Mechanisms of neurotoxicity

Programme Leader: Professor Giovanna Mallucci MD PhD

Summary of Research Interests

Neurodegenerative diseases, such as Alzheimer’s, Parkinson’s and Huntington’s disease, and also the rarer prion disorders, have enormous clinical and economic impact worldwide. They vary in clinical and pathological hallmarks, including aggregation of misfolded proteins, but in each there is irreversible loss of neurons, which cannot be cured. But before neuronal loss, there is there is synaptic impairment and loss, which potentially can be treated. The mechanisms controlling the causes and progression of synaptic loss to neuronal death are the focus of our research programme.

My background is in modelling prion diseases in mice, looking at mechanisms of neurotoxicity and developing new therapeutic approaches. We have shown that early synaptic changes in mice with prion disease can be reversed, resulting in recovery of synaptic and cognitive function and behavioural deficits, long term neuroprotection, and life long survival of affected animals. Thus neurodegeneration can be prevented by reversing early synaptic deficits.

This programme uses several model systems - mice (wild type and transgenic), primary neurons and the nematode C. elegans, to understand the early molecular events that cause synaptic toxicity and neuronal cell death in neurodegeneration. In parallel, we are looking at the mechanisms involved in synaptic repair processes.

Our main aims are to define:

1)    the earliest mechanistic impairments in the neuronal response to toxic proteins in neurodegenerative diseases
2)    how synaptic dysfunction and loss are triggered
3)    how synapse loss leads to neuronal loss
4)    how toxic responses can be reversed for neuroprotection
5)    how repair processes can be harnessed for therapy

In this way, we aim to define key pathways for modification in novel therapeutic approaches; to develop mouse models reflecting disease mechanisms; and to define new biomarkers for identifying susceptible individuals.

Background:  Modelling mechanisms of neurodegeneration in prion diseases

We previously defined a new therapeutic target for prion disease, the native prion protein, PrPC (1), which is converted to a protease resistant, infectious isoform, PrPSc, that accumulates in the brain during disease. We showed that by targeting PrPC, hence removing the substrate for ongoing prion replication, mice with prion infection can be cured at the stage of early synaptic dysfunction, when they have reversible neurophysiological, behavioural and morphological impairments (2,3). Reversing this early stage of disease allowed long-term neuroprotection and normal lifespan in the mice (2). We were able to do this both in transgenic mice in which PrP was deleted in the adult brain, and by using lentivirally- mediated RNA interference of PrP (4).

These findings set the scene for further analysis: What is the nature of the synaptic deficit that is reversible? What is the window for intervention? What are the mechanisms in neuroprotection? Which pathways are in common with other neurodegenerative disorders that could be modulated for therapy and identification of new biomarkers?

We hypothesize that common pathways govern synaptic and neuronal loss in neurodegenerative disorders, irrespective of specific disease entity. The reversal of early deficits that we have shown in prion disease has far-reaching implications for all neurodegeneration. The long term neuroprotection that results form this reversal strongly supports the concept of a window for intervention at the stage of synaptic dysfunction, in which neurons can be rescued from the early processes that lead to cell death.

Reversal of early morphological, behavioural and neurophysiological deficits in prion diseased mice:

Figure 1 Mice in which PrP is depleted throughout the brain at 8 weeks post infection survive long term after reversal of behavioural (2) and morphological changes (3).

Figure 2 A. Mice are allowed to explore two objects. They are then exposed to a novel object (test phase). Time spent actively exploring the novel object compared to the familiar one is a measure of object recognition memory. B. Prion-infected tg37 (blue bars) and NFH-Cre/tg37 mice (red bars) showed normal object recognition memory at 7 wpi, with preferential exploration of the novel object. This was significantly impaired in all mice by 8 wpi, but recovered in mice with Cre-mediated PrP depletion at 9 wpi.

Figure 3 H & E stained sections of hippocampi from prion-infected tg37 and NFH-Cre/tg37 mice have normal appearance at 6 wpi (panels A, D). Spongiosis develops in all animals (panels B, E) by 8 wpi when memory is impaired, but by 10 wpi this has reversed in mice with Cre-mediated depletion (panel F), in parallel with recovery of novel object memory.

Figure 4 Field excitatory post synaptic potential (EPSP) slope input-output curves show marked differences between the experimental groups. At 8 wpi both tg37 (blue squares) and NFH-Cre/tg37 mice (red squares) have smaller synaptic responses than age-matched uninoculated control mice of both genotypes (gray diamonds). In prion-infected tg37 mice at 9 wpi (blue circles) synaptic responses are further reduced. However, after Cre-mediated PrP depletion, synaptic responses return to control levels in NFH-Cre/tg37 mice at 9 wpi (red circles) and are sustained at this level up to 18 wpi (red triangles).

Figure 5 Treatment with anti-PrP shRNA expressing lentivirus prolongs survival in mice with established prion disease. Mice were infected with RML prions at 1 week of age and were treated with bilateral hippocampal injections of either a lentivirus targeting PrP (LV-MW1, n = 22) or LV-Empty (n = 18) at 8 wpi or with no virus (n = 18). RML-infected mice treated with no virus or with LV-Empty died within 91 and 101 days post infection (dpi) respectively; mice treated with LV-MW1 survived longer, living up to 129 dpi. (p < 0.0001 Student's t test, 2 tails, compared to both LV-Empty and untreated mice.

Figure 6

Current research goals

Synaptic dysfunction is a key process in neurodegeneration: it is an early, potentially rescuable stage in pathogenesis throughout the spectrum of these disorders. We have previously proved, in prion diseased mice, that this stage is indeed reversible, rescuing neurons in prion disease (2-4). Critically, the formation, remodelling and elimination of spines and synapses – plasticity - are continual physiological processes in the adult: there is an intrinsic capacity for regeneration that could be harnessed. Yet how plasticity is affected in neurodegeneration is also unknown.

We aim to understand the cellular and molecular mechanisms underlying early functional deficits in neurons and their recovery, and the processes driving synaptic repair.

We are using prion diseased mice ( these are the best mouse ‘model’ of neurodegenerative disease as these truly recapitulate the human disorder and, critically, have extensive neuronal loss) as a platform to understand fundamental mechanisms in synaptic and neuronal toxicity, and their rescue. We will use mechanistic insights to generate new mouse models for better prediction of therapeutic effects. In parallel, we have a strong focus on understanding repair pathways and how plasticity is affected in disease. Insights into the key pathways controlling plasticity allow us to modify these to cure impaired repair mechanisms. Combined new insights into these different processes will also give access to new biomarkers of disease for timely intervention in patients.