We compared adeno-associated virus (AAV) serotypes for expression levels of green fluorescent protein (GFP) in the adult rat hippocampus by biophotonic imaging. Preparations of AAV serotypes 8, 9, Rh10, and Rh43 incorporating cytomegalovirus (CMV) promoter-driven GFP were purified by a CsCl method. Neither AAV Rh10 nor AAV Rh43 produced greater levels of GFP than AAV8, which was used as a reference. For AAV9, there was an increase relative to AAV8. The CsCl-purified AAV8 displayed an astroglial transduction pattern in contrast to the expected neuronal expression of other AAVs. After preparing the same CMV-GFP plasmid in AAV8 with an iodixanol purification method, the expected neuronal pattern resulted. The astroglial expression with the CsCl AAV8 was probably due to relatively high levels of protein impurities. We compared the CMV promoter with the CMV/chicken beta-actin (CBA) promoter in the context of AAV8, both prepared by iodixanol, and found the CBA promoter to produce stronger GFP expression. At two doses of vectors optimized for serotype, promoter and purification, we did not observe serotype differences among AAV8, AAV9, or AAV Rh10. The purification method can therefore impact the transduction pattern as well as the results when comparing serotype strengths.

Improved spread of transduction in the central nervous system (CNS) was achieved from intravenous administration of adeno-associated virus serotype-9 (AAV9) to neonatal rats. Spinal lower motor neuron transduction efficiency was estimated to be 78% using the highest vector dose tested at a 12-week interval. The widespread expression could aid studying diseases that affect both the spinal cord and brain, such as amyotrophic lateral sclerosis (ALS). The protein most relevant to neuropathology in ALS is transactive response DNA-binding protein 43 (TDP-43). When expressed in rats, human wild-type TDP-43 rapidly produced symptoms germane to ALS including paralysis of the hindlimbs and muscle wasting, and mortality over 4 weeks that did not occur in controls. The hindlimb atrophy and weakness was evidenced by assessments of rotarod, rearing, overall locomotion, muscle mass, and histology. The muscle wasting suggested denervation, but there was only 14% loss of motor neurons in the TDP-43 rats. Tissues were negative for ubiquitinated, cytoplasmic TDP-43 pathology, suggesting that altering TDP-43's nuclear function was sufficient to cause the disease state. Other relevant pathology in the rats included microgliosis and degenerating neuronal perikarya positive for phospho-neurofilament. The expression pattern encompassed the distribution of neuropathology of ALS, and could provide a rapid, relevant screening assay for TDP-43 variants and other disease-related proteins.

Amyotrophic Lateral Sclerosis (ALS) is a progressive neuromuscular disease in which the premature death of motor neurons ultimately leads to fatal paralysis. The most pronounced pathological feature of ALS is the cytoplasmic deposition and aggregation of proteolytically cleaved fragments of the TAR DNA‐binding protein 43 (TDP‐43). This recent and pivotal identification of TDP‐43 in intracytoplasmic inclusions of neuronal and glial cells has revolutionized the current field of research to focus on the role of TDP‐43 in ALS pathogenesis. Previous data has demonstrated overexpression of TDP‐43 via an adeno‐associated virus vector (AAV9) in the substantia nigra of a rat yielded cytoplasmic localization of ubiquitinated TDP‐43, indicative of ALS‐like preinclusions (Klein et al., 2008). This study aims to further the understanding of ALS pathology by overexpressing TDP‐43 in either the red nucleus or forepaw motor cortex of Sprague‐Dawley rats via stereotaxic injection of an AAV9 expressing full‐length human TDP‐43. Utilizing photobeam tracking sensors and a cylinder test to monitor fluctuations of vertical and horizontal motor movements, data for TDP‐43 and GFP rats were compared to elucidate the influence of TDP‐43 overexpression in the rubrospinal tract and forepaw motor cortex. Preliminary results have not shown aberrant changes in locomotor activity thus far (4 weeks), although future analysis of the brain tissue may discern the neuropathological consequences of TDP‐43 overexpression in the red nucleus or forepaw motor cortex.