Brain Development in Experimental Hyperphenylalaninaemia: Disturbed Proliferation and Reduced Cell Numbers in the Cerebellum

Abstract

Rat littermates made hyperphenylalaninaemic by daily injections of α-methylphenylalanine plus phenylalanine exhibit only slight reductions in body weight but a considerably affected cerebellar development. Weight and size of the cerebellum are reduced. The DNA content (µg/mg wet weight and total cerebellar DNA) is decreased. Morphometric measurements reveal a smaller area of all cerebellar layers; the external granular layer being most, the white matter least affected. The cell packing density of the cells in the granular layer is decreased whereas Purkinje cells are lined up closer when compared with the controls. A small reduction in Purkinje cell number is accompanied by considerably reduced estimated total cell number of the internal granular layer. The incorporation of ³H-thymidine into cerebellar tissue is decreased at 5 days p. p., but increased at 15 days. No significant alteration is found in the kidney. Results are discussed in relation to a limited protein synthesis of developing neurons resulting from "cellular undernutrition" of other amino acids that compete with the high phenylalanine levels. A comparison is made with the effects of "systemic undernutrition" on cerebellar development.
Phenylketonuria (PKU) is one of the most striking clinical examples of impairing brain development by an amino acid imbalance. Experimental hyperphenylalaninaemia (hyperphe) can be elicited in suckling rats by the combined injection of phenylalanine and α-methylphenylalanine, an inhibitor of phenylalanine hydroxylase (Greengard et al 1976). This treatment brings about changes in the composition of serum and brain, which are similar to those found in human PKU (Delvalle et al 1978, Lane et al 1980), unspecific side effects of the inhibitor have not been reported (Kelly and Johnson 1978, Lane el al 1980).
Hyperphe is accompanied by reduced serum concentrations of other essential amino acids (Linneweh and Ehrlich 1960, 1962). Furthermore, high levels of phenylalanine were shown to restrict the carrier mediated transport of large neutral amino acids into the brain (Agrawal et al 1970, Aoki and Siegel 1970, Hughes and Johnson 1976, Neame 1961, Oja 1972). The resulting amino acid imbalance disturbs the normal function of the protein synthesis system of nerve cells which is reflected in increased RNAse activity (Roberts and Morelos 1976, 1980), polyribosome disaggregation (Copenhaver et al 1973, Hughes and Johnson 1977, Roberts and Morelos 1980, Taub and Johnson 1975), altered t-RNA-coupling to amino acids (Hughes and Johnson 1976, 1977, 1978, Taub and Johnson 1975), impaired incorporation of labelled amino acids into proteins (Agrawal et al 1970, Aoki and Siegel 1970, Berger et al 1980, Hughes and Johnson 1976, Oja 1972) and changed structure as well as pattern of neuronal proteins (Lane and Neuhoff 1980, Rodrigues and Borisy 1979). The most fascinating feature of hyperphe is its exclusive and specific effect on the differentiating brain; neither any other developing organ nor the adult brain have been shown to be influenced to a comparable extent. Neuropathological studies of PKU-brains are few. Microencephaly and reduced content and stainability of the white matter are the most striking features (Alvord et al 1950, Crome and Pare 1960, Malamud 1966). In a previous paper (Huether et al 1982) a desynchronisation of certain parameters of myelination and reduced myelin yields have been shown in brain and spinal cord of hyperphe rats. But in accordance with other reports (Figlewicz and Druse 1980) the gross chemical composition of isolated myelin was not found to be largely affected. This suggests that myelination is not likely to be the primary defect in PKU.
In this study the effect of experimental hyperphe on the developing cerebellum is investigated. Special attention has been given to biochemical and morphological parameters of proliferation and cell numbers which are known to be affected considerably by other metabolic disorders, such as undernutrition (Balázs et al 1979, Barnes and Altman 1973) and hormonal imbalances (Bohn and Lauder 1980, Cotterrell et al 1972, Gombos 1980, Gourdon et al 1973, Nicholson and Altman 1972).

Key words

Phenylketonuria - Hyperphenylalaninaemia - Cerebellum - Brain development - Undernutrition