Evidence that small molecule enhancement of ?-hexosaminidase activity corrects the behavioral phenotype in Dutch APP(E693Q) mice through reduction of ganglioside-bound A?.
ABSTRACT: Certain mutant Alzheimer's amyloid-? (A?) peptides (that is, Dutch mutant APP(E693Q)) form complexes with gangliosides (GA?). These mutant A? peptides may also undergo accelerated aggregation and accumulation upon exposure to GM2 and GM3. We hypothesized that increasing ?-hexosaminidase (?-hex) activity would lead to a reduction in GM2 levels, which in turn, would cause a reduction in A? aggregation and accumulation. The small molecule OT1001 is a ?-hex-targeted pharmacological chaperone with good bioavailability, blood-brain barrier penetration, high selectivity for ?-hex and low cytotoxicity. Dutch APP(E693Q) transgenic mice accumulate oligomeric A? as they age, as well as A? oligomer-dose-dependent anxiety and impaired novel object recognition (NOR). Treatment of Dutch APP(E693Q) mice with OT1001 caused a dose-dependent increase in brain ?-hex levels up to threefold over those observed at baseline. OT1001 treatment was associated with reduced anxiety, improved learning behavior in the NOR task and dramatically reduced GA? accumulation in the subiculum and perirhinal cortex, both of which are brain regions required for normal NOR. Pharmacological chaperones that increase ?-hex activity may be useful in reducing accumulation of certain mutant species of A? and in preventing the associated behavioral pathology.
Project description:Proneurogenic compounds have recently shown promise in some mouse models of Alzheimer's pathology. Antagonists at Group II metabotropic glutamate receptors (Group II mGluR: mGlu2, mGlu3) are reported to stimulate neurogenesis. Agonists at those receptors trigger ?-secretase-inhibitor-sensitive biogenesis of A?42 peptides from isolated synaptic terminals, which is selectively suppressed by antagonist pretreatment. We have assessed the therapeutic potential of chronic pharmacological inhibition of Group II mGluR in Dutch APP (Alzheimer's amyloid precursor protein E693Q) transgenic mice that accumulate Dutch amyloid-? (A?) oligomers but never develop A? plaques. BCI-838 is a clinically well-tolerated, orally bioavailable, investigational prodrug that delivers to the brain BCI-632, the active Group II mGluR antagonist metabolite. Dutch A?-oligomer-forming APP transgenic mice (APP E693Q) were dosed with BCI-838 for 3 months. Chronic treatment with BCI-838 was associated with reversal of transgene-related amnestic behavior, reduction in anxiety, reduction in levels of brain A? monomers and oligomers, and stimulation of hippocampal neurogenesis. Group II mGluR inhibition may offer a unique package of relevant properties as an Alzheimer's disease therapeutic or prophylactic by providing both attenuation of neuropathology and stimulation of repair.
Project description:Recent studies have implicated specific assembly subtypes of ?-amyloid (A?) peptide, specifically soluble oligomers (soA?) as disease-relevant structures that may underlie memory loss in Alzheimer disease. Removing existing soluble and insoluble A? assemblies is thought to be essential for any attempt at stabilizing brain function and slowing cognitive decline in Alzheimer disease. IV immunoglobulin (IVIg) therapies have been shown to contain naturally occurring polyclonal antibodies that recognize conformational neoepitopes of soluble or insoluble A? assemblies including soA?. These naturally occurring polyclonal antibodies have been suggested to underlie the apparent clinical benefits of IVIg. However, direct evidence linking anti-A? antibodies to the clinical bioactivity of IVIg has been lacking.Five-month-old female Dutch APP E693Q mice were treated for 3 months with neat IVIg or with IVIg that had been affinity-depleted over immobilized A? conformers in 1 of 2 assembly states. Memory was assessed in a battery of tests followed by quantification of brain soA? levels using standard anti-soA? antibodies.We provide evidence that NU4-type soA? (NU4-soA?) assemblies accumulate in the brains of Dutch APP E693Q mice and are associated with defects in memory, even in the absence of insoluble A? plaques. Memory benefits were associated with depletion from APP E693Q mouse brain of NU4-soA? and A11-soA? but not OC-type fibrillar A? oligomers.We propose that targeting of specific soA? assembly subtypes may be an important consideration in the therapeutic and/or prophylactic benefit of anti-A? antibody drugs.
Project description:Recent evidence suggests that high molecular weight soluble oligomeric Abeta (oAbeta) assemblies (also known as Abeta-derived diffusible ligands, or ADDLs) may represent a primary neurotoxic basis for cognitive failure in Alzheimer disease (AD). To date, most in vivo studies of oAbeta/ADDLs have involved injection of assemblies purified from the cerebrospinal fluid of human subjects with AD or from the conditioned media of Abeta-secreting cells into experimental animals. We sought to study the bioactivities of endogenously formed oAbeta/ADDLs generated in situ from the physiological processing of human amyloid precursor protein (APP) and presenitin1 (PS1) transgenes.We produced and histologically characterized single transgenic mice overexpressing APP(E693Q) or APP(E693Q) X PS1DeltaE9 bigenic mice. APP(E693Q) mice were studied in the Morris water maze (MWM) task at 6 and 12 months of age. Following the second MWM evaluation, mice were sacrificed, and brains were assayed for Abetatotal, Abeta40, Abeta42, and oAbeta/ADDLs by enzyme-linked immunosorbent assay (ELISA) and were also histologically examined. Based on results from the oAbeta/ADDL ELISA, we assigned individual APP(E693Q) mice to either an undetectable oAbeta/ADDLs group or a readily detectable oAbeta/ADDLs group. A days to criterion (DTC) analysis was used to determine delays in acquisition of the MWM task.Both single transgenic and bigenic mice developed intraneuronal accumulation of APP/Abeta, although only APP(E693Q) X PS1Delta9 bigenic mice developed amyloid plaques. The APP(E693Q) mice did not develop amyloid plaques at any age studied, up to 30 months. APP(E693Q) mice were tested for spatial learning and memory, and only 12-month-old APP(E693Q) mice with readily detectable oAbeta/ADDLs displayed a significant delay in acquisition of the MWM task when compared to nontransgenic littermates.These data suggest that cerebral oAbeta/ADDL assemblies generated in brain in situ from human APP transgenes may be associated with cognitive impairment. We propose that a DTC analysis may be a sensitive method for assessing the cognitive impact in mice of endogenously generated oligomeric human Abeta assemblies. ANN NEUROL 2010.
Project description:Lysosomal beta-hexosaminidase A (Hex A) is essential for the degradation of GM2 gangliosides in the central and peripheral nervous system. Accumulation of GM2 leads to severely debilitating neurodegeneration associated with Tay-Sachs disease (TSD), Sandoff disease (SD) and AB variant. Here, we present the X-ray crystallographic structure of Hex A to 2.8 A resolution and the structure of Hex A in complex with NAG-thiazoline, (NGT) to 3.25 A resolution. NGT, a mechanism-based inhibitor, has been shown to act as a chemical chaperone that, to some extent, prevents misfolding of a Hex A mutant associated with adult onset Tay Sachs disease and, as a result, increases the residual activity of Hex A to a level above the critical threshold for disease. The crystal structure of Hex A reveals an alphabeta heterodimer, with each subunit having a functional active site. Only the alpha-subunit active site can hydrolyze GM2 gangliosides due to a flexible loop structure that is removed post-translationally from beta, and to the presence of alphaAsn423 and alphaArg424. The loop structure is involved in binding the GM2 activator protein, while alphaArg424 is critical for binding the carboxylate group of the N-acetyl-neuraminic acid residue of GM2. The beta-subunit lacks these key residues and has betaAsp452 and betaLeu453 in their place; the beta-subunit therefore cleaves only neutral substrates efficiently. Mutations in the alpha-subunit, associated with TSD, and those in the beta-subunit, associated with SD are discussed. The effect of NGT binding in the active site of a mutant Hex A and its effect on protein function is discussed.
Project description:The GM2-gangliosidoses are neurological diseases causing premature death, thus developing effective treatment protocols is urgent. GM2-gangliosidoses result from deficiency of a lysosomal enzyme ?-hexosaminidase (Hex) and subsequent accumulation of GM2 gangliosides. Genetic changes in HEXA, encoding the Hex ? subunit, or HEXB, encoding the Hex ? subunit, causes Tay-Sachs disease and Sandhoff disease, respectively. Previous studies have showed that a modified human Hex µ subunit (HEXM) can treat both Tay-Sachs and Sandhoff diseases by forming a homodimer to degrade GM2 gangliosides. To this end, we applied this HEXM subunit in our PS813 gene editing system to treat neonatal Sandhoff mice. Through AAV delivery of the CRISPR system, a promoterless HEXM cDNA will be integrated into the albumin safe harbor locus, and lysosomal enzyme will be expressed and secreted from edited hepatocytes. 4 months after the i.v. of AAV vectors, plasma MUGS and MUG activities reached up to 144- and 17-fold of wild-type levels (n?=?10, p?<?0.0001), respectively. More importantly, MUGS and MUG activities in the brain also increased significantly compared with untreated Sandhoff mice (p?<?0.001). Further, HPLC-MS/MS analysis showed that GM2 gangliosides in multiple tissues, except the brain, of treated mice were reduced to normal levels. Rotarod analysis showed that coordination and motor memory of treated mice were improved (p?<?0.05). Histological analysis of H&E stained tissues showed reduced cellular vacuolation in the brain and liver of treated Sandhoff mice. These results demonstrate the potential of developing a treatment of in vivo genome editing for Tay-Sachs and Sandhoff patients.
Project description:Sandhoff disease (SD) is a lysosomal storage disease, caused by loss of ?-hexosaminidase (HEX) activity resulting in the accumulation of ganglioside GM2. There are shared features between SD and Parkinson's disease (PD). ?-synuclein (aSYN) inclusions, the diagnostic hallmark sign of PD, are frequently found in the brain in SD patients and HEX knockout mice, and HEX activity is reduced in the substantia nigra in PD. In this study, we biochemically demonstrate that HEX deficiency in mice causes formation of high-molecular weight (HMW) aSYN and ubiquitin in the brain. As expected from HEX enzymatic function requirements, overexpression in vivo of HEXA and B combined, but not either of the subunits expressed alone, increased HEX activity as evidenced by histochemical assays. Biochemically, such HEX gene expression resulted in increased conversion of GM2 to its breakdown product GM3. In a neurodegenerative model of overexpression of aSYN in rats, increasing HEX activity by AAV6 gene transfer in the substantia nigra reduced aSYN embedding in lipid compartments and rescued dopaminergic neurons from degeneration. Overall, these data are consistent with a paradigm shift where lipid abnormalities are central to or preceding protein changes typically associated with PD.
Project description:To find a new biomarker of Tay-Sachs disease and Sandhoff disease. The lyso-GM2 ganglioside (lyso-GM2) levels in the brain and plasma in Sandhoff mice were measured by means of high performance liquid chromatography and the effect of a modified hexosaminidase (Hex) B exhibiting Hex A-like activity was examined. Then, the lyso-GM2 concentrations in human plasma samples were determined. The lyso-GM2 levels in the brain and plasma in Sandhoff mice were apparently increased compared with those in wild-type mice, and they decreased on intracerebroventricular administration of the modified Hex B. The lyso-GM2 levels in plasma of patients with Tay-Sachs disease and Sandhoff disease were increased, and the increase in lyso-GM2 was associated with a decrease in Hex A activity. Lyso-GM2 is expected to be a potential biomarker of Tay-Sachs disease and Sandhoff disease.
Project description:BACKGROUND:GM2-gangliosidosis is a fatal neurodegenerative lysosomal storage disease (LSD) caused by deficiency of either β-hexosaminidase A (Hex-A) and β-hexosaminidase B (Hex-B) together, or the GM2 activator protein. Clinical signs can be variable and are not pathognomonic for the specific, causal deficiency. OBJECTIVES:To characterize the phenotype and genotype of GM2-gangliosidosis disease in an affected dog. ANIMALS:One affected Shiba Inu and a clinically healthy dog. METHODS:Clinical and neurologic evaluation, brain magnetic resonance imaging (MRI), assays of lysosomal enzyme activities, and sequencing of all coding regions of HEXA, HEXB, and GM2A genes. RESULTS:A 14-month-old, female Shiba Inu presented with clinical signs resembling GM2-gangliosidosis in humans and GM1-gangliosidosis in the Shiba Inu. Magnetic resonance imaging (MRI) of the dog's brain indicated neurodegenerative disease, and evaluation of cerebrospinal fluid (CSF) identified storage granules in leukocytes. Lysosomal enzyme assays of plasma and leukocytes showed deficiencies of Hex-A and Hex-B activities in both tissues. Genetic analysis identified a homozygous, 3-base pair deletion in the HEXB gene (c.618-620delCCT). CONCLUSIONS AND CLINICAL IMPORTANCE:Clinical, biochemical, and molecular features are characterized in a Shiba Inu with GM2-gangliosidosis. The deletion of 3 adjacent base pairs in HEXB predicts the loss of a leucine residue at amino acid position 207 (p.Leu207del) supporting the hypothesis that GM2-gangliosidosis seen in this dog is the Sandhoff type. Because GM1-gangliosidosis also exists in this breed with almost identical clinical signs, genetic testing for both GM1- and GM2-gangliosidosis should be considered to make a definitive diagnosis.
Project description:Mounting evidence suggests that soluble oligomers of amyloid-? (oA?) represent the pertinent synaptotoxic form of A? in sporadic Alzheimer's disease (AD); however, the mechanistic links between oA? and synaptic degeneration remain elusive. Most in vivo experiments to date have been limited to examining the toxicity of oA? in mouse models that also possess insoluble fibrillar A? (fA?), and data generated from these models can lead to ambiguous interpretations. Our goal in the present study was to examine the effects of soluble oA? on neuronal and synaptic structure in the amyloid precursor protein (APP) E693Q ("Dutch") mouse model of AD, which develops intraneuronal accumulation of soluble oA? with no detectable plaques in AD-relevant brain regions. We performed quantitative analyses of neuronal pathology, including dendrite morphology, spine density, and synapse ultrastructure in individual hippocampal CA1 neurons.When assessing neuronal morphology and complexity we observed significant alterations in apical but not in basal dendritic arbor length in Dutch mice compared to wild type. Moreover, Dutch mice exhibited a significant decrease in dendritic arborization with a decrease in dendritic length and number of intersections at 120 ?m and 150 ?m from the soma, respectively. We next examined synaptic parameters and found that while there were no differences in overall synaptic structure, Dutch mice displayed a significant reduction in the post-synaptic density (PSD) length of synapses on mushroom spines, in comparison to wild type littermates.The structural alterations to individual neurons in Dutch mice along with the changes in larger dendritic spines support the A? oligomer hypothesis, which postulates that the early cognitive impairments that occur in AD are attributed to the accumulation of soluble oA? first affecting at the synaptic level with subsequent structural disturbances and cellular degeneration.
Project description:GM2 gangliosidosis is a family of three genetic neurodegenerative disorders caused by the accumulation of GM2 ganglioside (GM2) in neuronal tissue. Two of these are due to the deficiency of the heterodimeric (?-?), "A" isoenzyme of lysosomal ?-hexosaminidase (HexA). Mutations in the ?-subunit (encoded by HEXA) lead to Tay-Sachs disease (TSD), whereas mutations in the ?-subunit (encoded by HEXB) lead to Sandhoff disease (SD). The third form results from a deficiency of the GM2 activator protein (GM2AP), a substrate-specific cofactor for HexA. In their infantile, acute forms, these diseases rapidly progress with mental and psychomotor deterioration resulting in death by approximately 4 years of age. After gene transfer that overexpresses one of the deficient subunits, the amount of HexA heterodimer formed would empirically be limited by the availability of the other endogenous Hex subunit. The present study used a new variant of the human HexA ?-subunit, ?, incorporating critical sequences from the ?-subunit that produce a stable homodimer (HexM) and promote functional interactions with the GM2AP- GM2 complex. We report the design of a compact adeno-associated viral (AAV) genome using a synthetic promoter-intron combination to allow self-complementary (sc) packaging of the HEXM gene. Also, a previously published capsid mutant, AAV9.47, was used to deliver the gene to brain and spinal cord while having restricted biodistribution to the liver. The novel capsid and cassette design combination was characterized in vivo in TSD mice for its ability to efficiently transduce cells in the central nervous system when delivered intravenously in both adult and neonatal mice. This study demonstrates that the modified HexM is capable of degrading long-standing GM2 storage in mice, and it further demonstrates the potential of this novel scAAV vector design to facilitate widespread distribution of the HEXM gene or potentially other similar-sized genes to the nervous system.