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Research Interests:

Molecular Biology of Neurodegenerative Disorders
   Alzheimer's Disease
      APP
      Presenilins
      Calcium signaling studies

Transgenic Mouse Models
      Triple Transgenic Model of AD
      Inclusion Body Myositis
      Targeted neuronal ablation

ALZHEIMER'S DISEASE (AD)
A major focus of research in our laboratory is aimed at elucidating the molecular pathogenesis of Alzheimer's disease (AD), the most common form of dementia worldwide. The majority of AD cases occur sporadically and begin in elderly individuals over 65-70 years of age. Some AD cases, however, are familial and transmitted in an autosomal dominant fashion. Familial AD (FAD) generally develops at a much earlier age of onset compared to sporadic AD. For instance, some FAD cases begin as early as 16 years of age! Other than the age of onset, both forms are quite similar pathologically and clinically. At the neuropathological level, the AD brain is characterized by two hallmark lesions, amyloid plaques and neurofibrillary tangles, that occur in selective brain regions such as the temporal lobe (see figure below).

APP
The beta -amyloid protein is derived from a much larger precursor protein called APP. The gene encoding this protein is located on chromosome 21. Liberation of the beta-amyloid protein from its precursor requires two proteolytic events to cleave beta-amyloid at its amino and carboxyl termini ; these are referred to as the beta- and gamma- secretase sites, respectively. Gamma-secretase mediated cleavage of APP yields also yields an intracellular fragment called the APP intracellular domain (AICD) that forms a transcriptively active complex.

Our lab has shown that AICD plays a physiologic role in regulating phosphoinositide-mediated calcium signaling. We showed that genetic ablation or pharmacological inhibition of gamma-secretase activity (and thereby AICD production) attenuated calcium signaling in a dose-dependent and reversible manner. This effect is manifested through the modulation of calcium stores in the endoplasmic reticulum. Likewise, we showed that cells lacking APP (and hence also AICD) exhibited similar calcium signaling deficits. Notably, these disturbances could be reversed by transfection with APP constructs containing an intact AICD, but not by constructs lacking this domain. Therefore, we believe that AICD regulates phosphoinositide-mediated calcium signaling through a gamma-secretase dependent signaling pathway, suggesting that the intramembranous proteolysis of APP may play a signaling role analagous to that of Notch.

Presenilins
The vast majority of early onset, autosomal dominant FAD cases are due to mutations in the presenilin-1 (PS1) gene, located on chromosome 14. Mutations in a closely related homolog, called presenilin-2 (PS2), found on chromosome 1 can also lead to FAD. Certain FAD mutations in PS1 cause AD as young as 16 years of age! The presenilins mediate an unusual intramembranous proteolytic activity known as gamma-secretase, of which two substrates are the Notch receptor and APP. FAD-linked mutations in the PS1 and PS2 genes produce well documented effects on gamma-secretase activity, leading to increased production of the longer, more pathologic forms of beta-amyloid.

Calcium signaling studies
In addition to their effects on gamma-secretase activity, mutations in the presenilins also produce highly consistent alterations in intracellular calcium signaling pathways. The following key findings have emerged from research in our lab:

• Mutations in PS1 and PS2 potentiate IP3-mediated calcium signaling

• Calsenilin, a calcium binding protein that associates with PS1 and PS2 can reverse the potentiation

• Calcium puffs, the fundamental unit of calcium release from the endoplamic reticulum, are increased in magnitide in mutant cells
  

• Calcium stores in the endoplasmic reticulum are elevated in mutant presenilin cells

• Capacitative calcium entry, a store-dependent refilling mechanism, is disrupted in mutant presenilin cells

• Genetic knockout of presenilins or pharmacologic inhibition of gamma-secretase activity greatly diminish IP3-mediated calcium signaling
  

 

TRIPLE TRANSGENIC AD MICE
The neuropathological correlates of Alzheimer’s disease (AD) include amyloid-ß (Aß) plaques and tau-laden neurofibrillary tangles. Although it has been possible to develop transgenic mice that develop one of these lesions, it has proven to be quite challenging to develop mice with both histopathological lesions in the same mouse, in AD-relevant brain regions such as the hippocampus and cortex. To better model AD neuropathology, my lab utilized a novel approach to create a triple transgenic model of AD (3xTg-AD). Rather than crossing independent lines, we microinjected two transgenes (ßAPP and tau) into single-cell embryos from homozygous PS1M146V knockin mice, generating mice with the same genetic background. Compared to crossbreeding, the approach we used offers several major advantages. The integration of the ßAPP and tau transgenes at the same genetic locus renders it unlikely that either transgene will independently assort in subsequent generations. Therefore, this tight linkage coupled to the ‘knockin’ of the PS1 mutation indicates that the 3xTg-AD mice breed as readily as any single transgenic line, particularly because these mice have also been bred to homozygosity. Thus, deriving a large colony is straightforward, cost-effective, and does not require extensive genotyping of the progeny. Moreover, the easy propagation of this transgenic line facilitates their crossing to other transgenic or gene-targeted mice to assess the impact of other genotypes on the neuropathological or physiological phenotype. Lastly, another advantage to this approach is that multiple transgenes are introduced into an animal without altering or mixing the background genetic constitution. Thus, an important confounding variable is avoided, which may be a crucial parameter for behavioral, electrophysiological, and vaccine-based experiments

The 3xTg-AD mice develop both plaque and tangle pathology in AD-relevant brain regions. The 3xTg-AD mice develop extracellular Aß deposits prior to tangle formation, consistent with the amyloid cascade hypothesis. Despite equivalent overexpression of the human ßAPP and human tau transgenes, Aß deposition develops prior to the tangle pathology, consistent with the amyloid cascade hypothesis. In addition, these mice exhibit deficits in synaptic plasticity, including long-term potentiation (LTP) that occurs prior to extracellular Aß deposition and tau pathology, but is associated with intracellular Aß immunoreactivity. These studies support the view that synaptic dysfunction is a proximal defect in the pathobiology of AD, preceding extracellular plaque formation and neurofibrillary pathology. As these 3xTg-AD mice phenocopy critical aspects of AD neuropathology, this model will be useful in pre-clinical intervention trials, particularly because the efficacy of anti-AD compounds in mitigating the neurodegenerative effects mediated by both signature lesions can be evaluated.

INCLUSION BODY MYOSITIS (IBM)
IBM is the most common muscle disease in individuals over the age of 55. It is an incurable disorder that leads to severe disability. IBM can occur sporadically or can be inherited.  Most IBM cases occur sporadically, with an unknown etiology. Surprisingly, IBM exhibits many shared pathogenic features with Alzheimer's disease. Although IBM patients are not cognitively impaired, their muscle fibers are characterized by the accumulation of many "dementia"-related proteins, most notably the beta-amyloid peptide. In IBM, the beta-amyloid peptide clearly accumulates intracellularly . Our lab has provided evidence that beta-amyloid can accumulate intraneuronally in the AD brain as well, although this still remains a controversial topic.

The role of beta-amyloid in the pathogenesis of IBM is still unresolved. There is clear evidence that expression of APP is elevated in IBM muscle fibers. To develop a transgenic mouse model of this common, age-related muscle disorder, we selectively targeted APP overexpression to skeletal muscle through use of the muscle creatine kinase gene promoter. We showed that the overexpression of APP led to the development of a histopathological and clinical features characteristic of human IBM, including motor deficits in aged-mice. Our findings were reported in PNAS. (Click here for the PDF).

Now that we have generated a mouse model that exhibits some of the major features of IBM, future research is aimed at addressing the following questions:

• Determine the molecular mechanisms by which APP mismetabolism in muscle triggers cellular degeneration.

• Determine whether it is the accumulation of APP per se or the failure to clear APP (or its proteolytic fragments) that underlie the muscle degeneration.

• Determine whether selective muscle types (e.g., fast versus slow twitch fibers) are preferentially affected in our mouse model and in the human condition.

• Test potential therapeutic targets, which is critical because there are no effective treatments for IBM.

• Study strategies that can augment or exacerbate the pathology, including the derivation of double transgenic mice.

TARGETED NEURONAL ABLATION
Lesioning experiments represent a common experimental paradigm in neurobiology, based on the premise that changes in behavior and/or physiological function can be elucidated after eliminating or inactivating a specific brain region. This is essentially the same premise that underlies the creation of gene knockout mice. Most brain lesioning experiments involve disruptive invasive procedures such as the physical removal or damage to particular brain regions or the use of pharmacological agents. My lab has been investigating whether transgenic mice can be derived to express cytotoxic genes in selective neuronal populations in an inducible fashion, thereby avoiding the untoward consequences of expression during developmental periods. We have had much success with this procedure and are able to selectively obliterate neuronal populations in particular brain regions without adversely affecting neighboring structures. We are utilizing these novel transgenic mice to study the effect of cell loss on behavior and synaptic physiology.