ABSTRACT In addition to glial cells, HIV-1 infection occurs in multipotent human neural precursor cells (hNPCs) and induces quiescence in NPCs. HIV-1 infection of the brain alters hNPC

stemness, leading to perturbed endogenous neurorestoration of the CNS following brain damage by HIV-1, compounding the severity of dementia in adult neuroAIDS cases. In pediatric neuroAIDS

cases, HIV-1 infection of neural stem cell can lead to delayed developmental milestones and impaired cognition. Using primary cultures of human fetal brain-derived hNPCs, we gained novel

insights into the role of a neural stem cell determinant, tripartite containing motif 32 (TRIM32), in HIV-1 Tat-induced quiescence of NPCs. Acute HIV-1 Tat treatment of hNPCs resulted in

at post mortem as well as by infection of hNPCs by HIV-1. We observed increased percentage of cells with nuclear localization of TRIM32 in the subventricular zone (SVZ) as compared with

age-matched controls. Our quest for probing into the mechanisms revealed that TRIM32 is targeted by miR-155 as downregulation of miR-155 by HIV-1 Tat resulted in upregulation of TRIM32

levels. Furthermore, miR-155 or siRNA against TRIM32 rescued HIV-1 Tat-induced quiescence in NPCs. Our findings suggest a novel molecular cascade involving miR-155 and TRIM32 leading to

HIV-1 Tat-induced attenuated proliferation of hNPCs. The study also uncovered an unidentified role for miR-155 in modulating human neural stem cell proliferation, helping in better

understanding of hNPCs and diseased brain. SIMILAR CONTENT BEING VIEWED BY OTHERS RETROVIRAL INFECTION OF HUMAN NEUROSPHERES AND USE OF STEM CELL EVS TO REPAIR CELLULAR DAMAGE Article Open

access 07 February 2022 IDENTIFICATION OF ANTIVIRAL RNAI REGULATORS, ILF3/DHX9, RECRUIT AT ZIKV STEM LOOP B TO PROTECT AGAINST ZIKV INDUCED MICROCEPHALY Article Open access 26 February 2025

MODULATION OF VIRUS-INDUCED NEUROINFLAMMATION BY THE AUTOPHAGY RECEPTOR SHISA9 IN MICE Article 20 April 2023 MAIN Failure to eliminate HIV-1 from brain limits its eradication from AIDS

patient. Despite success of antiretroviral therapies in reducing systemic viral load and severe dementia, milder forms of HIV-1-associated neurological disorder (HAND) still prevail because

of poor penetrance of combinatorial antiretroviral therapy (cART) drugs into the brain.1, 2, 3, 4 HIV-1 infects majority of brain cells, including neural precursor cells (NPCs).5, 6, 7, 8

Viral infection of NPCs impairs the ability of the brain for endogenous neurorestoration by differentiating existing NPCs, as viral proteins interfere with the self-renewing capability of

the precursors cells and perturbing their commitment toward neuronal lineage9, 10 in adult neuroAIDS cases. The severity of the NPCs being affected by HIV-1 is also a major concern in

pediatric ages, as HIV-1 infection can result in cognitive impairment as well as deranged brain development, brain atrophy and cerebrovascular abnormalities.11 In addition to this, the NPCs

are also considered as a reservoir for the latent virus.6 To gain insights into cellular cascades important for HIV-1 Tat-mediated quiescence of NPCs, we searched for cellular stemness

determinants and their association with HIV-1. We identified tripartite containing motif 32 (TRIM32), a HIV-1 Tat interacting protein,12 that also regulates stemness of NPCs.13, 14, 15

Hence, we explored the possibility of association of these two properties of TRIM32. TRIM32 is a member of TRIM family comprising 75 members,16 many of which including TRIM32 are critical to

innate immune response to viral replication.16, 17, 18, 19, 20, 21 TRIM32 has been implicated in limb girdle muscular dystrophy,22 Bardet–Biedl syndrome,23 epidermal carcinogenesis,24

Alzheimer’s disease25and stress-induced anxiety and depressive behavior.26 By interacting with Argonaute, TRIM32 collaborates with the miRNA machinery activating particular microRNAs13 and

regulates retinoic acid-mediated neuronal differentiation.27 As HIV-1 Tat and TRIM32 influence stemness of NPCs and are interacting proteins, we investigated whether TRIM32 mediates the

effects of HIV-1 Tat on stemness of NPCs. RESULTS HIV-1 IMPAIRS STEMNESS OF NEURAL PROGENITOR CELLS HIV-1 induces quiescence in human NPCs (hNPCs) as the HIV-1-infected brain autopsy tissue

showed lower Ki67 (a proliferation marker)-positive cells in hippocampal sections as compared with the controls.28 To analyze whether HIV-1 infection affected stemness of the NPCs, we

examined nestin positivity in the subventricular zone (SVZ) in brain sections from the frontal cortex that included the angle of lateral ventricle. Periventricular brain sections of

HIV-1-infected patients showed lower number of nestin-positive precursor cells as compared with age-matched controls. In control sections, nestin positivity was observed to be 12.9±4.9% in

the SVZ, whereas in HIV-1-infected brain sections, a significant decrement was observed in the percentage of nestin-positive cells (3±2.0%; _P_<0.05; Figure 1). NUCLEAR TRANSLOCATION OF

TRIM32 IN RESPONSE TO HIV-1 TAT Cellular localization of TRIM32 is crucial in determining stemness of the neural stem cells. TRIM32 translocates to nucleus following neuronal differentiation

signal where it ubiquitinates c-myc and arrests the proliferation so as to initiate neurogenesis.14 To examine TRIM32 localization in precursor cells, autopsied control adult and fetal

brain sections were probed for TRIM32. It was observed that TRIM32 was localized differentially in the SVZ. Microscopic examination revealed that most of the cells had TRIM32 localized to

the cytoplasm, others had TRIM32 localized to both cytoplasmic and nuclear TRIM32 and rarely the cells had TRIM32 within the nucleus (Supplementary Figure S2A). These cells were confirmed to

be neural stem cells by probing them for nestin in serial sections (Supplementary Figure S2B). In our culture system, TRIM32 was localized in both cytoplasm and nucleus in hNPCs. As nuclear

translocation of TRIM32 can arrest the proliferation in hNPCs, we investigated whether HIV-1 Tat affects the localization of TRIM32 by ICC following 24 h of exposure to HIV-1 Tat. Cells

were analyzed under two groups: (1) consisted cells with TRIM32 in both nucleus and cytoplasm and (2) cells with exclusively nuclear localization of TRIM32. The percentage of cells showing

cytoplasmic and nuclear localization of TRIM32 was 62.2% in control compared with 4.7% in Tat-treated test group (_P_<0.005). Furthermore, when TRIM32 nuclear localization was assayed, we

found that 37.8% of cells had TRIM32 in nucleus in control conditions, whereas in Tat-treated group this percentage increased to 95.3% (_P_<0.005; Figure 2a). The hNPCs were transfected

with HIV-1 Tat expressing pDs Red vector so that Tat-transfected cells could be easily visualized with Ds Red tag. ICC was performed to assess localization of TRIM32. TRIM32 colocalized with

HIV-1 Tat in the nucleus and cytoplasm. In cells where Tat had translocated completely into the nucleus, TRIM32 also localized entirely within the nucleus (Figure 2b), suggesting an

association between the two. Tat-induced nuclear localization of TRIM32 in hNPCs was further confirmed by western blotting in nuclear and cytosolic extracts from hNPCs treated with HIV-1 Tat

for 24 h. Nuclear extracts showed an increase of 43% (_P_<0.005) with concomitant decrease of 34.6% of TRIM32 in the cytosolic levels (_P_<0.0005) as compared with the control (Figure

2c). To validate our _in vitro_ findings, we probed for TRIM32 in SVZ region in human brain sections from HIV-1-infected subjects and age-matched control brain sections. In controls, TRIM32

was entirely cytoplasmic in 70.5% of the cells, but in HIV-1-infected brain sections this percentage was decreased to mere 5.0% (_P_<0.005). In control sections, there were only 7.9% of

cells with exclusive nuclear TRIM32, whereas in HIV-1-infected sections this percentage was a staggering 69.1% (_P_<0.05; Figure 2d). To confirm that the nuclear translocation of TRIM32

was correlated with viral infection in the brain, we also performed viral detection through p24 staining in serial section from a similar area of the same patient and observed substantial

staining for HIV-1 p24 antigen (Supplementary Figure S3). These _in vitro_ and _in vivo_ findings strongly suggest that HIV-1 infection drives nuclear translocation of TRIM32, thereby

affecting the stemness of NPCs. As nuclear translocation of TRIM32 can drive c-Myc ubiquitination and HIV-1 Tat mediates translocation of TRIM32, we also looked for c-Myc ubiquitination with

HIV-1 Tat. The hNPCs were transfected with HIV-1 Tat vector, and 12 h post transfection cells were treated with MG-132 (10 _μ_M) for 12 h and subjected to immunoprecipitation with anti

c-Myc and then immunoblotted with anti-ubiquitin. Not much to our surprise, we observed increased ubiquitination with HIV-1 Tat expression in hNPCs (Supplementary Figure S4). This result

further strengthened our hypothesis of HIV-1 Tat mediating proliferation arrest through c-Myc degradation. TRIM32 IS OVEREXPRESSED IN HNPCS DUE TO HIV-1 TAT Mouse NPCs overexpressing TRIM32

lose their precursor properties and commit toward neuronal lineage.13 Hence, we investigated the effect of HIV-1 Tat with treatment as well as with transient Tat transfection on expression

levels of TRIM32. Cells treated with Tat showed modest but significant upregulation of 17.07% in TRIM32 levels (data not shown). To compare the effects of endogenously expressed HIV-1 Tat

with that of exogenously added Tat (as the former would mimic the condition of an infected cell more closely), hNPCs were transfected with Tat-expressing vector and samples were processed

for whole-cell lysate. An upregulation in TRIM32 levels (approximately twofold) was observed (_P_<0.05; Figure 3a). In order to corroborate our findings, we also analyzed mean intensity

of TRIM32 in human brain sections in control as well as in infected samples particularly in SVZ area. A fold change of 2.56 was observed in TRIM32 intensity in infected cases as compared

with control (_P_<0.05). As TRIM32 can potentially arrest the hNPC proliferation, it is possible that HIV-1 Tat-mediated TRIM32 overexpression may lead to proliferation arrest in these

hNPCs, as shown subsequently. KNOCKDOWN OF TRIM32 REVERSES HIV-1 TAT-MEDIATED PROLIFERATION ARREST IN HNPCS In order to test the hypothesis of whether TRIM32 is mediating proliferation

arrest in hNPCs, TRIM32 was knocked down using siRNA against TRIM32 (Figure 4a). Cells were treated with HIV-1 Tat for 24 h with or without siRNA knockdown and immunostained for Ki67. The

percentage of proliferative cells in TRIM32 knockdown group was 54.2% as compared with 37.8% in the control group (_P_<0.0005). In Tat-treated group, the cell proliferation was

significantly reduced to 24.2% (_P_<0.005), but TRIM32 knockdown in Tat-treated group resulted in restoration of proliferation potential back to near normal levels of 35.1% (_P_<0.005;

Figure 4b). These results support our hypothesis that Tat is affecting the proliferation in hNPCs via upregulation of TRIM32 levels. POST-TRANSCRIPTIONAL REGULATION OF TRIM32 LEVELS TRIM32

mRNA transcript levels were analyzed in HIV-1 Tat-expressing hNPCs and then compared with control after 24 h of transfection. Surprisingly, we did not find any increase in transcript levels,

contrary to what we observed in protein levels (Figure 5a). The rationale for this discordance could be attributed to miRNA machinery-mediated regulation. The miRNA-mediated repression

leads to translational arrest of the miRNA transcript from where it can either remain repressed, degraded or even get translationally reactivated.29 Hence, we searched for TRIM32 targeting

miRNAs using miRanda database. The miR-155 and Let-7a were predicted to target TRIM32. Putative binding sites of miRNAs within TRIM32 transcript are shown (Figure 5b). The miR-155 is altered

in case of HIV-1-infected PBMCs and dendritic cells.30, 31 In addition, Let-7 family including Let-7a is observed to be downregulated in CD4+ T cells of chronically affected HIV patients.32

As both of these microRNAs were downregulated in HIV-1 cases, they were of special interest to us and were further investigated. In agreement with previous findings, miR-155 and Let-7a were

downregulated with HIV-1 Tat expression in our model system as well. Let-7a levels were downregulated by 2.56-fold (_P_<0.0005), whereas miR-155 levels were decreased by 35%

(_P_<0.05; Figure 5c). 3′UTR OF TRIM32 IS A POTENTIAL TARGET OF MIR-155 To study whether Let-7a and miR-155 bind to the 3′UTR of TRIM32 and regulate the protein levels of TRIM32, hNPCs

were transfected with inhibitors/mimics of Let-7a and miR-155 and compared with cells transfected with respective control. Samples were harvested for miRNA or whole-cell protein lysates

after 24 h. Quantitative PCRs (qPCRs) were carried to check the potency of the mimic and inhibitor used (Supplementary Figure S5). Western blot analysis after transfection showed that

lipofection of miR-155 inhibitor in hNPCs resulted in higher levels of TRIM32; however, Let-7a did not affect the TRIM32 levels. The miR-155 inhibition increased the TRIM32 levels up to

76.3% (_P_<0.05; Figure 6a). Similarly, transient transfection with miR-155 mimic but not Let-7a mimic lowered TRIM32 levels up to twofold (_P_<0.0005; Figure 6b), indicating that

TRIM32 3′UTR could be a direct target of miR-155. Transcript levels of TRIM32 were also analyzed with transfection of miR-155 mimic as well as miR-155 inhibitor in hNPCs but it did not show

any change in the mRNA levels, and this was contrary to what we observed at the protein level (data not shown). This observation further supports our hypothesis that miR-155 is repressing

the translation of TRIM32 mRNA rather than degrading it. To further validate that TRIM32 is a potential target of miR-155 but not of Let-7a, luciferase assay was performed. 3′UTR of TRIM32

was cloned downstream of luciferase gene in pMIReporter vector (Ambion, Austin, TX, USA). Cells were transfected with pMIR TRIM32 3′UTR as well as miR-155 or Let-7a mimic. Samples were

processed after 24 h of transfection. Normalized luciferase activity showed that with co-transfection of miR-155 and pMIR TRIM32 3′UTR, luciferase activity was reduced by twofold

(_P_<0.05), whereas the same remains unaffected when pMIR TRIM32 3′UTR was co-transfected with Let-7a mimic (Figure 6c). These results confirmed that miR-155 targets the TRIM32 3′UTR.

MIR-155 DIRECTLY REGULATES HIV-1 TAT-MEDIATED PROLIFERATION ARREST IN HNPCS As miR-155 suppresses TRIM32 expression and downregulation of TRIM32 induces proliferation in hNPCs, we asked

whether miR-155 directly influences proliferation of hNPCs. To test the above hypothesis, cells were transfected with miR-155 mimic or inhibitor and then immunostained for Ki67 to quantify

the proliferation rate. We observed that miR-155 mimic increased the proliferation from 36.5 to 47.7% (_P_<0.05), whereas inhibition of miR-155 led to a decreased rate of proliferation

from 35.5% to mere 18% (_P_<0.05; Figure 7a). Hence, it can be inferred that as miR-155 regulates TRIM32 levels, increased levels of miR-155 may result in higher proliferation rate in

hNPCs or vice versa. After confirming that miR-155 regulates TRIM32 levels and can affect the proliferation of the hNPCs, we wanted to assess the effects of miR-155 in Tat-treated hNPCs.

Transfection of miR-155 mimic increased the proliferation rate of hNPCs from 36.8 to 48.3% (_P_<0.005), whereas treatment of hNPCs with Tat decreased the proliferation rate to 21.4%

(_P_<0.005; Figure 7b). Interestingly, in HIV-1 Tat-treated group transfected with miR-155 mimic, the proliferation was restored to 34.9% (_P_<0.005). These results further confirmed

that Tat mediates its effect on TRIM32 via miR-155 dysregulation (Figure 7b). HIV-1 INFECTION OF HNPCS INDUCES INCREASE IN TOTAL TRIM32 LEVELS, CONCOMITANT DECREASE IN MIR-155 LEVEL AND

INCREASED NUCLEAR TRANSLOCATION OF TRIM32 To correlate our findings with viral infections as well, hNPCs were infected with vesicular stomatitis virus envelope G glycoprotein

(VSV-G)-pseudotyped HIV-1 for 24 h and samples were processed for either total protein, miRNA assay or nuclear protein extraction. Viral infection of hNPCs resulted in 61.3% upregulation of

TRIM32 levels in whole-cell protein extracts as compared with control (_P_<0.005; Figure 8a). Samples processed for miRNA assays also showed concomitant decrease of 2.38-fold in miR-155

levels in infected samples as compared with control (_P_<0.05; Figure 8b). In addition to these observations, we found that HIV-1-infected group showed an increase in nuclear levels of

TRIM32 (45.2%) as compared with control (_P_<0.05; Figure 8c). These results further substantiated our findings and confirmed that HIV-1 infection is perhaps driving the TRIM32

upregulation and nuclear translocation in hNPCs. DISCUSSION HAND is a major morbidity in HIV-1/AIDS survivors in adult and pediatric cases of neuroAIDS. This study focuses on a rather

neglected aspect of neuroAIDS and unravels how HIV-1 protein Tat possibly affects self-renewing capacity of multipotent NPCs in SVZ, an important neuroanatomical area for HIV-1 infection and

neurogenesis. The effect of HIV-1 brain viremia on NPCs translates to attenuated stemness of the precursor cells interfering with human brain’s regenerative capacity and contributes to

HAND. To study the mechanisms leading to quiescence in NPCs, a well-characterized model of human fetal brain-derived NPCs was utilized that provided us a distinctive system to study

proliferation of NPCs. Being derived from human fetal brain samples, this primary cell culture system has significant advantages over the cell lines. Our model provides a nontransformed cell

system that is supposedly closer to the control physiological conditions rather than transformed cell lines derived of NPCs. Furthermore, as it is derived from human nervous tissue, this

model system is apt for studies related to a human-specific virus like HIV-1 and better mimics conditions of neuroAIDS. A recent study from our laboratory demonstrated Erk(1/2)–p21–p53 cell

cycle axis is involved in the HIV-1 Tat-mediated proliferation arrest of precursor cells.33 The mechanism by which HIV-1 Tat disturbs normal functionality of NPCs is still unclear.

Henceforth, the role of TRIM32 (a neural stem cell determinant and HIV-1 Tat interacting partner) was questioned in the HIV-1 Tat-induced quiescence in hNPCs. TRIM32 translocates to the

nucleus upon neurogenesis signal where it ubiquitinates c-myc that results in its proteasomal-mediated degradation.14 TRIM32 thereby arrests the proliferation of neural stem cells upon

translocation into the nucleus, laying groundwork for initiation for neurogenesis. We observed that TRIM32 localization was perturbed by HIV-1 Tat where increased trafficking of TRIM32

molecules into the nucleus was observed to be closely associated with HIV-1 Tat. To validate the findings of our _in vitro_ observations, we utilized human brain tissues from HIV-1-infected

individuals and reconfirmed the increased nuclear translocation of TRIM32 molecules in the SVZ area of diseased brain tissues. Increased nuclear trafficking of TRIM32 leads to degradation of

c-myc, contributing to arrested proliferation of NPCs mediated by HIV-1 Tat. This was further confirmed with our experiments showing enhanced c-Myc ubiquitination with HIV-1 Tat in hNPCs.

The mechanism of nuclear translocation of TRIM32 is unknown. As TRIM32 does not contain any classical nuclear localization signal, post-translation modification such as ubiquitination is

predicted to be a signal for entry into the nucleus.14 Perhaps, HIV-1 Tat promotes such modifications that mediate entry of TRIM32 into the nucleus. Another possible mechanism could be of

direct pull of TRIM32 into the nucleus through interaction with HIV-1 Tat, as this viral protein has competent NLS signal. It will be an interesting query to address the mechanisms that

HIV-1 Tat exploits to induce increased trafficking of TRIM32 molecules into the nucleus. Overexpression of TRIM32 in NPCs is related to their attenuated proliferation.13 When a neural stem

cell undergoes mitosis, enrichment of TRIM32 in the daughter cell decides the fate of the progeny. Following asymmetric division of a mouse neural precursor cells, TRIM32 accumulation in one

of the progenies causes it to commit toward neuronal lineage, whereas the other progeny that has inherited lesser number of TRIM32 molecules retains its precursor fate. We observed higher

expression of TRIM32 protein induced by HIV-1 Tat that possibly mediated proliferation arrest. We further observed increased relative intensity in TRIM32 levels in human brain sections in

HIV-1 cases as compared with control. This hypothesis was further validated by TRIM32 knockdown in HIV-1 Tat-treated groups wherein HIV-1 Tat-mediated proliferation arrest in hNPCs was

inhibited. Interestingly, we also observed lack of increase at the transcript levels of TRIM32 following HIV-1 Tat exposure and subsequently proved that RNAi-mediated gene silencing is

involved in regulating TRIM32 protein levels. The role of microRNA (miR-155) in regulation of 3′UTR of the TRIM32 transcripts was elucidated through mimic/knockdown strategies and luciferase

reporter assays. Furthermore, evidence for the role of miR-155 in regulation of proliferation of hNPCs was also demonstrated. In addition, miR-155 could directly rescue the hNPCs from HIV-1

Tat-mediated proliferation arrest. Dysregulation of miR-155 is associated with aberrant proliferation in different tissues.34, 35, 36, 37 Enhanced expression of miR-155 leads to enhanced

proliferation, whereas downregulated levels of miR-155 is linked with decreased proliferation in various tissues. MiR-155 knockdown has been reported to decrease proliferation in

glioblastomas.38 However, literature showing role of miR-155 in the dysregulation of stemness of hNPCs was lacking till this study. MiR-155 negatively regulates SOX6 expression that

positively influences the expression of p21 in hepatocellular carcinoma.37 Enhanced levels of p21 have been reported in case of HIV-1 Tat-mediated proliferation arrest33 that would possibly

suggest that declined levels of miR-155 might upregulate SOX6 and result in HIV-1 Tat-induced enhancement in p21 expression in hNPCs. If and how SOX6 is regulated by TRIM32 is another open

question. The role of TRIM32 and HIV-1 Tat interaction in post-transcriptional regulation of TRIM32 is another interesting query. TRIM32 is an Argonaute interacting protein. It interacts

with three Argonaute proteins, Ago-1, Ago-2 and Ago-3.39 Hence, TRIM32 can be regarded as RISC interacting protein. NHL domain of TRIM32 is reported to influence miRNA activity.13 Enhanced

activity of a microRNA biomolecule could be a result of enhanced transcription or enhanced processing to form a functionally mature miRNA. How TRIM32 activates miRNAs is not known but NHL

domain of TRIM32 has been implicated to influence the miRNA activation. HIV-1 Tat interacts with the same NHL domain of TRIM32; NHL domain perhaps when hijacked by HIV-1 Tat culminates into

downregulated levels of miR-155. Another possibility is Tat somehow independently disrupts biogenesis of mature miR-155 and results in downregulated levels of miR-155 levels and hence

ensuing enhanced TRIM32 protein levels. As HIV-1 Tat was attenuating the proliferation, it was questioned whether Tat was inducing any lineage commitment (glial or neuronal). With acute

HIV-1Tat exposure, we observed that Tat did not enhance commitment of hNPCs toward any lineage (Supplementary Figure S6). The above observation indicated that hNPCs were merely quiescent

with HIV-1 Tat exposure. In fact, TRIM32 knockdown in HIV-1 Tat-treated hNPCs readily restored the proliferation. To further strengthen our findings, we infected hNPCs with VSV-G-pseudotyped

HIV-1 and found similar observations of increased whole-cell TRIM32 levels, concomitant decrease in miR-155 levels and increase in nuclear translocation of TRIM32 with HIV-1 infection.

TRIM32 overexpression has been linked to enhanced commitment toward neuronal lineage.13 On the other hand, HIV-1 Tat represses commitment toward neuronal lineage.9 TRIM32 utilizes different

domains to execute its diverse functions; for example, the one it utilizes to control proliferation might not be the same it uses to induce neuronal differentiation. Hence, it would be

interesting to investigate how HIV-1 Tat induces TRIM32 overexpression yet hinders neurogenesis, what are the pathways that TRIM32 overexpression triggers and the mechanism of interference

of HIV-1 Tat with these molecular switches resulting into aberrant neurogenesis; these lines of investigation are being currently pursued in our laboratory. The data shown here assert that

proliferation arrest in primary culture of hNPCs because of HIV-1 Tat can be rescued through TRIM32 knockdown that could be either mediated by a siRNA against TRIM32 or miR-155 mimic. We

hereby report a novel molecular cascade consisting of miR-155 and TRIM32 involved in HIV-1 Tat-mediated quiescence in hNPCs and show how the virus exploits the microRNA machinery to induce

proliferation arrest in hNPCs. MATERIALS AND METHODS THE HNPC ISOLATION Handling of human tissues was carried out in strict accordance with the guidelines of Institutional Human Ethics

Committee and Stem Cell and Research Committee of National Brain Research Centre, India, and the Indian Council of Medical Research (ICMR), India. The hNPCs were isolated from the

telencephalon region of 10–15-week-old fetuses as described earlier.9 Isolated cells were cultured in Poly D-lysine (Sigma-Aldrich, St. Louis, MO, USA)-coated flasks in Neurobasal media

(Invitrogen, San Diego, CA, USA) supplemented with Neural Survival Factor-1 (Lonza, Charles City, IA, USA), N2 supplement (Invitrogen), 25 ng/ml bovine fibroblast growth factor (bFGF)

(Sigma-Aldrich) and 20 ng/ml epidermal growth factor (EGF) (Sigma-Aldrich). Cells were passaged 9–10 times and then utilized for experiments after their characterizations. These NPCs were

tested for the expression of Nestin and SOX2 positivity (marker for stemness), which was found to be >99%, and the NPCs were negative for any lineage-specific markers particularly glial

fibrillary acidic protein (GFAP) or microtubule-associated protein 2 (MAP2). Their potential as neural stem cell was assessed by their ability to form neurospheres and differentiate into

neurons or astrocytes (analyzed by Tuj-1 and GFAP positivity). NPCs were differentiated into neuronal lineage by removing bFGF and EGF from the above mentioned media cocktail and

supplementing the same with platelet-derived growth factor (PDGF) (Sigma-Aldrich) and brain-derived neurotrophic factor (BDNF) (Sigma-Aldrich). NPCs were differentiated into astrocytes by

replacing the NPC media with minimum essential medium (MEM) (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma-Aldrich). For both neuronal and astrocytic lineage,

differentiation was carried out for 21 days. Depending upon exposure to defined media conditions, >95% of the differentiated cells were either Tuj-1 or GFAP positive (Supplementary Figure

S1). IMMUNOHISTOCHEMISTRY OF HUMAN BRAIN SECTIONS Paraffin-processed 5 _μ_m serial sections of control (non-HIV infected (_n_=4)) and HIV-1 infected without associated opportunistic

infections (_n_=4) were obtained from Human Brain Tissue Repository, National Institute of Mental Health and Neurosciences, Bangalore, India. Age-matched controls and HIV-1-infected tissues

from the frontal cortex–basal ganglia that included the lateral ventricle with SVZ were used in the study (age range: 34–40 years). In addition, a 23-week-old fetal tissue was included in

the study to compare the observations with that of adult control tissues. The control brain sections were from cases of accidental death who succumbed to road traffic accidents, who had no

prior history of neurological illness and who did not reveal any pathology other than related to trauma. Tissue sections were deparaffinized by immersing in xylene, followed by increasing

grades of alcohol. Sections were treated with methanol containing 3% hydrogen peroxidase for quenching endogenous peroxidase activity, followed by antigen retrieval with sodium citrate

buffer (pH 6.0). Sections were blocked using Power Block (BioGenex, Fremont, CA, USA). Anti-Nestin (Santa Cruz Biotechnology, Santa Cruz, TX, USA; 1 : 50) and anti-TRIM32 (Novus Biologicals,

Littleton, CO, USA; 1 : 250) were used to detect the respective antigens with overnight incubations at 4 °C. One-step Polymer-HRP Detection System (BioGenex) was used to develop the

staining. Sections were imaged under Olympus BX51 (Tokyo, Japan). Images from 8 to 10 random fields were captured from SVZ region for quantification. For measurement of TRIM32 intensity in

human brain sections in SVZ area, acquired images were converted to 8-bit grayscale. Intensity of the staining was assessed by determining mean pixel density by using ImageJ software (NIH,

Bethesda, MD, USA). Background staining was subtracted from the mean density obtained. All intensity measurements were performed by a person blinded to experimental groups. HIV-1 TAT

TREATMENT AND HIV-1 TAT TRANSIENT EXPRESSION Recombinant HIV-1 Tat Clade B (Diatheva, Fano, Italy) was used for treatment of hNPCs at a concentration of 100 ng/ml for 24 h. The concentration

of HIV-1 Tat chosen was in line with the existing literature that showed the effect of HIV-1 Tat on proliferation of NPCs,9, 33, 40 and is consistent with other studies.41, 42, 43, 44

Full-length HIV-1 Tat-expressing pcDNA 3.1 vector and pDS RED was a kind gift from Professor Udaykumar Ranga (JNCASR, Bangalore, India). The plasmids were transfected in 80% confluent hNPCs

using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Transfected cells were processed either for mRNA, miRNA or protein isolation according to the requirement of

the experiments. IMMUNOCYTOCHEMISTRY For immunocytochemistry, experiments were carried out in eight-well chamber slides (Nunc, Kamstrupvej, Denmark). The hNPCs were plated at a density of 20

 000 cells per well. Cells were fixed for 20 min with 4% paraformaldehyde, washed thrice with PBS, blocked and permeabilized using 10% normal goat serum (Vector Labs, Burlingame, CA, USA) or

4% bovine serum albumin (BSA) containing 0.5% Triton X-100. Cells were incubated with the following primary antibodies overnight at 4 °C: anti-Ki67 (Novacastra, Wetzlar, Germany; 1 : 1000)

and/or anti-TRIM32 (Gramsch Laboratories, Schwabhausen, Germany; 1 : 250), Tuj-1 (Promega, Madison, WI, USA; 1 : 3000), DCX (Abcam, Cambridge, UK, 1 : 1000) and GFAP (Dako, Glostrup,

Denmark; 1 : 1000 and Santa Cruz Biotechnology; 1 : 200). Later, the cells were washed thrice after incubation with primary antibody and incubated with appropriate secondary antibodies

tagged with Alexa Flour 594 and Alexa Flour 488 (Invitrogen). Slides were mounted with Hard set mounting media containing DAPI (Vector Labs). For each group, 5–7 images were captured from

random fields. Images were acquired using AxioImager.Z1 microscope (Carl Zeiss, Heidenheim, Germany). SMALL INTERFERING RNA-MEDIATED KNOCKDOWN OF TRIM32: FOR TRIM32 KNOCKDOWN

Endoribonuclease-prepared siRNAs (esiRNAs) against TRIM32 (Sigma-Aldrich) was used. A total of 1 pmol of esiRNA was transfected using RNAi Max (Invitrogen) according to the manufacturer’s

instructions. MISSION siRNA Universal Negative Control 1 (Sigma-Aldrich) was used as scrambled control. To check the efficiency of the knockdown at protein level, concentrations of 2.5, 5,

10 and 20 pmol were used. Then, 10 pmol concentration was selected as it showed highest level of knockdown of TRIM32 and was scaled down 10 times accordingly for experiments that were

carried out in eight-well chamber slides. Samples were processed for immunocytochemistry after 24 h of transfection. PREPARATION OF PROTEIN EXTRACTS AND WESTERN BLOTTING Samples were

processed for either whole-cell extract or nuclear and cytoplasmic extract. Whole-cell extract buffer consisted of 1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl (pH 8.0), 5 mM EDTA (pH 8.0),

1 mM EGTA (pH 8.0), 0.5% NP-40, 50 mM sodium fluoride, 1 mM sodium orthovanadate and protease inhibitor cocktail (Roche, Mannheim, Germany). The cytosolic extraction buffer comprised 0.2%

Triton X-100, HEPES buffer (pH 7.6), 10 mM magnesium chloride, 100 mM potassium chloride, 10 mM sodium butyrate and protease inhibitor cocktail. Cells were lysed with the cytosolic

extraction buffer and then centrifuged. Pellet so obtained was lysed with nuclear extraction buffer that contained 50 mM Tris (pH 7.5), 150 mM sodium chloride, 50 mM sodium fluoride, 1 mM

EDTA, 1 mM sodium orthovendate, 2% sodium dodecyl sulfate and protease inhibitor cocktail. Protein concentration was estimated by bicinchoninic acid (Sigma-Aldrich) and 4% copper sulfate.

Protein extract was separated on 12% SDS-PAGE and then blotted onto nitrocellulose membrane (MDI, Ambala, India). The membranes were blocked with 5% skimmed milk or 10% normal goat serum

(Vector Labs) in PBS/Tween-20. Blots were incubated overnight at 4 °C with the following primary antibodies: TRIM32 (Gramsch Laboratories; 1 : 2000), GAPDH (Santa Cruz Biotechnology; 1 : 10 

000), histone H3 (Millipore, Bedford, MA, USA; 1 : 3000), anti-HIV-1 Tat (a kind gift from Professor Udaykumar Ranga, JNCASR; 1 : 4000) and anti-p24 monoclonal antibody (NIH AIDS Reagent

Programme, Germantown, MD, USA; 1 : 3000). After 3 washes with TBS/Tween-20 (TBST), blots were probed with HRP-conjugated secondary antibodies (Vector Labs) for 1 h. Later, blots were washed

thrice with TBST and developed with chemiluminescence reagent (Millipore, Billerica, MA, USA). Blots were imaged using ChemiGenius Bio-imaging System (Syngene, Cambridge, UK). Densitometric

analysis of the protein bands were carried out using ImageJ software (NIH). QUANTITATIVE REAL-TIME PCR RNA was extracted from the samples using TRIZOL (Sigma-Aldrich) according to the

manufacturer’s protocol. cDNA was synthesized from the extracted RNA using high-capacity cDNA reverse transcription kit (Applied Biosystems, Austin, TX, USA) in accordance with the

manufacturer’s instructions. Real-time detection was performed with SYBER Green master mix (Applied Biosystems) using following primers for TRIM32 and GAPDH: TRIM32 forward

5′-GGCCACTGTACACTCCCTGT-3′ and TRIM32 reverse 5′-AGCCAAAGAGCCTGTGAAGA-3′, GAPDH forward 5′-GAAGGTGAAGGTCGGAGTC-3′ and GAPDH reverse 5′-GAAGATGGTGATGGGATTTC-3′. The cycling conditions used

were 95 °C for 3 min (1 cycle), 95 °C for 20 s, 62 °C for 30 s and 72 °C for 40 s (35 cycles). For miRNA isolation, RNA was isolated from the cells using miRNAeasy mini kit (Qiagen, Hilden,

Germany). cDNA was synthesized using miScript II RT kit (Qiagen) according to the manufacturer’s protocol. Specific primers for miR-155-5p and Let-7a-5p (Qiagen) were used to detect the

respective miRNAs. qPCR was performed to detect the levels of miRNAs of interest using miScript SYBER Green PCR kit (Qiagen) as per the manufacture’s protocol. qPCR was carried out using

ROTOR-GENE Q (Qiagen). Specificity of all the primers used was confirmed with single product peak in the melt curve analysis. MIRNA MIMIC AND INHIBITOR TRANSFECTION The hNPCs were plated at

80% confluency in T25 flasks. Then, 100 pmol of Syn-hsa-miR-155-5p miScript miRNA mimic or Syn-hsa-Let-7a-5p miScript miRNA mimic (Qiagen) was used to transfect hNPCs. All Stars Negative

Control siRNA (Qiagen) was used as a mimic control. Next, 100 pmol of Anti-hsa-miR-155-5p (Qiagen) and Anti-hsa-Let-7a-5p (Qiagen) were transfected in hNPCs to inhibit respective miRNAs.

miScript Inhibitor Negative Control (Qiagen) was used as control for miRNA inhibition experiments. Transfections were performed using RNAi MAX (Invitrogen). Opti-MEM (Invitrogen) was used as

transfection media. Cells were replaced with normal media after 4 h of transfection and samples were processed for protein extraction after 24 h. For immunocytochemistry assays of miR-155

mimic and inhibitor that was carried out in eight-well chamber slides, concentrations of the mimic and the inhibitor was scaled down 10 times accordingly. Efficacy of miRNA mimic and

inhibition was checked through qPCR using primers of respective miRNAs. MIRNA TARGET PREDICTION The miRanda database (www.microrna.org) was used to predict the miRNAs targeting TRIM32 3′UTR.

CLONING OF TRIM32 3′UTR AND LUCIFERASE ASSAY 3′UTR of TRIM32 was amplified using given primers: forward 5′-CAGTCTGATCGTCTTTACCAGTATG-3′ and reverse 5′-ATCCAAAGAAACACTACTACCATAAAAA-3′. The

1037 bp long amplified product contained the predicted miR-155 binding site and was cloned in pMIR-Reporter (Ambion). The product was cloned between _Spe_I and _Hin_dIII restriction sites

downstream of the luciferase coding region. The TRIM32 3′UTR luciferase reporter was transfected in HeLa cells with or without miR-155 or Let-7a mimic. Then, 5 _μ_g of the cloned product and

100 pmol of the mimic were used to transfect the cells using Lipofectamine 2000 as per the manufacturer’s protocol. Samples were collected for luciferase detection after 24 h. Luciferase

activity was measured with a Luciferase detection kit (Promega) as per the manufacturer’s protocol. Detection was performed using Sirius single tube luminometer (Berthold Detection Systems,

Pforzheim, Germany) and Luciferase units were normalized with the total protein. HIV-1 INFECTION OF HNPCS Viral stocks of VSV-G)-psuedotyped HIV-1 were prepared by co-transfection of pNL4-3

and VSV-G expressing plasmid into HEK 293T cells using Lipofectamine 2000 (Invitrogen). Medium was replaced with fresh complete DMEM 6 h post transfection. The supernatant containing virus

particles was collected 48 h post transfection. The collected virus supernatant was filtered through a 0.45 _μ_m pore size filter and the aliquots were then stored in −80 °C. P24 assay for

viral titer was done by _β_-galactosidase staining of HIV-1 using TZM-bl reporter cell line. HNPCs were infected with VSV-G-psuedotyped HIV-1 for 4 h at 37 °C by incubating the cells with

equal amounts of infectious virus (1 MOI) assessed by _β_-galactosidase staining using HIV-1 indicator TZMbl cells. The infected cells were harvested at 24 h post infection and subjected to

protein isolation or miRNA assay. UBIQUITINATION ASSAY Human neural precursor cells were transfected with HIV-1 Tat vector and Ubiquitin expressing vector (a kind gift from Dr. A Banerjea,

NII, New Delhi, India) using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. After 12 hours of transfection, MG-132 (Calbiochem, Merck Millipore, Billerica, MA,

USA; 10 _μ_M) was added to the media and cells were harvested after 12 h. Whole-cell extracts were prepared and 500 _μ_g of protein was incubated overnight with anti-c-myc (Santa Cruz

Biotechnology). Antigen antibody complexes were pulled out by incubating the lysate with Sepharose G beads (GE Healthcare, Buckinghamshire, UK). Beads were washed 6 times with IP buffer.

Immunoprecipitated protein was then separated on 8% SDS-PAGE gel and blot was probed with anti-ubiquitin (Dako) to detect ubiquitinated species of c-Myc. Input used was 20% of the lysate

used in IP. STATISTICAL ANALYSIS Data are represented as mean±S.D. All experiments were carried out three to five times independently. Significance of the comparison between control and

treated groups was computed using Student’s _t_-test. _P_<0.05 was considered as statistically significant. ABBREVIATIONS * TRIM32: tripartite containing motif 32 * hNPC: human neural

precursor cell * SVZ: subventricular zone REFERENCES * Maschke M, Kastrup O, Esser S, Ross B, Hengge U, Hufnagel A . Incidence and prevalence of neurological disorders associated with HIV

since the introduction of highly active antiretroviral therapy (HAART). _J Neurol Neurosurg Psychiatry_ 2000; 69: 376–380. Article  CAS  PubMed  PubMed Central  Google Scholar  * Neuenburg

JK, Brodt HR, Herndier BG, Bickel M, Bacchetti P, Price RW _et al_. HIV-related neuropathology, 1985 to 1999: rising prevalence of HIV encephalopathy in the era of highly active

antiretroviral therapy. _J Acquir Immune Defic Syndr_ 2002; 31: 171–177. Article  PubMed  Google Scholar  * Zhou L, Saksena NK . HIV associated neurocognitive disorders. _Infect Dis Rep_

2013; 5: e8. Article  PubMed  PubMed Central  Google Scholar  * Langford D, Marquie-Beck J, de Almeida S, Lazzaretto D, Letendre S, Grant I _et al_. Relationship of antiretroviral treatment

to postmortem brain tissue viral load in human immunodeficiency virus-infected patients. _J Neurovirol_ 2006; 12: 100–107. Article  PubMed  Google Scholar  * Kramer-Hammerle S, Rothenaigner

I, Wolff H, Bell JE, Brack-Werner R . Cells of the central nervous system as targets and reservoirs of the human immunodeficiency virus. _Virus Res_ 2005; 111: 194–213. Article  PubMed 

Google Scholar  * Rothenaigner I, Kramer S, Ziegler M, Wolff H, Kleinschmidt A, Brack-Werner R . Long-term HIV-1 infection of neural progenitor populations. _AIDS_ 2007; 21: 2271–2281.

Article  PubMed  Google Scholar  * Schwartz L, Civitello L, Dunn-Pirio A, Ryschkewitsch S, Berry E, Cavert W _et al_. Evidence of human immunodeficiency virus type 1 infection of

nestin-positive neural progenitors in archival pediatric brain tissue. _J Neurovirol_ 2007; 13: 274–283. Article  PubMed  Google Scholar  * Lawrence DM, Durham LC, Schwartz L, Seth P, Maric

D, Major EO . Human immunodeficiency virus type 1 infection of human brain-derived progenitor cells. _J Virol_ 2004; 78: 7319–7328. Article  CAS  PubMed  PubMed Central  Google Scholar  *

Mishra M, Taneja M, Malik S, Khalique H, Seth P . Human immunodeficiency virus type 1 Tat modulates proliferation and differentiation of human neural precursor cells: implication in

NeuroAIDS. _J Neurovirol_ 2010; 16: 355–367. Article  CAS  PubMed  Google Scholar  * Okamoto S, Kang YJ, Brechtel CW, Siviglia E, Russo R, Clemente A _et al_. HIV/gp120 decreases adult

neural progenitor cell proliferation via checkpoint kinase-mediated cell-cycle withdrawal and G1 arrest. _Cell Stem Cell_ 2007; 1: 230–236. Article  CAS  PubMed  Google Scholar  * Schwartz

L, Major EO . Neural progenitors and HIV-1-associated central nervous system disease in adults and children. _Curr HIV Res_ 2006; 4: 319–327. Article  CAS  PubMed  Google Scholar  * Fridell

RA, Harding LS, Bogerd HP, Cullen BR . Identification of a novel human zinc finger protein that specifically interacts with the activation domain of lentiviral Tat proteins. _Virology_ 1995;

209: 347–357. Article  CAS  PubMed  Google Scholar  * Schwamborn JC, Berezikov E, Knoblich JA . The TRIM-NHL protein TRIM32 activates microRNAs and prevents self-renewal in mouse neural

progenitors. _Cell_ 2009; 136: 913–925. Article  CAS  PubMed  PubMed Central  Google Scholar  * Hillje AL, Worlitzer MM, Palm T, Schwamborn JC . Neural stem cells maintain their stemness

through protein kinase C zeta-mediated inhibition of TRIM32. _Stem Cells_ 2011; 29: 1437–1447. CAS  PubMed  Google Scholar  * Hillje AL, Pavlou MA, Beckmann E, Worlitzer MM, Bahnassawy L,

Lewejohann L _et al_. TRIM32-dependent transcription in adult neural progenitor cells regulates neuronal differentiation. _Cell Death Dis_ 2013; 4: e976. Article  CAS  PubMed  PubMed Central

  Google Scholar  * Versteeg GA, Rajsbaum R, Sanchez-Aparicio MT, Maestre AM, Valdiviezo J, Shi M _et al_. The E3-ligase TRIM family of proteins regulates signaling pathways triggered by

innate immune pattern-recognition receptors. _Immunity_ 2013; 38: 384–398. Article  CAS  PubMed  PubMed Central  Google Scholar  * Carthagena L, Bergamaschi A, Luna JM, David A, Uchil PD,

Margottin-Goguet F _et al_. Human TRIM gene expression in response to interferons. _PLoS One_ 2009; 4: e4894. Article  PubMed  PubMed Central  Google Scholar  * Ozato K, Shin DM, Chang TH,

Morse HC 3rd . TRIM family proteins and their emerging roles in innate immunity. _Nat Rev Immunol_ 2008; 8: 849–860. Article  CAS  PubMed  PubMed Central  Google Scholar  * Rahm N, Telenti A

. The role of tripartite motif family members in mediating susceptibility to HIV-1 infection. _Curr Opin HIV AIDS_ 2012; 7: 180–186. Article  CAS  PubMed  Google Scholar  * Uchil PD, Hinz

A, Siegel S, Coenen-Stass A, Pertel T, Luban J _et al_. TRIM protein-mediated regulation of inflammatory and innate immune signaling and its association with antiretroviral activity. _J

Virol_ 2013; 87: 257–272. Article  CAS  PubMed  PubMed Central  Google Scholar  * Zhang J, Hu MM, Wang YY, Shu HB . TRIM32 protein modulates type I interferon induction and cellular

antiviral response by targeting MITA/STING protein for K63-linked ubiquitination. _J Biol Chem_ 2012; 287: 28646–28655. Article  CAS  PubMed  PubMed Central  Google Scholar  * Frosk P,

Weiler T, Nylen E, Sudha T, Greenberg CR, Morgan K _et al_. Limb-girdle muscular dystrophy type 2H associated with mutation in TRIM32, a putative E3-ubiquitin-ligase gene. _Am J Hum Genet_

2002; 70: 663–672. Article  CAS  PubMed  PubMed Central  Google Scholar  * Locke M, Tinsley CL, Benson MA, Blake DJ . TRIM32 is an E3 ubiquitin ligase for dysbindin. _Hum Mol Genet_ 2009;

18: 2344–2358. Article  CAS  PubMed  PubMed Central  Google Scholar  * Horn EJ, Albor A, Liu Y, El-Hizawi S, Vanderbeek GE, Babcock M _et al_. RING protein Trim32 associated with skin

carcinogenesis has anti-apoptotic and E3-ubiquitin ligase properties. _Carcinogenesis_ 2004; 25: 157–167. Article  CAS  PubMed  Google Scholar  * Yokota T, Mishra M, Akatsu H, Tani Y,

Miyauchi T, Yamamoto T _et al_. Brain site-specific gene expression analysis in Alzheimer's disease patients. _Eur J Clin Invest_ 2006; 36: 820–830. Article  CAS  PubMed  Google Scholar

  * Ruan CS, Wang SF, Shen YJ, Guo Y, Yang CR, Zhou FH _et al_. Deletion of TRIM32 protects mice from anxiety- and depression-like behaviors under mild stress. _Eur J Neurosci_ 2014; 40:

2680–2690. Article  PubMed  Google Scholar  * Sato T, Okumura F, Kano S, Kondo T, Ariga T, Hatakeyama S _et al_. TRIM32 promotes neural differentiation through retinoic acid

receptor-mediated transcription. _J Cell Sci_ 2011; 124: 3492–3502. Article  CAS  PubMed  Google Scholar  * Krathwohl MD, Kaiser JL . HIV-1 promotes quiescence in human neural progenitor

cells. _J Infect Dis_ 2004; 190: 216–226. Article  CAS  PubMed  Google Scholar  * Wilczynska A, Bushell M . The complexity of miRNA-mediated repression. _Cell Death Differ_ 2015; 22: 22–33.

Article  CAS  PubMed  Google Scholar  * Sun G, Li H, Wu X, Covarrubias M, Scherer L, Meinking K _et al_. Interplay between HIV-1 infection and host microRNAs. _Nucleic Acids Res_ 2012; 40:

2181–2196. Article  CAS  PubMed  Google Scholar  * Napuri J, Pilakka-Kanthikeel S, Raymond A, Agudelo M, Yndart-Arias A, Saxena SK _et al_. Cocaine enhances HIV-1 infectivity in monocyte

derived dendritic cells by suppressing microRNA-155. _PLoS One_ 2013; 8: e83682. Article  PubMed  PubMed Central  Google Scholar  * Swaminathan S, Suzuki K, Seddiki N, Kaplan W, Cowley MJ,

Hood CL _et al_. Differential regulation of the Let-7 family of microRNAs in CD4+ T cells alters IL-10 expression. _J Immunol_ 2012; 188: 6238–6246. Article  CAS  PubMed  Google Scholar  *

Malik S, Saha R, Seth P . Involvement of extracellular signal-regulated kinase (ERK1/2)-p53-p21 axis in mediating neural stem/progenitor cell cycle arrest in co-morbid HIV-drug abuse

exposure. _J Neuroimmune Pharmacol_ 2014; 9: 340–353. Article  PubMed  Google Scholar  * Zhang CM, Zhao J, Deng HY . MiR-155 promotes proliferation of human breast cancer MCF-7 cells through

targeting tumor protein 53-induced nuclear protein 1. _J Biomed Sci_ 2013; 20: 79. Article  CAS  PubMed  PubMed Central  Google Scholar  * Li T, Yang J, Lv X, Gao C, Xing Y, Xi T . miR-155

regulates the proliferation and cell cycle of colorectal carcinoma cells by targeting E2F2. _Biotechnol Lett_ 2014; 36: 1743–1752. Article  CAS  PubMed  Google Scholar  * Gracias DT,

Stelekati E, Hope JL, Boesteanu AC, Doering TA, Norton J _et al_. The microRNA miR-155 controls CD8(+) T cell responses by regulating interferon signaling. _Nat Immunol_ 2013; 14: 593–602.

Article  CAS  PubMed  PubMed Central  Google Scholar  * Xie Q, Chen X, Lu F, Zhang T, Hao M, Wang Y _et al_. Aberrant expression of microRNA 155 may accelerate cell proliferation by

targeting sex-determining region Y box 6 in hepatocellular carcinoma. _Cancer_ 2012; 118: 2431–2442. Article  CAS  PubMed  Google Scholar  * Liu S, Yin F, Zhang J, Wicha MS, Chang AE, Fan W

_et al_. Regulatory roles of miRNA in the human neural stem cell transformation to glioma stem cells. _J Cell Biochem_ 2014; 115: 1368–1380. Article  CAS  PubMed  Google Scholar  * Nicklas

S, Okawa S, Hillje AL, González-Cano L, Del Sol A, Schwamborn JC _et al_. The RNA helicase DDX6 regulates cell-fate specification in neural stem cells via miRNAs. _Nucleic Acids Res_ 2015;

43: 2638–2654. Article  CAS  PubMed  PubMed Central  Google Scholar  * Yao H, Duan M, Yang L, Buch S . Platelet-derived growth factor-BB restores human immunodeficiency virus

Tat-cocaine-mediated impairment of neurogenesis: role of TRPC1 channels. _J Neurosci_ 2012; 32: 9835–9847. Article  CAS  PubMed  PubMed Central  Google Scholar  * Yao H, Peng F, Dhillon N,

Callen S, Bokhari S, Stehno-Bittel L _et al_. Involvement of TRPC channels in CCL2-mediated neuroprotection against tat toxicity. _J Neurosci_ 2009; 29: 1657–1669. Article  CAS  PubMed 

PubMed Central  Google Scholar  * Hofman FM, Chen P, Incardona F, Zidovetzki R, Hinton DR . HIV-1 tat protein induces the production of interleukin-8 by human brain-derived endothelial

cells. _J Neuroimmunol_ 1999; 94: 28–39. Article  CAS  PubMed  Google Scholar  * Koller H, Schaal H, Freund M, Garrido SR, von Giesen HJ, Ott M _et al_. HIV-1 protein Tat reduces the

glutamate-induced intracellular Ca2+ increase in cultured cortical astrocytes. _Eur J Neurosci_ 2001; 14: 1793–1799. Article  CAS  PubMed  Google Scholar  * Malik S, Khalique H, Buch S, Seth

P . A growth factor attenuates HIV-1 Tat and morphine induced damage to human neurons: implication in HIV/AIDS-drug abuse cases. _PLoS One_ 2011; 6: e18116. Article  CAS  PubMed  PubMed

Central  Google Scholar  Download references ACKNOWLEDGEMENTS Technical assistance from Ms. Rabia Khatoon, Mr. DL Meena and Mr. Naushad Alam of NBRC, India, and Mr. Shivshankar HN,

Neuromuscular Laboratory, NRC, NIMHANS, India, during the study is greatly acknowledged. Availability of well-characterized autopsy brain sections from Human Brain Tissue Repository for

Neurobiological Studies, Department of Neuropathology, National Institute of Mental Health and Neurosciences, Bangalore, India, is greatly appreciated. We also acknowledge Mr. Bharat

Prajapati, NBRC, for critical analysis of the manuscript. Senior Research Fellowship to Ms. Mahar Fatima from CSIR, New Delhi, India, and project assistantship to Ms Rina Kumari from

National Brain Research Centre, Manesar, India, are greatly acknowledged. Financial support for the study from NBRC core funds to PS is greatly acknowledged. This project in the lab of JCS

is supported by the Schram-Stiftung (T287/21795/2011) and the Boehringer Ingelheim Foundation. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Cellular and Molecular Neuroscience, National

Brain Research Centre, Manesar (Gurgaon), Haryana, India M Fatima, R Kumari & P Seth * Luxembourg Centre for Systems Biomedicine (LCSB), University of Luxembourg, 7, Avenue des

Hauts-Fourneaux, L-4362, Campus Belval, Esch-sur-Alzette, Luxembourg J C Schwamborn * Department of Neuropathology, National Institute of Mental Health & Neurosciences, Bangalore, India

A Mahadevan & S K Shankar * Laboratory of Virology, National Institute of Immunology, New Delhi, India R Raja Authors * M Fatima View author publications You can also search for this

author inPubMed Google Scholar * R Kumari View author publications You can also search for this author inPubMed Google Scholar * J C Schwamborn View author publications You can also search

for this author inPubMed Google Scholar * A Mahadevan View author publications You can also search for this author inPubMed Google Scholar * S K Shankar View author publications You can also

search for this author inPubMed Google Scholar * R Raja View author publications You can also search for this author inPubMed Google Scholar * P Seth View author publications You can also

search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to P Seth. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no conflict of interest. ADDITIONAL

INFORMATION Edited by M Piacentini Supplementary Information accompanies this paper on Cell Death and Differentiation website SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE S1 (PPT 1498 KB)

SUPPLEMENTARY FIGURE S2 (PPT 4731 KB) SUPPLEMENTARY FIGURE S3 (PPT 1402 KB) SUPPLEMENTARY FIGURE S4 (PPT 881 KB) SUPPLEMENTARY FIGURE S5 (PPT 2058 KB) SUPPLEMENTARY FIGURE S6 (PPT 632 KB)

RIGHTS AND PERMISSIONS This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. The images or other third party material in this article

are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to

obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/4.0/ Reprints and permissions ABOUT

THIS ARTICLE CITE THIS ARTICLE Fatima, M., Kumari, R., Schwamborn, J. _et al._ Tripartite containing motif 32 modulates proliferation of human neural precursor cells in HIV-1

neurodegeneration. _Cell Death Differ_ 23, 776–786 (2016). https://doi.org/10.1038/cdd.2015.138 Download citation * Received: 24 January 2015 * Revised: 14 August 2015 * Accepted: 11

September 2015 * Published: 20 November 2015 * Issue Date: May 2016 * DOI: https://doi.org/10.1038/cdd.2015.138 SHARE THIS ARTICLE Anyone you share the following link with will be able to

read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing

initiative