Limits...
Lack of exon 10 in the murine tau gene results in mild sensorimotor defects with aging.

Gumucio A, Lannfelt L, Nilsson LN - BMC Neurosci (2013)

Bottom Line: Cognitive abilities or anxiety-like behaviours did not depend on exon 10 in tau, and neither pathological inclusions nor gene-dependent morphological abnormalities were found.Ablation of exon 10 in the murine tau gene alters alternative splicing and tau protein synthesis which results in mild sensorimotor phenotypes with aging.Presumably related microtubule-stabilizing genes rescue other functions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Public Health and Caring Sciences, Rudbeck Laboratory, Uppsala University, SE-75185 Uppsala, Sweden. Lars.Nilsson@medisin.uio.no.

ABSTRACT

Background: Complex species-specific, developmental- and tissue-dependent mechanisms regulate alternative splicing of tau, thereby diversifying tau protein synthesis. The functional role of alternative splicing of tau e.g. exon 10 has never been examined in vivo, although genetic studies suggest that it is important to neurodegenerative disease.

Results: Gene-targeting was used to delete exon 10 in murine tau on both alleles (E10-/-) to study its functional role. Moreover, mice devoid of exon 10 (E10+/-) on one allele were generated to investigate the effects of 1:1 balanced expression of 4R-/3R-tau protein, since equal amounts of 4R-/3R-tau protein are synthesized in human brain. Middle-aged E10-/- mice displayed sensorimotor disturbances in the rotarod when compared to age-matched E10+/- and wild-type mice, and their muscular grip strength was less than that of E10+/- mice. The performance of E10+/- mice and wild-type mice (E10+/+) was similar in sensorimotor tests. Cognitive abilities or anxiety-like behaviours did not depend on exon 10 in tau, and neither pathological inclusions nor gene-dependent morphological abnormalities were found.

Conclusion: Ablation of exon 10 in the murine tau gene alters alternative splicing and tau protein synthesis which results in mild sensorimotor phenotypes with aging. Presumably related microtubule-stabilizing genes rescue other functions.

Show MeSH

Related in: MedlinePlus

Tau mRNA and protein expression in E10+/− and wild-type mice. (A) Only 3R-tau mRNA (212 bp) was expressed at birth (P1) in wild-type mice (wt). At (P9), 3R-tau and 4R-tau mRNA (305 bp) was expressed, while at (P18) and in 2-month-old wt-mice, exon 10 was included and 4R tau mRNA was expressed. In contrast, 2-month-old E10+/− mice (E10) produced 3R-tau and 4R-tau mRNA in equal proportions. Signals depended on mRNA and reverse transcriptase in the cDNA synthesis reaction (−RT, E10). (B) Left; Western blot with a selective antibody for 3R tau resulted in a 38 kDa band in 2 months-old E10+/− mouse brain (E10). Right; Two additional bands were seen when films were overexposed. These bands likely represented tau-isoforms with N-terminal domains exon 2 and 3. In an age-matched wt-mouse there was only a small amount of 3R-tau protein. (C) Left; An antibody (Tau-1) that recognizes 3R-tau as well as 4R-tau generated two protein bands at 38 and 39 kDa in brains of 2-month-old E10+/− mice (E10). Right; Overexposure results in two additional double bands likely representing isoforms of tau with the N-terminal domains exon 2 and 3 of tau protein. Only a small amount of 3R tau protein was found in age-matched wild-type littermates (wt). Recombinant human tau protein representing all tau isoforms and a newborn wild-type mouse brain (P1) was included as a positive control. (D) Immunostaining with selective antibody 3RD showed that 3R-tau was mainly localized in axons of E10+/− mice, (E) but not present in an age-matched wild-type mice. (F-G) The non-selective antibody Tau-1, which recognizes both 3R-tau and 4R-tau, stained hippocampal layers which are rich in axons in both E10+/− (F) and wild-type mice (G). (H-I) No immunostaining in E10+/− (H) or wild-type mice in the absence of primary antibody (I).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4222861&req=5

Figure 2: Tau mRNA and protein expression in E10+/− and wild-type mice. (A) Only 3R-tau mRNA (212 bp) was expressed at birth (P1) in wild-type mice (wt). At (P9), 3R-tau and 4R-tau mRNA (305 bp) was expressed, while at (P18) and in 2-month-old wt-mice, exon 10 was included and 4R tau mRNA was expressed. In contrast, 2-month-old E10+/− mice (E10) produced 3R-tau and 4R-tau mRNA in equal proportions. Signals depended on mRNA and reverse transcriptase in the cDNA synthesis reaction (−RT, E10). (B) Left; Western blot with a selective antibody for 3R tau resulted in a 38 kDa band in 2 months-old E10+/− mouse brain (E10). Right; Two additional bands were seen when films were overexposed. These bands likely represented tau-isoforms with N-terminal domains exon 2 and 3. In an age-matched wt-mouse there was only a small amount of 3R-tau protein. (C) Left; An antibody (Tau-1) that recognizes 3R-tau as well as 4R-tau generated two protein bands at 38 and 39 kDa in brains of 2-month-old E10+/− mice (E10). Right; Overexposure results in two additional double bands likely representing isoforms of tau with the N-terminal domains exon 2 and 3 of tau protein. Only a small amount of 3R tau protein was found in age-matched wild-type littermates (wt). Recombinant human tau protein representing all tau isoforms and a newborn wild-type mouse brain (P1) was included as a positive control. (D) Immunostaining with selective antibody 3RD showed that 3R-tau was mainly localized in axons of E10+/− mice, (E) but not present in an age-matched wild-type mice. (F-G) The non-selective antibody Tau-1, which recognizes both 3R-tau and 4R-tau, stained hippocampal layers which are rich in axons in both E10+/− (F) and wild-type mice (G). (H-I) No immunostaining in E10+/− (H) or wild-type mice in the absence of primary antibody (I).

Mentions: Next we investigated E10+/− mice for expected deviations in tau mRNA expression and protein synthesis. RT-PCR analyses were performed with primers located within exon 9 and exon 11 to generate PCR products of 305 bp (4R-tau mRNA) and 212 bp (3R-tau mRNA) respectively. As expected, a 1-day-old wild-type mouse (wt; P1) expressed only 3R-tau mRNA, while a 9-days-old wild-type mouse (P9) generated equal amounts of 3R- and 4R-tau mRNA due to alternative splicing (Figure 2A). An 18-days-old wild-type mouse (P18) produced only 4R-tau mRNA. In contrast, a 2-months-old E10+/− mouse (E10) generated 3R- and 4R-tau mRNA in essentially equal proportions, while only 4R-tau mRNA was produced in an age-matched wild-type mouse (Figure 2A). Protein synthesis was examined with RD3 antibody, which is selective for 3R-tau, by western blot and immunohistochemistry. On western blot, all three isoforms of 3R-tau were found in E10+/− (E10) mouse brain, but the isoform devoid of exon 2 and 3 was the predominant (Figure 2B and Additional file 1: Figure S1). Tau-1, an antibody which recognizes both 3R- and 4R-tau, generated extra protein bands in E10+/− (E10) mice as compared to wild-type (wt) mice (Figure 2C). The results were consistent with 3R-tau protein synthesis in E10+/− (E10) mouse brain. Immunohistochemistry with RD3 antibody resulted in strong staining in the stratum radiatum in CA1-CA3 areas of the hippocampus and faint staining in the soma of pyramidal neurons in adult E10+/− mice (Figure 2D), but not in wild-type mice who did not express 3R-tau protein (Figure 2E). In contrast, the non-selective Tau-1 antibody showed a staining pattern consistent with tau being mainly confined to the axonal compartment [13] in both adult E10+/− (Figure 2F) and in wild-type mice (Figure 2G). There was no immunostaining in the absence of a primary antibody (Figure 2H-I).


Lack of exon 10 in the murine tau gene results in mild sensorimotor defects with aging.

Gumucio A, Lannfelt L, Nilsson LN - BMC Neurosci (2013)

Tau mRNA and protein expression in E10+/− and wild-type mice. (A) Only 3R-tau mRNA (212 bp) was expressed at birth (P1) in wild-type mice (wt). At (P9), 3R-tau and 4R-tau mRNA (305 bp) was expressed, while at (P18) and in 2-month-old wt-mice, exon 10 was included and 4R tau mRNA was expressed. In contrast, 2-month-old E10+/− mice (E10) produced 3R-tau and 4R-tau mRNA in equal proportions. Signals depended on mRNA and reverse transcriptase in the cDNA synthesis reaction (−RT, E10). (B) Left; Western blot with a selective antibody for 3R tau resulted in a 38 kDa band in 2 months-old E10+/− mouse brain (E10). Right; Two additional bands were seen when films were overexposed. These bands likely represented tau-isoforms with N-terminal domains exon 2 and 3. In an age-matched wt-mouse there was only a small amount of 3R-tau protein. (C) Left; An antibody (Tau-1) that recognizes 3R-tau as well as 4R-tau generated two protein bands at 38 and 39 kDa in brains of 2-month-old E10+/− mice (E10). Right; Overexposure results in two additional double bands likely representing isoforms of tau with the N-terminal domains exon 2 and 3 of tau protein. Only a small amount of 3R tau protein was found in age-matched wild-type littermates (wt). Recombinant human tau protein representing all tau isoforms and a newborn wild-type mouse brain (P1) was included as a positive control. (D) Immunostaining with selective antibody 3RD showed that 3R-tau was mainly localized in axons of E10+/− mice, (E) but not present in an age-matched wild-type mice. (F-G) The non-selective antibody Tau-1, which recognizes both 3R-tau and 4R-tau, stained hippocampal layers which are rich in axons in both E10+/− (F) and wild-type mice (G). (H-I) No immunostaining in E10+/− (H) or wild-type mice in the absence of primary antibody (I).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222861&req=5

Figure 2: Tau mRNA and protein expression in E10+/− and wild-type mice. (A) Only 3R-tau mRNA (212 bp) was expressed at birth (P1) in wild-type mice (wt). At (P9), 3R-tau and 4R-tau mRNA (305 bp) was expressed, while at (P18) and in 2-month-old wt-mice, exon 10 was included and 4R tau mRNA was expressed. In contrast, 2-month-old E10+/− mice (E10) produced 3R-tau and 4R-tau mRNA in equal proportions. Signals depended on mRNA and reverse transcriptase in the cDNA synthesis reaction (−RT, E10). (B) Left; Western blot with a selective antibody for 3R tau resulted in a 38 kDa band in 2 months-old E10+/− mouse brain (E10). Right; Two additional bands were seen when films were overexposed. These bands likely represented tau-isoforms with N-terminal domains exon 2 and 3. In an age-matched wt-mouse there was only a small amount of 3R-tau protein. (C) Left; An antibody (Tau-1) that recognizes 3R-tau as well as 4R-tau generated two protein bands at 38 and 39 kDa in brains of 2-month-old E10+/− mice (E10). Right; Overexposure results in two additional double bands likely representing isoforms of tau with the N-terminal domains exon 2 and 3 of tau protein. Only a small amount of 3R tau protein was found in age-matched wild-type littermates (wt). Recombinant human tau protein representing all tau isoforms and a newborn wild-type mouse brain (P1) was included as a positive control. (D) Immunostaining with selective antibody 3RD showed that 3R-tau was mainly localized in axons of E10+/− mice, (E) but not present in an age-matched wild-type mice. (F-G) The non-selective antibody Tau-1, which recognizes both 3R-tau and 4R-tau, stained hippocampal layers which are rich in axons in both E10+/− (F) and wild-type mice (G). (H-I) No immunostaining in E10+/− (H) or wild-type mice in the absence of primary antibody (I).
Mentions: Next we investigated E10+/− mice for expected deviations in tau mRNA expression and protein synthesis. RT-PCR analyses were performed with primers located within exon 9 and exon 11 to generate PCR products of 305 bp (4R-tau mRNA) and 212 bp (3R-tau mRNA) respectively. As expected, a 1-day-old wild-type mouse (wt; P1) expressed only 3R-tau mRNA, while a 9-days-old wild-type mouse (P9) generated equal amounts of 3R- and 4R-tau mRNA due to alternative splicing (Figure 2A). An 18-days-old wild-type mouse (P18) produced only 4R-tau mRNA. In contrast, a 2-months-old E10+/− mouse (E10) generated 3R- and 4R-tau mRNA in essentially equal proportions, while only 4R-tau mRNA was produced in an age-matched wild-type mouse (Figure 2A). Protein synthesis was examined with RD3 antibody, which is selective for 3R-tau, by western blot and immunohistochemistry. On western blot, all three isoforms of 3R-tau were found in E10+/− (E10) mouse brain, but the isoform devoid of exon 2 and 3 was the predominant (Figure 2B and Additional file 1: Figure S1). Tau-1, an antibody which recognizes both 3R- and 4R-tau, generated extra protein bands in E10+/− (E10) mice as compared to wild-type (wt) mice (Figure 2C). The results were consistent with 3R-tau protein synthesis in E10+/− (E10) mouse brain. Immunohistochemistry with RD3 antibody resulted in strong staining in the stratum radiatum in CA1-CA3 areas of the hippocampus and faint staining in the soma of pyramidal neurons in adult E10+/− mice (Figure 2D), but not in wild-type mice who did not express 3R-tau protein (Figure 2E). In contrast, the non-selective Tau-1 antibody showed a staining pattern consistent with tau being mainly confined to the axonal compartment [13] in both adult E10+/− (Figure 2F) and in wild-type mice (Figure 2G). There was no immunostaining in the absence of a primary antibody (Figure 2H-I).

Bottom Line: Cognitive abilities or anxiety-like behaviours did not depend on exon 10 in tau, and neither pathological inclusions nor gene-dependent morphological abnormalities were found.Ablation of exon 10 in the murine tau gene alters alternative splicing and tau protein synthesis which results in mild sensorimotor phenotypes with aging.Presumably related microtubule-stabilizing genes rescue other functions.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Public Health and Caring Sciences, Rudbeck Laboratory, Uppsala University, SE-75185 Uppsala, Sweden. Lars.Nilsson@medisin.uio.no.

ABSTRACT

Background: Complex species-specific, developmental- and tissue-dependent mechanisms regulate alternative splicing of tau, thereby diversifying tau protein synthesis. The functional role of alternative splicing of tau e.g. exon 10 has never been examined in vivo, although genetic studies suggest that it is important to neurodegenerative disease.

Results: Gene-targeting was used to delete exon 10 in murine tau on both alleles (E10-/-) to study its functional role. Moreover, mice devoid of exon 10 (E10+/-) on one allele were generated to investigate the effects of 1:1 balanced expression of 4R-/3R-tau protein, since equal amounts of 4R-/3R-tau protein are synthesized in human brain. Middle-aged E10-/- mice displayed sensorimotor disturbances in the rotarod when compared to age-matched E10+/- and wild-type mice, and their muscular grip strength was less than that of E10+/- mice. The performance of E10+/- mice and wild-type mice (E10+/+) was similar in sensorimotor tests. Cognitive abilities or anxiety-like behaviours did not depend on exon 10 in tau, and neither pathological inclusions nor gene-dependent morphological abnormalities were found.

Conclusion: Ablation of exon 10 in the murine tau gene alters alternative splicing and tau protein synthesis which results in mild sensorimotor phenotypes with aging. Presumably related microtubule-stabilizing genes rescue other functions.

Show MeSH
Related in: MedlinePlus