MicroRNA-195 rescues ApoE4-induced cognitive deficits and lysosomal defects in Alzheimer's disease pathogenesis

doi:10.1038/s41380-020-0824-3

. 2021 Sep;26(9):4687-4701.

doi: 10.1038/s41380-020-0824-3. Epub 2020 Jul 6.

MicroRNA-195 rescues ApoE4-induced cognitive deficits and lysosomal defects in Alzheimer's disease pathogenesis

Jiqing Cao^#^{1

2}, Min Huang^#^{1

2}, Lei Guo^{3

4}, Li Zhu^{1

2}, Jianwei Hou^{1

2}, Larry Zhang^{1

2}, Adriana Pero^{1

2}, Sabrina Ng^{1

2

5}, Farida El Gaamouch², Gregory Elder^{1

2}, Mary Sano^{1

6

7}, Alison Goate^{8

9}, Julia Tcw^{8

9}, Vahram Haroutunian^{6

7

8

10}, Bin Zhang^{3

4}, Dongming Cai^{11

12

13

14

15}

Affiliations

¹ James J Peters VA Medical Center, Research & Development, Bronx, NY, 10468, USA.
² Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
³ Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
⁴ Mount Sinai Center for Transformative Disease Modeling, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
⁵ Cornell University, Ithaca, NY, 14850, USA.
⁶ Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
⁷ Alzheimer Disease Rsearch Center, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
⁸ Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
⁹ Ronald M. Loeb Center for Alzheimer's disease, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
¹⁰ James J Peters VA Medical Center, MIRECC, Bronx, NY, 10468, USA.
¹¹ James J Peters VA Medical Center, Research & Development, Bronx, NY, 10468, USA. [email protected].
¹² Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. [email protected].
¹³ Mount Sinai Center for Transformative Disease Modeling, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. [email protected].
¹⁴ Alzheimer Disease Rsearch Center, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. [email protected].
¹⁵ Ronald M. Loeb Center for Alzheimer's disease, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. [email protected].

^# Contributed equally.

PMID: 32632205
PMCID: PMC7785685
DOI: 10.1038/s41380-020-0824-3

Free PMC article

MicroRNA-195 rescues ApoE4-induced cognitive deficits and lysosomal defects in Alzheimer's disease pathogenesis

Jiqing Cao et al. Mol Psychiatry. 2021 Sep.

Free PMC article

. 2021 Sep;26(9):4687-4701.

doi: 10.1038/s41380-020-0824-3. Epub 2020 Jul 6.

Authors

Affiliations

¹ James J Peters VA Medical Center, Research & Development, Bronx, NY, 10468, USA.
² Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
³ Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
⁴ Mount Sinai Center for Transformative Disease Modeling, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
⁵ Cornell University, Ithaca, NY, 14850, USA.
⁶ Department of Psychiatry, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
⁷ Alzheimer Disease Rsearch Center, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
⁸ Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
⁹ Ronald M. Loeb Center for Alzheimer's disease, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA.
¹⁰ James J Peters VA Medical Center, MIRECC, Bronx, NY, 10468, USA.
¹¹ James J Peters VA Medical Center, Research & Development, Bronx, NY, 10468, USA. [email protected].
¹² Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. [email protected].
¹³ Mount Sinai Center for Transformative Disease Modeling, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. [email protected].
¹⁴ Alzheimer Disease Rsearch Center, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. [email protected].
¹⁵ Ronald M. Loeb Center for Alzheimer's disease, Icahn School of Medicine at Mount Sinai, New York, NY, 10029, USA. [email protected].

^# Contributed equally.

PMID: 32632205
PMCID: PMC7785685
DOI: 10.1038/s41380-020-0824-3

Abstract

Our recent findings link the apolipoprotein E4 (ApoE4)-specific changes in brain phosphoinositol biphosphate (PIP₂) homeostasis to the susceptibility of developing Alzheimer's Disease (AD). In the present study, we have identified miR-195 as a top micro-RNA candidate involved in the ApoE/PIP₂ pathway using miRNA profiles in human ROSMAP datasets and mouse microarray studies. Further validation studies have demonstrated that levels of miR-195 are significantly lower in human brain tissue of ApoE4^+/- patients with clinical diagnosis of mild cognitive impairment (MCI) or early AD when compared to ApoE4^-/- subjects. In addition, brain miR-195 levels are reduced along with disease progression from normal aging to early AD, and cerebrospinal fluid (CSF) miR-195 levels of MCI subjects are positively correlated with cognitive performances as measured by mini-mental status examination (MMSE) and negatively correlated with CSF tau levels, suggesting the involvement of miR-195 in early development of AD with a potential impact on cognition. Similar differences in miR-195 levels are seen in ApoE4^+/+ mouse hippocampal brain tissue and cultured neurons when compared to ApoE3^+/+ counterparts. Over-expressing miR-195 reduces expression levels of its top predicted target synaptojanin 1 (synj1), a brain PIP₂-degrading enzyme. Furthermore, elevating miR-195 ameliorates cognitive deficits, amyloid plaque burden, and tau hyper-phosphorylation in ApoE4^+/+ mice. In addition, elevating miR-195 rescues AD-related lysosomal defects in inducible pluripotent stem cells (iPSCs)-derived brain cells of ApoE4^+/+ AD subjects while inhibiting miR-195 exacerbates these phenotypes. Together, our data uncover a novel regulatory mechanism of miR-195 targeted at ApoE4-associated brain PIP₂ dyshomeostasis, cognitive deficits, and AD pathology.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

**Fig. 1. MiR-195 is identified as a top miRNA candidate involved in *APOE*-regulated synj1 expression.**
a Venn diagram shows miR-195 as the only miRNA in common shared among 4 groups: miRNAs differentially expressed between *ApoE4*⁺ and *ApoE4*⁻ carriers in the human ROSMAP dataset, miRNAs differentially expressed between *ApoE4*⁺ and *ApoE4*⁻ in the mouse miRNA array studies, miRNAs negatively correlated with *synj1* mRNA in ROSMAP, and miRNAs predicted to target at *synj1* mRNA by multiMiR database. Numbers of miRNAs overlapping among subgroups are indicated (red numbers). b Log Fold of changes (LogFC) and p values of differences in miR-195 levels between *ApoE4*⁺ and *ApoE4*⁻ carriers, between female *ApoE4*⁺ and *ApoE4*⁻ carriers in ROSMAP dataset, as well as differences in miR-195 levels between mouse *ApoE4*⁺ and *ApoE4*⁻ treated neurons. c Analysis of correlation between miR-195 and *synj1* mRNA in human subjects of the ROSMAP database.

**Fig. 2. Reduction of brain miR-195 levels in human brain and CSF samples is associated with *ApoE4* genotype, disease progression, and cognitive decline.**
a Amounts of miR-195 (presented as Log₂ fold changes) in human parietal cortex tissue of *ApoE4*^+/− subjects (CDR0.5-1) were lower than those in *ApoE4*^−/− subjects. N = 17–18/group; log₂FC fold of changes: *ApoE4*^−/− 0.054 ± 0.113 versus *ApoE4*^+/− −0.570 ± 0.178, **p < 0.01 with independent-samples t-tests. b Pattern of reduction in miR-195 levels (presented as Log₂ fold changes) along with AD disease progression from normal aging to MCI and early AD. N = 12–19/group; log₂FC: 1.626 ± 0.696 in CDR 0 subjects versus 0.242 ± 0.104 in CDR 0.5 MCI patients; versus −0.663 ± 0.135 in CDR 1 AD subjects; *p < 0.05, ****p < 0.0001 with ANOVA tests. c Positive correlation between CSF miR-195 levels and MMSE scores (r = 0.455, p = 0.029; N = 23). d Negative correlation between CSF miR-195 and pTau levels (r = −0.408, p = 0.04; N = 23).

**Fig. 3. MiR-195 expression is reduced in hippocampal brain tissue and cultured primary neurons of *ApoE4* mice; modulating miR-195 levels regulates synaptojanin 1 expression.**
a Levels of miR-195 were reduced in 12-month old *ApoE4* hippocampal brain tissue (log₂FC: −0.283 ± 0.069) when compared to those in *ApoE3* mice (log₂FC: −0.036 ± 0.034). N = 11–13/group with both males and females; **p = 0.0096 with ANOVA tests. A nominal reduction in miR-195 levels was seen in *ApoE*^−/− brains with no statistical significance (log₂FC: −0.125 ± 0.067, p = 0.48). b Levels of miR-195 in *ApoE*^−/− neurons treated with *ApoE4*-CM were reduced (log₂FC = −0.314 ± 0.073,) when compared to levels of those treated with *ApoE3*-CM (log₂FC: 0.184 ± 0.094). N = 5/group; **p = 0.003 with independent-samples t-tests. c Differences in miR-195 expression levels between *ApoE3*-CM and *ApoE4*-CM treated neurons were abolished in the presence of RAP. The treatment of RAP in the presence of *ApoE3*-CM led to a reduction in miR-195 levels (log₂FC: −1.648 ± 0.125; p < 0.0001), whereas in *ApoE4*-CM treated conditions, miR-195 levels were much lower at baseline with a trend of improvement in the presence of RAP treatment (*ApoE4* CM + BSA log₂FC: −3.193 ± 0.144 versus *ApoE4* CM + RAP log₂FC: −2.678 ± 0.054; p = 0.052). N = 3/group; ****p < 0.0001 by One-Way ANOVA tests. d Synj1 protein levels were reduced with miR-195 over-expression but not miR-374 over-expression in *ApoE*^−/− hippocampal neurons in the presence of *ApoE4*-CM. N = 4/group; synj1 levels with miR-195: 62.87 ± 4.48% of controls, **p = 0.001; with miR-374: 102.4 ± 7.77% of controls, p = 0.93.

**Fig. 4. Over-expression of miR-195 rescues cognitive deficits and ameliorates AD-associated pathologies in *ApoE4* mouse models.**
a Preference index = (time exploring novel object)/(time exploring novel object + time exploring familiar object) and discrimination index = (time exploring novel object- time exploring familiar object)/(time exploring novel object + time exploring familiar object) in 4 groups of mice: *ApoE4*^+/+ scramble injection, *ApoE4*^+/+ miR-195 injection, *ApoE3*^+/+ scramble injection, and *ApoE3*^+/+ miR-195 injection. N = 19–23/group with both males and females; *p < 0.05 with ANOVA tests. b Levels of pTau in KI mouse hippocampus. N = 8/group with both males and females; *p < 0.05; **p < 0.01 with ANOVA tests. c Preference index and discrimination index in 8 groups of mice: *ApoE4*^+/+ FAD male scramble injection, *ApoE4*^+/+ FAD male miR-195 injection, *ApoE4*^+/+ FAD female scramble injection, *ApoE4*^+/+ FAD female miR-195 injection, *ApoE3*^+/+ FAD male scramble injection, *ApoE3*^+/+ FAD male miR-195 injection, *ApoE3*^+/+ FAD female scramble injection, and *ApoE3*^+/+ FAD female miR-195 injection. N = 6–10/group; *p < 0.05 with ANOVA tests. Levels of d pTau and e oligomer Aβ₄₂ in EFAD mouse hippocampus. N = 6/group with both males and females; *p < 0.05; ***p < 0.001; ****p < 0.00001 with ANOVA tests. f Amyloid plaque burden in EFAD mouse hippocampus. A representative image of brain section is shown. Amyloid plaque load is quantified by density measured as area of plaques per mm² of brain region, as well as total numbers of plaques/μm² in 4 groups of mice: *ApoE4*^+/+ FAD scramble injection, *ApoE4*^+/+ FAD miR-195 injection, *ApoE3*^+/+ FAD scramble injection, and *ApoE3*^+/+ FAD miR-195 injection. N = 3/group; *p < 0.05; **p < 0.01 with ANOVA tests.

**Fig. 5. Over-expression of miR-195 rescues lysosomal defects in *ApoE4* iPSC-derived brain cells.**
Representative immunofluorescence co-staining of lysosomes (Lysotracker: red fluorescence), a neuronal marker MAP-2 (green fluorescence) and DAPI (blue fluorescence) of iPSC-derived neuron and astrocyte co-culture of (a) *ApoE3*^+/+ normal aging (NA) and (b) *ApoE4*^+/+ AD subjects with various conditions: scramble control (ctrl), miR-195, and miR-195 inhibitor (miR inh). Alternatively, *ApoE3*^+/+ and *ApoE4*^+/+ N = 3–6/conditions. c Quantification of all lysosomes by size (measured by areas; μm²) of 60–90 neurons (MAP-2⁺) in each experimental condition, the distribution of lysosome sizes/cell (measured by diameters; 0–10 μm, 10–20 μm, 20–30 μm and >30 μm), as well as the number of lysosomes in each cell (grouped by 1–5, 6–10, 11–15, 16–20, and >20 lysosomes/cell). ****p < 0.00001 with ANOVA tests.

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References

1. Mayeux R. Epidemiology of neurodegeneration. Annu Rev Neurosci. 2003;26:81–104. - PubMed
1. Fagan AM, Watson M, Parsadanian M, Bales KR, Paul SM, Holtzman DM. Human and murine ApoE markedly alters A beta metabolism before and after plaque formation in a mouse model of Alzheimer’s disease. Neurobiol Dis. 2002;9:305–18. - PubMed
1. Jiang Y, Sun XC, Gui L, Tang WY, Zhen LP, Gu YJ, et al. Lack of association between apolipoprotein E promoters in epsilon4 carriers and worsening on computed tomography in early stage of traumatic brain injury. Acta Neurochir Suppl. 2008;105:233–6. - PubMed
1. Jordan J, Galindo MF, Miller RJ, Reardon CA, Getz GS, LaDu MJ. Isoform-specific effect of apolipoprotein E on cell survival and beta-amyloid-induced toxicity in rat hippocampal pyramidal neuronal cultures. J Neurosci: Off J Soc Neurosci. 1998;18:195–204. - PMC - PubMed
1. Ma J, Yee A, Brewer HB, Jr, Das S, Potter H. Amyloid-associated proteins alpha 1-antichymotrypsin and apolipoprotein E promote assembly of Alzheimer beta-protein into filaments. Nature. 1994;372:92–4. - PubMed

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[1] Mayeux R. Epidemiology of neurodegeneration. Annu Rev Neurosci. 2003;26:81–104. - PubMed

[2] Mayeux R. Epidemiology of neurodegeneration. Annu Rev Neurosci. 2003;26:81–104. - PubMed

[3] Fagan AM, Watson M, Parsadanian M, Bales KR, Paul SM, Holtzman DM. Human and murine ApoE markedly alters A beta metabolism before and after plaque formation in a mouse model of Alzheimer’s disease. Neurobiol Dis. 2002;9:305–18. - PubMed

[4] Fagan AM, Watson M, Parsadanian M, Bales KR, Paul SM, Holtzman DM. Human and murine ApoE markedly alters A beta metabolism before and after plaque formation in a mouse model of Alzheimer’s disease. Neurobiol Dis. 2002;9:305–18. - PubMed

[5] Jiang Y, Sun XC, Gui L, Tang WY, Zhen LP, Gu YJ, et al. Lack of association between apolipoprotein E promoters in epsilon4 carriers and worsening on computed tomography in early stage of traumatic brain injury. Acta Neurochir Suppl. 2008;105:233–6. - PubMed

[6] Jiang Y, Sun XC, Gui L, Tang WY, Zhen LP, Gu YJ, et al. Lack of association between apolipoprotein E promoters in epsilon4 carriers and worsening on computed tomography in early stage of traumatic brain injury. Acta Neurochir Suppl. 2008;105:233–6. - PubMed

[7] Jordan J, Galindo MF, Miller RJ, Reardon CA, Getz GS, LaDu MJ. Isoform-specific effect of apolipoprotein E on cell survival and beta-amyloid-induced toxicity in rat hippocampal pyramidal neuronal cultures. J Neurosci: Off J Soc Neurosci. 1998;18:195–204. - PMC - PubMed

[8] Jordan J, Galindo MF, Miller RJ, Reardon CA, Getz GS, LaDu MJ. Isoform-specific effect of apolipoprotein E on cell survival and beta-amyloid-induced toxicity in rat hippocampal pyramidal neuronal cultures. J Neurosci: Off J Soc Neurosci. 1998;18:195–204. - PMC - PubMed

[9] Ma J, Yee A, Brewer HB, Jr, Das S, Potter H. Amyloid-associated proteins alpha 1-antichymotrypsin and apolipoprotein E promote assembly of Alzheimer beta-protein into filaments. Nature. 1994;372:92–4. - PubMed

[10] Ma J, Yee A, Brewer HB, Jr, Das S, Potter H. Amyloid-associated proteins alpha 1-antichymotrypsin and apolipoprotein E promote assembly of Alzheimer beta-protein into filaments. Nature. 1994;372:92–4. - PubMed

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