilityand synaptogenesis in the ischemic brain. Furthermore, the in-creased expression of let-7i with stroke may explain why post-stroke therapy may not be so effective. As such, dampening theup-regulation of let-7i may prove to be an effective strategy toenhance the efficacy of P4 and other therapeutic candidates thatengage the BDNF system and, as such, could potentially extendthe “window of opportunity” for stroke therapy.

Materials and MethodsPrimary Cultures. Dissociated cortical neurons were prepared and maintainedas previously described (27). Briefly, cortices were removed from neonatalmouse brains (postnatal day 2 to 4, mixed gender) and dissociated with0.25% trypsin. Cortical neurons were then plated on glass coverslips orplastic culture dishes coated with poly-D-lysine (Sigma). The culture mediumused was Neurobasal (ThermoFisher Scientific), supplemented with Gluta-

max and B27 serum-free supplement (ThermoFisher Scientific). At day invitro (DIV) 3, 5 μM final concentration of 1-β-arabinofuranosylcytosine (38)(Sigma) was added to the neuronal cultures to prevent glial proliferation.Half of the medium was replaced with fresh medium every 4 d. For viabilityassay, cortical neurons were plated onto 96-well plates (Corning) at the con-centration of 1.2 × 105 cells per square centimeter. For immunocytochemistry,cortical neurons were plated onto 12-mm glass coverslips (Neuvitro) at thedensity of 4 × 104 cells per square centimeter. Treatments of primary corticalneurons started at DIV12.

Primary cortical astrocytes were prepared and maintained as previouslydescribed (39), with some modifications. Briefly, cortices of postnatal day2 to 4 mouse pups were dissociatedwith 0.25% trypsin and plated onto 75-cm2

tissue culture flasks. The culture medium used was DMEM (ThermoFisher Sci-entific), supplemented with 10% FBS (GE Healthcare Life Sciences) and10,000 U/mL penicillin-streptomycin (ThermoFisher Scientific). After reachingconfluence, mixed glial cultures were placed on the shaker for 48 h to dislodgemicroglia, resulting in cultures enriched with astrocyte population.

Treatment of Primary Cultures. To determine the miRNA regulation ofdownstream targets in primary cortical astrocytes, miRNA mimics and in-hibitors were transfected into these cells for 48 h. After transfection, totalRNA and proteins were isolated for gene and protein expression analysis.Mock transfection was used as the control for these experiments.

To study the effect of miRNA on P4-induced BDNF release from astrocytes,BDNF in situ ELISAs were performed. Expression ofmiRNAwas first manipulatedby transfection as described above. Twenty-four hours after transfection, 10 nMP4was added to primary cortical astrocytes for an additional 24 hwithout changingmedia containing transfection complexes. Vehicle controls were performed inparallel such that control cultureswereexposed to0.1%dimethyl sulfoxide (DMSO).The 10-nM concentration of P4 used in studies described herewas chosen because ithas been reported to elicit a maximal release of BDNF from astrocytes (18).

A

B

C

D

E

0

50

100

150

n.sn.s n.s

n.s

***

n.s#

***

n.s#

Day 2 Day 7 Day 14

mat

ure

BD

NF/

Gap

dh ra

tio(%

of s

ham

)

0

200

400

600

n.s n.sn.s

n.s n.s

***###

*n.s

Day 2 Day 7 Day 14

**##

#

Fig. 6. Combined treatment with progesterone (P4) and the let-7i inhibitor reversed ischemia-induced suppression of Pgrmc1 and BDNF expression in thepenumbra. (A) Representative immunoblots probed for Pgrmc1, pro-BDNF, and mature BDNF. (B) Quantitation graph of relative Pgrmc1 protein ratio toGapdh (n = 4 to 5 per group). (C) Quantitation graph of relative pro-BDNF protein ratio to Gapdh (n = 4 to 5 per group). (D) Quantitation graph of relativemature BDNF protein ratio to Gapdh (n = 4 to 5 per group). (E) Quantitation graph of relative let-7i expression in ischemic brain (n = 4 to 5 per group). n.s, notsignificant; **P < 0.01 and *P < 0.05 compared with sham; ###P < 0.01 between the two indicated groups, ##P < 0.05 between the two indicated groups, and#P < 0.05 compared with P4 + scrambled. Data are presented as the mean ± SEM.

Nguyen et al. PNAS | vol. 115 | no. 41 | E9673

NEU

ROSC

IENCE

Dow

nloa

ded

by g

uest

on

Dec

embe

r 1,

202

1

In experiments evaluating the effect of miRNA on P4-induced neuro-protection and the synaptogenic marker synaptophysin, we first transfectedmiRNAmimic and inhibitor into primary cortical astrocytes for 24 h. Afterward,P4 (10 nM) was added to these cultures for an additional 24 h to generate P4-treated astrocyte-derived conditioned media (P4-ACM). In parallel, treatmentof 0.1% DMSO was performed to generate DMSO-treated astrocyte-derivedconditioned media (DMSO-ACM), which served as vehicle controls. Beforeapplying to primary neurons, these conditioned media were filtered through a10-kDa cutoff column to eliminate residual P4 and miRNA mimic or inhibitor.In the neuroprotection assay, astrocyte-conditioned media were added toprimary cortical neurons with prior exposure to 1 h of oxygen–glucose dep-rivation (OGD), an in vitro model of ischemic-like insult. Based on our expe-rience, 1 h of OGD was enough to induce 50% neuronal cell death. BDNF(50 ng/mL) was directly added to different groups after OGD to serve aspositive control. Neuronal cultures exposed to normoxia were used as thecontrol for these datasets. Twenty-four hours after the applications of BDNF orconditioned media, a CellTiter-Glo Luminescent cell viability assay (Promega) wasperformed to measure neuroprotection. In the synaptogenic marker measure-ment assay, BDNF and astrocyte-derived conditioned media were directly addedto primary cortical neurons for 24 h. Synaptophysin expression and the numberof synaptophysin puncta in these neuronal cultures were assessed by immuno-cytochemistry, followed by confocal imaging and analysis using ImageJ (NationalInstitutes of Health) software (40).

Transfection. Transfection of miRNA mimics and inhibitors was performedusing theHiperfect transfection reagent (Qiagen) according to themanufacturer’s

instructions. Cells were transfected with miRNA mimics and inhibitors for 48 h.This duration was chosen since it resulted in an optimal effect on targets of in-terest. Synthetic miRNA mimics (Syn-mmu-let-7i-5p, Syn-mmu-let-7f-5p) and in-hibitors (Anti-mmu-let-7i-5p, Anti-mmu-let-7f-5p) were purchased from Qiagen.

Quantitative RT-PCR. Total RNA was isolated from primary cortical astrocytesand mouse brains using the MiRNeasy Mini Kit (Qiagen) according to themanufacturer’s instructions. Concentrations of extracted RNA were de-termined using absorbance values at 260 nm. The purity of RNA was assessedby ratios of absorbance values at 260 and 280 nm (A260/A280 ratios of 1.9 to2.0 were considered acceptable).

For miRNA expression measurements, total RNA (10 ng) was reversetranscribed into cDNA in a total volume of 15 μL using the microRNA cDNAArchive Kit (ThermoFisher Scientific) according to the manufacturer’s in-structions. The reaction mixture contained water, 2× quantitative PCRMaster Mix (Eurogentec), and 20× Assay-On-Demand for each target gene.A separate reaction mixture was prepared for the endogenous control, U6.The reaction mixture was aliquotted in a 96-well plate, and cDNA was addedto give a final volume of 20 μL. Each sample was analyzed in triplicate. Thecomparative cycle threshold (Ct) method (2−ΔΔCt) was used to calculate therelative changes in target miRNA expression.

For mRNA expression measurements, total RNA (1.6 μg) was reversetranscribed into cDNA in a total volume of 20 μL using the High-CapacitycDNA Archive Kit (ThermoFisher Scientific) according to the manufacturer’sinstructions. The reaction mixture contained water, 2× quantitative PCRMaster Mix (Eurogentec), and 20× Assay-On-Demand for each target gene.

Sham

holester

ol + sc

rambled

P4 + scram

bled

P4 + an

ti-let-

7i

Infa

rct s

ize

(% o

f con

tra.

hem

isph

ere)

A B

Fig. 7. Co-administration of let-7i antagomir (anti–let-7i) and progesterone (P4) reduces ischemic injury. (A) Representative images of serial coronal brainsections stained with triphenyltetrazolium chloride (TTC). (B) Quantification of infarct sizes of TTC-stained images (n = 4 per group). n.s, not significant; *P <0.01 compared with cholesterol + scrambled group. Data are presented as the mean ± SEM.

Late

ncy

to fa

ll (s

)

Fig. 8. Co-administration of let-7i antagomir (anti–let-7i) and progesterone (P4) enhances recovery ofmotor function/grip strength following stroke. Re-sults of wire suspension test at day 3, 7, and14 poststroke (n = 15 to 20 per group). n.s, not sig-nificant; ***P < 0.001 and **P < 0.01 compared withsham; ###P < 0.001, ##P < 0.01 compared with P4 +scrambled; and $$P < 0.01 compared with cholesterol +scrambled. Data are presented as the mean ± SEM.

E9674 | www.pnas.org/cgi/doi/10.1073/pnas.1803384115 Nguyen et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 1,

202

1

A separate reaction mixture was prepared for the endogenous control,gapdh. The reaction mixture was aliquotted in a 96-well plate, and cDNA(30 ng of RNA converted to cDNA) was added to give a final volume of 30 μL.Each sample was analyzed in triplicate. The comparative cycle threshold (Ct)method (2−ΔΔCt) was used to calculate the relative changes in targetgene expression.

PCR primers were purchased as Assay-On-Demand from ThermoFisherScientific. The assays were supplied as a 20 mix of PCR primers (900 nM)and TaqMan probes (200 nM). The let-7i (002221), U6 (001973), bdnf(Mm00432069_m1), gap-43 (Mm00500404_m1), gapdh (Mm99999915_g1), psd-95 (Mm00492193_m1), pgrmc1 (Mm00443985_m1), and syp (Mm00436850_m1)assays contained FAM (6-carboxy-fluorescein phosphoramidite) dye label at the5′ end of the probes and minor groove binder and nonfluorescent quencher atthe 3′ end of the probes.

CellTiter-Glo Luminescent Cell Viability Assay (Promega). The CellTiter-GloLuminescent Cell Viability Assay uses the level of adenosine triphosphate(41) as an indicator of metabolically active cells and is directly proportionalto the number of living cells (42, 43). The assay was performed according tothe manufacturer’s instructions. In brief, the cell plate was first equilibratedto room temperature for 30 min. A volume of the kit reagent equal to thevolume of cell culture present was then added to each well. The plate wasthen placed on an orbital shaker for 2 min to induce cell lysis, followed by10 min of incubation at room temperature. Luminescence was recordedusing a plate reader.

BDNF Immunoassay in Situ. To determine the amount of endogenous BDNFreleasedwith P4 treatment, we performed an ELISA in situ assay, as previouslydescribed (18). In brief, a 96-well Nunc MaxiSorp surface polystyrene flat-bottom immunoplate was precoated with an anti-BDNF monoclonal anti-body [diluted 1:1,000 in coating buffer (25 mM sodium bicarbonate and25 mM sodium carbonate, pH 9.7)]. After blocking nonspecific binding,primary cortical astrocytes were then plated, followed by appropriatetreatment applications. BDNF standards, ranging in concentration from

1.95 to 500 pg/mL, were added to parallel wells. At the end of hormonetreatment, cells were carefully washed with Tris-buffered saline containing0.2% Tween 20 (TBS-T). The plate was then incubated with the polyclonalanti-human BDNF antibody. The amount of specifically bound polyclonalantibody was then detected through the use of the anti–IgY-horseradishperoxidase (HRP) tertiary antibody, which, when exposed to the chromo-genic substrate (TMB reagent; Promega), changes color in proportion to theamount of BDNF present in the sample. The color intensity was quantified bymeasuring the absorbance at 450 nm with a Viktor3 ELISA plate reader(Perkin-Elmer). Only values within the linear range of the standard curve,and above the lowest standard, were considered valid. This method alloweddetection of as little as 2 pg/mL BDNF release in control cultures to ∼250 pg/mLin P4-treated cultures.

Oxygen–Glucose Deprivation.OGDwas performed according to an establishedprotocol, as described elsewhere, with minor modifications (44). Briefly,primary cortical neurons were carefully washed five times with HBSS(ThermoFisher Scientific) to remove residual glucose. Glucose-free DMEM(ThermoFisher Scientific) was then added to the cultures, and the plateswere transferred into a hypoxic chamber (0.1% oxygen) for 1 h. At the endof hypoxia, glucose-free DMEM was replaced with regular maintainingmedia. Reoxygenation was initiated by transferring the cells to a normoxic5% CO2 cell culture incubator.

Western Blotting. Primary cortical astrocytes andmouse brains were lysedwithradioimmunoprecipitation assay lysis buffer containing protease and phospha-tase inhibitors, as previously described (27). After homogenization, samples werecentrifuged at 100,000 × g for 30 min at 4 °C, and supernatants were collected.Total protein concentrations were determined using the Bio-Rad DC protein as-say kit (Bio-Rad Laboratories). Cell lysates were separated by SDS/PAGE andtransferred onto a polyvinylidene fluoride membrane (Bio-Rad Laboratories) byelectroblotting. Membranes were blocked with 5% skim milk in Tris-bufferedsaline containing 0.2% Tween 20 (TBS-T) for 1 h at room temperature, fol-lowed by overnight incubations of primary antibodies at 4 °C. The following

Sham Cholesterol + scrambledA

Day 2

Day 7

Day 14

P4 + scrambled

P4 + Anti-let-7i B

C

D

SYP/DAPI

Fig. 9. Inhibition of let-7i enhances progesterone (P4)’s effect on the expression of synaptophysin in the penumbra. (A) Representative confocal images ofpenumbra region staining for synaptophysin (SYP, red) and DAPI (blue). (Magnification: 60×.) (Scale bars: 30 μm.) (B) Quantification of average relative SYPpuncta present in each field (n = 3 per group). n.s, not significant; ***P < 0.001 compared with sham; ###P < 0.001 and ##P < 0.01 compared with P4 +scrambled. (C) Representative immunoblots probed for SYP protein. (D) Quantification graph of Syp signal, expressed as a ratio of Gapdh (n = 4 to 5 pergroup). n.s., not significant; ***P < 0.001, **P < 0.01, *P < 0.05 compared with sham; ##P < 0.01 and #P < 0.05 compared with P4 + scrambled.

Nguyen et al. PNAS | vol. 115 | no. 41 | E9675

NEU

ROSC

IENCE

Dow

nloa

ded

by g

uest

on

Dec

embe

r 1,

202

1

primary antibodies were used: rabbit polyclonal anti-PSD 95 (1:1,000, ab18258;Abcam), rabbit polyclonal anti-synaptophysin (1:1,000, ab14692; Abcam), rabbitmonoclonal anti-GAP43 (1:200,000, ab75810; Abcam), rabbit monoclonal anti-GAPDH (1:1,000, 14C10; Cell Signaling), rabbit polyclonal anti-BDNF (1:300,sc546; Santa Cruz), and goat polyclonal anti-Pgrmc1 (1:500, ab48012; Abcam).After washing three times with TBS-T, membranes were incubated with anti-goat IgG or anti-rabbit IgG conjugated with horseradish peroxidase (Millipore)for 1 h at room temperature. After triple washes with TBS-T, immunoreactivebands were visualized with the ECL detection system (ThermoFisher Scientific)and were captured using a luminescent image analyzer (Alpha Innotech).Densitometric analysis was conducted using ImageJ (National Institutes ofHealth) software (40).

Immunofluorescence. The cortical neurons were fixed in 4% paraformaldehyde(45) for 15 min, followed by incubation in 0.2% Triton X-100 in Tris-bufferedsaline (TBS) for 15 min at room temperature for permeabilization. Cultures werethen blocked with 5% donkey serum/1% BSA in TBS for 1 h at room tempera-ture and incubated with rabbit monoclonal anti-synaptophysin (1:500, ab32127;Abcam) for 48 h at 4 °C. After extensive rinsingwith TBS-Tween 20, cultures wereincubated with Alexa Fluor 647-conjugated secondary antibody (1:500; JacksonImmunoResearch Laboratories) for 2 h at room temperature. After extensivewashing with TBS to remove unbound secondary antibody, the coverslips weremounted onto glass slides (VWR Scientific) using Vectashield mounting mediumwith DAPI (Vector Laboratories). The slides were observed under a confocalfluorescence microscope (FV1200; Olympus) with a 60× objective.

Mouse brains were fixed in 4% paraformaldehyde overnight at 4 °C andsubsequently cryoprotected in 30% sucrose solution. The brains were thensectioned into 40-μm-thick coronal slices and subjected to immunostainingusing an established protocol described elsewhere, with some modifications(46). In brief, brain sections were blocked in 5% donkey serum/1%BSA/TBSsolution for 2 h at room temperature. In staining using mouse primary an-tibody, sections were subsequently blocked in F(ab) fragment donkey anti-mouse IgG (50 μg/mL; Jackson ImmunoResearch Laboratories) for 2 h atroom temperature to reduce background caused by secondary antibodybinding to endogenous mouse IgG in the tissue. After the blocking step,brain sections were then incubated in primary antibody solution at 4 °C for72 h. Primary antibodies used were as follow: mouse monoclonal anti-NeuN(1:500, ab104224; Abcam); rabbit polyclonal anti-GFAP (1:1,000, ab7260;Abcam); rabbit monoclonal anti-synaptophysin (1:500, ab32127; Abcam),and goat polyclonal anti-Pgrmc1 (1:200, ab48012; Abcam). Alexa Fluor 647,Alexa Fluor 594, or Rhodamine Red-conjugated secondary antibodies (JacksonImmunoResearch Laboratories) were used at 1:500 dilution. After immuno-staining, sections were mounted onto microscope slides with Vectashieldmounting medium (Vector Laboratories) and observed under a confocalfluorescence microscope (FV1200; Olympus) with a 63× objective.

Mice and Treatments. All procedures with animals were reviewed and ap-proved by the Institutional Animal Care and Use Committee of the Universityof North Texas Health Science Center. All institutional and federal guidelinesfor the care and the use of animals were followed. Female C57BL/6J mice(18 wk old) were purchased from The Jackson Laboratory. Animals werehabituated to housing conditions 1 wk before experiments.

All mice were first ovariectomized to deplete endogenous ovarian hor-mone levels. Two weeks after ovariectomy (OVX), P4 pellets were s.c.implanted into these animals to replenish their progesterone levels. Inparallel, different groups received cholesterol pellet implantations to serve asvehicle control. Oneweekafter pellet implantation, strokewas induced in thesemice using a middle cerebral artery occlusion (MCAo) procedure. In parallel,different groups received sham operation (nonstroke). Thirty minutes afterMCAo, 5 μg of either scrambled or let-7i inhibitor was injected into each an-imal brain via intracerebroventricular (ICV) injection. Experimental groups in-cluded sham-operated mice with cholesterol pellet implantation (sham),stroked mice with cholesterol pellet implantation and scrambled ICV injection(cholesterol + scrambled), stroked mice with P4 pellet implantation andscrambled ICV injection (P4 + scrambled), and stroked mice with P4 pelletimplantation and let-7i inhibitor ICV injection (P4 + scrambled).

Ovariectomy. Bilateral ovariectomy (OVX) was performed using a dorsalapproach under isoflurane anesthesia, as described elsewhere (47). Briefly,small incisions were made bilaterally to expose ovaries. The arteries adjacentto ovaries were ligated before removal of ovaries. Incisions were then closedusing a 4-0 Vicryl absorbable suture.

Transient Middle Cerebral Artery Occlusion. MCAo was performed to inducetransient focal cerebral ischemia, as previously described (48). In brief, mice

were anesthetized with isoflurane inhalation. A midline incision was madeon the neck. The left common carotid artery (CCA), external carotid artery(49), and internal carotid artery (ICA) were dissected from the connectivetissue. The left MCA was occluded by a 6-0 monofilament suture (DoccolCorporation) introduced via the internal carotid artery. After 45-min occlu-sion, the suture was withdrawn for reperfusion. In sham-operated animals,the monofilament was advanced to the MCA region and withdrawn im-mediately without MCA occlusion.

Intracerebroventricular Injection. Five micrograms of either scrambled or let-7iinhibitor (GE Healthcare Dharmacon) was suspended in 0.5 μL of PBS andinjected into lateral ventricles using a stereotaxic instrument, as previouslydescribed, with minor modifications (23). In brief, the solution was injectedusing a 5-μL Hamilton syringe attached to the Ultra Micro Pump UMP3 system(World Precision Instruments) at a flow rate of 0.2 μL/min. Coordinates usedfor ICV injection were anteroposterior (AP) −0.58 mm, mediolateral (ML)+1.2 mm, and dorsoventral (DV) −2.1 mm.

Assessment of Brain Tissue Damage: 2,3,5-Triphenyltetrazolium Chloride Staining.TTC staining was performed to assess ischemic injury among groups, as de-scribed in an established protocol (50). Briefly, 24 h after MCAo, mouse brainswere harvested and sectioned into 2-mm-thick coronal sections. These sectionswere immersed in 2% TTC solution for 30 min at 37 °C and then fixed in 10%formalin. The stained slices were photographed and subsequently measuredfor the surface area of the slices and the ischemic lesion (Image-Pro Plus 3.0.1;Silver Springs). Images of stained sections were captured, and infarct sizes wereanalyzed using ImageJ (National Institutes of Health) software (40).

Functional Recovery Assessment: Wire Suspension Test. In ordered to assessmotor function recovery with different treatments, a wire suspension test, atest of grip strength and endurance, was used, as described elsewhere (51). Inbrief, mice were allowed to suspend their bodies on a single wire that waselevated above a padded platform. The latency for animals to fall off thewire was recorded. Mice were trained 2 d before MCAo to establish abaseline across groups. Training was achieved with several rounds of ha-bituation and trials. In the actual testing phase, each mouse was tested threetimes, and average performance was taken as final values. Performances ofthese mice was evaluated at day 3, 7, and 14 poststroke.

Synaptophysin Optical Density Analysis and Puncta Quantification. For exper-iments using primary cortical neurons, mounted coverslips were imaged usinga confocal fluorescence microscope (FV1200; Olympus) with a 63× objective.Healthy cells that were at least two cell diameters from their nearestneighbor were identified and selected at random by eye by DAPI fluores-cence. Ten nonoverlapping fields per sample were imaged. Quantification ofSYP immunoreactivity (IR) was performed using ImageJ (National Institutesof Health) software (40). Average IR was calculated by dividing total IR valueby the number of cells presented in the captured image. Synaptophysinpuncta quantification was analyzed with a custom plug-in [written by BarryWark; available at https://elifesciences.org/articles/04047/figures#SD1-data(52)] for the ImageJ program. The details of this imaging and quantificationmethod can be found in a previous publication (53).

To quantify SYP fluorescence intensity and number of puncta in mousebrain, three independent coronal brain sections per animal were stained withSYP. Five-micrometer confocal scans were performed (optical section width,0.33 μm; 15 optical sections each) at 63× magnification, as previously de-scribed (54). Maximum projections of three consecutive optical sectionscorresponding to 1-μm sections were analyzed by using the ImageJ punctaanalyzer option to quantify for numbers of SYP puncta (≥5 optical sectionsper brain section and ≥15 total images per brain). Average SYP punctadensity per imaged area was calculated for each treatment group.

Statistical Analysis. In vitro data obtained from no fewer than three in-dependent experiments (where each independent experiment consisted ofbetween five to eight replicates) and in vivo data obtained from at least fouranimals per group (asmany as 20 animals per group for the functional recovery/motor function tests) were analyzed using an analysis of variance (ANOVA),followed by appropriate post hoc analyses for the assessment of group dif-ferences, and presented as a bar graph depicting the mean ± SEM, usingGraphPad Software. The parameters used to inform sample size consideredthe following: detecting an effect size of at least 30%, α = 0.05, the variance ofthe end point measured, and achieving a statistical power of at least 0.8.

E9676 | www.pnas.org/cgi/doi/10.1073/pnas.1803384115 Nguyen et al.

Dow

nloa

ded

by g

uest

on

Dec

embe

r 1,

202

1

ACKNOWLEDGMENTS. This work was supported in part by American HeartAssociation Grant 13SDG17050059 (to C.S.), National Institutes of Health

Grant AG027956 (to M.S.), and a fellowship (to T.N.) through Neurobiologyof Aging Training Grant T32 AG020494 (Program Director M.S.).

1. Mozaffarian D, et al.; American Heart Association Statistics Committee and StrokeStatistics Subcommittee (2015) Heart disease and stroke statistics–2015 update: Areport from the American Heart Association. Circulation 131:e29–e322, and erratum(2016) 133:e417.

2. Atif F, et al. (2013) Combination treatment with progesterone and vitamin D hor-mone is more effective than monotherapy in ischemic stroke: The role of BDNF/TrkB/Erk1/2 signaling in neuroprotection. Neuropharmacology 67:78–87.

3. Ishrat T, Sayeed I, Atif F, Stein DG (2009) Effects of progesterone administration oninfarct volume and functional deficits following permanent focal cerebral ischemia inrats. Brain Res 1257:94–101.

4. Zhao Y, et al. (2011) Progesterone influences postischemic synaptogenesis in theCA1 region of the hippocampus in rats. Synapse 65:880–891.

5. Kaur P, et al. (2007) Progesterone increases brain-derived neuroptrophic factor ex-pression and protects against glutamate toxicity in a mitogen-activated protein ki-nase- and phosphoinositide-3 kinase-dependent manner in cerebral cortical explants.J Neurosci Res 85:2441–2449.

6. Liu L, et al. (2010) The neuroprotective effects of Tanshinone IIA are associated withinduced nuclear translocation of TORC1 and upregulated expression of TORC1, pCREBand BDNF in the acute stage of ischemic stroke. Brain Res Bull 82:228–233.

7. Sato Y, et al. (2009) White matter activated glial cells produce BDNF in a stroke modelof monkeys. Neurosci Res 65:71–78.

8. Causing CG, et al. (1997) Synaptic innervation density is regulated by neuron-derivedBDNF. Neuron 18:257–267.

9. Coffey ET, Akerman KE, Courtney MJ (1997) Brain derived neurotrophic factor inducesa rapid upregulation of synaptophysin and tau proteins via the neurotrophin receptorTrkB in rat cerebellar granule cells. Neurosci Lett 227:177–180.

10. Heldt SA, Stanek L, Chhatwal JP, Ressler KJ (2007) Hippocampus-specific deletion ofBDNF in adult mice impairs spatial memory and extinction of aversive memories. MolPsychiatry 12:656–670.

11. McAllister AK (2007) Dynamic aspects of CNS synapse formation. Annu Rev Neurosci30:425–450.

12. Sheng M, Hoogenraad CC (2007) The postsynaptic architecture of excitatory synapses:A more quantitative view. Annu Rev Biochem 76:823–847.

13. Waites CL, Craig AM, Garner CC (2005) Mechanisms of vertebrate synaptogenesis.Annu Rev Neurosci 28:251–274.

14. Stroemer RP, Kent TA, Hulsebosch CE (1995) Neocortical neural sprouting, synapto-genesis, and behavioral recovery after neocortical infarction in rats. Stroke 26:2135–2144.

15. Liu HS, et al. (2011) Post-treatment with amphetamine enhances reinnervation of theipsilateral side cortex in stroke rats. Neuroimage 56:280–289.

16. Zhang L, et al. (2011) Delayed administration of human umbilical tissue-derived cellsimproved neurological functional recovery in a rodent model of focal ischemia.Stroke 42:1437–1444.

17. Zhang L, et al. (2013) Intravenous administration of human umbilical tissue-derivedcells improves neurological function in aged rats after embolic stroke. Cell Transplant22:1569–1576.

18. Su C, Cunningham RL, Rybalchenko N, Singh M (2012) Progesterone increases therelease of brain-derived neurotrophic factor from glia via progesterone receptormembrane component 1 (Pgrmc1)-dependent ERK5 signaling. Endocrinology 153:4389–4400.

19. Selvamani A, Sathyan P, Miranda RC, Sohrabji F (2012) An antagomir to microRNALet7f promotes neuroprotection in an ischemic stroke model. PLoS One 7:e32662.

20. Wendler A, Keller D, Albrecht C, Peluso JJ, Wehling M (2011) Involvement of let-7/miR-98 microRNAs in the regulation of progesterone receptor membrane component1 expression in ovarian cancer cells. Oncol Rep 25:273–279.

21. Di Y, et al. (2014) MicroRNAs expression and function in cerebral ischemia reperfusioninjury. J Mol Neurosci 53:242–250.

22. Caballero-Garrido E, et al. (2015) In vivo inhibition of miR-155 promotes recoveryafter experimental mouse stroke. J Neurosci 35:12446–12464.

23. Ni J, et al. (2015) MicroRNA let-7c-5p protects against cerebral ischemia injury viamechanisms involving the inhibition of microglia activation. Brain Behav Immun 49:75–85.

24. Pena-Philippides JC, Caballero-Garrido E, Lordkipanidze T, Roitbak T (2016) In vivoinhibition of miR-155 significantly alters post-stroke inflammatory response.J Neuroinflammation 13:287.

25. Sigler A, Murphy TH (2010) In vivo 2-photon imaging of fine structure in the rodentbrain: Before, during, and after stroke. Stroke 41(Suppl 1):S117–S123.

26. Franke H, Verkhratsky A, Burnstock G, Illes P (2012) Pathophysiology of astroglialpurinergic signalling. Purinergic Signal 8:629–657.

27. Sun F, et al. (2016) Pgrmc1/BDNF signaling plays a critical role in mediating glia-neuron cross talk. Endocrinology 157:2067–2079.

28. Jodhka PK, Kaur P, Underwood W, Lydon JP, Singh M (2009) The differences inneuroprotective efficacy of progesterone and medroxyprogesterone acetate corre-late with their effects on brain-derived neurotrophic factor expression. Endocrinology150:3162–3168.

29. Yousuf S, Atif F, Sayeed I, Wang J, Stein DG (2013) Post-stroke infections exacerbateischemic brain injury in middle-aged rats: Immunomodulation and neuroprotectionby progesterone. Neuroscience 239:92–102.

30. Stroemer RP, Kent TA, Hulsebosch CE (1998) Enhanced neocortical neural sprouting,synaptogenesis, and behavioral recovery with D-amphetamine therapy after neo-cortical infarction in rats. Stroke 29:2381–2393; discussion 2393–2395.

31. Chen J, Chopp M, Li Y (1999) Neuroprotective effects of progesterone after transientmiddle cerebral artery occlusion in rat. J Neurol Sci 171:24–30.

32. Toung TJ, Chen TY, Littleton-Kearney MT, Hurn PD, Murphy SJ (2004) Effects ofcombined estrogen and progesterone on brain infarction in reproductively senescentfemale rats. J Cereb Blood Flow Metab 24:1160–1166.

33. Murphy SJ, Traystman RJ, Hurn PD, Duckles SP (2000) Progesterone exacerbatesstriatal stroke injury in progesterone-deficient female animals. Stroke 31:1173–1178.

34. Wright DW, et al.; NETT Investigators (2014) Very early administration of pro-gesterone for acute traumatic brain injury. N Engl J Med 371:2457–2466.

35. Skolnick BE, et al.; SYNAPSE Trial Investigators (2014) A clinical trial of progesteronefor severe traumatic brain injury. N Engl J Med 371:2467–2476.

36. Johnson D, et al. (2017) Acute and subacute microRNA dysregulation is associatedwith cytokine responses in the rodent model of penetrating ballistic-like brain injury.J Trauma Acute Care Surg 83(Suppl 1):S145–S149.

37. Balakathiresan N, et al. (2012) MicroRNA let-7i is a promising serum biomarker forblast-induced traumatic brain injury. J Neurotrauma 29:1379–1387.

38. Spartalis E, et al. (2017) The “yin and yang” of platelet-rich plasma in breast re-construction after mastectomy or lumpectomy for breast cancer. Anticancer Res 37:6557–6562.

39. Roy Choudhury G, et al. (2015) Methylene blue protects astrocytes against glucoseoxygen deprivation by improving cellular respiration. PLoS One 10:e0123096.

40. Rueden CT, et al. (2017) ImageJ2: ImageJ for the next generation of scientific imagedata. BMC Bioinformatics 18:529.

41. Jala VR, et al. (2017) The yin and yang of leukotriene B4 mediated inflammation incancer. Semin Immunol 33:58–64.

42. Crouch SP, Kozlowski R, Slater KJ, Fletcher J (1993) The use of ATP bioluminescence asa measure of cell proliferation and cytotoxicity. J Immunol Methods 160:81–88.

43. Parker CG, et al. (2017) Ligand and target discovery by fragment-based screening inhuman cells. Cell 168:527–541.e29.

44. Ryou MG, et al. (2015) Methylene blue-induced neuronal protective mechanismagainst hypoxia-reoxygenation stress. Neuroscience 301:193–203.

45. Krebs CJ, et al. (2000) A membrane-associated progesterone-binding protein, 25-Dx, isregulated by progesterone in brain regions involved in female reproductive behav-iors. Proc Natl Acad Sci USA 97:12816–12821.

46. Jin K, et al. (2004) Increased hippocampal neurogenesis in Alzheimer’s disease. ProcNatl Acad Sci USA 101:343–347.

47. Singh M, Meyer EM, Millard WJ, Simpkins JW (1994) Ovarian steroid deprivation re-sults in a reversible learning impairment and compromised cholinergic function infemale Sprague-Dawley rats. Brain Res 644:305–312.

48. Li W, et al. (2014) PTEN degradation after ischemic stroke: A double-edged sword.Neuroscience 274:153–161.

49. Salvi V, et al. (2008) Metabolic syndrome in Italian patients with bipolar disorder. GenHosp Psychiatry 30:318–323.

50. Yang SH, et al. (2001) 17-beta estradiol can reduce secondary ischemic damage andmortality of subarachnoid hemorrhage. J Cereb Blood Flow Metab 21:174–181.

51. Hauben U, D’Hooge R, Soetens E, De Deyn PP (1999) Effects of oral administration ofthe competitive N-methyl-D-aspartate antagonist, CGP 40116, on passive avoidance,spatial learning, and neuromotor abilities in mice. Brain Res Bull 48:333–341.

52. Risher WC (2014) Astrocytes refine cortical connectivity at dendritic spines. eLife 3:e04047.

53. Ippolito DM, Eroglu C (2010) Quantifying synapses: An immunocytochemistry-basedassay to quantify synapse number. J Vis Exp 45:2270.

54. Kucukdereli H, et al. (2011) Control of excitatory CNS synaptogenesis by astrocyte-secreted proteins Hevin and SPARC. Proc Natl Acad Sci USA 108:E440–E449.

Nguyen et al. PNAS | vol. 115 | no. 41 | E9677

NEU

ROSC

IENCE

Dow

nloa

ded

by g

uest

on

Dec

embe

r 1,

202

1