Previous reports have provided evidence that insufficient or excessive maternal folic acid (FA) intake during pregnancy can alter neurodevelopment of the offspring by modulating prenatal neurogenesis. Furthermore, our earlier work in a mouse model confirmed long-term structural changes at the cellular level of either deficient or excessive FA supply by comparably reducing dendritic arborization of cortical projection neurons. Here, we report that excessive amounts of FA decrease arborization of deep layer projection neurons, but not upper layer neurons and that reduced complexity of deep layer neurons is not observed when folic acid is replaced by folinic acid, a stable reduced form of folate. In addition, deficiency of B₁₂, a vitamin that critically regulates folate metabolism, causes even more marked decreases in neuronal arborization in both deep and upper layer neurons and particularly in combination with FA excess. Furthermore, both FA excess and B₁₂ deficiency affect synaptic density and morphology. Our findings point to neurodevelopmental risks associated with insufficient amounts of prenatal B₁₂, particularly in association with high levels of FA intake, suggesting that the neurodevelopmental program is sensitive to an imbalance in the status of these interacting micronutrients.

Background

Proper cerebral cortical development depends on the tightly orchestrated migration of newly born neurons from the inner ventricular and subventricular zones to the outer cortical plate. Any disturbance in this process during prenatal stages may lead to neuronal migration disorders (NMDs), which can vary in extent from focal to global. Furthermore, NMDs show a substantial comorbidity with other neurodevelopmental disorders, notably autism spectrum disorders (ASDs). Our previous work demonstrated focal neuronal migration defects in mice carrying loss-of-function alleles of the recognized autism risk gene WDFY3. However, the cellular origins of these defects in Wdfy3 mutant mice remain elusive and uncovering it will provide critical insight into WDFY3-dependent disease pathology.

Methods

Here, in an effort to untangle the origins of NMDs in Wdfy3^lacZ mice, we employed mosaic analysis with double markers (MADM). MADM technology enabled us to genetically distinctly track and phenotypically analyze mutant and wild-type cells concomitantly in vivo using immunofluorescent techniques.

Results

We revealed a cell autonomous requirement of WDFY3 for accurate laminar positioning of cortical projection neurons and elimination of mispositioned cells during early postnatal life. In addition, we identified significant deviations in dendritic arborization, as well as synaptic density and morphology between wild type, heterozygous, and homozygous Wdfy3 mutant neurons in Wdfy3-MADM reporter mice at postnatal stages.

Limitations

While Wdfy3 mutant mice have provided valuable insight into prenatal aspects of ASD pathology that remain inaccessible to investigation in humans, like most animal models, they do not a perfectly replicate all aspects of human ASD biology. The lack of human data makes it indeterminate whether morphological deviations described here apply to ASD patients or some of the other neurodevelopmental conditions associated with WDFY3 mutation.

Conclusions

Our genetic approach revealed several cell autonomous requirements of WDFY3 in neuronal development that could underlie the pathogenic mechanisms of WDFY3-related neurodevelopmental conditions. The results are also consistent with findings in other ASD animal models and patients and suggest an important role for WDFY3 in regulating neuronal function and interconnectivity in postnatal life.

BACKGROUND: DATATOP was a study of early Parkinsons disease (PD) conducted in the 1980 s, before mandatory folic acid fortification in the United States. Our analysis of its baseline serum samples revealed a geometric mean vitamin B12 of 369 pg/mL and homocysteine (tHcy) of 9.5μmol/l. We also found that low B12 predicted greater worsening of ambulatory capacity (AC) and elevated tHcy (>15μmol/L) predicted greater declines in cognitive function. OBJECTIVE: We sought to measure B12 and tHcy in contemporary trial participants with early PD who had not started dopaminergic treatment and to determine whether these analytes were associated with clinical progression. METHODS: We measured B12 and tHcy from baseline and end-of-study blood samples from three recent clinical trials. RESULTS: Baseline geometric mean B12 levels for these studies ranged from 484- 618 pg/ml and for tHcy ranged from 7.4- 10μmol/L. Use of B12-containing supplements ranged from 41- 61%, and those taking supplements had higher B12 and lower tHcy. Those who began levodopa, but were not taking B12-supplements, had greater end-of-study tHcy. There was no association of baseline tHcy > 15μmol/L with annualized change in Montreal Cognitive Assessment and no association of baseline B12 tertiles with change in AC. CONCLUSIONS: In these longitudinal trials, B12 levels were higher than for DATATOP, due in large part to increased B12-supplement intake, while tHcy levels were similar. Initiation of levodopa was associated with increases of tHcy in those not taking a B12-containing supplement. These smaller studies did not replicate prior findings of low B12 and elevated tHcy with features of progression, possibly due to higher baseline B12.