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Abstract

Alzheimer’s disease (AD) is a chronic neurodegenerative disorder and the leading cause of dementia. The disease progression is associated with the build-up of amyloid plaques and neurofibrillary tangles in the brain. However, besides the well-defined lesions, the AD-related pathology includes neuroinflammation, compromised energy metabolism, and chronic oxidative stress. Likewise, the blood–brain barrier (BBB) dysfunction is suggested to be a cause and AD consequence. Accordingly, therapeutic targeting of the compromised BBB is a promising disease-modifying approach. We utilized a homozygous triple-transgenic mouse model of AD (3×Tg-AD) to assess the effects of L-norvaline on BBB integrity. We scrutinized the perivascular astrocytes and macrophages by measuring the immunopositive profiles in relation to the presence of β-amyloid and compare the results with those found in wild-type animals. Typically, 3×Tg-AD mice display astroglia cytoskeletal atrophy, associated with the deposition of β-amyloid in the endothelia, and declining nitric oxide synthase (NOS) levels. L-norvaline escalated NOS levels, then reduced rates of BBB permeability, amyloid angiopathy, microgliosis, and astrodegeneration, which suggests AD treatment agent efficacy. Moreover, results undergird the roles of astrodegeneration and microgliosis in AD-associated BBB dysfunction and progressive cognitive impairment. L-norvaline self-evidently interferes with AD pathogenesis and presents a potent remedy for angiopathies and neurodegenerative disorders intervention.

2.1. Norvaline Improves the BBB Integrity in the 3×Tg-AD Mice

Tibbling et al. (1977) pointed out that CNS albumin is derived from peripheral sources. Accordingly, the albumin index can indicate the rate of BBB disruption and facilitate the efficacy of routine clinical work []. Subsequently, several groups demonstrated elevated levels of CSF albumin in patients with AD [,]. We applied a traditional Evans blue assay to assess the albumin presence in the brain tissue. The mean measured supernatant optical density (OD620) per brain weight from WT control animals was 0.4 ± 0.02 (B), which accords with the results from other groups that utilized the same protocol []. Remarkably, the mean OD620 of the supernatant from seven-month-old 3×Tg-AD controls increased up to 0.62 ± 0.04 and still was substantially lower than OD620 = 0.9 obtained from 15-month-old 3×Tg-AD mice [], which points to gradual progressive severe age-dependent BBB defects in this particular AD model. Remarkably, L-norvaline treatment significantly (p < 0.01) improved the BBB integrity in the transgenic mice, as reflected by a reduction (by 31%) in the mean OD620 (0.43 ± 0.066). Of note, we did not observe any significant effect of the treatment upon the BBB permeability in the WT animals (). The two-way ANOVA test was used to reveal a significant (F(1,20) = 10.47 p = 0.0041) interaction (accounting for 14.21% of the total variance) between the genotype and the type of treatment, and their mutual effect upon the BBB permeability, which further indicates L-norvaline efficacy.

2.2.1. Norvaline Reduces Endothelial Amyloidosis

We applied an immunohistochemistry assay to analyze the effect of L-norvaline upon the rate of Aβ deposition in the 3×Tg-AD mice brain microvessels’ walls. Previously, we have shown a severe Aβ positivity decline in the cortices [] and hippocampi [] following the L-norvaline treatment. Here, we evidenced considerable levels of APP/Aβ in the cerebral microvessels’ ECs (A), and demonstrated a substantial reduction in the levels of Aβ positivity following the treatment (B).

2.2.2. Norvaline Diminishes the Rate of Astrodegeneration in the 3×Tg-AD Mice

Here, we analyze the GFAP immunopositive surface area and optical density (C) in the vicinity of hippocampal penetrating blood microvessels (A) (about 10 µm in diameter). The relative thickness of the astroglia end-feet (B) displayed significant genotype and treatment-related differences (D). The two-way ANOVA test revealed a significant (F(1,36) = 15.7 p = 0.0003) interaction (accounting for 13.94% of the total variance) between the genotype and the type of treatment, and their mutual effect upon the astroglia end-feet thickness. Remarkably, L-norvaline did not affect this parameter in WT mice but demonstrated a significant (p < 0.001) influence upon 3×Tg-AD animals with an increase (by about 42%) (D).

2.2.2. Norvaline Diminishes the Rate of Astrodegeneration in the 3×Tg-AD Mice

Imaris® software has been applied for large-scale analysis of 3D astrocyte morphology. We reconstructed individual cells in the CA1 area (A inset) and revealed a significant (by about 50%) increase (from 20.39 ± 1.55 to 29.79 ± 3.01) in the number of astrocytic protoplasmic processes following the treatment (E). The analysis of the staining intensity (C) and cell volume (D) also demonstrated an increase in the L-norvaline treated group; however, the effect did not reach statistical significance.

Abstract

Recently, a study published in “Toxicology In Vitro” (Kate Samardzic and Kenneth J. Rodgers) was entitled: “Cytotoxicity and Mitochondrial Dysfunction Caused by the Dietary Supplement L-Norvaline”. The title may be greatly overstated, and here we provide several arguments showing that norvaline is not as toxic as reported.