miR-449a mediated repression of the cell cycle machinery prevents neuronal apoptosis

Aberrant activation of the cell cycle of terminally differentiated neurons results in their apoptosis and is known to contribute to neuronal loss in various neurodegenerative disorders like Alzheimer's Disease. However, the mechanisms that regulate cell cycle–related neuronal apoptosis are poorly understood. We identified several miRNA that are dysregulated in neurons from a transgenic APP/PS1 mouse model for AD (TgAD). Several of these miRNA are known to and/or are predicted to target cell cycle–related genes. Detailed investigation on miR-449a revealed the following: a, it promotes neuronal differentiation by suppressing the neuronal cell cycle; b, its expression in cortical neurons was impaired in response to amyloid peptide Aβ42; c, loss of its expression resulted in aberrant activation of the cell cycle leading to apoptosis. miR-449a may prevent cell cycle–related neuronal apoptosis by targeting cyclin D1 and protein phosphatase CDC25A, which are important for G1-S transition. Importantly, the lentiviral-mediated delivery of miR-449a in TgAD mouse brain significantly reverted the defects in learning and memory, which are associated with AD.


Plasmid DNA constructs
a. psiCHECK-CDC25A3'-UTR.A 492-bp fragment of the human CDC25A 3'-untranslated region (UTR) containing the putative miR-449a binding site was PCR amplified from HEK293 human cell line and cloned into the XhoI and NotI sites of the psiCHECK2 vector upstream of the Renilla luciferase gene using CDC25 WT_UTR1 and CDC25WT_UTR2 primer pairs mentioned in Supp.Table S2.The mutant of the miR-449a binding site at 516-522 bp was generated by PCR, which was performed using CDC25mutUTR1 and CDC25mutUTR2 primers (Supp.Table S2).All the positive clones were confirmed by sequencing.It is important to note that miR-449a binding site in CDC25a-3'UTR is conserved in rat, mice and human (not shown here).b. psiCHECK-cyclin D1 3'-UTR.Since this construct has been described previously and was available in the laboratory (1), it was used for present studies.The mutant of the miR-449a binding site at 2076-2082 bp was generated by mutagenesis PCR, which was performed using D1mutUTR1 and D1mutUTR2 primers (Supp.Table S2).All the positive clones were confirmed by sequencing (1).It is important to note that miR-449a binding site in cyclin D1-3'UTR is conserved in rat, mice and human (not shown here).c. pAdTrack-CDC25A.For overexpression of CDC25A in primary cortical neurons, mouse CDC25A protein coding sequence along with C-terminus HA-tag was cloned in pAdTrack shuttle vector.Briefly, coding sequence for CDC25A was PCR amplified from mouse cDNA with CDC25A_adeno1 and CDC25A_adeno2 primers and cloned using the NotI and HindIII restriction sites of pAdTrack-CMV vector.CDC25A-HA-pAdTrack shuttle vector construct was digested with PmeI, and electroporated in Escherichia coli BJ5138 containing pAdEasy-1 vector.
The recombinant clones were digested with PacI before transfection in HEK293A cells.d. pAdTrack-Cyclin D1.For the overexpression of cyclin D1 in primary cortical neurons, cyclin D1 adenovirus was used which was previously prepared in the laboratory (2).

Thioflavin Staining:
The cryopreserved brain sections (10 µm thick) were thawed at room temperature followed by dehydration in 70% and 80% ethanol for one minute each.The slides were then dipped into the filtered solution of Thioflavin T (0.002% in 80% ethanol) for 8 min.
Slides were then washed in decreasing concentrations of ethanol from 80%, 70%, 50%, 50% and finally in distilled water for 2 min, air dried and mounted in DAPI containing mounting media (3,4).Brain sections encompassing the cortex region were stained for Thioflavin T (ThT).These ThT-stained brain sections were imaged with a 20x objective using a Zeiss microscope (Zeiss, Germany) for quantification.The intensity of ThT staining was quantified using Image J (NIH) (5).

Human Tissue lysate preparation for total RNA isolation:
Frontal cortex tissue from two AD patients and age-matched normal individuals were acquired from National Institute of Mental Health and Neurosciences brain bank to assess the level of these miRNA.Human brain samples acquisition and usage was approved under the Institutional Human Ethics Committee with IHEC serial #120/19.The human studies reported on this manuscript abide by the Declaration of Helsinki principles.There was no requirement of the patient consents since these tissues were acquired from a tissue bank.For RNA isolation, 20-25 mg of tissue was homogenised in Trizol reagent (cat.no.: 10296010 from Thermo Fisher Scientific, USA) and proceeded as in main text.

Supp. Figure S1
A. A flow chart showing the miRNAseq analysis pipeline and the tools used for the analysis.

B.
A Venn diagram showing miRNA that were significantly altered in TgAD neurons (fold change cut off < 0.6 and padj cutoff< 0.1 in the present study and were also previously reported to be aberrantly expressed in human AD brain and AD mouse brain/AD cell culture models.A list of miRNA that are common in all three datasets are listed on the right. C. RNA was isolated from the frontal cortex tissue of two AD patients or age matched healthy controls.miRNA expression was determined using qRT-PCR.Fold change in the expression of miRNA with respect to healthy individual is provided from three technical replicates for each of the two brains (mean ± SEM, n=3 of two biological samples, *p<0.05,**p<0.01,two tailed paired t-test, N=2).

Supp. Figure S2
A. miR-449a target prediction was done using indicated algorithms and databases.Venn diagram showing seven common targets predicted with miRDB, TargetScan and RNA22, which are indicated in the list (Supplementary Dataset S4).
B. Rat cortical neurons were transfected with anti-miR-449a or a control inhibitor.Total RNA was isolated and qRT-PCR was performed to determine the levels of CDC25A or cyclin D1 and fold change expression with respect to control inhibitor transfected neurons was determined (mean ± SEM, two tailed paired t-test, *p<0.05,N=3).
C. Rat cortical neurons were transduced with miR-449a or control lentivirus, treated with Aβ 42 .After 48h, samples were harvested for RNA and protein analysis.Western blotting was performed with indicated antibodies.CDC25A levels were normalized with respect to actin.
Right panel, RNA was isolated and qRT-PCR was performed to determine the levels of CDC25A and fold change expression with respect to control pLKO transduced neurons was determined (mean ± SEM, one-way ANOVA with Sidak's multiple comparison, *p<0.05,N=4).

D.
Rat cortical neurons were transduced with miR-449a or control lentivirus, treated or left untreated with Aβ 42 .After 48h, samples were harvested for RNA and protein analysis.Western blotting was performed with indicated antibodies.Cyclin D1 levels were normalized with respect to actin.
Right panel, RNA was isolated and qRT-PCR was performed to determine the levels of cyclin D1 and fold change expression with respect to control pLKO transduced neurons was determined (mean±SEM, one-way ANOVA with Sidak's multiple comparison, ***p<0.001,N=5).

E.
Cortical neurons cultured from WT or TgAD mice were transduced with miR-449a or control lentivirus for 48h.Subsequently, neurons were also infected with adenovirus expressing CDC25A or GFP (control).Western blotting was performed with indicated antibodies.Right Panel, Densitometry was performed to determine the level of PCNA and cl_caspase 3, which was normalized with respect to actin and fold change in expression with respect to pLKO transduced neurons was determined.Data represent mean±SEM of at least three replicates, *p<0.05,**p<0.01 with two tailed paired t-test, N=3.F. Cortical neurons from WT and TgAD mice were transduced with miR-449a or control lentivirus.The lysate was prepared and Western blotting was performed to determine the phosphorylated Retinoblastoma (pRb) and total Rb proteins.

Supp. Figure S3
A. Rat cortical neurons were transfected with anti-miR-449a or a control inhibitor.RNA was isolated and qRT-PCR was performed for miR-449a.Fold change in expression with respect to control inhibitor transfected neurons was determined (mean ± SEM, * p<0.05 by two tailed paired t-test, N=2).

B.
Average swimming speed (m/s) of mice from each group in Morris water maze probe test (Fig. 5D) is provided.No significant change in the average swimming speed was observed amongst the lentivirus (control or miR-449a) injected groups.Data represented as mean±SEM, N= 5-8 animals from each cohort, ns-not significant by one-way ANOVA with Tukey's multiple comparison.
C. Number of total arm entries in Y maze (Fig. 5E) is shown.No significant change was observed in the total number of arm entries of mice in each group.Data represented as mean ± SEM, N=6-8 animals from each cohort, not significant by one-way ANOVA with Tukey's multiple comparison.

Supp. Figure S4
Immunohistochemistry was performed on the brain sections of lentivirus-injected mice for the expression of GFP.Representative overview images showing GFP expression in the right hemisphere of the brain of TgAD mice injected with pLKO or miR-449a lentivirus in the cortex, image of age matched WT brain without any intervention is also provided (scale bar 100 μm).The images were stitched in the original settings in Zeiss microscope software.

.
Details of PCR primers used for cloning:

Table S3 .
Primers used for real time PCR:

Table S4 .
Details of Human brain tissue from NIMHANS Brain Bank: