Brian K. Kaspar

Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo

Neural stem cells exist in the developing and adult nervous systems of all mammals, but the basic mechanisms that control their behavior are not yet well understood. Here, we investigated the role of Sonic hedgehog (Shh), a factor vital for neural development, in regulating adult hippocampal neural stem cells. We found high expression of the Shh receptor Patched in both the adult rat hippocampus and neural progenitor cells isolated from this region. In addition, Shh elicited a strong, dose-dependent proliferative response in progenitors in vitro. Furthermore, adeno-associated viral vector delivery of shh cDNA to the hippocampus elicited a 3.3-fold increase in cell proliferation. Finally, the pharmacological inhibitor of Shh signaling cyclopamine reduced hippocampal neural progenitor proliferation in vivo. This work identifies Shh as a regulator of adult hippocampal neural stem cells.

Cell culture: Progenitor cells from human brain after death

Culturing neural progenitor cells from the adult rodent brain has become routine and is also possible from human fetal tissue, but expansion of these cells from postnatal and adult human tissue, although preferred for ethical reasons, has encountered problems. Here we describe the isolation and successful propagation of neural progenitor cells from human postmortem tissues and surgical specimens. Although the relative therapeutic merits of adult and fetal progenitor cells still need to be assessed, our results may extend the application of these progenitor cells in the treatment of neurodegenerative diseases.

IGF-I instructs multipotent adult neural progenitor cells to become oligodendrocytes.

Adult multipotent neural progenitor cells can differentiate into neurons, astrocytes, and oligodendrocytes in the mammalian central nervous system, but the molecular mechanisms that control their differentiation are not yet well understood. Insulin-like growth factor I (IGF-I) can promote the differentiation of cells already committed to an oligodendroglial lineage during development. However, it is unclear whether IGF-I affects multipotent neural progenitor cells. Here, we show that IGF-I stimulates the differentiation of multipotent adult rat hippocampus-derived neural progenitor cells into oligodendrocytes. Modeling analysis indicates that the actions of IGF-I are instructive. Oligodendrocyte differentiation by IGF-I appears to be mediated through an inhibition of bone morphogenetic protein signaling. Furthermore, overexpression of IGF-I in the hippocampus leads to an increase in oligodendrocyte markers. These data demonstrate the existence of a single molecule, IGF-I, that can influence the fate choice of multipotent adult neural progenitor cells to an oligodendroglial lineage.

Targeted retrograde gene delivery for neuronal protection.

The cellular heterogeneity and complex circuitry of the central nervous system make it difficult to achieve precise delivery of experimental and therapeutic agents. We report here an in vivo retrograde gene delivery strategy to target mature projection neurons using adeno-associated virus, a vector with low toxicity and the capacity for long-term gene expression. Viral delivery to axon terminal fields in the hippocampus and striatum resulted in viral internalization, retrograde transport, and transgene expression in specific projection neurons in entorhinal cortex and substantia nigra. Retrograde delivery of the anti-apoptotic gene Bcl2l (also known as Bcl-xL) protected entorhinal projection neurons from subsequent damage-induced cell death. Given the broad distribution of neurons affected by neurodegenerative diseases, gene delivery to both the terminal fields and the projection neurons through retrograde infection provides for strategic therapeutic intervention at both levels of the neural circuit. This approach may also facilitate experimental studies of defined neural circuits.

Synergy of insulin-like growth factor-1 and exercise in amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease of the neuromuscular system resulting in paralysis and ultimately death. Currently, no effective therapy is prescribed for patients; however, several therapeutic strategies are showing promise. Either exercise or treatment with adeno‐associated virus/insulin‐like growth factor–1 alone has therapeutic benefits in an amyotrophic lateral sclerosis transgenic mouse model. We show here that activity duration affects the therapeutic benefit associated with exercise, with 6‐ and 12‐hour exposure to a running wheel providing significant motor function benefits and increased survival. Remarkably, a combination of insulin‐like growth factor–1 gene delivery and exercise has profound effects on survival and function, indicative of synergistic effects with exercise and insulin‐like growth factor–1. Our results indicate that a drug treatment in combination with appropriate exercise may provide the most promising therapy for amyotrophic lateral sclerosis to date. Ann Neurol 2005

Virus-Delivered Small RNA Silencing Sustains Strength in Amyotrophic Lateral Sclerosis

Mutations in superoxide dismutase cause a subset of familial amyotrophic lateral sclerosis and provoke progressive paralysis when expressed in mice. After retrograde transport to the spinal cord following injection into muscles, an adeno‐associated virus carrying a gene that encodes a small interfering RNA was shown to target superoxide dismutase messenger RNA for degradation. The corresponding decrease in mutant superoxide dismutase in spinal motor neurons preserved grip strength. This finding provides proof of principle for the selective reduction of any neuronal protein and supports intramuscular injections of a small interfering RNA–encoding virus as a viable therapy for this type of familial amyotrophic lateral sclerosis. Ann Neurol 2005;57:773–776

Myocardial gene transfer and long-term expression following intracoronary delivery of adeno-associated virus.

Adeno‐associated viral vectors (AAV) can direct long‐term gene expression in post‐mitotic cells. Previous studies have established that long‐term cardiac gene transfer results from intramuscular injection into the heart. Cardiac gene transfer after direct intracoronary delivery of AAV in vivo, however, has been minimal in degree, and indirect intracoronary delivery, an approach used in an increasing number of studies, appears to be receiving more attention. To determine the utility of indirect intracoronary gene transfer of AAV, we used aortic and pulmonary artery cross clamping followed by proximal aortic injection of AAV encoding enhanced green fluorescent protein (AAV.EGFP) at 1011 DNase resistant particles (drp; high‐performance liquid chromatography (HPLC)‐purified) per rat. Gene expression was quantified by fluorescent microscopy at four time points up to 1 year after vector delivery, revealing 20–32% transmural gene expression in the left ventricle at each time point. Histological analysis revealed little or no inflammatory response and levels of transgene expression were low in liver and undetectable in lung. In subsequent studies in pigs, direct intracoronary delivery into the left circumflex coronary artery of AAV.EGFP (2.64–5.28 × 1013 drp; HPLC‐purified) resulted in gene expression in 3 of 4 pigs 8 weeks following injection with no inflammatory response in the heart. PCR analysis confirmed AAV vector presence in the left circumflex perfusion bed. These data indicate that intracoronary delivery of AAV vector is associated with transgene expression in the heart, providing a means to obtain long‐term expression of therapeutic genes. Copyright

Intramuscular AAV delivery of NT-3 alters synaptic transmission to motoneurons in adult rats

We examined whether elevating levels of neurotrophin‐3 (NT‐3) in the spinal cord and dorsal root ganglion (DRG) would alter connections made by muscle spindle afferent fibers on motoneurons. Adeno‐associated virus (AAV) serotypes AAV1, AAV2 and AAV5, selected for their tropism profile, were engineered with the NT‐3 gene and administered to the medial gastrocnemius muscle in adult rats. ELISA studies in muscle, DRG and spinal cord revealed that NT‐3 concentration in all tissues peaked about 3 months after a single viral injection; after 6 months NT‐3 concentration returned to normal values. Intracellular recording in triceps surae motoneurons revealed complex electrophysiological changes. Moderate elevation in cord NT‐3 resulted in diminished segmental excitatory postsynaptic potential (EPSP) amplitude, perhaps as a result of the observed decrease in motoneuron input resistance. With further elevation in NT‐3 expression, the decline in EPSP amplitude was reversed, indicating that NT‐3 at higher concentration could increase EPSP amplitude. No correlation was observed between EPSP amplitude and NT‐3 concentration in the DRG. Treatment with control viruses could elevate NT‐3 levels minimally resulting in measurable electrophysiological effects, perhaps as a result of inflammation associated with injection. EPSPs elicited by stimulation of the ventrolateral funiculus underwent a consistent decline in amplitude independent of NT‐3 level. These novel correlations between modified NT‐3 expression and single‐cell electrophysiological parameters indicate that intramuscular administration of AAV(NT‐3) can exert long‐lasting effects on synaptic transmission to motoneurons. This approach to neurotrophin delivery could be useful in modifying spinal function after injury.

Repression of Human Fibroblast Growth Factor 2 by a Novel Transcription Factor

Here we describe the cloning of the regulator of fibroblast growth factor 2 (FGF-2) transcription (RFT) using a yeast one-hybrid screening with a defined motif in FGF-2 promoter as a target sequence. Overexpression of human RFT (RFT-A) reduces FGF-2 RNA and protein levels in both normal and tumor cell lines. Its splice variants, RFT-A′ and RFT-B, have deletions in the putative DNA binding domain and fail to bind FGF-2 promoter and repress FGF-2 gene expression. The ratios of RFT isoforms differ between normal and tumor cells, with the splice variants dominating in tumor cells. Overexpression of RFT-A induces glioma cell death. Our data suggest that regulation of FGF-2 by RFT is important for cellular functions and may be impaired in certain tumors.