22 research outputs found
Intracranial injection of AAV expressing NEP but not IDE reduces amyloid pathology in APP+PS1 transgenic mice
The accumulation of β-amyloid peptides in the brain has been recognized as an essential factor in Alzheimer\u27s disease pathology. Several proteases, including Neprilysin (NEP), endothelin converting enzyme (ECE), and insulin degrading enzyme (IDE), have been shown to cleave β-amyloid peptides (Aβ). We have previously reported reductions in amyloid in APP+PS1 mice with increased expression of ECE. In this study we compared the vector-induced increased expression of NEP and IDE. We used recombinant adeno-associated viral vectors expressing either native forms of NEP (NEP-n) or IDE (IDE-n), or engineered secreted forms of NEP (NEP-s) or IDE (IDE-s). In a six-week study, immunohistochemistry staining for total Aβ was significantly decreased in animals receiving the NEP-n and NEP-s but not for IDE-n or IDE-s in either the hippocampus or cortex. Congo red staining followed a similar trend revealing significant decreases in the hippocampus and the cortex for NEP-n and NEP-s treatment groups. Our results indicate that while rAAV-IDE does not have the same therapeutic potential as rAAV-NEP, rAAV-NEP-s and NEP-n are effective at reducing amyloid loads, and both of these vectors continue to have significant effects nine months post-injection. As such, they may be considered reasonable candidates for gene therapy trials in AD
Recombinant AAV Gene Therapy and Delivery
Alzheimer\u27s disease (AD), first characterized in the early 20th century, is a common form of dementia which can occur as a result of genetic mutations in the genes encoding presenilin 1, presenilin 2, or amyloid precursor protein (APP). These genetic alterations can accelerate the pathological characteristics of AD, including the formation of extracellular neuritic plaques composed of amyloid beta peptides and the formation of intracellular neurofibrillary tangles consisting of hyperphosphorylated tau protein. Ultimately, AD results in gross neuron loss in the brain which is evidenced clinically as a progressive decline in mental capacity. A strong body of scientific evidence has previously demonstrated that the driving factor in the pathogenesis of AD is potentially the accumulation of Aß peptides in the brain. Thus, reduction of Aß deposition is a major therapeutic strategy in the treatment of AD. Recently it has been suggested that Aß accumulation in the brain is modulated, not only by Aß production, but also by its degradation. Several important studies have demonstrated that Aß degradation is modulated by several endogenous zinc metalloproteases shown to have amyloid degrading capabilities. These endogenous proteases include neprilysin (NEP), endothelin converting enzyme (ECE), insulin degrading enzyme (IDE) and matrix metalloprotease 9 (MMP9). In this investigation we study the effects of upregulating expression of several of these proteases through administration of recombinant adeno-associated viral vector (rAAV) containing both endogenous and synthetic genes for ECE and NEP on amyloid deposition in amyloid precursor protein (APP) plus presenilin-1 (PS1) transgenic mice. rAAV administration directly into the brain resulted in increased expression of ECE and NEP and a substantial decrease in amyloid pathology. We were able to significantly increase the area of viral distribution by using novel delivery methods resulting in increased gene expression and distribution.
These data support great potential of gene therapy as a method of treatment for neurological diseases. Optimization of gene transfer methods aimed at a particular cell type and brain region in the CNS can be accomplished using AAV serotype specificity and novel delivery techniques leading to successful gene transduction thus providing a promising therapeutic avenue through which to treat AD
Recombinant AAV Gene Therapy and Delivery
Alzheimer\u27s disease (AD), first characterized in the early 20th century, is a common form of dementia which can occur as a result of genetic mutations in the genes encoding presenilin 1, presenilin 2, or amyloid precursor protein (APP). These genetic alterations can accelerate the pathological characteristics of AD, including the formation of extracellular neuritic plaques composed of amyloid beta peptides and the formation of intracellular neurofibrillary tangles consisting of hyperphosphorylated tau protein. Ultimately, AD results in gross neuron loss in the brain which is evidenced clinically as a progressive decline in mental capacity. A strong body of scientific evidence has previously demonstrated that the driving factor in the pathogenesis of AD is potentially the accumulation of Aß peptides in the brain. Thus, reduction of Aß deposition is a major therapeutic strategy in the treatment of AD. Recently it has been suggested that Aß accumulation in the brain is modulated, not only by Aß production, but also by its degradation. Several important studies have demonstrated that Aß degradation is modulated by several endogenous zinc metalloproteases shown to have amyloid degrading capabilities. These endogenous proteases include neprilysin (NEP), endothelin converting enzyme (ECE), insulin degrading enzyme (IDE) and matrix metalloprotease 9 (MMP9). In this investigation we study the effects of upregulating expression of several of these proteases through administration of recombinant adeno-associated viral vector (rAAV) containing both endogenous and synthetic genes for ECE and NEP on amyloid deposition in amyloid precursor protein (APP) plus presenilin-1 (PS1) transgenic mice. rAAV administration directly into the brain resulted in increased expression of ECE and NEP and a substantial decrease in amyloid pathology. We were able to significantly increase the area of viral distribution by using novel delivery methods resulting in increased gene expression and distribution.
These data support great potential of gene therapy as a method of treatment for neurological diseases. Optimization of gene transfer methods aimed at a particular cell type and brain region in the CNS can be accomplished using AAV serotype specificity and novel delivery techniques leading to successful gene transduction thus providing a promising therapeutic avenue through which to treat AD
Characterization of HTT inclusion size, location, and timing in the zQ175 mouse model of Huntington's disease: an in vivo high-content imaging study.
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide repeat expansion in the huntingtin gene. Major pathological hallmarks of HD include inclusions of mutant huntingtin (mHTT) protein, loss of neurons predominantly in the caudate nucleus, and atrophy of multiple brain regions. However, the early sequence of histological events that manifest in region- and cell-specific manner has not been well characterized. Here we use a high-content histological approach to precisely monitor changes in HTT expression and characterize deposition dynamics of mHTT protein inclusion bodies in the recently characterized zQ175 knock-in mouse line. We carried out an automated multi-parameter quantitative analysis of individual cortical and striatal cells in tissue slices from mice aged 2-12 months and confirmed biochemical reports of an age-associated increase in mHTT inclusions in this model. We also found distinct regional and subregional dynamics for inclusion number, size and distribution with subcellular resolution. We used viral-mediated suppression of total HTT in the striatum of zQ175 mice as an example of a therapeutically-relevant but heterogeneously transducing strategy to demonstrate successful application of this platform to quantitatively assess target engagement and outcome on a cellular basis
Study 1: Distribution of HA expression in the hippocampus (A–D) and frontal cortex (E–H) following intracranial administration of rAAV vectors, detected using an anti-HA antibody.
<p>Panels A & E show NEP-n treated animals; panels B & F show NEP-m treated animals; panels C & G show NEP-s treated animals. Panels D & H show no positive staining in the contralateral uninjected left hippocampus and left anterior cortex, respectively. Scale bar = 120 µm.</p
Study 3: CD68 (A-C) and CD45 (D-F) immunostaining is observed throughout the hippocampus of study mice.
<p>In the hippocampus, no significant differences are observed in the amount of total CD68 staining between the NEP-n, NEP-s, and NEP-m groups, but CD45 staining in NEP-s treated mice is greater than CD45 staining in NEP-m treated mice. Scale bar = 50 µm. Panels G and H present ANOVA analysis of the ratio of CD68 to congophilic staining, and of the ratio of quantitated CD45 to congophilic staining, respectively, in the hippocampus of study mice. The (*) indicates significance compared to NEP-m mice with p<0.05, and the (<sup>∧</sup>) indicates significance compared to Tg control mice with p<0.05 or p<0.01 (°).Tg control (n = 9), NEP-n (n = 4), NEP-m (n = 7), NEP-s (n = 6).</p
Study 3: Aβ immunostaining is observed in mice throughout both the hippocampus (A, B and C) and anterior cortex (D, E and F).
<p>Aβ staining in the hippocampus of animals that received intracranial injections of rAAV- NEP-n (B) is reduced compared to staining in those animals that received injections of control vector rAAV- NEP-m (A). Aβ staining in the anterior cortex of mice that received intracranial injections of rAAV- NEP-n (E) or NEP-s (F) is also reduced compared to staining in mice that received control vector rAAV- NEP-m (D). Scale bar = 50 µm. Quantification of percent area of positive total Aβ staining is shown in and H. The (*) indicates significance compared to NEP-m mice with p<0.05; the (<sup>∧</sup>) indicates significance compared to Tg control mice with p<0.05. The (#) indicates significance compared to NEP-m mice with p<0.01. Tg control (n = 9), NEP-n (n = 4), NEP-m (n = 7), NEP-s (n = 6).</p
Study 2: Congophilic staining is observed in mice throughout both ipsilateral hippocampus (A, B and C) and ipsilateral anterior cortex (D, E and F).
<p>Congophilic staining in the hippocampus of animals that received intracranial injections of rAAV- IDE-n (B) or IDE-s (C) is unchanged compared to staining in those animals that received injections of control vector rAAV- GFP (A). Congophilic staining in the anterior cortex of mice that received intracranial injections of rAAV- IDE-n (E) or NEP-s (F) is also unchanged compared to staining in mice that received control vector rAAV- GFP(D). Scale bar = 200 µm. Quantification of percent area of positive total congophilic staining is shown in G and H (hemisphere ipsilateral to injection sites). No significant differences were found. n = 8/group.</p
Study 2: Aβ immunostaining is observed in mice throughout both the ipsilateral hippocampus (A, B and C) and ipsilateral anterior cortex (D, E and F).
<p>Aβ staining in the hippocampus of animals that received intracranial injections of rAAV- IDE-n (B) or IDE-s (C) is unchanged compared to staining in those animals that received injections of control vector rAAV- GFP (A). Aβ staining in the anterior cortex of mice that received intracranial injections of rAAV- IDE-n (E) or IDE-s (F) is also unchanged compared to staining in mice that received control vector rAAV- GFP (D). Scale bar = 120 µm. Quantification of percent area of positive staining is shown in the hippocampus (G) and in the anterior cortex (H). No significant differences were observed. n = 8/group.</p
Study 3: Congophilic staining is observed in mice throughout both the hippocampus (A, B and C) and anterior cortex (D, E and F).
<p>Congophilic staining in the hippocampus of animals that received intracranial injections of rAAV- NEP-n (B) is reduced compared to staining in those animals that received injections of control vector rAAV- NEP-m (A). Congophilic staining in the anterior cortex of mice that received intracranial injections of rAAV- NEP-n (E) or NEP-s (F) is also reduced compared to staining in mice that received control vector rAAV- NEP-m (D). Scale bar = 50 µm. Quantification of percent area of positive total Aβ staining is shown in G and H. The (*) indicates significance compared to NEP-m mice with p<0.05, the (<sup>∧</sup>) indicates significance compared to Tg control mice with p<0.05, and the (°) indicates significance compared to Tg control mice with p<0.01. Tg control (n = 9), NEP-n (n = 4), NEP-m (n = 7), NEP-s (n = 6).</p