Synaptosome microRNAs regulate synapse functions in Alzheimer’s disease

Synaptosomes preparations from postmortem brains

Increased levels of APP (c-terminal fragment) and p-tau proteins were detected in the AD cases compared to UC samples, especially in the cytosolic fraction (Supplementary Fig. 1). Next, these samples were processed for synaptosome preparation and downstream applications (Fig. 1a). Figure 1b showed a representative immunoblot for SNAP25, synaptophysin, and PSD95 and cytosolic/nuclear proteins elF1a and PCNA. Densitometry analysis showed significantly increased levels of SNAP25, synaptophysin, and PSD95 in the synaptosome fraction and reduced levels in the cytosolic fraction (Fig. 1c). SNAP25 and PSD95 were completely absent from the cytosolic fraction, however, synaptophysin was detected in the cytosolic fraction, which was also as reported by other researchers50. On the other hand, elF1a and PCNA protein levels were higher in cytosol. qRT-PCR analysis also showed increased expression of SNAP25, synaptophysin, and PSD95 genes in the synaptosomes relative to the cytosol and reduced expressions of elF1a and PCNA in the synaptosomes fraction relative to the cytosol (Fig. 1d). These results confirm a precise separation of cytosolic and synaptosomes fractions.

Fig. 1: Extraction and characterization of synaptosomes.figure 1

a Brief workflow of the current study. b Immunoblotting analysis of synaptic (SNAP25, synaptophysin and PSD95) and cytosolic (elF1a and PCNA) proteins in cytosolic fraction, synaptosomal fraction and leftover tissue debris of unaffected control postmortem brain tissues. c Densitometry analysis of synaptic and cytosolic proteins. Synaptic proteins levels (PSD95; P = 0.003), (SNAP25; P = 0.0061), (Synaptophysin; P = 0.026) were significantly higher in synaptosomes and cytosolic proteins (elF1a; P = 0.012) and (PCNA; P = 0.018) levels were significantly lower in synaptosomes relative to cytosol. d qRT-PCR analysis for mRNA fold change analysis of synaptic and cytosolic genes in cytosolic and synaptosomal fractions (n = 5). e TEM analysis of synapse assembly in synaptosomal fraction from unaffected control and AD patients’ postmortem brains (scale bar 500 nm magnification). Electron micrograph shows synapse components: Mt mitochondria, SV synaptic vesicles, PSD postsynaptic density, SC synaptic cleft. f Immunoblotting analysis of brain cells markers (Neuron-NeuN; Microglia-Iba1), excitatory synapse marker (VGLUT1) and inhibitory synapse marker (GABARA1) proteins in unaffected controls (n = 4) and AD (n = 4) synaptosomes. g Densitometry analysis of NeuN, Iba1, VGLUT1, and GABARA1 proteins in unaffected controls and AD synaptosomes. All blots are driven from the same experiment and were proceed parallelly (b, f). Values in the bar diagrams are mean ± SEM and error bars are equivalent throughout the figure (c, d, g).

Next, we processed the synaptosomes fraction from AD patients and UC for TEM analysis (Fig. 1e). The electron micrograph revealed the distinct synapse assembly and intact synaptosomes with all the components- mitochondria, synaptic vesicles, endosomes, postsynaptic density protein, and synaptic cleft. The mitochondrial structure and synaptic clefts were found to be distorted in AD postmortem brains and UC postmortem brains; however, mitochondrial distortion was more in AD cases. These results confirmed the purity and integrity of synapse and synaptosomes fraction.

Further, to confirm the brain cells specificity of synaptosomes, we checked the levels of cell type markers (NeuN-Neuron, Iba1-Microglia, GFAP-Astrocytes). We found significantly detectable levels of NeuN and Iba1 proteins (but not GFAP) in both UC and AD synaptosomes (Fig. 1f). NeuN level was found to be significantly reduced (P = 0.035) in AD synaptosomes relative to UC synaptosomes (Fig. 1g). We did not see any significant difference in Iba1 levels in AD vs UC synaptosome. These observations confirm the neuron specificity of synaptosomes.

We also characterized the synaptosomes as excitatory or inhibitory based on the levels of excitatory synapse marker Vesicular glutamate transporter 1 (VGLUT1) and inhibitory synapse markers Gamma-Aminobutyric Acid Type A Receptor Subunit Alpha1 (GABRA1). Immunoblots in Fig. 1f showed the levels of both markers in UC and AD synaptosomes. The levels of VGLUT1 (P = 0.004) and GABRA1 (P = 0.004) proteins were significantly reduced in AD synaptosomes relative to UC synaptosomes (Fig. 1g). These observations confirmed the presence of both types of synapses in synaptosomes fraction with their reduction in AD brains.

MicroRNAs expression in UC synaptosomes vs UC cytosol

The miRNA microarray data of synaptosomal and cytosolic fractions were analyzed by Transcription analysis console v.4. A total of 43 mature miRNAs were found to be differentially distributed in UC synaptosomal fraction and UC cytosolic fraction (Supplementary Table 4). As shown in Supplementary Table 4, the 20 Homosapiens (hsa) miRNAs were highly expressed in the synaptosomes and low in the cytosol. These observations indicate that highly expressed miRNAs in synaptosomes have functional importance in synapse function. The 23 hsa-miRNAs (Supplementary Table 4) were highly expressed in the cytosol and showed reduced expression in the synaptosomes, strongly suggesting that these miRNAs have cytosolic relevance in the healthy state.

MiRNAs were characterized on several selection criteria—fold change, standard deviation, P values, expression priority, transcript ID, chromosome location, strand specificity, start and stop codon, targeted, and validated gene symbols (Supplementary Table 4). Figure 2a shows the hierarchical clustering and heatmap of significantly distributed miRNAs with their ID numbers. As a result, 25 miRNAs were upregulated, and 23 miRNAs were downregulated significantly (Fig. 2b). Gene-filter analysis of the total miRNAs pool shows that 99.28% of miRNA population did not show a significant difference in the cytosol vs synaptosome compartments. Only 0.38% population of miRNAs is upregulated, and 0.35% miRNA population is downregulated (Fig. 2c). The scattered plot shows the average log2 fold changes values of miRNAs with different distributions in cytosol vs synaptosomes (Supplementary Fig. 2a) and the volcano plot shows the P values (−log10) of significantly deregulated miRNAs (Supplementary Fig. 2b). The top candidate miRNAs were selected for validation analysis.

Fig. 2: MiRNAs expression in synaptosome and cytosol in a healthy state.figure 2

a Hierarchical clustering and heatmap of significantly deregulated miRNAs in the synaptosome and cytosol of unaffected controls. (red color intensity showed the miRNAs upregulation and blue color intensity showed the miRNAs downregulation). b Total number of miRNAs deregulated in cytosol vs synaptosome in unaffected controls. (grayscale bar—total number of miRNAs; red scale bar—upregulated miRNAs; green scale bar—downregulated miRNAs). c Pi diagram showed the total miRNAs pool distribution and percentage of miRNAs population changed in cytosol and synaptosome in unaffected controls. d qRT-PCR-based validation analysis of significantly deregulated miRNAs in unaffected controls (n = 15). MiRNAs expression was quantified in terms of fold changes in unaffected controls synaptosomes compared to the cytosol. Each circle dot represents one sample. e Validation analysis of significantly deregulated mmu-miRNAs in WT mice (n = 7). MiRNAs expression was quantified in synaptosome relative to the cytosol. Each circle dot represents one animal. Values in the bar diagrams are mean ± SEM and error bars are equivalent throughout the figure (e, d).

Validation analysis of synaptosomal and cytosolic miRNAs in a healthy state

(i) UC postmortem brains: Validation analysis was performed on UC (n = 15) postmortem brains to distinguish synaptosomal and cytosolic miRNAs in the normal state. Out of the 43 deregulated miRNAs, only 33 miRNAs were successfully amplified by qRT-PCR using specific primers. The 18 miRNAs showed similar expression trends as obtained by Affymetrix data analysis. The remaining miRNAs did not concur with Affymetrix data. Overall, 24 miRNAs were significantly upregulated in the synaptosomes relative to the cytosol, and two miRNAs (miR-638 and miR-3656) were significantly downregulated in the synaptosomal fractions relative to the cytosolic fractions. Seven miRNAs did not show any significant changes (Fig. 2d).

(ii) WT mice brains: Further, we performed expression analysis of the above classified synaptosomal and cytosolic miRNAs in WT mice (n = 7). A total of 11 Mus musculus (mmu)-miRNAs were amplified, and out of them, nine were significantly upregulated and two were downregulated in WT mice synaptosome relative to the cytosol (Fig. 2e). The 11 miRNAs showed similar expression pattern as observed by primary screening and UC postmortem brain validation. Based on these observations, nine miRNAs were classified as synaptosomal miRNAs and two miRNAs as cytosolic miRNAs in the healthy state.

MicroRNAs expression in AD synaptosomes vs AD cytosol

Next, we compared the microarray data for miRNAs expression changes in AD synaptosomal fractions vs AD cytosolic fractions. A total of 39 mature miRNAs were found to be differentially distributed in AD synaptosome vs AD cytosol comparison as shown in Supplementary Table 5, and 28 hsa-miRNAs were highly expressed in the synaptosomes and low in the cytosol. The 11 out 39 miRNAs were highly expressed in the cytosol and showed reduced expression in the synaptosomes. The differential distribution of these miRNAs in the AD synaptosomes and AD cytosol suggests their functional relevance in the diseased state.

Figure 3a shows hierarchical clustering and a heatmap of significantly distributed miRNAs with their ID numbers. The 11 miRNAs were upregulated in the cytosol and 28 miRNAs were downregulated in the cytosol significantly (Fig. 3b). Gene-filter analysis of the total miRNAs pool shows that 99.41% of miRNA population did not show a significant difference in the cytosol vs synaptosome compartment, only, 0.59% of populations showed variable expression levels. The 0.17% of miRNAs are upregulated, and 0.42% of miRNAs population is downregulated (Fig. 3c). The scattered plot shows the average log2 fold changes values of significantly deregulated miRNAs (Supplementary Fig. 2c) and the volcano plot shows the P values (−log10) of significantly deregulated miRNAs in AD synaptosome vs AD cytosol (Supplementary Fig. 2d). Based on the miRNA(s) expression pattern in unaffected controls and AD samples, 22 miRNAs (37.3%) were expressed only in UC samples and 21 miRNAs (35.6%) were expressed only in AD samples. However, 16 miRNAs (27.1%) were commonly expressed in both conditions (Supplementary Fig. 3).

Fig. 3: MiRNAs expression in synaptosome and cytosol in AD.figure 3

a Hierarchical clustering and heatmap of significantly deregulated miRNAs in cytosol and synaptosome in AD samples. (red color intensity showed the miRNAs upregulation and blue color intensity showed the miRNAs downregulation) b Total numbers of miRNAs deregulated in cytosol and synaptosome in AD. (grayscale bar—total number of miRNAs; red scale bar—upregulated miRNAs; green scale bar—downregulated miRNAs). c Pi diagram showed the total miRNAs pool distribution and percentage of miRNA populations changed in cytosol and synaptosome. d qRT-PCR-based validation analysis of significantly deregulated miRNAs in AD samples (n = 27). MiRNAs expression was quantified in terms of fold changes in AD synaptosome compared to AD cytosol. Each circle dot represents one sample. e Validation analysis of significantly deregulated mmu-miRNAs in APP-Tg (n = 6) mice. MiRNAs expression was quantified in synaptosome relative to the cytosol. Each circle dot represents one animal. f Validation analysis of significantly deregulated mmu-miRNAs in Tau-Tg (n = 7) mice. MiRNAs expression was quantified in synaptosome relative to the cytosol. Values in the bar diagrams are mean ± SEM and error bars are equivalent throughout the figure (df).

Validation analysis of synaptosomal and cytosolic miRNAs in AD state

(i) AD postmortem brains: The top candidate miRNAs were selected for validation analysis. Validation analyses were performed on 27 AD postmortem brains to distinguish synaptosomal and cytosolic miRNAs in the diseased state. Out of the 39 deregulated miRNAs, 32 miRNAs were amplified by using specific primers. The 22 miRNAs showed a similar expression trend as obtained by Affymetrix data analysis. The remaining miRNAs either showed opposite trend to Affymetrix data or did not change significantly. This could be due to large number of samples that were used for validation of initial Affymetrix analysis and possible pathological (Braak stages) differences of samples, may be likely reasons for inconsistent expression of miRNAs in two systems. Overall, 27 miRNAs were significantly upregulated in the synaptosomes relative to the cytosol and no miRNA showed any significant downregulation. The five miRNAs did not show any significant changes in the synaptosomes relative to the cytosol (Fig. 3d).

(ii) APP-Tg mice: Next, we did synaptosomal and cytosolic miRNAs validation using APP-Tg mice (n = 6). The 13 mmu-miRNAs showed similar expression pattern as observed by primary screening and AD postmortem brain validation. MiR-103-3p, miR-185-5p, miR-24-3p, miR-502-3p, miR-320b, let-7d-5p, miR-124-3p, miR-140-3p, miR-17-5p, and miR-877-5p showed significant upregulation in the synaptosomes, while miR-138-5p, miR-3656, and miR-638 did not show any significantly changes in their expression (Fig. 3e).

(iii) Tau-Tg mice: Further, we did synaptosomes and cytosolic miRNAs validation using Tau-Tg mice (n = 7). The 13 mmu-miRNAs showed similar expression pattern as observed by primary screening and AD postmortem brain validation. MiR-103-3p, miR-185-5p, miR-24-3p, miR-502-3p, miR-320b, let-7d-5p, miR-124-3p, miR-140-3p, miR-17-5p, miR-877-5p, miR-320a, and miR-664a-3p showed significant upregulation in the synaptosomes, while miR-138-5p, miR-3656, and miR-638 did not show any significantly changes in their expression (Fig. 3f).

Based on these observations, 11 miRNAs were classified as synaptosomal miRNAs and two miRNAs as cytosolic miRNAs in the AD state.

MicroRNAs expression in AD cytosol vs UC cytosol

Next, we compared AD cytosolic vs UC cytosolic miRNAs. A total of 13 hsa-miRNAs were found to be significantly deregulated in the AD cytosol vs UC cytosol comparison Supplementary Table 6. Interestingly, expression levels of all miRNAs were reduced in AD cytosol as mentioned in Supplementary Table 6. Supplementary Fig. 4a shows the hierarchical clustering and heatmap of significantly deregulated miRNAs with their ID numbers. The 13 miRNAs were found to be downregulated significantly (Supplementary Fig. 4b). Gene-filter analysis of total miRNAs pool showed that 99.76% of miRNA population did not show a significant difference in the cytosol vs synaptosome compartment. Only, 0.24% of miRNA populations showed variable expression levels. All 0.24% miRNA population is downregulated (Supplementary Fig. 4c). The scattered plot showed the average log2 fold changes values of significantly deregulated miRNAs (Supplementary Fig. 5a) and volcano plot showed the p values (−log10) of significantly deregulated miRNAs in AD cytosol vs AD cytosol (Supplementary Fig. 5b). The top candidate miRNAs were selected for validation analysis.

Validation analysis of cytosolic miRNAs in AD and unaffected control

(i) AD and UC postmortem brains: Validation analysis of cytosolic miRNAs were performed on 15 UC and 27 AD postmortem brain samples. The 13 miRNAs candidates were selected for validation analysis. Opposed to the Affymetrix data, nine miRNAs were significantly upregulated in AD cytosol relative to UC cytosol and three miRNAs did not show significant changes (Supplementary Fig. 4d). Again, the differences in the validation data could be due to sample-to-sample pathological and genetic variations.

(ii) WT, APP-Tg, and Tau-Tg mice: We also performed the validation of cytosolic miRNAs in APP-Tg and Tau-Tg mice relative to WT mice. Other than the 13 cytosolic mmu-miRNAs, we also checked the expression of other potential mmu-miRNAs: miR-17-5p, let-7d-5p, miR-185-5p, miR-103-3p, miR-138-5p, miR-877-5p, miR-24-3p, miR-502-3p, miR-140-3p, miR-124-3p, and miR-3656. Most of the miRNAs were upregulated in the APP-Tg and Tau-Tg cytosol relative to WT cytosol. Only, miR-638 and miR-3656 were significantly downregulated in APP-Tg cytosol relative to WT (Supplementary Fig. 6).

MicroRNAs expression in AD synaptosomes vs UC synaptosomes

Lastly, we compared the microarray data for miRNAs expression changes in AD synaptosomes vs UC synaptosomes. A total of 11 miRNAs were found to be deregulated significantly in AD synaptosomes vs UC synaptosomes comparison as shown in (Supplementary Table 7). Four hsa-miRNAs- miR-502-3p, miR-500a-3p, miR-877-5p, and miR-664b-3p were highly expressed in AD synaptosomes relative to UC synaptosomes. The remaining seven hsa-miRNAs—miR-3196, miR-6511b-5p, miR-4508, miR-1237-5p, miR-5001-5p, miR-4492, and miR-4497 showed reduced expression in AD synaptosomes and were highly expressed in UC synaptosomes. The differential expression of these miRNAs in AD and UC synaptosomes suggests their importance in synapse function.

Figure 4a showed the hierarchical clustering and heatmap of significantly deregulated miRNAs with their ID numbers. The four miRNAs were upregulated, and seven miRNAs were downregulated significantly (Fig. 4b). Gene-filter analysis of total miRNAs pool showed that 99.83% of the miRNA population did not show a significant difference in the synaptosome compartments in AD vs UC. Only 0.17% miRNAs populations showed variable expression patterns. The 0.06% of miRNAs is upregulated and 0.11% of the miRNA population is downregulated (Fig. 4c). The scattered plot showed the average log2 fold changes values of significantly deregulated miRNAs (Supplementary Fig. 5c) and the volcano plot showed the P values (−log10) of significantly deregulated miRNAs in AD synaptosomes vs UC synaptosomes (Supplementary Fig. 5d). The top candidate miRNAs were selected for validation analysis.

Fig. 4: MiRNAs expression in synaptosome in AD and healthy state.figure 4

a Hierarchical clustering and heatmap of significantly deregulated miRNAs in synaptosome in AD and unaffected controls. (red color intensity showed the miRNAs upregulation and blue color intensity showed the miRNAs downregulation) b Total numbers of miRNAs deregulated in AD synaptosome vs UC synaptosome. (grayscale bar—total number of miRNAs; red scale bar—upregulated miRNAs; green scale bar—downregulated miRNAs). c Pi diagram showed the total miRNAs pool distribution and percentage of miRNAs population changed in AD synaptosome vs UC synaptosome. d qRT-PCR-based validation analysis of significantly deregulated miRNAs in AD (n = 27) and UC (n = 15) synaptosome. MiRNAs expression was quantified in terms of fold changes in AD synaptosome relative to UC synaptosome. Each circle dot represents one sample. e Multiple comparison analysis of synaptosomal miRNAs fold changes with Braak stages 2/3, Braak stages 4/5 and Braak stages 6 of AD samples. (**P < 0.01, ***P < 0.001, ****P < 0.0001). f Immunoblotting analysis of miRNAs biogenesis proteins (Ago2, Drosha, and Dicer) in the cytosol and synaptosomal of UC samples (n = 4). g Densitometry analysis of Ago2, Drosha and Dicer in cytosol relative to synaptosomes of UC samples. All blots are driven from the same experiment and were proceeded parallelly (f). Values in the bar diagrams are mean ± SEM and error bars are equivalent throughout the figure (d, g).

Based on the miRNAs’ expression pattern in cytosol and synaptosomes in AD vs UC samples, 15 miRNAs (68.2%) were expressed only in the cytosol, and seven miRNAs (31.8%) were expressed only in the synaptosomes. We did not see any miRNA that were commonly expressed in both conditions.

Validation analysis of synaptosomal miRNAs

(i) AD and UC postmortem brains: Validation analysis were performed on 15 UC and 27 AD postmortem brains. We checked synaptosomal expression of deregulated 16 miRNAs. However, only 14 hsa-miRNAs were amplified, the 12 hsa-miRNAs (miR-502-3p, miR-500a-3p, miR-877-5p, miR-664b-3p, miR-4508, miR-1237-5p, miR-5001-5p, miR-4497, miR-103a-3p, miR-124-3p, miR-24-3p, and let-7a-5p were significantly upregulated in the AD synaptosomes relative to UC synaptosomes, while two hsa-miRNAs (miR-3196 and miR-151-5p) did not show any significant changes (Fig. 4d).

(ii) WT, APP-Tg, and Tau-Tg mice: We also performed the validation of the above-mentioned miRNAs and other potential synaptosomal miRNAs in APP-Tg and Tau-Tg mice relative to WT mice. The 12 mmu-miRNAs, which were, amplified successfully included- miR-17-5p, let-7d-5p, miR-185-5p, miR-103-3p, miR-138-5p, miR-877-5p, miR-24-3p, miR-502-3p, miR-140-3p, miR-124-3p, miR-638, and miR-3656. In APP-Tg mice synaptosomes, seven miRNAs were significantly upregulated, four were significantly downregulated relative to WT synaptosomes and one miRNA showed no change (Supplementary Fig. 6). In Tau-Tg synaptosomes, nine miRNAs were significantly upregulated, and three miRNAs were significantly downregulated relative to WT synaptosomes (Supplementary Fig. 6).

Summarizing all validation analysis, only 12 miRNAs expression was consistent in different comparisons and sample settings. The ten miRNAs can be classified as synaptosomal miRNAs and two miRNAs as cytosolic miRNAs. The other miRNAs expression patterns were not aligned with Affymetrix data and qRT-PCR validation. This could be due to variation of the Braak stages of postmortem AD brains used for Affymetrix analysis and qRT-PCT validation.

Next, we examined the synaptosomal miRNAs expression patterns with AD samples Braak stages. Multiple comparison analyses showed that the expression of synaptosomal miRNAs were gradually increased with Braak stages. However, significant differences were found in miR-501-3p (P = 0.001), miR-502-3p (P < 0.0001), miR-877-5p (P = 0.010), and miR-103a-3p (P < 0.0001) fold changes at Braak stage 6 relative to Braak stage 2/3 (Fig. 4e). These results unveiled the strong connection of these miRNAs with AD progression.

Further, to determine the synaptosomal miRNAs synthesis at the synapse, we checked the levels of key miRNA biogenesis proteins (Ago2, Drosha, and Dicer) in the cytosol and synaptosome fractions. In Fig. 4f, immunoblots showed the levels of miRNA biogenesis proteins in UC cytosol and synaptosomes. Densitometry analysis showed very high levels of all three proteins in cytosol relative to synaptosomes (Fig. 4g). The presence of miRNA biogenesis proteins in synaptosomes confirmed that miRNAs might be synthesized at the synapse.

In silico ingenuity® pathway analysis of cytosolic and synaptosomal miRNAs in AD and healthy state

The deregulated miRNAs under different conditions were run for IPA analysis. The first comparison was cytosolic vs synaptosomal miRNAs in the healthy state. The top deregulated miRNAs were involved in several diseases, molecular and cellular functions, physiological system development and functions (Supplementary Data 1). However, we focused on the miRNA candidates which are involved in nervous system development and function in neurological diseases. Eleven miRNAs were identified which were significantly (P < 0.05) involved in many neurological diseases and dementia, including AD and MCI (Supplementary Fig. 7a). Next, we analyzed the mRNA target and seed sequences of these miRNAs to understand the molecular mechanism of miRNAs involved in neurological function (Supplementary Fig. 7b). The tumor suppressor gene (TP53) was the central gene that was targeted by many of these miRNAs. Other potential genes were BACE1, Smad2/3, Lypla1, Akt1, and SERBP1 pathway genes.

Similarly, we studied synaptosomal and cytosolic miRNAs function in AD cases. The top miRNA candidates were significantly (P < 0.05) involved in several nervous system development, function, and neurological diseases (Supplementary Data 2). However, our interest was neurological disorders and dementia, where eight miRNAs were detected which were involved in several neurological disorders, including AD (Fig. 5a). Further, miRNAs target predication analysis showed more than 20 genes that are targeted by these miRNAs (Fig. 5b). Next, we studied the biological roles of cytosolic miRNAs which were downregulated in AD compared to UC. The top five miRNAs were significantly involved in several diseases and molecular pathways (Supplementary Data 3). MiRNAs and diseased pathways showed integration with Amyotrophic lateral sclerosis (Supplementary Fig. 8a). Like other miRNAs, several genes were identified as a potential target for these five miRNAs (Supplementary Fig. 8b).

Fig. 5: Ingenuity pathway analysis of cytosolic and synaptosomal miRNAs in AD.figure 5

a In AD state, cytosolic and synaptosomal miRNAs expression network in various human diseases. Red nodes represent increased expression and green nodes represent a decreased expression of miRNAs. b MiRNAs target and seed sequences network of cytosolic and synaptosomal miRNAs in the AD state.

Lastly, we studied the biological functions of synaptosomal miRNAs which were deregulated in AD vs UC. The miR-500 family (miR-501-3p, miR-500a-3p) and miR-877-5p were identified to be significantly (P < 0.05) involved in several biological processes and disorders (Supplementary Data 4). MiRNA and disease interaction analysis showed a significant connection of miR-501-3p in GABAergic synapse function and other brain functions (Fig. 6a). The miRNAs target predication analysis showed more than 20 genes that are targeted by these miRNAs (Fig. 6b). The GABARA1 gene was identified as one of the potential common target of miR-501-3p and miR-502-3p (Supplementary Fig. 9). Further, gene ontology enrichment analysis of miR-502-3p showed that it involved in several biological processes, cellular components, and molecular functions. The most significant involvement was a response to external stimuli (P = 0.009) and nervous system development (P = 0.044). The most significant cellular component was GABAergic synapse (P = 0.028), and the molecular function was calmodulin binding (P = 0.020) (Supplementary Fig. 10).

Fig. 6: Ingenuity pathway analysis of synaptosomal miRNAs in AD.figure 6

a Synaptosomal miRNAs expression network in various human diseases. b MiRNAs target and seed sequences network of synaptosomal miRNAs in the AD and healthy state. Red nodes represent increased expression and green nodes represent a decreased expression of miRNAs. c Possible molecular mechanism of miR-501-3 and miR-502-3p in AD progression via negative modulation of GABAergic synapse. Inhibition of GABARA1 expression by the overexpression of these miRNAs could inhibits the GABAergic synapse function in AD.

Overall, IPA and gene ontology enrichment analyses showed that synaptosomal miRNAs are altered in several neurological disorders and participate in numerous cellular and molecular pathways related to brain function.

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