BRAIN

RESEARCH
ELSEVIER Brain Research 687 (1995) 211-213

Short communication

Decrease in cytosolic Aspartyl-aminopeptidase but not in

Alanyl-aminopeptidase activity in the frontal cortex of the aged rat
Concepcion Iribar *, M. Jose Esteban, J. Manuel Martinez, Jose M. Peinado
Departments of Biochemistry and Physiology, Institute of Neurosciences, University of Granada School of Medicine, Avenida de Madrid 11, 18010
Granada, Spain
Accepted 2 May 1995

Abstract

To test the neurotoxic hypothesis of excitatory amino acids, we evaluated the possible contribution to the free acidic amino acid pool
of Aspartyl-aminopeptidase activity in the frontal cortex of adult (3 month old) and aged rats (3 groups of animals aged 26, 29 and 33
months). Aspartyl-aminopeptidase activity showed a significant decrease in the oldest rats (29 and 33 months old) whereas the activity of
Alanyl-aminopeptidase, an unspecific enzyme, did not change with age. These data invalidate the idea that excess free acidic amino acids
are released by aminopeptidases in the aged rat but do provide evidence of age-related changes in this enzymatic activity. The possible
implications of our findings for general alterations in protein degradation are discussed.

Keywords: Aminopeptidase; Excitatory amino acid; Aging; /3-Amyloid peptide; Neuropeptide

Brain aminopeptidases (AP) have been implicated in the
enzymatic activation and degradation of several neuropep-
tides [18]. These two actions may regulate not only their
role as neuromodulators [2] but may also modify the free
amino acid pool through the release of N-terminal amino
acids, some of which, such as glutamate and aspartate, are
particularly active in the CNS [16,19]. Aspartyl-AP (Asp-
AP) (EC 3.4.11.7) is a cytosolic enzyme able to degradate
peptides selectively by cleaving glutamyl and Aspartyl
N-residues [12]. Therefore, changes in Asp-AP activity
may either contribute to or reflect modifications in excita-
tory amino acid turnover.
Because of their effects as neurotoxic agents, the excita-
tory amino acids aspartate and glutamate [19] have been
implicated not only in the mechanisms underlying normal
aging [16] but also in the pathogenesis of different neu-
rodegenerative diseases, such as hypoxic brain damage
[23], amyotrophic lateral sclerosis [22], Huntington's [27]
and Alzheimer's diseases [8].
In the present study, we analysed Asp-AP activity in
homogenates obtained from frontal cortex in 3 groups of
elderly rats and compared these activities to control values.
* Corresponding author. Fax: (34) (58) 244033.
The results were compared in each age group with the data
obtained for Alanyl-AP (AIa-AP) (EC 3.4.11.14), an en-
zyme that hydrolyzes Alanyl N-terminal and other neutral
amino acid residues [10] but not acidic amino acids. Be-
cause AIa-AP alone accounts for almost 80% of the total
soluble AP activity of cerebral cortex [17], it has been
named the 'major cortical AP'.
48 male Wistar albino rats were used, including a
3-month-old control group (n = 14), and 3 groups of aged
rats 26 (n = 14), 29 (n = 10) and 33 months (n = 10). In
all cases, the brains were perfused with saline through the
left ventricle under equithensin anesthesia, quickly re-
moved and cooled in dry ice. The brains were sliced and
the samples were dissected out from the frontal cortex
rostral to the corpus callosum genu. After being weighed,
the samples were homogenized in 10 vols. of 50 mM
Tris-HC1 buffer, pH 7.4, and ultracentrifuged (100,000 x
g, 30 min). The supernatant was decanted and immediately
analysed for AP activity.
Enzyme activity was measured fluorimetrically using
amino acyl-2-naphthylamide as an artificial substrate. Asp-
AP activity was assayed with Asp-2-naphthylamide and
AIa-AP with Ala-2-naphthylamide, according to the method
of Greenberg with slight modifications [1]. The fluores-
cence intensity of the 2-naphthylamine released was mea-

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212 C. lribar et aL / Brain Research 687 (1995) 211-213

Table 1 functionality of neurons and glial cells which can synthe-

Mean values±S.E.M, of Asp-AP and AIa-AP activity determined in
frontal cortex of 4 groups of aged rats ( n = 1 4 , n = 1 4 , n = 1 0 and
n = 10, respectively). Results for Asp-AP are expressed as p m o l / m i n / g
of tissue and for Ala-AP as / z m o l / m i n / g of tissue. * P < 0.05, * * P <
0.01.
Frontal cortex
Asp-AP Ala-AP
3 months 44.5 +5.9 314.0+ 16.2
26 months 50.0 + 2.6 289.2 4-10.6
29 months 18.7 + 3.8 * * 273.0 4- 37.3
33 months 21.83 + 5.0 * 299.5 + 38.8
sured at an emission wavelength of 412 nm with excitation
at 345 rim. The data were analysed statistically with the
Mann-Whitney test.
The results showed no statistical differences between
the 4 experimental groups in relation to Ala-AP activity.
Asp-AP activity was not modified in 26-month-old rats
compared with controls; however, the 2 oldest groups of
rats (29 and 33 months) showed a significant decrease of
nearly 50% in this enzymatic activity (Table 1, Fig. 1).
Ala-AP activity seems to be responsible for > 80% of
cortical AP activity [17], hydrolyzing different neuropep-
tides, such as enkephalins [25], endorphins [9] and dinor-
phins [5]. Because of its high activity, widespread presence
in the cortex and broad range of possible biological sub-
strates, it has been selected as a control for unspecific
cortical AP activity. No significant age differences in this
enzyme activity were found in this study. Our results in
normal aged rats are similar to those for Ala-AP activity in
Alzheimer's patients [17] for whom no significant changes
in this enzyme were reported.
Few data are availble for acidic AP activity in aging.
We may speculate that changes in enzymatic Asp-AP
activity are correlated with modifications in the number or
size this enzyme [5,13]. Although quantitative studies of
neuronal and glial populations during aging are controver-
sial [3], we recently found [21] that in the frontal cortex of
24-30-month-old rats there were no changes in neuronal
density although the number of glial cells did increase.
Evidently, the decrease in Asp-AP activity may be a
consequence of impaired neuronal functionality rather than
a reduction in the actual number of neurons. In fact,
endogenous glutamate content [4] as well as glutamate
uptake [20] in the frontal cortex decrease with age. Modifi-
cations in both acidic AP activity and amino acid levels
may reflect the same neuronal alteration.
Our results can also be discussed in the light of the
generic role of AP activity in protein degradation. In most
mammalian tissues, there is a general decrease in total
RNA synthesis with age, particularly mRNA [6]. The
effect of age on the expression of specific mRNA has been
reported, along with parallel changes in the protein activi-
ties which they encode [7]. A decrease in protein degrada-
tion rates with age has in fact been observed in several
tissues and cultured cells [6]. A decline in Asp-AP activity
may thus be responsible for the accumulation of proteins,
such as /3-amyloid protein and fl-amyloid peptide [26]
which contain Asp as the N-terminal amino acid. The
accumulation of this kind of protein seems to be responsi-
ble for characteristic lesions, such as senile plaques in
normal and pathological aging [11]. Therefore, it could
important to identify the proteases that degradate these
amyloid compounds [14,24] as this point the way toward
the development of useful therapeutic tool [15]. Neverthe-
less, it is also possible that changes of the enzymatic
activity may not modify the functionality of the process
that they control in vivo.
We propose Asp-AP activity as a possible marker of
neurodegeneration underlying the aging process.

FRONTAL CORTEX
Aminopeptidases Acknowledgements
3mo. 26 mo. ~ 29 too. ~ 33 mo.
This work was supported by DGICYT Grant PM 90-
200
0146 and the Junta de Andalucia. We thank K. Shashok
for revising the English style of the manuscript.

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