Survival and integration of transplanted postmitotic human neurons following experimental brain injury in immunocompetent rats

Object. Limitations regarding cell homogeneity and survivability do not affect neuronlike hNT cells, which are derived from a human teratocarcinoma cell line (Ntera2) that differentiates into postmitotic neurons with exposure to retinoic acid. Because NT2N neurons survive longer than 1 year after transplantation into nude mice brains, the authors grafted these cells into the brains of immunocompetent rats following lateral fluid-percussion brain injury to determine the long-term survivability of NT2N cell grafts in cortices damaged by traumatic brain injury (TBI) and the therapeutic effect of NT2N neurons on cognitive and motor deficits.Methods. Seventy-two adult male Sprague—Dawley rats, each weighing between 340 and 370 g, were given an anesthetic agent and subjected to lateral fluid percussion brain injury of moderate severity (2.2–2.5 atm in 46 rats) or to surgery without TBI (shamoperation, 26 rats). Twenty-four hours postinjury, 105 NT2N cells (24 injured animals) or 3 µl of vehicle (22 injured and 14 control animals) was stereotactically implanted into the periinjured or control cerebral cortex. Motor function was assessed at weekly intervals and all animals were killed at 2 or 4 weeks after their posttraumatic learning ability was assessed using a Morris water maze paradigm. Viable NT2N grafts were routinely observed to extend human neural cell adhesion molecule—(MOC-1)immunoreactive processes into the periinjured cortex at 2 and 4 weeks posttransplantation, although no significant improvement in motor or cognitive function was noted. Inflammation identified around the transplant at both time points was assessed by immunohistochemical identification of macrophages (ED-1) and microglia (isolectin B4).Conclusions. Long-term survival and integration of NT2N cells in the periinjured cortex of immunocompetent rats provides the researcher with an important cellular system that can be used to study maturation, regulation, and neurite outgrowth of transplanted neurons following TBI.

Detection of a Novel Intraneuronal Pool of Insoluble Amyloid β Protein that Accumulates with Time in Culture

The amyloid-β peptide (Aβ) is produced at several sites within cultured human NT2N neurons with Aβ1-42 specifically generated in the endoplasmic reticulum/intermediate compartment. Since Aβ is found as insoluble deposits in senile plaques of the AD brain, and the Aβ peptide can polymerize into insoluble fibrils in vitro, we examined the possibility that Aβ1-40, and particularly the more highly amyloidogenic Aβ1-42, accumulate in an insoluble pool within NT2N neurons. Remarkably, we found that formic acid extraction of the NT2N cells solubilized a pool of previously undetectable Aβ that accounted for over half of the total intracellular Aβ. Aβ1-42 was more abundant than Aβ1-40 in this pool, and most of the insoluble Aβ1-42 was generated in the endoplasmic reticulum/intermediate compartment pathway. High levels of insoluble Aβ were also detected in several nonneuronal cell lines engineered to overexpress the amyloid-β precursor protein. This insoluble intracellular pool of Aβ was exceptionally stable, and accumulated in NT2N neurons in a time-dependent manner, increasing 12-fold over a 7-wk period in culture. These novel findings suggest that Aβ amyloidogenesis may be initiated within living neurons rather than in the extracellular space. Thus, the data presented here require a reexamination of the prevailing view about the pathogenesis of Aβ deposition in the AD brain.