Research Interest

  •  Cell Biology
  • Molecular Biology
  • Molecular Mechanisms of axonal and synaptic degeneration
  • Neurodegeneration
  • Neuronal Signaling Pathways
  • Axonal Transport
  • Molecular Motors
  • Cytoskeleton’s dynamics

Pathologies of Interest

  • Alzheimer
  • Parkinson
  • Huntington
  • Motor Diseases
  • Prion Diseases

Dying Back Neuropathies and Axonal Transport

Neurons are highly specialized cells shaped by millions of years of evolution according to the function they perform. This specialization allows them to receive, process, transfer, and store information optimally. Typically, mature neurons have a cell body with several dendrites for receiving and processing information as well as a single axon as the output. Neurons are particularly vulnerable to transport defects, because unlike cell bodies and dendrites, axons effectively lack protein synthesis machinery. In this regard, molecules and membranous organelles required in axons and their synapses need to be synthesized in the cell body, packaged appropriately into membrane-bounded organelles, and delivered to proper compartments via a set of microtubule-dependent motor proteins (kinesins and dyneins) responsible for the neuronal process known as fast axonal transport (FAT). Interestingly, point mutations which compromise but not eliminate the function of molecular motors, kinesin-1 or cytoplasmic dynein, lead to two different “dying back neuropathies”, spastic paraplegia (Reid et al., 2002) and motor neuron disease (Hafezparast et al., 2003), respectively. Therefore, these biological evidences highlight the central role played by FAT in neuronal function, viability and neurodegeneration.

Mechanism Underlying Inhibition of FAT in Dying Back Neuropathies

Recently, we have shown deficits of FAT in several different progressive neurological disorders including Down’s syndrome, Alzheimer’s disease (AD), Parkinson’s disease (PD), Kennedy’s disease, Prion’s disease and Huntington’s disease (Busciglio et al., 2002, Pigino et al., 2003, Morfini et al., 2006, Salehi et al., 2006, Morfini et al., 2007, Morfini et al., 2009a, Morfini et al., 2009b, Pigino et al., 2009). Deficits in FAT show up early during the neuronal development preceding axonal degeneration and neuronal death (Pigino et al., 2003, Stokin et al., 2005). However, mutations in molecular motors are rare, usually embryonic lethal and certainly not associated to any of these late onset neurological disorders. Then what is the molecular basis of FAT inhibition in these late onset neuropathiesWe propose that deficits of fast axonal transport may arise as a consequence of improper motor regulation caused by imbalances in normal neuronal signaling pathways, that in turn affect functional properties of motor proteins leading to fast axonal transport inhibition (Morfini et al., 2009a). If these alterations in regulation of FAT lead to inhibition of FAT, the final outcome is the same as with mutations in motor proteins: reductions in the delivery of important material to axons and their synapses resulting in axonal and synaptic dysfunction, lack of trophic support and ultimately neuronal death.

Amyloid β is a Potent Inhibitor of Fast Axonal Transport and Neurotransmission

Recent data have shown that amyloid beta, the proteolytic peptide derived from the amyloid precursor protein (APP), in its oligomeric conformation (oAβ) accumulates progressively within neuronal processes of AD and DS brains (Takahashi et al 2004). Remarkably, we have recently showed that oAβ, inhibits FAT by a molecular mechanism involving abnormal activation of protein casein kinase 2 (CK2) and aberrant kinesin-1 phosphorylation (Pigino et al., 2009). However, the molecular basis for retrograde transport inhibition still remains to be determined. The robust inhibition of FAT by oAβ predicted failure of neurotransmission. Indeed, injection of oAβ in the presynaptic compartment of the squid giant synapse induced a profound inhibition of synaptic transmission (Moreno et al., 2009). Electron microscopy suggested that this failure resulted from a clear reduction in the availability of synaptic vesicles at the active zones, and a lack of neurotransmiter release, consistent with the idea that oAβ inhibits the anterograde transport of synaptic vesicles carrying neurotransmitters, essential components for synaptic transmission (Moreno et al., 2009). Furthermore, analysis of retrograde FAT of BDNF in mice expressing the double Swedish mutation in the APP gene, showed a dramatic reduction of BDNF transport from the periphery to the cell soma (Poon et al., 2009). This impairment in neurotrophic support triggers eventually specific neuronal mechanisms of program cell death (See Figure 1). Interestingly, these mice accumulates intracellular oligomeric amyloid beta in function of time (Takahashi et al., 2004). Figure 1. Dying backpattern of degeneration. A) In the intact nervous system, neurons and targets (orange ovals) are well matched, so activity and neurotrophic support (green arrows) are well coordinated. B) When the activity at a given synapse is compromised (i.e. by intracellular oAβ accumulation, red asterisk, inducing axonal transport inhibition of presynaptic components), the presynaptic terminals are retracted and neurotrophin return is reduced. Changes in gene expression may occur, but the perikaryon is still intact at this stage. C) When the number of functional synapses falls below a critical threshold, the remaining presynaptic terminals are typically shut down and retracted. Consequently, target-derived neurotrophin supplies are no longer sufficient to maintain the distal axon or to sustain neuronal viability. D) As the distal axon atrophies, the neuronal perikaryon begins to exhibit the characteristics of classical apoptotic cell death, including pycnotic nuclei, shrinkage of cell body, TUNEL staining, and blebbing of the plasma membrane, characteristic of neurons at the late stages of AD. The time from the earliest changes in synaptic function seen in B) to the clear activation of apoptotic pathways in D) may be months or years. Figure modified from Scott Brady et al., 2009.

Pathological Forms of Tau specifically Inhibit Fast Anterograde Axonal Transport. 

The classical vision of microtubule associated protein Tau’s function has been to provide dynamic stability to microtubules  (Wang, Yu et al., 1996, Conde and Caceres 2009). In recent years novel functions have been attributed to Tau including signaling functions (Morris, Maeda et al., 2011). Results gathered in collaboration with many different laboratories have demonstrated that different pathological forms of Tau can activate specific signaling pathways resulting in the activation of the protein phosphatase PP1 (Kanaan, Pigino et al., 2012) within the axonal compartment. PP1 can dephosphorylate the protein kinase GSK3βat its serine 9 resulting in its activation. The active form of GSK3β then phosphorylate the light chains of the anterograde motor Kinesin-1 inducing the release of the motor from its cargo. This way the anterograde transport is inhibited specifically without interfering with the retrograde transport. Vesicular trafficking experiments using extruded axoplasms isolated from the squid giant axon and different truncated forms of Tau allowed us to identify that in the first 18 amino acids resides a domain that can activate PP1. This particular domain was named PAD (Phsophatase Activating Domain). The identification of the PAD domain within the Tau molecule helped us to determine that the pathological forms of Tau induce a reduction of the anterograde transport via activation of PP1. The activation of PP1 is caused by the abnormal exposition of the Tau’s PAD domain which in turn results in the activation of GSK3β and the subsequent phosphorylation of the Kinesin-1 light chains and its release from the normal cargoes it binds and transport (Kanaan, Morfini et al., 2011, Kanaan, Pigino et al., 2012, Kanaan, Morfini et al., 2011).

Future Directions

Many important biological questions remain to be answered, which represent the driving force of my research effort: How does oAβ activates CK2? How does CK2 activation inhibit both anterograde and retrograde fast axonal transport? What is the molecular mechanism of oAβ-induced synaptic inhibition? How do point mutations in PS1 result in GSK3β activation? Is the activity or the expression of PS1 required for the regulation of GSK3β activity? What important axonal cargoes are specifically inhibited in their transport by oAβ and PS1 mutations? What is the functional consequence of kinesin-1 and dynein phosphorylation by CK2 and GSK3β? What is the role of axonal cytoskeleton on FAT inhibition in AD and other late onset neurological disorders?

Experimental Approach

To study regulatory mechanisms for FAT in real time, I will take advantage of the use of extruded axoplasm isolated from the squid, Loligo pealei, a unique ex vivo experimental system instrumental in the discovery of the major anterograde molecular motor kinesin-1, as well as key regulatory properties for FAT. This experimental system offers the unique possibility to explore in real time, biochemical properties of axons, as well as vesicle motility without cell body or dendrite process interferences. To address questions regarding the role of pathological proteins on synaptic function and transmission, I will take advantage of the use of the giant synapse from squid, working in collaboration with Dr. Rodolfo Llinas at the Marine Biological Laboratories in Woods Hole, MA.

To study molecular mechanism underlying axonal degeneration and synaptic dysfunction I will use a multidisciplinary experimental approach, which includes the use of primary hippocampal, cerebellar and cortical neurons from diverse transgenic mice models for familial Alzheimer’s disease (APP and PS-1), Parkinson’s disease (α-synuclein) and Prion disease.  Post-mortem brain tissue, human primary fibroblast from patients and human fetal primary neurons in culture will be used to address questions regarding the pathogenic mechanisms underlying these devastating disorders. Some of these studies will be perform in collaboration with Dr. Jorge Busciglio whom we share mutual scientific interest.


2011-2013. New Investigator Research Grant to Promote Diversity (NIRGD) Alzheimers Association. Mechanisms Underlying Oligomeric Abeta-inudced Axonal Transport Dysfunction. Role on grant: PI
2015-2017. PIP. Mecanismo molecular involucrado en el incremento de la producción del péptido Amiloide beta inducida por la deposición de Abeta: mecanismo patogénico de retroalimentación positiva en la enfermedad de Alzheimer. Role de la proteina quinasa PKC delta en la degeneracion axonal y sinaptica en la enfermedad de Parkinson. Director
2018-2020. PIP. Estudio de la relación entre regulación epigenética alterada de genes de memoria y pérdida de cholesterol como causa de problemas cognitivos asociados a la edad. Evalua los mecanismos por los cuales la perdida de colesterol durante el envejecimiento de neuronas hipocampales afecta la acetilación de histonas alterando la expresión de genes involucrados en el establecimiento de memoria. Co-Director

2019-2021. PRH-PIDRI. Mecanismo molecular asociado a la pérdida de conectividad sináptica en la enfermedad de Alzheimer: mecanismo patogénico del péptido amiloide β de 42 aminoácidos (Aβ-42). Rol del péptido amiloide β de 42 aminoácidos en la pérdida de conecciones sinápticas en neuronas afectadas por la enfermedad de Alzheimer. Director.

2018-2019. UNC-Primar-TP 2018-2021. Rol patológico del beta amiloide en la pérdida de conectividad sináptica en la enfermedad de alzheimer. Co-Director