Research

The major research interest of the LMVN involves understanding how retroviruses (RVs) cause paralytic and cognitive neurodegenerative diseases. (A link to a video that shows what happens clinically when laboratory mice are infected with a neurovirulent retrovirus is provided below.)

Retroviruses are mobile genetic elements whose structure includes a lipid bilayer, a protein capsid, and a positive strand RNA genome that encodes the protein machinery needed for infection and replication. Upon viral entry, RV RNA is reverse transcribed into DNA, which becomes integrated into the host cell genome and then expressed.

Infection is initiated on the surface of the host cell is where specific host membrane proteins act as receptors. The retrovirus must bind to one or more of these receptors in order to enter the host cell.
The virion envelope directly fuses with the plasma membrane which allows penetration of the host cell by the retrovirus.
ENV (red) binding to specific receptors (green) is significant for facilitating specific cell type targeting in the brain. However, it is the ENV expression within those targeted brain cells which initiates neurodegeneration.
Over the millennia, recurrent RV infection of germ cells has resulted in the accumulation of RV DNA in mammalian genomes with the potential to be expressed in all cells. In humans, endogenous RV sequences (ERVs) account for approximately 8% of the total genome (<2% encodes host cellular proteins). While certain ERVs are known to benefit the host, others have been implicated in the development of neurodegenerative diseases such as multiple sclerosis, sporadic amyotrophic lateral sclerosis, and schizophrenia. Similarly, exogenous RVs, like HIV-1 and HTLV-1, can cause progressive dementia and paralysis, NeuroAIDS and tropical spastic paraparesis, respectively. To understand how RVs cause neurodegeneration the LMVN studies virus-brain cell interactions in vitro and in vivo.

In LMVN the focus has been on studying model RVs derived from wild mice that cause paralytic disease in laboratory mouse strains when inoculated into neonatal pups.  This mimics the situation in the wild where mice pass viruses to their offspring in mother’s milk.  Depending on the sequences of the RV used, mice can show paralytic disease signs after 2 weeks to more than a year. By comparing viruses that cause disease fast or slow has allowed for the identification of those viral features and life cycle events are involved in causing neurodegeneration. Whether all RVs can alter neuronal function remains an open question which needs to be explored in greater detail given the number of ERVs that are present within the human genome.

Comparison of mouse brain tissue (stained with hematoxylin and eosin) from control mice (left) and mice infected with a highly neurovirulent virus called FrCasE (right), shows that the infected mice develop spongiform neurodegeneration (holes in the brain) that resembles pathology seen in prion diseases. These “holes” were shown to arise at synapses and represent swelling of the post synaptic terminals.  This vacuolation is postulated to arises due to excessive release of the neurotransmitter by the presynaptic terminal or excessive responsiveness by the post synaptic cells in a process known as excitotoxicity.  Other RVs cause similar disease with more limited spongiosis, owing to the slower nature of the disease, and more limited neuronal alterations arising in these mice.

The pathogenic timeline of the neurovirulent virus FrCasE in mice is highly stereotypic  which allows more careful dissection of the important infectious and developmental events that contribute to RV disease.  The rapid course of the disease makes it easier to study the process and quickly identify the important molecular and cellular events associated with the pathogenic process. As outlined, the presence of virus entering and infecting cells of the CNS can be detected by day six. The presence of virus within host cells occurs several days before the holes begin to form, at approximate ten days old. Clinical signs are observed by 15-16 days, and early within the fourth week animals begin to die if not euthanized beforehand.

In order to understand which cells play and important part in disease, our laboratory has pioneered the use of genetically engineer neural stem and progenitor cells to make mouse brain chimeras that reconstitute specific RV life cycle and host CNS developmental events.

Transplantation of genetically engineered neural stem cells (NSCs) in the developing brain results in widespread engraftment and integration into the detailed CNS architecture.
Engraftment results because the brain accepts the transplanted NSCs as a normal part of its development without activating innate or acquired immune system cells because the immune system itself is immature. Shown here, engrafted cells (purple/black) are widely distributed from the forebrain to the hind-brain in this adult animal after neonatal transplantation. By engineering these cells with various viruses or viral components we are learning how viral elements alter CNS development and brain circuit function. This has led to the following hypothesis regarding how viruses alter neuronal function relevant to paralytic disease.

The hypothesis for how RVs cause neurodegeneration argues that it is the infection of neural progenitor cells (NPCs), particularly those that serve as glial precursors, which results in the alteration of their differentiation and function.  One major area where these glial cells act is in regulating communication between neurons within circuits, particularly those involved in rhythm generation. Another area where they act is as mediators of communications between the vasculature (blood supply) and neurons in order to maintain appropriate energy and nutrient supplies.
When these forms of neuron-glia-vascular interactions are disrupted, it can affect the excitability of the neurons as well as that neuron’s ability to communicate with circuits that extend to cognitive and motor areas.

Our studies have shown that RVs disrupt neuron function and activity by affecting glial development and neuron-glial interactions. Further research is needed to understand how glia are specifically affected by different RVs inlcuding those from humans in order to develop rational therapeutic treatment strategies.

V I D E O S

1. ) Neurovirulent RV-Infected Laboratory Mice
https://www.youtube.com/watch?v=3kjeJx2FJUQ

Mice in the video can be seen to exhibit shaking (tremor), hunched posture (kyphosis), stilted-walking, and unkempt fur.  These characteristics indicate the mice have a paralytic neurodegenerative disease: loss of coordinated voluntary movement–motor/ muscle function due to injury or dysregulation of nerve cell function, which progesses over time, and ultimately leads to death.
In humans, similar clinical phenotypes are seen ALS, multiple sclerosis, tropical spastic paraparesis.

2.) Spontaneous Activity in the IC Visualized using VSD (controlled vs. virus-infected)

Virus-Infected: https://www.youtube.com/watch?v=tHg4maUFO9c
Controlled: https://www.youtube.com/watch?v=ndZLFhYg_ms

Neurons send signals to each other in the brain which in turn send signals to different muscles to initiate or change movement. Various neurons are being turned off and on at any given moment in a coordinated fashion to regulate everthing from thoughts to maintaining balance. When groups of neurons become active at the same time, this can be visualized by using voltate sensitive dyes (VSDs) that change their absorbance of certain wavelengths of light when illuminated, that can be detected, quantified and displayed by intesity plot videos.  Using this activity scanning technique brain regions showing hypoactivity (suppressed) are seen as blue whereas regions showing hyperactivity (stimulated) appear as yellow-red.  Green indicates that no coordinated neuronal activity can be distinguished.
The example video shows that in an area of the mouse brain known as the inferior colliculus, the paralytic RV causes a broad array of hyper and hypo activity when viewed using VSDs. The video example is of a brain slice examined prior to the time when animals show clinical neurological disease signs and symptoms, but at the same time at which spongiform histolopathological changes arise. In contrast, brains infected with a control RV or mock infected show little fluctuation in brain activity.

I M A G E S

To understand how RVs alter neuronal function we have begun to test different neuron types to see if and how they are affected by virus expression in the surrounding glia.  The image below is that of a rebound neuron (RN) after it was patched and filled with a fluorescent marker to show the cell body and processes.  Electrophysiological recordings of this and similar RNs showed that they were spontaneously active in FrCasE-infected brains. Remarkably, sustained neurons in the same physical environment were unaffected by the presence of virus (not shown).  The RN examples shown below shows altered dendritic architecture including swollen post-synaptic processes (green swellings).