Mark H. Tuszynski

A phase 1 clinical trial of nerve growth factor gene therapy for Alzheimer disease

Cholinergic neuron loss is a cardinal feature of Alzheimer disease. Nerve growth factor (NGF) stimulates cholinergic function, improves memory and prevents cholinergic degeneration in animal models of injury, amyloid overexpression and aging. We performed a phase 1 trial of ex vivo NGF gene delivery in eight individuals with mild Alzheimer disease, implanting autologous fibroblasts genetically modified to express human NGF into the forebrain. After mean follow-up of 22 months in six subjects, no long-term adverse effects of NGF occurred. Evaluation of the Mini-Mental Status Examination and Alzheimer Disease Assessment Scale-Cognitive subcomponent suggested improvement in the rate of cognitive decline. Serial PET scans showed significant (P < 0.05) increases in cortical 18-fluorodeoxyglucose after treatment. Brain autopsy from one subject suggested robust growth responses to NGF. Additional clinical trials of NGF for Alzheimer disease are warranted.

Neural stem cells constitutively secrete neurotrophic factors and promote extensive host axonal growth after spinal cord injury

Neural stem cells (NSCs) offer the potential to replace lost tissue after nervous system injury. This study investigated whether grafts of NSCs (mouse clone C17.2) could also specifically support host axonal regeneration after spinal cord injury and sought to identify mechanisms underlying such growth. In vitro, prior to grafting, C17.2 NSCs were found for the first time to naturally constitutively secrete significant quantities of several neurotrophic factors by specific ELISA, including nerve growth factor, brain-derived neurotrophic factor, and glial cell line-derived neurotrophic factor. When grafted to cystic dorsal column lesions in the cervical spinal cord of adult rats, C17.2 NSCs supported extensive growth of host axons of known sensitivity to these growth factors when examined 2 weeks later. Quantitative real-time RT-PCR confirmed that grafted stem cells expressed neurotrophic factor genes in vivo. In addition, NSCs were genetically modified to produce neurotrophin-3, which significantly expanded NSC effects on host axons. Notably, overexpression of one growth factor had a reciprocal effect on expression of another factor. Thus, stem cells can promote host neural repair in part by secreting growth factors, and their regeneration-promoting activities can be modified by gene delivery.

Neuroprotective effects of brain-derived neurotrophic factor in rodent and primate models of Alzheimer's disease

Profound neuronal dysfunction in the entorhinal cortex contributes to early loss of short-term memory in Alzheimers disease. Here we show broad neuroprotective effects of entorhinal brain-derived neurotrophic factor (BDNF) administration in several animal models of Alzheimers disease, with extension of therapeutic benefits into the degenerating hippocampus. In amyloid-transgenic mice, BDNF gene delivery, when administered after disease onset, reverses synapse loss, partially normalizes aberrant gene expression, improves cell signaling and restores learning and memory. These outcomes occur independently of effects on amyloid plaque load. In aged rats, BDNF infusion reverses cognitive decline, improves age-related perturbations in gene expression and restores cell signaling. In adult rats and primates, BDNF prevents lesion-induced death of entorhinal cortical neurons. In aged primates, BDNF reverses neuronal atrophy and ameliorates age-related cognitive impairment. Collectively, these findings indicate that BDNF exerts substantial protective effects on crucial neuronal circuitry involved in Alzheimers disease, acting through amyloid-independent mechanisms. BDNF therapeutic delivery merits exploration as a potential therapy for Alzheimers disease.

Guidelines for the conduct of clinical trials for spinal cord injury as developed by the ICCP panel : spontaneous recovery after spinal cord injury and statistical power needed for therapeutic clinical trials

The International Campaign for Cures of Spinal Cord Injury Paralysis (ICCP) supported an international panel tasked with reviewing the methodology for clinical trials in spinal cord injury (SCI), and making recommendations on the conduct of future trials. This is the first of four papers. Here, we examine the spontaneous rate of recovery after SCI and resulting consequences for achieving statistically significant results in clinical trials. We have reanalysed data from the Sygen trial to provide some of this information. Almost all people living with SCI show some recovery of motor function below the initial spinal injury level. While the spontaneous recovery of motor function in patients with motor-complete SCI is fairly limited and predictable, recovery in incomplete SCI patients (American spinal injury Association impairment scale (AIS) C and AIS D) is both more substantial and highly variable. With motor complete lesions (AIS A/AIS B) the majority of functional return is within the zone of partial preservation, and may be sufficient to reclassify the injury level to a lower spinal level. The vast majority of recovery occurs in the first 3 months, but a small amount can persist for up to18 months or longer. Some sensory recovery occurs after SCI, on roughly the same time course as motor recovery. Based on previous data of the magnitude of spontaneous recovery after SCI, as measured by changes in ASIA motor scores, power calculations suggest that the number of subjects required to achieve a significant result from a trial declines considerably as the start of the study is delayed after SCI. Trials of treatments that are most efficacious when given soon after injury will therefore, require larger patient numbers than trials of treatments that are effective at later time points. As AIS B patients show greater spontaneous recovery than AIS A patients, the number of AIS A patients requiring to be enrolled into a trial is lower. This factor will have to be balanced against the possibility that some treatments will be more effective in incomplete patients. Trials involving motor incomplete SCI patients, or trials where an accurate assessment of AIS grade cannot be made before the start of the trial, will require large subject numbers and/or better objective assessment methods.

Potential therapeutic uses of BDNF in neurological and psychiatric disorders

The growth factor brain-derived neurotrophic factor (BDNF) and its receptor tropomyosin-related kinase receptor type B (TRKB) are actively produced and trafficked in multiple regions in the adult brain, where they influence neuronal activity, function and survival throughout life. The diverse presence and activity of BDNF suggests a potential role for this molecule in the pathogenesis and treatment of both neurological and psychiatric disorders. This article reviews the current understanding and future directions in BDNF-related research in the central nervous system, with an emphasis on the possible therapeutic application of BDNF in modifying fundamental processes underlying neural disease.

Spontaneous corticospinal axonal plasticity and functional recovery after adult central nervous system injury

Although it is believed that little recovery occurs after adult mammalian spinal cord injury, in fact significant spontaneous functional improvement commonly occurs after spinal cord injury in humans. To investigate potential mechanisms underlying spontaneous recovery, lesions of defined components of the corticospinal motor pathway were made in adult rats in the rostral cervical spinal cord or caudal medulla. Following complete lesions of the dorsal corticospinal motor pathway, which contains more than 95% of all corticospinal axons, spontaneous sprouting from the ventral corticospinal tract occurred onto medial motoneuron pools in the cervical spinal cord; this sprouting was paralleled by functional recovery. Combined lesions of both dorsal and ventral corticospinal tract components eliminated sprouting and functional recovery. In addition, functional recovery was also abolished if dorsal corticospinal tract lesions were followed 5 weeks later by ventral corticospinal tract lesions. We found extensive spontaneous structural plasticity as a mechanism correlating with functional recovery in motor systems in the adult central nervous system. Experimental enhancement of spontaneous plasticity may be useful to promote further recovery after adult central nervous system injury.

The chondroitin sulfate proteoglycans neurocan, brevican, phosphacan, and versican are differentially regulated following spinal cord injury

Chondroitin sulfate proteoglycans (CSPGs) are extracellular matrix (ECM) molecules that are widely expressed throughout the developing and adult CNS. In vitro studies demonstrate their potential to restrict neurite outgrowth, and it is believed that CSPGs also inhibit axonal regeneration after CNS injury in vivo. Previous studies demonstrated that CSPGs are generally upregulated after spinal cord injury, and more recent reports have begun to identify individual proteoglycans that may play dominant roles in limiting axonal regeneration. The current study systematically examined the extended deposition patterns after CNS injury of four putatively inhibitory CSPGs that have not been extensively investigated previously in vivo: neurocan, brevican, phosphacan, and versican. After spinal cord injury, neurocan, brevican, and versican immunolabeling increased within days in injured spinal cord parenchyma surrounding the lesion site and peaked at 2 weeks. Neurocan and versican were persistently elevated for 4 weeks postinjury, and brevican expression persisted for at least 2 months. On the other hand, phosphacan immunolabeling decreased in the same region immediately following injury but later recovered and then peaked after 2 months. Combined glial fibrillary acidic protein (GFAP) immunohistochemistry and in situ hybridization demonstrated that GFAP astrocytes constituted a source of neurocan production after spinal cord injury. Thus, the production of several CSPG family members is differentially affected by spinal cord injury, overall establishing a CSPG-rich matrix that persists for up to 2 months following injury. Optimization of strategies to reduce CSPG expression to enhance regeneration may need to target several different family members over an extended period following injury.

INDUCTION OF BONE MARROW STROMAL CELLS TO NEURONS: DIFFERENTIATION, TRANSDIFFERENTIATION, OR ARTIFACT?

Differentiation of stem cells toward a neuronal lineage normally involves a gradually progressive restriction in developmental potential and is regulated by a diverse set of specific and temporally precise genetic events. However, recent studies have indicated that both rodent and human bone marrow stromal cells (MSCs) can be rapidly (within minutes to hours) induced to differentiate into neurons in vitro by relatively simple chemical means (using β‐mercaptoethanol [BME] or dimethylsulfoxide [DMSO] and butylated hydroxyanisol [BHA]; Woodbury et al. [ 2000 ] J. Neurosci. Res. 61:364–370). The ability to transdifferentiate an easily accessible cell source into neurons could have substantial potential for promoting neural repair. We therefore explored the potential of simple chemical methods to transdifferentiate other cell types, including primary rat fibroblasts, primary human keratinocytes, HEK293 cells, rat PC‐12 cells, and as positive control rat bone marrow stromal (BMS) cells. Surprisingly, all cells except for keratinocytes adopted at least partial “neuron‐like” pyramidal cell morphology with fine‐cellular extensions resembling neurites upon stimulation with BME or DMSO/BHA. However, time‐lapse microscopy indicated that the chemical exposure of MSCs did not result in new neurite growth but rather cellular shrinkage, with retraction of the majority of existing cell extensions, leaving only few, fine neurite‐like processes. To determine whether the chemically induced transdifferentiation resulted from simple cellular toxicity, MSCs were exposed to various stressors, including detergents, high‐molarity sodium chloride, and extremes of pH. In all cases, cellular shrinkage and adoption of pseudoneuronal morphology were observed. Concomitantly with cellular shrinkage, apparent increases in immunolabeling for the neuronal markers NSE and NeuN were detected in the cell soma that could not be confirmed by RT‐PCR. Furthermore, blockade of protein synthesis with cycloheximide did not prevent cells from adopting “neuron‐like” morphology after chemical induction. Thus, morphological changes and increases in immunolabeling for certain cellular markers upon “chemical induction” of MSCs are likely the result of cellular toxicity, cell shrinkage, and changes in the cytoskeleton and do not represent regulated steps in a complicated cellular differentiation process.

Long-Distance Growth and Connectivity of Neural Stem Cells after Severe Spinal Cord Injury

Neural stem cells (NSCs) expressing GFP were embedded into fibrin matrices containing growth factor cocktails and grafted to sites of severe spinal cord injury. Grafted cells differentiated into multiple cellular phenotypes, including neurons, which extended large numbers of axons over remarkable distances. Extending axons formed abundant synapses with host cells. Axonal growth was partially dependent on mammalian target of rapamycin (mTOR), but not Nogo signaling. Grafted neurons supported formation of electrophysiological relays across sites of complete spinal transection, resulting in functional recovery. Two human stem cell lines (566RSC and HUES7) embedded in growth-factor-containing fibrin exhibited similar growth, and 566RSC cells supported functional recovery. Thus, properties intrinsic to early-stage neurons can overcome the inhibitory milieu of the injured adult spinal cord to mount remarkable axonal growth, resulting in formation of new relay circuits that significantly improve function. These therapeutic properties extend across stem cell sources and species.

Combinatorial Therapy with Neurotrophins and cAMP Promotes Axonal Regeneration beyond Sites of Spinal Cord Injury

Previous attempts to promote regeneration after spinal cord injury have succeeded in stimulating axonal growth into or around lesion sites but rarely beyond them. We tested whether a combinatorial approach of stimulating the neuronal cell body with cAMP and the injured axon with neurotrophins would propel axonal growth into and beyond sites of spinal cord injury. A preconditioning stimulus to sensory neuronal cell bodies was delivered by injecting cAMP into the L4 dorsal root ganglion, and a postinjury stimulus to the injured axon was administered by injecting neurotrophin-3 (NT-3) within and beyond a cervical spinal cord lesion site grafted with autologous bone marrow stromal cells. One to 3 months later, long-projecting dorsal-column sensory axons regenerated into and beyond the lesion. Regeneration beyond the lesion did not occur after treatment with cAMP or NT-3 alone. Thus, clear axonal regeneration beyond spinal cord injury sites can be achieved by combinatorial approaches that stimulate both the neuronal soma and the axon, representing a major advance in strategies to enhance spinal cord repair.