SREBP2 gene therapy targeting striatal astrocytes ameliorates Huntington’s disease phenotypes

SREBP2 is the master transcription factor that activates the expression of most cholesterol biosynthesis genes.

“Cholesterol biosynthesis is regulated by sterol regulatory element binding protein 2 (SREBP2), the master transcription factor that activates the expression of most cholesterol biosynthesis genes.”

• cites [11] Jeon, 2012, SREBPs: Metabolic integrators in physiology and metabolism, Trends Endocrinol Metab sourcePartly supported

  The Introduction (page unavailable) calls SREBP-2 the main regulator of cholesterol metabolism, and the section on mammalian SREBP gene expression says it has a strong transcriptional activation domain. These passages support its central regulatory role, but our copy does not establish the more specific claim that it activates most cholesterol biosynthesis genes. (LLM, conf=0.82)

  Experimental — accuracy not yet measured

Steady-state levels of lanosterol, lathosterol, and desmosterol were reduced in the striata of R6/2-Tom mice compared with wt-Tom mice.

“As expected, the steady-state levels of all cholesterol precursors were reduced in the striata of R6/2-Tom mice compared with wt-Tom mice (Fig. 2H-J) 19,20,27,28 .”

• cites [19] Valenza, 2007, Cholesterol biosynthesis pathway is disturbed in YAC128 mice and is modulated by huntingtin mutation, Hum Mol Genet sourceNot checked — no full text of the source
• cites [20] Valenza, 2010, Cholesterol defect is marked across multiple rodent models of Huntington’s disease and is manifest in astrocytes, J Neurosci sourcePartly supported

  The Results report reduced lathosterol in YAC72 striatum, and the Figure 3 caption reports reduced sterols in R6/2 whole-brain fractions compared with wild-type mice. These findings support a general cholesterol-related defect in HD models, but this paper does not report reduced lanosterol, lathosterol, and desmosterol specifically in R6/2-Tom striata versus wt-Tom striata. No page numbers are available in our copy. (LLM, conf=0.85)

  Experimental — accuracy not yet measured
• cites [27] Arsic, 2003, Induction of functional neovascularization by combined VEGF and angiopoietin-1 gene transfer using AAV vectors, Mol Ther sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites [28] Ayuso, 2010, High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency, Gene Ther sourceNot checked — no full text of the source

Increasing hSREBP2 level specifically in the nucleus of astrocytes stimulates cholesterol biosynthesis in the striatum of HD mice.

“We found that increasing hSREBP2 level specifically in the nucleus of astrocytes stimulates cholesterol biosynthesis in the striatum of HD mice.”

Cognitive decline is completely rescued in HD mice after hSREBP2 expression in striatal astrocytes.

“As a consequence, synaptic transmission of both inhibitory and excitatory synapses is restored, the number of striatal MSNs expressing Drd2 receptors normalized, mutant HTT (muHTT) aggregation reduced, motor defects ameliorated, and cognitive decline is completely rescued.”

Robust hSREBP2 expression in striatal glial cells in HD mice activated the transcription of cholesterol biosynthesis pathway genes.

“Robust hSREBP2 expression in striatal glial cells in HD mice activated the transcription of cholesterol biosynthesis pathway genes, restored synaptic transmission, reversed Drd2 transcript levels decline, cleared muHTT aggregates and attenuated behavioral deficits.”

RNA transcripts of hSrebp2 were detected only in R6/2-hBP2 mice.

“As expected, RNA transcripts of hSrebp2 were detected only in R6/2-hBP2 mice (Fig. 1F).”

Western blot analysis with an antibody recognizing both endogenous and exogenous SREBP2 revealed an increase in total SREBP2 protein level in three out of four R6/2-hBP2 mice.

“Importantly, western blot analysis with an antibody that recognizes both the endogenous (mouse) and exogenous (human) SREBP2 revealed an increase in total SREBP2 protein level in three out of four R6/2-hBP2 mice (Fig. 1I).”

Motor defects are ameliorated in HD mice after hSREBP2 expression in striatal astrocytes.

“As a consequence, synaptic transmission of both inhibitory and excitatory synapses is restored, the number of striatal MSNs expressing Drd2 receptors normalized, mutant HTT (muHTT) aggregation reduced, motor defects ameliorated, and cognitive decline is completely rescued.”

The number of striatal MSNs expressing Drd2 receptors normalized in HD mice after hSREBP2 expression in striatal astrocytes.

“As a consequence, synaptic transmission of both inhibitory and excitatory synapses is restored, the number of striatal MSNs expressing Drd2 receptors normalized, mutant HTT (muHTT) aggregation reduced, motor defects ameliorated, and cognitive decline is completely rescued.”

Synaptic transmission of both inhibitory and excitatory synapses is restored in HD mice after hSREBP2 expression in striatal astrocytes.

“Robust hSREBP2 expression in striatal glial cells in HD mice activated the transcription of cholesterol biosynthesis pathway genes, restored synaptic transmission, reversed Drd2 transcript levels decline, cleared muHTT aggregates and attenuated behavioral deficits.”

• Also stated in: § Introduction · plain

  “As a consequence, synaptic transmission of both inhibitory and excitatory synapses is restored, the number of striatal MSNs expressing Drd2 receptors normalized, mutant HTT (muHTT) aggregation reduced, motor defects ameliorated, and cognitive decline is completely rescued.”

Robust hSREBP2 expression in striatal glial cells in HD mice reversed Drd2 transcript levels decline.

“Robust hSREBP2 expression in striatal glial cells in HD mice activated the transcription of cholesterol biosynthesis pathway genes, restored synaptic transmission, reversed Drd2 transcript levels decline, cleared muHTT aggregates and attenuated behavioral deficits.”

hSREBP2 expression in striatal astrocytes attenuates behavioral deficits in HD mice.

“Robust hSREBP2 expression in striatal glial cells in HD mice activated the transcription of cholesterol biosynthesis pathway genes, restored synaptic transmission, reversed Drd2 transcript levels decline, cleared muHTT aggregates and attenuated behavioral deficits.”

Mutant HTT aggregation is reduced/cleared in HD mice after hSREBP2 expression in striatal astrocytes.

“Robust hSREBP2 expression in striatal glial cells in HD mice activated the transcription of cholesterol biosynthesis pathway genes, restored synaptic transmission, reversed Drd2 transcript levels decline, cleared muHTT aggregates and attenuated behavioral deficits.”

• Also stated in: § Introduction · plain

  “As a consequence, synaptic transmission of both inhibitory and excitatory synapses is restored, the number of striatal MSNs expressing Drd2 receptors normalized, mutant HTT (muHTT) aggregation reduced, motor defects ameliorated, and cognitive decline is completely rescued.”

Recorded sIPSC events were GABA-mediated since they were completely abolished by administration of 10 μM bicuculline.

“The recorded events were GABA-mediated since they were completely abolished by administration of 10 µM bicuculline (Fig. 3G).”

The treatment was not able to significantly rescue paw-clasping behavior.

“As expected, R6/2-Tom mice had a worse performance compared with wt-Tom, but in this case the treatment was not able to significantly rescue this behavior (Fig. 5G).”

AAV2/5-gfaABC1D-hSREBP2-tdTomato did not influence transcript levels of genes involved in astrocytic homeostasis.

“Similarly, AAV2/5-gfaABC1D-hSREBP2-tdTomato did not influence transcript levels of genes involved in astrocytic homeostasis.”

11-week-old R6/2-Tom mice exhibited a severe hypokinetic phenotype as demonstrated by reduced global activity, total distance traveled, and number of rearings in the activity cage test.

“As expected, 11-week-old R6/2-Tom mice exhibited a severe hypokinetic phenotype as demonstrated by reduced global activity, total distance traveled, and number of rearings in the activity cage test (Fig. 5A-C).”

hSREBP2 over-expression in R6/2 mice did not rescue Dhcr7 transcript levels.

“Importantly, hSREBP2 over-expression in R6/2 mice rescued the transcript levels of Hmgcr, Mvk, Fdft1, and Cyp51 (Fig. 2B-E) but not of Dhcr7 (Fig. 2F).”

Images acquired with two-photon microscopy in the clarified striata of Drd2-wt-Tom mice confirmed distinct signals of tdTomato and eGFP.

“The images acquired with two-photon microscopy in the clarified striata of Drd2-wt-Tom mice confirmed distinct signals of tdTomato and eGFP, which labeled infected glial cells and DRD2-MSNs, respectively (Fig. 4E).”

Input resistance (Rin) was significantly higher in both the infused and the contralateral hemispheres of R6/2-hBP2 compared with wt-Tom mice.

“On the other hand, input resistance (Ri n), reflecting the number of ion channels expressed by the cell, was significantly higher in both the infused and the contralateral hemispheres of R6/2-hBP2 compared with wt-Tom mice, and unchanged in the infused striata of R6/2-hBP2 mice compared with the contralateral ones (Fig. 3C).”

Glial over-expression of hSREBP2 led to a normalization in the average frequency of sIPSCs in the infused hemisphere.

“In contrast, the average frequency of sIPSCs in MSNs in the R6/2-hBP2-contralateral hemisphere was significantly higher compared with wt-Tom mice, and glial over-expression of hSREBP2 led to a normalization in the average frequency of sIPSCs in the infused hemisphere (Fig. 3I), bringing it closer to that in the wt-Tom mice.”

PSD95 level was weakly but significantly increased (p = 0.0138) in R6/2-hBP2 mice compared with wt-Tom and R6/2-Tom animals.

“Regarding the post-synaptic site, our densitometric analyses of the immunoreactive bands indicated a weak but significant increase (p = 0.0138) in PSD95 level in R6/2-hBP2 mice compared with wt-Tom and R6/2-Tom animals (Fig. 2N and O), even though differences between wt-Tom and R6/2-Tom mice were not observed (Fig. 2I and K).”

SHANK3 levels were similar in all tested mice, including R6/2-hBP2 mice.

“Conversely, SHANK3 levels were similar in all the tested mice, including R6/2-hBP2 mice (Fig. 2N and O).”

Glial hSREBP2 over-expression completely reversed the increased SYP levels in R6/2-Tom mice.

“We found that R6/2-Tom mice exhibited higher expression levels of the pre-synaptic protein SYP than wt-Tom mice and that glial hSREBP2 over-expression completely reversed this parameter (Fig. 2L and M).”

mRNA levels of Hmgcr, Mvk, Sqs/Fdft1, Cyp51, and Dhcr7 were significantly reduced in R6/2 and R6/2-Tom mice compared with controls.

“The mRNA levels from all these genes were significantly reduced in R6/2 and R6/2-Tom mice compared with controls (Fig. 2B-F).”

Cm was similar between the contralateral and the infused hemispheres of R6/2-hBP2 mice.

“The comparison between R6/2-hBP2 and wt-Tom mice highlighted the fact that the membrane capacitance (Cm), which is proportional to cell size, was significantly lower in R6/2-hBP2 compared with wt-Tom MSNs, while it was similar between the contralateral and the infused hemispheres of R6/2-hBP2 mice (Fig. 3B).”

hSrebp2 mRNA was between 1.7-fold and 2.1-fold more abundant than the endogenous transcript in R6/2-hBP2 mice.

“Importantly, Fig. 1L shows that hSrebp2 mRNA was between 1.7-fold and 2.1-fold more abundant than the endogenous transcript in R6/2-hBP2 mice.”

• AssumptionRests on an assumption: No antibodies reliably distinguish human from mouse SREBP2.

mRNA levels of synapse-related genes Bdnf, Cplx2, Shank3, Homer, and Gap43 were not influenced by forced expression of hSREBP2 in glial cells.

“The mRNA levels of synapserelated genes such as Bdnf, Cplx2, Shank3, Homer, and Gap43, which were altered in R6/2 and R6/2-Tom mice, were not influenced by the forced expression of hSREBP2 in glial cells (Fig. S2G and H).”

Glial over-expression of hSREBP2 had a beneficial effect on muscle strength.

“When we measured the neuromuscular function of mice with the grip-strength test, we observed reduced muscle strength in R6/2-Tom mice compared with wt-Tom mice and, again, glial over-expression of hSREBP2 had a beneficial effect on this feature (Fig. 5F).”

Rin was unchanged in the infused striata of R6/2-hBP2 compared with the contralateral ones.

“On the other hand, input resistance (Ri n), reflecting the number of ion channels expressed by the cell, was significantly higher in both the infused and the contralateral hemispheres of R6/2-hBP2 compared with wt-Tom mice, and unchanged in the infused striata of R6/2-hBP2 mice compared with the contralateral ones (Fig. 3C).”

Cyp46a1 transcript level was not affected by exogenous hSREBP2.

“The transcript level of Cyp46a1, which encodes the enzyme that catalyzes the conversion of cholesterol to 24S-hydroxycholesterol (24S-OHC), was reduced in all R6/2 groups compared with controls but was not affected by exogenous hSREBP2 (Fig. 2G).”

ApoE and Lrp1 mRNA levels were similar in all conditions.

“Abca1 mRNA levels were increased in R6/2, R6/2-Tom, and R6/2-hBP2 mice with respect to wt mice (Fig. S2A), while ApoE and Lrp1 mRNA levels were similar in all conditions (Fig. S2B and C), indicating that hSREBP2 delivery in glial cells did not affect transcript levels of these genes.”

The number and size of EM48-positive aggregates were reduced in the infused striata of R6/2-hBP2 mice compared with the contralateral ones.

“Of note, the number and the size of EM48-positive aggregates were reduced in the infused striata of R6/2-hBP2 mice compared with the contralateral ones (Fig. 4B and C).”

At 11 weeks of age, R6/2-Tom mice were not able to discriminate between the novel object and the familiar one, in contrast to wt-Tom mice.

“Finally, to analyze the effect of treatment on cognitive decline, we performed the novel-object recognition (NOR) test: at 11 weeks of age, R6/2-Tom mice were not able to discriminate between the novel object and the familiar one, in contrast to the wt-Tom mice.”

Glial over-expression of hSREBP2 led to a partial rescue of the sEPSC frequency defect.

“In contrast, the average frequency of sEPSCs in MSNs from the contralateral hemisphere of R6/2-hBP2 mice was significantly lower compared with wt-Tom mice, and glial over-expression of hSREBP2 led to a partial rescue of this defect (Fig. 3F).”

Recorded sEPSC events were AMPA-mediated since they were completely abolished by administration of 10 μM NBQX.

“The recorded events were AMPA-mediated since they were completely abolished by administration of 10 µM NBQX (Fig. 3D).”

The average frequency of sIPSCs in MSNs in the R6/2-hBP2-contralateral hemisphere was significantly higher compared with wt-Tom mice.

“In contrast, the average frequency of sIPSCs in MSNs in the R6/2-hBP2-contralateral hemisphere was significantly higher compared with wt-Tom mice, and glial over-expression of hSREBP2 led to a normalization in the average frequency of sIPSCs in the infused hemisphere (Fig. 3I), bringing it closer to that in the wt-Tom mice.”

Transcript levels of genes related to energy and mitochondrial metabolism and autophagy were not affected by over-expression of hSREBP2.

“Transcript levels of genes related to pathways such as energy and mitochondrial metabolism and autophagy were also not affected by the over-expression of hSREBP2 (Fig. S3A-I).”

Glial over-expression of hSREBP2 led to a slight but significant rescue of these parameters.

“Glial over-expression of hSREBP2 led to a slight but significant rescue of these parameters (Fig. 5A-C).”

Quantification of the eGFP signal revealed a reduced number of DRD2-MSNs in the infused striata of Drd2-R6/2-Tom mice and in the contralateral striata of Drd2-R6/2-hBP2 compared with Drd2-wt-Tom mice.

“The subsequent quantification of the eGFP signal in the different experimental groups revealed a reduced number of DRD2-MSNs in the infused striata of Drd2-R6/2-Tom mice and in the contralateral striata of Drd2-R6/2-hBP2 compared with Drd2-wt-Tom mice (Fig. 4F and G).”

• AssumptionRests on an assumption: CLARITY allows an accurate measurement of the total number of MSNs expressing DRD2 in a large volume of the vector-injected striatum without dissociation.

The average amplitude of sIPSCs was not affected.

“The average amplitude of sIPSCs was not affected (Fig. 3H).”

Forced expression of hSREBP2 normalized NMDAR1 levels to that of wt-Tom mice.

“Finally, R6/2-Tom mice exhibited reduced NMDAR1 protein levels compared with wt-Tom mice, and forced expression of hSREBP2 normalized its levels to that of wt-Tom mice (Fig. 2N and O).”

VAMP1 levels were similar in all conditions.

“VAMP1 levels were similar in all conditions (Fig. 2L and M).”

hSREBP2 over-expression in R6/2 mice rescued transcript levels of Hmgcr, Mvk, Fdft1, and Cyp51.

“Importantly, hSREBP2 over-expression in R6/2 mice rescued the transcript levels of Hmgcr, Mvk, Fdft1, and Cyp51 (Fig. 2B-E) but not of Dhcr7 (Fig. 2F).”

Endogenous mSREBP2 transcripts are significantly reduced in R6/2, R6/2-Tom and R6/2-hBP2 mice compared with wt mice.

“Notably, we measured a significant reduction of the endogenous mSREBP2 transcripts in R6/2, R6/2-Tom and R6/2-hBP2 mice compared with wt mice (Fig. 1K).”

The number of DRD2-MSNs measured in the infused striata of Drd2-R6/2-hBP2 was similar to that quantified in Drd2-wt-Tom mice.

“Of note, the number of DRD2-MSNs measured in the infused striata of Drd2-R6/2-hBP2 was similar to that quantified in Drd2-wt-Tom mice (Fig. 4F and G), indicating that over-expression of hSREBP2 in astrocytes allows these neurons to regain DRD2 expression and, supposedly, functionality.”

• AssumptionRests on an assumption: CLARITY allows an accurate measurement of the total number of MSNs expressing DRD2 in a large volume of the vector-injected striatum without dissociation.

R6/2-Tom mice had a worse performance in the paw-clasping test compared with wt-Tom mice.

“As expected, R6/2-Tom mice had a worse performance compared with wt-Tom, but in this case the treatment was not able to significantly rescue this behavior (Fig. 5G).”

PSD95 levels did not differ between wt-Tom and R6/2-Tom mice.

“Regarding the post-synaptic site, our densitometric analyses of the immunoreactive bands indicated a weak but significant increase (p = 0.0138) in PSD95 level in R6/2-hBP2 mice compared with wt-Tom and R6/2-Tom animals (Fig. 2N and O), even though differences between wt-Tom and R6/2-Tom mice were not observed (Fig. 2I and K).”

R6/2-Tom mice had reduced muscle strength compared with wt-Tom mice in the grip-strength test.

“When we measured the neuromuscular function of mice with the grip-strength test, we observed reduced muscle strength in R6/2-Tom mice compared with wt-Tom mice and, again, glial over-expression of hSREBP2 had a beneficial effect on this feature (Fig. 5F).”

R6/2-Tom mice exhibited higher expression levels of pre-synaptic protein SYP than wt-Tom mice.

“We found that R6/2-Tom mice exhibited higher expression levels of the pre-synaptic protein SYP than wt-Tom mice and that glial hSREBP2 over-expression completely reversed this parameter (Fig. 2L and M).”

hSREBP2 delivery in glial cells did not affect lanosterol and lathosterol levels in the striata of R6/2-hBP2 mice.

“hSREBP2 delivery in glial cells, although unable to affect lanosterol and lathosterol levels (Fig. 2H-I), led to a significant increase in desmosterol in the striata of R6/2-hBP2 mice compared with R6/2-Tom mice (Fig. 2J), suggesting a preferential stimulation of the Bloch pathway of cholesterol biosynthesis which is more related to astrocytes 29 (Fig. 2A).”

SNAP25 levels were similar in wt-Tom and R6/2-Tom mice.

“SNAP25 levels were similar in wt-Tom and R6/2-Tom, while a 38% increase was found in R6/2-hBP2 tissues (Fig. 2L and M).”

No significant differences in the average amplitude of sEPSC from MSNs were found between groups.

“No significant differences in the average amplitude of sEPSC from MSNs were found between groups (Fig. 3E).”

R6/2-Tom mice showed anxiety-related behavior as they spent more time in the periphery compared with wt-Tom mice.

“R6/2-Tom mice showed anxiety-related behavior as they spent more time in the periphery compared with wt-Tom mice.”

Cyp46a1 transcript level was reduced in all R6/2 groups compared with controls.

“The transcript level of Cyp46a1, which encodes the enzyme that catalyzes the conversion of cholesterol to 24S-hydroxycholesterol (24S-OHC), was reduced in all R6/2 groups compared with controls but was not affected by exogenous hSREBP2 (Fig. 2G).”

hSREBP2 delivery increased 24S-OHC in the striata of R6/2-hBP2 mice.

“This was accompanied by an increase in 24S-OHC (Fig. 2K), consistent with the notion that cholesterol synthesis and catabolism are in balance in the HD brain 21 .”

Membrane capacitance (Cm) was significantly lower in R6/2-hBP2 compared with wt-Tom MSNs.

“The comparison between R6/2-hBP2 and wt-Tom mice highlighted the fact that the membrane capacitance (Cm), which is proportional to cell size, was significantly lower in R6/2-hBP2 compared with wt-Tom MSNs, while it was similar between the contralateral and the infused hemispheres of R6/2-hBP2 mice (Fig. 3B).”

R6/2-Tom mice exhibited reduced NMDAR1 protein levels compared with wt-Tom mice.

“Finally, R6/2-Tom mice exhibited reduced NMDAR1 protein levels compared with wt-Tom mice, and forced expression of hSREBP2 normalized its levels to that of wt-Tom mice (Fig. 2N and O).”

SNAP25 levels were increased by 38% in R6/2-hBP2 tissues.

“SNAP25 levels were similar in wt-Tom and R6/2-Tom, while a 38% increase was found in R6/2-hBP2 tissues (Fig. 2L and M).”

R6/2-hBP2 mice behaved similarly to wt-Tom mice in the activity cage test.

“Of note, R6/2-hBP2 mice behaved similarly to wt-Tom mice (Fig. 5E).”

The average frequency of sEPSCs in MSNs from the contralateral hemisphere of R6/2-hBP2 mice was significantly lower compared with wt-Tom mice.

“In contrast, the average frequency of sEPSCs in MSNs from the contralateral hemisphere of R6/2-hBP2 mice was significantly lower compared with wt-Tom mice, and glial over-expression of hSREBP2 led to a partial rescue of this defect (Fig. 3F).”

tdTomato did not influence the number and size of muHTT aggregates.

“As expected, tdTomato did not influence the number and the size of muHTT aggregates.”

Abca1 mRNA levels were increased in R6/2, R6/2-Tom, and R6/2-hBP2 mice with respect to wt mice.

“Abca1 mRNA levels were increased in R6/2, R6/2-Tom, and R6/2-hBP2 mice with respect to wt mice (Fig. S2A), while ApoE and Lrp1 mRNA levels were similar in all conditions (Fig. S2B and C), indicating that hSREBP2 delivery in glial cells did not affect transcript levels of these genes.”

hSREBP2 delivery in glial cells led to a significant increase in desmosterol in the striata of R6/2-hBP2 mice compared with R6/2-Tom mice.

“hSREBP2 delivery in glial cells, although unable to affect lanosterol and lathosterol levels (Fig. 2H-I), led to a significant increase in desmosterol in the striata of R6/2-hBP2 mice compared with R6/2-Tom mice (Fig. 2J), suggesting a preferential stimulation of the Bloch pathway of cholesterol biosynthesis which is more related to astrocytes 29 (Fig. 2A).”

The reduction in cholesterol biosynthesis in HD mouse brain is CAG-dependent.

“In the adult HD mouse brain, this dysfunction is evidenced by a reduced rate of de novo cholesterol biosynthesis, precedes the onset of motor symptoms, is CAG-dependent, as confirmed by studies in an allelic series of HD mice, and leads to a reduced content of total cholesterol (and of some of its metabolites and catabolites) at late symptomatic stages 21 .”

• cites [21] Shankaran, 2017, Early and brain region-specific decrease of de novo cholesterol biosynthesis in Huntington’s disease: A cross-validation study in Q175 knock-in mice, Neurobiol Dis sourceNot checked — no full text of the source

The reduction in cholesterol biosynthesis in HD mouse brain leads to reduced total cholesterol at late symptomatic stages.

“In the adult HD mouse brain, this dysfunction is evidenced by a reduced rate of de novo cholesterol biosynthesis, precedes the onset of motor symptoms, is CAG-dependent, as confirmed by studies in an allelic series of HD mice, and leads to a reduced content of total cholesterol (and of some of its metabolites and catabolites) at late symptomatic stages 21 .”

• cites [21] Shankaran, 2017, Early and brain region-specific decrease of de novo cholesterol biosynthesis in Huntington’s disease: A cross-validation study in Q175 knock-in mice, Neurobiol Dis sourceNot checked — no full text of the source

The reduction in cholesterol biosynthesis in HD mouse brain precedes the onset of motor symptoms.

“In the adult HD mouse brain, this dysfunction is evidenced by a reduced rate of de novo cholesterol biosynthesis, precedes the onset of motor symptoms, is CAG-dependent, as confirmed by studies in an allelic series of HD mice, and leads to a reduced content of total cholesterol (and of some of its metabolites and catabolites) at late symptomatic stages 21 .”

• cites [21] Shankaran, 2017, Early and brain region-specific decrease of de novo cholesterol biosynthesis in Huntington’s disease: A cross-validation study in Q175 knock-in mice, Neurobiol Dis sourceNot checked — no full text of the source

SREBP2 gene silencing in wild-type astrocytes has detrimental effects on HD neurons.

“SREBP2 gene silencing in wild-type (wt) astrocytes has detrimental effects on HD neurons 22 ,”

• cites [22] Valenza, 2015, Disruption of astrocyte-neuron cholesterol cross talk affects neuronal function in Huntington’s disease, Cell Death Differ sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

AAV-based delivery of SREBP2 to astrocytes counteracts key features of HD.

“We conclude that glial SREBP2 participates in HD brain pathogenesis in vivo and that AAV-based delivery of SREBP2 to astrocytes counteracts key features of HD.”

The N-terminal fragment of hSREBP2 (1-482 aa) is the active form.

“We obtained the AAV vector plasmid pZac-gfaABC1D-hSREBP2-tdTomato, expressing the active N-terminal fragment of hSREBP2 (1-482 aa) fused with tdTomato, starting from pZac2.1 gfaABC1D-tdTomato (from Addgene) and pcDNA3.1 hSREBP2(402)-eGFP (from T. Osborne) 61 .”

• cites [61] Glass, 2000, The pattern of neurodegeneration in Huntington’s disease: A comparative study of cannabinoid, dopamine, adenosine and GABA(A) receptor alterations in the human basal ganglia in Huntington’s disease, Neuroscience sourceNot checked — no full text of the source

AAV2/5 is known to be highly specific for glial cells.

“we employed a recombinant serotype of an adeno-associated virus (AAV2/5) that is known to be highly specific for glial cells 24 .”

• cites [24] Mangiarini, 1996, Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice, Cell sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Brain cholesterol is important for neuronal function.

“In neurons cholesterol plays important roles in synaptic transmission, as it is required for synaptic vesicle formation and function 3,4,5 and for optimal neurotransmitter release 6,7,8 .”

• cites [3] Mauch, 2001, CNS synaptogenesis promoted by glia-derived cholesterol, Science sourceNot checked — no full text of the source
• cites [4] Göritz, 2002, Role of glia-derived cholesterol in synaptogenesis: New revelations in the synapse-glia affair, J Physiol Paris sourceNot checked — no full text of the source
• cites [5] Goritz, 2005, Multiple mechanisms mediate cholesterol-induced synaptogenesis in a CNS neuron, Mol Cell Neurosci sourceNot checked — no full text of the source
• cites [6] Pfrieger, 2003, Role of cholesterol in synapse formation and function, Biochim Biophys Acta sourceNot checked — no full text of the source
• cites [7] Pfrieger, 2003, Cholesterol homeostasis and function in neurons of the central nervous system, Cell Mol Life Sci sourceNot checked yet
• cites [8] Pfrieger, 2003, Outsourcing in the brain: Do neurons depend on cholesterol delivery by astrocytes?, BioEssays sourceNot checked — no full text of the source

In adult HD mouse brain, the rate of de novo cholesterol biosynthesis is reduced.

“In the adult HD mouse brain, this dysfunction is evidenced by a reduced rate of de novo cholesterol biosynthesis, precedes the onset of motor symptoms, is CAG-dependent, as confirmed by studies in an allelic series of HD mice, and leads to a reduced content of total cholesterol (and of some of its metabolites and catabolites) at late symptomatic stages 21 .”

• cites [21] Shankaran, 2017, Early and brain region-specific decrease of de novo cholesterol biosynthesis in Huntington’s disease: A cross-validation study in Q175 knock-in mice, Neurobiol Dis sourceNot checked — no full text of the source

Forced expression of the N-terminal active fragment of SREBP2 in HD astrocytes reverses neurite outgrowth and synaptic defects in HD neurons.

“while the forced expression of the N-terminal active fragment of SREBP2 in HD astrocytes reverses neurite outgrowth and synaptic defects in HD neurons 22 .”

• cites [22] Valenza, 2015, Disruption of astrocyte-neuron cholesterol cross talk affects neuronal function in Huntington’s disease, Cell Death Differ sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Astrocyte-derived cholesterol is transferred to neurons via ApoE-containing lipoproteins.

“Of note, in adulthood astrocytes are believed to synthetize most cholesterol, which is transferred to the neurons via ApoE-containing lipoproteins 3,10 .”

• cites [3] Mauch, 2001, CNS synaptogenesis promoted by glia-derived cholesterol, Science sourceNot checked — no full text of the source
• cites [10] Nunes, 2018, Decreased content, rate of synthesis and export of cholesterol in the brain of apoE knockout mice, J Bioenerg Biomembr sourceNot checked — no full text of the source

In adulthood, astrocytes synthesize most brain cholesterol.

“Of note, in adulthood astrocytes are believed to synthetize most cholesterol, which is transferred to the neurons via ApoE-containing lipoproteins 3,10 .”

• cites [3] Mauch, 2001, CNS synaptogenesis promoted by glia-derived cholesterol, Science sourceNot checked — no full text of the source
• cites [10] Nunes, 2018, Decreased content, rate of synthesis and export of cholesterol in the brain of apoE knockout mice, J Bioenerg Biomembr sourceNot checked — no full text of the source

Glial SREBP2 participates in HD brain pathogenesis in vivo.

“We conclude that glial SREBP2 participates in HD brain pathogenesis in vivo and that AAV-based delivery of SREBP2 to astrocytes counteracts key features of HD.”

Reduced activity of SREBP2 in the presence of mutant HTT underlies the reduced cholesterol biosynthesis in HD, likely through sequestration of the SREBP2/importin b complex required for nuclear import.

“The underlying molecular mechanism implicates reduced activity of SREBP2 in the presence of mutant HTT 17,22 , likely through sequestration in the cytoplasm of the SREBP2/importin b complex required for nuclear import 23 .”

• cites [17] Valenza, 2005, Dysfunction of the cholesterol biosynthetic pathway in Huntington’s disease, J Neurosci sourceNot checked yet
• cites [22] Valenza, 2015, Disruption of astrocyte-neuron cholesterol cross talk affects neuronal function in Huntington’s disease, Cell Death Differ sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites [23] Di Pardo, 2020, Mutant huntingtin interacts with the sterol regulatory element-binding proteins and impairs their nuclear import, Hum Mol Genet sourceNot checked — no full text of the source

Enhanced cholesterol biosynthesis may participate in inducing clearance pathways.

“At the neuropathological level, increased hSREBP2 in the striatum of R6/2 mice reduced the number and size of muHTT aggregates, supporting the notion that enhanced cholesterol biosynthesis may participate in inducing clearance pathways, as we recently showed in the striata of HD mice following striatal infusion of cholesterol through osmotic mini-pumps 28 .”

• cites [28] Ayuso, 2010, High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency, Gene Ther sourceNot checked — no full text of the source

In these conditions, neuronal function and morphology are fully reestablished.

“In these conditions, we showed that neuronal function and morphology are fully reestablished 22 .”

• cites [22] Valenza, 2015, Disruption of astrocyte-neuron cholesterol cross talk affects neuronal function in Huntington’s disease, Cell Death Differ sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

Clearance may also occur by a non-cell autonomous mechanism because the transgene was delivered to astrocytes.

“The fact that in the current study the transgene was delivered to astrocytes suggests that clearance may also occur by a non-cell autonomous mechanism.”

hSREBP2 over-expression in glial cells carries out its beneficial effect by acting on different aspects of the disease.

“Mechanistically, our findings indicate that hSREBP2 over-expression in glial cells carries out its beneficial effect by acting on different aspects of the disease.”

In a similar gene therapy approach, the gene encoding for CYP46A1 was delivered into striatal neurons of HD mice by use of an AAVrh10 vector.

“In a similar gene therapy approach, the gene encoding for CYP46A1, the enzyme that converts cholesterol into the neuronal cholesterol catabolite 24S-OHC, was delivered into striatal neurons of HD mice by use of an AAVrh10 vector 55,56 .”

• cites [55] Zhao, 2015, Lanosterol reverses protein aggregation in cataracts, Nature sourceNot checked — no full text of the source
• cites [56] Upadhyay, 2018, Lanosterol suppresses the aggregation and cytotoxicity of misfolded proteins linked with neurodegenerative diseases, Mol Neurobiol sourceNot checked — no full text of the source

Similar data were obtained upon cholesterol delivery to the brain of this mouse model via brain-permeable polymeric nanoparticles or mini-pumps.

“Similar data were obtained upon cholesterol delivery to the brain of this mouse model via brain-permeable polymeric nanoparticles or mini-pumps 27,28 .”

• cites [27] Arsic, 2003, Induction of functional neovascularization by combined VEGF and angiopoietin-1 gene transfer using AAV vectors, Mol Ther sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites [28] Ayuso, 2010, High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency, Gene Ther sourceNot checked — no full text of the source

Cholesterol synthesis and catabolism are in balance in the HD brain.

“This was accompanied by an increase in 24S-OHC (Fig. 2K), consistent with the notion that cholesterol synthesis and catabolism are in balance in the HD brain 21 .”

• cites [21] Shankaran, 2017, Early and brain region-specific decrease of de novo cholesterol biosynthesis in Huntington’s disease: A cross-validation study in Q175 knock-in mice, Neurobiol Dis sourceNot checked — no full text of the source

HD neurons, which receive more cholesterol from hSREBP2 over-expressing astrocytes, increase DRD2 expression.

“Importantly, we show that HD neurons, which receive more cholesterol from hSREBP2 over-expressing astrocytes, increase DRD2 expression and are quantifiable through two-photon imaging.”

R6/2 mice do not show overt signs of neuronal loss.

“Although R6/2 mice (similar to other HD mouse models) do not show overt signs of neuronal loss 26 , the downregulation of Drd2 transcript in striatal MSNs is an established early mark of disease progression that has been proposed as a sensitive measure of the effects of therapeutics 37 .”

• cites [26] Zoumi, 2005, Spatial distribution and function of sterol regulatory element-binding protein 1a and 2 homo- and heterodimers by in vivo two-photon imaging and spectroscopy fluorescence resonance energy transfer, Mol Cell Biol sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

hSREBP2 delivery to astrocytes in vivo, by enhancing the cholesterol biosynthesis pathway, can revert molecular, biochemical, electrophysiological, and behavioral abnormalities associated with neuronal dysfunction typically observed in the R6/2 mouse model.

“Accordingly, we show that hSREBP2 delivery to astrocytes in vivo, by enhancing the cholesterol biosynthesis pathway, can revert molecular, biochemical, electrophysiological, and behavioral abnormalities associated with neuronal dysfunction typically observed in the R6/2 mouse model.”

Within the striatum, astrocyte-neuron signaling may be synapse-and cell-specific by acting at least on the DRD2-MSNs.

“These findings also suggest that within the striatum, the astrocyte-neuron signaling may be synapse-and cell-specific by acting at least on the DRD2-MSNs, as recently proposed 53 .”

• cites [53] Linetti, 2010, Cholesterol reduction impairs exocytosis of synaptic vesicles, J Cell Sci sourceNot checked — no full text of the source

The observed rescue in DRD2-MSNs, and in other synaptic features, may be favored by a closer contact between neurons and astrocytes, which is reduced in R6/2 astrocytes.

“The observed rescue in DRD2-MSNs, and in other synaptic features, may be favored by a closer contact between neurons and astrocytes, which is reduced in R6/2 astrocytes 54 .”

• cites [54] Thiele, 2000, Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles, Nat Cell Biol sourceNot checked — no full text of the source

Over-expression of hSREBP2 in astrocytes allows DRD2-MSNs to regain DRD2 expression.

“Of note, the number of DRD2-MSNs measured in the infused striata of Drd2-R6/2-hBP2 was similar to that quantified in Drd2-wt-Tom mice (Fig. 4F and G), indicating that over-expression of hSREBP2 in astrocytes allows these neurons to regain DRD2 expression and, supposedly, functionality.”

• AssumptionRests on an assumption: CLARITY allows an accurate measurement of the total number of MSNs expressing DRD2 in a large volume of the vector-injected striatum without dissociation.

The mechanisms by which stimulation of cholesterol biosynthesis counteracts synaptic dysfunction in HD mice do not involve major transcriptional changes.

“Our current data also suggest that the mechanisms by which stimulation of cholesterol biosynthesis counteracts synaptic dysfunction in HD mice do not involve major transcriptional changes since mRNA levels of key synaptic genes, which are known to be altered in HD, are unaffected by hSREBP2 delivery.”

hSREBP2 over-expression in glial cells rescues specific neurochemical features in MSNs.

“Importantly, hSREBP2 over-expression in glial cells rescues specific neurochemical features in MSNs.”

Unilateral AAV-delivery of SREBP2 also ameliorated motor defects in R6/2 mice.

“Unilateral AAV-delivery of SREBP2 also ameliorated motor defects in R6/2 mice, suggesting that this approach is able to produce the optimal content of newly synthetized cholesterol needed to partially restore motor-dependent circuits.”

Unilateral striatal AAV-delivery of SREBP2 in glial cells is sufficient to completely rescue cognitive decline in R6/2 mice.

“We show that unilateral striatal AAV-delivery of SREBP2 in glial cells is sufficient to completely rescue cognitive decline in R6/2 mice.”

hSREBP2 delivery preferentially stimulates the Bloch pathway of cholesterol biosynthesis.

“hSREBP2 delivery in glial cells, although unable to affect lanosterol and lathosterol levels (Fig. 2H-I), led to a significant increase in desmosterol in the striata of R6/2-hBP2 mice compared with R6/2-Tom mice (Fig. 2J), suggesting a preferential stimulation of the Bloch pathway of cholesterol biosynthesis which is more related to astrocytes 29 (Fig. 2A).”

• cites [29] Zentilin, 2001, Involvement of cellular double-stranded DNA break binding proteins in processing of the recombinant adeno-associated virus genome, J Virol sourceNot checked — no full text of the source

Cholesterol synthesis is downregulated in aging astrocytes.

“Moreover, cholesterol synthesis is downregulated in aging astrocytes 41,42 , suggesting that lack of cholesterol production in these cells may contribute to the synaptic and cognitive defects observed in aging.”

• cites [41] Cepeda, 2004, Increased GABAergic function in mouse models of Huntington’s disease: Reversal by BDNF, J Neurosci Res sourceNot checked — no full text of the source
• cites [42] DiFiglia, 1997, Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain, Science sourceNot checked — no full text of the source

Cholesterol produced from astrocytes in vivo is a critical player in memory recognition in the HD context.

“Here, we provide further evidence that cholesterol produced from astrocytes in vivo is also a critical player in memory recognition in the HD context.”

This approach is able to produce the optimal content of newly synthetized cholesterol needed to partially restore motor-dependent circuits.

“Unilateral AAV-delivery of SREBP2 also ameliorated motor defects in R6/2 mice, suggesting that this approach is able to produce the optimal content of newly synthetized cholesterol needed to partially restore motor-dependent circuits.”

R6/2 striatal MSNs exhibit altered synaptic transmission.

“As R6/2 striatal MSNs exhibit altered synaptic transmission 30,31 , we performed electrophysiological studies to evaluate whether synaptic dysfunction was reversed in R6/2-hBP2 mice.”

• cites [30] Miyagawa, 2017, Deletion of the virion host shut-off gene enhances neuronal-selective transgene expression from an HSV vector lacking functional IE genes, Mol Ther Meth Clin Dev sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites [31] Paolone, 2018, Personalized needles for microinjections in the rodent brain, J Vis ExpNot matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Genetic deletion of SCAP or SREBP2 in astrocytes of adult mice results in cognitive defects.

“The fact that genetic deletion of SCAP or SREBP2 in astrocytes of adult mice results in cognitive defects 38,39 confirms the role of astrocytes in memory and cognition in vivo, and that altered cross-talk between astrocytes and neurons at synaptic contacts may contribute to cognitive disturbances 40 .”

• cites [38] Birolini, 2020, EMBO Mol Med sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites [39] Pfrieger, 2011, Cholesterol metabolism in neurons and astrocytes, Prog Lipid Res sourceNot checked — no full text of the source

SREBP2 gene transfer to astrocytes may be a therapeutic option to fight core features of this disease.

“Thus, SREBP2 gene transfer to astrocytes may be a therapeutic option to fight core features of this disease.”

By targeting astrocytic cholesterol signaling in the striatum we were able to rescue biochemical, neuropathological, functional, and behavioral features in HD mice.

“By targeting astrocytic cholesterol signaling in the striatum we were able to rescue biochemical, neuropathological, functional, and behavioral features in HD mice.”

Our findings confirm the role of astrocytes as important regulatory cell types in brain function and behavior and in neurodegenerative disorders, including HD.

“In conclusion, our findings confirm the role of astrocytes as important regulatory cell types in brain function and behavior 40,57 and in neurodegenerative disorders, including HD 58,59 .”

• cites [40] Cepeda, 2003, Transient and progressive electrophysiological alterations in the corticostriatal pathway in a mouse model of Huntington’s disease, J Neurosci sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites [57] Lim, 2012, Mitochondrial dynamics and Parkinson’s disease: Focus on parkin, Antioxid Redox Signal sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites [58] Kacher, 2019, CYP46A1 gene therapy deciphers the role of brain cholesterol metabolism in Huntington’s disease, Brain sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.

• cites [59] Andrews, 1999, Huntington’s disease progression. PET and clinical observations, Brain sourceNot checked yet

  Checked before — its earlier verdict is held back until a new check runs.