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Mechanistic Studies of Antibody-Mediated Clearance of Tau Aggregates Using an ex vivo Brain Slice Model.

Krishnamurthy PK, Deng Y, Sigurdsson EM - Front Psychiatry (2011)

Bottom Line: Thus, clearance of neurofibrillary tangles and/or their precursors may reduce synaptic and neuronal loss associated with AD and other tauopathies.Additionally, tau and FITC-IgG were found together in an enriched lysosome fraction.In summary, antibody-mediated clearance of intracellular tau aggregates appears to occur via the lysosomal pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Neuroscience, New York University School of Medicine New York, NY, USA.

ABSTRACT
Recent studies have shown that immunotherapy clears amyloid beta (Aβ) plaques and reduces Aβ levels in mouse models of Alzheimer's disease (AD), as well as in AD patients. Tangle pathology is also relevant for the neurodegeneration in AD, and our studies have shown that active immunization with an AD related phospho-tau peptide reduces aggregated tau within the brain and slows the progression of tauopathy-induced behavioral impairments. Thus, clearance of neurofibrillary tangles and/or their precursors may reduce synaptic and neuronal loss associated with AD and other tauopathies. So far the mechanisms involved in antibody-mediated clearance of tau pathology are yet to be elucidated. In this study we have used a mouse brain slice model to examine the uptake and localization of FITC labeled anti-tau antibodies. Confocal microscopy analysis showed that the FITC labeled anti-tau antibody co-stained with phosphorylated tau, had a perinuclear appearance and co-localized with markers of the endosomal/lysosomal pathway. Additionally, tau and FITC-IgG were found together in an enriched lysosome fraction. In summary, antibody-mediated clearance of intracellular tau aggregates appears to occur via the lysosomal pathway.

No MeSH data available.


Related in: MedlinePlus

FITC labeled IgG from a high titer mouse and tau are present in enriched lysosome fractions. Immunoblotting analysis of enriched lysosome fractions from two separate JNPL3 mice brain preparations is shown, A and B respectively. Brain slices (400 μm) were incubated with FITC–IgG from a high titer Tau 379–408[pSer396, 404] immunized mouse for 2 h. Post treatment slices were processed to obtain an enriched lysosome fraction. Several fractions were obtained and the presence of lysosomes was confirmed in LAMP2 positive fractions A2, B1, B2, and B3. Mouse kidney lysate was used as a positive control. The kidney control is not a purified kidney preparation but a whole organ lysate, hence it has more background staining when compared to the enriched lysosome fractions from brain. Lysosome fractions were also immunoblotted for FITC–IgG. Fractions A2, B1, B2, and B3 were positive for FITC–IgG. To test if the lysosome fractions also contained tau, the FITC–IgG blot was reprobed with an antibody to total tau (DAKO A0024). Lysosome fractions A2, B1, B2, B3, and kidney lysate, all were positive for tau. Importantly, most of the FITC–IgG was detected in fractions with the highest levels of tau (A2 and B1). Overall, these findings are in accordance with the histological findings.
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Figure 3: FITC labeled IgG from a high titer mouse and tau are present in enriched lysosome fractions. Immunoblotting analysis of enriched lysosome fractions from two separate JNPL3 mice brain preparations is shown, A and B respectively. Brain slices (400 μm) were incubated with FITC–IgG from a high titer Tau 379–408[pSer396, 404] immunized mouse for 2 h. Post treatment slices were processed to obtain an enriched lysosome fraction. Several fractions were obtained and the presence of lysosomes was confirmed in LAMP2 positive fractions A2, B1, B2, and B3. Mouse kidney lysate was used as a positive control. The kidney control is not a purified kidney preparation but a whole organ lysate, hence it has more background staining when compared to the enriched lysosome fractions from brain. Lysosome fractions were also immunoblotted for FITC–IgG. Fractions A2, B1, B2, and B3 were positive for FITC–IgG. To test if the lysosome fractions also contained tau, the FITC–IgG blot was reprobed with an antibody to total tau (DAKO A0024). Lysosome fractions A2, B1, B2, B3, and kidney lysate, all were positive for tau. Importantly, most of the FITC–IgG was detected in fractions with the highest levels of tau (A2 and B1). Overall, these findings are in accordance with the histological findings.

Mentions: To examine antibody compartmentalization, we next prepared an enriched lysosome fraction from our FITC–IgG treated JNPL3 brain slices. Fractions were removed post separation and analyzed by western blot. Figure 3 indicates enriched lysosome fractions obtained from two separate mice, A and B respectively. Fractions (400 μl) were obtained from around and inclusive of the top visible band in the ultracentrifuge tube. Immunoblotting with an antibody to LAMP2, a marker of lysosomes, showed LAMP2 immunoreactivity in fractions A2, B1, B2, and B3. Fraction A1 is most likely only OptiPrep™ media without any lysosomes. Mouse kidney lysates were also run as a positive control. The kidney lysate is a whole tissue homogenate rather than a lysosome fraction (Figure 3). Immunostaining of the enriched lysosome fractions with an anti-FITC–IgG antibody showed more intense reactivity in fractions A2 and B1, indicating that our FITC conjugated antibody was taken up by the brain slices and present in the lysosome preparations (Figure 3). To test if the lysosome fractions also contained tau, the FITC–IgG blot was reprobed with an antibody to total tau (DAKO A0024). Lysosome fractions A2, B1, B2, and B3 were all positive for tau. Fraction A1 was weakly positive for tau, probably due to the fraction not being completely pure. And mouse kidney lysates were also tau positive since tau is known to be present in non-neuronal tissue (Gu et al., 1996; Figure 3).


Mechanistic Studies of Antibody-Mediated Clearance of Tau Aggregates Using an ex vivo Brain Slice Model.

Krishnamurthy PK, Deng Y, Sigurdsson EM - Front Psychiatry (2011)

FITC labeled IgG from a high titer mouse and tau are present in enriched lysosome fractions. Immunoblotting analysis of enriched lysosome fractions from two separate JNPL3 mice brain preparations is shown, A and B respectively. Brain slices (400 μm) were incubated with FITC–IgG from a high titer Tau 379–408[pSer396, 404] immunized mouse for 2 h. Post treatment slices were processed to obtain an enriched lysosome fraction. Several fractions were obtained and the presence of lysosomes was confirmed in LAMP2 positive fractions A2, B1, B2, and B3. Mouse kidney lysate was used as a positive control. The kidney control is not a purified kidney preparation but a whole organ lysate, hence it has more background staining when compared to the enriched lysosome fractions from brain. Lysosome fractions were also immunoblotted for FITC–IgG. Fractions A2, B1, B2, and B3 were positive for FITC–IgG. To test if the lysosome fractions also contained tau, the FITC–IgG blot was reprobed with an antibody to total tau (DAKO A0024). Lysosome fractions A2, B1, B2, B3, and kidney lysate, all were positive for tau. Importantly, most of the FITC–IgG was detected in fractions with the highest levels of tau (A2 and B1). Overall, these findings are in accordance with the histological findings.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198029&req=5

Figure 3: FITC labeled IgG from a high titer mouse and tau are present in enriched lysosome fractions. Immunoblotting analysis of enriched lysosome fractions from two separate JNPL3 mice brain preparations is shown, A and B respectively. Brain slices (400 μm) were incubated with FITC–IgG from a high titer Tau 379–408[pSer396, 404] immunized mouse for 2 h. Post treatment slices were processed to obtain an enriched lysosome fraction. Several fractions were obtained and the presence of lysosomes was confirmed in LAMP2 positive fractions A2, B1, B2, and B3. Mouse kidney lysate was used as a positive control. The kidney control is not a purified kidney preparation but a whole organ lysate, hence it has more background staining when compared to the enriched lysosome fractions from brain. Lysosome fractions were also immunoblotted for FITC–IgG. Fractions A2, B1, B2, and B3 were positive for FITC–IgG. To test if the lysosome fractions also contained tau, the FITC–IgG blot was reprobed with an antibody to total tau (DAKO A0024). Lysosome fractions A2, B1, B2, B3, and kidney lysate, all were positive for tau. Importantly, most of the FITC–IgG was detected in fractions with the highest levels of tau (A2 and B1). Overall, these findings are in accordance with the histological findings.
Mentions: To examine antibody compartmentalization, we next prepared an enriched lysosome fraction from our FITC–IgG treated JNPL3 brain slices. Fractions were removed post separation and analyzed by western blot. Figure 3 indicates enriched lysosome fractions obtained from two separate mice, A and B respectively. Fractions (400 μl) were obtained from around and inclusive of the top visible band in the ultracentrifuge tube. Immunoblotting with an antibody to LAMP2, a marker of lysosomes, showed LAMP2 immunoreactivity in fractions A2, B1, B2, and B3. Fraction A1 is most likely only OptiPrep™ media without any lysosomes. Mouse kidney lysates were also run as a positive control. The kidney lysate is a whole tissue homogenate rather than a lysosome fraction (Figure 3). Immunostaining of the enriched lysosome fractions with an anti-FITC–IgG antibody showed more intense reactivity in fractions A2 and B1, indicating that our FITC conjugated antibody was taken up by the brain slices and present in the lysosome preparations (Figure 3). To test if the lysosome fractions also contained tau, the FITC–IgG blot was reprobed with an antibody to total tau (DAKO A0024). Lysosome fractions A2, B1, B2, and B3 were all positive for tau. Fraction A1 was weakly positive for tau, probably due to the fraction not being completely pure. And mouse kidney lysates were also tau positive since tau is known to be present in non-neuronal tissue (Gu et al., 1996; Figure 3).

Bottom Line: Thus, clearance of neurofibrillary tangles and/or their precursors may reduce synaptic and neuronal loss associated with AD and other tauopathies.Additionally, tau and FITC-IgG were found together in an enriched lysosome fraction.In summary, antibody-mediated clearance of intracellular tau aggregates appears to occur via the lysosomal pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Neuroscience, New York University School of Medicine New York, NY, USA.

ABSTRACT
Recent studies have shown that immunotherapy clears amyloid beta (Aβ) plaques and reduces Aβ levels in mouse models of Alzheimer's disease (AD), as well as in AD patients. Tangle pathology is also relevant for the neurodegeneration in AD, and our studies have shown that active immunization with an AD related phospho-tau peptide reduces aggregated tau within the brain and slows the progression of tauopathy-induced behavioral impairments. Thus, clearance of neurofibrillary tangles and/or their precursors may reduce synaptic and neuronal loss associated with AD and other tauopathies. So far the mechanisms involved in antibody-mediated clearance of tau pathology are yet to be elucidated. In this study we have used a mouse brain slice model to examine the uptake and localization of FITC labeled anti-tau antibodies. Confocal microscopy analysis showed that the FITC labeled anti-tau antibody co-stained with phosphorylated tau, had a perinuclear appearance and co-localized with markers of the endosomal/lysosomal pathway. Additionally, tau and FITC-IgG were found together in an enriched lysosome fraction. In summary, antibody-mediated clearance of intracellular tau aggregates appears to occur via the lysosomal pathway.

No MeSH data available.


Related in: MedlinePlus