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Breakup of amyloid plaques

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Small Molecule EPPS Breaks Up Amyloid Plaques

One of the hallmarks of Alzheimer’s disease has been the generation of Amyloid-β (Aβ) oligomers, fibrils, and ultimately plaques. It is currently contended whether these plaques are a cause of Alzheimer’s disease and related mental deficits, or merely an effect. Researchers at the Korea Institute of Science and Technology have demonstrated in vivo formation and disaggregation of Aβ plaques. They previously reported small ionic molecules which could accelerate the formation of Aβ plaques. Six small molecules which inhibited aggregate formation were discovered at the same time. One of these molecules, 4-(2-hydroxyethyl)-1-piperazinepropanesulphonic acid (EPPS), works as a therapeutic in a Alzheimer’s mouse model. EPPS was found to be both orally available and cross the blood brain barrier where it directly binds to Aβ plaques. Double transgenic mice , APPswe/PS1-dE9 (amyloid precursor protein/presenilin protein 1) mice were administered EPPS in their drinking water for 3.5 months and compared to non-treated transgenic controls. EPPS treated mice both improved from their baseline and out-performed transgenic controls in both the Morris water maze and contextual fear response tests. Immunofluorescent staining of matched brain regions demonstrated elimination of Aβ plaques in the hippocampus of EPPS treated mice. Further study is required to completely understand the mechanism by which EPPS disaggregates the Aβ plaques. This study demonstrates the cause and effects Aβ plaque generation, and subsequent removal, has on Alzheimer’s disease related cognitive function. Should the effect transfer to humans, this could prove a significant discovery for the treatment of Alzheimer’s disease.

 

Kim, et al. (October, 2015) EPPS rescues hippocampus-dependent cognitive deficits in APP/PS1 ice by disaggregation of amyloid-b oligomers and plaques Nature Communications

 

EPPS  rescues hippocampus-dependent cognitive deficits in APP/PS1 mice by disaggregation of amyloid-β oligomers and plaques

Hye Yun Kim, Hyunjin Vincent Kim, Seonmi Jo, C. Justin Lee, Seon Young Choi, Dong Jin Kim & YoungSoo Kim

Nature Communications 2016; 6(8997)     http://dx.doi.org:/10.1038/ncomms9997

Alzheimer’s disease (AD) is characterized by the transition of amyloid-β (Aβ) monomers into toxic oligomers and plaques. Given that Aβ abnormality typically precedes the development of clinical symptoms, an agent capable of disaggregating existing Aβ aggregates may be advantageous. Here we report that a small molecule, 4-(2-hydroxyethyl)-1-piperazinepropanesulphonic acid (EPPS), binds to Aβ aggregates and converts them into monomers. The oral administration of EPPS substantially reduces hippocampus-dependent behavioural deficits, brain Aβ oligomer and plaque deposits, glial γ-aminobutyric acid (GABA) release and brain inflammation in an Aβ-overexpressing, APP/PS1 transgenic mouse model when initiated after the development of severe AD-like phenotypes. The ability of EPPS to rescue Aβ aggregation and behavioural deficits provides strong support for the view that the accumulation of Aβ is an important mechanism underlying AD.

 

During Alzheimer’s disease (AD) pathogenesis, amyloid-β (Aβ) monomers aberrantly aggregate into toxic oligomers, fibrils and eventually plaques. The concentration of misfolded Aβ species highly correlates with the severity of neurotoxicity and inflammation that leads to neurodegeneration in AD^{1, 2, 3}. Accordingly, substantial efforts have been devoted to reducing Aβ levels, including methods to prevent the production and aggregation of Aβ^{4, 5, 6, 7}. Although these approaches effectively prevent the de novo formation of Aβ aggregates, existing Aβ oligomers and plaques will still remain in the patient’s brain^{8, 9, 10}. Thus, the desirable effects of Aβ inhibitors may be expected when administered before a patient develops toxic Aβ deposits^{5, 6, 7}. However, in AD patients with mild-to-moderate symptoms, anti-amyloidogenic agents have not yielded expected outcomes, which may be due to the incomplete removal of pre-existing Aβ aggregates¹¹. As Aβ typically begins to aggregate long before the onset of AD symptoms, interventions specifically aimed at disaggregating existing plaques and oligomers may constitute a useful approach to AD treatment, perhaps in parallel with agents aimed at inhibiting aggregate formation^{8, 9, 10, 11, 12}.

 

Result highlights  

EPPS reduces Aβ-aggregate-induced memory deficits in mice

Figure 1: EPPS ameliorates Aβ-induced memory deficits in mice.

 

(a) Time course of the experiments. (b) Intracerebroventricular (i.c.v.) injection site brain schematic diagram. (c) Pretreated effects of EPPS on Aβ-aggregate-induced memory deficits observed by the % alternation on the Y-maze. EPPS, 0 (n=10), 30 (n=9) or 100 mg kg⁻¹ per day (n=10), was orally given to 8.5-week-old ICR male mice for 1 week; then, vehicle (10% DMSO in PBS, n=10) or Aβ aggregates (50 pmol per 10% DMSO in PBS; Supplementary Fig. 1A) were injected into the intracerebroventricular region (P=0.022). (d) Co-treated effects of EPPS on Aβ-aggregate-induced memory deficits observed by the % alternation on the Y-maze. Male, 8.5-week-old ICR mice received an injection of vehicle (n=9) or Aβ aggregates into the intracerebroventricular region, and then EPPS, 0 (n=10), 30 (n=10) or 100 mg kg⁻¹ per day (n=10), was orally given to these mice for 5 days. From the top, P=0.003, 0.006, 0.015. The error bars represent the s.e.m. One-way analysis of variance followed by Bonferroni’s post-hoc comparisons tests were performed in all statistical analyses. (*P<0.05, **P<0.01, ***P<0.001; other comparisons were not significant).

 

EPPS is orally safe and penetrates the blood–brain barrier

Orally administered EPPS rescues cognitive deficits in APP/PS1 mice

 

Figure 2: EPPS rescues hippocampus-dependent cognitive deficits.

http://www.nature.com/ncomms/2015/151208/ncomms9997/images_article/ncomms9997-f2.jpg

 

Figure 3: EPPS does not affect synaptic plasticity in mice.

http://www.nature.com/ncomms/2015/151208/ncomms9997/images_article/ncomms9997-f3.jpg

 

Figure 4: EPPS disaggregates Aβ plaques and oligomers in APP/PS1 mice.

APP/PS1 mice and WTs from the aforementioned behavioural tests were killed and subjected to brain analyses. EPPS, 0 (TG(−), male, n=15), 10 (TG(+), male, n=11) or 30 mg kg^-1 per day (TG(++), male,n=8), was orally given to 10.5-month-old APP/PS1 for 3.5 months and their brains were compared with age-matched WT brains (WT(−), male, n=16). (a) ThS-stained Aβ plaques in whole brains (scale bars, 1 mm) and the hippocampal region (scale bars, 200 μm) of each group. The mouse brain schematic diagram was created by authors (green and red boxes: regions of brain images, a and f, respectively). (b) Number or area of plaques normalized (%) to the level in 10.5-month-old TG mice. Plaque number: P-values compared with TG (male, 10.5-month-old) are all <0.0001 (#). P-values compared with TG(−) (male, 14-month-old) are all <0.0001 (*). Plaque area: P-values compared with TG (male, 10.5-month-old) are all <0.0001 (#). P-values compared with TG(−) (male, 14-month-old) are all <0.0001 (*). (c–e) Aβ-insoluble and -soluble fractions analyses from brain lysates. (c) Sandwich ELISA of Aβ-insoluble fractions. Hippocampus: all P<0.0001; cortex: P=0.004, 0.046. (d) Sandwich ELISA of Aβ-soluble fractions. (e) Dot blotting of the total Aβ (anti-Aβ: 6E10, also recognizes APP) and oligomers (anti-amyloidogenic protein oligomer: A11). (f) Histochemical analyses of Aβ deposition. Aβs were stained with the 6E10 antibody and ThS. Aβ plaques (first row): green; all Aβs (second row): red; 4,6-diamidino-2-phenylindole (DAPI): blue (as a location indicator). The third and bottom rows show merged images of plaques and Aβs, and plaques and Aβs with DAPI staining. Scale bars, 50 μm. (g) Western blotting analyses of APP expression in hippocampal and cortical lysates (detected at ~100 kDa by 6E10 antibody). Densitometry (see Supplementary Fig. 3A). Full version (see Supplementary Fig. 7). The error bars represent the s.e.m. One-way analysis of variance followed by Bonferroni’s post-hoc comparisons tests were performed in all statistical analyses (*P<0.05, **P<0.01, ***P<0.001, ^#P<0.05, ^##P<0.01,^###P<0.001; other comparisons were not significant).

 

EPPS removes Aβ plaques and oligomers in APP/PS1 mice

Collectively, these results indicate that EPPS rescues hippocampus-dependent cognitive deficits when orally administered to aged, symptomatic APP/PS1 TG mice.

Collectively, these results indicate that orally administered EPPS effectively decreases Aβ plaques and oligomers in APP/PS1 model mouse brains.

 

EPPS lowers Aβ-dependent inflammation and glial GABA release

Figure 5: EPPS lowers inflammation and glial GABA release.

EPPS disaggregates Aβ oligomers and fibrils by direct interaction and reduces cytotoxicity

Figure 6: EPPS disaggregates Aβ aggregates by selective binding.

 

(1) a small molecule, EPPS, converts neurotoxic oligomers and plaques into non-toxic monomers by directly binding to Aβ aggregates;

(2) orally administered EPPS produces a dose-dependent reduction of Aβ plaque deposits and behavioural deficits in APP/PS1 TG mice, even when administration was delayed until after the pathology was well established;

(3) the beneficial effect of EPPS probably operates through an Aβ-related mechanism rather by facilitating cognitive processes; and

(4) large doses of EPPS appeared to be well tolerated in initial toxicity studies^{6, 7, 33}.

Dr. T. Ronald Theodore
Email	rtheodore@integratedbiologics.com
URL	http://www.integratedbiologics.com
In Response To	Breakup of amyloid plaques
Submitted on	2016/05/18 at 3:33 am
Comment	Re: “EPPS rescues hippocampus-dependent cognitive deficits in APP/PS1 mice by disaggregation of amyloid-β oligomers and plaques” Kim et al, Nature Communications 8 December 2015 HEPES, Zwitterions, and the “Good” Buffers as Biological Response Modifiers In reference to the article “EPPS rescues hippocampus-dependent cognitive deficits in APP/PS1 mice by disaggregation of amyloid-β oligomers and plaques” Kim et al, Nature Communications 8 December 2015, we note some important omissions. Kim et al state specific effects of EPPS affecting Alzheimer’s disease. We would point out that EPPS is also referenced as HEPPS.1 HEPPS has been accepted as a “Good” buffer and a zwitterion. The authors attribute the effects of EPPS to anti-inflammatory action. The authors omit reference that EPPS (HEPPS) is a listed “Good” buffer and a zwitterion.1 The anti-inflammatory effects of zwitterions and “Good” buffers have been previously described.3,4 The effects of these zwitterions as biological response modifiers with effects on neurological diseases including Alzheimer’s have been previously noted.4,5 ( HEPES has been used preferentially based on Good’s original data showing HEPES has the highest ability to increase the rate of mitochondrial oxidative phosphorylation). Kim et al attribute the effects of EPPS to anti-inflammatory actions. The anti-inflammatory effects of the buffers are well known.3,4 We would suggest that anti-inflammatory effects of the buffers may be singular, synergistic or combined effects of other biological responses that have been noted including mitochondrial and other actions.4,5,6,7 Prior literature and data would certainly anticipate the findings of Kim et al. It is noted that all these zwitterionic buffers have effects on the neurological system. What is important is that further research to determine the effects of these zwitterionic buffers as biological response modifiers on neurological diseases including Alzheimer’s is continued. The ability of the zwitterionic buffers on brain and other organ injury are currently under review. T. Ronald Theodore Integrated Biologics, LLC rtheodore@integratedbiologics.com 1. Merck Index, 15th Edition, Feb 2015. 2. Norman E. Good et al., Hydrogen Ion Buffers for Biological Research, Biochemistry vol.5, No. 2, Feb. 1966. 3. “Effects of In-vivo Administration of Taurine and HEPES on the Inflammatory Response in Rats” Pharmacy and Pharmacology, vol. 46, No. 9, Sept. 1994. 4. Theodore et al., Zwitterionic Compositions and Methods as Biological Response Modifiers, US Patent No. 6,071,919. 5. Garvey et al., Phosphate and HEPES buffers potently affect the fibrillation and oligomerization mechanism of Alzheimer’s Aβ peptide, Biochemical and Biophysical Research Communications, 06/2011; 409(3):385-8. DOI: 10.1016/j.bbrc.2011.04.141. 6. Theodore et al., Pilot Ascending Dose Tolerance Study of Parenterally Administered 4-(2 Hydroxyethyl)-l-piperazine Ethane Sulfonic Acid (TVZ-7) in Dogs, Cancer Biotherapy & Radiopharmaceuticals, Volume 12, Number 5, 1997. 7. Theodore et al., Preliminary Evaluation of a Fixed Dose of Zwitterionic Piperazine (TVZ-7) in Clinical Cancer, Cancer Biotherapy and Radiopharmaceuticals, Volume 12, Number 5, 1997.