Growth factors influence the topography of axonal projections during nervous system development and facilitate axonal sprouting and regeneration after injury in the adult. However, in the absence of target reinnervation and reestablishment of synaptic activity, we hypothesized that continuing delivery of neurotrophins would be required to sustain regenerating axons for prolonged times points after neurotrophin-induced axon growth after spinal cord injury ( SCI) in the adult. Using tetracycline-inducible expression of brain-derived neurotrophic factor by genetically modified fibroblasts, we were able to extensively and significantly turn growth factor expression "on" or "off" in vitro and in vivo within sites of SCI. Notably, we find that transient growth factor delivery is sufficient to sustain regenerated axons for prolonged time periods within spinal cord lesion sites. Immunohistochemical analysis demonstrated an absence of neuronal targets or synapses within transient growth factor expressing grafts but the persistent presence of Schwann cells. Thus, the adult CNS appears capable of sustaining axons that have extended after transient growth factor delivery, an effect potentially attributable to the persistence of Schwann cells in lesion/graft sites.

Entorhinal cortex (EC), primary source of input into the hippocampus, is important for memory and learning. Alzheimer’s patients experience memory loss at early stages of the disease. The memory loss is associated with neuronal atrophy and death, which starts in EC. Previous evidence indicates that brain-derived neurotrophic factor (BDNF) could prevent cell loss and atrophy. The goal of this study was to test lower dosages of adeno-associated virus (AAV) 2-BDNF (1e11vp/ml, 3e11vp/ml and 3e11vp/ml x2 volume) and AAV9-BDNF (3e11vp/ml). Our hypothesis was the BDNF gene delivery into the EC will produce a neuroprotective effect preventing cell lose and cell body atrophy. A perforant pathway lesion was performed in rats that severed the projections from EC layer II neurons to dentate gyrus to induce cell death or atrophy in these neurons. Three days prior to the lesion, subject received treatments into the EC. Rats were euthanized two weeks after lesion, and the brains were analyzed using stereology to quantify the number of neurons and cell atrophy by measuring cell body size. Results showed that BDNF treatment produced a neuroprotective effect that ameliorated cell loss (p<0.01 – 0.05), while control groups had ~30% cell loss. Cell atrophy was decreased within the AAV9 treatment group (p<0.05). Quantification of pAKT cell numbers in ECLII showed no effect of BDNF treatment on pAKT activation after 2 weeks. BDNF does have the potential to prevent early cell lose and atrophy in AD. This might prove to prevent the early memory observed AD.

Neural stem cells (NSCs) can differentiate into both neurons and glia after transplantation into sites of spinal cord injury (SCI). The neuronal component of stem cell grafts has the potential to form functional synaptic relays across the lesion site. The glial component, on the other hand, may reform a blood–spinal cord barrier (BSCB), support neuronal function, and potentially contribute to remyelination. We performed a long-term, time-course study for 1.5 years focused on astrocyte survival, migration, differentiation, integration, and safety following transplantation of human NSCs into sites of C5 hemisection in immunodeficient rats. Subjects were sacrificed at 1, 3, 6, 12, and 18 months after transplantation.

Notably, NSCs that adopted a neuronal fate did not migrate from the lesion site. In contrast, transplanted cells that adopted astrocyte fates exhibited long-distance migration from the lesion site and through host white matter in rostrocaudal directions. NSCs migrated from the lesion slowly, at a mean rate of 2-3 mm per month, divided as they migrated, and gradually differentiated into astrocytes. After 1.5 years, astrocytes migrated 9 spinal cord segments, caudally to the mid-thoracic level, and rostrally into the brainstem. The migrated human astrocytes joined the endogenous population of astrocytes in the host spinal cord, extended endfeet towards cellular constituents of the blood-spinal cord barrier, and formed perivascular astrocytic networks connected by gap junctions, suggesting structural and potentially functional integration. No adverse consequences of this extended glial migration were detected. Thus, human astrocytes can migrate from NSC grafts transplanted into sites of SCI and safely integrate into the host central nervous system.

Adeno-associated viral (AAV) vectors are effective gene therapy delivery candidates, and the AAV-PhP.eB capsid is effective at transducing the CNS in mice when delivered intravenously (IV). Introduction of promoter sequences to the AAV-PhP.eB genome can restrict gene expression to specific cell types. More research is needed to identify cell-specific promoter sequences that restrict gene expression to specific CNS targets. Thus, the Ple394 and Ple394-HBB sequences were examined to see if they can restrict gene expression to cortical neurons. The HB9 sequence was examined to see it could restrict gene expression to spinal motor neurons. The Ple394 promoter resulted in strong cortical neuron gene expression and comparatively less gene expression in off-target brain and spinal cord regions. The Ple394 vector did not result in significantly different gene expression in the liver compared to mice that received saline. The Ple394-HBB promoter failed to cause cortical neuron-specific gene expression. The HB9 promoter resulted in spinal motor neuron-specific gene expression with relatively little expression in the brain. The HB9 vector didn’t result in significantly different gene expression in the liver compared to mice that received saline. In conclusion, introduction of the Ple394 and HB9 promoter sequences into the AAV-PhP.eB genome were able to restrict gene expression to cortical neurons and spinal motor neurons, respectively. Identification of promoters that can drive selective gene expression in the cerebral cortex or spinal motor neurons will allow for the IV delivery of AAV gene therapies to treat diseases such as Alzheimer’s disease or amyotrophic lateral sclerosis, respectively.

Fine motor behavior and circuit development of the motor cortex is associated with selective structural expansion in networks of functionally related neurons 1. Corticospinal neurons are amongst these that exhibit extensive structural plasticity when subjected to forelimb-specific behavioral tasks where these neurons undergo elaborate enrichment in dendritic boutons and an increase in apical and basilar spine density. In contrast, neighboring neurons not directly engaged in the motor behavior do not display the same structural modifications as a consequence of learning. We wanted to determine what drives this anatomical change on a molecular level. First, we identified the learning transcriptome by isolating C8-projecting corticospinal neurons that were subjected to skilled grasped training and purified mRNA-associated ribosomes. Our RNA sequencing revealed amyloid precursor protein as a functional network hub associated with motor learning. We determined if APP protein is upregulated in response to motor learning. Animals were subjected to either 1-day or 7-days of forelimb-skilled grasp training, motor cortices were isolated, and protein extracted. Western blot analysis indicated that holo APP protein is slightly upregulated in response to learning compared to untrained animals. APP proteolytic fragments showed a more robust upregulation in response to learning, and histology of these animals showed APP localization within layer 5 motor neurons suggesting APP protein plays a role during learning where this protein is proteolytically cleaved in response to this task.

An estimated 300,000 people suffer from spinal cord injury (SCI) in the US, costing about $9.7 billion annually (French et al., 2007). Quadriplegic patients, comprising nearly 60% of SCI cases (National Spinal Cord Injury Statistics Center), list the loss of motor control, especially hand function, as the primary impediment to their daily lives. To overcome this loss, it is necessary for the corticospinal tract (CST) – the main tract controlling voluntary motor movement – to regenerate and reform functional synapses. One method to reverse such dysfunctions is to use neural progenitor cell (NPC) grafts which recapitulate lost neural tissue and promote axon regeneration, ultimately serving as a relay for neural signals.

Previously, our lab demonstrated functional recovery and robust CST regeneration into rat embryonic day 14 (E14) NPC grafts placed into cervical and thoracic SCI sites (Kadoya et al., 2016). CST regeneration into a cervical graft has the potential to restore voluntary motor function, especially hand function, for quadriplegic patients. However, whether forelimb, hindlimb or both CST populations regenerate into a cervically placed NPC graft, is unknown. Because both populations are axotomized, we hypothesized that both forelimb and hindlimb projecting CST axons would regenerate into a mid-cervically placed NPC graft. Here, we used a Cre-mediated intersectional viral approach to selectively label either forelimb or hindlimb projecting CST axons. All rats were then subjected to a C3 dorsal column lesion and received an E14 spinal cord-derived NPC graft. Surprisingly, we found preferential regeneration of hindlimb CST axons into the C3 NPC grafts, with sparse forelimb axon regeneration. This creates a functional mismatch: hypothetically, to achieve optimal functional recovery, CST axons controlling the forelimbs instead of the hindlimbs would regenerate into the lesion to form functional synapses. An examination of mechanisms underlying this selective regeneration is necessary, and will contribute to optimizing corticospinal regeneration after SCI.

Spinal cord injury (SCI) is a widespread and drastic condition that causes permanent paralysis below the level of the lesion, as well as a myriad of associated problems. In the past, efforts to promote regeneration have remained largely unsuccessful. Recent work from the Tuszynski lab reported that grafts of spinal cord neural progenitor cells (NPCs) or neural stem cells (NSCs) grafted into sites of SCI support extensive corticospinal tract (CST) regeneration into the lesion site. This finding was significant because the CST is the most important motor system in humans and efforts to maximize regeneration of this system are key to developing translational human therapies. The regenerating transcriptome of the CST was identified, showing neurons underdoing active regeneration display a sustained upregulation of gene expression over time. We mapped our regenerating transcriptome signature onto the Broad Connectivity Map (CMAP). Our CST regeneration signature was queried against 1.3 million signatures, where a positive match showed a similar transcriptome profile to our CST signature. In silico analysis identified Thiorphan as a candidate drug that most closely resembles the transcriptome of our regenerating CST signature. In vitro, Thiorphan caused an increase in neurite outgrowth compared to control. When tested in a cervical contusion lesion, thiorphan treatment alone showed an increase in functional recovery after SCI compared to lesion-only controls. When used in combination with NPC graft, no additive benefit was seen. In conclusion, there may be a new drug-disease connection between Thiorphan and spinal cord injury.

Gene therapy is being explored for its potential to treat neurodegenerative diseases such as Alzheimer’s Disease and Huntington’s Disease through the delivery of therapeutic transgenes with Adeno-Associated Virus (AAV) vectors. The leading candidates for these treatments are AAV9 and AAV-PhP.eB because of their safety, efficacy in rodent and non-human primate studies, and their ability to cross the blood-brain barrier. These vectors have been tested in juvenile mice, but neurodegenerative diseases typically affect older individuals and these vectors have never been tested in aged mice. Therefore, we delivered AAV9-eGFP and AAV-PhP.eB-eGFP intrathecally or intravenously to mice and compared the virus’ transduction patterns in the brain across aging, using groups of aged 20-month-old mice versus juvenile 3-month-old mice. We found a reduction in gene expression with aging in mice treated with intravenous AAV9. However, there was no detectable change in gene expression across aging in mice receiving intravenous AAV-PhP.eB or intrathecal AAV9. There is a significant reduction associated with increased weight when mice are treated with intravenous AAV9 but not with intrathecal AAV9, indicating that the reduced efficacy of intravenous AAV9 in aged mice may be due to the larger size of these animals. These results suggest that the more promising delivery method for AAV9 gene therapy in older individuals is via the intrathecal route.

Neural stem cells or neural progenitor cells have been extensively studied in spinal cord injury (SCI) rat models to attempt to reconnect injured circuits to restore function. Attempts have shown significant success, and further studies have revealed corticospinal tract (CST) axon growth into the neural stem cell graft. The purpose of this study is to understand whether CST axons undergo regeneration, regenerative sprouting, or sprouting into a neural stem cell graft. The animals were grafted with E14 spinal neural progenitor cells in a C4 dorsal column lesion for this study. The rat models were then injected with a small volume and low concentration of a tracer, AAV8-CAG-rCOMET or biotinylated dextran amine, in the motor cortex of the brain to label a few individual axons in order to visualize their growth pathway. Through the visualization on the confocal microscope, the injured axons of the CST are shown to regenerate into the graft cells. There was minimal regenerative sprouting and no sprouting seen with this model. Thus, CST axons regenerate into a neural stem cell graft.