Dr. Arjun Krishnaswamy
Introduction: Our goal is to understand how internal states, such as mood or attention, modulate sensory processing and focus the brain on the most behaviorally relevant stimuli. We focus on the phenomenon of gain-control, in which internal states amplify or attenuate the sensitivity of sensory processing. As a model, we will use the mouse visual thalamus (LGN) and the serotonergic connections it receives from the dorsal raphe (DR), a critical center for satiety, reward, and arousal. Current models suggest that DR releases serotonin (5HT) onto neurons in visual thalamus to modulate thalamic drive to visual cortex in response to rewarded stimuli. However, the exact nature of the DR signal in LGN, the impact of DR signals on visual behavior, and the effects of abnormal DR-signaling –as occurs during autism (ASD) –are unclear.
Goal: To learn more, I propose to define how the DR-thalamic circuits shape sensory processing in awake, behaving animals that develop normally and those that develop ASD-like phenotypes.
Outline of Research
Aim#1: The precise timing and magnitude of DR signals to LGN determines its impact on visual processing and visual behavior. To understand how DR input activity varies with visual behavior, we will measure neural activity from LGN-projecting DR neurons using in vivo calcium imaging while mice perform a behavioral task; mice will be trained to choose the correct stimulus, given two alternatives, in order to obtain a reward. If DR controls LGN visual processing then it should be active during stimulus presentation and remain active until reward. Preliminary behavioral results indicate that mice can discriminate between two gratings and choose the grating of higher contrast.
Aim#2: DR might enhance or depress the gain of LGN visual responses, depending on how its activity affects geniculate circuitry in awake animals. To define the impact of DR on LGN visual processing, and correlate this to behavior, we will optogenetically stimulate DR inputs while mice perform our behavioral choice task. If DR increases LGN gain, then stimulating ChR2+ DR terminals should improve stimulus discrimination; if it decreases LGN gain, then stimulation should deteriorate performance.
Aim#3: The visual deficits, alterations in thalamus, and altered 5HT signaling in ASD point strongly to an underlying dysfunction in DR-thalamic signaling. To test this idea, we will monitor and perturb DR inputs to LGN in mice that lack Shank3. Shank3 null mice exhibit social and visual deficits that mimic ASD and recent studies show that DR activation in these mice rescues their social deficits. Briefly, we will image DR inputs in the LGN of Shank3 nulls while they learn and perform our visual behavioral test.
Projected Benefits and Application of Findings: These studies will be the first to define how DR contributes to behavioral sensitivity to visual stimuli in normally developing and ASD-mutant mice. Understanding how 5HT release from DR influences the visual sensitivity of LGN in behavior and ASD models will provide a framework to understand the debilitating sensory overload in ASD. The insights and reagents that come out of our studies hold the potential to inspire more effective diagnostics and therapies for ASD.
Timeline: We expect to finish the studies outlined in Aim#1 following release of funds on Oct 30th, 2018 by Spring 2019. Studies outlined in Aim#2 will take slightly longer due to time needed to expand a Pet1-Cre mouse colony using founders purchased from Jackson; we anticipate a complete dataset by Jan 2020. Studies on Shank3 null mice will begin in Fall 2020 (Aim#3), following purchase of the line and initial imaging studies (Aim#1). We anticipate a publication by Spring 2021 and another by summer 2021, putting us in a good position to leverage this early career grant into an operating grant by Fall 2021.
Targeting mitochondrial form and function in Autism Spectrum Disorder.
Dr. Tim Shutt
University of Calgary
Targeting mitochondrial form and function in Autism Spectrum Disorder.
Autism spectrum disorder (ASD) is a continuum of neurodevelopmental disorders with common symptoms affecting social development, behavior and language, which is now recognized in an estimated 1 in 66 Canadian children. Our understanding of ASD is complicated by the fact that a combination of diverse factors such as genetic background and environmental exposure are thought to contribute to its development. Notably, there is growing evidence for a link between ASD and mitochondrial dysfunction.
Mitochondria are best known as the cellular components that produce the bulk of the cell’s energy. Importantly, many of the same factors thought to cause ASD also result in mitochondrial dysfunction. Moreover, estimates suggest that 30-50% ASD patients show signs of mitochondrial dysfunction. These findings suggest that mitochondrial dysfunction underlie a significant proportion of ASD. Our preliminary data show that a well-established mouse model of ASD, the BTBR mouse, has mitochondrial dysfunction. That mitochondria are involved in ASD should not be surprising as they provide energy for the brain and play essential roles in brain development. Although the exact role of mitochondrial dysfunction in ASD remains undefined, targeting mitochondrial dysfunction is nonetheless a promising therapeutic approach.
One way to improve mitochondrial function is by changing the food source they are given. To this end, the high-fat low-carbohydrate ketogenic diet (KD), is a therapeutic approach that is beneficial in a growing list of neurological disorders with mitochondrial dysfunction. Importantly, the KD shows behavioural benefits in both ASD patients and animal models of ASD (including BTBR mice). In particular, our preliminary data show that the KD also ameliorates mitochondrial dysfunction in BTBR mice. Despite this exciting finding, we do not understand exactly how this occurs. Further highlighting the need for a better mechanistic understanding to develop alternative approaches, the KD is not without side effects and comes with implementation challenges, especially in children.
A key regulator of mitochondrial function is the structure of the dynamic mitochondrial network, which can range from fragmented spots to a reticular network, and is determined by the balance between fission and fusion events. Our preliminary data demonstrate not only that the KD also improves mitochondrial structure in BTBR mice, but also begin to delineate the molecular pathways through which the KD is improving mitochondrial structure by promoting mitochondrial fusion.
To understand how the KD improves mitochondrial structure and function, and to evaluate its applicability to ASD.
Outline of Research:
Our studies will exam how the ketogenic diet improves mitochondrial function by looking at mitochondrial structure, a key aspect for mitochondrial function. To this end, we will elucidate mechanisms through which the ketogenic diet improves mitochondrial structure and function. Specifically, we will determine how metabolic products generated by the KD regulate the structure of mitochondria (Aims 1 & 2). Finally, we will determine whether targeting this specific pathway, by supplementing the diet of BTBR mice with these metabolic products, improves mouse ASD-like behaviours (Aim 3).
Projected benefits and application of findings:
In addition to the stress that a child with ASD puts on a family, children with ASD also add a burden to the educational system and the economy. In 2001, the lifetime cost per Canadian with an autism condition was estimated at $2 million, with an overall Canadian annual cost estimated at more than $3 billion. While identification and treatment can reduce the impact of ASD, there is no cure, in part, because we do not understand the cause.
Our proposed studies will target improving mitochondrial dysfunction, which underlies a significant proportion of ASD. Specifically, understanding the underlying mechanisms by which the KD improves mitochondrial function will facilitate the development of novel therapies to restore mitochondrial morphology, and by extension mitochondrial dysfunction, while avoiding the complications of the KD. Finally, we will explore the benefits of ketone esters for ASD. Importantly, because ketone esters are clinically relevant and safe for children, they may be a preferable alternative to the KD, in particular for cases of ASD linked to mitochondrial dysfunction.
Neuroprotective strategies to prevent long-term cognitive deficits induced by CBI
Dr. Sophie Tremblay.
CHU Ste-Justine Research Centre
Cerebellar growth is highly vulnerable during the third trimester of pregnancy and could be affected by diverse insults leading to cerebellar growth failure and atrophy. Up to 19% of infants born extremely preterm (less than 28 weeks) will be affected by cerebellar injuries during their neonatology hospitalization. Nowadays, the cerebellum is recognized not only as a coordination centre where sensory input is processed in order to fine-tune movement and balance, but also plays a role in cognitive and affective processing, speech fluency, attention, spatial memory, and music learning. We have developed a novel translational mouse model of preterm cerebellar haemorrhage and inflammation that can serve as an entrée to explore long-term deficits induced by preterm cerebellar damage to address issues such as mechanism and neuroprotective strategies. It is critical to understand how we can minimize disruption of cerebellar development to maximize neurodevelopmental outcomes in extremely preterm infants.
In Objective 1, we will examine the role of innate immune system of the brain, microglial cells, in hindering the cerebellum’s resilience to insult and prevent long-term cognitive deficits. In the second Objective, we will quantify how microglial depletion preserves adult cerebellar structure and other brain region volumes affected by perinatal cerebellar injury. In Objective 3, we will test the potential therapeutic benefit of Etanercept, a FDA-approved drug known to modulate brain inflammation.
Outline of research
We have developed two mouse models of cerebellar insults: cerebellar haemorrhage using bacterial collagenase and perinatal inflammation induced using intraperitoneal lipopolysaccharides injection. As oftentimes these insults occur together, we have also modeled these in the early postnatal mouse (equivalent to the third trimester in the human). We are using a battery of pre-established behavioural and anatomical tests to examine the function and structure of the developing cerebellum up to adulthood. The overall focus of this proposal is to examine the role of microglial cells, our innate immune system in the brain, which may perpetuate damages to the cerebellum and means by which we may provide neuroprotection in the face of that damage.
At the mechanistic level, we will test the hypothesis that microglial activation after perinatal insults play a crucial role in the pathological responses to cerebellar injury leading to cerebellar atrophy, reduced total brain volumes and deficits in cerebellar-associated behaviours diagnosed in infants born extremely preterm. We will test the contribution of microglia to cerebellar haemorrhage and inflammation pathophysiology by using transgenic mouse pups expressing an inducible fusion protein only in brain microglia allowing a selective depletion in a timely manner in this specific cell population. In a parallel fashion we will explore how modulation of the brain’s immune and inflammation responses can provide neuroprotection from perinatal insults. Etanercept is known to reduce systemic inflammatory responses and to reduce neural injury induced by oxidative stress (in response to high level of oxygen). Despite the crucial role of microglia during brain development, their capacity to perpetuate inflammation after brain injury may lead to long-term consequences such as permanent neurodeficits. Modulation of their responses to injury may provide neuroprotection to the developing brain and contain injury progression. Furthermore, if microglial cell favours a release of pro-repair molecules, they may improve post-insult recovery, limit brain damage and potentially diminished long-term functional consequences of preterm brain injury.
Projected benefits and application of findings
The scientific community focus has been so far on protecting neonatal cerebral injury in infants born prematurely; preventing cerebellar insults is as important and will result in significant neurodevelopmental improvement. This work has the direct potential of providing new therapies for children with perinatal infections and CBH. Currently there is no therapeutic agent given routinely to prevent CBH damages or other CBI affecting extreme preterm infants. Achievement of our goals will lead to expand the repertoire of potential therapies available to protect the developing brain, especially the cerebellum, which could be applied to other preterm brain injuries.
The role of the cellular prion protein in alpha-synuclein propagation
Dr. Joel Watts
University of Toronto
More than 100,000 Canadians currently suffer from Parkinson’s disease (PD), and this number is predicted to increase dramatically in the coming years due to an ageing population. In PD, a protein called α-synuclein clumps together in the brain to form aggregates. These aggregates are believed to cause the death of brain cells, which produces the symptoms of PD. The progressive nature of PD is thought to result from the spread of α-synuclein aggregates between cells in the brain. Recently, it has been found that another brain protein, the prion protein (PrP), may facilitate the cell-to-cell spread of α-synuclein aggregates during PD.
The objective of this research is to determine whether the presence or absence of PrP on brain cells has any effect on the development of disease in mouse models of PD.
Outline of Research
We will use two different mouse models of PD in which the aggregation of α-synuclein in the brain can be stimulated by injecting the mice with pre-existing α-synuclein aggregates. These mice will be crossed with mice that have been genetically engineered so that they no longer produce PrP. The amount of α-synuclein pathology in the brain and the speed of disease development will be compared between mice that have normal amounts of PrP and mice that lack PrP.
Projected Benefits and Applications of Findings
These studies will determine whether PrP is a viable therapeutic target for PD. While there are treatments available that lessen the symptoms of PD, there are currently no drugs that target the root cause of the disease. If the absence of PrP in mice is found to prevent or delay the development of PD-like symptoms and pathology, reducing PrP levels in the brains of PD patients may be an effective means of halting the progression of the disease.