EVALUACIÓN Y SEGUIMIENTO DE LOS PIP

Neurotrophins play a critical role in the regulation of growth, development, survival, differentiation, and function of neurons in both central and peripheral nervous system and synaptic development and maintenance, thus they are likely players in autistic pathology. Each of the four major mammalian neurotrophins, brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin 3 (NT-3), and NT-4, binds to its receptors, tropomysin-related kinase (TrkA, TrkB, TrkC), and neurotrophin receptor (75NTR), and activates signaling pathways affecting transcription. Neurotrophins are synthesized and secreted by the target organs, bind to the receptors, and act locally to regulate target innervations and nerve function. The neurotrophin–receptor complexes are also internalized and transported to cell bodies where they promote neuronal survival and differentiation (Reichardt, 2006). Thus the activity of neurotrophins can be regulated by factors involved in the tran- scriptional and posttranscriptional steps, receptor binding, and transport.

During development, neurotrophins are involved in the regulation of neuronal density. Developmental expression of neurotrophins has been examined in the rat hippocampus, neocortex, and cerebellum (Das et al., 2001). The results of this study indicate brain region–specifi c neurotrophin expression and independent regulation of neurotrophin mRNAs and proteins.

The involvement of neurotrophins in neuronal plasticity is suggested by the observa- tions of altered brain levels of neurotrophins in response to social and physical stimula- tion in mice (Zhu et al., 2006) and learning in rats (Klinstova et al., 2004). Furthermore, exposure to environmental toxins such as lead (Bokara et al., 2008) or chlorpyrifos (Betancourt et al., 2007), resulting in increased oxidative stress, is associated with an increase in hippocampal BDNF (Chao et al., 2007), and maternal infection during pregnancy results in altered levels of brain NGF and BDNF (Marx et al., 1999).

3.2.1 EVIDENCEFOR ALTERED NEUROTROPHIN EXPRESSIONIN AUTISM

Analysis of archived neonatal blood samples by recycling immunoaffi nity chroma- tography showed an increase in BDNF and NT-4 in newborns that subsequently developed autism and mental retardation (Nelson et al., 2001). However, subsequent enzyme-linked immunosorbent assay (ELISA) analysis of these samples did not confi rm the increase (Nelson et al., 2006). Yet serum BDNF levels were elevated severalfold in older children with autism (Connolly et al., 2006). Similarly, increased levels of plasma BDNF were observed in children with early-onset autism (Enstrom et al., 2008). In the archived neonatal samples, the NT-3 levels were signifi cantly lower than in controls (Nelson et al., 2006). More recently, serum BDNF has been found to increase with age, while that of NT-3 and NT-4/5 decreased with age (Nelson et al., 2006). Serum BDNF levels were found to increase during the fi rst several years, which then decreased slightly in adults. They were signifi cantly lower in autism cases aged 0–9 years compared to age-matched controls (Katoh-Semba et al., 2007). In adult (25 years of age) autistic cases, serum levels of BDNF were lower than in controls (Hashimoto et al., 2006). An increase in serum BDNF and NT-4 levels in autism has been reported (Miyazaki et al., 2004). Serum levels of BDNF were lower in adult male patients with autism than controls (Hashimoto et al., 2006). BDNF hyperactivity may be associated with early brain overgrowth and increased prevalence of seizures in autism. These differences in the results of indi- vidual studies may refl ect an expected spectrum of changes in neurotrophin expres- sion in autism. However, irrespective of the direction, altered brain neurotrophin levels are likely to be associated with impaired brain structure and function.

Despite evidence pointing to the role of neurotrophins in the pathophysiology of autism discussed above, data on the levels of brain neurotrophins in autism have been limited to two studies, and NT-3 between control and autistic cases have not been directly compared. The only direct assay of brain neurotrophin levels was reported by Perry et al. (2001). Using an ELISA, they reported a threefold higher level of BDNF in the basal forebrain in autism with no changes in NGF in teen- age cases. NT-3 levels were not measured in this particular study. They interpreted this increase as an intrinsic component of the autism disease process. The results of the only other study of human brain neurotrophins in control subjects using an immunohistochemical approach indicated that all four neurotrophins (NGF, BDNF, NT-3, and NT-4) are expressed in the human hippocampus at all ages, with the higher number of immunoreactive perikarya present in pre- and perinatal ages than in adults (Quartu et al., 1999).

Our recent report (Sajdel-Sulkowska et al., 2009) is the fi rst to compare the levels of NT-3 between autistic and control brains. We examined the possible link between increased oxidative stress and brain neurotrophin levels in autism using cerebellar samples from the subset of autistic and control cases used in our previ- ous study on brain 3-nitrotyrosine levels (Sajdel-Sulkowska et al., 2008). We have selected the cerebellum because of its intimate involvement in autism indicated by increased cerebellar volume, decreased ratio of gray to white matter (Courchesne et al., 2001, 2003), and decrease in Purkinje cell number (Courchesne, 1991; Kemper and Bauman, 1993; Rivto et al., 1986; Whitney et al., 2008). NT-3 was

selected because of its high expression in the developing rat cerebellum (Das et al., 2001) and its critical role in migration and survival of cerebellar granule (Li et al., 2004) and Purkinje cells (Kawakami et al., 2000). The data presented in Figure 3.2 shows a statistically signifi cant NT-3 increase (40.3%) in the autistic cerebel- lum and support a concept of altered brain neurotrophin expression in autism. As stated above, NT-3 levels in human brain have not been previously quantifi ed; our data is thus the fi rst on the brain levels of NT-3 in autism. It is intriguing that the expression of this particular neurotrophin expression appears to decrease with age in the rodent brain (Das et al., 2001). Consequently, if a similar developmental pattern is present in human cerebellum, then prolonged and persistent elevation of NT-3 in autism could upset the balance of neurotrophic factors and affect brain growth and development. Specifi cally, overexpression of NT-3 could contribute to the cerebellar overgrowth observed in some autistic cases (Courchesne et al., 2001). Furthermore, persistent elevation of NT-3, specifi cally involved in neuronal differentiation (Ghosh and Greenberg, 1995), neurite fasciculation (Segal et al., 1995), and axonal targeting, may have other profound effects such as on synapse formation.

We also observed a positive correlation between cerebellar NT-3 and cerebellar 3-nitrotyrosine, which suggests an association between increased oxidative stress and altered brain neurotrophin levels in autism. This association is not perfect (r = .83), suggesting the presence of a subset of autistic cases with normal neurotro- phin levels. It is possible that subsets of autistic cases with unique clinical symptoms are related to specifi c molecular changes.

3.2.2 POSSIBLE MECHANISMS INVOLVEDIN ALTERED NEUROTROPHIN