Regeneración axonal asociada a la sobreexpresión de neurotrofina 3 en células de la glía envolvente olfatoria.

En este estudio se pretende evaluar el posible tratamiento de pacientes con lesión medular espinal. El tipo de lesión medular que se va a tratar es una contusión medular, ya que este es el tipo de daño medular más frecuente. Se propone la utilización de una terapia celular génica. Está basada en la modificación genética de células de la mucosa olfatoria para que sobreexpresen el factor neurotrófico neurotrofina 3. Estas células modificadas genéticamente, mediante un vector lentiviral, son implantadas en la zona de la lesión medular incluidas en el interior de un hidrogel. El hidrogel que se utiliza está compuesto por ácido hialurónico y quitosano, lo que hace que este hidrogel sea biodegradable y genere un soporte para la regeneración axonal. En este estudio se describen los análisis preclínicos in vitro, al igual que en modelos animales. Los modelos animales que utilizamos son ratas de la cepa Wistar y cerdos miniatura Yucatán. Estos animales son sometidos a dos cirugías para generar el modelo de lesión medular y posteriormente para aplicar el tratamiento. En estos modelos animales se evalúa directamente la regeneración axonal mediante cortes histológicos e indirectamente gracias a una ganancia de sus funciones motoras y sensoriales. Por último, se llevan a cabo los ensayos clínicos con pacientes, inicialmente en Fase I para valorar la factibilidad y la seguridad del tratamiento; y posteriormente, las Fase II y III, en las que se evaluará tanto la eficacia como la seguridad. Una vez completada la Fase III, se comienza el proceso de evaluación por las agencias reguladoras y comercialización del tratamiento y, por tanto, la Fase IV del mismo.pre-print673 K

Generation and characterization of monoclonal antibodies against pathologically phosphorylated TDP-43.

Inclusions containing TAR DNA binding protein 43 (TDP-43) are a pathological hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). One of the disease-specific features of TDP-43 inclusions is the aberrant phosphorylation of TDP-43 at serines 409/410 (pS409/410). Here, we developed rabbit monoclonal antibodies (mAbs) that specifically detect pS409/410-TDP-43 in multiple model systems and FTD/ALS patient samples. Specifically, we identified three mAbs (26H10, 2E9 and 23A1) from spleen B cell clones that exhibit high specificity and sensitivity to pS409/410-TDP-43 peptides in an ELISA assay. Biochemical analyses revealed that pS409/410 of recombinant TDP-43 and of exogenous 25 kDa TDP-43 C-terminal fragments in cultured HEK293T cells are detected by all three mAbs. Moreover, the mAbs detect pS409/410-positive TDP-43 inclusions in the brains of FTD/ALS patients and mouse models of TDP-43 proteinopathy by immunohistochemistry. Our findings indicate that these mAbs are a valuable resource for investigating TDP-43 pathology both in vitro and in vivo

26H10 rabbit mAb detects TDP-43 pathology in FTLD-TDP brain tissues.

26H10 rabbit mAb detects TDP-43 pathology in FTLD-TDP brain tissues.</p

Characteristics of patients with FTD/ALS.

Inclusions containing TAR DNA binding protein 43 (TDP-43) are a pathological hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). One of the disease-specific features of TDP-43 inclusions is the aberrant phosphorylation of TDP-43 at serines 409/410 (pS409/410). Here, we developed rabbit monoclonal antibodies (mAbs) that specifically detect pS409/410-TDP-43 in multiple model systems and FTD/ALS patient samples. Specifically, we identified three mAbs (26H10, 2E9 and 23A1) from spleen B cell clones that exhibit high specificity and sensitivity to pS409/410-TDP-43 peptides in an ELISA assay. Biochemical analyses revealed that pS409/410 of recombinant TDP-43 and of exogenous 25 kDa TDP-43 C-terminal fragments in cultured HEK293T cells are detected by all three mAbs. Moreover, the mAbs detect pS409/410-positive TDP-43 inclusions in the brains of FTD/ALS patients and mouse models of TDP-43 proteinopathy by immunohistochemistry. Our findings indicate that these mAbs are a valuable resource for investigating TDP-43 pathology both in vitro and in vivo.</div

Rabbit mAbs against pS409/410-TDP-43 detect TDP-43 pathology in brain tissues from FTLD and ALS patients and rNLS8 mice.

(A‒C) Representative images of immunohistochemical analysis using the indicated rabbit mAbs against pS409/410-TDP-43 in the frontal cortex of normal controls (A), FTLD-TDP type A patients (B), and FTLD-TDP type B patients (C), and in the motor cortex of ALS patients (C). Black arrows indicate neuronal cytoplasmic inclusions (NCI), and red arrows mark dystrophic neurites (DN). Inserts in B are higher magnifications of neuronal intranuclear inclusions (NCII). (D) Representative images of immunohistochemical analysis using the indicated rabbit mAbs against pS409/410-TDP-43 in the cortex of non-transgenic (nTg) and rNLS8 mice. Inserts are higher magnifications of NCI. (E) Representative images of immunohistochemical analysis using the indicated rabbit mAbs against pS409/410-TDP-43 in the cortex of AAV-2R and AAV-149R mice. Inserts are higher magnifications of inclusions. For all panels, scale bars are 20 μm.</p

Schematic representation of the methods and procedures to generate, screen and characterize monoclonal antibodies against pS409/410-TDP-43.

Schematic representation of the methods and procedures to generate, screen and characterize monoclonal antibodies against pS409/410-TDP-43.</p

The rabbit bleed specifically detects pS409/410-TDP-43.

(A) Immunoblot analysis of rTDP43 with or without CK1 treatment using the indicated sera and antibody. (B) Immunoblot analysis of HEK293T lysates expressing GFP, GFP-TDP-25, or GFP-TDP-25mut (S409A/S410A) using the indicated sera and antibodies. GAPDH was used as a loading control. (C) Representative images of immunohistochemical analysis using the indicated sera in the frontal cortex of human FTLD-TDP patients. Arrows mark TDP-43 inclusions. Scale bars are 20 μm.</p

Three rabbit mABs exhibit high specificity and sensitivity to pS409/410-TDP-43.

(A) ELISA analysis of phospho- and nonphospho-TDP-43 peptides using the indicated rabbit mAbs. (B) Dot blot analysis of rTDP43 treated with or without CK1 using the indicated rabbit mAbs. (C) Immunoblot analysis of HEK293T cell lysates expressing GFP, GFP-TDP-25 or GFP-TDP-25mut (S409/410A) using the indicated rabbit mAbs. GAPDH was used as a loading control.</p

26H10 rabbit mAb exhibits high specificity to pS409/410-TDP-43.

26H10 rabbit mAb exhibits high specificity to pS409/410-TDP-43.</p

Immunoreactivity of B cell clones against TDP-43 species as measured by ELISA immunoassay.

Immunoreactivity of B cell clones against TDP-43 species as measured by ELISA immunoassay.</p