Cohesive interpretation of published findings describing SIRT2 distribution and function is complicated by the existence of multiple isoforms of the protein. Three splice variants are predicted for mouse SIRT2 (Fig. A and B); these encode polypeptides of differing lengths with distinct amino-termini, and the two larger species have been documented in previous reports (10
). To identify and characterize the SIRT2 isoforms expressed in the brain, we performed 5′ RACE experiments and cloned three full-length SIRT2 cDNAs from mouse cortex RNA. The sequences of these cloned cDNAs, referred to here as SIRT2.1 (sirtuin 2, isoform 1) (calculated MW 43 kDa), SIRT2.2 (sirtuin 2, isoform 2) (39.5 kDa) and SIRT2.3 (sirtuin 2, isoform 3) (35.6 kDa), are identical to those predicted for the three alternatively spliced isoforms of mouse SIRT2, suggesting that all three proteins are expressed in the brain. Transfection of the cloned cDNAs into mouse neuroblastoma (Neuro-2a—N2a) cells demonstrated co-migration of SIRT2.1 and SIRT2.2 with the two endogenous SIRT2 species present in these cells (Fig. C); no endogenous protein species corresponding to SIRT2.3 was detectable in these studies.
Figure 1. Multiple protein isoforms are produced from the SIRT2 gene. (A) Diagram showing structural organization of the mouse SIRT2 gene (Entrez GeneID 64383). The locations of key features in the predicted coding sequence are indicated; patterned boxes denote (more ...)
We verified activity of all three isoforms cloned from the mouse brain by testing their ability to promote deacetylation of α-tubulin, a known protein substrate for SIRT2 (13
) in transfected cells (Fig. D). Our results show that overexpression of each cDNA isoform results in a decrease in the amount of acetylated α-tubulin present in the NP40-insoluble (i.e. MT-containing) fraction in transfected cells. As shown in Figure D, SIRT2.1-transfected cells also express a significant amount of SIRT2.2, presumably due to initiation of translation at the downstream start codon present in wild-type SIRT2.1 mRNA (Fig. A and B). Thus, it is possible that modulation of acetylated α-tubulin in SIRT2.1-transfected cells could be mediated by both SIRT2.1 and SIRT2.2, or by the SIRT2.2 deacetylase alone. To ensure that the observed decrease in acetylated α-tubulin in these studies does not result from increased HDAC6 expression, we also assessed HDAC6 levels in SIRT2-tranfected cells (Supplementary Material, Fig. S1
). HDAC6 levels were not increased in these studies; on the contrary, HDAC6 levels appear to be reduced in N2a cells in response to ectopic overexpression of SIRT2. Lastly, in our studies, the SIRT2.3 cDNA consistently yields lower expression levels than SIRT2.1 and SIRT2.2 constructs, yet the effects on α-tubulin acetylation are comparable for all three constructs. Although our experiments did not address the relative potencies of the different isoforms as MT deacetylases, these results suggest that even a modest increase in one or more SIRT2 proteins yields a marked reduction of acetylated α-tubulin in N2a cells.
Next, we used RNA interference-validated antibodies that detect endogenous SIRT2 (see Supplementary Material, Fig. S2
, and Materials and Methods) to determine the expression patterns of SIRT2 isoforms across multiple mouse tissues. As shown in Figure A, SIRT2 exhibits strikingly abundant and preferential expression in the CNS but is present at much lower levels in peripheral tissues (Fig. A) and in cultured cell lines (Fig. and data not shown). We further determined that SIRT2.1 (~43 kDa) and SIRT2.2 (~39 kDa) are readily detected in tissue extracts and that these isoforms exhibit distinct tissue- and cell type-specific expression patterns. The shorter SIRT2.2 isoform is expressed at extremely high levels in the CNS, whereas the larger SIRT2.1 species predominates in the skeletal muscle (Fig. A) and in immortalized cells in culture. Given that neurons contain significantly more acetylated α-tubulin than other cell types, we assessed whether preferential expression in the CNS is a general feature of tubulin deacetylases (Fig. A, lower panels). Our results show that the tissue distribution of HDAC6 is distinct from that of SIRT2 and confirm that, as has been reported previously (32
), HDAC6 expression is not specifically enriched in the brain.
Figure 2. Specific SIRT2 isoforms are abundantly and preferentially expressed in the CNS. (A). Upper panels: distribution of SIRT2 expression in tissues from adult (5-month-old) C57BL6/J mice. Samples from two animals are shown. To permit detection of SIRT2 in (more ...)
To verify that the SIRT2.2 species is a brain-enriched protein in mammals, we analyzed SIRT2 expression in human cortical tissue and found it identical to that observed in the mouse (Fig. B). Moreover, although we had not previously appreciated the SIRT2.3 isoform in immunoblots of mouse tissue extracts, the human samples clearly revealed the presence of a third distinct SIRT2 band migrating at the expected size for SIRT2.3. Because of the extremely close migration of SIRT2.2 and SIRT2.3, and the overwhelming abundance of SIRT2.2 in these samples, these species are not easily resolved in mouse extracts; however, the electrophoretic mobilities of human SIRT2.2 and SIRT2.3 isoforms differ slightly from the mouse and thus were more distinctly separated on SDS–PAGE. Lastly, although the SIRT2.3 isoform has not previously been reported for human SIRT2, mining of existing deep-sequencing data sets from human samples (publicly available at http://www.broadinstitute.org/igvdata/BodyMap/IlluminaHiSeq2000_BodySites/
) showed that all three splice variants are produced in the human brain (E.T. Wang and D.E. Housman, personal communication).
Our early studies of SIRT2 expression in different cell and tissue types revealed that endogenous SIRT2 is expressed at extremely low levels in immortalized cultured cells, including mouse N2a (Figs and ) and human SH-SY5Y neuroblastoma and U87-MG glioblastoma lines (not shown). In N2a cells, SIRT2 RNA and protein levels are enhanced upon differentiation to a neuron-like phenotype (Supplementary Material, Fig. S3A and B
). In cultured mouse embryonic cortical neurons, SIRT2 expression is low at plating and increases as the cultures mature (Supplementary Material, Fig. S3C and D
); these findings are in accordance with published studies showing that the gene is normally expressed postnatally (16
) and suggested that SIRT2 expression might be a marker of mature, fully developed neurons. To investigate this possibility, we assessed SIRT2 expression in the mouse cortex in late embryonic and early postnatal development and compared it with the pattern observed in adult animals (Fig. C). Our results confirm that SIRT2 is expressed at very low levels in the developing CNS, as has been reported previously (16
), and that accumulation of SIRT2.2 in the cortex is a relatively late postnatal event. Further, these studies show that acetylated α-tubulin levels increase dramatically in the early postnatal cortex (Fig. C) and then exhibit a modest reduction in samples from late postnatal and adult animals, when SIRT2 is abundant. In contrast, levels of the HDAC6 MT deacetylase remained largely similar throughout the developmental stages examined. These results suggest that high-level SIRT2 expression and preferential accumulation of SIRT2.2 are markers of the mature CNS.
In previous reports, SIRT2 has been alternately described as both an oligodendroglial (16
) and a neuronal (12
) protein; therefore, we next wished to assess the distribution of SIRT2 immunoreactivity in specific CNS cell types. In late-stage primary cultures of embryonic cortical neurons, SIRT2-specific antibodies label both neuronal perikarya and processes (Fig. D), and SIRT2 staining in neuronal processes is generally associated with MTs. In accordance with previous reports (15
), we detect robust SIRT2 labeling of CNPase-positive oligodendrocytes in primary mixed cortical cultures (Supplementary Material, Fig. S4A
), whereas GFAP-stained astrocytes in these cultures were at best weakly positive for SIRT2 (Supplementary Material, Fig. S4B
Embryonic cortical cultures cannot accurately represent mature CNS cells; we therefore extended our in vitro
findings to confirm neuronal expression of SIRT2 in adult mouse brain sections (Fig. ). Our results show that SIRT2 antibodies brightly label both fiber tracts and neuronal (i.e. NeuN-positive) cell bodies throughout the cortex (Fig. A) and in the cerebellum stain Purkinje cells (Fig. B), molecular layer neurons and fiber tracts (Fig. B–D). As expected, SIRT2 labeling overlapped with that of the oligodendroglial marker CNPase in fiber tracts (shown in the granule cell layer of the cerebellum, Fig. C); however, we did not detect robust SIRT2 labeling of oligodendroglial cell bodies (Fig. C, merged inset and Supplementary Material, Fig. S5
). Further, in accordance with previous reports (15
), in our studies SIRT2 staining did not coincide with that of the astrocyte marker GFAP (Fig. D and Supplementary Material, Fig. S5
). Overall, these results suggest that the SIRT2 species that is most abundant in CNS tissues is predominantly a neuronal protein.
Figure 3. SIRT2 in the adult brain is predominantly a neuronal protein. (A–D). Immunofluorescence microscopy of midsagittal sections from the adult (5-month-old) mouse brain, showing SIRT2 immunoreactivity (red in all panels) in perikarya of NeuN-positive (more ...)
Our previous studies demonstrated that pharmacologic inhibition of SIRT2 exerts neuroprotective effects in invertebrate and cell-based models of PD and HD (11
). Given that advent and progression of symptoms in these neurodegenerative diseases are age-dependent and that the sirtuin family of deacetylases is implicated in the regulation of longevity, we wished to determine whether SIRT2 expression levels vary with age. We therefore analyzed spinal cord extracts from young adult (4–5 month, n
= 9) and aged (19–22 month, n
= 11) C57BL6 mice, and these studies yielded two unexpected findings (Fig. A). First, although we had not previously detected appreciable amounts of the SIRT2.3 isoform in extracts from cultured cells or in tissues from young mice, a third protein species that likely represents SIRT2.3 is clearly present in the CNS of aged animals. Further, we detect significant accumulation of this faster migrating SIRT2 species in both the spinal cords (Fig. A and C) and cortices (not shown) of 19-month-old mice (Fig. C, P
= 0.001 for spinal cord SIRT2.3). We did not detect statistically significant changes in the levels of either SIRT2.1 or SIRT2.2 in these studies; however, age-dependent accumulation of the SIRT2.3 isoform results in a modest but significant increase in overall SIRT2 levels in the CNS of aged mice (Fig. C, P
= 0.014 for total spinal cord SIRT2). Because SIRT2 has been shown to function as an MT deacetylase, we assessed overall levels of acetylated α-tubulin in a subset of these animals (Fig. B). We detected a trend suggesting decreased α-tubulin acetylation in aged animals (from 0.94 ± 0.2 to 0.84 ± 0.06, Fig. C, right); however, this modest decrease was not statistically significant.
Figure 4. SIRT2 accumulates in the CNS of aged mice. (A–C). SIRT2 expression (A and C) and acetylated α-tubulin levels (B and C) in spinal cords from 4-month and 19-month C57BL6/J mice. (D and E) SIRT2 and HDAC6 expression (more ...)
To ensure that the observed age-dependent differences in SIRT2 levels were not limited to a particular group of animals, we next analyzed cortices from young (3 month, n
= 10) and aged (18 month, n
= 10) animals of a hybrid strain background (B6CBA) from an unrelated mouse colony. We again observed significant accumulation of SIRT2.3 in cortices from aged animals (Fig. D and E, P
= 0.001 for cortex SIRT2.3; P
= 0.003 for total cortex SIRT2). In contrast, we did not detect any significant age-dependent differences in levels of the MT deacetylase HDAC6 (Fig. D). Analysis of overall levels of acetylated α-tubulin in cortical samples from these animals again showed a modest decrease in aged animals that was not statistically significant (Supplementary Material, Fig. S6
); nonetheless, this small decrease was consistent with that observed in spinal cord samples. Finally, retrospective analysis (not shown) of immunoblot data for a different group of mice [generated in a separate study (34
)] revealed that this third SIRT2 isoform was readily detectable in all cortical samples from older animals (15–24 months, n
= 20) but not in those from young animals (3–5 months, n
= 8). Taken together, our results strongly suggest that accumulation of SIRT2.3 is an age-dependent marker in the CNS.
Although HDAC6 is generally considered to be the principal tubulin deacetylase in most cells, as noted above HDAC6 levels are low in the CNS compared with SIRT2 (30
) and are higher in some peripheral tissues [Fig. A and (32
)]. Further, HDAC6 knockout mice show increased α-tubulin acetylation in all tissues examined except the brain (32
). Intriguingly, although SIRT2 is expressed abundantly and preferentially in the CNS, it is not known whether endogenous SIRT2 modulates MT acetylation patterns in neurons. To investigate this possibility, we assessed SIRT2 and total and acetylated α-tubulin staining patterns in late-stage cultured primary neurons (Fig. A–C). Although SIRT2 generally co-localizes with α-tubulin staining in neurons (Fig. A), our results show non-uniform distribution of endogenous SIRT2 in neuronal perikarya and processes (Fig. A–C). Further, focal accumulation of this protein is associated with areas of reduced α-tubulin acetylation in both cell bodies (Fig. B) and neurites (Fig. C).
Figure 5. Focal accumulation of SIRT2 coincides with reduced MT acetylation in neurons. (A). SIRT2 antibodies label MTs in processes of cultured primary cortical neurons (DIV 10). Panels show SIRT2 (left), total α-tubulin (middle) and merged image (right) (more ...)
To extend these observations, we investigated whether the brain-enriched isoforms of SIRT2 are stably associated with neuronal MTs. Crude biochemical fractionation of endogenous SIRT2 proteins from cultured primary cortical neurons reveals that the SIRT2.2 isoform is preferentially associated with the NP40-insoluble (i.e. MT-containing) fraction, whereas SIRT2.1 is found almost exclusively in the soluble fraction (Fig. D). Moreover, on blots such as that shown in Figure D, we noted the presence of a faint third band migrating similarly to SIRT2.3 in the pellet fractions only. Because SIRT2.3 levels are low-to-undetectable in primary neurons, however, we were unable to confirm the fractionation profile of endogenous SIRT2.3 in these cells. As an alternative approach, we compared fractionation profiles of SIRT2.2 and SIRT2.3 in transfected N2a cells and found significant association of both isoforms with the insoluble fraction (Fig. E); again, endogenous SIRT2.1 in these samples was detected only in the soluble fraction, as has been previously reported (10
). Taken together, these results suggest that the brain-enriched SIRT2 isoforms—SIRT2.2 and SIRT2.3—are normally associated with MTs in neurons.