Transglutaminase 6 Is Colocalized and Interacts with Mutant Huntingtin in Huntington Disease Rodent Animal Models

Round 1

Reviewer 1 Report

In the manuscript, Schulze-Krebs and colleagues revealed that transglutaminase 6 (TG6) is colocalized and interacts with (mutant) huntingtin (mHTT) in Huntington disease rodent animal models. Also, they revealed the physical interaction between TG6 and mHTT by co-IP analysis and the contribution of its enzymatic activity for the total amounts of HTT aggregates. The results are potentially interesting but my major concern is that this study is lack of quantitative evaluation of TG6 levels and statistical analyses. I have several comments that should be addressed by the authors. Specific comments are below.

Major points.

1. Related to Figure 1A and Figure 2A: Does the expression level of TG6 differ between WT and BACHD mice? Also, is the level of TG6 increased in tgHD rats compared to WT rats? Is aging related to its expression levels? I think that the weakness of this study is lack of quantitative evaluation of TG6 levels and statistical analyses. Since quantification of immunohistochemistry is not reliable, the authors should verify this point by using western blotting or qPCR analyses.

2. Overall, the images are small and hard to see. For example, the authors claim that TG6 in BACHD tg mice at older age often displayed a characteristic ring-like (cytoplasmatic) staining pattern (Figure 1A). Also, they state that regional TG6 is colocalized with mutant huntingtin aggregates in cortical areas of mice (Figure 4A). However, higher magnification images should be provided to show the different structure and distribution.

3. For me, TG6 seems to bind (m)HTT from WT and tgHD mice at a similar level (Figure 6B). It means that a long stretch of polyglutamine (polyQ) is not involved in the interaction of TG6 and hungtintin. Then, what domain of hungtintin is involved in the interaction with TG6? This point should be clarified. Otherwise, it needs statistical analyses of repeated measurements.

Minor points.

1. Several different antibodies are used with each code number (e.g. 2B7, 2166, 1C2, 81D4, S830…) and I cannot follow them all. Please summarize them in a table with epitopes.
2. In Figure 7, representative images under microscopy should be provided for aggregates bearing cells in each group.

Author Response

Manuscript ID: ijms-1301688

Title: Transglutaminase 6 is colocalized and interacts with mutant huntingtin

in Huntington Disease rodent animal models

Authors: Anja Schulze-Krebs, et al.

Response Letter to reviewer comments

The authors of the MS entitled „Transglutaminase 6 is colocalized and interacts with mutant huntingtin in Huntington Disease rodent animal models” would like to thank both reviewers for their positive and constructive feedback.

We are grateful for the sorrowful and careful review provided by both reviewers. To address the comments/points the manuscript has been carefully revised by us and statements were discussed and/or cited more properly in our revised manuscript.

Overall, we again want to clarify that the manuscript was intended as one of the first reports aiming at describing a possible involvement of transglutaminase 6 (TG6) enzymatic activity for the pathogenesis of Huntington disease (HD) using different HD rodent animal models.

The TG6 isoform was detected only a few years ago and was found to play a possible role during neurodegenerative processes in different diseases. Our investigations were aimed at extending the knowledge about this isoform, which is mostly expressed in the brain, for HD. The results gathered in different HD rodent models indicate a possible contribution of TG6 enzymatic activity for the pathophysiology of HD, although a deep quantitative evaluation of TG6 protein levels was not made. This will be the goal/aim of further research.

Nonetheless the comments from the reviewers prompted us to include additional information describing a possible role of TG6 in the rodent brain. The present version of the manuscript has been carefully revised, and a point-by-point reply to all specific issues and comments compiled as follows.

Reviewer #1 Major points

We thank reviewer #1 for the detailed revision of our manuscript. A point-by-point reply to all specific issues and comments follows.

Reviewer #1 Point 1: “Related to Figure 1A and Figure 2A: Does the expression level of TG6 differ between WT and BACHD mice? Also, is the level of TG6 increased in tgHD rats compared to WT rats? Is aging related to its expression levels? I think that the weakness of this study is lack of quantitative evaluation of TG6 levels and statistical analyses. Since quantification of immunohistochemistry is not reliable, the authors should verify this point by using western blotting or qPCR analyses.”

In situ TG-based enzymatic activity is a histological technique that provides an image indicative of regional and spatial TG6-specific enzymatic activity using unfixed frozen brain tissue. Enzyme activity was visualized with a fluorescence microscope. However, despite the successful use of this technique, this method does not allow a quantitative analysis but is suitable for age-/gender- or genotype-specific analysis of the regional and spatial distribution of TG6, which was the aim of these experiments. Quantitative western blotting or qPCR analyses are reliable methods to study differences in expression levels, although they do not allow the analysis of enzymatic activity. A first attempt to evaluate TG-based enzymatic activity was already done by us, but showed no significant differences in BACHD mice [1].

Reviewer #1 Point 2: “Overall, the images are small and hard to see. For example, the authors claim that TG6 in BACHD tg mice at older age often displayed a characteristic ring-like (cytoplasmatic) staining pattern (Figure 1A). Also, they state that regional TG6 is colocalized with mutant huntingtin aggregates in cortical areas of mice (Figure 4A). However, higher magnification images should be provided to show the different structure and distribution.”

We provide the images within Figure 1 and Figure 4A at 100x max. magnification, enlarged the display of the figure, and provide an inlay, pin-pointing the “characteristic ring-like (cytoplasmatic) staining pattern often in the periphery of nuclei(Figure 1A; arrowhead; cf. inlay in Fig. 1A lower right panel).” We provide some more information within the text (see above, text in black italics), now more clearly relating this observation to Fig 1A lower right panel.

Reviewer #1 Point 3: “For me, TG6 seems to bind (m)HTT from WT and tgHD mice at a similar level (Figure 6B). It means that a long stretch of polyglutamine (polyQ) is not involved in the interaction of TG6 and hungtintin. Then, what domain of hungtintin is involved in the interaction with TG6? This point should be clarified. Otherwise, it needs statistical analyses of repeated measurements.”

We have now included the following sentences (italics, black letters) into the discussion (lines 589-ff), to further discuss and to clarify this point.

… modifications, albeit irrespective of the age and genotype in transgenic rats. In this context, it seems obvious that TG6 binds (m)HTT from transfected cell lines as well as in WT and tgHD rats at yet not defined levels (Fig. 6A+B). As coIP is not quantitative and since full-length as well as N-terminal fragments of HTT as well as mHTT were immuno-precipitated by TG6, we can conclude at this point that TG6 binds N-terminal HTT and mHTT regardless of the length of the polyQ-stretch. Thus, it should be kept in mind that (a) based on the direct evidence provided here, the length of the polyQ-stretch does not play in role in TG6 interaction (although the (m)HTT-ID-antibody mAB2166 has – according to manufacturer information - a higher affinity for HTT), that (b) purified (m)HTT-fragments are not available for qualitative/quantitative interaction studies at present, that (c) through using different antibodies (2166, S830, 2B7, EGFP) (cf. suppl. Table S1) all evidence accumulates that the site of interaction is found in exon 1, and that (d) all-in-all, it may well be that the affinity for mHTT by TG6 is higher though further studies are required.     In general, ...

Thus, to elucidate if TG6 is directly interacting with HTT co-immunoprecipitation studies were performed (Figure 6A+B). Although co-immunoprecipitation has become a standard technique, its protein-band output provides only a static and qualitative information on protein–protein interactions.

We used protein lysates of transiently double transfected SH-SY5Y cells (Fig. 6A) and brain protein lysates of wt or transgenic rats (Fig. 6B) and analyzed them for an interaction of full-length (m)HTT and exon 1 fragments with TG6. TG6 was co-immunoprecipitated from the soluble fractions of SH-SY5Y cells expressing either the full-length huntingtin variants or the exon 1 fragments, irrespective of the length of the polyglutamine stretch. Furthermore, TG6 was co-immunoprecipitated from brain lysates of wt and mHTT transgenic rats, suggesting a direct interaction between (m)HTT and TG6 also in vivo. Two different and well-established antibodies (S830, mEM48), which have their epitope in the N-terminus of mHTT and which are widely used to study mHTT aggregation, displayed immunopositive mHTT oligomerization products which 1) colocalize with the TG6 isoform and 2) were positive for TG-enzymatic reaction products.

Although, the misfolding and formation of (in)soluble huntingtin aggregates by mutated huntingtin (mHTT) is believed to be a key factor in the development of HD, little is known about the structural and conformational aspects of polyglutamine-induced misfolding and aggregation in this context. This is largely attributable to the fact that mHTT has proved difficult to purify in quantities, suitable for analyses, thus limiting the extent to which the protein can be conformationally characterized. A major reason for the lack of data specifically addressing interacting proteins is that the purification of recombinant huntingtin proteins, especially those of pathological length, has often proved to be technically challenging.

However, based on the information provided in this manuscript, we conclude, that the interacting domain of huntingtin with TG6 lies in the N-terminal part of (mutated) huntingtin, although the interaction itself seems to be irrespective of the length of the polyglutamine stretch. However, we cannot rule out the fact that the affinity of TG6 for mHTT may be higher compared to the huntingtin proteins displaying physiological polyglutamine stretches.

Reviewer #1 Minor points

1. The antibodies used in this study were summarized in a table (Supplementary table S1) and included in the revised manuscript.
2. Representative images for aggregate bearing cells were included (Supplementary figure S4).

Author Response File: Author Response.docx

Reviewer 2 Report

The authors of this manuscript aimed to investigate expression, distribution and activity of TGs in the brains of 2 different transgenic rodent models of Huntington’s disease (HD) and SH-SY5Y cells. The authors focused on analyzing the involvement of TG6, as this neuronal isoform is the least characterized in neurodegenerative disorders, yet it is the one abundantly expressed in the brain. First, the authors investigated the distribution of TG6 in the brain of 12- and 68 weeks old BACHD mice, displaying no obvious significant genotype- or age–dependent differences despite widespread immunoreactivity. The authors noted potentially greater immunoreactivity within large pyramidal cells of the motor cortex in older transgenic BACHD mice, although this was not quantified. The authors subsequently used a TG-isoform specific enzymatic activity assay to assess specific TG6 enzymatic activity in situ within the wt and transgenic BACHD mice. No obvious genotype-dependent differences regarding TG6-specific activity could be detected (which also applied to TG1, TG2 and TG3). The regional distribution of TG6 was also investigated in tgHD rats. The TG6 isoform had the highest expression compared to the other TG isoforms and was localised to neurons. The authors subsequently investigated the colocalisation of TG6 with huntingtin aggregates immunolabeled with either EM48 or S830 antibodies. In the knock-in zQ175 HD mouse model, regional colocalization of TG6 and mutant huntingtin aggregates in cortical areas of 10 month old animals was identified. TG6 immunostaining revealed marked differences between the two mouse genotypes, with TG6 labeling more prominent in wildtype mice and also more evidently localized to the periphery of the nucleus when compared to knock-in mice, leading the authors to suggest that mHTT impacts on TG6 distribution and/or turnover. These results were also confirmed in tgHD rats.

The authors subsequently moved towards investigating the occurrence of GGELs in full-length (m)HTT or exon1 fragments in protein lysates of transiently transfected SH-SY5Y cells. Isopeptide cross-links were found in full-length mutated huntingtin as well as in an exon 1 fragment bearing 97 polyglutamines, after immunoprecipitating isopeptide modified huntingtin using the 81D4 antibody. An additional study was conducted to elucidate if TG6 is directly interacting with HTT, and possibly responsible for its post-translational modification using co-immunoprecipitation studies in transiently double transfected SH-SY5Y cells and brain protein lysates of 55 weeks-old wt or transgenic rats. TG6 was co-immunoprecipitated from the soluble fractions of SH-SY5Y cells expressing either the full-length huntingtin variants or the exon 1 fragments, regardless of the length of the polyglutamine stretches. The authors also concluded that TG6 overexpression in double transfected SH-SY5Y cells accelerates the amount of huntingtin exon 1 GFP aggregates.

Overall, the authors have conducted a suitable study using HD rodent models and in vitro assays to suggest a role of TG6 in HD. The results suggest that TG6 activity on mHTT may be causative for the modification of mHTT fragments that would result to be more prone to aggregation. Furthermore, the aggregation process seems to depend more on the regional distribution and physical proximity of transglutaminases and mHTT, rather than on differences in total protein amounts and enzymatic activity. While this study is not without limitations, I personally feel that the authors have produced a manuscript which demonstrates a novel contribution to the understanding of transglutaminases as potential contributors to the pathogenesis of HD. I note a few comments below which would contribute to improvements in the manuscript and I ask that the authors specifically address each of my comments in their response.

Comment 1: Why wasn’t the R62 model used? While different rodent models were used in this manuscript, each model contains its strengths and limitations. If the authors could add a discussion about this, perhaps as an additional limitations section, that would add value to the paper.

Comment 2: On the theme of limitations, it would be worth discussing the overall limitations of the study. For example, the lack of translation of therapeutics from rodents to the clinic is still a major issue. The authors could discuss how these results need to be recapitulated in the human brain or larger animal models of disease. Furthermore, what are the limitations of using 5H-SY5Y cells?

Comment 3: In figure 1A, is the inset of the BACHD panel taken from the location of the arrow? If so, make this clear.

Comment 4: In Figure 1, move the location of the scale bar to a consistent location within all panels (i.e stick to the bottom right in all panels including insets). Please apply this principle to all figures throughout the manuscript.

Comment 5: For all figures, remove objective magnifications from the panels (ie 100x in Fig 1a inset, or 100x and 60x in all panels in Fig 2A and 2B). The magnifications do not add value because every microscope has a variable numerical aperture for each lens. The scale bars are far more informative so focus on clearly labeled scale bars being included in a consistent location on images in all figures.

Comment 6: For all figures, Please use consistent panel nomenclature. For example, in Figure 1A, each panel is named with the animal names “ie wt 12 weeks,” whereas in Fig 1B all of the panels are named “a” “b” “c” etc. In comparison, in Figure 2A, it was difficult to work out where each panel came from and it would have been helped to have “a” “b” “c” clearly for each panel at the top rather than hidden in the bottom left corner of each image. Please streamline the labels on each figure with a consistent font size, location, and nomenclature. It makes it much easier to understand and interpret each figure.

Minor comments:

Pp13, line 343: delete “s” from “neuropils”

Pp14, line 249: correct “modell” to “model”

Author Response

Manuscript ID: ijms-1301688

Title: Transglutaminase 6 is colocalized and interacts with mutant huntingtin

in Huntington Disease rodent animal models

Authors: Anja Schulze-Krebs, et al.

Response Letter to reviewer comments

The authors of the MS entitled „Transglutaminase 6 is colocalized and interacts with mutant huntingtin in Huntington Disease rodent animal models” would like to thank both reviewers for their positive and constructive feedback.

We are grateful for the sorrowful and careful review provided by both reviewers. To address the comments/points the manuscript has been carefully revised by us and statements were discussed and/or cited more properly in our revised manuscript.

Overall, we again want to clarify that the manuscript was intended as one of the first reports aiming at describing a possible involvement of transglutaminase 6 (TG6) enzymatic activity for the pathogenesis of Huntington disease (HD) using different HD rodent animal models.

The TG6 isoform was detected only a few years ago and was found to play a possible role during neurodegenerative processes in different diseases. Our investigations were aimed at extending the knowledge about this isoform, which is mostly expressed in the brain, for HD. The results gathered in different HD rodent models indicate a possible contribution of TG6 enzymatic activity for the pathophysiology of HD, although a deep quantitative evaluation of TG6 protein levels was not made. This will be the goal/aim of further research.

Nonetheless the comments from the reviewers prompted us to include additional information describing a possible role of TG6 in the rodent brain. The present version of the manuscript has been carefully revised, and a point-by-point reply to all specific issues and comments compiled as follows.

Reviewer #2 Major points

We thank reviewer #2 for the detailed revision of our manuscript. A point-by-point reply to all specific issues and comments follows.

Reviewer #2 Point 1: “Why wasn’t the R62 model used? While different rodent models were used in this manuscript, each model contains its strengths and limitations. If the authors could add a discussion about this, perhaps as an additional limitations section, that would add value to the paper.”

We now included a section into the discussion (2^nd and 4^th paragraphs), which read as follows

(2^nd paragraph in discussion):“Up to now, transgenic mouse models of HD are by far the most studied models for HD and are widely used and accepted [50, 51]. Although long and cost-intensive, research into disease mechanisms is an important basis for the development of novel, targeted therapies in translational research. Therefore, suitable animal models which allow predictions on the efficacy and safety of novel therapies are inevitable in this process. A successfully established animal model of a neurodegenerative disease adequately recapitulates the human disorder, both mimicking the symptomatology and developing adequate neuropathological lesions. Recently a transgenic minipig model for HD was established and characterized [52-54]. Although pig models are believed to be superior to mouse models with respect to recapitulation of human disease phenotypes, and technologies for generating genetically modified pig models have been well established, there are several reasons that hamper the broad applications of pigs [55].”

(4^th paragraph in discussion):” We chose the zQ175DN KI and not the R6/2 model, because they express the full-length protein in which the mouse Htt exon 1 has been replaced by the human HTT exon 1 sequence with a ~190 CAG repeat tract with spread nuclear and neuropil inclusions [57, 61]. In contrast to zQ175 DN KI mice, the R6/2 mice model human HD by expressing only a portion of the human HD gene under human gene promoter elements. The expression of this N-terminal exon 1-fragment of the huntingtin protein with its polyglutamine expansion is sufficient to produce a full HD-phenotype in a very short time. Therefore, knock in mice are very useful for studying Huntington disease pathogenesis of the juvenile HD-phenotype without hardly showing any survival rate limitation, especially in heterozygous animals.”

To further explain this: The zQ175DN KI express the full-length protein in which the mouse Htt exon 1 has been replaced by the human HTT exon 1 sequence with a ~190 CAG repeat tract with spread nuclear and neuropil inclusions [2, 3]. These mice are very useful for studying Huntington disease pathogenesis of the juvenile HD-phenotype without hardly any survival rate limitation in heterozygous animals. We believe that heterozygous knock-in models best mimic the human condition from a genetic perspective since they express the mutation in the appropriate genetic and protein context and published data support the suitability of this model for the evaluation of pathogenic events during HD [2].

In contrast to zQ175 DN KI mice, the R6/2 mice model human HD by expressing only a portion of the human HD gene under human gene promoter elements. The expression of this N-terminal exon 1-fragment of the huntingtin protein with its polyglutamine expansion is sufficient to produce a full HD-phenotype in a very short time. However, the fast progression of the HD-associated pathology limits the mean survival rate of this model to 2-3 months thus representing many features of a late stage phenotype after a very short time [4].

The BACHD transgenic mice express a neuropathogenic, full-length human mutant Huntingtin gene modified by a human mutant exon 1 sequence located on a bacterial artificial chromosome (BAC) with most of the aggregates in the neuropil and only sparse in cortex or striatum [5]. Overall, the immunohistochemical detection of mHtt-associated aggregates was very low in heterozygous animals compared to homozygous ones (personal observation), making it difficult to study aggregation-associated processes in heterozygous animals which mimic the human situation more closely.

The SD tgHD HD rat model was the first transgenic rat model bearing a human HD mutation with a high-end adult onset allele of 51 CAG repeats that exhibits progressive neurological, neuropathological and neurochemical phenotypes closely resembling the common late manifesting and slowly progressing type of disease [6] and only homozygous rats show many neuropil aggregates and nuclear inclusions in the striatum and cortex [7].

In our opinion the zQ175 DN KI mouse model better mimics the human genetic lesion by displaying a robust HD-linked phenotype with the associated molecular alterations, especially in heterozygous mice. We conclude that HD-animal models with only one affected allele reflect/mimic best the human disease condition, especially if they express the full-length protein.

Reviewer #2 Point 2: “On the theme of limitations, it would be worth discussing the overall limitations of the study. For example, the lack of translation of therapeutics from rodents to the clinic is still a major issue. The authors could discuss how these results need to be recapitulated in the human brain or larger animal models of disease. Furthermore, what are the limitations of using 5H-SY5Y cells?”

We thank reviewer#2 for this interesting aspect and have include it in our discussion (e.g. 2^nd + 4^th paragraph).

Cell lines are widely used in experiments that aim to understand disease mechanisms at a cellular level. The (Un-)differentiated SH-SY5Y cell line has been used widely in experimental neurological studies, including analysis of neuronal differentiation, metabolism, and function related to neurodegenerative processes, neurotoxicity, and neuroprotection [8-10]. Although studies have found that undifferentiated SH-SY5Y express only immature neuronal markers and lack mature neuronal markers the usage of this cellular model in the context of Parkinson disease (PD), Alzheimer disease (AD) and Huntington Disease is high as was could be shown by a systems genomics evaluation [9].

In HD medium spiny neurons (MSNs), which represent 90 to 97% of the striatal neuronal population are the most affected cells and striatal neurodegeneration usually starts a decade before symptom onset. However, the specific vulnerability of the striatal MSNs is still unexplained. In this regard, human induced pluripotent stem cell-derived MSNs of course would represent a powerful tool to study pathology-associated mechanisms. However, the differentiation protocols published so far shows a high heterogeneity of neuronal populations in vitro making the reproduction of published data challenging [11].

Although long and cost-intensive, research into disease mechanisms is an important basis for the development of novel, targeted therapies. Therefore, suitable animal models which allow predictions on the efficacy and safety of novel therapies are inevitable in this process. A successfully established animal model of neurodegenerative disease adequately recapitulates the human disease, both mimicking the symptomatology and developing adequate neuropathological lesions. Up to now, transgenic mouse models of HD are by far the most studied models for HD. However, translational failure of apparently promising translation research often has at least been attributed to an inadequate internal and external validity [12, 13]. In this context, a high internal and external validity, e.g. randomization, a blinded evaluation, sample size, gender, use of different models, hypothesis-driven design, statistics etc. is an inevitable base for successful translational research [12].

Recently a transgenic minipig model for HD was established and characterized [14-16]. However, for translational research a broad and comprehensive characterization of minipig model is needed, although it was already demonstrated that these pigs show an age-related pathology with a later onset of neurodegeneration [14]. Although pig models are believed to be superior to mouse models with respect to recapitulation of human disease phenotypes, and technologies for generating genetically modified pig models have been well established, there are several shortcomings that hamper the broad applications of pig models [17].

As already mentioned, using transgenic animal models for translational research in neurodegenerative diseases has both, benefits but also limitations, because neurodegenerative pathologies are often age-associated and limited to humans. Furthermore, genetically engineered rodents often recapitulate only aspects of the corresponding human disease, although, if well characterized and established, are useful tools to study disease-associated pathological mechanisms and for the testing of possible therapeutic strategies. However, effects either positive or negative of new treatment strategies in humans are sometimes difficult to predict on the basis of findings in rodents.

Our aim was, by using different HD-transgenic animal models and transfected SH-SY5Y cells, to take into account the broad mechanistic aspects of Huntington disease, allowing us comprehensive analyses of the HD-related phenotype. Up to know, specific TG6-inhibitors, which would facilitate the elucidation of the context within TG6 is involved during neurodegeneration, are still lacking and a TG6-knock out mouse model has only recently been available. These mice will allow more sophisticated studies in the future by crossbreeding them for example to the zQ175DN KI mice, thus allowing predictive studies with a high internal and external validity. In doing so, we hope to proof that TG6 is a beneficial target for translational research. Next to this, by modelling Huntington disease with induced pluripotent stem cells and/or cell-derived striatal MSNs would represent a powerful tool to investigate genetic disorders such as HD. However, this is still challenging and large differences were observed in differentiation protocols and reproducibility [11].

Reviewer #2 Point 3-5: “In figure 1A, is the inset of the BACHD panel taken from the location of the arrow? If so, make this clear. In Figure 1, move the location of the scale bar to a consistent location within all panels (i.e stick to the bottom right in all panels including insets). Please apply this principle to all figures throughout the manuscript. For all figures, remove objective magnifications from the panels (ie 100x in Fig 1a inset, or 100x and 60x in all panels in Fig 2A and 2B). The magnifications do not add value because every microscope has a variable numerical aperture for each lens. The scale bars are far more informative so focus on clearly labeled scale bars being included in a consistent location on images in all figures.

Thank you for these points. We have now removed all microscope-objective magnification and placed scale bars at similar positions, where ever possible.

Reviewer #2 Minor points

Mistakes in writing were corrected

Literature

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Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

The authors addressed all my concerns and I have no further points.