Functional recovery in a Friedreich's ataxia mouse model by frataxin gene transfer using an HSV-1 amplicon vector.

There is currently no effective treatment for Friedreich's ataxia (FA), the most common of the hereditary ataxias. The disease is caused by mutations in FRDA that drastically reduce expression levels of the mitochondrial protein frataxin. In FA animal models, a key difficulty is obtaining the precise levels of frataxin expression in the appropriate tissues to provoke pathology without early lethality. To develop strategies to circumvent these problems, conditional frataxin transgenic mice have been generated. We now show that frataxin expression can be eliminated in neurons from these loxP[frda] mice by infection with CRE-expressing herpes simplex virus type 1 (HSV-1) amplicon vectors. We have also achieved in vivo delivery by stereotaxic injection of these CRE-expressing vectors into the brainstem of loxP[frda] mice to generate a localized gene knockout model. These mice develop a behavioral deficit in the rotarod assay detectable after 4 weeks, and when re-injected with HSV-1 amplicon vectors expressing human frataxin complementary DNA (cDNA) exhibit behavioral recovery as early as 4 weeks after the second injection. To the best of our knowledge, this is the first proof of principle of recovery of neurological function by a therapeutic agent aimed at correcting frataxin deficiency.     

Activity-dependent neurotrophic factor: intranasal administration of femtomolar-acting peptides improve performance in a water maze

Activity-dependent neurotrophic factor (ADNF) is a glia-derived protein that is neuroprotective at femtomolar concentrations. A nine-amino acid peptide derived from ADNF (Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala; ADNF-9) captured the activity of the parent protein and has been reported to protect cultured neurons from multiple neurotoxins. Antibodies recognizing ADNF-9 produced neuronal apoptosis, and identified an additional, structurally related, glia-derived peptide, Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (NAP). Previous comparative studies have characterized s.c.-injected NAP as most efficacious in protecting against developmental retardation and learning impairments in apolipoprotein E-deficient mice. This study was designed to assess 1) neuroprotection after intranasal administration of ADNF-9 and NAP to rats treated with the cholinotoxin ethylcholine aziridium; and 2) bioavailability and pharmacokinetics after intranasal administration. Results showed significant improvements in short-term spatial memory, as assessed in a water maze, after daily intranasal administration of 1 microg of peptide (ADNF-9 or NAP) per animal. However, a 5-day pretreatment with ADNF-9 did not improve performance measured after cessation of treatment. Compared with rats treated with ADNF-9, NAP-pretreated animals exhibited a significantly better performance. Furthermore, NAP (and not ADNF-9) protected against loss of choline acetyl transferase activity. Significant amounts of (3)H-labeled NAP reached the brain, remained intact 30 min after administration, and dissipated 60 min after administration. This study revealed efficacy for ADNF-related peptides in rodent models for neurodegeneration. The small size of the molecules, the low dosage required, the noninvasive administration route, and the demonstrated activity in a relevant paradigm suggest NAP as a lead compound for future drug design.     

A peptide derived from activity-dependent neuroprotective protein (ADNP) ameliorates injury response in closed head injury in mice

Brain injury induces disruption of the blood-brain barrier, edema, and release of autodestructive factors that produce delayed neuronal damage. NAPSVIPQ (NAP), a femtomolar-acting peptide, is shown to be neuroprotective in a mouse model of closed head injury. NAP injection after injury reduced mortality and facilitated neurobehavioral recovery (P < 0.005). Edema was reduced by 70% in the NAP-treated mice (P < 0.01). Furthermore, in vivo magnetic resonance imaging demonstrated significant brain-tissue recovery in the NAP-treated animals. NAP treatment decreased tumor necrosis factor-alpha levels in the injured brain and was shown to protect pheochromocytoma (PC12 cells) against tumor necrosis factor-alpha-induced toxicity. Thus, NAP provides significant amelioration from the complex array of injuries elicited by head trauma.     

Blockage of VIP during mouse embryogenesis modifies adult behavior and results in permanent changes in brain chemistry

Vasoactive intestinal peptide (VIP) regulates growth and development during the early postimplantation period of mouse embryogenesis. Blockage of VIP with a VIP antagonist during this period results in growth restriction, microcephaly, and developmental delays. Similar treatment of neonatal rodents also causes developmental delays and impaired diurnal rhythms, and the adult brains of these animals exhibit neuronal dystrophy and increased VIP binding. These data suggest that blockage of VIP during the development of the nervous system can result in permanent changes to the brain. In the current study, pregnant mice were treated with a VIP antagonist during embryonic days 8 through 10. The adult male offspring were examined in tests of novelty, paired activity, and social recognition. Brain tissue was examined for several measures of chemistry and gene expression of VIP and related compounds. Glial cells from the cortex of freated newborn mice were plated with neurons and examined for VIP binding and their ability to enhance neuronal survival. Treated adult male mice exhibited increased anxiety-like behavior and deficits in social behavior. Brain tissue exhibited regionally specific changes in VIP chemistry and a trend toward increased gene expression of VIP and related compounds that reached statistical significance in the VIP receptor, VPAC-1, in the female cortex. When compared to control astrocytes, astrocytes from treated cerebral cortex produced further increases in neuronal survival with excess synaptic connections and reduced VIP binding. In conclusion, impaired VIP activity during mouse embryogenesis resulted in permanent changes to both adult brain chemistry/cell biology and behavior with aspects of autism-like social deficits.     

A neuronal microtubule-interacting agent, NAPVSIPQ, reduces tau pathology and enhances cognitive function in a mouse model of Alzheimer's disease

Neurofibrillary tangles composed of aggregated, hyperphosphorylated tau in an abnormal conformation represent one of the major pathological hallmarks of Alzheimer's disease (AD) and other tauopathies. However, recent data suggest that the pathogenic processes leading to cognitive impairment occur before the formation of classic tangles. In the earliest stages of tauopathy, tau detaches from microtubules and accumulates in the cytosol of the somatodendritic compartment of cells. Either as a cause or an effect, tau becomes hyperphosphorylated and aggregates into paired helical filaments that comprise the tangles. To assess whether an agent that modulates microtubule function can inhibit the pathogenic process and prevent cognitive deficits in a transgenic mouse model with AD-relevant tau pathology, we administered the neuronal tubulin-preferring agent, NAPVSIPQ (NAP). Three months of treatment with NAP at an early-to-moderate stage of tauopathy reduced the levels of hyperphosphorylated soluble and insoluble tau. A 6-month course of treatment improved cognitive function. Although nonspecific tubulin-interacting agents commonly used for cancer therapy are associated with adverse effects due to their anti-mitotic activity, no adverse effects were found after 6 months of exposure to NAP. Our results suggest that neuronal microtubule interacting agents such as NAP may be useful therapeutic agents for the treatment or prevention of tauopathies.     

NAP protects against cyanide-related microtubule destruction

The peptide NAP (NAPVSIPQ) was shown to protect neurons against a wide variety of insults. Particularly, NAP was shown to be neuroprotective in vitro against cyanide in hippocampal cultures and against oxygen-glucose deprivation in hippocampal and cortical neuronal cultures. Cyanide causes energy depletion in the cell and destroys the cytoskeleton, and NAP has been shown before to protect the microtubule cytoskeleton. The current study explored the effect of NAP on cyanide-induced microtubule destruction in cerebral cortical cultures. Sodium cyanide (6.8 mM) reduced the number of neurons containing intact microtubules as identified by bIII-tubulin immunostaining. When sodium cyanide was added together with NAP (10⁻¹⁴–10⁻¹² M), complete protection was observed. Although the primary site of action of cyanide is considered to be the mitochondria, the current results involve microtubule destruction by cyanide toxicity that is completely reversed by NAP treatment.