https://www.sciencedaily.com/releases/2016/11/161115150237.htm

Antibiotic restores cell communication in brain areas damaged by Alzheimer's-like disease in mice

November 15, 2016

University of British Columbia

New research has found a way to partially restore brain cell communication around areas damaged by plaques associated with Alzheimer's disease.
Ceftriaxone, an FDA-approved antibiotic to treat bacterial infections, were able to reduce synaptic disruption and clear the lines of neuronal communication in mice.

Published in Nature Communications

Demonstrate a possible target and a potential drug treatment to reduce damage to the brain that occurs in the early stages of Alzheimer's disease.

Amyloid plaques of beta-amyloid deposits develop in brain regions of patients with Alzheimer's disease,

These plaques are linked to the damage found in Alzheimer's disease because they prevent cell communication and are toxic to nerve cells.

The brain areas around these plaques show high levels of glutamate, a signaling molecule essential to communication between brain cells, accompanying high levels of hyperactivity in glia, the brain's support cells.

It is in this glutamate-rich environment that communication between neurons is changed or disrupted, causing neurons to die in the later stages of the disease.

By imaging the glial cells and glutamate itself around the plaques, the scientists were able to see that the cells were not able to 'remove' the glutamate accumulating in these brain areas.

Ceftriaxone was able to up-regulate glutamate transport

By restoring glutamate levels, neuronal activity was mostly restored.

The team's findings have implications for treatment of early symptoms of Alzheimer's disease.

This dysfunction in cell communication occurs at a very early stage in the disease, before memory impairment is detectable.

This makes the discovery particularly interesting, as it opens a window for an early intervention strategy to possibly prevent or delay neuron and memory loss.

Ceftriaxone is an antibiotic that is commonly administered before some types of surgery to prevent infections.

**University of British Columbia**.

1. J. K. Hefendehl, J. LeDue, R. W. Y. Ko, J. Mahler, T. H. Murphy, B. A. MacVicar. Mapping synaptic glutamate transporter dysfunction in vivo to regions surrounding Aβ plaques by iGluSnFR two-photon imaging. Nature Communications, 2016; 7: 13441 DOI:10.1038/ncomms13441

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http://www.nature.com/articles/ncomms13441

Mapping synaptic glutamate transporter dysfunction surrounding beta-amyloid plaques (by iGluSnFR two-photon imaging)

Amyloid-β (beta-amyloid) plaques are surrounded by regions of neuronal hyperactivity and glial hyperactivity.
imaging of the glutamate sensor iGluSnFR determine whether pathological changes in glutamate dynamics in the immediate vicinity of beta-amyloid deposits in APPPS1 transgenic mice could alter neuronal activity in this microenvironment.
In regions close to beta-amyloid plaques chronic states of high glutamate fluctuations are observed
and the timing of glutamate responses evoked by sensory stimulation exhibit slower decay rates in two cortical brain areas.

GLT-1 expression is reduced around beta-amyloid plaques
and upregulation of GLT-1 expression
and upregulation of GLT-1 activity by ceftriaxone
partially restores glutamate dynamics to values in control regions.

the toxic microenvironment surrounding beta-amyloid plaques results from enhanced glutamate levels

pharmacologically increasing GLT-1 expression and GLT-1 activity may be a new target for early therapeutic intervention.

extracellular accumulation of amyloid plaques

intracellular neurofibrillary tangles of protein

Positron emission tomography imaging

show that cerebral beta-amyloid plaque deposition is an early marker for the progression of preclinical to symptomatic Alzheimer disease

A current hypothesis is that soluble beta-amyloid adheres and combines with resident beta-amyloid plaques leading to progressive plaque growth.

Although plaques consist of insoluble beta-amyloid, each plaque may create a toxic microenvironment because soluble species continuously bind and unbind from its surface

The toxic microenvironment hypothesis is supported by observations of neuronal dysfunction and decreased spine density in the immediate regions surrounding beta-amyloid plaques
Hyperactive neurons, microglia and astrocytes with elevated intracellular calcium levels have been reported in the close proximity of beta-amyloid plaques
A shortcoming of the amyloid cascade hypothesis is that there are no clear connections between the onset of plaque deposition and the progression of cognitive impairment and neuronal loss
In contrast, there are stronger correlations with the levels of soluble beta-amyloid species and cognitive impairment as well as neuronal loss.

changes in glutamate dynamics in the microenvironment surrounding beta-amyloid deposits may establish an early link between beta-amyloid toxicity and neuronal dysfunction.
A genetically encoded fluorescent glutamate indicator, iGluSnFR, was used to detect the presence and time course of glutamate dynamics in vivo

It allows spatial and temporal resolution of local glutamate concentration on a subsecond timescale.

Rapid glutamate uptake from the synaptic cleft, principally by the astrocyte glutamate transporter excitatory amino-acid transporter 2 (EAAT-2 in humans or the homologue glutamate transporter 1 (GLT-1) in mice) permits fast synaptic transmission.

SLOW CLEARANCE of glutamate in the extracellular space in Alzheimer disease might underlie the reported upregulation of Ca2+ signalling in neurons and astrocytes in the microenvironment of beta-amyloid deposits
this UPREGULATION of Ca2+ signalling in neurons and astrocytes could contribute to excitotoxicity, which eventually leads to cell death.
EAAT-2 activity has been reported to be significantly REDUCED in early stages of Alzheimer disease correlating well with cognitive decline in Alzheimer disease patients
GLT-1 knockdown in an Alzheimer disease mouse model showed cognitive decline – supporting the theory that dysfunction of the astrocyte glutamate transporter is involved in Alzheimer disease pathogenesis

beta-amyloid is responsible for the loss of GLT-1 expression as a part of their toxicity to astrocytes
APPPS1 mice was treated with ceftriaxone – to determine whether GLT-1 expression and/or transporter activity could be upregulated around plaques and whether this would restore normal glutamate dynamics in this region.
ceftriaxone helps to partially restore glutamate dynamics and chronically elevated levels of glutamate.

it reduces the pathological impact of beta-amyloid deposits and surrounding prefibrillary forms of beta-amyloid.

ceftriaxone can alter GLT-1 function without changing overall protein levels

Upregulation of GLT-1 reduces changes in glutamate dynamics

ceftriaxone treatment reversed the selective decrease in GLT-1 expression within the 20 μm radius around amyloid deposits.

The major glutamate transporter is GLT-1.

Tplsm imaging of extracellular glutamate dynamics detected using iGluSnFR revealed impaired glutamate uptake from somatosensory or visual evoked responses in regions adjacent to visualized beta-amyloid plaques.
These synaptically evoked glutamate transients showed reduced glutamate clearance rates close to amyloid deposits, and chronic states of prolonged and elevated glutamate levels were detected in the direct vicinity of amyloid plaques.
The regions adjacent to plaques also showed reduced expression of the major glutamate transporter, GLT-1.
Treatment of APPPS1 mice with ceftriaxone restored GLT-1 expression in the plaque microenvironment

and the plaque-associated disturbances in glutamate clearance leading to chronically elevated glutamate concentration were significantly reduced.
These results indicate that beta-amyloid plaques are correlated to a regional reduction and dysfunction of GLT-1 that causes impaired glutamate clearance rates.
Functional changes to glutamate dynamics and thus possibly GLT-1 activity were detected and rescued at a distance of 40–60 μm (ROI3) from the beta-amyloid plaque edge by ceftriaxone treatment.

The dysfunction or mislocalization of GLT-1 may also contribute to the observed pathological alterations.

Alterations to glutamate signalling could be an important contributor to synaptic disruption and cognitive impairment in early Alzheimer disease.

Glutamate is the major excitatory neurotransmitter in the brain

The rapid kinetics of release of Glutamate and clearance of Glutamate from the extracellular space are critical for precisely timed synaptic communication.
!!! The principal pathway for glutamate clearance is uptake via the GLT-1 pathway that is expressed in astrocytes.
Uptake by astrocytes is essential for glutamate homeostasis
When Uptake by astrocytes for glutamate homeostasis is disrupted, this leads to high levels of glutamate in the extracellular space causing neuronal excitotoxicity and cell dysfunction.

EAAT2 is the human homologue of GLT-1

The reduction of EAAT2 (the human homologue of GLT-1) activity in the early stages of Alzheimer disease has been reported to be correlated with the cognitive decline seen in Alzheimer disease patients.

New scientific methods gave real-time measurements of extracellular glutamate dynamics.

APPPS1 mice develop amyloid plaques by 2 months of age in the neocortex
and beta-amyloid levels are at least five times higher than those of beta-amyloid
Though they are associated with neuronal dystrophy and robust astro- and microgliosis an overall or plaque-associated loss of neurons could not be shown in APPPS1 mice
we are looking at an early stage of dysfunction in various cell types in the microenvironment of amyloid deposits in this mouse model rather than a model for neuronal loss.
This makes the APPPS1 model a valuable tool to mimic early stages in Alzheimer disease in which detrimental downstream events can still be rescued.
We hypothesize that the soluble material, known to be mainly beta-amyloid in the case of APPPS1 mice, is present in relatively high concentrations in the microenvironment of amyloid deposits.
prefibrillar forms of beta-amyloid is detected in the microenvironment of beta-amyloid deposits.

toxic oligomeric species of beta-amyloid contribute to alterations in vivo glutamate dynamics

glutamate dynamics are changed in regions surrounding plaques,
The degree of change increases the closer the tissue is to the edge of the plaque.
The time constant of glutamate clearance rates is slower when approaching amyloid deposits,
chronic states of high, fluctuating glutamate concentrations are reached in the direct vicinity of amyloid plaques.

Hence, we investigated whether there was a reduction in GLT-1 expression that could lead to a reduction of glutamate reuptake in this area.

Histological staining of GLT-1 showed a significant reduction in the immediate vicinity of amyloid deposits adding evidence to the imaging data that
the chronically higher levels of glutamate within ROI1 are due to a reduction in glutamate uptake.

Other mechanisms unrelated to overall protein levels of GLT-1 such as receptor dysfunction were investigated by using iGluSnFR as a functional read-out.

!!! The beta-lactam antibiotic, ceftriaxone, has been widely used to upregulate GLT-1 transporters up to fivefold

and also has been shown to alter GLT-1 function and activity independent of overall protein levels

We thus aimed at reversing higher extracellular glutamate levels and slower glutamate decay rates by increasing glutamate reuptake via GLT-1 transporters.
The treatment of APPPS1 animals with ceftriaxone resulted in an increase in GLT-1 in histological staining in ROI1

and a partial rescue of the observed glutamate alterations measured by tplsm of iGluSnFR.

The treatment reduced the prolonged decay rates in ROI3 significantly to levels comparable to those in ROI4 and WT animals.

glutamate dynamics were restored within this region by the treatment.

the high average glutamate fluctuations in the direct plaque vicinity to WT levels could be reduced.

The continued lack of a stimulus-locked response in ROI1 might be caused by neuronal dysfunction, which is already also prevalent within this area.
It has been shown that a large number of neurons (up to 50%) in the primary visual cortex can display functional impairment without obvious behavioural deficits in transgenic mice.
This hints towards possible compensatory mechanisms that are able to sustain physiological activities even in circuits that contain a large number of dysfunctional neurons
when using wide-field imaging to record glutamate dynamics on a more global level no significant differences between APPPS1 and WT animals were found
the changes are very region specific and subtle and will be lost if not analysed with the necessary spatial resolution.

contribution of high glutamate concentrations to the reported excitotoxic microenvironment surrounding amyloid deposits.
!!! High levels of extracellular glutamate could mediate the reported upregulation of Ca2+ signalling and overall hyperactivity in the microenvironment of beta-amyloid deposits

and could contribute to the progressive synaptic disruption and cell death.
early changes in glutamate dynamics could be used to detect the early development of detrimental excitotoxic downstream effects.
studies in patients would require spatial and temporal resolution sufficient to determine changes in the environment around plaques.

changes in glutamate dynamics in the early stages of Alzheimer disease are part of a dysfunctional microenvironment that is directly linked to plaque deposition.
Even though we cannot conclude that higher levels of glutamate precede hyperactive states in different cell types

higher levels of glutamate contributes to the overall pathomechanism and excitotoxic effects described in the microenvironment of amyloid deposits.
higher levels of glutamate could be used as a novel biomarker to indicate cellular dysfunction aiming at early intervention that has the potential to stop or delay detrimental downstream effects caused by excitotoxicity.