A viral vector based in vivo model of tauopathy to explore therapeutic strategies against tau pathology

Tauopathies are neurodegenerative diseases whose common pathological feature is the intraneuronal accumulation of tau aggregates. Tau protein has various roles in the neuron, among which stabilization of microtubules (MT), regulation of synaptic activity or axonal transport. Subsequently, tau pathology exerts toxicity through mechanisms that likely involve the loss of normal function or the gain of toxic functions. In tauopathies, tau undergoes post-translational modifications (PTM), which are thought to promote the detachment from the MT, change its subcellular localization, and induce conformational changes that favor its assembly with other tau monomers, forming oligomers and fibrils. Furthermore, both normal and pathological forms of tau can be transferred from neuron to neuron. This process may have an important role in disease progression, by propagating the pathology along synaptically connected brain regions, which reflects the evolution of the symptoms. Therefore, the aggregation of tau protein and its propagation are promising targets for potential treatments. In the present thesis, our objective was to assess the efficacy of two treatments using a mouse model of tauopathy based on local overexpression of the 4R0N human tau (hTau) wild-type (WT) induced by intracerebral injection of an adeno-associated viral (AAV) vector. In the first part of the thesis, we overexpressed tau in the entorhinal cortex (EC) of WT mice to induce pathology and assess the effects of aggregation inhibitor compounds orally administered. Although we did not observe any effect on markers of tau aggregation, likely because the induced pathology was still at an early stage, the treatment was found to enhance phosphorylation of the Ser202/Thr205 residues in the EC and ipsilateral hippocampus (HPC). Additionally, we measured an increase in the density of microglial cells in the HPC, and significantly decreased levels of total hTau in the cerebrospinal fluid of the treated mice. These findings suggest a potential positive effect of this treatment on the clearance of hTau. In the second part of the thesis, we unilaterally overexpressed hTau in the mouse CA3 HPC and used the connectivity between both hemispheres to quantify the propagation of tau from neuron-to-neuron to the contralateral HPC. We found neurons labelled for hTau in the contralateral HPC, the number of which was increased in 5xFAD mice carrying the amyloid pathology. Next, to induce an antibody response against pathological tau, we subcutaneously injected a vaccine against the phospho-Ser396/404 tau epitope. The vaccine induced a potent antibody response in WT mice, but antibody titers remained low in the 5xFAD mice. In the treated WT mice, immunohistochemistry showed a decreased PHF-1 immunoreactivity in the HPC, indicating that the generated antibodies indeed recognized the phospho-Ser396/404 epitope. Remarkably, the vaccine decreased the number of neurons positive for hTau in the contralateral HPC, suggesting an effect of immunotherapy on tau propagation. Furthermore, the vaccine also showed potential neuroprotective effects, by decreasing the level of neurodegeneration in the dentate gyrus of treated mice as a function of the anti-phospho-tau antibody titer. In this work, using adapted AAV8-based models of local tau overexpression in the mouse brain, we reveal the effects of two treatments on different aspects of tau pathology related to the progression of the symptoms in tauopathies.

Transgenic SOD1 mice as a model for developing amyotrophic lateral sclerosis therapies

Sandrine Guillot

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by a loss of motor neurons in the brain (brainstem and cortex) and the spinal cord that leads to a motor neurological symptomatology. Approximately 10% of ALS cases have a familial form of ALS. Among the familial cases, 20% are caused by dominantly inherited mutations (around 100 different mutations) in the protein Cu/Zn superoxide dismutase (SOD1). To date, genetic ALS models on mutated SOD1 (SOD1G93A, SOD1G85R and SOD1G37R) transgenesis in rodents have been successful in recapitulating the main features of the human pathology, especially the loss of spinal or facial motoneurons, the increased astrogliosis, the activation of microglia and the cellular and molecular disruptions in motoneurons. The therapies toward a treatment or cure for ALS have been met with limited success. In the present study, SOD1G93A transgenic mice have been used to test new gene therapies to slow disease progression and pharmacological approach to obtain a better comprehension of the pathological process, more precisely, to define the involvement of microglial activation in the spread of the disease. For the approach by gene therapy, injection of viral vectors in a localized region of central nervous system (CNS) constitutes an excellent tool for the development of new therapies. In the present study, the viral-mediated expression of potential neuroprotective factors was explored in the lumbar spinal cord or facial nucleus of SOD1G93A transgenic mice. As HIV-1-derived lentiviral vectors can efficiently transduce neurons in CNS, these retroviral vectors were used to over-express the neurotrophic factor GDNF (glial cell line-derived neurotrophic factor) to supply a trophic support to lumbar and facial motoneurons, or RNAi molecules specifically targeting the SOD1G93A gene to inhibit the mutant SOD1 expression, the cause of the disease, in the lumbar spinal cord of SOD1G93A transgenic mice. For the study of the level of microglial involvement in the pathology, SOD1G93A transgenic mice had a daily pharmacological treatment with an inhibitor of microglial activation, the minocycline. The present thesis demonstrates that, on the one hand, at the difference of laboratories that delayed the disease progression by intramuscular injection of adenoviral vectors or adenoassociated vectors encoding for GDNF, the intraspinal injection of lentivirus encoding for GDNF did not lead to a protection of lumbar motoneurons and a delay of the disease. But this study showed a significantly rescue of facial motoneurons by the injection of lentivirus encoding for GDNF directly in facial nucleus. A selective vulnerability between motoneurons in facial nucleus and these in lumbar spinal cord has been therefore underlined. On the other hand, we showed that the therapeutic approach to inhibit the mutant SOD1 expression look as the most effective to delay the progression of the ALS pathology. Both studies of gene therapy, compared to others studies using the same therapeutic factors, demonstrated clearly that to optimize a therapeutic treatment, it seems better to act at muscular junction level with a retrograde transport form muscle to motoneuronal bodies of the therapeutic molecule or viral vector. Nevertheless, motoneuron-restricted silencing of mutant SOD1 would be useful for better understanding of neuronal and glial cellular mechanism mediating ALS-linked mutated SOD1 toxicity. Regarding the involvement of microglia in the pathological process, the inhibition of microglial activation by the minocycline treatment, suggests, on the one hand, that microglial activation do not initiate the pathological process and that this microgliosis might be a consequence and triggered by multiple disruptions. On the other hand, that minocycline could protect completely facial motoneurons and partially spinal motoneurons by its anti-apoptotic property. Therapeutic approaches aiming to act directly on the cause of the disease as well as a better comprehension of the role of non-neuronal cells in the pathological process may therefore open new perspectives for the treatment of ALS pathology.

EPFL2005

Adeno-Associated Virus-Mediated Gene Delivery for the Treatment of Amyotrophic Lateral Sclerosis and other Motor Neuron Disorders

Christopher Towne

The delivery of molecules and genes to the central nervous system (CNS) poses a major challenge for the treatment of neurodegenerative diseases. CNS disorders require long-term intervention and the presence of the blood-brain barrier (BBB) restricts the penetration of conventional drugs to the desired target cells. One approach towards stable delivery of therapeutic agents to the CNS is based on the transfer of DNA to target cells using viral vectors; a strategy known as gene therapy. Although viral vectors have provided encouraging results in CNS gene therapy trials for disorders such as Parkinson's disease, the small diffusion of the vector following direct injection into the brain region is not amenable to motor neuron (MN) disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy, that have cells distributed across fifty centimeters of spinal cord. The aim of this thesis was to explore non-invasive approaches for gene delivery to MNs of the spinal cord using adeno-associated viruses (AAV). AAV vectors are powerful gene transfer vehicles due to their low pathogenicity and ability to express genes in neurons for long periods of time. The first part of the thesis focused on ALS, an incurable paralytic disease resulting from a global death of MNs. Using a mouse model of ALS expressing a mutant form of superoxide dismutase 1 (SOD1) that causes the disease in humans, we have delivered genetic silencing instructions that act to degrade the SOD1 messenger RNA prior to its translation into the toxic protein. AAV serotype 6 was used to deliver the SOD1-silencing cassette through noninvasive peripheral routes to target relevant cell types in disease. The vector was administered intravenously following the hypothesis that AAV could infect MNs from the vasculature through traversing the peripheral axons that connect the MNs to the muscle fibers, a process known as retrograde transport. This resulted in infection of MNs across the spinal cord and brain stem, as well as almost total infection of skeletal muscle, another cell type implicated in ALS. Direct injections into multiple muscle groups were also performed, capitalizing on the retrograde transport capability of AAVs, to result in even higher levels of MN infection. These injections demonstrated that AAV serotype 6 could successfully deliver genes to MNs across the breadth of the spinal cord and resulted in protection of individual MN pools from ALS-mediated death. Curiously, however, despite the impressive infection profiles and neuroprotection at various spinal cord levels, neither of the techniques altered the disease course of the animals. These results stress the complexity of gene delivery and suggest that critical thresholds of SOD1-silencing and transduction across various cell types are required to rescue this particular disease model. The second component of the thesis concentrated purely on gene delivery to spinal cord neurons. AAV serotype 6 was injected directly into the muscles of Green African monkeys to determine whether the vector could undergo retrograde transport in larger animals. Indeed, efficient infection of MNs occurred, suggesting that this peripheral non-invasive delivery route is applicable to primates. These findings are not only relevant for human ALS therapy, but considering the similar dimensions of the injected monkeys with human newborns, directly relevant for treatment of infantile disease, spinal muscular atrophy. The last part of the thesis examined the ability of AAV serotype 6 to deliver genes to sensory neurons of spinal cord dorsal root ganglia in a mouse model of chronic pain. This study was initiated following the fortuitous observation of sensory neuron infection in the previous ALS mouse experiments. We examined various administration routes and found that AAV serotype 6 could efficiently deliver genes to sensory neurons in the context of chronic pain, demonstrating that the vector can be used to dissect mechanisms behind this disorder in rodents, and potentially serve as a vehicle for gene therapy towards intractable pain. In conclusion, the present thesis demonstrates that AAV serotype 6 is a powerful gene transfer vehicle for the CNS. Combined with future advances in AAV technology and delivery methods, the work presented here may one day facilitate gene therapy across the spinal cord for the treatment of devastating MN diseases, such as ALS and spinal muscular atrophy, as well as sensory neuron disorders, such as chronic pain.

EPFL2010

Application of anti-amyloid beta immunization using encapsulated cell technology in mouse models of Alzheimer's disease

Vanessa Laversenne

Alzheimer's disease (AD) is the most common form of dementia in the elderly. AD is characterized by the deposition of two aggregated proteins: Amyloid beta (Aß) and hyperphosphorylated tau. Accumulations of these proteins are thought to be the signature of a pathogenic process causing the loss of neurons and synaptic connections associated with severe cognitive impairments in AD patients. Other important factors are involved in the neurodegeneration occurring in AD, such as neuroinflammation. In the present thesis work, we are investigating the effects of passive anti-Aß immunization. Passive anti-Aß immunization consists in the delivery of antibodies targeting Aß, with the aim to prevent its accumulation and promote the removal of pathological amyloid species. Although the treatment has been shown to clear amyloid pathology in the brain of AD patients, it has so far failed to prevent or slow down cognitive decline. Hence, it is important to determine the effects of anti-Aß immunization when administered either before or during Aß deposition, and explore how the treatment affects the Alzheimerâs manifestations in mouse models combining Aß and tau pathologies. In order to continuously deliver antibodies in AD mouse models, we used encapsulated cell technology (ECT) to implant myoblasts genetically engineered and secrete the recombinant mAb11 mouse IgG2a antibody directed against aggregated forms of Aß. Using this approach, we investigated the following three topics of research:

We examined the effect of a preventive administration of anti-Aß antibodies in a slowly progressing mouse model with both Aß and tau (P301L) pathologies. We demonstrated that delivery of anti-Aß antibodies before the onset of the disease dramatically reduces Aß pathology. We also found that the treatment decreases hyperphosphorylated tau accumulation and recruits microglial around Aß plaques.
We next assessed the effects of passive anti-Aß immunization in another AD mouse model overexpressing both Aß and wild-type (WT) tau. In this second study, we administered the anti-Aß antibodies when both Aß and tau pathologies were already established. In the hippocampal formation, we showed that WT tau overexpression might affect the microglial response to ongoing pathology by reducing the tight interaction of these cells with the amyloid plaques. This effect, however, is counteracted by passive anti-Aß immunization, which was found to redirect microglia towards Aß deposits. In addition, passive anti-Aß immunization decreases tau spreading throughout the hippocampus, but fails to prevent neither tau phosphorylation, nor the hippocampal degeneration induced by local tau overexpression.
In parallel, we developed a human myoblast cell line secreting anti-Aß antibodies for translation of our ECT towards human application. Using C2C12 mouse myoblasts, our flat-sheet device allowed for long-term continuous antibody delivery in the plasma of subcutaneously implanted mice. We were able to generate antibody-secreting human cells using the immortalized C25 cell It was possible to differentiate these cells into myotubes and adapt them to long-term survival inside the capsules maintained in vitro. However, when implanted in the subcutaneous tissue of immunocompromised mice, myoblasts survived in the ECT device as a thin layer of cells and were not able to secrete recombinant antibodies in sufficient amounts to be considered for therapeutic purpose.

EPFL2019